CN121038800A - A composition comprising Lactobacillus curvaturei, Lactobacillus rhamnosus, Lactobacillus janniae, Lactobacillus gasseri, and 2'-fucosyllactose. - Google Patents

Abstract

This disclosure relates to a composition comprising Lactobacillus curvatureii, Lactobacillus rhamnosus, Lactobacillus janniae, Lactobacillus gasseri, and 2'-fucosylated lactose, for use as a supplement to support a healthy vaginal microbiota in pregnant women.

Description

Composition comprising Lactobacillus crispatus, lactobacillus rhamnosus, lactobacillus jensenii, lactobacillus gasseri and 2' -fucosyllactose

Technical Field

The present disclosure relates to compositions comprising selected lactobacillus and 2 '-fucosyllactose (2' -FL). The use of the composition to support the growth of vaginal microbiota and intra-vaginal beneficial microbiota is also contemplated.

Background

It is estimated that more than 10 million women worldwide each year suffer from urogenital infections (e.g., urinary Tract Infections (UTI), bacterial Vaginosis (BV), and yeast vaginitis). While antimicrobial agents can effectively provide clinical remedies, the use of such agents can result in adverse effects, such as disrupting the microbiota of the user and increasing resistance to antibiotic pathogens.

The mode of action of urogenital pathogens is known to involve the formation of microbiota in the gut. The intestinal microbiota then becomes a reservoir of urogenital pathogens that invade the urogenital tract. The urogenital microbiota then becomes a reservoir of vaginal infections (e.g., caused by yeasts and bacteria that cause vaginal diseases) and urinary tract infections (e.g., caused by organisms that cause urinary tract infections). Studies have shown that a specially selected probiotic Lactobacillus provided in pessaries inserted into the vagina can colonize (Reid et al, 1994) and compete with colonisation by enterococci and other urinary tract pathogens (Bruce & Reid, 1998). The use of Lactobacillus for the prevention and treatment of infections of the urogenital tract is also described in the art (Gardiner et al CLIN DIAGN Lab immunol. 2002 Jan; 9 (1): 92-96; EP 2509610). During menopause, the number of lactobacilli in the vagina decreases.

Since vaginal infection is an important disease mechanism leading to premature labor, it is particularly important to maintain a natural, healthy balance of the lactobacilli flora in the vagina during pregnancy. The lack of lactobacilli can disrupt the microbial balance within the vagina, potentially leading to bacterial vaginosis syndrome, which may be related to the conversion of the normally present lactobacilli in quantity and quality to a mixed flora of anaerobic bacteria. Bacterial vaginosis is characterized by the loss of lactobacillus and the concomitant increase in gram-variable and gram-negative rods, which mainly include gardnerella vaginalis and bacteroides, prasuvorexa and acinetobacter species. The loss of lactobacillus vaginalis also predisposes non-pregnant women to infection, resulting in endometritis or even pelvic inflammatory disease. During menopause, lactobacillus may be depleted and colonization by pathogenic microorganisms associated with bacterial vaginosis and urinary tract infections may be increased. In postmenopausal women, colpitis therapy can reduce E.coli colonization and increase the number of lactobacilli, thereby significantly reducing the incidence of recurrent urinary and genital tract infections.

Methods for selectively stimulating beneficial microbiota are well known. For example, administration of live beneficial bacteria (probiotics), administration of substrates on which beneficial microbiota grow (prebiotics), or combinations thereof. Human Milk Oligosaccharides (HMOs) are a non-digestible oligosaccharide (NDO) that forms a mixture of prebiotic molecules that provide a substrate for the beneficial microbiota. HMO is a part of breast milk that helps infants establish the beneficial microbiota required for optimal health. However, HMOs may also be used in adult subjects. The most abundant HMO in human milk is 2' -fucosyllactose.

There is a need for compositions that can be used to support healthy vaginal microbiota and help alleviate vaginal bacterial imbalance in pregnant women. In addition, probiotic compositions having beneficial effects on the vaginal microbiota are also of interest.

