JPH0975012A - Additive for artificial feed for silkworm and artificial feed raw material for silkworm - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a process for producing pheophytin a and/or b and obtain an additive for an artificial feed for silkworm and an artificial feed raw material for silkworm by using pheophytin a and/or b as active components. SOLUTION: A hydrophilic organic solvent solution of chlorophyll is mixed with water in the presence of an activation agent to obtain a mixture having a hydrophilic organic solvent concentration of 65-85wt.%. The mixture is extracted with a non-hydrophilic organic solvent and purified with an extractant to separate pheophytin a and/or b, which are used as active components for an artificial feed for silkworm.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing pheophytin a and / or b which is effective for artificial breeding of silkworms including domestic silkworms and wild silkworms, for example, silkworm, silkworm, eri silkworm, and pheophytin a and / or b. The present invention relates to an additive for artificial feed of silkworms and a raw material for artificial feed of silkworms, which comprises as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, breeding of silkworms has been carried out by using mulberry leaves or mulberry leaf powder, and it has been impossible to breed silkworms and produce cocoons by using other materials as feed. Various studies have been conducted on artificial diets for domestic silkworms, but artificial diets have not been put to practical use in the true sense that mulberry powder is not added. In addition, wild silkworms such as celestial silkworms are generally cultivated outdoors on trees such as Kunugi, Japanese oak, oak, azalea, Kakiyanagi, oak, etc., so breeding cannot be artificially controlled and protected from pests and diseases. Was also difficult. As for the silkworm, artificial feeds containing leaf powders of the above plants are commercially available. However, the cultivation of these feed trees and their management: From the viewpoint of practical breeding, because much labor is required for plant leaf harvesting. It has been desired to develop an artificial feed capable of exhibiting a breeding result which is not inferior to that of live leaves even though it does not contain plant leaf powder.

Next, pheophytins a and b have a structure in which Mg atoms are removed from chlorophyll a and b, respectively. For example, chlorophyll a or b contains concentrated HCl.
It is known that pheophytin a or b is obtained by dissolving it in a 1% acetone solution and leaving it for 1 hour to obtain pheophytin a or b, extracting this with ether, washing with water, transferring to acetone, and removing acetone. (Eg ID
Journal of Agricultural and Food Chemistry (J. Agr. Food Che
m.) 16, p. 81 (1968)). Also, in the same document, an acetone solution of pheophytin a or b and CuCl ₂ or ZnC
from the aqueous solution of l ₂ to Cu or Zn of pheophytin a or b
It is described to obtain a chelate compound and to use it as a green dye for keeping the apparent freshness of foods.

Regarding the pheophytin a, the present inventor has disclosed in Japanese Patent Laid-Open No. 5-130839 that water is added to a methanol extract of mulberry powder, cherry tree leaves, Japanese tea, rice or the like to adjust the methanol concentration to 70 to 80%, and precipitation is performed. The dark oily substance (CHL) obtained by dissolving the substance in petroleum ether and removing the solvent from it shows silkworm rearing results similar to those of mulberry leaves. Chlorophyll a, b and carotenes outer pheophytin a However, it is judged that chlorophyll a and b are effective substances because it is effective to remove pheophytin a and carotene from CHL subsequently, and it is rather effective that pheophytin a is effective. It is a statement that it is not an ingredient. And as a result of subsequent research, it became clear that this was wrong.

Furthermore, the present inventor added the chlorophyll a type by adding an aqueous solution containing an activator such as vitamin C, phenolic acid, phenol aldehyde, and a sulfur-containing compound to a chlorophyll extract containing a hydrophilic organic solvent from a chlorophyll-containing plant or algae. A chlorophyll a-type compound obtained by extracting the compound as a compound with a non-hydrophilic organic solvent has an excellent physiological activity such as growth promotion of silkworms (Japanese Patent Laid-Open No. 5-279368), and the above chlorophyll a The type compound is not a single substance, but it is blue-green (BG), yellow-green (YG),
It can be divided into 6 components showing yellow (Y), yellowish brown (DY), black (BGR) and dark green (DG) (Japanese Patent Laid-Open No. Hei 6)
-197704).

[0006]

DISCLOSURE OF THE INVENTION The present inventor has conducted earnest research with the aim of isolating the active ingredient contained in the above-mentioned chlorophyll a-type compound, and thereby completing an artificial diet for silkworms.

[0007]

Means for Solving the Problems and Embodiments of the Invention The present inventors tried to purify the chlorophyll a-type compound and succeeded in separating it into several fractions by a purification treatment using silica gel, Pheophytin a has a silkworm growth promoting action in the fraction.
The present invention has been completed by clarifying that they are b and b.

