HK1211847B - Selection and use of lactic acid bacteria preventing bone loss in mammals - Google Patents

Description

Selection and application of lactobacilli for preventing osteoporosis in mammals

Technical Field

The present invention relates generally to medicine, pharmacology and food supplements and, more particularly, to the selection and use of lactobacilli for preventing osteoporosis in mammals.

Background Art

More than 40 million Americans over the age of 50 (14 million of whom are men) suffer from low bone density, or osteoporosis, and the associated increased risk of fractures. People who suffer osteoporotic fractures are prone to depression, dependency, and increased mortality. While aging is the primary cause of osteoporosis, disease, disuse, and certain medications can also contribute to osteoporosis at any stage of life.

The skeleton is a highly organized system that supports the body's weight, houses bone marrow stroma and hematopoietic stem cells, and acts as a calcium reservoir. The structure of bone consists of an outer cortical, dense shell and an inner cancellous network. Exercise can increase trabecular bone mineral density (BMD), bone volume fraction (BVF), trabecular thickness, cortical BMD, and thickness. In contrast, disease, disuse, and certain medications (such as glucocorticoids) can reduce these parameters and lead to osteoporosis in both men and women. Osteoporosis is defined as a decrease in bone mass (more than 2.5 standard deviations (SD) below the mean) and altered bone microarchitecture (such as decreased trabecular thickness). The risk of fracture increases with decreased bone mass. Therefore, when diagnosed with osteoporosis, patients have a 16-fold increased risk of fracture compared to people with normal bone density. Fractures are associated with depression, dependency, and increased mortality (over 25% in older adults within 12 months), with hip fractures resulting in over 50,000 deaths annually (according to the National Osteoporosis Foundation (NOF)). While osteoporosis is less prevalent in men, over 30% of hip fractures occur in men, and the mortality rate is higher in men compared to women. Currently, the direct costs of osteoporosis exceed $20 billion in the United States and $30 billion in the European Union. Even more concerning, it is estimated that by 2020, over 61 million men and women over the age of 50 in the United States will have low bone density or osteoporosis (NOF statistics), making the search for effective, novel treatments a top priority. In fact, one-third of women over the age of 50 will experience osteoporosis-related fractures in their lifetime. Along with the associated increased risk of fractures, osteoporosis can negatively impact metabolism and insulin secretion. Despite the availability of treatments on the market, the number of people suffering from osteoporosis is on the rise in the United States and around the world. Several factors contribute to this, including a lack of awareness of the risk early in life, an increasingly aging population, and patient noncompliance due to unnecessary medication side effects. Furthermore, traditional osteoporosis treatments are not always effective. Currently, there are no alternative or natural treatments for people with low bone density or osteoporosis that can replace osteoporosis medications. Consequently, physicians are searching for new ways to increase their patients' bone density, and companies are working to improve pharmacological bone treatments.

Some people are more likely to develop osteoporosis than others, risk factors are;

*female

Elderly people

* Family history of osteoporosis or broken bones

*Short and thin people

* Certain race/ethnicity, such as Caucasian, Asian, or Hispanic/Latino

people, although African Americans are also at risk

*Broken Bones Historian

*People with low sex hormone levels

*Women with low estrogen levels, including menopausal women,

*Those who have lost their menstruation (amenorrhea)

*Men with low testosterone and estrogen levels

*diet

-Low calcium intake

-Low vitamin D intake

-Excessive intake of protein, sodium, and caffeine

*Lazy lifestyle

* Smoking

*alcoholism

* Certain medications such as steroids, anticonvulsants, etc.

* Certain diseases and conditions such as anorexia nervosa, rheumatoid arthritis, and gastrointestinal diseases,

Menopausal women are more susceptible to osteoporosis due to the decline in estrogen levels during menopause. Estrogen levels may even begin to decline during perimenopause (the period 2 to 8 years before menopause). Over time, this significant bone loss can lead first to osteopenia (low bone mass) and then to osteoporosis.

Diagnosis of type 1 diabetes (T1D) in children and adults is increasing. While medical advances have extended patients' lifespans, maintaining normal blood sugar levels remains difficult even with vigilant treatment. Consequently, more T1D patients (both men and women) are developing complications including osteoporosis. This means patients are entering aging/menopause with an already increased risk of fractures. Once fractures occur, they are difficult to heal, requiring prolonged hospitalizations, reducing quality of life and increasing mortality. Poor bone health also negatively impacts the entire body. Postmenopausal women with T1D diabetes have a higher incidence of osteoporotic fractures than women without diabetes. Children with T1D have lower bone density than children without diabetes. Maintaining bone health is therefore crucial to the overall quality of life of T1D patients and is important for maximizing medical/curative therapies involving bone marrow immune/progenitor cells due to the interconversion of bone marrow cells and osteoblasts.

Patients with type 2 diabetes (T2D) also have a higher risk of osteoporotic fractures than those without diabetes.

