RU2794803C1 - Purification method of mundticin p436 (ks) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for purification of P436(KS) mundticin is proposed, which includes cultivating the producer strain Enterococcus mundtii "GKPM - Obolensk" B-8925 for 12 hours in a bioreactor with a Brucella broth nutrient medium with the addition of meat extract and lactose and adding the sorbent CM-sephadex C25 10% of the volume into a bioreactor with a nutrient medium immediately before cultivation, followed by autoclaving of the biomass together with the sorbent at 0.5 atm for 15 min. Then the sorbent is taken, washed with phosphate-buffered saline with the addition of ethanol, transferred to a chromatographic column, followed by elution of the sorbed mundticin with a buffer, and the resulting one-stage purification product is further purified using HPLC by reverse-phase chromatography on a C18 column.

EFFECT: invention provides an increase in the purity and yield of P436 mundticin suitable for industrial use.

1 cl, 7 dwg, 5 tbl, 4 ex

Description

The invention relates to the field of biotechnology, biochemistry and the food industry, in particular to the preparation of a preparation used as a biopreservative in the food industry for decontamination of meat products from listeria, clostridia, enterococci and other gram-positive microorganisms involved in the spoilage of meat products.

BACKGROUND OF THE INVENTION

Mundticins are low molecular weight cationic thermostable class IIa peptides produced by Enterococcus mundtii that are weakly immunogenic and non-toxigenic for humans and animals.

The best-known mundticins are: mundticin ATO6 (Bennik et al. 1998) [1] isolated from the E. mundtii ATO6 strain obtained from endive chicory; mundticin KS (Kawamoto et al. 2002) [3] isolated from E. mundtii strain NFRI 7393 obtained from grass silage in Thailand; mundticin CRL35 (Saavedra et al. 2004, 2007) [5, 6] isolated from E. mundtii CRL35 strain obtained from Argentinean cheese; mundticin QU2 (K. Sonomoto et al. 2005) [7] - produced by E. mundtii QU2 strain isolated from soybeans. Despite different sources of isolation of producer strains, the peptides after post-translational modifications are almost identical. The presented works describe in detail the physicochemical, genetic and biological properties of mundticins, which make it possible to state that they are promising for use as biopreservatives. Despite this, there are a number of limitations for their implementation in practice, first of all, the low induction of the target peptide into the culture medium and its significant loss in the process of purification from ballast impurities. In addition, the producer strains themselves are poorly characterized and, therefore, do not meet the requirements for industrial strains.

Today, the fight against foodborne diseases associated with the contamination of food products with pathogenic microorganisms, in particular Listeria monocytogenes and Clostridium perfringens, remains relevant. Obtaining a drug, where the active ingredient is bacteriocin, which has a pronounced antilisteriosis activity, as well as an antimicrobial effect against a number of gram-positive microorganisms, in particular enterococci and clostridia, will prevent the risks of outbreaks of food poisoning in the population caused by listeria, clostridium and enterococci, and also increase the shelf life of products.

Several bacteriocins are known that have found application in the food industry, in particular: nisin - a commercial drug "Nisaplin", pediocin - Alta™ 2341 and Micocin®, a combination of three bacteriocins (carnocyclin A, carnobacteriocin BM1, piscicolin 126) produced by Carnobacterium maltaromaticum UAL307. Of these, only pediocin is recommended for use in meat products.

There are known "problem" places for the introduction of bacteriocins (including mundticins) into practice, such as the safety of producer strains, low yield and purity of the resulting product.

In particular, the method for producing mundticin CRL35 proposed by Farias et al. (1996) [8], using three steps: precipitation of mundticin with ammonium sulfate, gel filtration chromatography on Biogel P-6 and HPLC using a cation exchange column. The disadvantage of the method should be attributed to the fact that such a technique cannot be scaled up to obtain preparative quantities of the final product.

Also known is the purification method of mundticin proposed by Kawamoto et al. (2002) [3], using 3 stages: precipitation from the culture liquid with ammonium sulfate, followed by purification by cation exchange chromatography on SP-Toyopearl and post-purification on C18 SPE cartridge. The disadvantage of this method is the low yield of mundticin (25.6%) with a purity of not more than 50%.

