Ruminococcaceae species and Luminococcus lactaris strains for the treatment and prevention of Alzheimer's disease and aging

Abstract

The present invention relates to Ruminococcaceae species and a new strain of Luminococcus lactaris for use alone or in combination in the treatment and prevention of memory decline in an individual, particularly memory decline of aging or Alzheimer's disease-related origin. The present invention also provides a composition, particularly an oral composition, comprising a Ruminococcaceae species and a Luminococcus lactaris strain, and uses thereof.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a new strain of bacteria, a strain belonging to the family Lachnospiraceae, and a strain belonging to the species Ruminococcus lactaris, for use as a drug, particularly as a drug in the treatment and / or prevention of memory impairment or memory decline in an individual due to aging or Alzheimer's disease.

[0002] Memory impairment or memory decline is caused by a number of diseases, disorders, or conditions, including Alzheimer's disease (AD), aging, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction (POCD), attention deficit hyperactivity disorder, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS). Aging is characterized by a progressive decline in function in which the brain experiences significant changes in biological, psychological, neuroanatomical, and neurophysiological functions that are closely related to the decline in cognitive function. In fact, normal aging is associated with a decline in various memory abilities in several cognitive tasks involving both episodic memory, semantic memory, and the stimulation of short-term and long-term memory (Hedden et Gabrieli, 2004). These disorders may be related to a decline in the ability to retrieve recently processed information, a specific susceptibility of the hippocampus, a brain region that plays an important role in learning and memory in mammals, and the aforementioned neurodegenerative changes seen in aging (Dahan et al., 2020). Interacting environmental and genetic factors exacerbate the progression of aging in some segments of the elderly population, leading to mild cognitive impairment (MCI), a transitional state between normal aging and Alzheimer's disease (Mufson et al., 2016), and progressing to a state declared as Alzheimer's disease.

[0003] The latter is a neurodegenerative disease that affects approximately 40 million people worldwide, with its incidence increasing with age and affecting not only memory but also movement and language. Alzheimer's disease significantly reduces the cognitive abilities of patients. The gut microbiota (GM) has recently emerged as a central player in both healthy brain function and the etiology of diseases. Many studies have shown the association between the gut microbiota (GM) and aging / Alzheimer's disease (Holmes et al., 2020), corroborating the existence of changes in GM composition in elderly subjects (Jeffery et al., 2016) and Alzheimer's disease patients (Cattaneo et al., 2017). For example, the phenotypes of aging or Alzheimer's disease are known to be transmitted via the GM in studies where rodents were colonized by fecal microbiota transplantation (FMT) from aged rodents (D’Amato et al., 2020; Li et al., 2020), mouse models of Alzheimer's disease (Kim et al., 2021), or elderly human subjects (Rei et al., 2021). Furthermore, the phenotype of Alzheimer's disease is known to be improved by FMT from wild-type mouse donors in Alzheimer's disease mouse models (Sun et al., 2019), and probiotic treatment has been shown to be beneficial in Alzheimer's disease patients (Leblhuber et al., 2018). There is currently no effective treatment for the detrimental effects of aging and Alzheimer's disease. Therefore, there is an urgent need to find new ways to improve and / or maintain the cognitive abilities and quality of life of elderly subjects and Alzheimer's disease patients.

Summary of the Invention

[0004] In one embodiment, the present invention provides novel substances, particularly live biological bacterial preparations, and compositions for use as drugs in treating and / or preventing memory impairment, including age-related or Alzheimer's disease-related memory impairment in an individual, and methods of using these.

[0005] In one embodiment, the present invention presents that certain Ruminococcaceae species and Luminococcus lactaris strains have the unexpected ability to restore memory capacity in elderly or Alzheimer's disease individuals. As shown in the following examples and discussed elsewhere in this specification, the present specification presents that other bacteria such as Faecalicatena contorta, Faecalibacterium prausnitzii A2-165, and Roseburia intestinalis DSM14610 strains do not have such advantageous properties. The properties of the Ruminococcaceae species and Luminococcus lactaris strains of the present invention are shown in a mouse model of Alzheimer's disease obtained by fecal microbiota transplantation (FMT) of human Alzheimer's disease patients in mice and in an elderly aging mouse model. Accordingly, the present invention provides a composition comprising bacteria of at least one strain selected from Ruminococcaceae species and Luminococcus lactaris strains.

[0006] In one embodiment, the present invention provides a composition comprising bacteria of at least one strain selected from Luminococcus lactaris strains.

[0007] In one embodiment, the bacteria are each any one of the strains deposited with the CNCM under the accession numbers CNCM I-5830 and CNCM I-5831. In one embodiment, the bacteria are contained in a physiologically acceptable medium. In one embodiment, the composition further comprises prebiotics. In one embodiment, the composition further comprises a therapeutic agent. In one embodiment, the composition further comprises another compound, and the composition is formulated for use as a probiotic or animal feed. In one embodiment, the composition is administered in combination with one or more other therapies or therapeutic agents.

[0008] In another embodiment, the present invention presents that strains of the genus Ruminococcus and the genus Luminococcus can restore the functional decline of the hippocampus in animals with memory impairment, and as a result, can prevent and / or alleviate the memory impairment in the above animals. Such results were obtained by subjecting strains grown under anaerobic conditions collected from healthy young adult donors to fluorescence-activated cell sorting (FACS) for testing as anti-aging and Alzheimer's disease treatment and preventive drugs. From 400 classified strains, 67 strains were selected based on their identity and potential, and in vitro antioxidant activity and production of anti-inflammatory short-chain fatty acids. Next, interesting candidate strains based on these results (data not shown) were tested in vivo for their memory-enhancing effects and anti-Alzheimer's disease and anti-aging effects in FMT mouse models of human Alzheimer's disease patients and aging mice, respectively. From this newly generated strain library, an unidentifed new species of the genus Bacteroides, two bacteria characterized as species of the family Ruminococcaceae, and a Luminococcus lactaris strain genetically different from other members of its genus showed unexpected characteristics. Such characteristics of the Ruminococcaceae species and the Luminococcus lactaris strain were demonstrated in examples provided by the inventors in two different models: FM transplanted mice of Alzheimer's disease patients and aging mice. As shown in the examples, the Ruminococcaceae species and the Luminococcus lactaris strain according to the present invention have no adverse effects in individuals suffering from age-related or Alzheimer's disease-related memory impairment, and thus are safe as drugs for animals.

[0009] Accordingly, the present invention provides a method for treating or preventing memory impairment in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the Ruminococcaceae species and the Luminococcus lactaris strain, preferably the Luminococcus lactaris strain.

[0010] The present invention also provides a method for treating or preventing memory impairment caused by neurodegenerative diseases of the central nervous system in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis. The present invention also provides a method for treating or preventing neurodegenerative diseases of the central nervous system in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0011] In one embodiment, the present invention provides a method for treating or preventing memory impairment caused by neurodegenerative diseases of the central nervous system in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis, wherein the diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0012] In one embodiment, the present invention provides a method for treating or preventing neurodegenerative diseases of the central nervous system in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis, wherein the diseases are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0013] In one embodiment, the present invention provides a method for treating or preventing memory impairment caused by Alzheimer's disease in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0014] In one embodiment, the present invention provides a method for treating or preventing Alzheimer's disease in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably the bacteria of the strain Lactococcus lactis.

[0015] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at different disease stages in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably the bacteria of the strain Lactococcus lactis.

[0016] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at the preclinical stage in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably the bacteria of the strain Lactococcus lactis.

[0017] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at the mild cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably the bacteria of the strain Lactococcus lactis.

[0018] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at the mild cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae species and Lactococcus lactis strains, preferably bacteria of the Lactococcus lactis strain.

[0019] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at the moderate cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae species and Lactococcus lactis strains, preferably bacteria of the Lactococcus lactis strain.

[0020] In one embodiment, the present invention also provides a method for treating or preventing memory impairment caused by Alzheimer's disease at the severe cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae species and Lactococcus lactis strains, preferably bacteria of the Lactococcus lactis strain.

[0021] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at different disease stages in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae species and Lactococcus lactis strains, preferably bacteria of the Lactococcus lactis strain.

[0022] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the preclinical stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae species and Lactococcus lactis strains, preferably bacteria of the Lactococcus lactis strain.

[0023] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the mild cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0024] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the mild dementia stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0025] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the moderate dementia stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0026] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the severe dementia stage in a subject in need thereof by administering to the subject a composition comprising bacteria of one or more strains selected from the family Ruminococcaceae and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis.

[0027] In one embodiment, the bacterium is any one of the strains deposited with the CNCM under accession numbers CNCM I-5830 and CNCM I-5831, respectively. In one embodiment, the bacterium is included in a physiologically acceptable composition. In one embodiment, the composition further comprises prebiotics. In one embodiment, the composition further comprises a therapeutic agent. In one embodiment, the composition further comprises another compound, and the composition is formulated for use as a food. In one embodiment, the composition is formulated for use as a probiotic. In one embodiment, the composition is formulated for use as animal feed.

[0028] The present invention also provides a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Luminococcus lactaris for use as a medicament for the treatment or prevention of memory impairment or any of the other diseases listed above. In one embodiment, the strain is any one of the strains deposited with the CNCM under the accession numbers CNCM I-5830 and CNCM I-5831, respectively. In one embodiment, the bacterium is included in a physiologically acceptable composition. In one embodiment, the composition further comprises prebiotics. In one embodiment, the composition further comprises a therapeutic agent. In one embodiment, the composition further comprises another compound, and the composition is formulated for use as a food. In one embodiment, the composition is formulated for use as animal feed.

[0029] Furthermore, the present invention provides the use of a composition comprising one or more bacteria selected from the genus Ruminococcus and the strain Lactococcus lactis, preferably bacteria of the strain Lactococcus lactis, for the preparation of a medicament for the treatment or prevention of memory impairment or any of the other diseases mentioned above. In one embodiment, the bacteria are each one of the strains deposited at the CNCM under the accession numbers CNCM I-5830 and CNCM I-5831, preferably the strain CNCM I-5831. In one embodiment, the bacteria are included in a physiologically acceptable composition. In one embodiment, the composition further comprises prebiotics. In one embodiment, the composition further comprises a therapeutic agent. In one embodiment, the composition further comprises another compound and the composition is formulated for use as a food. In one embodiment, the composition is formulated for use as an animal feed.

