JP2002101869A - Agent for protecting from dehydration stress - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent for protecting from a dehydration stress, which does not deteriorate the flavor of a material to which the agent is added, and has a high protection effect even in a small amount. SOLUTION: This agent for protecting from the dehydration stress comprises sericin as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dehydration stress protectant which protects microorganisms, plants, animal cells, enzymes and the like from dehydration stress, and particularly to the fields of genetic engineering, biochemical industry, food industry, pharmaceutical industry and the like. The present invention relates to a dehydration stress protective agent having high utility in the present invention.

[0002]

2. Description of the Related Art Drying, low-temperature, freezing, and salt stress, which are typical stresses for cells, are dehydrated (water deficiency) in the cells.
They have a common aspect in bringing state,
Dehydration (water deficiency) is called stress. Especially "frozen"
Although the range of use is increasing for storage and preservation of microorganisms, cells, animal tissues, and the like, the cells suffer from dehydration stress during the process, and thus have a problem that the survival rate after lysis is reduced.

For example, preservation of microorganisms and cells of excellent strains and preservation of Escherichia coli into which a specific gene has been introduced are important in genetic engineering, and improving the survival rate of Escherichia coli after freezing and thawing has become an issue. ing. Therefore, in order to improve the survival rate of cells after freezing and thawing, a method of freezing cells with a solution containing a cryoprotectant has been studied.

A commonly used method is to use a medium containing 10 to 50% of animal serum as a protective component to prepare a composition for cryopreservation. However, animal serum is extremely expensive, which increases the storage cost when cryopreserving a large number of cell lines in large quantities, and studies have been made on additional components to replace serum. And glycerol, vitamin Es,
Methyl cellulose, trehalose and the like have been found to be effective. However, these methods also have problems such as improvement of the survival rate and unsuitability of the cryoprotectant. Japanese Patent Application Laid-Open No. 5-103586 also discloses a method for suppressing freeze-drying denaturation of oligosaccharides having a degree of polymerization of 3 to 10 and a saccharide mainly composed of reduced products thereof, in order to suppress denaturation of proteins caused by freezing of fish meat and the like. The use of a substance is disclosed. However, the flavor of the added food was also impaired, and it was difficult to add an amount sufficient to maintain the quality.

[0005]

That is, an object of the present invention is to provide a dehydration stress protective agent having a high protective effect even in a small amount without impairing the flavor of the additive.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that sericin, which is one of proteins, or a hydrolyzate thereof is effective as a dehydration stress protective agent, and reached the present invention. Things.

That is, the present invention is a dehydration stress protectant comprising sericin as an active ingredient. Preferably, sericin is natural sericin extracted from cocoons or raw silk. Or
Sericin is a hydrolyzate of natural sericin.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Sericin used in the present invention can be obtained from silkworm gland tissue, from cocoons or raw silk, but high-purity sericin (including hydrolyzate) derived from cocoons or raw silk can be easily obtained. Because you can get it,
Usually, it is preferably used. Sericin of the present invention, depending on the intended use, as it is solid, or dissolved in a solvent,
Preferably, an appropriate amount can be dissolved in water and used in an appropriate form such as an aqueous solution.

It can be used in the fields of genetic engineering and biochemical industry, cosmetics, foods, food additives, external medicines, quasi-drugs, pharmaceuticals and the like, as well as conventional dehydration stress protective agents. For example, the amount of sericin added to cosmetics, external medicines, quasi-drugs and the like is usually 0.1 to 20% by weight, preferably about 0.5 to 5% by weight. Sericin is usually added in foods in an amount of about 0.1 to 30% by weight, preferably about 0.5 to 10% by weight. However, sericin has no toxicity and is also excellent in water solubility, so that even if added or ingested in a large amount, no particular problem occurs, and the amount of sericin is not particularly limited. This is one of the features of the present invention. Sericin is not only toxic, but also tasteless and odorless, so that it does not impair the flavor of the added food or the like.

[0010] Dosage forms for cosmetics and external medicines include creams, emulsions, foundations, packs, lotions, gels, solutions, and sticks.

[0011] In addition, these may include appropriate components such as oils,
Humectants, thickeners, preservatives, emulsifiers, pigments, pH adjusters,
Other medicinal ingredients, ultraviolet absorbers, fragrances and the like may be added. It can also be administered orally as a pharmaceutical. Although the dose in this case is not particularly limited, for example, 10 mg to 100 g
Per day.

[0012]

Next, the present invention will be described in detail with reference to examples.

