CN102643865A - Application of aspergillus flavus in aspect of improving yield of alcohol - Google Patents

Abstract

The invention discloses the application of a strain of Aspergillus flavus in improving alcohol production. The present invention also protects a method for producing alcohol: (1) mix 1 kg of cornstarch with 3-6 kg of 50-60°C liquid molasses, add amylase after gelatinization, and incubate at 80-100°C for 1-1.5 hours; Then cool to 60-65°C and add glucoamylase, incubate at 60-70°C for 10-15 hours; filter and collect the filtrate, which is the saccharification liquid; liquid ketone is 9-4 parts by volume of alcohol waste liquid and 1-6 parts by volume CGMCC No. 3.951 is obtained by mixing Aspergillus flavus fermentation broth. (2) Inoculate Saccharomyces cerevisiae in the saccharification solution to obtain an initial fermentation system, and obtain alcohol after fermentation. Adopting the method of the invention to cultivate Aspergillus flavus can produce bacterial protein feed on the one hand, and can obtain fermented liquid on the other hand. The fermented liquid can be used to produce alcohol, and the alcohol production of brewer's yeast can be greatly improved while using the alcohol waste liquid. The invention has great economic and social benefits.

Description

Application of a strain of Aspergillus flavus in improving alcohol production

technical field

The invention relates to the application of a strain of Aspergillus flavus in improving alcohol production.

Background technique

Alcohol is an important industrial raw material, which is widely used in chemical industry, food, military industry, medicine and health and other fields. At the same time, alcohol is the most promising renewable energy to replace petroleum in whole or in part, and has wide application and development prospects. With an annual output of more than 2 million tons of alcohol, my country is the third largest alcohol producer in the world. With the growth of the national economy, the demand for alcohol will also increase. For every ton of alcohol produced, 13-15 tons of distillation waste liquid is discharged, and the national annual discharge exceeds 15 million tons, making it one of the most polluting industries in the food fermentation industry. Distillation waste liquid contains a large number of unused nutrients, such as protein, fat, cellulose, vitamin B group, glycerin, organic acids, fermentation residual carbohydrates, etc. Therefore, the recovery and reuse of distillation waste liquid has great application prospects .

At present, the treatment methods of distillation waste liquid at home and abroad include irrigation method, oxidation pond method, concentration method, yeast protein production-aerobic method, anaerobic method, anaerobic-aerobic method, physical and chemical method, etc., among which there are many researches and applications The main ones are anaerobic biological treatment and anaerobic-aerobic biological treatment. The Krupp Company of Germany was the first company to develop the reflux of potato lees filtrate for the alcohol fermentation process. This process is called "LBW". The key technology is to ensure low-temperature cooking and prevent the Maillard reaction from producing harmful substances. The solid granular protein feed (DDGS) production technology is also widely used in European countries, such as Germany, France, Norway, etc. The essence of this technology is to reuse part of the filtrate obtained from the solid-liquid separation of corn distiller's grains for alcohol production, and the other part after evaporation and concentration Mix with solids and dry to make DDGS.

To improve the reuse rate of distillation waste liquid, further efforts are needed to explore and research in many aspects. Breeding strains adapted to high concentrations of reuse inhibitors is one of the good ways to improve the reuse rate of alcohol waste liquid.

Contents of the invention

The purpose of the invention is to provide the application of a strain of Aspergillus flavus in improving alcohol production.

The invention protects a method for producing alcohol, comprising the steps of:

(1) Mix 1 kg of corn starch with 3-6 kg (such as 3-4.5 kg or 4.5-6 kg) of liquid diced at 50-60°C (such as 50-55°C or 55-60°C), and add it after gelatinization For amylase, incubate at 80-100°C (such as 80-90°C or 90-100°C) for 1-1.5 hours (such as 1-1.2 hours or 1.2-1.5 hours); then cool to 60-65°C (such as 60-63°C or 63-65°C) and add glucoamylase, incubate at 60-70°C (such as 60-65°C or 65-70°C) for 10-15 hours (such as 10-12 hours or 12-15 hours); filter and collect the filtrate, It is the saccharified liquid; the liquid diced is 9-4 parts by volume (such as 9-7 parts by volume or 7-4 parts by volume) alcohol waste liquid and 1-6 parts by volume (such as 1-3 parts by volume or 3-6 parts by volume) obtained by mixing Aspergillus flavus fermentation broth; the addition of the amylase is: adding 4-30U amylase (such as 4-18U or 18-30U) per gram of cornstarch; the addition of the glucoamylase is: Add 50-200U glucoamylase (such as 50-125U or 125-250U) per gram of cornstarch; the Aspergillus flavus is a strain whose CGMCC number is 3.951;

(2) Inoculating Saccharomyces cerevisiae in the saccharification solution of step (1) to obtain an initial fermentation system, and obtaining alcohol after fermentation.

