CN102643754B - Aspergillus oryzae and application thereof in aspect of improving yield of alcohol - Google Patents
Aspergillus oryzae and application thereof in aspect of improving yield of alcohol Download PDFInfo
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Abstract
The invention discloses an aspergillus oryzae and application thereof in the aspect of improving the yield of alcohol. The aspergillus oryzae provided by the invention has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 5725. The invention further provides a method for preparing alcohol. The method comprises the following steps of: (1) mixing 1kg of corn starch with 3-6kg of liquid D with the temperature of 50-60 DEG C, adding amylase after gelatinizing, and incubating for 1-1.5 hours at the temperature of 80-100 DEG C; cooling to 60-65 DEG C and adding saccharifying enzyme, and incubating for 10-15 hours at the temperature of 60-70 DEG C; and filtering and collecting filtrate which is a saccharified liquid, wherein the liquid D is obtained by mixing 9-14 parts by volume of alcohol waste liquid and 1-6 parts by volume of aspergillus oryzae fermentation liquid; and (2) inoculating saccharomyces cerevisiae in the saccharified liquid, and fermenting to obtain the alcohol. The aspergillus oryzae is cultivated by adopting the method, so that on the one hand, the microbial protein feed can be prepared, and on the other hand, the fermentation liquid can be obtained. The fermentation liquid can be used for preparing the alcohol, so that the alcohol waste liquid can be utilized, and the alcohol yield of the saccharomyces cerevisiae can be greatly improved. The aspergillus oryzae is good in economic benefit and social benefit.
Description
Technical field
The present invention relates to an Aspergillus oryzae and the application in improving alcohol output thereof.
Background technology
Alcohol is a kind of important industrial raw material, is widely used in the fields such as chemical industry, food, military project and medical and health.Alcohol is again the renewable energy source that is hopeful most all or part of petroleum replacing simultaneously, has widely application and development prospect.China produces alcohol per year more than 2,000,000 tons, is third place in the world large Alcohol Production state.Along with the growth of national economy, the demand of alcohol also can increase.1 ton of alcohol of every production will give off 13-15 ton slops, and the annual quantity discharged in the whole nation approximately surpasses 1,500 ten thousand tons, becomes one of industry that contaminate environment is the most serious in the food fermentation industry.Contain a large amount of unemployed nutritive ingredients in the slops, such as carbohydrate of protein, fat, Mierocrystalline cellulose, vitamin B complex, glycerine, organic acid, fermentation remnants etc., so the recycling of slops has great application prospect.
At present, the improvement method of slops has irrigation method, oxidation pond process, method of enrichment, production Yeast protein one aerobic method, anaerobic process, anaerobism one aerobic method, physico-chemical process etc. both at home and abroad, and what wherein research and application were more is anaerobic biological treatment and anaerobic-aerobic biological treatment.Germany gram captive one hundred company develops whole backflow of potato vinasse filtrate is used for the company of technology of alcohol the earliest, and this technique is called " LBW ", and its key problem in technology is to guarantee low temperature boiling, prevents that Maillard reaction from producing objectionable impurities.Solid particulate protein fodder (DDGS) production technology is in European countries, also be widely adopted such as Germany, France, Norway etc., this technical spirit is that the separating obtained filtrate part of maize alcohol stillage solid and liquid is back to Alcohol Production, after another part evaporation concentration with solid substance combination drying DDGS processed.
Improve the reclamation rate of slops and also need further make great efforts from many aspects exploratory development, the bacterial classification that seed selection adapts to reuse inhibition high density is one of good method that improves the alcohol slops reclamation rate.
Summary of the invention
The purpose of this invention is to provide an Aspergillus oryzae and the application in improving alcohol output thereof.
The invention provides an Aspergillus oryzae (Aspergillus oryzae), called after HSC, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 13rd, 2012 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5725.Aspergillus oryzae (Aspergillus oryzae) HSC CGMCC No.5725 is called for short aspergillus oryzae HSC.
The present invention also protects a kind of method for preparing the fermented liquid of aspergillus oryzae HSC, comprises the steps:
(1) aspergillus oryzae HSC is seeded to fermention medium, 20-37 ℃ (such as 20-30 ℃ or 30-37 ℃) shaking culture 30-60 hour (30-48 hour or 48-60 hour);
(2) culture system with completing steps (1) filters collection filtrate, is aspergillus oryzae HSC fermented liquid.
