Nutritional Supplements

Abstract

The present invention relates to the use of probiotics for one or more of: (i) preventing or reducing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality, and in particular to the use of Lacticaseibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019).

Description

[Technical field]

[0001] The present invention relates to the use of probiotics, in particular Lacticaseibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) or derivatives thereof, for one or more of the following purposes: (i) prevention or reduction of stress; (ii) prevention or reduction of anxiety; (iii) prevention or reduction of one or more symptoms of depression; (iv) reduction or reversal of certain changes caused by stress, anxiety or depression; (v) improvement of mood; (vi) increase in sense of relaxation; (vii) increase in activity. Methods of using the bacteria, as well as compositions and foods comprising the probiotic bacteria, are also provided. [Background technology]

[0002] Stress is the experience of emotional or physical tension in response to an event or thought process. Stress can be accompanied by physical and mental symptoms such as stomach aches, muscle tension, headaches, rapid breathing, increased heart rate, sweating, trembling, fatigue, changes in appetite, dizziness, confusion, feelings of impending doom, panic or irritability, difficulty concentrating, stomach discomfort or malfunction, irrational anger, restlessness, etc. Anxiety is often the result of stress and is associated with feelings of dissatisfaction, worry, nervousness, fear or apprehension regarding the current or future state of a subject.

[0003] Experiencing stress and anxiety is very common and not necessarily a bad thing. Short-term stress and anxiety can help you overcome difficult or dangerous situations. However, repeated, chronic or overwhelming stress and anxiety can cause serious health problems, both physical and mental. For example, chronic stress is associated with heart disease, high blood pressure, diabetes, depression and panic disorders.

[0004] The recent Covid-19 pandemic has significantly increased the proportion of people experiencing stress and anxiety in the general population, with estimates of as much as 30% suffering from defined stress, anxiety, or depression. Although various treatments are available for people suffering from stress and anxiety, there is growing appreciation that nutrition may be able to improve at least mild symptoms of stress and anxiety without the side effects associated with pharmacological treatments. Research into the gut-brain axis is providing evidence that dietary components that may benefit gut health may also influence aspects of mental well-being.

[0005] A growing body of literature links gut microbiota health to brain chemistry and behavior via multiple bidirectional pathways, including the immune system, neurotransmitter pathways, and the vast network of afferent and efferent nerves connecting the gut with the central nervous system, suggesting that enhancing the gut microbiota with probiotics may improve mood outcomes.

[0006] The microbiota-gut-brain axis mechanism encompasses a bidirectional relationship between the brain and the digestive system. Dysregulation of the microbiota-gut-brain axis has been increasingly revealed in association with various psychiatric disorders, including neurodevelopmental disorders, schizophrenia, anxiety disorders, and depression. See Younjung Lee and Yong-Ku Kim, 2021 (Current Psychiatry Reports, 23(5):22).

[0007] Zagorska et al., 2020 (Beneficial Microbes, 11(8):717-732) compiled literature on the potential impact of gut microbiota on psychiatric disorders through neural pathways that constitute the "gut-brain axis." In addition, they investigated the effects of the combination of probiotic and prebiotic intake and a dairy-rich diet on the gut microbiota and subsequent association with brain function. The authors reported that not all probiotic / prebiotic / dairy-rich dietary approaches show psychobiological effects on the central nervous system.

[0008] Taylor and Holscher 2020 (Nutritional Neuroscience, 23(3):237-250) summarized the findings of clinical studies using dietary interventions to improve depression, anxiety, and stress, and the role of the gastrointestinal microbiota in these disorders. The authors reported that probiotic intake improved psychological or biological indicators of depression, anxiety, and stress in people predisposed to mood disorders. Probiotics suppressed biological markers of stress in healthy people in a strain-dependent manner.

[0009] International Publication WO2018 / 220429 describes the use of Lactobacillus rhamnosus HN001 or a derivative thereof for treating or preventing postnatal depression and postnatal anxiety.

[0010] International Publication WO2018 / 002240 describes a study involving maternal nutritional supplementation of pregnant and lactating mice with a probiotic combination of Lactobacillus rhamnosus LPR and Bifidobacterium longum BL999. Levels of synaptophysin were found to be higher in mice treated with the probiotic compared to a control group that did not. Synaptophysin is a protein that allows for better adaptation in frightening situations, and is described in the application as regulating how fear is experienced in anxiety situations and helping to prevent future fear in similar situations.

[0011] There remains a need to provide compositions useful for reducing or preventing symptoms of stress, anxiety, and depression, and generally improving mood, enhancing relaxation, and increasing energy levels. It would be particularly advantageous to be able to provide these benefits through nutritional intake, thereby avoiding potential side effects associated with drug interventions.

[0012] SUMMARY OF THE PRESENT EMBODIMENT It is therefore an object of the present invention to address the above-mentioned needs. Summary of the Invention

[0013] The present invention relates to the use of Lacticaceobacterium rhamnosus strain HN001 (HN001) (previously classified as Lactobacillus rhamnosus HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019), or a derivative thereof, for one or more of the following purposes: (i) preventing or reducing stress; (ii) preventing or reducing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality.

[0014] According to another aspect of the present invention, (i) Lacticaceae Bacillus rhamnosus strain HN001 (HN001) and / or derivatives thereof; and / or (ii) Bifidobacterium animalis subsp. lactis strain HN019 (HN019) and / or its derivatives; and (iii) a physiologically acceptable diluent, adjuvant, carrier or excipient; A composition comprising: Compositions are provided for use in one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; and (vii) increasing vitality.

[0015] Another aspect of the present invention provides a method for one or more of (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality, comprising administering Lacticaceibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) and / or a derivative thereof to a subject in need thereof. The method may be a non-therapeutic method.

[0016] Also provided is the use of Lacticaceabacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) for the manufacture of a formulation for any one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; and (vii) increasing vitality.

[0017] A further aspect of the present invention provides a food ingredient, food product or dietary supplement containing, consisting of or consisting essentially of Lacticaseibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) for any one or more of: (i) reduction or prevention of stress; (ii) reduction or prevention of anxiety; (iii) prevention or reduction of one or more symptoms of depression; (iv) reduction or reversal of certain changes caused by stress, anxiety or depression; (v) improvement of mood; (vi) increased feelings of relaxation; (vii) increased vitality.

[0018] According to one embodiment, the aspect of the invention relating to HN001 does not include or is not directed to the treatment or prevention of postpartum depression or postpartum anxiety.

[0019] According to one embodiment, aspects of the invention relating to HN001 do not involve or are not intended to enhance calmness or reduce occasional anxiety.

[0020] According to another embodiment, aspects of the invention relating to HN001 do not include the combination of Lactobacillus rhamnosus LPR (ATCC 54193) and Bifidobacterium longum BL999 (ATCC BAA-9999) for reducing anxiety and chronic stress.

[0021] According to another embodiment, the aspect of the invention relating to HN001 does not include the combination of HN001 with glutamine and / or turmeric.

[0022] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows a schematic diagram of the experimental setup using the Chronic Unpredictable Mild Stress (CUMS) model. [Diagram 2] FIG. 2 shows images from the open field test (left image) and the elevated plus maze test (right image). [Diagram 3] FIG. 3 shows the changes in body weight of rats administered CUMS, CUMS-HN001, CUMS-HN019, CUMS-HN019 and CUMS-Flu compared to the control. [Figure 4] Figure 4. Western blot analysis of neuroendocrine hormone receptors corticotropin releasing factor type 1 (CRF1) and glucocorticoid receptor (GR) in brain tissue. Results are shown as the ratio of antibody staining intensity of the receptor (CFR1, left; GR, right) to the housekeeping protein GAPDH. *P<0.05. [Diagram 5]HE staining of brain and colon tissues is shown in Figure 5. Brain tissues included two regions of the hippocampus (dentate gyrus (DG) and CA1) and one region of the cerebral cortex (P2). [Figure 6] Figure 6 shows CUMS-induced changes in neurotransmitters and their metabolites in rat serum and brain normalized with probiotics. Serum and brain levels of 5-HT, DA, NE, Glu and GABA were detected at the end of the experiment (A, B, D, E, G, H, J, K, L). (C), (F) and (I) show the metabolites of monoamine neurotransmitters in the brain. Results are shown as mean ± SEM (n=10). #P<0.05 compared with CUMS group. [Figure 7] Figure 7 shows the levels of IL-6, IL-18, IL-1β and TNF-α in serum, brain and colon of CUMS rats during the experiment. These indicators were examined at the end of the experiment. The results are shown as mean ± SEM (n=10). *P<0.05 compared with the control group. #P<0.05 compared with the CUMS group. [Figure 8] Figure 8 shows four scatter plots of the correlation between TNF-α (A), IL-6 (B), IL-1β (C), and IL-18 (D) in the brain and colon. "rs" stands for Spearman correlation coefficient. [Figure 9] Figure 9 shows the effect of probiotic treatment on gut microbiota induced by CUMS. (A) Sobs index, (B) ACE index, (C) Chao1 index, (D) Shonnnon index. *P<0.05 compared with control group. #P<0.05 compared with CUMS group, &P<0.05 compared with CUMS-HN001&HN019 group. [Figure 10]Figure 10 shows the analysis of microbiota composition among the six groups: control, CUMS, CUMS-HN001, CUMS-HN019, CUMS-HN001&HN019, and CUMS-Flu. (A) is the unweighted PcoA, (B), (D), and (E) are the differences in microbiota abundance in phylum, family, and genus among the six groups. The area of ​​each color represents the abundance of different bacteria in the gut, and the colors of the bars in Figure 10B, Figure 10D, and Figure 10E are shown in the same order from top to bottom in the legend diagram to the right of the graph. The larger the area, the higher the abundance and vice versa. Data are presented as the average percentage value of each group (n=8 per group). (C) Comparison of the ratio of Firmicutes to Bacteroidetes among the six groups. [Figure 11] Figure 11 shows heat maps showing the correlation between phylum (A), family (B), and genus (C) and monoamine neurotransmitters and inflammatory factors. Red indicates positive correlation, and blue indicates negative correlation. *P<0.1, **P<0.05. [Figure 12-14] Figures 12, 13B-13E, 14A-14C are color versions of Figures 5, 10B-10E, 11A-11C, respectively. The rows in Figure 12 are labeled, from top to bottom, control, CUMS, CUMS-flu, CUMS-HN001, CUMS-HN019, and CUMS-HN001&HN019, respectively. The first three columns in Figure 12 are labeled, from left to right, DG, CA1, and P2, respectively. The colors in the bars in Figures 13B, 13D, and 13E are ordered from top to bottom in the same order as they are displayed in the legend to the right of the graphs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] According to one aspect of the present invention, there is provided a use of Lacticaceibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019), or a derivative thereof, for one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality.

