Biological degradation of ochratoxin A into ochratoxin ?

Abstract

The invention relates to the use of a microorganism of the genus Brevibacterium for the biological degradation of ochratoxin A, in which the microorganism is preferably Brevibacterium casei, Brevibacterium linens, Brevibacterium iodinum or Brevibacterium epidermidis. In addition, the invention relates to a method for the production of ochratoxin ? using said microorganism.

Claims

1. A method for the biological degradation of ochratoxin A, comprising: a. using at least one bacterium belonging to the genus Brevibacterium and selected from the group consisting of Brevibacterium casei DSM 20657, Brevibacterium casei DSM 9657, Brevibacterium casei DSM 20658, Brevibacterium casei RM101, Brevibacterium linens DMS 20425, Brevibacterium iodinum DSM 20626 and Brevibacterium epidermidis DSM 20660 in contact with an aqueous solution to obtain a first product, and b. placing the first product obtained in step (a) in contact with ochratoxin A to obtain a second product and incubating the second product at a temperature of between 10 and 50?C. for a period of between 1 hour and 20 days wherein the ochratoxin A is biologically degraded.

2. The method according to claim 1, wherein the aqueous solution of step (a) permits the survival of the bacterium.

3. The method according to claim 1, wherein the at least one bacterium belonging to the genus Brevibacterium is selected form the group consisting of Brevibacterium casei RM101 and Brevibacterium linens DSM 20425 and the biological degradation of ochratoxin A produces ochratoxin ?.

4. The method according to claim 1, wherein the first product of step (a) is placed in contact with a food product containing ochratoxin A.

5. The method according to claim 4, wherein the first product of step (a) is placed in contact with the food product by nebulization.

6. A method for the biological degradation of ochratoxin A, comprising: a. placing a biologically active molecule in contact with ochratoxin A to obtain a product, wherein the biologically active molecule is an enzyme with peptidase activity produced by a microorganism belonging to the genus Brevibacterium and selected from the group consisting of Brevibacterium casei DSM 20657, Brevibacterium casei DSM 9657, Brevibacterium casei DSM 20658, Brevibacterium casei RM101, Brevibacterium linens DSM 20425, Brevibacterium iodinum DSM 20626 and Brevibacterium epidermidis DSM 20660; and b. incubating the product obtained in step (a) at a temperature of between 10 and 50? C. for a period of between 30 minutes and 30 days, whereby the ochratoxin A is biologically degraded.

7. The method according to claim 6, wherein the at least one bacterium belonging to the genus Brevibacterium is selected from the group consisting of Brevibacterium casei RM101 and Brevibacterium linens DSM 20425 and the biological degradation of ochratoxin A produces ochratoxin ?.

8. The method according to claim 6, wherein step (b) the biologically active molecule is placed in contact with a food product containing ochratoxin A.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Chromatogram of the analysis of the supernatant of B. casei RM101 cultured in basal salt medium without a source of carbon but with OTA (40 mg/L). Fluorimetric detector, ?.sub.Ex (excitation wavelength)=330 nm; ?.sub.Em (emission wavelength)=460 nm.

(2) 1A. Shows the supernatant at time 0 (T.sub.0). Absorbance as measured in LU (luminescence units) is shown on the y-axis and time as measured in minutes (min) is shown on the x-axis.

(3) 1B. Shows the supernatant after 10 days of growth. The disappearance of the OTA peak and the appearance of the OT? peak can be observed. Absorbance as measured in LU (luminescence units) is shown on the y-axis and time as measured in minutes (min) is shown on the y-axis.

(4) FIG. 2. UV/VIS (ultraviolet/visible) spectrum of OT?, in the supernatant of B. casei RM101 cultured in basal salt medium without a source of carbon but with OTA (40 mg/L).

(5) 2A. Shows the ochratoxin ? spectrum obtained by diode array detector (DAD). Absorbance as measured in mAU (milli-absorption units) is shown on the y-axis and time as measured in minutes (min) is shown on the x-axis.

(6) 2B. Shows the ochratoxin ? spectrum obtained by fluorimetric detector (FLD). Absorbance as measured in LU (luminescence units) is shown on the y-axis and wavelength as measured in nanometers (nm) is shown on the x-axis. Excitation wavelength (?ex)=330 nm.

(7) FIG. 3. Mass spectrum of OT? in the supernatant of B. casei RM101 cultured in basal salt medium without a source of carbon but with OTA (40 mg/L).

(8) Ion abundance as measured is shown on the y-axis and mass-to-charge ratio (m/z) is shown on the x-axis.

EXAMPLES

(9) The invention will be illustrated below by a series of assays performed by the inventors, which reveal that microorganisms belonging to the genus Brevibacterium are highly effective in transforming the mycotoxin OTA into OT?. It is shown how OTA is fully degraded by Brevibacterium and how said degradation leads to synthesis of OT?.

