CN117062614A - Strains, compositions and methods of use - Google Patents

Abstract

The present invention relates to a composition comprising Pediococcus pentosaceus (Pediococcus pentosaceus) LB606R deposited under accession number DSM 33730; lactobacillus plantarum (Lactiplantibacillus plantarum) LB679R deposited under accession No. DSM 33731; or a combination thereof.

Description

Strains, compositions and methods of use

Technical Field

The present invention relates to novel lactic acid bacteria strains, which alone or in combination can be used as probiotics. The invention also relates to pharmaceutical compositions, supplement compositions and products for personal care comprising the strains, alone or in combination. In particular, the invention relates to the use of the strain for the prevention or treatment of candidiasis.

Background

The incidence of fungal infections has increased significantly over the past several decades, mainly due to the rise in antimicrobial resistance and the limited number of potent antifungal agents, which still have many side effects. Among fungi considered as human pathogens, some members of the candida genus are most commonly recovered from fungal infections (Silva et al 2012). The use of broad spectrum antibiotics, denture stomatitis, catheter and parenteral nutrition, the presence of immunosuppression, disruption of mucosal barriers, and the use of chemotherapy and radiotherapy, and dysfunctional microbiomes are among the most important contributors to the development of invasive fungal infections. Candida species contain more than 150 heterogeneous species, but only a few are associated with human candidiasis, as about 65% of candida species cannot grow at a temperature of 37 ℃ to enable them to thrive as human pathogens or symbionts. This genus consists of heterogeneous microbiota, and more than 17 different candida species are known to be causative agents of human infection; however, more than 90% of invasive infections are caused by candida species (Pfaller, MA. & dj. Diekema 2007).

Pathogenicity of candida species is due to certain virulence factors such as the ability to evade host defenses, hyphae formation, adhesion and biofilm formation (on host tissue or on medical equipment) and the production of tissue damaging hydrolases such as proteases, phospholipases and haemolysins (Silva et al 2012).

Biofilms are communities of organisms with remarkable degrees of organization, in which microorganisms form structured, coordinated and functional communities, embedded in self-produced extracellular matrix. Biofilm production is also associated with high levels of antibiotic resistance in the relevant organisms. The ability of candida species to form drug resistant biofilms is an important factor in causing human diseases, but it is also recognized that the more general provision of new medical practices (immunosuppressive therapy, invasive surgery, the use of broad spectrum antibiotics, and diseases caused by dysfunctional microbiota) is also of great importance.

Antifungal agents act by killing fungal cells (e.g., affecting substances in the cell wall, causing leakage and death of the cell contents) or by preventing fungal cells from growing and propagating. There are many classes of antifungal agents: polyenes, including amphotericin, nystatin and pimamycin; azoles including fluconazole, itraconazole, ketoconazole, miconazole, voriconazole, posaconazole and Luo Shakang azole (rosaconazole); echinocandins such as caspofungin and micafungin; and allylamines, including naftifine, terbinafine, morpholino drugs, amorolfine, and griseofulvin; and antimetabolite antifungal agents incorporating 5-fluorocytosine. Common to these drugs is the growing problem in developing resistance, lack of efficacy, and toxicity of the compounds.

Amphotericin B and fluconazole are the most widely used antifungal agents for the treatment of systemic fungal infections. The former is limited in its use due to its high toxicity to the human body. The latter is often used to treat candida infections, although many candida species exhibit fluconazole resistance.

Candida species are able to colonize host tissues as well as abiotic surfaces and develop there into a multi-layer biofilm structure. An important factor related to the adhesion capacity of candida species is the presence of specific proteins, i.e. adhesins, on their cell walls. In candida glabrata (c.glabra), a large class of adhesins is encoded by the Epa (epithelial adhesin) gene family. The overall structure of the Epa protein is similar to that of Als (lectin-like sequence) proteins of Candida albicans (C.albicans). ALS genes are an example of a family of genes that are involved in the pathogenic mechanisms of candida albicans and other candida species.

Biofilm formation capacity may confer ecological advantages to candida species, helping them survive as human symbiota and pathogens by allowing them to evade host immune mechanisms, resist antifungal therapy, and withstand competing pressures from other microorganisms. In addition to being a critical factor in the survival of these species, biofilm formation may also be responsible for the particular suitability of candida species for tissue colonization and colonization of indwelling medical devices.

The adhesion of candida albicans to host cells or polymers is an important and necessary first step in successful colonization and in the development of pathogenesis and infection. Candida albicans can adhere to, for example, acrylic dentures, either directly or through an intermediate layer, which is the first step in the development of the infection process. Several factors related to the adhesion of candida to different surfaces, including the surface structure, nature and composition of the biological material, hydrophobicity and roughness all affect the adhesion of candida.

In candida, so-called virulence factors include not only adhesion, hyphal formation and biofilm formation, but also the ability to destroy host tissues, which may be promoted by release of hydrolytic enzymes to the local environment. General secretion of enzymes such as proteases, phospholipases, lipases and hemolysins aid yeast in survival and replication within macrophages, thus enabling destruction of the epithelium.

Phospholipase (PL) hydrolyzes phospholipids to fatty acids and its production can lead to host cell membrane damage, promote cell damage or expose receptors that promote adhesion. Candida is known to produce lipases and phospholipases, which apparently promote a larger and stronger interface with the host mucosa, destroy the mucosa and support efficient invasion of the relevant tissues. Furthermore, hemolysin is known to be a putative virulence factor that promotes the invasion of candida disseminated pathogenic silk. Recently, several authors have reported that many candida species, including candida glabrata, exhibit different abilities to produce alpha-or beta-hemolysin (Luo et al 2001).

Candida species, including candida glabrata, can grow in a host by metabolic processes that degrade hemoglobin and extract elemental iron from the host cell (Negri M et al 2010). Hemolysin is known to be a putative virulence factor supporting pathogenicity of candida species, but little is known about the gene expression of hemolytic activity.

Antifungal resistance is both complex and versatile. It may be inducible in response to the compound or may be an irreversible genetic change due to prolonged exposure. In particular, these include alterations or even overexpression of the target molecule, active extrusion by efflux pumps, limited diffusion, tolerance and cell density, which are characteristic mechanisms by which fungi act against antifungal therapies. Planktonic cells generally rely on irreversible genetic changes to maintain a resistant phenotype, while biofilm cells are able to persist due to their physical presence and population density, which provides an almost inducible resistant phenotype, independent of defined genetic changes. Thus, candida is inherently less sensitive to commonly used antifungal agents due to its different characteristics.

Candida albicans is a binary symbiotic fungus, and is planted in the skin, mucous membrane, oral cavity and genital tract of healthy human beings. Candida albicans is also the primary opportunistic fungal pathogen, leading to disease manifestations such as disseminated candidiasis and chronic Cutaneous Mucosal Candidiasis (CMC).

Candida species belong to the normal microflora of the oral cavity, skin, gastrointestinal tract and vagina, causing a variety of clinical manifestations of overgrowth from skin mucosa to blood stream infections. Species of candida are once considered nonpathogenic, but soon become the leading cause of many human diseases. These pathological conditions are more recurrent throughout the year and are more difficult to treat, especially when the patient exhibits any level of immunosuppression or microbiome with dysfunctions, e.g., as the diversity of microorganisms decreases. These difficulties stem from the phenotype of candida being able to form biofilms and its high resistance to traditional antifungal therapies. Pathogenic fungi in the candida genus can cause superficial and severe systemic diseases and are now considered the primary causative agent of hospital acquired infections. Many candida infections involve the formation of biofilms on implanted devices such as indwelling catheters or prosthetic heart valves. The candida albicans biomembrane formed on the catheter material in vitro consists of microcolonies of saccharomycetes and hyphae which are closed by a matrix, and is arranged into a double-layer structure.