[0025] The American Psychological Association (APA) defines stress as a physiological or psychological response to an internal or external stressor. Stress involves changes that affect almost every system of the body, affecting the way a person feels and behaves. For example, symptoms may include palpitations, sweating, dry mouth, shortness of breath, fidgetiness, rapid speech, increased negative emotions (if already experienced), and prolonged stress fatigue. In severe stress, general adaptation syndrome appears. By causing these physical and mental changes, stress is directly involved in psychological and physiological disorders and diseases, affecting both physical and mental health and reducing quality of life.

[0026] Anxiety is defined by the APA as an emotion characterized by symptoms of fear and tension in anticipation of imminent danger, catastrophe, or misfortune. To respond to the perceived threat, the body often mobilizes: muscles tense, breathing quickens, and the heart beats faster.

[0027] While both stress and anxiety are emotional responses, stress is usually caused by an external trigger, which can be short-term, such as a deadline at work or an argument with a loved one, or long-term, such as not being able to work, experiencing discrimination, or a chronic illness. Anxiety, on the other hand, is defined by persistent, excessive worry that does not go away even in the absence of a stressor.

[0028] <Stress> As defined herein and by the American Psychological Association (APA), stress reduction means a reduction in one or more of the following symptoms: stomach aches, muscle tension, headaches, rapid breathing, shortness of breath, increased heart rate, palpitations, sweating, dry mouth, trembling, fatigue, fidgetiness, rapid speech, increased negative emotions (if already experienced), persistent stress fatigue, changes in appetite, dizziness, confusion, feelings of impending doom, panic, irritability, difficulty concentrating, intestinal discomfort or dysfunction, restlessness, irritability, irrational anger, and restlessness.

[0029] Stress may be mild "everyday" stress. Stress may be chronic stress, which is defined as a physiological or psychological response to an internal or external stressful event (i.e., a stressor). Stress may be acute stress.

[0030] <Anxiety> As defined herein and by the American Psychological Association (APA), reduction of anxiety is intended to mean a reduction in any one or more of dread, sense of impending danger, catastrophe, unhappiness, dissatisfaction, worry, nervousness, and fear or apprehension regarding a subject's current or future condition.

[0031] <Calmness> Equanimity is a mental state in which the mind is at peace, without confusion, agitation or agitation. It also refers to being in a state of peace, tranquility and tranquility. Equanimity is most easily achieved by the average person when they are relaxed, but it can also be achieved in a state of greater alertness and awareness.

[0032] <Depression> As defined herein and by the American Psychological Association (APA), major depressive disorder (depression) is a serious mood disorder typified by persistent feelings of sadness, emptiness, pessimism, and / or low mood that adversely affect daily activities such as eating and working. Symptoms include feelings of hopelessness, restlessness, guilt, worthlessness, or helplessness. Symptoms may also include loss of interest or pleasure in leisure activities, reduced energy, fatigue, restlessness, and may lead to difficulties with concentration, focus, memory, and decision-making. Depression may also disrupt normal appetite and / or weight, leading to suicide or attempted suicide. In addition, depression may be accompanied by digestive discomfort and other digestive problems, as well as other pains and suffering without a clear physical cause.

[0033] Postnatal depression (PND; also called perinatal depression or postpartum depression) shares many features with major depressive episodes, but PND is characterized by a set of features that make it a separate entity from major depressive disorder, in addition to the obvious association of PND with pregnancy / birth. For example, PND is associated with hormonal changes specific to the transition from pregnancy to the postpartum period. Animal model studies have shown that rats administered hormones that mimic this transition develop behavioral changes typical of PND. Furthermore, PND is associated with unique psychosocial stressors, such as infant temperament and the stress associated with caring for a newborn infant. In contrast to typical patients with major depressive disorder, PND patients show reduced activation in many neural regions, such as blunted amygdala responses to negative stimuli unrelated to the infant, or reduced emotional responses and regulation to the crying of their own infant.

[0034] <Lacticaceibacillus rhamnosus strain HN001 (HN001) and Bifidobacterium animalis subsp. lactis strain HN019 (HN019)> Lacticaceibacillus rhamnosus strain HN001 (HN001) and Bifidobacterium animalis subsp. lactis strain HN019 (HN019) are probiotics. The term "probiotic" is well known in the art and refers to a microbial cell preparation or a component of a microbial cell that has a beneficial effect on the health or well-being of the host. Means for determining the type of probiotic microorganism and its activity level are well known in the art and include, for example, evaluation using PBMC cytokine secretion assays. The term "prebiotic" is also well known in the art and refers to a food substance intended to promote the growth of probiotic bacteria in the intestine.

[0035] HN001 may be the only probiotic administered to or for administration to a subject. Alternatively, HN019 may be the only probiotic administered to or for administration to a subject. Alternatively, HN001 and HN019 may be the only prebiotic administered to or for administration to a subject. HN001 and HN019 may be administered in combination, sequentially, in any order, or together. HN001 and / or HN019 may also be administered individually or in combination with one or more additional probiotics and / or one or more prebiotics. Examples of probiotics suitable for co-administration include Lacticaceibacillus rhamnosus and / or other strains of Bifidobacterium animalis subspecies lactis, Lactobacillus acidophilus (e.g., Lactobacillus acidophilus (LAVRI-A1)), Lactobacillus reuteri (e.g., Lactobacillus reuteri ATCC 55730) or Bifidobacterium lactis (e.g., Bifidobacterium lactis strain BB12), Bifidobacterium bifidum, Bacillus coagulum, Saccharomyces boulardii, or combinations thereof. Examples of prebiotics suitable for co-administration include fructooligosaccharides, galactooligosaccharides, inulin, human milk oligosaccharides, and combinations thereof.

[0036] As described in International Publication WO 99 / 10476, HN001 is available at the Australian Government Analytical Laboratories (AGAL) under deposit number NM97 / 09514 dated 18 August 1997, and HN019 is available at AGAL under deposit number NM97 / 09513 dated 18 August 1997. This Budapest Treaty recognised depository is now called the National Metrology Institute of Australia (NMIA) rather than AGAL, ​​and is located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207.

[0037] HN001 is well known in the art, and its various properties and distinguishing features are described, for example, in WO99 / 10476 and WO2018 / 220429. HN019 is also well known in the art, and its various properties and distinguishing features are described, for example, in WO99 / 10476. HN001 is also described in Slykerman et al., 2017 (EBioMedicine 24:159-165) and Tay et al., 2020 (Nutrients 12:3530).

[0038] Lactobacillus rhamnosus strain HN001 (brand name HN001 (trademark)) and Bifidobacterium animalis subsp. lactis strain HN019 (brand name HN019 (trademark)) are both commercially available from New Zealand Milk Products (NZMP (trademark)), a materials and solutions brand of the dairy cooperative Fonterra. HN001 (trademark) and HN019 (trademark) are commercially available from NZMP, and are marketed in particular as Fonterra's SureStart (trademark) (early nutrition brand) and Nutiani (trademark) (health & wellness solutions brand). Lactobacillus rhamnosus strain HN001 is also sold under the names "LactoB HN001 (trademark)" and "Nutiani HN001 (trademark)". "HN001 (trademark)", "LactoB HN001 (trademark)" and "Nutiani H001 (trademark)" are trademarks of Fonterra TM Limited. Bifidobacterium animalis subsp. lactis strain (HN019) is sold under the names "BifidoB HN019 (trademark)" and "Nutiani HN019 (trademark)". "HN019 (trademark)", "BifidoB HN019 (trademark)" and "Nutiani HN019 (trademark)" are also trademarks of Fonterra TM Limited.

[0039] HN001 and / or HN019 are preferably administered in a form capable of growth. Additionally or alternatively, some of HN001 and HN019 may be killed, lysed, fractionated or attenuated.

[0040] <Derivatives of HN001 and / or HN019> As used herein, the term "derivatives" of HN001 and HN019 refers to mutants and homologues of HN001 and / or HN019, or killed or attenuated cells of HN001 and / or HN019, e.g., but not limited to, heat-killed, lysed, fractionated, autoclaved, irradiated, UV or light treated cells, as well as bacterial derived materials, including, but not limited to, bacterial cell wall compositions, bacterial cell lysates, lyophilized bacteria, bacterial derived probiotic factors, etc., where the derivatives retain probiotic activity. Methods for producing such derivatives or one or more probiotic factors, and particularly derivatives suitable for administration to a subject (e.g., included in a composition), are well known in the art.

[0041] <Target> The "subject" of the present invention is a mammal, preferably a human. The subject may also be a model animal, such as a rat, mouse, or guinea pig. The mammal may be a companion animal, such as a cat, dog, or horse, or may be a livestock animal, such as a pig, cow, horse, goat, sheep, deer, or poultry.

[0042] The subject may be a human infant, child, or adult, where an infant is under 12 months of age, a child is between 12 months of age and adolescence, and an adult is an older human, e.g., 16 years of age or older. For example, the subject infant or child may be between 0-3 months, 4-5 months, 6-9 months, 10-14 months, 15-18 months, 19-24 months, 25-30 months, or 31-42 months. The subject infant or child may be between 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days old. The infant or child may be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months of age. The infant or child may be up to 1 year of age, up to 2 years of age, up to 3 years of age, up to 4 years of age, up to 5 years of age, up to 6 years of age, up to 7 years of age, up to 8 years of age, up to 9 years of age, up to 10 years of age, up to 11 years of age, up to 12 years of age, up to 13 years of age, up to 14 years of age, or up to the age before the onset of puberty.

[0043] The subject may be a premature infant, ie, an infant born before 37 weeks of gestation. The subject may be pregnant or lactating, in which case the subject may be the woman or her offspring.