(10) Additionally, due to increasing use of these microorganisms in the food industry, they are highly, recommendable for use in degrading OTA on foods potentially contaminated with this mycotoxin.

(11) The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are only included for illustrative purposes and should not be interpreted as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the field of application thereof. Furthermore, although the invention examples have used some specific strains belonging to Brevibacterium, these examples are only included for illustrative purposes and should not be interpreted as limitations of the invention claimed herein, the results being extrapolable to any bacterium of the genus Brevibacterium spp., and preferably to the species Brevibacterium casei, Brevibacterium linens, Brevibacterium iodinum or Brevibacterium epidermidis.

Example 1

Detection of the OTA Degradation Capacity of Various Strains of Pseudomonas, Rhodococcus and Brevibacterium

(12) Due to the fact that soil bacteria are capable of transforming a wide variety of aromatic compounds, various species of Actinobacterias and Pseudomonas were initially cultured in liquid synthetic culture media such as BSM (basal salt medium) in the presence of OTA (10 ?g/L).

(13) 1.1. Bacterial Strains Used and Bacteria Culturing.

(14) In order to detect the capacity to degrade OTA, cultures of the strains Rhodococcus erythropolis CECT 3008, Rhodococcus erythropolis IGTS8, Pseudomonas putida DSMZ 291, Pseudomonas putida KT2442 and seven strains of various species of the genus Brevibacterium were used.

(15) Rhodococcus erythropolis CECT 3008 (DSMZ 43060) was obtained from the Spanish Type Culture Collection (CECT). Pseudomonas putida DSM 291.sup.T and six strains of Brevibacterium belonged to the German Collection of Microorganisms and Cell Cultures (DSMZ) (Brevibacterium epidermidis DSM 20660.sup.T, Brevibacterium iodinum DSM 20626.sup.T, Brevibacterium linens DSM 20425.sup.T, Brevibacterium casei DSM 20657.sup.T, Brevibacterium casei DSM 9657, Brevibacterium casei DSM 20658). The strain Brevibacterium casei, RM101, was isolated at the Institute of Industrial Fermentations IFI-CSIC, and was identified by 16S rDNA sequencing. Rhodococcus erythropolis IGTS8 and Pseudomonas putida KT2442 were supplied by Dr. Eduardo Diaz, of the CSIC Centre of Biological Research.

(16) All bacteria were cultured in Luria-Bertani (LB) liquid medium supplemented with 0.5% glucose and incubated at 30? C. under aerobic conditions. For the OTA degradation assay, the bacteria were cultured in basal salt medium (BSM) containing 0.2% of glycerol, 4 g of NaH2PO4-H2O, 4 g of K2HPO4-31-120, 2 g of NH4Cl, 0.2 g of MgCl2-6H2O, 0.001 g of CaCl2-2H2O, and 0.001 g of FeCl3-6H2O (Denome et al., 1994). Glycerol was not included in the experiments carried out to determine the potential use of OTA as a sole source of carbon by the bacteria analyzed.

(17) 1.2. OTA and OT? Reference Standard.

(18) OTA in solid form was acquired from Sigma (Sigma-Aldrich) and diluted in 99% methanol under sterile conditions to yield a stock solution of 500 ?g/mL. A reference standard solution of OT? (11.9 ?g/mL) was also acquired from LGC Standards (Germany) and diluted 1:2 in acetonitrile to yield a reference standard solution of 5.9 ?g/mL.

(19) 1.3. OTA Degradation Assay.

(20) Initially, some strains of Actinobacterias (Rhodococcus erythropolis CECT 3008, Rhodococcus erythropolis IGTS8 and Brevibacterium casei RM101) and Pseudomonas (P. putida DSMZ 291.sup.T and P. putida KT2442) were cultured in 25 mL of BSM medium spiked with OTA (approx. 10 ?g/L) at 30? C. under aerobic conditions until the exponential phase of growth. The culture supernatants were analyzed by HPLC to determine the OTA concentration present.

(21) In all degradation assays, the cells were separated from the supernatants by centrifuging at 3000?g for 10 min at 4? C. and the latter were analyzed by HPLC. In the case of Brevibacterium, settled cells were kept at ?80? C. for successive analyses. Controls of BSM medium with OTA and without bacteria were also prepared.

(22) 1.4. HPLC and Mass Spectrometry Quantitation of OTA and OT?

(23) The OTA concentration present in the supernatants, settled cells and settled cell wash solutions were quantitated according to the method disclosed in Del Prete V. et al., 2007. Journal of Food Protection, 70(9), pp. 2155-2160.

