CN101955887B - Penicillium producing raw amylase and raw amylase preparation prepared therefrom - Google Patents

Abstract

The invention discloses a penicillium producing raw amylase and a raw amylase preparation prepared therefrom. The present invention provides Penicillium sp. GXU20 CGMCC No.3690. The invention also provides the raw amylase preparation obtained by fermenting Penicillium GXU20. Both Penicillium GXU20 and raw amylase preparations can be used in the production of alcohol, which can avoid high-temperature cooking of raw materials and save a lot of energy; do not need to add α-amylase, saving costs; saccharification and fermentation are carried out in one container at the same time, reducing equipment costs. The invention is expected to solve the problem of high cost of alcohol production caused by high-temperature cooking starch.

Description

Penicillium producing raw amylase and raw amylase preparation prepared therefrom

technical field

The present invention relates to a kind of penicillium producing raw amylase and the raw amylase preparation thereof, in particular to a penicillium producing raw amylase, a liquid raw amylase preparation prepared by the strain and the preparation of raw tapioca starch Application of simultaneous saccharification and fermentation in the production of alcohol. the

Background technique

Starch is the most abundant polysaccharide stored in plants on earth. In the food industry, it is an economical raw material that can be used to produce sweeteners such as glucose, maltose, and fructose syrup, as well as antibiotics, amino acids, alcohol, and organic edible acids. Alcohol becomes anhydrous alcohol after dehydration, and anhydrous alcohol becomes fuel alcohol after denaturation treatment. Fuel alcohol has been used in many countries in the world. It is of great significance to alleviate the world energy crisis, improve the environment, and promote sustainable agricultural development.( Sanchez OJ, Cardona CA. Trends in biotechnologicalproduction of fuel ethanol from different feedstocks. Bioresource Technology 2008, 99: 5270-5295). the

In the traditional alcohol production process using starch as raw material, the raw material must first be cooked at a high temperature of about 90-110°C, and high-temperature-resistant α-amylase is added to cut the long-chain starch in the gelatinized raw material into short-chain dextrin, so that the mash Viscosity decreases, which is the gelatinization and liquefaction process of starch; short-chain dextrins cannot be used by yeast, so the liquefied raw materials need to be cooled to about 60°C, and glucoamylase is added to degrade short-chain dextrins into glucose. Is the saccharification process; finally the glucose is fermented by yeast to produce alcohol. Or after the raw material is gelatinized and liquefied, it is lowered to about 30°C, and saccharification enzyme and yeast are added at the same time to produce alcohol by saccharification and fermentation synchronously (Liang Yuchao, Li Kaimian, Research Progress in Cassava Alcoholic Fermentation, Guangxi Light Industry, 2007, 98: 16- 18). the

The above-mentioned ethanol production process is used when producing fuel alcohol with cassava starch and cornstarch as raw materials at present. Due to the high price of raw materials and the high-temperature cooking (gelatinization and liquefaction) in the traditional alcohol production process is a very energy-consuming process, it needs to consume about 35% of the energy required for the entire alcohol production process (Wang Chenxia, Du Fengguang, Li Gende, Starch Overview of Alcohol Production by Raw Meal Fermentation, Grain and Oils, 2008, 6: 11-13). The production cost of fuel alcohol is high, there is no market competitiveness, and production enterprises still need government subsidies to survive. Therefore, it is necessary to reduce the production cost of fuel alcohol. the

Raw amylase refers to a compound enzyme that can directly and effectively act on uncooked or treated raw starch granules, generally including α-amylase (α-1,4-D-Glucanohydrolase; EC 3.2. 1.1), β-amylase (β-amylase; EC 3.2.1.2) and glucoamylase (Glucoamylase; EC 3.2.1.3), etc. α-amylase randomly cuts the α-1,4 glycosidic bonds inside the starch molecule to generate short-chain dextrins; β-amylase hydrolyzes the α-1,4 glycosidic bonds between the starch molecules from the non-reducing end of the starch molecule, The product is maltose, which cannot hydrolyze α-1,6 glucosidic bonds, and when it acts on amylopectin, the product still has limit dextrin. Glucoamylase, commonly known as glucoamylase, can not only hydrolyze α-1, 4 glycosidic bonds, but also weakly hydrolyze α-1, 6 glycosidic bonds, and cut glucose one by one from the non-reducing ends of starch molecules, and the final product is glucose (Wang Jingyan, Zhu Shenggeng, Xu Changfa, Higher Education Press in Biochemistry, Beijing, 2002: 43). the

