FIELD OF THE INVENTION

The present invention to the identification of a novel isolated bacterial strain, the Limosilactobacillus reuteri strain which is characterized by a naturally occurring overproduction of riboflavin (vitamin B2) compared to other known strains of lactobacilli. Said strain of the Limosilactobacillus species is deposited under accession number LMG P-32020. In a further aspect, also the uses of said L. reuteri strain are disclosed herein. In particular, the use of said strain in food and feed industry, human and veterinary health, large-scale vitamin production, cosmetics and consumer care products is disclosed.

BACKGROUND TO THE INVENTION

Riboflavin (vitamin B2) is an essential water-soluble yellow-green fluorescent vitamin that participates in macronutrient and energy metabolism (fats, proteins and carbohydrates), has antioxidant functions and activates other vitamins such as folate and pyridoxine. Riboflavin is required by all life forms, and in humans it is important for reproductive function, lactation, successful pregnancy outcomes, development of children and adequate energy levels. The current average requirements are 0.5-1.4 mg riboflavin/day for children and 1.3 mg/day for adults as defined by the European Food Safety Authority (EFSA, 2017, EFSA-Q-2011-01222). According to Oregon State University, during pregnancy, women should have 1.4 milligrams per day, and when breastfeeding, 1.6 milligrams per day (https://lpi.oreqonstate.edu/mic/vitamins/riboflavin). Furthermore, riboflavin deficiency can lead to skin damage, sore throat, oedema of oral and mucous membranes, cheilosis and glossitis. Riboflavin intake can reverse these symptoms, and further improve efficiency of standard therapies against common viral and bacterial infectious diseases (Dey and Bishayi, 2016), such as Staphylococcus aureus infection. Based on the scientific opinion of the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) (2010), authorised health claims in the EU Register for foods which are a source of riboflavin include contribution to normal energy-yielding metabolism, maintenance of the normal function of the nervous system, maintenance of normal skin and mucous membranes, maintenance of normal red blood cells, maintenance of normal vision, contribution to normal metabolism of iron, protection of DNA, proteins and lipids from oxidative damage, reduction of tiredness and fatigue (Commission Regulation (EU) 432/2012 of 16/05/2012). Riboflavin is also used in animal feed to improve animal health, and it is used as food colouring with the food additive E number E101 in Europe. In large-scale industrial settings, riboflavin was traditionally produced by chemical synthesis. However, in the last decades microbial synthesis of riboflavin is increasingly implemented because of economic and environmental considerations. Riboflavin can be obtained from natural overproducer microorganisms, mostly filamentous fungi and yeasts ( Eremothecium ashbyii, Ashbya gossypii and Candida famata) and Bacillus subtilis mutants (Lim et al., 2001 ; Stahmann et al., 2000). While lactic acid bacteria have previously also been shown to produce riboflavin (Thakur et al, 2016), the lactic acid bacterial strains reported so far produce only limited amount of riboflavin, for example in a range of maximum 2.3-3 pg/ml of culture medium (Thakur and Tomar, 2015; Jayashree et al., 2010). Methods have been described to stimulate increased riboflavin production in lactobacilli. However, this requires defined conditions and still have only led to riboflavin concentrations of 5-6 pg/mL medium (Juarez del Valle et al., 2017). While recombinant Lactococcus lactis NZ9000 genetically modified for overexpression of ribG, ribB, ribA and ribH genes, was shown to produce as much as 24 pg riboflavin/ml medium, the use of genetically modified lactobacilli is currently not permitted in food products for human consumption for regulatory reasons. Riboflavin concentrations are typically around 26-30 mg/ml_ in industrial processes with e.g. Eremothecium ashbyii. Nevertheless an important consideration is that these concentrations are obtained with filamentous fungi and yeasts and not with lactobacilli that have a favourable safety profile in humans. Lactic acid bacteria are already widely implemented in the food industry or as probiotic formulations (e.g. Limosilactobacillus reuteri RC-14 and Lacticaseibacillus rhamnosus GG). Therefore, lactobacilli capable of enhanced vitamin production are of great interest for vitamin enrichment of food, feed and various probiotic formulations, supplements and even medicines. The riboflavin concentrations naturally occurring in previously described lactobacilli would not meet the needs of industrial production or food fermentation on a large scale, considering the current average requirements for children and adults.

Lactobacilli naturally overproducing riboflavin can thus prevent or treat riboflavin deficiency in humans and animals by providing a cost-effective and safe food fortification solution, or a probiotic or postbiotic product. Their administration to humans and animals can improve systemic and/or mucosal immune health, resistance to pathogens, macronutrient/energy metabolism and antioxidant function, and reproductive and offspring health.

SUMMARY OF THE INVENTION The present invention is based on the identification of novel Limosilactobacillus strains that show an increased production of riboflavin. In particular the inventors found that said Limosilactobacillus strains have alterations in genes that are associated with the riboflavin pathway. More specifically, the present invention is based on the identification of a novel isolated strain of the Limosilactobacillus reuteri (L reuteri) species. Said strain has been deposited with the Belgian Co-ordinated Collection of Micro-Organisms (BCCM), Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium) on October 9, 2020 with accession number LMG P- 32020, and herein further also indicated as L. reuteri AMBV339 or AMBV339 strain.

Typical for this novel isolated strain of the L. reuteri species is that it has a naturally occurring overproduction of riboflavin, also known as vitamin B2. In particular, the AMBV339 strain is capable of producing high levels of riboflavin, in particular as compared to other known strains of lactobacilli.

