TWI856019B - Selection and use of melatonin supporting bacteria to reduce infantile colic - Google Patents

Abstract

The invention herein provides certain strains of lactic acid bacteria selected for their ability of increasing melatonin levels for prophylaxis and/or treatment of colic (or other diseases associated with reduced levels of melatonin), including the ability to produce precursors and other important components for melatonin release, a method of selecting such strains, and products containing such strains. Moreover this invention relates to preparations comprising substrate components being specifically chosen to enhance the efficacy of such strains.

Description

Selection and application of melatonin-supporting bacteria to reduce infantile colic

The present invention provides certain lactic acid bacteria strains selected for their ability to increase melatonin levels for the prevention and/or treatment of colic (or other conditions associated with decreased melatonin levels), including the ability to produce precursors and other important components for melatonin release; methods for selecting these strains; and products containing these strains. In addition, the present invention relates to formulations comprising substrate components that are specifically selected to enhance the efficacy of these strains.

Despite its prevalence and distress, the nature and causes of infantile colic remain poorly understood. Different terms are used to describe the condition. These include "infant colic", "nocturnal colic" because the pain is primarily confined to the night, and "three-month colic" on the pretext that it disappears after about three months of age. Different authors have used different definitions. Wessels' definition of colic as episodes of crying for three hours or more per day, three days per week or more for at least three weeks is the most accepted in the literature. In 2006, the Rome III criteria were published, which modified this criteria to consider the diagnosis of "infantile colic" for infants who are irritable, fussy, or crying, that starts and stops without obvious cause, lasts 3 hours or more per day, and occurs at least 3 days per week but lasts at least 1 week (sometimes referred to as modified Wessel's criteria). Further refinements were made in the Rome IV criteria; (Benninga et al; Gastroenterology 2016;150:1443-1455). Either of these definitions (Rome III or Rome IV) is relevant to the present invention.

To date, the main possible etiological factors discussed for infantile colic have been divided into: psychosocial, gastrointestinal tract including microbial dysbiosis, and neurodevelopmental disorders.

Psychosocial factors include: changes in normal crying, the impact of atypical parenting behaviors, and manifestations of problems in parent-infant interactions.

Colic is associated with gastrointestinal disturbances due to the position of the baby's legs and facial distortion during the cry. Gastrointestinal factors are briefly described below:

Inappropriate feeding techniques such as bottle feeding, feeding in a horizontal position and not interrupting feedings are considered causative factors. Breastfeeding in the first six months has been found to be a protective factor. The risk of infantile colic is 1.86 times higher in infants who are not breastfed (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. Current treatment paradigms for colic include pharmacological and/or nonpharmacological approaches that, at best, reduce symptoms. Typical treatment interventions for colic offered to parents fall into four categories, including dietary, physical, behavioral, and pharmacological. Dietary manipulations include professional advice on various feeding techniques, or the use of hypoallergenic milk, soy, or lactose-free formulas, food supplements such as various probiotics and prebiotics, and early introduction of solids.

Most over-the-counter medications used to treat colic involve giving simethicone or dimethicone, a non-absorbable over-the-counter medication that reduces the size of gas bubbles in the intestines. Simethicone has a safe profile and is often recommended, although some studies have shown that simethicone is no more effective than a placebo for colic in infants.

There have been several reports of altered microbiota patterns in infants with colic, leading some researchers to conduct and publish different randomized controlled clinical trials (RCTs) evaluating 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 efficacy of this strain on infant colic. Fifty exclusively breastfed infants with colic diagnosed according to modified Wessel's criteria were randomly assigned to receive Lactobacillus reuteri DSM 17938 ( ¹⁰⁸ colony-forming units) or placebo daily for 21 days. Parent questionnaires monitored crying time and adverse reactions daily. Forty-six infants (Lactobacillus reuteri group: 25; placebo group: 21) completed the trial. On day 0, crying time expressed in minutes/day (median) was 370 vs 300; on day 21, it was 35.0 vs 90.0 (P = .022).

In 2013, Szajewska et al. (J Pediatr 2013;162:257-62) published a similarly designed study, an RCT of 80 infants aged <5 months, which determined that the treatment response rate was significantly higher in the probiotic group than in the placebo group. The median minutes/day was 180 vs. 180 on day 7, 75 vs. 128 on day 14, and 52 vs. 120 on day 21 (P<0.05). There are also other studies showing the effectiveness of Lactobacillus reuteri DSM 17938 in significantly reducing crying time in breastfed infants with colic. However, even more effective interventions are needed to reduce crying at higher rates in colic children, including in formula-fed children.

Therefore, there is a need to demonstrate new (and preferably improved) safe and effective compounds and compositions and techniques that can be used to treat infantile colic. The compositions and methods of the present invention respond to this need and provide products that can safely and effectively prevent and treat infantile colic (or symptoms associated with colic).

Other purposes and benefits will become more apparent from the following disclosures.

Neonatal hormonal immaturity is thought to be a major factor in early problems with intestinal motility development, and therefore also causes pain and crying in infants, such as colic. Melatonin is a hormone that has the ability to support biological rhythms and is important for many functions in humans and animals. The precursor of melatonin is 5-hydroxytryptamine, itself a neurotransmitter, which can be derived from the amino acid tryptophan.

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

The generation of synchronization between different diurnal rhythms and the subsequent release of 5-hydroxytryptamine and melatonin in infants may be important for avoiding intestinal motility disorders and pain such as infantile colic (IC).

5-HT increases intestinal smooth muscle contractions, and melatonin relaxes intestinal smooth muscle. Peak 5-HT concentrations can cause intestinal cramps if not balanced by other medications (of which melatonin is an important one). Both 5-HT and melatonin have diurnal rhythms. Although 5-HT is produced and the 5-HT diurnal rhythm develops when the baby is born, endogenous melatonin production and the melatonin diurnal rhythm do not begin in many babies until after the third month of life. This time coincides with the normal disappearance of infant colic.

It can be said that infantile colic is a symptom of melatonin deficiency. Therefore, it is recommended to give exogenous melatonin to infants with IC, but many doctors and parents are reluctant to give hormones to babies.

IC prevalence is estimated at 5% to 25% of all newborns, which can be burdensome and costly to society, parents, and the infants themselves in some ways.

Therefore, while some children appear to be able to produce and release the required melatonin from their food and/or their intestinal bacteria and are therefore less likely to develop colic, many others do not and require additional support to prevent or treat IC.

Therefore, there is a need to identify and develop products and methods to support melatonin production and release in children at risk for developing or suffering from IC.

The present invention is based on the discovery that bacteria, such as lactic acid bacteria, can be selected that are capable of producing adenosine. Lactic acid bacteria that produce adenosine have not been reported in the art, and such bacteria can be used, for example, to increase the production of arylalkylamine-N-acetyltransferase (AANAT), which is the rate-limiting enzyme in melatonin synthesis. Although the AANAT enzyme is found in the pineal gland, it is also produced at melatonin production sites outside the pineal gland, such as the gastrointestinal tract. Therefore, increased AANAT production (e.g., by increasing adenosine production) provides a means to increase melatonin production or increase melatonin levels in the gastrointestinal tract. Therefore, such bacteria can be used to treat or prevent infantile colic or other diseases associated with or characterized by reduced, low or deficient melatonin production or synthesis.

Therefore, the present invention provides a bacterial strain, such as a lactic acid bacteria strain capable of producing or inducing adenosine production, for producing adenosine in a subject (e.g., increasing adenosine levels or increasing or promoting adenosine production), or for producing melatonin in a subject (e.g., increasing melatonin levels or increasing or promoting melatonin production).

The present invention also provides a method for producing adenosine or melatonin in a subject (e.g., increasing adenosine or melatonin levels, or increasing or promoting adenosine or melatonin production), the method comprising the following steps: administering an effective amount of a bacterial strain capable of producing or inducing the production of adenosine, such as a lactic acid bacteria strain, to the subject.

The present invention also provides the use of bacterial strains capable of producing or inducing the production of adenosine, such as lactic acid bacteria strains, for preparing a drug or composition for producing adenosine or melatonin in a subject (e.g., increasing adenosine or melatonin levels, or increasing or promoting the production of adenosine or melatonin).

More generally, the present invention also provides bacteria, such as 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 the production or level of melatonin in a subject.

Therefore, in one aspect, the present invention provides a lactic acid bacteria strain capable of producing or inducing the production of melatonin, which is used to produce melatonin in a subject (e.g., increase melatonin levels or increase or promote the production of melatonin).

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

On the other hand, the present invention provides the use of a lactic acid bacteria strain capable of producing or inducing the production of melatonin for preparing a drug or composition for producing melatonin in a subject (e.g., increasing the level of melatonin or increasing or promoting the production of melatonin).

