RU2815940C2 - Selection and use of melatonin-providing bacteria for reduction of infant colic - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to strains Lactobacillus reuteri DSM 32846, Lactobacillus reuteri DSM 32847, Lactobacillus reuteri DSM 32849, Lactobacillus reuteri DSM 33198, producing adenosine. Said strains are used to treat or prevent infant colic, irritable bowel syndrome (IBS) or sleep disorders in a subject. Also disclosed is a combination product suitable for inducing adenosine production in a subject, comprising one of said strains and a component of the substrate, which is AMP.

EFFECT: invention can be effectively used to treat or prevent infant colic, IBS or sleep disorders in a subject.

9 cl, 5 dwg, 7 ex

Description

Field of technology to which the invention relates

The present invention provides certain strains of lactic acid bacteria selected for their ability to increase melatonin levels for the prevention and/or treatment of colic (or other diseases associated with low melatonin levels), including the ability to produce precursors and other important components for the release of melatonin, a method for selecting such strains and products containing such strains. In addition, this invention relates to preparations comprising substrate components specifically selected to enhance the effectiveness of such strains.

State of the art

Despite their importance in terms of prevalence and the suffering they cause, the nature and causes of infant colic remain poorly understood. There are also different terms used to describe this condition. These terms include terms such as "infantile colic", "evening colic" because the onset of pain is mainly limited to the evening, and "three-month colic" on the pretext that it disappears approximately three months after birth. Different authors use different definitions. The literature generally accepts Wessel's definition of colic as paroxysms of crying for three or more hours a day for three or more days a week for at least three weeks. In 2006, the Rome III criteria were published and modified this criterion to consider the diagnosis of infant colic to apply to infants with episodes of irritability, fussiness, or crying that start and stop without an obvious cause, lasting 3 hours or more. per day and occurring at least 3 days per week but not less than 1 week (sometimes called modified Wessel criteria). The Rome IV criteria represent a further refinement of the criteria (Benninga et al; Gastroenterology 2016; 150: 1443–1455). Either of these definitions (Rome III or Rome IV) applies to the present invention.

To date, the main discussed possible causative factors for infantile colic are divided into: psychosocial, gastrointestinal, including microbial dysbiosis, and neurodevelopmental disorders.

Psychosocial factors include variations in normal crying, behavioral effects of atypical parenting, and the manifestation of problems in parent-infant interactions.

Gastrointestinal disturbances due to the position of the infant's legs and grimacing during a crying episode may be implicated in colic. Below is a brief overview of gastrointestinal factors:

Incorrect feeding practices such as bottle feeding, horizontal feeding and failure to burp after feeding have been implicated as causative factors. Breastfeeding in the first six months has been found to be a protective factor. The risk of infantile colic was 1.86 times higher among non-breastfed infants (Saavedra MA, Dacosta JS, Garcias G, Horta BL, Tomasi E, Mendoca R. Infantile colic incidence and associated risk factors: a cohort study. Pediatr (Rio J) 2003;79(2):115-122).

There is currently no complete cure for colic. The current treatment paradigm for colic consists of pharmacological and/or non-pharmacological options that, at best, provide symptomatic relief. Typical therapeutic interventions for colic offered to parents fall into four categories, including dietary, physical, behavioral, and pharmacological. Dietary interventions include professional advice on different feeding methods or the use of hypoallergenic milk, soy-free or lactose-free formulas, nutritional supplements such as various probiotics and prebiotics, and the early introduction of solid foods.

Over-the-counter medication to treat colic generally involves administering simethicone or dimethylpolysiloxane, a non-absorbable, over-the-counter drug that reduces the size of intestinal gas bubbles. Simethicone has a safe profile and is often recommended despite the results of several studies demonstrating that simethicone's effectiveness for infant colic is no better than placebo.

There have been reports of altered microbiota structure in colicky infants, prompting several researchers to conduct and publish various randomized clinical controlled trials (RCTs) that assessed the ability of, for example, Lactobacillus reuteri DSM 17938 to reduce crying time in these infants. For example, Savino et al (Pediatrics 2010;126:e526-33) conducted an RCT in 2010 to test the effectiveness of this strain for infantile colic. Fifty exclusively breastfed colicky infants diagnosed according to modified Wessel criteria were randomly assigned to receive either L. reuteri DSM 17938 (10 ⁸ colony-forming units) or placebo daily for 21 days. Parent questionnaires tracked daily crying time and side effects. 46 infants (L. reuteri group: 25; placebo group: 21) completed the trial. Daily crying time in minutes/day (median) was 370 vs. 300 on day 0 and 35.0 vs. 90.0 (p = 0.022) on day 21.

In 2013, Szajewska et al (J Pediatr 2013; 162:257-62) published an RCT with a similar design in 80 infants <5 months of age, indicating that the response rate was significantly higher in the probiotic group compared to placebo . Minutes per day (median) were 180 vs. 180 on day 7, 75 vs. 128 on day 14, and 52 vs. 120 (p<0.05) on day 21. There have also been several other studies showing the effect of L. reuteri DSM 17938 on colicky infants, which was a significant reduction in crying time in breastfed infants. However, there is a need for even more effective interventions to reduce higher crying rates in colicky infants, including formula-fed infants.

Accordingly, there is currently a need for new (and preferably improved) safe and effective compounds, compositions and methods that will be useful for treating colic in infants. The compositions and methods of the present invention meet this need by providing products that can safely and effectively prevent and treat colic (or symptoms associated with colic) in infants.

Other purposes and advantages will become more apparent from the following disclosure.

Disclosure of the Invention

Hormonal immaturity in the newborn is considered a major factor in early intestinal motility problems and thus causes pain and crying in the infant, such as in infantile colic. Melatonin is a hormone that has the ability to maintain biological rhythms and has an important effect on many functions in humans and animals. Serotonin, also a neurotransmitter that can be derived from the amino acid tryptophan, is a precursor to melatonin.

Melatonin is a neurotransmitter that was previously thought to be secreted primarily by the pineal gland. However, there are extrapineal sites of melatonin production, such as the retina and the gastrointestinal tract.

Synchronization of production with different circadian rhythms and subsequent release of serotonin and melatonin in infants is potentially important for the prevention of intestinal motility disorders and pain such as infantile colic (IC).

Serotonin increases the contraction of intestinal smooth muscle, and melatonin relaxes intestinal smooth muscle. Peaks in serotonin concentrations can cause intestinal spasms if not balanced by other agents, of which melatonin is an important agent. Both serotonin and melatonin have circadian rhythms. While serotonin is produced and the serotonin circadian rhythm develops at birth, endogenous melatonin production and the melatonin circadian rhythm in many infants are not ready to begin until the 3rd month of life. This time coincides with the normal disappearance of infant colic.

We can say that infant colic is a condition of melatonin deficiency. Therefore, it has been proposed to give exogenous melatonin to infants with IC, but many clinicians and parents are reluctant to give hormones to infants.

Among all newborn infants, the prevalence of IC is estimated to range from 5% to 25%, which is burdensome and costly in many ways to society, parents, and the infant.

So while some babies appear to be able to produce and release the necessary melatonin from food and/or gut bacteria and are therefore less likely to develop infant colic, there are many others who are not able to do so and therefore need in additional support to prevent or treat IC.

Thus, there is a need to find and develop products and methods to support melatonin production and release in children at risk of developing IC or having IC.

The present invention is based on the discovery that it is possible to select bacteria, for example lactic acid bacteria, which are capable of producing adenosine. Lactic acid bacteria that produce adenosine are not described in the art, and such bacteria, for example, can be used to increase the production of arylalkyl amino-N-acetyltransferase (AANAT), the rate-limiting enzyme in melatonin synthesis. Although the AANAT enzyme is located in the pineal gland, it is also produced in extrapineal melatonin production sites, such as the gastrointestinal tract. Thus, increasing AANAT production (eg, by increasing adenosine production) provides a means to increase melatonin production or increase melatonin levels in the gastrointestinal tract. Such bacteria may thus be useful for the treatment or prevention of infant colic or other diseases associated with or characterized by reduced, low or deficient melatonin production or synthesis.

Thus, the present invention provides a bacterial strain, for example, a strain of lactic acid bacteria, that is capable of producing or inducing the production of adenosine, for use in the production of adenosine (for example, to increase adenosine levels or increase or stimulate the production of adenosine) in a subject, or for use in a product melatonin (eg, to increase melatonin levels or increase or stimulate melatonin production) in a subject.

The present invention also provides a method for producing adenosine or melatonin (e.g., increasing levels of adenosine or melatonin or increasing or stimulating production of adenosine or melatonin) in a subject, said method comprising the step of administering to said subject an effective amount of a bacterial strain, e.g., a strain of lactic acid bacteria, which is capable of produce or induce the production of adenosine.

The present invention also provides the use of a bacterial strain, for example a strain of lactic acid bacteria, that is capable of producing or inducing the production of adenosine, in the manufacture of a medicament or composition for use in the production of adenosine or melatonin (for example, to increase the level of adenosine or melatonin, or to increase or stimulate the production adenosine or melatonin) in the subject.

More generally, the present invention also provides bacteria, for example lactic acid bacteria, which are capable of producing or inducing the production of melatonin. Thus, such bacteria can be used to produce melatonin, for example, to increase melatonin production or levels in a subject.

Thus, in one aspect, the present invention provides a strain of lactic acid bacteria that is capable of producing or inducing melatonin production, for use in melatonin production (eg, to increase melatonin levels or increase or stimulate melatonin production) in a subject.

In another aspect, the present invention provides a method of producing melatonin (e.g., increasing melatonin levels or increasing or stimulating melatonin production) in a subject, the method comprising the step of administering an effective amount of a strain of lactic acid bacteria that is capable of producing or inducing melatonin production to the subject.

In another aspect, the present invention provides the use of a strain of lactic acid bacteria that is capable of producing or inducing melatonin production in the manufacture of a medicament or composition for use in melatonin production (eg, for increasing melatonin levels or increasing or stimulating melatonin production) in a subject.

As discussed above, such strains can be used in the treatment or prevention of infantile colic or other diseases associated with or characterized by reduced, low or insufficient production or synthesis or levels of melatonin.

Thus, the invention also relates to a strain of lactic acid bacteria that is capable of producing or inducing the production of melatonin, for use in the treatment or prevention of infantile colic or other diseases associated with or characterized by reduced, low or insufficient production or synthesis or levels of melatonin in a subject.

In another aspect, the present invention provides a method of treating or preventing infantile colic, or other disease associated with or characterized by reduced, low or deficient melatonin production or synthesis or levels, in a subject, said method comprising the step of administering an effective amount of a strain of lactic acid bacteria that is capable of produce or induce the production of melatonin in the specified subject.

In another aspect, the present invention provides the use of a strain of lactic acid bacteria that is capable of producing or inducing the production of melatonin in the manufacture of a medicament or composition for use in the treatment or prevention of infantile colic, or other disease associated with or characterized by reduced, low or insufficient production or synthesis or melatonin levels in the subject.

Such strains may be capable of producing or inducing the production of adenosine and thereby producing or inducing the production (eg, production downstream of the metabolic pathway) of melatonin, as described elsewhere herein.

Other objects and advantages of the present invention will become apparent to the reader, and it is intended that these objects and advantages fall within the scope of the present invention.

Brief description of drawings

Fig. 1 is a diagram showing the pathway of melatonin production.

Fig. 2 is a diagram showing how bacteria capable of producing adenosine according to the invention affect the melatonin production pathway.

Fig. 3 shows the measurement of 5'-nucleotidase activity in the supernatant of L reuteri bacterial cells.

Fig. 4 shows the production of melatonin in an intestinal enteroid model by adding supernatant of L reuteri bacterial cells.