Two key elements to strengthening bone and preventing osteoporosis are: 1) achieving maximum bone density and 2) preventing osteoporosis during adulthood and aging. 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 maintaining bone strength at sites where support is needed. When building and resorption activities are balanced, there is no net gain or loss of bone. However, when building decreases and/or resorption increases, osteoporosis ensue.

Increased osteoclast activity leads to bone resorption. Osteoclasts originate from hematopoietic stem cells. These cells give rise to cells of the monocyte/macrophage lineage, which, under appropriate conditions, develop into osteoclast precursors. Furthermore, osteoclast maturation is stimulated by signals such as RANKL (located on the surface of osteoblasts). Mature osteoclasts express enzymes involved in bone matrix degradation, including cathepsin K and TRAP5b.

Increased osteoblast activity leads to bone formation, which can be regulated at multiple levels, including: 1) selection of lineage, 2) maturation and 3) death. Because bone marrow stromal cells (BMSCs) produce osteoblasts, adipocytes and other cell types, selecting one lineage (adipocytes) may be at the expense of another lineage (osteoblasts). The reciprocal relationship between bone fat and mineral density supports this theory, which is identified by aging, limb unloading, cell culture models and type 1 diabetes (T1). The activity of osteoblasts can be further regulated by death/apoptosis. The increase in osteoblast death will lead to fewer bone-making cells, thus osteoporosis. The rapid bone adaptation to disuse/unloading is included in the embodiment, which leads to osteoporosis, increased bone marrow fat and increased osteocyte death. Aging also increases osteocyte apoptosis. Many factors contribute to the regulation of some or all aspects of osteoblast regulation (lineage, maturation, and death), including active factors such as TGFβ, bone morphogenetic proteins (BMPs), parathyroid hormone (PTH), and Wnts, as well as passive factors such as cytokines.

Bisphosphonates are one of the most common treatments for osteoporosis. These compounds bind to bone mineral, inhibiting bone breakdown by osteoclasts and effectively reducing fractures. However, many of these compounds must be taken on an empty stomach, which can cause gastric reflux and nausea, leading to reduced patient compliance. There are also concerns about how long these compounds remain in the bone and their long-term effects on bone remodeling and strength. Selective estrogen receptor modulators (SERMS) are another treatment option, but they pose some concerns regarding cancer. Studies have shown that hormone replacement therapy is effective in preventing or slowing the onset of osteoporosis, but continued use over many years may increase women's risk of breast cancer, increase the incidence of venous thrombosis (blood clots), worsen existing liver disease, increase the risk of endometrial cancer, and cause high blood pressure. Amgen has developed a drug (similar to osteoprotegerin) that works by modifying the RANKL/RANK system, thereby inhibiting osteoclast activity. Intermittent PTH therapy is an anabolic treatment, but this intravenous therapy is expensive and indicated only for patients with severe osteoporosis. Taken together, it's no surprise that many people diagnosed with low bone density remain confused about what to do. Many avoid taking medications due to concerns about long-term effects. While weight-bearing exercise and adequate calcium intake are two natural approaches, they often fall short of overcoming the effects of disease, medication, and aging.

Summary of the Invention

The main purpose of the present invention is to provide a method for finding lactic acid bacteria strains that can prevent osteoporosis, especially in menopausal women, diabetic patients, and people with osteopenia, such as young people with large energy intake and low exercise frequency.

One object of the present invention is to use a product comprising said strain in menopausal women for the prevention of osteoporosis.

One object of the present invention is to use a product comprising said strain for the prevention of osteoporosis in women who have had a hysterectomy.

Another object is to use a product containing said strain to prevent osteoporosis in men including but not limited to diabetics, young men with metabolic disorders and men with osteopenia.

Another object is to combine the product with therapeutic drugs for osteoporosis or bone formation in order to reduce the dosage of the drug and thus minimize side effects.

Another aim is to improve bone repair after fractures.

Therefore, a first aspect of the present invention provides a method for selecting a lactobacillus strain for preventing or treating osteoporosis, the method comprising selecting a lactobacillus strain having at least 95% identity with respect to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and the lactobacillus strain having the same nucleotides as the genome of Lactobacillus reuteri JCM1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment according to the first aspect, the method comprises selecting a Lactobacillus strain having at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM1112 (SEQ ID NO: 1), and the Lactobacillus strain has an identical nucleotide to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

A second aspect of the present invention provides a method for selecting a lactobacillus strain (such as a Lactobacillus reuteri strain) for preventing or treating osteoporosis, the method comprising selecting a Lactobacillus reuteri strain having the same nucleotide as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), wherein the nucleotide is located at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment of the method according to the first or second aspect, the Lactobacillus strain has at least two of said four nucleotides, such as at least three of said four nucleotides, all four of said four nucleotides.

