WO2011148221A1

WO2011148221A1 - Lactic acid bacteria for maturation of the enteric nervous system in infants

Info

Publication number: WO2011148221A1
Application number: PCT/IB2010/001542
Authority: WO
Inventors: Biliana Lesic; Michel Neunlist
Original assignee: Gervais Danone SA
Current assignee: Gervais Danone SA
Priority date: 2010-05-28
Filing date: 2010-05-28
Publication date: 2011-12-01
Anticipated expiration: 2012-11-28
Also published as: WO2011148352A1

Abstract

The invention relates to the use of lactic acid bacteria, for use in maturation of the enteric nervous system in newly born infants.

Description

LACTIC ACID BACTERIA FOR MATURATION OF THE ENTERIC NERVOUS

SYSTEM IN INFANTS

FIELD

The present invention relates to the field of infant nutrition, in particular to infant nutrition comprising lactic acid bacteria for use in maturation of the enteric nervous system in newly born infants.

BACKGROUND

The enteric nervous system (ENS) consists of a complex network of neurons organized in several plexuses, which interacts by means of neurotransmitters such as acetylcholine and nitric oxide. The ENS modulates the gastro-intestinal motility, and secretions of exocrine and endocrine origin independent of the central nervous system. Though in humans the ENS is for the most part developed before birth, the maturation of neurons and nerve plexuses continues after birth for a limited period of time. Especially in preterm infants the ENS is immature at birth, and there is a longer period after birth in which the ENS must further mature.

A mature ENS is required to ensure a normal pattern of intestinal motility in order to regulate digestion after birth. Gastro-intestinal motility is required for normal transit of the contents of the gastro-intestinal lumen and the mixing of nutrients with digestive secretions. Abnormal development of any of the participants in gastro-intestinal motility results in significant morbidity and mortality, in particular in preterm infants.

Enteral compositions which stimulate the maturation of the gastro-intestinal tract in newborn infants are known.

WO 2004/1 12509 discloses a composition for inducing a pattern of gut barrier maturation similar to that observed with breast feeding and able to improve gut barrier maturation, e.g. during neonatal stress.

EP 1 161 152 discloses a nutritional composition intended for specific gastro-intestinal maturation in premature mammals.

Indrio et al. (2008 J Pediatr 152:801-806) disclose an increased gastric emptying, reduced regurgitation in preterm infants receiving a probiotic strain of L. reuteri.

However there is still a further need for components that promote the maturation of the ENS just after birth, in particular in preterm infants.

SUMMARY OF THE INVENTION

The inventors found that in a new born rat model, representative for newborn human infants, the rectal administration of strains of lactic acid bacteria significantly increased weight and weight gain, and increased the length of the colon. Most strikingly the maturation of the enteric nervous system (ENS) was enhanced. This maturation of the ENS was prominent as an increased number of neurons per ganglion, an increased number of AcetylCholineTransferase-ImmunoReactive (ChAT-IR) nerves per ganglion, an increased number of neuronal Nitric Oxide Synthase-ImmunoReactive (nNOS-IR) nerves per ganglion and a decreased time of onset of gastro-intestinal motility compared to the control. Especially B. animalis strain CNCM 1-2494, disclosed in International patent application WO 02/02800, showed good results. These effects were not observed when as a control a buffer solution was administered and were different from the effects observed upon butyrate administration.

A composition with lactic acid bacteria is therefore advantageously used in infants for decreasing gastro-intestinal transit time, increasing gastro-intestinal motility, increasing gastro-intestinal muscle contractility, gastro-intestinal peristalsis preventing delayed gastric emptying, increasing coordinated contractions of the gastro-intestinal system, decreasing the time of meconium evacuation, decreasing the time at which gastro-intestinal motility starts after birth and/or permitting earlier enteral feeding (tolerance) after birth.

A composition with lactic acid bacteria is therefore advantageously used in infants for treating and/or preventing of an ENS related disorder selected from the group consisting of ileus, gastroparesis, abdominal distention, laparoschisis, dysmotility syndrome such as constipation, and intestinal obstruction such as atresia.

Since further maturation of the ENS, in particular the maturation of ganglions by increasing the number of neurons per ganglion, occurs only during a limited period after birth, administration early after birth of a composition comprising lactic acid bacteria will have long term beneficial effects on the ENS extending beyond the time the composition is administered and therefore will have a preventive effect on the occurrence of ENS related disorders such as constipation and irritable bowel syndrome later in life.

Since preterm infants have an even more immature ENS than term born infants, and suffer more from the consequences thereof, the present composition with lactic acid bacteria is advantageously used in preterm infants. Also Caesarean section delivered infants, having a delayed intestinal colonization, will especially benefit from the present invention.