Fig. 5A shows measurement of 5'-nucleotidase activity in the supernatant of L. reuteri DSM 33198 bacterial cells compared to DSM 17938 bacterial cells. FIG. 5B shows measurement of 5'-nucleotidase activity in bacterial cell supernatant of various L. reuteri bacterial cells compared to DSM 17938 bacterial cells.

Carrying out the invention and its preferred embodiments

Thus, as stated above, the present invention provides strains of bacteria, for example strains of lactic acid bacteria, which are capable of producing, stimulating, inducing or maintaining melatonin production, for example, to increase melatonin levels, especially in the gastrointestinal tract (GIT). Such strains may themselves produce melatonin, for example, secrete melatonin and thus directly provide or produce melatonin.

However, strains are also provided that indirectly lead to the production of melatonin, for example, by producing, stimulating or inducing the production of a component found in previous stages of the melatonin synthesis pathway (or a melatonin precursor) and thereby maintaining or increasing melatonin production. A preferred example is the production or induction of arylalkyl amino-N-acetyltransferase (AANAT), which is a rate-limiting enzyme in the conversion of serotonin to N-acetylserotonin, which in turn is converted to melatonin.

As stated above, another particularly preferred strain of lactic acid bacteria is one that can produce or induce the production of adenosine in a subject, especially in the gastrointestinal (GI) tract. Such adenosine can act on the corresponding cell surface adenosinosine receptors (eg, A2A receptors, A2B receptors, A3 receptors or A4 receptors, preferably A2A receptors) and lead to the production of increased levels of intracellular cAMP. A2A and other adenosine receptors are known to be present in cells of the gastrointestinal tract. Intracellular cAMP, in turn, is involved in the production of AANAT (see Fig. 2). Thus, increased intracellular cAMP levels may lead to increased AANAT levels and therefore increased melatonin levels. Adenosine, therefore, must preferably be supplied extracellularly in order to be able to bind to adenosine receptors on the cell surface. For this purpose, adenosine can be produced in bacterial cells and removed from the cell (or secreted). Alternatively, adenosine may be produced extracellularly, for example on the surface of bacterial cells or in the supernatant. Such extracellular production may, for example, be conveniently accomplished by the presence of a cell wall (or cell surface)-anchored 5'-nucleotidase enzyme (or ecto-5'-nucleotidase enzyme) which can convert the appropriate substrate to adenosine. Such extracellular production may equally result from the presence of the 5'-nucleotidase enzyme (or ecto-5'-nucleotidase enzyme) in both the cellular supernatant and the extracellular space. As shown herein, bacterial strains can provide, produce or release such a 5'-nucleotidase enzyme (or ecto-5'-nucleotidase enzyme) into the cellular supernatant or into the extracellular space, where it can then convert the corresponding substrate to adenosine. Suitable substrates include AMP.

Thus, when bacterial strains are referred to herein as being capable of producing adenosine, this means, inter alia, direct production of adenosine by the bacterial cells themselves, as well as production of adenosine by bacterial cells through cells having an active 5'-nucleotidase enzyme (which, for example , is present on the surface of the bacterial cell or is released into the cell supernatant or extracellular space) and thus converts or is capable of converting the corresponding substrate, for example, AMP, into adenosine. Such a substrate may be present in a conventional manner, for example endogenously in the environment, or may be provided in bacteria, for example through exogenous means.

Thus, such strains may result in increased melatonin levels, for example, compared to levels at which such strains are absent.

Melatonin is known to be produced in the gastrointestinal tract, and therefore production or increased production in this area should be physiologically effective. Preferably, the amount of melatonin produced is clinically or therapeutically significant. Thus, the strains of the invention can be used to treat any disease or condition associated with (or characterized by) reduced or reduced levels of melatonin, or associated with (or characterized by) melatonin deficiency, or they can be used to treat any disease or condition in which increased melatonin levels may benefit. Those skilled in the art will readily recognize such diseases or conditions (and certainly subjects suffering from such diseases or conditions) which will, for example, include diseases, conditions or subjects with low, reduced, for example, significantly reduced (or abnormal) levels of melatonin or melatonin deficiency, for example, compared with levels in a normal or healthy subject, for example, with levels in a normal or healthy subject of the same, equivalent or comparable age. A preferred example of such a disease is infantile colic. Preferred strains for such therapeutic purposes are strains that produce melatonin or adenosine.

Thus, in additional aspects of the invention, a strain of lactic acid bacteria is provided that is capable of producing or inducing the production of melatonin, for example, because the strain is capable of producing or inducing the production of adenosine, or a strain of lactic acid bacteria that is capable of producing or inducing the production of adenosine. Therapeutic use of such strains is also provided. Preferably, said strains contain a gene encoding a 5'-nucleotidase, or such strains have an active 5'-nucleotidase enzyme, for example to convert AMP (or other suitable substrate) to adenosine. Typical levels of adenosine production or levels of 5'-nucleotidase activity or melatonin production are described elsewhere herein.

Preferred strains of the present invention are L. reuteri strains, more preferably L. reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 (details of the depositories of these strains are provided elsewhere herein) and such strains, for example, isolated strains or biologically pure cultures, or preparations of such strains, constitute additional aspects of the invention, as do compositions (for example, pharmaceutical or nutritional, for example, food additives, or probiotic compositions, for example, with pharmaceutically or nutritionally acceptable diluents and/ or excipients) comprising said strains, or the therapeutic use of such strains, for example as described elsewhere herein, particularly for the treatment or prevention of infant colic (or other diseases as described elsewhere herein). In some embodiments, strain DSM 17938 is not used. Thus, the present invention provides Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 for therapeutic use, eg, for the treatment or prevention of diseases, as described elsewhere herein. The present invention further provides, for example, Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM33198 for use in the treatment or prevention of infant colic.

Thus, yet another additional aspect of the invention provides a method of treating or preventing infantile colic, or other diseases, as described herein, in a subject, which method includes the step of administering an effective amount of Lactobacillus reuteri strain DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 to the specified entity.

In another aspect, the present invention provides the use of Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 in the manufacture of a medicament or composition for use in the treatment or prevention of infant colic, or other disease as described elsewhere herein, from the subject.

Alternative and preferred embodiments and features of the invention, as described elsewhere herein, are equally applicable to the treatments and uses of the invention described herein and elsewhere herein.

L. reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 were selected for their ability to produce adenosine, for example, because they had a gene encoding a 5'-nucleotidase and an active 5'-nucleotidase enzyme that can convert the substrate AMP to adenosine, and is suitable not only for increasing adenosine levels (for example, compared to levels in which this trait is absent), but also for increasing melatonin levels, for example, by increasing AANAT levels as described elsewhere herein . The strains contain active 5'-nucleotidase enzyme anchored in the cell wall and also exhibit 5'-nucleotidase enzyme activity in their respective supernatants (see Fig. 3 and Fig. 5 A/B).

These strains of L. reuteri have been developed (in other words, modified, adapted or genetically altered) from natural strains, and have also been selected for one or more other improved properties, such as increased resistance to bile or increased adhesion to mucosal surfaces, e.g. , to the surface of the gastrointestinal tract. Thus, DSM 32846 and DSM 32847 have been modified to be more tolerant of bile acids and thus, for example, they survive in greater quantities in the gastrointestinal tract. DSM 32848 and DSM 32849 have been modified for better mucosal adhesion to improve colonization in the gastrointestinal tract and thus improve function, for example, according to the present invention. L. reuteri strain DSM 33198 has also been modified through a multi-step selection process, including a repeated freeze-drying procedure, to make it more tolerant and provide higher survival during production than its native isolate (parent strain). Thus, such strains do not correspond to strains found in nature, they have been forcibly genetically altered, and they are non-native strains. All Lactobacillus reuteri strains selected were DSM 32846, DSM 32847, DSM 32848, DSM 32849 and DSM 33198. Preferred strains were DSM 32846, DSM 32847, DSM 32849 or DSM 33198. More preferred strains were DSM 32846, DSM 32 847 or DSM 33198.

In one embodiment, Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848 and/or DSM 32849 are selected or used. Preferred strains are DSM 32846, DSM 32847, or DSM 32849. More preferred strains are DSM 32846 or DSM 32847, for example, DSM 32846 is a preferred strain in some embodiments. In some embodiments, strain DSM 33198 is selected or used.

The present invention provides the use of a bacterial strain, e.g., a strain of lactic acid bacteria, that is capable of producing adenosine or inducing adenosine production (e.g., increasing adenosine levels, or increasing or stimulating adenosine production), in the manufacture of a medicament or composition for use in producing adenosine in a subject.

Such methods and uses may be used to treat or prevent infantile colic or other diseases as described elsewhere herein, such as irritable bowel syndrome (IBS), sleep disorders, or neurodegenerative diseases or disorders, such as Alzheimer's disease or other types of dementia, such as other types of senile dementia. Such diseases are also examples of diseases associated with (or characterized by) a deficiency or decrease in melatonin, or which may benefit from elevated or increased levels of melatonin.

Gastrointestinal motility refers to the stretching and contraction of the muscles of the gastrointestinal tract to transmit digested food. The synchronized contraction of these muscles is called peristalsis.

The gastrointestinal tract contains many locally released factors, including hormones that regulate motility and secretion. Motility is determined by the movements of the digestive system and the movement of contents in it. When nerves and/or muscles in any part of the digestive tract, the motor function of the gastrointestinal tract, do not function with normal strength and coordination, a person develops symptoms associated with dysmotility.

Motility disorders have been described to cause a variety of symptoms in different areas of the intestine, such as in subjects with infantile colic.

Infant colic is a term used to describe babies who cry excessively for no apparent reason during the first three months of life. Colic is one of the most distressing problems of infancy. This is unpleasant for the baby, the parents, and the health care provider. The cause of colic is not fully understood.

Hormonal immaturity in newborns is considered a major factor in early developmental problems with intestinal motility and is also responsible for pain and crying in the infant, for example, with infant colic.

Melatonin is a hormone that has the ability to maintain biological rhythms and has important effects on many functions in humans and animals. The hormone influences gastrointestinal motility through selective receptors (melatonin receptors) expressed in smooth muscle and myenteric plexus cells of the gastrointestinal tract. Cyclic generation of electrical current is the main mechanism of contraction of smooth muscles of the gastrointestinal tract. The myoelectric activity of the gastrointestinal tract consists of two types of electrical potentials: slow wave and spike activity organized in the myoelectric migratory complex.

Serotonin is a precursor to melatonin, which is itself a neurotransmitter and can be derived from the amino acid tryptophan.

Melatonin has been shown to partially block the serotonin-induced reduction in food movement. This system, which balances melatonin and serotonin, regulates bowel activity.

In addition, a functional and balanced melatonin-serotonin system influences appetite and digestive processes through endocrine as well as paracrine effects in both the brain and gastrointestinal tract.

The two hormones appear to have opposing effects on motility, and the final effects are dose dependent. Melatonin and serotonin are also known to be involved in hypersensitivity processes and pain management.

Melatonin, also known as N-acetyl-5-methoxytryptamine, is a hormone and neurotransmitter previously thought to be secreted primarily by the pineal gland. But there are important extrapineal sites of melatonin production, such as the retina and the gastrointestinal tract. There are now reports showing that the gastrointestinal tract contains at least 400 times more melatonin than the pineal gland. Circadian fluctuations of gastrointestinal melatonin appear to be controlled not by light (like the pineal gland), but by food intake and food composition.

Synchronization between production and the various circadian rhythms and subsequent release of serotonin and melatonin in young children is important to prevent intestinal motility disorders and pain such as infantile colic (IC).

