Lactobacillus rhamnosus gg and animalia bifidum lactis for reducing release of proinflammatory cytokines

Abstract

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

Description

Technical Field

[0001] This disclosure generally relates to probiotics for reducing the release of pro-inflammatory cytokines. Specifically, this disclosure provides a composition comprising *Lacticaseibacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium animalis subsp. lactis* (DSM 15954) for reducing overall pro-inflammatory cytokines, such as interleukin-6 (IL-6), in a subject. Background Technology

[0002] Probiotics are defined as "live microorganisms that, when administered in adequate amounts, provide health benefits to the host" (FAO / WHO, 2001). *Lactobacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium animalis* subsp. *lactobacter* (DSM 15954) are two of the most studied probiotics. Positive effects of probiotics, used alone or in combination, have been found on mental health, immune health, metabolic health, and gastrointestinal health.

[0003] Both stress and anxiety are normal emotional responses. Stress is typically caused by external triggers, which can be short-term or long-term. Individuals under stress experience mental and physical symptoms such as irritability, anger, fatigue, muscle pain, digestive problems, and difficulty sleeping. Anxiety is defined as persistent, excessive worry that does not disappear even in the absence of a stressor. Anxiety can cause symptoms similar to stress: insomnia, difficulty concentrating, fatigue, muscle tension, and irritability. Both mild stress and mild anxiety usually respond well to similar coping mechanisms, such as physical activity, a nutritious diet, and good sleep hygiene.

[0004] Anxiety disorders differ from short-term anxiety in severity and duration: anxiety typically lasts for months and negatively impacts mood and function. Some anxiety disorders, such as agoraphobia (fear of public places or open spaces), may cause individuals to avoid pleasant activities or make it difficult to maintain a job. According to the latest data from the National Institute of Mental Health, 31% of Americans will experience an anxiety disorder in their lifetime. One of the most common anxiety disorders is generalized anxiety disorder. To diagnose generalized anxiety disorder, clinicians will screen for related symptoms, such as excessive, uncontrollable worry on most days over a six-month period. Generalized anxiety disorder may also be accompanied by physical symptoms of anxiety.

[0005] Decreased circulating levels of IL-6 and cortisol, coupled with a trend toward a reduced overall pro-inflammatory cytokine profile (i.e., a combination of IL-6, tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interferon-γ (IFN-γ),) may alleviate adverse symptoms in psychiatric indications such as stress and anxiety. It has been reported that anxious and non-anxious participants differ in IL-6 levels, but not in high-sensitivity C-reactive protein (hs-CRP) (another inflammatory marker). Notably, the psychosomatic pathways determining stress-related IL-6 production may differ significantly from those leading to CRP production. For example, previous research has shown that, unlike CRP, IL-6 may be directly influenced by the neurological and hormonal responses to stress, supporting the view that negative emotional states and inflammation are specific at both psychological and biological levels. Regarding cortisol, while short-term stress may be adaptive, maladaptive responses to stressors may exacerbate cortisol secretion and lead to a readily activated, highly sensitive physiological stress response. Ultimately, persistent or excessive stress responses may lead to chronic cortisol dysfunction and widespread inflammation. Furthermore, susceptibility to stress-induced inflammation is associated with dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, which involves IL-6 and cortisol. Summary of the Invention