Therefore, in a first aspect, the present application is thus directed to one or more isolated Limosilactobacillus bacterial strains that have a naturally occurring overproduction of riboflavin. In particular, the present application discloses one or more isolated Limosilactobacillus bacterial strains that have alterations in one or more genes or regulatory genetic sequences that are associated with the riboflavin production pathway. In a particular embodiment, said strain of the present invention has an alternation in one or more genes or upstream regulatory sequences thereof compared to said genes or regulatory sequences of the Limosilactobacillus reuteri type strain DSM20016. In another particular embodiment, said genes are selected from the genes for 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1 .99.12) and/or GTP cyclohydrolase II (EC 3.5.4.25), Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26) and/or 5-amino-6-(5-phosphoribosylamino)uracil reductase (EC 1.1.1.193), 6,7-dimethyl-8- ribityllumazine synthase (EC 2.5.1.78), Riboflavin synthase eubacterial/eukaryotic (EC 2.5.1.9), FMN adenylyltransferase (EC 2.77.2) and/or Riboflavin kinase (EC 2.7.1.26), RibT protein, riboflavin biosynthesis acetyltransferase (GNAT) family, or substrate-specific component RibU of riboflavin ECF transporter.

In particular, such regulatory genetic sequences are selected from the promoters, riboswitches or other regulatory sequences upstream of said genes or genetic operons they belong to. In a specific embodiment, the present application discloses one or more isolated Limosilactobacillus bacterial strains that have alterations in the regulatory region upstream of genes or genetic operon involved in riboflavin production, in particular the genes for Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26) and/or 5-amino-6-(5- phosphoribosylamino)uracil reductase (EC 1.1.1.193) also known as ribD, Riboflavin synthase eubacterial/eukaryotic (EC 2.5.1.9) also known as ribE, 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12) and/or GTP cyclohydrolase II (EC 3.5.4.25) also known as ribBA and/or 6,7-dimethyl-8-ribityllumazine synthase (EC 2.5.1 .78) also known as ribH.

In a very specific embodiment, said strain of the present invention comprises an alteration at base 931 ,824 in the genome assembly NC_009513.1 of said type strain, in particular said alteration is a guanine to thymine alteration.

More specifically, the isolated Limosilactobacillus bacterial strain that shows alterations in one or more genes or regulatory sequences upstream of said genes that are associated with the riboflavin production pathway is the Limosilactobacillus reuteri (L reuteri) species, said strain deposited at the BCCM under accession number LMG P-32020. In a further aspect, the present application provides an isolated bacterial strain of the Limosilactobacillus reuteri ( L reuteri) species, said strain deposited at the BCCM under accession number LMG P-32020.

In accordance with an embodiment of the present application, the bacteria of the isolated bacterial strain are in suspension, freeze-dried, spray-dried, in live or inanimate/postbiotic form, or in prebiotic or synbiotic formulations, provided that active components are not disrupted.

In another further embodiment, the present application provides a composition comprising an isolated bacterial strain of the L reuteri species deposited under accession number LMG P- 32020. In a further embodiment, said composition comprises one or more pharmaceutically acceptable excipients, aromatizing agents or carriers. In an even further embodiment, said composition can optionally comprise other probiotic bacteria.

In accordance with an embodiment of the present application, the composition comprises an amount of bacteria in the range between 10 ³ to 10 ¹¹ colony-forming units (CFU) for each gram of the composition.

The present application is based on the finding that the isolated bacterial strain L reuteri AMBV339 shows a naturally occurring overproduction of riboflavin. Therefore, in an embodiment, the isolated bacterial strain or the composition of the present application is for use in the treatment and/or prevention of diseases associated with reduced levels of riboflavin in a subject. In a further aspect, said diseases associated with reduced levels of riboflavin are selected from the group comprising diseases of the nervous system, skin diseases, respiratory diseases, diseases associated with reduced levels of red blood cells, eye diseases, diseases associated with disturbed metabolism of iron, diseases associated with oxidative stress, general fatigue or general illness, urogenital diseases, metabolic diseases (e.g. associated with reduced vitamin levels), headache or migraine, pregnancy, gastrointestinal diseases, immune diseases, cancer and associated, viral, fungal and bacterial infections. More specifically, the isolated bacterial strain or the composition of the present invention is for use in the treatment and/or prevention of diseases associated with reduced riboflavin levels.

Alternatively, said strains or compositions of the invention are suitable for use in the prevention or treatment of gut microbiome dysbiosis, the promotion of gut microbiome resilience, and/or the promotion of health-associated gut taxa.

In another aspect, the non-therapeutic use of the isolated bacterial strain or the composition according to the present invention is disclosed for the prevention and/or reduction of general fatigue. In still another aspect, the non-therapeutic use of the isolated bacterial strain orthe composition according to the present invention is disclosed in cosmetics or personal care products.

In accordance with another embodiment, the use of the isolated bacterial strain or the composition according to the present invention in a process for the production of riboflavin is disclosed. In particular, said process for the production of riboflavin is a fermentation process.

In another aspect, the use of the isolated bacterial strain orthe composition according to any of the embodiments of the invention as a probiotic or postbiotic, or in a prebiotic or synbiotic formulation, is disclosed. For example, It can be administered in oral or topical probiotic or postbiotic formulations.

In still another aspect, the use of the isolated bacterial strain or the composition according to any of the embodiments of the invention in the food or feed industry is disclosed.

In another aspect, the use of the isolated bacterial strain or the composition according to any of the embodiments of the invention as a colouring agent or as a starter culture in the food or feed industry is disclosed.

