Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K2035/11—Medicinal preparations comprising living procariotic cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus

Definitions

• the present invention relates generally to medicine, pharmacology and food supplements. More specifically the invention relates to selection and use of lactic acid bacteria for the prevention of bone loss in mammals.
• the skeleton is a highly organized system that supports the body's weight, houses mesenchymal and hematopoetic stem cells, and serves as a calcium reservoir.
• the structure of bone comprises an outer cortical dense shell and an inner trabecular bone meshwork.
• Exercise can increase trabecular bone mineral density (BMD), and bone volume fraction (BVF), trabeculi thickness, and cortical BMD and thickness.
• BMD trabecular bone mineral density
• BVF bone volume fraction
• trabeculi thickness trabeculi thickness
• cortical BMD and thickness cortical BMD and thickness.
• disease, disuse, and certain drugs can decrease these parameters and cause osteoporosis in both males and females.
• Osteoporosis is defined by a reduction in bone mass (more than 2.5 standard deviations (SD) below average) and altered bone micro-architecture (such as decreased trabeculi thickness).
• TID type 1 diabetes
• Type 2 Diabetes (T2D) patients are also at higher risk of osteoporotic fractures than non-diabetics.
• Bone remodeling occurs because bone is dynamic and constantly adapts to environmental cues to form or resorb bone.
• Targeted bone remodeling through the activities of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells) maintains blood calcium levels within a critical range while keeping bone strong at sites where support is needed.
• formation and resorption activities are in balance there is no net gain or loss of bone, however when formation is decreased and/or resorption is increased then bone loss ensues.
• Osteoclasts are derived from hematopoetic stem cells. These cells give rise to cells of the monocyte/macrophage lineage which, under the right conditions, develop into osteoclast precursors. Further signaling through factors such as RANKL (located on osteoblast surfaces) stimulate osteoclast maturation. Mature osteoclasts express enzymes involved in bone matrix degradation
• BMSC bone marrow stromal cells
• Osteoblast activity can be further regulated through death/apoptosis.
• An increase in osteoblast death will result in fewer bone making cells and therefore bone loss. Examples include the rapid bone adaptation to disuse/unloading, which results in bone loss, increased marrow adiposity, and increased bone cell death. Aging also increases bone cell apoptosis.
• Many factors contribute to modulating some or all aspects of osteoblast regulation (lineage, maturation, death) including: positive factors such as TGFP, bone morphogenic proteins (BMPs), parathyroid hormone (PTH), and Wnts and negative factors such as cytokines.
• Bisphosphonates are one of the most common treatments for osteoporosis. These compounds incorporate into the bone mineral and inhibit bone catabolism by osteoclasts and are effective at reducing fractures. However, many of these compounds need to be taken on an empty stomach and can cause gastric reflux and nausea resulting in reduced patient compliance. There is also concern about the length of time that these compounds reside in bone and their long-term impact on bone remodeling and strength. Selective estrogen receptor modifiers (SERMS) are another therapeutic treatment, but they still carry some concerns with regard to cancer.
• SERMS selective estrogen receptor modifiers
• Hormone replacement therapy has been studied as useful in preventing or slowing the occurrence of osteoporosis, but sustained use of hormone replacement over many years may increase women's risk of breast cancer, may increase incidence of venous thrombosis (blood clots), exacerbation of pre-existing liver diseases and an increased risk of endometrial cancer as well as hypertension.
• Amgen has a drug under development (that is similar to osteoprotegrin) that works by modifying the RA KL/RA K system and hence suppresses osteoclast activity.
• Intermittent PTH treatment is an anabolic treatment, but this intravenous treatment is expensive and only indicated for severe osteoporotic patients.
• a primary object of the present invention is to provide a method how to find a lactic acid bacterial strain that may prevent bone loss, especially in menopausal women, in diabetics, in osteopenic that includes for example young men with large energy intake and low exercise frequency.
• An object of the present invention is to use products containing such strains in menopausal women to prevent bone loss.
• An object of the present invention is to use products containing such strains in women who have had a hysterectomy to prevent bone loss.
• Another object is to use products containing such strains in men, including but not limited to diabetic, young men with metabolic disturbance and osteopenic men, to prevent bone loss.
• Another object is to use such product in combination with therapies for bone loss or bone formation in order to reduce the dose of such drugs to be able to minimize side effects.
• a first aspect of the invention provides a method for selecting a lactic acid bacterial strain for use in preventing or treating bone loss, comprising selecting a lactic acid bacterial strain having at least 95 % identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the method comprises selecting a lactic acid bacterial strain having at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• a second aspect of the invention provides a method for selecting a lactic acid bacterial strain, such as a Lactobacillus reuteri strain, for use in preventing or treating bone loss, comprising selecting a Lactobacillus reuteri harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain harbors at least two of said four nucleotides, such as at least three of said four nucleotides, such as all four of said nucleotides.
