WO2025098982A1

WO2025098982A1 - Lacticaseibacillus rhamnosus gg and bifidobacterium animalis subsp. lactis for reducing the release of proinflammatory cytokines

Info

Publication number: WO2025098982A1
Application number: PCT/EP2024/081166
Authority: WO
Inventors: Christopher Martoni; Anders DAMHOLT
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-11-06
Filing date: 2024-11-05
Publication date: 2025-05-15
Anticipated expiration: 2026-05-06
Also published as: WO2025098982A8

Abstract

P8093PC00 13 ABSTRACT The present disclosure generally relates to probiotics for reducing the release of proinflammatory cytokines. In particular, the present disclosure provides a composition comprising Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis that can reduce the overall pro-inflammatory cytokines in a subject such as, for example, interleukin-6 (IL-6). The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, with a composition comprising Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis.

Description

LACTICASEIBACILLUS RHAMNOSUS GG AND BIFIDOBACTERIUM ANIMALIS SUBSP. LACTIS

FOR REDUCING THE RELEASE OF PROINFLAMMATORY CYTOKINES

FIELD

The present disclosure generally relates to probiotics for reducing the release of proinflammatory cytokines. In particular, the present disclosure provides a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for reducing the overall pro-inflammatory cytokines in a subject such as, for example, interleukin-6 (IL-6).

BACKGROUND

Probiotics are "live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host" (FAO/WHO, 2001). Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) are two of the most studied probiotics. Alone, or in combination, the probiotics have been found to have positive effects in mental health, immune health, metabolic health, gastrointestinal health, and more.

Stress and anxiety are both normal emotional responses. Stress is typically caused by an external trigger which can be short or long-term. Individuals under stress experience mental and physical symptoms, such as irritability, anger, fatigue, muscle pain, digestive troubles, and difficulty sleeping. Anxiety is defined by persistent, excessive worries that don’t go away even in the absence of a stressor. Anxiety can lead to comparable symptoms as stress: insomnia, difficulty concentrating, fatigue, muscle tension, and irritability. Both mild stress and mild anxiety generally respond well to similar coping mechanisms, such as physical activity, a nutritious diet, and good sleep hygiene.

Anxiety disorders differ from short-term feelings of anxiety in their severity and in how long they last: The anxiety typically persists for months and negatively affects mood and functioning. Some anxiety disorders, such as agoraphobia (the fear of public or open spaces), may cause the person to avoid enjoyable activities or make it difficult to keep a job. According to the latest data from the National Institute of Mental Health, 31% of Americans will experience an anxiety disorder during their lifetimes. One of the most common anxiety disorders is generalized anxiety disorder. To identify generalized anxiety disorder, a clinician will look for symptoms such as excessive, hard-to-control worry occurring most days over six months. Generalized anxiety disorder may also be accompanied by the physical symptoms of anxiety.

Reduced circulating levels of IL-6 and cortisol, in concert with a trend of reduced overall pro-inflammatory cytokine profile (i.e. combination of IL-6, tumor necrosis factor-alpha (TNFa), interleukin-lbeta (IL-1P) and interferon-gamma (IFN-y)) may reduce unwanted symptoms within psychiatric indications such as stress and anxiety. It has been reported that anxious participants differed from non-anxious participants in IL-6, but not in high sensitivity C-reactive protein (hs-CRP), another marker of inflammation. Notably, the psychosomatic pathway that determines stress-related production of IL-6 is likely to differ in important ways from that leading to production of CRP. For example, previous research suggests that, unlike CRP, IL-6 may be subject to direct influence by neural and hormonal responses to stress and thus provide support for the notion of specificity at psychological and biological levels in the relation between negative emotional states and inflammation. Regarding cortisol, while short-term stress may be adaptive, maladaptive responses to stressors may intensify cortisol secretion and condition a sensitized physiologic stress response that is readily recruited. Ultimately, a prolonged or exaggerated stress response may perpetuate cortisol dysfunction and widespread inflammation. Additionally, vulnerability to stress-induced inflammation has been linked to a dysfunctional hypothalamus (or hypothalamic) - pituitary- adrenal (HPA) axis, involving both IL-6 and cortisol.