Specifically, the fermentation condition can be 20-40°C (such as 20-30°C or 30-40°C) and static culture for 30-60 hours (such as 30-45 hours or 45-60 hours).

In the initial fermentation system, the concentration of the Saccharomyces cerevisiae can be 1×10 ⁵ -3×10 ⁶ cfu/ml (such as 1×10 ⁵ -1.5×10 ⁶ cfu/ml or 1.5×10 ⁶ -3×10 ⁶ cfu/ml).

Specifically, the Saccharomyces cerevisiae can be inoculated into the saccharification solution in the form of a seed solution. The volume ratio of the seed liquid to the saccharification liquid is (1-30): (99-70), such as (1-15): (99-85) or (15-30): (85-70) .

Specifically, the Saccharomyces cerevisiae can be the strain numbered 31145 by CICC.

The preparation method of described Aspergillus flavus fermented liquid, comprises the steps:

(1) Inoculate the Aspergillus flavus into the fermentation medium, and shake and culture at 20-50°C (such as 20-35°C or 35-50°C) for 30-60 hours (30-45 hours or 45-60 hours);

(2) Filtrate the culture system that has completed step (1) and collect the filtrate, which is the Aspergillus flavus fermentation broth.

The fermentation medium is composed of a solvent and a solute; the solvent consists of 9.5-1 parts by volume (such as 9.5 to 5 parts by volume or 5 to 1 parts by volume) alcohol waste liquid and 0.5-9 parts by volume (such as 0.5 to 5 parts by volume) or 5 to 9 parts by volume) of water; the solute and its concentration in the fermentation medium are as follows: 20-200g carbon source, 5-300g nitrogen source, 0.1-10g (such as 0.1-1g, 1-5g or 5 -10g) KH ₂ PO ₄ , 0.1-10g (such as 0.1-5g, 1-5g or 5-10g) MgSO ₄ ·7H ₂ O, 0.1-4g (such as 0.1-2g, 1-2g or 2-4g) MnSO ₄ ·H ₂ O, 0.1-4g (such as 0.1-2g, 1-2g or 2-4g) CaCl ₂ , 0.1-4g (such as 0.1-2g, 1-2g or 2-4g) CuSO ₄ ·5H ₂ O, 0.1-4g (such as 0.1-2g, 1-2g or 2-4g) ZnSO ₄ ·7H ₂ O and 0.1-4g (such as 0.1-2g, 1-2g or 2-4g) FeSO ₄ ·4H ₂ O.

The 20-200g carbon source may be specifically composed of 10-100g glucose (such as 10-50g or 50-100g) and 10-100g (such as 10-50g or 50-100g) sugars containing sucrose. The 20-200g carbon source can specifically be 20-200g (such as 20-100g or 100-200g) of glucose or 20-200g (such as 20-100g or 100-200g) of sugars containing sucrose. The sucrose-containing sugar is specifically sucrose or molasses.

The 5-300g nitrogen source can be specifically composed of 2-100g (such as 2-50g or 50-100g) yeast extract, 2-100g (such as 2-50g or 50-100g) peptone and 1-100g (such as 1-50g or 50 -100g) of ammonium sulfate, or 2.5-150g (such as 2.5-70g or 70-150g) of yeast extract and 2.5-150g (such as 2.5-70g or 70-150g) of peptone, or 2.5-150g (such as 2.5- 70g or 70-150g) yeast extract and 2.5-150g (such as 2.5-70g or 70-150g) ammonium sulfate composition, can also be composed of 2.5-150g (such as 2.5-70g or 70-150g) peptone and 2.5-150g (such as 2.5- 70g or 70-150g) of ammonium sulfate. The 5-300g nitrogen source can specifically be 5-300g (such as 5-150g or 150-300g) yeast extract or 5-300g (such as 5-150g or 150-300g) peptone or 5-300g (such as 5-150g or 150-300g) ammonium sulfate.

The pH of the fermentation medium may be 4-6.5, such as 4-4.5, 4.5-5, 5-5.5, 5.5-6 or 6-6.5.

Specifically, the Aspergillus flavus can be inoculated into the fermentation medium in the form of seed solution.

The seed medium can be composed of water and solute; the solute and its concentration in the seed medium are as follows: glucose 10-40g/L, yeast powder 5-40g/L, peptone 10-40g/L and MnSO ₄ . _H2O 0.1-4g/L.

The pH of the seed medium may be 4-6.5.

The volume ratio of the seed liquid to the fermentation medium is (1-30): (99-70), such as (1-15): (99-85) or (15-30): (85-70 ).

The rotational speed of the shaking culture can be 100-200 rpm, such as 100-150 rpm or 150-200 rpm.

The initial concentration of the Aspergillus flavus in the fermentation medium may specifically be 1×10 ⁶ -1×10 ⁷ cfu/ml (such as 1×10 ⁶ -5×10 ⁶ cfu/ml or 5×10 ⁶ -1 ×10 ⁷ cfu/ml).