A third aspect of the present invention provides a method for selecting a lactobacillus strain for preventing or treating osteoporosis, the method comprising selecting a lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM1112 (SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), the mutation being selected from the group consisting of a C to T mutation in base pair 271391, a G to A mutation in base pair 453538, a G to A mutation in base pair 529228, and a C to T mutation in base pair 599338.

In an embodiment of the third aspect, the method comprises selecting a Lactobacillus strain having at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the method according to the third aspect, the Lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

The fourth aspect of the present invention is to provide a Lactobacillus strain for preventing or treating osteoporosis, wherein the Lactobacillus strain is selected according to the method described in the first, second or third aspect.

In an embodiment of the fourth aspect, the Lactobacillus strain selected for preventing or treating osteoporosis is Lactobacillus reuteri ATCC PTA 6475. This strain is publicly available from the American Type Culture Collection (10801 University Avenue, Manassas, VA, USA), where it was deposited on December 21, 2004, under the Budapest Treaty.

According to a fifth aspect, the present invention provides a lactobacillus strain for preventing or treating osteoporosis, wherein the lactobacillus strain has at least 95% identity with the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), wherein the nucleotides are located at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment of the fifth aspect, the Lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271 391, G in base pairs 453 538, G in base pairs 529 228, and C in base pairs 599 338.

In an embodiment of the fifth aspect, the Lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a sixth aspect, the present invention provides a lactobacillus strain, which has at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation relative to the genome of Lactobacillus reuteri JCM1112 (SEQ ID NO: 1), wherein the mutation is selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338.

In an embodiment of the sixth aspect, the Lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271 391, G to A in base pairs 453 538, G to A in base pairs 529 228, and C to T in base pairs 599 338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the sixth aspect, the Lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

According to a preferred embodiment of the fifth or sixth aspect, the lactobacillus strain is Lactobacillus reuteri ATCC PTA 6475.

A seventh aspect of the present invention provides a composition comprising a Lactobacillus strain, wherein the Lactobacillus strain is selected according to the method of the first, second or third aspect of the present invention.

According to an eighth aspect, a composition is provided comprising a lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) and having the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271 391, G in base pairs 453 538, G in base pairs 529 228, and C in base pairs 599 338.

In an embodiment of the eighth aspect, the lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment of the eighth aspect, the Lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a ninth aspect, the present invention provides a composition comprising a Lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the ninth aspect, the Lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271 391, G to A in base pairs 453 538, G to A in base pairs 529 228, and C to T in base pairs 599 338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the ninth aspect, the Lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, such as any of the four mutations.

In a preferred embodiment of the eighth or ninth aspect, the Lactobacillus strain is Lactobacillus reuteri ATCC PTA 6475.

In an embodiment of the eighth or ninth aspect, the composition is used to prevent or treat osteoporosis.

In another embodiment of the eighth or ninth aspect, the composition is used to prevent osteoporosis in menopausal women, women who have had a hysterectomy, diabetics, osteopenic individuals, osteoporotic individuals, and individuals with metabolic disorders.

In yet another embodiment of the eighth or ninth aspect, the composition is for use in improving bone repair after a bone fracture.

In an embodiment of the eighth or ninth aspect, the above-mentioned composition is combined with vitamin D for preventing or treating osteoporosis.

In another embodiment of the eighth or ninth aspect, the above-mentioned composition is used in combination with hormones (for hormone replacement therapy) to prevent or treat osteoporosis.

In an embodiment of the eighth or ninth aspect, the composition is a pharmaceutical composition (preferably comprising at least one pharmaceutically acceptable excipient), or a food product or food supplement (preferably comprising at least one food-grade excipient, which food-grade excipients are well known to those of ordinary skill in the art).

According to a tenth aspect, the present invention provides a use of a lactobacillus strain, wherein the lactobacillus strain has at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), wherein the nucleotides are located at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338, for preparing a pharmaceutical composition for preventing or treating osteoporosis.

In an embodiment of the tenth aspect, the Lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment of the tenth aspect, the Lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to an eleventh aspect, the present invention provides a use of a Lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), wherein the mutation is selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338, for preparing a pharmaceutical composition for preventing or treating osteoporosis.

In an embodiment of the eleventh aspect, the lactobacillus strain has at least 96% (e.g., 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the eleventh aspect, the Lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

In presently preferred embodiments of the tenth or eleventh aspect, the Lactobacillus strain is Lactobacillus reuteri ATCC PTA 6475.

A twelfth aspect of the present invention provides a method for treating or preventing osteoporosis, the method comprising administering to an individual a lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) and having the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), the nucleotides being located at at least one of the following four positions: C in base pairs 271-391, G in base pairs 453-538, G in base pairs 529-228, and C in base pairs 599-338.

In an embodiment of the method according to the twelfth aspect, the lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), and has the same nucleotides as the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1) at at least one of the following four positions: C in base pairs 271 391, G in base pairs 453 538, G in base pairs 529 228, and C in base pairs 599 338.