(24) For the determination and quantitation of OTA, a Hewlett-Packard HPLC Model I, Series 1100, chromatograph (Hewlett-Packard, Palo Alto, Calif.), equipped with degasser, quaternary pump, autosampler, DAD detector and fluorescence detector (FLD) was used. An Alltima C18 (5 ?m), 200 mm-4.6 mm column was used. The mobile phase was: eluent (A), acetonitrile; eluent (B), water (HPLC-grade)/acetonitrile/glacial acetic acid (89:10:1 v/v); eluent A:B=37:63 (v/v), in isocratic mode; flow rate, 1.3 mL/min; temperature, 30? C.; assay time, 20 min; FLD detector (?ex=330 nm, ?em=460 nm) and DAD detector (330 nm); injection volume, 100 ?L. The detection limit for OTA under these conditions was 0.02 ?g/L. The OTA reference standard was injected at two concentrations: 20 and 50 ?g/L and the OT? reference standard was injected at a concentration of 5.9 mg/L. The samples, diluted in eluent A:B, were injected directly for analysis by HPLC.

(25) The Brevibacterium settled cells were resuspended two times in 2 mL of absolute methanol for 1 hour to extract the OTA. After centrifuging at 3000?g for 15 min at 20? C., the supernatants were separated, collected in 5-mL vials and evaporated to dryness by nitrogen. To determine the OTA concentration, the dry residues were dissolved in HPLC mobile phase at the time of the chromatographic analysis.

(26) For the mass spectrometry assay, a Hewlett-Packard Series 1100 MSD chromatograph (Palo Alto, Calif.) coupled to a quadrupole-mass detector with an electrospray ionization (ESI) interface was used. The separation was carried out by direct injection. The ESI parameters used were: N2 flow rate at 10 L/min, N2 temperature 330? C.; nebulizer pressure, 40 psi; capillary voltage, 4000 V. The ESI was operated in negative-ion mode, using a mass range of between m/z 100 and 800 and variable voltage ramping for fragmentation.

(27) Table 1 lists the results obtained from the Actinobacterias and Pseudomonas species analyzed, capable of transforming a wide variety of aromatic compounds.

(28) TABLE-US-00001 Mean Decrease in OTA OTA OTA Strains [?g/L] [?g/L] (%) BSM + OTA (Control) 11.06 11.01 0 BSM + OTA (Control) 10.96 Rhodococcus erythropolis CECT 3008 8.37 7.88 28.47 (A) Rhodococcus erythropolis CECT 3008 7.38 (B) Rhodococcus erythropolis IGTS8 (A) 9.50 8.81 19.98 Rhodococcus erythropolis IGTS8 (B) 8.12 Brevibacterium casei RM101 (A) n.d. 0 100 Brevibacterium casei RM101 (B) n.d. Pseudomonas putida DSM 291.sup.T (A) 10.14 10.07 8.54 Pseudomonas putida DSM 291.sup.T (B) 10.00 Pseudomonas putida KT2442 (A) 7.72 8.18 25.70 Pseudomonas putida KT2442 (B) 8.64 The assays were performed two times (A) and (B); n.d.: not detected; .sup.Ttype strain.

(29) Table 1 lists the decrease in ochratoxin A (OTA) in BSM medium achieved by some species of Actinobacterias and Pseudomonas. The OTA concentration decrease observed is from 8 to 25% when analyzing the supernatants of cultures in the strains of Rhodococcus erythropolis and Pseudomonas putida. The small decrease observed appears to indicate that these bacteria have no OTA degradation mechanism and that OTA is likely adsorbed on the cell surface, in a similar manner to that described for lactic bacteria (Del Prete V. et al., 2007. Journal of Food Protection, 70(9), pp. 2155-2160) and yeasts (Cecchini F. et al., 2006. Food Microbiol. 23, pp. 411-417). Furthermore, in these strains the HPLC chromatographic analyses do not show the presence of OTA degradation products.

(30) In the case of the strain Brevibacterium casei RM101, OTA disappeared completely from the culture supernatants.

Example 2

Degradation of OTA by Brevibacterium

(31) Additional assays were performed using a larger number of Brevibacterium strains to confirm the observations with B. casei RM101 and to verify if this ability is a specific feature of the strain, species or genus being studied.

(32) Additionally, in order to confirm the degradation of OTA by Brevibacterium spp., the strains were cultured in BSM medium with a four-fold concentration of OTA (40 ?g/L). The Brevibacterium strains were cultured under aerobic conditions with agitation at 150 rpm for 10 days. These assays were performed in culture media containing OTA at a concentration of 40 ?g/L. OTA levels equivalent to 40 ?g/L are rarely found in foods or beverages. The degradation assays were performed as described in example 1.