The Aspergillus flavus can be used to produce alcohol, and can also be used to promote the production of alcohol by Saccharomyces cerevisiae; the Aspergillus flavus is a strain with a CGMCC number of 3.951.

The aspergillus flavus fermented liquid can be used to produce alcohol, and can also be used to promote the production of alcohol by Saccharomyces cerevisiae.

Specifically, the Saccharomyces cerevisiae can be the strain numbered 31145 by CICC.

Any of the alcohol waste liquids described above can be the alcohol waste liquid prepared in the laboratory, and can also be the waste alcohol waste liquid produced by the winery.

The specific method of preparing alcohol waste liquid in the laboratory can be as follows:

(1) Mix 1 kg of cornstarch with 3-6 kg of water at 50-60°C, add amylase after gelatinization, and incubate at 80-100°C for 1-1.5 hours; then cool to 60-65°C and add glucoamylase, 60 Incubate at -70°C for 10-15 hours; filter and collect the filtrate, which is the saccharification solution; the amount of amylase added is: 4-30 U amylase per gram of corn starch; the amount of glucoamylase added is: per gram Add 50-200U glucoamylase to cornstarch;

(2) Inoculate Saccharomyces cerevisiae into the saccharification liquid of step (1) to obtain an initial fermentation system, and culture it statically at 20-40° C. for 30-60 hours.

(3) Distill the culture system after step (2), and the remaining liquid is alcohol waste liquid.

In the initial fermentation system, the concentration of the Saccharomyces cerevisiae is 1×10 ⁵ -3×10 ⁶ cfu/ml (such as 1×10 ⁵ -1.5×10 ⁶ cfu/ml or 1.5×10 ⁶ -3×10 ⁶ cfu/ml).

Specifically, the Saccharomyces cerevisiae can be inoculated into the saccharification solution in the form of a seed solution. The volume ratio of the seed liquid to the saccharification liquid is (1-30):(99-70).

The Saccharomyces cerevisiae can specifically be the strain numbered 31145 by China Industrial Microorganism Collection Center.

The distillation may specifically be distillation at 50° C. for 20 minutes.

The method can be introduced into the alcohol preparation process, that is, on the basis of adding the fermentation liquid, the alcohol waste liquid is used as raw water to circulate.

The method provided by the invention can be used for producing alcohol, and can greatly increase the alcohol output of Saccharomyces cerevisiae while using the alcohol waste liquid. The invention has great economic and social benefits.

Description of drawings

Fig. 1 is the standard curve of peak area and alcohol concentration.

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. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

Amylase: 1U represents the amount of enzyme required to convert 1 mg of soluble starch into dextrin within 1 min at 70°C and pH 6.0; Beijing Aoboxing Biotechnology Co., Ltd., batch number 20110919.

Glucoamylase: 1U represents the amount of enzyme required to convert soluble starch into 1 mg of glucose within 1 min at 40°C and pH 4.6; Beijing Aoboxing Biotechnology Co., Ltd. (batch number 20110902).

The Saccharomyces cerevisiae used in the examples are all yeasts as follows: purchased from China Industrial Microorganism Collection Center ( http://www.china-cicc.org/ ), CICC No. 31145.

The Aspergillus flavus used in the examples are all the following Aspergillus flavus: purchased from China General Microorganism Culture Collection and Management Center (http://www.cgmcc.net/), CGMCC number is 3.951.

Embodiment 1, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

1. Mix 1 kg of corn starch with 3 kg of water at 50°C, add amylase after gelatinization (4 U amylase per gram of corn starch), incubate at 80°C for 1 hour; then cool to 60°C and add glucoamylase (per gram Add 50U glucoamylase to cornstarch) and incubate at 60°C for 10 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

2. Saccharomyces cerevisiae was inoculated into YPD medium, 30° C., 160 rpm shaking culture (shimming amplitude 25 mm) for 24 hours, to obtain seed liquid.

3. Inoculate 1 volume part of seed liquid into 99 volume parts of saccharification liquid to obtain the initial fermentation system (the concentration of Saccharomyces cerevisiae in the initial fermentation system is 1×10 ⁵ cfu/ml); the initial fermentation system was cultured at 20°C for 30 hours , to terminate the fermentation system.

4. Distill the fermentation system at 50°C for 20 minutes, and the remaining liquid is alcohol waste liquid.

2. Preparation of Aspergillus flavus fermentation supernatant

1. Inoculate the activated Aspergillus flavus into the seed culture medium, shake culture at 20° C. and 100 rpm (shake amplitude: 25 mm) for 18 hours, and obtain the seed liquid.

The seed medium (pH4.0) is composed of water and solutes; the solutes and their concentrations in the seed medium are as follows: glucose 10g/L, yeast powder 5g/L, peptone 10g/L and MnSO ₄ ·H ₂ O 0.1g/L.