Disclosure of Invention

The present disclosure provides compositions, uses, methods, and the like for supporting healthy vaginal microbiota in pregnant women. In particular, the present composition comprises lactobacillus crispatus (DSM 22566), lactobacillus rhamnosus (DSM 22560), lactobacillus jensenii (DSM 22567) and lactobacillus griseus (DSM 22583), and 2' -fucosyllactose.

Compositions comprising lactobacillus crispatus (DSM 22566), lactobacillus rhamnosus (DSM 22560), lactobacillus jensenii (DSM 22567) and lactobacillus griseus (DSM 22583) are known to support vaginal health and are commercially available under the brand name ASTARTETM. HMOs comprising 2' -fucosyllactose are known to provide support for the microbiota of infants and, in some cases, also for the microbiota of adults. However, 2' -fucosyllactose is not generally considered to be a good substrate for Lactobacillus (see e.g. Salli et al J. Agric. Food chem. 2021, 69, 170)182)。

While not wishing to be bound by theory, it is believed that the present composition provides a source of probiotics for pregnant women in a manner that supports healthy intestinal microbiota, resulting in a healthy repository of microorganisms beneficial to vaginal health. The present compositions may also be used in other female populations such as, for example, females with recurrent urogenital problems/infections, females who are taking antibiotics or other drugs that destroy the vaginal microbiota, or menopausal/postmenopausal females.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials equivalent or similar to those described herein can be used in the practice of the present disclosure, typical methods and materials are described. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range including the two ends, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

As used herein, the term "and/or" is intended to mean combined ("and") and exclusive ("or") use, i.e., "a and/or B" is intended to mean "a alone, or B alone, or a and B together.

As used herein, the term "effective amount," "effective concentration," or "effective dose" is defined as an amount, concentration, or dose of a bacterial strain sufficient to improve the overall health of an animal and deliver similar benefits as demonstrated in the examples. The absolute number of actual effective doses will depend on a number of factors, including the health of the subject in question and the other ingredients present. An "effective amount", "effective concentration" or "effective dose" of a bacterial strain may be determined by routine assays known to those of skill in the art.

As used herein, the term "isolated" refers to a bacterial strain described herein in a form or environment that does not exist in nature, i.e., the strain is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature.

As used herein, a bacterial "strain" refers to a bacterium that has its genes unchanged during growth or propagation and that is derived from a single isolate or pure culture. Probiotics are classified by their genus (e.g. bifidobacterium), species and subspecies (e.g. animal milk subspecies) and strain (e.g. DSM 15954 and/or BB-12 ^®). FAO/WHO indicates that the probiotic effect is strain specific, so that most of the probiotic properties of a particular strain cannot be generalized to other strains of the same species.

As used herein, the term "probiotic" refers to cultures, dead microorganisms, microbial fragments, and microbial extracts or supernatants of living or lyophilized microorganisms that, when applied to humans or animals, would beneficially affect the host (Hill et al (2014) expert consensus document, international probiotic and prebiotic science association. Consensus statement regarding the scope and proper use of the term probiotic （Hill et al. (2014) Expert Consensus Document, The International Scientific Association for Probiotics and Prebiotics. Consensus statement on the scope and appropriate use of the term probiotic））.

No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The invention has been described with reference to various embodiments, aspects, examples, etc. These elements are not intended to be read in isolation from each other. Thus, the present disclosure provides combinations of two or more of the described embodiments, aspects, examples, etc.

All embodiments described herein are intended to fall within the scope of the disclosed invention. These and other embodiments of the invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, which is to be read in light of the accompanying specification, the invention not being limited to any particular preferred embodiment disclosed.

The present disclosure provides a composition comprising lactobacillus crispatus (DSM 22566), lactobacillus rhamnosus (DSM 22560), lactobacillus jensenii (DSM 22567) and lactobacillus griseus (DSM 22583), and 2' -fucosyllactose.