Raw amylase is used for synchronous saccharification and fermentation of raw starch to produce alcohol. It can directly convert raw starch into fermentable sugar at low temperature (30-40°C), and the yeast present at the same time will hydrolyze the fermentable sugar produced Fermentation produces alcohol, so that saccharification and fermentation are carried out in the same reactor, which can greatly simplify the traditional alcohol production process, reduce equipment costs, and improve equipment utilization. More importantly, it can save a lot of energy consumed in the high-temperature cooking process and greatly reduce energy consumption. Therefore, the application of raw amylase has an attractive development prospect in the production of fuel alcohol, and the development of raw amylase is of great significance to reduce the production cost of fuel alcohol (Li S-Z, Chan-Halbrendt C. Ethanol production in China: Potential and technologies. Applied Energy 2009, 86: 162-169). the

There are many microorganisms that can produce raw amylase, and the reported microorganisms that can produce raw amylase are mainly fungi, such as Aspergillus sp., Rhizopus sp., and it has also been reported that amylase derived from bacteria can also act on raw starch (Sun H , Ge X, Wang L, et al. Microbial production of raw starchdigesting enzymes. African Journal of Biotechnology 2009, 8: 1734-1739). the

Contents of the invention

The object of the present invention is to provide a penicillium producing raw amylase and the raw amylase preparation prepared therefrom. the

The Penicillium sp. provided by the present invention, named GXU20, belongs to the genus Penicillium (Penicillium sp.), was isolated from the forest soil of Shiwanda Mountain in Guangxi, and was preserved in China Microorganisms on March 25, 2010 The General Microorganism Center of the Preservation Management Committee (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC No.3690. Penicillium sp. GXU20 CGMCC No.3690, referred to as Penicillium sp. GXU20. The product released by Penicillium GXU20 after acting on raw cassava flour is glucose, which can effectively improve the utilization rate of raw starch raw materials. the

The present invention also provides a medium for cultivating Penicillium GXU20, which is obtained by mixing wheat bran, bean cake powder, KH ₂ PO ₄ , MgSO ₄ 7H ₂ O, FeSO ₄ 7H ₂ O, CaCl ₂ and water; Each liter of the medium contains 30g of wheat bran, 25g of bean cake powder, 3g of KH ₂ PO ₄ , 0.2g of MgSO ₄ .7H ₂ O, 0.025g of FeSO ₄ .7H ₂ O, and 0.13g of CaCl ₂ . The raw material of the culture medium is cheap, the production cost is low and the preparation is easy.

The invention also provides a method for producing the raw amylase preparation, which comprises fermenting Penicillium GXU20 to obtain the raw amylase preparation. The fermentation conditions may be pH 4.0-6.0, 26-34°C, preferably pH 5.0, 28°C. the

The fermentation may include the following steps: inoculating the spore liquid of Penicillium GXU20 into the medium, culturing for 10-14 days at pH5.0-6.0, 26-32°C, 180rpm (centrifugal radius is 4.5-5.5cm);

Described fermentation specifically comprises the steps:

(1) Penicillium GXU20 is activated for 5-7 days;

(2) mixing the spores of the activated bacterial classification with water to obtain a spore liquid with a spore concentration of 1×10 ¹⁰ /mL;

(3) the spore liquid is inserted in the medium described in claim 2, pH 5.0, 28 ℃, 180rpm (centrifugal radius is 4.5-5.5cm) shaker culture 11 days. the

The raw amylase preparation prepared by any of the above methods also belongs to the protection scope of the present invention. the

The raw amylase preparation provided by the present invention: at pH 3.5-6.5, it has an activity of more than 60% of the highest enzyme activity, preferably at pH 4.0-6.0, and the optimum pH value is 4.5; at 35-60°C, it has the highest enzyme activity More than 60% of the activity, preferably 40-60°C, the optimum temperature is 50°C; the optimum action conditions are close to the fermentation conditions of high-temperature resistant yeast (pH 4.0, 40°C), and amylase is produced at pH 4.0 and 40°C The activity is as high as 216U/mL. the

Penicillium GXU20 and/or the raw amylase preparation can be used to produce alcohol. It can avoid high-temperature cooking of raw materials, save a lot of energy, reduce the addition of α-amylase, and carry out saccharification and fermentation in one container at the same time, reducing equipment costs. the

When the raw amylase preparation is used to produce alcohol, the pH value can be 3.5-6.5, and the temperature can be 35-60°C; when the raw amylase preparation is used, the pH value is preferably 4.5, and the temperature is preferably 50°C. the

The present invention also protects a method for producing alcohol, comprising the following steps: taking raw cassava flour as a substrate, adding the raw amylase preparation and Saccharomyces cerevisiae, pH4. 5.5cm) to ferment to obtain alcohol. the

Penicillium GXU20 and its raw amylase preparation provided by the present invention are mainly used in the synchronous saccharification and fermentation of raw starch to produce alcohol, which can avoid high-temperature cooking of raw materials and save a lot of energy; do not need to add α-amylase, saving costs; saccharification and fermentation Simultaneously in one container, reducing equipment costs. The invention is expected to solve the problem of high cost of alcohol production caused by high-temperature cooking starch. the