In an embodiment of the twelfth aspect, the Lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a thirteenth aspect, the present invention provides a method for treating or preventing osteoporosis, the method comprising administering to an individual a lactobacillus strain having at least 95% identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation relative to the genome of Lactobacillus reuteri JCM1112 (SEQ ID NO: 1), the mutation being selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338.

In an embodiment of the thirteenth aspect, the Lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1), provided that the Lactobacillus strain does not have at least one mutation selected from the group consisting of C to T in base pairs 271-391, G to A in base pairs 453-538, G to A in base pairs 529-228, and C to T in base pairs 599-338 relative to the genome of Lactobacillus reuteri JCM 1112 (SEQ ID NO: 1).

In an embodiment of the thirteenth aspect, the Lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

In a presently preferred embodiment of the twelfth or thirteenth aspect, the Lactobacillus strain is Lactobacillus reuteri ATCC PTA 6475.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the microbial communities that cluster in the jejunum and ileum;

FIG2 shows the inhibition of osteoporosis by Lactobacillus reuteri ATCC PTA 6475;

FIG3 shows the effects of different Lactobacillus reuteri strains on osteoporosis.

Detailed description of the invention and preferred embodiments

Chronic inflammatory diseases are often associated with systemic osteoporosis. In the abstract of grant number 1R21AT005472-01A1, granted by the National Institutes of Health (NIH), McCabe noted that therapies that improve overall gut health have the potential to benefit bone health. McCabe and Britton found that Lactobacillus reuteri therapy reduced ileal TNF levels and increased bone volume in healthy male mice, but not female mice, and suggested that Lactobacillus reuteri increased bone density in a sex-dependent manner by inhibiting intestinal inflammation and upregulating bone formation. They believe that Lactobacillus reuteri provides a new approach to increasing bone mass by using probiotics that can reduce intestinal inflammation. However, unlike certain specific strains selected for the present invention, this is a sex-dependent upregulation of bone formation, rather than prevention of osteoporosis, which is associated with the anti-inflammatory properties of Lactobacillus reuteri, which is different from certain specific strains selected for the present invention for the prevention of osteoporosis in men and women.

Probiotics can increase cortical bone thickness in chickens and reduce osteoporosis in aging mice. Narva et al., in "Effects of the bioactive peptide, valyl-prolyl-proline (VPP), and VPP-containing Lactobacillus helveticus fermented milk on osteoporosis in ovariectomized mice," describe the prevention of osteoporosis by Lactobacillus helveticus fermented milk, likely due to the action of the peptide valyl-prolyl-proline. Narva et al. further describe the positive acute effects of Lactobacillus helveticus fermented milk on calcium metabolism in "Effects of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women."

Yeo et al. in “Angiotensin I-converting enzyme inhibitory activity and isoflavone biotransformation by probiotics in prebiotic-supplemented soy milk” suggest that probiotics incorporated into prebiotic-supplemented soy milk may have potential as a dietary treatment for conditions such as osteoporosis.

Kim et al. in “Effects of a fermented milk product, Lactobacillus casei 393, on bone metabolism in ovariectomized mice” showed that Lactobacillus casei 393FMP has a preventive effect on osteoporosis in ovariectomized mice.

However, none of the above-mentioned prior arts, whether alone or in combination, provides any guidance on how to select specific probiotic strains for effectively preventing osteoporosis.

The present invention includes a method for selecting Lactobacillus strains effective for preventing osteoporosis in humans, as well as strains selected according to the method. As known in the art, products such as foods, nutritional supplements and formulations, pharmaceuticals, or medical devices containing whole cells or components derived from these strains can be prepared. These products typically include a known ingestible support to which the Lactobacillus strains are added, or a component derived therefrom.

Based on the prior art, one might naturally assume that a strain's ability to prevent osteoporosis is linked to its general effects on gut health or its anti-inflammatory properties. However, the inventors were surprised to discover that these properties were not predictable for their potential to prevent osteoporosis. Lactobacillus reuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659 are two nearly identical strains, both of which exhibit anti-inflammatory and gut health benefits. Therefore, one would naturally assume that these strains would have the same effects on osteoporosis. However, the inventors discovered that these strains do not exhibit the same osteoporosis-preventing effects. Based on this observation, they developed a new method for selecting Lactobacillus strains, such as Lactobacillus reuteri, that are effective in treating and/or preventing osteoporosis.

Lactobacilli specifically selected according to the methods provided herein are administered to humans to prevent osteoporosis.

