Method for obtaining a gramicidin s extract from microbial biomass and its use as an antimicrobial or antibiotic

Abstract

The invention relates to a method for obtaining a gramicidin S extract from spore-forming Bacillus belonging to the genus Aneurinibacillus, characterised by comprising a solid-liquid extraction step using a solvent selected from the group comprising lactic acid, acetic acid, a biosurfactant extract obtained from corn steep liquors, or combinations thereof, at a temperature equal to or less than 40 °C. The invention also relates to the use of the gramicidin S extract obtained as an antimicrobial agent in the cosmetic and agrochemical industries, and as an antibiotic in the pharmaceutical industry.

Description

[0001] PROCESS FOR OBTAINING A GRAMICIDIN SA EXTRACT FROM MICROBIAL BIOMASS AND ITS USE AS AN ANTIMICROBIAL OR ANTIBIOTIC DESCRIPTION

[0002] Technology sector

[0003] The present invention belongs to the field of obtaining biotechnologically produced antibiotics and antimicrobial agents.

[0004] The main object of the invention is a process for obtaining a gramicidin S extract from the microbial biomass of a gramicidin S-producing Bacillus such as Aneurinibacillus aneu nilyticus, belonging to the genus Aneurinibacillus, comprising a solid-liquid extraction step using a solvent selected from lactic acid, acetic acid, biosurfactant extract obtained from corn washing liquors or combinations thereof.

[0005] Background of the Invention

[0006] Gramicidin S is a polypeptide antibiotic effective against a variety of bacteria. It primarily attacks Gram-positive bacteria, excluding bacilli, although it also shows activity against some strains of Gram-negative bacteria such as Escherichia coli. Discovered more than eight decades ago by Georgyi Gause and Maria Brazhnikova in 1942, gramicidin S remains relevant in the fight against bacterial infections, especially now that bacteria are developing resistance to many conventional antibiotics.

[0007] Gramicidin S is a cationic cyclic decapeptide composed of ten amino acids comprising two sequences of valine (Val), ornithine (Orn), leucine (Leu), phenylalanine (Phe), and proline (Pro) (Val-Orn-Leu-Phe-Pro-Val-Orn-Leu-Phe-Pro) with a molecular weight of 1141 Da. However, this molecular weight can vary when some of these amino acids, primarily ornithine (Orn), are substituted by other amino acids such as lysine (Lys). For example, Alezeni and his team discovered in 2017 that Aneurinbacillus migulanus, Nagano strain, produces two additional gramicidin S analogs, with molecular weights of 1155 and 1169 Da, in addition to the known 1141 Da analog. These analogs substitute one or two ornithine residues with lysine. Other researchers (Hori and Kurotsu, 1997) also identified gramicidin S analogues in Bacillus brevis Nagano, where ornithine is substituted by Usine.The biotechnological production of gramicidin S involves the use of spore-forming Bacillus species such as Bacillus brevis (Fang, 1997) and Aneurinibacillus migulanus (Berditsch et al., 2007, 2017; Alenezi et al., 2017; Nesteruk and Syrov, 2020), which store gramicidin S in vacuoles. Therefore, extracting gramicidin S requires separating the microbial biomass from the fermentation medium, washing it, and then extracting the gramicidin S using various techniques. The following section reviews the different processes described in the literature for extracting gramicidin S from the microbial biomass of various spore-forming Bacillus species.

[0008] Furthermore, gramicidin S acts as an ionophore, meaning it facilitates the transport of ions across biological membranes. This ion-transporting capacity is closely related to its structure and its interaction with specific ions such as potassium (K+). + ) and sodium (Na + ). When gramicidin S interacts with K ions + or Na +Gramicidin S forms inclusion complexes in which the metal ion is encapsulated by the antibiotic structure, altering its molecular weight. These complexes can influence the biological activity of gramicidin S, as well as its ability to alter cell membrane permeability (Prosser et al., 1998), which can lead to variations in its antibiotic and antimicrobial activity. Ethanol is the most widely used and studied solvent for gramicidin S extraction. It was first used by Hotchkiss et al. in 1941, followed by Berditsch et al. (2007, 2015), Alenezi et al. (2017), and more recently Nesteruk and Syrov (2020) (PATENT EP3660141 A1). These latter authors have patented the biotechnological production and extraction of gramicidin S from a mutated strain Aneurinibacillus migulanus, VKPM B-10212 which has subsequently been extracted with ethanol.

