Intestinal health

Development and documentation of products for intestinal health
All ingested compounds
are exposed
to host digestion
and intestinal
bacteria
Drugs
Products for
intestinal health
• The major site of food
digestion by the host.
• Good probiotics retain
their viability.
• Drugs retain their
activity.
• Prebiotics and dietary
fibres undigested.
• Absorption of nutrient
and most drugs by
the host.
• Host has mechanisms
to slow down bacterial
growth in nutrient rich
digesta, but towards
distal part bacterial
growth accelerates.
• Site of action for
lactobacilli probiotics
and other products
preventing E. coli
and Salmonella
mediated diarrhoeas.
Some live fed bacteria lose
their viability and capability
to colonise the target
intestinal section.
Stomach
Active pharmaceuticals
may be modified bacteria,
potentially making them
inactive or even harmful.
Small
intestine
Colon
• Bacterial fermentation
of dietary compounds
unavailable for
the host.
• Dietary prebiotics
and fibres serve as
growth substrates for
beneficial saccharolytic
bacteria, thus
preventing putrefaction.
• Site of action for
bifidobacteria
probiotics.
Some oligosaccharides may
become digested too rapidly
in proximal colon leading to
overproduction of lactic acid
which causes discomfort.
Gastrointestinal tract of man is populated by
a bacterial population whose metabolic diversity
vastly exceeds that of the host itself. Accordingly,
all the dietary compounds within the reach of
the host can be metabolized also by the intestinal
microflora.
Bypass / faeces
Bacterial order Clostridiales dominates
human intestinal
microflora
It has been known for long that human intestine harbours a complex
microflora. In the past bacterial examination used culturing in the
laboratory, whereas DNA based methods are widely used today.
All methods have their inherent limitations. Culture based methods fail
to capture many fastidious bacteria, whereas modern molecular methods
alone provide little information on the activities of the bacteria.
We have carried out bacterial surveys based on 16S rDNA sequencing
to reveal the entire diversity of the human intestinal microbiota.
Approximately half of bacteria in the human colon belong to the order
Clostridiales. It should be noted that pathogenic clostridial species are
few and restricted only to the genus Clostridium. In fact, it appears
that most representatives of the Clostridial clusters are beneficial
for intestinal health providing nutrients for the intestinal epithelium, and,
act as a barrier against intestinal pathogens. Besides Clostridial clusters,
human colon harbours two abundant bacterial genera, Bacteroides
and Bifidobacterium. Bifidobacteria are saccharolytic and, therefore,
supposedly better for the intestinal health than Bacteroides species,
many of which ferment proteins leading to harmful end products.
Today there are many products targeted
to improve intestinal health. Probiotics,
live fed bacteria, are intended to balance
intestinal microflora either in the small
intestine or colon. Prebiotics are
carbohydrates that escape host digestion
and support the growth of beneficial
bacteria, mainly in the colon.
Antimicrobial medicines suppress
the growth of sensitive bacteria and
enrich those resistant to the drug
prescribed. This changes the structure
of the microbial community, and, thus,
the interplay between the man and
the resident microflora.
domain: Bacteria
phylum: Firmicutes
class: Clostridia
order: Thermoanaerobacteriales
order: Haloanaerobiales
order: Clostridiales
family: Lachnospiraceae
family: Peptostreptococcaceae
family: Eubacteriaceae
family: Peptococcaceae
family: Heliobacteriaceae
family: Acidaminoc occaceae
family: Syntrophomonadaceae
family: Clostridiaceae
genus: Acetivibrio
genus: Acidaminobacter
Clostridial
genus: Alkaliphilus
clusters I to XIX
genus: Anaerobacter
genus: Caloramator
genus: Caloranaerobacter
genus: Caminicella
genus: Dorea
genus: Natronincola
genus: Sarcina
genus: Sporobacter
genus: Tepidibacter
genus: Thermobrachium
genus: Tindallia
genus: Clostridium
Our methods detect diet and drug
mediated shifts in
the intestinal bacterial
composition
Under normal circumstances intestinal microflora of a human subject
is stable and changes little from day-to-day and month-to-month.
In contrast, each human subject has a characteristic intestinal
microflora, almost like a fingerprint. The bacterial profile reflects
personal eating habits, the structure and rate of secretion of intestinal
mucus, characteristics of the immunological defence, and possibly
also genetic background of the subject.
The balance of the intestinal microflora becomes most severely
disturbed if there are major changes in the dietary habits or if one
is treated with antibacterial drugs such as antibiotics. Colon
microflora is metabolically extremely active and, therefore, significant
changes in its composition and metabolism is likely to reflect to
the wellbeing of the host.
The accuracy of different methods to quantify changes in the total
bacterial community vary. If, for example, a method fails to detect
a major Clostridial cluster but captures everything else, the overall
outcome will be strongly biased. At Alimetrics we start a bacterial
survey by using methods that are totally unselective, thus capturing
all bacteria present. From there we can zoom in and, if we want,
specifically identify or type every single bacterial species.