[0044] Otherwise, the subject of the present invention may be a healthy individual, as determined, for example, by the Apgar scoring system (The American College of Obstetricians and Gynecologists, No. 644, October 2015) for newborns, or by the use of standard growth curves, such as the WHO Child Growth Standards (Acta Paediatrica, 2006, Suppl 450:76-85) for older subjects. The WHO Child Growth Standards can be used to convert weight, length, and head circumference to weight-for-age, length-for-age, and head circumference-for-age Z-scores. The subject of the present invention may suitably be a "term" baby, i.e., born after the end of the 37th week of gestation. In general, a "healthy" subject does not include low birth weight, very low birth weight, or premature babies, and a "premature baby" refers to an infant born before the end of the 37th week of gestation. However, the foregoing no longer applies when a subject who was a premature or low birth weight baby falls into the healthy infant range.

[0045] The reduction of stress, anxiety and depression, as well as the improvement of mood, increased relaxation and increased vitality (i.e., the technical effect of the present invention) is achieved by administering the probiotics HN001 and / or HN019, or their derivatives, to a subject, for example, a subject in need thereof. A "subject in need", as defined herein, may be one who self-determines to be in need by exhibiting one or more symptoms of stress, anxiety or depression, or who feels that he or she is in need of an increased mood or feeling more relaxed or increasing vitality levels. Alternatively, a "subject in need" may be formally diagnosed by a physician as having stress, anxiety or depression. Alternatively, the technical effect of the present invention may be realized even if the subject is not recognized as being in need. In particular, administering the probiotics HN001 and / or HN019, or their derivatives, to a subject may advantageously have a preventive effect on the onset of symptoms of stress, anxiety or depression.

[0046] A variety of routes and methods of administration are contemplated, although oral administration, in a suitable composition etc., is currently preferred. The term "oral administration" includes oral, buccal, enteral and intragastric administration.

[0047] Administration of HN001 and / or HN019 or derivatives thereof to a subject advantageously prevents or reduces stress, where the term "stress" is as defined herein. A subject may self-diagnose as having stress by exhibiting one or more symptoms of stress, as defined herein, or may be diagnosed by a physician as being in a state of stress or having symptoms of stress or a stress disorder.

[0048] Administration of HN001 and / or HN019 or derivatives thereof to a subject advantageously prevents or reduces anxiety, where the term "anxiety" is as defined herein. A subject may self-diagnose as having anxiety by exhibiting one or more symptoms of anxiety, as defined herein, or may be diagnosed by a physician as having an anxious state or symptoms associated with anxiety or an anxiety disorder.

[0049] Administration of HN001 and / or HN019 or a derivative thereof to a subject advantageously prevents or reduces one or more symptoms of depression, as provided herein under the definition of "depression." A subject may self-diagnose as having depression by exhibiting one or more symptoms of depression, as defined herein, or may be diagnosed by a physician as being depressed or having symptoms associated with depression.

[0050] The reduction in any one or more of stress, anxiety or depression symptoms can be determined qualitatively or quantitatively depending on the symptoms. The reduction in any one or more of stress, anxiety or depression symptoms can be self-determined by improvement in any one or more of stress, anxiety or depression, or the reduction can be determined by a physician.

[0051] Reduction in any one or more of the symptoms of stress, anxiety, or depression may result in any one or more of "improved mood," "increased relaxation," and "increased energy." Alternatively, "improved mood," "increased relaxation," and "increased energy" may be independent of reduction in the symptoms of stress, anxiety, or depression.

[0052] Improved mood, increased feelings of relaxation, and increased energy levels may be self-determined by the subject.

[0053] Administration of HN001 and / or HN019 or derivatives thereof may include: a) changes in the levels or activity of neurotransmitters or their metabolites; b) changes in stress hormone levels or activity; c) changes in the levels or activity of inflammatory factors; d) Morphological changes in brain tissue e) Morphological changes in the colon f) changes in the intestinal flora, g) Changes in body weight The present invention advantageously reduces or reverses certain changes caused by stress, anxiety or depression, such as changes in one or more of the following:

[0054] <Changes in levels or activity of neurotransmitters or their metabolites> Administration of HN001 and / or HN019 or derivatives advantageously reverses stress-induced decreases in any one or more of serotonin (5-HT), dopamine (DA) and norepinephrine (NE) and / or increases concentrations of glutamate and / or GABA (e.g., in serum and / or brain tissue). HIAA, DOPAC and HVA are metabolic products of 5-HT, DA and NE and are commonly increased in stressed individuals. Administration of HN001 and / or HN019 to individuals experiencing stress, anxiety or depression can adequately restore HIAA, DOPAC and HVA to levels found in non-stressed individuals.

[0055] <Changes in stress hormone levels or activity> Administration of HN001 and / or HN019 or their derivatives advantageously reduces the levels of one or more stress hormones, such as molecules involved in the hypothalamic-pituitary-adrenal (HPA) axis, such as any one or more of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTG), and glucocorticoid hormones, such as cortisol. CRH is a hormone produced in the hypothalamus of the brain in response to various stresses, such as physical and mental stress. Increased CRH production is associated with Alzheimer's disease and major depression. Short-term effects of CRH include appetite suppression, increased anxiety, and possibly inflammation.

[0056] Prevention or reduction of stress and / or anxiety in a subject through the use of HN001 and / or HN019 or their derivatives may occur via the HPA axis.

[0057] Thus, the present invention further provides the use of Lacticazei Bacillus rhamnosus (HN001) and / or Bifidobacterium animalis subsp. lactis (HN019) or a derivative thereof for reducing the levels of one or more molecules involved in the hypothalamic-pituitary-adrenal (HPA) axis, for example one or more of the glucocorticoid hormones such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTG) and cortisol.

[0058] <Changes in levels or activity of inflammatory factors> Inflammatory cytokines were found to be increased in stressed subjects. Among inflammatory cytokines, IL-6, TNF-α, IL-1β and IL-18 showed increased levels in stressed individuals. Administering Lacticazei Bacillus rhamnosus (HN001) and / or Bifidobacterium animalis subsp. lactis (HN019) or their derivatives favorably inhibited the increase of these inflammatory factors and effectively reversed the changes caused by stress. The reduction in the level of inflammatory cytokines can be preferably observed in serum, brain and colon samples.

[0059] Administration of HN001 and / or HN019 or derivatives advantageously reduces the levels and / or activity of pro-inflammatory cytokines such as any one or more of TNF-α, IL-6, IL-1β and IL-18.

[0060] <Morphological changes in brain tissue (hippocampus) and colon> The use of HN001 and / or HN019 or derivatives according to the invention advantageously protects and maintains the epithelial cell barrier from damage and loss of integrity, for example due to inflammation. The inflammation may be neuroinflammation (brain inflammation). Neuroinflammation is associated with many pathologies, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, encephalomyelitis, multiple sclerosis, and dementia. Other pathologies include vasculitis, stroke, delirium, and cognitive decline (e.g., spatial memory, executive function, processing speed, and other signs and symptoms of mild cognitive impairment (MCI)). Depression is also associated with increased neuroinflammation. Deterioration or impairment of the blood-brain barrier may allow pathogenic infection of nervous tissue, leading to meningitis, encephalitis, and brain abscess. Thus, strengthening the blood-brain barrier may prevent brain infection.

[0061] The use according to the invention of HN001 and / or HN019 or derivatives thereof may suitably be used to strengthen the blood-brain barrier (BBB), which may lead to preventing brain infections, for example by preventing pathogenic infection of nervous tissue which may lead to meningitis, encephalitis or brain abscess.

[0062] The use of HN001 and / or HN019 or derivatives thereof according to the invention may also be suitably used in the treatment of any one or more of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dementia; vasculitis; stroke; delirium; cognitive decline (e.g. spatial memory, executive function, processing speed and other signs and symptoms of mild cognitive impairment (MCI)).

[0063] Further advantageously, HN001 and / or HN019 or derivatives thereof may be used to reverse the effects of stress and / or anxiety on brain or colon cell morphology and / or to protect brain or colon tissue from stress- and / or anxiety-related cellular damage.

[0064] Administration of HN001 and / or HN019 or derivatives thereof may be advantageously used to protect or improve the integrity of the blood-brain barrier (BBB), which may lead to reduced brain inflammation and / or improved brain function, and may also be used to protect or improve the integrity of the intestinal barrier.

[0065] Protection of the integrity of the intestinal barrier may also be useful in the treatment or prevention of irritable bowel syndrome (IBS) and / or systemic inflammation.

[0066] Administration of HN001 and / or HN019 or derivatives thereof may also be advantageously used to improve digestive / gastrointestinal health (e.g., to reduce constipation or diarrhea and / or improve regularity).

[0067] Therefore, according to a further aspect of the present invention, HN001 and / or HN019 or their derivatives can be used in a method of treatment, such as, for example, any one or more of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, dementia; vasculitis; stroke; delirium; cognitive decline (e.g., spatial memory, executive function, processing speed, and other signs and symptoms of mild cognitive impairment (MCI)); protection or improvement of blood-brain barrier (BBB) ​​integrity (which can lead to reduced brain inflammation and / or improved brain function), and protection or improvement of gut barrier integrity; treatment or prevention of irritable bowel syndrome (IBS) and / or systemic inflammation; and / or treatment or prevention of digestive / gastrointestinal health. A method comprising administering a therapeutically effective amount of HN001 and / or HN019 or their derivatives to a subject in need thereof.

[0068] As used herein, "treat," "treatment," or "treating" refers to administering or providing a composition for prophylactic and / or therapeutic purposes.

[0069] As used herein, the terms "prophylactic treatment," "prevention," or "preventing" refer to treating a subject who does not yet exhibit symptoms of the disease or condition, but who is susceptible to or otherwise at risk of contracting a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.

[0070] <Changes in the intestinal flora> Further advantageously, HN001 and / or HN019 or derivatives thereof may be used to revert stress-induced changes in the gut microbiota to a non-stress profile, as it relates to abundance, (alpha and beta) diversity, richness, levels of dominance of the flora, etc.

[0071] Administration of HN001 and / or HN019 may suitably restore altered levels of any one or more of Firmicutes, Bacteroidetes, Verrucomicrobias, Proteobacteria and Actinobacteria to levels found in a non-stressed individual.

[0072] Administration of HN001 and / or HN019 may advantageously restore altered levels of one or more of Ruminococcaceae, Lactobacillaceae, and Lachnospiraceae to levels seen in non-stressed individuals.