As described above, such strains may be used to treat or prevent infantile colic, or other conditions associated with or characterized by decreased, low or deficient melatonin production or synthesis or levels.

Therefore, the present invention further provides lactic acid bacteria strains capable of producing or inducing the production of melatonin for use in treating or preventing infantile colic, or other diseases associated with reduced, low or deficient melatonin production or synthesis or levels in a subject.

In another aspect, the present invention provides a method for treating or preventing infantile colic or other diseases associated with or characterized by reduced, low or deficient melatonin production or synthesis or levels in a subject, the method comprising the steps of administering to the subject an effective amount of a lactic acid bacteria strain capable of producing or inducing the production of melatonin.

In another aspect, the present invention provides the use of a lactic acid bacterial strain capable of producing or inducing the production of melatonin in the preparation of a medicament or composition for treating or preventing infantile colic or other diseases associated with or characterized by reduced, low or deficient melatonin production or synthesis or levels in a subject.

Such strains may be capable of producing or inducing the production of adenosine, thereby producing or inducing the production (e.g., downstream production) of melatonin, as described elsewhere herein.

Other objects and advantages of the present invention will become apparent to the reader and are intended to be within the scope of the present invention.

[ Figure 1 ] is a schematic diagram showing the melatonin production pathway.

[ FIG. 2 ] is a schematic diagram showing how the bacteria of the present invention capable of producing adenosine affect the melatonin production pathway.

[ Figure 3 ] shows the measurement of 5'-nucleotidase activity in the supernatant of Lactobacillus reuteri bacterial cells.

[ Figure 4 ] shows the production of melatonin in an enteroid model of the intestine by adding supernatant of Lactobacillus reuteri bacterial cells.

[ Figure 5 ] shows the measurement of 5'-nucleotidase activity in the supernatant of Lactobacillus reuteri DSM 33198 bacterial cells.

Thus, as described above, the present invention provides bacterial strains, such as lactic acid bacteria strains, which are capable of producing or stimulating or inducing or supporting the production of melatonin, such as increasing the level of melatonin, particularly in the gastrointestinal (GI) tract. These strains can themselves produce melatonin, such as secreting melatonin, thereby directly providing or producing melatonin.

However, strains are also provided that indirectly cause melatonin production, such as by producing or stimulating or inducing the production of upstream components of the melatonin synthesis pathway (or precursors of melatonin), thereby supporting or increasing melatonin production. A preferred example is the production or induction of arylalkylamine-N-acetyltransferase (AANAT), which is the rate-limiting enzyme for the conversion of 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine, which is in turn converted to melatonin.

As mentioned above, another particularly preferred lactic acid bacteria strain is one that is able to produce or induce adenosine in a subject, particularly in the gastrointestinal (GI) tract. This adenosine can act on appropriate cell surface adenosine receptors (e.g., A2A receptors, A2B receptors, A3 or A4 receptors, preferably A2A receptors) and lead to the production of elevated levels of intracellular cAMP. A2A and other adenosine receptors are known to be present in cells found in the GI tract. Intracellular cAMP in turn participates in the production of AANAT (see Figure 2). Therefore, increased intracellular cAMP levels can lead to increased AANAT levels and therefore increased melatonin levels. Therefore, adenosine should preferably be provided extracellularly so that it can bind to cell surface adenosine receptors. To this end, adenosine can be produced inside the bacterial cell and transported extracellularly (or secreted). Alternatively, adenosine can be produced extracellularly, for example on the surface of the bacterial cell or in the supernatant. Such extracellular production can be conveniently performed, for example, by the presence of a cell wall (or cell surface) anchored 5'-nucleotidase (or extracellular 5'-nucleotidase), which can convert a suitable substrate into adenosine. Such extracellular production can also be performed by the presence of a 5'-nucleotidase (or extracellular 5'-nucleotidase) in the cell supernatant or in the extracellular space. As shown herein, a bacterial strain can provide or release such a 5'-nucleotidase (or an extracellular 5'-nucleotidase) into the cell supernatant or extracellular space, where it can then convert an appropriate substrate into adenosine. Suitable substrates include AMP.

Thus, when a bacterial strain is referred to herein as being capable of producing adenosine, this includes direct production of adenosine by the bacterial cell itself, and also includes production of adenosine by the bacterial cell via a cell that has an active 5'-nucleotidase (e.g. present on the bacterial cell surface or released into the cell supernatant or extracellular space) and thereby converts or is capable of converting a suitable substrate (e.g. AMP) into adenosine. Such a substrate may be naturally present, e.g. endogenously present in the environment, or may be provided to the bacterium, e.g. by exogenous means.

Such a strain could therefore lead to an increase in melatonin levels, for example compared to levels in the absence of such a strain.

Melatonin is known to be produced in the gastrointestinal tract, so 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 may be used to treat any disease or condition associated with (or characterized by) decreased or reduced levels of melatonin or any disease or condition associated with (or characterized by) melatonin deficiency, or any disease or condition that would benefit from an increase in melatonin levels. A preferred example of such a disease is infantile colic. Preferred strains for such therapeutic use are those that produce melatonin or adenosine.

Thus, a further aspect of the invention provides a lactobacillus strain capable of producing or inducing the production of melatonin (e.g. because the strain is capable of producing or inducing the production of adenosine), or a lactobacillus strain capable of producing or inducing the production of adenosine. The therapeutic use of these strains is also provided. Preferably, the strain has a gene encoding a 5'-nucleotidase, or such strain has an active 5'-nucleotidase, for example, converting an AMP substrate (or other suitable substrate) to adenosine. Exemplary levels of adenosine production or 5'-nucleotidase activity or levels of melatonin production are described elsewhere herein.

Preferred strains of the invention are Lactobacillus reuteri strains, more preferably Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 (deposit details of which are provided elsewhere herein), and these strains, e.g. isolated strains or biologically pure cultures or preparations of such strains, constitute further aspects of the invention, as well as compositions (e.g. pharmaceutical compositions or nutritional compositions such as food supplements, or probiotic compositions) comprising said strains, or therapeutic uses of such strains, e.g. as described elsewhere herein, in particular for the treatment or prevention of infantile colic (or other diseases described elsewhere herein). In some embodiments, the DSM 17938 strain is not used. Therefore, the present invention provides Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 for use in therapy, for example for the treatment or prevention of a disease, as described elsewhere herein. The present invention also provides Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 for use in the treatment or prevention of colic in infants.

Therefore, another aspect of the present invention provides a method for treating or preventing infantile colic or other diseases described elsewhere herein in a subject, the method comprising the step of administering to the subject an effective amount of Lactobacillus reuteri strain DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198.

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 preparation of a medicament or composition for treating or preventing infantile colic or other diseases as described elsewhere herein in a subject.

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

The strains Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848, DSM 32849 and/or DSM 33198 have been selected for their ability to produce adenosine (e.g., by having a gene encoding a 5'-nucleotidase, and an active 5'-nucleotidase that can convert an AMP substrate into adenosine), and are suitable for increasing not only adenosine levels (e.g., compared to levels in which the strain is not present), but also melatonin levels, e.g., by increasing the level of AANAT, as described elsewhere herein. The strains comprise an active cell wall-anchored 5'-nucleotidase, and also exhibit 5'-nucleotidase activity in their respective supernatants (see Figures 3 and 5A/B).

These Lactobacillus reuteri strains have been developed from naturally occurring strains (in other words, have been modified or engineered or evolved) and have also been selected for one or more other improved properties, such as increased resistance to bile or increased adhesion to mucosal surfaces, such as those of the gastrointestinal tract. Thus, DSM 32846 and DSM 32847 have evolved to be more tolerant to bile acid, and thus survive in greater numbers, for example, in the GI tract. DSM 32848 and DSM 32849 have evolved to adhere better to mucus, with the goal of better colonization in the GI tract, and thus, for example, better function according to the present invention. The Lactobacillus reuteri strain DSM 33198 has also been modified in a multi-step selection process including a repeated freeze drying process to allow it to be more tolerant and to give a higher survival rate in the production process than its natural isolate. Therefore, such a strain does not correspond to a naturally occurring strain and has been forced to evolve and is a non-natural strain. All Lactobacillus reuteri strains DSM 32846, DSM 32847, DSM 32848, DSM 32849 and DSM 33198 have been selected. Preferred strains are DSM 32846, DSM 32847, DSM 32849 or DSM 33198. More preferred strains are DSM 32846, DSM 32847 or DSM 33198.

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

The present invention provides the use of bacterial strains, such as lactic acid bacteria strains, that are capable of producing or inducing the production of adenosine (e.g., increasing adenosine levels, or increasing or promoting the production of adenosine) in the manufacture of a drug or composition for producing adenosine in a subject.

