This is the html version of the file http://www.danonevitapole.com/extranet/vitapole/EvenementsScientifiques.nsf/0/76610D5D88C53FE9C1256BF30038A599/$File/Symp3%20Barcelona.pdf.
G o o g l e automatically generates html versions of documents as we crawl the web.
To link to or bookmark this page, use the following url: http://www.google.com/search?q=cache:XEO5_QevduwJ:www.danonevitapole.com/extranet/vitapole/EvenementsScientifiques.nsf/0/76610D5D88C53FE9C1256BF30038A599/%24File/Symp3%2520Barcelona.pdf+ISBN+:+2-7420-0278-2&hl=en&gl=ca&ct=clnk&cd=1&client=firefox-a


Google is neither affiliated with the authors of this page nor responsible for its content.
These search terms have been highlighted: isbn 7420 0278 
Page 1
NUTRITION AND HEALTH COLLECTION
AN INITIATIVE OF THE DANONE RESEARCH CENTERS
Yoghurt: Eighty Years
of Active Research
for Health
Yoghurt: Eighty Years
of Active Research
for Health
Réf. DR0499
ISBN : 2-7420-0278-2
™xHSMHOCy002788z
A  source  of  life,  well-being  and  good  health,
yoghurt’s highly positive image has persisted down the
ages. At the dawn of the third millenium, its value has
been confirmed by epidemiological studies which have
shown its effectiveness in preventing serious illnesses.
This dairy product, which has been produced in
the same way since the beginning, is both authentic
and perfectly modern in its simplicity and effectiveness.
The aim of this book is to bring together a body
of scientific knowledge about yoghurt : its composition
and the role of the bacteria it contains, its use in cases
of milk intolerance, its curative action – particularly for
treating diarrhoea and its probable role in preventing
cancer of the colon.
This accessible and practical book is vital reading
for anyone working in the health field – bacteriologists,
public health officials and nutritionists – as well as for
anyone interested in maintaining good health.

Page 2
Yoghurt:
Eighty Years of Active Research
for Health

Page 3
Éditions John Libbey Eurotext
127, avenue de la République
92120 Montrouge, France
Tél. : 01 46 73 06 60
John Libbey & Company Ltd
13, Smiths Yard, Summerley Street
London SW18 4HR, England
Tel. : 1947 27 77
John Libbey CIC
Via L. Spallanzani, 11
00161, Rome, Italie
Tel. : 06 862 289
John Libbey Eurotext, Paris, 1999
ISBN : 2-7420-0278-2
Il est interdit de reproduire intégralement ou partiellement le présent ouvrage
sans autorisation de l’éditeur ou du Centre Français d’Exploitation du Droit de Copie (CFC),
20, rue des Grands-Augustins, 75006 Paris.

Page 4
ScientificCommittee
Juan-R.MALAGELADA,HospitalValld’Hebron,Spain
AlfredoMARTINEZ,UniversityofNavarra,Spain
JosephRAFTER,KarolinskaInstitute,Sweden
DennisSAVAIANO,PurdueUniversity,USA
JoseAntonioMATEOS,DanoneSA,Spain
Contributors
F.AZPIROZ,Barcelona
G.BOUDRAA,Oran
Y.BOUHNIK,Paris
P.BOURLIOUX,Paris
D.CARASSO,Barcelona
A.FAZEL,Paris
A.GONZALEZ,Pamplona
S.KERNEIS,Paris
A.MARCOS,Madrid
A.MARTINEZ,Pamplona
L.MORELLI,Parma
D.O’SULLIVAN,Minnesota
G.PERDIGON,Tucuman
B.POOL-ZOBEL,Karlsruhe
J.RAFTER,Stockholm
I.ROWLAND,Coleraine
J.ROBLES,Barcelona
M.E.SANDERS,Colorado
D.SAVAIANO,Indiana
J.SCHREZENMEIR,Kiel
F.SUAREZ,Minnesota
A.TOMKINS,London
M.TOUHAMI,Oran
R.VONK,Groningen
M.deVRESE,Kiel
ScientificCoordination
IreneLENOIR-WIJNKOOP,CIRDC,Paris
SummaryRedaction
FranciscoGUARNER,Barcelona

Page 5
CONTENTS
Introduction................................................................................................................
9
1. The functional properties of yoghurt.................................................................
11
Epidemiological studies..........................................................................................
11
The functional components of yoghurt..................................................................
12
2. The fate of lactic acid bacteria in the gastrointestinal tract...........................
17
Survival of lactic acid bacteria in the human body................................................
17
Intestinal transit......................................................................................................
19
The contribution of molecular biology ...................................................................
20
Key questions.........................................................................................................
21
3. The study of M-cells as targets of the interaction of bacteria with the
immune system.........................................................................................................
23
4. Yoghurt in the prevention and treatment of human diseases .......................
27
Diarrhoeic disorders...............................................................................................
27
Anorexia nervosa....................................................................................................
30
5. Yoghurt in lactose malabsorption and intolerance..........................................
33
Clinical significance of lactose malabsorption .......................................................
33
Lactose digestion and tolerance from live and heat-killed yoghurts.....................
36
Intestinal and colonic factors in lactose intolerance..............................................
37
6. The role of lactic acid bacteria in colon cancer prevention.............................
39
Early events associated with colon carcinogenesis ..............................................
39
Modulation of DNA damage..................................................................................
42
Late events associated with colon carcinogenesis...............................................
43
Mucosal immunostimulation by yoghurt in the prevention of colon cancer.........
45
Yoghurt: Eighty Years of Active Research for Health
7

Page 6
I
NTRODUCTION
In the beginning, yoghurt was sold in pharmacies.
Sincetheearlydays,anduptonow,researchinthisdomainhasbeencontinuous.
A lot of work has been done to define what is yoghurt in terms of microbiology,
process of fermentation, and the standard of identity of yoghurt.
Yoghurt is a defined product covered by different international standards of iden-
tity: Codex, I.D.F. and others have defined yoghurt as a milk fermented by specific cultures.
In most countries, L. bulgaricus and S. thermophilus together are the two basic yoghurt
cultures.
However, in other countries, other probiotic cultures may be added to these
basic cultures, and the product may still be called yoghurt.
What is important for all of us, to maintain the health benefits associated with
yoghurt, is that yoghurt must be alive and active during its shelflife. In other words, that
post-fermentation treatments that will reduce the amount of live and active cultures, should
not be part of the yoghurt definition.
Way back, a journalist asked me a question: “Do you think that yoghurt, such a
traditional product, still is and will be a modern food, a part of our diet and food habits in
the future?”
This is an excellent question, since yoghurt has been used throughout the ages,
centuries, thousands of years. But what is unique about this product, is that we are still
unraveling, discovering about its health benefits.
If we just try to answer this question: what are the basic principles that govern
the modern food behaviour we all have? I would say there are four major important aspects.
1) Our food pattern is toward a decrease in the energy intake: we were all eating
2,700-3,000 calories. Today the average is 1,800-2,200, so the energy intake has decreased.
2) Consumers are demanding more and more : “If I eat this food, is it good for
me? and why is it good for me?” Food is considered as a source of well being. I want to
take this food because I want to feel good, or because I want to have strong bones, or to
age successfully. Food is playing an important role in the well being and successfull ageing
of the population.
Yoghurt: Eighty Years of Active Research for Health
9

Page 7
3) The time we spend for preparation and intake of food has been reduced.
4) We eat everywhere: at home, in restaurants, in snack bars, everywhere we
go we eat.
How does yoghurt fit in this pattern? Perfectly well, in fact:
1) Calorie intake
As we are taking less and less energy each day, it becomes critical to derive
from our food the necessary amount of nutrients that we need in order not to suffer any
deficiency.
Yoghurt, given its content of calories, is a very rich product. It’s an excellent
source of protein needed for growth. If we want to have strong bones, it will deliver a good
quantity of calcium and phosphorus that are necessary. It’s also a good source of potassium
and selected vitamins.
2) Well being
The benefits of yoghurt in case of lactose intolerance are well known today.
Yoghurt allows people who cannot take milk, to benefit from all the nutrients present in
milk. A good percentage of the population – more than 50% in Spain probably – lack the
specific enzymatic machinery to be able to consume milk without any problem: for them
yoghurt is an alternative. That has been demonstrated.
Other researchers have explored the domain of diarrhoea: it has been shown –
notably by studies in Algeria – that yoghurt will help reduce the incidence of diarrhoea, even
may prevent the diarrhoea. This is specifically important for children because we don’t want
them to be malnourished. The World Health Organization has recommended that, when
possible, milk should be replaced by yoghurt, so to reduce the deficiencies associated with
diarrhoea.
Other researchers have been pioneers in demonstrating the impact of yoghurt
on specific immune response systems (this topic has been largely covered in the second
Danone Symposium in Bonn).
Yoghurt could also help prevent some sorts of risk associated with cancer. A
single product, yoghurt, is capable to do all that.
3) Convenience
Yoghurt is a very convenient type of food: you can take it and eat it wherever
you go.
Yoghurt does perfectly fit in the modern food pattern.
A lot of research has been done on yoghurt: the topic of lactose intolerance has
been largely addressed. As for immunity and cancer, what we have so far are hypotheses,
that need to be further established. For the future research we need rapid and reliable
methods, but what’s more, we need consensus on the methods that are needed to clearly
establish the link between yoghurt consumption and some health benefits.
Akram Fazel
Director of Research
Danone Dairy Division
Yoghurt: Eighty Years of Active Research for Health
10