That is, the present invention provides: An additive for a silkworm artificial feed, which contains at least one of pheophytin a and b as an active ingredient. 2. A raw material for artificial feed of silkworms containing at least one of pheophytin a and b. 3. A hydrophilic organic solvent solution of chlorophyll and water are mixed in the presence of an activator to adjust the concentration of the hydrophilic organic solvent to 65-8.
5% by weight, followed by extraction with a non-hydrophilic organic solvent, removing the solvent from the extract, and subjecting the concentrate to purification treatment using silica gel to isolate pheophytin a and / or b The method for producing pheophytin a and / or b. 4. The method for producing pheophytin a, wherein the activator is a polycarboxylic acid, a sulfur-containing amino acid, an inorganic acid or a water-soluble salt of a metal. 5. The method for producing pheophytin b, wherein the activator is a polyhydric phenol acid, a polyhydric phenol aldehyde, a sulfur-free amino acid, an oxycarboxylic acid or an oxyaldehyde. It is.

In the present invention, pheophytin a or b
May be synthetic, or a substance containing pheophytin a and / or b, for example, a plant containing pheophytin a and / or b, such as Phaeudonium communis, castor beetle, oak, oak, abemaki, karin, mateba shii, kamiyagi, oak, etc. The crude pheophytin extract may be obtained by crushing, grinding or crushing leaves of deciduous tree or evergreen tree, alfalfa, seaweed, kelp, etc., and pheophytin a and / or b may be obtained by conventional purification means.

Further, from the viewpoint of easy availability, the above-mentioned Japanese Patent Laid-Open No. 5-279368 by the present inventor.
It is also possible to partially use the method described in the issue. That is, a chlorophyll extract is obtained from a chlorophyll-containing plant or algae with a hydrophilic organic solvent, an activator-containing aqueous solution is added to this, and then the extract obtained by extraction with a non-hydrophilic organic solvent is subjected to purification treatment using silica gel. To obtain pheophytin a and / or b.

As the chlorophyll-containing plant or algae,
For example, plant leaves such as mulberry, cherry tree and tea, spinach, rice straw, spirulina, chlorella and the like can be mentioned. These are preferably crushed, ground or crushed before use. Examples of the hydrophilic organic solvent include lower alcohols such as methanol and ethanol, and acetone. Extraction of chlorophyll from chlorophyll-containing plants or algae should be performed at low temperature to room temperature.

Next, an aqueous solution containing an activator is mixed with the hydrophilic organic solvent extract of chlorophyll so that the concentration of the hydrophilic organic solvent becomes 65 to 85, preferably 70 to 80% by weight. Examples of the activator include the following. 1. Ascorbic acid, citric acid, malic acid, succinic acid,
Polycarboxylic acids such as fumaric acid, sulfur-containing amino acids such as cysteine and methionine, and inorganic acids such as hydrochloric acid. 2. Water-soluble salts of metals such as Ni, Mg and Mn, eg NiC
_{l 2, NiSO 4, Ni (} NO 3) 2 · 6H 2 O, MgCl 2, MgB
r ₂ , Mg (CH ₃ COO) ₂ , CuCl ₂ , CuSO _4, etc. 3. Protocatechuic acid, Protocatechu aldehyde,
Polyhydric phenol acids such as gallic acid and polyhydric phenol aldehydes. 4. In addition to oxycarboxylic acids such as glyoxylic acid and glycolic acid, and oxyaldehydes such as glyoxal, non-sulfur amino acids such as glycine, alanine, serine, threonine and 2-aminobutyric acid.

The activator is generally present as an aqueous solution thereof, but those which are soluble in a hydrophilic organic solvent such as protocatechuic acid or gallic acid may be dissolved in a chlorophyll-containing solution. The activator may be about 1.0 to 0.2% by weight of the expected chlorophyll,
The aqueous solution should have a solubility of pH 0.5 to 3.5. or,
When the active agent is classified as 1 or 2 above, pheophytin a is produced abundantly, and when the active agent is classified as 3 or 4 above, pheophytin b is produced abundantly. Furthermore, the use of multiple activators tends to produce pheophytin b.

Next, extraction is performed with a non-hydrophilic organic solvent.
Examples of the non-hydrophilic organic solvent include hexane, chloroform, petroleum ether and the like. At the time of this extraction,
When pigment substances such as carotene and cryptoxanthin are mixed, these pigments can be transferred to and removed from the aqueous layer by adding dioxane. The extract is separated, the solvent is removed therefrom, and subjected to purification treatment.