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

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

There are a number of locally released factors, including hormones, that regulate motility and secretion in the gastrointestinal tract. The activity of the digestive system and the transport of its contents determine said motility. When the nerves and/or muscles, gastrointestinal motor functions, in any part of the digestive tract, do not function with normal strength and coordination, individuals develop symptoms associated with motility disorders.

Disturbances in motor function have been described, for example, in subjects with infantile colic producing a variety of symptoms in various intestinal regions.

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. It is distressing for the baby, parents, and healthcare providers. The cause of colic is not fully understood.

Neonatal hormonal immaturity is thought to be a major factor in early problems with intestinal motility development, thus also causing pain and crying in infants, such as in infantile colic.

Melatonin is a hormone that supports biological rhythms and plays an important role in many functions in humans and animals. The hormone affects gastrointestinal motility through selective receptors (melatonin receptors) expressed on smooth muscle and gastrointestinal myometrium cells. The underlying basic mechanism of gastrointestinal smooth muscle contraction is loop-generated electrical current. Gastrointestinal myoelectric activity consists of two potentials: slow waves organized in migrating complex electromyography and spike activity.

The precursor of melatonin is 5-hydroxytryptamine, which is itself a neurotransmitter that can be derived from the amino acid tryptophan.

It has been shown that the 5-HT-induced reduction in food transit is partially blocked by melatonin. It is a counterbalancing system of melatonin and 5-HT in the regulation of intestinal activity.

In addition, a functional and balanced melatonin-5-hydroxytryptamine system influences appetite and digestive processes through endocrine and paracrine actions in the brain and gastrointestinal tract.

The actions of the two hormones appear to have opposite effects on exercise, and the final effect is dose-dependent. Melatonin and 5-hydroxytryptamine are also known to be involved in allergic reactions and pain transmission.

Melatonin, also known as N-acetyl-5-methoxytryptamine, is a hormone and neurotransmitter that was previously thought to be secreted primarily by the pineal gland. However, there are important sites of melatonin production outside the pineal gland, such as the retina and gastrointestinal tract. There are also reports showing that melatonin in the gastrointestinal tract is at least 400 times that of the pineal gland. The diurnal variation of gastrointestinal melatonin does not appear to be controlled by light exposure (as is the case with the pineal gland), but rather by food intake and food composition.

Synchrony between the generation and differentiation of diurnal rhythms and the subsequent release of serotonin and melatonin in infants is important for avoiding intestinal motility disorders and pain such as infantile colic (IC).

5-HT increases intestinal smooth muscle contractions, and melatonin relaxes intestinal smooth muscle. Peak 5-HT concentrations can cause intestinal cramps if not balanced by other agents (of which melatonin is an important one). Both 5-HT and melatonin have diurnal rhythms. Although 5-HT is produced and the 5-HT diurnal rhythm develops when the baby is born, endogenous melatonin production and the melatonin diurnal rhythm do not begin in many babies until after the third month of life. This time coincides with the normal disappearance of colic in infants. It has been shown that infants with colic have low melatonin in the morning early in life, whereas infants without colic have high melatonin (Cengiz et al 2015, Turkish journal of family medicine and primary care; 9(1)/10-15). These infants (e.g., infants with various forms of infantile colic associated with a lack of melatonin or melatonin balance, such as lack of melatonin levels in the morning, such as before 6am, 8am, 10am, or 12pm) are examples of subjects that may be treated using the present invention.

Thus, while some children appear to be able to endogenously produce and release the required melatonin from their food and/or their gut bacteria, and are therefore less likely to develop colic, many children are not, and therefore require additional support to prevent or treat IC. These infants (e.g., infants with melatonin deficiency caused by, for example, an underdeveloped gut microbiota or infants with nutritional or feeding problems) are also examples of subjects that may be treated using the present invention.

It can be said that infantile colic is a symptom of melatonin deficiency. This is particularly important for children who are under stress or who are not able to cope with stress (e.g., for children born before the full gestational time or who do not have fully functional food metabolism, etc.). Such infants (e.g., melatonin-deficient infants born before the full gestational time, infants delivered by caesarean section, or infants who do not have fully functional food metabolism) are also examples of subjects that can be treated using the present invention.

Another example of a condition involving the balance of melatonin function is irritable bowel syndrome (IBS), which is a common condition characterized by recurrent abdominal pain or discomfort, along with disrupted bowel habits without an identifiable cause. Melatonin is thought to show positive effects on pain perception and bowel habits (Siah KTH et al, 2014, World J Gastroenterol; 20(10): 2492-2498). Therefore, subjects with IBS, particularly forms of IBS associated with a lack of melatonin or melatonin balance, are also examples of subjects that can be treated using the present invention.

Melatonin deficiency is investigated primarily in the pineal gland, blood circulation, saliva, cerebrospinal fluid, and by measuring the metabolite 6-hydroxymelatonin sulfate in urine.

There are more than 20 types of enteroendocrine cells in the intestine, which makes it the largest endocrine organ in the human body. Enteric chromaffin (EC) cells are a type of enteroendocrine and neuroendocrine cell. They are located next to the epithelial cells lining the luminal lining of the digestive tract and play a crucial role in gastrointestinal regulation, especially intestinal motility and secretion. EC cells regulate neuronal signaling in the enteric nervous system (ENS) by secreting the neurotransmitter serotonin and other peptides, and they further express adenosine receptors and have active synthesis of melatonin.

A common symptom of insufficient melatonin signaling is sleep disturbance, and melatonin is one of the most popular natural health supplements of our time. Melatonin is often referred to as the "dark hormone" because its synthesis and secretion are enhanced by darkness and inhibited by light in certain parts of the body. Sleep disturbances are very common in children and can become chronic and persist for many years without appropriate treatment (Esposito et al. 2019, J Transl Med, 17: 77). Even elderly people have been reported to experience sleep disturbances. A decrease in the nighttime melatonin peak is regularly observed with aging. Therefore, subjects who have or suffer from a sleep disorder or sleep condition, particularly a form of sleep disorder or sleep condition associated with a lack of melatonin or melatonin balance, are also examples of subjects that can be treated using the present invention.

A decrease in melatonin levels has also been repeatedly described in neurodegenerative diseases, in particular in Alzheimer's disease and other types of senile dementia (Blumenbach Johann Friedrich, 2012, The Scientific World Journal Volume 2012, Article ID 640389). Therefore, subjects with such neurodegenerative diseases (particularly forms of such neurodegenerative diseases associated with a lack of melatonin or melatonin balance) are also examples of subjects that can be treated using the present invention.

The rate of melatonin formation depends on the activity of arylalkylamine-N-acetyltransferase (AANAT) in the pineal gland (Figures 1 and 2), but also on the activity of melatonin-producing sites outside the pineal gland, such as the gastrointestinal tract.

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

One object of the present invention is to use adenosine-producing lactic acid strains to prevent and/or treat infantile colic.

Another object of the present invention is to use adenosine-producing lactic acid strains to prevent and/or treat IBS.

Another object of the present invention is to use adenosine-producing lactic acid strains to prevent and/or treat sleep disorders.

Another object of the present invention is to use adenosine-producing lactic acid strains to prevent and/or treat neurodegenerative diseases, such as Alzheimer's disease and other types of dementia, such as other types of Alzheimer's disease.

The present invention also relates to methods for selecting specific bacterial strains, including lactic acid bacteria strains, capable of producing adenosine, and the use of such strains to deliver beneficial effects to infants suffering from colic or at risk of colic. Another object of the present invention is to provide new bacterial strains capable of producing adenosine.

An important metabolic process in the human body is purine metabolism, in which purines are metabolized and degraded by specific enzymes. An example of those enzymes is ecto-5'-nucleotidase (CD73), which is considered to be a key enzyme in the generation of adenosine.

The inventors unexpectedly discovered that some specific probiotic strains are able to produce adenosine.

Thus, the present invention includes novel methods for selecting specific bacterial strains, including lactic acid bacteria strains, that are effective in producing adenosine. The purpose of selecting specific bacterial strains is to use them to treat certain diseases, such as infantile colic (or other diseases described elsewhere herein).

The present invention provides a method for selecting bacterial strains, in particular lactic acid bacteria strains, which can be used as probiotics and for treatment, for example as a medicament or as a food supplement.

One aspect of the present invention therefore provides a method for selecting a bacterial strain, preferably a lactic acid bacterial strain, capable of producing adenosine, wherein the method comprises: a) screening a bacterial strain, such as a lactic acid bacterial strain, based on the presence of a gene encoding a 5'-nucleotidase (e.g. a cell wall-anchored 5'-nucleotidase); and/or b) screening a bacterial strain, such as a lactic acid bacterial strain, based on the presence of an active 5'-nucleotidase (e.g. a cell wall-anchored 5'-nucleotidase), or a supernatant thereof.

