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NUTRAfoods
2004 3(3) 37-49
RESEARCH
D(-)-LACTIC ACID-PRODUCING BACTERIA
Safe to use in infant formulas
Eammonn Connolly
BioGaia AB, Stockholm, Sweden
Bo Lönnerdal
University of California, Davis, USA
INTRODUCTION
Lactobacillus reuteri (L. reuteri), which produces a
mixture of both D(-)- and L(+)-lactic acids when it
ferments sugars, is indigenous to the human GI
tract and the commercial probiotic strain was isolated in the late 1980´s from human milk. The clinical benefits of using this strain include the treatment and prevention of acute diarrhoea and gastrointestinal infections in infants and small children and recent evidence suggests the potential
benefits of L. reuteri in newborn infants to direct
the maturation of the immune system and thereby
prevent allergy. The immune system seems to be
programmed by intestinal bacterial colonisation
in the first days and months of life and imbalances can lead to allergy and other chronic disorders. Thus, there is a good rationale to investigate
and use this strain, other L. reuteri strains and
other D(-)-lactic acid-producing Lactobacilli in
infant formulas as probiotic ingredients.
There is no evidence in the available scientific literature to suggest that healthy infants (or any
healthy human) would be affected detrimentally
by supplementation of the diet with Lactobacilli
that produce D(-)-lactic acid. The medical literature
contains many examples of D(-)-lactic acidosis in
man, but these cases are almost all related to short
bowel syndrome (SBS) in the patients studied.
Even though there is evidence that Lactobacilli may,
in fact, be helpful in the treatment of SBS bacterial
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overgrowth, there may be a weak
case for exercising caution in giving exogenous Lactobacilli to SBS
patients. Such caution is, however,
not warranted in normal healthy
infants.
Key words
D-lactic acid
Lactobacilli
Lactobacillus reuteri
ATCC 55730
Safety
Probiotic
The main concern is that ‘traditional’ views from CODEX on the use
of D(-)-lactic acid as an additive to
infant foods will lead, without due
examination of the facts, to a
unwarranted restriction of the use,
in infant formulas, of Lactobacilli
that produce both D(-)- and L(+)lactic acids. Such a restriction
would deprive many infants of a
potentially beneficial supplementation that may influence their
health.
The objective of this paper is to
provide a review of the facts for
the scientific experts before any
new CODEX Infant Formula
Directive is finalised and to ensure
that infant formulas containing
probiotics with demonstrated safety profiles be sanctioned for use
from birth, whether or not these
probiotics are producers of D(-)lactic acid.
Eammon Connolly, PhD
Vice President Research
BioGaia AB
Box 3242
SE-103 64 Stockholm, Sweden
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+46.8.555 293 18
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+46.8.555 293 01
email [email protected]
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NUTRAfoods 2004, 3(3)
RESEARCH
What is D-lactic acid?
L(+)-lactic acid and D(-)-lactic acid
are optical isomers of 2-hydroxypropanoic acid. The two optical
isomers can occur in the pure form
or as the racemate DL-lactate,
which contains a mixture of both
forms (1).
Metabolism of D(-)-lactic acid
in man
In mammals, L(+)-lactic acid is
formed through the reduction of
pyruvate by lactate dehydrogenase
(LDH) and is a major metabolic
intermediate. Mammalian tissues
have only L-LDH which cannot
metabolise the D(-)-lactic acid isomer. D(-)-lactic acid is, however,
normally found in the blood (2)
and urine (3). In normal adults,
lactic acid levels in serum can
range from 0.5-6.0 mM for L(+)lactic acid and 0.02-0.25 mM for
D(-)-lactic acid (2,4,5). This D(-)lactic acid is derived either
through the methyl-glyoxal metabolic pathway (6,7) or from the
gastrointestinal tract where it is
produced by the normal commensal bacterial flora.
Despite the lack of D-LDH, most of
the data in the literature indicates
that D(-)-lactic acid is efficiently
metabolised in mammals (8,9).
This metabolism probably occurs
through D-2-hydroxyacid dehydrogenase, a mitochondrial enzyme that converts D(-)-lactic acid to
pyruvate and which is found in a
variety of animal tissues (10,11).