[0006] This disclosure provides a method for preventing, alleviating, or improving stress and / or anxiety symptoms in a subject in need using a composition comprising *Lactobacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium lactis* subsp. *ani* (DSM 15954). A further provision provides a composition comprising *Lactobacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium lactis* subsp. *ani* (DSM 15954) for use in preventing, alleviating, or improving stress and / or anxiety symptoms. Specifically, *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *ani* (DSM 15954) can be used to prevent, alleviate, or improve stress and / or anxiety symptoms in a subject in need, wherein the prevention, alleviation, or improvement of stress and / or anxiety symptoms includes reducing levels of IL-6, TNF-α (tumor necrosis factor-α), IL-1β, IFN-γ (interferon-γ), and / or cortisol, thereby reducing inflammation, for example, by reducing levels of interleukin-6 (IL-6). In one embodiment, *Lactobacillus rhamnosus* GG and / or *Bifidobacterium animalis* subsp. *lactobacter* (DSM 15954) can be used to prevent, alleviate, or improve stress and / or anxiety symptoms in subjects in need, wherein the prevention, alleviation, or improvement of stress and / or anxiety symptoms includes reducing levels of IL-6, TNF-α, IL-1β, and IFN-γ, thereby reducing inflammation. In another embodiment, *Lactobacillus rhamnosus* GG and / or *Bifidobacterium animalis* subsp. *lactobacter* (DSM 15954) can be used to prevent, alleviate, or improve stress and / or anxiety symptoms in subjects in need, wherein the prevention, alleviation, or improvement of stress and / or anxiety symptoms includes reducing levels of IL-6, TNF-α, and IL-1β, thereby reducing inflammation. This disclosure provides uses, methods, and compositions for improving the quality of life of subjects suffering from stress and / or anxiety. This disclosure also provides the use of Lactobacillus rhamnosus GG and / or Bifidobacterium animalis subsp. lactis (DSM 15954) as dietary supplements for the prevention, reduction or improvement of stress and / or anxiety symptoms in subjects in need.

[0007] Although the methods and uses described herein are particularly relevant to humans, animals (such as pets, such as cats, dogs and horses) are also included within the scope of this invention. Attached Figure Description

[0008] Figure 1 The levels of the cytokine IL-6 in the blood at three time points were shown in the placebo group, the single probiotic strain (Bif-038) group, and the combined probiotic strain (LGG™ / BB-12™) group.

[0009] Figure 2The changes in blood cytokine IL-6 relative to baseline at three time points are shown in the placebo group, the single probiotic strain (Bif-038) group, and the combined probiotic strain (LGG™ / BB-12™) group.

[0010] Figure 3 Blood cortisol levels were shown at three time points in the placebo group, the single probiotic strain (Bif-038) group, and the combined probiotic strain (LGG™ / BB-12™) group.

[0011] Figure 4 The changes in blood cortisol levels relative to baseline at three time points are shown in the placebo group, the single probiotic strain (Bif-038) group, and the combined probiotic strain (LGG™ / BB-12™) group. Detailed Implementation

[0012] This disclosure provides a method for preventing, alleviating, or improving stress and / or anxiety symptoms in a subject in need using a composition comprising *Lactobacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium animalis* subsp. *lactobacter* (DSM 15954). It further provides a composition comprising *Lactobacillus rhamnosus* GG (DSM 33156) and *Bifidobacterium animalis* subsp. *lactobacter* for use in preventing, alleviating, or improving stress and / or anxiety symptoms. This disclosure provides uses, methods, and compositions for improving the quality of life of subjects suffering from stress and / or anxiety.

[0013] This disclosure provides a method for preventing, alleviating, or improving stress and / or anxiety symptoms in a subject in need using a composition comprising *Lactobacillus rhamnosus* GG (DSM 33156), *Bifidobacterium animalis* subsp. *lactobacter* (DSM 15954), and *Bifidobacterium adolescentis* Bif-038 (DSM 29103). Further, such compositions for use in preventing, alleviating, or improving stress and / or anxiety symptoms are provided. This disclosure provides uses, methods, and compositions for improving the quality of life of subjects suffering from stress and / or anxiety.

[0014] *Lactobacillus rhamnosus* GG (DSM 33156) is available from Chr. Hansen A / S, Denmark, under the trade name LGG™. *Bifidobacterium animalis* subsp. *lactamase* is available from Chr. Hansen A / S, Denmark, under the trade name BB-12™. *Bifidobacterium adolescentis* Bif-038 is available from Chr. Hansen A / S and deposited on July 16, 2014, at DSMZ (Germany Center for Microbiology and Cell Culture Collection, 20 7B Indhofenstrasse, Braunschweig, D-38124) with accession number DSM 29103. *Lactobacillus rhamnosus* GG (DSM 33156) used in this study can be replaced with *Lactobacillus rhamnosus* deposited under ATCC 53103.