These biofilms are resistant to a range of antifungal agents currently in clinical use, including amphotericin B and fluconazole, and appear to exist in a variety of drug resistance mechanisms.

Lactic acid bacteria are part of the human intestinal, oral and vaginal microbiota. Lactic acid bacteria play an important role in protecting the human body from infection by producing acids and acidifying, e.g. the vagina, by other antimicrobial products such as hydrogen peroxide H2O2, antimicrobial peptides or biosurfactants.

Many topical, vaginal, oral and systemic medications may kill lactic acid bacteria. Thus, treatment of an infection with an antibiotic may increase the risk of the body being exposed to repeated acquisition of the infection or to new infections caused by resistant microorganisms (e.g. candidiasis).

Antifungal agents have been used successfully for decades to prevent mucosal and invasive fungal infections. However, antifungal prophylaxis has not been completely successful due to drug side effects (nausea, vomiting and diarrhea) and the emergence of resistant strains. The biofilm phenotype and stage of candida are more resistant to all these antifungal agents than their planktonic counterparts. The limited effectiveness and the progressive emergence of antifungal drug resistance are alarming, and alternative therapies are therefore urgently needed.

In view of the limitations of currently available antimicrobial compounds, the use of probiotic microorganisms to combat candidiasis is an attractive alternative to the treatment or prevention of candidiasis.

Lactic acid bacteria for intravaginal or oral use have been marketed in the form of probiotic preparations in health food shops or dairy products for over 50 years. These products include pessaries containing lyophilized lactic acid bacteria species. These products are essentially ineffective in treating infections because they do not colonize the vagina or gastrointestinal tract with exogenous lactobacillus bacteria, do not prevent pathogen biofilm formation, or lack in vivo antimicrobial activity. Typically, traditional probiotics are used to maintain a healthy intestinal flora, but are not known to have any targeted or specific antibacterial mechanisms.

Commercial lactobacillus has proven incapable of preventing the formation of pathogen biofilms, nor has commercial lactobacillus been antimicrobial active against persistent candida infections. Most commercial probiotic strains are not systematically selected for their antimicrobial activity, but for their ability to scale up (Kwak, Y-k.et al 2017).

The ability of candida species to form biofilms results in a high recurrence rate typical of candidiasis and, unfortunately, this makes repeated antifungal therapy unavoidable. Thus, lactic acid bacteria capable of preventing, inhibiting or reducing the development of biofilms by candida species would be advantageous.

Furthermore, there is a need for lactic acid bacteria for use in the treatment of infections, wherein the lactic acid bacteria retain antimicrobial activity in co-cultures, the ability to acidify and prevent biofilm formation in the presence of candida species.

The present invention provides novel probiotic strains and compositions that target at least one virulence mechanism of candida, including antimicrobial activity determined as growth inhibition, biofilm attachment or formation inhibiting or preventing candida's ability to secrete virulence factors such as phospholipase, protease and hemolysis factors.

Disclosure of Invention

The object of the present invention therefore relates to novel lactic acid bacteria and to compositions, topical compositions, vaginal compositions, ocular compositions, otic compositions or oral compositions comprising said novel lactic acid bacteria, and their ability to prevent or treat candidiasis and candidiasis infections.

In particular, it is an object of the present invention to provide new lactic acid bacteria strains and compositions comprising the new lactic acid bacteria strains which solve the problems of biofilm formation in the prior art, in particular of candida pathogens, thereby preventing infections.

Accordingly, one aspect of the invention relates to a composition comprising pediococcus pentosaceus (Pediococcus pentosaceus) LB606R deposited under accession No. DSM 33730; lactobacillus plantarum (Lactiplantibacillus plantarum) LB679R deposited under accession No. DSM 33731; or a combination thereof.

Another aspect of the invention relates to an isolated bacterial strain selected from Pediococcus pentosaceus LB606R as deposited under accession number DSM 33730; and/or one or more bacterial strains selected from lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

Yet another aspect of the invention relates to a composition comprising one or more bacterial strains selected from one or more Pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus (lactplantibiotic) strains for use as a medicament.

Yet another aspect of the invention relates to a composition comprising one or more bacterial strains selected from one or more Pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in the prevention and/or treatment of candidiasis in a human or animal.

Yet another aspect of the invention relates to a composition comprising one or more bacterial strains selected from one or more Pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in preventing, inhibiting or treating biofilm formation.

Yet another aspect of the invention relates to a method for preventing biofilm formation in an environment, wherein the biofilm comprises candida species, the method comprising the steps of: administering to the environment an effective amount of a lactic acid bacteria having antimicrobial activity, wherein the environment is a home, workplace, laboratory, industrial environment, hospital environment, aquatic environment, medical device, dental device, epithelial cell, mucosa, animal body, or human body.

Another aspect of the invention relates to food or feed ingredients and personal hygiene products comprising the composition according to claims 1-6.

Drawings

FIG. 1 shows co-aggregation of Candida albicans strain L26 and LB 606R. Control of candida albicans L26 (fig. 1 a) is shown by combination and copolymerization with LB606R (fig. 1 b), and

FIG. 2 shows the growth inhibition of Candida albicans strain L26 by CFS of LB606R (FIG. 2 a) and LB679R (FIG. 2 b) at different concentrations of LB606R (FIG. 2 a) and LB679R (FIG. 2 b).

The present invention will be described in more detail below.

Detailed Description

Thus, the inventors of the present invention have found specific lactic acid bacteria having antimicrobial activity against candida albicans.

It is believed that the lactic acid bacteria of the present invention are capable of colonizing the mucosa and/or preventing biofilm formation by species of the genus candida pathogenic and/or have antimicrobial activity against species of the genus candida pathogenic, in particular candida albicans.

The lactic acid bacteria may be selected from Pediococcus strains and/or Lactobacillus strains, preferably from Pediococcus pentosaceus LB606R deposited as DSM 33730 at DSMZ German micro-organisms and cell culture Collection Co., ltd (German Collection of Microorganisms and Cell Cultures GmbH) and Lactobacillus plantarum deposited as DSM 33731 at 14/12/2020.

Lactic acid bacteria selected from Pediococcus strains (preferably selected from Pediococcus pentosaceus LB606R deposited as DSM 33730 at DSMZ German microorganisms and cell culture Collection, inc. 12/14/2020).

In an embodiment of the invention, the composition may be a pharmaceutical composition comprising one or more lactic acid bacteria according to the invention and a pharmaceutically acceptable carrier and/or diluent.

Preferably, the pharmaceutical composition comprises 10 per gram ³ To 10 ¹³ Lactic acid bacteria according to the invention in individual colony forming units. More particularly, the pharmaceutical composition comprises 10 per gram ⁶ To 10 ¹² Lactic acid bacteria of individual colony forming units. More particularly, the pharmaceutical composition comprises 10 per gram ⁷ To 10 ¹¹ Lactic acid bacteria of individual colony forming units.

The pharmaceutical composition may be in the form of a suspension, spray, gel, cream, lotion, powder, capsule, oil, lavage solution, ovule, vaginal plug, suppository, lozenge, tablet, microcapsule product or in the form of a food supplement or food item.