Thus, D(-)-lactic acid has been
shown to be metabolised in rats
(12,13), rabbits (14), dogs (15),
ruminants (16), baboons (17) and
man (8,9,18).
D(-)-lactate is a component of lactate-Ringers solutions used for
intravenous fluid replacement and
it has been shown that large amounts of D(-)-lactic
acid administered intravenously are metabolised
rapidly (19).
Further, normal subjects can metabolise D(-)-lactic
acid efficiently albeit at 70% of the rate of the L(+)lactic acid isomer (8,9).
Contrary to the view apparently held by CODEX
(20), there are no data in the literature to suggest
that the metabolism of D(-)-lactic acid should be
impaired or slower in the human infant compared
to the adult. Mollet (21) states ‘Because of liver
immaturity, newborn infants fail to completely
metabolise ingested or in situ produced D-lactic
acid.’ This statement is unfounded, not only since
there is no data to demonstrate that the capacity of
the infant liver to metabolise D(-)-lactic acid is
lower than that of humans of any age, but also
since there is no investigation related to in situ produced D(-)-lactic acid which is probably directly
metabolised locally by other enteric bacteria (see
below).
Metabolism of D(-)-lactic acid in bacteria
The type of lactic acid produced by a Lactobacillus
is a characteristic of the species and does not vary
within the species.
Table 1 provides a list of common Lactobacillus
species and the isomeric form of lactic acid they
produce (22). There are other enterobacteria that
produce D(-)-lactic acid, examples being Eubacteria,
Streptococcus bovis, Megasphera elsdenii and Mitsuoka
multiaciad (23,24).In bacteria, the two isomeric
forms of lactate, D(-)- and L(+)-lactate, are formed
by distinct stereo-specific NAD-dependent lactate
dehydrogenases (LDHs). The presence of one or
both of these enzymes determines which of the isomers it produces during the fermentation process.
The genes encoding for the D-LDH have been
identified in L. plantarum, L. bulgaricus, L. helviticus
and L. johnsonii and have been deleted in a modified L. johnsonii strains La1 and N312 (25). This targeted genetic engineering of the strains has been
carried out to provide these strains that do not produce D(-)-lactic acid as food supplements.
However, the need for such manipulated strains is
unclear and the safety documentation needed to
ensure that such strains do not pose a threat to
human health remains formidable (21,25).
The gastrointestinal tract of man
NUTRAfoods
2004, 3(3)
RESEARCH
The foetus lives in a sterile world until birth. In the
first days of life, the gut of the infant is rapidly
colonised with, amongst many other bacteria,
Lactobacillus spp. (26,27). Thus some infants can
reach levels of around 10 6 CFU Lactobacilli per
gram of faeces by the third day of life and such levels remain throughout life.
Many of the naturally occurring Lactobacilli in the
human gastrointestinal (GI) tract belong to the
group of DL-lactic acid producers. Reuter (28) has
described species that are indigenous to the human
GI tract. L. reuteri, L. gasseri (both DL-producers)
and L. ruminis (L(+)-lactic acid producer) were
shown to be the predominant indigenous
Lactobacillus species in infants as well as in adults.
Further, transient species derived from intake of
fermented foods include amongst many others, L.
plantarum, L. buchneri and L. brevis (28). Thus it is
perfectly natural to have DL-lactic acid producers
in the intestine of humans throughout life and
even though these bacteria do produce D(-)-lactic
acid, this does not pose any threat to the human
host. To emphasise this point, there are no reports
in the literature of D(-)-lactic acidosis in healthy
humans of any age.
What happens to D(-)-lactic acid
in the human GI tract?
D(-)-lactic acid from ingested food or from the fermentation of sugars by Lactobacilli spp. is a substrate
that is utilised by other bacterial species in the distal
gut. For example, the acids produced by Lactobacilli
are utilised by the butyrate-producing bacteria, particularly Megasphera elsdenii and Mitsuoka multiaciad
(24). Megasphera elsdenii has D(-)-lactate dehydrogenase and a lactate racemase, which is induced by
D(-)-lactic acid (29). Thus, it is clear that whichever
lactic acid isomer is present in the gut, it will become
a substrate for lactate utilisers in this ecosystem and
little will be absorbed over the gut wall.