In accordance with another embodiment, the present application provides a process for the production of riboflavin. Said process is characterized in that the bacterial strain or the composition according to an embodiment of the present application, is incubated in an aqueous medium under conditions that allow that production of riboflavin.

In a further embodiment, said process for the production of riboflavin comprises the steps of: a) providing a bacterial strain or composition according to an embodiment of the present invention, b) incubating said bacterial strain or said composition in aqueous cultivation medium under conditions that allow the production of riboflavin; and optionally c) isolating and purifying the riboflavin from the aqueous cultivation medium.

BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. Fig. 1: Regulatory intergenic region upstream of the riboflavin operon of L. reuteri ( ribD , ribE, ribBA and ribH genes) with a guanine to thymine mutation in riboflavin- overproducing L. reuteri AMBV339 compared to non-riboflavin overproducing L. reuteri strains AMBV336, AMBV337, AMBV368, AMBV369 and AMBV371, and the type strain L. reuteri DSM20016.

Fig. 2: Estimation of production of riboflavin by L. reuteri AMBV339.

(A) Fluorescence characteristic of riboflavin quantified in L. reuteri AMBV339 culture, cell-free culture supernatant and cell pellet compared to a selection of other L. reuteri strains and the commercial probiotic strains Lacticaseibacillus rhamnosus GG and L. reuteri RC-14] p<0.05 indicates statistically significant higher riboflavin fluorescence of the AMBV339 condition compared to all other strains. (B & C) Quantification of riboflavin concentrations in cultures of L. reuteri AMBV339 and other vaginal L. reuteri AMBV336 and AMBV368, and the commercial probiotics L. reuteri RC-14 and L. rhamnosus GG based on (A) fluorescence and (B) absorbance of cultures plotted against a standard curve of riboflavin dilutions. Measured values for the riboflavin standard curve in MRS are depicted as grey circles with corresponding riboflavin concentrations depicted on the x-axis. Fluorescence and absorbance measurements of L reuteri AMBV339 (A), AMBV336 (T), AMBV368 (¨), RC-14 (■) and L rhamnosus GG (·) are plotted within the riboflavin standard curve.

Fig. 3. (A) Growth and (B) fluorescence characteristic of riboflavin quantified in L. reuteri AMBV339 cultures in food matrices compared to L. reuteri AMBV336 and L. reuteri RC- 14; (C) Fluorescence characteristic of riboflavin quantified in L. reuteri AMBV339 cocultured with starter Streptococcus thermophilus. p<0.05 indicates statistically significant higher riboflavin fluorescence of the AMBV336, AMBV339 or RC-14 condition compared to the growth medium condition.

Fig. 4. Longitudinal survival of L. reuteri AMBV339 in (A) gastric juice at different pH, or

(B) in different parts of a simulated gastrointestinal tract inoculated with human fecal microbiota; (C) Bray-Curtis dissimilarities between the microbial community composition of the L. reuteri AMBV339 in feces condition and feces as such (0.9% NaCI and coconut milk). For the simulated gastrointestinal tract L reuteri AMBV339 in a 0.9% NaCI solution (or saline) or L reuteri AMBV339 culture in coconut milk was added. Data is presented as means with standard deviation. DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound. The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

The present invention is based on the identification of a novel L reuteri strain, that was isolated from a healthy human vaginal sample.

This strain is characterized by an alteration in one or more genes or regulatory genetic sequences that are associated with the riboflavin production pathway. Specific for the novel isolated bacterial strain is that it exhibits natural riboflavin-overproducing properties.

In the context of the present invention, the term alteration is meant to be a modification of the genomic sequence compared to the herein disclosed comparative strains such as the other AMBV strains or the type strain. In particular, such modification may constituted a mutation, deletion, insertion,... in the genomic sequence of the strain.

In particular, the inventors identified that the L reuteri AMBV339 strain comprises riboflavin synthesis pathway genes that are required for riboflavin production. More specifically, growth of the L reuteri AMBV339 strain in de Man-Rogosa-Sharpe (MRS) medium or the tested food matrices showed that the AMBV339 cultures exhibited a yellow-green fluorescence characteristic for the presence of riboflavin, which was not present in the other L reuteri strains that were tested. The riboflavin concentration in the supernatant of the L reuteri AMBV339 strain was quantified at approximately 18-40 pg riboflavin/ml using fluorescence-, absorbance- and HPLC/UV-based methods. According to the prior art related to riboflavin production by lactic acid bacteria, these riboflavin concentrations are at least 3-20 times higher than the maximum riboflavin production levels previously reported for other lactic acid bacteria strains, depending on the detection method used.

Due to their fermenting and probiotic properties, lactobacilli are often used in food/feed applications and supplements. Indeed, also the L reuteri AMBV339 strain has a Qualified Presumption of Safety (QPS) status, indicating that it can be directly used in feed, food and health applications. Furthermore, the AMBV339 strain also is efficiently growing and yielding high biomass overnight under the tested conditions.

The present application thus provides the isolated bacterial L reuteri AMBV339 strain, typically characterized by a high level of riboflavin production. Further, also a composition comprising the L reuteri AMBV339 strain is provided. The L reuteri AMBV339 strain has been deposited with the Belgian Co-ordinated Collection of Micro-Organisms (BCCM) on October 9, 2020 with the accession number LMG P-32020. The isolated AMBV339 strain or a composition comprising said strain is thus capable of producing riboflavin or vitamin B2 in high quantities.