Serotonin increases the contraction of intestinal smooth muscle, and melatonin relaxes intestinal smooth muscle. Peaks in serotonin concentrations can cause intestinal spasms if not balanced by other agents, of which melatonin is very important. Both serotonin and melatonin have circadian rhythms. Although serotonin is produced and the serotonin circadian rhythm develops at birth, endogenous melatonin production and the melatonin circadian rhythm in many infants are not ready to begin until the 3rd month of life. This time coincides with the normal disappearance of infant colic. Colicky infants have been shown to have low morning melatonin levels, while non-colicky infants have high morning melatonin levels (Cengiz et al, 2015, Turkish journal of family medicine and primary care; 9 (1)/10- 15). Such infants (eg, infants with forms of infantile colic associated with a deficiency of melatonin or melatonin balance, e.g., deficient melatonin levels in the morning, e.g., at or before 6 a.m., 8 a.m., 10 a.m., or 12 p.m.) are examples of subjects who can be treated using the present invention.

Thus, although some infants appear to be able to produce and release the necessary melatonin endogenously, from food and/or gut bacteria, and are therefore less likely to develop infantile colic, there are many other infants who do not. and therefore they need additional support to prevent or treat IC. Such infants (eg, infants with melatonin deficiency caused by, for example, an incompletely developed gut microbiome, or infants suffering from nutritional problems or feeding problems) are also examples of subjects who can be treated with the present invention.

We can say that infant colic is a condition of melatonin deficiency. This is especially important for children in stressful situations or for children who are less able to cope with stress, for example, for children born before full gestational age or with not fully functional nutritional metabolism, etc. Such infants (eg, melatonin-deficient infants born before full gestation, by cesarean section, or without fully functional nutritional metabolism) are also examples of subjects that can be treated using the present invention.

Another example of a condition related to the balance of melatonin function is irritable bowel syndrome (IBS), which is a common condition characterized by recurring abdominal pain or discomfort coupled with irregular bowel movements in the absence of an identifiable cause. Melatonin has been suggested to have a positive effect on pain perception and bowel rhythm (Siah KTH et al, 2014, World J Gastroenterol; 20 (10): 2492-2498). Thus, subjects suffering from IBS, particularly forms of IBS associated with melatonin deficiency or melatonin imbalance, are also examples of subjects who can be treated using the present invention.

Melatonin deficiency has mainly been studied in the pineal gland, bloodstream, saliva, cerebrospinal fluid and, by measuring the metabolite 6-sulfatoxymelatonin, in urine.

The intestine contains more than 20 types of enteroendocrine cells, making it the largest endocrine organ in the human body. Enterochromaffin cells (ECs) are a type of enteroendocrine and neuroendocrine cells. They are located next to the epithelium lining the lumen of the digestive tract and play a crucial role in the regulation of the gastrointestinal tract, especially intestinal motility and secretion. EC cells modulate neuronal signaling in the enteric nervous system (ENS) through the secretion of the neurotransmitter serotonin and other peptides, they additionally express adenosine receptors and have active melatonin synthesis.

Sleep disturbance is one of the common signs of insufficient melatonin signaling, and melatonin is one of the most popular natural health supplements today. Melatonin is often called the “darkness hormone” because its synthesis and secretion in some parts of the body is enhanced by darkness and suppressed by light. Sleep disorders are common in children and without appropriate treatment can become chronic and last for many years (Esposito et al.2019, J Transl Med, 17:77). Even older people have been reported to have disrupted sleep. The nocturnal peak of melatonin regularly decreases with age. Thus, subjects having or suffering from sleep disorders or sleep disorders, particularly forms of sleep disorders or sleep disorders associated with melatonin deficiency or melatonin imbalance, are also examples of subjects who can be treated using the present invention.

Decreased melatonin levels have also been repeatedly described in neurodegenerative disorders, especially Alzheimer's disease and other types of senile dementia (Blumenbach Johann Friedrich, 2012, The Scientific World Journal Volume 2012, Article ID 640389). Thus, subjects having such neurodegenerative disorders, especially those forms of neurodegenerative disorders that are associated with melatonin deficiency or melatonin imbalance, are also examples of subjects that can be treated using the present invention.

The rate of melatonin production depends on the activity of the enzyme arylalkylamine-N-acetyltransferase (AANAT) (Fig. 1 and Fig. 2) in the pineal gland, but also at extrapineal sites of melatonin production, for example, in the gastrointestinal tract.

Adenosine increases intracellular cAMP levels through the adenosine receptor, which increases the production of AANAT, the rate-limiting enzyme in melatonin synthesis.

The purpose of the present invention is to use adenosine-producing strains of lactic acid bacteria for the prevention and/or treatment of infant colic.

Another object of the present invention is the use of adenosine-producing strains of lactic acid bacteria for the prevention and/or treatment of IBS.

Another object of the present invention is to use adenosine-producing strains of lactic acid bacteria for the prevention and/or treatment of sleep disorders.

Another object of the present invention is to use adenosine-producing strains of lactic acid bacteria for the prevention and/or treatment of neurodegenerative disorders, such as Alzheimer's disease and other types of dementia, such as other types of senile dementia.

This invention also relates to a method for selecting specific bacterial strains, including lactic acid bacteria strains, capable of producing adenosine, and the use of such strains to provide beneficial effects for an infant suffering from colic or at risk of colic. New bacterial strains that are capable of producing adenosine represent a further object of the invention.

Purine metabolism, in which purines are metabolized and broken down by certain enzymes, is one of the important metabolic processes in the human body. Ecto-5'-nucleotidase (CD73), which is considered a key enzyme in adenosine production, is one example of such enzymes.

The inventors unexpectedly discovered that certain specific strains of probiotic bacteria are capable of producing adenosine.

The present invention thus includes a new method for selecting specific bacterial strains, including lactic acid bacteria strains, that are effective in producing adenosine. The purpose of selecting specific strains of bacteria is for their use in the treatment of specific diseases, such as infantile colic (or other diseases, as described elsewhere in this document).

The present invention provides a method for selecting strains of bacteria, especially strains of lactic acid bacteria, that are useful as probiotics and in therapeutics, for example as pharmaceuticals or nutritional supplements.

Thus, one aspect of the present invention provides a method for selecting a bacterial strain, preferably a strain of lactic acid bacteria, capable of producing adenosine, the method comprising:

a) screening a bacterial strain, for example a strain of lactic acid bacteria, for the presence of a gene encoding a 5'-nucleotidase, for example a cell wall-anchored 5'-nucleotidase; and/or

b) screening a bacterial strain, for example a strain of lactic acid bacteria, or its supernatant for the presence of an active 5'-nucleotidase enzyme, for example a 5'-nucleotidase enzyme anchored in the cell wall.

Such 5'-nucleotidase (5'NT) enzymes may also be referred to as ecto-5'-nucleotidase or CD73 (cluster of differentiation 73) enzymes. Strains that are capable of producing adenosine can then be selected.

Alternatively, the present invention provides a method for selecting a bacterial strain, preferably a lactic acid bacteria strain, by screening a bacterial strain, for example a lactic acid bacteria strain, for its ability to produce adenosine and selecting a strain having such ability.

Once an appropriate strain has been selected using the method of the present invention, it can then be used to produce, eg locally, adenosine in a subject.

A bacterial strain, for example a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the methods of the present invention, in which the specified strain is capable of producing adenosine, for example, producing adenosine locally, represents another additional aspect of the invention.

In addition, the strains selected by the present invention provide therapeutic use for infantile colic (or other diseases as described elsewhere herein).

Thus, a further aspect of the present invention provides a bacterial strain, for example a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the methods of the present invention, wherein said strain has a 5'-nucleotidase gene or an active 5'-nucleotidase enzyme and is capable of producing adenosine, for use in products, such as topical products, of adenosine in a subject.

Alternative embodiments of the invention provide a bacterial strain, for example a strain of lactic acid bacteria, wherein said strain has a 5'-nucleotidase gene or an active 5'-nucleotidase enzyme and is capable of producing adenosine, for use in the production, for example, topical production, of adenosine in a subject. Preferred features of this strain and its use are described elsewhere herein.

As will be stated elsewhere herein, a particular embodiment of the invention is the prevention and/or treatment of infantile colic (or other diseases as described elsewhere herein).

In addition, methods of treating or methods of producing, for example, local production, adenosine in a subject are provided, these methods include administering a bacterial strain, for example, a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the selection method of the invention, or introducing a bacterial strain, for example, a strain of lactic acid bacteria, wherein said strain has a 5'-nucleotidase gene or an active 5'-nucleotidase enzyme and is capable of producing adenosine in said subject in an amount effective to produce, for example, locally, adenosine in said subject. Preferred features of this strain and its use, for example, in infantile colic (or other diseases as described elsewhere herein), are described elsewhere herein.

In addition, the present invention provides the use of a bacterial strain, for example a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the selection method of the invention, in which the said strain has a 5'-nucleotidase gene or an active 5'-nucleotidase enzyme and is capable of produce adenosine, in the manufacture of a composition or medicinal product for use in a product, for example, a local product, adenosine in a subject. Alternative embodiments provide the use of a bacterial strain, for example a strain of lactic acid bacteria, wherein said strain has a 5'-nucleotidase gene or an active 5'-nucleotidase enzyme and is capable of producing adenosine, in the manufacture of a composition or medicament for use in a product, for example, a topical product , adenosine in the subject. Preferred features of this strain and its use, for example, in infantile colic (or other diseases as described elsewhere herein), are described elsewhere herein.

Articles or compositions, e.g., pharmaceutical compositions, probiotic compositions, or dietary/nutraceutical compositions, comprising bacterial strains (e.g., including one or more bacterial strains) as described herein (e.g., bacterial strains capable of producing or inducing the production of adenosine, melatonin etc.), and the use of said articles or compositions in the methods and applications as described herein further form further aspects of the invention.

The United Nations Food and Drug Organization defines probiotics as “live microorganisms that, when administered in adequate quantities, confer a health benefit on the host.” A number of different bacteria are currently used as probiotics, for example lactic acid producing bacteria such as Lactobacillus and Bifidobacterium strains.

Alternative and preferred embodiments and features of the invention, as described elsewhere herein, are equally applicable to the treatments, applications and articles of the invention.

As described above, the present invention relates to the selection and use of bacterial strains that are capable of producing adenosine.

These strains, which are effective in producing adenosine, can be used for local production of adenosine in a subject, for example, a mammal, preferably a human.

Thus, as stated above, the present invention provides various methods for selecting or screening bacterial strains capable of producing adenosine.

Some of the methods include the step (eg, step a)) of screening for the presence of a gene encoding a 5'-nucleotidase, eg, a cell wall-anchored 5'-nucleotidase. Such screening can be carried out using any suitable method, and strains positive for the 5'-nucleotidase gene are selected. Such methods are conveniently carried out in vitro, for example, they may be genetic or nucleic acid-based methods for detecting the presence of a gene encoding a 5'-nucleotidase, for example, a cell wall-anchored 5'-nucleotidase (or an identifying fragment thereof), the sequences of which known in the art. For example, a PCR protocol (or other nucleic acid-based method) can be easily developed based on known nucleic acid sequences encoding enzymes, or, alternatively, the genome sequences of candidate strains can be carefully studied to identify the specified a gene encoding a 5'-nucleotidase, e.g., a cell wall-anchored 5'-nucleotidase, e.g., based on homology to known 5'-nucleotidase sequences, including, e.g., the presence of the LPXTG motif (SEQ ID NO: 1), as discussed below.

An exemplary gene to be detected in the methods of the invention is the cell wall-anchored (ecto)-5'-nucleotidase gene from L. reuteri (e.g., GenBank accession number: AEI56270.1, cell wall protein with anchoring domain, containing the LPXTG motif [Lactobacillus reuteri SD2112]), or the corresponding homolog/5'-nucleotidase from other bacterial species, for example, other lactic acid bacteria. An example procedure is described in the experimental section “Examples”.