A series of studies on the metabolism of organic
acids by the complex variety of bacteria in the gastrointestinal tract has been summarised by Hove
(9). It has been shown that addition of large doses
of a purely L(+)-lactic acid producing bacteria,
Bifidobacterium bifidum could, in fact affect the other
colonic bacterial populations such as to increase
the level of D(-)-lactic acid produced. A high level
Table 1 Production of lactic acid
isomers by Lactobacillus spp.
Producers of only D(-)-lactic acid
Lactobacillus delbrueckii subsp. delbrueckii
Lactobacillus delbrueckii subsp. lactis
Lactobacillus delbrueckii subsp.bulgaricus
Lactobacillus jensenii
Lactobacillus vitulinus
Producers of only L(+)-lactic acid
Lactobacillus agilis
Lactobacillus amylophilus
Lactobacillus animalis
Lactobacillus bavaricus
Lactobacillus casei
Lactobacillus mali
Lactobacillus maltaromicus
Lactobacillus murinus
Lactobacillus paracasei subsp. paracasei
Lactobacillus paracasei subsp. tolerans
Lactobacillus ruminis
Lactobacillus salivarius
Lactobacillus sharpeae
Lactobacillus rhamnosus
Producers of racemate DL-lactic acid
Lactobacillus acidophilus
Lactobacillus amylovorus
Lactobacillus aviarius subsp. aviaries
Lactobacillus brevis
Lactobacillus buchneri
Lactobacillus crispatus
Lactobacillus curvatus
Lactobacillus fermentum
Lactobacillus gasseri
Lactobacillus graminis
Lactobacillus hamsteri
Lactobacillus helviticus
Lactobacillus homohiochii
Lactobacillus pentosus
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillus sake
(22,66)
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RESEARCH
of L(+)-lactic acid in the colon is
expected to induce an increased
D(-)-lactate level through the activity of bacterial DL-lactate racemase
(9). A similar effect could be
obtained by the addition of a DLlactic acid producer like L. acidophilus (9).
Thus, ingestion of L(+)-lactic acid
producing bacteria can also lead to
elevations of D(-)-lactic acid in the
human gastrointestinal tract.
The induction of even quite significant fluctuations in the production
of D(-)-lactic acid in situ in the
colon through ingestion of excesses of lactulose, does not induce
changes in D(-)-lactate in the circulation and stable, low levels of this
isomer are detected in blood of
healthy humans after such provocation (9). Thus, in healthy
humans, D(-)-lactic acidosis has
not been reported and is difficult
to induce experimentally.
D-lactic acidosis in humans
The phenomenon of D-lactic acidosis is a rare condition in man but
has been known in ruminants for
many years (16,23). It was first
described in humans by Oh et al
(30) and since then there have been
many reports in the literature of
cases of D-lactic acidosis, their origin and treatment (31-37). This literature has been reviewed in detail
and these reviews contain almost
all the available data on this syndrome to date (8,9).
Uribarri et al (8) defined D-lactic
acidosis as a serum D(-)-lactic acid
level of greater than 3 mmol/L (10100-fold normal levels) and they
reviewed 29 cases from as many
independent reports.
Twenty-seven of the 29 cases were
subjects with short bowel syndrome either due to surgical resection of the intestine or intestinal
bypass for the treatment of obesity. One case was
the result of accidental placement of an enteral
feeding line in the colon instead of the stomach
and the remaining subject probably had malabsorption as a result of pancreatic insufficiency (8).
The typical clinical symptom of D(-)-lactic acidosis,
altered mental status, varying in degree from mild
drowsiness to coma, accompanied every case
whilst other symptoms included slurred speech,
ataxia and disorientation (8). Further, hyperventilation has been observed in infants/small children
(38,39).
Hove (9) reviewed 31 cases, some common to the
Uribarri review. Twenty-eight of these cases had
short bowel. Two cases were mentally retarded and
the D(-)-lactic acidosis was related to a inborn error
of metabolism.
