NZ331816A - Nutrient medium comprising peptone, yeast extract, cottonseed flour, glucose and palm oil - Google Patents

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. The medium for use in fermentation comprised of peptone, autolyzed yeast extract, cottonseed flour, glucose (dextrose), palm oil, cholesterol, CaCl2.2H2O, and MgCl2.6H2O.

Description

New Zealand Paient Spedficaiion for Paient Number 331 816 Intellectual Property Office of New Zealand IP Summary Report Page: 1 of 1 Date: 17 May 2000 Time: 15:02:37 (iprlp02 2.00.21) (51) Classification: C12N1/14 IPC Edition: IPC Status: 70 Accepted 331816 Version number: 4 IP type: Patent PCT Inward Client Ref: P396327 DCC (86) International Application number: US97/10343 Date actions completed: Application Accepted 17 May 2000 Next renewal date: 17 June 2001 (87) WO Publication number. Elected: Y 98/58049 (22) NZ Filing date: 17 June 1997 Date entered National phase: 09 September 1998 (71) Applicant: AGRAQUEST, INC., 1530 Drew Ave, Davis, California 95616, United States of America (72) Inventors: Heins, Sherry D Ewing, Duane D Man-one, Pamela G Contact: A J PARK, 6th Floor, Huddart Parker Building, 1 Post Office Square, Wellington, New Zealand Primary Examiner: GEORGE WARDLE Journal: 1452 Office title: Nutrient medium comprising peptone, yeast extract, cottonseed flour, glucose and palm oil (54) Applicant title: Nutrient medium for increasing cell yield in fermentation <57) Abstract: Patent 331816 A nutrient medium used in fermentation for increasing the yield of cells or microornanism is provided. The formulation provided increases the yield of the fungus Lane idium giganteum two-to three-fold over known media. The medium for use in fermentation comprised of peptone, autolyzed yeast extract, cottonseed flour, glucose(dextrose), palm oil, cholesterol, CaClj.2H20, and MgCI2.6H,0.

" End of report ** WO 98/58049 PCT/LS97/10343 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 tungus Lagenidium giganieum grown in fcnncntcrs is used as a I5 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 ccll growth are provided in the medium used during the fermentation process. Accordingly, the yield obtained from fermentation depends, in part, 20 on the composition of the medium.

There arc 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 Ardaminc pH, 2.0 g glucose, ! mL com oil, 0.S g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) 25 "Ground and aerial application of the sexual and asexual stages of Lagenidium giganteum (oomycetes: Lagenidiales) for mosquito control."./. Am. Mos. Control Asxoc. 2(2): 182-189).

SUBSTITUTE SHEET (RULE 28) Printed from Mimosa 17:27:40 r WO 98/58049 «"T A Q 1 §i\ PCT/ljS97/10343 331816 Another formulation comprises 2.0 g autolyzed yeast extract, 1.0 g proflo, 0.5 g fish meal, 2 mM CaCl32H20, ImM MgCl;6H,0, 0.05 g cholesterol and 2 mL cottonseed oil. (Kerwin, James L. and Washino, Robert K.. (1988) "Field evaluation of Lagenidium giganteum (Oomycetes: Lagenidiales) and description of a natural epizootic involving a 5 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 com oil, 1 g linseed oil, and 0.075 g CaCl22H20. (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 10 glucose and 1.5 g com oil. (Lord, Jeff.ey C. and Roberts, Donald W. (1986) "The effects of culture medium quality and host passage on zoosporogensis and infectivity of Lagenidium giganteum (Oomycetcs: Lagenidiales)," 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 15 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 20 A medium for use in fermentation consisting essentially of 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 CaCl, • 2H,0; 0.2 g per liter MgClj • 6H20 and, 25 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.

Preferably, the amount of autolyzed yeast is 3.0 g per liter of autolyzed yeast extract. 1 'I*'cuxliUAL PROPgfyo^n Of N2. 19 APR 2000 .RECEIVED 3 DESCRIPTION OF THE PRFFFRRFD F.MR OOIMF.NTS 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 5 invention is useiul 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 ot microorganisms in specialized vessels. "Nutrient medium" ("medium") refers to a solid 10 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; 7.0 to 7.75 g per liter glucose (dextrose); 2.5 gper liter palm oil; 0.2 g per liter cholesterol; 0.6 g per liter CaClt • 2H,0; and 20 0.2 g per liter MgCl, • 6H20.

Deionizcd 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 25 Lagenidium giganteum, this medium increases yield at least two to three fold over knowr. 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 30 construed as limiting the invention in any way.