• a third aspect of the invention provides a method for selecting a lactic acid bacterial strain for use in preventing or treating bone loss, comprising selecting a lactic acid bacterial strain having at least 95% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the method comprises selecting a lactic acid bacterial strain having at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain does not harbor at least two of said four mutations, such as at least three of said four mutations, such as anyone of said four mutations.
• a fourth aspect of the invention provides a lactic acid bacterial strain, selected according to the method according to the first, second or third aspect, for use in the prevention or treatment of bone loss.
• the lactic acid bacterial strain selected is L reuteri ATCC PTA 6475, for use in the prevention or treatment of bone loss. This strain is available to the public at the American Type Culture Collection (10801 Univ. Blvd.,
• the invention provides a lactic acid bacterial strain having at least 95 % identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338, for use in the prevention or treatment of bone loss.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain harbors at least two of said four nucleotides, such as at least three of said four nucleotides, such as all four of said nucleotides.
• the invention provides a lactic acid bacterial strain having at least 95% identity to the genome of J reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain does not harbor at least two of said four mutations, such as at least three of said four mutations, such as anyone of said four mutations.
• the lactic acid bacterial strain is L reuteri ATCC PTA 6475.
• a seventh aspect of the invention provides a composition comprising a lactic acid bacterial strain selected according to the method according to the first, second, or third aspect of the invention.
• a composition comprising a lactic acid bacterial strain having at least 95 % identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain harbors at least two of said four nucleotides, such as at least three of said four nucleotides, such as all four of said nucleotides.
• the invention provides a composition comprising a lactic acid bacterial strain having at least 95% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain does not harbor at least two of said four mutations, such as at least three of said four mutations, such as anyone of said four mutations.
• the lactic acid bacterial strain is L reuteri ATCC PTA 6475.
• the composition is for use in the prevention or treatment of bone loss.
• the composition is for use in preventing bone loss in menopausal women, women who have had hysterectomy, diabetics, osteopenic individuals, osteoporotic individuals, and individuals with metabolic disturbance.
• the composition is for use in improving bone repair after fracture.
• the above-described composition in combination with vitamin D is for use in preventing or treating bone loss.
• composition in combination with a hormone is for use in preventing or treating bone loss.
• the above-described composition is a pharmaceutical composition (optionally comprising at least one pharmaceutically acceptable excipient), or a food product or a food supplement (optionally comprising at least one food- grade excipient, as known to a person of ordinary skill in the art).
• the invention provides a use of a lactic acid bacterial strain having at least 95 % identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions; C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338, for the manufacture of a pharmaceutical composition for the prevention or treatment of bone loss.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain harbors at least two of said four nucleotides, such as at least three of said four nucleotides, such as all four of said nucleotides.
• the invention provides a use of a lactic acid bacterial strain having at least 95% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338, for the manufacture of a pharmaceutical composition for the prevention or treatment of bone loss.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain does not harbor at least two of said four mutations, such as at least three of said four mutations, such as anyone of said four mutations.
• the lactic acid bacterial strain is L reuteri ATCC PTA 6475.
• a twelfth aspect of the invention provides a method for the treatment or prevention of bone loss, comprising administering, to an individual, a lactic acid bacterial strain having at least 95 % identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harboring an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), and harbors an identical nucleotide relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1) in at least one of the following four positions: C in base pair 271 391, G in base pair 453 538, G in base pair 529 228, and C in base pair 599 338.
• the lactic acid bacterial strain harbors at least two of said four nucleotides, such as at least three of said four nucleotides, such as all four of said nucleotides.
• the invention provides a method for the treatment or prevention of bone loss, comprising administering, to an individual, a lactic acid bacterial strain having at least 95% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain has at least 96%, such as 97%, such as 98%, such as 99% identity to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactic acid bacterial strain does not harbor at least one mutation relative to the genome of L reuteri JCM 1112 (SEQ ID NO: 1), selected from the group of four mutations consisting of C to T in base pair 271 391, G to A in base pair 453 538, G to A in base pair 529 228, and C to T in base pair 599 338.
• the lactic acid bacterial strain does not harbor at least two of said four mutations, such as at least three of said four mutations, such as anyone of said four mutations.
• the lactic acid bacterial strain is L reuteri ATCC PTA 6475.
• Figure 1 shows microbial community clustering in jejunum and ileum.