Faecal bacteriological analysis in SBS individuals
with D(-)-lactic acidosis has shown a large increase
in the levels of Lactobacilli normally resident in the
GI tract.
This elevation of the levels of these bacteria is
caused by abnormally rapid intestinal transit time
leading to malabsorption in the small intestine and
an excessive supply of carbohydrates and nutrients
to the colon. These events are all consequences of
the underlying disturbance in the function of the
gastrointestinal tract.
Dominant species found in SBS patients are
Lactobacillus acidophilus, L. fermentum, L. plantarum,
and L. salivarius (40-42). These types of organisms
are usually dominant in short bowel syndrome
(SBS) patients even without any symptoms of acidosis (38).
In SBS subjects, Kaneko et al (43) showed that the
D-lactic acid producer L. delbrueckii subsp lactis
was involved in eliciting the acidosis whilst Satoh
et al. (36) showed a predominance of the DL-producers L. buchneri and L. fermentum in two SBS
patients.
Bongaerts and his colleagues have studied D-lactic
acidosis in infants and children (age 1-3 years)
with SBS and have found that D-lactic acidosis
occurs 2-3 weeks after oral feeding concomitant
with a large increase in Lactobacilli in the faeces
(38,39,42).
L. acidophilus and L. fermentum were increased in
the faeces of SBS subjects (1010–1012 CFU/g faeces)
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compared to that of control adults or children (108
– 109 CFU/g faeces (42).
Other evidence indicates that short bowel patients
that have been treated with antibiotics can develop
abnormal Lactobacilli populations and consequent
D(-)-lactic acidosis (23,40,44,45).
Thus, an underlying short bowel syndrome is a
pre-requisite for the development of D(-)-lactic acidosis in humans. This conclusion is made by
Uribarri et al (8) who conclude that the development of D(-)-lactic acidosis requires the following
conditions in man:
1) carbohydrate malabsorption with increased
delivery of nutrients to the colon,
2) colonic bacterial flora of the type that produces
d-lactic acid,
3) ingestion of large amounts of carbohydrate,
4) diminished colonic motility, allowing time for
nutrients in the colon to undergo bacterial fermentation, and
5) impaired d-lactate metabolism
The review by Hove (9) arrived at the same conclusion.
Unusual exceptions to this are patients undergoing
continuous ambulatory peritoneal dialysis (CAPD)
who have been shown to have abnormally high
levels of serum D(-)-lactate (due to the high lactate
concentration in the dialysis solution) (4,46).
Significantly, there is no evidence or report in the
literature of any healthy infant in the first year of
life or indeed any healthy human with D-lactic
acidosis.
Is orally administered D(-)-lactic acid toxic
in human adults?
Many fermented foods including yoghurt, kefir,
pickles and sauerkraut as well as cheese, meats
and sausages contain D(-)-lactic acid often derived
from the lactic acid bacteria involved in the fermentation. Yoghurt, for example, contains high
levels of D(-)-lactic acid derived from L. dulbrueckii
subsp. bulgaricus which produces large amounts of
only D(-)-lactic acid during the fermentation
process. Yoghurt also contains L(+)-lactic acid produced by the other yoghurt culture bacterium
Streptococcus thermophilus.
De Vrese and colleagues (47) examined the elimi-
nation of an oral aqueous solution
of DL-lactic acid and found that a
level of 2.2 mmol/kg body weight
of D(-)-lactic acid could be effectively eliminated, did not induce
D(-)-lactic acidosis and was not
harmful in man. The same group
(48) further showed that ingestion
of D(-)-lactic acid after an oral load
from a ‘real’ meal of yoghurt further diminshed any risk of D(-)lactic acidosis.
It is well-established and obvious,
of course, that ingestion of yoghurt
(and other fermented foods) with
high levels of D(-)-lactic acid does
not pose a threat to human health.
Is orally administered
D(-)-lactic acid toxic
in human newborn infants?
Three short-term studies in human
infants have been cited in the literature as addressing this issue (20).