[0073] Administration of HN001 and / or HN019 may advantageously restore altered levels of one or more of Lactobacillus, Eubacterium, Coprostanoligenes, and Lachnospira to levels seen in non-stressed individuals.

[0074] Administration of HN001 and / or HN019 may advantageously restore the ratio of Firmicutes to Bacteroidetes (F / B ratio) to that found in unstressed individuals.

[0075] Alpha diversity, as measured by richness, can be measured using the Sobs index, Chao1 index, and ACE index. Diversity can be measured using the Shannon index. Beta diversity can be measured using PCoA.

[0076] <Weight change> The use according to the invention of HN001 and / or HN019 or their derivatives may also be suitably used to reverse changes in weight (either gain or loss) caused by either stress, anxiety or depression.

[0077] HN001 and / or HN019 may be administered in the form of a composition which may include a physiologically acceptable diluent, adjuvant, carrier or excipient.

[0078] Therefore, according to another aspect of the present invention, (i) Lacticaceae Bacillus rhamnosus strain HN001 (HN001) and / or its derivatives; (ii) Bifidobacterium animalis subsp. lactis strain HN019 (HN019) and / or derivatives thereof; and (iii) a physiologically acceptable diluent, adjuvant, carrier or excipient; A composition comprising: Compositions are provided for use in one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; and (vii) increasing vitality.

[0079] According to a further aspect of the present invention, there is provided a method for any one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality, comprising administering to a subject a composition as described above or administering Lacticaseibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) or a derivative thereof.

[0080] According to a further aspect of the present invention, there is provided the use of Lacticaceibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) in the manufacture of a formulation for any one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing feelings of relaxation; (vii) increasing vitality. The manufacturer can prepare a suitable formulation.

[0081] The uses, compositions or methods of the invention may also be used to reduce the levels of one or more molecules involved in the hypothalamic-pituitary-adrenal (HPA) axis, for example any one or more glucocorticoid hormones such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTG), and cortisol, and / or to reduce the levels and / or activity of pro-inflammatory cytokines such as TNF-α and / or IL-6 cytokines.

[0082] The increase in CRH in response to stress is the first step in what is known as the hypothalamic-pituitary-adrenal (HPA) axis, which is characterized by the hypothalamus releasing CRH (in response to stress), which binds to CRH receptors in the pituitary gland and stimulates the release of adrenocorticotropic hormone (ACTH). ACTH leaves the brain through the circulatory system, binds to receptors in the adrenal cortex, and stimulates the release of glucocorticoid hormones, such as cortisol in humans.

[0083] When blood cortisol concentrations reach a certain level, cortisol exerts a negative feedback on the release of CRH from the hypothalamus and ACTCH from the pituitary gland, restoring the system to homeostasis. However, repeated exposure to stressors can repeatedly and persistently activate the HPA axis, leading to habituation of the system. Chronic HPA activation has a variety of potential consequences, including increased risk of type 2 diabetes, neurodegenerative and inflammatory diseases, as well as depression and anxiety disorders and immune dysfunction.

[0084] Advantageously, according to the present invention, stress-induced CRH response is blunted by HN001, HN019, and HN001+HN019. Moreover, ACTH and cortisol (other components of the HPA axis) are also reduced to near non-stressed control levels by administration of HN001, HN019, and HN001+HN019. These findings are supported by data on the expression of neurotransmitters in the brain. The stress-related increase in the cell receptor for CRH was found to be blunted by probiotics, while the stress-related decrease in glucocorticoid receptors was reduced by probiotics.

[0085] The composition preferably comprises a physiologically acceptable diluent, adjuvant, carrier or excipient, which may comprise a food product. Alternatively, HN001 and / or HN019 and / or derivatives thereof may be administered in the form of a food or dietary supplement.

[0086] Thus, a further aspect of the present invention also provides a food ingredient, food or dietary supplement comprising, consisting of or consisting essentially of Lacticaseibacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) for one or more of: (i) reduction or prevention of stress; (ii) reduction or prevention of anxiety; (iii) prevention or reduction of any one or more symptoms of depression; (iv) reduction or reversal of certain changes caused by stress, anxiety or depression; (v) improvement of mood; (vi) increased feelings of relaxation; (vii) increased vitality.

[0087] A food product may be any edible consumer product capable of carrying bacteria or bacterial derivatives. Examples of such consumer products include, but are not limited to, cultured milk, yogurt, cheese, milk and yogurt drinks or powdered milk, baby food, fruit juices, rice pudding, rusks, purees, cereals, porridge, confectionery products, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, sports supplements including dairy and non-dairy based sports supplements, food additives such as protein sprinkles, dietary supplements such as daily supplement tablets, weaning foods and yogurt, prepared foods such as powdered or liquid maternal formulas, including hypoallergenic forms of such compositions.

[0088] The food products can be selected from maternal supplements, nutritional products, infant formulas, follow-on formulas, and growing-up formulas. As used herein, "infant formula" refers to a composition for infants aged 0 days to 6 months, and "follow-up formula" refers to a composition for infants aged 6 months to 1 year. As used herein, the term "growing formula" refers to a composition for infants and children aged 1 year and above. Growing formulas include growing milk powder or GUMP. Those skilled in the art will understand the age ranges for the different compositions: "infant formula", "follow-up formula" and "growing formula" may be different for different children depending on the individual's development. As used herein, the term "maternal formula" refers to a composition consumed by pregnant or lactating women. A food product may also be a nutritional product, which refers to a product that is specially processed or formulated and presented to meet specific dietary requirements that exist due to a particular physical or physiological condition and / or specific diseases and disorders. The product may be a concentrated or ready-to-drink liquid or may be provided as a concentrated powder.

[0089] Food products such as dairy-based beverages (e.g., milk drinks and yogurt drinks) may typically contain, in addition to HN001 and / or HN019 or derivatives thereof, a protein source (e.g., a milk protein source), a lipid source, a carbohydrate source.

[0090] The composition or food or supplement may also contain other nutrients such as proteins, carbohydrates, vitamins, minerals, or amino acids.The composition may be in a form suitable for oral use, such as tablets, hard or soft capsules, aqueous or oily suspensions, or syrups; or in a form suitable for parenteral use, such as aqueous propylene glycol solutions, or buffered aqueous solutions.The amount of active ingredient in the nutritional supplement composition depends largely on the specific needs of the subject.The amount also varies depending on the route of administration and the possibility of co-use with other probiotic factors or agents, as recognized by those skilled in the art.

[0091] The composition or food or supplement of the present invention can be provided as a capsule. The capsule can comprise standard pharma- ceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated according to conventional procedures by compressing a mixture of active ingredients with solid carriers and lubricants. Examples of solid carriers include starch and sugar bentonite. The active ingredient can also be administered in the form of a hard-shelled tablet or capsule, which includes a binder, such as lactose or mannitol, a conventional filler, and tableting. The pharmaceutical composition can also be administered by parenteral route. Examples of parenteral dosage forms include aqueous solutions of the active agent, isotonic saline or 5% glucose in water, or other well-known pharma-ceutically acceptable excipients. Cyclodextrins, or other solubilizers known to those skilled in the art, can be utilized as excipients for the delivery of therapeutic agents.

[0092] As known to those skilled in the art, flavors, colorants, and other additives, carriers, or excipients may also be included in the composition or food or supplement of the present invention. It will be understood that such compositions or foods may include a wide range of additives or carriers, for example, to improve or preserve the viability of bacteria or to enhance the therapeutic effect of HN001 and HN019 or their derivatives. For example, additives such as cryoprotectants such as maltodextrin, as well as surfactants, wetting agents, humectants, adhesives, dispersants, stabilizers, penetrants, and so-called stress additives (such as potassium chloride, glycerol, sodium chloride, and glucose) to improve the vitality, growth, replication, and viability of bacterial cells may be included. Additives also include compositions such as unrefined corn oil that help maintain the viability of microorganisms in long-term storage, and "inverse" emulsions that include an outer mixture of oil and wax and an inner mixture of water, sodium alginate, and bacteria.

[0093] The composition or food or supplement of the present invention may include live HN001 and / or live HN019, preferably in a form and amount that allows growth. Methods for producing such compositions are well known in the art. The composition may include a carbohydrate source, such as a disaccharide, including sucrose, fructose, glucose, or dextrose. Preferably, the carbohydrate source is one that can be utilized aerobically or anaerobically by HN001 and / or HN019. Preferably, the composition is capable of supporting the growth capacity of HN001 and HN019 for a period of about 2 weeks or more, preferably about 1 month or more, about 2 months or more, about 3 months or more, about 4 months or more, about 5 months or more, more preferably about 6 months or more, and most preferably at least about 2 years to about 3 years or more.

[0094] Methods for preparing the compositions of the present invention are well known in the art and standard microbiological and pharmaceutical techniques can be used.

[0095] In one embodiment, useful compositions herein comprise, consist essentially of, or consist of at least about 0.1, 0.2, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5, 99.8, or 99.9% by weight of HN001 and / or HN019, and / or derivatives thereof. Useful ranges can be selected between the following values: For example, about 0.1 to about 50%, about 0.2 to about 50%, about 0.5 to about 50%, about 1 to about 50%, about 5 to about 50%, about 10 to about 50%, about 15 to about 50%, about 20 to about 50%, about 25 to about 50%, about 30 to about 50%, about 35 to about 50%, about 40 to about 50%, about 45 to about 50%, about 0.1 to about 60%, about 0.2 to about 60%, about 0.5 to about 60%, about 1 to about 60%, about 5 to about 60%, about 10 to about 60%, about 1 5 to about 60%, about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60%, about 45 to about 60%, about 0.1 to about 70%, about 0.2 to about 70%, about 0.5 to about 70%, about 1 to about 70%, about 5 to about 70%, about 10 to about 70%, about 15 to about 70%, about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, about 0.1 to about 80%, about 0.2 to about 80%, about 0.5 to about 80%, about 1 to about 80%, about 5 to about 80%, about 10 to about 80%, about 15 to about 80%, about 20 to about 80%, about 25 to about 80%, about 30 to about 80%, about 35 to about 80%, about 40 to about 80%, about 45 to about 80%, about 0.1 to about 90%, about 0.2 to about 90%, about 0.5 to about 90%, about 1 to about 90%, about 5 to about 90%, about 10 to about 90%, about 15 to about 90 %, about 20 to about 90%, about 25 to about 90%, about 30 to about 90%, about 35 to about 90%, about 40 to about 90%, about 45 to about 90%, about 0.1 to about 99%, about 0.2 to about 99%, about 0.5 to about 99%, about 1 to about 99%, about 5 to about 99%, about 10 to about 99%, about 15 to about 99%, about 20 to about 99%, about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99%, and about 45 to about 99%.