In accordance with an embodiment of the present invention, the bacteria of the isolated bacterial AMBV339 strain can be provided in suspension, freeze-dried, spray-dried in live or postbiotic, prebiotic of synbiotic form, provided that the active components are not inactivated. In the context of the present invention, by means of the term “live form”, “probiotic” and “synbiotic” form, reference is made to a form wherein the bacteria are alive. In the context of the present invention, by means of the term “postbiotic form” or “prebiotic form”, reference is made to a form wherein the bacteria are not alive, such as in the case of inanimate applications or tyndalized versions of the bacteria or their molecular products.

In the context of the invention, the following definitions are used:

Probiotic = live microorganisms that, when administered in adequate amounts, confer a health benefit on the host

Postbiotic = A preparation of inanimate microorganisms and/or their components that confers a health benefit on the host

Prebiotics = a substrate that is selectively utilized by host microorganisms conferring a health benefit

Synbiotic = a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host

Also a composition comprising the isolated bacterial stain AMBV339 is provided. The preparation of the compositions of the invention can be implemented by freeze-drying or spraydrying of bacterial cultures, mixing the dried bacteria both in suspension with water or with further suitable excipients and optionally with addition of further active principles. As used herein, a “composition”, refers to any mixture of two or more products or compounds (e.g. agents, modulators, regulators, etc.). It can be a solution, a suspension, liquid, powder or a paste, aqueous or non-aqueous formulations or any combination thereof. In the context of the present invention, the compositions are preferably pharmaceutical or cosmetic compositions, comprising one or more pharmaceutically excipients, or diluents, such as suitable sugars, copolymers PEG/PPG, or cryoprotectants.

In one embodiment, said composition comprises one or more pharmaceutically acceptable excipients, aromatizing agents or carriers. Examples of excipients that can be selected in such compositions are rubber, xanthan, carboxymethyl cellulose, silicone, Vaseline, white soft paraffin, magnesium stearate, maltodextrin, mannitol, starch, glucose, trehalose, glycerine, propylene glycol, lactose, and similar.

The compositions may also comprise aromatizing agents; such as thyme or any extract thereof. In accordance with an embodiment of the present invention, the carriers provide an improvement of the bioavailability, the stability and/or the endurance of the microorganism.

The compositions may thus further comprise one or more carriers in order to improve the bioavailability, the stability and the endurance of the microorganism. The carrier may also improve the adhesion of the bacterial strain to a suitable surface, for example the mucosal surface. Such a carrier can for example by exopolysaccharides produced by S. salivahus or lactobacilli. Further, the carrier may be a heat-sensitive polymer able to increase the viscosity and thus the adhesiveness by increasing the temperature or Gantrex for example. In another embodiment, the carrier can be hydroxypropyl methylcellulose (HPMC).

In accordance with an embodiment of the present invention, the composition comprises an amount of bacteria that is preferably in the range between 10 ³ to 10 ¹¹ CFU for each gram of the composition.

In a further aspect, the present invention relates to the isolated bacterial strain AMBV339 or the composition comprising said strain according to the invention for use as a medicament in human or veterinary medicine. In particular, the isolated bacterial strain or the composition according to the invention is for use in the treatment and/or prevention of diseases associated with reduced levels of riboflavin in a subject. In another embodiment, the isolated bacterial strain or the composition of the present invention is for use in the treatment and/or prevention of diseases for which a boost of riboflavin is desirable in a subject. In a further aspect, the diseases associated with reduced levels of riboflavin are selected from the group comprising diseases of the nervous system, skin diseases, respiratory diseases, diseases associated with reduced levels of red blood cells, eye diseases, diseases associated with disturbed metabolism of iron, diseases associated with oxidative stress, general fatigue or general illness, urogenital diseases, metabolic diseases (e.g. associated with reduced vitamin levels), headache or migraine, pregnancy, gastrointestinal diseases, immune diseases, cancer and associated, viral, fungal and bacterial infections. More specifically, the isolated bacterial strain or the composition of the present invention is for use in the treatment and/or prevention of diseases associated with reduced riboflavin levels.

In another aspect, the present invention relates to a method of treatment of a disease in a subject by administration of the isolated bacterial strain AMBV339 or the composition comprising said strain to a subject. In particular, the disease can be selected from diseases for which a boost of riboflavin is desirable. In a further aspect, said diseases associated with reduced levels of riboflavin are selected from the group comprising diseases of the nervous system, skin diseases, respiratory diseases, diseases associated with reduced levels of red blood cells, eye diseases, diseases associated with disturbed metabolism of iron, diseases associated with oxidative stress, general fatigue or general illness, urogenital diseases, metabolic diseases (e.g. associated with reduced vitamin levels), headache or migraine, pregnancy, gastrointestinal diseases, immune diseases, cancer and associated, viral, fungal and bacterial infections. More specifically, the invention is related to a method of treatment of a disease associated with reduced riboflavin levels in a subject by the administration of the isolated bacterial strain AMBV339 or the composition comprising said strain to the subject.

The terms “treatment”, “treating”, “treat” and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” covers any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptoms, i.e. arresting its development; or (c) relieving the disease symptoms, i.e. causing regression of the disease or symptom. In the context of the present invention, the terms “prevention” and the like refer to preventing a disease or conditions from happening. In the context of the present invention, the term “immunomodulation” refers to the process of altering an immune response to a desired level and/or direction.

