Fermentation process

Abstract

The present invention relates to a process for the preparation of a product by fermentation with a strain of Bacillus subtilis RBI. The invention relates also to the product obtainable by such fermentation process and its use as an antifungal. The present invention further relates to the strain of Bacillus subtilis RBI.

Description

[0001] “FERMENTATION PROCESS’’

[0002] DESCRIPTION

[0003] Technical Field of the Invention

[0004] The present invention relates to a process for the preparation of a product by fermentation with a strain of Bacillus subtilis RBI and to the fermentation product obtainable by such fermentation process. The thus obtained fermentation product is useful as an antifungal.

[0005] Further, the invention relates to the strain of Bacillus subtilis RBI, used in the fermentation process.

[0006] Background

[0007] Fungal infections in pets or cattle or fungal infections, in particular cutaneous, vaginal, or systemic, in humans, are widely spread pathologies, which are normally treated by antimycotics or antibiotics.

[0008] The continued emergence and development of pathogenic microorganisms resistant to antibiotics and antimycotics represents a global growing concern, and the pledge in the discovery of novel antimicrobics and antifungals will be remarkable until a sound solution will be found.

[0009] Biological control has become an interesting alternative to the conventional methods for controlling microbic infections, using directly antagonist organisms or metabolites thereof to halt the propagation of harmful pathogens.

[0010] The genus Bacillus is known for producing a wide range of bioactive secondary metabolites, including lipopeptides, polyketides, lantibiotics, siderophores, lytic enzymes, and peptides. Further, the most of Bacillus species, such as B. subtilis, for example Bacillus subtilis B25, Bacillus subtilis subsp. inaquosorum strain, B. amyloliquefaciens B. licheniformis, and Bacillus subtilis SH21, are generally recognized as safe microorganisms for the application in the food industry, for example to prevent pathologies in crops, and they are considered important agents of biologic control in the inhibition of antibiotic multi-resistant food pathogens, for example usable for the conservation of food goods.

[0011] In particular, Bacillus subtilis, non-pathogenic bacterium widely distributed in nature, is considered an extremophile microorganism due to its adapting capacity to severe environmental conditions, such as high or low temperatures, pH, salinity, and pressure, all adverse conditions for the most of the living species.

[0012] The need to provide a product with effective, eventually enhanced, antifungal activities, and a method to obtain it, is still strongly felt.

[0013] Summary of the InventionObject of the present invention is a process for the preparation of a fermentation product comprising the following steps:

[0014] a) providing an inoculum of Bacillus subtilis RB 1 ;

[0015] b) incubating the inoculum;

[0016] c) inoculating a culture medium selected from LB, LB1-S, LB2-S, and GMSM-S with the incubated inoculum;

[0017] d) fermenting to obtain a suspension comprising the fermentation products;

[0018] e) centrifuging the suspension so to obtain a solid fraction, also called precipitate, and a liquid fraction, also called supernatant;

[0019] f) isolating the liquid fraction to obtain the fermentation product.

[0020] Object of the present invention is also the fermentation product obtainable, or obtained, by the fermentation process of the invention and its use as an antifungal.

[0021] A further object of the invention is the strain of Bacillus subtilis RB 1 deposited at DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number “DSM 35197” on 08 / 10 / 2024 under the Budapest Treaty.

[0022] Brief Description of Figures

[0023] Figure 1 shows the growth inhibition of fungus Trichophyton interdigitale ATCC 9533 due to the product of the invention, the control - (negative control, CTRL-) and the control + (positive control, CTRL+, amphotericin) in Agar tests using the GMSM-S culture medium with a glucose concentration of 20 g / L in A-B duplicate.

[0024] Figure 2 shows the growth inhibition of fungus Trichophyton interdigitale ATCC 9533 due to the product of the invention, the control - (negative control, CTRL-) and the control + (positive control, CTRL+, amphotericin) in Agar tests using the GMSM-S culture medium with a glucose concentration of 50 g / L in A-B duplicate.

[0025] Figure 3 shows the growth inhibition of fungus Microsporum canis ATCC 36299 due to the product of the invention, the control - (negative control, CTRL-) and the control + (positive control, CTRL+, amphotericin) in Agar tests using the GMSM-S culture medium with a glucose concentration of 50 g / L in A-B duplicate.

[0026] Figure 4 shows the growth inhibition of fungus Microsporum canis ATCC 36299 due to the product of the invention, the control - (negative control, CTRL-) and the control + (positive control, CTRL+, amphotericin) in Agar tests using the GMSM-S culture medium with a glucose concentration of 20 g / L in A-B duplicate.