[0096] In one embodiment, a composition, food, ingredient or dietary supplement useful herein comprises, consists essentially of, or consists of at least about 0.001, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 grams by weight of HN001 and / or HN019 and / or derivatives thereof. Useful ranges may be selected between the following values: For example, about 0.01 to about 1 gram, about 0.01 to about 10 grams, about 0.01 to about 19 grams, about 0.1 to about 1 gram, about 0.1 to about 10 grams, about 0.1 to about 19 grams, about 1 to about 5 grams, about 1 to about 10 grams, about 1 to about 19 grams, about 5 to about 10 grams, and about 5 to about 19 grams.

[0097] In one embodiment, a food or supplement useful herein comprising HN001 and / or HN019, and / or derivatives thereof, may further comprise about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or 99.9% by weight fresh whole milk or milk derivative, useful ranges may be selected between the following values: For example, about 0.1 to about 50%, about 0.2 to about 50%, about 0.5 to about 50%, about 1 to about 50%, about 5 to about 50%, about 10 to about 50%, about 15 to about 50%, about 20 to about 50%, about 25 to about 50%, about 30 to about 50%, about 35 to about 50%, about 40 to about 50%, and about 45 to about 50%.Milk derivatives include recombined, powdered or fresh skim milk, recombined or reconstituted whole milk or skim milk powder, skim milk concentrate, skim milk retentate, concentrated milk, ultrafiltered milk, milk protein concentrate (MPC), milk protein isolate (MPI), calcium depleted milk protein concentrate. (MPC), reduced fat milk, reduced fat milk protein concentrate (MPC), casein, caseinates, milk fat, cream, butter, ghee, anhydrous milk fat (AMF), buttermilk, butterserum, betaserum, hard milk fat fraction, soft milk fat fraction, sphingolipid fraction, milk fat globule membrane fraction, milk fat globule membrane lipid fraction, phospholipid fraction, complex lipid fraction, colostrum, colostrum fraction, colostrum protein concentrate (CPC), colostrum whey, immunoglobulin fraction from colostrum, whey (sweet whey, lactic acid whey, mineral acid whey, or reconstituted whey powder), whey protein isolate, whey protein concentrate, compositions derived from any milk or colostrum processing stream, ultrafiltration or microfiltration in any milk or colostrum processing stream The composition may be selected from compositions derived from the retentate or permeate obtained by, compositions derived from the non-adsorbed or adsorbed fraction obtained by chromatographic (including, but not limited to, ion permeation chromatography and gel permeation chromatography) separation of any milk or colostrum processing stream, derivatives of any of these milk derivatives including extracts prepared by multi-stage fractionation, differential crystallization, solvent fractionation, supercritical fractionation, near-critical fractionation, distillation, centrifugal fractionation, or fractionation with modifiers (e.g., soaps or emulsifiers), hydrolysates of any of these derivatives, fractions of hydrolysates, and any combination of any two or more of these derivatives including combinations of hydrolysates and / or non-hydrolysates. It should be understood that the source of these derivatives may be milk or colostrum, or combinations thereof.

[0098] It will be apparent that the concentration of HN001 and / or HN019 or derivatives thereof in a composition formulated for administration may be lower than the concentration in a composition formulated for, for example, distribution or storage, and that the concentration of a composition formulated for storage and then formulated into a composition suitable for administration must also be sufficient to enable the composition for administration to be sufficiently concentrated so that it can be administered in a therapeutically effective dose.

[0099] When used in combination with other therapeutic agents, the administration of the composition useful herein and the other therapeutic agent can be simultaneous or sequential. Simultaneous administration includes administration of a single dosage form containing all components, or administration of separate dosage forms that are substantially simultaneous. Sequential administration includes administration according to different schedules, preferably such that there is an overlap in the period during which the composition useful herein and the other therapeutic agent(s) are provided.

[0100] Suitable agents that can be administered separately, simultaneously, or sequentially with the compositions useful herein include one or more probiotic agents, one or more prebiotic agents, one or more phospholipids, one or more gangliosides, other suitable agents known in the art, and combinations thereof.Useful prebiotics include galactooligosaccharides (GOS), short-chain GOS, long-chain GOS, fructooligosaccharides (FOS), human milk oligosaccharides (HMO), short-chain FOS, long-chain FOS, inulin, galactan, fructan, lactulose, and any mixture of two or more thereof.Other useful agents include dietary fiber, such as fully or partially insoluble or indigestible dietary fiber.

[0101] In one embodiment, the compositions useful herein comprise or are administered simultaneously or sequentially with milk components such as whey protein, whey protein fractions (including acidic or basic whey protein fractions or combinations thereof), glycomacropeptides, lactoferrin, iron-lactoferrin, functional lactoferrin variants, functional lactoferrin fragments, vitamin D or calcium, or combinations thereof. Useful milk component-containing compositions include compositions such as food, beverages, food additives, beverage additives, dietary supplements, nutritional products, medical foods or dietary supplements. Milk fractions enriched with these components may also be employed.

[0102] It should be understood that the additional therapeutic agents listed above (both food-based and pharmaceutical) may also be employed in the methods according to the invention, and are administered in the methods separately, simultaneously, or sequentially with the compositions useful herein.

[0103] The composition, food, ingredient or dietary supplement useful herein may further comprise a pharma- ceutically acceptable carrier. The composition may be or be formulated as a food, beverage, food additive, beverage additive, dietary supplement, nutritional product, medical food, enteral nutrition product, parenteral nutrition product, meal replacement, cosmeceutical, nutraceutical, medicament, or pharmaceutical. The supplement containing HN001 and / or HN019 or their derivatives may be in the form of a tablet, caplet, pill, hard or soft capsule, or lozenge. The supplement consisting of HN001 and / or HN019 or their derivatives may be in the form of a sachet, powder, dispensing powder, granule, suspension, elixir, liquid, or any other form that can be added to food or beverage, including, for example, water, milk, or fruit juice. The compositions or foods or supplements useful herein may further comprise one or more ingredients, such as antioxidants, for example, to prevent or reduce deterioration of the compositions during storage or after administration. Examples of compositions or foods or supplements useful herein include any edible consumer product that can retain bacteria or bacterial derivatives, including heat-killed, autoclaved, lysed, UV or light-treated, irradiated, fractionated, or other killed or attenuated bacteria. Examples of suitable edible consumer products include aqueous products, baked products, confectionery products such as chocolate, gels, ice cream, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy products such as yogurt and cheese, drinking milk such as dairy and non-dairy based beverages, sports supplements including milk powders, dairy and non-dairy based sports supplements, fruit juices, food additives such as protein sprinkles, dietary supplements such as daily supplement tablets, weaning foods and yogurt, and formulas such as infant formulas, follow-up formulas, or growth formulas in powdered or liquid form. Suitable nutritional supplement compositions useful herein may be provided in similar forms.

[0104] It will be understood that different compositions of the present invention can be formulated with a view to administration to a specific group of subjects.For example, the formulation of a composition suitable for administration to a pregnant mother (e.g., for indirect administration to a subject that is a fetus, or a subject that is a breast-feeding newborn, infant, or child) can be different from the formulation of a composition that is administered directly to a subject.It should also be understood that the formulation of a composition that is administered prophylactically, i.e., before symptoms appear, can be different from the formulation of a composition that is formulated for a subject that already shows symptoms of stress and anxiety.

[0105] At least a portion of the probiotics are preferably alive in the composition, and more preferably reach the intestine alive, where they can persist and multiply to become effective, and may be effective by interacting with resident bacteria and / or the host.

[0106] The compositions, foods, ingredients and supplements useful in the present invention are administered in an amount effective to: (i) reduce or prevent stress; (ii) reduce or prevent anxiety; (iii) prevent or reduce one or more symptoms of depression; (iv) reduce or reverse certain changes caused by stress, anxiety or depression; (v) improve mood; (vi) increase relaxation; (vii) increase vitality. An amount sufficient to accomplish this is defined as a "therapeutically effective amount." Amounts effective for this purpose depend on many factors known to those skilled in the art, such as the severity of stress or anxiety, the weight and general condition of the patient.

[0107] In preventive applications, the compositions, foods and supplements according to the present invention are administered to patients who are susceptible to or otherwise at risk of stress or anxiety in an amount sufficient to at least partially reduce the risk of developing disease.Such an amount is defined as a "prophylactically effective amount".Again, the exact amount administered depends on many patient-specific factors, such as the patient's health condition and weight.

[0108] Generally, the probiotics contained in the compositions, foods or supplements according to the present invention are administered in a therapeutically and / or prophylactically effective amount.

[0109] Such probiotics are theoretically effective at any concentration, given that the bacteria will colonize and grow in the intestine if present in a viable form.

[0110] These expressions of bacterial load include the possibility that the bacteria may be alive, inactivated, dead, or may exist as fragments such as DNA or cell wall material, or as metabolites. In other words, the bacterial load is expressed as the colony forming capacity (cfu) of that amount of bacteria as if all the bacteria were alive, regardless of whether the bacteria are actually alive, inactivated, dead, fragmented, or a mixture of any or all of these.

[0111] HN001 and / or HN019 or their derivatives, or foods or supplements containing them, may be administered daily, where daily administration refers to an amount taken once or in several divided doses per day (24 hours).

[0112] The oral compositions are formulated to allow administration of a sufficient amount of HN001 and / or HN019 or derivatives thereof to establish a population in the gastrointestinal tract of a subject when ingested. The established population may be transient or permanent.

[0113] Theoretically, one colony forming unit (cfu) would be sufficient to establish a population of HN001 and / or HN019 in a subject, but since effects depend on a viable live population of probiotic bacteria, administering more units to a subject would improve efficacy.

