CN102643754A - 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 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 alternative oil simultaneously, has to use widely and development prospect.China produces alcohol per year more than 2,000,000 tons, is third place in the world big ethanol produce 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 surpasses 1,500 ten thousand tons approximately, 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, like the remaining glucide of protein, fat, Mierocrystalline cellulose, vitamin B complex, glycerine, organic acid, fermentation 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 filtrating is used for the company of technology of alcohol the earliest, and this technology 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; Like Germany, France, Norway etc. also by extensive employing; This technical spirit is that the separating obtained filtrating part of maize alcohol stillage solid and liquid is back to ethanol produce, after another part evaporation concentration with solid substance combination drying system DDGS.
Improve the reclamation rate of slops and also need further make great efforts exploratory development from many aspects, 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 ℃ (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 filtrating, is aspergillus oryzae HSC fermented liquid.
Said fermention medium is made up of solvent and solute; Said solvent can be alcohol slops, also can be made up of alcohol slops and water; Said solute and the concentration in fermention medium thereof are following: 5-100g/L (like 5-50g/L or 50-100g/L) glucose, 0.1-10g/L (like 0.1-5g/L or 5-10g/L) KH
2PO
4And 0.1-10g/L (like 0.1-5g/L or 5-10g/L) MgSO
47H
2O.
In the said solvent, the shared volume ratio of said alcohol slops is (like 10%-50% or 50%-100%) more than 10%.
Said aspergillus oryzae HSC specifically can be seeded to said fermention medium with the mode of seed liquor.The volume proportion of said seed liquor and said fermention medium is (1-20): (99-80), and like (1-10): (99-90) or (10-20): (90-80).
The thalline that specifically can contain the 0.2g dry weight in the said seed liquor of every 100ml.
The rotating speed of said shaking culture can be 100-200rpm, like 100-160rpm or 160-200rpm.
More than the aspergillus oryzae HSC fermented liquid for preparing of arbitrary said method all belong 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 ℃ (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.
Said fermention medium is made up of solvent and solute; Said solvent can be alcohol slops, also can be made up of alcohol slops and water; Said solute and the concentration in fermention medium thereof are following: 5-100g/L (like 5-50g/L or 50-100g/L) glucose, 0.1-10g/L (like 0.1-5g/L or 5-10g/L) KH
2PO
4And 0.1-10g/L (like 0.1-5g/L or 5-10g/L) MgSO
47H
2O.
In the said solvent, the shared volume ratio of said alcohol slops is (like 10%-50% or 50%-100%) more than 10%.
Said aspergillus oryzae HSC specifically can be seeded to said fermention medium with the mode of seed liquor.The volume proportion of said seed liquor and said fermention medium is (1-20): (99-80), and like (1-10): (99-90) or (10-20): (90-80).
The thalline that specifically can contain the 0.2g dry weight in the said seed liquor of every 100ml.
The rotating speed of said shaking culture can be 100-200rpm, like 100-160rpm or 160-200rpm.
More than the solid particulate protein fodder for preparing of arbitrary said method all belong 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.
Said aspergillus oryzae HSC fermented liquid can be used for producing alcohol, also can be used for promoting yeast saccharomyces cerevisiae to produce alcohol.
Said 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 (like 3-4.5 kilogram or 4.5-6 kilogram) 50-60 ℃ (as 50-55 ℃ or 55-60 ℃); Add glycase after the gelatinization, 80-100 ℃ (as 80-90 ℃ or 90-100 ℃) hatched 1-1.5 hour (as 1-1.2 hour or 1.2-1.5 hour); Be cooled to 60-65 ℃ (as 60-63 ℃ or 63-65 ℃) then and add saccharifying enzyme, 60-70 ℃ (as 60-65 ℃ or 65-70 ℃) hatched 10-15 hour (as 10-12 hour or 12-15 hour); Filter and collect and filtrate, be saccharification liquid; Said liquid fourth obtains 9-4 parts by volume (like 9-7 parts by volume or 7-4 parts by volume) alcohol slops and the said aspergillus oryzae HSC fermented liquid mixing of 1-6 parts by volume (like 1-3 parts by volume or 3-6 parts by volume); Said diastatic add-on is: every gram W-Gum adds 4-30U glycase (like 4-18U or 18-30U); The add-on of said saccharifying enzyme is: every gram W-Gum adds 50-200U saccharifying enzyme (like 50-125U or 125-250U);
(2) in the saccharification liquid of step (1), inoculate yeast saccharomyces cerevisiae, obtain initial fermentation system, obtain alcohol after the fermentation.