(Method for Producing Sericin Powder) 1 kg of silk fabric made of raw silk was put in 0.2% aqueous sodium carbonate (pH 11).
-12) Treatment in 50 l at 95 ° C. for 2 hours to extract sericin hydrolyzate. The obtained extract was treated with an average pore size of 0.
After filtering through a 2 μm filter to remove aggregates, the filtrate was dialyzed using a dialysis membrane having a molecular weight cutoff of 3500 to remove permeated substances, and then fractionate non-permeated substances to obtain a concentration of 0%. A clear and colorless sericin hydrolyzate extract of 0.2% was obtained. The extract was concentrated by an evaporator to a sericin concentration of about 2%, and then lyophilized to obtain 100 g of a hydrolyzate of sericin having a purity of 90% or more.

[0014]

Example 1 Cryoprotective Effect of Sericin on Cells 1 E. coli JM109 strain was inoculated into 5 ml of M9 + 2% casamino acid liquid medium and cultured with shaking at 37 ° C. overnight. The culture solution was dispensed into Eppendorf tubes in an amount of 80 μl each, and a sericin solution was added as follows. In control experiments, only sterile water was added. <Frozen as 2% sericin solution> Culture solution 80μl 10% sericin solution 20μl (final concentration 2%) <Frozen as 0.5% sericin solution> Culture solution 80μl 2.5% sericin solution 20μl (final concentration 0.5%) <0% sericin solution Frozen (control)> Culture medium 80 μl Sterile water 20 μl To measure the number of viable cells before freezing (day 0), dilute each sample 10 ⁴ , 10 ⁵ , 10 ⁶ with sterile water and add 100 μl L
Plated on B agar medium. After overnight culture at 37 ° C,
The number of colonies that grew was counted. The rest of each sample was frozen at -30 ° C. After freezing for one day, the mixture was left to stand at room temperature for 10 minutes to thaw, diluted and plated on 100 μl of LB agar medium as in day 0. After culturing overnight at 37 ° C., the number of grown colonies was measured, and the survival rate of the cells was calculated on the basis of day 0 as 100%. The results are shown in FIG.

As a result, as a control, Escherichia coli suspended in sterilized water and subjected to freezing treatment had a survival rate of 16.7% on the first day, whereas sericin was added to a final concentration of 0.5%. The survival rate when frozen in water was 43.0%. The survival rate when sericin was added to a final concentration of 2% and frozen was 44.4%.

[0016]

Example 2 Cryoprotective effect of sericin on cells 2 Escherichia coli JM109 strain was inoculated into 5 ml of M9 + 2% casamino acid liquid medium and cultured with shaking at 37 ° C. overnight. Dispense 100 μl of the culture solution into Eppendorf tubes and centrifuge (10,000 rpm ×
3min). The culture supernatant was removed, and the cells were treated with 100 μl of a 2% sericin solution, 100 μl of a 0.5% sericin solution, and 100 ml of sterilized water.
Each was resuspended in μl. Hereinafter, similarly to Method I, -30
The survival rate after freezing at 5 ° C. for 5 days was calculated. The results are shown in FIG.

As a result, as a control, E. coli suspended in sterilized water and subjected to freezing treatment exhibited a survival rate of 25.0% on the fifth day.
In contrast, the viability when suspended in a 0.5% sericin solution and frozen was 46.3%. In addition, the viability when suspended in a 2% sericin solution and frozen was 39.9%.

[0018]

Example 3 Cryoprotective Effect of Sericin on Cells 3 Budding yeast S288C was inoculated into 5 ml of SD liquid medium and incubated at 30 ° C. for 2 hours.
Shaking culture was performed for a day. The culture solution was dispensed into Eppendorf tubes in an amount of 100 μl each, and the cells were collected by centrifugation (6,000 rpm × 3 min). Remove the culture supernatant, 100% 2% sericin solution
The cells were resuspended in 100 µl of sterilized water. Hereinafter, the survival rate after freezing at −30 ° C. for 5 days was calculated in the same manner as in Method I. The results are shown in FIG.

As a result, as a control, the viability of the yeast suspended in sterile water and subjected to the freezing treatment was 47.7% on the fifth day, whereas the yeast suspended in the 0.5% sericin solution was frozen. The survival rate was 65.8%. In addition, the viability when suspended in a 2% sericin solution and frozen was 88.0%.