[0009] Nesteruk and Syrov (2020) mention the use of lactic acid in the development of their patent, but not in the extraction process; rather, it serves as a nutritional supplement during fermentation. In fact, these authors refer to lactic acid within the methodology section, which covers the fermentation conditions and medium. They conclude that the optimal medium for producing gramicidin S with the studied compound should consist of: 30 mL of glycerol; 24.6 mL of lactic acid; 2 g of K₂HPO₄; 5 g of NaCl; 0.2 g of MgSO₄·7H₂O; 12 g of ammonium oxalate; up to 10 mg of yeast autolysate; up to 20 mg of casein hydrolysate; and 2 mL of Laprol. Regarding the gramicidin S extraction process proposed by Nesteruk and Syrov (2020), it is carried out in the absence of lactic acid. The biomass obtained is then filtered through a ceramic filter unit using ultrafiltration and washed with deionized water to remove impurities.The concentrated biomass is then spray-dried at 180 °C. The dried biomass is subsequently washed with acetone at 40 °C to remove colored impurities. The acetone is removed by purging with nitrogen, followed by an additional acetone rinse and vacuum drying at 45 °C. Gramicidin S is then extracted using ethanol, adjusting the pH to 3.0–4.0 with dilute hydrochloric acid (HCl:ethanol = 1:2). This extraction is repeated several times, removing the extracts with pressurized nitrogen. After ethanol removal, the biomass is vacuum-dried at approximately 47 °C. The dried biomass is then dissolved in water and decolorized using activated carbon, followed by filtration in a filter press and a final ethanol rinse to minimize product loss through carbon sorption. After these processes, the resulting extract is transferred to a crystallizer, diluted with deionized water, and a concentrated sodium chloride solution is added.It is heated to a temperature of 55 to 65 °C. After clarification, the solution is cooled to a temperature below 5 °C. The crystals formed are separated in a centrifuge and dried under vacuum at a maximum temperature of 65 °C.

[0010] On the other hand, Alenezi et al. (2017) proposed the use of ethanol with hydrochloric acid, ethanol being the most studied solvent in the literature for the extraction of gramicidin S in combination with other separation processes. Other authors have also considered the use of solvents other than ethanol for the extraction and purification of gramicidin, such as acetone or ether (Hotchkis and Dubos, 1940, 1941), primarily for the extraction of linear gramicidin composed of 15 amino acids with a molecular weight of around 1882 Da, depending on the variability of some of its amino acids. Dammak et al. (2017) also considered the extraction of gramicidin S from microbial biomass using ethyl acetate.

[0011] A search of Scopus without any restrictions, using the keywords "Gramicidin S" and "Lactic acid," yields four publications: Tran et al., 2022; MacPhee et al., 2005; Demain, 2004; and Haynes et al., 1974, none of which are related to the extraction of gramicidin S from microbial biomass. Similarly, a search using the keywords "Gramicidin S" and "Acetic acid" returns 18 publications, including: Sarabando et al., 2023; Ishikawa and Tanabe, 2023; Hiraoka et al., 2022; Frikha et al., 2017; Dammak et al., 2017; Tuin et al., 2009; and Mogi et al., 2009. Hiraoka et al., 2006; Smirnova et al., 2003; Yang et al., 1999; Helle et al., 1992; Bulgakova et al., 1988; Hancock and Wong, 1984; Tochikubo et al., 1981; Shorts et al., 1975; and Kotelnikova and Slovjeva, 1974.Of all the publications mentioned, the one most related to the extraction of gramicidin S using solvents is the work published by Dammak et al. in the journal Biomed Research International in 2017, which includes the extraction of antimicrobial compounds, among which gramicidin S was detected, from Paludifilum haiophilum using ethyl acetate.