[0027] Figure 5 shows the growth inhibition of fungus Candida albicans due to the product of theinvention obtained by the strain of Bacillus subtilis RB 1 , the comparison obtained by the strain of Bacillus subtilis M12, the control - (negative control, CTRL-) and the control + (positive control, CTRL+, amphotericin) in Agar spot tests using the GMSM culture medium a-b duplicate.

[0028] Detailed Description of the Invention

[0029] It was surprisingly found that, by the fermentation process of the invention, it is possible to obtain an extracellular product due to the metabolism of the strain of Bacillus subtilis RBI, released in the external environment, able to inhibit the growth of pathogenic fungi and dermatophytes, and bacteria. Therefore, it was possible to obtain an effective, possibly enhanced, antifungal product.

[0030] In the present invention, the following terms mean:

[0031] “LB culture medium” or “LB ” wherein LB means Luria-Bertani, a culture medium comprising from 7.5 to 12.5 g / L, preferably 10 g / L, of tryptone; from 3 to 7 g / L, preferably 5 g / L, of yeast extract; from 8 to 12 g / L, preferably 10 g / L, of sodium chloride;

[0032] the media can be in a solid form in Agar or in a liquid form in water forming a broth;

[0033] “LB1 -S culture medium” or “LB1 -S” a culture medium comprising from 7.5 to 12.5 g / L, preferably 10 g / L, of peptone, more preferably rice peptone; from 3 to 7 g / L, preferably 5 g / L, of yeast extract, and from 8 to 12 g / L, preferably 10 g / L, of sodium chloride;

[0034] “LB2-S culture medium” or “LB2-S” a culture medium comprising from 7.5 to 12.5 g / L, preferably 10 g / L of peptone, more preferably rice peptone; from 35 to 45 g / L, preferably 40 g / L, of glucose; from 3 to 7 g / L, preferably 5 g / L, of yeast extract, and from 8 to 12 g / L, preferably, 10 g / L, of sodium chloride;

[0035] “LB3-S culture medium” or “LB3-S” a culture medium comprising from 7.5 to 12.5 g / L, preferably 10 g / L, of peptone, more preferably rice peptone; from 35 to 45 g / L, preferably 40 g / L, of glucose; from 2.5 to 3.5 g / L, preferably 3 g / L, of NaNCL; from 3 to 7 g / L, preferably 5 g / L, of yeast extract, and from 8 to 12 g / L, preferably 10 g / L, of sodium chloride;

[0036] “LB4-S culture medium” or “LB4-S” a culture medium comprising from 7.5 to 12.5 g / L, preferably 10 g / L of peptone, more preferably rice peptone; from 35 to 45 g / L, preferably 40 g / L, of glucose; from 2.5 to 3.5 g / L, preferably 3 g / L, of NalLCl; from 3 to 7 g / L, preferably 5 g / L, of yeast extract, and from 8 to 12 g / L, preferably 10 g / L, of sodium chloride;

[0037] “GMSM-S culture medium” or “Glucose Mineral Salt Medium-S” or “GMSM-S” a culture medium comprising from 20 g / L to 50 g / L, preferably 50 g / L, of glucose; from 2.2 to 3.2 g / L, preferably 2.7 g / L, of NH4CI; from 3.5 to 4.5 g / L, preferably 4 g / L, of NaNCL; from 6.5 to 7.5 g / L, preferably 7 g / L, of Na2HPO4; from 0.30 to 0.36 g / L, preferably 0.33 g / L, of MgSO4- 7H2O; from 0.08 to 0.12g / L, preferably 0.10 g / L, of CaC12-2H2O; from 0.03 to 0.05 g / L, preferably 0.04 g / L, of FeSC ; from 0.14 to 0.20 g / L, preferably 0.17 g / L, of MnSCU- H2O.

[0038] Object of the present invention is a process for the preparation of a fermentation product comprising the following steps:

[0039] a) providing an inoculum of Bacillus subtilis RB 1 ;

[0040] b) incubating the inoculum;

[0041] c) inoculating a culture medium selected from LB, LB1-S, LB2-S, and GMSM-S with the incubated inoculum;

[0042] d) fermenting to obtain a suspension;

[0043] e) centrifuging the suspension so to obtain a solid fraction, also called precipitate, and a liquid fraction, also called supernatant;

[0044] f) isolating, preferably by filtration, the liquid fraction to obtain the fermentation product. Step a) can be preceded by a step a’) comprising the following sub-steps:

[0045] al) keeping a strain of Bacillus subtilis RBI in a broth, preferably in a LB medium in the form of a broth, with the adding of glycerol and stored at a temperature from -90 °C to -70 °C, preferably -80 °C;

[0046] a2) activating the strain of Bacillus subtilis RB 1 in a broth, preferably in a LB medium in the form of a broth;

[0047] a3) incubating the strain of Bacillus subtilis RBI overnight at a temperature from 30 °C to 26 °C, preferably 28 °C, preferably under stirring from 130 to 150 rpm, more preferably 140 rpm, for example in an orbital shaker;

[0048] a4) transferring an aliquot of the strain of Bacillus subtilis RBI incubated in step a3) in an agar LB plate and incubating at a temperature from 30 °C to 26 °C, preferably 28 °C, obtaining a colony; and

[0049] a5) transferring the colony obtained in step a4) and further subsequent inoculation in a broth, preferably in a LB medium in the form of a broth, to obtain an inoculum.