[0030] The present invention also provides a method for preventing and / or treating memory impairment due to age-related memory decline or neuropsychiatric diseases and / or neurodegenerative diseases, disorders or conditions, the method comprising administering to a patient in need thereof a composition comprising a Ruminococcus species and / or a Lactococcus lactis strain, preferably a Lactococcus lactis strain, and / or a culture extract thereof, and a carrier or excipient.

[0031] The present invention presents that memory impairment is caused by neuropsychiatric and / or neurodegenerative diseases, disorders or conditions. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis and / or aging. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease and / or aging. In some embodiments, the memory impairment is caused by agnosia, amnesia, traumatic brain injury, dementia, or attention deficit / hyperactivity disorder. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease at different disease stages. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from preclinical stage Alzheimer's disease. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease at the mild cognitive impairment stage. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease at the mild dementia stage. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease at the moderate dementia stage. In some embodiments, the neuropsychiatric and / or neurodegenerative diseases, disorders or conditions are selected from Alzheimer's disease at the severe dementia stage.

[0032] In one embodiment, the present invention presents that memory impairment can be measured by a fear conditioning task. In another embodiment, the memory impairment may be measured by a novel object exposure assay. In some embodiments, the memory impairment is evaluated by an object recognition task. In some embodiments, verbal short-term memory is tested by a digit span task in which an individual is exposed to numbers of various digit lengths for various times and is then asked to recall those numbers later. In one embodiment, non-verbal short-term memory may be tested by various motor or spatial memory tasks such as a spatial information test. In these tasks, the subject is exposed to various motor tasks or spatial orientation and is then asked to recall or reconstruct them later. In one embodiment, procedural memory can be tested by various tasks such as a mirror tracing test, a mirror reading task, a weight sampling task, a speed reading of repeated nonwords, and a resolving random-dot stereograms. In one embodiment, the memory impairment can be evaluated using various forms of classical conditioning tasks such as blink reflex conditioning that combines the onset of sound or light with an air puff. In one embodiment, the subject is tested particularly for the ability to classify strings as grammatical or ungrammatical. In one embodiment, declarative memory is tested by recall of facts, various types of matching tests, retention tests over minutes to days or years, language learning and recall tasks, and many other similar tests. Accordingly, the present invention also provides a method for generating a mouse model showing symptoms of a neuropsychiatric disease and / or a neurodegenerative disease, the method including the step of transplanting the intestinal flora obtained from the fecal sample of a human patient suffering from a neuropsychiatric disease and / or a neurodegenerative disease into a mouse.

[0033] In one embodiment, the present invention provides a method for generating a mouse model exhibiting symptoms of a neuropsychiatric disorder and / or a neurodegenerative disorder, the method comprising transplanting an intestinal microbiota obtained from a fecal sample of a human patient suffering from a neuropsychiatric disorder and / or a neurodegenerative disorder into a mouse, wherein the neuropsychiatric disorder and / or the neurodegenerative disorder is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.

[0034] In one embodiment, the present invention provides a method for generating a mouse model exhibiting symptoms of Alzheimer's disease, the method comprising transplanting an intestinal microbiota obtained from a fecal sample of a human patient suffering from Alzheimer's disease into a mouse.

[0035] The present invention further provides a mouse model generated by the above-described method.

[0036] The present invention further provides the use of the above composition for the treatment and / or prevention of memory decline due to aging. Regarding the figures in particular, it is emphasized that the content shown is illustrative and is for the purpose of exemplary consideration of different embodiments of the present invention only. They are presented to provide what is considered to be the most useful and understandable explanation of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to show the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The description together with the drawings will make some forms of the present invention apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

FIG. 1A-1D

FIG. 2A-2C

FIG. 3A-3C

Mode for Carrying Out the Invention

[0038] Definitions To make the present invention more readily understandable, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout this specification. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology, pharmacology within the skill of the art. Such techniques are well explained in the literature.

[0039] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise specified or apparent from the context, as used herein, the term "or" is inclusive and is understood to mean "or" and "and" both.

[0040] As used herein, the term "and / or" is construed as a specific disclosure of each of the two specified features or components, whether or not it includes the other. Thus, the term "and / or" as used in expressions such as "A and / or B" is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in expressions such as "A, B, and / or C" is intended to include aspects of A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0041] As used herein, the terms "for example" and "i.e." are used merely by way of illustration and without intended limitation and should not be construed as referring only to the items expressly listed in the specification.

[0042] Terms such as "or more", "at least", "more", for example, "at least one", although not limited, are understood to include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or numbers greater than the specified value. Also included are any larger or smaller numbers in between. Conversely, the expression "~ or less" includes each value less than the recited value. For example, "100 or fewer compounds" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 compounds. Also included are any lesser number or fraction therebetween.

[0043] Terms such as "plurality", "at least two", "two or more", "at least the second", etc. are not limited, but are understood to include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Also included are any larger or smaller numbers in between.

[0044] Throughout this specification, variations such as "comprising", "comprises", or "comprised of" are to be understood to mean the inclusion of the specified element, integer, or step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, or steps, or group of elements, integers, or steps. When an aspect is described in the specification using the term "comprising", it is understood that similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" excludes elements, steps, or components not specified in the claims. The term "consisting essentially of" limits the scope of the claim to the specified materials or steps of the claimed invention, "and those that do not materially affect the basic novel characteristics".

[0045] Unless otherwise specified or apparent from the context, as used herein, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, the expressions "about" or "approximately" may mean within the range of one or more standard deviations, in accordance with the practice in the art. The expressions "about" or "approximately" may mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Further, especially with respect to biological systems or processes, these terms may mean up to one order of magnitude or up to five-fold the value. When a particular value or composition is indicated in the present invention, unless otherwise stated, the expressions "about" or "approximately" should be considered to be within the acceptable error range of that particular value or composition.

[0046] The term "substantially" does not exclude "completely". For example, a composition that is "substantially free of" Y may possibly be completely free of Y. If desired, the term "substantially" may be omitted from the definition of the invention.

[0047] As described herein, any concentration range, percentage range, ratio range, integer range is understood to include any integer value within the stated range, and, if necessary, fractions thereof (such as one-tenth or one-hundredth of an integer), unless otherwise specified.

[0048] The units, prefixes, and symbols used herein are provided in the form recognized in the International System of Units (SI). Numerical ranges include the numerical values defining the range.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Juo's "The Concise Dictionary of Biomedicine and Molecular Biology" 2nd ed (2001), CRC Press; "The Dictionary of Cell & Molecular Biology" 5th ed (2013), Academic Press, and "The Oxford Dictionary Of Biochemistry And Molecular Biology", Cammack et.al. eds., 2nd ed, (2006), Oxford University Press provide general dictionaries for many of the terms used in the present invention to those of ordinary skill in the art.

[0050] "Administering" refers to the physical introduction of an agent to a subject using any of a variety of methods and delivery systems well known to those of ordinary skill in the art. Exemplary routes of administration of the compositions disclosed herein include oral, rectal, intravenous, intramuscular, subcutaneous, intraperitoneal, or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a route of administration other than enteral and topical administration, and typically by injection, including, but not limited to, intravenous, intramuscular, arterial, intrathecal, intranodular, intralesional, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, the composition is administered by a non-injection route, such as orally. Other non-injection routes include topical, epidermal, or mucosal routes of administration, such as nasal, vaginal, rectal, sublingual, or topical administration. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0051] As used herein, terms such as "treatment", "treating", etc. refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or the adverse effects caused by the disease. The term "treatment" as used herein also encompasses any treatment of memory impairment or memory decline, or diseases, disorders, or conditions that cause memory impairment or memory decline, particularly in mammals including humans, and includes the following: (a) preventing the occurrence of a disease in a subject who has a predisposition to the disease or is at risk of developing the disease but has not yet been diagnosed with the disease; (b) suppressing the disease, i.e., arresting its progression; (c) alleviating the disease, i.e., causing the disease to regress. Preferred embodiments of "treatment" are further discussed below. In some embodiments, "treating" refers to administering a therapeutic agent to a patient who is suspected of having or already has memory impairment or memory decline. It also refers to reducing, eliminating, or at least partially suppressing one or more symptoms caused by the disease and / or the disease and / or its complications, and bringing about a beneficial effect. "Prevention" refers to administering to a patient who is susceptible to or at risk of a particular disease. Everyone in the general population is at risk of memory impairment or memory decline. For example, everyone is at risk of Alzheimer's disease. Some individuals have an increased genetic risk of memory impairment or memory decline (e.g., Alzheimer's disease). Prevention can eliminate or reduce the risk or delay the onset of the disease. The delay in onset or progression can be measured based on the standard time of disease progression in a similar population or individual.

[0052] The term "combination" refers to a fixed combination in one dosage unit form, or to the administration of the compounds of the invention and optionally a combination partner (e.g., another agent as described below, which may also be referred to as a "therapeutic agent" or "agent") either simultaneously or separately within a time interval, particularly where these time intervals enable the combination partner to exhibit a synergistic effect, e.g., a multiplicative effect, of any of the combination administrations. The single components may be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) may be reconstituted or diluted to the desired dosage prior to administration. Terms such as "co-administration" or "combined administration" as used herein are meant to encompass the administration of a selected combination to a single subject (e.g., a patient) in need thereof, and are intended to include treatment regimens where the agents need not necessarily be administered by the same route or simultaneously.

[0053] The terms "reducing" and "decreasing" are used herein with the same meaning and indicate any change to a value lower than the original value. "Reducing" and "decreasing" are relative terms and require a comparison of a pre-measured value and a post-measured value. "Reducing" and "decreasing" include complete depletion. Similarly, the term "increasing" indicates any change to a value higher than the original value. "Increasing", "higher" and "lower" are relative terms and require a comparison of a pre-measured value and a post-measured value, and / or a comparison between reference standards. In some embodiments, the reference value is obtained from the values of the general population, which may be a general population of patients. In some embodiments, the reference value is obtained from a quartile analysis of the general patient population.