Some methods include the step (eg, step b)) of screening for the presence of an active (functional) 5'-nucleotidase enzyme, eg, a cell wall-anchored 5'-nucleotidase enzyme. Such a step can be carried out using any suitable method, for example, using an enzymatic assay. The enzyme 5'-nucleotidase catalyzes the following reaction: AMP + H ₂ O adenosine + phosphate (AMP is adenosine monophosphate), and an assay to measure this reaction can easily be used to determine the presence of active 5'-nucleotidase enzyme (or 5'-nucleotidase activity). In other words, when referring herein to an active or functional 5'-nucleotidase enzyme, it is understood that it is an enzyme that is capable of catalyzing this reaction under suitable conditions, for example, when supplied with a suitable substrate, such as AMP. If desired, the activity of the 5'-nucleotidase enzyme can be quantified, for example, in such an enzyme assay, for example, by measuring the amount or concentration of phosphate or adenosine or other appropriate downstream product in the adenosine breakdown pathway that is formed in the reaction and can be measured or determined quantitatively. The activity can be conveniently measured in a sample of bacterial cells or supernatant of bacterial cells.

Methods for performing such analysis should be well known to one skilled in the art. For example, suitable kits are commercially available, such as the Crystal Chem 5'-nucleotidase assay kit (Crystal Chem, catalog no. 80229, Downers Grove, IL, USA), in which the activity of the 5'-nucleotidase enzyme is measured by the production of a dye (quinone dye ), which is formed as a subsequent product in the metabolic pathway of the breakdown of AMP to adenosine (i.e. as a result of adenosine production). AMP is provided as a substrate. An illustrative method is described in the Examples section. The corresponding procedure can also be readily obtained from other reagent suppliers, such as Sigma, in combination with their 5'-nucleotidase enzyme reagent.

Strains with high or significant levels of 5'-nucleotidase enzyme production (or 5'-nucleotidase activity) are preferred, for example strains that have a 5'-nucleotidase activity of at least or more than 2 units/L (units per liter) and/or are capable of produce adenosine at a level of at least or more than 2 µmol l ^-1 ⋅min ^-1 (µmol per liter per minute). Thus, in preferred embodiments, a strain is selected having a 5'-nucleotidase activity of at least or greater than 3, 4, 5, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 , 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 units/L, and/or capable of producing adenosine at levels of at least or greater than 3, 4, 5, 8, 9, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 µmol l ^-1 ⋅ min ^-1 . In some embodiments, such values may represent upper limits. Such values typically refer to 5'-nucleotidase activity values measured on a sample of bacterial cells, for example on the surface of bacterial cells or in the supernatant of bacterial cells in culture, preferably the supernatant. Such values typically refer to adenosine levels (preferably, extracellular adenosine levels) measured in a sample of bacterial cells, for example, in the supernatant of bacterial cells in culture. Such values typically refer to 5'-nucleotidase activity values (or adenosine levels) when measured at a concentration of 10 ⁹ bacteria/ml or in the supernatant from such a culture. One unit is defined as the amount of enzyme required to produce 1 μmol of product/min (eg, 1 μmol of product/liter/min). The concentration in units per liter will then correspond to the enzyme concentration required to increase the concentration by 1 µM/min (e.g. 1 µM/liter/min). A suitable and exemplary assay for measuring this activity is shown in the Examples using a 5'-nucleotidase kit (No. 80229) from Crystal Chem Inc. Thus, in preferred embodiments, the above units and values refer to the units and values when measured using this kit and/or the conditions set forth in the examples, especially at a concentration of 10 ⁹ bacteria/ml, or in the supernatant from such a culture, or equivalent analysis. Thus, such methods will be conveniently performed in vitro.

Methods including at least step b) will be preferred since the presence of a gene encoding a 5'-nucleotidase does not always indicate the presence of an active 5'-nucleotidase enzyme. In the methods, applications and lactic acid bacteria of the invention, adenosine production or adenosine activity must occur extracellularly, that is, outside or on the surface of the lactic acid bacteria, so that it may, for example, be present in the supernatant or other extracellular fluid produced by the lactic acid bacteria. Thus, the presence of active 5'-nucleotidase enzyme on the cell surface, for example as cell wall-anchored 5'-nucleotidase, or extracellular to the bacterial cell, for example in the supernatant, may be a useful feature since the reaction producing adenosine, occurs outside the cell, such as on the surface of a bacterial cell.

Thus, the production of adenosine at good levels (eg, extracellular adenosine produced by the enzyme 5'-nucleotidase) can also serve as an indicator of the presence of the 5'-nucleotidase gene or the presence of an active 5'-nucleotidase enzyme. Thus, the breeding methods of the invention may also include the steps of selecting an adenosine-producing strain. Strains with high or significant levels of adenosine production, e.g., extracellular adenosine, are preferred, for example, strains that produce adenosine at a level that represents a therapeutically effective level for the subject, or at a level at which increased (and especially therapeutically effective) ) melatonin levels. Such values generally refer to adenosine values measured in the supernatant of strains in culture or on the cell surface of strains in culture (in vitro), and some illustrative specific values are given above.

However, in some embodiments, it is appropriate to refer to the values, levels or amounts of adenosine produced in a subject, for example, locally, for example, at the site of administration, for example, in the gastrointestinal tract. For example, preferred strains may promote increased adenosine production, e.g., increased in vivo production, e.g., local production (e.g., in the gastrointestinal tract), of adenosine, e.g., compared to an appropriate control as described elsewhere herein, such as adenosine levels when no strains are administered, or the baseline or natural adenosine level in a particular subject.

In all aspects of the invention described herein, local production may refer to the production or levels of adenosine at the site of administration, for example, to the production or levels of adenosine in the gastrointestinal tract (eg, when administered orally). In one aspect, local production of adenosine may have further systemic effects and may increase levels of adenosine or melatonin in the circulatory system, such as in the blood or plasma or other sites outside the gastrointestinal tract.

Thus, strains that can cause increased local levels of adenosine (or melatonin or 5'-nucleotidase activity), for example, in the gastrointestinal tract of a subject, are preferred, particularly when such an effect is observed when the strains are administered orally. Preferably, said increases are measurable or significant increases, such as statistically or clinically significant increases. For example, preferred strains are those that can cause an increase of at least or up to 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8 fold in adenosine levels (or melatonin or 5'-nucleotidase activity), for example, local levels of adenosine (or melatonin or 5'-nucleotidase activity). Any suitable comparison may be used, eg, an increase over the levels observed when no strain was administered, or levels when a control composition, eg, a control composition not containing the corresponding strain, was administered.

Appropriate methods for measuring levels of adenosine production (or melatonin or 5'-nucleotidase activity) are well known to one skilled in the art. Thus, in some embodiments of the invention, the selection method will include the step of detecting or determining the amount or level (eg, concentration) of adenosine produced by the candidate strain.

Optionally, levels of adenosine production or 5'-nucleotidase activity (or, indeed, any other suitable property of the strains described herein) can be conveniently compared with positive or negative control strains. A suitable positive control strain would be DSM 17938, which is shown in the Examples to produce significant levels of adenosine/5'-nucleotidase activity, for example, in the supernatant of bacterial cells. Some strains will produce higher, sometimes significantly higher, levels of adenosine/5'-nucleotidase activity than DSM 17938, for example in the supernatant of bacterial cells. Thus, strains capable of producing higher (increased) levels or significantly higher (increased) levels of 5'-nucleotidase activity than DSM 17938, for example in the supernatant of bacterial cells, when assessed in vitro, form a further additional aspect of the invention. DSM 32846, DSM 32847, DSM 32849 and DSM 33198 are exemplary strains (see Fig. 3 and Fig. 5 A/B). On the other hand, strains capable of producing or inducing the production of higher (increased) levels, or significantly higher (increased) levels, of adenosine than DSM 17938, for example in the supernatant of bacterial cells, when assessed in vitro, form a further additional aspect of the invention. For example, strains capable of producing or inducing the production of higher (increased) levels or significantly higher (increased) levels of adenosine in a subject, e.g., higher, etc., local levels of adenosine in a subject than DSM 17938, when assessed in vivo, for example, form another aspect of additional inventions, especially where such an effect is observed when the strains are administered orally.

In general, for some embodiments, strains that have one or more improved (eg, significantly improved) properties compared to DSM17938 are preferred.

Preferably, said increases (adenosine production or 5'-nucleotidase activity) are measurable or significant increases, such as statistically or clinically significant increases. For example, strains that can cause an increase of at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or higher adenosine levels, such as local or in vitro adenosine levels, or 5'-nucleotidase activity levels (eg, when assessed in vitro) compared to levels in DSM 17938, represent preferred strains. On the other hand, strains are preferred which can cause at least or up to 1.5-, 2-, 2.5-, 3-, 3.5-, 4-, 4.5-, 5-, 6 -, 7-, 8-, 9-, or 10-fold increase in adenosine levels, e.g., local or in vitro adenosine levels or 5'-nucleotidase activity levels, compared to DSM 17938 levels. In vitro levels can be conveniently measured as described elsewhere herein, for example, on bacterial cultures. Preferably, such an increase is an increase in the extracellular level of adenosine or 5'-nucleotidase activity, for example, when measured in vitro, for example, in the supernatant of bacterial cultures.

A suitable negative control strain may be a strain that does not contain a gene encoding a 5'-nucleotidase, or does not contain an active 5'-nucleotidase enzyme (or does not have 5'-nucleotidase activity, as described elsewhere herein).

Because of the subsequent use of strains that are selected by the methods of the invention, after the selection or isolation of adenosine-producing strains, other embodiments will involve additional steps of cultivating or propagating or producing such strains, and optionally compiling said cultured or propagating or producing strains into a composition including said strain, for example, in a pharmaceutical or nutritional composition, for example, as described elsewhere herein, or possibly storing such strains for future use, for example, by lyophilization or freeze-drying. In one embodiment, the bacterial strains are provided in lyophilized form.

The selection steps of the methods of the invention (and, of course, the therapeutic methods as described herein) must be carried out in a suitable culture medium (or in vivo environment) that supports adenosine production. Preferred culture media (or in vivo environment) will contain a suitable carbon source that will support adenosine production by the strain, preferably together with a suitable substrate for adenosine production by a 5'-nucleotidase enzyme, for example AMP.

Although such assays are conveniently performed in vitro, another possibility would be to evaluate the strains in an appropriate ex vivo assay, for example using an appropriate intestinal enteroid model (see, for example, the enteroid model and assays described in Example 5, in which it is possible to evaluate, e.g. , melatonin levels).

Thus, while any suitable in vitro or ex vivo models for assessing strains, for example, to assess the induction of adenosine or melatonin production by strains, preferred in vitro or ex vivo models for assessing strains are intestinal enteroid models, preferably human intestinal enteroid models, which , for example, can be used to assess melatonin levels. Such models preferably include enteroendocrine cells, for example, as described in Example 5.

Preferred strains of the invention are capable of producing or inducing the production of at least or up to 10, 20, 30, 40, 50, 60 or 70 pg/ml melatonin. In some embodiments, such values may represent upper limits. Such values are typically referred to the levels of melatonin production induced by said strains when measured in an appropriate in vitro or ex vivo assay, for example referred to the levels of melatonin produced by enteroendocrine cells (preferably human cells), or produced in an intestinal enteroid model comprising enteroendocrine cells (eg, with % enteroendocrine cells as described elsewhere herein), preferably in a model involving human cells, when said cells come into contact with strains of the invention or with a supernatant obtained from said strains. A suitable and illustrative assay for measuring this activity is exemplified using a human intestinal enteroid model comprising enteroendocrine cells, e.g., at least 20% or 30%, e.g., 20-40%, 30-40%, or up to 40% enteroendocrine cells. cells. Thus, in preferred embodiments, the above values refer to the values measured using this assay (or an equivalent assay, e.g., with the above % enteroendocrine cells) and/or the conditions described in the Examples section.

