KR20120117160A - Method and product of freeze drying of photorhabdus temperata m1021 - Google Patents

Abstract

PURPOSE: A freeze-drying method for photorhabdus temperata M1021 (deposit number: kacc91627p) and freeze-drying powder are provided to increase survival rate of photorhabdus temperate M1021 strain and to facilitate formulation. CONSTITUTION: A freeze-drying method for photorhabdus temperata M1021 (deposit number: kacc91627p) is processed by adding skim milk, sodium alginate, starch, or preserver of glucose. The skim milk is added in 1-10% concentration. The skim milk is added in 7 % concentration. The sodium alginate is added in 0.5-2% concentration. The starch is added in 1-7 % concentration. The glucose is added in 0.5-1.5% concentration. The freeze-drying is processed at -60 to -80 deg. Celsius for 48-72 hours. The biological agricultural chemical includes freeze-drying powder of photorhabdus temperata M1021 strain. The biological agricultural chemical is the agricultural chemical for Spodoptera frugiperda prevention.

Description

Freeze drying method and freeze drying powder. (Method and product of freeze drying of Photorhabdus temperata M1021}

The present invention relates to a method of freeze-drying a novel strain of Poturaddus temperata M1021 (Accession No. KACC91627P). More specifically, it relates to a freeze drying method characterized by adding a preservative.

In addition, the present invention relates to a freeze-dried powder of the strain of Porturadops temperata M1021 produced by the freeze-drying method as described above and to a biological pesticide comprising the same.

Organic chemical synthetic insecticides have been widely used to control pests, but due to decades of continuous use and abuse, the emergence of abnormal or resistant pests of pest groups, toxic expression of non-targeted insects including humans, and environmental pollution It is causing many side effects. As a result of various problems caused by pesticide residue toxicity and environmental pollution around the world, an international agreement was reached to refrain from using highly toxic organic synthetic pesticides in order to protect human health. Under these international agreements, the world reduced production by 50% of the chemical synthetic organophosphorus and chlorine pesticides used in the past 10 years in 2004, and again reduced production of organophosphorus and organochlorine insecticides by 50% by 2010. It is in the phase of implementation, agreeing to international agreements to be made.

However, after the consultation on the reduction and production of toxic pesticides, many researchers around the world have tried to develop environmentally friendly pesticides, but have not been able to develop new and safe insecticides to replace the organophosphorus and organochlorine pesticides that have been used. The resolution of the World Pesticide Reduction Conference is difficult to keep. If a safe insecticide is not developed and produced soon, there will be a big problem at home and abroad due to the lack of pesticides for pest control as well as agricultural pesticides related to food and agricultural production. It is expected.

In recent years, microorganisms have been able to reduce or replace the use of chemical fertilizers or pesticides, microorganisms capable of biological control techniques that are less harmful to livestock, do not harm crops, and have less damage to the environment, such as soil ecosystems. As evidenced by several researchers, there is a growing interest and interest in biological pesticides that can complement the chemical pesticides.

The use of biological pesticides using microorganisms in biological pesticides is expected. Lyophilization methods can be used for the use of such microorganisms in biopesticide formulations. Freeze drying is one of drying methods used to preserve microorganisms for a long time, to preserve active substances that are relatively unstable according to conditions such as temperature in an aqueous solution state such as enzymes or functional proteins, or to preserve the original shape and flavor of food. .

On the other hand, Lepidoptera (Lepidopter) butterfly acids and moths acids and contain, species belonging to the neck are the egg, larva, pupa, to complete transformation after the adult stage 4, of Lepidoptera larvae herbivorous then large with respect to the wide range of crops It is a representative pest that causes economic damage. In particular, the larva of the bee-flying moth (Galleria mellonella) belonging to the butterfly neck is a pest that affects the beehive. It is in a state of condition and eventually collapses, causing huge economic losses to honey farmers. In addition, tobacco castor moth belonging to the butterfly neck is a photogenic and omnivorous pest with more than 100 species of host, which are inherently high resistance to drugs and low susceptibility. Are known to be very difficult representative heating pests (Choi et al., Kor. J. Appl. Entomol., 1996, 35: 249-253; Bae et al., Kor. J. Appl. Entomol., 2003, 42 : 225-231). Night moth leaves moths penetrate into chestnut trees to cool chestnut flesh. Perch moths are the main pests of rice, and rice leaves are dried vertically and eat the leaves in them, so that the grains of grain do not grow well. It is damaging economic crops. Therefore, there is a need for the development of a low toxicity environmentally friendly microbial agent with excellent insecticidal ability to biologically control against lepidopteran pests that cause economic losses to farms as described above.