[0054] The term "viable bacteria" means that when bacteria are cultured in an appropriate medium and conditions, the integrity of the cells is maintained and cell processes occur or can occur. Viable bacteria can be reseeded in an appropriate medium and grown under appropriate conditions. Viable bacteria may be preserved prior to administration, preferably by freezing with liquid nitrogen, slow freezing or lyophilization and subsequent storage at a temperature in the range of +4°C to -80°C.

[0055] The term "phylogenetically related" means that the strains have sequences that are at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical.

[0056] The term "neurodegenerative disease" means a disease caused by the progressive loss of the structure or function of neurons in a process known as neurodegeneration. Such nerve damage may ultimately be accompanied by cell death. Neurodegenerative diseases may include amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, and the like.

[0057] Luminococcus is a genus of bacteria in the class Clostridia. These are anaerobic Gram-positive intestinal bacteria. The strain "Luminococcus lactaris" has recently been reclassified as Mediterraneibacter lactaris, but these two classifications are equivalent in the context of the present invention.

[0058] The novel strains referred to in the present invention have been deposited in accordance with the provisions of the Budapest Treaty. The depositor and owner of the strains described and / or claimed in this patent application have agreed from the outset to make all of the above strains available for use throughout the entire term of the patent.

[0059] The bacteria of the present invention The present invention for the first time identifies the ability of certain bacteria, namely the Ruminococcaceae species or the Luminococcus lactaris strain, to be used as a drug for treating and / or preventing memory impairment associated with aging or Alzheimer's disease in an individual.

[0060] The inventors have unexpectedly identified that the Ruminococcaceae species and / or the Luminococcus lactaris strain described below have the ability to prevent and / or restore memory activity in the memory-related brain regions of the hippocampus in individuals suffering from memory impairment associated with aging or Alzheimer's disease. These new strains of the Ruminococcaceae species and Luminococcus lactaris are part of a strain library composed of fecal samples from young healthy donors.

[0061] The strain according to the present invention can prevent and / or restore memory impairment associated with aging or Alzheimer's disease and the activity level of the hippocampus, particularly in individuals suffering from memory impairment associated with aging or Alzheimer's disease. The above-mentioned memory ability and hippocampal activity may return to normal levels. This is because the memory ability observed after administration of the strain according to the present invention is similar to the memory ability observed in mice not suffering from memory impairment associated with aging or Alzheimer's disease.

[0062] The Ruminococcaceae species strain of the present invention The Ruminococcaceae species is a member of the Bacillota phylum. Bacteria of this family are commonly present in the digestive tract systems of many animals, including humans, and are involved in the breakdown of fiber and the synthesis of short-chain fatty acids (SCFAs) such as butyrate. The short-chain fatty acid butyrate is very important in the beneficial effects on intestinal physiology, whole-body function, and human health (Macfarlane and Macfarlane, 2011) and is involved in the production of microbial inhibitors.

[0063] The family Ruminococcaceae, particularly Butyrivibrio fibrisolvens, is known to have anti-inflammatory and protective effects in mouse models of acute and chronic colitis, i.e., in inflammatory diseases. Indeed, oral administration of live Butyrivibrio fibrisolvens MDT-1 increased the production rate of butyrate measured as its concentration in feces and alleviated the formation of aberrant crypt foci (Ohkawara et al., 2005) (a pre-stage of colorectal cancer) in dextran sulfate sodium-induced experimental colitis (Ohkawara et al., 2006), and delayed and decreased the occurrence of 3-methylcholanthrene-induced tumors in mice (Ohkawara et al., 2007). Another member of the family Ruminococcaceae, Butyrivibrio crossotus, is a butyrate-producing bacterium, and a decrease in its abundance has been shown to occur at the end-of-life of centenarians (Luan et al., 2020), suggesting its association with longevity. Also, a low abundance of other Ruminococcaceae species has recently been shown to be responsible for a higher probability of amyloid and p-tau positivity in the population of Alzheimer's disease patients (Verhaar et al., 2022). This showed a negative correlation between the presence of these bacteria and two Alzheimer's disease pathological markers.

[0064] In one aspect of the present invention, a novel bacterium of the family Ruminococcaceae is provided herein, and its representative culture has been deposited on March 2, 2022, under the CNCM accession number I-5830, at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, for use as a drug.

[0065] In certain embodiments, the Ruminococcaceae bacterial strain of the present invention is phylogenetically related to the Butyrivibrio proteoclasticus strain and the Butyrivibrio hungatei strain.

[0066] In another aspect, for use as a drug, a culture extract of the above strain is provided herein, and the culture extract is selected from the group consisting of a strain culture supernatant, cell debris, cell wall, and protein extract.

[0067] As disclosed in Example 1 below, the 16S rRNA gene sequence of the Ruminococcaceae species CNCM I-5830 has the sequence shown as SEQ ID NO: 1 in the Examples section.

[0068] In certain embodiments, the invention also provides a strain having a 16S rRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence (SEQ ID NO: 1) of the above strain for use as a drug.

[0069] The Luminococcus lactaris strain of the present invention Luminococcus lactaris is a member of the class Clostridia. Bacteria belonging to Clostridium Cluster IV (Ruminococcaceae) are numerically abundant in the human large intestine and typically account for 10-40% of the total bacterial 16S rRNA sequences (Lay et al., 2005). Some members of this family are known to be important species in the degradation of complex carbohydrates in the human large intestine, meaning that they produce secondary products that are used downstream by many other bacteria and can thus be utilized to modulate the overall gut microbiota composition. Also, some bacteria belonging to this family have been shown to assist in the recovery from diarrheal disease caused by Vibrio cholerae (Hsiao et al., 2014) and reduce the mortality rate due to graft-versus-host disease after allogeneic blood / bone marrow transplantation (Jenq et al., 2015). Additionally, it has recently been shown that the low prevalence of the member of another Ruminococcus genus, Ruminococcus torques species, is caused by the high probability of amyloid positivity in the Alzheimer's disease patient population (Verhaar et al., 2022). This indicates a negative correlation between the presence of this bacterium and this Alzheimer's disease pathological marker.

[0070] In another aspect of the present invention, a novel bacterium of the species Lactococcus lactis is provided herein, and a representative culture thereof has been deposited on March 2, 2022, under accession number CNCM I-5831, at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, for use as a drug.

[0071] In one aspect of the present invention, the Lactococcus lactis strain is phylogenetically related to Lactococcus lactis ATCC 29176. In another aspect, for use as a drug, a culture extract of the above strain is provided herein, and the above culture extract is selected from the group consisting of a strain culture supernatant, cell debris, cell wall, and protein extract.

[0072] As disclosed in Example 1 below, the 16S rRNA gene sequence of Lactococcus lactis CNCM I-5831 has the sequence shown in the Examples section as SEQ ID NO: 2.

[0073] In certain embodiments, the present invention also provides a strain having a 16S rRNA sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16S rRNA sequence of SEQ ID NO: 2 for use as a drug.

[0074] Other strains Other strains useful in the compositions and methods of the present invention, such as one or more derivatives of the strains deposited under accession numbers CNCM I-5830 and / or CNCM I-5831, can be identified using any suitable method or strategy.

[0075] Derivative strains of the present invention may be daughter strains (descendants) or strains cultured (subcloned) from the original strain. Derivative strains of the present invention may be modified, for example, at the genetic level, without losing biological activity and without removing biological activity. In particular, the derivative strains of the present invention have therapeutic activity. The derivative strain has a therapeutic activity equivalent to that of one or more of the strains deposited under accession numbers CNCM I-5830 and / or CNCM I-5831. Preferred is a strain for use in preventing and / or restoring memory impairment and hippocampal activity levels associated with aging or Alzheimer's disease, particularly in individuals suffering from memory impairment associated with aging or Alzheimer's disease.

[0076] The composition of the present invention In one embodiment, the present invention provides a composition comprising one or more strains of the Ruminococcaceae species and Luminococcus lactaris of the present invention. In other words, the present invention provides a composition comprising either a Ruminococcaceae species strain or a Luminococcus lactaris strain. The present invention also provides a composition comprising a Ruminococcaceae species strain and a Luminococcus lactaris strain. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier or excipient.

[0077] In a preferred embodiment, the composition according to the present invention comprises, optionally together with a pharmaceutically acceptable carrier or excipient, a novel bacterium of the Ruminococcaceae species deposited with the CNCM under accession number CNCM I-5830, or a bacterium of the Luminococcus lactaris strain deposited with the CNCM under accession number CNCM I-5831.

[0078] In a more preferred embodiment, the composition of the present invention comprises, optionally together with a pharmaceutically acceptable carrier or excipient, a novel bacterium of the Ruminococcaceae species strain deposited with the CNCM under accession number CNCM I-5830, and a bacterium of the Luminococcus lactaris strain deposited with the CNCM under accession number CNCM I-5831.

[0079] In certain embodiments, the composition of the present invention comprises a culture extract of the above strain, and the culture extract is selected from the group consisting of a strain culture supernatant, cell debris, cell wall, and protein extract.

[0080] In one embodiment, the composition according to the present invention targets the gastrointestinal tract, particularly the intestinal tract. Accordingly, the composition according to the present invention is selected from oral, rectal or parenteral compositions. The composition of the present invention is preferably an oral or rectal composition, more preferably an oral composition. Such compositions may be in the form of suspensions, tablets, pills, capsules, granules or powders.

[0081] Advantageously, the composition according to the present invention for oral administration can be provided with a coating agent resistant to gastric juice, thereby ensuring that the strain of the present invention contained in the composition can pass through the stomach without being damaged. Release of the strain can thus occur only in the colon.

[0082] In one embodiment, the composition of the present invention comprises a carrier or excipient, preferably a pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause harmful, allergic, or other undesirable reactions when administered to a mammal, particularly a human, if necessary. Pharmaceutically acceptable excipients refer to non-toxic solid, semi-solid, or liquid fillers, diluents, capsule encapsulating materials, or any type of formulation auxiliary material. Pharmaceutically acceptable carriers or excipients that can be used in the composition according to the present invention are well known to those skilled in the art and may vary depending on the disease to be treated and the route of administration. In embodiments of the present invention, the carrier results in improved bioavailability, stability and / or durability of the bacteria. In one embodiment, the carrier or excipient improves the bioavailability, stability and durability of the bacteria or their secondary metabolites.