Some strains will be able to produce or induce higher, sometimes significantly higher, levels of melatonin than DSM 17938, for example from enteroendocrine cells or in intestinal enteroid models as described above. Thus, strains capable of producing or inducing higher (increased) levels or significantly higher (increased) levels of melatonin than DSM 17938, for example, when assessed as described above, for example, using enteroendocrine cells or an intestinal enteroid model, form yet another additional aspect of the invention. Exemplary strains may result in an increase of at least or up to 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5. 5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, or 10 times in melatonin production levels compared to DSM 17938 levels and represent are the preferred strains.

The screening methods of the invention may thus optionally include the additional step of selecting a strain that is capable of producing or inducing melatonin production, for example, using a methodology as described above, or any other suitable methodology. Selected strains may preferentially induce melatonin production at levels as described above.

In embodiments in which more than one bacterial strain is screened using the methods of the invention, the amount of adenosine or 5'-nucleotidase (or melatonin) activity generated may optionally be quantified, and the bacterial strain, e.g., a lactic acid bacteria strain, with the highest 5'-nucleotidase enzyme activity or level, or sufficiently high 5'-nucleotidase enzyme activity or level, e.g., with activities or levels as described elsewhere herein, e.g., with activities or levels higher (preferably significantly higher), than DSM17938, or a strain that produces the greatest amount or level of adenosine or melatonin, or a sufficiently high amount or level of adenosine or melatonin, e.g., with amounts or levels as described elsewhere herein, e.g., with amounts or levels higher ( preferably significantly higher) than DSM17938.

Thus, another aspect of the invention is to provide a method for selecting bacterial strains, preferably lactic acid bacteria strains, effective in producing adenosine (or melatonin), comprising:

a) screening strains of lactic acid bacteria for the presence of a gene encoding a 5'-nucleotidase, for example cell surface-anchored 5'-nucleotidase, and;

b) quantifying the activity of the 5'-nucleotidase enzyme and optionally

c) selecting a strain of lactic acid bacteria that has the highest or sufficiently high activity of the 5'-nucleotidase enzyme.

Methods comprising certain steps as described herein also include, where appropriate, methods comprising those steps.

As stated above, a bacterial strain, for example a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the methods of the invention, in which said strain is capable of producing adenosine, and, preferably, in turn producing, or promoting the production of, for example, increasing the production of melatonin represents another additional aspect of the invention.

Any suitable bacterial strain, for example, a probiotic bacterial strain, for example, any probiotic bacteria, can be subjected to the selection method of the invention and any suitable bacterial strain, for example, a probiotic bacterial strain, which is capable of producing adenosine can be used in the methods or applications of the present invention, for example , in therapeutic methods or applications described herein.

Preferred bacterial strains are lactic acid bacteria, for example Lactobacillus or Bifidobacterium. Particularly preferred bacterial strains are Lactobacillus reuteri, in particular Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848 and DSM 32849, which were deposited under the Budapest Treaty in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (MascheroderWeg 1b, Inhoffenstr. 7B , D-38124 Braunschweig)) July 4, 2018, and Lactobacillus reuteri DSM 33198 which was deposited under the Treaty of Budapest in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (MascheroderWeg 1b, Inhoffenstr. 7B, D-38124 Braunschweig)) 9 July 2019. These bacterial strains or isolated bacterial strains (and other strains, e.g. L.reuteri strains, especially related L.reuteri strains, with one or more characteristics, e.g. the ability to produce adenosine and/or 5'-nucleotidase activity, and/ or melatonin or to act on their products, in one or more deposited strains) form preferred aspects of the invention and may, for example, be used to treat diseases or conditions as described elsewhere herein, or for any other uses as described in this document. In some embodiments, L. reuteri strain DSM 17938 is not used.

Thus, in yet another additional aspect, the invention provides the use of such strains for the treatment or prevention of one or more diseases as described herein.

In another aspect, the invention provides a method for improving the use and effect of treating specific diseases as described herein, comprising maximizing, increasing or improving the 5'-nucleotidase activity of selected strains, for example, strains of lactic acid bacteria, which will preferably lead to increased or improved adenosine production . This can be achieved by culturing the bacteria under specific growth conditions to provide a high adenosine yield (or a higher, e.g., significantly higher adenosine yield, e.g., compared to the yield observed in the absence of certain growth conditions). In one embodiment of the invention, the specific growth conditions may be, for example, the cultivation of lactic acid bacteria in a normal nutrient medium with the addition of an appropriate substrate for the production of adenosine by the action of the 5'-nucleotidase enzyme, for example, AMP, or appropriate components from which it can be obtained AMPs such as ADR and/or ATP.

In a further similar embodiment of the invention, in order to maximize 5'-nucleotidase activity, a higher concentration of bacteria, for example lactic acid bacteria, can be administered. For example, the concentration may be 2-10 times higher than the usual dose.

In a further similar embodiment of the invention, the 5'-nucleotidase gene can be overexpressed or induced in bacterial strains using appropriate methods, for example, insertion into a plasmid vector of the 5'-nucleotidase gene under the control of an appropriate promoter, for example, an inducible promoter. Alternatively, the 5'-nucleotidase gene can be overexpressed by inserting one or more additional copies of the gene into the bacterial cell, for example, by inserting into the chromosome of the bacterial cell under the control of an appropriate promoter, for example, an inducible or native promoter.

As described above, the strains of the invention, or strains selected, produced, obtained, or that can be obtained by the methods of the invention, have therapeutic uses. Thus, a further aspect of the invention provides the adenosine-producing strains of the invention, or strains selected, obtained or that can be obtained by the breeding methods of the invention, for use in the production, for example, topical production, of adenosine, and thus preferably melatonin, in a subject. In preferred embodiments of the invention, said adenosine production, etc. and, preferably, melatonin is used to treat infantile colic (and other diseases as described herein) that would benefit from production or increased production, for example, local production, of adenosine and thus melatonin.

Administration of bacterial strains to subjects (animals/mammals) in need of treatment in the methods of treatment and use of the invention is carried out in pharmaceutically, therapeutically or physiologically effective amounts. Thus, these methods and applications may include the additional step of identifying a subject in need of treatment.

Treatment of a disease or conditions in accordance with the present invention (eg, treatment of a pre-existing disease) includes the cure of the specified disease or conditions or any reduction or alleviation of the disease (eg, reducing the severity of the disease) or symptoms of the disease.

The methods and uses of the present invention are suitable for the prevention of diseases or conditions, as well as for the treatment of diseases or conditions. Thus, prophylactic treatment is also within the scope of the invention. It is for this reason that in the methods and applications of the present invention, treatment or therapy also includes prophylaxis or prevention, where necessary. Such preventive (or protective) aspects can be conveniently performed in healthy or normal subjects as described herein, and can include both complete prevention and substantial prevention. Similarly, a significant warning may include a scenario in which the severity of a disease or symptoms of a disease are reduced (e.g., measurably or significantly reduced) compared to the severity or symptoms that would be expected if treatment were not given.

In yet another further aspect, the invention provides a strain or article for therapeutic uses, as defined elsewhere herein, wherein said use further includes administration of at least one additional agent, such as an additional therapeutic or nutritional agent. For example, the agents may be substrate components that increase or enhance adenosine production (eg, a component that can increase or enhance adenosine production), or sources of such components.

Thus, in yet another further embodiment of the invention involving adenosine-producing bacteria, administration of said strains or articles further comprises administration of a substrate component, for example, AMP and/or a material or agent that produces this component. The preferred substrate will be AMP, or an AMP source.

In such embodiments, a substrate component or other additional component may be added to the bacterial preparation immediately prior to administration to the subject. Alternatively, it can be added to the bacterial preparation at the end of the manufacturing process, for example at the end of fermentation, after which the bacteria can be preserved for future use, for example by lyophilization or freeze-drying. It can be entered separately as described below.

In such embodiments, the additional therapeutic agent may be any additional agent that will be useful in treating the disease in question.

These additional agents can be administered together with the strains or products of the invention (for example, in the form of a combination preparation) or can be administered separately. In addition, these additional agents can be administered simultaneously with the strains or products according to the invention or at different points in time, for example, sequentially. Suitable modes and times of administration can be readily determined by one skilled in the art depending on the adjunctive agent being considered.

The present invention also provides a composition (or combination product or kit) comprising:

(i) a bacterial strain, for example a strain of lactic acid bacteria, selected, produced, obtained or which can be obtained using the selection method of the invention (or a bacterial strain capable of producing or inducing the production of adenosine as otherwise defined herein), in which said bacterial strain has a gene encoding a 5'-nucleotidase attached to the cell surface, or an active 5'-nucleotidase enzyme and is capable of producing adenosine; And

(ii) one or more substrate components or agents that increase or improve adenosine production, or a source of such components or agents.

Exemplary components or means have been described above. AMP or an AMP source is a preferred component.

The invention also provides a composition (or combination product or kit) suitable for increasing melatonin or adenosine production in a subject, comprising:

(i) a first bacterial strain, for example a strain of lactic acid bacteria, capable of producing or inducing the production of melatonin or adenosine, as described herein; And

(ii) an additional source of melatonin and/or adenosine, or one or more substrate components or agents for increasing or enhancing the production or induction of melatonin and/or adenosine production, or a source of such components or agents, optionally a strain of lactic acid bacteria.

A preferred component is AMP or a source of AMP.

In kits or combination products of the invention, components (i) and (ii) are typically provided in separate compartments or vials or as separate components of the kit or product. Kits may include additional components, which may also be contained in separate compartments or vials. Preferably, the kits (or combination products) are intended for use in the methods or applications of the present invention as described herein, for example, for use in the treatment or prevention of other diseases described herein, particularly infantile colic. Optionally, instructions for use of the kits or articles in the methods and uses of the invention may be provided.

The term "subject" as used herein includes mammals, especially humans, especially infants (eg, when the disease to be prevented or treated is colic). In preferred embodiments of the invention, the selected strain, for example a strain of lactic acid bacteria, is administered to a human. For diseases other than colic, any subject is suitable, for example a human subject, for example an adult subject or a child subject or an infant subject suffering from the specified disease or suspected of suffering from the specified disease or being at risk of the specified disease.

A preferred subject of the present invention is an infant less than or up to 12 months of age, preferably less than or up to 5 or 6 months of age, preferably less than or up to 3 or 4 months of age. Such subjects are particularly suitable when the disease to be treated or prevented is infantile colic. Subjects may thus be treated from birth or during the first few weeks (eg, during the first 1, 2, 3, or 4 weeks) or during the first few months (eg, during the first 1, 2, 3, or 4 months ) life.

As described elsewhere herein, preferred subjects are those who suffer from infantile colic (or one of the other diseases as described herein), or believe or are suspected to suffer from infantile colic (or one of the other diseases as described herein). Since the therapeutic applications of the invention can also be used to prevent disease, suitable subjects include those at risk of suffering from infantile colic (or one of the other diseases as described herein).

By way of example, subjects would include infants who were exclusively breastfed and infants who were exclusively bottlefed (nonbreastfed infants), or infants who were breastfed and on artificial feeding.

Other subjects that may be exemplified include infants, particularly colicky infants, suspected of being colicky, or at risk of developing colic, who do not yet produce or release melatonin or are otherwise unable to produce or release melatonin, e.g. infants in whom the melatonin circadian cycle has not yet established or begun, or infants who are unable to produce or release essential melatonin from their food and/or their gut bacteria, or include infants who have low, reduced, e.g., significantly reduced ( or abnormal) melatonin levels or melatonin deficiency, for example, compared with levels in a normal or healthy subject, for example, with levels in a normal or healthy subject of the same, equivalent or comparable age.