Description of drawings

Figure 1 is the colony morphology of Penicillium GXU20 on the separation plate. the

Figure 2 is the starch degradation circle of Penicillium GXU20 on the separation plate. the

Figure 3 is the colony morphology of Penicillium GXU20 on the PDA plate. the

Fig. 4 is a micrograph of Penicillium GXU20. the

Figure 5 shows the effect of pH on the production of raw amylase by Penicillium GXU20. the

Figure 6 is the effect of temperature on the production of raw amylase by Penicillium GXU20. the

Figure 7 is the effect of different carbon sources and nitrogen sources on the production of raw amylase by Penicillium GXU20; A: the effect of different carbon sources on the production of raw amylase by Penicillium GXU20; B: the effect of different nitrogen sources on the production of raw amylase by Penicillium GXU20 Influence. the

Figure 8 is the concentration of wheat bran and soybean cake powder most suitable for Penicillium GXU20 to produce raw amylase; A: the most suitable concentration of wheat bran for Penicillium GXU20 to produce raw amylase; B: the most suitable soybean cake powder for Penicillium GXU20 to produce raw amylase concentration. the

Figure 9 is a comparison of the ability of Penicillium GXU20 to produce amylase before and after medium optimization. the

Figure 10 is the product identification of penicillium GXU20 liquid raw amylase preparation hydrolyzed raw cassava flour; A: sugar standard, G1 is glucose, G2 is maltose, G3 is maltotriose; B: hydrolysis for 5 minutes; C: hydrolysis for 10 minutes; D : hydrolyzed for 30min; E: hydrolyzed for 4h. the

Fig. 11 is the optimum action pH curve of Penicillium GXU20 liquid raw amylase preparation. the

Figure 12 is the optimum action temperature curve of Penicillium GXU20 liquid raw amylase preparation. the

Fig. 13 is the hydrolysis effect of Penicillium GXU20 liquid raw amylase preparation on different concentrations of raw cassava flour. the

Figure 14 is an electron micrograph of cassava flour particles treated with Penicillium GXU20 liquid raw amylase preparation. the

Fig. 15 is the measurement result of Penicillium GXU20 liquid raw amylase preparation in the simultaneous saccharification and fermentation of raw cassava flour to produce alcohol. the

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. % in the following examples, unless otherwise specified, are mass percentages. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. The rotational speeds in the following examples are all rotational speeds at a centrifugal radius with a radius of 4.5-5.5 cm. the

Basic fermentation medium: raw cassava flour 10g, (NH ₄ ) ₂ SO ₄ 2.5g, tryptone 2g, K ₂ HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g, FeSO ₄ 7H ₂ O 0.0255g, CaCl ₂ 0.13g, dilute to 1L with distilled water; adjust the pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; sterilize with damp heat at 121°C for 20min.

Disodium hydrogen phosphate-citric acid buffer at pH 4.0: Mix 7.71 ml of 0.2 mol/L Na ₂ HPO ₄ aqueous solution and 12.29 ml of 0.1 mol/L citric acid aqueous solution.

In embodiment 1, embodiment 2, embodiment 3, raw amylase activity assay adopts 3,5-dinitrosalicylic acid (3,5-dinitrosalicylate, DNS) method (Miller GL.Use of dinitrosalicyclic acidreagent for determination of reducing sugar.Analytical Chemistry1959, 31:426-428), the specific steps are as follows:

1. Dissolve glucose in sterile distilled water to make glucose standard solutions of different concentrations; add 1mL DNS to 500μL glucose standard solution, develop color in a boiling water bath for 5min, cool to room temperature, and measure the light absorption value at 540nm; get light absorption Value and glucose concentration of the standard curve, the function of the standard curve is y=2.7489x-0.0168 (R ² =0.9994) (y is the light absorption value, x is the glucose concentration).

2. Suspend 2 g of raw cassava powder in 100 mL of disodium hydrogen phosphate-citric acid buffer solution at pH 4.0 to obtain raw cassava powder suspension; add 450 μL of raw cassava powder suspension to a 2 mL EP tube, and then add 50 μL of liquid raw cassava powder For amylase preparation, react at 40°C for 30 minutes, shake continuously during the period, so that the enzyme and the substrate fully contact, add 1mL DNS after the reaction, develop color in a boiling water bath for 5 minutes, cool to room temperature, centrifuge at 1,000rpm for 1 minute, and measure the light absorption value at 540nm , Enzyme activity was calculated based on the standard curve and light absorbance values. Enzyme activity definition: the amount of enzyme needed to hydrolyze raw cassava flour at pH 4.0 and 40°C to release 1 mg of reducing sugar (equivalent to an equivalent amount of glucose) in 1 hour is one enzyme activity unit (1U). the

Embodiment 1, the acquisition of Penicillium GXU20

1. Acquisition of strains

1. Collection of soil samples

Soil was collected from a shallow soil layer of about 8-20 cm in Shiwandashan Forest, Fangchenggang City, Guangxi, China. the

2. Isolation and screening of strains

(1) Prepare separation medium with distilled water; each liter of separation medium contains: raw cassava flour 10g, NaNO ₃ 3g, KH ₂ PO ₄ 1g, FeSO ₄ 7H ₂ O 0.001g, MgSO ₄ 7H ₂ O 0.5g , agar 15g; pH5.5; other components except cassava flour were mixed and sterilized at 121°C for 20 minutes by damp heat. Raw cassava flour was sterilized by Co ⁶⁰ irradiation. When the components are cooled to 45°C, add them aseptically, mix well, and pour onto plates.