Described fermention medium is comprised of solvent and solute; Described solvent can be alcohol slops, also can be comprised of alcohol slops and water; Described solute and the concentration in fermention medium thereof are as follows: 5-100g/L (such as 5-50g/L or 50-100g/L) glucose, 0.1-10g/L (such as 0.1-5g/L or 5-10g/L) KH
2PO
4And 0.1-10g/L (such as 0.1-5g/L or 5-10g/L) MgSO
47H
2O.
In the described solvent, the shared volume ratio of described alcohol slops is (such as 10%-50% or 50%-100%) more than 10%.
Described aspergillus oryzae HSC specifically can be seeded in the mode of seed liquor described fermention medium.The volume proportion of described seed liquor and described fermention medium is (1-20): (99-80), and such as (1-10): (99-90) or (10-20): (90-80).
The thalline that specifically can contain the 0.2g dry weight in the described seed liquor of every 100ml.
The rotating speed of described shaking culture can be 100-200rpm, such as 100-160rpm or 160-200rpm.
The aspergillus oryzae HSC fermented liquid that above arbitrary described method prepares all belongs to protection scope of the present invention.
The present invention also protects a kind of method for preparing the solid particulate protein fodder, comprises the steps:
(1) aspergillus oryzae HSC is seeded to fermention medium, 20-37 ℃ (such as 20-30 ℃ or 30-37 ℃) shaking culture 30-60 hour (30-48 hour or 48-60 hour);
(2) culture system with completing steps (1) filters the collection thalline, is the solid particulate protein fodder.
Described fermention medium is comprised of solvent and solute; Described solvent can be alcohol slops, also can be comprised of alcohol slops and water; Described solute and the concentration in fermention medium thereof are as follows: 5-100g/L (such as 5-50g/L or 50-100g/L) glucose, 0.1-10g/L (such as 0.1-5g/L or 5-10g/L) KH
2PO
4And 0.1-10g/L (such as 0.1-5g/L or 5-10g/L) MgSO
47H
2O.
In the described solvent, the shared volume ratio of described alcohol slops is (such as 10%-50% or 50%-100%) more than 10%.
Described aspergillus oryzae HSC specifically can be seeded in the mode of seed liquor described fermention medium.The volume proportion of described seed liquor and described fermention medium is (1-20): (99-80), and such as (1-10): (99-90) or (10-20): (90-80).
The thalline that specifically can contain the 0.2g dry weight in the described seed liquor of every 100ml.
The rotating speed of described shaking culture can be 100-200rpm, such as 100-160rpm or 160-200rpm.
The solid particulate protein fodder that above arbitrary described method prepares all belongs to protection scope of the present invention.
Aspergillus oryzae HSC can be used for producing alcohol, also can be used for promoting yeast saccharomyces cerevisiae to produce alcohol.
Described aspergillus oryzae HSC fermented liquid can be used for producing alcohol, also can be used for promoting yeast saccharomyces cerevisiae to produce alcohol.
Described yeast saccharomyces cerevisiae specifically can be Chinese industrial microbial strains preservation center and is numbered 31145 bacterial strain.
The present invention also protects a kind of method of producing alcohol, comprises the steps:
(1) 1 kg corn starch is mixed with the liquid fourth of 3-6 kilogram (such as 3-4.5 kilogram or 4.5-6 kilogram) 50-60 ℃ (such as 50-55 ℃ or 55-60 ℃), add amylase after the gelatinization, 80-100 ℃ (such as 80-90 ℃ or 90-100 ℃) hatches 1-1.5 hour (such as 1-1.2 hour or 1.2-1.5 hour); Then be cooled to 60-65 ℃ (such as 60-63 ℃ or 63-65 ℃) and add saccharifying enzyme, 60-70 ℃ (such as 60-65 ℃ or 65-70 ℃) hatches 10-15 hour (such as 10-12 hour or 12-15 hour); Filter and collection filtrate, be saccharified liquid; Described liquid fourth is mixed to get aspergillus oryzae HSC fermented liquid as described in 9-4 parts by volume (such as 9-7 parts by volume or 7-4 parts by volume) alcohol slops and the 1-6 parts by volume (such as 1-3 parts by volume or 3-6 parts by volume); Described diastatic add-on is: every gram W-Gum adds 4-30U amylase (such as 4-18U or 18-30U); The add-on of described saccharifying enzyme is: every gram W-Gum adds 50-200U saccharifying enzyme (such as 50-125U or 125-250U);
(2) in the saccharified liquid of step (1), inoculate yeast saccharomyces cerevisiae, obtain initial fermentation system, obtain alcohol after the fermentation.