• Figure 2 shows the suppression of bone loss by L. reuteri ATCC PTA 6475.
• Figure 3 shows the effect on bone loss in different J. reuteri strains.
• Probiotics can increase chicken cortical bone thickness and reduce bone loss in aging mice.
• Narva et al. described in "Effects of bioactive peptide, valyl-prolyl-proline (VPP), and lactobacillus helveticus fermented milk containing VPP on bone loss in ovariectomized rats. " that L. helveticus fermented milk prevent bone loss, the effect might be due to the peptide valyl-prolyl-proline.
• Narva et al. further described in “The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women” that fermentation of milk with Lactobacillus helveticus had a positive acute effect on calcium metabolism.
• Kim et al. showed in "Effects of a Lactobacillus casei 393 fermented milk product on bone metabolism in ovariectomised rats" that J. casei 393 FMP had a preventative effect on bone loss in ovariectomised rats.
• the present invention herein comprises a method for selecting lactic acid bacterial strains effective for preventing bone loss in humans and strains that have been selected according to the presented method.
• Products such as foods, nutritional additives and formulations, pharmaceuticals or medical devices containing whole cells or components derived from these strains may be formulated as is known in the art, and generally include an ingestible support as known plus the lactic acid bacterial strain, or its derived component.
• Lactobacillus reuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659 are two almost identical strains, which are both anti-inflammatory and improve overall intestinal health. It is natural to assume that these strains therefore would have the same effect on bone loss as well.
• Lactic acid bacteria specifically selected by the method presented herein may be administered to humans to prevent bone loss.
• L. reuteri ATCC PTA 6475 and ATCC PTA 4659 differ in four S Ps, which are important for the bacteria's ability to prevent bone loss. These SNPs are shown in Walter et al. (Walter et al. Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm; PNAS, vol. 108 p. 4645-4652), which is hereby fully incorporated by reference. For the SNP analysis, sequencing results were mapped onto a reference genome (L. reuteri JCM 1112, GenBank accession no AP007281, SEQ ID NO: 1). Seven SNPs were found in L.
• L. reuteri ATCC PTA 4659 and three of them were also found in L. reuteri ATCC PTA 6475 (SNP 4 located at bp 567 368, SNP 6 located at bp 968 088, and SNP 8 located at bp 1 358 460, referring to the reference genome, L. reuteri JCM 1112, GenBank accession no AP007281, SEQ ID NO: 1).
• SNP 1, SNP 2, SNP 3 and SNP 5, respectively constitute the genomic differences between L. reuteri ATCC PTA 6475 and L. reuteri ATCC PTA 4659. Said four SNPs are located at:
• SNP 1 is located in a gene coding for a conserved hypothetical protein (L. reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224225), SNP 2 is located in a gene coding for a chloride channel protein (L. reuteri JCM 1112:
• SNP 3 is located in a gene coding for an ATP synthase gamma subunit (L. reuteri JCM 1112:
• the SNPs involved in this invention are the ones that match L. reuteri ATCC PTA 6475, the sequence of which has identical nucleotides as L. reuteri JCM 1112 in the positions of SNP 1, SNP 2, SNP 3 and SNP 5 ). Listed below are the nucleotides that differ between the strains L. reuteri ATCC PTA 6475 and 4659:
• SNP 1 a gene coding for a hypothetical protein, where nucleotide 267 has been changed in ATCC PTA 4659 from a C (as in ATCC PTA 6475 and JCM 1112) to a T.
• SNP 2 the gene coding for the chloride channel protein, where nucleotide 373 has been changed in ATCC PTA 4659 from a G (as in ATCC PTA 6475 and JCM 1112) to an A.
• SNP 5 the gene coding for the HexB protein, where nucleotide 1966 has been changed from a C (as in ATCC PTA 6475 and JCM 1112) to a T.
• strains are sought that, in at least one of these SNPs, harbor the same nucleotides as L. reuteri ATCC PTA 6475 for the above mentioned SNPs.
• the microbiota plays an important role in bone loss; many patients suffering from bone loss have a disturbed intestinal microbiota. Lactic acid bacteria that are able to reestablish the normal microbial community in the GI tract are surprisingly more effective in preventing bone loss.
• the present invention discloses a unique method of selection, selecting strains effective for preventing bone loss. The ability to reestablish the total gut microbial
• composition is surprisingly also important for the function in preventing bone loss.
• the inventors have found out that strains capable of reestablishing an altered microbial community to normal and/or harboring at least one of the four specific S Ps are effective for preventing bone loss.