The purification treatment using silica gel refers to a silica gel as an adsorbent or a purifying means mainly using this, for example, a mixture of an adsorbent other than silica gel such as diatomaceous earth, sucrose and cellulose is appropriately used. Good.
Further, the purification treatment using silica gel is preferably performed by silica gel column chromatography. For example, a silica gel column is loaded with the above extract or its concentrate, and a non-hydrophilic organic solvent such as petroleum ether or chloroform and acetone or methanol are used. Adsorption and elution with a mixed solution of a suitable hydrophilic organic solvent gives a fraction containing pheophytin a and b, from which the solvent can be removed to obtain the target substance.

In the present invention, pheophytins a and b include chelate compounds having a silkworm feeding-inducing action such as Ni or Mg chelate compounds. And that the Mg chelate compound of pheophytin a or b is different from chlorophyll a or b,
Each UV / VIS absorption spectrum has similar characteristics to those of pheophytin a or b, and chlorophyll a
Or b, the former both have the same silkworm feeding-inducing effect as pheophytin a or b, whereas the latter chlorophyll a or b has no such effect. Therefore, it is considered that the Mg chelate compound of pheophytin a or b is different in the coordination position of Mg from chlorophyll a or b, respectively. The chelate compound can be obtained, for example, by the method of ID Jones mentioned above. That is, purified or crude pheophytin a or b
An aqueous solution of a water-soluble metal salts that form chelates, such as NiSO ₄ and MgCl ₂ in acetone solution added, in a stream of N _2, 10
Leave it for -15 hours, extract with petroleum ether, and extract with N
After drying with a ₂ SO ₄ , the solvent is removed and the product can be obtained by purification by sugar column chromatography.

The present invention relates to pheophytin a and / or b
The present invention relates to an additive for a silkworm feed containing as an active ingredient (hereinafter, these may be collectively simply referred to as pheophytin), which includes known feeds for a silkworm such as B ₆ and nicotinic acid amide. , Various vitamins such as calcium pantothenate, sodium ascorbate, β-sitosterol,
A mixture of nutritional components such as inositol, antibacterial substances such as chloromycetin, and antifungal agents such as oxyquinoline may be used. The degree of blending can be appropriately determined, but the pheophytin content may be, for example, 2 to 8% by weight, preferably 1 to 4% by weight.

Further, the present invention relates to a silkworm artificial feed material containing pheophytin. The silkworm artificial feed material of the present invention, although slightly different for domestic silkworms and wild silkworms, known feed materials, such as starch, defatted soybean powder, agar, cellulose powder and the like, but of course this It is not limited to. The content of pheophytin in the feed raw material is 0.9
˜1.4 wt%, preferably 0.6-0.9 wt%.

Pheophytin is more effective in b than in a and stable in both, and does not affect other additives or feed materials at all. Therefore, the additive for silkworm feed of the present invention is also used. It is stable, and the artificial silkworm feed of the present invention and the artificial silkworm feed having a plate shape of an appropriate shape by adding an appropriate amount of water to the feed raw material are also stable and induce feeding to silkworms. And, the production promoting effect can be sufficiently exerted.

Experimental Example 1 As a chlorophyll-containing material, methanol was added to 120 g of dried mulberry leaf powder (sold from the Kyoto Institute of Technology and Textiles Farm) to extract chlorophyll, and the extract was filtered. It was set to 1,100 ml.
An aqueous solution prepared by dissolving 10.0 g of citric acid (CA) in 370 ml of water was added thereto (methanol concentration 75%), and then extracted repeatedly with 250 ml of petroleum ether three times, and the combined extract was dehydrated with sodium sulfate, The solvent was distilled off to obtain a dark brown oily substance (referred to as ML-CA), which was subjected to silica gel thin layer chromatography (TLC) for adsorption (solvent petroleum ether 70: acetone 30),
The silica gel layer was scraped off for each colored part, extracted for each part with the same solvent as above, the extract was concentrated and subjected to silica gel TLC again to obtain four colored adsorption fractions,
After extraction with a solvent from each fraction, the solvent was removed to obtain four components (from the upper part of TLC adsorption, 1, 2 and
UV / VIS absorption maximum values were measured for (hereinafter, referred to as 3, 4 and the same). The results are as follows.

[0021]

[Table 1] From this result, the component of fraction 1 is pheophytin a
(Kozo Hayashi, published by Yokendo: plant pigments, page 463 (1988), values 667.0, 609.5, 560.0, 534.
0, 505.0, 471.0, 408.5). The IR was also in agreement with pheophytin a (data not shown).