Page 8
CHAPTER I
T
HE FUNCTIONAL PROPERTIES
OF YOGHURT
Functional foods hold a great promise for future trends in human nutrition, and
yoghurt is certainly one of the most exciting players in this field. Modern consumers are
interested in foods which will keep them healthy and prevent disease. However, the inter-
action between food and health is a very complex one. Research aimed at identifying spe-
cific effects of food on health and at a better understanding of the mechanisms involved
in these effects will be required to provide consumers with a real understanding of the
value of food. Proof of efficacy is a huge challenge facing both nutritional science and the
food industry.
Compared to milk, yoghurt has some interesting properties when considered
from a physiological point of view. Yoghurt certainly seems to have a beneficial effect on
gastric emptying time. Yoghurt also contributes to a better digestion of lactose. The avail-
ability of calcium is increased after yoghurt ingestion. A beneficial influence on the micro-
flora of the gastrointestinal tract has been shown by a number of studies in recent years.
Finally, yoghurt offers interesting effects on the immune system. All of these effects can
reveal themselves in an overall positive influence on human health.
E
PIDEMIOLOGICAL STUDIES
Epidemiology is often considered as the first approach used to identify a rela-
tionship between diet and health. Most modern epidemiology studies that have been car-
ried out with yoghurt are in the area of cancer. Between 30 to 60 per cent of the environ-
mental risk of cancer can be attributed to diet, and therefore dietary habits represent a
major influence on cancer incidence rates. A case-control study performed in France found
Yoghurt: Eighty Years of Active Research for Health
11

Page 9
a negative association between yoghurt consumption and the risk of breast cancer, based
on data from 1,010 breast cancer patients and 1,950 controls. Again, a study performed in
the Netherlands showed lower consumption of fermented milk among 133 breast cancer
patients as compared to 289 population controls. In Los Angeles County, a case-control
study showed that calcium intake was associated with decreased risk of colon cancer and
it was also shown that yoghurt consumption was protective. Finally, a large epidemiological
study in the Netherlands showed a weak inverse association between colorectal cancer
and the consumption of fermented milk and dietary calcium. It can be concluded that epi-
demiological evidence suggests that yoghurt consumption may have beneficial effects on
health, at least in the area of cancer.
T
HE FUNCTIONAL COMPONENTS OF YOGHURT
The main nutritional and functional characteristics of yoghurt have been sum-
marised in table I. Certainly, yoghurt is an important source of high quality protein, calcium,
and vitamins. In addition, a variety of functional components that may be active at a level
beyond the basic nutritional value of yoghurt have been identified in recent years. The list
includes bioactive peptides, conjugated linoleic acid, sphingolipids, butyrate and probiotic
bacteria.
Table I. Nutritional and functional components of yoghurt.
Functional peptides are produced during the fermentation process from casein
and other proteic fractions of milk. As part of a larger protein, they are inactive but become
bioactive when released. Their size ranges from 3 to 64 aminoacids and they tend to be
hydrophobic. These peptides can resist digestive hydrolysis within the gut. When absorbed
as intact bioactive peptides, they have a physiological effect, either locally in the gastroin-
testinal tract or systemically once entering the circulatory system. Very few human studies
Yoghurt: Eighty Years of Active Research for Health
12

Page 10
on these peptides are currently available, and our knowledge on their biological effects
relies mainly on data obtained by in vitro and animal studies. Table II shows a list of some
of the functional peptides that have been isolated from milk proteins. Effects on the immune
system were described. Some of these peptides are able to modulate gastrointestinal motil-
ity. Cardiovascular effects include antihypertensive and antithrombotic activity. Other pep-
tides act on the nervous system as opiate agonists or antagonists.
Table II. Bioactive peptides from milk proteins.
Other interesting components of yoghurt or milk fat are the conjugated linoleic
acids (CLA). This is a collective term to describe one or more positional and geometric
isomers of the essential fatty acid, linoleic acid. Linoleic acid is converted into CLA by
intestinal bacteria, or bacteria found in the ruminant. Animal fat is the principal dietary source
of CLA, including milk fat and meat. Between 75 to 90% of the CLA in dairy foods is of
the cis-9, trans-11 isomeric form. CLA is absorbed from the gut and makes its way to cell
membranes, fat tissue and blood lipids. In vitro and experimental animal studies indicate
that CLA inhibits the development of a variety of tumors, particularly mammary tumors.
Diet supplementation with CLA at 0.1 to 1% decreases the carcinogen-induced formation
of some cancers in experimental animals. However, there is no direct evidence that CLA
protects against cancer in humans, including colon or mammary cancer. An interesting
effect which has been observed is that CLA can decrease body fat and increase the muscle
mass in mice.
The sphingolipids are a group of phospholipids that are found in milk fat globular
membrane. The sphingomyelin is the most common species in milk, at concentrations
ranging from 39 to 119 mg per litre, which are physiologically active concentrations. Dairy
products are the main dietary source of sphingolipids. Interestingly, they can enter the
aqueous phase of milk through processing effects on the milk fat globular membrane, which
means that they can also be found in low-fat dairy products. The physiological effects of
Yoghurt: Eighty Years of Active Research for Health
13

Page 11
sphingolipids include anticarcinogenic effects that have been shown in animal research.
They are thought to influence cell regulation and thereby influence carcinogenesis. They
may inhibit the growth and metastasis of tumour cells and transform precancerous cells
towards normal cells. They have been shown to increase calcium release from intracellular
stores, thereby eliciting calcium protective effects against colon cancer.
Butyrate is another important component of milk and milk fat. It is a four carbon,
short-chain fatty acid that is found uniquely in milk fat, at an average of 3 to 4% by weight.
No other common food contains butyrate. However, the primary source of butyrate in the
colon is the bacterial fermentation of fibre and not dietary butyrate. A variety of important
physiological effects of butyrate have been described. Butyrate inhibits epithelial cell pro-
liferation and induces differentiation and programmed cell death. It is associated with down-
regulation or inactivation of the expression of oncogenes. It may also inhibit tumour inva-
siveness and metastasis. In experimental animal studies, butyrate may protect against colon
cancer.
Finally, the bacteria associated with yoghurt fermentation as well as additional
lactic acid bacteria added as probiotic bacteria are thought to promote health via a variety
of mechanisms. Oral probiotics can be described as “living microorganisms which, upon
ingestion in certain numbers, exert health benefits beyond basic inherent nutrition”. Pri-
marily, probiotic bacteria are associated with fermented dairy products. In more recent
years, the trend has been to isolate probiotic candidates from the intestinal habitat, the
rational being that they may have the opportunity to influence the gastrointestinal tract
microflora. Some strains such as Lactobacillus acidophilus and Lactobacillus casei have
both an intestinal source and the ability to survive in the intestine, and they are also asso-
ciated with fermented dairy foods. Other organisms are primarily associated with fermented
dairy foods, including Lactobacillus bulgaricus and Streptococcus thermophilus. Some other
strains have an intestinal source but they are not associated with traditionally
Table III. Activities of probiotic bacteria.
Yoghurt: Eighty Years of Active Research for Health
14

Page 12
fermented dairy products, such as bifidobacteria and some E. coli strains. The activities of
probiotic bacteria are summarised in table III, and they will be discussed in more detail in
the following chapters. They can play an interesting and potential role in intestinal health,
including anti-cancer effects. They modulate the immune system and have an influence on
milk tolerance, both lactose intolerance as well as milk allergy. Vaginal and urinary tract
infections may be prevented by probiotic bacteria both with intravaginal suppository appli-
cations as well as with oral consumption. Gastritis can be treated with some probiotics.
Pathogen translocation may be prevented by probiotics which provide a barrier effect. Arte-
rial hypertension as well as hypercholesterolaemia are also targets for probiotic applications.
Of course, only one or a few strains have been specifically identified to play a role in each
of these particular indications.
There are important issues surrounding the development of products containing
probiotic bacteria, including the essential one of defining what the active principle is, and
what the mechanisms leading to a beneficial health effect are. We also need to define what
doses of specific probiotic strains are required to elicit specific health benefits in target
populations. Well-conducted human studies are warranted.
Yoghurt: Eighty Years of Active Research for Health
15

Page 13
CHAPTER II
T
HE FATE OF LACTIC ACID BACTERIA
IN THE GASTROINTESTINAL TRACT
S
URVIVAL OF LACTIC ACID BACTERIA IN THE HUMAN BODY
Lactic acid bacteria can influence physiology and health through direct or indirect
effects occurring in the gastrointestinal tract. For instance, lactic acid bacteria can deliver
active constituents such as enzymes to target segments of the intestine. Knowledge of
their pharmacokinetics is needed to determine the optimal conditions of their consumption
to obtain a physiological effect. It is usually accepted that live bacteria in the human gas-
trointestinal tract need to reach a minimal concentration of 10
5
units per mL in the small
intestine or 10
7
units per mL in the colon to express potential probiotic activities.
Host factors known to influence the pharmacokinetics of lactic acid bacteria are
gastric acid secretion, intraluminal bile acids, gastrointestinal motility, microbial interactions
and the immune system, which controls the intestinal flora by IgA secretion, among other
mechanisms. The vehicle is an important point in the delivery of viable bacteria. The survival
rate of lactic acid bacteria is substantially higher when they are administered in yoghurt
rather than in a liquid medium with the same pH.
Pharmacokinetics of probiotics are also studied by in vitro models. For instance,
in vitro experiments can provide information on bacterial resistance to acid and bile, adhe-
sion properties of a strain to epithelial cells, etc., though these data differ greatly from those
from in vivo pharmacodynamic experiments. Recently, multicompartimental dynamic mod-
els which reproduce the environment and motility of the gastrointestinal tract have been
developed. They may predict results of in vivo situations and may provide a useful screening
tool. However, human studies are the best way to establish the pharmacokinetics of lactic
Yoghurt: Eighty Years of Active Research for Health
17