Antibiotic treatment changes the composition intestinal microflora
Bacterial group
Clostridial cluster IV
Clostridial cluster XIVa
Clostridial cluster XVIII
Eubacterium
Bacteroides
Bifidobacterium
Other groups
Baseline
During amoxicillin
Change
7%
37%
7%
1%
19%
3%
26%
8%
15%
6%
0%
54%
0%
17%
+1
–22
–1
–1
+35
–3
–9
Relative abundance
Independent Research Partner in
Microbial Management
20
30
40
50
60
70
60
70
60
70
Percent guanine + cytosine
colon microflora
in human subjects
We used %G+C profiling to characterize
microbial community in the gastrointestinal
tract of three people that changed to the
Atkins diet. Blue lines indicate microbial
Relative abundance
Atkins diet alters
community structure just before the diet
change and the red ones two weeks later.
20
30
40
50
Relative abundance
Percent guanine + cytosine
20
30
40
50
Percent guanine + cytosine
We measure bacteria and
bacterial
metabolites
that are
true
indicators
of human
health
The use of molecular techniques has enabled us
to detect the species richness and shifts caused
by interventions in intestinal microbial communities. We constantly learn about the activities of
individual bacteria and entire communities. It is
obvious that bacterial functions in the human
digestive tract affect the health of intestine itself.
However, growing evidence suggests that a wide
range of bacterial metabolites and modified drugs
are absorbed, and hence, mediate health effects
to the entire human body. The range of microbial
health indicators grows parallel to the advancement of science of intestinal microbiology.
Our scientists are actively involved in the discovery process and
include established health indicators into our research service
portfolio. We help our clients to discover new health products
which beneficially affect the recognised health indicators, and,
to benchmark their present products against the competing ones.
Butyric acid
Many gastrointestinal bacteria,
e.g. Clostridial clusters IV and XIVa,
produce butyric acid. Butyric acid is highly
beneficial as it is a preferred energy source
for intestinal epithelial cells and inhibits
growth of aberrant cells thus likely
suppressing the development of bowel
cancer.
Sulphate reducing bacteria
A taxonomically heterogeneous but
metabolically homogenous group of
strictly anaerobic bacteria use sulphate in
their anaerobic respiration. This leads to
formation of hydrogen sulphide which
is highly toxic to all eukaryotic cells.
Putrefaction products
Colon bacteria preferably metabolise
carbohydrates. Once the carbohydrates
are depleted protein fermentation starts.
Threfore, the presence of slowly degraded
dietary carbohydrates such as prebiotics
and dietary fibres supporting saccharolytic
metabolism inhibits putrefaction.
Protein fermentation produces toxic and
potentially carcinogenic compounds such
as indole, skatole, cresol and ammonia.
Probiotic bacteria
Species of lactic acid producing bacteria,
Lactobacillus and Bifidobacterium have
gained a good reputation of being
beneficial for intestinal health, and
therefore, they are essential elements
in many probiotic products. However,
according to our experience, many of
the characteristics are not species,
but strain specific.
Pathogens
Recognised pathogen is a bacterium with
proven adverse effects on the health of
the host. Pathogens produce toxins that
damage intestinal epithelium of the host,
utilise substrates leaking from degrading
tissues, and, actually penetrate the tissues.
Colonisation of pathogenic strains of
Escherichia coli and Salmonella enterica
is aided by their adherence on intestinal
epithelium, whereas Campylobacter
jejuni is able to move upstream with
a strong flagellum.
Laboratory simulations provide
good
statistical
power and
possibility
to test
multiple
compounds
Targeted laboratory simulations provide
a rational way to start developing
second generation health products.
With Alimetrics it is possible to start
screening from tens or hundreds of
different bacterial strains, pharmaceuticals, product preforms or process
variants. Exact selection criteria
chosen depend on the product type
and the intended physiological target.
Test panels and simulations are
always adjusted to reveal the product
characteristics agreed in the
confidential discussions with the
client. All simulations are designed
to accurately mimic physicochemical
and microbial conditions prevailing in
the target site. Typical outcome of
the simulation project is a ranking of
the tested products according to their
potential to access the target site in
active form and to produce the desired
physiological effect.
Authentic simulation material
Simulation of pathogen attachment
Intestinal tract with food digest is a complex habitat
Intestinal mucus plays an important role in bacterial
for the resident microflora. The range of carbon
colonisation. Intestinal epithelium is constantly
sources, nutrients and cofactors available for
covered with mucus produced by goblet cells.
bacterial growth is vast and beyond our knowledge.