[0015] Preferably, the nucleotide sequence of *Lactobacillus rhamnosus* GG has at least 99.00% identity with SEQ ID NO. 1, more 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%, or at least 99.80% identity with SEQ ID NO. 1, 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%, or at least 99.98% identity with SEQ ID NO. 1, and even more preferably at least 99.99% identity with SEQ ID NO. 1 or is identical to SEQ ID NO. 1.

[0016] In some embodiments, the composition comprises the probiotic *Lactobacillus rhamnosus* GG, and the nucleotide sequence of *Lactobacillus rhamnosus* GG differs from the nucleotide sequence of SEQ ID NO. 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Preferably, the nucleotide sequence of *Lactobacillus rhamnosus* GG differs from the nucleotide sequence of SEQ ID NO.1 by no more than 5000, 4000, 3000, 2750, 2500, 2250, 2000, 1750, 1500, 1250, 1000, 750, 500, 400, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, or 15 nucleotides.

[0017] The nucleotide sequence of SEQ ID NO. 1 can be found in the ncbi database ( https: / / www.ncbi.nlm.nih.gov / The genome of *Lactobacillus rhamnosus* GG was obtained using reference number GCF_028475085.1 as ASM2847508v1. The reference database version was accessible on March 7, 2024.

[0018] In the context of two or more nucleotide sequences, the term "[certain]% sequence identity" refers to the relationship between two polynucleotide sequences determined through sequence comparison (alignment). As used herein, "sequence identity" is determined over the entire length of the sequence. "Sequence identity" means that two or more sequences have a given percentage of common nucleotides when compared and aligned. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignment (if any) is handled through a specific mathematical model, algorithm, or computer program.

[0019] The percentage of sequence identity of a nucleotide sequence can be readily calculated by any method known to those skilled in the art. In a preferred embodiment, the “percentage of identity” between two sequences (e.g., polynucleotide or amino acid sequences) is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87:2264-68, 1990), a modified version of which is described in Karlin and Altschul Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 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. [Journal of Molecular Biology] 215:403-10, 1990. In the presence of gaps between two sequences, Gapped BLAST® can be used, for example, as described in Altschul et al., Nucleic AcidsRes. [Nucleic Acid Research] 25(17):3389-3402, 1997. When using 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 appropriately adjusted in a manner that can be understood by one of ordinary skill in the art.

[0020] Another local alignment technique that can be used is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981) J. Mol. Biol. [Journal of Molecular Biology] 147:195-197). A general global alignment technique that can be used is based on the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443-453).

[0021] The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0022] As used in this article, a bacterial "strain" refers to a bacterium that remains genetically unchanged during growth or reproduction. This includes multiple identical strains. A "wild-type strain" refers to a non-mutated form of bacteria found in nature.

[0023] The term "derived strain" should be understood as a strain derived from a parent strain through, for example, genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. In certain embodiments, a derived strain is a functionally equivalent mutant, for example, a mutant having properties substantially the same as or improved upon the parent strain (e.g., probiotic properties). Such derived strains are part of this disclosure. The term "derived strain" includes strains obtained by subjecting the strains of this disclosure to any conventionally used mutagenesis treatment, including treatment with chemical mutagens (e.g., ethanemethanesulfonate (EMS) or N-methyl-N'-nitro-nitroguanidine (NTG)) or UV light.

[0024] The probiotics of the present invention can be conveniently administered via probiotic compositions. The term "probiotic composition" means any composition containing probiotics. Probiotic compositions containing strains according to the present disclosure can be administered in the form of food or dietary supplements. For example, the compositions can be incorporated into dairy products (e.g., milk), and particularly fermented dairy products, optionally in combination with other lactic acid bacteria (e.g., yogurt starter cultures), or incorporated into other foods (e.g., snack bars) or beverages (e.g., fruit juices).