This 5'-nucleotidase (5'NT) is also called endo-5' nucleotidase or CD73 (cluster of differentiation 73). Strains that are able to produce adenosine can then be selected.

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

Once a suitable strain has been selected using the methods of the invention, it can be used to produce, for example, localized adenosine in a subject.

A bacterial strain, such as a lactic acid bacteria strain, selected, produced, obtained or obtainable by the method of the invention, wherein the strain is capable of producing adenosine in a subject, such as locally producing adenosine, is another aspect of the invention.

Also provided is the therapeutic use of the strain selected according to the present invention in infantile colic (or other diseases described elsewhere herein).

Therefore, another aspect of the present invention provides a bacterial strain, such as a lactic acid bacteria strain, selected, produced, obtained or obtainable by the method of the present invention (wherein the strain has a 5'-nucleotidase gene or an active 5'-nucleotidase and is capable of producing adenosine) for use in producing, such as locally producing, adenosine in a subject.

An alternative embodiment of the invention provides a bacterial strain, such as a lactic acid bacteria strain, wherein the strain has a 5'-nucleotidase gene or an active 5'-nucleotidase and is capable of producing adenosine, the bacterial strain being used to produce, for example locally, adenosine in a subject. Preferred features of the strain and its use are described elsewhere herein.

As described elsewhere herein, a specific embodiment of the present invention is to prevent and/or treat infantile colic (or other conditions described elsewhere herein).

Also provided are methods of treatment or methods of producing, e.g., locally producing adenosine in a subject, comprising administering to the subject a bacterial strain, e.g., a lactic acid bacteria strain, selected, produced, obtained, or obtainable by the selection method of the invention, in an amount effective to produce, e.g., locally produce adenosine in the subject, wherein the strain has a 5' nucleotidase gene or an active 5'-nucleotidase and is capable of producing adenosine. Preferred features of the strain and its therapeutic use, e.g., in infantile colic (or other diseases described elsewhere herein), are described elsewhere herein.

The present invention also provides the use of a bacterial strain, such as a lactic acid strain, selected, produced, obtained or obtainable by the selection method of the present invention in the preparation of a composition or drug for producing (e.g., locally producing) adenosine in a subject, wherein the strain has a 5' nucleotidase gene or an active 5' nucleotidase and is capable of producing adenosine. Alternative embodiments provide the use of a bacterial strain, such as a lactic acid bacteria strain, in the preparation of a composition or drug for producing, such as locally producing adenosine in a subject, wherein the strain has a 5' nucleotidase gene or an active 5'-nucleotidase and is capable of producing adenosine. Preferred features of the strain and its therapeutic use, such as in infantile colic (or other diseases described elsewhere herein), are described elsewhere herein.

Products or compositions, such as pharmaceutical compositions, probiotic compositions or dietary/nutritional compositions, comprising a bacterial strain as described herein (e.g. comprising one or more bacterial strains) (e.g. a bacterial strain capable of producing or inducing adenosine, melatonin, etc.) and the use of said products or compositions in methods and uses as described herein constitute further aspects of the present invention.

The Food and Agriculture Organization of the United Nations defines probiotics as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host". Today, many different bacteria are used as probiotics, such as lactic acid-producing bacteria, such as strains of Lactobacillus and Bifidobacterium .

Alternative and preferred embodiments and features of the invention as described elsewhere herein also apply to the methods of treatment, uses and products of the invention.

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

The strain that effectively produces adenosine can be used to locally produce adenosine in a subject (e.g., a mammal, preferably a human).

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

Certain methods include a step of screening for the presence of a gene encoding a 5'-nucleotidase (e.g., a cell wall anchoring 5'-nucleotidase) (e.g., step a)). Such screening may be performed using any suitable method and strains that are positive for the 5'-nucleotidase gene may be selected. Such methods may conveniently be performed in vitro, for example, by genetic or nucleic acid based methods to detect the presence of a gene encoding a 5'-nucleotidase, such as a cell wall anchoring 5'-nucleotidase (or a recognition fragment thereof), which sequence is known in the art. For example, PCR protocols (or other nucleic acid-based techniques) can be readily designed to accomplish this based on known nucleic acid sequences encoding enzymes, or alternatively, the genomic sequence of candidate strains can be carefully examined to identify genes encoding 5'-nucleotidase (e.g., cell wall anchoring 5'-nucleotidase), for example, based on homology to known 5'-nucleotidase sequences, including, for example, the presence of the LPXTG motif as discussed below.

An exemplary gene to be detected in the methods of the present invention is a cell wall anchoring (intracellular) 5'-nucleotidase gene from Lactobacillus reuteri (e.g. Genbank Accession No.: AEI56270.1, LPXTG-motif cell wall anchoring domain protein [Lactobacillus reuteri SD2112]), or an appropriate homolog/5' nucleotidase from other bacterial species, such as other lactic acid bacteria. Exemplary techniques are described in the Experimental Examples.

腺苷+磷酸(AMP是腺苷一磷酸)和測量該反應的測定法可以容易地用於確定活性5'-核苷酸酶的存在情況(或5'-核苷酸酶活性)。換句話說，本文提及的活性或功能性5'-核苷酸酶是能夠在合適的條件下例如當提供合適的底物例如AMP時催化該反應的酶。如果需要，對該5'-核苷酸酶的活性進行定量，例如通過測量在所述反應產生和可以被測量或定量的磷酸或腺苷或腺苷的其它適當的下游產物的量或濃度。可以方便地對細菌細胞樣品或來自細菌細胞的上清液進行活性測量。 Some methods include a step of screening for the presence of an active (functional) 5'-nucleotidase (e.g., a cell wall-anchored 5'-nucleotidase) (e.g., step b)). Such a step can be performed using any suitable method, for example, using an enzyme assay. 5'-nucleotidase catalyzes the following reaction: AMP + _H2O

Adenosine + phosphate (AMP is adenosine monophosphate) and assays to measure the reaction can be readily used to determine the presence of active 5'-nucleotidase (or 5'-nucleotidase activity). In other words, an active or functional 5'-nucleotidase as referred to herein is an enzyme that is capable of catalyzing the reaction under appropriate conditions, such as when a suitable substrate, such as AMP, is provided. If desired, the activity of the 5'-nucleotidase is quantified, such as by measuring the amount or concentration of phosphate or adenosine or other appropriate downstream products of adenosine that are produced in the reaction and can be measured or quantified. Activity measurements can be conveniently performed on bacterial cell samples or supernatants from bacterial cells.

Methods for performing such assays are well known to those skilled in the art. For example, suitable kits are commercially available, such as the Crystal Chem 5'-Nucleotidase Assay Kit (Crystal Chem, catalog #80229, Downers Grove, IL, USA), in which 5'-nucleotidase activity is measured by the production of a dye (quinone dye) that is formed as a downstream product of the conversion of AMP to adenosine (i.e., as a result of adenosine production). AMP is provided as a substrate. Exemplary techniques are described in the Examples section. Suitable methods are also readily available from other reagent suppliers, such as Sigma, and their 5'-nucleotidase reagents.

Strains with high or significant 5'-nucleotidase production levels (or 5'-nucleotidase activity) are preferred, for example strains with at least or greater than 2 units/L (units per liter) of 5'-nucleotidase activity and/or capable of producing adenosine at at least or greater than 2 μmol L ^-1 min ^-1 (μmol per minute per liter). Thus, in preferred embodiments, strains are selected that have 5'-nucleotidase activity at a level 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 are capable of producing adenosine at a level 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, these values may represent upper limits. These values usually refer to the value of 5'-nucleotidase activity measured in a sample of bacterial cells, for example on the surface of bacterial cells, or in the supernatant of cultured bacterial cells, preferably in the supernatant. These values usually refer to the level of adenosine (preferably extracellular adenosine level) measured in a sample of bacterial cells, for example in the supernatant of cultured bacterial cells. When measured with a concentration of 10 ⁹ bacteria / ml or in the supernatant from such a culture, these values usually refer to the value of 5'-nucleotidase activity (or adenosine level). One unit is defined as the amount of enzyme required to produce 1 μmol product / minute (e.g. 1 μmol product / liter / minute). The concentration in units/liter then corresponds to the enzyme concentration required to increase the concentration by 1 μM/minute (e.g. 1 μM/liter/minute). Suitable and exemplary assays for measuring this activity are shown in the Examples using the 5'-nucleotidase kit (#80229) from Crystal Chem Inc. Therefore, in preferred embodiments, the above units and values refer to the units and values when measured using the conditions described in the kit and/or the Examples, particularly at a concentration of 10 ⁹ bacteria/ml or supernatant from the culture or an equivalent assay. Therefore, these methods will be conveniently performed in vitro.