A strain of Enterococcus mundtii ST4SA is known [Patent US 8444998], which produces a peptide - bacteriocin ST4SA (m.v. 3400 Da), which has antimicrobial properties, however, the high values of the bactericidal activity of the drug presented by the authors refer only to a sensitive test strain of lactobacilli - Lactobacillus casei LHS. Activity against Listeria was evaluated only for one representative of this genus, the non-pathogenic strain of Listeria innocua LMG 13568.

Known strain Enterococcus mundtii PPHS-5-13, producing a substance of peptide nature (bacteriocin) with antilisteriosis activity, not resistant to vancomycin [patent RU 2532227 C1]. The disadvantage of the strain is that bacteriocin itself has not been characterized, and the strain is not sufficiently annotated in terms of such indicators as the presence of virulence factors and biogenic amines.

Closest to the proposed invention is a strain of Enterococcus faecium 1073, producing bacteriocin E-1073 (m.v. 3256 Da), isolated from the intestines of a broiler chicken, identified and investigated in the SSC PMB. Under submerged culture conditions, this strain produced bacteriocin E-1073 with activity against L. monocytogenes at the level of 800-1600 AU/ml into the culture liquid. Such a low yield of the target product required the additional use of specific biosynthesis activators - competitive bacteria and signal peptides, which generally complicates the technology for obtaining bacteriocins.

The technical result of the invention is to obtain a strainEnterococcus mundii- producer mundticin P436, which has antimicrobial activity against listeria, clostridia and enterococci, as well as increasing the purity and yield of mundticin P436, suitable for industrial use.

The technical result is achieved by the proposed strainEnterococcus mundii - producer mundticin P436, which has antimicrobial activity,- deposited in the museum of the SSC PMB with the assignment of the number "GKPM - Obolensk" V-8925.

The Enterococcus mundti strain was isolated from the infraorbital sinuses of a bird. As a result of PCR analysis with specific primers, it was found that it contains bacteriocin - mundticin. Phenotypically, its manifestation is characterized by inhibition of test strains L. monocytogenes and Cl. perfringens (Table 1 (see the graphic part)).

Whole genome sequencing of the strain was carried out, followed by assembly and annotation of the genome. Whole genome sequencing was performed on Illumina MiSeq instrument (San Diego, CA USA, Illumina, Inc.) and MGISeq-2000 (Shenzhen, China, MGI Tech Co., Ltd). The de novo genome was assembled using the SPAdes v. 1.13.1. Final annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline. The search for virulence and antibiotic resistance factors was carried out using the ResFinder 4.1 software. and VirulenceFinder 2.0 (Center for Genomic Epidemiology (http://www.genomicepidemiology.org). Bacteriocin genes were searched using the Bagel 4 and BLAST programs. Biogenic amines were searched using the BLAST program. Prophages were searched using the Prophage Hunter program (https: //doi.org/10.1093/nar/gkz380).

The genome of the strain E. mundtii "GKPM - Obolensk" B-8925 was placed in the NCBI database under the number JAIZFT (strain SCPM-OB-8925). The strain does not contain genes for virulence and antibiotic resistance. Does not contain genes for biogenic amines and prophages. Contains the mundticin gene (figure 1, table 2, table 3 (see the graphic part)).

When cultivated on nutrient agar No. 1, the E. mundtii strain "GKPM - Obolensk" B-8925 forms S-shaped colonies, 0.1-0.2 mm in diameter, with the formation of a yellowish pigment. Gram microscopy shows Gram-positive cocci, singly or in pairs. Catalase- and oxidase-negative. In semi-liquid agar, they are immobile. Facultative anaerobe, optimum cultivation temperature 37°C. Ferments arginine, mannitol, L-arabinose, melibiose, raffnose, sorbitol, melicitose, does not ferment sorbose (EN-KOKKUS test, Erba Lachema, Czech Republic)

Thus, the strain meets the safety criteria, including the QPS (Qualified List of Presumption of Safety) requirements of the European Food Safety Authority (EFSA) and can be used as a producer strain under industrial conditions.