[0050] Step b) of inoculum incubation preferably occurs overnight at a temperature from 30 °C to 26 °C, preferably 28 °C, preferably under stirring from 130 to 150 rpm, more preferably 140 rpm, for example in an orbital shaker.

[0051] The culture media of the invention were proven particularly advantageous even with respect to other culture media.

[0052] The culture medium of step c) is preferably GMSM-S, more preferably GMSM-S with glucose in a concentration from 20 g / L to 50 g / L, even more preferably GMSM-S with glucose ina concentration of 50 g / L.

[0053] Preferably, in step c) the culture medium was inoculated with an inoculum in an exponential phase.

[0054] According to a preferred aspect, in step c) the inoculum amount ranges from 1% to 4% v / v, more preferably is equal to 2% v / v, and even more preferably the inoculum concentration ranges from 5xl07to 5xl08CFU / mL.

[0055] The fermentation of step d) is preferably an in-batch fermentation, more preferably in a shake flask or bioreactor, even more preferably in a bioreactor. A usable bioreactor is the Applikon Minibio bundle, preferably with a work volume of 0.8 L.

[0056] The fermentation of step d) preferably occurs for a time ranging from 48 to 96 hours, more preferably of 72 hours.

[0057] The pH of step d) is preferably kept in neutral conditions from 6 to 7, more preferably at 6.5, even more preferably by adding of 1 M NaOH.

[0058] The fermentation temperature of step d) is preferably kept in a range from 35 to 25 °C, preferably at 28 °C.

[0059] The stirring speed of step d) preferably ranges from 200 to 500 rpm.

[0060] In step d), the percentage of oxygen dissolved in the culture medium preferably ranges from 40 to 60%, more preferably is 50%, more preferably reached by a continuous air stream of 2 L / min. The measurement of the dissolved oxygen can be carried out by luminescence, preferably based on the phase displacement between excitation and emission, directly correlated to the oxygen partial pression; more preferably it is carried out by a titanium membrane sensor such as LumiSens Optical DO AppliSens DO2.

[0061] After the fermentation of step d) a suspension is obtained which can be subjected to centrifugation (step e)) thus to obtain a solid fraction, also called precipitate, and a liquid fraction, also called supernatant containing the fermentation products. Subsequently, the liquid fraction containing the fermentation products can be isolated (step f)), preferably by filtration, more preferably by means of a filter with 0.22 pm pores.

[0062] Object of the present invention is also the fermentation product obtainable, or obtained, by the above-described fermentation process of the invention.

[0063] Said fermentation product can preferably comprise the proteolytic enzymes as reported below in Table 1:Table 1

[0064] Bacillolysine

[0065] Neutral metalloproteinases

[0066] Serine proteases

[0067] Beta-glucanases

[0068] Chitosanases

[0069] Beta-N-acetylglucosaminidases

[0070] Muramidases / Lytic Transglycosylases

[0071] Peptidoglycan beta-N-acetylmuramidases

[0072]

[0073] The enzymes listed in Table 1 can comprise:

[0074] - proteolytic enzymes such as Bacillolysine, Neutral metalloproteinases, Serine proteases which degrade the proteins of the cell wall and membrane, compromising the fungal cellular integrity;

[0075] - enzymes for the degradation of the cell wall, such as:

[0076] - Beta-glucanases breaking the P-glucanes, the main components of the fungal cell wall, causing cellular lysis;

[0077] - Chitosanases degrading chitin and chitosan, essential for the fungal cell wall; - Beta-N-acetylglucosaminidases destroying N-acetylglucosamine, weakening the fungal cell wall;

[0078] - enzymes with a specific activity, such as:

[0079] - Muramidases / Lytic Transglycosilases acting on the structural components of the fungal cell wall;

[0080] - Peptidoglycan beta-N-acetylmuramidases degrading key elements of the cell wall, compromising the fungal vitality.

[0081] Object of the invention is a fermentation product comprising the proteolytic enzymes as reported in Table 1.