SUMMARY

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, with a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954). Further provided is a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety. In particular, the Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis (DSM 15954) may be used in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a (Tumor Necrosis Factor-alpha), IL-ip, IFN-y (Interferon-gamma) and/or cortisol, thereby reducing inflammation, such as reducing the level of interleukin-6 (IL-6). In one embodiment, the Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis (DSM 15954) maybe used in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a, IL- 1P and IFN-y, thereby reducing inflammation. In another embodiment, the Lacticaseibacillus rhamnosus GG and/ or Bifidobacterium animalis subsp. lactis (DSM 15954) may be used in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a and IL-ip, thereby reducing inflammation. The present disclosure provides uses, methods, and compositions for improving the quality of life of a subject with stress and/or anxiety. The present disclosure also provides the use of the the Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis (DSM 15954) as a dietary supplement for preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof.

Although the methods and uses described herein are of particular relevance for humans, animals such as pets, e.g. cats, dogs and horses, are also included within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the level of cytokine IL-6 in the blood at three time points for the placebo, single probiotic strain (Bif-038), and combination probiotic strain (LGG™/BB-12™) groups. Figure 2 shows the change from baseline of cytokine IL-6 in the blood at three time points for the placebo, single probiotic strain (Bif-038), and combination probiotic strain (LGG™/BB-12™) groups.

Figure 3 shows the blood cortisol level at three time points for the placebo, single probiotic strain (Bif-038), and combination probiotic strain (LGG™/BB-12™) groups.

Figure 4 shows the change from baseline of blood cortisol level at three time points for the placebo, single probiotic strain (Bif-038), and combination probiotic strain (LGG™/BB-12™) groups.

DETAILED DESCRIPTION

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, with a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954). Further provided is a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety. The present disclosure provides uses, methods, and compositions for improving the quality of life of a subject with stress and/or anxiety.

The present disclosure provides a method of preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof, with a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156), Bifidobacterium animalis subsp. lactis (DSM 15954), and B. adolescentis Bif-038 (DSM29103). Further provided are such compositions for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety. Further provided are such compositions for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety. The present disclosure provides uses, methods, and compositions for improving the quality of life of a subject with stress and/or anxiety.

Lacticaseibacillus rhamnosus GG (DSM 33156) is available from Chr. Hansen A/S, Denmark under the tradename LGG™. Bifidobacterium animalis subsp. lactis is available from Chr. Hansen A/S, Denmark under the tradename BB-12™. B. adolescentis Bif-038 is available from Chr. Hansen A/S, Denmark and deposited with DSMZ- Deutsche Sammlung van Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 207B, D-38124 Braunschweig, on July 16, 2014 under the accession No. DSM 29103. The Lacticaseibacillus rhamnosus GG (DSM 33156) used herein maybe substituted with Lactobacillus rhamnosus deposited as ATCC 53103.

It is preferred that the nucleotide sequence of the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1, preferably at least 99.10%, at least 99.20%, at least 99.30%, at least 99.40%, at least 99.50%, at least 99.60%, at least 99.70%, at least 99.80% identity to SEQ ID NO.l, more preferably at least 99.90%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98% identity to SEQ ID NO. 1, even more preferably at least 99.99% identity to SEQ ID NO. 1 or is identical to SEQ ID NO. 1.

In certain embodiments, the composition comprises the probiotic Lacticaseibacillus rhamnosus GG and the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides from the nucleotide sequence of SEQ ID NO.l. Preferably, the nucleotide sequence of the Lacticaseibacillus rhamnosus GG differs in no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15 nucleotides from the nucleotide sequence of SEQ ID NO.l.

The nucleotide sequence of SEQ ID NO. 1 is available under reference number GCF_028475085.1 in the ncbi database (https://www.ncbi.nlm.nih.gov/) as Lacticaseibacillus rhamnosus GG genome assembly ASM2847508vl. Reference is made to the database in the version accessible on 07 March 2024.