The composition may be provided with a therapeutically effective amount of lactic acid bacteria, in particular:

-a therapeutically effective amount of one or more bacterial strains selected from pediococcus strains, most preferably a therapeutically effective amount of pediococcus pentosaceus LB606R deposited under accession number DSM 33730; or alternatively

-a therapeutically effective amount of one or more bacterial strains selected from the group of lactobacillus strains, most preferably a therapeutically effective amount of lactobacillus plantarum LB 679R deposited under accession No. DSM 33731; or alternatively

-combinations thereof.

In the context of the present invention, pediococcus strains such as Pediococcus pentosaceus LB606R deposited under accession number DSM 33730 may be more preferred than Lactobacillus strains such as Lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

In the context of the present invention, the term "therapeutically effective amount" or "effective amount" may refer to the amount of a compound in a composition or formulation that, when administered as part of a desired dosage regimen (to a human or animal, preferably a human), alleviates symptoms, ameliorates conditions or slows the onset of a disease condition according to clinically acceptable criteria for the condition or disorder to be treated or cosmetic purpose, e.g., at a reasonable risk of benefit ratio applicable to any medical treatment.

For practical use, the microorganisms of the present invention may be formulated into a suitable administration form, such as a gel, cream, capsule, tablet, lavage solution, pouch, any food, etc. The unit dose may comprise 10 to 10 of each individual strain ¹³ The preferred dosage per unit dose is in excess of 10 per cell ⁵ Individual cells. More preferred dosages are in excess of 10 per unit dose ⁶ Individual cells.

The bacterial cultures may be stabilized in lyophilized, freeze-dried or microencapsulated form and may be prepared according to conventional methods.

To prepare the aqueous formulation for lavage and irrigation, a two-phase system may be used. For example, a vial with a reservoir containing lyophilized microorganisms is dissolved in a suitable liquid carrier contained in the vial prior to use.

To prepare creams and gels, lactic acid bacteria are stabilized in the formulation. Formulations with low water activity or in the form of microcapsules are preferred.

In an embodiment of the present invention, one or more lactic acid bacterial strains according to the present invention may be provided in a pharmaceutical composition for the prevention and/or treatment of vaginal infections.

In yet another embodiment of the present invention, one or more lactic acid bacteria strains according to the present invention may be provided in a pharmaceutical composition for preventing and/or treating urinary tract infections.

In a further embodiment of the invention, one or more lactic acid bacteria strains according to the invention may be provided in a pharmaceutical composition for the prevention and/or treatment of local, skin, eye, ear or mucosal infections.

In yet another embodiment of the present invention, one or more lactic acid bacteria strains according to the present invention may be provided in a pharmaceutical composition for preventing and/or treating oral infections.

In other embodiments of the invention, the composition according to the invention and/or the one or more lactic acid bacteria are used in combination with a medical device.

Yet another embodiment of the invention relates to a method for treating or preventing a microbial imbalance in the mucosa of a human or animal comprising administering an effective amount of a lactic acid bacterium according to the invention.

In the context of the present invention, the term "preventing" may be art-recognized and, when used in connection with a condition such as local recurrence, may be well understood in the art and includes administration of a composition that reduces the frequency of symptoms of a medical condition in a subject or delays the onset thereof relative to a subject not receiving the composition. Thus, preventing an infection includes, for example, reducing the number of detectable pathogenic microorganisms in a population of patients receiving prophylactic treatment relative to an untreated control population, and/or delaying the occurrence of detectable lesions in a treated population relative to an untreated control population, e.g., by a statistically and/or clinically significant amount.

As used herein, the term "treating" or "treatment" includes reversing, reducing or preventing symptoms, clinical signs and underlying pathology of a disorder in a manner that ameliorates or stabilizes the disorder in a subject.

As used herein, and as is well understood in the art, "treatment" may be a method for obtaining beneficial or desired results, including clinical results. For the purposes of the present subject matter, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilization of (i.e., not worsening) the state of the disease, prevention of disease, delay or slowing of disease progression, and/or amelioration or palliation of the disease state.

The reduction may be at least 10% reduction in severity of the complication or symptom, such as at least 20% reduction in severity of the complication or symptom, for example at least 30% reduction, such as at least 40% reduction, for example at least 50% reduction, such as at least 60% reduction, for example at least 70% reduction, such as at least 80% reduction, for example at least 90% reduction, such as at least 95% reduction, for example at least 98% reduction, such as at least 99% reduction, for example 100% reduction.

The term "lactic acid bacteria" includes species from the families lactobacillaceae, balloon bacteriaceae, bifidobacteriaceae, sarcobacteriaceae, enterococcaceae, leuconostoc and streptococcaceae. These are considered non-pathogenic and are commonly used as probiotics to improve the gastrointestinal flora and treat gastrointestinal symptoms. The lactic acid bacteria according to the invention are preferably selected from lactic acid bacteria strains which inhibit, prevent or reduce biofilm formation. Particularly the biofilm formation of one or more yeasts. More particularly, biofilm formation by pathogenic candida species. Even more particularly, biofilm formation by candida albicans.

The inventors of the present invention have surprisingly found that certain strains of lactic acid bacteria have been shown to be capable of inhibiting, preventing or reducing biofilm formation.

Accordingly, a preferred aspect of the invention relates to a composition comprising pediococcus pentosaceus LB606R deposited under accession No. DSM 33730; lactobacillus plantarum LB679R deposited under accession No. DSM 33731; or a combination thereof.

Preferably, the composition according to the invention comprises Pediococcus pentosaceus LB606R deposited under accession number DSM 33730.

The composition according to the invention may comprise living or non-living cells of the lactic acid bacteria according to the invention.

In embodiments of the invention, one or more pediococcus strains (such as pediococcus pentosaceus LB606R deposited under accession No. DSM 33730) and/or one or more lactobacillus strains (such as lactobacillus plantarum LB 679R deposited under accession No. DSM 33731) may be provided as living cells, as non-living cells, as a lysate, as a fraction, as a ferment, as a metabolite, as an extract, as a derivative, as an analogue, or as a mutant of one of the pediococcus strains or lactobacillus strains according to the invention.

Preferably, one or more pediococcus strains (such as pediococcus pentosaceus LB606R deposited under accession number DSM 33730) and/or one or more lactobacillus strains (such as lactobacillus plantarum LB 679R deposited under accession number DSM 33731) may be provided as living cells, as non-living cells or as lysates.

Embodiments of the present invention relate to a composition comprising a live/live probiotic bacterial strain according to the present invention for use in the treatment, alleviation, inhibition, prevention and/or prevention of the growth of pathogenic microorganisms.

More preferably, the present invention may provide the use of a composition as defined herein for the treatment, alleviation, inhibition, prevention of one or more pathogenic bacterial infections in a human or animal.

In embodiments of the invention, animals may include, but are not limited to, primates, farm animals, sports animals (sport animals), rodents, and pets. More particularly, animals may include mice, rats, hamsters, and guinea pigs; a rabbit; a dog; a cat; sheep; pig; piglets; sow; poultry; a turkey; broiler chicken; mink; a goat; cattle; a horse; and non-human primates such as apes and monkeys.

More preferably, the present invention may provide the use of a composition as defined herein for preventing the growth of pathogenic microorganisms.

The "reduction" in growth may be "statistically significant" as compared to the growth period in the absence of the bacterial strain of the present invention, and may include a 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 99, or 100 percent reduction.

In embodiments of the invention, growth inhibition may be determined as a reduction in growth of at least 25 percent. Preferably, growth inhibition is determined to be at least 50 percent reduction in growth, more preferably growth inhibition is determined to be at least 80 percent reduction in growth, more preferably growth inhibition is determined to be at least 90 percent reduction in growth.