• ATCC PTA 4659 are both anti-inflammatory strains, but J. reuteri ATCC PTA 6475 is much more effective when used for prevention of bone loss, L. reuteri ATCC PTA 4659 is not selected according to this invention.
• Specific lactic acid bacterial strains selected according to the present invention may be used for preventing bone loss in general and the embodiments below are not intended to limit the scope of this invention, but to exemplify preferred embodiments.
• Vitamin D is crucial to bone health and people with low levels of vitamin D have lower bone density or bone mass. People that do not get enough vitamin D may lose bone, since vitamin D is required to absorb calcium. The inventors have seen that an altered microbiota will lead to vitamin D deficiency and bone loss, administration of lactic acid bacteria selected according to the present invention will reestablish the microbiota and thereby increase the intestinal vitamin D absorption and restore the levels of vitamin D. It is also an option to combine vitamin D with the selected strains in order to get an even more efficient method/product for preventing bone loss.
• T1D patients suffer from complications such as bone loss. Patients suffering from T1D will as a result of the condition have an altered microbiota. Administration of lactic acid bacteria selected according to the present invention will reestablish the microbiota and prevent bone loss.
• High bone density during youth and adulthood can help prevent diseases like osteoporosis later in life. This is due to the fact that high bone density will allow a higher degree of bone loss before reaching a bone density within the osteoporosis zone.
• Specific lactic acid bacteria selected according to the present invention prevents bone loss in healthy recipient as well as those suffering from bone loss.
• Administration of selected lactic acid bacteria may be combined with hormone replacement therapy Such a combination would make it possible to reduce the amount of hormones and thereby reduce the side effects, such as reducing the risk of cancer.
• Lactic acid bacteria selected for preventing bone loss would preferably be
• osteoporosis and administration of selected lactic acid bacteria will prevent bone loss and thus preventing low bone density and osteoporosis.
• the inventors have seen that estrogen depletion alters the gut microbiota, treatment with lactic acid bacteria selected according to the present invention will reestablish the microbiota in people suffering from decreased estrogen levels, including but not limited to menopausal women and women who had hysterectomy, consequently preventing bone loss.
• Lactic acid bacterial strains selected according to the present invention may also be used to improve fracture repair.
• mice were non-ovx mice that received a vehicle control gavage three times per week.
• Ovx mice received a vehicle control gavage three times per week.
• Ovx + L. reuteri 6475 were mice that received 300 ⁇ of overnight L. reuteri 6475 three times per week for four weeks.
• mice were euthanized and tissue samples from the stomach, duodenum, jejunum, ileum, proximal and distal colons were isolated and saved for microbial ecology analysis. DNA extraction
• Murine intestinal tissue was placed in MoBio Ultra Clean Fecal DNA Bead Tubes (catJ 12811-100-DBT) containing 360 ⁇ Buffer ATL (Qiagen cat.#19076) and lysed on a Mini-Beadbeater-8 (BioSpec Products) for 1 minute at full speed.
• DNA was extracted from murine intestinal tissue using Qiagen DNeasy Blood and Tissue kit (cat.# 69504). The tissue was further disrupted by adding 40 ⁇ proteinase K (Qiagen, cat.# 19133) and incubating at 55°C for 1 hour. DNA was extracted using the Qiagen DNeasy Blood and Tissue kit
• Bacterial 16S sequences were amplified for 454 sequencing from murine intestinal tissue using the V3-V5 barcoded primer set and amplification protocol developed by the Broad Institute for the Human Microbiome Project. Barcoded forward primers were synthesized by IDT DNA Technologies and the reverse primer was synthesized by Sigma. Barcoded forward primers were diluted to a working concentration of 4 ⁇ in 96 well plates; the reverse primer was added to each well to a final concentration of 4 ⁇ . Triplicate reactions in a 25 ⁇ volume were prepared containing 400 ⁇ g murine intestinal DNA, 2 ⁇ 1 4 ⁇ primers, and 0.15 ⁇ Accuprime HiFi Taq polymerase in IX Accuprime Buffer II (Invitrogen, cat.# 12346086). Reactions were amplified in an Eppendorf Pro aluminum plate thermal cycler with a 2 minute 95°C denaturation, followed by 30 cycles of 95°C x 20 sec, 50°C x 30 sec, 72°C x 5min.
• 16S amplification products were purified using Ampure Agencourt XP beads
• the eluent was separated from the beads on the magnetic stand, transferred to a new 1.7ml microfuge tube, and quantified using the using the Quant-It dsDNA high-sensitivity assay kit (Invitrogen, cat# Q33120). Equal amounts of each sample were then pooled into one tube for 454 sequencing.
• Table 1 ANOSIM analysis at species level using Bray-Curtis dissimilarity matrix, ** indicates statistical significance.
