WO1998058049A1 - Nutrient medium for increasing cell yield in fermentation - Google Patents
Nutrient medium for increasing cell yield in fermentation Download PDFInfo
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- WO1998058049A1 WO1998058049A1 PCT/US1997/010343 US9710343W WO9858049A1 WO 1998058049 A1 WO1998058049 A1 WO 1998058049A1 US 9710343 W US9710343 W US 9710343W WO 9858049 A1 WO9858049 A1 WO 9858049A1
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- per liter
- medium
- fermentation
- yield
- cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Definitions
- This invention relates to a novel medium for use in fermentation which provides an increased cell yield compared to that of known media. More particularly, the present invention produces at least a two to three-fold increase in the yield of the fungus Lagenidium giganteum compared to the yield obtained with known media. In addition to increasing yield of cells, L. giganteum grown in novel medium containing lecithin exhibits increased effectiveness against mosquitoes.
- Fermentation is the process of growing microorganisms or cells in specialized vessels. The cells or organisms may then be purified and used for a variety of purposes. For instance, the fungus Lagenidium giganteum grown in fermenters is used as a biocontrol agent for mosquitoes.
- Optimal growth of the microorganism during fermentation depends on several factors including available nutrients, oxygen concentration, pH, temperature, and degree of mixing. Nutrients necessary for cell growth are provided in the medium used during the fermentation process. Accordingly, the yield obtained from fermentation depends, in part, on the composition of the medium.
- Lagenidium giganteum There are several published nutrient media currently used in the fermentation of Lagenidium giganteum. All use deionized water added to a final volume of 1 L, and all are sterilized.
- One formulation comprises 2.0 g Ardamine pH, 2.0 g glucose, 1 mL corn oil, 0.5 g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) "Ground and aerial application of the sexual and asexual stages of Lagenidium giganteum (oomycetes: Lagenidiales) for mosquito control.” J. Am. Mos. Control Assoc.
- Yet another fermentation medium comprises 1.25 g glucose, 1.25 g peptone, 1.25 g autolyzed yeast extract, 2 g corn oil, 1 g linseed oil, and 0.075 g CaCl 2 2H 2 O.
- the fourth published medium contains 1.25 g yeast extract, 1.2 g glucose, 3.2 g powdered wheat germ, hemp seed extract to provide 250 mg/L of soluble protein, 1.25 g bactopeptone, 3 g glucose and 1.5 g corn oil.
- a medium for use in fermentation consisting essentially of 3.6 g per liter peptone
- This medium provides increased yields of Lagenidium giganteum compared to prior art media, and, yield and infectivity of the organism is further increased when lecithin is included in the medium.
- DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to an improved medium for fermentation.
- the medium increases yield at least approximately two to three fold over known media.
- the invention is useful in large scale production of Lagenidium giganteum, a biocontrol agent for mosquitoes.
- fertilization refers to the process of growing cells or microorganisms in specialized vessels.
- Nutrient medium (“medium”) refers to a solid or liquid substrate that will support the growth of an organism.
- the nutrient medium is prepared as follows:
- Deionized water is added to a final volume of 1 L and the pH is adjusted to 6.5.
- the constituents are heated until dissolved and then the medium is sterilized by autoclaving at 121°C, 15 p.s.i., for 30 minutes.
- this medium increases yield at least two to three fold over known media.
- the nutrient medium is prepared by adding up to 2.0 g per liter of lecithin to the above formulation.
- Example 1 The following example is provided only for illustrative purposes, and is not to be construed as limiting the invention in any way.
- Example 1
- Medium #1 and Medium #2 yielded approximately the same number of cells per mL of medium in each experiment.
- the novel nutrient medium consistently increased the number of cells/mL in comparison to either Medium #1 or Medium #2.
- the average yield of Lagenidium giganteum was increased approximately three and half fold when grown in the novel nutrient medium.
- Example 1 Having established that the novel medium formulation of Example 1 increases cell yield over known media, the effect of varying amounts of dextrose and yeast extract and adding 1.0 g or 2.0 g lecithin to the basal novel medium was examined. All media were homogenized with a large probe at 70% speed for 10-15 seconds to ensure components were in solution. Using EmReagents color Phast®, the pH of all media was adjusted to 6.5 and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with 50 mL of medium, inoculated, cultured and harvested as described in Example 1. Results are summarized in Table 2 and Table 3 Table 2
- Lagenidium giganteum was grown in novel media described in Example 2 which contained no lecithin, 0.1000 % by weight lecithin or 0.2000 % by weight lecithin. Culturing conditions were as described in Example 1. The concentration of cells was calculated and their ability to kill mosquitoes measured at concentrations of 5,000; 2,500; 1,250 and 675 cells/mL. Results summarized in Table 3 are averages of duplicate experiments.