Described fermentation condition specifically can be 20-40 ℃ (such as 20-30 ℃ or 30-40 ℃) and leaves standstill 30-60 hour (such as 30-45 hour or 45-60 hour) of cultivation.
In the described initial fermentation system, the concentration of described yeast saccharomyces cerevisiae can be 1 * 10
5-3 * 10
6Cfu/ml is (such as 1 * 10
5-1.5 * 10
6Cfu/ml or 1.5 * 10
6-3 * 10
6Cfu/ml).
Described yeast saccharomyces cerevisiae specifically can be seeded in the mode of seed liquor described saccharified liquid.The volume proportion of described seed liquor and described saccharified liquid is (1-30): (99-70), and such as (1-15): (99-85) or (15-30): (85-70).
Described yeast saccharomyces cerevisiae specifically can be Chinese industrial microbial strains preservation center and is numbered 31145 bacterial strain.
Above arbitrary described alcohol slops can be the alcohol slops of laboratory preparation, also can be brewery and produces the discarded alcohol slops of alcohol.
The laboratory prepares the concrete grammar of alcohol slops can be as follows:
(1) 50-60 ℃ of water of 1 kg corn starch and 3-6 kilogram is mixed, add amylase after the gelatinization, hatched 1-1.5 hour for 80-100 ℃; Then be cooled to 60-65 ℃ and add saccharifying enzyme, hatched 10-15 hour for 60-70 ℃; Filter and collection filtrate, be saccharified liquid; Described diastatic add-on is: every gram W-Gum adds 4-30U amylase; The add-on of described saccharifying enzyme is: every gram W-Gum adds the 50-200U saccharifying enzyme;
(2) yeast saccharomyces cerevisiae is inoculated in the saccharified liquid of step (1), obtains initial fermentation system, 20-40 ℃ leaves standstill and cultivated 30-60 hour.
(3) with the culture system distillation of completing steps (2), remaining liq is alcohol slops.
In the described initial fermentation system, the concentration of described yeast saccharomyces cerevisiae is 1 * 10
5-3 * 10
6Cfu/ml is (such as 1 * 10
5-1.5 * 10
6Cfu/ml or 1.5 * 10
6-3 * 10
6Cfu/ml).
Described yeast saccharomyces cerevisiae specifically can be seeded in the mode of seed liquor described saccharified liquid.The volume proportion of described seed liquor and described saccharified liquid is (1-30): (99-70).
Described yeast saccharomyces cerevisiae specifically can be Chinese industrial microbial strains preservation center and is numbered 31145 bacterial strain.
Described distillation specifically can be 50 ℃ of distillation 20min.
Described method can be introduced alcohol preparation technology, namely on the basis of adding described fermented liquid alcohol slops is circulated as raw material.
Adopt method of the present invention to cultivate aspergillus oryzae HSC, can produce animal feeding-stuff containing somatic protein on the one hand, can obtain fermented liquid on the one hand.Described fermented liquid can be used for producing alcohol, greatly improves the alcohol output of yeast saccharomyces cerevisiae when utilizing alcohol slops.The present invention has great economic benefit and social benefit.
Description of drawings
Fig. 1 is the typical curve of peak area and ethanol concn.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Amylase: 1U represents and transforms the 1mg Zulkovsky starch under 70 ℃, pH6.0 condition in the 1min and generate the required enzyme amount of dextrin; Beijing extensive and profound in meaning star biotechnology limited liability company, lot number 20110919.
Saccharifying enzyme: 1U represents and transforms Zulkovsky starch under 40 ℃, pH4.6 condition in the 1min and generate the required enzyme amount of 1mg glucose; Beijing extensive and profound in meaning star biotechnology limited liability company (lot number 20110902).
