KR19990087785A - Nutritional medium to increase cell yield during fermentation - Google Patents

Abstract

It is an object to provide a nutrient medium for use in fermentation to increase the yield of cells or microorganisms. The provided formulations increase the yield of Lagenidium giganteum bacteria 2-3 times more than known media.

Description

Nutritional medium to increase cell yield during fermentation

Fermentation is the process of culturing microorganisms or cells in specialized containers. Cells or microorganisms can be purified and used for various purposes. For example, Lagenidium giganteum bacteria cultured in fermenters are used as biological control against mosquitoes.

Optimal growth of microorganisms during fermentation depends on several factors including available nutrients, oxygen concentration, pH, temperature and degree of mixing. Nutrients necessary for cell growth are supplied in the medium used during fermentation. Therefore, the yield obtained from fermentation depends in part on the composition of the medium.

There are several published nutritional media currently used for fermentation of Lagenidium giganteum. Deionized water is added to make a final volume of 1L, and all are sterilized. One formulation contains 2.0 g Ardamine pH, 2.0 g glucose, 1 mL corn oil, 0.5 g cholesterol and 2 mM Ca ²⁺ (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): 182-189).

Other preparations include 2.0 g magnetized yeast extract, 1.0 g proflo, 0.5 g fish meal, 2 mM CaCl ₂ 2H ₂ O, 1 mM MgCl ₂ 6H ₂ O, 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 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 magnetized yeast extract, 2 g corn oil, 1 g linseed oil, and 0.075 g CaCl ₂ 2H ₂ O (US Pat. No. 4,687,744). The fourth published medium contains 1.25 g yeast extract, 1.2 g glucose, 3.2 g powdered malt, hemp seed extract providing 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: Lagenidiales)," J. Invertebr. Pathol. 48: 355-361)

When used in fermentation, the above-mentioned published media preparations all yield about the same number of cells and infect mosquitoes that can be infected at about the same rate. Therefore, improved fermentation medium is needed to increase the yield and infectivity of biological control agents such as Lagenidium giganteum.

The present invention relates to a novel medium for use in fermentation which provides increased cell yield compared to known media. More specifically, the present invention produces at least 2 to 3 times increased yield compared to the yield obtained with known media in the yield of Lagenidium giganteum bacteria. In addition to increasing cell yield, L.giganteum cultured in new medium containing lecithin shows increased anti-mosquito effects.

3.6 g / L peptone; 3.0 g / L Magnetized Yeast Extract; 3.6 g / L peptone; 1.5 to 3.0 g / L magnetized yeast extract; 1.6 g / L cottonseed flour such as ProFlo ^R (Traders Protein, Memphis, TN); 2.0 to 7.75 g / L glucose (dextrose); 2.5 g / L palm oil; 0.2 g / L cholesterol; 0.6 g / L CaCl ₂ · 2H ₂ O; A medium used for fermentation containing 0.2 g / L MgCl ₂ · 6H ₂ O and, optionally, 0.0 to 2.0 g / L of lecithin as an essential component. This medium provides an increased yield of Lagenidium giganteum compared to the medium of the prior art, and when lecithin is included in this medium, the yield and infectivity of microorganisms are further increased.

The present invention relates to an improved medium for fermentation. The medium increases yield at least approximately 2-3 times greater than known medium. The present invention is useful for the large scale production of Lagenidium giganteum, a biological control against mosquitoes.

Justice

The term "fermentation" as used herein refers to the process of culturing cells or microorganisms in specialized containers. "Nutritional medium" ("medium") refers to a solid or liquid substance that supports the growth of an organism.

In a preferred embodiment of the invention, the nutrient medium is formulated as follows:

3.6 g / L peptone;

1.5 to 3 g / L magnetized yeast extract;

1.6 g / L cottonseed flour;

2.0 to 7.75 g / L glucose (dextrose);

2.5 g / L palm oil;

0.2 g / L cholesterol;

0.6 g / L CaCl ₂ · 2H ₂ O; And

0.2 g / L MgCl ₂ .6H ₂ O.

Deionized water is added to a final volume of 1 L and the pH adjusted to 6.5. The components are heated until dissolved and then sterilized by autoclaving the medium at 121 ° C., 15 p.s.i. for 30 minutes. When used for fermentation of Lagenidium giganteum, this medium increases at least 2 to 3 times the yield compared to known media.

In another preferred embodiment, 2.0 g / L of lecithin is added to the preparation.

The following examples are for illustrative purposes only and should not be construed to limit the invention in any way.

Example 1

Comparison of shake flasks of growth rate of Lagenidium giganteum in different media

Growth rates in the new nutrient medium were compared side by side with the other two media by shake flask experiments.

