JPH0587236B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for extracting intracellular active ingredients, and more particularly to a method for extracting intracellular active ingredients from filamentous fungi using chitosanase. Filamentous fungi, along with yeast, have been used for a long time in the production of foods, medicines, etc. through fermentation methods. There is a growing demand for methods for extracting active ingredients more efficiently. [Prior art] Chemical, physical, and enzymatic methods (Japanese Unexamined Patent Application Publication No. 1989-1999) have been used to crush and dissolve cell walls as a pretreatment for extracting active ingredients from filamentous fungi.
75281, 60-164477, Special Publication No. 61-7318)
It has been known. [Problems to be Solved by the Invention] However, unlike bacteria such as Escherichia coli, filamentous fungi have solid cell walls, and therefore it is not easy to crush and dissolve them. Therefore, the high heat treatment that has been proposed in the past,
Decomposition of cell walls by strong acid, strong alkali treatment, solvent treatment, etc., ultrasonic treatment, mechanical treatment (ball mill,
Methods for disrupting cell walls, such as those using homogenizers, bantam mills, etc., do not sufficiently decompose or disrupt the cell walls, and inevitably lead to the destruction of active components within the microbial cells. Under these circumstances, methods that use enzymes to degrade cell walls are attracting attention. Although enzymes originating from various microorganisms are known to be involved in the lysis of filamentous fungal cell walls, it is difficult to say that these enzymes are necessarily satisfactory. In particular, when extracting the intracellular active ingredients from filamentous fungi that have chitosan in their cell walls, commercially available cellulase, chitinase, β-
When cell wall lytic enzymes such as 1,3-glucanase are used, extraction is not carried out efficiently, which poses a major obstacle to the utilization of the active ingredients inside the filamentous fungi. The present invention relates to a method for efficiently extracting active components inside filamentous fungi using the cell wall lytic enzyme chitosanase. [Means for Solving the Problems] As a result of repeated studies on a method for efficiently extracting the active components inside filamentous fungi, the present inventors found that they can extract them under mild conditions by using the cell wall lytic enzyme chitosanase. The present inventors have discovered that the active ingredients within the microorganism can be efficiently extracted without causing consumption and destruction of the active ingredients, leading to the completion of the present invention. That is, the present invention relates to a method for extracting intracellular active ingredients from a filamentous fungus having chitosan in its cell wall, which is characterized by using a cell wall lytic enzyme, chitosanase. As the chitosanase used in the present invention, any chitosanase derived from a microorganism that produces chitosanase can be used regardless of its origin. Furthermore, the microorganisms may originate from variants or mutant strains of these microorganisms. For example, there is chitosanase described in Japanese Patent Application No. 61-207780. As chitosanase, the culture solution of the enzyme-producing bacteria or its culture filtrate may be used, and furthermore, the filtrate may be dialyzed after salting out ammonium sulfate, or the crude enzyme obtained by adding a solvent such as ethanol or acetone. Various forms can be used, such as a powder, a solution of the powder in water or a buffer solution, and an active fraction fractionated by gel filtration, ion exchange, or the like. Next, the filamentous fungi may be those that have chitosan in their cell walls, such as filamentous fungi belonging to the order Mucorales, specifically filamentous fungi belonging to the genus Rhizopus, genus Mucor, genus Mortiherella, etc. Examples include filamentous fungi belonging to the order Moriliales, such as filamentous fungi belonging to the genus Trichoderma. In carrying out the present invention, first, filamentous fungi are cultured on a suitable medium, such as a solid medium (agar medium), to form spores, and the spores are transferred to a liquid medium to proliferate the fungi. Accumulate useful active ingredients in the fields of food, medicine, etc. The obtained bacterial cells are suspended in an aqueous solution or a buffer solution either as they are or after being washed, and cell wall lytic enzymes containing chitosanase prepared in advance are added thereto in any order, and the cells are heated at 25 to 45°C, preferably 30 to 40°C. So 4~
