EP0954562A1 - Nutrient medium for increasing cell yield in fermentation - Google Patents
Nutrient medium for increasing cell yield in fermentationInfo
- Publication number
- EP0954562A1 EP0954562A1 EP97930011A EP97930011A EP0954562A1 EP 0954562 A1 EP0954562 A1 EP 0954562A1 EP 97930011 A EP97930011 A EP 97930011A EP 97930011 A EP97930011 A EP 97930011A EP 0954562 A1 EP0954562 A1 EP 0954562A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- per liter
- medium
- fermentation
- yield
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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, e.g. protozoa; 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
-
- 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, e.g. protozoa; 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
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.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US1997/010343 WO1998058049A1 (en) | 1996-03-15 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP0954562A1 true EP0954562A1 (en) | 1999-11-10 |
| EP0954562A4 EP0954562A4 (en) | 2000-05-10 |
Family
ID=22261083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP97930011A Withdrawn EP0954562A4 (en) | 1997-06-17 | 1997-06-17 | Nutrient medium for increasing cell yield in fermentation |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0954562A4 (en) |
| JP (1) | JP2002503090A (en) |
| KR (1) | KR19990087785A (en) |
| AU (1) | AU733497B2 (en) |
| CA (1) | CA2256519A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015002678A (en) * | 2011-10-21 | 2015-01-08 | 株式会社カネカ | Culturing method of microorganisms, and method for producing pha by microorganisms |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0494592A1 (en) * | 1991-01-10 | 1992-07-15 | W.R. Grace & Co.-Conn. | A process and method for production and use of pathogenic fungal preparation for insect control |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL7704040A (en) * | 1976-04-28 | 1977-11-01 | Merck & Co Inc | PROCEDURE FOR PREPARING NEW ANTI-BIOTICS. |
| US4687744A (en) * | 1982-09-30 | 1987-08-18 | The Regents Of The University Of California | Artificial culture of the sexual stage of lagenidium giganteum |
-
1997
- 1997-06-17 JP JP53335898A patent/JP2002503090A/en active Pending
- 1997-06-17 EP EP97930011A patent/EP0954562A4/en not_active Withdrawn
- 1997-06-17 CA CA002256519A patent/CA2256519A1/en not_active Abandoned
- 1997-06-17 KR KR1019980707266A patent/KR19990087785A/en not_active Application Discontinuation
- 1997-06-17 AU AU33942/97A patent/AU733497B2/en not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0494592A1 (en) * | 1991-01-10 | 1992-07-15 | W.R. Grace & Co.-Conn. | A process and method for production and use of pathogenic fungal preparation for insect control |
Non-Patent Citations (1)
| Title |
|---|
| See also references of WO9858049A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002503090A (en) | 2002-01-29 |
| CA2256519A1 (en) | 1998-12-23 |
| KR19990087785A (en) | 1999-12-27 |
| AU733497B2 (en) | 2001-05-17 |
| AU3394297A (en) | 1999-01-04 |
| EP0954562A4 (en) | 2000-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10757946B2 (en) | Microbial inoculant formulations | |
| US20070004022A1 (en) | Industrial fermenting production process of Hirsutella hepiali Chen & Shen of anamorphic fungi related to Chinese Cordyceps Sinensis | |
| CN111235039B (en) | Culture medium for culturing metarhizium anisopliae with high toxicity, preparation and application thereof | |
| CN106148216B (en) | A kind of streptomycete and its method for producing mibemycin A3 | |
| CN108060096A (en) | A kind of probiotic combinations and its application in litopenaeus vannamei seed rearing | |
| CN1730647A (en) | Bacterial strain and its formulation for preventiong sting type trophi insect | |
| US5691191A (en) | Medium for the cultivation of lagenidium giganteum | |
| CN1570078A (en) | Bacterial wilt biocontrol bacterium ANTI-8098A and its culturing medium, culturing method and biocontrol uses | |
| CN1167790C (en) | High-toxicity gene engineering bacteria WG001 of Bacillus thuringiensis and its production process and product | |
| CN1498865A (en) | Micro ecological agent for supporting water in fishing use and its prepn. method | |
| CN115747130B (en) | Culture medium for promoting destruxin Mr006 to produce spores, preparation and application thereof | |
| AU733497B2 (en) | Nutrient medium for increasing cell yield in fermentation | |
| US3073749A (en) | Preparation of microbial insecticide | |
| CN113528350B (en) | Rhizopus strain, distiller's yeast and rice wine and production method of distiller's yeast | |
| JP4434927B2 (en) | Method for producing γ-aminobutyric acid-containing food, and yeast having high γ-aminobutyric acid production ability | |
| EP0346545B1 (en) | Maintenance of the viability of microorganisms for use in microbial inoculants | |
| JP4384497B2 (en) | Novel composition for fast and sufficient sporulation of fungi and method therefor | |
| CN1222190A (en) | Nutrient medium for increasing cell yield in fermentation | |
| JP3857238B2 (en) | Process for producing a mushroom composition for eating and drinking | |
| KR100208968B1 (en) | A novel saccharomyces cerevisiae ns9 which is used for producing yeast extract contained high density of nucleic acid | |
| NZ331816A (en) | Nutrient medium comprising peptone, yeast extract, cottonseed flour, glucose and palm oil | |
| Št̆astná et al. | Production, long term preservation and synchronous germination of aerial spores of Streptomyces | |
| JP2003079363A (en) | Culture medium for separating soy sauce lactic bacterium having low turbidity, method for separating soy sauce lactic bacterium having low turbidity by using the medium, and method for producing soy sauce having high clarity by using the lactic bacterium | |
| CN115353981B (en) | Liquid fermentation culture method of trephine hirsutum and active metabolite | |
| CN114276936B (en) | Eurotium cristatum strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 19980914 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| A4 | Supplementary search report drawn up and despatched |
Effective date: 20000323 |
|
| AK | Designated contracting states |
Kind code of ref document: A4 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| 17Q | First examination report despatched |
Effective date: 20021028 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20030308 |
|
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: WD Ref document number: 1025123 Country of ref document: HK |