"probiotic" may be defined as viable microorganisms that, when administered or consumed in sufficient amounts, provide health benefits to the host.

The non-living cells according to the invention may be provided as lysates, as fractions, as fermentates, as metabolites, as extracts, as derivatives, as analogues, or as mutants of one of the Pediococcus or Lactobacillus strains according to the invention.

The "lysate", "derivative", "analogue", "fraction" or "extract" may be obtained from dead or killed lactic acid bacteria. These lysates, fractions, derivatives, analogues and extracts preferably have the property of being able to bind or co-aggregate with pathogenic bacteria, thereby preventing the growth of pathogens and/or biofilm formation, wherein the term "lysate" and the term "extract" particularly refer to a solution or suspension of cells and/or metabolites of a microorganism according to the invention in an aqueous medium and comprise, for example, macromolecules such as DNA, RNA, proteins, peptides, lipids, carbohydrates and the like as well as cell debris. The lysate preferably comprises a cell wall or cell wall component comprising binding receptors for co-aggregation. Methods of producing lysates are well known to those skilled in the art and include, for example, using a French press or enzymatic cleavage, a ball mill with glass or iron beads. The cells may be opened enzymatically, physically or chemically. Examples of enzymatic cell lysis may include individual enzymes as well as enzyme mixtures, such as proteases, proteinase K, lipases, glycosidases; chemical cleavage may be induced by ionophores, detergents such as SDS, acids or bases; physical methods may also be implemented by using high pressure such as French press, osmotic pressure, temperature or alternating cold and hot. Furthermore, it is of course possible to combine chemical, physical and enzymatic methods. The "extract" may be any of these cellular components, metabolites, ferments or cellular fractions.

Mutants of one of the Pediococcus strains or Lactobacillus strains according to the invention may be provided by a method of obtaining mutants of one of the following strains: pediococcus pentosaceus LB606 deposited under accession number DSM 33730 with the German collection of microorganisms and cell cultures or Lactobacillus plantarum LB679R deposited as DSM 33731, comprising the use of the deposited strain as starting material and the application of mutagenesis, wherein the obtained mutant retains or enhances the probiotic and/or antifungal and/or antimicrobial properties and/or the ability to inhibit candidiasis.

In embodiments of the invention, the composition may be free or substantially free of viable microorganisms. The composition may comprise cellular material, including dead cells in the form of lysates or ferments. In other embodiments of the invention, the composition may comprise supernatant from fermentation and cellular material having other functional effects.

In an embodiment of the invention, the lactic acid bacteria according to the invention are capable of co-aggregating with or inhibiting candida strains.

The microorganism according to the invention is preferably in isolated or purified form, wherein the term "isolated" particularly means that the lactic acid bacteria originate from its medium, including its natural medium.

The term "inhibit" or "inhibit" as used herein refers to killing a microorganism, such as an undesired microorganism, or controlling the growth of the microorganism. Including inhibiting biofilm formation, which may be the inhibition of initial attachment of microorganisms or the inhibition of further biofilm formation by inhibiting the growth of biofilm-forming microorganisms.

In an embodiment of the invention, the composition and/or lactic acid bacteria according to the invention may inhibit biofilm formation.

The lactic acid bacteria may preferably be one or more pediococcus strains, such as pediococcus pentosaceus LB606R deposited under accession No. DSM 33730; and/or one or more lactobacillus strains, such as lactobacillus plantarum LB 679R deposited under accession No. DSM 33731.

A preferred embodiment of the invention relates to an isolated bacterial strain selected from pediococcus pentosaceus LB606R deposited under accession number DSM 33730; and/or one or more bacterial strains selected from lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

Embodiments of the invention relate to an isolated bacterial strain selected from pediococcus pentosaceus LB606R deposited under accession number DSM 33730; and/or one or more bacterial strains selected from lactobacillus plantarum LB 679R deposited under accession number DSM33731, wherein the strains exhibit antimicrobial activity.

The microorganism according to the invention may preferably be in an isolated form, wherein the term "isolated" particularly means that the lactic acid bacteria originate from its medium, including its natural medium, e.g. isolated from other species.

Antimicrobial activity can normalize dysfunctional vaginal microflora and/or inhibit biofilm formation. In particular, the isolated bacterial strain selected from Pediococcus pentosaceus LB606R deposited under accession number DSM 33730 and/or the bacterial strain or strains selected from Lactobacillus plantarum LB 679R deposited under accession number DSM 33731 may inhibit biofilm formation by one or more yeasts. More particularly, the isolated strain may inhibit biofilm formation by pathogenic candida species. Even more particularly, the isolated strain may inhibit biofilm formation by candida albicans.

The "dysfunctional microflora" or "dysfunctional vaginal microflora" can be determined by neogold scoring (Nugent score) or by diagnosis, which evaluates pH, presence of species of the genus lactobacillus compared to a mixed flora consisting of gardnerella vaginalis (Gardnerella vaginalis), bacteroides species or candida species, or using the amersl standard of bacterial vaginosis (Amsel criterion), which includes pH, evaluation of presence of clue cells, white secretions, and amine odor after mixing with KOH.

The antimicrobial activity may be treatment of candidiasis in humans or animals; or inhibit biofilm formation on a surface as defined herein.

In an embodiment of the invention, the composition inhibits biofilm formation. In particular, the compositions of the invention inhibit biofilm formation by one or more yeasts. More particularly, the compositions of the present invention inhibit biofilm formation by pathogenic candida species. Even more particularly, the compositions of the present invention inhibit biofilm formation by candida albicans.

In this context, inhibition of biofilm formation may be at least 25% biofilm inhibition, such as at least 50% biofilm inhibition, for example at least 75% biofilm inhibition, such as at least 85% biofilm inhibition, for example at least 90% biofilm inhibition, for example at least 95% biofilm inhibition, for example at least 98% biofilm inhibition.

Bacterial lactobacillus plantarum (Lactiplantibacillus plantarum) has previously been called lactobacillus plantarum (Lactobacillus plantarum) and may be a member of the genus lactobacillus (lactplantibilitis) and is common in many fermented foods as well as anaerobic plant matter.

The inventors of the present invention identified and isolated specific strains of Pediococcus pentosaceus (LB 606R/DSM 33730) and Lactobacillus plantarum (LB 679R/DSM 33731) and surprisingly identified one or more specific activities provided by the specific strains, such as inhibition of biofilm formation and/or vaginal microflora for the treatment of dysfunctions.

In embodiments of the invention, the compositions according to the invention may be used to inhibit biofilm formation on surfaces. Preferably, the surface may be a surface in a hospital and/or a surface of a medical device.

In yet another embodiment of the invention, the composition according to the invention may be used for inhibiting biofilm formation in or on the human or animal body.

In the context of this document, biofilm formation may be produced by one or more microorganisms. In particular, the one or more microorganisms may be yeasts. The one or more yeasts may be one or more pathogenic candida species. The one or more pathogenic candida species may be one or more candida albicans.

In an embodiment of the invention, the composition (or lactic acid bacteria) according to the invention may be formulated or used in a medicament, a drug, a food, a feed product, a disinfectant or a personal care product.

The disinfectant according to the invention can be used for treating surfaces to avoid biofilm formation on said surfaces. The surface may be a medical or non-medical surface. The medical surface may be a surface in a hospital or a surface of a medical device. The medical device may comprise a medical device implanted in or used in a human or animal body or a medical device used primarily outside the human or animal body.