Shake gently for 12 hours, preferably 6-8 hours. Here, any known cell wall lytic enzymes other than chitosanase can be used. For example, enzymes such as β-1,3-glucanase, cellulase, and chitinase may be used alone or in combination with chitosanase. can.
Note that the origin of these enzymes does not matter. [Examples] Next, the present invention will be explained using Examples, but these are not intended to limit the scope of the present invention in any way. Example 1 Mortierella Isabelina
isaberina) IFO 8187 was cultured at 25° C. for one week on a MY agar medium (consisting of 3 g of yeast extract, 5 g of polypeptone, 3 g of malt extract, 10 g of glucose, 1 g of water, and 20 g of agar) to form spores.
Add physiological saline to this to prepare a spore suspension,
Add this spore suspension to 200 g of glucose per 1 water.
Urea 6g, KH ₂ PO ₄ 9g, MgSO ₄ 7H ₂ O 1g,
NaC0.3g, malt extract 0.6g, yeast extract 0.6g,
Peptone 0.3g, _FeSO4・_7H2O0.03g , _CaC2・
2H ₂ O0.004g, CuSO ₄・5H ₂ O0.0006g, ZnSO ₄・
The cells were transplanted into a liquid medium consisting of 0.003 g of 7H ₂ O, 0.003 g of MnC ₂ 4H ₂ O, and PH 5.5, and cultured with aeration at 30°C for 5 days to accumulate fats and oils containing γ-linolenic acid within the bacterial cells. . Next, centrifugation and washing (0.2M acetic acid buffer) were repeated twice from this culture broth to obtain wet bacterial cells. 1 kg of this wet bacterial body was added to 0.2 M acetic acid buffer (PH
5.6) Suspend Bacillus pamilus BN- in 5000ml.
262 (FERM P-8814) (Japanese Patent Application No. 61-207780) (8×10 ³ U) was added and gently stirred at 35° C. for 7 hours. After the reaction is complete, the bacterial cells are separated by suction filtration, and the cells are separated under vacuum.
Drying was performed at 40°C for 12 hours. This dried bacterial body is
- 2500 ml of hexane was added and stirred at room temperature for 3 hours to extract fats and oils from the bacterial cells into n-hexane. Furthermore, this n-hexane solution was concentrated under reduced pressure.
105 g of γ-linolenic acid-containing fat and oil was obtained. As a comparative example, commercially available cell wall lytic enzymes cellulase (1.6×10 ⁸ U), chitinase (manufactured by Sigma) (4×10 ⁴ U) and β-glucuronidase (manufactured by Sigma) (4×10 ⁶ U) were used as comparative examples. ), the yield of γ-linolenic acid-containing oil and fat was 63 g, and when cellulase (1.6 × 10 ⁸ U) and β-glucuronidase (4 × 10 ⁶ U) were added, the yield was 48 g. When no enzyme was added, the amount was 35 g (see Table 1). Example 2 Chitosanase (4×
10 ³ U) and chitinase (manufactured by Sigma) (4×10 ² U)
When used in combination with γ-linolenic acid, 124 g of γ-linolenic acid-containing fat and oil was obtained. The chitosanase crude enzyme powder used in this example was prepared as follows. The culture solution of Bacillus pamilus BN-262 (FERM P-8814) was centrifuged (6000 rpm) to remove bacterial cells and obtain a culture supernatant. Ammonium sulfate was added to this to make it 80% saturated, and the resulting salting out precipitate was thoroughly dialyzed with cellophane tubes and lyophilized to obtain a crude enzyme powder.

〔Effect of the invention〕

According to the present invention, intracellular active ingredients can be efficiently and easily extracted from filamentous fungi that have chitosan in their cell walls. Therefore, the method of the present invention can be widely used for producing substances useful as foods, medicines, etc.

Claims (1)

[Scope of Claims] 1. A method for extracting intracellular active ingredients from a filamentous fungus having chitosan in its cell wall, which comprises using a cell wall lytic enzyme chitosanase. 2. The method according to claim 1, wherein the filamentous fungus having chitosan in its cell wall belongs to the order Mucorales or the order Moriliales. 3. Chitosanase is Bacillus pumilus BN-262 (FERM P-
8814)
The method described in section.