Also, the technical result of the invention is achieved by the fact that a method for purifying mundticin P436 is proposed, which consists in introducing the sorbent CM-sepharose C25 - up to 10% by volume, into a bioreactor with nutrient broth (Brucella broth (Himedia, Mumbai, India) with the addition of meat extract (5 g/l) and lactose (2 g/l)) before cultivating the producer strain for 12 hours, followed by autoclaving the culture liquid at 0.5 ATM for 10 minutes, selecting the sorbent on filtration sieves, washing and packing it into a chromatographic column with subsequent elution of the adsorbed mundticin with a buffer of the following composition: 0.4 M NaCl, 10%/V glycerol, 20%/V ethyl alcohol, 0.01 M acetate buffer, pH 5.0, then the obtained product of one-stage purification is further purified using HPLC by the reversed- phase chromatography on a C18 column.

The invention is illustrated by the following graphics.

Table 1. Mundticin P436 MIC for tested strains.

Figure 1. Mundticin gene P436 of producer strain SCPM-O-B-8925

Table 2. Sequence comparison of Mundticin P436 with the most closely related Mundticin ATO6.

Figure 2. Graph of growth curve and culture parameters of E. mundtii strain B-8925.

Figure 3. Chromatogram of P436 eluate after one-step purification.

Table 4. Analytical chromatography data of P436 eluate after one-step purification.

Figure 4. Mundticin P436 after two-step purification.

Table 5. Chromatographic data of purified mundticin P436.

Figure 5. Mass spectrum of a purified sample of Mundticin P436.

Figure 6. Antimicrobial activity of mundticin P436 using the "spot method" on the test strain L. monocytogenes ATCC 19111.

Figure 7. Samples of mundticin P436 on phoresis, flooded with test culture
L. monocytogenes ATCC 19111.

Example 1 Cultivation of the Strain

Inoculum preparation. 24-hour colonies of E. mundtii B-8925 are inoculated into a test tube with 5 ml of nutrient medium (Brucella broth (Himedia, Mumbai, India) supplemented with meat extract (5 g/l) and lactose (2 g/l)) and cultivated at a temperature of 37 °C, carrying out successive transfers of the culture into test tubes every two hours. The culture from the tube of the fourth passage is transferred into a flask with 300 ml of nutrient medium and cultivated in an incubator (IS-971 RF, Korea) at a temperature of 32°C and a rotation speed of 110 rpm. A 4-hour culture is used to inoculate the culture in the bioreactor.

Preparation of a sorbent for cultivation. CM Sephadex C-25 (Sigma, C25120, Sweden) is used as a sorbent. Preparation of the sorbent is as follows: dry powder is poured with deionized water and allowed to stand until complete swelling. Next, the sorbent is transferred to the Na ⁺ - form, washed successively with six volumes of 1M HCl, ten volumes of deionized water, six volumes of 1M NaOH, and then finally washed with deionized water to neutral pH values (5 - 7) and sterilized in an autoclave at 1 atm for 10 min. The sorbent prepared in this way is introduced in a volume of 10% of the volume into a bioreactor with a nutrient medium immediately before cultivation.

Deep cultivation. Sartorius Biostat B plus (Germany) with a working volume of 8 liters is used as a bioreactor. Cultivation is carried out at a temperature of 32°C, with stirring 120-160 rpm. with pH control (Hamilton EasyFerm Plus pH Sensors 225mm) and dO ₂ (Hamilton OxyFerm™ DO Sensor 225mm). Cultivation time 12 hours. At the end of cultivation, 2.5 l of deionized water with 10 ml of 1M HCl is added to the bioreactor and stirring is continued for another 30 minutes, after which the biomass together with the sorbent is pumped into a bottle and autoclaved at 0.5 atm for 15 minutes. Next, the sorbent is collected on a filtration sieve passing through the biomass (Fig.2 Graph of the growth curve and cultivation parameters of the strain E. mundtii B-8925).