2. Inoculate the seed solution obtained in step 1 into the fermentation medium (the volume ratio of the seed solution to the fermentation medium is 1:99), the initial concentration of Aspergillus flavus spores is 1×10 ⁶ cfu/ml, Shake culture at 100rpm (shake amplitude 25mm) for 30h.

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 1 volume part alcohol waste liquor and 9 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (10g glucose and 10g sucrose), nitrogen source (2g yeast extract, 2g peptone and 1g ammonium sulfate), 0.1g KH ₂ PO ₄ , 0.1g MgSO ₄ ·7H ₂ O, 0.1g MnSO ₄ ·H ₂ O, 0.1g CaCl ₂ , 0.1 g CuSO ₄ .5H ₂ O, 0.1 g ZnSO ₄ .7H ₂ O and 0.1 g FeSO ₄ .4H ₂ O.

3. Filter the culture system in step 2 with four layers of gauze, and collect the filtrate (Aspergillus flavus fermented liquid) and thalline respectively.

4. Dry the thallus obtained in step 3 to a constant weight, which is the thalline protein feed, and 1.78 g thalline protein feed is obtained per 100 ml of culture system.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

1. Application of Aspergillus flavus in reuse of alcohol waste liquid

(1) Mix 1 kg of corn starch with 3 kg of liquid A at 50°C (composed of 9 parts by volume of alcohol waste liquid and 1 part by volume of Aspergillus flavus fermentation liquid), add amylase after gelatinization (add 4U of starch per gram of corn starch enzyme), incubate at 80°C for 1 hour; then cool to 60°C, add glucoamylase (50 U glucoamylase per gram of cornstarch), incubate at 60°C for 10 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

(2) Inoculate Saccharomyces cerevisiae into YPD medium and culture in YPD medium to obtain seed solution.

(3) Inoculate 1 volume part of seed liquid into ⁹⁹ volume parts of saccharification liquid to obtain the initial fermentation system (the concentration of S. After 30 hours, the fermentation system was terminated.

(4) The culture system completed in step (3) was centrifuged at 4000 rpm (centrifugal radius: 13.5 cm) for 10 min, and the supernatant was collected and filtered with a bacterial filter (0.22 μm) to obtain a cell-free filtrate.

2. Control test A

(1) Mix 1 kg of corn starch with 3 kg of alcohol waste liquid at 50°C, add amylase after gelatinization (add 4U amylase per gram of corn starch), incubate at 80°C for 1 hour; then cool to 60°C, add glucoamylase (add 50U glucoamylase per gram of cornstarch), incubate at 60°C for 10 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

3. Controlled experiment B

(1) Mix 1 kg of corn starch with 3 kg of water at 50°C, add amylase after gelatinization (add 4U amylase per gram of corn starch), incubate at 80°C for 1 hour; then cool to 60°C, add glucoamylase (per gram of corn starch) Add 50 U glucoamylase to 1 gram of corn starch), incubate at 60°C for 10 h; filter with four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

4. Determination of alcohol content in cell-free filtrate

The alcohol content in the cell-free filtrate was detected by HPLC. HPLC parameters: instrument: Agilent 1200; chromatographic column: Rezex ROA and corresponding guard column; detector is differential detector; sample volume 20 μL; mobile phase is 0.005MH ₂ SO ₄ aqueous solution; flow rate: 0.6ml/min; column temperature : 65°C. A standard curve was prepared by using alcohol standard substance. The retention time of alcohol was 22.02 min. The standard curve of peak area and alcohol concentration is shown in Figure 1 (the standard curve equation is: Y=141027.5X-11941.4; R ² =0.9991). Calculate the alcohol content in the cell-free filtrate from a standard curve.

The alcohol content in the cell-free filtrate obtained in step 1 is 40.161 mg/ml, which is increased by 9.11 compared with the alcohol content in the cell-free filtrate obtained in step 2, and increased compared with the alcohol content in the cell-free filtrate obtained in step 3 up 9.39%.

Embodiment 2, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

1. Mix 1 kg of corn starch with 6 kg of water at 60°C, add amylase after gelatinization (30U amylase per gram of corn starch), incubate at 100°C for 1.5h; then cool to 65°C, add glucoamylase (per gram of corn starch) Add 200U glucoamylase to 1 gram of cornstarch), incubate at 70°C for 15 hours, filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

2. Saccharomyces cerevisiae was inoculated into YPD medium, 30° C., 160 rpm shaking culture (shimming amplitude 25 mm) for 24 hours, to obtain seed liquid.

3. Inoculate 30 parts by volume of seed liquid into 70 parts by volume of saccharification liquid to obtain an initial fermentation system (the concentration of Saccharomyces cerevisiae in the initial fermentation system is 3×10 ⁶ cfu/ml); culture the initial fermentation system at 40°C for 60 hours , to terminate the fermentation system.

