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Functional Foods: Probiotics and Prebiotics

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The main role of diet is to provide nutrients to meet host physiological requirements. As research behind diet and health has evolved, so has the concept of ‘functional foods’ become popular. Foods which are touted as being ‘functional’ are thought to exert certain positive properties over and above their normal nutritional value. While not universally popular and sometimes plagued by inadequate research/claims, the concept is certainly commercially successful, e.g. The Institute of Grocery Distributors (http://www.igd.com) estimates that the functional food market in the UK in 2007 will have annual sales worth around £1800m. This shows an exponential rise from the 1996 figure of £134m. Examples of functional foods include organic and inorganic micronutrients, vitamins, anti-oxidants, dietary fibre, some proteins (e.g. lactoferrin), certain bioactive peptides and polyunsaturated fatty acids.
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by kadam m. on October 22nd, 2010 at 10:13 am
today we all wants heathy diet which is given gy this
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V O L 2 8 N O 2 S E P T E M B E R 2 0 0 7
I S S N 0 9 6 5 - 0 9 8 9
Functional foods: probiotics and prebiotics
Glenn R Gibson
Food Microbial Sciences Unit, Department of Food Biosciences, The University of Reading, Reading, UK
Introduction
Functional Foods and the Gut
bacteroides), it is also the case that some
genera/species may offer health promoting
The main role of diet is to provide nutrients
Recent years have seen a major change in
attributes. For example, bifidobacteria and
to meet host physiological requirements. As
how activities of the human gastrointestinal tract
lactobacilli are thought to exert powerful anti-
research behind diet and health has evolved, so
are perceived. This has been driven by increased
pathogenic effects and are mainly responsible
has the concept of ‘functional foods’ become
knowledge of the gut microflora composition and
for ‘colonisation resistance’ in the gut. Moreover,
popular. Foods which are touted as being
activities. This has been helped by a shift away
the same genera have been attributed with other
‘functional’ are thought to exert certain positive
from traditional microbiological culture methods
beneficial aspects: such as protection from
properties over and above their normal
(Figure 1) to the use of molecular markers of
bowel tumours and metabolism of cholesterol
nutritional value. While not universally popular
culture identity. The colon is the most heavily
and other lipids in the gut1. Whilst many of the
and sometimes plagued by inadequate
populated region of the gastrointestinal tract
health promoting aspects have yet to be
research/claims, the concept is certainly
and, because of this resident microbiota, is one
definitively proven in humans, it would appear
commercially successful, e.g. The Institute of
of the most metabolically active organs in the
that there is value in eliciting a change away
Grocery Distributors (http://www.igd.com)
body. The concept of modulating activities
from a gut flora dominated by potentially
estimates that the functional food market in the
directed towards improving gut microbial
harmful bacteria towards a more benign, or
UK in 2007 will have annual sales worth around
function has a long history, as diet can have a
beneficial, composition.
£1800m. This shows an exponential rise from
major effect on the gut microflora activities1.
the 1996 figure of £134m. Examples of
Whilst some indigenous bacteria can be
Probiotics
functional foods include organic and inorganic
pathogenic (e.g. proteolytic clostridia and
micronutrients, vitamins, anti-oxidants, dietary
The most frequently used dietary method of
fibre, some proteins (e.g. lactoferrin), certain
influencing the gut flora composition is that of
bioactive peptides and polyunsaturated fatty
probiotics, whereby live microbial additions are
acids.
made to appropriate food vehicles, usually
The concept has now moved markedly
fermented milks2. The concept was expounded
towards gastrointestinal function, in particular
in a scientific note by Metchnikoff2. He
the impact of gut bacteria. Possibly this is driven
hypothesised that longevity in Bulgarian
by the ubiquity of gastrointestinal disorders but
peasants was associated with their elevated
also the fact that diet is an important controlling
intake of ‘soured milks’, i.e. dairy based drinks
factor with regard to indigenous microbiota
containing live bacteria. This was the basis of
activities. The gut microflora contains
what is now recognised as the probiotic concept.