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) used among the embodiment is following yeast: available from Chinese industrial microbial strains preservation center (
Http:// www.china-cicc.org/), be numbered 31145.
The acquisition of embodiment 1, aspergillus oryzae and evaluation
One, the acquisition of aspergillus oryzae
From near the soil sampling brewery.
1, take by weighing 5.0g soil in the aseptic enrichment medium of 45ml, add an amount of granulated glass sphere jolting number minute after, put into 30 ℃ of constant incubator enrichment culture 24h.
Enrichment medium: peptone 5g/L, yeast extract paste 5g/L, sucrose 20g/L, Zulkovsky starch 10g/L, K
2HPO
42g/L, ZnSO
40.4g/L, CuSO
40.05g/L, MgSO
40.01g/L all the other are water, adding penicillin when being cooled to 50 ℃ after the sterilization, to make its concentration be 1U/ml.
2, nutrient solution is carried out gradient dilution with aseptic 0.9% physiological saline, get 10
-4, 10
-5, 10
-6Three gradients, employing dilution flat band method separates at the isolation and purification culture base.In 30 ℃ of constant incubators, cultivate 24h.
Isolation and purification culture base: 2g agar is added alcohol slops and is settled to 100mL with alcohol slops.
3, with each single bacterium colony of aseptic inoculation ring picking respectively at diluting in aseptic 0.9% physiological saline, be inoculated in respectively afterwards and carry out single bacterium colony on the isolation and purification culture base and cultivate, repeated multiple times after until in the plate colonial morphology consistent.
4, with each single bacterium colony of aseptic inoculation ring picking respectively at diluting in aseptic 0.9% physiological saline, be inoculated into multiple sieve substratum with 10% (volume ratio) afterwards, 48h is cultivated in jolting under 30 ℃, pH5.5,160rpm condition.
Sieve again substratum: 50% (volume ratio) alcohol slops, 5g/L glucose, 2g/L KH
2PO
4, 2g/L MgSO
47H
2O, all the other are water.
5, thalline in the fermented liquid is filtered, weigh after the oven dry, determine the mould that in alcohol slops, can well grow.
6, carry out preliminary classification according to colony morphology characteristic and microscopic examination.
Obtain a strain bacterial strain, called after HSC.
Two, the evaluation of aspergillus oryzae
1, morphological specificity
Bacterial strain is very fast in the growth of isolation and purification culture base, and bacterium colony just becomes white, rear yellowing or yellow-green colour, and the back is colourless.Can observe at microscopically, mycelia is flourishing, and barrier film is arranged, and the mycelia top directly produces sporophore, and conidium is concatenated.With reference to " fungi identification handbook ", this bacterial strain tentatively is defined as Aspergillus.
2, Molecular Identification
Carry out the analysis of 26SrDNS ITS region sequence, sequencing result is seen the sequence 1 of sequence table.Sequencing result and ncbi database Blast are compared, determined that this bacterium and aspergillus oryzae sequence (HQ285587.1) similarity are very high, its homology reaches 99%, can determine that this Pseudomonas is in aspergillus oryzae (Aspergillus oryzae).
Aspergillus oryzae provided by the invention (Aspergillus oryzae) HSC, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 13rd, 2012 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5725.Aspergillus oryzae (Aspergillus oryzae) HSC CGMCC No.5725 is called for short aspergillus oryzae HSC.
Embodiment 2, the aspergillus oryzae HSC application in the alcohol slops recycling
One, the preparation of alcohol slops
1, the water of 1 kg corn starch with 3 kilograms 50 ℃ is mixed, add amylase (every gram W-Gum adds 4U amylase) after the gelatinization, hatch 1h for 80 ℃; Then be cooled to 60 ℃ and add saccharifying enzyme (every gram W-Gum add 50U saccharifying enzyme), hatch 10h for 60 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
2, yeast saccharomyces cerevisiae is seeded in the YPD substratum, 30 ℃, 160rpm shaking culture (shimmy amplitude 25mm) 24h obtain seed liquor.