The term “sequence identity of [a certain] %” in the context of two or more nucleotide sequences refers to a relationship between the sequences of two polynucleotides, as determined by sequence comparison (alignment). As used herein, “sequence identity” is determined across the entire length of a sequence. “Sequence identity” means that the two or more sequences have nucleotides in common in the given percentage when compared and aligned. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model, algorithms, or computer program.

Percent sequence identity of nucleotide sequences can be readily calculated by any of the methods known to one of ordinary skill in the art. In preferred embodiments, the “percent identity” of two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. Where gaps exist between two sequences, Gapped BLAST ® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.

Another local alignment technique which may be used, for example, is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) J. Mol. Biol. 147:195-197). A general global alignment technique which may be used, for example, is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) J. Mol. Biol. 48:443-453), which is based on dynamic programming.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

A bacterial "strain" as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied. The multiplicity of identical bacteria is included. "Wild type strain" refers to the non-mutated form of a bacterium, as found in nature.

The term "derived strain" should be understood as a strain derived from a mother strain by means of e.g. genetic engineering, radiation and/or chemical treatment, and/or selection, adaptation, screening, etc. In specific embodiments the derived strain is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding probiotic properties) as the mother strain. Such a derived strain is a part of the present disclosure. The term "derived strain" includes a strain obtained by subjecting a strain of the disclosure to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-Nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. The present probiotics may conveniently be administered via a probiotic composition. By the term "a probiotic composition" is meant any composition which comprises probiotic bacteria, A probiotic composition comprising strains according to the disclosure may be administered in the form of a food product or a dietary supplement. The composition may, for example, be incorporated in a dairy product, such as milk, and in particular a fermented dairy product, optionally in combination with other lactic acid bacteria, for example with yogurt ferments, or in other food products such as a snack bar, or beverages such as juice.

The probiotic composition can also be provided as a dietary supplement in the form of a powder, tablet, such as a lozenge or effervescent tablet, pastille, capsule, chewing gum, in individual sachets or as a component of a more general composition such as oil drops, an emulsion or a paste, or in any other suitable carrier determined by those of skill in the art to be an effective carrier for live microorganisms.

Probiotic bacteria are live microorganisms which can present a challenge during formulation and storage of probiotic compositions. Probiotic bacteria are especially sensitive towards temperature, moisture content, and oxygen and other ingredients in a formulation matrix. It is preferred that the bacteria of the disclosure remain viable after prolonged storage in order for the bacteria to impart their beneficial effect upon administration of the present probiotic compositions to the individual in need thereof.

By the term "viable" is meant that the cell is alive and capable of forming a colony in a petri dish during pour plating or spread plating. The number of viable probiotic bacteria is determined as the number of colony forming units (CFU) by pour plate or spread plate methods with incubation under conditions suitable for growth of the probiotic strain(s). By this method cells capable of growing and forming colonies will be counted. When a number is given in the present specification and claims, it should be understood as CFU/g unless the context indicates otherwise. In some embodiments, the present probiotic composition comprises at least 10⁹ CFU/unit at end of shelflife (EOS). The end of shelflife may be at least 3 months, such as at least 6 months, at least 9 months, at least 12 months, at least 18 months, or at least 24 months.

Using a low water activity ensure a better survival of the probiotic bacteria during storage of the composition. Water activity (aw) is defined as the partial vapor pressure of water in a composition at a specified temperature divided by the standard state partial vapor pressure of water at the same temperature. Water activity thus acts as a measure of the amount of free (i.e. unbound) water in a composition. It may be calculated as: aw = P / Po where p is the partial vapor pressure of water in the composition and Po is the vapor pressure of pure water at the same temperature. In probiotic compositions it is generally preferred that the water activity (aw) is in the range of 0.1-0.2. The probiotic bacteria to be used in the present probiotic compositions are generally frozen or freeze- dried. In order to obtain a high viability the bacteria are mixed with a cryoprotectant before they are frozen or freeze-dried.