[0025] Probiotic compositions may also be provided as dietary supplements in the form of powders, tablets (e.g., lozenges or effervescent tablets), soft lozenges, capsules, chewing gum, individual sachets, or as a component of more general compositions (e.g., oil drops, emulsions, or pastes) or any other suitable carrier that a person skilled in the art has determined to be an effective carrier of live microorganisms.

[0026] Probiotics are live microorganisms, which can present challenges during the formulation and storage of probiotic compositions. Probiotics are particularly sensitive to temperature, moisture content, oxygen, and other components in the formulation matrix. Preferably, the bacteria disclosed herein remain viable after prolonged storage so that they can exert their beneficial effects when the probiotic compositions of the present invention are administered to individuals in need.

[0027] The term "viable" means that the cells are alive and capable of forming colonies in a petri dish during pour or spread plating. The number of viable probiotics is determined by pour or spread plating after incubation under conditions suitable for the growth of the probiotic strain, and is expressed in colony-forming units (CFU). This method allows for the counting of cells capable of growth and colony formation. Unless the context otherwise requires, the figures given in this specification and claims should be understood as CFU / g. In some embodiments, the probiotic composition of the present invention contains at least 10 at the end of shelf life (EOS). 9 CFU / unit. The expiration date can 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.

[0028] Using low water activity ensures better survival of probiotics during composition storage. Water activity (aw) is defined as the partial vapor pressure of water in a composition at a specific temperature divided by the standard state partial vapor pressure of water at the same temperature. Therefore, water activity can be used as a measure of the amount of free (i.e., unbound) water in a composition. It can be calculated as:

[0029] aw = P / P0

[0030] Where p is the partial vapor pressure of water in the composition, and P0 is the vapor pressure of pure water at the same temperature. In probiotic compositions, a water activity (aw) range of 0.1-0.2 is generally preferred. The probiotics used in the probiotic compositions of this invention are typically frozen or freeze-dried. To obtain high activity, the bacteria are mixed with a cryoprotectant before freezing or freeze-drying.

[0031] The term "cryoprotectant" refers to a substance that improves survival during freezing and / or drying and enhances the storage stability of bacteria. Cryoprotectants as used herein typically comprise sugars. Sugars can be monosaccharides, disaccharides, oligosaccharides, or polysaccharides, or mixtures of at least two sugars. Compositions can even contain three, four, or more sugars. In some embodiments, the composition comprises a mixture of at least one monosaccharide or disaccharide and at least one oligosaccharide. In other embodiments, the composition comprises a mixture of at least one monosaccharide or disaccharide and at least one polysaccharide.

[0032] Monosaccharides that can be used in the probiotic compositions disclosed herein include glucose (also known as dextrose), fructose, ribose, and galactose. Disaccharides that can be used in the probiotic compositions disclosed herein particularly include sucrose, trehalose, maltose, and lactose. The composition may contain one or more monosaccharides or disaccharides, such as one, two, three, or even more different sugars.

[0033] In some embodiments, the probiotic compositions disclosed herein comprise at least one oligosaccharide. Oligosaccharides are sugar polymers containing three to nine monosaccharides. Fructose oligosaccharides (FOS) are found in many vegetables and consist of short-chain fructose molecules. Galacto-oligosaccharides (GOS) are also naturally occurring and consist of short-chain galactose molecules. These compounds can only be partially digested by humans. The composition may contain one, two, or even more different oligosaccharides.

[0034] In some embodiments, the probiotic compositions disclosed herein comprise at least one polysaccharide. Polysaccharides are polymeric carbohydrate molecules composed of more than ten monosaccharide units linked by glycosidic bonds, and hydrolysis yields the monosaccharides or oligosaccharides that constitute them. Their structures range from linear to highly branched. Examples of polysaccharides that can be used in the probiotic compositions disclosed herein are maltodextrin, cyclodextrin, alginate, pectin, chitosan, starch, and inulin. The compositions may contain one, two, three, or even more different polysaccharides.