Since the present invention relates to administration to infants which have an immature ENS and hence implicitly an impaired gastro-intestinal motility, the present composition is preferably administered rectally.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions comprising at least one strain of lactic acid bacteria, preferably strains selected from the group consisting of Lactobacillus and Bifidobacterium, more preferably Bifidobacterium, for use in infants for obtaining improvements of the enteric nervous system, in view of improving its maturation and/or of treating and/or preventing enteric nervous system related disorders. Lactic acid bacteria used

Lactic acid producing bacteria used in the present invention are preferably provided as a mono- or mixed culture. Lactic acid bacteria as referred to in the present invention consists of the genera Bifidobacterium, Lactobacillus, Carnobacterium, Enter ococcus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Preferably the lactic acid bacteria used in the present invention comprises bacteria of the genus Lactobacillus and/or Bifidobacterium. Bifidobacterium is the dominant genus in the intestinal microbiota of breast-fed infants and is preferred over Lactobacillus.

Bifidobacteria are Gram-positive, anaerobic, rod-shaped bacteria. The

Bifidobacterium species used in the present invention preferably have at least 95 % identity of the 16 S rRNA sequence when compared to the type strain of the respective Bifidobacterium species, more preferably at least 97% identity as defined in handbooks on this subject, for instance Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory Press. The Bifidobacteria preferably used are also described by Scardovi V., Genus Bifidobacterium, p.1418 - p.1434, Bergey's manual of systematic Bacteriology, Vol. 2, Sneath, P.H.A., N.S. Mair, M.E. Sharpe and J.G. Holt (ed.), Baltimore: Williams & Wilkins, 1986, 635 p. Preferably the lactic acid producing bacteria used for fermentation comprises or is at least one Bifidobacterium selected from the group consisting of B. breve, B. infantis, B. bifidum, B. catenulatum, B. adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis, B. angulatum, B. pseudocatenulatum, B. thermacidophilum and B. Longum, more preferably B. breve, B. infantis, B. bifidum, B. catenulatum, B. longum, even more preferably B. animalis and B. breve, most preferably B. breve DN 156 007 (CNCM 1-2219 deposited on May 31^th 1999) disclosed in International patent application WO 01/01785, B. breve DN-156 032 (CNCM 1-4321 , deposited on May 19, 2010), B. animalis ATCC SD5220, B. animalis DN 173 010 (CNCM 1-2494 deposited on June 20^th, 2000) disclosed in International patent application WO 02/02800.

Lactobacilli are Gram-positive, anaerobic, rod-shaped bacteria. The present Lactobacillus species preferably have at least 95 % identity of the 16 S rRNA sequence when compared to the type strain of the respective Lactobacillus species, more preferably at least 97% identity as defined in handbooks on this subject for instance Sambrook J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory Press. Preferably the lactic acid producing bacteria used for fermentation comprises at least one, more preferably at least two Lactobacillus species selected from the group consisting of L. casei, L. reuteri, L. paracasei, L. rhamnosus, L. acidophilus, L. johnsonii, L. lactis, L. salivarius, L. crispatus, L. gassed, L. helveticus, L. zeae, L. fermentum and L. plantarum, more preferably L. casei, L. paracasei, L. rhamnosus, L. johnsonii, L. acidophilus, L. plantarum, L. fermentum and even more preferably L. plantarum strain DN 121 0304 (CNCM 1-4318 deposited on May 19^th 2010).

The present invention also provides new strains of lactic acid producing bacteria which are particularly suitable for use in the compositions of the invention. These are strains DN-156 032 (CNCM 1-4321) and DN 121 0304 (CNCM 1-4318), which have been deposited according to the Budapest Treaty, on May 19, 2010 at Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, Paris which is either CNCM (or ATCC (American Type Culture Collection)

B. breve strain DN-156 032 (CNCM 1-4321) has the following fermentation profile when tested on an API 20A gallery: Fermentation of D-glucose, D- mannitol, D-lactose, D-saccharose, D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-mannose, D-melezitose, D-raffinose, sorbitol. It also has the following characteristics: no spores formed, Gram + staining, and no coccoid forms.

L. plantarum strain DN 121 0304 (CNCM 1-4318) has the following fermentation profile when tested on an API 50 CH gallery: L-arabinose, D-xylose, D- galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, D- saccharose, D-trehalose, D-melezitose, D-raffinose, gentiobiose, potassium gluconate.

Preferably the present composition comprises lactic acid producing bacteria in an amount of at least lxl 0² cfu per g dry weight of the final composition, more preferably at least lxl 0⁴ cfu, even more preferably at least lxlO⁵ cfu. Preferably the present composition comprises lactic acid bacteria in an amount less than l xlO^{1 1} cfu per g based on dry weight of the final composition, more preferably less than lxl 0¹⁰ cfu, even more preferably less than lxl 0⁹ cfu per g dry weight.