Lactobacillus reuteri ATCC PTA6475 and Lactobacillus reuteri ATCC PTA 4659 differ in four SNPs that are important for the bacteria's ability to prevent osteoporosis. These SNPs were shown by Walter et al. (Walter et al., Host-microbe symbiosis and the Lactobacillus reuteri paradigm in the vertebrate gastrointestinal tract; Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, pp. 4645-4652), which is hereby incorporated by reference in its entirety. For SNP analysis, sequencing results were mapped to a reference genome (Lactobacillus reuteri JCM1112, GenBank Accession No. AP007281, SEQ ID NO: 1). Seven SNPs were found in Lactobacillus reuteri ATCC PTA 4659, three of which were also found in Lactobacillus reuteri ATCC PTA 6475 (SNP 4 at bp 567-368, SNP 6 at bp 968-088, and SNP 8 at bp 1-358-460, with respect to the reference genome, Lactobacillus reuteri JCM 1112, GenBank accession number AP007281, SEQ ID NO: 1), and the remaining four unique SNPs (hereinafter referred to as SNP 1, SNP 2, SNP 3, and SNP 5, respectively, for the purposes of this article) constitute the genomic differences between Lactobacillus reuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659. The four SNPs are located at:

-bp 271 391 (SNP 1),

-bp 453 538 (SNP2),

-bp 529 228 (SNP3), and

-bp 599 338 (SNP5),

(Regarding the reference genome, Lactobacillus reuteri JCM 1112, GenBank Accession No. AP007281, SEQ ID NO: 1).

SNP 1 is located in the gene encoding a conserved hypothetical protein (Lactobacillus reuteri JCM1112: http://www.ncbi.nlm.nih.gov/protein/183224225), SNP 2 is located in the gene encoding a chloride channel protein (Lactobacillus reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224386), SNP 3 is located in the gene encoding the ATP synthase γ subunit (Lactobacillus reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224455), and SNP 5 is located in the gene encoding the DNA mismatch repair protein HexB (Lactobacillus reuteri JCM 1112: http://www.ncbi.nlm.nih.gov/protein/183224511). The SNPs involved in the present invention are SNPs that match those of Lactobacillus reuteri ATCC PTA 6475, and their sequences have the same nucleotides as those of Lactobacillus reuteri JCM1112 at positions SNP1, SNP2, SNP3, and SNP5. The following are the different nucleotides between Lactobacillus reuteri ATCC PTA 6475 and 4659:

SNP 1) is for a gene encoding a hypothetical protein, in which nucleotide 267 is changed from a C in ATCC PTA 4659 (as in ATCC PTA 6475 and JCM 1112) to a T.

SNP 2) is for a gene encoding a chloride channel protein, wherein nucleotide 373 is changed from G to A in ATCC PTA4659 (as in ATCC PTA6475 and JCM 1112).

SNP 3) is for the gene encoding the gamma subunit of ATP synthase, wherein nucleotide 296 is changed from G to A in ATCC PTA4659 (as in ATCC PTA6475 and JCM 1112).

SNP 5) In the gene encoding the HexB protein, nucleotide 1966 is changed from C (eg, ATCC PTA6475 and JCM1112) to T.

In the selection method of the present invention, a strain is sought in which at least one of these SNPs has the same nucleotide sequence as the above-mentioned SNP of Lactobacillus reuteri ATCC 6475 PTA.

The microbiome plays a key role in osteoporosis; many patients with osteoporosis have a disturbed gut microbiome. Lactobacilli, which are able to reestablish a normal microbial community in the gastrointestinal tract, are surprisingly more effective in preventing osteoporosis.

The present invention discloses a unique method for selecting strains effective for preventing osteoporosis. The ability to reconstitute the overall gut microbiome is surprisingly important for preventing osteoporosis. The inventors discovered that strains capable of reconstituted altered microbial communities to normal and/or possessing at least one of four specific SNPs are effective for preventing osteoporosis.

The ability to prevent osteoporosis is unique to certain strains, and not all Lactobacillus strains possess this unique ability. Using anti-inflammatory ability as a selection criterion for effective strains is insufficient, as the inventors have clearly shown that this effect is independent of anti-inflammatory properties. Both Lactobacillus reuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659 are anti-inflammatory strains, but when used to prevent osteoporosis, Lactobacillus reuteri ATCC PTA 6475 is more effective, while Lactobacillus reuteri ATCC PTA 4659 is not selected according to the present invention. In general, specific Lactobacillus strains selected according to the present invention can be used to prevent osteoporosis. The following examples are not intended to limit the scope of the invention but rather to illustrate preferred embodiments.

Vitamin D is crucial for bone health; people with low vitamin D levels have lower bone density and bone mass. Because vitamin D is required for calcium absorption, people who don't get enough vitamin D may develop osteoporosis. The inventors have discovered that altered microbiota can lead to vitamin D deficiency and osteoporosis. Administration of lactobacilli selected according to the present invention can reshape the microbiota, thereby increasing intestinal vitamin D absorption and restoring vitamin D levels. Combining vitamin D with the selected strains is also an option, resulting in a more effective method/product for preventing osteoporosis.

T1D patients suffer from complications such as osteoporosis. Patients with T1D will have an altered microbiome due to this condition. Administration of lactobacilli selected according to the present invention will reconstitute the microbiome and prevent osteoporosis.