(2) Take 1g of soil sample and put it into a 150mL Erlenmeyer flask, add 19mL of sterile water, stir on a magnetic stirrer for 30min, and take a gradient dilution of the suspension (10 ^-2 , 10 ^-3 , 10 ^-4 and 10 ^-5 ), spread 50 μL each on the separation medium plate, and incubate at 28°C.

(3) After 3-5 days, observe the growth of the colony, select a gradient with an appropriate number of colonies and spread it on 8 separate plates in large quantities, and culture at 28°C. the

(4) After 5 days, stain with I ₂ /KI, and if there is a transparent hydrolysis circle around the colony, it means that the strain secretes amylase. Pick the fungus with obvious hydrolysis circle, transfer to a new separation plate, and purify into a single colony.

(5) Prepare the basic fermentation medium with pH 5.5. the

(6) Inoculate the target strain obtained in step (4) into the basic fermentation medium (liquid), culture at 28°C and 180rpm for 5-7 days, take the supernatant to measure the activity of raw amylase, and screen out the highest enzyme activity strain GXU20. the

See Figure 1 for the morphology of the strain GXU20 on the isolation medium, and Figure 2 for the degradation circle of the raw starch of the strain GXU20 on the isolation medium. the

2. Identification of strains

The colony morphology of the bacterial strain on the PDA plate is shown in Figure 3. The bacterial strain grows faster on the PDA medium, and the diameter of the colony can reach 4-5cm in 7 days. Turquoise, no pigment on the back of the culture medium. the

The microscopic photo of the strain is shown in Figure 4. There are many spores under the optical microscope. The conidia are oval or round. . the

According to the morphological characteristics of the strain, the strain GXU20 was identified as Penicillium with reference to the "Handbook of Fungal Identification". The Penicillium GXU20 was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number was CGMCC No.3690. the

Embodiment 2, the optimization of the culture condition that Penicillium GXU20 produces raw amylase

1. Preparation of spore liquid

1. Sterilize the PDA medium at 121°C for 20 minutes. the

2. Wash the spores of Penicillium GXU20 that have been subcultured and activated for 5-7 days on the PDA plate with sterile water to make a spore suspension with a concentration of 1×10 ¹⁰ spores/mL.

2. Optimization of pH value

1. Prepare basic fermentation media with different pH values (4.0, 4.5, 5.0, 5.5 or 6.0). the

2. The spore liquid of Penicillium GXU20 was inoculated into the culture medium in step 1 at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm for 5 days. the

3. Collect the crude enzyme solution as the solution to be tested, and measure the activity of raw amylase. the

The results are shown in Fig. 5, the optimal culture pH value of Penicillium GXU20 producing raw amylase is 5.0. the

3. Optimization of temperature

1. Prepare a basic fermentation medium with a pH of 5.0. the

2. Inoculate the spore liquid of Penicillium GXU20 into the culture medium of step 1 at an inoculation amount (volume percentage) of 1%, and place them at different temperatures (26°C, 28°C, 30°C, 32°C or 34°C) ) on a shaker at 180rpm for 5 days. the

3. Collect the crude enzyme solution as the solution to be tested, and measure the activity of raw amylase. the

The results are shown in Figure 6. The optimum culture temperature for the production of raw amylase by Penicillium GXU20 is 28°C. the

4. Determination of the optimum carbon and nitrogen sources for the culture medium

1. Preparation of various culture media

Medium containing various carbon sources (pH5.0): replace the basic fermentation culture with other carbon sources (potato flour, sweet potato flour, corn flour, rice flour, buckwheat flour, soluble starch, glutinous rice flour or wheat bran) of equal mass raw cassava flour in the base to obtain medium containing various carbon sources. the

Medium containing various nitrogen sources (pH5.0): raw cassava flour 10g, nitrogen source (urea, sodium nitrate, ammonium sulfate, ammonium chloride, tryptone, yeast extract, beef extract or bean cake powder) 10g, _K2 HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g, FeSO ₄ 7H ₂ O 0.0255g, CaCl ₂ 0.13g, dilute to 1L with distilled water; adjust pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; Bacteria 20min.