Preferably the present composition, when present as a liquid, comprises lactic acid producing bacteria in an amount of at least l xlO³ cfu per 100 ml of the final composition, more preferably at least lxlO⁵ cfu, even more preferably at least lxlO⁶ cfu. Preferably the present composition, when present in liquid form, comprises lactic acid producing bacteria in an amount of less than 1x10 cfu per 100 ml of the final composition, more preferably less than 1x10 1 1 cfu, even more preferably less than 1x108 cfu per 100 ml.

Preferably the present composition, when present as a supplement, such as human milk fortifier, depository, pill, tablet or enema, comprises lactic acid bacteria in an amount of at least 1x10 cfu per g dry weight, more preferably at least 1x10 cfu, even more preferably at least lxl 0⁶ cfu. Preferably the present composition, when present as a supplement, such as human milk fortifier, depository, pill, tablet or enema, comprises lactic acid bacteria in an amount of less than lxlO¹² cfu per g based on dry weight of the final composition, more preferably less than 1x10 1 1 cfu, even more preferably less than 1x108 cfu per g dry weight. In one embodiment the cells are preferably inactivated. Living bacterial cells are preferably inactivated by methods selected from the group consisting of heat treatment, UV treatment, sonication, treatment with oxygen, treatment with bactericidals such as ethanol, ultra high pressure application, high pressure homogenisation and/or use of a cell disruptor. Preferably the lactic acid producing bacteria are heat killed after fermentation of the milk-derived product. Preferable ways of heat killing are pasteurization, sterilization, ultra high temperature treatment, spray cooking and/or spray drying at temperatures bacteria do not survive. Cell fragments are preferably obtained by heat treatment, sonication, treatment with bactericides such as ethanol, ultra high pressure application, high pressure homogenisation and/or use of a cell disruptor. Preferably intact cells of bacteria are removed from the fermented product by physical elimination such as filtration or centrifugation, for example centrifugation at 1 h at 3000 g, with the intact cells remaining in the pellet or retentate and the cell fragments remaining in the supernatant and/or filtrate, respectively. The inactivation and/or physical removal of living cells is such that the amount of living lactic acid producing bacteria is below detection limit as used by conventional plating techniques known in the art. This detection limit is less than 10 cfu living cells of lactic acid producing bacteria based on g dry weight composition.

Inactivation of living cells has the advantage that, after production, the final nutritional composition can be pasteurised and/or sterilised, consequently reducing the chance of contamination with harmful micro-organisms, such as E. sakazakii. This is especially of importance for preterm infants and/or caesarean delivered infants since they are more prone to infections.

So the present invention enables liquid, ready-to-use formula to be prepared and stored at room temperature. Furthermore, the dose of bioactive components received by each infant can be more easily controlled, since no further growth in a liquid product occurs, nor growth in the intestinal tract of the infant. The latter is a variable factor depending on the individual's intestinal environment, and thereby leads to variations in the extent of beneficial effects in individual infants.

Additional advantages are that the nutritional composition can be stored more easily and with reduced costs, since no special precautions have to be taken to maintain the viability of lactic acid producing bacteria at an acceptable level. This is especially the case in products with a water activity above 0.3. Also no post-acidification occurs in stored products with a high water activity and/or in infant formula in the period after reconstitution with water and before consumption. Adverse effect relating to coagulation of proteins and adverse taste are avoided in this way.

Preferably the present nutritional composition comprises inactivated lactic acid producing bacteria and/or bacterial fragments derived from lactic acid producing bacteria obtained from more than lxl 0² cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably lxlO⁴ cfu, even more preferably lxl 0⁵ cfu. Preferably the inactivated bacteria or bacterial fragments are obtained from less than lxl 0" cfu lactic acid producing bacteria per g based on dry weight of the final composition, more preferably lxl0¹⁰ cfu, even more preferably lxl 0⁹ cfu.

Optionally other lactic acid bacteria may be present in the composition.

Preferably the present composition is a fermented product.

Compositions

The composition used in the present invention are preferably nutritional and/or pharmaceutical compositions and suitable for administration to infants. The present composition is preferably enterally administered. Enteral administration includes oral administration, administration via a tube, which can end in the stomach or the duodenum, and rectal administration.

In case the composition is administered orally or via a tube, the composition is preferably a nutritional formula, preferably an infant formula. The present composition can be advantageously applied as a complete nutrition for infants. The present composition preferably comprises a lipid component, protein component and carbohydrate component and is preferably administered in liquid form. The present invention includes dry food (e.g. powders) which is accompanied with instructions as to mix said dry food mixture with a suitable liquid (e.g. water). Oral administration or administration via a tube has the advantage that the lactic acid bacteria also may have the beneficial effect in the small intestine, when they pass the gastro-intestinal tract.