4. Distill the fermentation system at 50°C for 20 minutes, and the remaining liquid is alcohol waste liquid.

2. Preparation of Aspergillus flavus fermentation supernatant

1. Inoculate the activated Aspergillus flavus into the seed culture medium, shake culture at 40° C. and 200 rpm (shake amplitude: 25 mm) for 40 hours, and obtain the seed liquid.

The seed medium (pH 6.5) consists of water and solutes; the solutes and their concentrations in the seed medium are as follows: glucose 40g/L, yeast powder 40g/L, peptone 40g/L and MnSO ₄ ·H ₂ O 4g/L.

2. Inoculate the seed solution obtained in step 1 into the fermentation medium (the volume ratio of the seed solution to the fermentation medium is 30:70), the initial concentration of Aspergillus flavus spores is 1×10 ⁷ cfu/ml, Shake culture at 200rpm (shake amplitude 25mm) for 60h.

Fermentation medium (pH6.5) is made up of solvent and solute; Said solvent is made up of 9.5 parts by volume alcohol waste liquor and 0.5 volume part water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g glucose and 100g sucrose), nitrogen source (100g yeast extract, 100g peptone and 100g ammonium sulfate), 10g KH ₂ PO ₄ , 10g MgSO ₄ ·7H ₂ O, 4g MnSO ₄ ·H ₂ O, 4g CaCl ₂ , 4g CuSO ₄ · 5H ₂ O, 4g ZnSO ₄ .7H ₂ O and 4g FeSO ₄ .4H ₂ O.

3. Filter the culture system in step 2 with four layers of gauze, and collect the filtrate (Aspergillus flavus fermented liquid) and thalline respectively.

4. Dry the thallus obtained in step 3 to a constant weight, which is the thalline protein feed, and 2.3 g thalline protein feed is obtained per 100 ml of culture system.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

1. Application of Aspergillus flavus in reuse of alcohol waste liquid

(1) Mix 1 kilogram of cornstarch with 6 kilograms of liquid B at 60 °C (composed of 4 parts by volume of alcohol waste liquid and 6 parts by volume of Aspergillus flavus fermentation broth), add amylase after gelatinization (add 30 U of starch per gram of cornstarch) enzyme), incubate at 100°C for 1.5h; then cool to 65°C, add glucoamylase (200U glucoamylase per gram of cornstarch), incubate at 70°C for 15h; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

(2) Inoculate Saccharomyces cerevisiae into YPD medium and culture in YPD medium to obtain seed solution.

(3) Inoculate 30 parts by volume of seed liquid into 70 parts by volume of saccharification liquid to obtain the initial fermentation system (the concentration of Saccharomyces cerevisiae in the initial fermentation system is 3×10 ⁶ cfu/ml); the initial fermentation system was cultured statically at 40°C After 60 hours, the fermentation system was terminated.

(4) The culture system completed in step (3) was centrifuged at 4000 rpm (centrifugal radius: 13.5 cm) for 10 min, and the supernatant was collected and filtered with a bacterial filter (0.22 μm) to obtain a cell-free filtrate.

2. Control test A

(1) Mix 1 kg of corn starch with 6 kg of alcohol waste liquid at 60°C, add amylase after gelatinization (30U amylase per gram of corn starch), incubate at 100°C for 1.5h; then cool to 65°C, add saccharification Enzyme (add 200U glucoamylase per gram of corn starch), incubate at 70°C for 15 hours; filter with four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

3. Controlled experiment B

(1) Mix 1 kg of corn starch with 6 kg of water at 60°C, add amylase after gelatinization (30U amylase per gram of corn starch), incubate at 100°C for 1.5h; then cool to 65°C, add glucoamylase ( Add 200U glucoamylase per gram of cornstarch) and incubate at 70°C for 15 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

3. Determination of alcohol content in cell-free filtrate

Same as step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 40.161mg/ml, which is 11.2% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 10.1%.

Embodiment 3, the application of Aspergillus flavus in the reuse of alcohol waste liquid

The alcohol waste liquid in this example was obtained from Jilin Fuel Ethanol Co., Ltd., and the specific components are shown in Table 1.

Table 1 The test report of the quality inspection center of Jilin Fuel Ethanol Co., Ltd. alcohol waste liquid

8:00 13:00 Polysaccharide (g/L) 34.20 35.65 Maltotriose (g/L) 2.58 2.61 Maltose (g/L) 16.79 16.35 Glucose (g/L) 11.58 11.90 Fructose (g/L) 5.43 5.85 Arabinose (g/L) 9.69 10.03 Succinic acid (g/L) 7.25 7.62 Lactic acid (g/L) 33.44 33.31 Glycerol (g/L) 61.33 64.35

Acetic acid (g/L) 3.97 4.21 Methanol 9g/L) 46.7 49.55 Ethanol (g/L) none none

1. Preparation of Aspergillus flavus fermentation supernatant

1. Inoculate the activated Aspergillus flavus into the seed culture medium, shake culture at 30° C. and 150 rpm (shake amplitude: 25 mm) for 30 hours, and obtain the seed liquid.

