Patent Application
20080138875
1. Field of the Invention
In the field of the present invention, the medicinal fungi Ganoderma lucidum has been commercially cultured in solid state using substrates such as agricultural waste, cereals and wood. Cultures in solid state take 2 to 8 months and yields are low.
In recent years, submerged fermentation cultures in Erlenmeyer's flasks escalated to bioreactors have surfaced as a more efficient alternative for the production of mycelia biomass. However, the cost still remains high and the demand for these medicinal fungi is increasing.
2. Description of Prior Art
In the prior art, the most efficient production of mycelia biomass for Ganoderma lucidum has been reported by Tang & Zong (see Tang, Y. J., Zhong, J. J., Enzyme and Microbial Technology, 31:20-28 (2002); Tang, Y. J., Zhong, J. J., Biotechnology Letters, 24:1023-1026 (2002)). These authors reported submerged fermentation cultures with a yield of 22.1 g/L, and a culture time of 14-17 days, using submerged cultures or liquid fermentation.
The present invention overcomes the limitations of solid and liquid fermentation, and submerged cultures. In addition, the present invention provides a method for the production of biomass from fungi mycelia, wherein said method reduces the cost more than 60 times. Moreover, the method of the present invention increases the yield per day of biomass. Furthermore, said biomass has a better or comparable exo-polysaccharides, intra-polysaccharides, and ganoderic acid composition.
The present invention provides method to produce fungi biomass from mycelia comprising inoculating differentiated cells or mycelia into a culture medium which contains at least NaNO3, KH2PO4, MgSO4, KCl, and a source of carbon; and, incubating said culture medium with differentiated cells or mycelia in a container with transparent walls.
The present invention also provides a tridimensional closed system, wherein the tridimensional closed system has internal surfaces with all the characteristics of a mirror, and wherein the tridimensional closed system houses, while incubating, one or more containers with transparent walls containing the culture medium with differentiated cells or mycelia, and wherein the tridimensional closed system has a source of light with a determined wave length; and wherein the incubation is made under agitation, and wherein the tridimensional closed system has a mechanism to monitor temperature, and a mechanism to regulate temperature.
In one aspect of the present invention the source is a light emitting diode (LED) which emits blue light.
In one further aspect of the present invention, the tridimensional closed system comprises a tridimensional enclosure that is constituted by one or more than one compartment, wherein each compartment has at least one source of light, and wherein the source of light of each compartment is a LED, and wherein the LED of each compartment emits a light with a determined wave length.
In another aspect of the present invention, biomass produced after incubating the container/s with transparent walls containing the culture medium with differentiated mycelia or cells, inside the tridimensional closed system, is purified and dried.
Objectives and additional advantages of the present invention will become more evident in the description of the figures, the detailed description of the invention and the claims.
The present invention provides a method to produce biomass comprising:
wherein the container (1) with transparent walls containing the culture medium is agitated (5)(which indicates agitating motion), and wherein the closed system has a mechanism to monitor temperature and a mechanism to regulate temperature, and wherein the source of light (4) is at least one light emitting diode (LED) (4) which emits a light with a determined wave length, and wherein the wave length can be any wave length from the light spectrum.
The present invention also provides a tridimensional closed system (
wherein the closed system has a mechanism to agitate (5) the container (2) with transparent walls, and wherein the tridimensional enclosure has a mechanism to monitor temperature and a mechanism to regulate temperature, and wherein the source of light (4) is at least one LED (4) which emits light with a determined wave length, and wherein the wave length can be any wave length from the light spectrum.
The definition of the tridimensional closed system of the present invention, in which all the internal walls of said system have all the characteristics of a mirror, includes all kind of enclosures with room or space, (e.g., incubators, bioreactors) to house any type of container for culturing cells, bacteria, mycelia etc.
The tridimensional closed system of the present invention may also have an attached mechanism to agitate the container or containers with transparent walls.
In one embodiment of the present invention the container with transparent walls is an Erlenmeyer flask (1A) (
In another embodiment of the present invention, the tridimensional enclosure is constituted by a single compartment (
In another embodiment of the present invention, the tridimensional enclosure is constituted by more than one compartment (
The present invention further provides a method to produce biomass from fungi mycelium comprising:
wherein the solid culture medium contains at least agar, and a source of carbon in a concentration between 20 and 100 grams/Liter; and wherein, the pH of the liquid culture medium is adjusted between 4.5 and 7.0.
In an additional aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the step of filtering and purifying of the biomass produced in the second liquid culture medium is performed by putting the biomass in a mesh that filters the excess liquid culture medium, and then, drying said biomass.
In a more preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the solid culture medium contains at least agar, and a source of carbon in a concentration of 50 grams/Liter; and wherein, the pH of the liquid culture medium is adjusted to 5.5±0.1.
In another preferred embodiment of the present invention, regarding the method to produce biomass from fungi mycelium, the source of carbon is a source selected from the group of barley flour, oats flour, rice flour, wheat flour, corn flour, and a mix of cereals. However, in a more preferred embodiment of the present invention, regarding the method to produce biomass from fungi mycelium, the source of carbon is barley flour or oats flour.