The present invention advantageously provides an enteral food composition wherein preferably the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrates provides 15 to 90% of the total calories. Preferably, in the present composition, the lipid provides 35 to 50% of the total calories, the protein provides 7.5 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. For calculation of the % of total calories for the protein, the total of energy provided by proteins, peptides and amino acids needs to be taken into account. Protein is measured as the amount of nitrogen determined by Kjeldahl x 6.38 for milk protein and x 6.25 for non-milk protein.

The present composition preferably comprises at least one lipid selected from the group consisting of animal lipid (excluding human lipids) and vegetable lipids. Preferably the present composition comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil, algae oil, fungal oil, and bacterial oil. The present composition excludes human milk.

The protein used in the nutritional preparation is preferably selected from the group consisting of non-human animal proteins (preferably milk proteins), vegetable proteins (preferably soy protein and/or rice protein), hydrolysates thereof, free amino acids and mixtures thereof. The present composition preferably contains casein, whey, hydrolysed casein and/or hydrolysed whey protein. The use of hydrolysed proteins advantageously improves the absorption of the dietary protein component by the immature intestine of the infant.

The present composition preferably comprises digestible carbohydrates selected from the group consisting of sucrose, lactose, glucose, fructose, corn syrup solids, starch and maltodextrins, more preferably lactose.

The present composition preferably has a viscosity between 1 and 60 mPa.s, preferably between 1 and 20 mPa.s, more preferably between 1 and 10 mPa.s, most preferably between 1 and 6 mPa.s. The low viscosity ensures a proper administration of the liquid, e.g. a proper passage through the whole of a nipple or a tube. Also this viscosity closely resembles the viscosity of human milk. Furthermore, a low viscosity results in a normal gastric emptying and a better energy intake, which is essential for infants who need the energy for optimal growth and development. The present composition is preferably prepared by admixing a powdered composition with water. Normally infant formula is prepared in such way. The present invention thus also relates to a packaged powder composition wherein said package is provided with instruction to admix the powder with a suitable amount of liquid, thereby resulting in a liquid composition with a viscosity between 1 and 60 mPa.s. The viscosity of the liquid is determined using a Physica Rheometer MCR 300 (Physica Messtechnik GmbH, Ostfilden, Germany) at a shear rate of 95 s^"1 at 20 °C.

Stool irregularities (e.g. hard stools, insufficient stool volume, and diarrhoea) are an important problem in infants. It was found that stool problems may be reduced by administering the non-digestible oligosaccharides present in liquid food with an osmolality between 50 and 500 mOsm/kg, more preferably between 100 and 400 mOsm/kg.

In view of the above, it is also important that the liquid food does not have an excessive caloric density, however still provides sufficient calories to feed the subject. Hence, the liquid food preferably has a caloric density between 0.1 and 2.5 kcal/ml, even more preferably a caloric density of between 0.5 and 1.5 kcal/ml, most preferably between 0.6 and 0.8 kcal/ml.

Preferably the infant formula is especially adapted to be suitable for feeding preterm infants or small for gestational age (SGA) infants. In such a formula for preterm or SGA infants the enteral composition of the present invention preferably comprises lipid providing 35 to 50% of the total calories, protein providing 10 to 20% of the total calories, and digestible carbohydrate providing 30 to 50 % of the total calories. The lipid preferably comprises at least 15 % medium chain fatty acyl chains, having a length of from 6 to 12 carbon atoms, based on total fatty acids. Such medium chain fatty acyl chains are better digested by preterm or SGA infants. In one embodiment the present composition is administered as a human milk fortifier (HMF). HMF are compositions that are added to human milk, e.g. obtained either from the infants own mother or from a donor, in order to supplement the milk with extra nutrients. Human milk fortifiers are usually present as dose units which can be added to the human milk, mixed and subsequently the fortified human milk is administered to the infant. Feeding an infant with a HMF comprising lactic acid bacteria is especially advantageous for preterm or small for gestational age (SGA) infants. This administration is enterally and may be orally via a bottle with a teat or by tube feeding. Preferably the dose units are sachets comprising 0.5-10 g based on dry weight, more preferably 1-8 g, even more preferably 2-4 g. Preferably the human milk fortifier is a reconstitutable powder. A reconstitutable powder is a powder which can be dissolved in an aqueous liquid. Powdered human milk formulae have the advantage that they minimize the volume displacement of human milk.

Preferably the HMF is administered in a quantity of 0.1 to 20 g dry weight per day, more preferably 0.5 to 10 g dry weight per day.

Thus in one aspect the invention concerns a container comprising a human milk fortifier according to the invention in the form of a powder in an amount of from about 0.5 g to about 10 g per unit dose.

In one embodiment the human milk fortifier is present in the form of a concentrated liquid. Use of concentrated liquid human milk fortifiers may reduce the risk of microbial contamination associated with the preparation of an infant feeding. This is especially important in case of preterm infants.

