Cinnabarin synthesis by Pycnoporus sanguineus strains and antimicrobial activity against bacteria from food products

Síntese de cinabarina por Pycnoporus sanguineus e sua atividade antimicrobiana sobre bactérias isoladas de alimentos

CINNABARIN SYNTHESIS BY PYCNOPORUS SANGUINEUS STRAINS AND ANTIMICROBIAL ACTIVITY AGAINST BACTERIA FROM FOOD PRODUCTS

Elza de Fátima Albino Smânia¹, Artur Smânia Júnior1^* , Clarice Loguercio-Leite²

¹Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; ²Departamento de Botânica.

Submitted: March 31, 1998; Returned to authors for corrections: July 21, 1998;

Approved: September 08, 1998.

ABSTRACT

Among three strains of Pycnoporus sanguineus, MIP 89007 produced more cinnabarin than MIP 95001 and MIP 95002. The antimicrobial activity of cinnabarin was tested against 11 species of bacteria isolated from food. Bacillus cereus and Leuconostoc plantarum were the most sensitive to cinnabarin, being inhibited by 0.0625 mg/ml. Klebsiella pneumoniae was the least sensitive (>4.0 mg/ml).

Key words: Pycnoporus sanguineus, cinnabarin, antimicrobial activity, fungus, bacteria.

INTRODUCTION

Cinnabarin is an antibiotic substance produced by Pycnoporus sanguineus (Polyporaceae). This compound is an orange pigment which has a basic phenoxazin-3-one structure, with a carbonyl group at C-1, an amino group at C-2 and an hydroxyl group at C-9 (1,3). Production of this substance in vitro occurs between the 18^th and the 23^rd day of incubation (7). Moreover, the synthesis of cinnabarin is increased significantly when the pH of the broth media is adjusted to 9.0, with incubation at 25ºC, under light (8).

As this substance has a cromophore orange colour and can transfer this colour to food products, the substance was tested against bacteria obtained from foods.

In this study, the production of cinnabarin by three P. sanguineus strains was investigated, and the antimicrobial activity of this substance against bacterial species from food sources was tested.

MATERIALS AND METHODS

Fungal strains: Three isolates, MIP 89007, MIP 95001 and MIP 95002, were obtained from carpophores of P. sanguineus collected in Criciúma, Florianópolis and Imbituba, respectively, in the state of Santa Catarina, southern Brazil. The fungus was cultured on potato dextrose agar, and the isolates were maintained in the same medium (slant) and stored at 4ºC.

Bacteria: Staphylococcus aureus ATCC 25923 strain was used as a bacteria indicator in the antimicrobial dosage. For determinations of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cinnabarin, the following bacteria, isolated from foods, were used: Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Leuconostoc mesenteroides, Leuconostoc plantarum, Pseudomonas aeruginosa, Salmonella sp., Salmonella typhimurium and Staphylococcus aureus.

Production of cinnabarin by Pycnoporus sanguineus: In this experiment, cinnabarin synthesis by P. sanguineus MIP 89007, MIP 95001 and MIP 95002 was studied. Each isolate was inoculated into 6 Roux flasks containing 150 ml of potato dextrose broth. In 3 flasks, the pH of the media was adjusted to 7.0 and in the other 3, to 9.0. All cultures were incubated, without shaking, at 25ºC, under light. After 20 days of incubation, the cultures were filtered in Whatman nº 1 paper, tared previously. The mycelial masses were weighed and an aliquot of each pellet was left at 105ºC for approximately 20 h, for dry weight determination. The remains of the mycelial masses were homogenized in a blender and filtered, and the cinnabarin was extracted from supernatants with ethyl acetate. The solvent was evaporated and the residues were maintained in vacuum until the moment of use. The cinnabarin content of the extracts was determined, indirectly, by method described in Smânia et al., 1997 (8). The experiments were carried out in triplicate.

Cinnabarin quantification: The agar diffusion method was used to determine the calibration curve for cinnabarin as described by Smânia et al. (8), and to test the antimicrobial activity of the extracts. An overnight culture of S. aureus ATCC 25923 was diluted to a final concentration of approximately 10⁶ CFU/ml, and the bacterial suspension was spread over the surface of Mueller-Hinton agar, containing five wells of 7 mm diameter. The wells were filled with each one of the ethyl acetate extracts (range 0.5 to 30 mg), in separate experiments. The plates were incubated at 36ºC for 20 h. A zone of 9 mm or more in diameter of growth inhibition was defined as a positive result. The concentration of cinnabarin in each extract was estimated by the formula y = 9.2949x + 12.3926, where y = diameter of the inhibition zone, and x = amount of cinnabarin (8).