In diseases other than colic, exemplary subjects include subjects who have low, reduced, e.g., significantly reduced (or abnormal) melatonin levels or deficient melatonin, e.g., compared to levels in a normal or healthy person. subject, for example with levels in a normal or healthy subject of the same, equivalent or comparable age.

Other exemplar subjects include infants who have some difficulty or problem with feeding or diet that, for example, results in low or insufficient melatonin levels. Such subjects may include infants born before full gestation, such as premature infants or infants born before 36 weeks of gestation, or infants who do not have adequate nutritional metabolism, or infants who have feeding problems or nutritional problems.

Other exemplary subjects include subjects, preferably infants, with hormonal immaturity (in other words, suboptimal, subnormal, undeveloped, deficient, low or immature hormone production), for example, compared to a normal infant or subject of the same age, for example, melatonin immaturity, especially those subjects who have intestinal motility deficiency as a result of immaturity of the said hormone. Such hormonal immaturity, such as melatonin immaturity or decreased or deficient melatonin, may, for example, be associated with some infants suffering from infantile colic. Other preferred diseases (and therefore subjects) to be treated are diseases or conditions associated with melatonin deficiency (or subjects suffering from these diseases).

In all such subjects, if appropriate or necessary, melatonin levels for each of these subjects can be readily measured using methods readily available and known in the art. For example, melatonin levels can be easily measured in serum/blood or saliva samples. Commercial kits for such measurements are also available.

Any suitable mode of administration may be used. It is convenient to carry out the specified administration orally, rectally or through a tube. Thus, the bacteria can be introduced into the gastrointestinal tract or into the oral cavity, if desired or appropriate. The bacteria may be included in an infant formula or dietary supplement or oil drops, as well as in a pharmaceutical composition.

Appropriate dosages of the strains, articles and compositions of the invention, as defined herein, can be readily selected depending on the disease (or condition) being treated, the route of administration and the composition in question.

For example, the dosage and mode of administration are selected such that the bacteria administered to a subject in accordance with the present invention can lead to an increase in the production, for example, local production, of adenosine (or melatonin) and cause the desired therapeutic effect or health benefit in infantile colic (or for another disease being treated). Thus, preferably, said dosage is a therapeutically effective dosage that is appropriate for the type of mammal and condition being treated, and is administered to, for example, a subject in need thereof. For example, daily doses may be used, for example a single daily dose of 10 ⁴ to 10 ¹² , for example 10 ⁵ to 10 ⁹ , or 10 ⁶ to 10 ⁸ , or 10 ⁸ to 10 ¹⁰ , or 10 ¹⁰ to 10 ¹² total CFU of bacteria.

In one embodiment, the daily dose (for example, 10 ⁵ to 10 ⁹ or 10 ⁶ to ^{10 8} or 10 8 to 10 ¹⁰ or 10 ¹⁰ to 10 ¹² CFU) is divided into several administrations, for example, 2-8 administrations (for example, 3- 5 administrations) or in connection with each feeding (breastfeeding and/or bottle feeding) of the subject. In one embodiment, a higher dose, e.g., 2 to 10 or 10 to 100 times the dose after the initial period, and/or a more frequently prescribed dose is administered as an initial dose for 3 to 14 days (e.g. 5-10 days, e.g. 7 days) to achieve faster relief in the subject.

The term “increase” or “enhance” or “increased” (or equivalent terms) as described herein includes any measurable increase or increase compared to the corresponding control. Appropriate controls can be readily determined by one skilled in the art and may include levels when no strains are present, or levels in untreated or placebo-treated subjects, or levels in healthy or normal subjects, such as age-matched subjects, or the same subject before treatment. Preferably, the increase will be significant, eg clinically or statistically significant, eg with a probability value ≤0.05.

Preferably, such increases (and, of course, other increases, improvements or benefits as indicated elsewhere herein) are measurable increases, etc. (as appropriate), more preferably they represent significant, clinically significant or statistically significant increases, for example, with a probability value ≤0.05 compared to the corresponding control level or value (for example, compared to an untreated or placebo-treated subject or compared with a healthy or normal subject or the same subject before treatment).

The term “reduce” or “reduce” (or equivalent terms) as described herein includes any measurable decrease or reduction compared to an appropriate control. Appropriate controls can be readily determined by one skilled in the art and may include levels when no strains are present, or levels in untreated or placebo-treated subjects, or levels in healthy or normal subjects, such as age-matched subjects, or the same subject before treatment. Preferably, the decrease or reduction will be significant, eg clinically or statistically significant, eg with a probability value of ≤0.05.

Thus, preferably, such reductions (and, of course, other reductions, reductions or negative effects, as mentioned elsewhere in this document) are measurable reductions, etc., (if necessary), more preferably, they are significant reductions, preferably clinically significant or statistically significant reductions, for example with a probability value ≤0.05, compared with an appropriate control level or value (for example, compared with an untreated or placebo-treated subject or compared with a healthy or normal subject or the same subject before treatment).

As used throughout the application, the term referring to a particular component or step is used in the sense that it refers to “at least one,” “at least a first,” “one or more,” or “plurality” of such components or stages unless an upper limit is specifically stated thereafter.

In addition, when the terms “comprise”, “comprises”, “has” or “having” or other equivalent terms are used herein, in some more specific embodiments these terms include the term “consists of” or “consists essentially of” or other equivalent terms. Listings “consisting of” various components and features discussed herein may also refer to listings “including” various components and features.

Other objects and advantages will be more apparent from the following non-limiting examples and the accompanying claims.

Examples

Example 1

Method for identifying strains containing a gene encoding a 5'-nucleotidase located on the cell surface

Bacteria were cultured on MRS plates for 16 hours at 37°C in an anaerobic atmosphere. 10 bacterial colonies were collected with a sterile plastic loop and suspended in 100 microliters of sterile water (PCR quality). (Alternative: obtain DNA from bacterial culture using a suitable method.)

The presence of the 5'-nucleotidase gene can be tested by PCR, for example, using PuReTaq Ready To Go PCR Beads (GE HealthCare) and any of the primer pairs described below (0.4 mM each). The bacterial suspension or DNA preparation (0.5 µl) should be added to the PCR mixture and the PCR reaction should be carried out by running the 95°C program, 5 min; 30x (95°C, 30 sec; 55°C, 30 sec; 72°C, 30 sec); 72°C, 10 min. PCR products could be separated and visualized using standard agarose gel electrophoresis, and sequences could be determined using standard Sanger sequencing (using the forward primer used for PCR).

The gene can be detected using any of the following primers:

Primer pair 1 (product size 233 bp)

LrNuc1f: GGAACTTTGGGAAACCATGA(SEQ ID NO:2)

LrNuc1r:CGGGCAACTTTACCATCACT(SEQ ID NO:3)

Primer pair 2 (product size 212 bp)

LrNuc2f: TACTCGTGAAAATGCCGTTG(SEQ ID NO:4)

LrNuc2r: GTGCCCCTGTCATTTCAACT(SEQ ID NO:5)

Primer pair 3 (product size 232 bp)

LrNuc3f: AGCTTTACCAAATTGACCCTGA(SEQ ID NO:6)

LrNuc3r: TTGATATTAGGCGCATCCTTTT(SEQ ID NO:7)

Sequences

Gene encoding cell surface anchored 5'-nucleotidase, GenBank accession number: AEI56270.1, cell wall protein with anchor domain carrying LPXTG motif [Lactobacillus reuteri SD2112] (SEQ ID NO:8)

ATGAAGAATAATAGTTCAAAATATTGTTTATTGTTAGGGACAGCGCTGTTAGGACTATATTTCCAAGCTAATAGTGTTCATGCGGATGCGACTGGTATCACAGCTAATGGAGAAACTACCCATAGCAATGTTACTCCAATGGTTCAGACTAATAAGGATGAGGCAAGTACACCGCAAACAACTACTGATTGGTCTGACCCGGCCAAATATCAAAGTGACATTCCAGTTCAGATTTTAGGAATCAATGACT TGCATGGTGGGTTAGAAACGACTGGATCAGCTACGATTGGAGATAAGACTTATTCGAATGCCGGAACAGTTGCACGCCTAGCTGGTAACCTTGATGCGGCGGAGGAAAGTTTTAAGAACGCTAATCCGACGGGAAGCTCAATTCGGGTAGAAGCCGGAGATATGGTTGGGGCTTCTCCAGCAAATTCTGCTCTTCTCCAAGACGAATCAACCATGCATGCTTTAGACGCAATGCATTTTGAAATAGGAACTTTGG GAAACCATGAGTTCGATGAAGGTTTAGCTGAGTATATGCGGATTGTTAATGGTGGTGAACCTACTAAACAATATAATGAAGCTGAGATGGCCTATCCTCATGTGAAAACAGGGATTAATTCATTACTGCCAATGTTGTAAATAAATCTGATGGTCAAATCCCATTTGGAATGCAACCATACTTGATTAAAGAAATTCATACTAGTGATGGTAAAGTTGCCCGGATCGGATTTATTGGGATTGAAACTACT TCCCTACCAATTTTAACCTTATACGATAATTACAAAGATTATGATGTTTTAGACGAGGCTGAAACAATTGCAAAATATGATCAAATTTTACGCAAAAAAGGTGTTAACGCAATTGTAGTTCTTGCCCATACAGGGGTTTCAACTGATAAAGATGGCAGCACTAAAGGTAATGCTGTTGATATCATTAAGAAGCTTTACCAAATTGACCCTGATAATTCTGTCGACCTTTTATATTGCTGGTCACTCCCACCAATATG CTAATGCTACTGTTGGAAGTGTAAAATTAGTGCAAGCCATTTACACGGGTAAAGCTTACGATGATATTATCGGTTACATCGATCCAACAACTAATGATTTTGCGCCCAATAGTCTCGTTTCACATGTCTTTCCGGTACTATCTGAAAAGGATGCGCCTAATATCAAAACGGATGCAAATGTTACAGCAATTGTTGAAGATGCGAACAACCGAGTAGCACCGATTATTAACAAGAAAATAGGGGAAGCTGCT ACAACAGGCGATATTCTTGGACGACTTCATAATACTCCTACTCGTGAAAATGCCGTTGGTGAATTAGTTGTCGATGGTCAATTATATGCCGCTCATAAAGTAGGCTTACCAGCTGATTTTGCGATGACTAATACAGGGGGCGTTCGTGCAGATCTGCATGTTAATCCTGATCGTTCCATTACATGGGGGAGTGCCCAAGCAGTTCAACCATTTGGTAATATTTTGCGGGTAGTTGAAATGACAGGGGCACAAA TCGTTGAAGCCTTGAATCAACAATACGACGAAGATCAAGCTTACTACTTACAGATTTCCGGGCTACATTATACTTATACTGACCAAAACGATCCTAACCAACCATATAAGGTCGTTCAAGTTTATGACCAACATAATCAACCGCTTGATATGAATAAGACTTACAATGTTGTTATTAATGACTTTTTAGCAGGTGGCGGAGATGGCTTTTCTGCATTTAAGGGTACTAAAGTTGTCGGGATTGTTGGTC AAGATACAGACGCGTTTATTGACTATATTACTGATATGACTAATGATGGTAAACCAATTACTGCGCCAACAATGAACCGTAAGATTTACTTGACTGCTGAACAAGTAGCGAAGGCTGACTCAGATTCACAGTTACAAACAGGAACTAATCAGAACACTCAAAACGATGCTAATTCCCAGACTGAAGGAAATCAGCTTCAAGAAGTTCCGAGCCAACGGTATCTCCAACAGTAACCTTGCCAACAACAGCTGGTCAA CCCGCCGAAACTGTTACACTACATGCTCAATCTAAGCAACAAACCGTAGCTGCTAATAATCAATTAATTAATTTGACGCCTACATCAATTAATGGCCAAAAACAAAAAGCAGCTGACCAGCAAGCAGCTTTACCACAAACTAGTAACGATGAAGATCTTGCATTACTTCTTCTCGGAAGTTCATTAATGGCAGCAACCGGATTGACAATTATTGATCGCAAGCGTAAACATGCTTAA