A method comprising 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. In this method, the use of the invention and the lactic acid bacteria, adenosine production or adenosine activity should occur extracellularly, i.e. on the outside or surface of the lactic acid bacteria, whereby it can be, for example, present in the supernatant or other extracellular fluid produced by the lactic acid bacteria. Thus, the presence of an active 5'-nucleotidase on the cell surface (e.g. in the form of a cell wall-anchored 5'-nucleotidase) or from outside the bacterial cell (e.g. in the supernatant) can be a useful feature, so that the reaction to produce adenosine occurs extracellularly, for example on the surface of the bacterial cell.

Thus, the production of good levels of adenosine (e.g., extracellular adenosine produced by a 5'-nucleotidase) can also be an indicator of the presence of a 5'-nucleotidase gene or an active 5'-nucleotidase. Thus, the selection method of the invention can also involve a step of selecting strains that produce adenosine. Strains with high or significant levels of adenosine, e.g., extracellular adenosine, are preferred, e.g., strains that produce adenosine at levels that are therapeutically effective in a subject or at levels at which increased melatonin levels (and preferably therapeutically effective levels) are observed. These values generally refer to adenosine values measured in the supernatant of a cultured strain or on the surface of cells of a cultured strain (in vitro) and some exemplary specific values are provided above.

However, in some embodiments, appropriate refers to the value, level or amount of adenosine produced in a subject, e.g., locally, e.g., at the site of administration, e.g., in the gastrointestinal tract. For example, a preferred strain may result in increased adenosine production, e.g., increased production in vivo, e.g., local production of adenosine (e.g., in the gastrointestinal tract), e.g., compared to a relevant control described elsewhere herein (e.g., the level of adenosine when the strain is not administered, or the basal or natural level of adenosine in a particular subject).

In all aspects of the invention described herein, local production can refer to the production or level of adenosine at the site of administration, such as the production or level of adenosine in the gastrointestinal tract (e.g., when administered orally). In one aspect, local production of adenosine can have systemic downstream effects and may increase the level of adenosine or melatonin in the circulatory system, such as in the blood or plasma, or elsewhere outside the gastrointestinal tract.

Thus strains are preferred that are capable of causing an increase in local levels of adenosine (or melatonin), for example in the gastrointestinal tract of a subject, particularly when such an effect is observed when the strain is administered orally. Preferably, the increase is a measurable or significant increase, for example statistically or clinically significant. For example, strains that produce at least or up to 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increases in adenosine (or melatonin) levels, for example local increases in adenosine (or melatonin) levels are preferred. Any appropriate comparison may be used, for example an increase compared to levels observed when the strain is not administered, or levels when a control formulation, for example a control formulation not containing the relevant strain, is administered.

Suitable methods for measuring the level of adenosine (or melatonin) production are well known to those skilled in the art. Therefore, in some embodiments of the present invention, the selection method will include a step of detecting or determining the amount or level (e.g., concentration) of adenosine produced by the candidate strain.

Optionally, it may be convenient to compare the adenosine production level or 5'-nucleotidase activity to a positive or negative control strain. A suitable positive control strain may be DSM 17938, which has been 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. Therefore, strains that are able to produce higher (increased) levels or significantly higher (increased) levels of 5' nucleotidase activity than DSM 17938 (e.g. in the supernatant of bacterial cells, for example when assessed in vitro) constitute another aspect of the invention. Exemplary strains are DSM 32846, DSM 32847, DSM 32849 and DSM 33198 (see Figure 3 and Figure 5A/B). Alternatively, strains capable of producing or inducing the production of higher (increased) levels or significantly higher (increased) levels of adenosine compared to DSM 17938 constitute a further aspect of the present 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, such as higher local levels of adenosine in a subject, constitute a further aspect of the present invention compared to DSM 17938, in particular when such an effect is observed when the strain is administered orally.

Preferably, the increase (in adenosine production or 5' nucleotidase activity) is a measurable or significant increase, e.g., statistically or clinically significant. For example, strains that produce at least or up to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more increase in adenosine levels, e.g., local levels of adenosine or 5' nucleotidase activity levels (e.g., when assessed in vitro) compared to DSM 17938 levels are preferred. Alternatively, strains that can produce at least or up to a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increase in adenosine levels (e.g. local adenosine levels) or 5'-nucleotidase activity levels compared to the levels of DSM17938 are preferred. Preferably, this increase is an increase in the extracellular level of adenosine or 5'-nucleotidase activity, for example as measured in the supernatant of a bacterial culture, for example in vitro.

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 (or does not have 5'-nucleotidase activity as described elsewhere herein).

Due to downstream applications of strains selected by the methods of the present invention, after selecting or isolating adenosine-producing strains, other embodiments will involve the further steps of culturing or propagating or producing such strains, and optionally formulating the cultured or propagated or produced strains into compositions comprising the strains, such as pharmaceutical or nutritional compositions, such as described elsewhere herein, or possibly storing these strains for future use, such as by lyophilization or freeze drying. In one embodiment, the bacterial strains are provided in lyophilized form.

The selected steps of the methods of the invention (and indeed the treatment methods as described herein) generally need to be carried out in a suitable culture medium (or in vivo environment) that supports adenosine production. Preferably, the culture medium (or in vivo environment) will contain a suitable carbon source that will support the production of adenosine by the strain, preferably together with a suitable substrate (such as AMP) for the production of adenosine by 5'-nucleotidase.

While such assays can conveniently be performed in vitro, another option is to assess the strains in a suitable ex vivo assay, such as an enteroid model using a suitable intestine (see, e.g., the enteroid model and assay described in Example 5, where, for example, melatonin levels can be assessed).

Therefore, a preferred in vitro or ex vivo model for evaluating strains is an enteroid model of the intestine, preferably an enteroid model of the human intestine. Such a model preferably comprises intestinal endocrine 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. These values generally refer to the level of melatonin production induced by the strain when measured in a suitable in vitro or ex vivo assay, for example, to the level of melatonin produced by enteroendocrine cells (preferably human cells) or in an enteroid model of the intestine comprising enteroendocrine cells (preferably a model comprising human cells) when the cells are contacted with the strain of the invention or supernatant removed from the strain (e.g., expressed as % of enteroendocrine cells, as described elsewhere herein). Suitable and exemplary assays for measuring this activity are shown in the Examples using an intestinal model of the human intestine comprising intestinal endocrine cells, such as at least 20% or 30%, such as 20-40%, 30-40% or up to 40% intestinal endocrine cells. Therefore, in preferred embodiments, the above values refer to values measured using the assay (or an equivalent assay, such as using the above intestinal endocrine cells) and/or the conditions described in the Examples.

Some strains are capable of producing or inducing higher, sometimes significantly higher, levels of melatonin than DSM 17938, for example from enteroendocrine cells or an enteroid model of the intestine as described above. Therefore, strains capable of producing or inducing higher (increased) levels or significantly higher (increased) levels of melatonin compared to DSM 17938 (e.g. when assessed as described above, for example using enteroendocrine cells or an enteroid model of the intestine) constitute another aspect of the invention. Exemplary strains may produce at least or up to 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increase in melatonin production levels compared to DSM 17938 levels, and are preferred.

Therefore, the screening method of the present invention may optionally include a further step of selecting a strain that is capable of producing or inducing the production of melatonin, for example using the method described above. The selected strain can preferably induce the production of melatonin at the level described above.

In embodiments where the methods of the invention are used to screen more than one bacterial strain, the amount of adenosine or 5'-nucleotidase activity (or melatonin) produced can be optionally quantified, and bacterial strains, such as lactic acid bacteria strains, having the highest activity or level of 5'-nucleotidase, or sufficiently high activity or level of 5'-nucleotidase (e.g., having an activity or level described elsewhere herein, such as having an activity or level higher (preferably significantly higher) than DSM17938) can be selected; or strains producing the highest amount or level of adenosine or melatonin, or sufficiently high amount or level of adenosine or melatonin (e.g., having an amount or level described elsewhere herein, or having an amount or level higher (preferably significantly higher) than DSM17938) can be selected.

Therefore, another aspect of the present invention is to provide a method for selecting a bacterial strain, preferably a lactic acid bacteria strain, that effectively produces adenosine (or melatonin), comprising: a) screening the lactic acid bacteria strain for the presence of a gene encoding a 5'-nucleotidase, such as a 5'-nucleotidase anchored on the cell surface, and; b) quantifying the 5'-nucleotidase activity and; optionally c) selecting the lactic acid bacteria strain with the highest or sufficiently high 5'-nucleotidase activity.

Methods comprising certain steps as described herein also include, where appropriate, methods consisting of these steps.