The seed medium (pH 5.5) consists of water and solutes; the solutes and their concentrations in the seed medium are as follows: glucose 25g/L, yeast powder 25g/L, peptone 25g/L and MnSO ₄ ·H ₂ O 2g/L.

2. Inoculate the seed solution obtained in step 1 into the fermentation medium (the volume ratio of the seed solution to the fermentation medium is 15:85), the initial concentration of Aspergillus flavus spores is 5×10 ⁶ cfu/ml, and the Shake culture at 150rpm (shake amplitude 25mm) for 45h.

Fermentation medium (pH5.5) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (50g Glucose and 50g sucrose), nitrogen source (50g yeast extract, 50g peptone and 50g ammonium sulfate), 5g KH ₂ PO ₄ , 5g MgSO ₄ ·7H ₂ O, 2g MnSO ₄ ·H ₂ O, 2g CaCl ₂ , 2g CuSO ₄ · _5H2O , 2g _ZnSO4 · _7H2O and 2g _FeSO4 · _4H2O .

3. Filter the culture system in step 2 with four layers of gauze, and collect the filtrate (Aspergillus flavus fermented liquid) and thalline respectively.

4. Dry the thallus obtained in step 3 to a constant weight, which is the thallus protein feed, and 2.96 g thallium protein feed is obtained per 100 ml of culture system.

2. Application of Aspergillus flavus in reuse of alcohol waste liquid

1. Application of Aspergillus flavus in reuse of alcohol waste liquid

(1) Mix 1 kg of corn starch with 4.5 kg of 55°C liquid acrylic (composed of 7 parts by volume of alcohol waste liquid and 3 parts by volume of Aspergillus flavus fermentation broth), add amylase after gelatinization (add 18U starch per gram of corn starch enzyme), incubate at 90°C for 1.2h; then cool to 63°C, add glucoamylase (125U glucoamylase per gram of cornstarch), incubate at 65°C for 12h; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

(2) Inoculate Saccharomyces cerevisiae into YPD medium and culture in YPD medium to obtain seed solution.

(3) Inoculate 15 parts by volume of seed liquid into 85 parts by volume of saccharification liquid to obtain an initial fermentation system (the ^{concentration} of S. After 45 hours, the fermentation system was terminated.

(4) The culture system completed in step (3) was centrifuged at 4000 rpm (centrifugal radius: 13.5 cm) for 10 min, and the supernatant was collected and filtered with a bacterial filter (0.22 μm) to obtain a cell-free filtrate.

2. Control test A

(1) Mix 1 kg of corn starch with 4.5 kg of alcohol waste liquid at 55°C, add amylase after gelatinization (add 18U amylase per gram of corn starch), incubate at 90°C for 1.2h; then cool to 63°C, add saccharification Enzyme (add 125U glucoamylase per gram of the mixture), incubate at 65°C for 12 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

3. Controlled experiment B

(1) Mix 1 kg of corn starch with 4.5 kg of water at 55°C, add amylase after gelatinization (add 18U amylase per gram of corn starch), incubate at 90°C for 1.2h; then cool to 63°C, add glucoamylase ( Add 125U glucoamylase per gram of the mixture), incubate at 65°C for 12 hours; filter through four layers of gauze and collect the filtrate, which is the saccharification solution.

Steps (1) to (4) are the same as step 1 (2) to (4).

3. Determination of alcohol content in cell-free filtrate

Same as step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 24.35mg/ml, which is 6.8% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 5.9%.

Embodiment 4, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 1.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 9.5 parts by volume alcohol waste liquid and 0.5 volume part water; Described solute and its concentration in fermentation medium are as follows: carbon source (10g glucose and 10g molasses), nitrogen source (2g yeast extract, 2g peptone and 1g ammonium sulfate), 10g KH ₂ PO ₄ , 10g MgSO ₄ ·7H ₂ O, 4g MnSO ₄ ·H ₂ O, 4g CaCl ₂ , 4g CuSO ₄ · 5H ₂ O, 4g ZnSO ₄ .7H ₂ O and 4g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 1.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 20.65 mg/ml, which is 5.9% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 Increased by 5.0%.

Embodiment 5, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 2.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH6.5) is made up of solvent and solute; Said solvent is made up of 9.5 parts by volume alcohol waste liquor and 0.5 volume part water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g glucose and 100g molasses), nitrogen source (100g yeast extract, 100g peptone and 100g ammonium sulfate), 0.1g KH ₂ PO ₄ , 0.1g MgSO ₄ ·7H ₂ O, 0.1g MnSO ₄ ·H ₂ O, 0.1g CaCl ₂ , 0.1 g CuSO ₄ .5H ₂ O, 0.1 g ZnSO ₄ .7H ₂ O and 0.1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 2.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 2,

The alcohol content in the cell-free filtrate obtained in step 1 is 35.45 mg/ml, which is 10.9% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 9.1%.