Minimal inhibitory and minimal bactericidal concentrations: The cinnabarin used in this study was obtained from the culture of strain MIP 89007, which was extracted and purified according to the procedure of Smânia et al (6). The antimicrobial activity of this substance was tested against 11 bacterial species obtained from food, by determinations of minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) (6). Test strains were cultured overnight at 36ºC in Mueller-Hinton broth, diluted to a final concentration of approximately 10⁶ CFU/ml and inoculated into tubes containing serial dilutions of cinnabarin and incubated at 36ºC for 20 h. The MIC was defined as the lowest concentration of antibiotic, expressed in mg/ml, able to completely inhibit the growth of each bacterial strain. For the MBCs, a subculture of each tube without apparent growth was made on Mueller-Hinton agar. The MBC was the lowest concentration of the antibiotic which yielded a >99.9% reduction in the number of colony forming units.

RESULTS

Production of cinnabarin by P. sanguineus: The biomasses for the three isolates of P. sanguineus are shown in Fig. 1. In average, at pH 7.0, the MIP 89007 produced 4.6 g/l, MIP 95001 5.2 g/l and MIP 95002 5.1 g/l of mycelial mass. When the media had the pH adjusted to 9.0, the yields were 6.3, 5.7 and 5.7 g/l, respectively. Fungal growth was similar for the 3 isolates when the cultures were carried out at either pH 7.0 or at pH 9.0 (p > 0.1). With respect to cinnabarin synthesis (Fig. 2), the culture of MIP 89007 at pH 7.0 had a yield of 21.6 InU/l, MIP 95001 a yield of 4.2 InU/l, and MIP 95002 a yield of 2.0 InU/l, and at pH 9.0 these yields were 29.3 InU/l, 11.8 InU/l and 3.1 InU/l, respectively. Synthesis of cinnabarin by MIP 89007 and MIP 95001 was significantly increased (p < 0.01) when the pH of the broth medium was adjusted to 9.0. At both pH values, MIP 89007 was the best producer of cinnabarin.

Figure 1 -
Production of biomass by three strains of Pycnoporus sanguineus. Error bars indicate standard deviation (triplicate samples).

Figure 2-
Cinnabarin synthesis by three strains of Pycnoporus sanguineus. Error bars indicate standard deviation (triplicate samples). 1 InU = 1 inhibitory unit = amount (mg) which gives an inhibition equivalent to 1 mg cinnabarin, by diffusion test.

Antibacterial activity: The results obtained for the MICs and the MBCs are shown in Table 1. B. cereus and L. plantarum were inhibited by the lowest concentration of cinnabarin (0.0625 mg/ml). The Gram-negative bacteria presented the highest values for MIC and MBC. The same MBCs and MICs were detected in E. faecalis, L. plantarum, Salmonella sp., S. typhimurium, and S. aureus. For K. pneumoniae and L. mesenteroides, the CMB was above the highest concentration used (4 mg/ml). In five of the eleven species the MBC/MIC ratio was 1, in four it was 2, and in the remaining two, this ratio was not determined.

^a Expresed mg/ml

DISCUSSION

P. sanguineus MIP 89007 strain, when cultured at 25ºC under light, for 20 days (7,8), is a poor cinnabarin producer. For this reason, new cultures were obtained from carpophores collected in several geographical regions of Santa Catarina State. Among the new isolates, MIP 95001 and MIP 95002 were chosen for this study because they produced strong orange-coloured cultures, suggesting good cinnabarin production. However, the new isolates produced less amounts of cinnabarin than MIP 89007. A better production by the new isolates was expected since these secondary metabolites are usually produced for self defence (9), and MIP 95001 and MIP 95002 were recently obtained from nature. Results previously reported for MIP 89007 indicated a better cinnabarin production at a non-neutral pH (9.0) (8), so the new isolates were also cultivated at two pHs. The influence of pH on secondary metabolite synthesis has also been reported by authors using other non-Basidiomycete fungi species. Shipanova et al. (5) evaluated the relationship between pH and biosynthesis of the antibiotic produced by Fusidium coccineum (Deuteromycetes) and observed that basic pH favoured the synthesis. Monascus purpureus (Ascomycetes) increased production of mycelial mass and pigment ankaflavina when cultivated in acidic pH and in darkness (2).