[0012] On the other hand, an advanced search in PubMed using the keywords "Gramicidin S" and "Lactic acid" yields a total of 2 publications, while a search using "Gramicidin S" and "Acetic acid" yields a total of 3 publications. The most relevant to the extraction of gramicidin S is the work by Ninomiya et al. in 2024, which describes the use of acetic acid in conjunction with other solvents to detect peptides by mass spectrometry. In this study, they dissolved gramicidin S in acetic acid, among other solvents, after it had already been extracted. Therefore, in this case, acetic acid was not used as an extraction agent for gramicidin S from microbial biomass, but rather as a solvent for analysis.

[0013] More recently, the extraction of gramicidin S from Aneurinibacillus microbial biomass using phosphate buffer saline has been reported (López-Prieto et al., 2012; Lvova et al., 2024), but the extraction of gramicidin S using biosurfactants and more specifically biosurfactant extracts extracted from corn washing liquors has not been considered to date.

[0014] Furthermore, in 2023, Moldes et al. presented a paper at the Congress organized by the American Society of Chemists in San Francisco (USA) entitled “Promising green solvents for the production of Gramicidin S biosurfactant extract from Aneurinibacillus aneurinilyticus isolated from corn steep liquor” which includes the use of ethanol or phosphate buffer saline commonly known as PBS or ethanol, without including any mention of the extracting agents comprising lactic acid, acetic acid or biosurfactants for the extraction of Gramicidin S.

[0015] Explanation of the invention

[0016] A process is proposed for obtaining a gramicidin S extract from spore-forming Bacillus, using the biomass of Aneurinibacillus aneuriniliticus as a model. The process is based on a physical extraction involving the use of biodegradable and biocompatible solvents, including lactic acid, acetic acid, a biosurfactant extract obtained from corn washing liquors, or combinations thereof.

[0017] Detailed explanation of the invention

[0018] In this invention, a gramicidin S extract has been obtained from the microbial biomass of Aneurinibacillus aneuriniliticus, belonging to the genus Aneurinibacillus, which also includes other gramicidin S-producing species such as Bacillus brevis and Aneurinibacillus migulanus. This microorganism was isolated from corn washings and is stored in the Spanish type culture collection in Valencia for the exclusive use of the University of Vigo.

[0019] The culture medium used for microbial biomass growth consisted of tryptic soy broth (TSB) or Sabouraud dextrose broth (SDB). Fermentation was carried out at 37 °C with stirring at 150 rpm.

[0020] After the microbial biomass grew, it was mechanically separated by filtration and / or centrifugation and washed with water until colorless. Gramicidin S was then extracted using lactic acid, acetic acid, and / or biosurfactants extracted from corn washing liquors. This extract was obtained following the protocol established in patent WO2014 / 044876. Preferably, the biosurfactant extract obtained from corn washing liquors was extracted using ethyl acetate.

[0021] The extraction process was carried out for between 5 minutes and 24 hours with agitation that allowed for complete homogenization of the solvent with the microbial biomass. The process was performed at various temperatures up to 40 °C, and it was observed that polymerization and denaturation of gramicidin S began to occur at 40 °C. Preferably, the process temperature is ambient temperature.

[0022] The solvents used ranged from aqueous solutions of the solvents (lactic acid, acetic acid, and / or biosurfactant extract) at 1% v / v, either separately or in combination, to the use of the pure solvents alone or in combination. It was observed that 1% v / v solutions of the tested solvents (lactic acid, acetic acid, and / or biosurfactant extract) resulted in yields comparable to those obtained with pure ethanol.

[0023] Table 1 shows the extraction conditions tested in which the presence of gramicidin S was confirmed. The extractions were carried out in aqueous solution.