Page 14
acid bacteria. Bacterial recovery in faeces after ingestion of a specific strain is a simple
non-invasive method. Intestinal intubation is a better approach to investigate the fate of
bacteria in the gastrointestinal tract, since samples can be collected at different levels of
the intestinal lumen. These methods are subjected to the reliability of the bacteriological
techniques used to detect and identify the test strain. As mentioned further on in this
chapter, the use of molecular biology techniques allows the detection of specific strains
and the distinction of the given strain from endogenous strains of the same species. The
use of transit markers such as spores of Bacillus stearothermophilus is a valuable tool to
investigate the potential ability of lactic acid bacteria to colonise the intestinal tract. Per-
sistance of the given strain for a longer period than that of the transit marker suggests
actual colonization.
Using a triple lumen tube that was introduced orally up to the terminal ileum in
human volunteers, Streptococcus thermophilus was detected in the jejunum, and Lactoba-
cillus bulgaricus both in the jejunum and ileum. However, Lactobacillus casei and a Bifido-
bacterium sp. were detected throughout the entire intestinal tract and in the faeces
(table IV). After ingestion of yoghurt, duodenal aspirates showed viable Streptococcus ther-
mophilus and Lactobacillus bulgaricus, but after ingestion of heated yoghurt no viable bac-
teria of these species were obtained from the duodenal lumen. About 1 to 10% of Lacto-
bacillus acidophilus ingested in fermented milk were found to survive up to the terminal
ileum, and so did 30% of ingested Bifidobacterium bifidum. Exogenous bifidobacteria inges-
ted in a fermented dairy product reached a mean fecal concentration of 10
9
colony forming
units per gram of faeces and remained at this high level during the ingestion period. How-
ever, after cessation of oral administration, the kinetics of the bifidobacteria gradually drop-
ped and paralleled those of the bacillus used as a transit marker. This observation indicates
that the bifidobacteria strain did not colonise the human colon. So far, colonisation of the
human intestine with exogenous lactic acid bacteria has not been proven. However,
Table IV. Detection of ingested exogenous microorganisms in the intestinal tract.
Yoghurt: Eighty Years of Active Research for Health
18

Page 15
in this study, the concentration of bifidobacteria in faeces during the period of consumption
achieved levels above those required to obtain a significant metabolic activity provided by
a probiotic.
I
NTESTINAL TRANSIT
The fate of lactic acid bacteria within the gastrointestinal tract is influenced by
the secretory and motility functions of the gut. Stable isotope methods are currently used
to assess the gastrointestinal function. The most obvious advantage of methodologies
based on stable isotopes is safety. They are environmentally safe. The use of stable iso-
topes is feasible even with pregnant women and new-born children. Simultaneous and
repeated use of several tracers are possible in the same subject. Most tests are based on
the exhalation of
13
C and are easy to perform and painless to the patient. Lactose-
13
C-ureide
measures intestinal transit time. Lactose-ureides are barely absorbed and after oral inges-
tion they will pass through the small intestine into the caecum. Bacteria in the colon hydro-
lyse the molecule; all individuals harbour bacteria capable of splitting the urea bond that is
followed by the breakdown of urea to CO
2
and ammonia. The
13
C label can therefore be
measured in breath or urine. This test can be used to measure oro-caecal transit time and
is an alternative to the lactulose hydrogen breath test. Gastric emptying is also measured
using
13
C- labelled substrates. Lactase deficiency can be studied with the oral administration
of
13
C-lactose. Bacterial overgrowth in the small intestine can be evaluated by tests based
on breath exhalation of
13
CO
2
after oral ingestion of either
13
C-glucose or
13
C-xylose.
Small and large bowel transit can be studied with relative simplicity and tolera-
bility using the standard lactulose breath test to measure oro-caecal transit time and the
radio-opaque pellet method to measure colonic transit time. The lactulose breath test is
based on the oral administration of lactulose, a sugar which is not absorbed in the small
bowel, but reaches the caecum and is fermented by the flora. Hydrogen produced by its
fermentation can be detected in end-expiratory breath samples. Oro-caecal transit time is
defined as the interval between ingestion of lactulose and first breath sample showing an
increase in hydrogen concentration above a baseline of at least 3 parts per million, and this
being maintained in three subsequent breath samples. This test presents a good correlation
with scintigraphic measurements of small bowel transit. The radio-opaque pellet method
is used for the measurement of colonic transit time. Gelatine capsules containing cubic
radio-opaque pellets are administered three times a day at 8-hour intervals for three con-
secutive days. On day 4, an abdominal X-ray film is taken. The pellets are identified in the
different colonic segments and counted. Markers located to the right of the vertebral spi-
nous process and above a line from the fifth lumbar vertebrae to the pelvic outlet are
assumed to be in the right colon. Markers to the left of the vertebral spinous process and
above a line from the fifth lumbar vertebrae to the anterior superior iliac crest are assumed
to be in the left colon. Markers located below the two aforementioned lines are assumed
to be in the rectosigmoid colon. Colonic transit times in each segment and throughout the
Yoghurt: Eighty Years of Active Research for Health
19

Page 16
entire colon are calculated by a standard formula. This method is accurate and gives a good
correlation with the radioscintigraphic method.
T
HE CONTRIBUTION OF MOLECULAR BIOLOGY
Probiotic properties are strain related, rather than species related. Conventional
methods to detect the presence of a given probiotic strain in samples from a feeding trial
are time-consuming and often ambiguous. Specific primers based on sequences of the 16S
ribosomal RNA gene will provide a rapid method for the detection and identification of
lactobacilli isolated from faecal samples by means of polymerase chain reaction (PCR) ampli-
fications. In a multinational European Union project, strain-specific primers were developed
and successfully used to detect bacterial cells by PCR analysis. Faecal samples were taken
from human volunteers fed with specific probiotic lactobacilli. Colony forming units grown
in Lactobacillus selective media were used for the analysis. The detection analysis was
done in a double-blind fashion. A universal band was used as control for the amplification
protocol and a strain specific band to detect the probiotic strain. The technique allowed the
identification of all volunteers treated with the probiotic strains, without any misidentifica-
tion of the untreated volunteers. These results clearly suggest that PCR-based detection
of enteric strains will be of great value for future human research with probiotics.
The challenge facing us at present is to understand the diversity of the species
and strains comprising the genera Bifidobacterium and Lactobacillus, that may potentially
have probiotic attributes. It is necessary to identify a subset of strains with probiotic poten-
tial. The advent of molecular tools has shown tremendous promise for unraveling the mys-
teries of the true diversity of lactic acid bacteria within the human intestine. These tools
comprise genetic fingerprinting, specific probes, molecular speciation and techniques for
the in situ analysis of specific microbial groups in the intestine. Essentially, molecular biol-
ogy tools for studying the gastrointestinal ecology can be divided into three different types:
(a) tracking tools used to track the passage of microbes through the intestine;
(b) molecular speciation tools which are clear and effective as opposed to the
classical approach to speciation, which is often subjected to contention and debate, and
(c) in situ tools which give a direct picture of the activity of micro-organisms in
the intestine without having to culture them on plates.
Using PCR with universal primers targeting the bacterial 16S ribosomal RNA
gene, a fragment of the gene can be amplified and isolated. Thereafter, the fragment is
restricted by a restriction enzyme to obtain a characteristic sequence from a polymorphic
area of the gene (figure 1). The restriction fragment can be used as a fingerprint for a
particular strain. This PCR-based technique was used to track the fate of a bifidobacteria
strain in six volunteers. The fingerprint of the ingested probiotic strain was different from
the resident strains in the six individuals. During the eight-day feeding trial, the ingested
strain quickly attained dominance over endogenous bifidobacteria among all individuals up
to about sixty percent. When cessation of feeding occurred there was a wash-over effect,
Yoghurt: Eighty Years of Active Research for Health
20

Page 17
and after another eight days the probiotic strain disappeared, which is an indication that
this probiotic strain was transient in these individuals.
Figure 1. Restriction fragment length polymorphism of the 16S rRNA gene. (From O’Sullivan.)
The molecular tools for tracking, speciation and in situ analysis will be used to
give us a better understanding of the microbial ecology of the gastrointestinal tract. This
will allow a directed approach to probiotic strain selection.
K
EY QUESTIONS
Some questions are currently important for our knowledge of the fate of lactic
acid bacteria in the digestive tract (for this purpose, the term lactic acid bacteria includes
the genera lactobacillus and bifidobacterium, and also lactococcus and some streptococci).
The first question is how can we differentiate efficiently the resident lactic acid bacteria
from the in-transit lactic acid bacteria? According to the data so far, lactic acid bacteria
ingested with fermented milks cannot colonise the gut. They are in-transit flora. However,
efficient methodologies to differentiate ingested bacteria from endogenous strains are nee-
ded to clarify this point.
It is generally accepted that lactic acid bacteria must survive within the gastro-
intestinal tract in order to be efficient for a particular probiotic activity. How do they survive?
Doesthereexistonlyatransitordoesthereexistalsoacolonisationinthemucosa?Perhaps,
Yoghurt: Eighty Years of Active Research for Health
21