2. The spore liquid of Penicillium GXU20 was inoculated into various culture media in step 1 at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm for 7 days. the

3. Collect the crude enzyme solution as the solution to be tested, and measure the activity of raw amylase. the

The results are shown in Figure 7. The optimum carbon source for Penicillium GXU20 to produce raw amylase was wheat bran, and the optimum nitrogen source was bean cake flour. the

5. Determination of the optimum concentration of wheat bran and bean cake powder

1. Determination of the optimum concentration of wheat bran

(1) Preparation of various culture media

Medium containing various concentrations of wheat bran (pH5.0): wheat bran (5g, 10g, 15g, 20g, 25g, 30g, 35g or 40g), ammonium sulfate 2.5g, tryptone 2g, K ₂ HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g, FeSO ₄ 7H ₂ O 0.0255g, CaCl ₂ 0.13g, dilute to 1L with distilled water; adjust pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; sterilize at 121℃ for 20min.

(2) The spore liquid of Penicillium GXU20 was respectively inoculated into various media in step (1) at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm for 7 days. the

(3) Collect the crude enzyme solution as the solution to be tested, and carry out the determination of the activity of raw amylase. the

The results are shown in Figure 8A. The optimal concentration of wheat bran for Penicillium GXU20 to produce amylase was 3% (g/100mL). the

2. Determination of the optimum concentration of bean cake powder

(1) Preparation of various culture media

Medium containing various concentrations of bean cake powder (pH5.0): raw cassava flour 10g, bean cake powder (10g, 15g, 20g, 25g, 30g or 35g), K ₂ HPO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g , FeSO ₄ ·7H ₂ O 0.0255g, CaCl ₂ 0.13g, dilute to 1L with distilled water; adjust the pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; sterilize with moist heat at 121°C for 20min.

(2) The spore liquid of Penicillium GXU20 was respectively inoculated into various media in step (1) at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm for 7 days. the

(3) Collect the crude enzyme solution as the solution to be tested, and carry out the determination of the activity of raw amylase. the

The results are shown in Figure 8B. Penicillium GXU20 produced the most suitable concentration of bean cake powder for raw amylase was 2.5% (g/100mL). the

6. Comparison of enzyme production of Penicillium GXU20 in basic fermentation medium and optimized fermentation medium

1. Preparation of various culture media

(1) Prepare a basic fermentation medium with a pH of 5.0. the

(2) Prepare an optimized fermentation medium with pH 5.0: wheat bran 30g, bean cake powder 25g, KH ₂ PO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g, FeSO ₄ 7H ₂ O 0.025g, CaCl ₂ 0.13g Make up to 1L with distilled water; adjust the pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; sterilize with moist heat at 121°C for 20min.

2. The spore liquid of Penicillium GXU20 was inoculated into various mediums in step 1 at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm. the

3. During the cultivation process, the crude enzyme solution was taken regularly every day as the solution to be tested, and the raw amylase activity was measured. the

The results are shown in Figure 9. Penicillium GXU20 in the basic fermentation medium produced the highest peak of amylase on the fifth day, but the enzyme production activity began to decline from the sixth day, while the highest enzyme production peak of the optimized fermentation medium was on the 11th day, and 11-16 days, the enzyme activity remains stable. The results showed that the optimized fermentation medium was more practical than the basic fermentation medium. First, the raw materials of wheat bran and bean cake powder were cheaper than tryptone and ammonium sulfate. Second, the enzyme activity remained stable after the enzyme production reached its peak, making it easier to prepare enzyme preparations. After using the optimized fermentation medium, the amylase activity of Penicillium GXU20 fermentation broth increased to about 6 times of the original. the

The formulation of the optimized fermentation medium is as follows: wheat bran 30g, bean cake powder 25g, KH ₂ PO ₄ 3g, MgSO ₄ 7H ₂ O 0.2g, FeSO ₄ 7H ₂ O 0.025g, CaCl ₂ 0.13g and dilute to 1L with distilled water ; Adjust the pH value with 2M HCl aqueous solution or 2M NaOH aqueous solution; sterilize with damp heat at 121°C for 20min.

Embodiment 3, the preparation of liquid raw amylase preparation

1. Prepare optimized fermentation medium

Prepare an optimized fermentation medium with pH 5.0. the

2. Preparation of spore liquid

(1) Sterilize the PDA medium at 121°C for 20 minutes. the

(2) The spores of Penicillium GXU20 passaged and activated for 5-7 days on the PDA plate were washed with sterile water to prepare a spore suspension with a concentration of 1×10 ¹⁰ spores/mL.