3,1 parts by volume seed liquor is seeded in the 99 parts by volume saccharified liquids, (concentration of yeast saccharomyces cerevisiae is 1 * 10 in the initial fermentation system to obtain initial fermentation system
5Cfu/ml); 20 ℃ of initial fermentation systems are left standstill cultivation 30h, obtain stopping fermentation system.
4, will stop 50 ℃ of distillations of fermentation system 20min, remaining liq is alcohol slops.
Two, the preparation of aspergillus oryzae fermentation supernatant
1, aspergillus oryzae HSC is cultivated in the PDA substratum, obtain seed liquor (every 100ml seed liquor contains the thalline of 0.2g dry weight).
2, the seed liquor that step 1 is obtained is seeded in the fermention medium (volume proportion of seed liquor and fermention medium is 1: 99), 20 ℃, 100rpm shaking culture (shimmy amplitude 25mm) 30h.
Fermention medium is comprised of solvent and solute; Described solvent is mixed to get 9 parts by volume water and 1 parts by volume alcohol slops; Described solute and the concentration in fermention medium thereof are as follows: 5g/L glucose, 0.1g/L KH
2PO
4, 0.1g/L MgSO
47H
2O.
3, with the culture system of step 2 with four layers of filtered through gauze, collect respectively filtrate (aspergillus oryzae fermented liquid) and thalline.
4, the thalline that step 3 is obtained is dried to constant weight, is animal feeding-stuff containing somatic protein, and per 100 milliliters of culture systems have obtained the 1.78g animal feeding-stuff containing somatic protein.
Three, the application of aspergillus oryzae aspect the alcohol slops recycling
1, the application of aspergillus oryzae aspect the alcohol slops recycling
(1) 1 kg corn starch is mixed with 3 kilograms 50 ℃ liquid first (being comprised of with 1 parts by volume aspergillus oryzae fermented liquid 9 parts by volume alcohol slopss), add amylase (every gram W-Gum adds 4U amylase) after the gelatinization, hatch 1h for 80 ℃; Then be cooled to 60 ℃, add saccharifying enzyme (every gram W-Gum adds the 50U saccharifying enzyme), hatch 10h for 60 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
(2) yeast saccharomyces cerevisiae is seeded in the YPD substratum, in the YPD substratum, cultivates, obtain seed liquor.
(3) 1 parts by volume seed liquor is seeded in the 99 parts by volume saccharified liquids, (concentration of yeast saccharomyces cerevisiae is 1 * 10 in the initial fermentation system to obtain initial fermentation system
5Cfu/ml); 20 ℃ of initial fermentation systems are left standstill cultivation 30h, obtain stopping fermentation system.
(4) the culture system 4000rpm with completing steps (3) centrifugal (centrifugal radius is 13.5cm) 10min collects supernatant liquor and with biofilter (0.22 μ m) filtration, obtains cell-free filtrate.
2, controlled trial first
(1) alcohol slops of 1 kg corn starch with 3 kilograms 50 ℃ mixed, add amylase (every gram W-Gum adds 4U amylase) after the gelatinization, hatch 1h for 80 ℃; Then be cooled to 60 ℃, add saccharifying enzyme (every gram W-Gum adds the 50U saccharifying enzyme), hatch 10h for 60 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, controlled trial second
(1) water of 1 kg corn starch with 3 kilograms 50 ℃ is mixed, add amylase (every gram W-Gum adds 4U amylase) after the gelatinization, hatch 1h for 80 ℃; Then be cooled to 60 ℃, add saccharifying enzyme (every gram W-Gum adds the 50U saccharifying enzyme), hatch 10h for 60 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, the ethanol content in the cell-free filtrate is measured
Adopt the ethanol content in the HPLC detection cell-free filtrate.HPLC parameter: instrument: Agilent 1200; Chromatographic column: Rezex ROA and corresponding guard column; Detector is the differential detector; Sample size 20 μ L; Moving phase is 0.005MH
2SO
4The aqueous solution; Flow velocity: 0.6ml/min; Column temperature: 65 ℃.Adopt standards of alcohol product production standard curve, the retention time of alcohol is 22.02min, and the typical curve of peak area and ethanol concn sees that (the typical curve equation is Fig. 1: Y=141027.5X-11941.4; R
2=0.9991).By the ethanol content in the typical curve calculating cell-free filtrate.