Page 18
some lactic acid bacteria can come close to the mucosa and perhaps this close interaction
with the mucosa is responsible for the function that we observe. Other bacteria may be
metabolically active in the gut lumen during the intestinal transit. If this were true, we would
need to know at which level they are active. Is it at the level of the small intestine or is it
in the colon? From stomach to rectum the number of lactic acid bacteria in transit declines
gradually. In the colon, we may need a large bacterial load to get an effect in the presence
of the resident microflora.
To be efficient and exert a physiological function, lactic acid bacteria must be
present in a sufficient number. This is the third key question, how many lactic acid bacteria
must be alive and active to exert a physiological function and at which level of the gastro-
intestinal tract? We should be able to determine the number of living lactic acid bacteria,
their location in the gut during transit and their viability. Molecular techniques give informa-
tion on the presence of strain-specific DNA, but these methodologies do not tell us if the
bacteria are alive.
Finally, we need to define the most appropriate method of answering each of
these questions. Some investigations may be properly conducted using in vitro or animal
experiments, but which kind of experiments should be done in humans? Current method-
ologies, as described in this chapter, may provide some answers. However, it is often
difficult to compare experiments from one laboratory to another one. We need to stand-
ardise the current methods and we also need a consensus on their validity and implications.
Certainly, new developments will also be required.
Yoghurt: Eighty Years of Active Research for Health
22

Page 19
CHAPTER III
T
HE STUDY OF M-CELLS AS TARGETS
OF THE INTERACTION OF BACTERIA
WITH THE IMMUNE SYSTEM
The human body is in permanent contact with bacteria. Most interactions with
bacteria are positive and beneficial, like, for example the relationship of the microflora with
the intestinal mucosa, but interactions with pathogen bacteria may lead to an infectious
disease. Some of these pathogens enter the body through the digestive tract. There are
two types of protection against infection. One type of protection is non-specific and includes
non-immune mechanisms such as the physical barrier provided by the mucus layer (glyco-
calix and mucins), and the secretion of chemicals with bactericidal activity (lysozyme, lac-
toferrin, defensins) by Paneth cells in the intestinal epithelium. The second type of protec-
tion is specific for a particular pathogen and includes antibody-mediated and cell-mediated
immune responses against a specific antigen.
To develop a specific immune response, contact between the pathogen and the
host is required. In the intestinal epithelium, there are specialised sites to sample antigens
and microbes. These are M-cells. These cells are present in special structures called Peyer’s
patches. The interaction between microbes and the M-cells is facilitated by the fact that
there are almost no goblet cells around to secrete mucus and there is no chemical defence
against the micro-organisms. After sampling by M-cells, antigens or micro-organisms are
internalised and directed to the underlying lymphoid follicle. Lymphoid follicles consist of a
germinal centre containing mostly B-lymphocytes surrounded by CD4 positive T-lympho-
cytes. Macromolecules or particles are actively transported by M-cells and delivered to the
lymphoid tissue. However, several pathogens including bacteria and viruses are able to
exploit this pathway and use M-cells to cross the epithelial barrier and induce infection.
Yoghurt: Eighty Years of Active Research for Health
23

Page 20
Follicle-associated epithelium (FAE) is lacking in immunodeficient mice. M-cells
are absent, suggesting that the lymphoid cells of the follicle are essential for differentiation
of the epithelium. When isolated lymphoid follicles from donor mice were injected in an
intestinal segment without Peyer’s patches from a recipient mouse, microscopic formation
of a true Peyer’s patch was observed in the same place 9-11 days after injection (figure 2).
Morphological and histochemical characteristics of the epithelium in de novo – formed
patches were similar to those of normal FAE. This experiment suggested that M-cells can
originate from crypt cells and that lymphoid tissue has a key role in the differentiation of
these cells.
Figure 2. De novo formation of FAE-like structures in the intestinal mucosa after injection of
Peyer’s patch lymphocytes. (From Kerneis, Kraehenbuhl and Pringault.)
An in vitro system for coculture of a human colonic epithelial cell line (Caco-2) with Peyer’s
patch lymphocytes was developed using a transwell device (figure 3). Phenotypic conver-
sion of enterocytes into cells that express M-cells properties was observed after a few days
of coculture. Lymphoid cells were found to enter the epithelial cell monolayer. The presence
of lymphoid cells did not affect the monolayer, since transepithelial resistance did not
change. In some areas morphological changes were observed, such as a decrease in the
thickness of the apical surface, sometimes with complete disappearance of the typical
Yoghurt: Eighty Years of Active Research for Health
24

Page 21
Figure 3. In vitro system for coculture of colonic epithelial cells and Peyer’s patch lymphocytes.
(From Kerneis and Pringault.)
enterocyte staining and with real modification of the surface. Transformed cells were able
to transport inert particles, such as latex beads. These are fluorescent, and their adhesion
and transport by the monolayer can therefore be monitored. In coculture at 4
o
C, beads
were not transported, but when the temperature of the system was raised to 37
o
C, a high
rate of passage of the beads was observed, indicating that the cells were actively trans-
porting the beads.
The interaction between M-cells and microorganisms was investigated using the
in vitro coculture system. Some bacteria like E. coli are able to interact with the apical
surface of M-cells without being transported, whereas certain pathogens interacting with
M-cells are transported by different mechanisms, and use this path to cross the epithelial
barrier. Interestingly, Vibrio cholerae incubated with Caco2 cells only interacted with the
apical surface of cells but was not internalised inside the cells. However, in the coculture
system with Caco-2 cells and lymphocytes (i.e. with transformed M-cells), Vibrio cholerae
was found inside the monolayer. This finding was confirmed by transmission electron micro-
scopy that revealed the presence of vibria in cytoplasmic vacuoles. Bacteria like Shigella
flexneri and Vibrio cholerae were efficiently transported by this system, but Listeria mon-
ocytogenes was transported at a lower rate, suggesting that there are at least two different
mechanisms. Adhesion of Vibrio cholerae to apical surfaces was similar in the single mon-
olayer culture of Caco-2 cells compared to the coculture system. However, other pathogens
Yoghurt: Eighty Years of Active Research for Health
25

Page 22
did not adhere to Caco-2 cells in a single culture but showed high adherence to the apical
surface of the same cells in the coculture system, suggesting the expression of new surface
molecules that allow the interaction after coculture.
The coculture system is a useful model to learn about the nature of the interac-
tions between microorganisms and M-cells, including the characterisation of surface recep-
tors. This model also gives information on the nature of lymphoid components which are
able to induce conversion of enterocytes into M-cells.
Yoghurt: Eighty Years of Active Research for Health
26

Page 23
CHAPTER IV
Y
OGHURT IN THE PREVENTION AND
TREATMENT OF HUMAN DISEASES
Lactic acid bacteria are able to modulate the immune system according to a large
number of studies. Some of these microorganisms are capable of crossing over the gas-
trointestinal tract, and they can interact with other bacteria from the microflora and other
cells within the intestinal tract. Some of the immunomodulatory properties consist of an
increase in the production of intestinal immuno-globulines by mucosal B-cells. In vitro, lactic
acid bacteria increase the release of gamma-interferon. Most research in this field has been
carried out in animal or in vitro models, regarding both cellular or humoral immunity. How-
ever, there are still lots of questions to be answered about the effects of yoghurt intake in
humans. Some data suggest that yoghurt is useful in the prevention and treatment of some
pathologic conditions, such as diarrhoeal diseases, certain tumours and allergies.
D
IARRHOEIC DISORDERS
Despite the introduction of oral rehydration, many children die from diarrhoea
every year, and many more suffer episodes of malnutrition which is precipitated by diar-
rhoeal disease. In 1990, the WHO predicted a reduction of the number in children dying
from diarrhoea from 5 million to 1.5 million per year. However, estimates at the present
timearethatatleast3 millionchildrendieeveryyearfromdiarrhoea-relatedillness.Between
two and ten episodes of diarrhoea per year is the normal experience for many children in
sub-Saharan Africa and in poor areas of Asia.
Fermented food products may decrease the impact of diarrhoea on public health
in Third World countries. Management objectives to reduce death by diarrhoea include
several aspects: first of all, to reduce the number of germs in the diet; secondly, to reduce
Yoghurt: Eighty Years of Active Research for Health
27