pathogenic, benign and beneficial microbial
A recent definition of probiotics was given as ‘a
species. A predominance of the former can lead
live microbial feed supplement that is beneficial
towards gut upset which can be both acute (e.g.
to health’3.
gastroenteritis) and chronic (e.g. inflammatory
Over the years, many species of micro-
bowel disease). Functional foods directed
organisms have been used. They consist not
towards the gut microbiota would serve to
Figure 1. Studies on the complex microbial ecology of
only of lactic acid bacteria (e.g. lactobacilli,
the intestinal tract have moved away from cultural
influence the composition of activities towards
streptococci, enterococci, lactococci,
procedures towards molecular biological methods.
a more positive metabolism.
bifidobacteria) but also Bacillus spp., E. coli and
Courtesy of Dr Kieran Tuohy (University of Reading).

Vol 28 No 2
fungi/yeasts such as Saccharomyces spp. and
these carbohydrates have a specific colonic
microbiota (in some exceptional cases this may
Aspergillus spp. The most common probiotics
fermentation directed towards bifidobacteria1,5.
be nearer to 8g/d). A possible side effect of
belong to the genera Lactobacillus (e.g. L. casei,
Bifidobacteria are able to break down and utilise
prebiotic intake is intestinal discomfort from gas
L. acidophilus, L. rhamnosus, L. johnsonii,
fructo-oligosaccharides due to their possession
production. However, bifidobacteria and
L. reuteri ) and Bifidobacterium (e.g. B. bifidum,
of a ?-fructofuranosidase enzyme, providing a
lactobacilli cannot produce gas as part of their
B. longum, B. breve). To be effective, probiotics
competitive advantage in a mixed culture
metabolic process. Therefore, at a rational dose,
must be capable of being prepared in a viable
environment like the human gut11.
of up to 20g/d, gas distension should not occur.
manner and on a large scale (e.g. for industrial
Galacto-oligosaccharides (GOS) are another
If gas is being generated, then the carbohydrate
purposes), whilst during use and under storage
class of prebiotics that are manufactured and
is not acting as an authentic prebiotic. This is
the probiotic should remain viable and stable, be
marketed in Europe as well as Japan. These
perhaps because dosage is too high and the
able to survive in the intestinal ecosystem and
consist of a lactose core with one or more
prebiotic effect is being compromised i.e.
the host should gain beneficially from
galactosyl residues linked via ?1?3, ?1?4 and
bacteria other than the target organisms are
harbouring the probiotic. The strains used
?1?6 linkages12. They have found application in
becoming involved in the fermentation5.
should be generally regarded as safe.
infant formula foods.
Probiotics are marketed as functional foods,
Recent documents have suggested that FOS
Possible Health Benefits
whereby they are ingested for their purported
and GOS are accepted prebiotics that fulfil
positive advantages in the digestive tract and/or
current selection criteria13, 14.
Several different avenues are being explored
systemic areas like the liver, vagina or blood-
A prebiotic dose of 5 grams/day should be
for pre/probiotics. These are largely mediated by
stream. Consumers should be provided with an
sufficient to elicit a positive effect upon the gut
affecting an increase in beneficial bacteria within
independent assessment of physiological,
microbial and safety aspects of these live
microbial products – especially if they can
improve health. Probiotic trials should use the
best methodologies available. For probiotics to
exert beneficial properties, they must have a high
viability in the product and have robust survival
properties in the gut, which is their first point of
contact4. Moreover, they should not adversely
affect immune up-regulation, produce toxins,
disrupt colonocyte function or have the ability to
transfer antibiotic resistance to the normal gut
microflora. Food vehicles include live yoghurts,
fermented dairy drinks, freeze-dried supplements
(capsules, pills, liquid suspensions, sprays),
cheese, fromage frais and fruit juices. Both
single and multiple strain products are available.
Prebiotics
An alternative, or additional, approach is the
prebiotic concept. A prebiotic is ‘a non-digestible
food ingredient that beneficially affects the host
by selectively stimulating the growth and/or
activity of one or a limited number of bacteria in
the colon, that can improve the host health’5.