Preferably the HMF comprises besides the protein and fat component also digestible carbohydrates. The HMF preferably comprises at least 10 % digestible carbohydrates based on total calories, more preferably at least 20 %, even more preferably at least 30 % based on total calories. Preferably the HMF does not comprise more than 70 % digestible carbohydrates based on total calories, even more preferably not more than 50 % based on total calories. Preferably the present HMF comprises at least 10 wt.% digestible carbohydrate based on dry weight, more preferably at least 20 wt.%, even more preferably at least 35 wt.% based on dry weight of the HMF. Preferably the HMF does not comprise more than 75 wt.% digestible carbohydrate based on dry weight, more preferably not more than 60 wt.%, even more preferably not more than 50 wt.% based on dry weight. Suitable sources of digestible carbohydrates are lactose and maltodextrin. Lactose advantageously resembles the carbohydrates source in human milk. Maltodextrin advantageously decreases the overall osmolality of the HMF.

A low osmolality of the present HMF will advantageously not increase the osmolality of the human milk after fortification. A too high osmolality of the fortified human milk increases the risk on necrotizing enterocolitis (NEC). NEC is especially a problem in preterm and/or SGA infants. Therefore the present fortified human milk when fortified with the present HMF preferably has an osmolality between 300 and 480 mOsm/kg.

With an immature ENS, gastro-intestinal motility and transit are lowered, or even impaired in severe cases Therefore in an especially preferred embodiment the present invention is administered rectally or anally, for instance in the form of a suppository or enema.

Infants

In the present invention, "infants" relate to human subjects with an age of 0 to 36 months, preferably 0 to 12 months. The present composition is administered to human subjects with the age of 0-36 months, more preferably an age of 0 to 12 months, even more preferably 0 to 6 months, most preferably from 0 to 2 months. The younger the infant is, the more effect on ENS maturation will be.

The present invention preferably relates to a method for feeding premature infants and/or small for gestational age (SGA) babies. A premature infant relates to an infant born before the standard period of pregnancy is completed, i.e. before or on 37 weeks from the beginning of the last menstrual period of the mother. SGA babies are those whose birth weight lies below the 10th percentile for that gestational age. They have usually been the subject of intrauterine growth restriction (IUGR). Premature and/or SGA infants include low birth weight infants (LBW infants), very low birth weight infants (VLBW infants), and extremely low birth weight infants (ELBW infants). LBW infants are defined as infants with a weight less than 2500 g. VLBW infants as infants with a weight which is less than 1500 g, and ELBW infants as infants with a weight less than 1000 g. Preterm infants are very vulnerable, especially in the first week after birth, and have an immature ENS.

The present invention preferably relates to the enteral administration of the composition of the present invention to infants delivered via caesarean section. A caesarean section (c-section) is a surgical procedure where an infant is delivered through an incision made in the mother's abdominal wall, and then through the wall of the uterus. A caesarean section is usually performed when it is safer for the mother or the infant than a vaginal delivery. Other times, a woman may choose to have a caesarean section rather than deliver her infant vaginally. C-section infants have a delayed and impaired colonization of the intestine after birth. Therefore the further maturation of the ENS is impaired in such infants. Most preferably the present composition is for use in preterm and/or SGA infants who are delivered by C-section.

Application

The present composition is preferably enterally administered, more preferably orally. The present composition is therefore preferably a liquid. The term "enteral" here used also encompasses a rectal or anal administration. In a further preferred embodiment the present composition is administered rectally or anally to the infant in a small volume, e.g. by "inoculating" the infant. Preferably, the present composition is administered to the infant as an enema with a syringe, pipette or tube, or in the form of a pill, tablet or suppository.

Hence, the present invention also provides a suppository, pill or table suitable for rectal administration to an infant, wherein said suppository, pill or tablet contains lactic acid bacteria.

This procedure has the advantage that it does not interfere with the normal breast-feeding practice and the lactic acid bacteria will reach their side of action, the colon, directly without having to make use of the gastro-intestinal tract with immature or even impaired motility or transit. The present invention also provides a method for administering to the infant within the first month after birth, preferably within two weeks after birth, more preferably within one week after birth, most preferably within 48 hours after birth.

The composition is preferably suitable for administration directly after birth. Hence, in a further preferred embodiment, the present invention provides a container comprising a liquid composition with a volume between 0.5 and 50 ml, which contains the present composition. The liquid with the present lactic acid bacteria can be suitably used in the present method. Preferably the liquid has a volume between 0.5 and 25 ml. This volume is preferably small, because it otherwise could interfere with the appetite and drinking behaviors of the infant.

Similarly and encompassed by this invention is a container with a reconstitutable dry composition containing the present composition, wherein the container has a volume of between 0.5 and 50 ml. This container is preferably accompanied with instruction to reconstitute the powder in a small volume of liquid, e.g. water.