(33) TABLE-US-00002 TABLE 2 Decrease in ochratoxin A (OTA) in BSM medium achieved by Brevibacterium. Decrease of OTA OTA Strains (?g/L).sup.a (%) BSM + OTA (Control) 39.81 0 Brevibacterium casei DSM 20657.sup.T n.d. 100 Brevibacterium casei DSM 9657 n.d. 100 Brevibacterium casei DSM 20658 n.d. 100 Brevibacterium casei RM101 n.d. 100 Brevibacterium linens DSM 20425.sup.T n.d. 100 Brevibacterium iodinum DSM20626.sup.T n.d. 100 Brevibacterium epidermidis DSM 20660.sup.T n.d. 100 OTA (?g/L).sup.a determined in the supernatants of three independent cultures; n.d.: not detected; .sup.Ttype strain.

(34) The OTA degradation assays show a complete disappearance of the toxin in all strains of the various Brevibacterium species analyzed. OTA added to the medium at a concentration of 40 ?g/L was not detected by HPLC in any of the culture supernatants (Table 2). Nevertheless, after methanol extraction, the presence of OTA was also not detected in either the settled cells or the wash solutions.

(35) These results corroborate the previous results and indicate that all Brevibacterium strains are capable of degrading OTA. The Brevibacterium genus comprises a heterogeneous group of nine coryneform species which are capable of degrading insecticides (DTT, DDE, etc.), as well as of producing extracellular proteases. These bacteria can be found in different habitats, including among them soil, poultry, fish, and human skin, and in foods.

Example 3

Mechanism of OTA Degradation by Brevibacterium

(36) The strains B. casei RM101 and B. linens DSM 20425.sup.T were used, in order to confirm the capacity of these strains to degrade OTA at a higher concentration (40 mg/L), 1000-fold the concentration used in the previous assay, and to confirm the capacity of these strains to use OTA as a sole source of carbon. In this case the strains were cultured in BSM medium wherein glycerol (0.2%) and OTA (40 mg/L) were or were not present to thereby study the possible use of OTA as a sole source of carbon. The degradation assays were performed as described in example 1.

(37) It has been possible to elucidate the mechanism of OTA degradation carried out by Brevibacterium spp. by analyzing the HPLC and mass spectrometry data of these strains, B. casei RM101 and B. linens DSM 20425.sup.T.

(38) TABLE-US-00003 TABLE 3 Decrease in ochratoxin A (OTA) and production of ochratoxin ? (OT?) by Brevibacterium casei RM101 and Brevibacterium linens DSM 20425.sup.T in BSM medium with different composition. BSM medium with: Decrease in Glycerol OTA OTA OTA OT? Strains (0.2%) (40 mg/L) (mg/L) (%) (mg/L) B. casei ? ? ? ? ? RM101 + ? ? ? ? ? + n.d. 100 25.07 + + n.d. 100 26.22 B. linens ? ? ? ? ? DSM 20425 + ? ? ? ? ? + n.d. 100 26.19 + + n.d. 100 26.79 n.d.: not detected; .sup.Ttype strain.

(39) The results obtained (Table 3) again indicate that both Brevibacterium strains fully degrade the OTA present in the culture medium. The assay carried out using BSM medium without the traditional source of carbon (glycerol) showed that the strains B. casei RM101 and B. linens DSM 20425.sup.T are capable of using OTA as a sole source of carbon.

(40) In parallel, an analysis of the respective chromatograms of the culture supernatants indicates the absence of the OTA peak and the appearance, in the elution profile, of a new peak with a different retention time and spectrum (FIG. 1). The amide bond present in OTA can be enzymatically hydrolyzed by a CPA to yield L-phenylalanine and OT?. The UV/VIS spectrum of the resulting compound (FIG. 2) is identical to the one seen in the reference standard corresponding to OT?. Furthermore, the results of the HPLC-MS assay make it possible to confirm the identity of the degradation product as OT?, given that a molecular ion peak [M-H].sup.? was observed at m/z 255 in mass spectrometry (MW OT?=256) (FIG. 3).

(41) The two Brevibacterium strains studied, B. casei RM101 and B. linens DSM 20425.sup.T, were capable of growing in the absence of glycerol, as observed by increased cloudiness in the culture medium. In a culture medium containing OTA as a sole source of carbon, the bacterial population grew more slowly compared to the cultures grown in medium with glycerol and OTA; nevertheless, it should be mentioned that OTA was fully degraded in all cases. These results indicate the presence of a carboxypeptidase-type enzyme in Brevibacterium spp. strains, an enzyme that hydrolyzes the amide bond present in the OTA molecule. The L-phenylalanine released may be used as a source of carbon during bacterial growth. Table 3 shows the concentration of OT? recovered from the culture supernatants. The two strains studied, B. casei RM101 and B. linens DSM 20425.sup.T, yield equivalent amounts of OT? regardless of whether the BMS medium contains glycerol or not. The OT? amounts detected correspond to the theoretical concentration produced by complete hydrolysis of OTA added to the medium.