[0114] The applicants have reported a dose of 1.9 x 10 per kg of body weight per day. 9 cfu of HN001 and / or 6.5 × 10 per kg body weight per day7 It has been determined that a dose of HN019 cfu is sufficient to establish a population in the gastrointestinal tract of a human subject. Thus, in one example, the composition formulated for administration contains at least about 1.9×10 9 cfu HN001 and / or 6.5 x 10 7 It will be sufficient to provide cfu of HN019 and / or their derivatives.

[0115] Methods for determining the presence of a population of gut microbiota, such as HN001 and / or HN019, in the gastrointestinal tract of a subject are well known in the art. In some embodiments, the abundance of HN001 and / or HN019 can be determined directly, for example, by analyzing one or more samples from a subject and determining the presence or amount of HN001 and / or HN019 in the sample. In other embodiments, the abundance of HN001 and / or HN019 can be determined indirectly, for example, by observing one or more of the following: (i) reduction or prevention of stress; (ii) reduction or prevention of anxiety; (iii) prevention or reduction of one or more symptoms of depression; (iv) reduction or reversal of certain changes caused by stress, anxiety or depression; (v) improvement of mood; (vi) increased sense of relaxation; (vii) increased energy. Combinations of such methods are also envisioned.

[0116] The efficacy of the compositions useful in the present invention can be evaluated both in vitro and in vivo. For example, the compositions may be tested for one or more of the following: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing relaxation; (vii) increasing vitality. In vivo studies can be performed by feeding or injecting the compositions into animal models (such as mice) or administering them to human subjects to evaluate the effect on any of the above characteristics (i) to (v). Based on the results, the appropriate dosage range and route of administration can be determined.

[0117] The method of calculating the appropriate dosage may depend on the nature of the active agent in the composition. For example, if the composition contains live HN001 and / or HN019, the dosage may be calculated with reference to the number of live bacteria present. For example, the dosage may be established with reference to the number of colony forming units (cfu) administered per day. In examples where the composition contains one or more derivatives of HN001 and / or HN019, the dosage may be calculated with reference to the amount or concentration of the derivatives of HN001 and / or HN019 present. For example, in the case of a composition containing HN001 and / or HN019 cell lysate, the dosage may be calculated with reference to the concentration of HN001 and / or HN019 cell lysate present in the composition.

[0118] As a general example, the dosage of HN001 and / or HN019 per kg of body weight per day is approximately 1×10 6 cfu ~ approx. 1 × 10 12 cfu, for example, about 1×10 6 cfu ~ approx. 1 × 10 11 cfu / kg / day: approx. 1×10 6 cfu ~ approx. 1 × 10 10 cfu / kg / day: approx. 1×10 6 cfu ~ approx. 1 × 10 9cfu / kg / day: approx. 1×10 6 cfu ~ approx. 1 × 10 8 cfu / kg / day; or approximately 1 × 10 6 cfu ~ approx. 1 × 10 7 cfu / kg / day. The dosage is about 5×10 6 cfu ~ approx. 4 × 10 8 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 3 × 10 8 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 2 × 10 8 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 1 × 10 8 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 9 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 8 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 7 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 6 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 5 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 4 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 3 × 10 7 cfu / kg / day: approx. 5×10 6 cfu ~ approx. 2 × 10 7 cfu / kg / day; or approximately 5 × 10 6 cfu ~ approx. 1 × 10 7 It may be cfu / kg / day.

[0119] In certain embodiments, the regular dosage does not need to vary with the body weight or other characteristics of the subject. In such an example, the daily dosage of HN001 and / or HN019 is about 1×10 6 cfu ~ approx. 1 × 10 13 cfu, preferably about 1×10 6 cfu ~ approx. 1 × 10 12 cfu / day: approx. 1 x 106 cfu ~ approx. 1 × 10 11 cfu / day: approx. 1 x 10 6 cfu ~ approx. 1 × 10 10 cfu / day: approx. 1 x 10 6 cfu ~ approx. 1 × 10 9 cfu / day: approx. 1 x 10 6 cfu ~ approx. 1 × 10 8 cfu / day: approx. 1 x 10 6 cfu ~ approx. 1 × 10 7 The daily dose of HN001 and / or HN019 may be about 2×10 6 cfu ~ approx. 2 × 10 13 cfu, preferably about 2×10 6 cfu ~ approx. 2 × 10 12 cfu / day: approx. 2×10 6 cfu ~ approx. 2 × 10 11 cfu / day: approx. 2×10 6 cfu ~ approx. 2 × 10 10 cfu / day: approx. 2×10 6 cfu ~ approx. 2 × 10 9 cfu / day: approx. 2×10 6 cfu ~ approx. 2 × 10 8 cfu / day: approx. 2×10 6 cfu ~ approx. 2 × 10 7 The daily dose of HN001 and / or HN019 may be about 3×10 6 cfu ~ approx. 3 × 10 13 cfu, preferably about 3×10 6 cfu ~ approx. 3 × 10 12 cfu / day: approx. 3×10 6 cfu ~ approx. 3 × 10 11 cfu / day: approx. 3×10 6 cfu ~ approx. 3 × 10 10 cfu / day: approx. 3×10 6 cfu ~ approx. 3 × 10 9 cfu / day: approx. 3×10 6 cfu ~ approx. 3 × 10 8 cfu / day: approx. 3×10 6 cfu ~ approx. 3 × 10 7 The daily dose of HN001 and / or HN019 may be about 4×106 cfu ~ approx. 4 × 10 13 cfu, preferably about 4×10 6 cfu ~ approx. 4 × 10 12 cfu / day: approx. 4×10 6 cfu ~ approx. 4 × 10 11 cfu / day: approx. 4×10 6 cfu ~ approx. 4 × 10 10 cfu / day: approx. 4×10 6 cfu ~ approx. 4 × 10 9 cfu / day: approx. 4×10 6 cfu ~ approx. 4 × 10 8 cfu / day: approx. 4×10 6 cfu ~ approx. 4 × 10 7 The daily dose of HN001 and / or HN019 may be about 5×10 6 cfu ~ approx. 5 × 10 13 cfu, preferably about 5×10 6 cfu ~ approx. 5 × 10 12 cfu / day: approx. 5 × 10 6 cfu ~ approx. 5 × 10 11 cfu / day: approx. 5 × 10 6 cfu ~ approx. 5 × 10 10 cfu / day: approx. 5 × 10 6 cfu ~ approx. 5 × 10 9 cfu / day: approx. 5 × 10 6 cfu ~ approx. 5 × 10 8 cfu / day: approx. 5 × 10 6 cfu ~ approx. 5 × 10 7 The daily dose of HN001 and / or HN019 may be about 6×10 6 cfu ~ approx. 6 × 10 13 cfu, preferably about 6×10 6 cfu ~ approx. 6 × 10 12 cfu / day: approx. 6×10 6 cfu ~ approx. 6 × 10 11 cfu / day: approx. 6×10 6 cfu ~ approx. 6 × 10 10 cfu / day: approx. 6×10 6 cfu ~ approx. 6 × 10 9 cfu / day: approx. 6×10 6 cfu ~ approx. 6 × 108 cfu / day: approx. 6×10 6 cfu ~ approx. 6 × 10 7 The daily dose of HN001 and / or HN019 may be about 7×10 6 cfu ~ approx. 7 × 10 13 cfu, preferably about 7×10 6 cfu ~ approx. 7 × 10 12 cfu / day: approx. 7×10 6 cfu ~ approx. 7 × 10 11 cfu / day: approx. 7×10 6 cfu ~ approx. 7 × 10 10 cfu / day: approx. 7×10 6 cfu ~ approx. 7 × 10 9 cfu / day: approx. 7×10 6 cfu ~ approx. 7 × 10 8 cfu / day: approx. 7×10 6 cfu ~ approx. 7 × 10 7 cfu / day.

[0120] Preferably, the dosage of HN001 and / or HN019 per day per kg of body weight is about 7×10 7 cfu ~ approx. 7 × 10 10 5×10 7 cfu: ~ approx. 5 × 10 10 cfu, preferably about 5×10 7 cfu ~ approx. 4 × 10 10 cfu / day: approx. 5 × 10 7 cfu ~ approx. 3 × 10 10 cfu / day: approx. 5 × 10 7 cfu ~ approx. 2 × 10 10 cfu / day: approx. 5 × 10 7 cfu ~ approx. 1 × 10 10 cfu / day: approx. 5 × 10 7 cfu ~ approx. 9 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 8 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 7 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 6 × 10 9 cfu / day: approx. 5 × 10 7cfu ~ approx. 5 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 4 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 3 × 10 9 cfu / day: approx. 5 × 10 7 cfu ~ approx. 2 × 10 9 cfu / day; or approximately 5 × 10 7 cfu ~ approx. 1 × 10 9 cfu / day.

[0121] One effective dose of lyophilized HN001 and / or HN019 is approximately 6×10 9 It was determined that the cfu.

[0122] It will be understood that the composition is preferably formulated to allow administration of an effective amount of HN001 and / or HN019, or one or more derivatives thereof. The dose of the composition administered, the duration of administration, and the general administration regime may vary between patients, depending on variables such as the severity of the subject's symptoms, the type of disease being treated, the method of administration selected, and the age, sex, and / or overall health of the patient. Furthermore, as mentioned above, the appropriate dose may vary depending on the nature of the active agent in the composition and the method of formulation. For example, when the composition includes live HN001 and / or HN019, the dose may be calculated with reference to the number of live bacteria present. For example, as described in the examples herein, the dose may be set with reference to the number of colony forming units (cfu) to be administered per day. In examples where the composition includes one or more derivatives of HN001 and / or HN019, the dose may be calculated with reference to the amount or concentration of the derivatives of HN001 and / or HN019 to be administered per day. For example, in the case of a composition comprising HN001 and / or HN019 cell lysate, the dosage can be calculated with reference to the concentration of HN001 and / or HN019 cell lysate present in the composition.

[0123] It will be understood that the preferred composition is formulated to provide an effective amount in a convenient form and amount.In certain embodiments, such as but not limited to those in which regular dose does not need to vary according to subject's weight or other characteristics, the composition can be formulated for unit dose.It should be understood that administration can include one daily administration or multiple separate distributed administrations as appropriate.For example, as presented in the examples herein, the effective amount of HN001 and / or HN019 can be formulated into capsules for oral administration.

