A kind of trichoderma dark viride producing high temperature resistant ferulic acid esterase and high temperature resistant cellulase and application thereof

Abstract

The invention discloses a Trichoderma atroviride AWS26 producing high-temperature-resistant ferulic acid esterase and high-temperature-resistant cellulase and an application thereof. The preservation number of the Trichoderma atroviride AWS26 is CGMCC No. 8673. The Trichoderma dark green of the present invention has the advantages of low nutritional requirements, fast reproduction, easy extraction and separation of fermentation products, and short fermentation cycle, and ferulic acid esterase and cellulase composite enzyme can be obtained by fermentation, and the composite enzyme is resistant to high temperature, Good acid-base stability.

Description

technical field

[0001] The invention belongs to the technical field of microbes, and in particular relates to a Trichoderma viride producing high-temperature-resistant ferulic acid esterase and high-temperature-resistant cellulase and an application thereof. Background technique

[0002] Ferulic acid esterase (E.C.3.1.1.73, ferulic acid esterase, FAE), also known as cinnamic esterase, is a new type of enzyme preparation. Ferulic acid ester and lignin ferulic acid ester are used as substrates to destroy the ester bond between ferulic acid and polysaccharide, release ferulic acid from the plant cell wall, make the remaining polysaccharide main chain easy to be degraded, and effectively improve the content of a large amount of lignin. Nutritional value of by-products of cellulose and ferulic acid. Ferulic acid has a variety of physiological functions such as scavenging free radicals, anti-ultraviolet radiation, anti-thrombosis, lowering blood lipids, preventing and treating co...

Examples

Embodiment 1

[0045] The present embodiment illustrates the screening method of Trichoderma dark green AWS26, and concrete steps are as follows:

[0046] Step 1, sampling and sample processing:

[0047] 10 soil samples were taken from the high-temperature fermented compost in Dahan Village, Yunlong District, Xuzhou City, Jiangsu Province. Weigh 5g of solid soil sample, put it into a triangular flask filled with glass beads and 50mL of sterilized saline, vibrate at 160r / min for 30min to make a suspension, and then put the triangular flask into a constant temperature shaking water bath at 60-70°C In the pot, shake at 100r / min for 20-30min to obtain a soil-like turbid solution.

[0048] Step 2, enrichment culture:

[0049] Take 2-4mL of the soil sample turbid solution obtained in step 1, add it to a 250mL Erlenmeyer flask containing 30-60mL enrichment medium 1, and place it in a constant temperature shaker at 45-50°C and a speed of 160-200r / min After culturing for 5-7 days, an enriched cult...

Embodiment 2

[0076] Example 2 Trichoderma dark viride producing high temperature resistant ferulic acid esterase and high temperature resistant cellulase is applied to the degradation of feed materials

[0077] Dry corn straw powder 10g, bran 5g, ammonium sulfate 1g, soybean meal powder 1g, ammonium nitrate 0.4g, NaCl 0.5g, potassium dihydrogen phosphate 0.2g, MgSO4 0.05g, add according to the solid-liquid mass ratio 1:1.3 Ionized water, put the above feed into the Erlenmeyer flask, put it into the constant temperature and humidity incubator after sterilization and inoculation, adjust the temperature to 26-30°C, pH6.0. The inoculated strain is the activated strain AWS26 of the present invention. During the fermentation process, the Erlenmeyer flask was turned every day, and after 10 days of fermentation, the content changes of cellulose, hemicellulose and lignin were detected. The test results showed that among the fermentation raw materials, the degradation rate of lignin was 77.4%, the ...

Embodiment 3

[0078] Example 3 Trichoderma dark viride producing thermostable ferulic acid esterase and thermostable cellulase is applied to ferment wheat bran to prepare ferulic acid

[0079] Add 50 g of bran, 1 g of ammonium sulfate, 2 g of peptone, 0.2 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, and 75 mL of water into a 500 mL Erlenmeyer flask, mix and sterilize. After PDA medium slant culture and inoculation of the Trichoderma dark green strain of the present invention, 30-35 ℃ culture 3-4d, then add sterile water 10ml in the slant and fully suspend spore, add 2ml spore suspension in the sterilized medium, After mixing well, culture at 30-35°C for 3-5 days, and turn over 2-3 times in the middle. Add deionized water to the fermented bran koji, stir well to disperse the bran koji, place it on a vibrating shaker at a speed of 100r / min, and extract at 50°C for 2h. Filter, centrifuge at 8000r / min for 20min, take the supernatant with a saturation of 80% (NH 4 ) 2 SO ...