Page 24
the incidence of diarrhoea; thirdly, to reduce the duration of diarrhoeal episodes, and
fourthly, to reduce the severity of diarrhoea. As a result of these strategies, a lower rate
of fatalities is expected, and also an improved nutritional outcome of diarrhoea sufferers.
Food fermentation induces a low pH and inhibits the growth of diarrhoeal patho-
gens in food. Fermentation of cereals in Ghana by a traditional procedure showed a pH
reduction from 6 down to below 4. The food children eat in poor communities is grossly
contaminated. Porridges are usually prepared in the morning and left for up to twelve hours
later before being consumed by the children. The number of coliform bacteria in food at
0,6 and 12 hours after cooking was estimated by microbiological culture. Among samples
of unfermented food – this is food that is freshly prepared and boiled – and fermented food,
it was found that there were quite a lot of organisms in the freshly – prepared, unfermented
food. It is a fallacy to think that all bacteria are destroyed by cooking. Six hours later, the
number of food samples that were free of bacteria was greater in fermented food than in
unfermented food. Twelve hours later, there were many more coliforms in the unfermented
food as opposed to the fermented food. Thus, fermentation protects the growth of poten-
tially harmful microorganisms in food.
In a study performed in Mexico, either a probiotic mixture including Lactobacillus
reuteri or a placebo was given to children of a low socio-economic group for 14 weeks.
Regular administration of the Lactobacillus reduced the incidence of diarrhoea from 0.42
to 0.27 episodes per child. Prevalence was reduced from 35 in the placebo group to 24 in
the treatment group. No difference was found in the severity of the diarrhoea episodes
among both groups. In this study, etiology of the diarrhoea was not investigated or consid-
ered for stratification. Most of the previous studies investigated the effect of probiotics in
diarrhoea induced by rotavirus, since this is the main cause of diarrhoea among children in
Europe, and a positive effect was established. There is not much evidence, so far, on the
effect of probiotics in the prevention of diarrhoea induced by parasites or bacteria, but the
Mexican study may suggest a positive health effect in this area.
A study in Pakistan looked at the effect of yoghurt on acute diarrhoea. Children
in the control group were treated with a commercial preparation, known to be effective in
diarrhoea. Traditional yoghurt was given to another group. At 48 to 72 hours after onset,
traditional yoghurt had a clinical advantage over the most expensive commercial prepara-
tion.
Intestinal permeability can be assessed by the lactulose/mannitol test. After oral
ingestion of the sugars, urine is collected during a 5-hour period to measure the urinary
excretion of both sugars, and the lactulose/mannitol concentration ratio is calculated. In
healthy control children the ratio is low, about 0.2, but it is high in children with intestinal
mucosal damage, which occurs with diarrhoeal diseases. A group of Tanzanian children
with severe diarrhoea were rehydrated, and then allocated to receive one of the two types
of diet: either a control diet, which was the normal porridge made out of freshly prepared
cereals, or a special porridge that had been fermented. Then, the impact of diet on intestinal
permeability was measured by the lactulose/mannitol test. After feeding, there was an
Yoghurt: Eighty Years of Active Research for Health
28

Page 25
improvement in intestinal permeability in both groups as compared to the baseline. Inter-
estingly, the improvement was much greater in children receiving fermented food than in
controls. Metabolic products of fermentation, such as butyrate and propionate, have trophic
properties in the intestinal epithelium and they might account for the positive effect.
The effect of yoghurt as a treatment of acute diarrhoea in children was recently
investigated in Oran (Algeria). Children admitted for acute diarrhoea were randomised to
receive either an infant milk formula or yoghurt. They were stratified depending on the
presence of rotavirus in their stools. Yoghurt was prepared with the same infant milk for-
mula by fermentation with freeze-dried starters of Streptococcus thermophilus and Lacto-
bacillus bulgaricus. Clinical characteristics were comparable between the two groups at
inclusion. Rotavirus was found in 35 out of the 46 children in the infant milk group and 37
out of 47 in the yoghurt group. The mean duration of diarrhoea was higher in the milk group
(66 hours) than in the yoghurt group (44 hours). At 48 hours after inclusion, total remission
was achieved in 64% of children receiving yoghurt, but only in 35% of children receiving
milk. The beneficial effects were most apparent in subgroups of children with rotavirus:
68% cured in the yoghurt group versus 27% in milk group.
A second study compared the clinical efficacy of yoghurt feeding in children with
persistent diarrhoea. Persistent diarrhoea denotes an episode of diarrhoea that begins
acutely but persists beyond the expected time period for the usual self-limited disease.
After a two-day period of observation, 78 children with persistent diarrhoea were randomly
assigned to receive either milk or yoghurt for a period of 5 days. Both groups were similar
on inclusion in terms of age, nutritional status, severity of diarrhoea and lactose hydrogen
breath test. Clinical failure was defined as a 5% loss of body weight in 24 hours, absence
of body weight gain during 3 consecutive days or persistence of diarrhoea after 5 days.
Clinical failure rate was 43% in the group of children receiving milk, but only 12% in children
receiving yoghurt. The advantage of yoghurt feeding was already apparent at 24 and
48 hours after inclusion. These results indicate that yoghurt can be used satisfactorily for
nutritional rehabilitation in children with persistent diarrhoea.
Another study compared the value of fermented milk in the prevention of diar-
rhoea. The trial concerned Algerian infants of less than 5 months old living in an urban area,
without previous history of diarrhoea and regularly fed with infant formulas but no breast
milk. At entry, each mother was offered a batch of unlabeled boxes containing either infant
formula or fermented infant formula (Streptococcus thermophilus and Bifidobacterium bre-
vis). Fermented infant formula was similar to infant formula except for its taste, low pH
and the presence of galactose and lactic acid resulting from partial hydrolysis of lactose. A
total of 98 infants entered the trial, 49 per group. At inclusion, both groups were comparable
in terms of age (a mean of 45 days in both groups). After 21 days follow-up, body weight
gain was similar in both groups. In the formula group, 25 infants suffered from diarrhoea,
as opposed to 17 in the fermented formula group. The total episodes of diarrhoea were 45
in the formula group and 17 in the fermented formula group. Calculated rates were 4 epi-
sodes per child per year with the infant formula and 2 episodes per child per year in children
Yoghurt: Eighty Years of Active Research for Health
29

Page 26
receiving the fermented formula. These results confirm the preventive effect of fermented
milk on diarrhoea. They further suggest that the fermented formula is well tolerated and
can induce a clinically significant positive effect in well nourished, healthy children of less
than 5 months old.
Fermentation technology has an enormous benefit to offer in the prevention and
treatment of diarrhoeal diseases. Further clinical studies looking at fermented vs non-
fermented foods, with or without probiotics, are needed. The specific role of probiotics as
part of the treatment of diarrhoea remains to be defined.
A
NOREXIA NERVOSA
Malnutrition has always been associated with developing countries. However,
in spite of the existing resources nowadays in industrialised countries, there is an increasing
number of cases of malnutrition. Unfortunately, eating disorders are becoming more and
more frequent. In this sense, anorexia nervosa is a syndrome that affects a considerable
part of the female population during adolescence. These patients exhibit an altered behav-
iour with abnormal dietary patterns. They have phobias about their food intake, especially
towards foods containing fat or high levels of carbohydrates. These patients chiefly eat fruit
and vegetables. In addition, they may use some compensatory means to avoid weight gain,
such as self-induced vomiting, and compelling use of drugs (diuretics, laxatives and also
anorexigens). This behaviour involves weight loss leading to a final situation of malnutrition.
The production and release of cytokines is known to be affected by nutrient
intake. A study carried out in patients with anorexia nervosa aimed at investigating the
possible beneficial effect of yoghurt intake on cytokine production. A cross-over study was
designed including 27 anorexia nervosa patients divided into two groups. Patients were
recruited upon hospital admission and followed for 20 weeks. During the first 10-week
period, a group of patients received yoghurt and the other group received fresh milk. During
the second 10-week period, patients were changed to the alternative treatment (either
yoghurt or milk). The patients received either 3 yoghurts per day or an iso-caloric volume
of milk. Diet was similar in both groups of patients throughout the 20-weeks of study.
Cytokine production by peripheral blood mononuclear cells was assessed in vitro after phy-
tohaemaglutinin stimulation.
The group who started nutritional therapy with yoghurt showed a rapid and sig-
nificant increase in the release of gamma-interferon that persisted during the milk intake
period. The group who started nutritional recovery with milk showed a decrease of gamma-
interferon production, but there was an increase during the second period (shift to yoghurt)
just at the end of the study, between the 16th and 20th week. Thus, both groups showed
an increase in gamma-interferon secretion during the period of yoghurt intake. Regarding
interleukin-2, there were no significant changes in patients who started with yoghurt. How-
ever, the group that started nutritional therapy with milk showed significant interleukin-2
production during the period of yoghurt consumption (20th week). A similar result was
Yoghurt: Eighty Years of Active Research for Health
30

Page 27
found when looking at the release of tumour necrosis factor alpha (TNFa). A certain degree
of previous nutritional recovery may be needed to observe a positive effect of yoghurt on
the production of interleukin-2 and TNFa. Production of interleukin-6 showed an early
response during yoghurt consumption, that persisted during the first weeks of consumption
of milk but finally dropped. Data on interleukin-1 were similar to those observed with inter-
leukin-6.
In summary, modulation of cytokine production in patients with anorexia nervosa
can be promoted by yoghurt intake. The effects vary according to the cytokine tested. In
general, inclusion of yoghurt at an early stage of nutritional therapy may be of value in
improving a deficient immunocompetence.
Yoghurt: Eighty Years of Active Research for Health
31