[0082] Said product can further preferably comprise the lipopeptides as reported below in Table 2:

[0083] Table 2

[0084] Lipopeptides Amount (mg / L)

[0085] Surfactin (C12) 661.9 + 36.2

[0086] Surfactin (C13) (A) 763.9 + 8.0

[0087] Surfactin (C14) (B) 794.0 + 55.1

[0088]

[0089] Surfactin (C15) (c) 811.8 + 43.3

[0090] Fengycin C17 841.3 + 9.5

[0091] Plipastatin A2 200.1 + 1.7

[0092] Plipastatin Bl 134.9 + 0.5

[0093]

[0094] Object of the invention is a fermentation product comprising a lipopeptide content as reported in Table 2.

[0095] A further object of the invention is therefore the use of such fermentation product as an antifungal. The fermentation product obtained according to the invention was proven effective against fungal pathogens such as Candida albicans, in particular the product obtained from the LB2-S or GMSM-S culture medium, and Malasezzia furfur, in particular when the product is obtained from the GMSM-S culture medium, and dermatophyte fungi such as Microsporum canis, in particular when the product is obtained from the GMSM-S culture medium, and Trichophyton interdigitale, in particular when the product is obtained from the GMSM-S culture medium.

[0096] The fermentation product of the invention can be used as an antifungal in products for personal care comprising detergents, shampoos, creams, lotions, and treatments for skin and scalp.

[0097] The fermentation product of the invention can be used as an antifungal even in cosmetic products, for example in products for make-up and skin care.

[0098] The fermentation product of the invention can be used as an antifungal in agriculture, for example in pesticide products and in products to prevent and control fungal diseases in crops.

[0099] Further, the fermentation product of the invention can be used as an antifungal in the food industry, for example as an antifungal preservative in food products and beverages.

[0100] A further object of the invention is the fermentation product for use as a medicament, in particular as antifungal or antibacterial.

[0101] The fermentation product according to the invention was proven effective as an antibacterial, preferably against infections from S. aureus, more preferably when the product is obtained from the LB, LB1-S or GMSM-S culture medium, even more preferably GMSM-S.

[0102] Object of the invention is the fermentation product for use in the prevention and / or treatment of fungal infections, for example in pets or cattle.

[0103] Object of the invention is the fermentation product for use in the prevention and / or treatment of fungal infections, for example cutaneous, vaginal, or systemic, in humans.

[0104] A further aspect of the present invention is a method comprising the application or the administration of a product according to the invention for the prevention and / or treatmentof fungal infections in a human or animal in need thereof.

[0105] The invention further relates to compositions comprising the product obtained according to the invention and at least an acceptable excipient and / or vehicle.

[0106] A further object of the invention is the strain of Bacillus subtilis RBI. The strain of Bacillus subtilis herein named “RBI” was deposited at DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number “DSM 35197” on 08 / 10 / 2024 under the Budapest Treaty.

[0107] Such endophytic strain can be isolated from stems of Robinia pseudoacacia.

[0108] All the features and embodiments described in the present invention can be combined between each other.

[0109] The following below Examples further illustrate the invention.

[0110] EXAMPLES

[0111] Example 1 - Fermentation process of the invention

[0112] The strain of Bacillus subtilis RB 1 was kept in a LB medium in the form of a broth and glycerol was added as a cryoprotective in order to be subsequently stored at -80 °C for its maintenance (step al).

[0113] To activate the strain, sterile aliquots withdrawn from -80 °C were transferred in 10 mL of LB broth (step a2) and incubated overnight at 28 °C under stirring at 140 rpm in an orbital shaker (step a3). Then, one aliquot was transferred in an agar LB plate (step a4) and incubated at 28 °C and then stored at 4 °C in a fridge for a subsequent use as a preparation of inoculum (step a5).

[0114] A colony from this plate was transferred (using a loop) and inoculated in 20 mL of LB broth; the inoculum was incubated (step b) overnight in an orbital shaker at 140 rpm at 28 °C. The bacterial growth was then monitored by the medium optical density (OD) measurement at 600 nm.

[0115] The following media were tested: LB, LB1-S, LB2-S, and GMSM-S.

[0116] 250 mL flasks containing 50 mL of the tested media were inoculated with a concentration of ~lxl07CFU / mL of Bacillus subtilis RBI.

[0117] The samples were incubated for 48 hours at 28 °C, under stirring at 140 rpm (step c).

[0118] The strain growth was analyzed following the OD (optical density) at 600 nm and the maximum growth rate (pmax), the generation time (g) of Bacillus subtilis RBI in the different analyzed media were calculated by means of this value.

[0119] The in-batch fermentations (step d) were carried out in a 1 L bioreactor (Applikon Minibio bundle) with a work volume of 0.8 L.

[0120] The fermentations were carried out for 72 hours. The pH was kept at 6.5 using a peristalticpump to feed 1 M NaOH.