[0035] For example, cryoprotectants can contain a mixture of disaccharides (such as sucrose or glucose) and polysaccharides (such as maltodextrin). Adding oligosaccharides or polysaccharides (such as FOS, GOS, inulin, and other polysaccharides) helps reduce water activity and has the additional advantage that oligosaccharides and polysaccharides are not as sweet as monosaccharides and disaccharides, and they also add fiber to the composition. Polyols (sugar alcohols) have the general formula HOCH2(CHOH). nCH20H. Because they are lower in calories and sweetness than sugars, they are often added to foods. Furthermore, they are not broken down or metabolized into acid by bacteria in the mouth, thus not causing tooth decay. The composition may further comprise at least one polyol, such as erythritol, inositol, isomaltitol, mannitol, maltitol, sorbitol, or xylitol, or mixtures 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 peptides, proteins, protein hydrolysates, or mixtures thereof. Examples of peptides and proteins used herein are casein, pea protein, whey protein, albumin, soy protein, glutamic acid, or gelatin, and any isolates or hydrolysates thereof. Other additives may also be present, such as antioxidants, like ascorbate, sodium citrate, or propyl gallate.

[0036] A combination of several probiotic species or strains may 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 species and strains. In the examples, the probiotic composition may contain two, three, four or five different strains.

[0037] In addition to probiotics, the probiotic composition may also include one or more other active ingredients, such as one, two, three, four or more active ingredients selected from the group consisting of: vitamins, such as vitamins A, D, E, K2, C, B2, B6, B12, biotin, niacin, folic acid; minerals, such as zinc, selenium, chromium, copper, calcium, chloride; and plant extracts, such as cranberry extract / cranberry juice, royal jelly. To achieve therapeutic effects, the probiotic composition is expected to be formulated with at least 10 6 CFU (e.g., at least 10) 7 CFU, preferably at least 10 8 CFU, usually in 10 9 CFU and 10 12 The corresponding amount of probiotics (between CFU) is administered daily for at least one week, and preferably for a longer period of time, such as at least two weeks, at least four weeks, at least six weeks, at least nine weeks, and preferably at least twelve weeks.

[0038] In this study, the probiotic composition contained *Lactobacillus rhamnosus* GG and *Bifidobacterium animalis* subsp. *lactobacter* as active ingredients. Probiotics may be used as the sole active ingredient. Alternatively, the probiotic composition described herein may contain other target compounds, such as other bacterial strains, vitamins, prebiotics, fiber, or other compounds that may have health benefits.

[0039] Other bacteria can be selected from the following groups: *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. diacetylactis*, *Lactobacillus casei subsp. casei*, *Streptococcus thermophilus*, *Bifidobacterium longum*, and *Lactobacillus*. Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus acidophilus. Therefore, the composition may further comprise one or more lactic acid bacteria strains selected from the group consisting of: Lactobacillus acidophilus preserved with DSM 13241, Lactobacillus rhamnosus preserved with ATCC 55826, Lactobacillus reuteri preserved with ATCC 55845, Lactobacillus paracasei subsp. paracasei preserved with ATCC 55544, Lactobacillus paracasei preserved with LMG-17806, Streptococcus thermophilus preserved with DSM 15957, Lactobacillus fermentum preserved with NM02 / 31074, and Lactobacillus paracasei subsp. paracasei preserved with CCTCC.

[0040] As used herein, the terms “therapeutic effective amount” or “effective amount” mean the amount of an active ingredient that elicits a biological or pharmaceutical response in a subject, including at least a partial reduction, prevention, or relief of symptoms of the treated condition. Example

[0041] Example 1

[0042] Sixty-two otherwise healthy adults who had scores of 20-40 on the Beck Depression Scale-II (BDI-II) during the screening and baseline periods presented with depressive symptoms.

