EP-4737581-A1 - NOVEL SPERMIDINE AND AGMATINE PRODUCING BACILLUS STRAINS

EP4737581A1EP 4737581 A1EP4737581 A1EP 4737581A1EP-4737581-A1

Abstract

Inventors

SPECKMANN, Bodo
BREMGES, Andreas
TOM DIECK, HEIKE
EHRING, Ellen
Ochrombel, Ines
KLEINBÖLTING, Jessica
Wiegräbe, Iris
VOGEL, Marie-Luise

Assignees

Evonik Operations GmbH

Dates

Publication Date: 20260506
Application Date: 20241031

Claims (16)

Microorganisms and microbial preparations selected from the following group: a) Bacillus subtilis DSM 35086; b) Bacillus subtilis DSM 35087; c) Bacillus subtilis DSM 35085; d) Bacillus subtilis DSM 33561; e) Bacillus pumilus DSM 35088; f) Bacillus pumilus DSM 35089; g) Bacillus pumilus DSM 35090; h) Bacillus pumilus DSM 35091; i) Bacillus pumilus DSM 35093; j) Bacillus altitudinis DSM 35092; k) A mutant of any of the strains according to (a) to (j), wherein the mutant has a sequence identity to the genomic DNA of said strain of at least 95 %, preferably of at least 97, 98 or 99 %, more preferably of at least 99.5, 99.8 or 99.9 %, in particular of at least 99.95, 99.98 or 99.99 %; l) A preparation of any of the strains according to (a) to (k), wherein the preparation is preferably an optionally concentrated or dried fermentation broth, ferment, supernatant of a fermentation broth, cell-lysate or cell-extract of said strains, wherein the preparation may be a cell-free preparation or may contain living or dead cells or cell debris of such a microorganism or any combination thereof.
Microorganisms and microbial preparations according to claim 1, wherein the microorganisms are able to produce at least 2.5 mg/L, preferably at least 3, 4 or 5 mg/L, more preferably at least 5, 7 or 8 mg/L, above all at least 9 or 10 mg/L, spermidine in an appropriate fermentation medium, in particular in LBG medium supplemented with 0.5 g/L L-arginine and 0.5 g/L L-methionine, and/or wherein the mutants of the deposited strains are preferably able to produce at least 90 %, more preferably at least the same amount of spermidine in comparison to the parent strain, from which they are derived.
Microorganisms or microbial preparations according to claim 1 or 2, wherein the microorganisms are able to produce at least 2 mg/L, preferably at least 5 or 10 mg/L, more preferably at least 20 or 30 mg/L, above all at least 35 or 40 mg/L, agmatine in an appropriate fermentation medium, in particular in LBG medium supplemented with 0.5 g/L L-arginine and 0.5 g/L L-methionine, and/or wherein the mutants of the deposited strains are preferably able to produce at least 90 %, more preferably at least the same amount of agmatine in comparison to the parent strain, from which they are derived.
Composition containing at least one microorganism or microbial preparation according to any of claims 1 to 3, wherein the composition is preferably selected from feed, food, cosmetical, agricultural, cleaning and laundry compositions.
Composition according to claim 4, wherein the composition contains at least one, preferably at least two or three, further substances selected from carriers, further probiotics, prebiotics, enzymes, vitamins, minerals, amino acids, peptides, plant extracts and algal extracts.
Composition according to any of claims 4 or 5, wherein the composition is a composition for oral administration, preferably selected from food and feed compositions, food and feed supplements, food and feed additives, pet foods, dietary supplements, nutraceuticals and functional foods and preferably contains at least one further food or feed additive, in particular selected from proteins, carbohydrates, fats, feed concentrates, silage, mashfeed, immune modulators, milk replacers, acid-based products and/or medicines, in particular antibiotics.
Composition according to any of claims 4 or 5, wherein the composition is a cosmetical composition, preferably for topical application, and preferably contains at least one further additive selected from surfactants, emulsifiers, phospholipids, sphingoid bases, free fatty acids, hyaluronic acid, collagens, protein hydrolysates, polymers, light stabilizers, anti-perspirants, deodorants, cosmetic oils, emollients, antioxidants, preservatives, thickeners, viscosity regulators, stabilizers, hydrotropes, solids, fillers, film formers, conditioners, insect repellents, self-tanning agents, odor absorbers, solvents, perfumes and dyes.
