Penicillium vulpinum fungi strain and method for preparing levo 7-hydroxyl butylphthalide employing fungi strain

Abstract

The invention discloses a penicillium vulpinum fungi strain and a method for preparing levo 7-hydroxyl butylphthalide ((S)-(-)-butyl-7-hydroxyl phthalide) employing the fungi strain. The fungi strain disclosed by the invention is a penicillium vulpinum fungi strain NCC3421 (penicillium vulpinum) and is preserved at the China General Microbiological Culture Collection Center (CGMCC for short); and the preservation number is CGMCC No.9094. Under the culture condition provided by the method, the fermentation unit of the levo 7-hydroxyl butylphthalide reaches over 1600mg / L; the fermented product is relatively single in components, and easy to separate and purify, and can be applied to industrialized production of the levo 7-hydroxyl butylphthalide.

Description

technical field

[0001] The invention relates to the technical field of microbes, in particular to a fungal strain of Penicillium vulpeides and a method for preparing L-7-hydroxybutylphthalide ((S)-(-)-3-butyl-7-hydroxyphthalide) using the strain . Background technique

[0002] L-7-Hydroxybutylphthalide was first isolated from Penicillium vulpes by Japanese scholar Makino M et al [Heterocycles, 1998, 48(9): 1931-1933]. This compound is used alone or in combination with other phthalide compounds. Prevention and treatment of diabetes (US20090192218Al), depression and anxiety and other disorders related to impaired neurotransmission (US20100184852A1), and diseases related to angiogenesis (US20060246157A1). The currently reported ways to obtain 7-hydroxybutylphthalide in the world include: extraction from Rhizoma Chuanxiong, chemical synthesis, and separation from the culture solution of Penicillium vulpeides.

[0003] Patent US20060246157A1 discloses that the active compound L...

Examples

Embodiment 1

[0046] Example 1, the isolation and identification of Penicillium vulpeides NCC3421

[0047] (a) Isolation of strain NCC3421

[0048] The medium used for the isolation of fungal strain NCC3421 is PDA (Beijing Shuangxuan);

[0049] Take 1 gram of soil sample (collected from the mountainous soil in Baoxing County, Ya'an City, Sichuan Province, China), put it into a 50 mL centrifuge tube filled with 9 mL of sterile water, and shake fully. Take 1mL of the suspension and put it into a test tube filled with 9mL of sterile water, and mix well. Serial serial dilutions were carried out until 10 -10 . Take 0.2 mL of each gradient to plate. The coated plate was cultured statically at 26°C, and a fungus was obtained from it, which was named NCC3421.

[0050] (b) Identification of strain NCC3421—morphological characteristics and molecular identification

[0051] The spore liquid of strain NCC3421 was inoculated in PDA medium, cultured at 25°C for 7 days, and observed under scanning e...

Embodiment 2、5

[0065] The preparation of embodiment 2,5L tank fermentation 7-hydroxybutylphthalide

[0066] (a) Preparation of the seed solution of Penicillium voxenum strain NCC3421

[0067] The Penicillium voxenum strain NCC3421 was inoculated into the seed medium, and cultured at 26 °C and 200 rpm for 72 h to obtain the seed solution. The preparation method of the seed medium is: 20.0 grams of corn starch, 10.0 grams of glucose, 2.0 grams of hot-pressed soybean cake powder, 6.0 grams of malt powder, 3.0 grams of yeast powder, 2.0 grams of sodium chloride, 1.0 grams of magnesium sulfate heptahydrate, and tap water Dissolve and dilute to 1000 ml, pH 6.8, and sterilize at 121 °C for 30 min.

[0068] (b) Preparation of the fermentation broth of Penicillium vulpeides strain NCC3421

[0069] The seed solution was inoculated into a fermenter with 5 L of fermentation medium at an inoculum volume of 5% by volume. The tank temperature was 26 °C, the tank pressure was 0.05±0.01 Mpa, the ventilatio...

Embodiment 3

[0090] Embodiment 3, the preparation of shake flask fermentation 7-hydroxybutylphthalide

[0091] (a) Preparation of seed solution of Penicillium vulpecum strain NCC3421

[0092] The Penicillium voxenum strain NCC3421 was inoculated into the seed medium, and cultured at 26 °C and 200 rpm for 72 h to obtain the seed solution. The preparation method of the seed medium is: 20.0 grams of corn starch, 10.0 grams of glucose, 2.0 grams of hot-pressed soybean cake powder, 6.0 grams of malt powder, 3.0 grams of yeast powder, 2.0 grams of sodium chloride, 1.0 grams of magnesium sulfate heptahydrate, and tap water Dissolve and dilute to 1000 ml, pH 6.5, and sterilize at 121 °C for 30 min.