High bone density in youth and adulthood can help prevent osteoporosis in later life. This is because before reaching bone density in the osteoporotic zone, high bone density will allow for a higher degree of osteoporosis. Therefore, the object of the present invention is to help individuals obtain maximum bone density to prevent osteoporosis in later life by administering the lactobacillus strains selected according to the present invention to youth and adults. The specific lactobacillus selected according to the present invention can prevent osteoporosis in healthy recipients as well as those suffering from osteoporosis.

Administration of selected lactobacilli can be combined with hormone replacement therapy. Such a combination allows for a reduction in the amount of hormones used, thereby reducing side effects, such as a lower risk of cancer.

Lactobacillus selected for preventing osteoporosis is preferably administered to menopausal women and men with osteopenia who are susceptible to osteoporosis. Administration of the selected lactobacillus can prevent osteoporosis, thereby preventing low bone density and osteoporosis.

The inventors have learned that estrogen depletion alters the intestinal microbiota and that treatment with lactobacilli selected according to the present invention will reconstitute the microbiota in patients with reduced estrogen levels, thereby preventing osteoporosis.

The lactic acid bacteria strains selected according to the present invention may also be used to improve fracture repair.

To reduce the side effects of medications such as bisphosphonates and hormone replacement therapy used to treat osteoporosis, it may be possible to combine the medication with the administration of selected lactobacilli, thereby reducing the dosage and minimizing side effects.

Example 1

Study of the ability of Lactobacillus reuteri ATCC PTA 6475 to reconstitute an altered microbial community in ovariectomized mice.

Controls (non-ovariectomized), ovariectomized, and ovariectomized mice fed L. reuteri had significant changes in the gut microbial community.

Experimental groups and tissue collection

To measure the effects of ovariectomized mice (ovx) and ovariectomized mice treated with Lactobacillus reuteri 6475, we compared three experimental animal groups. Control mice without ovariectomized mice received a vehicle control diet three times per week. Ovariectomized mice received a vehicle control diet three times per week. Ovariectomized mice treated with Lactobacillus reuteri 6475 received 300 μl of overnight Lactobacillus reuteri 6475 three times per week for four weeks. At the end of the experiment, mice were euthanized, and tissue samples from the stomach, duodenum, jejunum, ileum, proximal colon, and distal colon were isolated and stored for microbial ecology analysis.

DNA extraction

The intestinal tissue of the mouse was placed in a MoBio Ultra Clean Fecal DNA Magnetic Bead Test Tube (cat.#12811-100-DBT). The magnetic bead test tube contained 360 μl of Buffer ATL (Qiagen cat.#19076) and dissolved by stirring at full speed for 1 minute on a Mini Magnetic Bead Mixer-8 (BioSpec Products). DNA was extracted from the mouse intestinal tissue using the Qiagen DNeasy Blood and Tissue Kit (cat.#69504). The tissue was further disrupted by adding 40 μl of Proteinase K (Qiagen, cat.#19133) and incubating at 55°C for 1 hour. DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (cat.#69504). The yield of DNA was quantified using a Nanodrop 1000.

polymerase chain reaction amplification

Bacterial 16S sequences were amplified for 454 sequencing from mouse intestinal tissue using the V3-V5 barcoded primer set and an amplification protocol developed by the Broad Institute of the Human Microbiome Project. Barcoded forward primers were synthesized by IDT DNA Technology, and reverse primers were synthesized by Sigma. Barcoded forward primers were diluted to a working concentration of 4 μM in a 96-well plate; reverse primers were added to each well at a final concentration of 4 μM. Reactions were prepared in triplicate in a 25 μl volume, including 400 μg of mouse intestinal DNA, 2 μl of 4 μM primers, and 0.15 μl of Accuprime HiFi Taq polymerase (Invitrogen, cat. #12346086) in 1X Accuprime buffer II. Reactions were amplified in an Eppendorf Pro aluminum plate thermal cycler with a 2-minute denaturation cycle at 95°C, followed by 30 cycles of 95°C x 20 seconds, 50°C x 30 seconds, and 72°C x 5 minutes.

Amplification of product purification

16S amplified products were purified using Ampure Agencourt XP magnetic beads (Beckman Coulter, cat#A63880). First, triplicate reactions for each sample were mixed in a 1.7 ml microcentrifuge tube and Ampure XP magnetic beads were added at a 0.7 volume ratio. After vortexing, the mixed sample was incubated at room temperature for 10 minutes and then separated by magnetic beads (Invitrogen, cat#123-21D) on a magnetic stand. The beads were washed twice with 200 μl of 70% ethanol according to the manufacturer's instructions. The beads were dried at 37°C for 5 minutes, and the DNA was eluted with 20 μl of 10 μM Tris buffer (Tris)/0.1 μM ethylenediaminetetraacetic acid (EDTA). The eluate was separated from the beads on a magnetic stand and transferred to a new 1.7 ml microcentrifuge tube for quantification using the Quant-It dsDNA High Sensitivity Assay Kit (Invitrogen, cat#Q33120). Equal amounts of each sample were then pooled into 454 sequencing tubes.