Pharmaceutical composition containing at least one microorganism or microbial preparation according to any of claims 1 to 3 and a pharmaceutically acceptable carrier.
Composition according to any of claims 4 to 8, wherein the composition comprises at least one precursor of spermidine or agmatine, wherein the precursor of spermidine or agmatine is selected from L-arginine, L-ornithine, L-methionine, putrescine or sources thereof and is preferably L-arginine or an arginine source, in particular dipeptides comprising L-arginine, proteins or protein hydrolysates, preferably prepared from wheat, wheat germs or soy, or an L-arginine derivative like L-arginine alpha-ketoglutarate or L-arginine ethylester.
Composition according to any of claims 4 to 9, wherein the composition comprises living cells of at least one microorganism according to any of claims 1 to 3, preferably in an amount of from 1×10 2 to 1×10 12 CFU, more preferably in an amount of 1×10 3 to 1×10 10 CFU, above all in an amount of 1×10 4 to 1×10 9 CFU, wherein the microorganisms are preferably present exclusively or mainly as endospores.
Composition according to any of claims 4 to 10, wherein the composition is a coated composition and/or contained in a capsule, wherein the coated composition comprises preferably an enteric coating, in particular comprising methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, sodium alginate or zein or combinations thereof and wherein the capsule is preferably a bile-resistant capsule.
Composition according to any of claims 4 to 11 for use in the treatment, prevention or mitigation of a disease selected from dementia, Alzheimer's disease, Parkinson's disease, arthritis, hypertension, arterial stiffness, heart failure, myopathy, sepsis or cardiovascular diseases and/or for use in increasing the life span, promoting longevity, improving male or female fertility, promoting hair or nail growth, improving immunogenicity after vaccination, improving mitochondrial respiration.
Product comprising at least one microorganism or microbial preparation according to any of claims 1 to 2, wherein the product is preferably a dietary supplement, agricultural product or a personal care article, wherein the personal care article is preferably a disposable absorbent article, in particular selected from sanitary napkins, panty liners, tampons, diapers, incontinent pads, breast pads, perspiration pads, human or animal waste management devices and interlabial pads.
Non-medical use of a microorganism or microbial preparation according to any of claims 1 to 3 or of a composition according to any of claims 3 to 11 for improving skin firmness, elasticity, viscoelasticity, resilience or hydration, for reducing or preventing wrinkles or skin irritation or for promoting hair and nail growth.
Use of a microorganism of the species Bacillus pumilus or Bacillus altitudinis and/or of a microorganism according to any of claims 1 to 3 for producing spermidine or agmatine, wherein the microorganism is preferably selected from the strains Bacillus subtilis DSM 35086, Bacillus subtilis DSM 35087, Bacillus subtilis DSM 35085, Bacillus subtilis DSM 33561, Bacillus pumilus DSM 35088 Bacillus pumilus DSM 35089, Bacillus pumilus DSM 35090, Bacillus pumilus DSM 35091, Bacillus pumilus DSM 35093 and Bacillus altitudinis DSM 35092 and wherein production is preferably carried out in presence of a precursor of spermidine or agmatine, wherein the precursor of spermidine or agmatine is preferably selected from L-arginine, L-ornithine, L-methionine, putrescine or sources thereof and is preferably L-arginine or an arginine source, in particular dipeptides comprising L-arginine, proteins or protein hydrolysates, preferably prepared from wheat, wheat germs or soy, or an L-arginine derivative like L-arginine alpha-ketoglutarate or L-arginine ethylester.
Use of a microorganism of the species Bacillus pumilus or Bacillus altitudinis and/or of a microorganism according to any of claims 1 to 3 in a process of extracting or enriching spermidine from food sources, in particular from wheat germs, soy, seeds or mushrooms.