In the first of these studies (49), 40
full-term infants were given a feed
formula containing 0.4% DL-lactic
acid between 2-4 weeks of age. No
effect was observed on the rate of
weight gain during this time.
In the second study on infants,
healthy babies were fed milk formula acidified with 0.4-0.5% DLlactic acid for periods of 10 days
during the first 3 months of life
(50). There was evidence of acidosis in one third of the infants and
this was coupled to a decrease in
body weight gain as a result of
reduced food intake.
The third study was also performed by Droese & Stolley (51).
In this study, 0.35% DL-lactic acid
(80% D(-)-lactic acid and 20% L(-)lactic acid) was given to healthy
babies between the 10 th and 20 th
day of life.
A 3-fold increase in urinary excretion of L(+)-lactic acid and a 12-
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NUTRAfoods 2004, 3(3)
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fold increase in excretion of D(-)lactic acid was observed which
could be normalised by removing
the DL-lactic acid from the diet.
It was concluded that D(-)-lactic
acid appeared to be less well
metabolised than the natural L(+)lactic acid form and the excretion
of both acids indicated that the
infant could not fully utilise the
0.35% level of lactic acid in the
diet. A number of babies could not
tolerate lactic acid (both forms)
with rapid body weight loss, diarrhoea and increased blood pH as a
consequence, which could be
reversed when DL-lactic acid was
no longer given.
These studies examine the effect of
feeding high doses of lactic acid
mixtures to infants early in life.
They do not specifically address
the stereo-isomers of lactic acid
given and can only conclude,
therefore, that addition of high levels of lactic acid to infant foods is
not recommendable. These few
studies are the basis of the CODEX
recommendations (see below) and
although CODEX have asked for
more specific studies on the stereoisomers of lactic acid, no new data
have appeared since 1964.
Why give D(-)-lactic acid
producing bacteria to infants?
Lactobacillus reuteri is found in the
gut of all the mammalia studied
(52) and is one of the few species
of Lactobacilli indigenous to the
gastrointestinal tract of man (28).
L. reuteri is present in human milk
(53) and the commercial strain of L.
reuteri ATCC 55730 developed and
marketed by BioGaia (Sweden)
was originally isolated from the
milk of a nursing Peruvian mother
in the late 1980´s (52). The bacterium can thus be considered a component of the natural flora of the
new-born infant, providing balanced colonisation
of the infant gastro-intestinal tract. Indeed, natural
colonisation of expressed milk with commensal
bacteria has been known for some time and these
bacteria are transferred to the infant gut (54).
The potential effects of these bacteria in the infant
are only just being appreciated. The gut of human
infants is rapidly colonized after birth and this provides a protective barrier against pathogenic bacteria and other microbes. The growth of a series of
pathogens can be inhibited by L. reuteri through
the action of reuterin and other antimicrobial
agents released by the bacterium (52). Two doubleblind, placebo controlled clinical studies examined
the effects of L. reuteri in children from 6 mo of age,
hospitalised with acute diarrhoea. In the first study
on acute infectious diarrhoea (of both viral and
bacterial origin), the children received 1010-10 11
CFU/d (n=19) or placebo (n=21) until the diarrhoea resolved. L. reuteri significantly reduced the
duration of diarrhoea from 2.9 to 1.7 days compared to placebo (60). In the second study, the
duration of 2.5 days of rotavirus-induced diarrhoea for placebo (n=25) was reduced to 1.9 days
with a dose of 107 CFU/d (n=20) and to 1.5 days at
1010 CFU of L. reuteri/d (n=21) (61). Thus, L. reuteri
confers a clinically relevant protective effect
against pathogens and consequent diarrhoea.
Recent clinical evidence confirms this protective
effect of L. reuteri in infants. Weizman et al (62) performed a double-blind, placebo controlled trial in
infant formulas supplemented with either L. reuteri
ATCC 55730 (approx 10 8 CFU/day) or B. lactis
(Bb12) for 12 weeks and followed the incidence of
spontaneous infections in children between 4-10
mo of age.