The term "a cryoprotectant" denotes a substance that is able to improve the survival during freezing and/or drying and to improve the storage stability of bacteria. The cryoprotectant used herein generally comprises a saccharide. The saccharide may be a mono-, di-, oligo- or polysaccharide, or a mixture of at least two saccharides. The composition may even comprise three, four or more saccharides. In some embodiments, the composition comprises a mixture of at least one mono- or disaccharide and at least one oligosaccharide. In other embodiments, the composition comprises a mixture of at least one mono- or disaccharide and at least one polysaccharide.

Monosaccharides useful in the probiotic composition of the present disclosure include glucose (also known as dextrose), fructose, ribose, and galactose. Disaccharides useful in the probiotic composition of the present disclosure include among other sucrose, trehalose, maltose, and lactose. The composition may comprise one or more mono- or disaccharides, such as one, two or three or even more different saccharides.

In some embodiments the probiotic composition of the disclosure comprises at least one oligosaccharide. An oligosaccharide is a saccharide polymer containing three to nine monosaccharides. Fructo-oligosaccharides (FOS), which are found in many vegetables, consist of short chains of fructose molecules. Galactooligosaccharides (GOS), which also occur naturally, consist of short chains of galactose molecules. These compounds can be only partially digested by humans. The composition may comprise one, two or even more different oligosaccharides.

In some embodiments the probiotic composition of the disclosure comprises at least one polysaccharide. Polysaccharides are polymeric carbohydrate molecules composed of more than ten monosaccharide units bound together by glycosidic linkages and on hydrolysis give the constituent monosaccharides or oligosaccharides. They range in structure from linear to highly branched. Examples of polysaccharides to be used in a probiotic composition of the disclosure are maltodextrin, cyclodextrin, alginate, pectin, chitosan, starch and inulin. The composition may comprise one, two, three or even more different polysaccharides.

As an example, the cryoprotectant may comprise a mixture of a disaccharide, such as sucrose or glucose, and a polysaccharide, such as maltodextrin. The addition of oligo- or polysaccharides such as FOS, GOS, inulin and other polysaccharides can assist in reduction of the water activity and has the further advantage that oligo- and polysaccharides are not quite as sweet as mono- and disaccharides and further that they add fibers to the composition. Polyols (sugar alcohols) have the general formula HOCH2(CHOH)_nCH2QH. They are commonly added to foods because of their lower caloric content and less sweetness than sugars. Furthermore they are not broken down by bacteria in the mouth or metabolized to acids, and thus do not contribute to tooth decay. The composition may further comprise at least one polyol such as erythriol, inositol, isomalt, mannitol, maltitol, sorbitol, or xylitol, or a mixture thereof. Preferred polyols are xylitol, sorbitol and mannitol. The composition may comprise one, two, three or even more different polyols. The cryoprotectant may further comprise a peptide, protein, protein hydrolysate or a mixture thereof. Examples of peptides and proteins to be used herein are casein, pea, whey, albumin, soy protein, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives, e.g. antioxidants such as ascorbate, sodium citrate, propyl gallate may also be present.

Combinations of several species or strains of probiotic bacteria can be used, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more of species and strains. In embodiments, two, three, four, or five different strains may be present in a probiotic composition.

In addition to the probiotic bacteria, one or more other active ingredients, for example one, two, three, four or more active ingredients selected from the group consisting of vitamins such as vitamin A, D, E, K2, C, B2, B6, B12, biotin, niacin, folic acid; minerals such as zinc, selenium, chromium, copper, calcium, chloride; and vegetable extracts such as cranberry extract/juice, royal jelly could be included in the probiotic composition. It is contemplated that in order to obtain a therapeutical effect, the probiotic composition should be administered daily for at least one week, and advantageously for a longer period such as at least 2 weeks, at least 4 weeks, at least 6 weeks, at least 9 weeks, preferably at least 12 weeks, in an amount corresponding to at least 10⁶ CFU, such as at least 10⁷ CFU, preferably at least 10⁸ CFU, generally between 10⁹ CFU and 10¹² CFU of probiotic.