[0093] (b) Preparation of fermentation broth of Penicillium vulpeides strain NCC3421

[0094] The seed solution was inoculated into a 250 mL shake flask containing 40 mL of fermentation medium at an inoculum volume of 5% by volume, and fermented for 144 h at 26 °C with a rotation speed of 200 rpm. ...

technical field

[0001] The invention relates to the technical field of microbes, in particular to a fungal strain of Penicillium vulpeides and a method for preparing L-7-hydroxybutylphthalide ((S)-(-)-3-butyl-7-hydroxyphthalide) using the strain . Background technique

[0002] L-7-Hydroxybutylphthalide was first isolated from Penicillium vulpes by Japanese scholar Makino M et al [Heterocycles, 1998, 48(9): 1931-1933]. This compound is used alone or in combination with other phthalide compounds. Prevention and treatment of diabetes (US20090192218Al), depression and anxiety and other disorders related to impaired neurotransmission (US20100184852A1), and diseases related to angiogenesis (US20060246157A1). The currently reported ways to obtain 7-hydroxybutylphthalide in the world include: extraction from Rhizoma Chuanxiong, chemical synthesis, and separation from the culture solution of Penicillium vulpeides.

[0003] Patent US20060246157A1 discloses that the active compound L...

Examples

Embodiment 1

[0046] Example 1, the isolation and identification of Penicillium vulpeides NCC3421

[0047] (a) Isolation of strain NCC3421

[0048] The medium used for the isolation of fungal strain NCC3421 is PDA (Beijing Shuangxuan);

[0049] Take 1 gram of soil sample (collected from the mountainous soil in Baoxing County, Ya'an City, Sichuan Province, China), put it into a 50 mL centrifuge tube filled with 9 mL of sterile water, and shake fully. Take 1mL of the suspension and put it into a test tube filled with 9mL of sterile water, and mix well. Serial serial dilutions were carried out until 10 -10 . Take 0.2 mL of each gradient to plate. The coated plate was cultured statically at 26°C, and a fungus was obtained from it, which was named NCC3421.

[0050] (b) Identification of strain NCC3421—morphological characteristics and molecular identification

[0051] The spore liquid of strain NCC3421 was inoculated in PDA medium, cultured at 25°C for 7 days, and observed under scanning e...

Embodiment 2、5

[0065] The preparation of embodiment 2,5L tank fermentation 7-hydroxybutylphthalide

[0066] (a) Preparation of the seed solution of Penicillium voxenum strain NCC3421

[0067] The Penicillium voxenum strain NCC3421 was inoculated into the seed medium, and cultured at 26 °C and 200 rpm for 72 h to obtain the seed solution. The preparation method of the seed medium is: 20.0 grams of corn starch, 10.0 grams of glucose, 2.0 grams of hot-pressed soybean cake powder, 6.0 grams of malt powder, 3.0 grams of yeast powder, 2.0 grams of sodium chloride, 1.0 grams of magnesium sulfate heptahydrate, and tap water Dissolve and dilute to 1000 ml, pH 6.8, and sterilize at 121 °C for 30 min.

[0068] (b) Preparation of the fermentation broth of Penicillium vulpeides strain NCC3421

[0069] The seed solution was inoculated into a fermenter with 5 L of fermentation medium at an inoculum volume of 5% by volume. The tank temperature was 26 °C, the tank pressure was 0.05±0.01 Mpa, the ventilatio...

Embodiment 3

[0090] Embodiment 3, the preparation of shake flask fermentation 7-hydroxybutylphthalide

[0091] (a) Preparation of seed solution of Penicillium vulpecum strain NCC3421

[0092] The Penicillium voxenum strain NCC3421 was inoculated into the seed medium, and cultured at 26 °C and 200 rpm for 72 h to obtain the seed solution. The preparation method of the seed medium is: 20.0 grams of corn starch, 10.0 grams of glucose, 2.0 grams of hot-pressed soybean cake powder, 6.0 grams of malt powder, 3.0 grams of yeast powder, 2.0 grams of sodium chloride, 1.0 grams of magnesium sulfate heptahydrate, and tap water Dissolve and dilute to 1000 ml, pH 6.5, and sterilize at 121 °C for 30 min.

[0093] (b) Preparation of fermentation broth of Penicillium vulpeides strain NCC3421

[0094] The seed solution was inoculated into a 250 mL shake flask containing 40 mL of fermentation medium at an inoculum volume of 5% by volume, and fermented for 144 h at 26 °C with a rotation speed of 200 rpm. ...

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