Cell surface anchored 5'-nucleotidase protein, GenBank accession number: AEI56270.1, cell wall protein with anchor domain carrying LPXTG motif [Lactobacillus reuteri SD2112] (SEQ ID NO:9)

MKNNSSKYCLLLGTALLGLYFQANSVHADATGITANGETTHSNVTPMVQTNKDEASTPQTTTDWSDPAKYQSDIPVQILGINDLHGGLETTGSATIGDKTYSNAGTVARLAGNLDAAEESFKNANPTGSSIRVEAGDMVGASPANSALLQDESTMHALDAMHFEIGTLGNHEFDEGLAEYMRIVNGGEPTKQYNEAEMAYPHVKTGINIITANVV NKSDGQIPFGMQPYLIKEIHTSDGKVARIGFIGIETTSLPILTLYDNYKDYDVLDEAETIAKYDQILRKKGVNAIVVLAHTGVSTDKDGSTKGNAVDIIKKLYQIDPDNSVDLYIAGHSHQYANATVGSVKLVQAIYTGKAYDDIIGYIDPTTNDFAPNSLVSHVFPVLSEKDAPNIKTDANVTAIVEDANNRVA PIINKKIGEAATTGDILGRLHNTPTTRENAVGELVVDGQLYAAHKVGLPADFAMTNTGGVRADLHVNPDRSITWGSAQAVQPFGNILRVVEMTGAQIVEALNQQYDEDQAYYLQISGLHYTYTDQNDPNQPYKVVQVYDQHNQPLDMNKTYNVVINDFLAGGGDGFSAFKGTKVVGIVGQDTDAFIDYIT DMTNDGKPITAPTMNRKIYLTAEQVAKADSDSQLQTGTNQNTQNDANSQTEGNQLQEVPSQPVSPTVTLPTTAGQPAETVTLHAQSKQQTVAANNQLINLTPTSINGQKQKAADQQAALPQTSNDEDLALLLLGSSLMAATGLTIIDRKRKHA

Example 2

Procedure for 5'-nucleotidase activity assay

The Crystal Chem 5'-nucleotidase assay kit (Crystal Chem, Elk Grove Village, IL, USA) was used to determine the 5'-nucleotidase activity of bacterial cells and fermentation supernatants. Briefly the procedure was as follows.

Reagents CC1 and CC2 were added in two steps to samples containing bacteria or supernatants. Reagent 1 contains AMP, which has been converted to adenosine by the enzyme 5'-nucleotidase from bacteria. Adenosine was further hydrolyzed to inosine and hypoxanthine by the components of reagent 1. In the second step, hypoxanthine was converted to uric acid and hydrogen peroxide, which was used to produce a quinone dye, which was measured kinetically at 550 nm on a spectrophotometer. Activity was determined by calculating the change in absorbance between 3 and 5 minutes and comparing with the value from the calibrator sample.

Example 3

5'-nucleotidase activity in a Lactobacillus reuteri strain

Experimental data showing 5'-nucleotidase activity in Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849, and in Lactobacillus reuteri DSM 17938 (deposited under the Budapest Treaty in the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen G mbH (Mascheroder Weg 1b , D-38124 Braunschweig) January 30, 2006) were obtained using the method described above in Example 2. The results are presented in FIG. 3.

Example 4

Strain selection

All new strains in example 3 above, i.e. Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849 demonstrated 5'-nucleotidase activity in the bacterial supernatant. The analysis was carried out on the supernatant of bacterial cultures with a concentration of 10 ⁹ bacteria per ml.

Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849 have been developed/developed for improved properties. DSM 32846 and DSM 32847 have been modified to be more tolerant of bile acids and thus survive in greater quantities in the gastrointestinal tract. DSM 32848 and DSM 32849 have been modified to better adhere to mucosa in order to better colonize the gastrointestinal tract and thus function better in accordance with the present invention.

All Lactobacillus reuteri strains selected were DSM 32846, DSM 32847, DSM 32848 and DSM 32849. The preferred strains were DSM 32846, DSM 32847 and DSM 32849.

Example 5

Effect of probiotic bacterial strains on melatonin production in a human intestinal enteroid model

Background

Human intestinal enteroids (HIE), derived from intestinal crypt stem cells, are biologically relevant in an in vitro model of intestinal epithelium. HIEs contain all types of intestinal epithelial cells; however, like the human intestine, HIEs are normally produced by enteroendocrine cells (EECs) in small quantities (~1%), which limits their studies. To increase the number of EECs in HIEs, lentiviral transduction was used to stably construct jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIE). In this genetically engineered cell line, the number of enteroendocrine cells present in culture increases from ≤1% to ~40%.

Materials and methods

Human intestinal enteroids

HIE cultures were obtained from crypts isolated from jejunal tissues of adult bariatric surgery patients. These stabilized cultures were obtained from Baylor College of Medicine as part of a clinical research design and a Texas Medical Center for Digestive Diseases Clinical Study. Three-dimensional HIE cultures were prepared from tissue samples and maintained in culture. To increase the number of EECs in HIEs, lentiviral transduction was used to stably construct jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIE). In this genetically engineered cell line, the number of enteroendocrine cells present in culture increases from ≤1% to ~40%. All enteroids were expanded and differentiated into a 96-well monolayer according to standard protocols. Additional information about this enteroid line, its culture methods, and general assay methodology are described in Chang-Graham et al. (Chang-Graham et al., Cellular and Molecular Gastroenterology and Hepatology, 2019, S2352-345X (19) 30049-9, in press, online publication available).

Bacterial strains and preparations

In this experiment, two different strains of Lactobacillus reuteri (DSM 17938 and DSM 32846) were studied for their ability to increase melatonin production in the intestine.

This study used conditioned media (supernatant) from Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 32846, grown in Lactobacillus Defined Media 4 (LDM4) until they reached the stationary phase, see below for details.

A single colony of the bacterial strain was picked from the MRS agar plate and used to inoculate 10 ml of MRS broth and incubated in a tightly sealed conical tube in a water bath at 37°C. After eight hours, the culture was diluted 1:10, optical density at 600 nm was recorded using a spectrophotometer, then 25 ml of pre-warmed LDM4 was inoculated at an initial OD of 0.1 (calculated below) and placed in a water bath to incubate for 16 hours.

○ OD = 0.307. Dilution 1:10 = OD = 3.07

25 ml * (0.1 OD) = 3.07x x = 0.814 ml

○ 814 µl combined with 24.19 ml LDM4

After 16 hours, the culture was removed. The final OD was 2.6. Cells were pelleted by centrifugation at 4700 rpm and the supernatant was transferred to a new 50 ml conical tube. Supernatants were stored at -20°C overnight.

Chemicals used

AMP = Adenosine 5'-monophosphate, 99% (Acros Organics) Code: 102790259, Lot: A0395274.

10 mL of stock solution (2 mM stock solution = 6.9 mg/mL (FW = 347.224)) was prepared in MiliQ water and sterilized by filtration through a 0.22 μM filter.

Experimental conditions

Control treatments on induced enteroids:

• LDM4 (culture medium),

• LDM4 + 20 µM AMP,

• LDM4 + 200 µM AMP

Experimental treatment of DSM 32846 and DSM 17938 supernatant on induced enteroids:

• DSM 32846 supernatant only

• DSM 32846 supernatant + 20 µM AMP

• DSM 32846 supernatant + 200 µM AMP

• DSM 17938 supernatant only

• DSM 17938 supernatant + 20 µM AMP

• DSM 17938 supernatant + 200 µM AMP

Before the supernatant or other additives were ready to be applied to the enteroids, the pH was neutralized and the supernatant was filter sterilized. The pH was adjusted to 7.0 with 10-30 μl of 10 M sodium hydroxide solution, and the pH was measured by placing 2 μl of the supernatant on pH paper (Fisher Brand - Cat. No. 13-640-510, range 6, 0-8.0). The supernatant was sterilized by filtration using a 0.22 μm filter (Thermo Scientific Nalgene Rapid Flow 0.22 μM filters, code: 00158).

100 μl (total volume) of treatment material was placed on the enteroids. Incubation on enteroids lasted 1 hour at 37°C in a tissue culture incubator with 5% CO ₂ . After incubation, the supernatant was removed and stored at -20°C until further analysis.

Melatonin was detected using ELISA (Eagle Biosciences Inc, Amherst, NH, USA). The only step that was performed before following the standard protocol was to thaw the supernatants and allow them to reach room temperature (approximately 20-21 degrees Celsius).

results

Human intestinal enteroids (HIE) are biologically relevant as an in vitro model of intestinal epithelium. As shown in FIG. 4, DSM 32846 alone does not have a significant effect, but when added with AMP, it shows a pronounced effect on melatonin production in HIE. Higher concentrations of AMP resulted in even higher melatonin production. DSM 17938 has increased melatonin production compared to the control, but less than DSM 32846. DSM 32846 is therefore a good candidate strain for use in intestinal melatonin production.

Example 6

5'-nucleotidase activity in Lactobacillus reuteri strains

Experimental data showing 5'-nucleotidase activity in Lactobacillus reuteri DSM 33198 were obtained using the method described in Example 2 above. The results are shown in FIG. 5A, where they are normalized to the 5'-nucleotidase activity of DSM17938 (DSM17938 activity = 1) from the same experiment. The same type of normalization to the 5'-nucleotidase activity of DSM17938 (DSM17938 activity = 1) was also performed for the results of Fig. 3 and this is shown in FIG. 5B, allowing easy comparison of the fold change in 5'-nucleotidase activity relative to DSM17938 for different bacterial strains.

Example 7

Strain selection

The new strain in example 6 above, i.e. Lactobacillus reuteri DSM 33198 exhibits high 5'-nucleotidase activity in the bacterial supernatant. The analysis was carried out on the supernatant of a bacterial culture with a concentration of 10 ⁹ bacteria per ml.

Lactobacillus reuteri DSM 33198 was developed for improved properties. L. reuteri strain DSM 33198 was modified through a multi-step selection process, including repeated freeze-drying, to make it more tolerant and provide higher survival during production compared to its native isolate.

Lactobacillus reuteri DSM 33198 was selected.

