Methods for producing phycotoxins

• US 8,957,207 B2
• Filed: 09/23/2011
• Issued: 02/17/2015
• Est. Priority Date: 03/24/2009
• Status: Active Grant

• Current Assignee: Proteus Corporation
• Original Assignee: Proteus Corporation
• Inventors: Lagos Gonzlez, Marcelo Santiago
• Primary Examiner(s): Hui, San-Ming
• Assistant Examiner(s): LEE, ANDREW P
• Application Number:
  US13/243,119
• Publication Number:
  US 20120053344A1
• Time in Patent Office: 1,243 Days
• US Class Current: 544/251
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07D 473/00 Heterocyclic compounds containing purine ring systems
• CPC Class Codes: C07D 487/14 Ortho-condensed systems
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12P 17/182 Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system alloxazine or isoalloxazine, e.g. riboflavine C12P25/00

Abstract

Methods for producing phycotoxins from natural sources, wherein the phycotoxins have a definite compositional profile are described herein. In one embodiment, the phycotoxins are produced by cyanobacteria. In one embodiment, the phycotoxins are produced by continuously culturing cyanobacteria under strictly controlled conditions in order to produce a definite compositional profile. In another embodiment, organic nutrients are added to the culture that allows for higher concentrations of neosaxitoxin and saxitoxin or gonyaulatoxins 2 and 3 per weight of the algae. The phycotoxins are isolated primarily from the bacteria but can also be isolated from the culture medium. In one embodiment, the cyanobacteria produce only neosaxitoxin and saxitoxin in a ratio of about 6:1, 5:1, 4:1, or 3:1. In a preferred embodiment, the amount of saxitoxin is less than 20% by weight of the total amount of neosaxitoxin and saxitoxin produced. In another embodiment, the cyanobacteria produce only GTX2 and GTX 3.

Claims

1. A method for the continuous production of phycotoxins from cyanobacteria, the method comprising

(a) inoculating a culture medium with the cyanobacteria, wherein the cyanobacteria is selected from the group consisting of Cylindrospermopsis raciborskii;

Aphanizomenon flos-aquae;

Aphanizomenon (APh) issatschenkoi (usaceb) Proskina-Lavrenco;

Aphanizomenon gracile (Lemm) Lemm;

Aphanizomenon elenkinii var. Gracile Kashtanova;

Anabaena circinalis;

Lyngbya wollei;

and combinations thereof,

wherein the culture medium comprises MgSO₄.7H₂O, NaNO₃, K₂HPO₄, H₃BO₃, H₂SeO₄, biotin, vitamin B12, thiamine HCl, CuSO₄.5H₂O, ZnSO₄.7H₂O, CoCl₂.6H₂0, NaMoO₄.2H₂O, Na₂EDTA, FeCl₃.6H₂O, NaHCO₃, and MnCl₂.H₂O, about 2 to about 3.5 mM arginine, about 1 to about 2.2 mM methionine, and about 0.7 to about 1.3 mM allantoic acid;

(b) culturing the cyanobacteria in the culture medium at a temperature from 15 to 30°

C. under permanent natural or artificial illumination;

(c) collecting a volume of culture medium ranging from 20 to 40% of the total volume every 1 to 3 days and replacing the removed volume with fresh culture medium; and

(d) harvesting the cyanobacteria to isolate the phycotoxins.

2. The method of claim 1, wherein the cyanobacteria is Aphanizomenon gracile (Lemm) Lemm.

3. The method of claim 1, wherein the cyanobacteria are cultured in a continuous light cycle.

4. The method of claim 1, wherein the cyanobacteria are cultured at a temperature from about 15°

C. to about 25°

C.

5. The method of claim 1, wherein the cyanobacteria are cultured at a temperature of about 22±

2°

C.

6. The method of claim 1, wherein the culture medium has a final arginine concentration of about 2.8 mM.

7. The method of claim 1, wherein the culture medium has a final methionine concentration of about 1.7 mM.

8. The method of claim 1, wherein the culture medium has a final allantoic acid concentration of about 1.0 mM.