Ethanol content in the cell-free filtrate that step 1 obtains is 30.04mg/ml, compares with the ethanol content in the cell-free filtrate that step 2 obtains and has improved 6.25%, compares with the ethanol content in the cell-free filtrate that step 3 obtains and has improved 4.57%.
Embodiment 3, the application of aspergillus oryzae in the alcohol slops recycling
One, the preparation of alcohol slops
1, the water of 1 kg corn starch with 6 kilograms 60 ℃ is mixed, add amylase (every gram W-Gum adds 30U amylase) after the gelatinization, hatch 1.5h for 100 ℃; Then be cooled to 65 ℃, add saccharifying enzyme (every gram W-Gum adds the 200U saccharifying enzyme), hatch 15h for 70 ℃, four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
2, yeast saccharomyces cerevisiae is seeded in the YPD substratum, 30 ℃, 160rpm shaking culture (shimmy amplitude 25mm) 24h obtain seed liquor.
3,30 parts by volume seed liquor are seeded in the 70 parts by volume saccharified liquids, (concentration of yeast saccharomyces cerevisiae is 3 * 10 in the initial fermentation system to obtain initial fermentation system
6Cfu/ml); 40 ℃ of initial fermentation systems are left standstill cultivation 60h, obtain stopping fermentation system.
4, will stop 50 ℃ of distillations of fermentation system 20min, remaining liq is alcohol slops.
Two, the preparation of aspergillus oryzae fermentation supernatant
1, aspergillus oryzae HSC is cultivated in the PDA substratum, obtain seed liquor (every 100ml seed liquor contains the thalline of 0.2g dry weight).
2, the seed liquor that step 1 is obtained is seeded in the fermention medium (volume proportion of seed liquor and fermention medium is 20: 80), 37 ℃, 200rpm shaking culture (shimmy amplitude 25mm) 60h.
Fermention medium is comprised of solvent and solute; Described solvent is alcohol slops; Described solute and the concentration in fermention medium thereof are as follows: 100g/L glucose, 10g/L KH
2PO
4, 10g/L MgSO
47H
2O.
3, with the culture system of step 2 with four layers of filtered through gauze, collect respectively filtrate (aspergillus oryzae fermented liquid) and thalline.
4, the thalline that step 3 is obtained is dried to constant weight, is animal feeding-stuff containing somatic protein, and per 100 milliliters of culture systems have obtained the 2.3g animal feeding-stuff containing somatic protein.
Three, the application of aspergillus oryzae aspect the alcohol slops recycling
1, the application of aspergillus oryzae aspect the alcohol slops recycling
(1) 1 kg corn starch is mixed with 6 kilograms 60 ℃ liquid second (being comprised of with 6 parts by volume aspergillus oryzae fermented liquids 4 parts by volume alcohol slopss), add amylase (every gram W-Gum adds 30U amylase) after the gelatinization, hatch 1.5h for 100 ℃; Then be cooled to 65 ℃, add saccharifying enzyme (every gram W-Gum adds the 200U saccharifying enzyme), hatch 15h for 70 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
(2) yeast saccharomyces cerevisiae is seeded in the YPD substratum, in the YPD substratum, cultivates, obtain seed liquor.
(3) 30 parts by volume seed liquor are seeded in the 70 parts by volume saccharified liquids, (concentration of yeast saccharomyces cerevisiae is 3 * 10 in the initial fermentation system to obtain initial fermentation system
6Cfu/ml); 40 ℃ of initial fermentation systems are left standstill cultivation 60h, obtain stopping fermentation system.
(4) the culture system 4000rpm with completing steps (3) centrifugal (centrifugal radius is 13.5cm) 10min collects supernatant liquor and with biofilter (0.22 μ m) filtration, obtains cell-free filtrate.
2, controlled trial first
(1) alcohol slops of 1 kg corn starch with 6 kilograms 60 ℃ mixed, add amylase (every gram W-Gum adds 30U amylase) after the gelatinization, hatch 1.5h for 100 ℃; Then be cooled to 65 ℃, add saccharifying enzyme (every gram W-Gum adds the 200U saccharifying enzyme), hatch 15h for 70 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, controlled trial second
(1) water of 1 kg corn starch with 6 kilograms 60 ℃ is mixed, add amylase (every gram W-Gum adds 30U amylase) after the gelatinization, hatch 1.5h for 100 ℃; Then be cooled to 65 ℃, add saccharifying enzyme (every gram W-Gum adds the 200U saccharifying enzyme), hatch 15h for 70 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, the ethanol content in the cell-free filtrate is measured
With 3 of the step 3 of embodiment 2.