Infants given either of the probiotics showed less
febrile episodes and fewer GI illnesses and infections than the placebo group and the authors noted
that L. reuteri ATCC 55730 was found to be superior to both placebo and supplementation with B.
lactis in this respect.
Further, supplementation with L. reuteri was the
only regimen effective in maintaining the gastrointestinal health of the infants as shown by significant reduction in the number of visits to the doctor
and, more importantly, a significant reduction in
the prescription of antibiotics to the L. reuteri supplemented infants (62 and personal communication).
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Maturation of the infant immune system also
appears to be related to early colonisation of the
infant gastrointestinal tract with lactic acid bacteria
(26,55).
Insufficient colonization of infants with these bacteria may be associated with the increased incidence of allergy in Western societies (27,56) and
this has prompted attempts to reintroduce such
bacteria to infants.
There is convincing data that L. rhamnosus ATCC
53103 supplementation of infants prevents the
development of allergy (57) an effect which is
maintained in later life (58). Further, L. reuteri
together with L. rhamnosus has been shown to the
beneficial in the management of atopic dermatitis
in children (59).
Thus, there is ample support to justify the use of L.
reuteri ATCC 55730, a natural component of human
milk, in infant formulas to improve the health and
development of the infant and to mimic expressed
mother´s milk as closely as possible.
Do supplements with D(-)-lactic
acid producing bacteria induce
or affect D-lactic acidosis in adults or infants?
In the first case of D(-)-lactic acidosis described in
humans, Oh et al (30) noted that the short bowel
syndrome patient in question had taken supplements of L. acidophilus prior to the onset of the
problem and the authors concluded that this may
have been relevant.
Short bowel syndrome patients with bacterial
overgrowth that did not respond to antibiotic therapy were treated with various alternative therapies
including supplementation with the DL-lactic acid
producer L. plantarum (63). A dose of 1010 CFU L.
plantarum was given once per day to one patient
who showed significant recovery within 2-3 weeks.
The removal of the L. plantarum supplement after 2
months led to a reoccurrence of the bacterial overgrowth (63). The authors concluded that probiotic
therapy in short bowel syndrome may be effective
in controlling symptoms related to bacterial overgrowth, the main cause of D(-)-lactíc acidosis in
man.
No data exist in the literature describing D(-)-lactic
acidosis in healthy humans of any age for any reason, despite extensive use of probiotic Lactobacillus
supplements around the world.
Clinical safety with Lactobacillus
reuteri (ATCC 55730)
Safety in infants and children
L. reuteri ATCC 55370 is a
Lactobacillus strain that has been
shown to be safe in numerous
studies in adults, immuno-compromised adults, children, infants
and even premature infants.
The clinical safety program sponsored by BioGaia AB with L. reuteri
ATCC 55730 (also called SD2112),
has examined many infants and
children after administration of
this probiotic. The data are summarised here:
In Shornikova et al (60,61), children
(ages 6 to 36 months) with infectious diarrhoea were treated with
Lactobacillus reuteri ATCC 55730. In
one of the studies, children
received 10 10 to 10 11 CFU of L.
reuteri once per day, and in the
other 107 or 1010 CFU. In neither of
the studies could any adverse
effects of L. reuteri supplementation on weight gain, consumption
of oral rehydration solution or
electrolyte, or on acid-base balance, be detected.
Supplementation with higher
doses of L. reuteri led to elevations
of the total faecal Lactobacillus
count to levels between 106 - 107
CFU/g faeces, whilst lower doses
did not induce marked changes
(60,61).
In short bowel syndrome patients
where bacterial overgrowth and Dlactic acidosis are apparent, the
levels of Lactobacilli reach significantly higher levels of 10 10- 10 12
CFU/g faeces (42) and thus L.
reuteri supplementation, even at
high doses does not induce bacterial overgrowth. Shornikova (60)
reported no abnormality between
the L reuteri treated group compared to placebo group regarding
blood pH or its correction with
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NUTRAfoods 2004, 3(3)
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fluid replacement in infants with
acute diarrhoea. D(-)-lactic acidosis
is a form of acidosis and is characterised by a reduced blood pH (8).