The composition according to the invention and/or the one or more isolated bacterial strains of the invention may be used for the treatment of one or more infections caused by: surgical wounds, decubitus ulcers, infections from catheters, stents, heart circulatory devices, prostheses, prosthetic inserts, otology, orthopedic and dental prostheses, screws and nails, oral infections, as well as oral and vaginal mucosal infections, topical infections, otitis media, sinusitis, pharyngitis, laryngitis and pneumonia.

In embodiments of the invention, the composition according to the invention and/or the one or more isolated bacterial strains of the invention may be effective against antibiotic-resistant microorganisms. In particular for antibiotic resistance infections.

The use of the composition (or lactic acid bacteria) according to the invention as a disinfectant may reduce the risk of yeast infections, in particular pathogenic candida infections, even more in particular candida albicans infections.

Embodiments of the present invention relate to one or more bacterial strains selected from one or more Pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains for use as a medicament.

In an embodiment of the invention, the composition consists essentially of one or more bacterial strains selected from one or more pediococcus strains and/or one or more bacterial strains selected from one or more lactobacillus strains for use as a medicament.

In the context of the present invention, the term "consisting essentially of … …" relates to those features or steps which limit the scope of the claims to the specified features or steps as well as those features or steps which are not mentioned and do not materially affect the basic and novel characteristics of the claimed invention.

A preferred embodiment of the present invention relates to a composition comprising one or more bacterial strains selected from one or more pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in the prevention and/or treatment of candidiasis in a human or animal.

Preferably, the prevention and/or treatment of candidiasis in a human or animal may be the prevention and/or treatment of vaginal candidiasis; prevention and/or treatment of oral candidiasis; and/or the prevention and/or treatment of intestinal candidiasis.

The present invention may relate to a composition consisting essentially of one or more bacterial strains selected from one or more Pediococcus strains and/or one or more bacterial strains selected from one or more Lactobacillus strains for use in the prevention and/or treatment of candidiasis in a human or animal.

A preferred embodiment of the present invention relates to a composition comprising one or more bacterial strains selected from one or more pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in preventing, inhibiting or treating biofilm formation.

In embodiments of the invention, the biofilm formation may be yeast biofilm formation, such as pathogenic candida species biofilm formation; in particular the formation of candida albicans biofilm.

In other embodiments of the invention, the composition consists essentially of one or more bacterial strains selected from one or more pediococcus strains and/or one or more bacterial strains selected from one or more lactobacillus strains for use in the prevention and/or treatment of candidiasis in a human or animal.

In an embodiment of the invention, the composition according to the invention may reduce virulence of yeasts such as pathogenic candida species, in particular candida albicans.

In the context of the present invention, the term "virulence" relates to the virulence, toxic or harmful nature or characteristic of yeast/pathogenic candida (in particular candida albicans) to the life or well-being of a human or animal.

The composition according to the invention may preferably consist essentially of one or more bacterial strains selected from Pediococcus strains.

The one or more bacterial strains selected from the genus pediococcus may preferably comprise pediococcus pentosaceus LB606R deposited under accession number DSM 33730.

The composition according to the invention may consist essentially of one or more bacterial strains selected from the group consisting of lactobacillus strains.

The one or more bacterial strains selected from the genus lactobacillus may preferably comprise lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

The composition according to the invention may consist essentially of one or more bacterial strains selected from Pediococcus strains and Lactobacillus strains.

The one or more bacterial strains selected from the genus Pediococcus may preferably comprise Pediococcus pentosaceus LB606R deposited under accession number DSM 33730, and the one or more bacterial strains selected from the genus Lactobacillus may preferably comprise Lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

A preferred embodiment of the present invention relates to a method for preventing biofilm formation in an environment, wherein the biofilm comprises candida species, the method comprising the steps of: administering to the environment an effective amount of a lactic acid bacteria having antimicrobial activity, wherein the environment is a home, workplace, laboratory, industrial environment, aquatic environment, medical device, dental device, epithelial cell, mucous membrane, human or animal body.

The composition may be formulated for oral, topical, enteral or genital use.

Preferably, the composition according to the invention is formulated for oral or genital use. Even more preferably, the composition according to the invention is formulated for genital use.

In embodiments of the invention, the genital use may be a female genital use, such as a vaginal use, and/or a male genital use. Preferably, the genital use may be vaginal use.

During topical use of the compositions of the present invention, the compositions may be applied to the skin, mucous membranes or nails of a human or animal.

In embodiments of the invention, the compositions may be formulated as suspensions, sprays, gels, creams, lotions, powders, capsules, oils, lavages, ovules, vaginal plugs, suppositories, lozenges, tablets, microencapsulated products or in the form of food supplements or foods.

The lactic acid bacteria may include one or more Pediococcus strains; one or more bacterial strains selected from one or more lactobacillus strains; or a combination thereof. The one or more pediococcus strains may preferably comprise pediococcus pentosaceus LB606R deposited under accession number DSM 33730. The one or more lactobacillus strains may preferably comprise lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

Preferred embodiments of the present invention relate to a food or feed ingredient, or a food or feed product, comprising a composition according to the present invention.

Preferred embodiments of the present invention relate to personal hygiene products comprising the composition according to the present invention.

The composition according to the invention may be supplemented with further components to improve the effect of the composition, or to stabilize the composition, or both.

In an embodiment of the invention, the composition further comprises an antifungal agent. Such additional antifungal agents may further improve the composition's activity in preventing or inhibiting biofilm formation.

Aspects of the invention relate to medical devices comprising a composition according to the invention, or a vaginal microbiome graft according to the invention, or an isolated bacterial strain according to the invention.

The composition according to the invention may be formulated into a medical device. Thus, the composition according to the invention comprising one or more lactic acid bacteria according to the invention may be used as a medical device.

The medical device according to the invention may be formulated as a suspension, spray, gel, cream, lotion, powder, capsule, ointment, oil, lavage solution, ovule, vaginal plug, suppository, lozenge, tablet, microcapsule product.

In other embodiments of the invention, the composition further comprises one or more prebiotics. The prebiotic may improve the viability of the added lactic acid bacteria and/or naturally occurring microflora.

In embodiments of the invention, the composition comprising lactic acid bacteria may further comprise a prebiotic.

Prebiotics are non-digestible food components that enhance the growth of specific microorganisms. A "synbiotics" is a composition comprising at least one probiotic and at least one prebiotic. Such compositions are understood to promote the growth of beneficial bacteria (e.g., probiotics). Thus, a potent synbiotics is based on a combination of a specific probiotic strain with a carefully selected prebiotic. They may bring important health benefits to humans or animals.

In embodiments of the present invention, the compositions according to the present invention may comprise intergrown compositions.

According to other embodiments of the present invention, a probiotic composition comprising a probiotic microorganism and at least one or more active ingredients may be provided.

Prebiotics refer to chemical products that induce the growth and/or activity of commensal microorganisms (e.g., bacteria and fungi) to aid in the health of the host. Prebiotics are non-digestible carbohydrates that are not digested or partially digested by the host and stimulate the growth and/or activity of beneficial bacteria that colonize the host.

Some oligosaccharides that may be used as prebiotics are fructo-oligosaccharides (FOS), xylo-oligosaccharides (XOS), polydextrose, pectin, galacto-oligosaccharides (GOS) or Human Milk Oligosaccharides (HMO). In addition, disaccharides such as lactulose or lactose and some monosaccharides such as tagatose may also be used as prebiotics.

The other active ingredients (or other ingredients) may not be limited in any way.