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- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
A nutrient medium used in fermentation for increasing the yield of cells or microorganism is provided. The formulation provided increases the yield of the fungus Lagenidium giganteum two-to three-fold over known media.
Description
NUTRIENT MEDIUM FOR INCREASING CELL YIELD IN FERMENTATION
FIELD OF THE INVENTION This invention relates to a novel medium for use in fermentation which provides an increased cell yield compared to that of known media. More particularly, the present invention produces at least a two to three-fold increase in the yield of the fungus Lagenidium giganteum compared to the yield obtained with known media. In addition to increasing yield of cells, L. giganteum grown in novel medium containing lecithin exhibits increased effectiveness against mosquitoes.
BACKGROUND OF THE INVENTION Fermentation is the process of growing microorganisms or cells in specialized vessels. The cells or organisms may then be purified and used for a variety of purposes. For instance, the fungus Lagenidium giganteum grown in fermenters is used as a biocontrol agent for mosquitoes.
Optimal growth of the microorganism during fermentation depends on several factors including available nutrients, oxygen concentration, pH, temperature, and degree of mixing. Nutrients necessary for cell growth are provided in the medium used during the fermentation process. Accordingly, the yield obtained from fermentation depends, in part, on the composition of the medium.
There are several published nutrient media currently used in the fermentation of Lagenidium giganteum. All use deionized water added to a final volume of 1 L, and all are sterilized. One formulation comprises 2.0 g Ardamine pH, 2.0 g glucose, 1 mL corn oil, 0.5 g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) "Ground and aerial application of the sexual and asexual stages of Lagenidium giganteum (oomycetes: Lagenidiales) for mosquito control." J. Am. Mos. Control Assoc. 2(2): 182-
Another formulation comprises 2.0 g autolyzed yeast extract, 1.0 g proflo, 0.5 g fish meal, 2 mM CaCl22H2O, ImM MgCl26H2O, 0.05 g cholesterol and 2 mL cottonseed oil. (Kerwin, James L. and Washino, Robert K. (1988) "Field evaluation of Lagenidium giganteum (Oomycetes: Lagemdiales) and description of a natural epizootic involving a new isolate of fungus." J. Med. Entomol. 25(6): 452-460) Yet another fermentation medium comprises 1.25 g glucose, 1.25 g peptone, 1.25 g autolyzed yeast extract, 2 g corn oil, 1 g linseed oil, and 0.075 g CaCl22H2O. (U.S. Patent No. 4,687,744). The fourth published medium contains 1.25 g yeast extract, 1.2 g glucose, 3.2 g powdered wheat germ, hemp seed extract to provide 250 mg/L of soluble protein, 1.25 g bactopeptone, 3 g glucose and 1.5 g corn oil. (Lord, Jeffrey C. and Roberts, Donald W. (1986) "The effects of culture medium quality and host passage on zoosporogensis and infectivity of Lagenidium giganteum (Oomycetes: Lagemdiales)," J. Invertebr. Pathol. 48:355-361)
When used in fermentation, the above-referenced published medium formulations all yield approximately the same number of cells and infect susceptible mosquitoes at approximately the same rate. Thus, in order to increase the yield and infectivity of biocontrol agents like Lagenidium giganteum, there is a need for an improved fermentation medium.
SUMMARY OF THE INVENTION A medium for use in fermentation consisting essentially of 3.6 g per liter peptone;
3.0 g per liter autolyzed yeast extract; 3.6 g per liter peptone; 1.5 to 3.0 g per liter autolyzed yeast extract; 1.6 g per liter cottonseed flour, such as ProFlo® (Traders Protein, Memphis, TN); 2.0 to 7.75 g per liter glucose (dextrose); 2.5 g per liter palm oil; 0.2 g per liter cholesterol; 0.6 g per liter CaCl2 • 2H2O; 0.2 g per liter MgCl2 • 6H2O and, optionally, 0.0 to 2.0 g per liter of lecithin. This medium provides increased yields of Lagenidium giganteum compared to prior art media, and, yield and infectivity of the organism is further increased when lecithin is included in the medium.
DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to an improved medium for fermentation. The medium increases yield at least approximately two to three fold over known media. The invention is useful in large scale production of Lagenidium giganteum, a biocontrol agent for mosquitoes.