Karvonen et al. (64) studied the
safety of doses of L. reuteri
between 10 5 and 10 9 CFU/day
given to 90 healthy neonates from
birth for 30 days. There was no
evidence of adverse events during
the trial in any infant. Total faecal
Lactobacillus counts after 30 days of
supplementation at all doses were
on average between 10 6 –10 8
CFU/g faeces and were not different from infants that had received
placebo (BioGaia; data on file).
The same clinic recently performed
two further double-blind, placebocontrolled safety trials. In the first,
20 healthy, term newborn infants
received L. reuteri at a dose of 108
CFU/day from birth for 28 days.
The total Lactobacillus count in the
faeces was unchanged between
placebo and supplemented infants
at around 107 CFU/g. In the second study, uncomplicated premature infants (n=27) were given L.
reuteri at doses of 10 7 and 10 9
CFU/day for 28 days from birth.
No abnormally high levels of faecal Lactobacilli were observed at
either dose (Karvonen et al. submitted for publication).
In both these well-controlled clinical trials, there was no evidence of
any adverse events and certainly
no evidence of acidosis in the
infants. Further, in an ongoing
placebo-controlled, double blind
trial, a total of over 100 infants are
given L. reuteri supplementation
(10 8 CFU/day) from birth and
through the first year of life
(BioGaia; study ongoing).
To date, all these infants have
passed 12 months of age and there
have been no reports of adverse
events and no indications of acidosis. Blood from
supplemented infants in this trial has been
analysed for D(-)-lactic acid and it was found that
all infants tested had normal, low levels ere were
no abnormal elevations compared to placebo-treated infants (Connolly et al submitted for publication).
Recent data (65) demonstrate the safety of supplementation of infant formulas with L. reuteri ATCC
55730 (approx 108 CFU/day for 12 weeks) in day
care infants (65 given L. reuteri) from 4 months of
age. The infants consumed normal volumes of formula and grew normally and there were no
adverse events recorded in this double-blind,
placebo-controlled trial.
In conclusion, in well-controlled clinical trials
where L. reuteri supplementation of more than 160
infants from birth and a further 65 from 4 months
of age have been examined, no adverse events
have been observed. Faecal levels of total
Lactobacilli indicate that no overgrowth of
Lactobacilli was induced by the administration of L.
reuteri. Since such an overgrowth is a pre-requisite
for D(-)-lactic acidosis in man, none of these
infants had D(-)-lactic acidosis.
Conclusions on the available evidence
1. There is no existing evidence to show that the
normal gastrointestinal tract flora can induce D(-)lactic acidosis in the healthy human adult or
infant. D(-)-lactic acidosis only occurs in subjects
with a disturbed gastrointestinal function following bowel resection.
2. Well-controlled clinical safety studies where the
DL-lactic acid producing probiotic bacterium, L.
reuteri has been given to over 160 human newborn
term and preterm infants clearly indicate that clinical signs of acidosis do not occur after L. reuteri
administration at any dose tested.
3. Bacterial overgrowth and a disturbed gastrointestinal microflora in the large bowel is a pre-requisite for D(-)-lactic acidosis in humans. There is no
evidence that exogenous probiotics can induce
Lactobacillus overgrowth or imbalance in the bacterial flora of healthy newborn infants.
4. Exposure of infants to L. reuteri does not result
in abnormal levels of D(-)-lactic acid in the blood.
NUTRAfoods
5. There is no valid reason to exclude the supplementation of indigenous human Lactobacillus spp to
the newborn human infant on the basis of the
stereo-isomers of lactic acid these bacteria produce.
CODEX
The CODEX recommendations (Appendix 1) cannot
be applied to the use of D(-)-lactic acid producing
Lactobacilli as ingredients in infant formulas as
there is no scientific basis for such a restriction.
There is, on the other hand, strong evidence to
indicate the safety and efficacy of such additives
for infants. CODEX must clarify this discrepancy
so that the use of potentially beneficial probiotics is
not prevented or discouraged.
The CODEX Standard for Infant Formula is currently being revised (CODEX draft revision; ALINORM 03/26). In this draft under “Optional
Ingredients” the restriction “Only L(+) producing
lactic acid cultures may be used” again appears.