As described previously (6), cinnabarin is active on some bacteria species obtained from humans. The bacteria isolated from foods, were slightly more resistant than those from human sources. These results agree with those previously described, with the Gram-positives being more susceptible than the Gram-negatives. Antimicrobial activity of pigments has also been investigated by food industries and the same results were reported. Monascus purpureus (Ascomycetes) produce hexaketide pigments, and among these, red pigments are used by many Asiatic countries to colour and flavour foods (2). Another pigment used for this purpose is the ß-carotene synthesised by Phycomyces blaskeleanus (Zygomycetes) (4).

The results of this study demonstrate that, among the tested strains, the P. sanguineus MIP 89007 strain is the best producer of cinnabarin and presents antibacterial activity against bacteria from foods.

ACKNOWLEGMENTS

This study was supported by the Federal University of Santa Catarina (FUNPESQUISA/94).

RESUMO

Síntese de cinabarina por Pycnoporus sanguineus e sua atividade antimicrobiana sobre bactérias isoladas de alimentos

Entre três estirpes de Pycnoporus sanguineus, MIP 89007 produziu maior quantidade de cinabarina que MIP 95001 e MIP 95002. A atividade antimicrobiana dessa substância foi testada contra 11 espécies de bactérias associadas a fontes alimentares. Bacillus cereus e Leuconostoc plantarum foram os microrganismos inibidos com a menor concentração de cinabarina (0.0625 mg/ml), e Klebsiella pneumoniae foi a espécie menos sensível (>4.0 mg/ml).

Palavras-chave: Pycnoporus sanguineus, cinabarina, atividade antimicrobiana, fungo, bactéria.

REFERENCES

• ¹
  Achenbach, H.; Blümm, E. Investigation of the pigments of Pycnoporus sanguineus - picnosanguin and new phenoxazin-3-ones. Arch. Pharm. (Weinheim), 324:3-6, 1991.
• ²
  Chen, M.H.; Johns, M.R. Effect of pH and nitrogen source on pigment production by Monascus purpureus. Appl. Microbiol. Biotechnol., 40:132-138, 1993.
• ³
  Gripenberg, J. Fungus pigment. I. Cinnabarin, a colouring matter from Trametes cinnabarina Jacq. Acta Chem. Scand., 12:590-595, 1951.
• ⁴
  Margalith, P. Enhancement of carotenoid synthesis by fungal metabolites. Appl. Microbiol. Biotechnol., 38:664-666, 1993.
• ⁵
  Shipanova, I.; Bartoshevich, Y.; Sibeldina, L.; Zaslavskaya, P.; Michtchenko, A. Relationship between intracellular pH and antibiotic biosynthesis in Fusidium coccineum. Appl. Microbiol. Biotechnol., 43:514-517, 1995.
• ⁶
  Smânia, A. Jr.; Delle Monache, F.; Smânia, E.F.A.; Gil, M.L.; Benchetrit, L.C.; Cruz, F.S. Antibacterial activity of a substance produced by the fungus Pycnoporus sanguineus(Fr.) Murr. J. Ethnopharmacol., 45:177-181, 1995a.
• ⁷
  Smânia, A. Jr.; Smânia, E.F.A.; Cruz, F.S.; Benchetrit, L.C. Growth and production phases of Pycnoporus sanguineus Rev. Microbiol. (São Paulo), 26:302-306, 1995b.
• ⁸
  Smânia, E.F.A.; Smânia, A. Jr.; Loguercio-Leite, C.; Gil, M.L. Optimal parameters for cinnabarin synthesis by Pycnoporus sanguineus. J. Chem. Biotechnol., 70:57-59, 1997.
• ⁹
  Stone M.J.; Williams, D.H. On the evolution of functional secondary metabolites. Mol. Microbiol., 6:29-34, 1992.

*

Corresponding author. Mailing address: Departamento de Microbiologia e Parasitologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, CEP 88040-900, Florianópolis, SC, Brasil. FAX [phone]

Publication Dates

• Publication in this collection
  27 May 1999
• Date of issue
  Oct 1998

History

• Accepted
  08 Sept 1998
• Reviewed
  21 July 1998
• Received
  31 Mar 1998