As described above, a bacterial strain, such as a lactic acid bacteria strain, selected, produced, obtained or obtainable by the method of the present invention (wherein the strain is capable of producing adenosine, and preferably in turn produces or supports the production of, for example, increased production of, melatonin) is another aspect of the present invention.

The selection method of the invention can be performed on any suitable bacterial strain, such as a probiotic strain, such as any probiotic, and any suitable bacterial strain, such as a probiotic strain, capable of producing adenosine can be used in the methods or uses of the invention, such as in the treatment methods or uses described herein.

Preferred bacterial strains are lactic acid bacteria, such as Lactobacillus or Bifidobacterium . Particularly preferred bacterial strains are Lactobacillus reuteri, in particular strains Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849, which were deposited on July 4, 2018 under the Budapest Treaty with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Mascheroder Weg 1b, D-38124 Braunschweig); and Lactobacillus reuteri DSM 33198, which was deposited on July 9, 2019 under the Budapest Treaty with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Mascheroder Weg 1b, D-38124 Braunschweig). In some embodiments, Lactobacillus reuteri strain DSM 17938 is not used. Thus, a further aspect of the invention provides the use of such a strain for treating or preventing one or more diseases described herein.

Another object of the present invention is to provide a method for improving the therapeutic use and effect of a specific disease described herein, comprising maximizing, increasing or improving the 5'-nucleotidase activity of a selected strain, such as a lactic acid bacterium, preferably resulting in increased or improved adenosine production. This can be achieved by culturing the bacteria under specific growth conditions to obtain a high yield of adenosine (or a higher, such as a significantly higher yield of adenosine, such as compared to the yield observed in the absence of specific growth conditions). In one embodiment of the present invention, the specific growth conditions can be, for example, culturing lactic acid bacteria in a normal growth medium supplemented with a suitable substrate (such as AMP) or a suitable upstream component of AMP such as ADP and/or ATP for the production of adenosine by 5' nucleotidase.

In another related embodiment of the present invention, in order to maximize the activity of 5'-nucleotidase, a higher concentration of bacteria, such as lactic acid bacteria, may be applied. Exemplary concentrations may be 2 to 10 times the conventional dosage.

In another related embodiment of the present invention, the 5'-nucleotidase gene can be overexpressed or induced in a bacterial strain by an appropriate method, such as inserting a plasmid vector having the 5'-nucleotidase gene under the control of a suitable promoter, such as an inducer promoter. Alternatively, the 5'-nucleotidase gene can be overexpressed by inserting one or more additional gene copies into a bacterial cell, such as into a chromosome of a bacterial cell, under the control of a suitable promoter, such as an inducer or natural promoter.

As described above, the strains of the invention, or strains selected, produced, obtained or obtainable by the methods of the invention, can be used for treatment. Therefore, another aspect of the invention provides a strain producing adenosine of the invention, or a strain selected, obtained or obtainable using the selection method of the invention, for producing, for example, locally, adenosine in a subject, and thereby preferably melatonin. In a preferred embodiment of the invention, the production of adenosine, etc. and preferably melatonin is used to treat infantile colic (and other diseases as described herein), which would benefit from the production or increased production of adenosine and further melatonin, for example production.

In the treatment methods and uses of the present invention, the bacterial strain is administered to a subject (animal/mammal) in need of treatment in a pharmaceutically, therapeutically, or physiologically effective amount. Therefore, the methods and uses may involve an additional step of identifying a subject in need of treatment.

Treatment of a disease or condition according to the present invention (e.g., treatment of an existing disease) includes cure of the disease or condition, or any reduction or alleviation of the disease or disease symptoms (e.g., reduction in the severity of the disease).

The methods and uses of the present invention are applicable to the prevention of diseases or conditions as well as the treatment of diseases or conditions. Therefore, preventive treatment is also included in the present invention. Therefore, in the methods and uses of the present invention, treatment or therapy also includes prevention or prevention when appropriate.

These prevention (or) aspects may be conveniently performed on healthy or normal subjects as described herein, and may include complete prevention and significant prevention. Similarly, significant prevention may include a situation where the severity of a disease or disease symptom is reduced (e.g., measurably or significantly reduced) compared to the severity or symptom that would be expected if no treatment were given.

Another aspect of the invention provides a strain or product for therapeutic use as defined elsewhere herein, wherein the use further comprises administering at least one additional agent, such as an additional therapeutic agent or nutrient. Exemplary agents may be substrate components that increase or enhance adenosine production (components that can increase or enhance adenosine production), or sources of such components.

Therefore, in another embodiment of the present invention, when it comes to bacteria that produce adenosine, the administration of the strain or product also includes the administration of a substrate component such as AMP and/or a substance or agent that produces the component. The preferred substrate is AMP, or a source of AMP.

In such an embodiment, the substrate component or other additional components can be added directly to the bacterial preparation before administration to the subject. Alternatively, it can be added to the bacterial preparation at the end of the manufacturing process, such as at the end of fermentation, after which the bacteria can be stored for future use, such as by freeze drying or lyophilization. Alternatively, it can be administered separately as described below.

In such embodiments, the additional therapeutic agent can be any other agent useful in treating the target disease.

The other agent can be administered together with the strain or product of the present invention (e.g., as a combined preparation) or can be administered separately. In addition, the other agent can be administered at the same time as the strain or product of the present invention or at different time points, such as sequential administration. A person skilled in the art can easily determine the appropriate dosing regimen and timing based on the other target agent.

The present invention also provides a composition (or a combination product or a kit) comprising: (i) a bacterial strain selected, produced, obtained or obtainable by the selection method of the present invention, such as a lactic acid bacteria strain (or a bacterial strain capable of producing or inducing the production of adenosine as otherwise defined herein), wherein the bacterial strain has a gene encoding a cell surface-anchored 5'-nucleotidase or an active 5'-nucleotidase and is capable of producing adenosine; and (ii) one or more substrates that increase or enhance adenosine production, or a source of these components or agents.

Exemplary components or agents are summarized above. A preferred component is AMP or a source of AMP.

The present invention also provides a composition (or combination product or kit) suitable for increasing the production of melatonin or adenosine in a subject, comprising: (i) a first bacterial strain, such as a lactic acid bacteria strain, capable of producing or inducing the production of melatonin or adenosine, as described herein; and (ii) another source of melatonin and/or adenosine, or one or more substrate components or agents that increase or enhance the lactic acid bacteria strain's ability to produce or induce melatonin and/or adenosine, or sources of these components or agents, as appropriate.

The preferred component is AMP or a source of AMP.

In the kit or combination product of the present invention, components (i) and (ii) are generally provided in separate compartments or containers, or as separate components of the kit or product. The kit may contain further components, which may also be in separate compartments or containers. Preferably, the kit (or combination product) is used in the methods or uses of the present invention as described herein, for example for the treatment or prevention of other diseases described herein, in particular infantile colic. Instructions for the kit or product used in the methods and uses of the present invention may optionally be provided.

The term "subject" as used herein includes mammals, particularly humans, especially infants. In a preferred embodiment of the present invention, the selected strain, such as a lactic acid bacteria strain, is administered to a human.

Preferred subjects according to the invention are infants younger than or at most 12 months, preferably younger than or at most 5 or 6 months, preferably younger than or at most 3 or 4 months old. These subjects are particularly suitable when the disease to be treated or prevented is infantile colic. Thus, the subjects can be treated from birth, or within the first weeks of life (e.g. within the first 1, 2, 3 or 4 weeks) or within the first months of life (e.g. within the first 1, 2, 3 or 4 months).

As described elsewhere herein, preferred subjects are those who suffer from colic or are believed or suspected to suffer from colic. Because the therapeutic uses of the invention may also be used to prevent disease, appropriate subjects include subjects who are at risk for colic.

Exemplary subjects would include infants who are exclusively (only) breastfed, and infants who are exclusively (only) formula-fed (non-breastfed infants), or infants who have been both breastfed and formula-fed.

Other exemplary subjects include infants, particularly infants who have colic, are suspected of having colic, or are at risk for developing colic, who do not yet produce or release melatonin or are otherwise unable to produce or release melatonin, such as infants in whom a diurnal melatonin cycle has not yet developed or initiated, or who are unable to produce or release the required melatonin from their food and/or their intestinal bacteria; or infants who have low, reduced (or abnormal) or melatonin-deficient levels, such as compared to levels in normal or healthy subjects (e.g., levels in normal or healthy subjects of the same, equivalent, or comparable age).

Other exemplary subjects include infants who have some type of nutritional or dietary problem or difficulty, such as one that results in low or insufficient melatonin levels. These subjects may include infants born before full gestational time, such as premature infants or infants born before 36 weeks of gestation, or infants who lack full functional food metabolism, or infants who have feeding problems or other nutritional deficiencies or who have a poor or suboptimal diet.