[0124] The composition, food or supplement may be intended for use by humans, pets or livestock. The term "intended for use by" means that there is a specific adaptation to the nutritional requirements of the target mammalian population. Those skilled in the art know ingredients that are useful in such nutritional compositions in addition to the probiotics according to the present invention, so as to be suitable as a supplement or nutritionally complete composition.

[0125] According to one embodiment, aspects of the invention relating to HN001 do not include the treatment or prevention of postpartum depression or postpartum anxiety.

[0126] According to one embodiment, aspects of the invention related to HN001 do not include increasing calmness or reducing occasional anxiety.

[0127] According to another embodiment, aspects of the invention related to HN001 do not include the combination of Lactobacillus rhamnosus LPR (ATCC 54193) and Bifidobacterium longum BL999 (ATCC BAA-9999) for reducing anxiety and chronic stress.

[0128] According to another embodiment, the aspect of the invention relating to HN001 does not include the combination of HN001 with glutamine and / or turmeric.

[0129] Various aspects of the present invention will now be illustrated, in a non-limiting manner, with reference to the following examples.

[0130] <Example> Example 1: Chronic Unpredictable Mild Stress (CUMS) model to test the effects of Lacticaceibacillus rhamnosus (HN001) and Bifidobacterium animalis subsp. lactis (HN019) A series of experiments was conducted using the chronic unpredictable mild stress (CUMS) rodent model. Rats were exposed daily to one of two randomly selected mild physiological stressors: 12 hours of water shortage 12 hours of food shortage 8 hours of damp sawdust · 24-hour reversed light / dark cycle Pinch the tail for 2 minutes 1 hour of 60Hz noise; 15 minutes of cage shaking; 5-10 minutes of swimming in 4°C water -2 hours of detention

[0131] Such exposure induces depressive symptoms and produces a variety of physiological and behavioral effects in rats that are thought to resemble anxiety and depression in humans.

[0132] Six groups of rats were evaluated. 1. Control rats: rats that were not subjected to stress treatment and were raised normally without nutritional or drug intervention. 2. CUMS rats: rats that underwent the CUMS protocol but no nutritional or drug intervention. 3. CUMS HN001: rats that underwent the CUMS protocol and were given HN001. 4. CUMS HN019: rats that underwent the CUMS protocol and were given HN019. 5. CUMS HN001+HN019: rats undergoing the CUMS protocol and receiving HN001 and HN019. 6. CUMS Fluox: Participants underwent the CUMS protocol and were administered fluoxetine, a type of antidepressant known as a selective serotonin reuptake inhibitor (SSRI), which is notable for having fewer side effects than other antidepressants.

[0133] As shown in Figure 1, the experiment was performed on groups of rats (n=20 each) that were allowed to acclimate for 7 days. A baseline assessment, including blood sampling, was then performed. For the next 40 days, rats in groups 2-6 were exposed to CUMS treatment to induce stress- and anxiety-related depressive symptoms. At this point, the assessment and blood sampling were repeated in all groups, and CUMS treatment with the addition of probiotics or fluoxetine intervention for another 40 days was resumed in groups 2-6.

[0134] The rats were then subjected to tests to evaluate their behavior, including the open field test and the elevated plus maze (see Figure 2), which are standard methods for measuring stress and anxiety characteristics in rats.

[0135] Stressed rats became timid and tended to avoid open and illuminated areas. Rats receiving CUMS showed reduced exploratory behavior in both the open field and elevated plus maze tests, but these responses were nearly reversed in CUMS rats given HN001, HN019, or both, to the same extent as rats given antidepressants and to the same extent as control rats not receiving CUMS.

[0136] <Result> Example 2: Body weight The rat group exposed to CUMS showed reduced weight gain compared to the control rats not exposed to CUMS, indicating that CUMS treatment affected the rats physiologically. The reduced weight gain in the rats exposed to CUMS was ameliorated by administration of HN001, HN019 or both (Figure 3). Also, no difference in weight gain was observed between the CUMS groups.

[0137] Example 3: Open field test In the first behavioral test, the open field test, all CUMS rats after the first 6 weeks (CUMS treated but no probiotic / drug intervention) were less active, indicating that they were too anxious to explore their local environment; this is an expected response to CUMS. However, after the probiotic / drug intervention, rats exposed to CUMS but not to probiotics or fluoxetine continued to show low activity in the open field test, while all three probiotic groups showed an increase in activity comparable to the control group and to CUMS rats treated with the antidepressant fluoxetine. See table below.

[0138] [Table 1]

[0139] [Table 2]

[0140] Example 4: Elevated Plus Maze Similar results were obtained in the second behavioral test, the elevated plus maze: before the intervention, all CUMS rats exhibited stress-related behaviors, whereas after the intervention, the probiotic-fed rats improved these behaviors almost as well as control rats not exposed to CUMS, and similarly to the addition of fluoxetine.

[0141] [Table 3]

[0142] [Table 4]

[0143] The results of the first and second behavioral tests indicate that rats exposed to CUMS exhibited depressive-like behavior, and that treatment with HN001 and / or HN019 was able to attenuate this behavior.

[0144] Example 5: Probiotics attenuate elevated inflammatory factors in serum, brain and colon. A series of blood markers were evaluated. Results showed that CUMS increased the levels of the cytokines TNFα, IL-6, IL-1β and IL-18 in serum, brain tissue and colon compared to the control group (P<0.05). Treatment with probiotics and fluoxetine was associated with a reduction in cytokine levels (see Tables 5 and 6 below, and Figure 7).

[0145] [Table 5]

[0146] [Table 6]

[0147] Example 6: Correlation analysis of inflammatory factors in the brain and colon To further validate that gut inflammation is associated with brain inflammation, we assessed the correlation between gut inflammatory factors and inflammatory factors in the brain. A clear positive correlation was found between TNF-α in the brain and colon (rs=0.493, p=0.000). There was also a positive correlation between IL-6 (rs=0.323, p=0.013), IL-1β (rs=0.260, p=0.049), and IL-18 (rs=0.272, p=0.039) in the brain and colon. See Figure 8.

[0148] Example 7: Effects on neuroendocrine hormones - Corticotropin releasing factor (CRH) and adrenocorticotropic hormone (ACTH) content Examination of components of the HPA axis demonstrated that, similar to fluoxetine, treatment with HN001, HN019, or HN001+HN019 reduced specific neuroendocrine hormone and hormone receptor induced stress levels following CUMS treatment. See Tables 7 and 8 below.

[0149] [Table 7]

[0150] [Table 8]

[0151] Example 8: Western blot analysis of neuroendocrine hormone receptors - Corticotropin releasing factor type 1 (CRF1) and glucocorticoid receptor (GR) in brain tissue Western blot analysis showed that ingestion of both or either of the probiotic strains, as well as fluoxetine, reversed CUMS-induced changes in HPA axis-related neuroendocrine receptor expression in the brain. Results are shown in Figure 4 as the ratio of antibody staining intensity of receptors (CFR1, left; GR, right) vs. housekeeping protein GAPDH. *P<0.05.

[0152] Example 9: Probiotics normalize morphological changes in the hippocampus and colon Brain and colon tissues were stained with HE method. Brain tissues included two regions of the hippocampus (dentate gyrus (DG) and CA1) and one region of the cerebral cortex (P2). The results are shown in Figure 5 and show that in the control group, the neurons in the hippocampal regions (dentate gyrus (DG) and CA1) and cerebral cortex (P2) were normal, well-ordered, tightly packed cells. The cells showed the expected morphology, uniform cytoplasmic staining, without increased interstitial or abnormal changes. In contrast, the corresponding tissues from CUMS rats showed some disorganized, irregularly shaped cells, nuclear flattening, indistinct nucleoli, and heterogeneous cytoplasmic staining. However, CUMS-associated damage was greatly reduced in tissues from all four intervention groups (fluoxetine, HN001, HN019, HN001+HN019).

[0153] In colon tissue, the intestinal villi of the control group were well-ordered, the cell morphology was normal, and no obvious damage was observed. Compared with the control group, the intestinal villi of the CUMS group were disorganized and obvious damage was observed. The thickness of the intestinal mucosa was significantly thinner than that of the control group. This CUMS-related damage was significantly reduced by the ingestion of the probiotic strain, as was the case with fluoxetine.

[0154] Example 10: Effects of probiotics on neurotransmitters and their metabolites in serum and brain CUMS significantly decreased the contents of 5-HT, DA, and NE in serum and brain, and increased the concentrations of Glu and GABA (Fig. 6A-L). HIAA, DOPAC, and HVA, which are metabolites of 5-HT, DA, and NE, were increased in rat brain after CUMS stimulation. Administration of HN001 and / or HN019 could reverse the effects of CUMS on these neurotransmitters.

[0155] Example 11: Changes in the microbiota by probiotic treatment To assess whether probiotics alter the gut microbiota in a similar way to CUMS, we performed 16S rRNA sequencing of fecal samples. Alpha diversity, as measured by richness (Sobs index, Chao1 index, ACE index; see Figure 9A-C) and diversity (Shannon index; see Figure 9D), was decreased after CUMS.

[0156] Interestingly, compared with the CUMS group, the Sobs, ACE, Chao1 and Shannon indexes were decreased in the other groups administered HN001 and / or HN019. Furthermore, the Shannon index in the combination group was significantly decreased compared with the monotherapy group, indicating that the diversity of the gut microbiota was significantly altered. This result may be explained by the change in the dominant gut microbiota after the intervention. Further analysis determined the dominant microbiota associated with anxiety- and depression-like behaviors.

[0157] Beta diversity analysis was evaluated by PCoA to assess the differences in microbial composition among the six groups (Figure 10A). PCoA results showed that the microbial community of the CUMS group exhibited a different composition compared with that of the control rats. Furthermore, treatment with HN001, HN019, and HN001&HN019 induced significant differences in the PCoA plot compared with that of the CUMS rats. In particular, the gut microbiota profile of rats treated with HN001&HN019 showed significant divergence from that of the CUMS rats.

[0158] According to the heatmap of species abundance clusters, at the phylum level (Fig. 11B), Firmicutes, Bacteroidetes, Verrucomicrobias, Proteobacterias and Actinobacterias were the dominant phyla. The relative abundances are shown in Fig. 10. CUMS decreased the relative abundance of Bacteroidetes and increased the relative abundance of the other bacteria mentioned above. Treatment with HN001, HN019 and HN001&HN019 alleviated the changes in phyla, and the changes in the combined group of bacteria were more pronounced. Meanwhile, after CUMS stimulation, the ratio of Firmicutes to Bacteroidetes (F / B ratio) increased, and administration of HN001 and / or HN019 decreased the F / B ratio.