Ethanol content in the cell-free filtrate that step 1 obtains is 21.76mg/ml, compares with the ethanol content in the cell-free filtrate that step 2 obtains and has improved 11.2%, compares with the ethanol content in the cell-free filtrate that step 3 obtains and has improved 10.1%.
Embodiment 4, the application of aspergillus oryzae aspect the alcohol slops recycling
Alcohol slops in the present embodiment is available from Jilin Fuel Ethanol Co.,Ltd, and concrete component sees Table 1.
The Product Quality Verification Centers examining report of table 1 Jilin Fuel Ethanol Co.,Ltd alcohol slops
| 8:00 | 13:00 | |
| Polysaccharide (g/L) | 34.20 | 35.65 |
| Trisaccharide maltose (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 |
| Pectinose (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 |
| Methyl alcohol 9g/L) | 46.7 | 49.55 |
| Ethanol (g/L) | Nothing | Nothing |
One, the preparation of aspergillus oryzae fermentation supernatant
1, aspergillus oryzae HSC is cultivated in the PDA substratum, obtain seed liquor (every 100ml seed liquor contains the thalline of 0.2g dry weight).
2, the seed liquor that step 1 is obtained is seeded in the fermention medium (volume proportion of seed liquor and fermention medium is 10: 90), 30 ℃, 160rpm shaking culture (shimmy amplitude 25mm) 48h.
Fermention medium is comprised of solvent and solute; Described solvent is mixed to get 5 parts by volume water and 5 parts by volume alcohol slopss; Described solute and the concentration in fermention medium thereof are as follows: 50g/L glucose, 5g/L KH
2PO
4, 5g/L MgSO
47H
2O.
3, with the culture system of step 2 with four layers of filtered through gauze, collect respectively filtrate (aspergillus oryzae fermented liquid) and thalline.
4, the thalline that step 3 is obtained is dried to constant weight, is animal feeding-stuff containing somatic protein, and per 100 milliliters of culture systems have obtained the 2.96g animal feeding-stuff containing somatic protein.
Two, the application of aspergillus oryzae in the alcohol slops recycling
1, the application of aspergillus oryzae aspect the alcohol slops recycling
(1) liquid third (7 parts by volume alcohol slopss with 3 parts by volume aspergillus oryzae fermented liquids be made of) of 1 kg corn starch with 4.5 kilograms 55 ℃ is mixed, add amylase (every gram W-Gum adds 18U amylase) after the gelatinization, hatch 1.2h for 90 ℃; Then be cooled to 63 ℃, add saccharifying enzyme (every gram W-Gum adds the 125U saccharifying enzyme), hatch 12h for 65 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
(2) yeast saccharomyces cerevisiae is seeded in the YPD substratum, in the YPD substratum, cultivates, obtain seed liquor.
(3) 15 parts by volume seed liquor are seeded in the 85 parts by volume saccharified liquids, (concentration of yeast saccharomyces cerevisiae is 1.5 * 10 in the initial fermentation system to obtain initial fermentation system
6Cfu/ml); 30 ℃ of initial fermentation systems are left standstill cultivation 45h, obtain stopping fermentation system.
(4) the culture system 4000rpm with completing steps (3) centrifugal (centrifugal radius is 13.5cm) 10min collects supernatant liquor and with biofilter (0.22 μ m) filtration, obtains cell-free filtrate.
2, controlled trial first
(1) alcohol slops of 1 kg corn starch with 4.5 kilograms 55 ℃ mixed, add amylase (every gram W-Gum adds 18U amylase) after the gelatinization, hatch 1.2h for 90 ℃; Then be cooled to 63 ℃, add saccharifying enzyme (every gram mixture adds the 125U saccharifying enzyme), hatch 12h for 65 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, controlled trial second
(1) water of 1 kg corn starch with 4.5 kilograms 55 ℃ is mixed, add amylase (every gram W-Gum adds 18U amylase) after the gelatinization, hatch 1.2h for 90 ℃; Then be cooled to 63 ℃, add saccharifying enzyme (every gram mixture adds the 125U saccharifying enzyme), hatch 12h for 65 ℃; Four layers of filtered through gauze are also collected filtrate, are saccharified liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, the ethanol content in the cell-free filtrate is measured
With 3 of the step 3 of embodiment 2.