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

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

Any suitable mode of administration may be used. Conveniently, the administration is oral, rectal or by tube feeding. Thus, the bacteria may be administered to the gastrointestinal tract or oral cavity as required or appropriate. The bacteria may be contained in infant formula or food supplements or oil drops, as well as in pharmaceutical preparations.

Depending on the disease (or condition) to be treated, the mode of administration and the formulation involved, the appropriate dosage of the strains, products and compositions of the present invention as defined herein can be easily selected.

For example, the dose and dosing regimen are selected so that the bacteria administered to a subject according to the present invention can result in increased production of adenosine (or melatonin), e.g., local production, and produce the desired therapeutic effect or health benefit in infantile colic (or other disease to be treated). Thus, preferably, the dose is a therapeutically effective dose that is appropriate for the type of mammal and condition being treated and is administered, e.g., to a subject in need thereof. For example, a daily dose of 10 ⁴ -10 ¹² , e.g., 10 ⁵ -10 ⁹ or 10 ⁶ -10 ⁸ or 10 ⁸ -10 ¹⁰ or 10 ¹⁰ -10 ¹² total CFU of the bacteria may be used, e.g., a single daily dose.

In one embodiment, the daily dose (e.g., 10 ⁵ -10 ⁹ or 10 ⁶ -10 ⁸ or 10 ⁸ -10 ¹⁰ or 10 ¹⁰ -10 ¹² CFU) is divided into several doses, such as 2-8 doses (e.g., 3-5 doses) or is associated with the subject's feeding (breast milk and/or formula). In one embodiment, a higher dose, such as 2 to 10 times or 10 to 100 times the dose after the initial period, and/or a more frequently administered dose is administered as an initial dose for 3-14 days (e.g., 5-10 days, e.g., 7 days) to achieve faster relief in the subject.

0.05。 As used herein, the term "increase" or "enhance" or "higher" (or equivalent terms) includes any measurable increase or elevation when compared to an appropriate control. Appropriate controls will be readily determined by those skilled in the art and may include levels in the absence of the strain, 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, such as clinically or statistically significant, such as a probability value.

0.05.

0.05，當與適當的對照水準或值比較時(例如，與未治療或用安慰劑治療的受試者相比或與健康或正常受試者或治療前相同受試者相比)。 Preferably, such increases (and indeed other increases, improvements or positive effects as mentioned elsewhere herein) are measurable increases etc. (as appropriate), more preferably they are significant increases, preferably clinically significant or statistically significant increases, e.g. a probability value of

0.05 when compared to an appropriate control level or value (e.g., compared to untreated or placebo-treated subjects or compared to healthy or normal subjects or to the same subjects before treatment).

0.05。 The term "reduction" or "reduction" (or equivalent terms) as used herein includes any measurable reduction or decrease when compared to an appropriate control. Appropriate controls can be readily determined by a person skilled in the art, and may include levels in the absence of the strain, or levels in untreated or placebo-treated subjects, or levels in healthy or normal subjects, such as age-matched subjects or the same subjects before treatment. Preferably, the reduction or decrease will be significant, such as clinically or statistically significant, such as a probability value.

0.05.

0.05，當與適當的 對照水準或值比較時(例如，與未治療或用安慰劑治療的受試者相比或與健康或正常受試者或治療前相同受試者相比)。 Thus, preferably, such reductions (and indeed other reductions, decreases or adverse effects as mentioned elsewhere herein) are measurable reductions etc. (as appropriate), more preferably they are significant reductions, preferably clinically significant or statistically significant reductions, e.g. a probability value of

0.05 when compared to an appropriate control level or value (e.g., compared to untreated or placebo-treated subjects or compared to healthy or normal subjects or to the same subjects before treatment).

Other objects and advantages will become more fully apparent from the following non-limiting embodiments and the appended claims.

Embodiment

Embodiment 1

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

Grow bacteria on MRS plates at 37°C in an anaerobic atmosphere for 16 hours. Collect 10 bacterial colonies using a sterile plastic loop and suspend in 100 μl sterile water (PCR quality). (Alternative: prepare DNA from bacterial cultures using a suitable method).

The presence of the 5'-nucleotidase gene can be checked by PCR, for example by 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 microliters) should be added to the PCR mixture, and the PCR reaction should be run by running the program at 95°C, 5 minutes, 30x (95°C, 30s; 55°C, 30s; 72°C, 30s); 72°C, 10 minutes. The PCR products can be separated and visualized by using standard agarose gel electrophoresis and the sequence determined by standard Sanger sequencing (using the forward primer used for PCR).

Genes can be detected using any of the following primers:

Primer pair 1 (product size 233bp)

LrNuc1f：GGAACTTTGGGAAACCATGA

LrNuc1r:CGGGCAACTTTACCATCACT

Primer pair 2 (product size 212bp)

LrNuc2f: TACTCGTGAAAATGCCGTTG

LrNuc2r:GTGCCCCTGTCATTTCAACT

Primer pair 3 (product size 232bp)

LrNuc3f: AGCTTTACCAAATTGACCCTGA

LrNuc3r: TTGATATTAGGCGCATCCTTTT

Sequence

Gene encoding a cell surface anchoring 5'-nucleotidase, Genbank accession number: AEI56270.1, LPXTG-motif cell wall anchoring domain protein [Lactobacillus reuteri SD2112]

Cell surface anchoring 5'-nucleotidase protein, Genbank accession number: AEI56270.1, LPXTG-motif cell wall anchoring domain protein [Lactobacillus reuteri SD2112]

Embodiment 2

Methods for analyzing 5'-nucleotidase activity

The Crystal Chem 5’-Nucleotidase Assay Kit (Crystal Chem, Elk Grove Village, IL, USA) was used to measure the 5’-nucleotidase activity of bacterial cells and fermentation supernatants. Briefly, the procedure was as follows.

In two steps, reagents CC1 and CC2 are added to samples containing bacteria or supernatant. Reagent 1 contains AMP that is converted to adenosine by 5'-nucleotidase from bacteria. Adenosine is further hydrolyzed to inosine and hypoxanthine by components in reagent 1. In the second step, hypoxanthine is converted to uric acid and hydrogen peroxide, which are used to generate a quinone dye that is measured kinetically at 550 nm in a spectrophotometer. Activity is determined by calculating the change in absorbance between 3 and 5 minutes and comparing it to the value of the calibration sample.

Embodiment 3

5'-Nucleotidase Activity in Lactobacillus reuteri Strains

Experimental data were generated using the method described in Example 2 above, which show 5'-nucleotidase activity in Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849 and in Lactobacillus reuteri DSM 17938 (deposited on January 30, 2006 under the Budapest Treaty at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Mascheroder Weg 1b, D-38124 Braunschweig)). The results are shown in Figure 3.

Embodiment 4

Select strain

All the novel strains in Example 3 above, namely Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849, showed 5'-nucleotidase activity in the bacterial supernatant. The supernatant of a bacterial culture at a concentration of 10 ⁹ bacteria per ml was analyzed.

Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849 have been developed/evolved for improved performance. DSM 32846 and DSM 32847 have been evolved to be more resistant to bile acid and thus survive in greater numbers in the gastrointestinal tract. DSM 32848 and DSM 32849 have been evolved to adhere better to mucus with the aim of better colonization in the gastrointestinal tract and thus better function according to the invention.

Select all strains of Lactobacillus reuteri DSM 32846, DSM 32847, DSM 32848 and DSM 32849. Preferred strains are DSM 32846, DSM 32847 and DSM 32849.

Embodiment 5

Effects of probiotic strains on melatonin production in an enteroid model of the human intestine

background

1%增加至約40%。 Human intestinal enteroids (HIE), derived from intestinal crypt stem cells, are biologically relevant in vitro models of intestinal epithelial cells. HIE contain all intestinal epithelial cell types; however, similar to the human intestine, HIE typically produce enteroendocrine cells (EECs) in low numbers (approximately 1%), which limits their study. To increase the number of EECs in HIE, lentiviral transduction was used to stably engineer jejunal HIE (tetNGN3-HIE) with expression of doxycycline-induced neural element-3 (NGN3), a transcription factor that drives EEC differentiation. In this genetically engineered cell line, the number of enteroendocrine cells present in culture increased from

1% to approximately 40%.