Definitions
As used herein, the term "fermentation" refers to the process of growing cells or microorganisms in specialized vessels. "Nutrient medium" ("medium") refers to a solid or liquid substrate that will support the growth of an organism.
In a preferred embodiment of this invention, the nutrient medium is prepared as follows:
3.6 g per liter peptone;
1.5 to 3 g per liter autolyzed yeast extract; 1.6 g per liter cottonseed flour;
2.0 to 7.75 g per liter glucose (dextrose);
2.5 g per liter palm oil;
0.2 g per liter cholesterol;
0.6 g per liter CaCl2 • 2H2O; and 0.2 g per liter MgCl2 • 6H2O.
Deionized water is added to a final volume of 1 L and the pH is adjusted to 6.5. The constituents are heated until dissolved and then the medium is sterilized by autoclaving at 121°C, 15 p.s.i., for 30 minutes. When used in the fermentation of Lagenidium giganteum, this medium increases yield at least two to three fold over known media.
In another preferred embodiment, the nutrient medium is prepared by adding up to 2.0 g per liter of lecithin to the above formulation.
The following example is provided only for illustrative purposes, and is not to be construed as limiting the invention in any way.
Example 1
Shake flask comparison of growth rates of Lagenidium giganteum in different media
Growth rate in the novel nutrient medium was compared with two other media in side by side shake flask experiments. Medium #1:
1.25 g glucose (dextrose)
1.25 g peptone 1.25 g autolyzed yeast extract
2.0 g corn oil
1.0 g palm oil
0.03 g cholesterol
0.4 g CaCl2 .2H2O 0.2 g MgCl2 . 6H2O
Medium #2:
1.2 g peptone
1.2 g autolyzed yeast extract 3.0 g glucose (dextrose) 0.5 g cholesterol
Novel Nutrient Medium:
3.6 g peptone 3.0 g autolyzed yeast extract
1.6 g Proflo cottonseed extract
2.0 g glucose (dextrose)
2.5 g palm oil
0.2 g cholesterol 0.6 g CaCl2 • 2H2O
0.2 g MgCl2 • 6H2O
When preparing each of the media, all ingredients were combined and deionized water was added to a final volume of 1 L. The pH was adjusted to 6.5. Contents were heated in a microwave until dissolved and then sterilized at 121 C°, 15 psi for 30 minutes. For each medium, nine 250 mL flasks were each filled with 50 mL of medium. A disk of Lagenidium giganteum (California strain) taken from a petri dish was used to inoculate each flask. The flasks were shaken at 120 φm, 29 C° in an orbital temperature controlled
shaker for 7 days. Cells were harvested by centrifuging the fungal mass at 5,200 m for 20 minutes at 18 C°. The centrifuged cell mass was weighed and cell counts made with a hemacytometer. Mean cell counts were recorded. Results are summarized in Table 1.
Table 1
Medium #1 and Medium #2 yielded approximately the same number of cells per mL of medium in each experiment. The novel nutrient medium consistently increased the number of cells/mL in comparison to either Medium #1 or Medium #2. The average yield of Lagenidium giganteum was increased approximately three and half fold when grown in the novel nutrient medium.
Example 2
Shake flask comparison of novel medium with lecithin added
Having established that the novel medium formulation of Example 1 increases cell yield over known media, the effect of varying amounts of dextrose and yeast extract and adding 1.0 g or 2.0 g lecithin to the basal novel medium was examined. All media were homogenized with a large probe at 70% speed for 10-15 seconds to ensure components were in solution. Using EmReagents color Phast®, the pH of all media was adjusted to 6.5 and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with 50 mL of medium, inoculated, cultured and harvested as described in Example 1. Results are summarized in Table 2 and Table 3
Table 2
Average Cell
Yield (cells/mL) without lecithin:
3.6 x lO5
Average Cell
Yield (cells/mL) with lecithin:
4.63 x 105
As shown in Table 2, for media without lecithin, the average cells /mL yield is 3.6 x 105. With lecithin, yield increases to 4.63 x 105 cells/mL.
Example 3
Infectivity of Lagenidium giganteum grown in various media
Lagenidium giganteum was grown in novel media described in Example 2 which contained no lecithin, 0.1000 % by weight lecithin or 0.2000 % by weight lecithin. Culturing conditions were as described in Example 1. The concentration of cells was calculated and their ability to kill mosquitoes measured at concentrations of 5,000; 2,500;
1,250 and 675 cells/mL. Results summarized in Table 3 are averages of duplicate experiments.