--->

LIST OF SEQUENCES

<110> BIOGAIA AB

<120> SELECTION AND USE OF MELATONIN SUPPORTING BACTERIA TO REDUCE

INFANTILE COLIC

<130> 69.139115/02

<150> GB 1905470.9

<151> 2019-04-17

<150> GB 1812079.0

<151> 2018-07-24

<160> 9

<170> Patent In version 3.5

<210> 1

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223>Motif

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> any amino acid

<400> 1

Leu Pro Xaa Thr Gly

15

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LrNuc1f

<400> 2

ggaactttgg gaaaccatga

20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LrNuc1r

<400> 3

cggggcaactt taccatcact

20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LrNuc2f

<400> 4

tactcgtgaa aatgccgttg

20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer LrNuc2r

<400> 5

gtgcccctgt catttcaact

20

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer LrNuc3f

<400> 6

agctttacca aattgaccct ga

22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223>Primer LrNuc3r

<400> 7

ttgatattag gcgcatcctt tt

22

<210> 8

<211> 2259

<212> DNA

<213> Lactobacillus reuteri SD112

<400> 8

atgaagaata atagttcaaa atattgttta ttgttaggga cagcgctgtt aggactatat

60

ttccaagcta atagtgttca tgcggatgcg actggtatca cagctaatgg agaaactacc

120

catagcaatg ttactccaat ggttcagact aataaggatg aggcaagtac accgcaaaca

180

actactgatt ggtctgaccc ggccaaatat caaagtgaca ttccagttca gattttagga

240

atcaatgact tgcatggtgg gttagaaacg actggatcag ctacgattgg agataagact

300

tattcgaatg ccggaacagt tgcacgccta gctggtaacc ttgatgcggc gggaggaaagt

360

tttaagaacg ctaatccgac gggaagctca attcgggtag aagccggaga tatggttggg

420

gcttctccag caaattctgc tcttctccaa gacgaatcaa ccatgcatgc tttagacgca

480

atgcattttg aaataggaac tttgggaaac catgagttcg atgaaggttt agctgagtat

540

atgcggattg ttaatggtgg tgaacctact aaacaatata atgaagctga gatggcctat

600

cctcatgtga aaacagggat taatatcatt actgccaatg ttgtaaataa atctgatggt

660

caaatcccat ttggaatgca accatacttg attaaagaaa ttcatactag tgatggtaaa

720

gttgcccgga tcggatttat tgggattgaa actacttccc taccaatttt aaccttatac

780

gataattaca aagattatga tgttttagac gaggctgaaa caattgcaaa atatgatcaa

840

attttacgca aaaaaggtgt taacgcaatt gtagttcttg cccatacagg ggtttcaact

900

gataaagatg gcagcactaa aggtaatgct gttgatatca ttaagaagct ttaccaaatt

960

gaccctgata attctgtcga cctttatatt gctggtcact cccaccaata tgctaatgct

1020

actgttggaa gtgtaaaatt agtgcaagcc atttacacgg gtaaagctta cgatgatatt

1080

atcggttaca tcgatccaac aactaatgat tttgcgccca atagtctcgt ttcacatgtc

1140

tttccggtac tatctgaaaa ggatgcgcct aatatcaaaa cggatgcaaa tgttacagca

1200

attgttgaag atgcgaacaa ccgagtagca ccgattatta acaagaaaat aggggaagct

1260

gctacaacag gcgatattct tggacgactt cataatactc ctactcgtga aaatgccgtt

1320

ggtgaattag ttgtcgatgg tcaattatat gccgctcata aagtaggctt accagctgat

1380

tttgcgatga ctaatacagg gggcgttcgt gcagatctgc atgttaatcc tgatcgttcc

1440

attacatggg ggagtgccca agcagttcaa ccatttggta atattttgcg ggtagttgaa

1500

atgacagggg cacaaatcgt tgaagccttg aatcaacaat acgacgaaga tcaagcttac

1560

tacttacaga tttccggggct acattatact tatactgacc aaaacgatcc taaccaacca

1620

tataaggtcg ttcaagttta tgaccaacat aatcaaccgc ttgatatgaa taagacttac

1680

aatgttgtta ttaatgactt tttagcaggt ggcggagatg gcttttctgc atttaagggt

1740

actaaagttg tcgggattgt tggtcaagat agacgcgt ttattgacta tattactgat

1800

atgactaatg atggtaaacc aattactgcg ccaacaatga accgtaagat ttacttgact

1860

gctgaacaag tagcgaaggc tgactcagat tcacagttac aaacaggaac taatcagaac

1920

actcaaaacg atgctaattc ccagactgaa ggaaatcagc ttcaagaagt tccgagccaa

1980

ccggtatctc caacagtaac cttgccaaca acagctggtc aacccgccga aactgttaca

2040

ctacatgctc aatctaagca acaaaccgta gctgctaata atcaattaat taatttgacg

2100

cctacatcaa ttaatggcca aaaacaaaaa gcagctgacc agcaagcagc tttaccacaa

2160

actagtaacg atgaagatct tgcattactt cttctcggaa gttcattaat ggcagcaacc

2220

ggattgacaa ttattgatcg caagcgtaaa catgcttaa

2259

<210> 9

<211> 752

<212>PRT

<213> Lactobacillus reuteri SD112

<400> 9

Met Lys Asn Asn Ser Ser Lys Tyr Cys Leu Leu Leu Gly Thr Ala Leu

1 5 10 15

Leu Gly Leu Tyr Phe Gln Ala Asn Ser Val His Ala Asp Ala Thr Gly

20 25 30

Ile Thr Ala Asn Gly Glu Thr Thr His Ser Asn Val Thr Pro Met Val

35 40 45

Gln Thr Asn Lys Asp Glu Ala Ser Thr Pro Gln Thr Thr Thr Asp Trp

50 55 60

Ser Asp Pro Ala Lys Tyr Gln Ser Asp Ile Pro Val Gln Ile Leu Gly

65 70 75 80

Ile Asn Asp Leu His Gly Gly Leu Glu Thr Thr Gly Ser Ala Thr Ile

85 90 95

Gly Asp Lys Thr Tyr Ser Asn Ala Gly Thr Val Ala Arg Leu Ala Gly

100 105 110

Asn Leu Asp Ala Ala Glu Glu Ser Phe Lys Asn Ala Asn Pro Thr Gly

115 120 125

Ser Ser Ile Arg Val Glu Ala Gly Asp Met Val Gly Ala Ser Pro Ala

130 135 140

Asn Ser Ala Leu Leu Gln Asp Glu Ser Thr Met His Ala Leu Asp Ala

145 150 155 160

Met His Phe Glu Ile Gly Thr Leu Gly Asn His Glu Phe Asp Glu Gly

165 170 175

Leu Ala Glu Tyr Met Arg Ile Val Asn Gly Gly Glu Pro Thr Lys Gln

180 185 190

Tyr Asn Glu Ala Glu Met Ala Tyr Pro His Val Lys Thr Gly Ile Asn

195 200 205

Ile Ile Thr Ala Asn Val Val Asn Lys Ser Asp Gly Gln Ile Pro Phe

210 215 220

Gly Met Gln Pro Tyr Leu Ile Lys Glu Ile His Thr Ser Asp Gly Lys

225 230 235 240

Val Ala Arg Ile Gly Phe Ile Gly Ile Glu Thr Thr Ser Leu Pro Ile

245 250 255

Leu Thr Leu Tyr Asp Asn Tyr Lys Asp Tyr Asp Val Leu Asp Glu Ala

260 265 270

Glu Thr Ile Ala Lys Tyr Asp Gln Ile Leu Arg Lys Lys Gly Val Asn

275 280 285

Ala Ile Val Val Leu Ala His Thr Gly Val Ser Thr Asp Lys Asp Gly

290 295 300

Ser Thr Lys Gly Asn Ala Val Asp Ile Ile Lys Lys Leu Tyr Gln Ile

305 310 315 320

Asp Pro Asp Asn Ser Val Asp Leu Tyr Ile Ala Gly His Ser His Gln

325 330 335

Tyr Ala Asn Ala Thr Val Gly Ser Val Lys Leu Val Gln Ala Ile Tyr

340 345 350

Thr Gly Lys Ala Tyr Asp Asp Ile Ile Gly Tyr Ile Asp Pro Thr Thr

355 360 365

Asn Asp Phe Ala Pro Asn Ser Leu Val Ser His Val Phe Pro Val Leu

370 375 380

Ser Glu Lys Asp Ala Pro Asn Ile Lys Thr Asp Ala Asn Val Thr Ala

385 390 395 400

Ile Val Glu Asp Ala Asn Asn Arg Val Ala Pro Ile Ile Asn Lys Lys

405 410 415

Ile Gly Glu Ala Ala Thr Thr Gly Asp Ile Leu Gly Arg Leu His Asn

420 425 430

Thr Pro Thr Arg Glu Asn Ala Val Gly Glu Leu Val Val Asp Gly Gln

435 440 445

Leu Tyr Ala Ala His Lys Val Gly Leu Pro Ala Asp Phe Ala Met Thr

450 455 460

Asn Thr Gly Gly Val Arg Ala Asp Leu His Val Asn Pro Asp Arg Ser

465 470 475 480

Ile Thr Trp Gly Ser Ala Gln Ala Val Gln Pro Phe Gly Asn Ile Leu

485 490 495

Arg Val Val Glu Met Thr Gly Ala Gln Ile Val Glu Ala Leu Asn Gln

500 505 510

Gln Tyr Asp Glu Asp Gln Ala Tyr Tyr Leu Gln Ile Ser Gly Leu His

515 520 525

Tyr Thr Tyr Thr Asp Gln Asn Asp Pro Asn Gln Pro Tyr Lys Val Val

530 535 540

Gln Val Tyr Asp Gln His Asn Gln Pro Leu Asp Met Asn Lys Thr Tyr

545 550 555 560

Asn Val Val Ile Asn Asp Phe Leu Ala Gly Gly Gly Asp Gly Phe Ser

565 570 575

Ala Phe Lys Gly Thr Lys Val Val Gly Ile Val Gly Gln Asp Thr Asp

580 585 590

Ala Phe Ile Asp Tyr Ile Thr Asp Met Thr Asn Asp Gly Lys Pro Ile

595 600 605

Thr Ala Pro Thr Met Asn Arg Lys Ile Tyr Leu Thr Ala Glu Gln Val

610 615 620

Ala Lys Ala Asp Ser Asp Ser Gln Leu Gln Thr Gly Thr Asn Gln Asn

625 630 635 640

Thr Gln Asn Asp Ala Asn Ser Gln Thr Glu Gly Asn Gln Leu Gln Glu

645 650 655

Val Pro Ser Gln Pro Val Ser Pro Thr Val Thr Leu Pro Thr Thr Ala

660 665 670

Gly Gln Pro Ala Glu Thr Val Thr Leu His Ala Gln Ser Lys Gln Gln

675 680 685

Thr Val Ala Ala Asn Asn Gln Leu Ile Asn Leu Thr Pro Thr Ser Ile

690 695 700

Asn Gly Gln Lys Gln Lys Ala Ala Asp Gln Gln Ala Ala Leu Pro Gln

705 710 715 720

Thr Ser Asn Asp Glu Asp Leu Ala Leu Leu Leu Leu Gly Ser Ser Leu

725 730 735

Met Ala Ala Thr Gly Leu Thr Ile Ile Asp Arg Lys Arg Lys His Ala

740 745 750

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Claims (11)

1. An adenosine-producing bacterial strain, wherein said strain is Lactobacillus reuteri DSM 32846. 2. An adenosine-producing bacterial strain, wherein said strain is Lactobacillus reuteri DSM 32847. 3. An adenosine-producing bacterial strain, wherein said strain is Lactobacillus reuteri DSM 32849. 4. An adenosine-producing bacterial strain, wherein said strain is Lactobacillus reuteri DSM 33198. 5. Use of the strain according to any one of paragraphs. 1-4 for the treatment or prevention of infantile colic in a subject. 6. Use of the strain according to any one of paragraphs. 1-4 for the treatment or prevention of irritable bowel syndrome (IBS) in a subject. 7. Use of the strain according to any one of paragraphs. 1-4 for the treatment or prevention of sleep disorders in a subject. 8. Application according to any one of paragraphs. 5-7, wherein the subject is an infant under 12 months of age, preferably under 6 or 5 months, or preferably under 3 or 4 months, and/or wherein the subject is breastfed or formula-fed or a combination thereof. 9. A combination product suitable for inducing adenosine production in a subject, comprising: (i) a bacterial strain according to any one of paragraphs. 1-4 and (ii) the substrate component, which is AMP.