[0043] The study included a 12-week intervention period (3 groups):

[0044] • 5 billion CFU of Bifidobacterium adolescentis Bif-038

[0045] • 1 billion CFU of Lactobacillus rhamnosus LGG™ and Bifidobacterium BB-12™

[0046] • Placebo

[0047] After 12 weeks, blood markers of inflammation (IL-6, TNF-α, IL-1β, and IFN-γ) and blood and hair cortisol levels were assessed and compared with baseline and placebo.

[0048] Figure 1 The levels of IL-6 are shown in Table 1 and... Figure 2 The changes in blood IL-6 relative to baseline are shown.

[0049] Table 1 – Changes in IL-6 relative to baseline

[0050]

[0051] P-value: The Mann-Whitney test was used to analyze the change relative to baseline.

[0052] Figure 3 The levels of blood cortisol are shown in Table 2. Figure 4 The changes in blood cortisol relative to baseline are shown.

[0053] Table 2 – Changes in Cortisol (Blood) Relative to Baseline

[0054]

[0055] P-value: Generalized linear mixture model (GLMM) was used to analyze the change relative to baseline, with treatment as a factor and adjusted for baseline values ​​(values ​​have been logarithmically transformed).

[0056] Compliant Solution Set

[0057] Table 3 – Changes in inflammation scores of major pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) relative to baseline

[0058]

[0059] P-value: Analyzes change relative to baseline using the Mann-Whitney test.

[0060] Table 4 – Inflammation scores of all measured pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, and IFN-γ) relative to Baseline changes

[0061]

[0062] P-value: Analyzes change relative to baseline using the Mann-Whitney test.

[0063] Table 5 – Changes in Cortisol (blood and hair) relative to baseline

[0064]

[0065] P-value: Generalized linear mixture model (GLMM) was used to analyze the change relative to baseline, with treatment as a factor and adjusted for baseline values ​​(values ​​have been logarithmically transformed).

[0066] Compliant Solution Set

[0067] describe:

[0068] Promising findings of this study include evidence that probiotic supplementation has anti-inflammatory effects. Compared to the placebo group, supplementation with Bifidobacterium adolescentis was associated with a significant reduction in IL-6 and blood cortisol, and there was a strong trend indicating a greater decrease in TNF-α, IFN-γ, and IL-1β. Furthermore, when assessing three major pro-inflammatory cytokines (IL-6... TNF-α IL-1β or four measured pro-inflammatory cytokines (IL-6) TNF-α IL-1β When calculating the product score of IFN-γ, the decrease in *Bifidobacterium adolescentis* was statistically significant compared to the placebo group, and there was a strong trend indicating a greater decrease in *Lactobacillus rhamnosus* GG and *Bifidobacterium animalis* subsp. lactis compared to the placebo group. Furthermore, both blood and hair cortisol levels showed more significant reductions in *Lactobacillus rhamnosus* GG and *Bifidobacterium animalis* subsp. lactis compared to the placebo group. The anti-inflammatory effects of *Bifidobacterium adolescentis*, *Lactobacillus rhamnosus* GG, and *Bifidobacterium animalis* subsp. lactis have important therapeutic implications, as administration of these strains may offer greater benefit to individuals exhibiting excessive inflammation or suffering from inflammation-related conditions. Project List

[0069] 1. The use of a composition comprising Lactobacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for the prevention, relief or improvement of stress and / or anxiety symptoms in a subject in need.

[0070] 2. As described in Project 1, the dose of Lactobacillus rhamnosus GG is at least approximately 500 million CFU / day.

[0071] 3. As described in Project 1, the dosage of Bifidobacterium animalis subsp. lactis is at least approximately 500 million CFU / day.

[0072] 4. As described in items 1-3, wherein the composition is applied for at least 42 days.

[0073] 5. As described in items 1-3, wherein the composition is applied for at least 84 days.

[0074] 6. A composition comprising Lactobacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM 15954) for use in alleviating or preventing symptoms of stress and / or anxiety.