Accordingly, the present inventors have made efforts to develop a biological pesticide that can effectively control butterfly neck pests, and as a result, a novel strain, Poturaddus temperata M1021 (accession number: KACC91627P), has been identified and the strain is effectively used as a biopesticide. The present invention has been completed to find the most suitable freeze-drying method for and to provide it as a biological pesticide formulation.

It is an object of the present invention to provide a freeze-drying method that is most suitable for the novel strain of Portuabdus temperata M1021.

In addition, an object of the present invention is to provide a freeze-dried powder of the strain of Porturadops temperata M1021 prepared by the above freeze-drying method and a biological pesticide comprising the same.

In order to achieve the above object, the present invention is in the form of milk milk, sodium alginate, starch, starch (glucose), at least one selected from the group consisting of glucose, Porturabdus temperata M1021 (Accession Number: KACC91627P) Provide a lyophilization method.

In another aspect, the present invention provides a freeze-dried powder of Porturadops temperata M1021 provided by the above freeze-drying method.

In still another aspect, the present invention provides a biopesticide comprising such a lyophilized powder.

According to the present invention, it is possible to control lepidopteran pests by using the Potorapis temperata M1021 strain as an environmentally-friendly microbial pesticide preparation, and in particular, by inventing a freeze-drying method most suitable for the novel strains of the strains, Portofrapus temperata M1021. Increasing the survival rate and facilitating formulation can increase the availability of biopesticides.

FIG. 1 shows the effect of a preservative on the survival of Photolabdus temperata M1021 after lyophilization. (A: Scheme milk, B: Starch C: Sodium alginate, D: Sodium glutamate E: Glucose)
FIG. 2 shows the effect of Schememilk on the viability of the frozen precursor powder Photorapdus temperata M1021 at 4 ° C. (Skimmilk was added to the 25-fold cell suspension. The added concentrations were: ■: 0%, ○: 1%, Δ: 5%, ▲: 7%, and ●: 10%, respectively.)
Figure 3 shows the insecticidal effect of photolabdus temperata M1021 freeze drying. (Dose concentrations are: 2.02 × 10 μs, ◇: 2.02 × 10 ³ ▲: 2.02 × 10 ² ◆: 2.02 × 10 ¹ ●: 2.02 × 10 ^0, Δ: 2.02 × 10 ⁻¹ , ○: Control group. )
4 shows the insecticidal effect upon treatment of lyophilized Photorapdus temperata M1021 (left control, right treatment).

Hereinafter, the present invention will be described in detail.

The present invention provides a lyophilization method of photolabdus temperata M1021 (Accession Number: KACC91627P).

Photolapdus temperata M1021 (Accession No .: KACC91627P) used in the present invention was isolated and identified from an insect pathogenic nematode, and 16s rDNA sequences (1493bp) of the strain were compared with known strains. Photorhabdus temperata) and 99% homology. This strain is a strain that is not completely identical to the existing strain, and is a novel strain belonging to the species. According to the analysis result, the strain of the present invention is named photolabdus temperata M1021, and as of January 4, 2011, It was deposited with the accession number KACC 91627P at the National Institute of Agricultural Science.

Looking at the morphological and biochemical characteristics of the strains of the present invention, it shows catalase, protease, and lipase enzyme activity as Gram-negative bacillus, and the growth temperature is active at 28 ° C to 30 ° C, and growth is inhibited as the culture temperature is increased. It may be characterized by.

The present invention adds at least one preservative selected from the group consisting of scheme milk, sodium alginate, starch, and glucose for efficient freeze drying of the photolabose temperata M1021.