[0024] Table 1. Extraction conditions of gramicidin S

[0025] Concentration, Temperature, Time, Extractant (minimum, maximum)

[0026] % v / v % v / v °C min 5' 10- Ci- o- 5(T Lactic acid 1 85.5 15-40 ' ¿n.ín ' bO; 120 5- 0- 20- 80- 50- Acetic acid 1 99.7 15-40 ' ¿í don '

[0027] 60; 120 ExtraCtO 4 \. n. on. ry

[0028]

[0029] biosurfactant 1 5 15-40 'í

[0030] bU; IzU Lactic acid + ' in - 90' 80 o - Acetic acid 1 85.5-100 15-40 ' n ' bO; 120 Lactic acid +

[0031] extract. 5; 10; 20; 30; 50; biosurfactant 1 85.5-100 15-40 60; 12Q

[0032] Acetic acid +

[0033] e Cx.tUraQctIo. 85 5-100 15-40 5' ' 10' 20' 30' 50' biosurfactant 60; 120 Acetic acid +

[0034] lactic acid +. r,rr 5; 10; 20; 30; 50;

[0035] ,. 1 85.5-100 15-40 „... extract 60; 120 biosurfactant

[0036] For the determination and characterization of gramicidin S, mass spectrometry coupled with high-performance liquid chromatography (UPLC) was used, in addition to Fourier transform infrared spectroscopy (FTIR). Commercial gramicidin S (Byosinth) and gramicidin S extract produced from Aneurinibacillus aneurinilyticus using ethanol or ethanol combined with HCl as solvents were used as controls.

[0037] Figure 1 shows the general protocol followed, and Figure 2 shows the mass spectra obtained by extraction with acetic acid (Fig. 2A), lactic acid (Fig. 2B), and biosurfactant extract (Fig. 2C), corresponding to the gramicidin S extracts protected by this invention. Furthermore, Figure 3A shows the mass spectra of commercial gramicidin S, and Figure 3B shows the mass spectra of the gramicidin S extract produced with ethanol and HCl. Additionally, as an example, Figure 4A shows the FTIR of the gramicidin S extract obtained with 1% v / v acetic or lactic acid, and Figure 4B shows the FTIR of gramicidin S, where the characteristic trifluoroacetic acid (TFA) band can be seen between 1000 and 1250 cm⁻¹. -1 in comparison to the conventional gramicidin extract obtained with ethanol and HCl. The gramicidin S extract produced under the present invention is free of trifluoroacetic acid (TFA).

[0038] Brief description of the drawings

[0039] To complement the description provided and for a better understanding of the characteristics of the invention, a set of drawings is included as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0040] Fig. 1. Schematic of the proposed process for obtaining gramicidin S extract from Aneurinibacillus aneurinilyticus biomass. This scheme begins with a fermentation process in TSB or SDB medium inoculated with Aneurinibacillus aneurinilyticus at 24–168 h and 37 °C. After fermentation, the microbial biomass is recovered by centrifugation and washed with water. The washed microbial biomass is then subjected to a solid-liquid extraction process using a solvent selected from lactic acid, acetic acid, and / or a biosurfactant extract obtained from corn washing liquors with ethyl acetate. After extraction, the biomass is separated by centrifugation or filtration, yielding a gramicidin S extract dissolved in the extractant matrix. This extract can be used directly in its dissolved state or lyophilized to obtain a powdered extract.

[0041] Fig. 2. Mass spectrum of gramicidin S extracts extracted with: A) lactic acid (1% v / v); B) acetic acid (1% v / v) and C) biosurfactant extract (1% v / v) obtained from corn washing liquors by extraction with ethyl acetate.

[0042] Fig. 3. Mass spectra of: A) commercial gramicidin S and B) gramicidin S extract obtained with the ethanol+HCl mixture

[0043] Fig. 4. Fourier transform infrared (FTIR) spectrum obtained from the samples of: A) gramicidins S extract obtained with solvents comprising acetic acid and lactic acid and B) from the samples of commercial gramicidin S and from that extracted with ethanol and HCl (B).