Badge # 1:

1.25 g glucose (dextrose)

1.25 g peptone

1.25g Magnetized Yeast Extract

2.0g corn oil

1.0g palm oil

0.03g cholesterol

0.4 g CaCl ₂ · 2H ₂ O

0.2 g MgCl ₂ · 6H ₂ O

Badge # 2:

1.2g peptone

1.2g Magnetized Yeast Extract

3.0 g glucose (dextrose)

0.5g cholesterol

New Nutritional Medium:

3.6g peptone

3.0g Magnetized Yeast Extract

1.6g Proflo Cotton Seed Extract

2.0 g glucose (dextrose)

2.5g palm oil

0.2g cholesterol

0.6 g CaCl ₂ · 2H ₂ O

0.2 g MgCl ₂ · 6H ₂ O

When preparing each medium, all components are combined to add deionized water to a final volume of 1 L. pH is adjusted to 6.5. The contents are heated in microwave until dissolved and sterilized at 121 ° C., 15 psi for 30 minutes. For each medium, nine 250 mL flasks were each filled with 50 mL of medium. Disks of Lagenidium giganteum (California strain) taken from Petri dishes were used to inoculate each flask. The flasks were shaken at 120 rpm, 29 ° C. in an orbital temperature controlled shaker for 7 days. Cells were harvested by centrifugation of the bacterial mass at 5,200 rpm for 20 minutes at 18 ° C. Centrifuged cell mass was weighed and cell counts were measured with a hemocytometer. The average cell number was recorded. The results are summarized in Table 1.

Badge # 1 Badge # 2 New nutritional medium Increase in cells / mL when using new medium Experiment # 1 1.2-2.0 × 10 ⁶ cells / mL 1.2-2.0 × 10 ⁶ cells / mL 4.4 × 10 ⁶ cells / mL 2.2x Experiment # 2 6.25 × 10 ⁵ cells / mL 7.5 × 10 ⁵ cells / mL 1.38 × 10 ⁶ cells / mL 1.84-2.2 times Experiment # 3 2.97 × 10 ⁵ cells / mL 3.3 × 10 ⁵ cells / mL 4.75 × 10 ⁵ cells / mL 1.4-1.6 times Experiment # 7 9.77 × 10 ⁴ cells / mL none 9.38 × 10 ⁵ cells / mL 9.6 times Experiment # 8 1.93 × 10 ⁵ cells / mL none 7.30 × 10 ⁵ cells / mL 3.7 times

Medium # 1 and medium # 2 yielded approximately equal cell numbers per mL of medium in all experiments. The new nutrient medium consistently increased the number of cells / mL compared to medium # 1 or medium # 2. The average yield of Lagenidium giganteum increased approximately 3.5-fold when cultured in fresh medium.

Example 2

Comparison of shake flasks in new medium with lecithin

Demonstrating that the novel media formulation of Example 1 increases cell yield over known media, changing the amount of dextrose and yeast extract and adding 1.0 g or 2.0 g of lecithin to the new media base Was tested. All media was homogenized with large probes at 70% speed for 10-15 seconds to dissolve the components. The pH of all media was adjusted to 6.5 using EmReagent color Phast ^R and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with 50 mL of medium, inoculated, incubated and harvested as in Example 1. The results are summarized in Tables 2 and 3.

As shown in Table 2, the average cell / mL yield is 3.6 × 10 ⁵ in a lecithin-free medium. In the presence of lecithin, the yield increases to 4.63 × 10 ⁵ cells / mL.

Example 3

Infectivity of Lagenidium giganteum incubated in various media

Lagenidium giganteum was incubated in the novel medium described in Example 2, containing no lecithin, containing 0.1000% lecithin or 0.2000% lecithin. Culture conditions are as described in Example 1. Calculate the concentration of the cell, and the ability to kill mosquitoes 5,000; 2,500; Measurements were made at concentrations of 1250 and 675 cells / mL. The results summarized in Table 3 are the average of two experiments.

% Mortality at 5,000 cells / mL % Mortality at 2,500 cells / mL % Mortality at 1250 cells / mL % Mortality at 675 cells / mL Lecithin-Free Badge 66 67 61 51 Medium containing lecithin 87 87 89 74

These results illustrate that Lagenidium giganteum cultured in new medium kills more mosquitoes than cells cultured in medium without lecithin.

Claims (4)

(a) 3.6 g / L peptone; (b) 1.5 to 3 g / L magnetized yeast extract; (c) 1.6 g / L cottonseed flour; (d) 2.0 to 7.75 g / L glucose (dextrose); (e) 2.5 g / L palm oil; (f) 0.2 g / L cholesterol; (g) 0.6 g / L CaCl ₂ 2H ₂ O; And (h) a medium for use in fermentation, with 0.2 g / L MgCl ₂ 6H ₂ O as an essential component. The medium according to claim 1, further comprising a lecithin of 2.0 g / L or less. The medium according to claim 1, which is used for culturing Lagenidium giganteum. The medium according to claim 2, which is used for culturing Lagenidium giganteum.