Thus, the prebiotic approach advocates the
administration of non-viable entities. Dietary
carbohydrates, such as fibres, are candidate
prebiotics, but most promise has been realised
with non-digestible oligosaccharides, because
of their selective metabolism. In particular, the
ingestion of fructo-oligosaccharides (FOS) has
been shown to stimulate bifidobacteria in the
lower gut. As prebiotics exploit non-viable food
ingredients, their applicability in diets is wide
Figure 2. Model system of the large intestine used for in vitro studies on probiotic and prebiotic functionality.
ranging. A further approach is synbiotics, where
Courtesy of Dr Kieran Tuohy (University of Reading).
The system consists of 3 vessels of increasing size, aligned in series such that a sequential feeding of growth medium
probiotics and prebiotics are combined5.
occurs. The vessels are pH regulated to reflect in vivo differences. Thus, vessel 1 has a high availability of substrate,
The prebiotic activity of fructose-containing
bacteria grow quickly and is operated at an acidic pH, similar to events in the proximal colon. In contrast, the final
vessel resembles the neutral pH, slow bacterial rate and low substrate availability which is characteristic of more distal
oligosaccharides has been confirmed in both
regions. After inoculation with faeces, an equilibration period is allowed such that the bacterial profiles respond to
laboratory and human trials6-10. This is because
their imposed conditions. Then, candidate pro/prebiotics are added and the fermentation profiles monitored.
2

Vol 28 No 2
the gut flora. At Reading, a model system of the
the liver. However, this has not been
scientific principles and research that provide
human colon is in operation (Figure 2) whereby
unequivocally proven, and there are
reliable information on efficacy – effect as well
probiotic and prebiotic efficacy can be
contrasting data from human volunteer trials.
as mechanisms involved.
researched before moving onto human studies.
G Vitamin synthesis: bifidobacteria can
The health evidence is variable with the
synthesise various vitamins, largely of the B
following being examples:
group. The physiological value of this in the
lower bowel is questionable, however.
References
G Improved tolerance to lactose: it is thought
G Irritable bowel syndrome: IBS is a major
that probiotics may help in this regard,
drain on general practitioners’ time, and
1. Gibson, G.R., and Roberfroid, M.B. (eds.) (1999)
through their ?-galactosidase activity.
some evidence has implicated a
Colonic Microbiota, Nutrition and Health. Kluwer
G Protection from gastroenteritis: the most
‘dysfunctional’ gut flora. This may be
Academic Publishers, Dodrecht.
compelling evidence for the success of
addressed through pro/prebiotics. Evidence
2. Fuller, R. (1989) Probiotics in man and animals.
Journal of Applied Bacteriology 66, 365–378.
probiotics and prebiotics probably lies in
is very contradictory, however.
3. Salminen, S., Bouley, C., Boutron-Ruault, M-C., et
their ability to improve resistance to
G Improved digestion and gut function: an
al. (1998) Functional food science and
pathogens. Lactic acid excreting
active gut flora helps to adequately digest the
gastrointestinal physiology and function. British
microrganisms are known for their inhibitory
food that enters the adult colon each day.
Journal of Nutrition 80, S147–S171.
properties. There are a number of potential
4. Collins, M.D. and Gibson, G.R. (1999) Probiotics,
G Food allergy: it has been suggested that gut
prebiotics and synbiotics: Dietary approaches for the
mechanisms for probiotic micro-organisms
flora modulation may down-regulate gut
modulation of microbial ecology. American Journal
to reduce intestinal infections1. Firstly,
inflammation and hypersensitivity that would
of Clinical Nutrition 69, 1052–1057.
metabolic end products such as acids
otherwise lead to atopic eczema.