Ethanol content in the cell-free filtrate that step 1 obtains is 24.35mg/ml, compares with the ethanol content in the cell-free filtrate that step 2 obtains and has improved 6.8%, compares with the ethanol content in the cell-free filtrate that step 3 obtains and has improved 5.9%.
Claims (10)
1. aspergillus oryzae (Aspergillus oryzae) HSC, its deposit number is CGMCC No.5725.
2. a method for preparing the fermented liquid of aspergillus oryzae HSC comprises the steps:
(1) the described aspergillus oryzae HSC of claim 1 is seeded to fermention medium, 20-37 ℃ shaking culture 30-60 hour;
(2) culture system with completing steps (1) filters collection filtrate, is aspergillus oryzae HSC fermented liquid.
3. method as claimed in claim 2, it is characterized in that: described fermention medium is comprised of solvent and solute; Described solvent is that alcohol slops or described solvent are comprised of alcohol slops and water; The concentration of described solute in fermention medium is as follows: 5-100g/L glucose, 0.1-10g/L KH
2PO
4With 0.1-10g/L MgSO
47H
2O.
4. the aspergillus oryzae HSC fermented liquid for preparing of claim 2 or 3 described methods.
5. a method for preparing the solid particulate protein fodder comprises the steps:
(1) the described aspergillus oryzae HSC of claim 1 is seeded to fermention medium, 20-37 ℃ shaking culture 30-60 hour;
(2) culture system with completing steps (1) filters the collection thalline, is the solid particulate protein fodder.
6. method as claimed in claim 5, it is characterized in that: described fermention medium is comprised of solvent and solute; Described solvent is that alcohol slops or described solvent are comprised of alcohol slops and water; The concentration of described solute in fermention medium is as follows: 5-100g/L glucose, 0.1-10g/L KH
2PO
4With 0.1-10g/L MgSO
47H
2O.
7. the solid particulate protein fodder for preparing of claim 5 or 6 described methods.
8. the described aspergillus oryzae HSC of claim 1 or the described aspergillus oryzae HSC of the claim 4 fermented liquid application in producing alcohol.
9. the described aspergillus oryzae HSC of claim 1 or the described aspergillus oryzae HSC of claim 4 fermented liquid are promoting yeast saccharomyces cerevisiae to produce the application in the alcohol.
10. a method of producing alcohol comprises the steps:
(1) the liquid fourth of 50-60 ℃ of 1 kg corn starch and 3-6 kilogram is mixed, add amylase after the gelatinization, hatched 1-1.5 hour for 80-100 ℃; Then be cooled to 60-65 ℃ and add saccharifying enzyme, hatched 10-15 hour for 60-70 ℃; Filter and collection filtrate, be saccharified liquid; Described liquid fourth is mixed to get 9-4 parts by volume alcohol slops and the described aspergillus oryzae HSC of 1-6 parts by volume claim 4 fermented liquid; Described diastatic add-on is: every gram W-Gum adds 4-30U amylase; The add-on of described saccharifying enzyme is: every gram W-Gum adds the 50-200U saccharifying enzyme;
(2) in the saccharified liquid of step (1), inoculate yeast saccharomyces cerevisiae, obtain initial fermentation system, obtain alcohol after the fermentation.
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| CN1884476A (en) * | 2006-06-06 | 2006-12-27 | 李怀宝 | Aspergillus oryzae bacteria and its application |
| CN101942396A (en) * | 2010-09-06 | 2011-01-12 | 华南理工大学 | Aspergillus oryzae HG76 and application thereof |
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| CN1884476A (en) * | 2006-06-06 | 2006-12-27 | 李怀宝 | Aspergillus oryzae bacteria and its application |
| CN101942396A (en) * | 2010-09-06 | 2011-01-12 | 华南理工大学 | Aspergillus oryzae HG76 and application thereof |
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