The present composition is preferably administered at least once a day to the infant in need thereof. However, the frequency of administration and the dosage of said composition depend, for example, upon the delivery vehicle and the infant to whom the composition is administered. It is within the level of knowledge of one of skill in the art to determine the frequency of administration and the dosage of said composition according the infant to whom it is administered.

Preferably the composition is administered daily from birth until the infant is at least one week old, more preferably at least two weeks old. Preferably the composition is administered at least once a day to the infant for at least a period of one week, more preferably at least two weeks.

The inventors observed that the ENS of newborns was improved when lactic acid bacteria were administered. Therefore in one embodiment the present composition comprising at least one strain of lactic acid bacteria is used for use in maturation of the ENS in infants. In particular it is used to increase the number of neurons per ganglion, increase the percentage of ChAT per neuron, and/or increasing the percentage of nNOS per neuron.

Therefore in one embodiment the composition comprising at least one strain of lactic acid bacterium is for use in infants in decreasing gastro-intestinal transit time, increasing gastro-intestinal motility, increasing gastro-intestinal muscle contractility, gastrointestinal peristalsis preventing delayed gastric emptying, increasing coordinated contractions of the gastro-intestinal system, decreasing the time of meconium evacuation, decreasing the time at which gastro-intestinal motility starts after birth and/or permitting earlier enteral feeding (tolerance) after birth.

Therefore in one embodiment the composition comprising at least one strain of lactic acid bacterium is for use in infants in treating and/or preventing of a enteric nervous system related disorder selected from the group consisting of ileus, gastroparesis, abdominal distention, laparoschisis, dysmotility syndrome, in particular constipation, and intestinal obstruction. The disorder ileus is a disruption of the normal propulsive gastrointestinal motor activity often due to non-mechanical causes and includes for example post operative ileus, paralytic ileus, physiological ileus, acute colonic pseudo-obstruction and meconium ileus. Meconium ileus is a condition where the meconium, the first stool of an infant composed of materials ingested during the time the infant spends in the uterus becomes thickened and congested in the ileum. No meconium is passed, and abdominal distension and vomiting appear soon after birth. Normally meconium should be completely passed by the end of the first few days of postpartum life. Meconium ileus in context of the present invention also relates to the meconium plug syndrome, a disorder of the colon characterized by delayed passage of meconium and intestinal dilatation in which a tenacious mass of mucus prevents the meconium from passing. Intestinal obstruction includes atresia and/or stenosis of small intestine.

Since further maturation of the ENS, in particular the maturation of the ganglions by increasing the number of neurons per ganglion, occurs only during a limited period birth, administration early after birth of a composition comprising lactic acid bacteria will have long term beneficial effects on the ENS extending beyond the time the composition is administered and therefore will have a preventive effect on the occurrence of ENS related disorders such as constipation and irritable bowel syndrome later in life, i.e. when the infant has reached an age above 12 months, more preferably when it has reached an age above 3 years, even more preferably above 10 years.

Advantageously the lactic acid bacteria strains selected from the group consisting of Bifidobacterium breve DN 156 032 (CNCM 1-4321), Bifidobacterium animalis ATCC SD5220, Bifidobacterium animalis CNCM 1-2494, Lactobacillus plantarum CNCM I- 4318 and Bifidobacterium breve CNCM 1-2219, more preferably Bifidobacterium animalis CNCM 1-2494 and Lactobacillus plantarum CNCM 1-4318, were shown to increase the production of vaso-intestinal peptide (VIP) levels produced by enteric nervous system cells in an in vitro model in coculture with a monolayer of enteral epithelial cells (data not shown). Increased VIP levels advantageously result in decreased intestinal barrier (IEB) permeability. This is especially advantageous for the use in new born infants, who have an immature intestinal barrier function, and more particularly for the use in preterm infants with an even more impaired IEB function, even further decreased by stress. Increased VIP levels also advantageously result in an improved function of the ENS.

EXAMPLE 1

Pregnant Sprague-Dawley rats were obtained at 13-14 days of gestation. Rats were accustomed to laboratory conditions for at least 1 week before delivery and individually housed in cages on a 12: 12-h light-dark cycle with free access to food and water. Mothers and their pups (12-16 pups/litters) were kept in the same conditions during the whole experiment.

Newborn rats received from day 7 to day 17 after birth an enema (20 μΐ/g body weight) comprising Bifidobacterium animalis Bi07 (Danisco; ATCC SD5220) or Bifidobacterium animalis CNCM 1-2494. The dose was 2.10⁷ cfu/day, administered in a volume of 20 μΐ/g bodyweight. As a positive and negative control a butyrate solution (2.5 mM) and PBS (IX) was used respectively, and given at 20 μΐ/g body weight. At day 27, newborn rats were sacrificed.