5. Gibson, G.R. and Roberfroid, M.B. (1995) Dietary
excreted by these micro-organisms may
G Immune regulation: a stimulation of the non-
modulation of the human colonic microbiota:
lower the gut pH to levels below those at
specific immune response through non-
introducing the concept of prebiotics. Journal of
Nutrition
125, 1401–1412.
which pathogens are able to effectively
pathogenic means may help improve
6. McCartney, A.L. and Gibson, G.R. (1998) The
compete. Also, many lactobacilli and
resistance to infection.
application of prebiotics in human health and
bifidobacteria are able to excrete natural
G Mineral bioavailability: a reduced pH in the
nutrition, In: Proceeding Lactic 97. Which Strains?
antibiotics which can have a broad spectrum
bowel because of a lactic fermentation is
For Which Products? Adria Normandie, pp. 59-73.
of activity. Moreover, there is competition for
thought to better sequester calcium and
7. Wang, X. and Gibson, G.R. (1993) Effects of the in
vitro fermentation of oligofructose and inulin by
nutrients and colonisation sites15. This
perhaps magnesium.
bacteria growing in the human large intestine.
inhibitory effect also has relevance for more
Journal of Applied Bacteriology 75, 373–380.
chronic diseases thought to have an
Conclusions
8. Williams, C., Witherly, S.A. and Buddington, R.K.
involvement of pathogens.
(1994) Influence of dietary neosugar on selected
bacterial groups of the human faecal microbiota.
G Reduced toxins: stimulating a more beneficial
The incidence of acute and chronic gut
Microbial Ecology in Health and Disease 7, 91–97.
community should reduce toxin levels,
disorders continues to rise, with many diseases
9. Kleessen, B., Sykura, B., Zunft, H-J. and Blaut, M.
perhaps including carcinogens, some of
being untreatable. The functional food industry’s
(1997) Effects of inulin and lactose on fecal
which act systemically as well as locally. In
perception of the importance of gut microbiology
microflora, microbial activity and bowel habit in
humans, colorectal cancer is thought to have
in human health and nutrition has led to a major
elderly constipated persons. American Journal of
a bacterial origin, with around 10 different
increase in probiotic and prebiotic-based
Clinical Nutrition 65, 1397–1402.
10. Gibson, G.R., Beatty, E.R., Wang, X. and
carcinogens described that have been
products. Not all products will be reliable in
Cummings, J.H. (1995) Selective stimulation of
attributed to microbial events16. Dietary
terms of their efficacy, however, and it is
bifidobacteria in the human colon by oligofructose
strategies that lead to a reduced
important that these are not allowed to skew an
and inulin. Gastroenterology 108, 975–982.
accumulation of such products may be
important area of human health and the
11. Imamura, L., Hisamitsu, K. and Kobashi, K. (1994)
possible. Dietary fibres and resistant starches
functional food concept generally. Moreover,
Purification and characterization of
?-fructofuranosidase from Bifidobacterium infantis.
may be fermented in the large gut to increase
claims on particular products cannot be
Biological and Pharmacological Bulletin 17, 596–602.
faecal bulk and reduce the residence time of
extrapolated to others, e.g. if one probiotic strain
12. Playne, M.J. and Crittenden, R. (1996)
such materials in the gut. Moreover,
elicits a particular positive effect, it cannot be
Commercially available oligosaccharides. Bulletin
probiotics and prebiotics may modify the
assumed that this is applicable to others (even
International Dairy Foundation 313, 10–22.
activities of enzymes that are involved in
of the same species). A further issue is public
13. Gibson, G.R., Probert, H.M., van Loo, J.A.E., et al.
(2004) Dietary modulation of the human colonic
carcinogenesis.
acceptance, with dietary response to change
microbiota: Updating the concept of prebiotics.
G Cholesterol reduction: the lipid hypothesis
being weak – it is estimated that only about 8%
Nutrition Research Reviews 17, 259–275.
purports that dietary saturated fatty acids
of UK citizens consume at least 5 pieces of
14. Gibson, G.R. and Rastall, R.A. (eds.) (2006)
lead to an increase in blood cholesterol
fruit/vegetables per day, and this is a well
Prebiotics: Development and Application. John
levels. This may have the effect of depositing
understood health message.
Wiley & Sons Ltd., Chichester.