Said fermentation condition specifically can be 20-40 ℃ (as 20-30 ℃ or 30-40 ℃) and leaves standstill 30-60 hour (as 30-45 hour or 45-60 hour) of cultivation.
In the said initial fermentation system, the concentration of said yeast saccharomyces cerevisiae can be 1 * 10
5-3 * 10
6Cfu/ml is (as 1 * 10
5-1.5 * 10
6Cfu/ml or 1.5 * 10
6-3 * 10
6Cfu/ml).
Said yeast saccharomyces cerevisiae specifically can be seeded to said saccharification liquid with the mode of seed liquor.The volume proportion of said seed liquor and said saccharification liquid is (1-30): (99-70), and like (1-15): (99-85) or (15-30): (85-70).
Said yeast saccharomyces cerevisiae specifically can be Chinese industrial microbial strains preservation center and is numbered 31145 bacterial strain.
More than arbitrary said alcohol slops can be the alcohol slops of prepared in laboratory, also can be brewery and produce alcohol depleted alcohol slops.
The concrete grammar of prepared in laboratory alcohol slops can be following:
(1) 50-60 ℃ of water of 1 kg corn starch and 3-6 kilogram is mixed, add glycase after the gelatinization, hatched 1-1.5 hour for 80-100 ℃; Be cooled to 60-65 ℃ and add saccharifying enzyme then, hatched 10-15 hour for 60-70 ℃; Filter and collect and filtrate, be saccharification liquid; Said diastatic add-on is: every gram W-Gum adds 4-30U glycase; The add-on of said saccharifying enzyme is: every gram W-Gum adds the 50-200U saccharifying enzyme;
(2) yeast saccharomyces cerevisiae is inoculated in the saccharification 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 said initial fermentation system, the concentration of said yeast saccharomyces cerevisiae is 1 * 10
5-3 * 10
6Cfu/ml is (as 1 * 10
5-1.5 * 10
6Cfu/ml or 1.5 * 10
6-3 * 10
6Cfu/ml).
Said yeast saccharomyces cerevisiae specifically can be seeded to said saccharification liquid with the mode of seed liquor.The volume proportion of said seed liquor and said saccharification liquid is (1-30): (99-70).
Said yeast saccharomyces cerevisiae specifically can be Chinese industrial microbial strains preservation center and is numbered 31145 bacterial strain.
Said distillation specifically can be 50 ℃ of distillation 20min.
Said method can be introduced alcohol preparation technology, promptly on the basis of adding said 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.Said fermented liquid can be used for producing alcohol, improves the alcohol output of yeast saccharomyces cerevisiae when utilizing alcohol slops greatly.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 like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.
Glycase: 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 Ltd, 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 Ltd (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 penicillium mould during sterilization postcooling to 50 ℃, to make its concentration be 1U/ml.
2, nutrient solution is carried out gradient dilution with aseptic 0.9% saline water, get 10
-4, 10
-5, 10
-6Three gradients, employing dilution flat band method separates on the separation and purification substratum.In 30 ℃ of constant incubators, cultivate 24h.
Separation and purification substratum: 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% saline water, be inoculated in respectively afterwards and carry out single bacterium colony on the separation and purification substratum and cultivate, after the repeated multiple times in plate colonial morphology consistent.
4, with each single bacterium colony of aseptic inoculation ring picking respectively at diluting in aseptic 0.9% saline water, be inoculated into multiple sieve substratum with 10% (volume ratio) afterwards, 48h is cultivated in jolting under 30 ℃, pH5.5,160rpm condition.
Sieve substratum again: 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, confirm 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 grown on the separation and purification substratum comparatively fast, and bacterium colony just becomes white, back 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 is tentatively confirmed as Aspergillus.
2, Molecular Identification
Carry out the analysis of 26SrDNS ITS region sequence, sequencing result is seen the sequence 1 of sequence table.With sequencing result and ncbi database Blast comparison, confirm that this bacterium and aspergillus oryzae sequence (HQ285587.1) similarity are very high, its homology reaches 99%, can confirm 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.