Experimental Example 2 Chlorella tablets as a chlorophyll-containing material (CL: trade name: Noachlorella; sold by KK Kyoto Nutrition Research Institute) 2
After crushing 0 g, ethanol was used as a hydrophilic organic solvent, and 18 g of ascorbic acid (VC) was used as an activator, which was extracted with petroleum ether in the same manner as in Example 1. The extract was dehydrated and then concentrated to darkness. Brown oil (this is CL-V
850 mg). This CL-VC was subjected to silica gel TLC (solvent, petroleum ether 7) as in Experimental Example 1.
0: Acetone 30), and UV / VIS spectra of the obtained four components (referred to as 1, 2, 3, and 4 from the upper part of TLC adsorption) were measured. The results are as follows.

[0023]

[Table 2] From this result, the component of fraction 1 is pheophytin a
It became clear that The IR was also in agreement with pheophytin a.

Experimental Example 3 Commercially available spinach (HO) was dried and then pulverized 2
Methanol was added to 7 g to extract chlorophyll, the extract was filtered, and methanol was added to the filtrate to bring the total amount to 640 ml.
And To this, 1.7 g of gallic acid (GA) powder was added, 270 ml of water was further added, and the mixture was well stirred to dissolve GA to complete the activation (methanol concentration 70%). It was then extracted twice with 250 ml petroleum ether. The extracts were combined, dehydrated with sodium sulfate, and then the solvent was distilled off to obtain 1,250 mg of a dark brown oily substance (HO-GA), which was treated with silica gel TLC (solvent, petroleum ether 70: Acetone 30), scrape off the silica gel layer for each colored portion, extract this with the same solvent as above, concentrate the extract, and subject it again to silica gel TLC, and UV / VIS
Absorption maximum value is 656, 600, 556, 522, 43
A component of 3, 412, 369, 328 nm (measurement solvent n-hexane: acetone = 9: 1) was obtained, and the reference value was 655.
5,600,556.5,522,432.5,412,
370, 324.5 nm (measurement solvent ether) [Helvetika Kimika Acta (Vol. 63), 1060]
Page (1980)], and this is pheophytin b.
Was found. The IR value was also in agreement with the literature value.

Experimental Example 4 Spirulina (SP, trade name: Spirulina "family drug", KK marketed drug) as tablets containing chlorophyll 7.0 g
, Methanol as a hydrophilic organic solvent, and 8.0 g of citric acid (CA) and cysteine (CS) as activators.
Was a dark brown oily substance obtained by the same procedure as in Experimental Example 1 except that petroleum ether was used as the non-hydrophilic organic solvent and the methanol concentration was 70%.
). This SP-CA-CS was subjected to silica gel TCL in the same manner as in Experimental Example 1 to obtain fractions, and each fraction was again subjected to silica gel TCL to separate each component 1 and 2 into two fractions (each. 1a, 1b, 2a, 2b). The UV / VIS absorption maximum values of the obtained 6 components were measured and the following results were obtained.

[0026]

[Table 3] As a result, it was revealed that the component of fraction 2a was pheophytin b. The IR was also in agreement with pheophytin b.

Experimental Example 5 90 parts by weight of mulberry leaf dry powder (ML) was extracted with methanol, the extract was filtered, and methanol was added to the filtrate to make 350 parts by volume, which was used as a hydrophilic organic solvent for chlorophyll. did. A 10% NiCl ₂ aqueous solution was prepared, and citric acid (CA) was added to this to make a 1.0% aqueous solution. This aqueous solution was added to the above methanol solution to adjust the methanol concentration to 70%, extraction was performed with petroleum ether, and the solvent was removed from the extract to obtain 700 mg of an oily substance (hereinafter referred to as ML-CA-Ni). ML-CA-Ni was subjected to silica gel TLC in the same manner as in Experimental Example 1, and the UV / VIS absorption maximum value (measurement solvent n-hexane: acetone = 9: 1) was 6.
56, 600, 556, 522, 433, 413, 37
A component of 1,325 nm was obtained, and this value was in agreement with the literature value of pheophytin b.

Experimental Example 6 To 80 ml of the acetone solution containing ₂₀₀ mg of ML-CA obtained in Experimental Example 1 was added 20 ml of an aqueous solution containing 750 mg of MgCl ₂ and 80 mg of ascorbic acid (to prevent oxidation), and the mixture was stirred well and then in a stream of N _2. It was left for 10 hours. The reaction product was extracted with petroleum ether, the extract was dried over NaSO ₄ ,
After filtration and concentration of the filtrate, purification by sucrose column chromatography gave 140 mg of a green substance. UV / VI of this product
The maximum S absorption value was in agreement with pheophytin a, and it was confirmed from the flame reaction that Mg was contained.