The present compositions comprise an effective amount of a probiotic. For example, each of the foregoing Lactobacillus species may be present in a concentration range of 0.05 x 10 ⁹ CFU/g to 30 x 10 ⁹ CFU/g, preferably 0.5 x 10 ⁹ CFU/g to 25 x 10 ⁹ CFU/g. According to one embodiment, the present composition comprises lactobacillus crispatus (DSM 22566) in the concentration range of 3 x 10 ⁹ to 22 x 10 ⁹ CFU/g, lactobacillus rhamnosus (DSM 22560) in the concentration range of 3 x 10 ⁹ to 22 x 10 ⁹ CFU/g, lactobacillus jensenii (DSM 22567) in the concentration range of 0.7 x 10 ⁹ to 6 x 10 ⁹ CFU/g, lactobacillus griseus (DSM 22583) in the concentration range of 1 x 10 ⁹ to 8 x 10 ⁹ CFU/g.

The present compositions may comprise the ingredients listed in the following table:

*2' -fucosyllactose having a particle size measured using a sieve compliant with DIN ISO 3310-1:

Percentage passing through #230 mesh (63 μm) — <15%.

The percentage of passing through #100 mesh (150 μm) is not less than 60%.

The percentage passing through #45 mesh (355 μm) — was 90%.

Percentage passing through #20 mesh (850 μm) -100%.

The present compositions may comprise additional components. For example, at least one other bacterial strain, vitamin, mineral, prebiotic, fiber, or combination thereof. The other component may be, for example, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), human Milk Oligosaccharides (HMO), or a combination thereof.

The present composition may comprise biotin. For example, the composition may comprise at least about 1 μg, at least about 2 μg, at least about 5 μg, at least about 10 μg, at least about 15 μg, at least about 17 μg biotin.

The present composition may comprise at least one other bacterial strain, such as lactobacillus lactis subspecies diacetyl (Lactococcus lactis subsp. Lactis biovar. Diacetylactis), lactobacillus lactis subspecies milk fat (Lactococcus lactis subsp. Cremoris), lactobacillus lactis subspecies (Lactococcus lactis subsp. Lactis), any strain belonging to the genus lactobacillus (including but not limited to lactobacillus acidophilus, lactobacillus casei subspecies, lactobacillus delbrueckii subsp. Bulgaricus, lactobacillus fermentum, lactobacillus grignard, lactobacillus helveticus, lactobacillus lactis, lactobacillus rhamnosus, lactobacillus salivarius), any strain belonging to the genus bifidobacterium (including but not limited to bifidobacterium adolescentis, bifidobacterium hornensis, bifidobacterium animalis subsp. Animalis, bifidobacterium bifidum, bifidobacterium breve (Bifidobacterium catenulatum), bifidobacterium odontovorum, bifidobacterium longum subsp. Longum, bifidobacterium pseudocatenulatum, or lactobacillus ananas, or a strain belonging to the genus lactobacillus, or any combination of the genera lactobacillus, bifidobacterium, or any of the genus bifidobacterium, such as described herein may be suitable.

The present composition may comprise at least one strain of bacteria selected from the group consisting of bifidobacterium animalis subspecies lactis deposited with DSM 15954, lactobacillus acidophilus deposited with DSM 13241, lactobacillus rhamnosus deposited with ATCC 53103, lactobacillus paracasei subspecies paracasei deposited with ATCC 55544, lactobacillus paracasei deposited with LMG-17806, streptococcus thermophilus deposited with DSM 15957, lactobacillus fermentum deposited with NM02/31074, lactobacillus paracasei subspecies paracasei deposited with cctccc M204012, and suitable combinations thereof.

The present compositions may comprise any form of probiotic suitable for administration to a subject. In a preferred embodiment, the composition may comprise bacteria in dry form, which may be obtained by freeze-drying, spray-drying, freeze-drying or the like.

If the bacteria are freeze-dried, they are typically mixed with a cryoprotectant prior to freeze-drying. The term "cryoprotectant" as used herein refers to a substance that is capable of increasing the survival rate of bacteria during freezing and/or drying and improving their storage stability. The cryoprotectant used herein preferably comprises a saccharide and/or sugar alcohol, such as inositol.