The prebiotic may be a compound that can be metabolized by a probiotic. Preferably, the prebiotic is indigestible or indigestible by the mammal. Prebiotics are well known in the art and, when used in the present invention, there are no particular restrictions on the prebiotic itself.

In embodiments of the present invention, at least one prebiotic may be added to the composition. Preferably, the at least one prebiotic may be selected from the following compounds and compositions: non-digestible carbohydrates, beta-glucans, mannooligosaccharides, inulin, fructooligosaccharides, human Milk Oligosaccharides (HMO), galactooligosaccharides (GOS), lactulose oligosaccharides, fructooligosaccharides (FOS), cellobiose, cellodextrins, cyclodextrins, maltitol, lactitol, glucosyl sucrose. Optionally, oligomannose and/or inulin may be preferred.

HMOs may include lacto-N-tetraose, lacto-N-fucopyranose, lacto-N-triose, 3 '-sialyllactose, lacto-N-neofucopyranose, sialic acid, L-fucose, 2-fucosyllactose, 6' -sialyllactose, lacto-N-neotetraose and 3-fucosyllactose.

D-and L-fucose are believed to enhance the natural defenses of the skin, stimulate the epidermal immune defenses and/or prevent and/or treat autoimmune diseases of the skin. Thus, in embodiments of the invention, the composition may comprise D-and/or L-fucose.

In yet another embodiment of the invention, the composition comprises L-fucose in a concentration in the range of 1mM to 1000mM, such as in the range of 10mM to 500mM, for example in the range of 25mM to 250 mM.

The composition according to the invention may also comprise at least one active ingredient.

In an embodiment of the present invention, the composition may comprise at least one additional probiotic microorganism selected from the group consisting of bacteria, yeasts or moulds.

The at least one additional probiotic microorganism may be selected from, but is not limited to, bifidobacterium lactis (Bifidobacterium lactis) DSM10140, bifidobacterium lactis LKM512, bifidobacterium lactis DSM 20451, bifidobacterium bifidum (Bifidobacterium bifidum) BB-225, bifidobacterium adolescentis (Bifidobacterium adolescentis) BB-102, bifidobacterium breve (Bifidobacterium breve) BB-308, bifidobacterium longum (Bifidobacterium longum) BB-536 from Zaidanhojin Nihon Bifizusukin Senta (bifidobacterium center of japan), bifidobacterium NCIMB 41675 described in EP 2823822. Bifidobacterium bifidum BB-225, bifidobacterium adolescentis BB-102, bifidobacterium breve BB-308, bifidobacterium lactis HN019 (Howaru), bifidobacterium bifidum BB-02, bifidobacterium bifidum BB-06, bifidobacterium longum KC-1, bifidobacterium longum 913, bifidobacterium lactis BI-04, bifidobacterium lactis BI-07 (obtainable from DuPont Nutrition Biosciences ApS), bifidobacterium lactis DN 173010 (obtainable from group Danone), bifidobacterium lactis BB-12 (obtainable from chr.hansen a/S), bifidobacterium breve M-16V (Morinaga) and/or lactobacillus having a probiotic effect, and may be any of the following strains; lactobacillus rhamnosus LGG (chr. Hansen), lactobacillus gasseri (Lactobacillus gasseri) LN40, lactobacillus gasseri EB01TM, lactobacillus rhamnosus PB01TM, lactobacillus rhamnosus LN113, lactobacillus fermentum LN99, lactobacillus acidophilus (Lactobacillus acidophilus) NCFM, lactobacillus bulgaricus (Lactobacillus bulgaricus) 1260, lactobacillus paracasei (Lactobacillus bulgaricus) Lpc-37, lactobacillus rhamnosus HN001 (obtainable from DuPont Nutrition Biosciences ApS), streptococcus thermophilus (Streptococcus thermophilus) 715 and streptococcus thermophilus ST21 (obtainable from DuPont Nutrition Biosciences ApS), lactobacillus paracasei subspecies CRL431 (ATCC 55544), lactobacillus paracasei strain F-19 (from medicharm, inc.), lactobacillus paracasei fti L26 (DSM Food Specialties, the Netherlands) and Lactobacillus paracasei CRL431 (Chur.Hansen), lactobacillus acidophilus PTA-4797, lactobacillus salivarius (L.salivarius) Ls-33 and Lactobacillus curvatus (L.Curvatus) 853 (DuPont Nutrition Biosciences ApS), lactobacillus pentosus CECT 7504, lactobacillus plantarum 299v (Probi AB), lactobacillus plantarum LMC1 (DSM 32252), lactobacillus paracasei LMC4 (DSM 32254), lactobacillus rhamnosus LMC6 (DSM 32256 (DSM 32257), lactobacillus paracasei LMC8 (DSM 32258), lactobacillus casei subspecies rhamnosus LC705 (described in Fl patent 92498, valio Oy), lactobacillus rhamnosus LC705 (DSM 7061), propionic acid bacteria (Propionic acid) such as Propionibacterium freudenreichii subspecies Shellmanii (Propionibacterium freudenreichii ssp. Shaman ii) PJS (DSM 7067) (described in detail in FI patent 92498, valio Oy), nitromonas (Nitrosomonas eutropha) D23 (ABIome), human staphylococci (Staphylococcus hominis) strains A9, C2, AMT3, AMT4-C2, AMT4-Gl and/or AMT4-D12 (All from Matrisys Bioscience), staphylococcus epidermidis (Staphylococcus epidermidis) strains M034, M038, all, AMT1, AMT5-C5 and/or AMT5-G6 (All from Matrisys Bioscience), lactobacillus plantarum YUN-V2.0 (BCCM LMG P-29456), lactobacillus pentosus YUN-V1.0 (BCCN LMG P-29455), lactobacillus rhamnosus YUN-S1.0 (CM LMG P-61) and/or any combination thereof.

Additionally, the at least one additional probiotic microorganism may be selected from the group consisting of: weissella viridis (Weissella viridescens) LB10G (DSM 32906), lactobacillus paracasei LB113R (DSM 32907), lactobacillus plantarum LB244R (DSM 32996), lactobacillus paracasei LB116R (DSM 32908), lactobacillus brevis LB152G (DSM 32995), lactobacillus paracasei LB28R (DSM 32994), enterococcus faecium (Enterococcus faecium) LB276R (DSM 32997), leuconostoc mesenteroides (Leuconostoc mesenteriodes) LB349R (DSM 33093), lactobacillus plantarum LB316R (DSM 33091), lactobacillus plantarum LB356R (DSM 33094), lactobacillus plantarum LB312R (DSM 33098); and/or any combination thereof or mutant strains thereof and/or cell lysates and/or soluble metabolites of any of these probiotic strains.

In embodiments of the invention, the probiotic bacterial strain may be used as a stable form of the viable isolated microorganism. Suitable stabilization methods are known to those skilled in the art and include lyophilization, spray drying, or freeze drying involving different cryoprotectants.

In other embodiments of the invention, the strain may be used as a live isolated strain.

Preferably, the strain may be used as a viable isolated stable strain. Even more preferably, the strain may be used as a live isolated strain stabilized by lyophilization. Even more preferably, the strain may be used as a live isolated strain that is stable by lyophilization and that comprises a cryoprotectant and is incorporated into a formulation suitable for oral, vaginal or topical use.

The composition according to the application may comprise a combination of at least one bacterial strain and at least one further probiotic microorganism, wherein the at least one further probiotic microorganism may be selected from, but is not limited to, strains selected from the genus lactobacillus.