In the present studies the probiotic composition comprises Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis as the active ingredients. The probiotics may be used as the only active ingredient. Alternatively, the probiotic composition as described herein may comprise further compounds of interest such as other bacterial strains, vitamins, prebiotics, fibers, or other compounds which may have a beneficial health effect.

Other bacterium may be selected from the group consisting of Bifidobacterium lactis, Lactobacillus rhamnosus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc lactis, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacety lactis, Lactobacillus casei subsp. casei, Streptococcus thermophilus, Bifidobacterium longum, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus. Thus, the composition may further comprise one or more strain(s) of a lactic acid bacterium selected from the group comprising Lactobacillus acidophilus deposited as DSM 13241, Lactobacillus rhamnosus deposited as ATCC 55826, Lactobacillus reuteri deposited as ATCC 55845, Lactobacillus paracasei subsp. paracasei deposited as ATCC 55544, Lactobacillus paracasei deposited as LMG- 17806, Streptococcus thermophilus deposited as DSM 15957, Lactobacillus fermentum deposited as NM02/31074, Lactobacillus paracasei subsp. paracasei deposited as CCTCC.

The term “therapeutically effective amount” or “effective amount” as used herein means that amount of active ingredient that elicits the biological or medicinal response in a subject which includes at least partial reduction, prevention, or alleviation of the symptoms of the condition being treated.

EXAMPLES

EXAMPLE 1

62 otherwise healthy adults experiencing depressive symptoms based on a Beck Depression Inventory (BDI-II) score of 20-40 at screening and baseline.

Study incorporated a 12-week intervention period (3-arms):

• 5 billion CFU B. adolescentis Bif-038

• 1 billion CFU L. rhamnosus LGG™ and Bifidobacterium BB-12™

• Placebo

Blood markers for inflammation (IL-6, TNF-a, IL-ip and IFN-y) and blood and hair cortisol levels were assessed after 12 weeks and compared to baseline and placebo.

Figure 1 show the levels of IL-6. Table 1 & figure 2 shows the change from baseline of blood IL-6.

Table 1- Change from baseline in IL-6

P-Value: Change from baseline using Mann Whitney Test

Figure 3 show the levels of blood cortisol. Table 2 & figure 4 shows the change from baseline of blood cortisol.

Table 2- Change from baseline in Cortisol (Blood)

P-Value: Change from baseline using Generalized Linear Mixed Models (GLMM) with treatment as factor and adjusted for baseline value (values log transformed)

*Per Protocol Set Table 3 - Change from baseline in inflammation score of primary pro-inflammatory cytokines (IL-6, TNF-a and IL-1 (3)

P-Value: Change from baseline using Mann Whitney Test

Table 4 - Change from baseline in inflammation score of all measured pro-inflammatory cytokines (IL-6, TNF-a, IL- 16 and IFN-y)

P-Value: Change from baseline using Mann Whitney Test

Table 5 - Change from baseline in cortisol (Blood and Hair)*

P-Value: Change from baseline using Generalized Linear Mixed Models (GLMM) with treatment as factor and adjusted for baseline value (values log transformed) ‘Per Protocol Set

Description:

A promising finding from this study included evidence of the anti-inflammatory effects of probiotic supplementation. Compared to the placebo group, supplementation with B. adolescentis was associated with significantly greater reductions in IL-6 and blood cortisol and strong trends suggesting greater reductions in TNF-a, IFN-y and IL-1 p. Moreover, when assessing the multiplicative score of the three primary inflammatory cytokines (IL-6‘ TNF-a‘ IL-1 p ) or the four measured inflammatory cytokines (IL-6‘ TNF-a‘ IL-1 p‘ IFN-y), there was a statistically significant greater reduction in the B. adolescentis group compared to the placebo group, and a strong trend suggesting a greater reduction in the Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis group compared to the placebo group. Additionally, when compared to placebo, the Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis group showed significantly greater reductions in both blood and hair cortisol. The anti-inflammatory effects of B. adolescentis, Lacticaseibacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis have important treatment implications as their administration may have larger benefits for people exhibiting excess inflammation or having inflammatory-related conditions.