Page 28
CHAPTER V
Y
OGHURT IN LACTOSE
MALABSORPTION AND INTOLERANCE
C
LINICAL SIGNIFICANCE OF LACTOSE MALABSORPTION
Lactose is a disaccharide composed of glucose and galactose, that is mainly
found in milk and dairy products. To be absorbed in the small intestine, lactose must be
hydrolysed into glucose and galactose by a specific enzyme, lactase or beta-galactosidase
which is present in the brush border of the small intestinal epithelium. In the early 60’s, it
was demonstrated that most of the world adult population have a genetically-determined
decrease in lactase activity after weaning, and this was the key that opened different areas
of investigation regarding lactose malabsorption. Thus, prevalence of lactase deficiency is
relatively high, with variations between different geographical areas: 10% in Northern
Europe, 25% in the United States, 50% in Mediterranean countries and above 50% in Africa
and South America (figure 4). Lactose malabsorption is the result of lactase deficiency and
means that a fraction of the ingested lactose is not absorbed in the small intestine and will
therefore end up in the colon. This can lead to gastrointestinal symptoms such as diarrhoea,
flatulence, abdominal bloating and pain. Lactose intolerance is the symptomatic complex
that may result from lactose malabsorption, but lactose malabsorption does not necessarily
imply lactose intolerance.
To illustrate the concrete implications of this phenomenon, let’s examine the
following example. If 12.5 g of lactose (the amount present in one cup of milk) are malab-
sorbed and reach the colon, the colonic bacteria will ferment the disaccharide, and will
produce 120 mmols of organic acids that will associate with electrolytes
leading to an osmotic load that will induce severe diarrhoea. Moreover, fermentation
Yoghurt: Eighty Years of Active Research for Health
33

Page 29
Figure 4. Prevalence of lactose malabsorption. (From Martinez.)
of 12.5 g of lactose will produce 2,600 ml of CO
2
and approximately 4 litres of hydrogen.
However, studies using labelled lactose have shown that such an amount of gases is not
produced, which means that the lactose is either hydrolysed by a remnant lactase or con-
sumed by the colonic bacteria. Bacteria ferment the lactose, and the products can be absor-
bed into the colon. Thus, even when lactose is malabsorbed, fermentation and absorption
of the products can prevent diarrhoea. Moreover bacteria not only produce hydrogen, but
also consume it. The balance between the production and consumption of gas determines
the amount of gas present in the colon lumen.
Therefore it is difficult to know the quantity of lactose that will induce symptoms.
In double-blind studies, ingestion of up to 18 g of lactose induced a similar rate of symptoms
in lactose malabsorbers to those in controls. It was only after ingestion of 50 g of lactose
(4 cups of milk) that 80% of the subjects complained of symptoms.
In addition to people with diagnosed lactase deficiency, some subjects with
apparently normal lactase activity will describe themselves as being lactose intolerant. In a
group of 30 subjects self-described as severe lactose intolerants, it was found that only
21 subjects were lactose malabsorbers. Regardless of the lactose absorption status, they
were included in a double-blind crossover study in which they consumed one cup of either
regular milk or lactose-hydrolysed milk with breakfast for one week. There was no signifi-
cant difference in any of the symptoms (bloating, flatulence, abdominal pain and diarrhoea)
between those taking regular milk and those taking hydrolysed milk in the malabsorber
group. Another important point was that the symptoms were mild. In general people who
Yoghurt: Eighty Years of Active Research for Health
34

Page 30
identify themselves as severe lactose intolerants can drink a cup of milk without experi-
encing significant problems.
Nevertheless, self-described lactose intolerant subjects will behave as though
they are diagnosed lactose malabsorbers by eliminating dairy products from their diet, con-
sequently loosing the nutritional benefits of dairy food. In order to avoid the nutritional
consequences of the exclusion of dairy foods from the regular diet, several studies have
demonstrated that yoghurt is a good alternative for lactose maldigesters. Further investi-
gations have also stressed that the presence of living lactic acid bacteria in yoghurt is
essential to overcome symptoms and signs of lactose malabsorption, as mentioned later
in this chapter.
A critical aspect in this context is the possible loss in terms of calcium intake.
Given the relationship between osteoporosis and calcium intake, a National Institute of
Health Consensus Conference in the United States recommended a daily intake of
1,500 milligrams of calcium for post-menopausal women. Dairy products are the main nat-
ural rich source of calcium (table V), however 4 to 5 servings of dairy products per day
would need to be consumed to obtain this amount of calcium which could cause lactose
malabsorbers to develop symptoms. A study was conducted with 31 lactose malabsorbers
and 31 lactose absorbers, 50% being pre- and 50% being post-menopausal women. In a
double-blindrandomisedfashion,thedietwassupplementedwithmilk,cheeseandyoghurt,
provided either as conventional products (34-g lactose/day) or as lactose hydrolysed prod-
ucts (2-g lactose/day). There was no significant difference for symptoms in the absorbers
group. In the malabsorbers group, lactose hydrolysed products induced similar symptom
scores to those for conventional products for abdominal pain and bloating, but there was
a significant increase in flatus frequency with the conventional products. However, the
general well being of the subjects was not affected, and there were no significant differ-
ences between the two groups. In conclusion, this study suggests that symptoms resulting
Tableau V. Lactose and calcium: content of various foods.
Yoghurt: Eighty Years of Active Research for Health
35

Page 31
from lactose malabsorption do not represent a major impediment to the ingestion of dairy-
rich diets, containing around 1,500 milligrams of calcium per day.
Figure 5. Lactose malabsorption as assessed by hydrogen breath test
(HK = heat-killed yoghurt; LY = Live yoghurt; D= day). (A. Martinez)
L
ACTOSE DIGESTION AND TOLERANCE FROM LIVE
AND HEAT-KILLED YOGHURTS
Preliminary data were presented from human studies using the same product
in three different countries with essentially the same protocol. Yoghurt as a live culture
compared to heat-killed yoghurt was tested in a randomised crossover protocol in lactose
malabsorbers from Germany, Spain and the United States. Both products contained the
same amount of lactose. One group of subjects consumed 500 g of live yoghurt per day
for 14 days. This was followed by a two-week wash out period. Subjects followed the
same protocol with the heat-killed product. The other group started with the heat-killed
product, then after the wash out period they consumed live yoghurt for another 14-day
period. Lactose digestion was determined by the hydrogen breath test on specific test
days. Gastrointestinal symptoms were recorded throughout the study by a validated ques-
tionnaire.
In all three studies, hydrogen production increased after ingestion of the heat-
killed product, but there was no increase in breath hydrogen after ingestion of live yoghurt
(figure 5). This finding clearly indicates that live yoghurt as compared to heat-killed yoghurt
improved lactose digestion in lactose malabsorbers. Gastrointestinal symptom scores were
Yoghurt: Eighty Years of Active Research for Health
36

Page 32
lower during the period of consumption of live yoghurt than during consumption of heat-
killed yoghurt. In addition, yoghurt consumption over several weeks increased lactose tol-
erance in malabsorbers. Finally, there was a trend towards less hydrogen production after
lactose ingestion at the end of the study, suggesting that repeated exposure to lactose
from either live or heat-killed yoghurts induced adaptation of the organism.
I
NTESTINAL AND COLONIC FACTORS IN LACTOSE
INTOLERANCE
Live yoghurt can influence lactose maldigestion and alleviate complaints of lac-
tose intolerance. Nevertheless, all the mechanisms involved in the apparition of symptoms
are not known. Further research is needed to identify which factors play a role in the
generation of the symptomatic complex associated with lactose intolerance, since this
knowledge will lead to the development of new healthy foods based on dairy products.
A recent investigation has pointed out that some subjects are free of symptoms
despite the fact that they suffer from lactose malabsorption. Lactase deficiency is usually
assessed by the lactose-hydrogen breath test. Lactose reaching the colon is fermented by
bacteria and an increase in hydrogen production can be detected in breath samples. How-
ever, the increase in breath hydrogen results from two processes: malabsorption of lactose
in the small intestine and fermentation in the colon by bacteria, so, the absence of a peak
of hydrogen in breath might be due to changes in the fermentation process rather than the
actual absorption of lactose. Therefore, false negative hydrogen tests are common. A
method has been developed based on the ingestion of
13
C labelled lactose. Absorption is
assessed by the levels of
13
C labelled glucose in serum. The label with the stable isotope
discriminates between endogenous and ingested glucose.
The
13
C-lactose test was validated in a population of healthy Chinese medical
students with a genetic background of low lactase activity: 73 subjects ingesting 25 g of
lactose were screened; 68 tested hydrogen positive, and 5 hydrogen negative. The
13
C-lac-
tose test was then applied to 5 hydrogen negative subjects and 12 hydrogen positive sub-
jects by the oral administration of 25 g of
13
C-lactose. Interestingly, there was no significant
difference in peak serum levels of
13
C-glucose between hydrogen positive and hydrogen
negative subjects, suggesting that those individuals who tested hydrogen negative were
true malabsobers with a false negative breath test. The cut-off value of
13
C-glucose in serum
was estimated as 2.0 mmol/l, 1 hour after lactose ingestion. Subjects with values below
this level would be lactose malabsorbers.
The
13
C-lactose test was also performed on 48 Dutch medical students. Again,
correlation of the outcome of the hydrogen breath test with the
13
C-lactose test showed
areas with low concordance. It was found that 25% of the Dutch students showed serum
13
C-glucose levels below the cut-off value, whereas only 17% of the students had a positive
Yoghurt: Eighty Years of Active Research for Health
37