technical field

[0001] The invention belongs to the technical field of microbes, and in particular relates to a Trichoderma viride producing high-temperature-resistant ferulic acid esterase and high-temperature-resistant cellulase and an application thereof. Background technique

[0002] Ferulic acid esterase (E.C.3.1.1.73, ferulic acid esterase, FAE), also known as cinnamic esterase, is a new type of enzyme preparation. Ferulic acid ester and lignin ferulic acid ester are used as substrates to destroy the ester bond between ferulic acid and polysaccharide, release ferulic acid from the plant cell wall, make the remaining polysaccharide main chain easy to be degraded, and effectively improve the content of a large amount of lignin. Nutritional value of by-products of cellulose and ferulic acid. Ferulic acid has a variety of physiological functions such as scavenging free radicals, anti-ultraviolet radiation, anti-thrombosis, lowering blood lipids, preventing and treating co...

Examples

Embodiment 1

[0045] The present embodiment illustrates the screening method of Trichoderma dark green AWS26, and concrete steps are as follows:

[0046] Step 1, sampling and sample processing:

[0047] 10 soil samples were taken from the high-temperature fermented compost in Dahan Village, Yunlong District, Xuzhou City, Jiangsu Province. Weigh 5g of solid soil sample, put it into a triangular flask filled with glass beads and 50mL of sterilized saline, vibrate at 160r / min for 30min to make a suspension, and then put the triangular flask into a constant temperature shaking water bath at 60-70°C In the pot, shake at 100r / min for 20-30min to obtain a soil-like turbid solution.

[0048] Step 2, enrichment culture:

[0049] Take 2-4mL of the soil sample turbid solution obtained in step 1, add it to a 250mL Erlenmeyer flask containing 30-60mL enrichment medium 1, and place it in a constant temperature shaker at 45-50°C and a speed of 160-200r / min After culturing for 5-7 days, an enriched cult...

Embodiment 2

[0076] Example 2 Trichoderma dark viride producing high temperature resistant ferulic acid esterase and high temperature resistant cellulase is applied to the degradation of feed materials

[0077] Dry corn straw powder 10g, bran 5g, ammonium sulfate 1g, soybean meal powder 1g, ammonium nitrate 0.4g, NaCl 0.5g, potassium dihydrogen phosphate 0.2g, MgSO4 0.05g, add according to the solid-liquid mass ratio 1:1.3 Ionized water, put the above feed into the Erlenmeyer flask, put it into the constant temperature and humidity incubator after sterilization and inoculation, adjust the temperature to 26-30°C, pH6.0. The inoculated strain is the activated strain AWS26 of the present invention. During the fermentation process, the Erlenmeyer flask was turned every day, and after 10 days of fermentation, the content changes of cellulose, hemicellulose and lignin were detected. The test results showed that among the fermentation raw materials, the degradation rate of lignin was 77.4%, the ...

Embodiment 3

[0078] Example 3 Trichoderma dark viride producing thermostable ferulic acid esterase and thermostable cellulase is applied to ferment wheat bran to prepare ferulic acid

[0079] Add 50 g of bran, 1 g of ammonium sulfate, 2 g of peptone, 0.2 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, and 75 mL of water into a 500 mL Erlenmeyer flask, mix and sterilize. After PDA medium slant culture and inoculation of the Trichoderma dark green strain of the present invention, 30-35 ℃ culture 3-4d, then add sterile water 10ml in the slant and fully suspend spore, add 2ml spore suspension in the sterilized medium, After mixing well, culture at 30-35°C for 3-5 days, and turn over 2-3 times in the middle. Add deionized water to the fermented bran koji, stir well to disperse the bran koji, place it on a vibrating shaker at a speed of 100r / min, and extract at 50°C for 2h. Filter, centrifuge at 8000r / min for 20min, take the supernatant with a saturation of 80% (NH 4 ) 2 SO ...

Images