In a further aspect, the isolated bacterial strain or composition of the present invention can be used in the non-therapeutic reduction and/or prevention of general fatigue. Thus, the present application also provides the non-therapeutic use of the isolated bacterial strain or composition of the invention for the prevention and/or reduction of general fatigue or weakness. The term “non-therapeutic” and the like refers to obtaining a desired physiological effect in a non-disease context, and thus before any signs of a disease are present in the subject. In another aspect, the use of the isolated bacterial strain or composition of the present invention is disclosed as a supplemental intake of riboflavin. In another aspect, the non-therapeutic use of the isolated bacterial strain or composition of the present invention in cosmetics or personal care products is disclosed. In a specific embodiment, the non-therapeutic use of the isolated bacterial strain or composition of the present invention in cosmetics is disclosed. The form of the cosmetics of the present invention is not particularly limited, and it is possible to provide the cosmetics as emulsion, liquid, cream, solid, or paste. The cosmetics can also be provided as agent, gel, powder, mousse or spray. For example, the cosmetics can be selected from skin cosmetics, makeup or hairdressing cosmetics. Skin cosmetics can include cleaning products, anti-transpiration products, hydration products, UV resistance products, etc. Hair-dressing cosmetics can include hair cleaning products, such as shampoo, hair coloring products, products for improving hair-growing, hair rinse products or hair conditioning products. The cosmetic products can also include cleansing gels, cleansing cream, cleaning foam, cleaning lotion, liquid soaps, hand soaps, bath oil, saving cream. Makeup cosmetics may include foundation powder, face powder, lipstick, lip gloss, eyeliner, eye cream, mascara, rouge, manicure oil and other products, nail polish.

In a specific embodiment, the non-therapeutic use of the isolated bacterial strain or composition of the present invention in personal care products is disclosed. Said personal care products may include but are not limited to personal hygiene and cleansing products, oral care products such as tooth paste, eye care products, ear care products, sanitary products.

One aspect of the present application discloses the use of the isolated bacterial strain or composition according to the invention as a probiotic or postbiotic. Probiotics are defined as live microorganisms that exert beneficial effects on the host when administered in adequate amounts. In the context of the present invention, for postbiotics is intended any factor resulting from the metabolic activity of a probiotic or any released molecule capable of conferring beneficial effects to the host in a direct or indirect way. The term “postbiotic”, “fermented product” or “fermented supernatant” in the context of the present invention may thus comprise also any factor resulting from the metabolic activity of the isolated bacterial strain of the present invention, capable of conferring beneficial effects to the host in a direct or indirect way.

In a further aspect, the use of the isolated bacterial strain or composition according to the invention in the food or feed industry is provided; in particular in the production of dairy and nondairy fermentation products, dietary supplements, dietary food additives and/or nutraceuticals. Said food industry can thus encompass fermented food products (dairy-based worth, soy). Said food industry can also include the bioreactors and processing environments used in the production of food products, wherein the bacterial strain or the composition of the present invention can be added to the food products during production. In a specific embodiment, the non-therapeutic use of the isolated bacterial strain or composition of the present invention in food supplementation is thus disclosed. The isolated bacterial strain or composition can thus be provided as a food supplement, alternatively it may suitably be used as a colouring agent in the food or feed industry.

In still another aspect, the use of the isolated bacterial strain or the composition of the present invention is disclosed in a process for the production of riboflavin. Since the isolated bacterial strain of the present invention is characterized for its high levels of naturally occurring riboflavin production, it is highly suitable for the production of riboflavin.

In a further embodiment, the present application also discloses a process for the production of riboflavin. In said process the bacterial strain or the composition according to any of the disclosed embodiments is incubated in aqueous medium under conditions that allow the production of riboflavin from a given substrate. The aqueous medium is typically an aqueous medium comprising for instance salts, substrate(s), and a certain pH. The medium is also referred to as production medium.

The process may thus comprise the steps of: (a) providing a bacterial strain or a composition according to any of the disclosed embodiments, (b) incubating said bacterial strain or said composition in aqueous cultivation medium under conditions that allow the production of riboflavin; and optionally (c) isolating and purifying the riboflavin from the aqueous cultivation medium.

“Fermentation” or “production” or “fermentation process” as used herein may be the use of growing bacterial cells using media, conditions and procedures known to the skilled person.

The produced riboflavin may be recovered from the cells by any suitable means. Recovering means for instance that the produced riboflavin may be separated from the production medium. Optionally, the thus produced fermentation product may be further processed, e.g. purified.

In connection with the process according to the present invention, the growing step of the bacteria can be performed in an aqueous medium, i.e. the growth medium, supplemented with appropriate nutrients for growth normally under aerobic conditions. The cultivation may be conducted, for instance, in batch, fed-batch, semi-continuous, or continuous mode.

The terms “production” or “productivity” are art-recognized and include the concentration of riboflavin formed with a given time and a given fermentation volume (e.g. kg product per hour per liter). The term “efficiency of production” includes the time required for a particular level of production to be achieved. The term “yield” is art-recognized and includes the efficiency of the production of riboflavin.

The isolated bacterial strain or the composition according to the present invention can be in any form suitable to be administered topically, orally, or through the respiratory tract. Further, the bacterial strain or composition according to the present invention can be in a pharmaceutical form selected from, but not limited to, a spray, cream, lotion, gel, an ointment, a solution, a suspension, an emulsion, a capsule, a tablet, a powder, a granule, drops, inhaler, tooth paste, mouth wash. The bacterial strain or composition can further be formulated in such a manner that it can be administered through the respiratory tract by a nebulizer, with or without propellants. Finally, and in a preferred embodiment, the bacterial strain or composition can also be formulated in such a manner that it can be administered via vaginal administration. For example, the bacterial strain or composition of the present invention can be provided as a vaginal wash solution, vaginal soap, vaginal cream, tampon or vaginal pill.