Materials and methods

Enteroids of the human intestine

1%增加至約40%。根據標準方案，所有類腸以96孔單層形式 繁殖和分化。關於該類腸細胞系的更多資訊、如何培養以及測定的一般方法描述於Chang-Graham等人(Chang-Graham et al.,Cellular and Molecular Gastroenterology and Hepatology,2019,S2352-345X(19)30049-9，印刷中，線上出版可用)中。 HIE cultures were generated from crypts isolated from jejunal tissue of adult patients undergoing bariatric surgery. These established cultures were obtained from the Baylor College of Medicine at the Texas Medical Center Digestive Diseases Center Study Design and Clinical Research Core. Three-dimensional HIE cultures were prepared from tissue samples and maintained in culture. To increase the number of EECs in HIE, lentiviral transduction was used to stably engineer jejunal HIE (tetNGN3-HIE) with expression of doxycycline-induced neural element-3 (NGN3), a transcription factor that drives EEC differentiation. In this genetically engineered cell line, the number of enteroendocrine cells present in the culture increased from

1% to approximately 40%. All enteroids were propagated and differentiated in 96-well monolayer format according to standard protocols. More information about this enteroid cell line, how to culture it, and general methods for assays 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, available online).

Bacterial strains and preparations

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

Conditioned media (supernatants) from Lactobacillus reuteri DSM 17938 and Lactobacillus reuteri DSM 32846 grown in Lactobacillus Defined Media 4 (LDM4) until they reached stationary phase were used in this study, see below for more details.

Single colonies of bacterial strains were picked from MRS agar plates and used to inoculate 10 mL of MRS broth and incubated in a sealed conical tube in a 37°C water bath. After 8 hours, a 1:10 dilution of the culture was performed and the optical density at 600 nm was read using a spectrophotometer, then inoculated with 25 mL of pre-warmed LDM4 at a starting OD of 0.1 (calculated below) and incubated in a water bath for 16 hours.

○OD=0.307. 1:10 dilution=OD is 3.07

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

○ Combine 814μL with 24.19mL LDM4

After 16 hours, remove the culture. The final OD is 2.6. Pellet the cells by centrifugation at 4700 rpm and transfer the supernatant to a new 50 mL conical tube. Store the supernatant at -20 °C overnight.

Chemicals used

AMP=adenosine 5'-monophosphate, 99% (Acros Organics) Code: 102790259, Batch number: A0395274.

Prepare 10 mL of stock solution in MiliQ water (2 mM stock solution = 6.9 mg/ml (FW = 347.224)) and sterilize by filtering through a 0.22 μM filter.

Experimental setup

Control treatments for induced enteroids: ￭LDM4 (culture medium), ￭LDM4+20uM AMP, ￭LDM4+200uM AMP

Experimental treatment of induced enteroids with supernatants from DSM 32846 and DSM 17938:

￭ Only DSM 32846 supernatant

￭DSM 32846 supernatant + 20uMAMP

￭DSM 32846 supernatant + 200uMAMP

￭ Only DSM 17938 supernatant

￭DSM 17938 supernatant + 20uMAMP

￭DSM 17938 supernatant + 200uMAMP

Before preparing the supernatant or other additives for application to enteroids, neutralize the pH and filter-sterilize the supernatant. Adjust the pH to 7.0 using 10-30 μL of 10M sodium hydroxide solution and measure the pH by applying 2 uL of the supernatant to a pH test strip (Fisher Brand-Cat. 13-640-510 range 6.0-8.0). Filter-sterilize the supernatant using a 0.22 uM filter (Thermo Scientific Nalgene Rapid Flow 0.22 uM filter code: 00158).

100uL (total volume) of treatment was placed on the enteroids. The enteroids were incubated for 1 hour at 37°C in a 5% _CO2 tissue incubator. After incubation, the supernatant was removed and stored at -20°C until downstream analysis.

Melatonin was detected by ELISA (Eagle Biosciences Inc, Amherst, NH, USA). The only step completed before following the standard protocol was thawing the supernatant and allowing it to reach room temperature. (approximately 20-21 degrees Celsius).

result

Human enteroids (HIE) are biologically relevant as an in vitro model of the intestinal epithelium. As shown in Figure 4, DSM 32846 alone had no significant effect, but when supplemented with AMP it showed a clear effect on melatonin production in HIE. Higher concentrations of AMP resulted in even higher melatonin production. DSM 17938 had increased melatonin production compared to the control, but less than DSM 32846. Therefore, DSM 32846 is a good candidate strain for melatonin production in the intestine.

Embodiment 6

5'-Nucleotidase Activity in Lactobacillus reuteri Strains

Experimental data showing 5'-nucleotidase activity in Lactobacillus reuteri DSM 33198 were generated using the method described in Example 2 above. The results are shown in Figure 5A where they have been normalized to the 5'-nucleotidase activity of DSM17938 (DSM17938 activity = 1) from the same experiment. Based on the results of Figure 3, the same type of normalization to the 5'-nucleotidase activity of DSM17938 (DSM17938 activity = 1) has also been performed and this is shown in Figure 5B, making it easy to compare the fold change in 5'-nucleotidase activity relative to DSM17938 for different bacterial strains.

Embodiment 7

Select strain

The novel strain in Example 6 above, Lactobacillus reuteri DSM 33198, showed high 5'-nucleotidase activity in the bacterial supernatant. The supernatant of a bacterial culture at a concentration of 10 ⁹ bacteria per ml was analyzed.

Lactobacillus reuteri DSM 33198 has been developed for improved properties. Lactobacillus reuteri DSM 33198 has been modified in a multi-step selection process, including repeated freeze-drying procedures, making it more resistant to the production process and providing a higher survival rate than its natural isolate.

Choose Lactobacillus reuteri DSM 33198.

【Biological material storage】

Domestic biomaterials

1. Food Industry Development Institute 2019/11/20; BCRC 910955

2. Food Industry Development Institute 2019/11/20; BCRC 910956

3. Food Industry Development Institute 2019/11/20; BCRC 910957

4. Food Industry Development Institute 2019/11/20; BCRC 910958

5. Food Industry Development Institute 2019/11/20; BCRC 910959

Foreign biomaterials

1. Germany; DSMZ; 2018/07/04; DSM 32846

2. Germany; DSMZ; 2018/07/04; DSM 32847

3. Germany; DSMZ; 2018/07/04; DSM 32848

4. Germany; DSMZ; 2018/07/04; DSM 32849

5. Germany; DSMZ; 2018/07/09; DSM 33198

Claims (12)

A Lactobacillus reuteri strain capable of producing or inducing the production of melatonin, wherein the strain is used to produce melatonin in a subject, wherein the strain is used to treat a disease associated with melatonin deficiency or reduction; wherein the strain has 5'-nucleotidase activity, and/or is capable of producing or inducing the production of adenosine, and/or is capable of producing or inducing the production of melatonin, the level of which is increased compared to Lactobacillus reuteri DSM 17938, wherein the strain is Lactobacillus reuteri DSM 32846 (BCRC 910955), DSM 32847 (BCRC 910956), DSM 32849 (BCRC 910958), or DSM 33198 (BCRC 910959). The strain as claimed in claim 1, wherein the strain is used to treat infantile colic. A strain as claimed in claim 1 or 2, wherein the strain is Lactobacillus reuteri DSM 32846. A strain as claimed in claim 1 or 2, wherein the strain causes an increase in 5'-nucleotidase activity and/or adenosine levels and/or melatonin levels of between 1.5 and 5 times, between 2 and 5 times, between 3 and 5 times, or between 4 and 5 times compared to the levels of DSM 17938. The strain of claim 1 or 2, wherein the strain has a 5'-nucleotidase activity greater than 8 units/L, and/or is capable of producing or inducing the production of adenosine at a level greater than 8 μmol L ^-1 min ^-1 , and/or is capable of producing or inducing the production of melatonin at a level greater than 20 pg/ml. The strain of claim 1 or 2, wherein the subject is an infant up to 12 months, or 6 months, or 5 months, or 4 months or 3 months old; and/or wherein the subject is breastfed or formula fed or a combination thereof. A strain as claimed in claim 1 or 2, wherein the strain is further administered in combination with an AMP. A bacterial strain, wherein the strain is Lactobacillus reuteri DSM 32846 (BCRC 910955), DSM 32847 (BCRC 910956), DSM 32849 (BCRC 910958) or DSM 33198 (BCRC 910959). A strain as claimed in claim 8, wherein the strain is Lactobacillus reuteri DSM 32846. Use of a strain as claimed in claim 8 or claim 9 for the manufacture of a medicament for treating infantile colic. A combination product suitable for increasing melatonin or adenosine production in a subject, comprising: (i) a Lactobacillus reuteri strain capable of inducing melatonin production or producing adenosine, wherein the strain is Lactobacillus reuteri DSM 32846 (BCRC 910955), DSM 32847 (BCRC 910956), DSM 32849 (BCRC 910958), or DSM 33198 (BCRC 910959); and (ii) a substrate component that increases or enhances the ability of the Lactobacillus reuteri strain to induce melatonin production and/or produce adenosine, wherein the substrate component is AMP. The combination product of claim 11, wherein the strain is Lactobacillus reuteri DSM 32846.