Preservation of biological materials

The lactic acid bacteria according to the application comprise in particular microorganisms or analogues, fragments, derivatives, fermentates, lysates, mutants or combinations obtained from the microorganisms deposited at the German collection of microorganisms and cell cultures at 12.14.2020: pediococcus pentosaceus LB606R deposited as DSM 33730 and Lactobacillus plantarum deposited as DSM 33731.

It should be noted that the embodiments and features described in the context of one aspect of the application also apply to other aspects of the application.

All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

The application will now be described in further detail in the following non-limiting examples.

Examples

Example 1 screening and identification of strains

Sample of

For the identification and selection of microorganisms according to the application, a new strain collection of isolated Lactic Acid Bacteria (LAB) was established. Samples from different sources, such as homemade kimchi, kimchi and healthy human donor samples (vagina, oral cavity, anus, skin, human milk, baby diapers) were collected, and at least 1200 new lactic acid bacteria strains were isolated. Samples were collected on Man Rogosa Sharp (MRS, sigma-Aldrich) broth and agar and incubated anaerobically at 37℃overnight or until colonies formed. The isolates were plated and subcultured until isolated colonies were obtained. Isolated colonies were stored at-80 ℃ in MRS broth containing 25% glycerol for future use. The strain was identified using 16S rRNA sanger sequencing standard method.

Example 2 agar spot assay

Isolated +1200LAB (example 1) were screened for their ability to inhibit the growth of candida albicans, a pathogen associated with candidiasis.

Candida strains were obtained from BEI resources:

candida albicans strain P57055, catalog number 29439 (blood isolate)

Candida albicans, strain L26, catalog number 29445 (vaginal isolate)

Candida albicans, strain 19F, catalog No. 29449 (vaginal isolate).

Competition between LAB and candida strains was determined according to the method described in the following publications: dowarah, r., et al 2018, selection and characterization of probiotic lactic acid bacteria and its impact on growth, nutrient digestibility, health and antioxidant status in weaned pig. Plos ONE,13 (3), khare, a., & Tavazoie, s. (2015), multifactorial Competition and Resistance in a Two-Species Bacterial systems, plos Genetics,11 (12), 1-21.

Lactic Acid Bacteria (LAB) strains were incubated overnight at 37℃in Man Rogosa Sharp (MRS, sigma-Aldrich) broth. Candida albicans was incubated overnight at 37℃in YPD broth (20 g/L peptone, 10g/L yeast extract, 20g/L glucose). Candida albicans cultures with OD600 of about 0.5 were diluted to 10-1 or 10-2 and 1mL were plated on Mueller-Hinton agar plates to form lawn. From the LAB culture, 20. Mu.L was spotted on top of the Mueller-Hinton plate. After overnight incubation at 37 ℃ under aerobic conditions, the zone of inhibition was measured.

EXAMPLE 3 copolymerization set

Determination of Co-aggregation according to known methods Cisar, J.O.et al (1979), "Specificity of Coaggregation Reactions between Human Oral Streptococci and Strains of Actinomyces Viscosus or Actinomyces Naeslundii.," Infection and Immunity (3): 742-52.

LAB and Candida albicans were grown overnight at 37 ℃. MRS broth was used for LAB and YPD broth for Candida albicans. Cultures were harvested by centrifugation at 6000rpm for two minutes. The supernatant was discarded and the pellet was washed once with 500 μl Phosphate Buffered Saline (PBS) and then resuspended in 1mL PBS. From Candida albicans cultures and each LAB culture, 0.5, 0.25, 10-1, 10-2 and 10-3 dilutions were prepared in PBS. Undiluted LAB was mixed with each candida albicans dilution and undiluted candida albicans was mixed with each LAB dilution. Likewise, diluted candida albicans was mixed with diluted LAB. From LAB and candida albicans dilutions, 200 μl was mixed in the wells of a 48 well microtiter plate and incubated on a shaker at 200rpm at room temperature. After 24 hours, the wells were observed for the presence of co-aggregation and self-aggregation.

The formation of co-aggregates was scored visually from 1-5 using the following scale:

1 no aggregation

2 visual initial aggregation

3 forming <0.5mm aggregates

4 forming aggregates >0.5mm and <1mm

5 formation of agglomerates >1mm

EXAMPLE 4 prevention of Candida biofilm

The effect of lactic acid bacteria on preventing candida biofilm formation was determined as follows: gottschick et al (2016) Screening of compounds against Candida biofilms. PLos One 11 (4) doi.org/10.1371/journ.fine.0154086.

Candida precultures were prepared in YPD broth and incubated for 24 hours at 37 ℃. The preculture was diluted in PBS to od600=0.05 for the final biofilm culture. Lactic acid bacteria were prepared in a similar manner, pre-incubated with MRS, diluted to od600=0.5 in PBS, and mixed 1:1, then in Nunc ^TM MicroWell ^TM 96 hole micro-well plate (Thermo)Scientific) in culture. After 30min the mixture was removed from the wells, washed once with PBS to remove non-adherent cells and incubated in YPD for 20 hours at 37 ℃. The supernatant was removed and the biofilm was washed twice with sterile PBS buffer. OD600 measured compared to controls without biofilm and control biofilm for each microorganism grown as a single biofilm culture.

Inhibition of biofilm formation by Lactic Acid Bacteria (LAB) was determined as:

((OD 600[ biofilm of candida ] -OD600[ mixed biofilm culture of LAB test strain and candida ])/[ biofilm of candida ]) x 100%

The selected strains were compared to commercially available consumer products.

Product A: gynoLact, available from Vitabalans of Denmark, comprises 3 strains of Lactobacillus (Lactobacillus acidophilus, lactobacillus casei, lactobacillus rhamnosus). Precultures for testing were prepared by crushing a tablet into MRS medium and anaerobic culturing at 37 ℃ overnight.

Product B: vivag Capsule, available from Orkla Care A/S of Denmark, comprises 2 strains of Lactobacillus (Lactobacillus gasseri EB01TM and Lactobacillus rhamnosus PB01 TM). Precultures for testing were prepared by crushing a tablet into MRS medium and anaerobic culturing at 37 ℃ overnight.

Each product was prepared similarly to candida and LAB strains for experiments. The LAB strains selected have a significantly better ability to prevent candida biofilm formation.

Example 5 pore diffusion assay

Overnight cultures of LAB and candida albicans were prepared. At 37 ℃, LAB was grown in MRS broth and candida was grown in YPD broth. A10-1 dilution was prepared from Candida cultures each having an OD600 of about 0.5 and 1mL was plated on top of Mueller-Hinton agar plates. Wells were created in the plates and 50 μl of LAB culture was transferred into the wells. After overnight incubation at 37 ℃, the zone of inhibition surrounding the wells was checked.

EXAMPLE 6 Hydrogen peroxide formation

To investigate whether LAB strains produce hydrogen peroxide (H2O 2), hydrogen peroxide assays were performed according to the method described by Pendharkar et al (2013). MRS agar plates containing 0.25mg/mL of 3,3', 5' -Tetramethylbenzidine (TMB) and 0.01mg/mL horseradish peroxidase were prepared. After autoclaving, both TMB and peroxidase are added to the agar. The bottles containing TMB and peroxidase were stirred using a magnetic stirrer before pouring the agar into the plates. From the overnight LAB culture, 20 μl was spotted on top of the agar. Plates were incubated at 37 ℃ for three days under anaerobic conditions and then exposed to air for 30 minutes. The production of H2O2 was observed as blue colonies. The H2O2 production score was between 0 and 2.