Embodiment 6, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH5.5) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (50g glucose and 50g molasses), nitrogen source (50g yeast extract, 50g peptone and 50g ammonium sulfate), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ • 5H ₂ O, 1 g ZnSO ₄ •7H ₂ O and 1 g FeSO ₄ •4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 28.90mg/ml, which is 7.58% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 6.23%.

Embodiment 7, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 1.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquor and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (20g glucose), nitrogen source (2.5g yeast extract and 2.5g peptone), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1 g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 1.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 28.05 mg/ml, which is 7.25% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 6.02%.

Embodiment 8, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 2.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.5) is made up of solvent and solute; Said solvent is to be made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g glucose), nitrogen source (150g yeast extract and 150g peptone), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO _{4.7H 2} O and 1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 2.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 2.

The alcohol content in the cell-free filtrate obtained in step 1 is 34.55 mg/ml, which is 10.60% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 9.21%.

Embodiment 9, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquor and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (20g sucrose), nitrogen source (70g yeast extract and 70g peptone), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1gCuSO ₄ ·5H ₂ O, 1g ZnSO ₄ • 7H ₂ O and 1 g FeSO ₄ • 4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 25.25mg/ml, which is 7.65% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 6.23%.

Embodiment 10, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 1.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH6.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g sucrose), nitrogen source (2.5g yeast extract and 2.5g ammonium sulfate), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O , 1 g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 1.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 30.45 mg/ml, which is 9.55% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 7.90%.

Example 11, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 2.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g sucrose), nitrogen source (150g yeast extract and 150g ammonium sulfate), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ .7H ₂ O and 1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 2.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 2.

The alcohol content in the cell-free filtrate obtained in step 1 is 27.22 mg/ml, which is 8.50% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 6.89%.

Embodiment 12, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.5) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g glucose), nitrogen source (70g yeast extract and 70g ammonium sulfate), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ .7H ₂ O and 1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 24.05 mg/ml, which is 6.55% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 5.40%.

Embodiment 13, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 1.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.5) is made up of solvent and solute; Said solvent is to be made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g molasses), nitrogen source (70 g peptone and 70 g ammonium sulfate), 1 g KH ₂ PO ₄ , 1 g MgSO ₄ 7H ₂ O, 1 g MnSO ₄ H ₂ O, 1 g CaCl ₂ , 1 g CuSO ₄ 5H ₂ O, 1 g ZnSO _{4.7H 2} O and 1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 1.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 33.35mg/ml, which is 9.45% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 8.22%.

Embodiment 14, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 2.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquor and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (20g molasses), nitrogen source (150 g peptone and 150 g ammonium sulfate), 1 g KH ₂ PO ₄ , 1 g MgSO ₄ 7H ₂ O, 1 g MnSO ₄ H ₂ O, 1 g CaCl ₂ , 1 g CuSO ₄ 5H ₂ O, 1 g ZnSO ₄ • 7H ₂ O and 1 g FeSO ₄ • 4H ₂ O.

Others are the same as step 2 of embodiment 2.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 2.

The alcohol content in the cell-free filtrate obtained in step 1 is 31.55 mg/ml, which is 7.90% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 6.75%.

Example 15, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g molasses), nitrogen source (5g yeast extract), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 33.80mg/ml, which is 9.58% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 8.46%.

Example 16, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 1.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH6.5) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (10g glucose and 10 g sucrose), nitrogen source (2.5 g peptone and 2.5 g ammonium sulfate), 1 g KH ₂ PO ₄ , 1 g MgSO ₄ ·7H ₂ O, 1 g MnSO ₄ ·H ₂ O, 1 g CaCl ₂ , 1 g CuSO ₄ ·5H ₂ O, 1 g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 1.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 1.

The alcohol content in the cell-free filtrate obtained in step 1 is 24.1 mg/ml, which is 5.95% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 4.78%.

Example 17, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 2.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.5) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (100g glucose and 100g sucrose), nitrogen source (5g peptone), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ • 7H ₂ O and 1 g FeSO ₄ • 4H ₂ O.

Others are the same as step 2 of embodiment 2.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 2.

The alcohol content in the cell-free filtrate obtained in step 1 is 23.5mg/ml, which is 5.85% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 4.65%.