One, the preparation of alcohol slops
1, the water of 1 kg corn starch with 3 kilograms 50 ℃ is mixed, add glycase (every gram W-Gum adds 4U glycase) after the gelatinization, hatch 1h for 80 ℃; Be cooled to 60 ℃ and add saccharifying enzyme (every gram W-Gum add 50U saccharifying enzyme) then, hatch 10h for 60 ℃; Four layers of filtered through gauze are also collected filtrating, are saccharification 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 saccharification liquid, (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 made up of solvent and solute; Said solvent obtains 9 parts by volume water and the mixing of 1 parts by volume alcohol slops; Said solute and the concentration in fermention medium thereof are following: 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 filtrating (aspergillus oryzae fermented liquid) and thalline respectively.
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, aspergillus oryzae is in the application of alcohol slops aspect utilizing again
1, aspergillus oryzae is in the application of alcohol slops aspect utilizing again
(1) 1 kg corn starch is mixed with 3 kilograms 50 ℃ liquid first (being made up of with 1 parts by volume aspergillus oryzae fermented liquid 9 parts by volume alcohol slopss), add glycase (every gram W-Gum adds 4U glycase) after the gelatinization, hatch 1h for 80 ℃; Be cooled to 60 ℃ then, 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 filtrating, are saccharification 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 saccharification liquid, (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 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 glycase (every gram W-Gum adds 4U glycase) after the gelatinization, hatch 1h for 80 ℃; Be cooled to 60 ℃ then, 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 filtrating, are saccharification 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 glycase (every gram W-Gum adds 4U glycase) after the gelatinization, hatch 1h for 80 ℃; Be cooled to 60 ℃ then, 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 filtrating, are saccharification liquid.
Step (1) to (4) with (2) of step 1 to (4).
3, the ethanol content in the cell-free filtrate is measured
Adopt HPLC to detect the ethanol content in the cell-free filtrate.HPLC parameter: instrument: Agilent 1200; Chromatographic column: Rezex ROA and corresponding protection post; 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 alcohol standard substance production standard curve, the RT 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).Calculate the ethanol content in the cell-free filtrate through typical curve.
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 alcohol slops utilizes again
One, the preparation of alcohol slops
1, the water of 1 kg corn starch with 6 kilograms 60 ℃ is mixed, add glycase (every gram W-Gum adds 30U glycase) after the gelatinization, hatch 1.5h for 100 ℃; Be cooled to 65 ℃ then, 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 filtrating, are saccharification 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 saccharification liquid, (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 made up of solvent and solute; Said solvent is an alcohol slops; Said solute and the concentration in fermention medium thereof are following: 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 filtrating (aspergillus oryzae fermented liquid) and thalline respectively.
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, aspergillus oryzae is in the application of alcohol slops aspect utilizing again
1, aspergillus oryzae is in the application of alcohol slops aspect utilizing again
(1) 1 kg corn starch is mixed with 6 kilograms 60 ℃ liquid second (being made up of with 6 parts by volume aspergillus oryzae fermented liquids 4 parts by volume alcohol slopss), add glycase (every gram W-Gum adds 30U glycase) after the gelatinization, hatch 1.5h for 100 ℃; Be cooled to 65 ℃ then, 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 filtrating, are saccharification 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 saccharification liquid, (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 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 glycase (every gram W-Gum adds 30U glycase) after the gelatinization, hatch 1.5h for 100 ℃; Be cooled to 65 ℃ then, 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 filtrating, are saccharification 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 glycase (every gram W-Gum adds 30U glycase) after the gelatinization, hatch 1.5h for 100 ℃; Be cooled to 65 ℃ then, 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 filtrating, are saccharification 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%.
Alcohol slops in the present embodiment is available from Jilin Fuel Ethanol Co.,Ltd, and concrete component is seen table 1.
The quality inspection center 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 |
| SANMALT-S (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 |
| USP Kosher (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) | Do not have | Do not have |
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 made up of solvent and solute; Said solvent obtains 5 parts by volume water and the mixing of 5 parts by volume alcohol slopss; Said solute and the concentration in fermention medium thereof are following: 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 filtrating (aspergillus oryzae fermented liquid) and thalline respectively.