454 sequencing and sequence analysis

454 sequencing was performed using a GS primary (Roche) using titanium chemistry. In addition to determining the sequences that had passed the assay using the standard filters of the GS primary, we used a modified amplicon processing algorithm to reduce the number of sequences that were falsely discarded. 16S rRNA sequences were aligned to the E. coli 16S sequence by the Ribosomal Database Project staff at MSU and sorted from nucleotide positions 617 to 900 in E. coli 16S. Subsequent processing and analysis (including diversity metrics) were performed using MOTHUR v.1.21 (http://www.mothur.org/wiki/). ANOSIM (analysis of similarities) and principal coordinate analysis were performed using the software package PAST. The degree of dissimilarity between two or more microbial communities was measured using the Bray-Curtis method with accompanying figures and tables. For these analyses, we chose an operational taxonomic unit (OUT) cutoff of 0.03, which was considered to be a community observed at the species level. From these data, we conclude that treatment of ovariectomized mice with Lactobacillus reuteri ATCC PTA 6475 produces significant shifts in the microbial communities of the ileum and jejunum that are associated with improved bone health (OVX and Lactobacillus in Table 1).

Table 1

organize comparison R value (p value) jejunum Wild type - ovariectomized - ovariectomized and fed with Lactobacillus 0.3367(0.0183)* Wild type - ovariectomized 0.0443(0.3633) Wild type - ovariectomized and fed with Lactobacillus 0.6078(0.0250)* Ovariectomy - Ovariectomy and lactobacillus feeding 0.3297(0.0712) ileum Wild type - ovariectomized - ovariectomized and fed with Lactobacillus 0.2068(0.0084)* Wild type - ovariectomized 0.1710(0.1180) Wild type - ovariectomized and fed with Lactobacillus 0.2540(0.0290)* Ovariectomy - Ovariectomy and lactobacillus feeding 0.2209(0.0206)*

Table 1: ANOSIM analysis at the species level using the Bray-Curtis dissimilarity matrix.

**Indicates statistical significance

A three-way comparison of wild type, ovariectomized, and ovariectomized and treated with Lactobacillus showed significant changes in the microbial community (Table 1). These differences were largely caused by the dramatic changes in the microbial community after treatment with Lactobacillus reuteri. Principal coordinate analysis of the microbial communities from the wild type control group (triangle △), the ovariectomized group (circle ○), and the ovariectomized group treated with Lactobacillus reuteri (square □) was used to observe how the microbial communities clustered in the jejunum and ileum. Figure 1 shows that the ovariectomized mice treated with Lactobacillus reuteri from a cluster of microbial communities were significantly different from the microbial communities in the jejunum and ileum of wild type and ovariectomized mice. Several OTUs classified as Clostridiales are the main bacterial communities, and these OUTs cause the microbial communities of Lactobacillus reuteri treated ovariectomized mice to be different from those of the other two groups.

Example 2

Study of the ability of Lactobacillus reuteri ATCC PTA 6475 to reconstitute an altered microbial community in ovariectomized mice.

The experiment was carried out according to Example 1, but Lactobacillus reuteri ATCC PTA 4659 was used instead of Lactobacillus reuteri ATCC PTA 6475.

Lactobacillus reuteri ATCC PTA 4659 treatment failed to reconstitute ovariectomized mice relative to controls.

Example 3

Identification of specific SNPs

Illumina sequence of the Lactobacillus reuteri genome

The Lactobacillus reuteri strains ATCC PTA4659 and 6475 used in this study were cultivated in MRS medium, and genomic DNA was prepared using the Genomic-Tip system. DNA was fragmented by 20 minutes of sonication (130W) to obtain fragments averaging 500 bp, which were then further purified and concentrated using QIAquick PCR purification spin columns (Qiagen). Removal of 3′ extensions and addition of 5′ extensions resulted in blunt ends of the genomic fragments. Adenine fragments were added to the 3′ termini using terminal transferase, and the resulting fragments were ligated to Solexa adapters. The products were separated by agarose gel electrophoresis, and fragments between 150 and 200 bp were excised from the gel. DNA fragments were extracted from the agarose gel slices using the QIAquick Gel Extraction Kit (Qiagen). The adapter-modified DNA fragments were enriched by 18-cycle PCR using Solexa universal adapter primers. The DNA fragment pool was quantified and then diluted to a 10 nM working stock for cluster generation. Adapter-ligated fragments (2 nM) were denatured in 0.1 M sodium hydroxide solution for 5 minutes, then further diluted to a final concentration of 9 pM in 1 ml of pre-chilled hybridization buffer and concentrated, and introduced onto the Solexa flow cell using the Cluster Station. Following isothermal amplification, bacterial clusters were denatured into single strands by 0.1 M sodium hydroxide and metered across the flow cell using the Solexa Cluster Station. Sequencing primers complementary to the Solexa adapters were added to the single strands of each bacterial cluster. Once hybridized and excess primers were eluted, the flow cell was ready for sequencing. The Solexa Genome Analyzer II was programmed to provide 36 continuously flowing fluorescent labels, with the 3′-OH blocking nucleotides and polymerase on the surface of the flow cell, resulting in a fixed read length of 36 bp. After each base incorporation step, the reactants were eluted from the flow cell surface and then imaged by a microscope objective. In this experiment, 300 tile images (“slices”) were acquired per flow cell channel, with each tile image containing an average of 30,000 bacterial clusters.