• L. reuteri ATCC PTA 4659 treatment is not able to restore ovx communities toward control.
• L. reuteri strains used in this study were ATCC PTA 4659 and 6475 grown in MRS media (Difco) and genomic DNA prepared by using the Qiagen Genomic-Tip System. DNA was fragmented by 20 min sonication (130 W) to obtain an average fragment size of 500 bp, then further purified and concentrated with QIAquick PCR Purification Spin Columns (Qiagen). Treatment to remove 3' over- hangs and fill in 5' overhangs resulted in blunt-ended genomic fragments. An adenine residue was added by terminal transferase to the 3' end, and the resulting fragments were ligated to Solexa adapters.
• the products were separated by agarose gel electrophoresis, and the band between 150 and 200 bp was excised from the gel.
• the DNA fragments were extracted from the agarose slice using a QIAquick Gel Extraction Kit (Qiagen). Adapter-modified DNA fragments were enriched by an 18-cycle PCR using Solexa universal adapter primers.
• the DNA fragment library was quantitated, and then diluted to a 10-nM working stock for cluster generation.
• Adapter-ligated fragments (2 nM) were denatured in 0.1 M NaOH for 5 min, then further diluted to a final 9 pM concentration in 1 mL of prechilled hybridization buffer, and introduced onto the Solexa flow cell using the Cluster Station.
• clusters were made single-stranded by 0.1 M NaOH denaturation, metered across the flow cell by the Solexa Cluster Station.
• a sequencing primer complementary to one Solexa adapter was added to prime the single strands of each cluster. Once hybridized and with excess primer removed by a wash, the flow cell was ready for sequencing.
• the Solexa Genome Analyzer II was programmed to provide up to 36 sequential flows of fluorescently labeled, 3'-OH blocked nucleotides and polymerase to the surface of the flow cell, thus producing a fixed 36-bp read length. After each base incorporation step, the flow cell surface was washed to remove reactants and then imaged by microscope objective. The experiments collected 300 tiled images ("tiles”) per flow cell lane, each containing on average 30,000 clusters.
• the two lanes' sequencing results were mapped onto the reference genome L. reuteri JCM 1112T (GenBank accession no AP007281) separately.
• the mapping software Maq version 0.6.6 http://maq.sourceforge.net/maq-man.shtml was used to perform the mapping (default parameters).
• SNPs were identified and validated by the MAQ software, and classified into coding SNP and intergenetic SNPs. Coding SNPs were identified as synonymous and nonsynonymous. The SNPs were finally verified by PCR amplification of the surrounding region, followed by Sanger sequence determination.
• strains effective for prevention of bone loss is based on the ability to restore altered microbial communities. Based on the results of examples 1 and 2, L. reuteri ATCC PTA 6475 is selected based on the fact that this strain has the ability to restore altered microbial communities. L. reuteri ATCC PTA 4659 is not selected based on the results of example 2.
• strains effective for prevention of bone loss is based on example 3 and 4 and 5, strains harboring at least one of the four sought SNPS as well as the capacity to restore altered microbial communities is selected. Based on these criteria L. reuteri ATCC PTA 6475 is selected.
• ovariectomized (ovx) BALB/c mice were used as a mouse model for bone loss. Mice (12 weeks old) were ovariectomized and divided into two groups were the first group was treated with L. reuteri ATCC PTA 6475 three times a week during four weeks. BALB/c that had not been ovariectomized were used as a control group. Distal femur bone volume fraction (BV/TV) and bone TRAP5 RNA (relative to HPRT) were measured. Mice treated with L. reuteri ATCC PTA 6475 showed the same bone volume fraction as the control group. Further it was to be seen that TRAP5 (a marker of osteoclast function) is returned to baseline (control group) upon L. reuteri ATCC PTA 6475 treatment.
• TRAP5 a marker of osteoclast function
• Figure 2 shows that the suppression of bone loss by L. reuteri ATCC PTA 6475 is nearly 100% and that the expression of TRAP5 is returned to baseline.
• the selected L. reuteri ATCC PTA 6475 is superior to the non-selected L. reuteri ATCC PTA 4659 in suppressing bone loss.
• Distal femur bone volume fraction (BV/TV) was measured by ⁇ CT.
• L. reuteri ATCC PTA 6475 suppressed bone loss and was indistinguishable from control mice (Fig.3).
• L. reuteri ATCC PTA 4659 did not suppress bone loss to a sufficient level that reached statistical significance (p ⁇ 01).
• L. reuteri ATCC PTA 4659 is not as effective as the selected strain L. reuteri ATCC PTA 6475.

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