[0159] At the family level, the relative abundance of Ruminococcus and Lactobacillus increased and Lachnospira decreased in the CUMS group, and HN001 and / or HN019 reversed the family changes, especially the joint intervention. The genus Lactobacillus, the Eubacterium_coprostanoligenes group, and the Lachnospira-NK4A136 group, all belonging to the phylum Firmicutes, increased and decreased, respectively, and these changes were also ameliorated by supplementation with HN001 and / or HN019. The relative ratios of the microbiota were altered after CUMS administration and after HN001 and / or HN019 treatment.

[0160] <Correlation analysis between intestinal microbiota, monoamine neurotransmitters, and inflammatory factors> Deficiency of monoamine neurotransmitters and imbalance of inflammatory factors are important in the development of depression. Spearman's correlation analysis was performed to clarify the association between the altered microbiota and depression-related traits including neurotransmitters and inflammatory factors. As shown in Figure 9, Firmicutes, Verrucomicron, Proteobacteria, Pasteurisibacteria, Actinobacteria and Eurychota were positively correlated with serum, brain and colon inflammatory factors and negatively correlated with serum NE, DA and 5-HT levels. Meanwhile, Epsilonbacteraeota and Bacteroidetes were positively correlated with serum neurotransmitter concentrations and negatively correlated with inflammatory mediators. Apart from that, clear correlations were identified between the family and genus microbiota and these indicators. In particular, the families Lachnospiraceae, Ruminococcus, Christensenellaceae, and Peptostreptococaceae, as well as the genus Ruminococcus, Eubacterium coprostanoligenes group, Ruminococcus_UCG014, Ruminococcus_NK4A214_group, and Ruminococcus-1, all of which belong to Firmicutes, showed positive and negative correlations with inflammatory factors and serum neurotransmitters, respectively. The families Muribaculaceae and Prevotellaceae, which belong to Bacteroidetes, showed negative correlations with inflammatory factors and positive correlations with serum 5-HT, NE, and DA, which were consistent with Bacteroidetes. These results suggest that the altered microbiota, especially those specifically affected by HN001 and / or HN019, simultaneously showed strong associations with neurotransmitters and inflammatory cytokines, indicating an important role for the gut microbiota in regulating neurotransmitters and inflammation.

[0161] These results indicate that the probiotics HN001 and HN019, alone or in combination, can reduce stress and anxiety in rat models via the HPA axis. Interestingly, probiotics also appear to have anti-inflammatory effects, as probiotic intake was associated with a reduction in TNFα and IL-6 cytokine levels, which were elevated in CUMS rats that did not receive probiotic treatment. Probiotics were also able to reverse the effects of CUMS on brain and colon cell morphology, protecting these tissues from stress-related cell damage.

[0162] Compared to pharmaceutical treatments for mild stress and anxiety, probiotics generally have no side effects and can be consumed through a wide range of dairy and non-dairy foods, as well as supplements. The experiments show similar effects to SSRIs commonly prescribed for conditions such as anxiety, obsessive-compulsive disorder or post-traumatic stress disorder.

[0163] Throughout the description and claims, the words "comprise" and "contain," as well as variations of these words, such as "comprising" and "comprises," mean "including but not limited to" and do not exclude other elements, integers or steps. Furthermore, the singular encompasses the plural unless the context requires otherwise: in particular where the indefinite article is used, the specification is understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0164] Preferred features of each aspect of the invention may be the same as those described in relation to any of the other aspects.Within the scope of this application, the various aspects, embodiments, examples and alternatives described in the preceding paragraphs, claims and / or the following description and drawings, in particular their individual features, are expressly intended to be taken independently or in any combination, i.e., all embodiments and / or features of any embodiment may be combined in any manner and / or combination, except where such features are incompatible.

[0165] Reference to a range of numerical values ​​disclosed herein (e.g., 1-10) is intended to include references to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), as well as any range of rational numbers within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and thus all subranges of every range explicitly disclosed herein are expressly disclosed herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the recited lower and upper limits are to be considered as being expressly intended in this application as well.

Claims

1. 1. Use of Lacticazemi bacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) or a derivative thereof for any one or more of: (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing relaxation; (vii) increasing energy, The use of HN001 is not intended to increase calmness, reduce occasional anxiety, or treat or prevent postpartum depression or postpartum anxiety in a subject. use.

2. Lacticase Bacillus rhamnosus strain HN001 (HN001) or a derivative thereof; and / or b. Bifidobacterium animalis subsp. lactis strain HN019 (HN019) or a derivative thereof; and c. a physiologically acceptable diluent, adjuvant, carrier or excipient; Including, (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing relaxation; (vii) increasing energy, The use of HN001 is not intended to increase calmness, reduce occasional anxiety, or treat or prevent postpartum depression or postpartum anxiety in a subject. composition.

3. (i) reducing or preventing stress; (ii) reducing or preventing anxiety; (iii) preventing or reducing one or more symptoms of depression; (iv) reducing or reversing certain changes caused by stress, anxiety or depression; (v) improving mood; (vi) increasing relaxation; (vii) increasing energy, Lacticazemi bacillus rhamnosus strain HN001 (HN001) and / or Bifidobacterium animalis subsp. lactis strain HN019 (HN019) or a derivative thereof, comprising administering to a patient in need thereof The use of HN001 is not intended to treat or prevent postpartum depression, postpartum anxiety or stress in a subject; method.

4. The reduction or reversal of the changes caused by said stress, anxiety or depression a) changes in the levels of neurotransmitters or their metabolites; b) changes in stress hormone levels; c) changes in the levels of inflammatory factors; d) morphological changes in brain tissue; e) morphological changes in the colon; f) changes in the intestinal flora; g) Weight change including one or more of the following: The use, composition or method of any one of claims 1 to 3.

5. The HN001 and / or HN019 or derivatives thereof are orally administered to a patient in need thereof. The use, composition or method of any one of claims 1 to 3.

6. said administration reduces the level of one or more molecules involved in the hypothalamic-pituitary-adrenal (HPA) axis, for example, the level of any one or more of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTG), and glucocorticoid hormones such as cortisol; said administration optionally reduces inflammation through a reduction in inflammatory cytokines such as TNFα, IL-6, IL-1β, IL-18, etc.; The use, composition or method of any one of claims 1 to 3.

7. The reduction in the level of one or more molecules involved in the HPA axis or inflammatory cytokines is in any one or more of serum, brain, or intestinal tissue; 7. The use, composition or method of claim 6.

8. said administration of HN001 and / or HN019 or derivatives reverses stress-induced decreases in any one or more of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) or metabolites, and / or increases glutamate and / or GABA concentrations to levels found in non-stressed individuals; The use, composition or method of any one of claims 1 to 3.

9. said administration of HN001 and / or HN019 or a derivative restores stress-induced changes in the gut microbiota back to a stress-free profile, said profile being in terms of abundance, (alpha and beta) diversity, richness, and levels of dominant microbiota; The use, composition or method of any one of claims 1 to 3.

10. administration of HN001 and / or HN019 or derivatives reverses the effects of stress and / or anxiety on brain or colon cell morphology and / or protects brain or colon tissue from stress- and / or anxiety-related cellular damage; The use, composition or method of any one of claims 1 to 3.

11. Administration of HN001 and / or HN019 or derivatives protects and maintains the epithelial cell barrier from damage and loss of integrity due to, for example, inflammation; The use, composition or method of any one of claims 1 to 3.

12. administration of HN001 and / or HN019 or derivatives protects or improves the integrity of the blood-brain barrier (BBB), optionally resulting in reduced brain inflammation and / or improved brain function; The use, composition or method of any one of claims 1 to 3.

13. administration of HN001 and / or HN019 or a derivative protects or improves the integrity of the intestinal barrier, and optionally said use is in the treatment or prevention of irritable bowel syndrome (IBS) and / or systemic inflammation; The use, composition or method of any one of claims 1 to 3.

14. The stress is chronic stress. The use, composition or method of any one of claims 1 to 3.

15. The reduction in stress and / or anxiety occurs through the HPA axis. The use, composition or method of any one of claims 1 to 3.

16. The reduction may include a reduction in any one or more of the following: stomach pain, muscle tension, headache, rapid breathing, shortness of breath, increased heart rate, palpitations, sweating, dry mouth, trembling, fatigue, fidgetiness, rapid speech, increased negative emotions (if already experienced), prolonged duration of stress fatigue, changes in appetite, dizziness, confusion, feelings of impending doom, panic, irritability, difficulty concentrating, bowel discomfort or dysfunction, irrational anger, restlessness; The use, composition or method of any one of claims 1 to 3.

17. The reduction in anxiety includes a reduction in fear, a sense of impending danger, catastrophe, unhappiness, dissatisfaction, worry, nervousness, and fear or apprehension regarding the subject's current or future condition; The use, composition or method of any one of claims 1 to 3.

18. The HN001 gene is designated by Genebank accession number NZ_ABWJ00000000 and is available under AGAL deposit number NM97 / 09514 dated August 18, 1997, and the HN019 gene is available under AGAL deposit number NM97 / 09513. The use, composition or method of any one of claims 1 to 3.

19. The HN001, HN019 or derivative thereof is in a propagable form and / or is killed, lysed, fractionated or attenuated; 19. The use, composition or method of claim 18.

20. the HN001 and / or HN019 or derivatives thereof are administered in a food or supplement; The food product is selected from cultured milk, yogurt, cheese, dairy drinks and milk powder; and / or the food product is selected from maternal supplements, nutritional products, infant formulas, follow-up formulas and growth formulas. The use, composition or method of any one of claims 1 to 3.

21. The food or supplement further contains probiotics and / or prebiotics.

21. The use, composition or method of claim 20.

22. The food or supplement is administered to a human subject at a concentration of at least about 1.9 x 10 per day to form a population in the digestive tract of the subject. ９ cfu of HN001 and / or 6.5 × 10 ７ administered daily to provide 100 cfu of HN019; 21. The use, composition or method of claim 20.

Images