As used herein, the term “subject” refers to any living organisms. The subject can thus be a mammal, a fish or a bird. Preferably, it refers to a human subject or a non-human mammal. Even more preferably, the subject is a human subject. In another particular embodiment, the subject is a production or farm animal, or a pet. The production or farm animal can be selected from the group comprising a pig, sheep, a goat, a cow, a horse, a chicken, a goose, a turkey, or a rabbit. The pet can be selected from a cat or a dog.

EXAMPLES

MATERIALS AND METHODS

Whole-genome sequencing of the L reuteri AMBV339strain and gene annotation The strain was grown overnight in de Man-Rogosa-Sharpe (MRS) medium (Carl Roth, Karlsruhe, Germany). In duplicate, 1 .5 ml culture was transferred to a sterile tube and ampicillin was added at 100 pg/ml. After 1 h of incubation at 37°C, the culture was centrifuged for 3 min at 12.000 g. The resulting cell pellets were washed three times with sterile NaCI-EDTA (0.8766g NaCI and 0.292 g EDTA in 0.5 L distilled water) and resuspended in 100 pi NaCI-EDTA. Then, 100 pi of lysozyme (10 mg/ml) and 1 pi of RNase (20 mg/ml) were added for 1 h at 37°C. Next,

229 pi NaCI-EDTA, 50 pi 10% SDS and 20 pi Proteinase K (20 mg/ml) were added, vortexed and incubated for 1 h at 55°C. After incubation, 200 pi of a cold protein precipitation solution (6 ml of 5M CH3COOK, 1.15 ml Glacial acetic acid and 2.85 ml distilled water) was added and vortexed at maximum speed for 20 sec and incubated on ice for 5 min. Then, the solution was centrifuged for 3 min at 4°C and 12.000 g. The supernatant was transferred to a clean 1.5 ml tube and centrifuged for 3 min at 4°C and 12.000 g. The resulting supernatant was transferred to a clean 1.5 ml tube and DNA was precipitated with 600 pi of ice-cold isopropanol. After a centrifugation step of 3 min at 4°C and 12.000 g the supernatant was discarded and the pellet was resuspended with 600 pi fresh 70% EtOH. After a centrifugation step of 3 min at 4°C and 12.000 g the supernatant was discarded and the pellet was left to air-dry until all the ethanol evaporated. The DNA pellet was dissolved in 50 pi distilled water and incubated for 5 min at 55°C.

Whole-genome sequencing was performed using the Nextera XT DNA Sample Preparation kit (lllumina, San Diego, CA, USA) and the lllumina MiSeq platform, using 2 x 250 cycles, at the Laboratory of Medical Microbiology (University of Antwerp, Antwerp, Belgium). Gene annotation was performed using the Pathosystems Resource Integration Center (PATRIC, www.patricbrc.org) and presence of riboflavin genes was determined based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathways (https://www.qenome.ip/keqq/pathwav.html).

Riboflavin quantification based on fluorescence

Quantitative analysis of riboflavin production in culture medium based on fluorescence was performed according to a modified method previously described. Lactobacilli were either inoculated in MRS broth as growth medium (Difco) and incubated statically overnight at 37°C, or inoculated in commercially obtained food matrices (coconut milk, soy milk, whole and semi- skimmed cow milk, butter milk, apple juice or MRS broth as control) and incubated statically for 96h at 37°C.

To obtain cell-free culture supernatant, the bacterial culture in MRS was subsequently centrifuged (2486 g, 10 min) and the supernatant was filter sterilized (through a 0.2 pm filter) to obtain cell-free culture supernatant. The resulting cell pellet was washed twice in phosphate- buffered saline (PBS) and resuspended in fresh MRS. For fluorescence measurements, the fluorescence of 100 pi of the whole cell culture, cell-free supernatant or cell pellet was measured in a 96-well plate using the Synergy HTX Multi-mode microplate reader (BioTek) with the 485/20 excitation filter and 520/20 emission filter. Serial dilutions of riboflavin powder in MRS obtained commercially were used to obtain a standard curve as reference for quantification. Statistical analysis was performed with a two-way ANOVA.

Riboflavin quantification based on absorbance

Quantitative analysis of riboflavin production in culture medium based on absorbance at 444 nm was performed according to Sauer et al. (1996). Lactobacilli were inoculated in MRS medium and incubated statically overnight at 37°C. The culture was subsequently centrifuged (2486 g, 10 min) and the supernatant was filter sterilized (through a 0.2 pm filter) to obtain cell-free culture supernatant. Then, 0.2 ml of 1 M NaOH were added to 0.8 ml of cell-free culture supernatant, and 0.4 ml of the resulting solution was neutralized with 1 ml of 0.1 M potassium phosphate buffer (pH 6.0). The absorbance at 444 nm was measured in a 96-well plate using the Synergy HTX Multi-mode microplate reader (BioTek). Serial dilutions of riboflavin powder in MRS obtained commercially were used to obtain a standard curve as reference for quantification. Statistical analysis was performed with a two-way ANOVA. Riboflavin quantification with HPLC-UV

Riboflavin concentration was measured in cell-free culture supernatant samples with a standard isocratic method for HPLC-UV based on that from the European Pharmacopoeia methodology for vitamin B12. Lactobacilli were either inoculated in MRS broth as growth medium (Difco) and incubated statically overnight at 37°C, or inoculated in commercially obtained food matrices (coconut milk, butter milk or MRS broth as control) and incubated statically for 96 h at 37°C. To obtain cell-free culture supernatant, the bacterial culture was subsequently centrifuged (2486 g, 10 min) and the supernatant was collected. For HPLC-UV measurements, samples were diluted with both medium and mobile phase from the HPLC, and tested in duplicate with 2 standard series. Samples and control were also spiked with riboflavin.