[0083] The composition of the present invention may further contain prebiotics. Prebiotics can assist in the growth of probiotics before they become non-replicating. "Prebiotics" means non-digestible food substances that promote the growth of healthy and beneficial microorganisms and / or probiotics in the intestine. They are not decomposed in the stomach and / or upper intestine of the person who ingests them and are not absorbed in the digestive tract, but they are fermented by the gastrointestinal microbiota and / or probiotics. Preferably, these can be selected from the group consisting of oligosaccharides (optionally including fructose, galactose, mannose), dietary fiber (especially soluble fiber), soy fiber, inulin, or mixtures thereof. Preferred prebiotics are fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, arabinoxylooligosaccharides, manooligosaccharides, soy oligosaccharides, glycosyl sucrose, lactosucrose, lactulose, palatinose oligosaccharides, maltooligosaccharides, gums and / or their hydrolysates, pectin and / or their hydrolysates.

[0084] The composition of the present invention may be administered by any method suitable for deposition in the digestive tract of the subject being treated, preferably in the small intestine and / or colon. In particular, the composition can be administered by enteral or parenteral routes, preferably oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal routes. Preferably, the composition of the present invention is administered or adapted to be administered by rectal or oral routes.

[0085] In one embodiment, the composition is administered via the oral route. In the case of oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules, etc., and liquid preparations such as syrups, elixirs, concentrates, etc. A non-toxic solid carrier or diluent may be used, which includes, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. In the case of compressed tablets, a binder, which is a drug that imparts adhesiveness to the powder material, is also necessary. For example, saccharides such as starch, gelatin, lactose or dextrose, natural or synthetic rubbers, etc. can be used as binders. A disintegrant may also be necessary in the tablet if it is desired to make the tablet easily crushable. Disintegrants include starch, clay, cellulose, algin, rubber, cross-linked polymers, etc. Furthermore, lubricants and glidants may also be included in the tablet to prevent adhesion of the tablet material to the surface during the manufacturing process and to improve the flow characteristics of the powder material during production. Colloidal silicon dioxide is most commonly used as a lubricant, and compounds such as talc or stearic acid are most commonly used as lubricants. Thickeners such as corn starch, agar, natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, guar, xanthan, etc. may also be added to the composition. Preservatives such as methyl paraben, propyl paraben, benzyl alcohol, ethylenediaminetetraacetate, etc. may also be included in the composition.

[0086] The composition formulated for oral administration may be a gastric-resistant oral dosage form that allows the active compound contained in the composition to pass through the stomach and be released into the intestine. Materials that can be used in enteric coating agents include, for example, alginic acid, cellulose acetate phthalate, plastics, waxes, shellac, and fatty acids (e.g., stearic acid or palmitic acid). The composition according to the present invention may be formulated to release the active ingredient substantially immediately after administration, or at any predetermined time or period. In one embodiment, the release of the strain may occur only in the upper intestinal tract for the first time.

[0087] In other embodiments, the composition may be a food composition or a dietary supplement. "Food composition" means any composition containing food ingredients such as macronutrients, micronutrients, vitamins and / or minerals. Food compositions are intended for consumption by humans or animals and may be liquid, paste or solid. Examples of food compositions include, but are not limited to, dairy products such as cheese, butter, cream, yogurt, fermented milk, ice cream, cooked products such as bread, biscuits and cakes, fruit products such as fruit juice, fruit compote, fruit paste, soy foods, starch-based foods, edible oil compositions, spreads, breakfast cereals, prepared milk powder for infants, food bars (such as cereal bars, breakfast bars, energy bars, nutritional bars, etc.), chewing gum, beverages, beverage supplements (powders added to beverages), and the like. As used herein, the term "dietary supplement" refers to a composition that is formulated and administered separately from other foods to supplement the nutrition of a subject, i.e., a human or animal. This dietary supplement may be in any suitable form well known to those skilled in the art, preferably in the form of a therapeutic diet or an oral nutritional supplement. The food compositions of the present invention may contain ingredients normally added to foods during manufacture, such as proteins, carbohydrates, fats, nutrients, seasonings, flavors. Examples of the above carbohydrates are monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; oligosaccharides; and polysaccharides such as dextrin, sugar alcohols such as conventional sugar and xylitol, sorbitol, erythritol such as cyclodextrin. Examples of flavors are natural flavors (thaumatin, stevia extract (such as rebaudioside A, glycyrrhizin, etc.) and / or synthetic flavors (saccharin, aspartame, etc.). For example, if the food composition of the present invention is a drink or beverage, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and other plant extracts, etc.

[0088] In another preferred embodiment, the composition is administered by the rectal route. Suitable rectal route forms include, but are not limited to, suppositories and enemas. In particular, the bacteria may be incorporated into any known suppository base by methods well known in the art. Examples of such bases include cocoa butter, polyethylene glycol (Carbowax), polyethylene sorbitan monostearate, and mixtures thereof with other compatible materials to improve the melting point or dissolution rate.

[0089] In one embodiment, the composition of the present invention comprises one or more additional therapeutic agents. In one embodiment, the therapeutic agent is used to treat or prevent a disease, disorder, or condition underlying memory impairment, but does not necessarily affect memory itself. In one embodiment, the disease, disorder, or condition underlying memory impairment is selected from Alzheimer's disease, aging, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, or attention deficit / hyperactivity disorder, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. In a preferred embodiment, the disease, disorder, or condition underlying memory impairment is selected from Alzheimer's disease, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, or attention deficit / hyperactivity disorder. In one embodiment, the composition further comprises a therapeutic agent used for symptomatic treatment of Alzheimer's disease, such as an acetylcholinesterase inhibitor and an N-methyl-D-aspartic acid (NMDA) receptor antagonist. In one embodiment, the composition further comprises a therapeutic agent used as a therapeutic agent based on the etiology of Alzheimer's disease, such as a secretase inhibitor, a microglia-directed therapeutic agent, an amyloid binder, an amyloid immunotherapeutic agent, and a tau therapeutic agent comprising an immunotherapeutic agent. In one embodiment, the composition further comprises a therapeutic agent used as a microbiota-based therapeutic agent for Alzheimer's disease, such as probiotics, prebiotics, oligosaccharides and polysaccharides, or transplanted fecal microbiota.

[0090] In some embodiments, the composition comprises a population of microorganisms. For example, in some embodiments, the composition comprises, as a member of the microbial population, a strain of the Ruminococcaceae species of the present invention and / or a strain of Luminococcus lactaris of the present invention. For example, in some embodiments, the strain of the Ruminococcaceae species of the present invention and / or the strain of Luminococcus lactaris is present in combination with one or more (e.g., at least 2, 3, 4, 5, 10, 15, or 20) other strains of other genera that can symbiotically exist in the intestinal endosome. For example, in some embodiments, the composition comprises a strain of the Ruminococcaceae species of the present invention and / or a strain of Luminococcus lactaris in combination with strains of different genera. In some embodiments, the population of microorganisms comprises a single organism, e.g., two or more strains obtained from a human GM / fecal sample. In some embodiments, the population of microorganisms is not found together in nature. For example, in some embodiments, the population of microorganisms comprises strains obtained from GM / fecal samples of at least two different organisms. In some embodiments, the two different organisms are of the same species, e.g., from two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal. In alternative embodiments, the composition of the present invention comprises 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer different bacterial species. In certain embodiments, the composition comprises 4 or fewer different bacterial species. In certain embodiments, the composition comprises 3 or fewer different bacterial species. In certain embodiments, the composition comprises 2 or fewer different bacterial species. In certain embodiments, the composition comprises a strain of the Ruminococcaceae species of the present invention and / or a strain of Luminococcus lactaris and no other bacterial species. In a preferred embodiment, the composition of the present invention comprises a single strain of the Ruminococcaceae species and / or the Luminococcus lactaris species. Such a composition may contain only minimal amounts or biologically irrelevant amounts of other strains or species. In some embodiments, the composition "substantially" does not contain other bacterial species, or "substantially does not contain" bacterial species of other species.In some embodiments where the composition of the present invention comprises two or more strains, different species or genera, the individual strains of different species or genera may be for separate, simultaneous or sequential administration. For example, the composition may comprise all of two or more strains, species, genera, or the strains, species, genera may be stored separately and administered separately, simultaneously, or sequentially. In some embodiments, two or more strains, species, genera are stored separately but are mixed together prior to use.

[0091] The composition for use according to the present invention may or may not require marketing approval. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the above strain is lyophilized. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the above strain is spray-dried. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the strain is lyophilized or spray-dried and is in a live state. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the strain is lyophilized or spray-dried and is viable. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the strain is lyophilized or spray-dried and can colonize the intestinal tract partially or completely. In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the strain is lyophilized or spray-dried, is viable, and can colonize the intestinal tract partially or completely.

[0092] Cultivation method The strains for use in the present invention can be cultured using standard microbiological techniques as detailed, for example, in "Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press"; "Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press", "Strobel (2009) Methods Mol Biol. 581: 247-61".

[0093] Therapeutic use One or more Ruminococcaceae species and / or Lactococcus lactis strains of the present invention may be for use, alone or in combination, in the treatment and / or prevention of neuropsychiatric diseases or disorders that cause memory impairment. In one embodiment, the Ruminococcaceae species and / or Lactococcus lactis strains of the present invention may be for use in the treatment or prevention of age-related memory impairment such as Alzheimer's disease or vascular dementia. In some embodiments, the memory impairment is due to dementia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, attention deficit / hyperactivity disorder, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. In some embodiments, the memory impairment is due to dementia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, attention deficit / hyperactivity disorder. In some embodiments, the memory impairment is short-term memory loss. In some embodiments, the memory impairment is long-term memory loss. In some embodiments, Alzheimer's disease is genetic. In some embodiments, the compositions of the present invention may be useful as nootropics (e.g., memory enhancers).