Description

The present invention concerns novel spermidine and agmatine producing bacterial strains belonging to the species Bacillus subtilis, Bacillus pumilus and Bacillus altitudinis and preparations thereof as well as food, feed, pharmaceutical, cosmetical, agricultural and laundry compositions containing such strains or preparations thereof and concerns further generally the use of strains of the species Bacillus pumilus and Bacillus altitudinis to produce spermidine or agmatine, in particular in food, feed, pharmaceutical, cosmetical, agricultural, cleaning and laundry compositions. Spermidine (spd) is a biologically active polyamine with beneficial functions for animals, humans, and plants (Madeo, Eisenberg et al. 2018). It mediates and mimics the effects of (intermittent) fasting via e.g., the induction of autophagy. The induction of spermidine levels during fasting has recently been described to be indispensable for triggering autophagy and extension of animals' life span (PMID 39117797, 39212197). In addition to supporting human health, spermidine has also been ascertained to promote growth of plants and aid in detergent formulations (Xie, Wu et al. 2014) Human applications of spermidine include its use as food ingredient or dietary supplement to promote longevity, cardio- and neuroprotection, as well as its use in skincare and hair care products. Dietary sources of spermidine include e.g. wheat germs, soy, seeds, and mushrooms. The usefulness of these sources is limited by the fact that they contain only small amounts of spermidine and by the presence of dietary restrictions, food intolerances, practice of exclusion diets or simply by their limited availability in normal diets. In order to achieve a repeated or continuous supply of spermidine from these dietary sources their regular application is mandatory, which further limits their usability. Beyond nutrition, the gut microbiota is a relevant source of spermidine, and finding bacterial taxa that produce it is a promising approach to make it available for the applications mentioned above. While few taxa -Bacteroides, Fusobacterium, Bacillus subtilis OKB105(Xie, Wu et al. 2014), Bacillus amyloliquefaciens SQR9, and Heyndrickxia coagulans YF1, Heyndrickxia-coagulans DSM 1, and Bifidobacterium infantis- have been identified or claimed to produce spermidine (Chen, Liu et al. 2017, Zou, Zhao et al. 2022, Lee, Kwon et al. 2023, Suzuki, Fujiwara et al. 2023)the state-of-the-art demonstrates a lack of robust, defined bacterial strains that produce and secrete high levels of spermidine in the context of relevant technical and/or physiological conditions, substrates, and matrices. CN 112111536 discloses the genetic engineering and use of recombinant bacteria to synthesize spermidine. Similarly, Escherichia coli and Bacillus amyloquifaciens strains have been bioengineered to produce spermidine(Caffaratti, Plazy et al. 2022, Zou, Zhao et al. 2022). Spermidine production by non-GMO microbial strains has been reported, but levels of spermidine found in supernatants of cultured strains do not exceed 5-6 mg/I(Lee, Kwon et al. 2023). Therefore, there is a need to provide new microbial strains and/or species that can produce high yields of spermidine under practically relevant conditions and from available sources. By applying bioinformatic and cell culture techniques, novel spermidine producing strains of the species Bacillus subtilis, Bacillus pumilus and Bacillus altitudinis could be identified which produce high amounts of spermidine, in particular higher amounts than the benchmark strain Heyndrickxia coagulans DSM 1. While for strains of the species Bacillus subtilis it was known before that they are able to produce spermidine, it was unknown and surprising that strains of the species Bacillus pumilus and Bacillus altitudinis are also able to produce spermidine in significant amounts. This was the more surprising, because the strains which could be identified as effective spermidine producers according to the invention do not possess the genes encoding for the expected spermidine transporter proteins PotA, PotB, PotC and PotD, which were expected to be necessary for an effective transport and thus accumulation of spermidine into the surrounding medium. Furthermore, it turned out that the identified strains are besides spermidine also producing agmatine as a beneficial co-product in relatively high amounts. The subject matter of the invention has some significant advantages compared to the state of the art: the microorganisms of the invention can be applied in the form of dietary supplements or functional foods, optionally combined with L-arginine or sources thereof such as proteins or protein hydrolysates in a form that is suitable for any type of allergen requirements, exclusion diets or particular nutritional needs, to provide the health benefits of spermidine via its sustained production in the gastrointestinal tract. As the microorganisms of the invention can engr