3. Preparation of liquid raw amylase preparation

(1) Put 50mL optimized fermentation medium into a 250mL shake flask. the

(2) The spore liquid of Penicillium GXU20 was inoculated into the optimized fermentation medium at an inoculum amount (volume percentage) of 1%, and cultured at 28° C. and 180 rpm for 11 days. the

(3) Centrifuge culture at 13,000rpm, remove bacterial body, collect supernatant as solution to be tested, carry out raw amylase activity assay. the

The raw amylase activity of the solution to be tested was 216U/mL. the

The solution to be tested is the liquid raw amylase preparation, and the experiments of Example 4, Example 5 and Example 6 are carried out. the

Embodiment 4, the enzymatic characteristic of liquid raw amylase preparation

1. Product analysis of raw cassava flour hydrolyzed by liquid raw amylase preparation

1. Preparation of raw cassava flour suspension

Suspend 2 g of raw cassava flour in 100 mL of disodium hydrogen phosphate-citric acid buffer at pH 4.0 to obtain raw cassava flour suspension. the

2. Product analysis

Add 6mL raw cassava flour suspension into the finger bottle, then add 2mL liquid raw amylase preparation, place it at 40°C, and react on a shaking table at 150rpm. Take 500μL samples at 5min, 10min, 30min and 4h respectively, boil in boiling water for 5min to inactivate raw amylase, cool; centrifuge at 12,000rpm for 5min, take the supernatant, and detect the sugar in the supernatant by HPLC (standard samples are glucose, maltose , maltotriose). the

The results are shown in Figure 10. The results show that when liquid raw amylase acts on raw cassava flour, even in a very short reaction time (within 5 minutes), the released product is only glucose, and the glucose tends to increase with time. Therefore, it can be deduced that the raw starch glucoamylase in the liquid raw amylase preparation of Penicillium GXU20 is mainly responsible for the hydrolysis. the

2. Optimum pH value and optimum temperature for liquid raw amylase preparation

1. The most suitable pH value

Detect the difference in enzyme activity of liquid raw amylase preparations under different pH conditions: use different pH disodium hydrogen phosphate-citric acid buffer (pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0) instead The disodium hydrogen phosphate-citric acid buffer solution of pH4.0, other is the raw amylase activity assay method with embodiment 3; Enzyme activity definition: under the condition of 40 ℃, hydrolyze raw cassava flour, 1h releases 1mg reducing sugar (equivalent to The amount of enzyme required for the amount of glucose) is one enzyme activity unit (1U). the

The results of raw amylase activity determination are as follows: 85.04U/mL (pH 3.0), 164.30U/mL (pH3.5), 216.00U/mL (pH4.0), 271.78U/mL (pH4.5), 256.44U/mL mL (pH5.0), 231.66U/mL (pH5.5), 210.86U/mL (pH6.0), 159.83U/mL (pH6.5), 127.07U/mL (pH 7.0). Taking the highest enzyme activity as 100%, the ratio of the enzyme activity at other pH values to the highest enzyme activity is the relative enzyme activity. The abscissa is the pH value, and the relative enzyme activity is the ordinate, as shown in Figure 11. The results showed that the optimum pH of Penicillium GXU20 liquid raw amylase preparation was about 4.5 at 40℃, and the relative enzyme activity was 79% at pH 4.0. the

2. Optimum working temperature

Detect the difference in enzyme activity of liquid raw amylase preparations under different temperature conditions: use different temperatures (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C), and other With the raw amylase activity assay method of embodiment 3; Enzyme activity definition: under the condition of pH4.0 hydrolysis raw cassava flour, 1h releases the required enzyme amount of 1mg reducing sugar (equivalent to equivalent glucose) as one enzyme activity unit (1U). the

The results of raw amylase activity determination are as follows: 71.64U/mL (20°C), 81.43U/mL (25°C), 126.03U/mL (30°C), 168.87U/mL (35°C), 216.00U/mL (40°C ℃), 235.27U/mL (45℃), 248.70U/mL (50℃), 232.69U/mL (55℃), 212.07U/mL (60℃). Taking the highest enzyme activity as 100%, the ratio of the enzyme activity at other temperatures to the highest enzyme activity is the relative enzyme activity, with the temperature as the abscissa and the relative enzyme activity as the ordinate, as shown in Figure 12. The results showed that the optimum action temperature of Penicillium GXU20 liquid raw amylase preparation was about 50°C at pH 4.0, and the relative enzyme activity was 87% at 40°C. the

3. Substrate specificity of liquid raw amylase preparations

Detect the difference in enzyme activity of liquid raw amylase preparations under different substrate conditions: use different substrates of the same quality (raw cassava flour, raw corn flour, raw potato flour, raw sweet potato flour, raw glutinous rice flour, raw rice flour, raw Buckwheat flour or soluble starch) instead of raw cassava flour, other raw amylase activity assay method with embodiment 3; Enzyme activity definition: pH4.0, hydrolysis substrate under 40 ℃ condition, 1h releases 1mg reducing sugar (equivalent to The amount of enzyme required for the amount of glucose) is one enzyme activity unit (1U). the

Protein concentrations of liquid raw amylase preparations were determined using the Micro BCA protein assay reagent (Pierce, Rockford, IL). the