The saccharide may be a monosaccharide, disaccharide, oligosaccharide or polysaccharide, or a mixture of at least two saccharides. Useful monosaccharides include, for example, glucose (also known as dextrose), fructose, ribose, and galactose, and useful disaccharides include, for example, sucrose, trehalose, maltose, and lactose. The composition may comprise one or more mono-or disaccharides, such as one, two or three or even more different saccharides.

The cryoprotectant may comprise a mixture of disaccharides (such as sucrose) and polysaccharides (such as maltodextrin).

The cryoprotectant may further comprise peptides, proteins, protein hydrolysates or mixtures thereof. Examples of peptides and proteins to be used are casein, pea, whey, albumin, glutamic acid or gelatin, and any isolate or hydrolysate thereof. Other additives may also be present, for example antioxidants such as sodium ascorbate, sodium citrate, trisodium citrate dihydrate and cysteine hydrochloride. Skim milk powder and yeast extract may also be ingredients.

Currently, there are about 200 known HMOs of varying structures. They can be divided into fucosylated, sialylated and neutral core HMOs. The composition of HMOs in breast milk varies from mother to mother and during lactation. 80% of all the major oligosaccharides in women are 2' -fucosyllactose, which is present in human breast milk at a concentration of about 2.5 g/L, other abundant oligosaccharides including lactose-N-tetraose, lactose-N-neotetraose and lactose-N-fucose pentasaccharide. It has been found that the concentration of each individual HMO varies during different phases of lactation (colostrum, transitional milk, mature milk and late milk) and depends on various factors such as the genetic secretion status and length of gestation of the mother.

In general, HMOs are derived from lactose and can be modified with four monosaccharides (N-acetyl-D-glucosamine, D-galactose, sialic acid, and/or L-fucose) to form oligosaccharides.

The present composition comprises 2' -fucosyllactose. The composition may be formulated as a combination of probiotic bacterial strain and HMO or as a separate composition. The composition may comprise other Human Milk Oligosaccharides (HMOs) such as, for example, 3-fucosyllactose, 3 '-sialyllactose, 6' -sialyllactose, lacto-N-tetraose, lacto-N-neotetraose and combinations thereof. HMOs may be any suitable combination. For example, 2' -fucosyllactose may be combined with any one, two, three, four or five of 3-fucosyllactose, 3' -sialyllactose, 6' -sialyllactose, lacto-N-tetraose and lacto-N-neotetraose. In one embodiment, the HMO mixture comprises 2' -fucosyllactose, 3' -sialyllactose, 6' -sialyllactose, and lactose-N-tetraose.

The present compositions may comprise any suitable amount of 2' -fucosyllactose, such as, for example, at least about 0.001 g, at least about 0.01 g, at least about 0.05 g, at least about 0.1 g, at least about 0.5 g by weight. The composition may comprise, for example, less than about 20g, less than about 10g, less than about 5g, less than about 4 g, less than about 3g, less than about 2g, less than about 1.5 g of 2' -fucosyllactose.

Compounding the composition with HMO can be problematic. It has been found that by controlling the Particle Size Distribution (PSD) of the HMO, a more repeatable and consistent composition can be obtained. While not wishing to be bound by theory, it is believed that a slightly narrower PSD may increase the flowability of the HMO, enabling it to be more effectively mixed with other ingredients. Furthermore, it is believed that a PSD within a certain range provides a better solubility curve. The particle size of the HMO can be determined using standard methods, such as using a sieve tower that separates the powder into different fractions after a predetermined time using a predetermined amplitude. The screen used in this method may be a screen conforming to DIN ISO 3310-1.

Preferably, the 2' -fucosyllactose (or other HMO) used in the present composition has the following particle size characteristics:

Percentage passing through #230 mesh (63 μm) -less than about 20%, less than about 18%, less than about 16%, less than or equal to about 15%.

Percentage of passing through #100 mesh (150 μm) -greater than about 75%, greater than about 70%, greater than about 65%, greater than or equal to about 60%.

Percentage of passing through #45 mesh (355 μm) -greater than about 95%, greater than about 92%, greater than or equal to about 90%.