[0121] The fermentation temperature was kept at 28 °C. The stirring speed of the bioreactor ranged from 200 to 500 rpm.

[0122] The stirring was provided from two Rushton stirrers. A continuous air stream of 2 L / min was supplied and the bioreactor was aerated through a diffuser. In these conditions, the percentage of oxygen dissolved in the medium was 50% v / v during the fermentation. The culture medium was inoculated with an inoculum in an exponential phase. The inoculum size for the in-batch fermentation for the production of antifungal fermented product from Bacillus subtilis was 2% ((v) / (v)). The inoculum ODeoonm was 0.3 (corresponding to a concentration of ~ IxlO8CFU / mL).

[0123] Example 2 - Analysis of the profile of the antifungal peptides and the bioactive enzymes of the fermentation product of the strain of Bacillus subtilis RBI of the invention

[0124] For the determination of peptides and lipopeptides, the samples were diluted with water, homogenizing 1 mL of each product in 9 mL of MilliQ water. Subsequently, the samples (control with glucose in a concentration of 50 g / L and fermentation product obtained from GMSM-S with glucose in a concentration of 50 g / L contacted for 6 h) were injected in the LC-ESLMS-TOF system. The analysis was performed in triplicate. The concentration of the identified lipopeptides is reported in Table 3:

[0125] Table 3

[0126] Lipopeptides (mg / L) Control 50 g / L GMSM 50 g / L

[0127] Surfactin (C12) n.d. 661.9 + 36.2

[0128] Surfactin (C13) (A) n.d. 763.9 + 8.0

[0129] Surfactin (C14) (B) n.d. 794.0 + 55.1

[0130] Surfactin (C15) (c) n.d. 811.8 + 43.3

[0131] Fengycin C17 n.d. 841.3 + 9.5

[0132] Plipastatin A2 n.d. 200.1 + 1.7

[0133] Plipastatin Bl n.d. 134.9 + 0.5

[0134]

[0135] No lipopeptide was detected in the control sample. However, in the fermentation product obtained from GMSM-S with glucose in a concentration of 50 g / L (GMSM-S 50 g / L), the lipopeptides show an effective antibacterial activity at low concentrations towards gram-positive and gram-negative bacteria.

[0136] The identification of the proteins in the fermentation product of GMSM-S 50 g / L was performed by LC / MS-TOF. The proteins were precipitated with trichloroacetic acid (10%) andwashed twice with cold acetone. Finally, the proteins were reduced with DTT, alkylated with iodoacetamide, and digested with trypsin. After the digestion, the peptides were purified and concentrated with the Amicon Ultra-0.5 mL (3 kDa) centrifuge filters and analyzed with LC / MS- TOF. The identification of the peptides from the raw data was performed with the Spectrum Mill MS Proteomics software and research in the UniProt (SwissProt) database with a taxonomic restriction to Bacillus spp.

[0137] Example 3 - Test of antifungal and antibacterial activity of the fermented products of the invention

[0138] Tested strains:

[0139] The strains used in the experiments belong to the American Type Culture Collection (ATCC): Staphylococcus aureus ATCC 6538; Candida albicans IHEM 2894; Trichophyton interdigitale ATCC 9533; Microsporum canis ATCC 36299; and M alas ezzia furfur ATCC 14521.

[0140] Experimental protocol:

[0141] Staphylococcus aureus ATCC 6538 was cultivated on Tryptic Soy Agar (TSA), while Candida albicans IHEM 2894 and Maias ezzia furfur ATCC 14521 on Sabouraud Dextrose Agar (SDA) at 37 °C for 16-20 hours.

[0142] For the bacterial strain an inoculum with a cellular density of 5xl07CFU / mL in physiological solution (0.9% NaCl) was prepared. For the fungal strains, an inoculum with a cellular density of IxlO7CFU / mL in physiological solution was prepared.

[0143] Subsequently, 1 mL of the final bacterial suspension was used to inoculate 14 mL of TSA (Tryptic Soy Agar) with the adding of 0.7% agar, while 1 mL of the fungal suspension was used to inoculate 14 mL of SDA (Sabouraud Agar) with the adding of 1% Agar, only in the case of M. furfur olive oil was added to SDA to favor the fungal growth on Agar. Sterile wells were created on Agar plates and filled with 30 pL of the solutions to be tested, in duplicate.

[0144] LB, LB1-S or LB2-S were used as positive control in a non-fermented GMSM-S culture medium with glucose in a concentration of 50 g / L or 20 g / L:

[0145] - 30 pL of a Chloramphenicol solution for the bacterial inhibition of S. aureus,

[0146] - 30 pL of a Fluconazole solution for the inhibition of C. albicans,

[0147] - 30 pL of an Amphotericin B solution for the inhibition of dermatophyte fungi M. furfur, M. canis, T. interdigitale.