Table 1 shows the results of raw amylase activity determination of liquid raw amylase preparations under different substrate conditions. the

The substrate specificity of table 1 Penicillium GXU20 liquid raw amylase preparation

Substrate Specific enzyme activity (U/mg) Relative enzyme activity (%) Raw Tapioca Flour 71.8±1.03 100±1.4 raw cornmeal 151.7±6.9 211±9.7 Raw rice flour 148.3±3.67 206±5.2 raw potato flour 68.8±0.28 96±0.4 Raw sweet potato powder 45.5±0.76 63±1.0 Raw buckwheat flour 39.1±0.75 54±1.0 Raw glutinous rice flour 24.4±0.19 34±0.3 soluble starch 2.8±0.06 4±0.09

Taking the enzyme activity on raw cassava flour as 100%, the ratio of the enzyme activity on other substrates to the enzyme activity on raw cassava flour is the relative enzyme activity. The enzymatic activity of Penicillium GXU20 liquid raw amylase preparation on various substrates from strong to weak order is: corn, rice, cassava, potato, sweet potato, buckwheat, glutinous rice, soluble starch. Penicillium GXU20 liquid raw amylase preparation has strong enzyme activity on corn and rice, indicating that Penicillium GXU20 liquid raw amylase preparation has application potential in other starch processing industries in addition to its application in the processing of raw cassava starch. the

Embodiment 5, utilize liquid raw amylase preparation to hydrolyze raw cassava flour

1. Hydrolysis of raw cassava flour by liquid raw amylase preparation

Set 5 groups of treatment, each group of treatment sets 12 repetitions: add raw cassava flour, liquid raw amylase preparation and pH4.0 disodium hydrogen phosphate-citric acid buffer solution in the Erlenmeyer flask of 150mL (each group handles each group See Table 2 for the addition amount of the points) to obtain a reaction system; place the reaction system in a shaker at 40° C. and 150 rpm after mixing; take 2 bottles for each group every 12 hours, detect the hydrolysis percentage of raw cassava starch, and calculate the average value. the

Table 2 The addition of each component in each group of treatments

Calculation method of raw tapioca starch hydrolysis percentage (Shariffa YN, Karim AA, Fazilah A, ZaidulISM Enzymatic hydrolysis of granular native and mildly heat-treated tapioca and sweet potato starches at sub-gelatinization temperature. FoodHydrocolloid 2009, 23: 434-440): The remaining raw cassava flour after the action was washed with distilled water for 3 times, dried in an oven at 40°C to constant weight, weighed, and the weight was W ₂ (g); Raw cassava flour, after washing 3 times with distilled water, is also placed in 40 ℃ oven and dried to constant weight, weighs, and weight is W ₁ (g); Raw cassava starch hydrolysis percentage is calculated according to the following formula: hydrolysis percentage (%) =(W ₁ -W ₂ )×100/W ₃ ; W ₃ is the total starch content in the unhydrolyzed raw cassava flour (the starch content per 1g of raw cassava flour is 0.76g after testing).

Take time as the abscissa, and the hydrolysis percentage of raw cassava starch as the ordinate, as shown in Figure 13. The results showed that Penicillium GXU20 raw amylase could effectively hydrolyze different concentrations of raw cassava flour. For raw cassava flour with a concentration of 15%, the percentage of hydrolysis reached 95% after being treated with liquid raw amylase preparation for 72 hours. the

2. Electron microscope observation of cassava flour particles treated with Penicillium GXU20 liquid raw amylase preparation

The raw cassava flour before treatment and the residual raw cassava flour of the above-mentioned number 3 treatment in each time period were sent to the School of Materials Engineering, Guangxi University for fixed gold spraying, and the morphological changes of raw cassava flour particles before and after treatment were observed by electron microscope. The electron microscope model is S-3400N, produced by Hitachi Corporation. the

The surface of raw cassava flour particles before treatment was smooth (see Figure 14A), while the surface of raw cassava flour particles treated with Penicillium GXU20 liquid raw amylase preparation was rough, forming many small pits of different sizes, and a large hole was visible inside ( Wherein the raw cassava flour processed by enzymolysis for 12 hours with the above-mentioned number as the third group is shown in Fig. 14B). It shows that after raw amylase hydrolyzes the surface of cassava flour granules, the small holes formed facilitate the penetration of raw amylase into the interior of starch granules, and then better hydrolyze raw starch. It can be seen that the raw amylase of Penicillium GXU20 liquid raw amylase preparation has a strong degradation effect on raw cassava flour. the

Embodiment 6, utilize liquid raw amylase preparation synchronous saccharification fermentation raw tapioca flour (150g/L) to produce alcohol

Angel Saccharomyces cerevisiae dry yeast was purchased from Hubei Angel Yeast Co., Ltd.; activated according to the package instructions. the