[0094] As used herein, the term "memory" refers to acquired information stored in and retrievable from the brain, or the ability to encode, store, register, access, and / or retrieve acquired information. In some embodiments, the terms "memory" and "memory ability" are used interchangeably in the present invention to refer to the ability to encode, store, register, access, and / or retrieve information. In some embodiments, memory may include short-term memory and / or long-term memory. In some embodiments, short-term memory may refer to the ability to hold a small amount of information in the brain for a short period of time (e.g., 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, etc.). In some embodiments, short-term memory can be formed rapidly and can persist for a relatively short period of time (e.g., 1 second or more, 1 minute or more, 1 hour or more, 1 day or more, etc.). In some embodiments, long-term memory may be the stage of the Atkinson-Shiffrin memory model in which useful knowledge can be retained indefinitely. In some embodiments, long-term memory is formed more slowly and can persist for a relatively long period of time (e.g., 1 day or more, 1 week or more, 1 month or more, 1 year or more, etc.) compared to short-term memory. In some embodiments, newly acquired information is initially stored in the brain in a vulnerable state and tends to be gradually forgotten by the subject. In the process of memory consolidation, the vulnerable state of the acquired information can be converted into a relatively stable state in the brain, and accordingly, the acquired information may be less likely to be forgotten by the subject. In some embodiments, the process of memory consolidation can occur naturally over time or along with the reacquisition of the same acquired information (or related information).

[0095] As used herein, the term "memory impairment" can refer to a condition in which a subject has abnormally difficultly encoding, storing, registering, accessing, and / or retrieving information that the subject has acquired. In one embodiment, the treatment or prevention of memory impairment can be measured as proposed by the models used in the examples. Clinical and animal studies have shown that explicit memory and spatial memory, the most vulnerable cognitive domains, are dependent on the hippocampus. Accordingly, methods for assessing the activity and / or neural function of a subject's hippocampus can be used to evaluate memory impairment. In some embodiments, memory impairment is evaluated by an object recognition task. In some embodiments, verbal short-term memory is tested by a digit span task in which an individual is exposed to various numbers of digits for various lengths of time and then asked to recall those digits some time later. In one embodiment, non-verbal short-term memory may be tested by various motor or spatial memory tasks such as a spatial information test. In these tasks, the subject is exposed to various motor tasks or spatial orientations and then asked to recall or reconstruct them later. Procedural memory is the memory underlying motor performance or skills. In humans, separating procedural memory from declarative memory is not a straightforward task. This is because humans can develop declarative memory strategies for motor performance. However, this type of memory can be tested by various tasks such as a mirror drawing test, a mirror reading task, a weight sampling task, rapid reading of repeated nonsense, resolution of random dot stereograms, etc. Additionally, various classical conditioning tasks such as blink reflex conditioning combined with applying the onset of sound or light to the eye with an air puff can be used to test procedural memory function. This type of task has many forms and variations, and they have been used for various disorders and conditions. There are many other tests related to human procedural memory, such as those used to test the classification ability included in procedural memory. Here, the subject is tested, among other things, on their ability to classify strings as grammatical or ungrammatical. Declarative memory can be tested by many types of tests, including fact recall, paired associate tests, retention tests over minutes to days or years, language learning and recall tasks, and many other such tests.The number and type of tests are very numerous and depend on whether the impairment is in the autobiographical (episodic) memory system or in the semantic (factual) memory system of the world.

[0096] In a preferred embodiment, the memory impairment is thus evaluated by one or more of an object recognition task, a digit span task, a motor or spatial memory task (such as a spatial information test), a mirror drawing test, a mirror writing reading task, a weight sampling task, rapid reading of repeated nonsense, resolution of random dot stereograms; a classical conditioning task (e.g., eyelid reflex conditioning); the ability to classify strings as grammatical or ungrammatical; recall of facts; various types of paired-associate tests; retention tests over periods from minutes to days or years; language learning and recall tasks, and combinations thereof.

[0097] In one embodiment, the subject is identified for treatment based on a diagnosis of Alzheimer's disease or risk of Alzheimer's disease. In another embodiment, the subject is identified for treatment based on age. In another embodiment, the subject is identified based on having symptoms of an impairment of memory ability (evaluated in one embodiment by the method of the preceding paragraph) together with other neuropsychological test results, magnetoencephalogram (MEG) test results, brain image evaluations (e.g., computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET)) results, etc., or any combination thereof.

[0098] The bacteria or bacterial composition may be administered one or two times, over a long period, in a continuous mode for a certain period, or intermittently, with interruptions or in cycles. Combinations of bacteria may be administered simultaneously (e.g., as part of the same composition) or separately, e.g., sequentially. Usually, the bacteria or bacterial composition is administered in an effective amount, over an appropriate period, such as an amount sufficient to colonize the digestive tract of the subject. In one embodiment, the effective amount includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an effective amount, in terms of dosage and period, necessary to achieve a desired therapeutic result, such as reduction of memory impairment as evaluated in the preceding section. The therapeutically effective amount of the (bacterial) composition can vary depending on factors such as the disease state, age, sex, weight of the subject, and the ability of the (bacterial) composition to elicit a desired response in the individual. The dosing schedule can be adjusted to provide an optimal therapeutic response. A "prophylactically effective amount" refers to an effective amount, in terms of dosage and period, necessary to achieve a desired prophylactic result. Generally, prophylactic dosages are used for subjects in the early or pre - symptomatic stages of a disease or disease symptom, and thereby the prophylactically effective amount may be less than the therapeutically effective amount. In some embodiments, the composition is administered to a subject at risk of memory impairment. A "subject at risk" is a subject who is more likely to develop memory impairment compared to the general population.

[0099] The appropriate range of the therapeutically or prophylactically effective amount of the bacteria or bacterial composition, or the probiotic amount, is determined by those skilled in the art as described herein and is not limited, but per unit dosage, at least, or about 10 0 、10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、or 10 14 colony - forming units (CFU) of bacteria, particularly 10 per unit dosage2 ~10 10 contain bacteria of CFU (the amount of drug administered to a patient once). In some embodiments, the dosage of viable bacteria, whether in vegetative or spore form, is from about 0.1 to about 1000 mg per single dose or per composition, such as from about 0.5 mg to about 5 mg, from about 1 mg to about 1000 mg, from about 2 mg to about 200 mg, from about 2 mg to about 100 mg, from about 2 mg to about 50 mg, from about 4 mg to about 25 mg, from about 5 mg to about 20 mg, from about 10 mg to about 15 mg, from about 50 mg to about 200 mg, from about 200 mg to about 1000 mg, or more than about 1, 2, 3, 4, 5 or 5 g; or from 0.001 mg to 1 mg, 0.5 mg to 5 mg, 1 mg to 1000 mg, 2 mg to 200 mg, or 2 mg to 100 mg, or 2 mg to 50 mg, or 4 mg to 25 mg, or 5 mg to 20 mg, or 10 mg to 15 mg, or 50 mg to 200 mg, or 200 mg to 1000 mg, or more than about 1, 2, 3, 4, 5 or 5 g per single dose or per composition. The dosage value can vary depending on the severity of the condition being improved. For any particular subject, a specific dosing schedule may be adjusted over a period of time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition. For example, a single bolus may be administered, doses divided into multiple administrations may be given over a period of time, or the dosage may be proportionally reduced or increased according to the urgency of the situation. The bacteria or composition of the present invention may be administered daily or more frequently, such as more than once a day. "Probiotics" refers to live microorganisms that, when ingested in appropriate amounts, confer a health benefit on the host. These microorganisms are often similar to the microorganisms that normally inhabit the human intestine, but may be found in only very small amounts (e.g., as evident from intestinal dysbiosis), or they may need to process prebiotics or other substrates to promote the growth of other microorganisms.

[0100] In one embodiment, the appropriate daily dosage of the strain according to the present invention is 10 7 ~10 12 viable cells / ml (vc / ml) as the drug, more preferably 10 9 ~10 10vc / ml, for example, 10 as a daily dose 9 is equivalent to vc / ml.

[0101] In one embodiment, the strain of the present invention is a live biological preparation (LBP) whose activity is in the intestine. The LBP bacteria according to the present invention means bacteria that can be ingested alive in an appropriate amount and bring beneficial effects to human health. In a preferred embodiment, these strains are administered alive to the intestine. The strains of the present invention may be administered to the intestine of an individual to be treated by different methods, i.e., by oral, rectal, or parenteral routes. The bacteria according to the present invention are preferably administered by the oral or rectal route, more preferably by the oral route.

[0102] In one embodiment, the strain of the present invention may be used as a feed additive. The feed additive may be administered alone in an edible carrier or in combination with other feed additives. Further, the feed additive may be directly mixed with animal feed and used in animals as a coating agent or as another oral preparation. When the feed additive is administered separately from the animal feed, it can be combined with an acceptable edible carrier known in the art and can be immediately prepared as an immediate release or sustained release formulation. Such edible carriers can be solid or liquid, such as corn starch, lactose, sucrose, peanut oil, olive oil, sesame oil, and propylene glycol. The feed or feed additive of the present invention can be administered to various animals including mammals (e.g., pet dogs), poultry, and fish.

[0103] In one embodiment, the strain of the present invention is used in combination with other therapeutic agents for treating a disease or disorder underlying memory impairment or for treating other diseases. In some embodiments, the strains of the present invention are used simultaneously. In some embodiments, the strains of the present invention and other therapeutic agents are used sequentially (before and / or after). In some embodiments, one or more of the therapeutic agents are added to the composition comprising the bacterium of the present invention. In some embodiments, the bacterium of the present invention is used in combination with other bacteria. They may be used as separate compositions in combination or used in the same composition. As used herein, "simultaneously" means that the therapeutic agents and compositions of the present invention are administered at the same time or as part of the same treatment plan or have been administered. "Sequentially" means that the doses of the therapeutic agents and compositions of the present invention are administered at the same time or as part of the same treatment plan or have been administered, and "separately" means that all doses of the therapeutic agents and compositions of the present invention are administered or have been administered in sequence as part of the same treatment plan. In the case of these embodiments, "treatment plan" refers to the prescription of a treatment program that includes the administration of both the therapeutic agents and compositions of the present invention to a subject, i.e., a physician actively prescribes both the therapeutic agents and compositions of the present invention simultaneously for the treatment or prevention of memory impairment or a disease, disorder, or condition causing memory impairment.

[0104] In one embodiment, the bacterium of the present invention is administered together with other therapeutic agents used for the treatment of a disease underlying memory impairment. In one embodiment, the therapeutic agent is used in the prevention or treatment of Alzheimer's disease. In one embodiment, the bacterium of the present invention is administered together with other therapeutic agents used to "slow down" aging. In one embodiment, such treatment is calorie restriction. Accordingly, the present invention encompasses the use of the compositions of the present invention for the treatment or prevention of Alzheimer's disease or memory impairment associated with aging.

