JP2815383B2 - Production method of silkworm body fluid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a silkworm body fluid as a cell culture additive that has an insect cell growth activity and has good preservability.

[Conventional technology]

Since silkworm varieties are well preserved, it is possible to obtain a homogenous body fluid, and since the body fluid is also known to promote cell proliferation, it is promising as a culture additive derived from insects. However, when the silkworm's body fluid is collected by cutting the silkworm's feet,
Immediately, the oxidation reaction by phenol oxidase starts, causing browning and the production of substances harmful to cells such as dopaquinone. Therefore, at present, in order to obtain silkworm body fluid, after cutting the silkworm's feet and collecting the body fluid little by little, heat treatment is performed quickly to avoid oxidation reaction by enzymes, then centrifugation is performed, and the supernatant is added to culture We use as thing. But,
In this method, it is difficult to cut the silkworm's feet, and it requires heating under certain conditions immediately after collecting the body fluid, so it is difficult to process a large amount of it. There were difficulties such as being done.

[Problems to be solved by the invention]

The present invention solves the above-mentioned problems in the prior art, and suppresses the oxidation reaction of silkworm humor temporarily or for a long period of time, so that it has good preservation properties and has a cell culture additive having an insect cell growth action. An object of the present invention is to provide an efficient method for producing a silkworm body fluid.

[Means for solving the problem]

Under these circumstances, the present inventors have added antioxidants to the silkworm body and conducted a study focusing on its preservability and its effect on cultured cells. As a result, the antioxidant was added to the silkworm body and mixed. After grinding, the supernatant is collected and used as silk humor, which, like the silk humor cut off from the silkworm's feet, inhibits the oxidative reaction of the silk humor for a long time, and furthermore, the cell proliferation action The present invention was also found to give good results.

That is, the present invention provides a method for producing a silkworm body fluid comprising adding an antioxidant to a silkworm body itself or a frozen or vacuum freeze-dried substance, mixing, grinding, and centrifuging to collect a supernatant. Is the way.

In the present invention, in order to produce a silkworm body fluid, the silkworm may be a living silkworm itself, or a frozen silkworm or a vacuum freeze-dried silkworm can be used.

Next, an antioxidant is added to the silkworm body itself or a frozen or vacuum freeze-dried one, followed by mixing and grinding.

The antioxidant is added to the silkworm body fluid as a phenol oxidase inhibitor and can be used alone or in combination of two or more such as L-ascorbic acid, L-ascorbate and thiosulfate.

It is desirable that the antioxidant is prepared in a phosphate buffer solution, contained in the prepared solution, and added to the silkworm body.

At this time, the phosphate buffer containing the antioxidant is preferably not more than 10% of the weight of the silkworm living body, and the concentration range of the antioxidant to be contained in the silkworm body fluid is as follows. The higher the concentration, the longer the time during which the oxidative reaction of the body fluid is suppressed, but since the oxidative reaction does not occur at a certain concentration or more, the amount of L-ascorbic acid or L-ascorbate is 1 to 1 with respect to the silkworm body fluid. In the case of thiosulfate, it is preferably in the range of 0.05 to 0.2%.

In adding, mixing and grinding a phosphate buffer solution containing an antioxidant to the silkworm body, it is desirable to grind while cooling the vessel of the grinding machine so that the temperature of the silkworm fluid does not rise.

Then, the ground material is centrifuged and the supernatant is collected to obtain silkworm body fluid.

The silkworm body fluid obtained by such a production method may be frozen and stored as it is, or may be dried and stored by a freeze-drying method. The body fluid obtained in this way is, for example, 60
After heat treatment at 15 ° C for 15 minutes, freeze-drying or cryopreservation enables longer-term storage.

In addition, the silkworm body fluid obtained by the production method of the present invention may be preferably used as a cell culture additive after thawing or dissolving with water, and the oxidation reaction by phenol oxidase is suppressed.

Example 1 Sodium thiosulfate was added to a phosphate buffered saline solution containing 75 mM sodium monohydrogen phosphate, sodium dihydrogen phosphate, PH 7.2, and 2.75 mM sodium chloride so that the concentration of sodium thiosulfate became 1%. A phosphate buffer was prepared.

28 ml of the phosphate buffer solution prepared as described above was added to 300 silkworm living bodies aged 5 days and 6 days after mixing, and the mixture was ground for 1 minute while cooling the container with a grinder.
Next, the ground material was centrifuged at 5 ° C. at 10,000 rpm for 15 minutes, and the supernatant was collected to obtain 250 ml of a silkworm body fluid containing a buffer solution. This silkworm body fluid was immediately dried by a freeze dryer to obtain 21.3 g of silkworm body fluid powder.

It took about 24 hours to obtain silkworm body fluid powder from body fluid collection, during which time no browning reaction occurred. In addition, 0.1 g of the silkworm body fluid powder was dissolved in 1 ml of pure water, but the browning reaction could be suppressed for 8 hours or more. It was clear that browning due to oxidase was suppressed.

Example 2 A phosphate buffer solution containing an antioxidant was prepared by adding L-ascorbic acid to the same phosphate buffer solution as in Example 1 so that L-ascorbic acid would be 2.0%.

Next, 22 ml of the phosphate buffer prepared above was added to -25 ° C in advance.
10 g of dried silkworm body obtained by freezing at 10 ℃ and vacuum drying at 10 ℃
(Approximately 11 heads) while grinding for 1 minute with a grinder while cooling
The supernatant was collected by centrifugation at 10,000 rpm for 15 minutes to obtain 15 ml of a body fluid, which was stored at 4 ° C.

Next, using the Bm strain (cultured cell line derived from silkworm), based on a Grace insect medium containing 5% fetal bovine serum, the above silkworm body fluid was heated at 60 ° C. for 15 minutes and centrifuged. The growth of cells was observed when 3% of the supernatant from which the precipitate was removed was added.

As a control, a control group in which cell growth was attempted in an insect medium of Grace containing only 5% fetal bovine serum without adding silkworm body fluid as a control was used.

For comparison, from the same number of silkworms as the test plot, 6 ml of the silkworm body fluid produced by the method of using the phosphate buffer containing an antioxidant in the same manner and cutting the silkworm's feet to collect the silkworm body fluid was used. Cell growth was observed in the same manner as in the test and control groups.

The results are shown in Table 1. The silkworm body fluid produced by the method of the present invention had a cell growth effect and was comparable to that obtained by cutting and collecting the silkworm's feet.

[Effects of the Invention] The production method of the present invention is for grinding silkworms themselves or frozen or vacuum freeze-dried and collecting silkworm body fluid. Compared with the above, the yield is remarkably good, and it can be manufactured in a labor-saving manner.

In addition, the addition of an antioxidant suppresses the oxidation reaction, so that it is possible to obtain a large amount of bodily fluids that are comparable to conventional ones as culture additives, and that lyophilization and other treatments are possible. Therefore, there is an advantage that silkworm fluid can be distributed and industrially used.

Claims (1)

(57) [Claims] 1. A method for producing a silkworm body fluid, comprising adding an antioxidant to the silkworm body itself or a frozen or vacuum freeze-dried substance, mixing, grinding, and then centrifuging to collect a supernatant. Method.