In one preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the fungi mycelium is from a fungus from a phyllum selected from the group consisting of basidiomycetes phyllum, and Ascomycetes phyllum. However, in a more preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the fungi mycelium is a fungus from the basidiomycetes phyllum.
More specifically, regarding the method to produce biomass from fungi mycelium, the fungi mycelium is preferably selected from a group consisting of Ganoderma lucidum, Ganoderma australe, Auricularia fuscosuccinea, Auricularia aurícula, Oudemansiella canarri, Agaricus blazei also known as Agaricus brasiliensis, Agaricus bisporus, Lentinula edodes, Grifola frondosa, Flammulina velutipes, Volvariella volvaceae, Amauroderma sp., Schysophyllum communis, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus citrinopileatus, Pleurotus djamor, Pleurotus sp., Psilocybe cubensis, Coprinus sp., Polyporus sanguineus, Hericium erinaceus, Morchella sculenta, Gibberella fujikuroi, and Mycosphaerella fijiensis; However, the fungi mycelium is even more preferably from the fungus Ganoderma lucidum.
In another aspect of the present invention, regarding the method to produce biomass from fungi mycelium, both, the first liquid culture medium and the second liquid culture medium, contain at least NaNO3 in a concentration of more than 59 mg/Liter, KH2PO4 in a concentration between 20 and 80 mg/Liter, MgSO4 in a concentration between 10 and 60 mg/Liter; KCl in a concentration between 5 and 40 mg/Liter, and source of carbon in a concentration between 20 and 100 gramos/Liter, and wherein, the pH of the liquid culture medium is adjusted between 4.5 and 7.0.
In a more preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the first liquid culture medium and the second liquid culture medium, contain NaNO3 in a concentration of 80 mg/Liter, KH2PO4 in a concentration of 30 mg/Liter, MgSO4.H2O in a concentration of 20 mg/Liter, KCl in a concentration of 10 mg/Liter, and a source of carbon in a concentration of 50 grams/Liter; and wherein, the pH of the liquid culture medium is adjusted to 5.5±0.1.
In another aspect of the present invention, regarding the method to produce biomass from fungi mycelium, incubating in the first incubator is for at least 15 days at a temperature between 15 and 35° C.; incubating in the second incubator is for at least 15 days at a temperature between 15 and 35° C.; and incubating in the third incubator is for at least 9 days at a temperature between 15 and 35° C.
In a preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, incubating in the first incubator, in the second incubator and in the third incubator is at room temperature or at 25±1° C.
In another aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the third incubator is a tridimensional closed system comprising:
wherein the third incubator is a tridimensional closed system with a mechanism to monitor temperature and a mechanism to regulate temperature.
In one aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the third incubator is a tridimensional closed system, wherein the tridimensional enclosure is constituted by at least one compartment, and wherein the tridimensional enclosure has a source of light to the inside of said compartment, and wherein the source of light is a LED that emits a determined wave length, and wherein the wave length can be any wave length from the light spectrum.
In a preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the wave length emitted by the LED is between 425-475 nm.
In a more preferred aspect of the present invention, regarding the method to produce biomass from fungi mycelium, the wave length emitted by the LED is between 425-475 nm for Ganoderma lucidum.
In addition, the present invention provides a culture medium for the production of biomass from the culture of fungi mycelium comprising at least NaNO3, KH2PO4, MgSO4, KCl, and a source of carbon.
In a preferred aspect of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, said culture medium contains at least NaNO3 in a concentration of more than 59 mg/Liter, KH2PO4 in a concentration between 20 and 80 mg/Liter, MgSO4 in a concentration between 10 and 60 mg/Liter; KCl in a concentration between 5 and 40 mg/Liter, and source of carbon in a concentration between 20 and 100 gramos/Liter, and wherein, the pH of the liquid culture medium is adjusted between 4.5 and 7.0.
In a more preferred aspect of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, said culture medium contains NaNO3 in a concentration of 80 mg/Liter, KH2PO4 in a concentration of 30 mg/Liter, MgSO4.H2O in a concentration of 20 mg/Liter, KCl in a concentration of 10 mg/Liter, and a source of carbon in a concentration of 50 grams/Liter; and wherein, the pH of the liquid culture medium is adjusted to 5.5±0.1.
In one preferred embodiment of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, the source of carbon is a source selected from the group of barley flour, oats flour, rice flour, wheat flour, corn flour, and a mix of cereals. However, in a more preferred embodiment of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, the source of carbon is barley flour or oats flour.
In one preferred aspect of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, the fungi mycelium is from a fungus from a phyllum selected from the group consisting of basidiomrycetes phyllum, and Ascomycetes phyllum. However, in a more preferred aspect of the present invention, regarding the culture medium for the production of biomass from fungi mycelium, the fungi mycelium is a fungus from the basidiomycetes phyllum.