Quantification of bacterial growth

Bacterial growth in food matrices was quantified by preparing serial tenfold dilutions of bacterial cultures in sterile PBS and plating them out on MRS agar. The plates were incubated at 37°C for 1-2 days and colonies corresponding to each serial dilution were subsequently counted. Bacterial growth was expressed as colony-forming units (CFU)/ml culture.

Survival and microbiome modulation testing of L. reuteri AMBV339 in a simulated gastrointestinal tract system

Live L reuteri AMBV339 washed and resuspended in saline (0.9% NaCI) at ~1.5c10 ^L 9 CFU/ml or L reuteri AMBV339 cultured in coconut milk were added to a simulated human gastrointestinal system set-up cfr. Mortele et al. (2019). Briefly, the system consisted of vessels representing the stomach (37°C, volume: 34 ml, pH 2-4, reagents added: 16 ml), small intestine (37°C, volume: 50 ml, pH 7.5, reagents added: 15 ml) and different parts of the large intestine (37°C, anaerobic, volume: 65 ml, pH: 5.8, reagents added: 55 ml). The small and large intestine parts were inoculated with human fecal microbiota. Samples were taken from the system at regular time points to assess L reuteri AMBV339 survival via plating out on MRS agar, and to analyze the fecal microbiome composition using 16S rRNA sequencing with subsequent bioinformatics analysis in R studio.

RESULTS

Genes of the riboflavin production pathway are present in Umosilactobacillus reuteri AMBV339

The whole genome of the L reuteri AMBV339 strain has been sequenced and annotated. All genes of the riboflavin synthesis pathway required for riboflavin production and possibly transport have been detected in the genome of L. reuteri AMBV339, including those for:

• 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1.99.12) / GTP cyclohydrolase II (EC 3.5.4.25) • Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26) / 5-amino-6- (5-phosphoribosylamino)uracil reductase (EC 1.1.1.193)

• 6,7-dimethyl-8-ribityllumazine synthase (EC 2.5.1 .78)

• Riboflavin synthase eubacterial/eukaryotic (EC 2.5.1 .9)

• FMN adenylyltransferase (EC 27.7.2) / Riboflavin kinase (EC 2.7.1 .26)

• RibT protein, riboflavin biosynthesis acetyltransferase (GNAT) family

• Substrate-specific component RibU of riboflavin ECF transporter

A naturally present genetic alteration compared to several other non-riboflavin-overproducing L reuteri isolates such as L reuteri AMBV336 and L reuteri AMBV337, as well as the type strain L reuteri DSM20016, has been detected in L reuteri AMBV339 using a bioinformatics-based alignment of the strain genomes. The genetic alteration was found to be specifically in the regulatory region upstream of the gene cluster responsible for riboflavin synthesis comprising the sequences for Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26) / 5-amino-6-(5-phosphoribosylamino)uracil reductase (EC 1.1.1.193), Riboflavin synthase eubacterial/eukaryotic (EC 2.5.1.9), 3,4-dihydroxy-2-butanone 4-phosphate synthase (EC 4.1 .99.12) / GTP cyclohydrolase II (EC 3.5.4.25) and/or 6,7-dimethyl-8-ribityllumazine synthase (EC 2.5.1.78) as depicted in Figure 1. In the genome assembly NC_009513.1 (https://www.ncbi.nlm.nih.gov/nuccore/NC 009513) of the strain L. reuteri DSM20016 (type strain of the species; https://lpsn.dsmz.de/species/limosilactobacillus-reuteri), the location ofthis genetic alteration is at base 931 ,824 (guanine in L reuteri DSM20016 to thymine in L reuteri AMBV339).

Riboflavin is produced by L reuteri AMBV339 in high concentrations in laboratory medium

High levels of fluorescence characteristic of riboflavin (excitation at approx. 440 nm and emission at approx. 520 nm) were observed in L reuteri AMBV339 whole culture grown overnight in MRS and the corresponding cell-free culture supernatant (Fig. 2A). The fluorescence was much lower in the cell pellet, suggesting that riboflavin is mostly excreted. Compared to L reuteri AMBV339, the fluorescence detected in whole cultures, cell-free culture supernatants and cell pellets of a selection of other vaginal L reuteri strains AMBV336, AMBV337, AMBV368, AMBV369 and AMBV371 isolated from the same participant, and the commercial probiotic strains L reuteri RC-14 and Lacticaseibacillus rhamnosus GG was negligible, indicating low levels or no riboflavin production.

Initial quantification of riboflavin production in L reuteri AMBV339 supernatant was performed against a standard curve based on serial dilutions of pure riboflavin in MRS. Two complementary methods were implemented: fluorescence-based detection of riboflavin in solution with excitation at approx. 440 nm and emission at 520 nm and quantitative analysis of riboflavin in solution using absorbance at 444 nm. With both methods, riboflavin in L reuteri AMBV339 supernatant was quantified at approximately 40 pg/ml, while the riboflavin levels in culture supernatants of other vaginal L reuteri AMBV336 and AMBV368, and the commercial probiotics L reuteri RC- 14 and L rhamnosus GG, were close to zero (Fig. 2B & C).

These results were validated using standard HPLC-UV for vitamin detection, which quantified riboflavin in the supernatant of overnight L reuteri AMBV339 culture in MRS at approximately 18.7 pg/ml. No riboflavin was detected in culture supernatants of L rhamnosus GG, while the other L reuteri tested had the following riboflavin concentrations in their overnight culture supernatant: L reuteri AMBV336 0.933 pg/ml, L reuteri AMBV337 0.604 pg/ml, L reuteri L reuteri AMBV368 0.953 pg/ml, L reuteri AMBV369 0.706 pg/ml, L reuteri AMBV370 0.953 pg/ml, L reuteri AMBV371 0.585 pg/ml and L reuteri RC-14 0.423 pg/ml.

L reuteri AMBV339 grows and produces riboflavin in various milk food matrices

Growth (Fig. 3A) and fluorescence as read-out for riboflavin production (Fig. 3B) of L reuteri AMBV339 was assessed in a number of commercially available food matrices: coconut milk, soy milk, whole and semi-skimmed cow milk, butter milk, apple juice or MRS broth as control, and compared to L reuteri AMBV336 and L reuteri RC-14.

All L reuteri strains grew well in coconut milk, soy milk, whole and semi-skimmed cow milk, butter milk and MRS, and their growth was negligible in apple juice (Fig. 3A). L reuteri AMBV339 reached the highest colony-forming units (CFU)/ml in coconut milk (6,07E+10 CFU/ml), soy milk (5,00E+10 CFU/ml), and whole cow milk (4.20E+10 CFU/ml), followed by butter milk (1 ,50E+08 CFU/ml), MRS (6.70E+08 CFU/ml) and semi-skimmed milk (1.07E+07 CFU/ml). Thus, growth of L reuteri AMBV339 in food matrices was similar to the growth of the commercial probiotic strain L reuteri RC-14, especially in coconut milk, soy milk and whole cow milk.

High levels of fluorescence reflective of riboflavin production were detected in L reuteri AMBV339 cultures in all food matrices (coconut milk, soy milk, whole and semi-skimmed cow milk, butter milk), except for apple juice, which is likely due to lack of growth in apple juice. The fluorescence of L reuteri AMBV336 and L reuteri RC-14 in the same food matrices was significantly lower, indicating low levels or no riboflavin production.

Highest riboflavin production by L reuteri AMBV339 relative to the baseline was detected in coconut milk (Fig. 3B). The corresponding fluorescence values were similar to those in MRS, therefore riboflavin production by L reuteri AMBV339 in coconut milk is expected to lie in the range of 18-40 pg/ml. Highest absolute fluorescence was detected in L reuteri AMBV339 cultures in butter milk.

Also when L reuteri AMBV339 was co-cultured in coconut milk with the widely used dairy starter Streptococcus thermophilus, it retained its high fluorescence values indicative of riboflavin production (Fig. 3C). Consequently, it is possible to use L reuteri AMBV339 not only as such, but also in combination with other starter cultures for riboflavin production in fermented foods. L reuteri AMBV339 cells resuspended in a 0.9% NaCI solution or L reuteri AMBV339 culture in coconut milk were also inoculated into a simulated human gastrointestinal tract. L reuteri AMBV339 survived throughout the simulated human gastrointestinal tract (Fig. 4A). Specifically, it survived the conditions of gastric juice at pH 2, 3 and 4 for 90 minutes, as shown in Fig. 4A, resulting in 10 ⁶ - 10 ¹¹ CFU/ml. This indicates that L reuteri AMBV339 can survive the three different acidic conditions of the stomach up to 90 minutes. Also in the small and large intestine, an average of 10 ⁵ CFU/ml L reuteri AMBV339 survived when delivered in coconut milk after 72 hours in the simulated gastrointestinal tract and an average of 10 ⁵ CFU/ml survived when delivered in a 0.9% NaCI solution (Fig. 4B).

Consistent modulation of the fecal microbiome composition was observed due to the addition of L reuteri AMBV339 to the simulated gastrointestinal tract, reflected in increased dissimilarity of the microbiome of feces with added L reuteri AMBV339 compared to feces as such (Fig. 4C). This microbiome modulating effect of L reuteri AMBV339 could have potential beneficial properties on species that can profit from the addition of a riboflavin-overproducing strain such as L reuteri AMBV339.

To conclude, high production of riboflavin by the healthy human isolate L reuteri AMBV339 was demonstrated in laboratory conditions and in food matrices. The genome sequence of L reuteri AMBV339 was shown to contain all genes of the riboflavin synthesis pathway with a mutation in the regulatory genetic sequence, suggesting its natural ability to overproduce riboflavin. Fluorescence-, absorbance- and HPLC-UV-based measurements against riboflavin standard curves quantified natural riboflavin production in L reuteri AMBV339 cultures and cell-free supernatant at approximately 18-40 pg/ml, while riboflavin levels in cultures of all other tested L reuteri strains and L rhamnosus GG were close to zero. Riboflavin production levels by L reuteri AMBV339 are thus significantly higher than those reported for natural production by other lactobacilli in literature (2.3-3 pg/ml). Furthermore, L reuteri AMBV339 grew well in a range of food matrices (coconut milk, soy milk, whole and semi-skimmed cow milk, butter milk) at levels comparable to the commercial probiotic L reuteri RC-14. In all of these food matrices, significant riboflavin production by L reuteri AMBV339 was detected, with highest riboflavin production relative to baseline estimated in coconut milk in the range of 18-40 pg/ml, and highest absolute levels of riboflavin reached in butter milk. Finally, L reuteri AMBV339 survived in simulated conditions of the human gastrointestinal tract and provided fecal microbiome modulation. REFERENCES

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