LIST OF ITEMS

1. Use of a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof.

2. The use of Item 1 wherein the dosage of Lacticaseibacillus rhamnosus GG is at least about 0.5 billion CFU per day.

3. The use of Item 1 wherein the dosage of Bifidobacterium animalis subsp. lactis is at least about 0.5 billion CFU per day.

4. The use of Items 1 -3 wherein the composition is administered for at least 42 days.

5. The use of Items 1-3 wherein the composition is administered for at least 84 days.

6. A composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for use in the reduction or prevention of the symptoms of stress and/or anxiety.

7. The composition of Item 6, wherein said composition comprises at least about 1 billion CFU of the probiotic.

8. The composition of Items 6 or 7, wherein the composition further comprises B. adolescentis Bif038.

9. A method of reducing or preventing the symptoms of stress and/or anxiety in a subject, said method comprising administering to said subject an effective amount of a composition comprising Lacticaseibacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954).

10. The method of Item 9, wherein the dosage of B. adolescentis Bif038 is at least about 1 billion CFU per day.

11. The method of Item 10, wherein the dosage of probiotic is at least about 1 billion CFU per day.

Claims

1. Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof.

2 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof according to claim 1, wherein the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1 and/or the Bifidobacterium animalis subsp. lactis is the strain deposited as DSM 15954.

3 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to claim 1 or 2, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a, IL-ip, IFN-y and/or cortisol, thereby reducing inflammation.

4 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to claims 1 or 2, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a and IL-ip.

5 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to claims 1 to 4, wherein said preventing, reducing, or ameliorating the symptoms of stress and/or anxiety comprises reducing the levels of IL-6, TNF-a, IL-ip and IFN-y.

6 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to any of claims 1 to 5, wherein the dosage of Lacticaseibacillus rhamnosus GG is at least about 0.5 billion CFU per day.

7 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to any one of claims 1 to 6, wherein the dosage of Bifidobacterium animalis subsp. lactis is at least about 0.5 billion CFU per day.

8 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to any of claims claim 1 to 7, wherein the composition is administered for at least 42 days.

9 The Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof according to any of claims 1 to 7, 1 wherein the composition is administered for at least 84 days.

10. A composition comprising Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety in a subject in need thereof.

11. The composition comprising Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety , in a subject in need thereof according to claim 10, wherein said composition comprises at least about 1 billion CFU of the probiotic.

12. The composition comprising Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 for use in preventing, reducing, or ameliorating the symptoms of stress and/or anxiety , in a subject in need thereof according to claim 10 or 11, wherein the composition further comprises B. adolescentis Bif038.

13. A method of reducing or preventing the symptoms of stress and/or anxiety in a subject, said method comprising administering to said subject an effective amount of a composition comprising Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. Lactis deposited as DSM 15954 wherein the Lacticaseibacillus rhamnosus GG has at least 99.00% identity to SEQ ID NO. 1.

14. The method of claim 13, wherein the dosage of Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 fBifO38) is at least about 1 billion CFU per day.

15. The method of claim 13, wherein the dosage of Lacticaseibacillus rhamnosus GG and/ or Bifidobacterium animalis subsp. Lactis deposited as DSM 15954 is at least about 1 billion CFU per day.

16. The method of claims 13-15, wherein the Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 are administered for at least 42 days, preferably at least 84 days.

17. Use of the Lacticaseibacillus rhamnosus GG and/or Bifidobacterium animalis subsp. lactis deposited as DSM 15954 as a dietary supplement for preventing, reducing, or ameliorating the symptoms of stress and/or anxiety, in a subject in need thereof according to any of the previous claims.