Example 18, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH4.0) is made up of solvent and solute; Described solvent is to form with 5 volume parts alcohol waste liquor and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (50g glucose and 50 g sucrose), nitrogen source (5 g ammonium sulfate), 1 g KH ₂ PO ₄ , 1 g MgSO ₄ 7H ₂ O, 1 g MnSO ₄ H ₂ O, 1 g CaCl ₂ , 1 g CuSO ₄ 5H ₂ O, 1 g ZnSO _{4.7H 2} O and 1 g FeSO ₄ .4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 21.85 mg/ml, which is 5.45% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 4.20%.

Example 19, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH6.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g sucrose), nitrogen source (300g yeast extract), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 32.25 mg/ml, which is 9.55% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 8.30%.

Example 20, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH5.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g sucrose), nitrogen source (300g peptone), 1g KH ₂ PO ₄ , 1g MgSO ₄ ·7H ₂ O, 1g MnSO ₄ ·H ₂ O, 1g CaCl ₂ , 1g CuSO ₄ ·5H ₂ O, 1g ZnSO ₄ ·7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 30.15 mg/ml, which is 9.05% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 7.76%.

Example 21, the application of Aspergillus flavus in the reuse of alcohol waste liquid

1. Preparation of alcohol waste liquid

Same as Step 1 of Example 3.

2. Preparation of Aspergillus flavus fermentation supernatant

Fermentation medium (pH5.0) is made up of solvent and solute; Said solvent is made up of 5 volume parts alcohol waste liquid and 5 volume parts water; Described solute and its concentration in fermentation medium are as follows: carbon source (200g sucrose), nitrogen source (300 g ammonium sulfate), 1 g KH ₂ PO ₄ , 1 g MgSO ₄ 7H ₂ O, 1 g MnSO ₄ H ₂ O, 1 g CaCl ₂ , 1 g CuSO ₄ 5H ₂ O, 1 g ZnSO ₄ 7H ₂ O and 1 g FeSO ₄ ·4H ₂ O.

Others are the same as step 2 of embodiment 3.

3. Application of Aspergillus flavus in reuse of alcohol waste liquid

Same as Step 3 of Example 3.

The alcohol content in the cell-free filtrate obtained in step 1 is 29.85mg/ml, which is 8.96% higher than the alcohol content in the cell-free filtrate obtained in step 2, and compared with the alcohol content in the cell-free filtrate obtained in step 3 An increase of 7.60%.

Claims (7)

1. A method for producing alcohol, comprising the steps of: (1) Mix 1 kg of cornstarch with 3-6 kg of 50-60°C liquid diced, add amylase after gelatinization, incubate at 80-100°C for 1-1.5 hours; then cool to 60-65°C and add glucoamylase , incubate at 60-70°C for 10-15 hours; filter and collect the filtrate, which is the saccharification solution; the liquid is obtained by mixing 9-4 parts by volume of alcohol waste liquid and 1-6 parts by volume of Aspergillus flavus fermentation broth; The addition amount of said amylase is: add 4-30U amylase per gram of cornstarch; the addition amount of said glucoamylase is: add 50-200U glucoamylase per gram of cornstarch; said Aspergillus flavus is a bacterial strain whose CGMCC number is 3.951 ; (2) Inoculating Saccharomyces cerevisiae in the saccharification solution of step (1) to obtain an initial fermentation system, and obtaining alcohol after fermentation. 2. the method for claim 1 is characterized in that: the preparation method of described aspergillus flavus fermented liquid comprises the steps: (1) Inoculate the Aspergillus flavus into the fermentation medium, and shake and culture at 20-50° C. for 30-60 hours; (2) Filtrate the culture system that has completed step (1) and collect the filtrate, which is the Aspergillus flavus fermentation broth. 3. The method according to claim 2, characterized in that: said fermentation medium is made up of solvent and solute; said solvent is made up of 9.5-1 parts by volume alcohol waste liquid and 0.5-9 parts by volume of water; said solute Its concentration in the fermentation medium is as follows: 20-200g carbon source, 5-300g nitrogen source, 0.1-10g KH ₂ PO ₄ , 0.1-10g MgSO ₄ ·7H ₂ O, 0.1-4g MnSO ₄ ·H ₂ O , 0.1-4g CaCl ₂ , 0.1-4g CuSO ₄ ·5H ₂ O, 0.1-4g ZnSO ₄ ·7H ₂ O and 0.1-4g FeSO ₄ ·4H ₂ O. 4. The application of Aspergillus flavus in the production of ethanol; the Aspergillus flavus is a bacterial strain whose CGMCC number is 3.951. 5. The application of Aspergillus flavus fermented liquid in the production of ethanol; said Aspergillus flavus is the bacterial strain whose CGMCC number is 3.951. 6. The application of Aspergillus flavus in promoting the production of alcohol by Saccharomyces cerevisiae; the Aspergillus flavus is a bacterial strain whose CGMCC number is 3.951. 7. The application of Aspergillus flavus fermented liquid in promoting the production of ethanol by Saccharomyces cerevisiae; the Aspergillus flavus is the bacterial strain whose CGMCC number is 3.951.

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