[0044] Realizations of the invention

[0045] In a preferred embodiment, the process of the present invention for obtaining a gramicidin S extract from microbial biomass of Aneurinibacillus aneurinilyticus comprises a solid-liquid extraction step using lactic acid, acetic acid and / or biosurfactant extract obtained according to patent WO2014 / 044876 with ethyl acetate at a concentration of 1% v / v, the extraction time being 10 minutes, at room temperature, with stirring at 300 rpm.

[0046] In another embodiment of the invention, the process can be carried out without agitation.

[0047] In another embodiment of the invention, the process can be carried out with combinations of lactic acid (1% v / v), acetic acid (1% v / v) and / or biosurfactant extract (1% v / v) the latter obtained as claimed in patent WO2014 / 044876 and extracted with ethyl acetate, for obtaining the gramicidin S extract.

[0048] The gramicidin S extract obtained after extraction with lactic acid comprises, in addition to lactic acid, gramicidin S and gramicidin S derivatives.

[0049] Furthermore, the gramicidin S extract extracted with acetic acid contains acetic acid, gramicidin S and gramicidin S derivatives.

[0050] The gramicidin S extract produced with the biosurfactant extract, obtained in turn from corn washing liquors, in addition to biosurfactant contains gramicidin S and gramicidin S derivatives.

[0051] Gramicidin S extracts extracted with combinations of acetic acid, lactic acid and / or biosurfactant extract are composed of the extracting agents used, in the selected proportion.

[0052] In another embodiment of the invention, the gramicidin S extract obtained is subjected to precipitation following standard protein precipitation methods. In another embodiment, the gramicidin S extract obtained can be lyophilized to obtain the powdered extract.

[0053] With respect to this invention, the gramicidin S extract was produced by a novel extraction process, and said extract contains components different from those obtained with ethanol, including lactic acid, biosurfactant extract or acetic acid used as extractants and which form part of the formula, for example antibiotic, containing different proportions of gramicidins S, which forms a cluster around 1141 Da and gramicidins S analogues comprising common masses, after the proposed extraction processes, around the following molecular mass clusters: 1113 Da, 1127 Da, 1135 Da, 1149 Da, 1155 Da, 1163 Da, 1177 Da, 1192 Da, 1207 Da, 1221 Da, in different proportions (see Fig. 2 and 3).Therefore, formulations comprising extracts such as the one of the present invention can be considered to have varying proportions of gramicidin S and its derivatives, which could result in different therapeutic effects, although with the same qualitative differences. Some authors indicate that biotechnologically produced gramicidin S extracts have different antimicrobial activity depending on the proportion of these components. For example, Alezeni et al. (2017) discovered that the microbial biomass of Aneurinibacillus migulanus produces two additional gramicidin S analogs with masses of 1155 and 1169 Da, in addition to the recognized gramicidin S variant with a mass of 1141 Da. In these analogs, one or two ornithine residues are substituted by usine in the peptide sequence.These authors observed that those strains of Aneurinibacillus migulanus that showed lower efficacy in inhibiting plant pathogens had a lower proportion of the gramicidin S derivative containing Usina.

Claims

CLAIMS 1. Process for obtaining a gramicidin S extract from speculated Bacillus belonging to the genus Aneurinibacillus characterized in that it comprises a solid-liquid extraction step using a solvent selected from the group comprising lactic acid, acetic acid, biosurfactant extract obtained from corn washing liquors or combinations thereof, at a temperature equal to or less than 40 °C.

2. The process according to claim 1, wherein the biosurfactant extract obtained from corn washing liquors is obtained by extraction with ethyl acetate.

3. The process according to claim 1 or 2, wherein the extraction temperature is ambient temperature.

4. The process according to any of claims 1 to 3, wherein the process further comprises a freeze-drying step of the extract obtained in the solid-liquid extraction step.

5. Use of the extract obtained by the process according to any of claims 1 to 4, as an antimicrobial agent in the cosmetic or agrochemical industry.

6. Extract obtained by the process according to any of claims 1 to 4, for use as an antibiotic.

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