SUBSTITUTE SHEET (RULE 26) Printed from Mimosa 17:27:40 4 Example 1 Shake flask comparison of growth rates of Ixi%enidium gifcmteum 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 10 1.25 g autolyzed yeast extract 2.0 g corn oil 1.0 g palm oil 0.03 g cholesterol 0.4 g CaClj • 2H,0 15 0.2 gMgClj.eHjO Medium #2: 1.2 g peptone 1.2 g autolyzed yeast extract 20 3.0 g glucose (dextrose) 0.5 g cholesterol Novel Nutrient Medium: 3.6 g peptone 25 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 30 0.6 g CaCl, • 2HjO 0.2 g MgCl] *6HjO When preparing each of the medio, 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 35 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 rpm, 29 C° in an orbital temperature controlled SUBSTITUTE SHEET (RULE 2(1) Printed from Mimosa 17:27:40 shaker for 7 days. Cells were harvested by centrifuging the fungal mass at 5,200 rpm 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 it 1 Medium #2 Novel Nutrient Medium Fold Increase in cells/mL when Novel Medium used Exp't# 1 1.2 - 2.0 x 10" cells/raL 1.2-2.0 x 10° cells/mL 4.4 x 10° cells/mL 2.2 fold Exp't #2 6.7.5 x 10^ cells/mL 7.5 x 10^ cells/mL 1.38 x 10°cells/mL 1.84-2.2 fold vxp't #3 2.97 x IOJcells/mL 3.3 x 10Jcells/mL 4.75 x 103 cells/mL 1.4-1.6 fold Exp't #7 9.77 x 104 cells/mL not done 9.38 x 10Jceils/mL 9.6 fold Exp't #8 1.93 x IOJcells/mL not done 7.30 x 103 cells/mL 3.7 fold 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 10 the novel nutrient medium.

F.xnmple 2 Shakf. flask comparison nf navel medium with lecithin nAdfd Having established that the novel medium formulation of Example 1 increases cell yield over known media, the cffcct of varying amounts of dextrose and yeast extract and adding 1.0 g or 2.0 g Iccithin to the basal novel medium was examined. All media were 20 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 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa 17:27:40 Table 2 Dextrose %Wt Yeast extract %Wt Lecithin %Wt Cell Yield (cells/mL) 0.8750 0.1250 0.0000 2.0 xlO5 0.8750 0.1250 0.0000 3.0 x 10' 0.6875 0.3125 0.0000 4.8 x 103 0.5000 0.5000 0.0000 4.1 x 10" 0.5000 0.5000 0.0000 4.13 x 10J 0.5875 0.3125 0.1000 .4 x 10* 0.5875 0.3125 0.1000 4.05 x 10J 0.3000 0.5000 0.2000 7.4 x 10' 0.3000 0.5000 0.2000 4.9 x 103 0.6750 0.1250 0.2000 4.5 x 10J 0.6750 01250 0.20(0 4.1 x 10' 0.4875 0.3125 0.2000 3.6 x I05 Average Cell Yield (cells/mL) without lecithin: 3.6 xlO5 Average Cell Yield (cells/mL) with lecithin: 4.63 x I05 As shown in Table 2, for media without lecithin, the average cells /mL yield is 3.6 x 10s. With lecithin, yield increases to 4.63 x 10: cells/mL.

Example 3 Infectivity of Lavenidium vioanteum crown 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; SUBSTITUTE SHEET (RULE 28) Printed from Mimosa 17:27:40

Claims (6)

WO 98/58049 7 PCT/US97/10343 1^250 and 675 cells/mL. Result1; summarized in Table 3 are averages of duplicate experiments. Table 3 % Mortality at 5,000 cells/mL % Mortality at 2,500 cells/mL % Mortality at 1,250 cells/mL % Mortality at 675 cells/mL Medium without lecithin 66 67 61 51 Medium with lecithin 87 87 89 74 These results illustrate that Lagenidium giganteum grown in the novel media killed more mosquitoes than cells grown in media without added lecithin. SUBSTITUTE SHEET (RULE 26) Printed from Mimosa 17:27:40 WO 98/58049 PCT/US97/10343 33181 f? 10 We claim:

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 floiir, (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 CaCl,2H20; and (h) 0.2 g per liter MgCl26HjO.

2. The medium according to claim I, further comprising up to 2.0 g per liter 15 lecithin.

3. The medium according to claim 1, for use in culuiring Lagenidium giganteum.

4. The medium according to claim 2, for use in culturing Lagenidium giganteum.

5. The medium according to claim I, where the amount of autolyzed yeast extract is 3.0 g per liter of autolyzed yeast extract.

6. A medium according to claim 1 substantially as herein described with or without reference to any example thereof. END OF CLAIMS iNitLLECTUAL PROPERTY OFRCeI OF N2. 1 J 3 APR 2000 RECEIVED