Page 33
hydrogen breath test. In addition, only 13% of the students manifested complaints of lac-
tose intolerance. Subjects with low serum
13
C-glucose levels and no complaints after con-
sumption of 25 g of
13
C-lactose are considered as non-symptomatic malabsorbers. Like-
wise, in Chinese population of malabsorbers, it was found that students with symptomatic
complaints after the consumption of lactose showed similar
13
C-glucose levels to those
students without complaints.
The study demonstrated that some subjects with poor absorption of lactose do
not developsymptoms afterachallenge withoral lactose.Thisfinding suggeststhatbesides
digestion, there must be other factors involved in the genesis of symptoms after lactose
ingestion. These factors are presumably related to the fermentation process in the colon.
Yoghurt: Eighty Years of Active Research for Health
38

Page 34
CHAPTER VI
T
HE ROLE OF LACTIC ACID BACTERIA
IN COLON CANCER PREVENTION
E
ARLY EVENTS ASSOCIATED WITH COLON CARCINOGENESIS
Many studies support the role of the human intestinal microflora in tumour devel-
opment in the colon. By definition, probiotic bacteria can beneficially affect the host by
improving its intestinal microbial balance. Hence, probiotics could have anti-cancer effects.
In general, in vitro studies and in vivo studies in laboratory animals give strong support to
the protective role of probiotic bacteria in colon cancer progression. Human studies reported
so far are interesting, but they are certainly not as supportive as the laboratory studies.
Although there is no real direct evidence to show the protective effect of probiotic bacteria
in human tumour development of the colon, there clearly is a lot indirect evidence.
Colon cancer develops through several distinct stages, from normal epithelium
to hyper-proliferating epithelium, adenoma, carcinoma and metastasis. In the last ten years,
huge progress has been made in identifying the genes that are responsible for or contribute
to the progression of these sequential stages. On the other hand, epidemiology indicates
that there are dietary factors associated with increased risk, for example fat, and with
decreased risk including lactic acid bacteria, fibre and fruit (table VI). Much effort in current
studies is directed towards environment interactions with gene expression/suppression.
The question today is how dietary components, including lactic acid bacteria, interact with
genes that contribute to tumour development. For instance, butyrate, a metabolite of
yoghurt, has been shown to directly affect the expression of genes which are important in
tumour development in the colon.
Yoghurt: Eighty Years of Active Research for Health
39

Page 35
Table VI. Diet and colon carcinogenesis. Epidemiology.
Some hypotheses on the relationship between diet and colon cancer are cur-
rently under investigation. The diet-derived components that may influence colon cancer
are generally divided into initiators or promoters. Initiators are genotoxic compounds which
can induce DNA damage and may give rise to genetic alterations. Most genotoxic agents
are detected with short-term assays, for example the Comet assay, and are called muta-
gens. Mutagens are genotoxins that have been shown to induce tumours in animals, and
in this sense they are also carcinogens. Where human colon cancer is concerned, however,
we do not know which are the actual mutagens involved. Free-radicals produced in the
colon or coming from dietary components could contribute to genotoxicity. Fecapentaenes
produced by the intestinal microflora are also strongly mutagenic and are believed to be
involved in producing human colon cancer. Another group of compounds, the heterocyclic
amines, are formed when burning meat at a high temperature. They are mutagenic and
carcinogenic in animals. Another hypothesis concerns detoxified mutagens. Environmental
contaminants like polyaromatic hydrocarbons or benzopyrenes are detoxified in the liver,
like many foreign compounds, and excreted in bile as conjugates. Bacterial enzymes in the
intestine can deconjugate them and liberate the mutagenic moiety which could contribute
to tumour development.
Promoters are not genotoxic; they do not damage genes. They contribute to
tumour progression by other methods, for instance by inducing cell proliferation. At high
cell proliferation rates, a damaged cell will have a greater statistical chance of developing
into a tumour. In addition, cells that are highly proliferative are much more sensitive to
genotoxic agents. Fat is a well-known tumour promoter, because it contributes to a hyper-
proliferating effect in the colonic epithelium that is believed to be mediated by bile acids.
Dietary fat increases the secretion of bile containing primary bile acids that may leak down
in to the colon. Secondary bile acids produced by colonic bacteria are believed to be tumour
promoters, since they are cytotoxic to epithelial cells and induce proliferation. Fibre may
Yoghurt: Eighty Years of Active Research for Health
40

Page 36
protect by inducing a low pH that prevents the enzymatic transformation of primary bile
acids into secondary bile acids, and calcium may protect against the cytotoxic effect of
secondary bile acids on epithelial cells.
Probiotics can act on early events of the process. First of all, probiotic bacteria
can alter enzyme activity of the indigenous flora and avoid the production of genotoxic
compounds. Secondly, probiotic bacteria may interfere with mutagens and prevent geno-
toxic damage to the colonic epithelial cells. A considerable amount of studies clearly show
that probiotics can produce an in vivo decrease of faecal enzymes which play a part in the
conversion of pro-carcinogens into carcinogens. Indeed, it was the observation that probi-
otic bacteria influence these enzyme activities that opened up the interest in probiotics as
anti-carcinogenic agents. In animal models, several strains of probiotic bacteria are very
efficient in decreasing the activity of several enzymes that are known to be active by pro-
ducing potential initiators of colon cancer.
On the other hand, a huge amount of studies have shown that probiotics are
able to inhibit mutagenic activity. In vitro studies of mutagenic compounds and lactic acid
bacteria actually demonstrate that they can decrease genotoxic activity, i.e. they prevent
damage to DNA sequences. Some other studies have shown that lactic acid bacteria have
the ability to physically bind mutagenic compounds, such as heterocyclic amines derived
from cooked meat. Absorption of mutagens from the gut can be suppressed. There are
strain differences, and there are different effects with different mutagens. A few studies
have shown that some lactic acid bacteria strains can have direct effects on colonic cells
in culture. These strains lower growth rate and induce differentiation in a colon carcinoma
cell line. Growth arrest or growth differentiation can be associated with protection.
Dietary intervention studies in human volunteers have shown that several strains
of lactic acid bacteria can decrease the enzymes that may be involved in carcinogen gen-
eration. A continuous consumption of the active strain is necessary to maintain the effect.
Still, it is very important to confirm that these enzymes play a essential role in human colon
carcinogenesis. Healthy subjects on a standardised diet consumed fried beef patties twice
daily for 3 days and a significant increase in mutagenicity was observed in urinary and faecal
samples. Simultaneous supplementation of the diet with a strain of Lactobacillus acidophi-
lus in fermented milk showed no increase in the excretion of urinary and faecal mutagens.
Likewise, another study showed that oral administration of Lactobacillus casei suppressed
the urinary excretion of mutagens arising after the ingestion of fried ground beef in humans.
Further studies have shown the effects of certain lactic acid bacteria on soluble
faecal bile acids. Consumption for six weeks of a fermented milk product containing Lac-
tobacillus acidophilus markedly decreased bile acid levels in the aqueous phase of faeces
in colon cancer patients. This change can be associated with reduced cytotoxicity and
reduced epithelial cell proliferation. In fact, an Italian study has shown that the abnormally
high proliferative activity that is observed in the colonic crypts of patients with adenoma
was returned to normal after a three months administration of Lactobacillus acidophilus and
Bifidobacterium bifidum. Thus, lactic acid bacteria can be seen to influence the levels of
Yoghurt: Eighty Years of Active Research for Health
41

Page 37
soluble bile acids in the colon, which could indirectly reduce proliferative activity in the
epithelium.
However, two recent studies on the effect of lactic acid bacteria supplements
on risk markers for colon cancer, failed to confirm these findings. Yoghurt enriched with
Bifidobacterium longum significantly increased the excretion of lactic acid bacteria in the
stools of human volunteers, but there was no effect on risk markers such as oro-anal transit
time, stool weight and pH, faecal short chain fatty acids, faecal bile acids and faecal neutral
sterols. Again, another study looked at the effect of a prebiotic (a fructo-oligosaccharide)
on faecal bifidobacteria and a number of risk markers for colon cancer. Prebiotic ingestion
led to a significant increase in faecal bifidobacterial counts, but no effect was observed on
faecal pH, activities of nitroreductase, azoreductase or beta-glucuronidase.
M
ODULATION OF DNA DAMAGE
Damage in DNA sequences of important genes, such as proto-oncogenes,
tumour suppressor genes and DNA repair genes, will cause an increased cell proliferation
and a high probability of cell transformation. The Comet assay is an interesting method to
use for monitoring DNA damage. Isolated cells are embedded in agarose, where the cells
are lysed and the DNA is liberated and subjected to electrophoresis. Thereafter, DNA is
stained with fluorescent dyes and damage can be recognized by a comet shape and quan-
tified in different ways. This method can be used in animals, in in vivo studies, and also in
cultured cells.
In rats fed with carcinogens (dimethyl-hydrazine, DMH or methyl-nitro-nitroso-
guanidine, MNNG) and pretreated with lactic acid bacteria, such as L. acidophilus, L. gasseri,
B. longum, B. brevis or Streptococcus thermophilus, there was a significant decrease in the
genotoxic effects on colonic epithelial cells compared to those in rats receiving the carci-
nogens only. However, heated lactic acid bacteria were not protective for either carcinogen.
The effects were strain specific, as observed with different strains of Lactobacillus del-
brueckii, sub-species bulgaricus, and Streptococcus thermophilus. Some of the strains were
active but others had no effect.
In vitro incubation of colonic cells with MNNG and different cellular fractions of
the effective lactic acid bacteria strains showed that whole cell preparations, cell wall frac-
tions and isolated peptidoglycans of the cell wall induced a significant reduction in geno-
toxicity, but that cytoplasm fractions had no effect. In addition, genotoxicity by MNNG on
isolated colon cells was prevented by both the supernatant and pellet of Lactobacillus acid-
ophilus culture in logarithmic growth. However, heat treated pellets had no effect, suggest-
ing that anti-genotoxic activity was mediated by relatively short-lived products generated
by viable bacteria. For instance, acetate can prevent DNA damage induced by hydrogen
peroxide in colon cells in vitro. Another protective compound was found to be the aminoacid
cysteine, that is released from cell wall proteins by proteases.
Yoghurt: Eighty Years of Active Research for Health
42