The term "preservative" of the present invention refers to protecting the cells from various physicochemical external impacts such as nutrient depletion, osmotic pressure, and especially protecting the cells from ice crystals formed during freezing.

“Skim milk” used in the present invention is a portion remaining after centrifugation of fat in milk, and the scheme milk of the present invention is added at a concentration of 1% to 10% (w / v) to prevent death of the strain by freeze drying. It can be used as a preservative. Preferably, it may be added at a concentration of 7% (w / v), but is not limited thereto.

In addition, the "sodium alginate" of the present invention is a food additive for increasing the adhesiveness and viscosity of foods, enhancing the emulsion stability, and improving the physical properties and feel of foods, and the formula (C6H7O6Na) n. It is characterized by that.

 The present invention relates to a method for freeze-drying Poturadops temperata M1021 by adding sodium alginate at a concentration of 0.5% to 1.5% (w / v). This addition is most preferably but not limited to 1.5% (w / v) and the addition of starch as a preservative is also preferably at a concentration of 1% to 7% (w / v), more preferably 5% (w / v). It may be added as, but is not limited thereto.

In the present invention, "glucose" is a representative aldohexose (monosaccharide having 6 carbons and an aldehyde group) and is a central compound of carbohydrate metabolism, characterized by being able to synthesize 38 ATPs per molecule. In the present invention, glucose may be added as a preservative, and may be preferably added at a concentration of 0.5% to 1.5% (w / v). At higher concentrations, glucose may have difficulty in powder formation.

In the present invention, in performing the freeze-drying method, the Poturadap temperata M1021 is preferably freeze at -60 ° C to -80 ° C, more preferably -70 ° C, and freezes for 48 to 72 hours. Can be performed.

In another embodiment of the present invention, the above freeze drying method is performed to provide a freeze-dried powder of the Poturadap temperata M1021 strain.

For the powdering step in the present invention, powdering methods known in the art can be used without limitation. For example, the powdering may be performed to pass 90% or more of the sieve of 300 mesh. When pulverizing, a pin miller, an airjet miller, or a hammer miller can be used. Especially, in the case of some finely ground particles after coarse pulverization using a pin miller, It can be milled using an jet miller. Thus pulverization can be prepared in a formulation selected from the group consisting of a hydrate (WP), a solid (GM), a granule (WG), a granule (GR), powder (DP) and seed treatment hydration (WS). In addition to the above formulation, any formulation that can be used as a biopesticide is possible.

The hydrating agent is a formulation of the pesticide that is hydrated when diluted in water as a powder phase, the solid agent is a formulation that is mixed or adsorbed microbial culture medium to the solid material means a formulation that does not correspond to powder, granules and hydrating agents. In addition, the granular hydrating agent is a granular formulation of a pesticide used in dilution with water, the granules are granulated formulation of the pesticide used as a raw state, the powder is a powdered formulation of the pesticide used in its original state as the seed treatment Hydrating means a formulation of pesticide that is hydrated and used as a suspension prior to treatment on the seed as a powder.

The present invention may also include a dry powder obtained by the drying method and a biopesticide containing the same. The dry powder may be prepared as a biopesticide in a formulation selected from the group consisting of a hydrate (WP), a solid (GM), a granule (WG), a granule (GR), powder (DP) and seed treatment hydration (WS). have.

Hereinafter, the present invention will be described in detail by Examples and Test Examples. However, the following Examples and Test Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Test Examples.

Example  1. Materials and Methods.

1.1 Use Media and Reagents

Scheme milk, starch, sodium alginate, dextrose, sodium-glutamate and the like were purchased from Sigma. Tryptic Soy Broth (TSB), Bacto-trypton, and Bacto-yeast extract, which are microbial culture media, were purchased from Difco and other reagents were used as special reagents.

1.2 Strains and Cultures Used

Insect pathogenic microorganisms were used for Portrapads temperata M1021 isolated from entomopathogenic nematodes and identified by 16S rDNA sequencing (GenBank accession number HQ647119). Strains were incubated for 48 hours at 28 ° C. using TSB (Difco, France) agar medium, followed by shaking culture (28 ° C., 200 rpm) by inoculating single colonies in 100 ml TSB liquid medium prior to inoculation. This culture was incubated for 48 hours at 28 ℃ using 1L TSB liquid medium and then used to prepare a lyophilized sample.