[0075] 7. The composition according to item 6, wherein the composition contains at least about 1 billion CFU of probiotics.

[0076] 8. The composition according to item 6 or 7, wherein the composition further comprises Bifidobacterium adolescentis Bif038.

[0077] 9. A method for alleviating or preventing stress and / or anxiety symptoms in a subject, the method comprising administering to the subject an effective amount of a composition comprising Lactobacillus rhamnosus GG (DSM 33156) and Bifidobacterium animalis subsp. lactis (DSM15954).

[0078] 10. The method described in Project 9, wherein the dose of Bifidobacterium adolescentis Bif038 is at least about 1 billion CFU / day.

[0079] 11. The method according to Project 10, wherein the dosage of probiotics is at least about 1 billion CFU / day.

Claims

1. A strain of Lactobacillus rhamnosus GG and / or Bifidobacterium animalis subsp. lactis for use in the prevention, relief or improvement of stress and / or anxiety symptoms in subjects in need.

2. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium animalis* subsp. *lactobacter* as claimed in claim 1 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, wherein the *Lactobacillus rhamnosus* GG has at least 99.00% identity with SEQ ID NO. 1, and / or the *Bifidobacterium animalis* subsp. *lactobacter* is a strain deposited under DSM 15954.

3. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, as described in claim 1 or 2, wherein the prevention, alleviation or improvement of stress and / or anxiety symptoms comprises reducing the levels of IL-6, TNF-α, IL-1β, IFN-γ and / or cortisol, thereby reducing inflammation.

4. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, as described in claim 1 or 2, wherein the prevention, alleviation or improvement of stress and / or anxiety symptoms includes reducing the levels of IL-6, TNF-α and IL-1β.

5. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, as described in claims 1 to 4, wherein the prevention, alleviation or improvement of stress and / or anxiety symptoms includes reducing the levels of IL-6, TNF-α, IL-1β and IFN-γ.

6. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, as described in any one of claims 1 to 5, wherein the dose of *Lactobacillus rhamnosus* GG is at least about 500 million CFU / day.

7. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *anis* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, as described in any one of claims 1 to 6, wherein the dose of *Bifidobacterium lactis* subsp. *anis* is at least about 500 million CFU / day.

8. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954, as described in any one of claims 1 to 7, for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, wherein the composition is administered for at least 42 days.

9. The *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954, as described in any one of claims 1 to 7, for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need, wherein the composition is administered for at least 84 days.

10. A composition comprising *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in subjects in need.

11. The composition of claim 10 comprising *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in a subject in need, wherein the composition comprises at least about 1 billion CFU of probiotics.

12. The composition according to claim 10 or 11 comprising *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 for use in preventing, alleviating or improving stress and / or anxiety symptoms in a subject in need, wherein the composition further comprises *Bifidobacterium adolescentis* Bif038.

13. A method for alleviating or preventing stress and / or anxiety symptoms in a subject, the method comprising administering to the subject an effective amount of a composition comprising *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *ani* preserved in DSM 15954, wherein the *Lactobacillus rhamnosus* GG has at least 99.00% identity with SEQ ID NO.

1.

14. The method of claim 13, wherein the dose of *Lactobacillus rhamnosus* GG and / or *Bif038* subsp. *animal* preserved in DSM 15954 is at least about 1 billion CFU / day.

15. The method of claim 13, wherein the dose of *Lactobacillus rhamnosus* GG and / or *Bifidobacterium animalis* subsp. *lactamase* preserved in DSM 15954 is at least about 1 billion CFU / day.

16. The method according to claims 13-15, wherein the *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 are administered for at least 42 days, preferably at least 84 days.

17. The use of *Lactobacillus rhamnosus* GG and / or *Bifidobacterium lactis* subsp. *animal* preserved in DSM 15954 as a dietary supplement for the prevention, relief or improvement of stress and / or anxiety symptoms in subjects in need.

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