[0148] As negative control the sterile non-fermented GMSM-S culture media with glucose in a concentration of 50 g / L or 20 g / L were used.

[0149] The plates were then kept at 4 °C for 30 minutes to facilitate the complete absorption of thesample in the Agar, followed by a night incubation at 37 °C.

[0150] The dermatophyte fungi were cultivated on Potato Dextrose Agar (PDA) plates at 28 °C for 15 days. Once the fungi reached a suitable growth step, fungi circular discs with a diameter of 5 mm were prepared. A fungus disc was placed at the center of a 25 mL PDA plate. Subsequently, sterile wells were created and filled with 30 pL of the test solutions, in duplicate. Finally, the plates were incubated at 28 °C for 15 days.

[0151] After the incubation period, the plates were examined to verify the presence of an inhibition halo (a light circular area around the Agar wells), indicating the antibacterial or antifungal activity. The antimicrobial and antifungal activity was considered positive when the halo diameter was higher than 6 mm.

[0152] Results of the antifungal and antibacterial test

[0153] The effects of the inhibitory activity of the fermentation products of the invention are illustrated in Table 4. The results show that the fermentation products with the media LB, LB1-S, and GMSM-S show an antimicrobial activity against S. aureus. In particular, the fermentation products obtained from LB2-S and GMSM-S showed a growth inhibition even against the pathogenic fungus C. albicans.

[0154] Table 4 provides the measurements of the inhibition halos of the fermentation products against the selected pathogenic strains.

[0155] Table 4

[0156] C. albicans S'. aureus

[0157] Sample

[0158] Inhibition halo (mm)

[0159] Positive control 26 33

[0160] Negative control 0 0

[0161] LB ___ 12

[0162] LB1 ___ 12

[0163] LB2 12 ___ GMSM 33 12

[0164]

[0165] The results indicate that the fermentation products obtained from LB, LB1-S, and GMSM-S showed an effective antimicrobial activity against S. aureus, with an inhibition area of 12 mm.

[0166] Further, the fermentation product obtained from GMSM-S showed also a high antifungal activity against C. albicans, showing an inhibition area of 33 mm.Further, the fermentation products of the invention showed a higher inhibitory effect of the fungal growth with respect to the positive control (Fluconazole), thereby showing an enhanced efficacy with respect to the commonly used antifungal drug.

[0167] Example 4 - Antifungal tests of the fermented products of the invention starting from GMSM

[0168] A fermentation of strain according to Example 1 was carried out using the GMSM-S culture medium, and testing two concentrations of glucose in the culture medium, i.e. 50 g / L or 20 g / L. Subsequently, the culture medium was used to test the fungal strains of Example 3, by the protocol of Example 3.

[0169] It is possible to observe halos with a width of 3.8 and 3.7 cm for the product of the invention and of 2.5 and 2.4 cm for the control + (fluconazole) in the Agar tests of Candida albicans IHEM 2894 ATCC using the GMSM-S culture medium with a glucose concentration of 50 g / L.

[0170] It is possible to observe halos with a width of 3.7 and 3.8 cm for the product of the invention and of 2.5 and 2.6 cm for the control + (fluconazole) in the Agar tests of Candida albicans IHEM 2894 ATCC using the GMSM-S culture medium with a glucose concentration of 20 g / L.

[0171] It is possible to observe a fungus growth inhibition for the product of the invention with respect to the control - (negative) and a fungus growth inhibition for the product of the invention more evident with respect to the control + (positive, amphotericin) in the Agar tests of Trichophyton interdigitale ATCC 9533 using the GMSM-S culture medium with a glucose concentration of 20 g / L, as shown in Figure 1.

[0172] It is possible to observe a fungus growth inhibition for the product of the invention with respect to the control - (negative) and a fungus growth inhibition for the product of the invention more evident with respect to the control + (positive, amphotericin) in the Agar tests of Trichophyton interdigitale ATCC 9533 using the GMSM-S culture medium with a glucose concentration of 50 g / L, as shown in Figure 2.

[0173] It is possible to observe a fungus growth inhibition for the product of the invention with respect to the control - (negative) and a fungus growth inhibition for the product of the invention more evident with respect to the control + (positive, amphotericin) in the Agar tests of Microsporum canis ATCC 36299 using the GMSM-S culture medium with a glucose concentration of 50 g / L, as shown in Figure 3.

[0174] It is possible to observe a fungus growth inhibition for the product of the invention with respect to the control - (negative) and a fungus growth inhibition for the product of the invention more evident with respect to the control + (positive, amphotericin) in the Agar tests ofMicrosporum canis ATCC 36299 using the GMSM-S culture medium with a glucose concentration of 20 g / L, as shown in Figure 4.