Immunofluorescence staining

The mesenteric plexus of the proximal colon tissue was immuno-stained for Hu (protein used to label specifically neuronal cells), ChAT, and nNOS (neuronal nitric oxide synthase), by anti-HuC/HuD, anti-ChAT and anti-nNOS antibodies.

Segments of colon (2 cm from the caecum and 1 cm long) were fixed in 0.1 M phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) at room temperature for 3h at 4°C. Whole mounts of longitudinal muscle and myenteric plexus (LMMP) were obtained by removing the circular muscle by microdissection. Whole mounts of LMMP were first permeabilized with PBS /0.1% sodium azide /4% horse serum (HS) /triton IX (TX) for 3h at room temperature.

Tissues were then incubated sequentially with the primary antibodies for 3h and the secondary antibodies for lh in the following order: goat anti-ChAT (1 :200; Millipore, USA) and anti-goat Cy3 (carboxymethylindocyanine) (1 :500; Jackson Immuno Research, USA); rabbit anti-nNOS (1 : 1000; Alexis, USA) and anti-rabbit Cy5 (7-amino-4- indodicarbocyanin) (1 :500; Jackson Immuno Research, USA) and mouse anti-HuC/HuD (1 :200; Invitrogen, USA) and anti-mouse FITC (Flurorescein isothiocyanate) (1 :500; Jackson Immuno Research, USA).

Pictures were then acquired with a digital camera (model DP71, Olympus, France) coupled to a fluorescence microscope (Olympus IX 50) and analyzed with the Cell B software (Soft Imaging System, Olympus). To determine the general coding, an average of 500 neurons was analyzed for each animal and condition. The data are expressed as the percent of ChAT-IR or nNOS-IR neurons normalized to the total number of Hu-IR neurons. For neuronal density measurement, we counted 500 neurons per experimental condition and animal with the objective 40X.

Statistical analysis

The results are expressed as mean +/- standard error of the mean (s.e.m.). Statistical differences were determined by paired t-test or one way or two ways analysis of variance (ANOVA), followed by post hoc test, as appropriate, p values of 0.05 or less were considered statistically significant.

Results

The body weight was significantly increased at day 21 in rats having received the enema with Bifidobacteria, compared to rats having received the buffer. No such effect on growth was observed with butyrate enemas. See Table 1.

Also the length of the colon was increased in rats which had been exposed to the Bifidobacteria compared to the placebo. See Table 1. Butyrate did not have this effect. Table 1 : Effect of lactic acid bacteria enemas on bod wei ht ain and colon len th in newborn rats

^*p < 0.05 ANOVA compared to placebo

The number of neurons per ganglion as determined by Hu staining was increased in rats receiving the Bifidobacteria strains comprising enemas, in particular strain CNCM 1-2494, compared to the placebo, see Table 2.

Also the quality of the neurons was improved. The percentage of ChAT neurons was increased in rats receiving the lactic acid bacteria enemas, compared to the placebo, most significantly in rats receiving CNCM 1-2494, and the percentage of nNOS per neuron was increased in rats receiving the lactic acid bacteria enemas, compared to the placebo, see Table 2. Butyrate showed another effect. It did not have a significant effect on the number of neurons per ganglion, but did also increase ChAT/Hu and nNOS/Hu. Table 2: Effect of lactic acid bacteria enemas on the ENS in new born rats

^* p < 0.05 ANOVA, Bonferoni post hoc test

^** p = 0.07 Mann Whitney

^*** p < 0.05 ANOVA, Bonferoni post hoc test

^**** p < 0.05 Mann Whitney

These results are indicative for a beneficial effect of lactic acid bacteria, especially Bifidobacteria, in particular B. lactis, more preferably strain Bi 07 and CNCM I-

2494, most preferably CNCM 1-2494, on the maturation of ENS of the gut, in particular on

ChAT and NOS and in the establishment of digestive function. The effects observed cannot be explained by the production of short chain fatty acids alone, since the butyrate control showed a different physiological effect.

EXAMPLE 2

Pregnant Sprague-Dawley rats were at 13-14 days of gestation. After delivery, mothers and their pups were kept in the same conditions during the whole experiment. Newborn rats received from day 7 to day 17 after birth an enema (20 μΐ/g body weight) comprising Bifidobacterium animalis Bi07 or Lactobacillus plantarum CNCM I- 4318. Two daily doses were tested, being 10⁵ and 10⁷ cfu/day. As a negative control a PBS (IX) solution was used and given at 20 μΐ/g body weight.

At day 21 , rats were sacrificed and the colon was dissected, proximal and distal colon samples were stored for analysis. Proximal colon refers to the ascending and transverse colon while distal colon refers to the descending colon.

The mesenteric plexus of the proximal and distal colon tissue was immuno- stained for VIP, according to the method as described in Example 1, using anti-VIP antibodies.

Results are shown in Tables 3 and 4. The VIP level is expressed in ng/mL^g total proteins. Standard deviation (SD) is also indicated.

Enemas comprising lactic acid bacteria enhanced the VIP level in the proximal and distal colon. This effect was the highest with strain CNCM 1-4318. Table 3: Effect of lactic acid bacteria enemas on VIP level in roximal colon in new born rats

All pairwise multiple comparison procedure (Duncan's method)

*p value < 0.05 compared to PBS

Table 4: Effect of lactic acid bacteria enemas on the ENS in distal colon in newborn rats

All pairwise multiple comparison procedure (Duncan's method)

*p value < 0.05 compared to PBS

These results are indicative for a beneficial effect of lactic acid bacteria, on maturation of the ENS resulting in a more mature protected barrier function. Such an improved barrier function in newborns is beneficial as it may result in les infections and diseases as necrotizing enterocolitis

Claims

1) Composition comprising at least one strain of lactic acid bacteria, preferably strains selected from the group consisting of Lactobacillus and Bifidobacterium, more preferably Bifidobacterium, for use in infants for treating and/or preventing of an enteric nervous system related disorder selected from the group consisting of ileus, gastroparesis, abdominal distention, dysmotility syndrome such as constipation, laparoschisis, and intestinal obstruction.

2) Composition comprising at least one strain of lactic acid bacteria, preferably strains selected from the group consisting of Lactobacillus and Bifidobacterium, more preferably Bifidobacterium, for use in infants for obtaining an improvement of the enteric nervous system, said improvement being selected from the group consisting of decreasing gastro-intestinal transit time, increasing gastro-intestinal motility, increasing gastro-intestinal muscle contractility, gastro-intestinal peristalsis preventing delayed gastric emptying, increasing coordinated contractions of the gastro-intestinal system, decreasing the time of meconium evacuation, decreasing the time at which gastro-intestinal motility starts after birth and permitting earlier enteral feeding after birth.

3) Composition comprising at least one strain of lactic acid bacteria, preferably strains selected from the group consisting of Lactobacillus and Bifidobacterium, more preferably Bifidobacterium, for use in infants for obtaining an improvement of the maturation of the enteric nervous system.

4) Composition according to claim 3, wherein said improvement of the maturation of the enteric nervous system is selected from the group consisting of increasing the number of neurons per ganglion, increasing the percentage of ChAT neurones per ganglion, and increasing the percentage of nNOS neurons per ganglion.

5) Composition according to any one of the preceding claims, for use in preventing the occurrence later in life, when the infant has reached an age above 12 months, of an enteric nervous system related disorder selected from the group consisting of constipation and irritable bowel syndrome.

6) Composition according to any one of the preceding claims, wherein the infant has an age below 6 months, more preferably below 2 months.

7) Composition according to any one of the preceding claims, which is in a form to be administered rectally.

8) Composition according to any one of the preceding claims, wherein the infant is a premature infant or an infant delivered via Caesarean section, more preferably a premature infant, most preferably a premature infant delivered via Caesarean section.

9) Composition according to any one of the preceding claims, which is administered within the first month, preferably within the first week, after birth. 10) Composition according to any one of the preceding claims, which is administered during at least one week for at least once a day.

1 1) Composition according to any one of the preceding claims, wherein the lactic acid bacterial strain is selected from the group consisting of Bifidobacterium breve DN 156 032 (CNCM 1-4321), Lactobacillus plantarum DN 121 0304 (CNCM 1-4318), Bifidobacterium animalis ATCC SD5220, Bifidobacterium animalis DN 173 010 (CNCM I- 2494), and Bifidobacterium breve DN 156 007 (CNCM 1-2219), more preferably from the group consisting of Bifidobacterium animalis DN 173 010 (CNCM 1-2494) and Lactobacillus plantarum DN 121 0304 (CNCM 1-4318).

12) Composition according to any one of the preceding claims, wherein the composition comprises at least 10⁵ cfu lactic acid bacteria based on g dry weight.

13) Composition according to any one of the preceding claims, wherein the lactic acid bacteria is inactivated.

14) Composition according to any one of the preceding claims, wherein the composition is an infant milk formula, a human milk fortifier and/or a depository.

15) Composition comprising lactic acid bacteria, wherein the lactic acid bacteria strain is selected from the group consisting of Bifidobacterium breve DN 156 032 (CNCM 1-4321), Lactobacillus plantarum DN 121 0304 (CNCM 1-4318), Bifidobacterium animalis ATCC SD5220, Bifidobacterium animalis DN 173 010 (CNCM 1-2494), and Bifidobacterium breve DN 156 007 (CNCM 1-2219), more preferably from the group consisting of Bifidobacterium animalis DN 173 010 (CNCM 1-2494) and Lactobacillus plantarum DN 121 0304 (CNCM 1-4318), for use in rectal administration in infants, preferably preterm infants.