Example 7 challenge assay Using LAB cell-free supernatant

The candida albicans strain and LAB were incubated overnight at 37 ℃. Candida albicans was incubated in YPD broth and LAB strains were incubated in MRS broth. Cell-free supernatants (CFSs) were prepared from LAB cultures using syringe and 0.2 μm filter. Candida albicans cultures with OD600 of about 0.5 were diluted 10-2. From each candida albicans dilution, 100 μl was challenged in 96-well microtiter plates with 100 μl CFS dilutions of 100, 75, 50, 25, 10 and 0% CFS. CFS dilutions were prepared using MRS broth. Plates were incubated in an oCellscope at 37 ℃ and phase contrast microscopy and image analysis were used as growth measurements (oCelluScope, bioSense Solution, denmark). Growth kinetics measurements were performed every 20 minutes for 8 hours.

Example 8 Co-culture assay

LAB and Candida albicans were grown overnight at 37 ℃. LAB was grown in MRS broth and Candida albicans was grown in YPD broth. All cultures were adjusted to an OD600 of about 0.4. Bacteria were harvested from 4mL of each culture by centrifugation at 4500rpm for 10 minutes. The supernatant was discarded and the pellet was resuspended in 2mL PBS and then re-centrifuged at 4500rpm for 10 minutes. After draining the supernatant, the pellet was resuspended in 4mL of PBS (4.5 mL for candida albicans). From these solutions, 0.5mL was transferred to 25mL BHI broth. Candida albicans was mixed with each LAB strain, and a single culture of LAB and candida albicans was also prepared. Serial dilutions were made and plated on BHI plates to count CFU after 0, 6 and 24 hours. The mixed culture and candida albicans single culture were plated on BHI agar plates supplemented with 32 μg/mL chloramphenicol to allow candida albicans growth, but not LAB growth.

Results

All LABs isolated as described in example 1 were screened using the methods described in examples 2-8, and both strains were identified as having antimicrobial effect on candida albicans in all methods used.

These two identified strains were deposited by Lactobacillus ApS from Copenhagen, denmark at 12.14.2020 with Germany microorganisms and cell culture Collection Co., ltd (DSMZ). The strain was deposited as follows:

Pediococcus pentosaceus LB606R as deposited under DSM 33730

Lactobacillus plantarum LB679R deposited as DSM 33731

Lactobacillus plantarum is named according to the taxonomic changes published by the International journal of evolution microbiology 2020, 4, 15. Previously known as lactobacillus plantarum.

Table 1: spot and diffusion on lawn were measured and inhibition zone was measured in mm.

The copolymerization set of strains LB606R and LB679R was determined to be 3-4. An example of co-aggregation between candida albicans strain L26 and LB606R is shown in figure 1.

Table 2: reduction of candida albicans biofilm (strains L26 and 19F).

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Dilution of the cell-free supernatants of both LB606R and LB679R to 25% concentration significantly inhibited candida albicans growth. The data for candida albicans strain L26 are shown in figures 2a and 2 b. In the co-culture assay, both strains were able to defeat all 3 candida albicans test strains.

Fig. 1: co-aggregation of Candida albicans strain L26 and LB 606R. FIG. 1a shows control of Candida albicans L26 with combination and copolymerization with LB606R (FIG. 1 b).

Fig. 2: CFS of LB606R (fig. 2 a) and LB679R (fig. 2 b) inhibited growth of candida albicans strain L26.

Reference to the literature

Silva,S.,M.Negri,M.Henriques,R.Oliveira,DW.Williams and J.Azeredo(2012)Candida glabrata,Candida parapsilosis and Candida tropicalis:biology,epidemiology,pathogenicity and antifungal resistance.FEMS Microbiology Reviews,Volume 36(2):288–305,https://doi.org/10.1111/j.1574-6976.2011.00278.x

Pfaller,MA,JD Diekema(2007)Epidemiology of invasive candidiasis:apersistent public health problem.Clin Microbiol Rev 20:133-163.

Luo,G.,Samaranayake,LP,and JY.Yau(2001)Candida species exhibit differential in vitro hemolytic activities.J.Clin.Microbiol.39:2971-2974.

Negri M et al 2010.Mycophatologia 169:175-182.Doi:10.1007/s11046-009-9246-0

Dowarah,R.,et al.(2018)Selection and characterization of probiotic lactic acid bacteria and its impact on growth,nutrient digestibility,health and antioxidant status in weaned piglets.PLoS ONE,13(3)

Kwak,Y-K.Et al.(2016)Persistence of Lactobacilli in postmenopausal women–a double-blind,randomized,pilot study.Gynecol.Obstet Invest 82:144-150.Doi:10.1159/000446946

Gottschick et al.(2016)Screening of compounds against Gardnerella vaginalis biofilms.PLos One 11(4).doi.org/10.1371/journal.pone.0154086

Khare,A.,&Tavazoie,S.(2015).Multifactorial Competition and Resistance in a Two-Species Bacterial System.PLoS Genetics,11(12),1–21。

Marshall,V.M.(1979)J.Appl.Bacteriol.47pp 327-328.

doi.org/10.1111/j.1365-2672.1979.tb01762.x。

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Claims (13)

1. A composition comprising pediococcus pentosaceus (Pediococcus pentosaceus) LB606R deposited under accession No. DSM 33730; lactobacillus plantarum (Lactiplantibacillus plantarum) LB679R deposited under accession No. DSM 33731; or a combination thereof.

2. The composition of claim 1, wherein the composition inhibits biofilm formation.

3. The composition of any of the preceding claims, wherein the composition is formulated for oral use, topical use, anal use, or genital use.

4. A composition according to any preceding claim, wherein the composition further comprises one or more prebiotics.

5. Composition according to any one of the preceding claims, wherein the one or more Pediococcus (Pediococcus) strains, such as Pediococcus pentosaceus LB606R deposited under accession number DSM 33730, and/or the one or more lactobacillus (lactobacillus plantarum) strains, such as lactobacillus plantarum LB 679R deposited under accession number DSM 33731, may be provided as living cells, as non-living cells, as lysate or as supernatant.

6. The composition of claim 5, wherein the non-living cells are provided as a lysate of one of the pediococcus strain or the lactobacillus strain, as a fraction, as a ferment, as a metabolite, as an extract, as a derivative, as an analogue, or as a mutant.

7. The composition of claim 5, wherein the lysate comprises a cell wall or cell wall component comprising a binding receptor.

8. An isolated bacterial strain selected from pediococcus pentosaceus LB606R deposited under accession number DSM 33730; and/or one or more bacterial strains selected from lactobacillus plantarum LB 679R deposited under accession number DSM 33731.

9. A composition comprising one or more bacterial strains selected from one or more pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains for use as a medicament.

10. A composition comprising one or more bacterial strains selected from one or more pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in the prevention and/or treatment of candidiasis in a human or animal.

11. A composition comprising one or more bacterial strains selected from one or more pediococcus strains; and/or one or more bacterial strains selected from one or more lactobacillus strains, for use in preventing, inhibiting or treating biofilm formation.

12. The composition of claim 11, wherein the biofilm formation may be yeast biofilm formation, such as pathogenic Candida (Candida) species biofilm formation; in particular Candida albicans (Candida albicans) biofilm formation.

13. A method for preventing biofilm formation in an environment, wherein the biofilm comprises candida species, the method comprising the steps of: administering to the environment an effective amount of a lactic acid bacteria having antimicrobial activity, wherein the environment is a home, a workplace, a laboratory, an industrial environment, an aquatic environment, a medical device, a dental device, epithelial cells, mucous membranes, a human or animal body.