[0175] The fermentation product obtained according to Example 1 with the GMSM-S culture medium at a glucose concentration in the medium of 50 g / L was subjected to a lyophilization process. The thus obtained lyophilizate was used in MIC (minimum inhibitory concentration) quantitative studies to determinate the minimum concentration at which the fermented product of the invention exerts an inhibitory activity against a specific fungus. The following MICs emerged from the studies:

[0176] Trichophyton interdigitale ATCC 9533 - MIC: 46.52 mg / mL;

[0177] Microsporum canis ATCC 36299 - MIC: 11.63 mg / mL.

[0178] In this case, a fungus growth inhibition with respect to the negative control was observed, with an even more evident inhibition with respect to the antibiotic used as positive control.

[0179] It is possible to observe halos with a width of 2.1 and 2.2 cm for the product of the invention and of 1.7 and 1.9 cm for the control + (amphotericin) in the Agar tests of Malasezzia furfur ATCC 14521 using the GMSM-S culture medium with a glucose concentration of 50 g / L.

[0180] It is possible to observe halos with a width of 2 cm for the product of the invention and of 1.7 cm for the control + (amphotericin) in the Agar tests of Maias ezzia furfur ATCC 14521 using the GMSM-S culture medium with a glucose concentration of 20 g / L.

[0181] It is possible to verify from the halos, that the same antifungal activity with a lower amount of glucose in the fermentation medium was obtained.

[0182] Example 5 - Quantitative test (MIC Test) of the antifungal power of the fermented products of the invention, according to Example 1

[0183] Both the fermentation product obtained from GMSM-S 20g / L, and the non-fermented GMSM-S 20g / L culture medium were lyophilized, using 40 mL for each.

[0184] The fermented medium was dissolved in 7 mL of PBS, while the non-fermented medium, used as a control, was dissolved in 8.5 mL of PBS. Both the samples were then sterilized with UV rays for 30 minutes.

[0185] 1.3027 g of fermented antifungal broth were obtained, while 1.7544 g were obtained from the lyophilization process of the non-fermented medium. With these amounts, different concentrations were prepared to carry on with quantitative studies of MIC (minimum inhibitory concentration), i.e. the minimum concentration at which the fermented product of the invention exerts an inhibitory activity against a specific bacterium or fungus.

[0186] Two 96- wells plates were prepared.The first plate contained the fermentation product obtained from GMSM-S 20 g / L, and the non-fermented GMSM-S 20 g / L culture medium, with the adding of RPMI 2x medium and Candida albicans IHEM 2894 ATCC.

[0187] The second plate contained the fermentation product obtained from GMSM-S 50 g / L, and the non-fermented GMSM-S 50 g / L culture medium, with the adding of RPMI 2x medium without Candida albicans IHEM 2894 ATCC.

[0188] 12 different concentrations for each sample were tested, obtained by 1:2 dilution starting from the lyophilized samples, specifically: 93.05 mg / mL, 46.52 mg / mL, 23.26 mg / mL, 11.63 mg / mL, 5.81 mg / mL, 2.90 mg / mL, 1.45 mg / mL, 0.72 mg / mL, 0.36 mg / mL, 0.18 mg / mL, 0.09 mg / mL, 0.045 mg / mL.

[0189] The same procedure was carried out for the fermentation product obtained from GMSM-S 50 g / L, and the non-fermented GMSM-S 50 g / L culture medium, to compare the antifungal efficacy of the two tested fermentation conditions.

[0190] A suspension of Candida albicans IHEM 2894 ATCC was prepared by dissolution in 6 mL of RPMI 2X medium, adjusting the ODeoonm between 0.12 and 0.15 nm to obtain a microbic concentration of 1.5xl06CFU / mL.

[0191] Subsequently, 1 mL of this suspension was withdrawn and it was diluted in 9 mL of medium to obtain a 1:10 dilution (1.5xl05CFU / mL).

[0192] From the diluted suspension, 100 microliters were withdrawn and distributed in each well, afterwards adding 100 microliters of the fermentation product obtained from GMSM-S 50 g / L or the non-fermented GMSM-S 50 g / L culture medium in the several wells.

[0193] The same procedure was repeated with the fermentation product obtained from GMSM-S 20g / L or the non-fermented GMSM-S 20g / L culture medium within the wells.

[0194] The MIC measurement was performed with a multi- well reader Multiscan FC, using the OD at 450 nm, according to the EUCAST protocol.