Page 38
Carcinogen induced DNA damage can be prevented in the rat by prebiotic car-
bohydrates. A group of rats treated with saccharose and the carcinogen MNNG showed
higher DNA damage than a group of animals receiving lactulose with the carcinogen. Lac-
tulose is fermented in the gut and butyrate could be responsible for the observed protection.
In fact, colonic cells incubated with butyrate showed less DNA damage induced by hydro-
gen peroxide than cells incubated without butyrate. Similar effects were also observed after
inducing genotoxicity with MNNG. Further studies suggested that protection by butyrate
could be mediated by the release of mucus. Butyrate induces the secretion of mucus and
it has been shown that mucus itself decreases the genotoxic effects of chemicals by a
scavenging mechanism.
Another protective mechanism is the induction of chemopreventive enzymes
against oxidative stress, notably the enzyme glutathion-S-transferase (GST), which is abun-
dant in colonic epithelial cells and can be induced by dietary factors. Some non-digestible
carbohydrates can induce GST activity in colonic cells. Interestingly, GST is also induced by
the microflora. Colon cells from germ-free rats showed lower levels of GST than cells from
conventional rats.
L
ATE EVENTS ASSOCIATED WITH COLON CARCINOGENESIS
Early events in colon carcinogenesis are related to the initiation phase. They are
molecular events where the carcinogens interact with DNA causing damage, mutations and
chromosome aberrations. Late events are related to the promotion phase, and are usually
recognised by morphological cell changes that can be observed under the microscope or
eventually as macroscopic changes in the colonic mucosa. Protective effects of probiotics
at this stage consist of the suppression of precancerous changes and the suppression of
tumours in animals.
Aberrant crypt foci (ACF) are induced in the colonic mucosa of rats and mice by
colon carcinogens such as azoxymethane (AOM), dimethyl-hydrazine (DMH) or imidazo-
quinolines (IQ), which are the carcinogens formed in fried meat. In these animals, ACF do
not develop spontaneously but only after exposure to a carcinogen. Aberrant crypts are
characterised by increased size and a thicker area around the crypt. Some ACF may pro-
gress to adenomas and malignant carcinomas. They are also found in human colonic
mucosa, but their precise significance in humans has not been fully established.
Numerous animal studies have clearly documented the protective effect of dif-
ferent probiotic strains and of some prebiotics (lactulose, inulin, fructo-oligosaccharides)
against the induction of ACF by exposure to carcinogens. Some studies have tested the
effects of probiotics, prebiotics or both throughout the whole process of initiation and pro-
motion of tumours. In such studies, animals are fed with the probiotic/prebiotic for about
a week, dosed thereafter with the carcinogen, and then they continue the probiotic/prebiotic
treatment for the remainder of the study. Other studies have concentrated on the effects
on promotional events by giving the carcinogen first and then feeding the probiotic/prebiotic
Yoghurt: Eighty Years of Active Research for Health
43

Page 39
2 or 3 weeks later, up to the end of the study. Induction of ACF is usually assessed after
2-4 months. In a recent study, three probiotic strains (B. longum, L. acidophilus, L. casei)
were tested after the carcinogen (promotion phase design). There was a decrease in the
number of ACF in probiotic-treated groups as compared to controls, suggesting that there
had been protection by the probiotic during the early promotional phase. Another study
with B. longum and lactulose showed that both the probiotic strain and lactulose on their
own significantly decreased the number of ACF, but the combination of the two was much
more effective (figure 6). Interestingly, the activity of gluthation-S-transferase in the colon
was found to be significantly higher in animals treated with B. longum, lactulose or both,
than in controls. Another study has shown that the combination of B. longum and inulin is
extremely effective at reducing the number of ACF after exposure to AOM. Again, the
protective effects were being exerted at the promotional phase, because treatments were
administered after the carcinogen.
Figure 6. Effect of B. longum and lactulose on ACF induction by AOM in rats
(
1
p < 0.05 vs control;
2
p < 0.05 vs BL or Lac alone) (From Challa et al.)
Therehavebeenanumberofstudiesshowingthesuppressionoftumoursexper-
imentally induced in animals. A very early study found a decrease in the number of colon
tumours and also a corresponding decrease in bacterial enzyme activity in animals fed with
a L. acidophilus strain. In another interesting study, rats received IQ over a period of about
nine months. When compared with the incidence of tumours in rats that were given IQ
alone, the rats simultaneously receiving a freeze-dried preparation of B. longum showed a
significant decrease in liver tumours (approx. 50%) a decrease in mammary tumours, and
no incidence of colon tumours. In this study, treatment with B. longum completely sup-
pressed the induction of colon tumours by IQ. This is particularly interesting because IQ is
found in the human diet, and is thought to be a human colon carcinogen. Lactobacillus GG
was also shown to significantly reduce the incidence of colon tumours in animals exposed
to DMH, when the probiotic was given during the initiation and promotion phases. More-
over, the stage of the tumours in the control group was further advanced since some had
Yoghurt: Eighty Years of Active Research for Health
44

Page 40
penetrated the colonic wall, whereas none in the probiotic group had. However, no signif-
icant effect was observed when the probiotic was given only after the promotional phase.
In summary, animal studies have shown that probiotics and non-digestible car-
bohydrates can decrease precancerous lesions in the colon of rats. Combinations of probi-
otics and prebiotics seem to be highly effective. Probiotics can also suppress tumours in
the colon, and indeed might suppress tumours in other tissues of rats as shown by one
experiment. They may be acting at the early promotional stage of carcinogenesis.
M
UCOSAL IMMUNOSTIMULATION BY YOGHURT
IN THE PREVENTION OF COLON CANCER
A group of mice treated with yoghurt did not develop colonic tumours after
exposure to DMH. Yoghurt as well as L. bulgaricus and S. thermophilus are known to
enhance immunity. The role of mucosal immunity as a mechanism in the prevention of
tumourigenesis was investigated in the mouse. Treatment with yoghurt was associated
with an increase of CD4
+
T lymphocytes in the intestinal mucosa as shown by histological
studies. The effect of yoghurt was observed in normal mice and in mice exposed to DMH.
By contrast, exposure to DMH without further treatment enhanced counts of CD8
+
T lym-
phocytes in intestinal tissue, but the effect of DMH was blunted by treatment with yoghurt.
Interestingly, the amount of mucosal IgG-secreting cells was increased in the colon of mice
exposed to the carcinogen, and again yoghurt treatment prevented an increase in the num-
ber of these cells. Moreover, the amount of IgA-secreting cells was markedly increased in
colon samples from mice treated with yoghurt. Further studies also suggested that yoghurt
consumption increases the number of apoptotic cells in animals exposed to DMH.
Taken together, these data suggest that yoghurt increases gut mucosal immun-
ity in the host by increasing CD4
+
cells and shifts the response towards an anti-inflammatory
pattern with predominance of IgA over IgG production. Yoghurt may promote mechanisms
of cell apoptosis.
Yoghurt: Eighty Years of Active Research for Health
45

Page 41
Achevé d’imprimer par
Corlet, Imprimeur, S.A.
14110 Condé-sur-Noireau (France)
N
o
d’Imprimeur : 40046 - Dépôt légal : août 1999
Imprimé en U.E.

Page 42
NUTRITION AND HEALTH COLLECTION
AN INITIATIVE OF THE DANONE RESEARCH CENTERS
Yoghurt: Eighty Years
of Active Research
for Health
Yoghurt: Eighty Years
of Active Research
for Health
Réf. DR0499
ISBN : 2-7420-0278-2
™xHSMHOCy002788z
A  source  of  life,  well-being  and  good  health,
yoghurt’s highly positive image has persisted down the
ages. At the dawn of the third millenium, its value has
been confirmed by epidemiological studies which have
shown its effectiveness in preventing serious illnesses.
This dairy product, which has been produced in
the same way since the beginning, is both authentic
and perfectly modern in its simplicity and effectiveness.
The aim of this book is to bring together a body
of scientific knowledge about yoghurt : its composition
and the role of the bacteria it contains, its use in cases
of milk intolerance, its curative action – particularly for
treating diarrhoea and its probable role in preventing
cancer of the colon.
This accessible and practical book is vital reading
for anyone working in the health field – bacteriologists,
public health officials and nutritionists – as well as for
anyone interested in maintaining good health.