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 alcohol slops utilizes again
1, aspergillus oryzae is in the application of alcohol slops aspect utilizing again
(1) liquid third (7 parts by volume alcohol slopss with 3 parts by volume aspergillus oryzae fermented liquids be made up of) of 1 kg corn starch with 4.5 kilograms 55 ℃ is mixed, add glycase (every gram W-Gum adds 18U glycase) after the gelatinization, hatch 1.2h for 90 ℃; Be cooled to 63 ℃ then, 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 filtrating, are saccharification 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 saccharification liquid, (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 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 glycase (every gram W-Gum adds 18U glycase) after the gelatinization, hatch 1.2h for 90 ℃; Be cooled to 63 ℃ then, add saccharifying enzyme (every gram mixture adds the 125U saccharifying enzyme), hatch 12h for 65 ℃; Four layers of filtered through gauze are also collected filtrating, are saccharification 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 glycase (every gram W-Gum adds 18U glycase) after the gelatinization, hatch 1.2h for 90 ℃; Be cooled to 63 ℃ then, add saccharifying enzyme (every gram mixture adds the 125U saccharifying enzyme), hatch 12h for 65 ℃; Four layers of filtered through gauze are also collected filtrating, are saccharification 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 said 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 filtrating, is aspergillus oryzae HSC fermented liquid.
3. method as claimed in claim 2 is characterized in that: said fermention medium is made up of solvent and solute; Said solvent is that alcohol slops or said solvent are made up of alcohol slops and water; Said solute and the concentration in fermention medium thereof are following: 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 said methods.
5. a method for preparing the solid particulate protein fodder comprises the steps:
(1) the said 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 is characterized in that: said fermention medium is made up of solvent and solute; Said solvent is that alcohol slops or said solvent are made up of alcohol slops and water; Said solute and the concentration in fermention medium thereof are following: 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 said methods.
8. said aspergillus oryzae HSC of claim 1 or the said aspergillus oryzae HSC of the claim 4 fermented liquid application in producing alcohol.
9. said aspergillus oryzae HSC of claim 1 or the said 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 glycase after the gelatinization, hatched 1-1.5 hour for 80-100 ℃; Be cooled to 60-65 ℃ and add saccharifying enzyme then, hatched 10-15 hour for 60-70 ℃; Filter and collect and filtrate, be saccharification liquid; Said liquid fourth obtains 9-4 parts by volume alcohol slops and the mixing of the said aspergillus oryzae HSC of 1-6 parts by volume claim 4 fermented liquid; Said diastatic add-on is: every gram W-Gum adds 4-30U glycase; The add-on of said saccharifying enzyme is: every gram W-Gum adds the 50-200U saccharifying enzyme;
(2) in the saccharification liquid of step (1), inoculate yeast saccharomyces cerevisiae, obtain initial fermentation system, obtain alcohol after the fermentation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695340A (en) * | 2016-03-04 | 2016-06-22 | 北京工商大学 | Aspergillus oryzae and application thereof |
| CN108865911A (en) * | 2018-09-21 | 2018-11-23 | 侯军 | It is a kind of for improve wine brewing distillation yield leavening and preparation method thereof |
| CN114231573A (en) * | 2021-12-27 | 2022-03-25 | 南京中医药大学 | Method for producing essence of heaven by using aspergillus oryzae genetic engineering bacteria |
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|---|---|---|---|---|
| CN1869195A (en) * | 2006-06-06 | 2006-11-29 | 李怀宝 | Aspergillus oryzae bacteria and its application |
| 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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2012
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1869195A (en) * | 2006-06-06 | 2006-11-29 | 李怀宝 | Aspergillus oryzae bacteria and its application |
| 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 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695340A (en) * | 2016-03-04 | 2016-06-22 | 北京工商大学 | Aspergillus oryzae and application thereof |
| CN105695340B (en) * | 2016-03-04 | 2020-08-21 | 北京工商大学 | Aspergillus oryzae and application thereof |
| CN108865911A (en) * | 2018-09-21 | 2018-11-23 | 侯军 | It is a kind of for improve wine brewing distillation yield leavening and preparation method thereof |
| CN114231573A (en) * | 2021-12-27 | 2022-03-25 | 南京中医药大学 | Method for producing essence of heaven by using aspergillus oryzae genetic engineering bacteria |
| CN114231573B (en) * | 2021-12-27 | 2024-04-09 | 南京中医药大学 | Method for producing Tianjing by using aspergillus oryzae genetically engineered bacteria |
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