The inclusion here of specific lactic acid producing
cultures under “Optional Ingredients” prohibits
the use of D(-)-lactic acid producing organisms as
ingredients. This is totally unwarranted on the
basis of available clinical evidence and disagrees
with current medical expert opinion, including the
EU directives for infant formulas.
The data described in this position paper provide
clear evidence that supplementation of infant formula with Lactobacilli does not represent any risk
in the healthy infant. The currently ongoing
CODEX revision of The Codex Standard for Infant
Formula: CODEX STAN 72-1981 should correct
this misinterpretation and prevent unfounded discrimination of safe and efficacious Lactobacilli
ingredients in infant formulas, Lactobacilli that are
naturally transferred to the infant via their mother´s milk.
Appendix 1
Codex recommendations for infant formulas
must be based on the available evidence
The Codex Standard for Infant Formula
(CODEX STAN 72-1981) allows “L(+)-lactic
acid” and “L(+)-lactic acid producing cultures”
as pH-adjusting agents in the manufacture of
infant formulas.
2004, 3(3)
RESEARCH
Although these restrictions
apply only to live bacteria as
fermentation aids for the adjustment of pH during the manufacture of an infant formula, this
clause is being misinterpreted as
a hinder for the addition of D(-)lactic acid producing bacterial
species as freeze-dried ingredients added to the final infant
formula product.
Such ingredients do not produce
any lactic acid in the infant formula product as they are freezedried and metabolically inactive.
The rationale for the recommendation (20) is based on the studies of Jacobs & Christian, (49)
and Droese & Stolley (50,51)
described above. The CODEX
Expert Committee meetings
concluded as follows:
“However, human studies
determining the maximum load
of lactate are not available.
There is some evidence that
babies in their first three months
of life have difficulties in utilizing small amounts of DL and
D(-) lactic acids.” The Expert
Committee also desired further
work on “metabolic studies on
the utilization of D(-) and DLlactic acid in infants.” This conclusion on D(-)-lactic acid use is
unfounded based on the clinical
evidence available. More importantly, however, even though
the addition of lactic acid as
such to infant formulas has been
questioned, this same reasoning
cannot be applied directly to the
use of probiotic ingredients that
do not produce lactic acid in the
infant formula.
During expert reviews at the
WHO meeting in Cordoba
(Health
and
Nutritional
45
46
NUTRAfoods 2004, 3(3)
RESEARCH
Properties of Probiotics in Food
including Powder Milk with
Live Lactic Acid Bacteria;
October 2001) and at the
FAO/WHO Working Group
Meeting in Ontario (Guidelines
for the evaluation of probiotics
in food; May 2002) there was no
discussion of D(-)-lactic acid,
presumably since this was not
considered an issue in safety
assessments of lactic acid bacteria for foods, even for infants.
The European Community has
also addressed this issue via its
Scientific Committee for Food.
Their conclusions can be seen on
http://europa.eu.int/comm/fo
od/fs/sc/scf/outcome_en.html.
Importantly, the experts at EU
made a clear distinction
between a technical aid used for
acidifying infant formulas (for
use in the production process)
and optional ingredients that
are added to mimic human milk
and to replace needed nutrients/probiotics. EU experts recommended that these ingredients should be evaluated case by
case based on the safety and
efficacy data available.
Finally, the issue has been raised
by the independent panel of
experts at the International
Scientific Association for
Probiotics and Prebiotics
(ISAPP) and there were no concerns for the use of D(-)-lactic
acid producing Lactobacilli as
ingredients in infant foods (see
ISAPP 2003 at www.isapp.net/
MeetingInfo.htm).
SUMMARY
This is a position paper addressing
the safety of adding probiotic bacteria in infant formulas. It reviews
the literature and describes the facts with regard to
the CODEX restriction on adding D(-)-lactic acid to
infant formulas and discusses the relevance of this
restriction on the substance itself, in relation to the
perceived restriction on the use of D-lactic acidproducing Lactobacilli ingredients added to infant
formulas.
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49