[0105] In one embodiment, pharmacological treatment for Alzheimer's disease can be divided into two categories: symptomatic therapies such as acetylcholinesterase inhibitors and N-methyl-D-aspartic acid (NMDA) receptor antagonists, and etiology-based treatments such as secretase inhibitors, amyloid binders, and tau therapies. In one embodiment, strategies for the prevention of Alzheimer's disease by non-pharmacological therapies are brought about by lifestyle interventions such as exercise, mental challenges, and social interactions as well as calorie restriction and a healthy diet. In one embodiment, the bacteria of the present invention are used in combination with any of the listed examples of treatment methods for Alzheimer's disease. In some embodiments, the therapeutic agent and the bacteria are in the same composition.

[0106] Treatment method The present invention also relates to a method for treating and / or preventing memory impairment in a subject in need thereof by administering to the subject a composition comprising one or more strains of bacteria selected from the genus Ruminococcaceae and the strain Luminococcus lactaris.

[0107] In one embodiment, the memory impairment is caused by aging. In one embodiment, the memory impairment is caused by a neuropsychiatric and / or neurodegenerative disease, disorder, or condition. Preferably, the neuropsychiatric and / or neurodegenerative disease, disorder, or condition is selected from Alzheimer's disease, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, attention deficit / hyperactivity disorder, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. More preferably, the neuropsychiatric and / or neurodegenerative disease, disorder, or condition is selected from Alzheimer's disease, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive dysfunction, or attention deficit / hyperactivity disorder.

[0108] The present invention also provides a method for treating or preventing Alzheimer's disease in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Lactospiraceae and Lactococcus lactis strains, preferably one or more bacteria selected from Lactococcus lactis strains.

[0109] The present invention also provides a method for treating or preventing Alzheimer's disease at different disease stages in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Lactospiraceae and Luminococcus lactis strains, preferably one or more bacteria selected from Luminococcus lactis strains.

[0110] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the preclinical stage in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Lactospiraceae and Luminococcus lactis strains, preferably one or more bacteria selected from Luminococcus lactis strains.

[0111] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the mild cognitive impairment stage in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Lactospiraceae and Luminococcus lactis strains, preferably one or more bacteria selected from Luminococcus lactis strains.

[0112] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at the mild dementia stage in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Lactospiraceae and Luminococcus lactis strains, preferably one or more bacteria selected from Luminococcus lactis strains.

[0113] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at a moderate dementia stage in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably one or more bacteria selected from the strain Lactococcus lactis.

[0114] In one embodiment, the present invention also provides a method for treating or preventing Alzheimer's disease at a severe dementia stage in a subject in need thereof by administering to the subject a composition comprising one or more bacteria of a strain selected from the genus Ruminococcaceae and the strain Lactococcus lactis, preferably one or more bacteria selected from the strain Lactococcus lactis. Preferably, the subject is a warm-blooded animal, more preferably a human.

[0115] In one embodiment, the bacteria are each one of the strains deposited with the CNCM under accession numbers CNCM I-5830 and CNCM I-5831. In one embodiment, the bacteria are included in a physiologically acceptable composition. In one embodiment, the composition further comprises prebiotics. In one embodiment, the composition further contains a therapeutic agent. In one embodiment, the composition further contains another compound, whereby the composition is formulated for use as a food.

[0116] The mouse model of the present invention In a further aspect, the present invention provides a novel mouse model referred to as a "human Alzheimer's disease-like" or "human Alzheimer's disease model". This mouse receives the gut microbiota of a patient suffering from human Alzheimer's disease. This is obtained by transplanting the gut microbiota obtained from fecal samples of Alzheimer's disease patients according to the protocol described in Rei et al, 2021 in RjOrl:SWISS mice, preferably male RjOrl:SWISS mice.

[0117] Prior to FMT, the mice are preferably treated with a protocol of intestinal lavage consisting of administering the broad-spectrum antibiotics used in Rei et al, 2021 or preferably oral administration of a laxative solution.

[0118] In one embodiment, the laxative solution contains polyethylene glycol. In one embodiment, the laxative solution contains 50-90% by weight of polyethylene glycol. In a preferred embodiment, the laxative solution contains 60-80% by weight of polyethylene glycol.

[0119] In one embodiment, the polyethylene glycol is PEG3350. In one embodiment, the laxative solution is the COLOPEG solution, especially the COLOPEG solution macrogol 3350, sold in particular by LABORATOIRES BOUCHARA-RECORDATI.

[0120] In one embodiment, the laxative solution is administered to the mice by a forced feeding cannula. In one embodiment, the laxative solution is preferably administered at least 2 times, more preferably at least 3 times, and even more preferably 5 times. In one embodiment, the laxative solution is preferably administered 2 times at intervals of at least 30 minutes, more preferably 3 times at intervals of at least 30 minutes, and even more preferably 5 times at intervals of at least 30 minutes. In one embodiment, the laxative solution is administered in an amount of 100-500 μl per administration, preferably 200-300 μl per administration. In one embodiment, before administration of the laxative solution, the mice are fasted for at least 1 hour, preferably about 2 hours.

[0121] In one embodiment, fecal samples from AD-afflicted patients are suspended in an aqueous solution before injection into the mice. In one embodiment, the feces are diluted in an aqueous solution at a volume ratio of 1 / 40 to 1 / 10, preferably about 1 / 20 by volume in the aqueous solution. In one embodiment, the aqueous solution of feces is administered to the mice by a gavage cannula. In one embodiment, the aqueous solution of feces is administered in an amount of 100 - 500 μl, preferably 150 - 300 μl, per administration. In one embodiment, the aqueous solution of feces is preferably administered at least 2 times, more preferably at least 3 times. In one embodiment, the aqueous solution of feces is preferably administered at least 2 times at one-day intervals, more preferably at least 3 times at one-day intervals.

[0122] The animals of the mouse model thus obtained experience memory loss typical of the symptoms of Alzheimer's disease about 28 days after FMT. As shown in Example 2 below, the animals show memory impairment in the isotropic version novel object location (ISO-NOL) task, the isotropic version novel object recognition (ISO-NOR) task, and the fear conditioning task, and show down-regulation of synaptic plasticity genes and up-regulation of neuroinflammation-related markers.

[0123] The present invention thus further provides a method for generating a mouse model showing symptoms of a neuropsychiatric disease and / or a neurodegenerative disease. The above method includes the step of transplanting the gut microbiota obtained from fecal samples of human patients suffering from a neuropsychiatric disease and / or a neurodegenerative disease into the mice.

[0124] In one embodiment, the above neuropsychiatric disease and / or neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis. The present invention thus further provides a method for generating a mouse model exhibiting symptoms of Alzheimer's disease, said method comprising the step of transplanting the gut microbiota obtained from a fecal sample of a human patient suffering from Alzheimer's disease into a mouse. In one embodiment, the method of the invention further comprises, prior to the step of transplanting the gut microbiota obtained from a fecal sample of a human Alzheimer's patient, the step of performing gut lavage on the mouse, preferably by administration of a broad-spectrum antibiotic or oral administration of a laxative solution.

[0125] The present invention thus also further provides a mouse model produced by the method described above.

[0126] Examples Example 1: Isolation and culture of strains from fecal samples of healthy young donors, and phylogenetic characterization of the CNCM I-5830 strain of the family Ruminococcaceae and the Luminococcus lactaris I-5831 strain

[0127] To obtain strains to be tested for their anti-Alzheimer's disease and anti-aging effects, a method for species-targeted isolation and culture of gut microbiota strains from fecal samples of healthy young human donors was used under anaerobic conditions using flow cytometry. The antibodies used targeted Faecalibacterium praunitzii (ATCC 27766 and ATCC 27768) and Roseburia intestinalis (DSM 14610) strains. This method can separate not only the strains of the targeted species but also the strains of other species, and thus it was used as a means to construct a collection of strains from young adult donors to be tested for their anti-Alzheimer's disease / anti-aging potential. This resulted in a collection of a large number of individual strains belonging to various families. The culturable candidate strains were then cultured individually, and 16S sequencing of their extracted DNA enabled their phylogenetic identification.

[0128] (A) Species-targeted isolation of gut microbiota strains from fecal samples of healthy young donors Fecal samples were collected from healthy young donors (18 - 40 years old) without pathological diseases or drug prescriptions and without antibiotic use in the three months prior to collection. Table 1 shows the age and gender of the donors used for fecal sample collection.

Table 1

[0129] This isolation resulted in a collection of numerous strains. Some were Faecalibacterium praunitzii and Roseburia intestinalis strains, while the rest belonged to other families. This was probably due to the nature of the non-exclusive epitopes recognized by the antibodies and some non-specific binding. Table 2 shows the donor and flow cytometry information regarding the isolation of strains 1006-B-1-182_18-4, 1006-B-1-182_21-1 (CNCM I-5831), 1006-M-1-013_15-5 (CNCM I-5830).

Table 2

[0130] (B) DNA extraction and cryopreservation of Faecalicatena contorta 1006 - B - 1 - 182_18 - 4, Ruminococcaceae sp. CNCM I - 5830, and Luminococcus lactaris strain CNCM I - 5831 DNA extraction and cryopreservation of bacteria were performed on strains 1006 - B - 1 - 182_18 - 4, 1006 - M - 1 - 013_15 - 5 (CNCM I - 5830), and 1006 - B - 1 - 182_21 - 1 (CNCM I - 5831) to enable their subsequent 16S DNA sequencing and culturing, respectively.

[0131] Colonies of the selected strains 1006 - B - 1 - 182_18 - 4, 1006 - M - 1 - 013_15 - 5 (CNCM I - 5830), and 1006 - B - 1 - 182_21 - 1 (CNCM I - 5831) were visually selected on mGAM - CRI selection plates and sub - cultured on mGAM plates. DNA samples were collected with 2 - 4 strain colonies in Instagen matrix (Biorad) and extracted from isolated colonies of different strains according to the supplier's "DNA preparation for PCR" instructions. Similar numbers of colonies were collected in a small amount of 16% glycerol in PBS and stored at - 80 °C for cryopreservation of the strains and their subsequent cultures.