Example 2 . Preparation of Lyophilized Samples.

The cells to be used for lyophilization were centrifuged (10,000 rpm, 4 ° C., 20 min) in a culture solution of Forturabdus temperata M1021 incubated for 48 hours in this culture, and then only the cells were recovered. After washing several times, the same saline was suspended 25 times (25 ⅹ) of the culture solution. In order to confirm the effect of preservatives affecting the viability of the cells during lyophilization, Scheme Milk, Starch, Sodium Glutamate were prepared to 15% (w / v), respectively, and Sodium Alginate was 4% (w) due to its high viscosity. / v) and used.

The final concentration of preservative added to 7 ml of suspension was 1, 3, 5, 7% (w / v) for starch and sodium glutamate, 1, 5, 7, 10% (w / v) for skim milk, and dextrose 0.5, 1% (w / v) and sodium alginate were added in 0.5, 1, 1.5% (w / v) portions. The mixture was added uniformly by the addition of a preservative and then completely frozen at −70 ° C., followed by freeze drying for 3 days using a lyophilizer (LABCONCO, USA).

After the lyophilization was completed, the sample was weighed and then crushed uniformly to be refrigerated (4 ° C).

Test Example 1 . Survival Rate of Bacteria by Freezing

As a result of experiments, the survival rate of Poturadap temperata M1021 was found to be 3.03 × 10 ⁶ colony forming unit (CFU) / ml before freezing and 2.63 × 10 ³ CFU / ml after freezing. The survival rate of the bacteria was only 0.09%.

Therefore, in order to improve the survival rate of the bacteria by freezing and find an optimized freeze-drying method, the survival rate of the bacteria by the addition of a preservative was examined.

Test Example 2 . Survival Rate of Bacteria by Preservative Type and Concentration

After each preservative is taking a Fortuna drawer Douce tempera other respectively by 0.1 g of freeze-dried sample of the M1021 strain containing various concentrations completely suspended in sterile physiological saline for 10 ㎖, step-by-step using a sterile physiological saline (10 ^1? 10 ⁶ Dilution). 100 μl of each diluted sample was smeared on a TSB agar plate and incubated at 28 ° C. for 48 hours to determine the number of bacteria.

In order to minimize cell loss caused by lyophilization, polymers such as skim milk, starch, sodium alginate and sodium glutamate were used.

In case of skim milk, as the concentration increased, the survival rate of S. aureus M1021 strain in lyophilized sample gradually increased, showing the highest survival rate of 63% at 7% (w / v). It decreased sharply (FIG. 1A).

At 5% of starch, glucose showed 3.53 and 2.11% survival rate at 1% (w / v), respectively, about 4 and 21 times, respectively. This result shows that the survival rate of the bacteria in the freeze-dried strain of Poturadap temperata M1021 can be increased, but the cell protection ability is relatively low when freeze-dried compared to the sample milk added sample. (Fig. 1B)

Sodium alginate is known to be a high molecular material that is widely used for the preservation and encapsulation of microorganisms. When the concentration of sodium alginate was 1.5% (w / v), the survival rate was 3.4% compared to the control (no addition). (Figure 1C)

On the other hand, in the case of sodium glutamate used in the comparative example there was no cell protection effect according to lyophilization. (Figure 1D)

When glucose was added more than 1.5% (w / v), it was difficult to form a normal powder after freeze-drying, and the survival rate was evaluated at the final concentration of 1% (w / v). (Fig. 1E)

Comparative Example 1 . Sodium Glutamate  Survival rate of bacteria when preservative is added

 In general, sodium glutamate, widely used as a preservative for lyophilization, has been found to be an unsuitable preservative for lyophilization of the novel strain of Poturadaps temperata M1021.

As shown in FIG. 1D, when 1-10% (w / v) of sodium glutamate was added, the survival rate of the strain was 0.01% when 10% was added, and the maximum survival rate was 0.13% in the non-treated group.