Example 2 Purification of Mundticin P436. Collected on a CM Sephadex C-25 filtration sieve in a volume of 800 ml, washed successively with six volumes of deionized water, 2.5 l of 0.1 M phosphate-buffered saline pH 7.4 with 10% ethanol, and transferred to a chromatographic column XK 50/60 (28988964, GE Healthcare). Chromatography is carried out on an AKTA Purifier 100 chromatograph (GE Healthcare) with detection at a wavelength of A280 and a flow rate of 12 ml/min. The sorbent is washed with 0.1 M phosphate-buffered saline pH 7.4 with 10% ethanol until reaching the baseline and then eluted with 100% buffer B (0.4 M NaCl, 20% ethanol, 10% glycerol (141339.1211, Panreac) ). In the collected fraction determine the activity. Also, to analyze the purity and yield of mundticin, an aliquot of the active fraction was analyzed by HPLC reverse phase chromatography on a Partisphere rtf c18, 5μm 150×4.6 column (4522-0202, Whatman). The elution is carried out in a gradient mode of 0→100%. Buffer A dH2O + 0.1% TFA, buffer B ACN + 0.1% TFA (figure 3, table 4). The output of the obtained mundticin is 150-270% (from the control cultivation, without adding a sorbent) and the purity is not lower than 55-72% in one stage.

Example 3 Preparation of Mundticin P436 with a purity of ≥90%. To obtain highly purified mundticin P436, after one-step chromatography, the pH of the active eluate is adjusted to 10.6 with 1 M NaOH solution with stirring, centrifuged, the supernatant pH is adjusted to 7.4 with 1 M HCl solution, and reverse-phase chromatography is carried out on a Reprosil-PurBasic C18 column, 5 μm 250× 4.6 (Dr Maisch GmbH, Germany). The elution is carried out in a gradient mode of 0→100%. Buffer A dH2O + 0.1% TFA, Buffer B ACN + 0.1% TFA. The active fraction is collected, evaporated on a Laborota 4000 rotary evaporator (Heidolph, Germany), the volume is adjusted to 1/10 of the original volume with deionized water, and SPE (solid phase extraction) is performed on a Strata C18-E column (Phenomenex, USA). The eluate used was acetonitrile with 2% formic acid. The purity of the resulting mundticin is determined chromatographically by HPLC analysis by reverse phase chromatography on a Partisphere rtf c18, 5μm 150×4.6 column (4522-0202, Whatman). The elution is carried out in a gradient mode of 0→100%. Buffer A dH2O + 0.1% TFA, buffer B ACN + 0.1% TFA (figure 4, table 5) and mass spectral method MALDI-TOF MS on a microflex LRF mass spectrometer, Bruker Daltonik, in linear mode . Mass spectra were obtained and analyzed using the FlexControl and FlexAnalisys programs (FIG. 5).

Example 4. Determination of the antibacterial activity of P436 by the spot method. The method is based on the procedure described by Ennahar et al. 2000 [2]. Briefly: 20 ml of “lower” agar, consisting of Antibiotic Assay Medium No. 1 with the addition of BactoAgar to 1%, is poured into a Petri dish. After solidification of the “lower” agar, a suspension of the test strain ( L. monocytogenes ATCC 19111, E. faecalis ATCC 29212, Cl. perfringens ATCC 13124 ) to a final concentration of 0.5 McFarland units, mix and layer 5 ml on plates with "lower" agar. Leave the plates until the agar solidifies and dry slightly. 10 μl of two-fold dilutions of the studied solution of mundticin P436 are applied to the dishes prepared in this way. Cups are incubated at 37°C. Accounting for the results is carried out after 16-18 hours, for 1 UE take the zone of clear growth inhibition of the test strain in the maximum dilution equal to 2 mm (Fig.6). Also, in the purified mundticin P436, the molecular weight and antimicrobial activity were determined in 16% tricine SDS-PAGE electrophoresis (according to the protocol of Schagger and Von Jagow, 1987) with Spectra Multicolor Low Range protein markers (26628, Termo Scientific) using the Mini Protean Tetra system apparatus ( BioRad). To determine the antibacterial activity of the strip containing mundticin P436, the gel was poured with agar with a test microorganism and incubated at 37°C for 18 h (Fig.7).

l, где с - концентрация, А - оптическая плотность при длине волны 280, l - длина оптического пути кюветы (1 см - пренебрегается),