Example 1 Breeding test of silkworms The basic feed materials used are as follows.

[Table 4]

As the varieties of silkworms, "Kinshu x Kanewa" or "Harumine x Kanetsuki" is used, the silkworm species (disperse) are washed with 70% methanol, and the liquid is wiped off with sterilized dry filter paper. Put the obtained seeds in a petri dish, place this in a casten, introduce formalin gas into the casten, leave it for 1 day, take out the petri dish from the casten, cover it, leave it at 25 ° C for 10 days, and hatch it Was used for sweeping. 5 parts by weight of the above basic feed material (1) Pheophytin a 0.075,
(2) b0.067, (3) Pheophytin aMg chelate compound 0.06, or (4) Pheophytin a0.05.
And 0.02 parts by weight of pheophytin b. To each of 5 parts by weight of water, 8.2 parts of water was added and kneaded well to form discs (thickness 7 mm, diameter 7 cm). Sterilization was performed at 40 ° C for 40 minutes, and after cooling, hatched ant silkworms were put in and swept. Place the rearing container in a constant temperature bath at 28 ° C for rearing, clean up, and remove the fish and stop eating on the 7th day.
On the 8th day, the body weight of the silkworm, 3rd instar, was measured. Table 4 shows the results of the breeding results obtained.

[0031]

[Table 5]

Example 2 Breeding test of silkworms The basic feed materials used are as follows.

[Table 6] Pheophytin a or b0.
Mixing 06 parts by weight, adding 28.0 parts by weight of water and kneading well, 40 g is made into a strip shape (thickness 5.0 mm x width 15.0 mm x length 40.0 mm) and put in a 100 ml triangular Kolben.
It was sterilized by heat in an autoclave. After cooling, the eggs were sterilized (sterilization was carried out by removing the film with a proteolytic enzyme prosthesis and then immersing it in 3% formalin water for 20 minutes).
Put 10 pieces each, and this is 30 ℃ for 1st and 25 ℃ for 2nd
The animals were continuously sterilized without changing the feed. After the second instar, the body weight of the sleeping silkworm was measured. Table 5 shows the results.

[0033]

[Table 7]

Example 3 Rearing test of the late 5th era of silkworm (after 3 days) The composition of the feed raw material used is as follows.

[Table 8] Using the raw material, silkworms after 5th and 3rd day were bred by artificial feed processed and molded by a conventional method. The results are shown in Table 6. The cocoons obtained were 5th and 3rd day after the artificial feed prepared by using the mulberry leaf powder in an amount of 4 times without the presence of pheophytin b in the above feed material. In comparison with the cocoons obtained from the silkworms reared, the quality was not inferior.

[Table 9]

[0035]

EFFECTS OF THE INVENTION Pheophytins a and b exhibit excellent silkworm growth effects by themselves. Therefore, an artificial feed prepared from a feed material containing the same does not contain expensive mulberry powder at all. The silkworm eats this smoothly and grows. However, considering the quality of the obtained cocoons and the like, it is preferable that mulberry powder is also present. Therefore, about 30 to 10% by weight of mulberry powder has been conventionally used in feed raw materials. The consumption of feed increased dramatically after the silkworm reached the fifth age, and the amount of mulberry powder digested also increased, which increased the cost of artificial rearing of silkworms.
Practical application has become difficult. According to the present invention, it is possible to reduce the amount of mulberry powder used in artificial feed for, for example, domestic silkworms by at least 1/4 of the conventional amount, and the economical effect thereof is extremely large.

Claims (5)

[Claims] 1. An additive for artificial feeding of silkworms containing at least one of pheophytin a and b as an active ingredient. 2. A silkworm artificial feed material comprising at least one of pheophytin a and b. 3. A solution of chlorophyll in a hydrophilic organic solvent and water are mixed in the presence of an activator to adjust the concentration of the hydrophilic organic solvent to 65 to 85% by weight, followed by extraction with a non-hydrophilic organic solvent, A method for producing pheophytin a and / or b, characterized in that the concentrate obtained by removing the solvent from the extract is subjected to a purification treatment using silica gel to isolate pheophytin a and / or b. 4. The method for producing pheophytin a, wherein the activator is a polycarboxylic acid, a sulfur-containing amino acid, an inorganic acid or a water-soluble salt of a metal. 5. The method for producing pheophytin b, wherein the activator is a polyhydric phenol acid, a polyhydric phenol aldehyde, a sulfur-free amino acid, an oxycarboxylic acid or an oxyaldehyde.