In comparison, Scheme Milk, Starch, Sodium Alginate and Glucose showed up to 63%, 3.53%, 3.4% and 2.11%, respectively. It was found to be a suitable preservative for lyophilization of the lobdus temperata M1021 strain.

Test Example 3 . Freeze drying Insecticidal  evaluation

A freeze-dried powder sample of Porturadops temperata M1021 containing 7% (w / v) scheme milk was used to inject toxic to honeybee larvae larvae. The degree of death of the larvae was measured over time.

Fortuna drawer Douce When the scanning all were dead, at least 2.02ⅹ10 ¹ cells in the tempera other jusaryang the 2.02ⅹ10 ^three cells 24 hours after injection than when the M1021 strain most of the larvae is shown to death within 2 days (Figure 3) . In addition, 50% of the larvae died at 4 days after injection, even at very low concentrations of 2.02ⅹ10 ⁰ cells, so the LD (lethal dose) value for the bee-larged moth larvae of the Poturadap temperata M1021 Found to be very low.

After the death of the bee-larged moth larvae, the body became red and lost swelling due to the death of the beetrapoddus temperata M1021. (FIG. 4) This is due to the anthraquinone pigment produced by the strains of P. luminescens. It was confirmed that the high insecticidal power was exhibited by the treatment of the freeze-dried strain according to the present invention as a feature of only Dus temperata strain.

Test Example 4 . Evaluation of the stability of the strain according to the storage period.

The stability of the strains of Potorab dos Temperata M1021 strains during storage of the lyophilized samples was tested by the addition of skim milk, which showed the highest survival rate.

After 8 weeks at low temperature (4 ℃), the survival rate of the bacteria was measured. When the number of bacteria before and after lyophilization was expressed as 100, all of them were killed in 2 weeks when no skim milk was added. appear.

For the scheme milk concentrations of 5% and 10% (w / v), after 4 weeks of lyophilization, the survival rates were 24.38% and 36.74%, respectively. However, more than 75% of the strains of Scheme Milk 7% (w / v) were maintained for up to 4 weeks of storage, but the number of viable cells continued to decrease with increasing storage period. At 8 weeks of storage, the survival rate was 18.53%.

Agricultural Biotechnology Research Institute KACC91627P 20110104

Claims (11)

Method of freeze-drying Poturadap temperata M1021 (Accession Number: KACC91627P), characterized in that at least one preservative selected from the group consisting of milk, sodium alginate, starch, glucose is added.
The freeze drying method of claim 1, wherein the scheme milk is added at a concentration of 1% to 10% (w / v).
The freeze drying method of claim 2, wherein the scheme milk is added at a concentration of 7% (w / v).
The freeze-drying method of claim 1, wherein the sodium alginate is added at a concentration of 0.5% to 2% (w / v).
The freeze-drying method of claim 1, wherein the starch is added at a concentration of 1% to 7% (w / v).
The method of claim 1, wherein the glucose is added at a concentration of 0.5% to 1.5% (w / v).
The freeze drying method according to any one of claims 1 to 6, wherein the freeze drying is performed at -60 ° C to -80 ° C for 48 to 72 hours.
A freeze-dried powder of the Poturarabdu temperata M1021 strain prepared by the method of any one of claims 1 to 6.
A biopesticide comprising the lyophilized powder of claim 8.
10. The biopesticide according to claim 9, wherein the biopesticide is a pesticide for controlling butterfly neck pests.
The method of claim 10, wherein the butterfly neck pests are beetle moth (Galleria mellonella), Chinese cabbage moth (Plutella xylostella), tobacco moth (Spodoptera litura), moths of the branch (Alcis angulifera), moth pattern leaf moth ( Adoxophyes orana, Persimmon Leaf Moth (Ptycholoma lecheana), Chestnut Leaf Moth (Cydia Kurokoi), Peach Pure Moth (Grapholita molesta), Silver Moth (Lyonetia prunifoliella), Peach Heart Moth (Carposina sasakii), Sparoptera exigua), diaphania indica, Cnaphalocrocis medinalis, Chilo suppressalis, and Helicoverpa armigera.

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