SNP analysis

The results of bidirectional sequencing were mapped to the control genome of Lactobacillus reuteri JCM 1112T (GenBank accession number AP007281). Mapping was performed using the mapping software Maq version 0.6.6 (http://maq.sourceforge.net/maq-man.shtml) (default parameters). SNPs were identified and verified using MAQ software and classified as coding SNPs and intergeneric SNPs. Coding SNPs were identified as synonymous or nonsynonymous. After Sanger sequencing, SNPs were finally verified by PCR amplification of the surrounding regions.

Example 4

Strain selection method

Selection of strains effective in preventing osteoporosis was based on their ability to reconstitute altered microbial communities. Based on the results of Examples 1 and 2, Lactobacillus reuteri ATCC PTA 6475 was selected based on its ability to reconstitute altered microbial communities. Lactobacillus reuteri ATCC PTA 4659 was not selected based on the results of Example 2.

Example 5

Strain selection method

Selection of strains effective in preventing osteoporosis is based on the presence of specific SNPs. As shown in Example 3, Lactobacillus reuteri ATCC PTA 6475 was selected because it possesses all four required SNPs. Lactobacillus reuteri ATCC PTA 4659 was not selected because it lacked these SNPs.

Example 6

Strain selection method

Selection of strains effective in preventing osteoporosis was based on Examples 3, 4, and 5, selecting strains that possess at least one of the four desired SNPs and have the ability to reconstitute and alter microbial communities. Based on these criteria, Lactobacillus reuteri ATCC PTA 6475 was selected.

Example 7

Lactobacillus reuteri ATCC PTA 6475 inhibits ovariectomy-induced osteoporosis

In this study, ovariectomized (ovx) BALB/c mice were used as a mouse model of osteoporosis. Mice (12 weeks old) were ovariectomized and divided into two groups. The first group was treated with Lactobacillus reuteri ATCC PTA 6475 three times a week for four weeks. Non-ovariectomized BALB/c served as the control group. Distal femoral bone volume fraction (BV/TV) and bone TRAP5 RNA (relative to HPRT) were measured. Bone volume fraction in mice treated with L. reuteri ATCC PTA 6475 was the same as in the control group. Furthermore, TRAP5 (a marker of osteoclast function) was observed to return to baseline (control group) following treatment with L. reuteri ATCC PTA 6475.

FIG2 shows that the inhibition of osteoporosis by Lactobacillus reuteri ATCC PTA 6475 was close to 100%, and the expression of TRAP5 returned to the baseline.

Example 8

The selected Lactobacillus reuteri ATCC PTA 6475 was superior to the unselected Lactobacillus reuteri ATCC PTA 4659 in inhibiting osteoporosis.

In this study, we gavage-fed animals with strains of Lactobacillus reuteri ATCC PTA 6475 and Lactobacillus reuteri ATCC PTA 4659, and also provided these strains continuously in drinking water for 28 days. Distal femoral bone volume fraction (BV/TV) was measured by μCT. Lactobacillus reuteri ATCC PTA 6475 inhibited osteoporosis to an extent indistinguishable from control mice (Figure 3). Lactobacillus reuteri ATCC PTA 4659 did not inhibit osteoporosis to a level sufficient to achieve statistical significance (p < .01). Lactobacillus reuteri ATCC PTA 4659 was not as effective as the selected strain of Lactobacillus reuteri ATCC PTA 6475.

Claims (6)

1. Use of Lactobacillus reuteri ATCC PTA 6475 in the preparation of compositions for the prevention or treatment of osteoporosis. 2. The use according to claim 1, for the prevention of osteoporosis in menopausal women, women who have undergone hysterectomy, patients with diabetes, individuals with osteopenia, individuals with osteoporosis, and individuals with metabolic disorders. 3. The use according to claim 1, for improving bone repair after fracture. 4. The use according to claim 1, wherein the composition is used in combination with vitamin D. 5. The use according to claim 1, wherein the composition is used in combination with a hormone. 6. The use according to any one of claims 1-5, wherein the composition is a pharmaceutical composition.