[0020]

Example 4 Cryoprotective Effect of Sericin on Enzyme 1 LDH Cryoprotective Activity Test Sericin was used to measure the protective activity of the enzyme against inactivation by freeze-thawing. Lactate dehydrogenase (LDH) was used as a model enzyme. Lactate dehydrogenase (LDH) is an enzyme that works in glycolysis to produce L-lactic acid from pyruvate, and is known to be sensitive to freezing and easily deactivated by freeze-thaw treatment. . (K. Goller, EGalinski J
ournal of Molecular Catalysis B: Enzymatic 7 (1
999) 37-45) The reaction of lactate dehydrogenase (LDH) is shown below. (reaction)

(Preparation of enzyme solution) Commercial LDH [5,000 U / ml]
(From Oriental Yeast, from Pig heart), 50mM
The mixture was diluted with a potassium phosphate buffer to prepare an enzyme solution of about 250 units. The prepared enzyme solution was dialyzed overnight at 4 ° C. to completely remove ammonium sulfate and the like contained in a commercially available enzyme solution. The dialyzed enzyme solution was diluted with a 50 mM potassium phosphate buffer to prepare about 4 units of an LDH solution.

(Measurement of LDH activity) After mixing a potassium phosphate buffer and an LDH solution and preincubating at 25 ° C, Na-Pyruvate and NADH were added quickly, and the change in absorbance at 340 nm was measured using a spectrophotometer [Beckman, DU640]. ] For 5 minutes. The composition of the reaction solution for LDH activity measurement and the measurement conditions of the spectrophotometer are shown below (Reaction solution composition) (Measurement conditions) Wavelength; 340 nm, optical path length; 1 cm, temperature; 25 ° C

The activity of LDH was determined from the change in NADH (the change in absorbance at 340 nm) according to the following formula. (Calculation formula) (ΔA / min · V · D) / (ε · d · v) = IU / ml ΔA / min = Absorbance change per minute at 340 nm V = final liquid volume (3.17 ml) D = final Dilution ratio Molecular absorption coefficient of NADH at ε = 340 nm (6.3 × 10 ³ l ·
mole ^-1 · cm ^-1 ) d = optical path length (1 cm) v = enzyme solution volume (0.02 ml)

(Measurement of LDH cryoprotective activity of sericin) Sericin was added to the LDH solution (about 4 units / ml) prepared by the above method so that the concentrations became 0.01% and 0.05%. As a control, bovine serum albumin (BSA) used as a protein cryoprotectant [Sigma, Albumin bovinefraction V]
Added at a concentration of 0.01% and 0.1%, or not added (potassium phosphate buffer only)
Was prepared. Samples of the prepared LDH solution are summarized below. LDH [4unit / ml] LDH [4unit / ml] + 0.01% sericin LDH [4unit / ml] + 0.05% sericin LDH [4unit / ml] + 0.01% BSA LDH [4unit / ml] + 0.05% BSA

Next, 100 μl of each prepared sample was dispensed into a 1.5 ml test tube and frozen with liquid nitrogen for 1 minute. Then, melt at 30 ° C for 5 minutes, and use LDH as described above.
Activity was measured. The LDH activity before freezing was set to 100% for each sample, and the remaining activity of LDH after repeated freezing / thawing treatment was determined. FIG. 4 shows the results.

By repeated freezing / thawing, the LDH residual activity of the sample to which sericin was not added was remarkably reduced, and became approximately 3% after eight freeze-thaw treatments, whereas the sample to which sericin was added was not used. Had about 80% residual activity.

[0027]

As described above, sericin is effective in relieving stress when cells and enzymes are frozen, and can utilize the excellent cryoprotective activity of sericin against various cryoprotectants.

[Brief description of the drawings]

FIG. 1 is a graph showing the cryoprotective effect of sericin on cells (Escherichia coli) of Example 1. (Graph 1)

FIG. 2 is a graph showing the cryoprotective effect of sericin on cells (Escherichia coli) of Example 2. (Graph 2)

FIG. 3 is a graph showing the cryoprotective effect of sericin on cells (yeast) of Example 3. (Graph 3)

FIG. 4 is a graph showing the cryoprotective effect of sericin on the enzyme of Example 4. (Graph 4)

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/42 A61K 47/42 (C12N 1/04 (C12N 1/04 C12R 1:19) C12R 1:19 ) (C12N 1/04 (C12N 1/04 C12R 1: 645) C12R 1: 645) F term (reference) 4B050 CC07 KK18 KK20 4B065 AA26X AA72X BD09 BD12 BD39 BD42 BD50 CA42 4C076 BB31 CC18 EE41 FF57 4C083 AD451 AD452 CC02 EE10 4H011 BB19 BB23 CA04 CB08 CB14 CD03 CD06 DH11

Claims (3)

[Claims] 1. A dehydration stress protectant comprising sericin as an active ingredient. 2. The agent for protecting dehydration stress according to claim 1, wherein the sericin is natural sericin extracted from cocoons or raw silk. 3. The method according to claim 1, wherein the sericin is a hydrolyzate of natural sericin.