Percentage passing through #20 mesh (850 μm) -100%.

Preferably, the compositions of the present disclosure are administered orally. Thus, the compositions are generally in a form suitable for oral administration. The composition may be a solid or liquid composition. The composition may be in unit dosage form. For example, the composition may be a capsule, lozenge, pill, tablet, soft gel, pouch, stick, bar powder, or more generally a composition such as an oil droplet, emulsion, or paste, or any other suitable carrier that one of skill in the art would determine to be an effective carrier for a living organism.

The composition may be encapsulated, for example, using a suitable polymer matrix, to improve the long term stability and storage of the composition. Those skilled in the art will appreciate that any suitable encapsulation material or matrix and encapsulation methods and techniques known to those skilled in the art may be used.

The composition may be included in a dietary supplement or pharmaceutical composition, or may be part of a feed product or a food product, such as a fermented dairy product, e.g. yoghurt.

Those of skill in the art will appreciate that the compositions disclosed herein may be administered at desired dosage levels and dosing regimens, depending on the circumstances and the subject's condition. Suitable dosage regimens may be determined based on the teachings of the application. Dosage regimens may be adjusted to provide optimal support for the subject's vaginal microbiota. It will be appreciated that the exact amount and rate of administration will depend on many factors, such as the age, weight, general health, sex, and dietary requirements of the subject. Based on the teachings herein, one of ordinary skill in the art can determine appropriate dosage regimens for a particular situation by routine experimentation and experimentation.

In exemplary embodiments, the composition may be administered daily for at least 1 day. Alternatively, the composition may be administered one or more times per day for at least 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.

The present compositions may comprise any suitable amount of 2' -fucosyllactose, such as, for example, at least about 0.001 g per day, at least about 0.01 g per day, at least about 0.05 g per day, at least about 0.1 g per day, at least about 0.5 g per day. For example, the composition may comprise less than about 20 g per day, less than about 10g per day, less than about 5g per day, less than about 4 g per day, less than about 3 g per day, less than about 2 g per day, less than about 1.5 g per day of 2' -fucosyllactose.

The present disclosure provides a method of supporting a vaginal microbiota of a pregnant subject. The method comprises providing to the subject an effective amount of the present composition. For example, the subject may be provided with the present composition once a day for a week or more, preferably a month or more.

The present disclosure provides the use of the present compositions for supporting the vaginal microbiota of a pregnant subject.

Claims (9)

1. A composition comprising Lactobacillus curvatureii (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus janniae (DSM 22567), Lactobacillus gasseri (DSM 22583) and 2'-fucosylated lactose, for use as a supplement to support a healthy vaginal microbiota in pregnant women. 2. The composition for use according to claim 1, wherein the composition comprises at least 0.5 x ^10⁹ CFU/g of Lactobacillus curvaturei (DSM 22566), Lactobacillus rhamnosus (DSM 22560), Lactobacillus jansii (DSM 22567), and Lactobacillus gasseri (DSM 22583). 3. The composition for use according to claim 1 or 2, wherein the composition comprises at least 0.1 g of 2'-fucosyllactose. 4. The composition for use according to any one of the preceding claims, wherein the 2'-fucosylated lactose has the following particle size distribution as measured using a sieve conforming to DIN ISO 3310-1: less than about 20% passing through #230 mesh (63 μm), more than about 65% passing through #100 mesh (150 μm), more than about 92% passing through #45 mesh (355 μm), and 100% passing through #20 mesh (850 μm). 5. The composition for use according to any one of the preceding claims, wherein the composition is an oral dosage form. 6. The composition for use according to any one of the preceding claims, wherein the composition is in capsule form. 7. The composition for use according to any one of the preceding claims, wherein the composition is in dry powder form. 8. The composition for use according to claim 1 or 2, wherein the composition comprises at least 0.1 g of 2'-fucosylated lactose per day. 9. A method for supporting the vaginal microbiota of a pregnant woman, the method comprising administering to the woman an effective amount of the composition according to any one of the preceding claims.