Table 3
These results illustrate that Lagenidium giganteum grown in the novel media killed more mosquitoes than cells grown in media without added lecithin.
Claims
1. A medium for use in fermentation, consisting essentially of: (a) 3.6 g per liter peptone; (b) 1.5 to 3 g per liter autolyzed yeast extract;
(c) 1.6 g per liter cottonseed flour;
(d) 2.0 to 7.75 g per liter glucose (dextrose);
(e) 2.5 g per liter palm oil;
(f) 0.2 g per liter cholesterol; (g) 0.6 g per liter CaCl22H2O; and
(h) 0.2 g per liter MgCl26H2O.
2. The medium according to claim 1 , further comprising up to 2.0 g per liter lecithin.
3. The medium according to claim 1, for use in culturing Lagenidium giganteum.
4. The medium according to claim 2, for use in culturing Lagenidium giganteum.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/616,738 US5691191A (en) | 1996-03-15 | 1996-03-15 | Medium for the cultivation of lagenidium giganteum |
| PCT/US1997/010343 WO1998058049A1 (en) | 1996-03-15 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
| NZ331816A NZ331816A (en) | 1997-06-17 | 1997-06-17 | Nutrient medium comprising peptone, yeast extract, cottonseed flour, glucose and palm oil |
| CA002256519A CA2256519A1 (en) | 1997-06-17 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
| EP97930011A EP0954562A4 (en) | 1997-06-17 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
| KR1019980707266A KR19990087785A (en) | 1997-06-17 | 1997-06-17 | Nutritional medium to increase cell yield during fermentation |
| AU33942/97A AU733497B2 (en) | 1997-06-17 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
| JP53335898A JP2002503090A (en) | 1997-06-17 | 1997-06-17 | Nutrient media for increasing cell yield in fermentation |
| TW087117128A TW411365B (en) | 1996-03-15 | 1998-10-15 | Nutrient medium for increasing cell yield in fermentation |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/616,738 US5691191A (en) | 1996-03-15 | 1996-03-15 | Medium for the cultivation of lagenidium giganteum |
| PCT/US1997/010343 WO1998058049A1 (en) | 1996-03-15 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1998058049A1 true WO1998058049A1 (en) | 1998-12-23 |
Family
ID=26792571
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1997/010343 Ceased WO1998058049A1 (en) | 1996-03-15 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
Country Status (3)
| Country | Link |
|---|---|
| US (1) | US5691191A (en) |
| TW (1) | TW411365B (en) |
| WO (1) | WO1998058049A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7842479B2 (en) | 2004-05-21 | 2010-11-30 | The Board Of Regents Of The University Of Nebraska | Methods for altering acetic acid production and enhancing cell death in bacteria |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100379075B1 (en) * | 2002-03-07 | 2003-04-08 | Jinis Biopharmaceuticals Co | Method for producing low cholesterol animal food product and food product therefrom |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4141986A (en) * | 1976-04-28 | 1979-02-27 | Merck & Co., Inc. | Antibiotics 890A2 and 890A5 |
| US4687744A (en) * | 1982-09-30 | 1987-08-18 | The Regents Of The University Of California | Artificial culture of the sexual stage of lagenidium giganteum |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0494592B1 (en) * | 1991-01-10 | 1996-09-18 | W.R. Grace & Co.-Conn. | A process and method for production and use of pathogenic fungal preparation for insect control |
-
1996
- 1996-03-15 US US08/616,738 patent/US5691191A/en not_active Expired - Fee Related
-
1997
- 1997-06-17 WO PCT/US1997/010343 patent/WO1998058049A1/en not_active Ceased
-
1998
- 1998-10-15 TW TW087117128A patent/TW411365B/en not_active IP Right Cessation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4141986A (en) * | 1976-04-28 | 1979-02-27 | Merck & Co., Inc. | Antibiotics 890A2 and 890A5 |
| US4687744A (en) * | 1982-09-30 | 1987-08-18 | The Regents Of The University Of California | Artificial culture of the sexual stage of lagenidium giganteum |
Non-Patent Citations (1)
| Title |
|---|
| See also references of EP0954562A4 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7842479B2 (en) | 2004-05-21 | 2010-11-30 | The Board Of Regents Of The University Of Nebraska | Methods for altering acetic acid production and enhancing cell death in bacteria |
Also Published As
| Publication number | Publication date |
|---|---|
| TW411365B (en) | 2000-11-11 |
| US5691191A (en) | 1997-11-25 |
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