15. Mackey, B.M. and Gibson, G.R. (1997) Escherichia
cholesterol in the arterial wall, leading to
Next, legislation is loose and open to abuse
coli 0157 – from farm to fork and beyond. Society
atherosclerosis and possibly coronary heart
from manufacturers launching untested
for General Microbiology Quarterly 24, 55–57.
disease. Some studies have hypothesised a
products. This will be tightened up in time and is
16. Reddy, B.S. (1998) Prevention of colon cancer by
role for the lactic microflora in systemically
needed. However, if food law and claim hurdles
pre- and probiotics: evidence from laboratory
reducing blood lipid values17. It has been
are set too high, a degree of reluctance among
studies. British Journal of Nutrition 80, S219–S223.
17. Gilliland, S.E., Nelson, C.R. and Maxwell, C.
suggested that some probiotics can degrade
manufacturers with good products may ensue.
(1985) Assimilation of cholesterol by Lactobacillus
cholesterol in the gut as well as produce
For the full value to be realised, it is imperative
acidophilus. Applied and Environmental Microbiology
metabolites that interfere with its synthesis in
that developments are based upon sound
49, 377–381.
3


Vol 28 No 2
Chromogenic media: bacteriology in colour
Alistair Brown
Senior Research Scientist, Oxoid, UK
Introduction
Gradually, as more became known about the
and extent of confirmatory testing required
biochemical distinctions between different
compared to the original Nutrient Agar, their
Laboratory managers are often faced with
genera and species (i.e. their ability to degrade
overall specificity remains comparatively limited.
difficult decisions in terms of allocation of
certain substances or produce specific
Despite their limitations, many of these types of
resources, not only with regard to staffing and
biochemical substances as end-points of
media remain useful microbiological tools and
prioritisation of work tasks, but also procurement
metabolism), so culture media evolved to
are still extensively used today.
of the most effective tools for the job within tight
incorporate additional components, such as
Given the widespread use of fluorogenic and
budgetary constraints. These decisions can have
specific carbohydrate sources and a suitable pH
chromogenic substrates in biochemistry, it is
a significant effect on the efficiency of sample
indicator, to aid differential identification.
somewhat surprising that their application to
processing and turnaround times to the reporting
Inhibitory agents (e.g. bile salts, certain dyes
microbiology did not really take off until the
of results. By its very nature, microbiological
and other compounds) also became commonly
1980s. The catalyst for research in this field was
bench work is often very labour intensive, time
used to reduce or eliminate growth of unwanted
the desire of water microbiologists to develop a
consuming and requires skill and experience.
organisms. Thus, media could be designed to be
Whilst new, rapid methods of sample analysis,
selective as well as differential. A classic
such as real-time PCR and other automated
example of this is MacConkey Agar (Figure 1),
molecular techniques, inevitably generate an air
which contains lactose and neutral red for the
of excitement amongst scientists and represent
differentiation of lactose fermenting organisms,
important advances in scientific technology, the
along with bile salts for the inhibition of bile-
importance and relevance of advances in culture
sensitive species. These properties make it
methods in routine microbiology must not be
useful in the identification of intestinal pathogens
forgotten or ignored. The introduction of new
such as salmonellas (of importance in clinical,
culture media is a crucial factor that can have a
food and water microbiology), which are
significant impact on cost-effective, accurate and
generally non-lactose fermenting (NLF), as well
timely results in food, water, clinical and
as commensals such as Escherichia coli that are
industrial laboratories worldwide.
able to ferment lactose (a key indicator of faecal
contamination, also of importance in food, water
Culture media
and industrial microbiology).
In many cases, the specificity of conventional
For over a century, since Robert Koch’s early
selective media has been improved by the
Figure 1. MacConkey agar inoculated with
Escherichia coli (ATCC 25922) and Salmonella
work with “the mixture of nutrient liquid and
addition of antibiotic supplements to inhibit
enterica subsp. enterica serovar Enteritidis (ATCC
gelatin”, solid culture media have been used for
unwanted organisms. One example of this is
13076). Lactose fermenters (including E. coli and
other coliforms) appear as pink to red colonies, while
the cultivation of an ever-expanding array of
mannitol salt agar with oxacillin for the detection
non-lactose fermenters (NLFs) (including Salmonellas
micro-organisms. It was the work of Walther
and isolation of meticillin-resistant
and Shigellas) produce colourless or straw colonies
Hesse (1846-1911) and his wife Fanny (née
Staphylococcus aureus (MRSA) (Figure 2). Many
with orange to yellow halos.
Eilshemius) (1846-1934) that established the
variants of S. aureus are halophilic and able to
use of agar as a superior gelling agent for solid
ferment mannitol. Only those resistant to
media1. The main problem with such general
oxacillin (the first widely-used surrogate marker
purpose nutrient media was the inability to
for meticillin resistance) are able to survive the
distinguish between pathogenic and non-
presence of this antibiotic. Such organisms
pathogenic organisms by morphological
appear as yellow colonies with yellow haloes on
characteristics alone. At a time when many new
this medium. However, owing to other, less
strains were being discovered, microbial
clinically significant staphylococci (e.g. S.
identification and taxonomy were in their relative
haemolyticus) that also possess these qualities,
infancy and the need to establish biochemical
a significant number of false positives are
profiles of bacterial species (and thus provide a
encountered. This has a significant effect on
means of differentiation) became apparent.
increasing the volume of confirmatory testing
However, this involved extensive further testing
required. Also, there are emerging variants of
of individual colonies of bacterial growth and was
MRSA that are unable to ferment mannitol,
highly labour intensive. Furthermore, the work
yielding false negative results.
Figure 2. Mannitol salt agar with oxacillin after 48
hours incubation at 37°C following inoculation with
was highly skilled and experience was in the
Although conventional selective, differential
meticillin-resistant Staphylococcus aureus (MRSA)
hands of a few.
media have effected a reduction in the volume
(ATCC 43300).
4


Vol 28 No 2
rapid screening method for the faecal indicator
Table 1. Examples of commercially available hydrolase substrates (modified from Bovill and Druggan,
E. coli 2. Since the work of Feng and Hartman3, who
2005).
pioneered the use of 4-methylumbelliferone-?,
D-glucuronide for the detection of E. coli in
Enzyme
Chromogenic substrate
water and food samples, an explosion of
Aminopeptidase
A wide variety of substrates are available, containing single amino acids
through to longer peptide lengths.
research and development in the field of
Esterase (carboxilic)
A range of substrates containing various fatty acid chain lengths: C2, C4, C6,
chromogenic culture media has ensued.
C9, C10, C12, C14, C15, C16 and C18.
Esterase (inorganic)
Phosphate, phosphodiester, venom phosphodiester, sulphate.
Chromogenic substrates
Glycosidase
?-L-arabinoside, ?-D-cellobioside, ?- and ?-L-fucoside, ?-D-fucoside,
?- and ?-galactosaminide, ?- and ?-D-galactoside, ?- and
A chromogenic substrate may be defined as
?-D-glucosaminide, ?- and ?-D-glucoside, ?-D-gluconoride, ?-lactoside,
?- and ?-D-maltoside, ?- and ?-mannoside, ?-L-rhamnoside, ?-xyloside.
“a compound or substance that contains a
Others
Substrates for lysozyme and phosphoinositol phospholipase C.
colour-forming group”4. Commercially
synthesised chromogenic substrates (or
chromogens, for short) are available for the
detection of many hydrolase enzymes, including
glycosidases, peptidases, phosphatases and
esterases (Table 1). This group of enzymes
includes many gene products specific to certain
genera (or in some cases species) of bacteria
and their detection can often be an invaluable aid
to differentiation and identification. This can
often significantly reduce the amount of work
Figure 3. Formation of indigo blue by enzymic hydrolysis of indoxyl acetate in the presence of oxygen.
required to confirm the identity and significance
of the suspect colony.
Glycosidases exhibit specificity not only for
the sugar type, but also for its steric conformation
(D- or L-) and the conformation of the glycosidic
bond (?- or ?-)2. For example, ?-D-glucoside
chromogens are specific for detecting ?-D-
glucosidase activity and their usefulness in
bacterial differentiation is well documented5-7.
Indoxyl chromophores
Detection of specific hydrolase activity can
be achieved by attachment of a chromophore
(the “colour-forming group”) to the target
substrate, such that hydrolysis of the substrate
yields a specific colour, dependant on the type of
chromophore used. Various types are available,
but the most commonly used in solid culture
media are derivatives of indoxyl. In its simplest
form, indoxyl is a colourless, water soluble
compound that rapidly oxidises in air to form
indigo blue, a coloured, insoluble, dimeric
compound (Figure 3).
In practice, indoxyl generally gives a weak
colour when used in culture media, but the
indoxyl ring may be modified by the addition of
one or more halogens at certain locations on the
ring. This results in changes in absorbance in
the visible spectrum and consequently yields
different coloured end-products. Examples of
commonly used indoxyl-based chromophores
and their respective colours are shown in Figure 4.
Furthermore, subtle changes to these colours
and their intensities can be achieved by the
Figure 4. Examples of indoxyl chromophores, showing chemical structures and resultant colour formation. The
addition of halogens at specific positions on the indoxyl ring affects the resultant colour formation in colonies of
inclusion of other components in the medium
bacterial growth possessing the hydrolase enzyme necessary to release the chromophore from the specific
(e.g. cations, peptones, inducers, etc.).
chromogenic substrate.
5

Vol 28 No 2
This technology has also been applied to the
differentiation of Candida spp. by the use of
indoxyl chromogens to detect the presence of
hexosaminidase and alkaline phosphatase. This
is illustrated in Figure 7.
The solubility of the initial substrates and the
insolubility of the end-products are
characteristics that make the indoxyl group of
chromophores particularly suitable for use in
Figure 5. Venn diagram of colour reactions.
solid culture media. This is because colouration
is restricted to the cellular mass, enabling
colonies of a species possessing the relevant
hydrolase to be easily recognised in a mixed
culture. A drawback of the indoxyl chromogens
is their reliance on oxidation, making them
unsuitable for detection of anaerobic bacteria.
Alternative chromogens have been described
that overcome this problem, notably the metal
chelators (e.g. esculin, 8-hydroxyquinoline,
dihydroxyflavones and alizarin)2,8.
Other chromophores
Other chromophores, such as nitrophenol
and nitroaniline, are available in a variety of
substrate forms. Indeed, ortho-nitrophenol-?-D-
Figure 6. Differentiation of organisms commonly isolated from urine samples on a chromogenic medium by their
galactoside (ONPG) is still widely used in
ability to produce either, both or neither ?-galactosidase or ?-glucosidase.
biochemical differentiation of bacteria. However,
nitrophenol and nitroaniline substrates suffer
two drawbacks: they have a low extinction
coefficient, often resulting in poor sensitivity due
to insufficient colour production; and the end-
product is highly soluble, rendering them better
suited to liquid (broth) assays than to solid
media. Nitroaniline (and other amine-containing)
substrates are particularly suitable for the detection
of amino-peptidases when linked to a peptide.
However, these require the addition of a developer
(usually dimethylaminocinnamaldehyde) to illicit
the observed colour reaction (formation of the
Schiff base) and this makes them impractical for
use in solid culture media.
Benefits of chromogenic media
Figure 7. Enzyme-dependant colour production and consequent differentiation of clinically significant Candida
spp. on a chromogenic medium for the detection of hexosaminidase and alkaline phosphatase activity.
A wide range of chromogenic media are
Chromogenic Culture Media
to form green colonies (e.g. Enterococcus spp.).
commercially available for the detection of many
Expression of both enzymes results in dark,
organisms of significance in food, water, clinical
Incorporation of more than one chromogen
blue-purple colonies and is indicative of
and industrial microbiology (e.g. Listeria,
in a medium can improve both its specificity and
Klebsiella, Enterobacter or Serratia spp. (KES
Salmonella, Bacillus cereus, clostridia, Candida,
differential properties. A medium containing both
group). Staphylococci (with the main exception
enterococci, staphylococci, E. coli and
5-bromo-4-chloro-3-indoxyl ?-D-
of S. saprophyticus) and streptococci do not
coliforms). The main benefits of these over
glucopyranoside (X-Gluc) and 6-chloro-3-indoxyl
produce either enzyme and grow as white or
conventional media are their improved sensitivity
?-D-galactopyranoside (red-Gal) is useful for the
colourless colonies. Differentiation of these
and specificity. In some cases improved
differentiation of potential urinary tract
genera is rapidly established by a catalase test
sensitivity may lead to a reduction in incubation
pathogens, as illustrated in Figures 5 and 6.
(staphylococci are positive for this enzyme,
time (e.g. chromogenic agars for MRSA
Organisms that express ?-galactosidase
streptococci negative). Proteus spp. may be
detection), allowing a faster turnaround time to
cleave the red-Gal substrate to produce pink/red
differentiated by inclusion of tryptophan, forming
reporting of results. These properties also make
colonies (e.g. E. coli), while expression of
tan coloured colonies due to the tryptophan
them ideally suited as high-volume screening
?-glucosidase results in cleavage of the X-Gluc
deaminase (TDA) reaction.
media owing to the resultant reduction in
6

Vol 28 No 2
confirmatory testing required. From a laboratory
certain circumstances, but can be a major
References
manager’s perspective, the key benefits of
disadvantage, especially in many scenarios, where
chromogenic media can be summarised, as
further phenotypical characterisation is required
1. Bridson, E.Y. The development, manufacture and
control of microbiological culture media. Unipath
follows:
(e.g. antimicrobial sensitivity patterns). The main
Ltd. 1994.
advantage of molecular systems is a faster time to
2. Bovill, R., Druggan, P. The use of chromogenic
G Ease of use and interpretation:
result. The impact of this in real terms can be
enzyme substrates in microbial identification.
– minimal training required;
measured only by the efficiency of the reporting
Culture 2005; 26(2): 5–8.
– allow for more appropriate use of
system itself and in certain cases there may be no
3. Feng, P.C., Hartman, P.A. Fluorogenic assays for
immediate confirmation of Escherichia coli. 1982;
experienced staff;
time benefit at all.
Appl. Environ. Microbiol. 43: 1320–1329.
G Improved performance:
Overall, chromogenic media represent a cost
4. Azaid, A., Hughes, H.G., Porceddu, E., Nicholas, F.
– greater confidence in results compared to
effective way of achieving improved sensitivity
Glossary of biotechnology and genetic engineering.
conventional media;
and specificity of results without the expense of
1999. Series title: FAO Research and Technology
– faster results;
automated molecular techniques. Further
Papers – 7. ISBN: 9251043698. Available from:
http://www.fao.org/docrep/003/X3910E/X3910E00.
– reduced volume of follow-up work;
applications of chromogenic technology are
HTM, cited 27th April 2007.
G Cost effectiveness:
being found all the time and are limited only by
5. Bascomb, S. Enzyme tests in bacterial identification.
– reduced confirmatory testing outweighs
the quest to find more differentially useful
Methods Microbiol. 1987; 19: 105–160.
extra cost of media;
substrates.
6. Kaufhold, A., Lütticken, R., Schwien, U. Few-
– significantly cheaper than PCR and other
minutes test for the identification of group A
streptococci and enterococci with chromogenic
automated molecular methods.
substrates. 1989. Zentralbl. Bakteriol. 272: 191–195.
Acknowledgements
7. Manafi, M., Kneifel, W., Bascomb, S. Fluorogenic
Although molecular techniques are rapidly
and chromogenic substrates used in bacterial
gaining recognition and credibility for certain
This article is adapted from one previously
diagnostics. Microbiol. Reviews 1991; Sept:335–348.
applications, owing to their expense they are
published by the author in The Biomedical Scientist.
8. Perry, J., Morris, K., James, A., Oliver, M., Gould, F.
Evaluation of novel chromogenic substrates for the
generally only cost-effective for a very large
I would especially like to thank Marie Blackman,
detection of bacterial b-glucosidase. Journal Appl.
throughput of samples or where detection is not
Bryan DeCaux, Stephen Dimmer, Fiona Macrae,
Microbiol. 2007; 102: 410–415.
achievable by conventional means. These
Brian Nation, Frances Presland, Jane Ramsay,
techniques do not allow subsequent culturing of
Peter Stephens and Jane Williams for their
the organism. This is not necessarily a problem in
assistance and support in preparing this article.
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Vol 28 No 2
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