This serves as a barrier against intestinal pathogens,
We believe that it is not possible to mimic this
but attachment to mucus is most likely also the step
nutrient base by providing intestinal bacteria with
preceding the penetration of intestinal pathogens
a mixture of defined man-made chemicals.
into epithelial cells. We simulate the adherence
Therefore, we mainly use authentic intestinal
of intestinal pathogens on mucus. For this purpose
extracts as substrates to simulate intestinal
we have a selection of mucus samples from various
fermentations. The most common source of extract
animals and man. The attachment simulation is
is swine fed with the relevant human diet.
a powerful tool for studying products aimed to
inhibit the initial step of pathogenesis.
We use fresh, authentic and complete microbial
community as an inoculum in our simulations
because missing bacteria and bacterium-tobacterium interactions would significantly affect
the outcome of the fermentation processes.
Statistical considerations
It is important to use sufficient replication in every
Physicochemical aspects
phase of the simulation to achieve sufficient
Physicochemical conditions have a major impact on
statistical power. Most commonly we aim at
bacterial growth and metabolism. Therefore, we
experimental arrangement that is able to
carefully mimic authentic conditions prevailing in
demostrate 5% effects in each parameter for
the target site to make sure that the resident bacteria
the tested products at 95% confidence level.
meet the same growth factors and constraints that
However, the detection limit is always adjusted
they would meet in the authentic environment.
to meet the demands of our clients.
Simulation is a practical and easy-to-replicate test arrangement that can be used to rank
multiple product candidates by using a set of relevant quantifiable parameters.
Why to test products in simulators?
•
In practice it is not possible to test many products in clinical trials
•
Due to the large subject-to-subject variation, testing in vivo
requires a very high number of study subjects for acceptable
detection limit
•
In vitro simulations have a low variation, and therefore,
small effects of products can be reliably detected
•
Validated simulations are more likely to reveal the true mode of
action of products than clinical studies
•
Screening in simulators is rapid
•
Less animal and human studies are required, which offers ethical
and financial benefits
•
The outcome is a rationally developed and well-documented
product which has a good possibility to succeed
Intestinal responses can be verified
and
documented
in clinical trials
Simulation systems provide a high throughput
screening tool for ranking of product
candidates. However, even the best in vitro
simulations are always compromises
mimicking accurately only selected aspects
of the true system. Therefore, the most
promising candidates from the screening
process should be tested in live animals
and finally in clinical studies using human
subjects. Careful trial design with proper
power calculations, accurate analytical
capabilities and adequate data analysis are
crucial for successful product documentation
in clinical trials.
We arrange small to medium size clinical trials.
For the clinical studies arranged by us or by
the client we provide the analysis of faecal
microbial communities, any single bacterial
group or species, and microbial metabolites.
Furthermore, we offer all analyses common
in clinical practice.
Shifting microbial fermentation from putrefactive
to saccharolytic The results of the colon
simulation study are visualized in principal
component analysis where points that cluster
together are positively correlated while the
points that locate on opposite sides of the
origin (O) are negatively correlated. The results
reveal that both xylan and polydextrose (PDX)
significantly shift microbial fermentation towards
saccharolytic metabolism thus reducing
the production of harmful protein fermentation
products. This can be concluded from
the measured drop in pH and reduction of
branched chain fatty acids and skatole.
Alimetrics can manage complete clinical trial packages:
•
Optimising the trial design to fulfil the needs of the client
•
Arranging ethical committee approval
•
Recruiting subjects and running the study
•
Analysing more than one hundred clinically relevant
parameters from faeces, serum and urine
•
Carrying out the data management, statistical analyses
and mathematical modelling
•
Providing the customer with conclusive report and
marketing material
Alimetrics Ltd
Alimetrics is an independent contract research company that provides tailored
project-type services for the life sciences, including food, feed and healthcare
industries. Alimetrics designs, executes and reports small projects and
comprehensive research programmes sponsored by the client. Along with
simple analyses, Alimetrics provides leading edge technologies in the area
of microbiology, biological simulations and biomathematics. We are proud
of our short delivery times, punctuality, high scientific standards, good
reproducibility, and the value we have created for our multinational clients.
Alimetrics research technology packages
for several product groups
for different target hosts
Host specific technology packages are
composed of many advanced methods
Bacterial attachment
HUMANOMETRICS
Probiotics
Batch simulations
Mycotoxin binding
VFA analysis
RUMINOMETRICS
16S rDNA based surveys
Statistical designs
Enzymes
Microbial community analysis
AVIAMETRICS
Prebiotics
Data analysis
Identification of bacteria
SWINOMETRICS
Dynamic simulations
Bacterial enumeration
Pharma products
Dry matter digestibility
AQUAMETRICS
Design of specific Q-PCR assays
Putrefaction analysis
Specific Q-PCR analyses
Contact information
Alimetrics Ltd
Helsinki, Finland
E-mail:
[email protected]
Phone:
+358 9 50605 651
Fax:
+358 9 50605 652
www.alimetrics.com