[0132] Strains 013 - HF_15 - 5 and 182 - OH_21 - 1 were respectively deposited at the "Collection Nationale de Cultures de Micro - organismes" (CNCM) microbial bank under CNCM I - 5830 and CNCM I - 5831. Table 2 shows the correspondence between flow cytometry - related information and the CNCM identification codes of CNCM I - 5830 strain and CNCM I - 5831 strain.

[0133] (C) Phylogenetic Characterization of the Ruminococcaceae Species CNCM I-5830 Strain and Luminococcus lactaris CNCM I-5831 Strain The 16S rRNA sequences of the Ruminococcaceae species and Luminococcus lactaris were analyzed.

[0134] Using DNA samples of Faecalicatena contorta 1006-B-1-182_18-4, Ruminococcaceae species CNCM I-5830, and Luminococcus lactaris CNCM I-5831 strain, PCR amplification of the 16S rRNA gene was performed using the 27F-YM and 1391R primer sets. The obtained products were sent to Eurofins (Cologne, Germany) for sequencing.

[0135] From the sequencing results, pairwise sequence similarities were calculated for the 16S rRNA genes available via the GGDC web server using the method recommended by Meier-Kolthoff et al. (2013). The phylogenetic tree was inferred using the DSMZ phylogenetic analysis pipeline adapted for single genes (Meier-Kolthoff et al. 2014) at the GGDC web server available at https: / / urldefense.com / v3 / __http: / / ggdc.dsmz.de / __;!!JFdNOqOXpB6UZW0!-aXbUPgCyD4K36hsYEddOSdpKKIRdOy_uYRXwty43D1t06alsnxh1HrZbIc3mvcMKQ$ by Meier-Kolthoff et al. (2022).

[0136] Multiple sequence alignments were created using MUSCLE (Edgar 2004, Nucleic Acids Res 32:1792-1797). Maximum likelihood (ML) and maximum parsimony (MP) trees were inferred from the alignments using RaxML (Stamatakis 2014, Bioinformatics 30:1312-1313) and TNT (Goloboff et al. 2008, Cladistics 24:774-786), respectively. For ML, fast bootstrap with the autoMRE bootstrap criterion (Pattengale et al. 2010, J Comput Biol 17:337-354) and subsequent search for the best tree were used. For MP, 1000 bootstrap replicates were used in combination with tree bisection-reconnection branch swapping and 10 random sequence addition replicates. Sequences were checked for compositional bias using the χ 2 test implemented in PAUP* (Swofford 2002, version 4.0 b10. Sinauer Associates, Sunderland).

[0137] Full-length 16S rRNA sequencing of the isolated strains was determined.

[0138] The DSMZ phylogenetic pipeline analysis of the 16S rRNA gene sequence of strain 182-0H_18-4 enabled its classification as Faecalicatena contorta (data not shown). A similar analysis of the 16S rRNA gene sequence of strain 013-HF_15-5 CNCM I-5830 (SEQ ID NO: 1) showed that it was phylogenetically closest to a group of uncharacterized strains that are a new strain (Figure 1A). The closest known strains are Butyrivibrio proteoclasticus and Butyrivibrio hungatei. It was thus named based on the closest known higher phylogeny, i.e., the Ruminococcaceae. This strain was thus classified as a Ruminococcaceae species. The results of a nucleotide BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) against the 013-HF_15-5 CNCM I-5830 16S rRNA gene sequence showed that all 10 bacterial species most closely related to the 16S rRNA gene sequence of the CNCM I-5830 strain were uncharacterized strains (Figure 1B).

[0139] For the 16S rRNA gene sequence of the 182-OH_21-1 CNCM I-5831 strain (SEQ ID NO: 2), the DSMZ phylogenetic analysis showed that it was a novel strain belonging to the phylogenetic branch of the Ruminococcus lactaris strain (Figure 1C). The nucleotide BLAST analysis of the 16S rRNA gene sequence of this strain showed that it was different from other members of the genus and had the closest sequence similarity of 99.01% with the Ruminococcus lactaris strain ATCC 29176 (Figure 1D).

[0140] SEQ ID NO: 1: "013-HF_15-5": Partial sequence (1055 bp) of the 16S rRNA gene of the Ruminococcaceae species CNCM I-5830

[0141] Accession number 2: "182 - OH_21 - 1 strain": Partial 16S rRNA gene sequence (1238 bp) of Lactococcus lactaris CNCM I - 5831

[0142] Example 2: Effect of Ruminococcaceae species CNCM I-5830 or Luminococcus lactaris CNCM I-5831 on memory ability in mouse models of Alzheimer's disease and aging The Faecalicatena contorta, Ruminococcaceae species, and Luminococcus lactaris strains according to the present invention were tested for their ability to directly affect Alzheimer's disease-related memory impairment in Alzheimer's disease model mice.

[0143] (A) Mice transplanted with GM from Alzheimer's disease-afflicted human donors (human Alzheimer's disease-like mice) Mice were transplanted with GM from either young human controls or Alzheimer's disease D-afflicted patients. Recruitment of Alzheimer's disease-afflicted donors was carried out at the Sainte Perine Hospital (APHP, Paris, France). The selection criteria included being diagnosed with Alzheimer's disease based on clinical examinations and having a score of 24 or less on the Mini-Mental State Examination (MMSE). The exclusion criteria included the absence of other forms of dementia (especially vascular dementia), not having used antibiotics during the month prior to collection, and the absence of any pathological conditions and / or drug prescriptions. Fecal samples collected from young individuals (18 to 35 years old) were selected from the Diagmicoll and CoSImmGEn cohorts of healthy volunteers at the Institut Pasteur (IP) ICAReB platform. GM from both young and Alzheimer's disease patient donors was prepared according to the protocol described in (Rei et al., 2021). Collection was carried out at the Sainte Perine Hospital for Alzheimer's disease-afflicted subjects and at home for young donor individuals.

[0144] Experiments were performed using adult (10 - 12 weeks old) male RjOrl:SWISS mice purchased from Janvier labs (St Berthevin, France). Animals before human FMT were treated according to the intestinal lavage protocol and then underwent FMT according to the protocol of Rei et al. (2021).

[0145] Strain treatment (described below) to evaluate the effect on memory ability in a human GM model of Alzheimer's disease was initiated on day 28 after FMT. This timing is when mice have previously been shown to exhibit the phenotype of memory loss in Alzheimer's disease, with associated markers such as memory impairment in the isotropic version of the novel object location (ISO - NOL) task, the isotropic version of the novel object recognition (ISO - NOR) task, and the fear conditioning task, as well as down - regulation of synaptic plasticity genes and up - regulation of neuroinflammation - related markers being shown.

[0146] (B) Growth conditions of the strain Subsequent strain cultures of Faecalicatena contorta, Ruminospira species, and isolates of Lachnococcus lactaris were performed using a reducing sterile liquid medium within an anaerobic chamber. Initial bacterial inoculation for the cultures was from the cryoprotected 16% glycerol bacterial stock described in Example 1B. TGV medium was used for bacterial culture and it was composed of 3% tryptone peptone, 2% yeast extract (BD Difco), 1% D(+) glucose, 2 mg / l resazurin sodium salt, 0.5 mg / l menadione, 1 mg / l thiamine hydrochloride, 1 mg / l nicotinic acid, 0.5 mg / l riboflavin, 0.1 mg / l p-aminobenzoic acid, 1 mg / l calcium pantothenate, 0.5 ml / l vitamin B12 from a 10 ug / ml solution in ultrapure water, 2.5 ml / l biotin solution from a 0.2 mg / 100 ml solution in ultrapure water (Sigma), 0.5 mg / l pyridoxamine dihydrochloride, 0.05% L-cysteine hydrochloride monohydrate, 0.05 mg / l folic acid (Merck) supplemented with a 2.5% hemin solution made from 2 mg / l hemin chloride, 21.76 mg / l potassium dihydrogen phosphate (Merck), 67.2 mg / l disodium hydrogen phosphate (VWR) in ultrapure water. When the bacterial culture reached the stationary phase, the bacteria were centrifuged at 6000 g for 5 minutes, resuspended in sterile PBS, centrifuged again and aliquoted into 16% glycerol in sterile PBS. The vehicle solution consisted of 16% glycerol in PBS. Samples were stored at -80 °C until use.

[0147] (C) Treatment of "human Alzheimer's disease-like mice" with the purified strain Mice previously established as human Alzheimer's disease-like mice (see above) were gavaged daily with the strain at a volume of 300 μl per animal at 1 - 5 x 10 9Received either the dose of live cells / ml or vehicle treatment for 2 weeks (Figure 2A). Strains or vehicle samples (previously stored at -80 °C) were thawed in a 37 °C water bath for 3 minutes, and mouse treatment was performed by enteral nutrition using a flexible feeding tube (FTP-18-38, Instech Laboratories, USA). Control mice consisted of animals transplanted with young human FM. The FMT procedure was the same as the process described above.

[0148] (D) Effect of treatment on memory ability evaluated in the fear conditioning task model The memory ability (learning and memory impairment) of treated mice was determined in the contextual fear conditioning task model according to the protocol of Rei, D. et al., 2015. Briefly, on the 12th day of strain treatment, mice were individually placed in the fear apparatus for 3 minutes, and then a procedure consisting of three electric shocks (0.8 mA, 2 seconds, shock interval 28 seconds) delivered through the grid floor of the fear conditioning activity area was initiated. The animals were returned to their home cages after 15 seconds. The next day, the mice were returned to the fear conditioning activity area for 3 minutes, and the duration of "freezing", defined as no movement other than breathing, was measured by an experimenter blinded to the treatment.