[0195] For the fermentation product obtained from GMSM-S 20 g / L an inhibition percentage of 100% at 5.81 mg / mL, an inhibition percentage of 98% at 23.26 mg / mL, an inhibition percentage of 96% at 93.05 mg / mL were found.

[0196] For the fermentation product obtained from GMSM-S 50 g / L an inhibition percentage (MIC90) at 11.63 mg / mL was found.

[0197] Therefore, a fungus growth inhibition with respect to the negative control and an even more marked inhibition with respect to the antibiotic used as a control was observed.

[0198] Example 6 - Comparative test of the antifungal power of the fermented productsobtained with the strain of Bacillus subtilis RBI (according to the invention) and a strain of Bacillus subtilis M12 (not according to the invention)

[0199] The strains of Bacillus subtilis RB 1 (according to the invention) and one strain of Bacillus subtilis M12 (comparative, known also as BSM12 or MBPL BSM12, commercially available) were pre-cultivated in LB medium for 24 h (28 °C, 140 rpm) and then inoculated (~107cells) in duplicate in the same GMSM-S medium.

[0200] A fermentation in the same operative conditions was then carried out, according to Example 1.

[0201] The supernatants were recovered by centrifugation and sterile filtration 0.22 pm.

[0202] The antifungal activity was assessed by agar spot test against Candida albicans IHEM 2894 using positive (Amphotericin B) and negative (physiological solution) controls.

[0203] Both the strains show antifungal activity, but with quantitative differences significative in favor to the strain of the invention as shown in Fig. 5 and Table 5:

[0204] Table 5

[0205] Halo diameter

[0206] Strain Repetition (cm)

[0207] RBI a 4.1

[0208] RBI b 4.2

[0209] M12 a 3.5

[0210] M12 b 3.5

[0211]

[0212] Therefore, the strain RBI produces an average halo of ~ 4.15 cm, while the comparative strain M12 produces an average halo of ~ 3,5 cm, corresponding to an increment of antifungal activity of about +18-20% in the same experimental conditions for the strain RBI.

[0213] The test validity is confirmed by the positive answer of the standard antifungal control and the absence of inhibition in the negative control.

[0214] The strain RBI shows a higher ability of producing active extracellular antifungal metabolites against pathogenic yeasts with respect to a comparative strain M12 in the same experimental conditions.

[0215] The experimental data demonstrate that, cultivating two strains of the same species in the same medium and in the same conditions, the strain RBI produces an antifungal activity significatively higher with respect to the comparative strain M12.

Claims

CLAIMS1. A process for the preparation of a fermentation product comprising the following steps:a) providing an inoculum of Bacillus subtilis RBI deposited at DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 35197 on 08 / 10 / 2024;b) incubating the inoculum;c) inoculating a culture medium selected from Luria-Bertani culture medium, Luria- Bertani\S culture medium, Luria-Bertani2-S culture medium, and Glucose Mineral Salt Medium-S culture medium with the incubated inoculum;d) fermenting to obtain a suspension;e) centrifuging the suspension so to obtain a solid fraction and a liquid fraction;f) isolating the liquid fraction to obtain the fermentation product.

2. A process according to claim 1, wherein the culture medium is the Glucose Mineral Salt Medium-S culture medium.

3. A process according to claim 2, wherein the Glucose Mineral Salt Medium-S culture medium comprises glucose in a concentration from 20 g / L to 50 g / L.

4. A process according to claim 2, wherein the Glucose Mineral Salt Medium-S culture medium comprises glucose in a concentration of 50 g / L.

5. A fermentation product obtainable by the process of claims 1-4.

6. A fermentation product according to claim 5, comprising the following proteolytic enzymes:Bacillolysine,Neutral metalloproteinases,Serine proteases,Beta-glucanases,Chitosanases,Beta-N-acetylglucosaminidases,Muramidases / Lytic transglycosilases,Peptidoglycan beta-N-acetylmuramidases.

7. A fermentation product according to claim 6, further comprising the following lipopeptides:Lipopeptides Amount (mg / L)Surfactin (C12) 661.9 + 36.2Surfactin (C13) (A) 763.9 + 8.0Surfactin (C14) (B) 794.0 + 55.1Surfactin (C15) (c) 811.8 + 43.3Fengycin C17 841.3 +9.5Plipastatin A2 200.1 + 1.7Plipastatin Bl 134.9 + 0.

58. Non-therapeutic use of the fermentation product according to claims 5-7 as an antifungal.

9. A fermentation product according to claims 5-7 for use as a medicament.

10. A fermentation product according to claims 5-7 for use in the prevention and / or treatment of fungal or bacterial infections.

11. A fermentation product for use according to claim 10, in detergent products for human or animal use.

12. A strain of Bacillus subtilis RBI deposited at DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 35197 on 08 / 10 / 2024.

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