- коэффициент экстинкции, постоянная величина, высчитанная на основании данных базы Bactibase и равная 3,27 (14105/4308,55). При переводе результата активности в спот-методе на полученную концентрацию эталонного образца получили значение 1УЕ = 1,25 нг мундтицина Р436 для тест-культуры L. monocytogenes ATCC 19111. Converting the activity of mundticin P436 from EU to mg/ml.To convert the activity from UE to mg/ml, a “reference” sample P436 is obtained (two-stage purification of mundticin P436) and its activity is checked against the “standard” strainL. monocytogenes ATCC 19111. The concentration in mg/ml of the obtained reference sample is calculated by the formula: C= A/

l, where c is the concentration, A is the optical density at a wavelength of 280, l is the length of the optical path of the cuvette (1 cm is neglected),

- extinction coefficient, a constant value calculated on the basis of Bactibase data and equal to 3.27 (14105/4308.55). When translating the result of activity in the spot method to the obtained concentration of the reference sample, the value 1U = 1.25 ng of mundticin P436 for the test culture was obtainedL. monocytogenes ATCC 19111.

List of used literature

1. Bennik M.H., Vanloo B., Brasseur R., Gorris L.G.M., Smid E.J. (1998). A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms. BBA-Biomemranes 1373, Issue 1: 47-58.

2. Ennahar, S., Deschamps, N., & Richard, J. (2000). Natural variation in susceptibility of Listeria strains to class IIa bacteriocins. Current Microbiology, 41, 1-4.

3. Kawamoto S., Shima J., Sato R., Eguchi T., Ohmomo S., Shibato J., Horikoshi N., Takeshita K., Sameshima T. (2002) Biochemical and genetic characterization of Mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393. Appl Environ Microbiol 68: 3830-3840.

4. Patrzykat A., Friedrich C.L., Zhang L., Mendoza V. and Hancock R.E.W. (2002) Sublethal Concentrations of Pleurocidin-Derived Antimicrobial Peptides Inhibit Macromolecular Synthesis in Escherichia coli. Antimicrob Agents CH 46 No. 3:605-614.

5. Salvucci E., Saavedra L. and Sesma F. (2007) Short peptides derived from the NH2-terminus of subclass IIa bacteriocin enterocin CRL35 show antimicrobial activity. J Antimicrob Chemoth 59: 1102-1108

6. Saavedra, L., Minahk, C., de Ruiz Holgado, A.P. and Sesma, F. (2004) Enhancement of the enterocin CRL35 activity by a synthetic peptide derived from the NH2-terminal sequence. Antimicrob Agents Chemother 48, 2778-2781

7. Zendo T., Eungruttanagorn N., Fujioka S., Tashiro Y., Nomura K., Sera Y., Kobayashi G., Nakayama J., Ishizaki A., Sonomoto K. (2005). Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean. J Appl Microbiol. 2005; 99(5):1181-90. doi: 10.1111/j.1365-2672.2005.02704.x.

8. Farias M.E., Faria R.N., de Ruiz Holgado A.P., Sesma F. (1996) Purification and N-terminal amino acid sequence of enterocin CRL 35, a 'pediocin-like'bacteriocin produced by Enterococcus faecium CRL 35. Lett Appl Microbiol 22 :417-419.

Claims (1)

Method for purification of P436(KS) mundticin, including cultivation of the producer strain Enterococcus mundtii "GKPM - Obolensk" B-8925 for 12 hours in a bioreactor with a nutrient medium Brucella broth with the addition of meat extract 5 g/l and lactose 2 g/l and the introduction of a sorbent CM-sephadex C25 10% of the volume into a bioreactor with a nutrient medium immediately before cultivation, followed by autoclaving of the biomass together with the sorbent at 0.5 atm for 15 min, the selection of the sorbent on filter sieves, washing with phosphate-buffered saline with the addition of ethyl alcohol up to 10 % by volume; then it is transferred to a chromatographic column, followed by elution of the adsorbed mundticin with a buffer consisting of: 0.4 M NaCl, 10% glycerol, 20% ethanol, 0.01 M acetate buffer, pH 5.0; and the resulting one-step purification product was further purified using HPLC by reverse-phase chromatography on a C18 column.

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