Set up 12 repetitions: add 22.5mL liquid raw amylase preparation (216U/mL), 15g raw cassava flour, 5mL 6% urea aqueous solution (containing 0.3g urea), 5mL activated Saccharomyces cerevisiae ( Contains about 0.1g of dry yeast), 67.5mL of sterile distilled water; adjust the pH to 4.0 with 2M HCl aqueous solution; seal the bottle mouth and place it in a shaker at 40°C and 150rpm (to make the substrate fully contact with the enzyme, which is beneficial to the enzyme’s hydrolysis of raw cassava flour); take 2 bottles every 12 hours, measure the volume, determine the amount of reducing sugar, residual starch, and alcohol production. the

Determination of residual reducing sugar: the content of reducing sugar in the fermentation broth (%, g/100mL) was determined by DNS method. the

Determination of residual starch: take 1mL of the fermentation broth whose reducing sugar content has been determined, add 1mL of 12M concentrated hydrochloric acid, and then add 4mL of distilled water to make the total volume 6mL, the final concentration of hydrochloric acid in the solution is 2M, take a boiling water bath for 15min, cool to room temperature, and use 2M NaOH aqueous solution was neutralized to neutral, the amount of reducing sugar was determined by DNS method, and the residual amount of starch was calculated (%, g/100mL). the

Determination of alcohol concentration: Add 100mL of distilled water to each bottle of the remaining fermentation broth after sampling and measuring reducing sugar and residual starch, heat and distill 100mL of distillate, measure the alcohol content of the distillate with an alcohol meter, and measure the temperature at the same time Alcohol concentration corrected to 20°C. the

Calculation of fermentation rate: fermentation rate (%)=alcohol (g) * 100/theoretical alcohol (g) produced by correspondingly consumed tapioca starch produced by fermentation. the

The results are shown in Figure 15. The results showed that after the addition of Penicillium GXU20 liquid raw amylase preparation, the raw cassava flour was degraded into glucose, and the yeast fermented the glucose into alcohol at the same time. The post-production wine volume can reach 53.3g/L, the fermentation rate is 92%, and the reducing sugar and residual starch content is very low (<0.5%, g/100mL), which meets the requirements of the residual sugar content in the starch alcohol industry, indicating that the fermentation has been carried out completely ,thorough. These data all indicate that the raw amylase produced by Penicillium GXU20 can be used in the process of synchronous saccharification and fermentation of raw cassava flour to produce alcohol, and has broad application prospects. the

Claims (11)

1. mould (Penicillium sp.) GXU20, its deposit number is CGMCC No.3690.

2. a method of producing living diastase is fermentation mould claimed in claim 1 (Penicillium sp.) GXU20, obtains living diastase.

3. method as claimed in claim 2, it is characterized in that: the condition of described fermentation is pH4.0-6.0,26-34 ℃.

4. method as claimed in claim 3 is characterized in that: the condition of described fermentation is pH5.0,28 ℃.

5. method as claimed in claim 2, it is characterized in that: described fermentation comprises the steps: the spore liquid of mould claimed in claim 1 (Penicillium sp.) GXU20 is seeded in the substratum, and pH5.0-6.0,26-32 ℃, 180rpm were cultivated 10-14 days;

Described substratum is with wheat bran 30g, soybean cake powder 25g, KH ₂PO ₄3g, MgSO ₄7H ₂O 0.2g, FeSO ₄7H ₂O0.025g and CaCl ₂0.13g being mixed to cumulative volume with water is that 1L obtains.

6. method as claimed in claim 2, it is characterized in that: described fermentation comprises the steps:

(1) with mould claimed in claim 1 (Penicillium sp.) GXU20 activation 5-7 days;

(2) spore of bacterial classification with activation mixes with water, and getting spore concentration is 1 * 10 ¹⁰The spore liquid of individual/mL;

(3) spore liquid is accessed in the substratum, pH 5.0,28 ℃, 180rpm shaking table cultivated 11 days;

Described substratum is with wheat bran 30g, soybean cake powder 25g, KH ₂PO ₄3g, MgSO ₄7H ₂O 0.2g, FeSO ₄7H ₂O0.025g and CaCl ₂0.13g being mixed to cumulative volume with water is that 1L obtains.

7. the living diastase that arbitrary described method prepares in the claim 2 to 6.

8. the described mould of claim 1 (Penicillium sp.) GXU20 and/or the described living diastase of claim 7 application in producing alcohol.

9. application as claimed in claim 8 is characterized in that: when using described living diastase, the pH value is 3.5-6.5, and temperature is 35-60 ℃.

10. application as claimed in claim 9 is characterized in that: when using described living diastase, the pH value is 4.5, and temperature is 50 ℃.

11. a method of producing alcohol, comprising the steps: to give birth to Tapioca Starch is substrate, adds the described living diastase of claim 7 and Angel yeast saccharomyces cerevisiae, and pH4.0,40 ℃, 150rpm fermentation obtain alcohol.

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