[0149] Vehicle-treated human Alzheimer's disease-like mice (veh., huAD) showed memory deterioration compared to young human FM-transplanted animals of the control vehicle (veh., huY). On the other hand, human Alzheimer's disease-like mice treated with the Ruminococcaceae species strain CNCM I-5830 (CNCM I-5830, huAD) or the Lactococcus lactis strain CNCM I-5831 (CNCM I-5831, huAD) showed complete recovery of their memory impairment in the fear conditioning task compared to vehicle huY and vehicle huAD mice (Figure 2B). Interestingly, the Faecalibacterium prausnitzii reference strain A2-165 (F.prau.A2-165, huAD), despite its known anti-inflammatory effects (Bellais et al., 2020), its protective effects in intestinal bowel disorder (IBD) (Martin et al, 2014) and its recent involvement in the etiology of Alzheimer's disease (Ueda et al., 2021), the Roseburia intestinalis strain DSM14610 (R.intest., huAD), particularly through the secretion of butyric acid (Duncan et al 2004), its known anti-inflammatory effects (Shen et al., 2018), its protective effect in IBD (inflammatory bowel disease) (Luo W. et al, 2019), and its decreased presence in Alzheimer's disease patients (Zhuang et al., 2018), and the Faecalicatena contorta 18-4 (F.contorta 18-4, huAD), despite being the highest acetic acid SCFA producer in our library of strains isolated from young human donors (data not shown), these other strains did not show improvement in the fear conditioning memory impairment of the treated animals compared to vehicle human Alzheimer's disease-like (veh., huAD) animals when administered as a therapeutic to human Alzheimer's disease-like mice (Figure 2B).These results demonstrate the ability of the Ruminococcaceae species strain CNCM I-5830 and the Luminococcus lactaris strain CNCM I-5831 to fully restore memory in an Alzheimer's disease model, as well as the specificity of this effect.

[0150] (E) Effect of treatment on memory ability evaluated in the novel object exposure assay The previously obtained results regarding the anti-Alzheimer's disease and memory-promoting effects of the Ruminococcaceae species and Luminococcus lactaris strains of the present invention have been confirmed at the hippocampal neural circuitry level. Indeed, the measurement of the hippocampal ability to respond to a novel object can be considered a surrogate indicator of the tonus of neural activity related to hippocampal memory (Rei et al., 2021; Takeuchi et al., 2016). This evaluation is performed by measuring the increase in the number of hippocampal neurons positive for the immediate early gene c-fos after the exposure of animals to a novel situation. Our previous characterization of a human Alzheimer's disease-like model has shown that it is caused by a disorder in the hippocampal ability to respond to novel object exposure. Following the novel object exposure of strain-treated human Alzheimer's disease-like model mice, it was used herein as an additional method to evaluate the anti-Alzheimer's disease / memory-promoting effect of the strains of the present invention.

[0151] The novel object exposure assay was performed on the 14th day of strain treatment (Figure 2A) according to the experimental procedure described in Rei, D., et al., 2021. Briefly, animals were exposed to a new situation (an activity area with a rough plastic floor) for 3 minutes and then returned to their home cages. Ninety minutes after this novel object exposure, corresponding to the peak of c-fos expression after the behavioral stimulus, the mice were perfused intracardially with paraformaldehyde, their brains were harvested, and the number of c-fos positive cells in the CA1 subregion of the hippocampus was quantified.

[0152] In animals transplanted with FM from young human donors and treated with vehicle, exposure to a novel situation (veh., huY, nov.expo.: +) led to an increase in the number of c-fos positive neurons in the hippocampal CA1 compared to controls not exposed to the novel situation (veh., huY, nov.expo.: -). On the other hand, this response to novelty was lost in vehicle-treated human Alzheimer's disease-like mice (veh., huAD, nov.expo.: +) compared to vehicle HuY animals. Furthermore, treatment of human Alzheimer's disease-like mice with either the F. praunitzii A2-165 strain (F.prau A2-165, huAD, nov.expo.: +) or Faecalicatena contorta (F.contorta 18-4, huAD, nov.expo.: +) resulted in a moderate improvement or no improvement, respectively, in the number of c-fos activated neurons induced by novel object exposure when compared to vehicle-treated human Alzheimer's disease-like mice. In contrast, treatment with the Ruminococcaceae species strain CNCM I-5830 (CNCM I-5830, huAD, nov.expo.: +) or the Lactococcus lactis strain CNCM I-5831 (CNCM I-5831, huAD, nov.expo.: +) completely restored the ability of the hippocampus to respond to novelty when compared to vehicle huY (veh., huY, nov.expo.: - / +) and human Alzheimer's disease-like (veh., huAD, nov.expo.: +) animals (Figure 2C).

[0153] In conclusion, it is clear that the Ruminococcaceae species and Lactococcus lactis according to the present invention have the ability to completely restore memory in a model of Alzheimer's disease.

[0154] (F) Effects of the Ruminococcaceae species CNCM I-5830 and Lactococcus lactis CNCM I-5831 on memory ability in an aged mouse model of aging (Figure 3) The aged mice were male RjOrl:SWISS mice, 18 - 20 months old, purchased from Janvier labs (St Berthevin, France). At this age, the animals have previously been shown to exhibit a complete impairment in age - related memory impairment in ISO - NOL, ISO - NOR, and fear conditioning tasks and in the induction of neuronal activation after exposure to novel situations in the novel object exposure assay (Rei et al., 2021). They also exhibit some changes in markers related to memory impairment, such as a decrease in hippocampal neurogenesis and signs of neuroinflammation. According to this model, it was used to measure the anti - aging / memory - promoting effects of the Ruminococcaceae strain CNCM I - 5830 and Luminococcus lactaris CNCM I - 5831 of the present invention. The control young animals were similar to the mice (10 - 12 weeks old) used as controls for human Alzheimer's disease - like mice.

[0155] The isolation and growth conditions of the strains, the strain treatment, as well as the fear conditioning task and the novel object exposure assay were the same as in the previous experiment in human Alzheimer's disease - like mice described in Example 2 (Figure 3A).

[0156] Vehicle - treated aged (A) mice (veh., A) showed memory impairment in the fear conditioning task model when compared to vehicle - treated control young (Y) adult mice (veh., Y). Treatment of aged animals with the Ruminococcaceae strain CNCM I - 5830 (CNCM I - 5830, A) and Luminococcus lactaris CNCM I - 5831 (CNCM I - 5831, A) resulted in a complete recovery of their learning and memory ability compared to vehicle - treated control (veh., A), as the freezing levels of the animals treated in the task were indistinguishable from the scores shown by the control young animals (Figure 3B).

[0157] The effect of strain treatment in aged mice was also evaluated at the hippocampal neural circuit level using a novel object exposure assay. The number of neurons activated by exposure to a novel situation and positive for c-fos in the hippocampal CA1 region was dramatically reduced in aged mice (veh., A, nov. exp.: +) compared to young control animals not exposed to the novel situation (Y, nov. exp.: -). Treatment of aged mice (CNCM I-5830, A, nov. exp.: +) with the strain Lactobacillaceae CNCM I-5830 resulted in an increase in the ability of the hippocampus to respond to novel objects compared to vehicle-treated animals. On the other hand, this ability was fully restored in aged mice treated with the strain Luminococcus lactaris CNCM I-5831 (CNCM I-5831, A, nov. exp.: +) when compared to vehicle-treated young and aged control animals (Figure 3C).

[0158] In conclusion, the Lactobacillaceae bacteria and Luminococcus lactaris strains according to the present invention are clearly shown to reverse memory impairment in models of Alzheimer's disease and aging.

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Claims

1. A composition comprising a Ruminococcus lactalis strain and / or its culture extract, and a carrier or excipient, for use in the treatment and / or prevention of memory impairment resulting from age-related memory loss or neuropsychiatric and / or neurodegenerative diseases, disorders, or conditions.

2. The composition for use according to claim 1, wherein the Ruminococcus lactalis strain is phylogenetically related to Ruminococcus lactalis ATCC 29176.

3. The composition for use according to any one of claims 1 to 2, wherein the neuropsychiatric and / or neurodegenerative disease, disorder, or condition causing memory impairment is selected from neurodegenerative diseases of the central nervous system, particularly Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, disease, agnosia, amnesia, traumatic brain injury, dementia, postoperative cognitive impairment, or attention deficit / hyperactivity disorder.

4. The composition for use according to claim 1, wherein the neuropsychiatric and / or neurodegenerative disease, disorder, or condition causing memory impairment is Alzheimer's disease.

5. A composition for use according to claim 1, comprising the Ruminococcus lactalis strain deposited on March 2, 2022, under acceptance number CNCM I-5831, at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15.

6. The composition for use according to claim 1, wherein the culture extract is selected from the group consisting of strain culture supernatant, cell fragments, cell walls, and protein extracts.

7. The composition for use according to claim 1, wherein the bacteria or their culture extract is in either an activated or inactivated form, is a suspension, freeze-dried, or spray-dried.

8. The composition for use according to claim 1, wherein the bacteria or culture extract is provided as a tablet, capsule, powder, granule or liquid formulation.

9. The composition for use according to claim 1, wherein the bacteria or culture extract is formulated for oral administration in an oral dosage form, preferably covered with a coating material resistant to gastric juice.

10. The composition for use according to claim 1, wherein the bacteria or culture extract is formulated for rectal administration.

11. The composition for use according to claim 1, wherein the bacteria or culture extract is formulated as a drug or LBP drug.

12. The composition for use according to claim 1, wherein the bacteria or culture extract is prepared as a probiotic or dietary supplement.

13. The composition for use according to claim 1, wherein the Ruminococcus lactalis strain is combined with a Lacnospiraceae strain.

14. The composition for use according to claim 13, wherein the aforementioned Lacnospiraceae strain is a Lacnospiraceae strain deposited on March 2, 2022, under acceptance number CNCM I-5830, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15.

15. The bacteria of the aforementioned Lacnospiraceae species strain deposited on March 2, 2022, under receipt number CNCM I-5830, to Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS Cedex 15, and under receipt number CNCM I-5831, on March 2, 2022, to Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur A composition for use according to claim 13 or 14, comprising the bacterium of the Ruminococcus lactalis strain deposited in Roux, 75724 Paris Cedex 15.

16. The composition for use according to claim 1, wherein the memory impairment is assessed by one or more object recognition tasks; digit span tasks; motor or spatial memory tasks such as spatial information tests; mirror drawing tests; mirror letter reading tasks; weight sampling tasks; rapid reading of repetitive nonwords; resolution of random dot stereograms; classical conditioning tasks such as blink reflex conditioning; the ability to classify strings as grammatical or ungrammatical; fact recall; various types of mating tests; tests for retention from minutes to days or years; language learning and recall tasks; and combinations thereof.

17. A method for generating a mouse model exhibiting symptoms of Alzheimer's disease, comprising the step of transplanting a gut microbiota obtained from a fecal sample of a human patient with Alzheimer's disease into the mouse.

18. A mouse model generated by the method described in claim 17.

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