More specifically, regarding the culture medium for the production of biomass from fungi mycelium, the fungi mycelium is preferably selected from a group consisting of Ganoderma lucidum, Ganoderma australe, Auricularia fuscosuccinea, Auricularia aurícula, Oudemansiella canarii, Agaricus blazei also known as Agaricus brasiliensis, Agaricus bisporus, Lentinula edodes, Grifola frondosa, Flammulina velutipes, Volvariella volvaceae, Amauroderma sp., Schysophyllum communis, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus citrinopileatus, Pleurotus djamor, Pleurotus sp., Psilocybe cubensis, Coprinus sp., Polyporus sanguineus, Hericium erinaceus, Morchella sculenta, Gibberella fujikuroi, and Mycosphaerella fijiensis; However, the fungi mycelium is even more preferably from the fungus Ganoderma lucidum.
While the description presents the preferred embodiments of the present invention, additional changes can be made in the form and disposition of the parts without distancing from the basic ideas and principles comprised in the claims.
Materials and Methods
Microorganism and Maintaining Medium
The strain of Ganoderma lucidum (donated by Dr. S. T. Chang to Applicants—Laboratory of Vegetal Biologia, Universidad de Antioquia) was kept on a médium made of dextrose potato agar in Petri plates, incubated at 26° C. for 10 days and stored a 4° C.
Inóculo
Petri plates with the strain of Ganoderma lucidum were activated at a temperature of 26° C. during 24 hours prior to inoculation. Then, discs of 1 cm of diameter of mycelia and agar were carried to Erlenmeyer's flasks of 250 ml with 62.5 ml culture medium, under aseptic conditions in a cabin of laminar horizontal flow (Laserquim®).
Culture Medium
The used culture medium corresponds to the protocol developed for the fungi culture by the Laboratory of Vegetal Biology. Such culture consists of a basic medium with salts: NaNO3, 80 mg/l; KH2PO4, 30 mg/l; MgSO4.7H2O, 20 mg/l; KCl, 10 mg/l, supplemented with barley flour or oats flour 50 g/l.
Conditions of Culture Medium
The culture medium was adjusted to a pH of 5.5±0.1 (pH-meter HI-221, Hanna Instruments®), then, it was sterilized (Pressure Sterilizer® 1941x) at 15 psi, 121° C. for 15 minutes. Then, the culture medium was inoculated with 1 cm discs of mycelia and agar under constant agitation at 100 rpm during 9 days at 25±1° C., under different light wave lengths.
Study Design to Evaluate LEDs Effects
To maximize the illumination of the culture medium, a box of 55 cm3 was made with all its internal surfaces covered with flat mirrors. A central perforation was made on the superior wall of the box in order to install different LEDs. A thermocouple (UNI-T®) was installed inside the box to monitor the temperature. The box was put covering an orbital agitator which upper surface was also covered with a flat mirror, and over which nine Erlenmeyer's flasks of 250 ml were put on. The Erlenmeyer's flasks contain the culture of Ganodeema lucidum.
Analytic Methods
A completely random design was used with 9 experimental units per treatment for a total of 45 experimental units. The results were analyzed with the Statgraphics® 4.0. statistical package. The statistical analysis includes ANOVA y Post-ANOVA tests.
Results
The culture medium and the temperature remain constant, to evaluate the effect of different wave length emitting LEDs over the biomass production. The source of carbon for the described culture medium was barley flour. Table 1 illustrates a comparison of the used culture medium with other culture mediums in the literature.
For Ganoderma lucidum, the yield/day in the Vegetal Biology Laboratory was 3.225 g/Liter, compared with the best result, 1.564 g/Liter, published in the literature. In addition, with the protocol used by Applicants in the Vegetal Biology Laboratory, the efficiency cost-wise, in the production of the mycelia biomass, increased more than 69 times (445 Colombian pesos or 0.19 USDollars/1 gram (Tang & Zhong, 2002) compared with 6.37 pesos or 0.0027 USDollars/1 gram in the Vegetal Biology Laboratory and Plant Biotechnology Laboratory).
Moreover, the mycelia biomass produced by Applicants in the Vegetal Biology Laboratory—Plant Biotechnology Laboratory has a better or comparable exo-polysaccharides (EPS), intra-polysaccharides (IPS), and ganoderic acid (AG) composition (EPS: 2.705±0.15 g/L, IPS: 2, 33±0.182 g/L, AG: 369, 67±12,73 mg/L), when compared with the best results in the literature as shown in the review by Wagner et al. (see Wagner, R., Mitchell, D. A, Sassaki, G. L., Lopes de Almeida Amazonas A. M., Berovic, M., Food Technology and Biotechnology, 41(4): 371-382 (2003)), which analyzes the Ganoderma lucidum mycelia biomass composition for exo-polysaccharides, intra-polysaccharides, and ganoderic acid (EPS: 1.71 g/L, IPS: 2.49 g/L, AG: 582 mg/L).
Culture Medium
The culture medium consisting of a basic medium with salts: NaNO3, 80 mg/l; KH2PO4, 30 mg/l; MgSO4.7H2O, 20 mg/l; KCl, 10 mg/l, supplemented with barley flour or oats flour 50 g/l; is been used by Applicants to maintain the following fungi strains in the Laboratory of Vegetal Biology:
