CN105524772A - Compound ethanol enzyme with acid proteases and method for preparing compound ethanol enzyme - Google Patents
Compound ethanol enzyme with acid proteases and method for preparing compound ethanol enzyme Download PDFInfo
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- CN105524772A CN105524772A CN201410520390.5A CN201410520390A CN105524772A CN 105524772 A CN105524772 A CN 105524772A CN 201410520390 A CN201410520390 A CN 201410520390A CN 105524772 A CN105524772 A CN 105524772A
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention discloses a compound ethanol enzyme with acid proteases and a method for preparing the compound ethanol enzyme. The compound ethanol enzyme and the method have the advantages that the high-activity acid proteases and plant extract with abundant plant-based enzymes and nutrient substances are particularly compounded on the basis of scientific compound food-grade enzyme preparations, so that the compound ethanol enzyme is provided with complete enzyme systems; activators, protective agents and antioxidants are further added into the high-activity acid proteases and the plant extract, the enzyme activity can be completely released by the aid of the activators and can be stabilized by the aid of the protective agents and the antioxidants, and hops extract and novasil which are used for inhibiting bacteria contamination and promoting saccharification of leaven and quick proliferation and fermentation of saccharomyces cerevisiae are further scientifically compounded; starch, proteins, fat, cellulose, hemi-cellulose, pectin and the like in unprocessed grain can be sufficiently and effectively hydrolyzed, accordingly, the raw material utilization rate and the liquor yields can be effectively increased, and the alcohol contents can be increased by 13.63%, 36.25% and 17.44% respectively as compared with commercially available special compound ethanol enzymes; the liquor yields of raw materials can be respectively increased by 17.41%, 38.81% and 20.04%, and the ethanol production cost can be obviously reduced.
Description
Technical field
The present invention relates to prozyme, be specifically related to a kind of alcohol prozyme containing aspartic protease and preparation method thereof.
Background technology
Produce the raw material mainly non-food crop such as the food crop such as corn, wheat and Ipomoea batatas (doing), cassava (doing), cane molasses (tangerine water) of alcohol.In the fruit or rhizome of these plants, except a certain amount of starch-polysaccharides, also have part non-starch polysaccharide, pectin, protein, ash, VITAMIN and vitamin H etc.Alcohol factory liquefying to raw material, saccharification time the Gao Wen α – amylase that uses and saccharifying enzyme, high efficiency conversion can be carried out to starch-polysaccharides, but cannot be hydrolyzed for materials such as non-starch polysaccharide, pectin, protein, ash, when lean on fermentation, the endogenous enzyme of yeast self secretion is hydrolyzed utilization.But, due to environment factor and yeast itself, this decomposition be slowly, inefficient.The developing into of modern enzyme engineering technology makes full use of above-mentioned substance and provides feasible technique means, a NEW TYPE OF COMPOSITE zymin that alcohol prozyme designs for these features existed in Alcohol Production just.
Employing thick mash fermentation produces alcohol, can shorten fermentation period, reduces energy consumption, improves plant factor, is a kind of zymamsis technology having huge applications and be worth, and along with the maturation of enzyme production technology, never stops both at home and abroad to the research of thick mash fermentation production alcohol.Such as, large quantity research shows, adds appropriate phytase, aspartic protease, cellulase, zytase respectively, can further improve alcohol output in High Concentration Mash Fermentation of Corn Alcohol process, increases liquor ratio of raw material.Therefore, research and development are suitable for the alcohol prozyme of Alcohol Production is one of important means reducing Alcohol Production cost.Under alcohol thick mash fermentation condition, fermenting speed must be accelerated for shortening fermentation period, and maize raw material is except containing except much starch, also containing a certain amount of robust fibre, crude protein and grease, combines closely, affect Starch Hydrolysis speed with starch granules.Therefore, add suitable alcohol prozyme, raw material mix can be destroyed further on the one hand, give full play to the effect of saccharifying enzyme, thus increase fermentable sugar, improve liquor ratio of raw material; The sufficient nutritional factor such as nitrogenous source, lipid acid can be provided for yeast on the other hand, promote yeast growth breeding, yeast concn during increase Primary Fermentation, thus accelerate fermenting speed, improve yeast alcohol tolerance.
In nearly ten years, Alcohol Production technology achieves huge progress, makes the sector become more lucrative.But rate of profit is still very low.Bacterial contamination is the one of the main reasons that spirit yield and quality decline.In order to efficiency of controlling cost, suppress the bacterium in zymamsis of crucial importance.In the production of alcohol fuel, the general interpolation microbiotic during the fermentation that passes through reaches this object as process auxiliaries.At present, the growing concern to making pathogenic bacteria generation microbiotic multiple resistance in agricultural and technical field application microbiotic, has created the strong request adopting a kind of safe natural substitute.Fermentation common property thing is that the economical efficiency of grain distillery has made important contribution.Increasing Alcohol Production business does not get profit containing microbiotic because claiming in their fermentation common property thing.Be devoted to promote that the bio-ethanol manufacturer of renewable energy source especially may cause the disagreement of the process auxiliaries of environmental hazard to be perplexed by using.Certain areas have implemented strict regulation, as the end of the year 2005, are used as the mixture of animal-feed in European Union, and its fermentation common property thing has not allowed antibiotic remains.
To sum up, after zymotechnique and mode are determined, the focal issue improving alcohol output and quality is the application of alcohol prozyme and prevents the pollution of fermenting process miscellaneous bacteria (milk-acid bacteria).Chinese patent CN101245339B discloses a kind of nutritive salt of alternative yeast and alcohol and the special saccharification nourishing complex enzyme of alcohol fuel, and the yeast of cultivation is healthy and strong, and yeast count is many, enzymic activity is strong, yeast logarithmic phase is long simultaneously, suppresses varied bacteria growing, reduces acidity; Shorten fermentation time; Also increase fermentable sugars, the utilization ratio of raw material can be improved, improve alcoholic strength, bring considerable economic benefit to enterprise.The present invention relates to alcohol yeast nourishing complex enzyme, its one-tenth is grouped into and is by ratio of weight and the number of copies: proteolytic enzyme 2-3 part; Phytase 1-2 part; Amylase 1-2 part; Cellulase 2-3 part; Starch debranching enzyme 1-2 part.In use, need only be sprinkled in the mash of distiller's yeast culture tank by 0.5 ‰ (W/W) by raw material in Alcohol Production, operation sequence is simple in the present invention.Chinese patent CN103571878A discloses a kind of recombiner for jerusalem artichoke powder producing fuel ethyl alcohol by ferment, and it includes: Thermostable α-Amylase 8-10 part; Prozyme: saccharifying enzyme 80-100 part, Pullulanase 30-40 part, cellulose enzyme 15-20 part, aspartic protease 8-15 part, polygalacturonase 15-25 part, inulinase 20-30 part, beta-glucanase 5-10 part, 'beta '-mannase 3-6 part, zytase 5-15 part, phytase 3-5 part; High temperature resistant brewer's dried yeasts 80-90 part.The invention has the beneficial effects as follows: adopt this recombiner to carry out fermentation of inulin and produce fuel alcohol; the advantage such as simple to operate, good stability, fermentation period are short, transformation efficiency is high, production cost is low; utilize jerusalem artichoke to ferment to realizing further, large-scale production alcohol fuel has certain directive significance.The defect of above-mentioned publication is: the combination 1) for the prozyme of Alcohol Production being simple commercial enzyme preparation, and enzyme system is incomplete; 2) do not solve the enzyme activity of compound enzymic preparation and the problem of storage stability thereof, prozyme application is limited; 3) fundamentally solution prevents living contaminants from causing the problem of alcohol output and Quality Down.
Therefore, prepare that a kind of enzyme system is complete, enzyme activity is high, storage-stable, effectively prevent from the fermenting alcohol prozyme of the significantly improved alcohol output of omnidistance living contaminants and quality be extremely urgent.
Summary of the invention
Technical problem solved by the invention is that to overcome proteinase activity in existing alcohol prozyme low, prozyme enzyme system is incomplete, resistance of oxidation is low, enzyme lives loss greatly, without bacteriostatic action or the weak defect of bacteriostatic action, with food-grade enzyme preparations such as high vigor aspartic proteases for main raw material, science compound botanical extract, antioxidant, Flos lupuli (Flos Humuli Lupuli) extract, de-mycin, protective material, activator, ethanol-tolerant active dry yeast etc., prepare a kind of enzyme system comprehensive, enzyme activity is stablized, the alcohol prozyme containing aspartic protease that enzyme effect is high, this prozyme can effectively improve ethanol production and quality, be applicable to various raw material and prepare alcohol or alcohol fuel.
In order to achieve the above object, the present invention is by the following technical solutions:
Containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 2 0-30 part, proteolytic enzyme 15-25 part, plant milk extract 10-15 part, hemicellulase 10-12 part, Flos lupuli (Flos Humuli Lupuli) extract 10-12 part, polygalacturonase 8-10 part, esterase 8-10 part, high temperature resistant brewer's dried yeasts 7-10 part, de-mycin 5-10 part, phytase 5-7 part, protective material 4-6 part, activator 4-6 part, antioxidant 0.6-1.2 part;
The zymin Homogeneous phase mixing that described amylase is made up of following mass percent forms: 70% saccharifying enzyme, 10% Pullulanase, 10% fungal alpha-amylase, 5% beta-amylase, 5% mesophilicα-diastase;
Containing various plants enzymes such as proteolytic enzyme, amylase, hemicellulase, esterase, oxydo-reductase in described plant milk extract;
Also containing the nutritive substance such as vegetable polysaccharides and monose, plant amylum, vegetable-protein in described plant milk extract, not only can be the plant enzyme that brewed spirit prozyme provides comprehensively, enriches, also can be used as the fermentation medium components preparing aspartic protease, improve proteinase activity and microorganism enzymatic productivity;
Described plant milk extract adopts the extract at low temperature technology such as ultrasonic cleaning, microwave-assisted supersound extraction and pulsed electric field extraction, ultrafiltration and concentration, effectively improves raw material availability, plant enzyme activity and productive rate; Effectively ensure that the food safety of plant milk extract;
The preparation method of described plant milk extract is: by Fructus Hordei Germinatus and wheat malt 5-7:3-5 Homogeneous phase mixing in mass ratio, be crushed to granularity 0.5-1mm, obtain pulverizing Fructus Hordei Germinatus, then by pawpaw, pineapple, Fructus Fici respectively in Ultrasonic Cleaners at power 200W, ultrasonic cleaning 5-10min under frequency 30KHz condition, drain, granularity 0.5-1mm is crushed under room temperature, and 5-7:2-4:1-3 Homogeneous phase mixing in mass ratio, add mixture quality 1.5-3 pulverizing Fructus Hordei Germinatus doubly and obtain raw mixture, add raw mixture quality 1-3 water doubly, be 3-4 by citric acid adjust ph, at power 150-300W, microwave extraction is carried out under frequency 2000Hz condition, wherein, each microwave exposure total time 60-80s, carry out compartment irradiation: irradiation 10s, interval 10s, control temperature 20-35 DEG C, irradiation like this 10 times, simultaneously at power 200-300W, ultrasonic-assisted extraction is carried out under frequency 30-40KHz condition, insulation 1-3h, then, at power 200-400W, microwave extraction is carried out under frequency 2000Hz condition, wherein, each microwave exposure total time 90-105s, carry out compartment irradiation: irradiation 15s, interval 10s, control temperature 40-60 DEG C, irradiation like this 10 times, simultaneously at power 300-500W, ultrasonic-assisted extraction is carried out under frequency 40-50KHz condition, last Temperature fall is to room temperature, in strength of electric field 25-35kV/cm, burst length 400-600 μ s, carry out pulsed electric field (PEF) under pulse-repetition 200-300Hz condition and extract 15-20min, extracting liquid filtering obtains the first filtrate, adds the water rinse of filter residue 2 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:1-2 Homogeneous phase mixing in mass ratio, namely mixed solution ultrafiltration and concentration, lyophilize, pulverize at low temperature obtain plant milk extract,
Preferably, in described Ultrasonic Cleaners, scavenging solution is the sodium hydrogen carbonate solution of 0.3-0.5%.
The zymin Homogeneous phase mixing that described proteolytic enzyme is made up of following mass percent forms: aspartic protease 80%, neutral protease 10%, 10% proline protein enzyme;
Preferably, described aspartic protease is for starting strain with bacterial strain Aspergillus usamii (Aspergillususamil) CCTCCNO:M2013601 of high yield aspartic protease, liquid submerged fermentation under specifically fermentation condition and obtaining, and the composite plant milk extract of above-mentioned preparation of science in fermention medium;
Preferably, the preparation method of described aspartic protease comprises the following steps: the bacterial classification of intact Aspergillus usamii CCTCCNO:M2013601 through slant strains activation, one-level, secondary, three grades of liquid seeds enlarged culturing to seeding tank, by seeding tank liquid seeds with 5% inoculum size access fermentation tank culture medium, culture temperature 31-32 DEG C, stirring velocity 200-400r/m, ventilation 1:1-2, incubation time 10-12h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 26-30 DEG C, stirring velocity 400-600r/m, ventilation 1:1-3, constant temperature culture 8-10h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 23-25 DEG C, stirring velocity 500-700r/m, ventilation 1:2-4, incubation time 22-31h, then, adds access fermentor tank by seeding tank liquid seeds with 3% inoculum size, finally slowly be warming up to 31-32 DEG C with 1-2 DEG C/h temperature rise rate, stirring velocity 200-400r/m, ventilation 1:1-2, constant temperature culture 10-12h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 23-25 DEG C, stirring velocity 500-700r/m, ventilation 1:2-4, constant temperature culture 22-31h; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid acid proteolytic enzyme;
Described slant medium consists of: glucose 20g, agar 20g, Chinese herbal medicine extract 5-10g, casein food grade 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, distilled water l000mL, pH value 5.8,121 DEG C of sterilizing 20min.
Described one-level, secondary, three grades of seed culture mediums consist of: wheat bran 60-80g, Semen Maydis powder 50-60g, soybean cake powder 35-40g, trehalose 10-15g, fish meal 6-10g, ammonium chloride 10-12g, calcium chloride 5-10g, casein food grade 5-10g, Chinese herbal medicine extract 5-10g, magnesium sulfate 2-4g, dipotassium hydrogen phosphate 1-3g, pure water l000mL, pH value 5-7,121 DEG C of sterilizing 20min;
Described seed tank culture base consists of: wheat bran 60-80g, Semen Maydis powder 50-60g, soybean cake powder 35-40g, trehalose 10-15g, Chinese herbal medicine extract 10-15g, fish meal 6-10g, ammonium chloride 10-12g, calcium chloride 5-10g, casein food grade 5-10g, magnesium sulfate 2-4g, dipotassium hydrogen phosphate 1-3g, pure water l000mL, pH value 5-7,121 DEG C of sterilizing 20min;
Described seeding tank fermented liquid cell concentration is 7.0x10
8-8.0x10
8individual/ml;
Described fermentation tank culture medium consists of: wheat bran 60-80g, Semen Maydis powder 50-60g, plant milk extract 40-60g, soybean cake powder 35-40g, Chinese herbal medicine extract 20-30g, trehalose 10-30g, fish meal 6-10g, ammonium chloride 10-12g, calcium chloride 5-10g, casein food grade 3-5g, magnesium sulfate 2-4g, dipotassium hydrogen phosphate 1-3g, saltpetre 1-2g, zinc sulfate 0.1-0.2g, pure water l000mL, pH value 5-7,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine extract is as follows:
Count by weight, take Radix Astragali 60-70 part, Radix Angelicae Sinensis 50-60 part, Radix Codonopsis 40-45 part, Radix Glycyrrhizae 40-45 part, Herba Houttuyniae 35-45 part, Divine Comedy 35-45 part, radix bupleuri 10-15 part, root of large-flowered skullcap 10-15 part; Said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 3-6 times of weight in container, control temperature 70 DEG C-90 DEG C keeps 2-4h, then 45-60 DEG C is cooled to, the mixing enzyme preparation adding mixture gross weight 5-10% carries out enzymolysis, be 5.5-6.8 by lactic acid adjust ph, enzymolysis 2-4h, finally add the mixture of mixture 0.5-3 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:1.5, control temperature to 60 DEG C-78 DEG C of maintenance 3-4h, filter, obtain the first filtrate; Add the water of filter residue 1-3 times of weight, control temperature 85 DEG C-95 DEG C keeps 1-3h, is then cooled to 25-35 DEG C, filters, obtains the second filtrate; First filtrate and the second filtrate are merged according to mass ratio 2-4:1-3, filter vacuum concentrates postlyophilization, pulverizes and obtains Chinese herbal medicine extract;
Described mixed enzyme is dextranase, zytase, pentosanase, polygalacturonase 5:3:1:0.5 Homogeneous phase mixing in mass ratio.
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms: 35% heatproof beta-glucan prozyme, 35% beta-glucan prozyme, 10% mannase, the zytase of 10%, the pentosanase of 10%.
Described Flos lupuli (Flos Humuli Lupuli) extract can effectively suppress the Growth and reproduction of gram-positive microorganism and not affect the normal fermentation of distillery yeast, to the bacterial flora in alcoholic fermentation process as milk-acid bacteria has very effective resistance.By suppressing the formation of lactic acid and acetic acid under inhibition concentration, thus helping avoid alcohol output and lose and increase quantity of reflux.
The preparation method of described Flos lupuli (Flos Humuli Lupuli) extract is: put by hops in Ultrasonic Cleaners in 200W, 40KHz cleans 10-15min, drain, pulverize immediately after-18--22 DEG C of freezing 20-40min, freezing bed thickness 3-5cm, grinding particle size 0.5-3mm, add and pulverize hops weight 1-3 ethanol doubly and the mixture of propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 2-4:1-3, be 3.0-3.5 by lactic acid adjust ph, at power 150-300W, frequency 2000Hz, microwave extraction 20-30min is carried out under temperature 20-35 DEG C of condition, simultaneously at power 200-300W, ultrasonic-assisted extraction is carried out under frequency 30-40KHz condition, insulation 1-3h, then, microwave extraction 15-20min is carried out under power 200-400W, frequency 2000Hz condition, simultaneously at power 300-500W, ultrasonic-assisted extraction is carried out under frequency 40-50KHz condition, last Temperature fall is to room temperature, in strength of electric field 35-45kV/cm, burst length 400-600 μ s, carry out pulsed electric field (PEF) under pulse-repetition 200-300Hz condition and extract 20-30min, namely extracting solution concentrating under reduced pressure, vacuum lyophilization, pulverize at low temperature obtain Flos lupuli (Flos Humuli Lupuli) extract,
Described hops is fresh mature hops, compression film clips or compressing grains hops, preferred fresh mature hops; When adopting pellet hop, need not clean;
Preferably, described ultrasonic cleaning employing mass percent concentration is the sodium hydrogen carbonate solution of 0.6-0.8% is clean-out system;
Preferably, the mass ratio of described ethanol and propyl alcohol mixing is 3:2;
Preferably, described microwave extraction adopts intermittent type to extract, i.e. microwave exposure 20s stops 10s, so circulates;
Described alcohol concn >=75%.
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms: 80% lipase, 15% acid phosphatase, 5% phosphoesterase.
Described de-mycin is a kind of alumino-silicate derivatives being different from ordinary silicon aluminate produced by du pont company, is synthetics, has latticed multilayer or chain-like structure, and divalence or Tricationic and oxygen or hydroxyl form octahedral covalent structure; Silicon-dioxide then forms tetrahedral covalent structure with oxygen or hydroxyl.There is strong selectivity adsorption, the multiple toxin such as energy active adsorption raw material (starchy material such as corn, wheat) middle aflatoxin, vomitoxin, zearalenone, T-2 toxin, and the nutritions such as amino acid, VITAMIN, trace element can not be adsorbed, can effectively prevent bulk toxin to the harm of saccharification in alcohol production process, fermented bacterium, promote its healthy propagation and fermentation, improve product enzyme, produce alcohol ability.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 15-25 part, calcium chloride 10-15 part, sodium sulfate 10-15 part, magnesium chloride 5-10 part.
Described protective material is made up of the raw material of following parts by weight: ganoderan 20-30 part, trehalose 20-30 part, NaCl10-20 part, (NH
4)
2sO
410-15 part, halfcystine 10-15 part.
Described antioxidant is any or several combination in grape pip procyanidin, rosemary extract and apricot leaf extract;
Preferably, described antioxidant is grape pip procyanidin, rosemary extract and apricot leaf extract 2-4:1-3:1-2 Homogeneous phase mixing in mass ratio;
Described grape pip procyanidin, rosemary extract and apricot leaf extract are commercial commodity.
The preparation method of the above-mentioned alcohol prozyme containing aspartic protease, comprises the steps:
First by described protective material, activator micronizing; immediately add de-mycin, Flos lupuli (Flos Humuli Lupuli) extract, plant milk extract Homogeneous phase mixing 20-30min successively; then amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, phytase Homogeneous phase mixing is added successively; finally add high temperature resistant brewer's dried yeasts and antioxidant successively, after mixing, pack and obtain alcohol prozyme.
Another object of the present invention is the application of alcohol prozyme in Alcohol Production containing aspartic protease.
Using method:
1. prozyme action condition: adapt to pH value 2-6, optimum pH 3.5-5.5; Adaptive temperature 30-55 DEG C, optimal reactive temperature 30-45 DEG C.
2. using method: 1) at saccharifying and saccharification pulvis (commodity saccharifying enzyme or saccharifying koji) Homogeneous phase mixing, add together; 2) by prozyme and 40 DEG C of water 1:10 Homogeneous phase mixing in mass ratio, adjusted to ph is 3.5-5.5, and activation 30-50min, result of use is best.
3. usage quantity: be the 0.01-0.15% of raw grain dry weight.
Aspergillus usamii 801-2 provided by the invention produces the Aspergillus usamii 801 of aspartic protease successively through ultraviolet compounded lithium chloride mutagenesis, nitrosoguanidine mutagenesis, ethyl sulfate mutagenesis by a strain of Laboratories Accession, and to mutant strain separation, screening, finally through leavening property test screen, the Aspergillus usamii 801-2 producing high vigor aspartic protease is obtained to strain excellent.
The bacterial strain of the high vigor aspartic protease of product provided by the invention is specially Aspergillus usamii (Aspergillususami) l801-2.This bacterial strain is preserved in China typical culture collection center on November 24th, 2013 and (is called for short CCTCC, address is: China. Wuhan. and Wuhan University's postcode: 430072), preserving number is CCTCCNO:M2013601, and Classification And Nomenclature is Aspergillus usamii 801-2Aspergillususamil801-2.
Aspergillus usamii of the present invention (Aspergillususamil) 801-2 (CCTCCNO:M2013601) has following microbial characteristic:
1, morphological feature:
Aspergillus usamii (Aspergillususamil) 801-2, biology morphology is for comprising conidium, born of the same parents' stalk, top capsule, producing several parts such as born of the same parents' structure.Conidial head is spherical to Radiation, and diameter 150-450 μm, conidiophore betides matrix.Born of the same parents obstruct stem 1000-3000 (length) × 12-20 (diameter) μm, yellow or tawny, and wall is level and smooth; Spherical or the almost spherical of top capsule, diameter 35-50 μm, surface can be educated comprehensively; Produce born of the same parents' structure double-deck, metulae 15-20 (length) × 3-4.0 (diameter) μm, bottle stalk 6-8 (length) × 2-4 (diameter) μm; conidium is spherical or subsphaeroidal, less, diameter 3.5-5.0 μm; brown, wall is coarse.
2, feature is cultivated:
Aspergillus usamii (Aspergillususamil) 801-2 bacterial strain grows rapidly on wort agar substratum, 32 DEG C of 4 days colony diameter 82mm; Quality velvet shape or be slightly with cotton-shaped; Conidium structure is a large amount of, and brown-black, has a small amount of transudate; Bacterium colony reverse side yellowish.
3, physiological and biochemical property:
Aspergillus usamii (Aspergillususamil) 801-2 can at potato, Semen Maydis powder, Zulkovsky starch, the upper growth such as molasses, optimum pH 5-6, optimum growth temperature 31-32 DEG C, the suitableeest product enzyme temperature 23-25 DEG C.
The triage techniques route of bacterial strain Aspergillus usamii (Aspergillususamil) 801-2 of the present invention is: preparation → mutagenic treatment → plate isolation → primary dcreening operation → multiple sieve → sieve → expansion experiment (leavening property mensuration) again again of starting strain → slant culture → spore suspension.
By mutagenesis screening scheme, mutant strain step-sizing is eliminated, finally to strain excellent through leavening property test screen, obtain a plant height and produce enzyme performance bacterial strain Aspergillus usamii 801-2, aspartic protease enzyme work after 72-96 hour of fermenting can reach 14000U/mL, the thermostability of enzyme is analyzed, at crude enzyme liquid is placed in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C respectively, lives every 10 minutes sampling and measuring enzymes.At 40 DEG C, 45 DEG C, 50 DEG C, 60 minutes enzymes are lived and are not declined.At 55 DEG C and 60 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 90% in 95%, 60 minutes.At 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 75% of constitutive enzyme work for 80%, 60 minutes.Aspartic protease adaptive response temperature 30-55 DEG C, optimal reactive temperature 40-50 DEG C, similarity condition is issued to the same enzyme tolerable temperature ratio bacterium that sets out that lives and on average improves 8-12 DEG C, adapts to pH value range 2-6, optimum pH 2.5-3.5.
Beneficial effect:
Alcohol prozyme of the present invention, on the basis of science formulated food level zymin, composite especially high vigor aspartic protease, makes prozyme enzyme system more complete containing the plant milk extract enriching plant enzyme and nutritive substance, with the addition of again the activator that enzyme activity is discharged completely and the protective material making enzymic activity more stable and antioxidant, and science compositely suppresses miscellaneous bacteria, promotes the Flos lupuli (Flos Humuli Lupuli) extract of saccharifying koji and yeast saccharomyces cerevisiae fast breeding and fermentation, de-mycin simultaneously, can be abundant, starch in effective hydrolysis raw grain, protein, fat, Mierocrystalline cellulose, hemicellulose, pectin etc., disintegration raw grain mesenchymal cell wall, release content, obtain more sugar fermentation and amino acid, lipid acid, VITAMIN, the nutritive substances such as mineral substance, increase fermenting carbon source, promote that bacterium active fermentation produced by drinks, improve raw material availability and the yield of liquor, alcohol output can be significantly improved, with commercially available corn alcohol special composite enzyme, cassava alcohol special composite enzyme and potato alcohol special composite enzyme are compared, alcohol prozyme of the present invention can effectively improve liquor ratio of raw material and ethanol concn, compared with control group, alcoholic strength improves 13.63% respectively, 36.25% and 17.44%, liquor ratio of raw material improves 17.41%, 38.81% and 20.04% respectively.Be in particular in:
1. plant milk extract of the present invention with, enzyme system comprehensive brewer's barley bud, wheat malt abundant containing plant enzyme, pawpaw, pineapple, Fructus Fici for raw material, adopt the extract at low temperature technology such as ultrasonic cleaning, microwave-assisted supersound extraction and pulsed electric field extraction, ultrafiltration and concentration, effectively improve raw material availability, plant enzyme activity and productive rate; Effectively ensure that the food safety of plant milk extract; Containing various plants enzyme systems such as proteolytic enzyme, amylase, hemicellulase, esterase, oxydo-reductase in the plant milk extract of preparation; Also containing the nutritive substance such as vegetable polysaccharides and monose, plant amylum, vegetable-protein in described plant milk extract, not only can be the plant enzyme that brewed spirit prozyme provides comprehensively, enriches, also can be used as the fermentation medium components preparing aspartic protease, improve proteinase activity and microorganism enzymatic productivity.
2. aspartic protease of the present invention with the bacterial strain Aspergillus usamii CCTCCNO:M2013601 of high yield aspartic protease for starting strain, according to its optimum growth temperature and the suitableeest product enzyme temperature, refinement sporogony and the processing parameter in enzymatic production stage, adopt the zymotechnique of gradient cooling and gradient increased temperature, added inoculation simultaneously, especially the zymotechnique of gradient cooling and gradient increased temperature significantly improves the anti-stress ability of starting strain, causes the enzymatic productivity of bacterial classification to manifest to greatest extent.And the present invention implements full optimization to substratum composition, with the addition of the root of large-flowered skullcap, the radix bupleuri with anti-heat stress original work, with the addition of and there is adjustment and repair body function, the immunologic function of enhancing body, the Radix Astragali with Tiny ecosystem regulatory function, Radix Codonopsis etc., further enhancing the body function of microorganism under same yeasting, adaptation of virus and collaborate, and then enhance the metabolic function of microorganism, such that acid protease activity that the present invention produces is high, tolerable temperature is higher, stability is strong, be suitable for suitability for industrialized production.
3. the interpolation of fermention medium plant milk extract in aspartic protease preparation process of the present invention, makes fermented liquid aspartic protease enzyme activity increase substantially, is increased to 19000-22000U/mL, amplification 57.14% by 13000-14000U/mL; Fermented liquid crude enzyme liquid can withstand higher temperatures, and at 40 DEG C, 45 DEG C, 50 DEG C, 60 minutes enzymes are lived and do not declined.At 55 DEG C and 60 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 90% in 95%, 60 minutes.At 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 75% of constitutive enzyme work for 80%, 60 minutes.Aspartic protease adaptive response temperature 30-55 DEG C, optimal reactive temperature 40-50 DEG C, adapt to pH value range 2-6, optimum pH 2.5-3.5.More be applicable to traditional saccharifying koji alcohol preparation technology, thoroughly can decompose the high molecular weight protein in raw material, improve alcohol output.
4. in alcohol prozyme of the present invention, Flos lupuli (Flos Humuli Lupuli) extract adopts extract at low temperature technology, remain the content of hop acid in hops to greatest extent, can effectively suppress the Growth and reproduction of gram-positive microorganism and not affect the normal fermentation of distillery yeast, to the bacterial flora in alcoholic fermentation process as milk-acid bacteria has very effective resistance.By suppressing the formation of lactic acid and acetic acid under inhibition concentration, thus helping avoid alcohol output and lose and increase quantity of reflux.
5. in alcohol prozyme of the present invention, the science of high temperature resistant brewer's dried yeasts is composite, and Alcohol Production saccharification stage enzymolysis product concentration can be made slowly to reduce, and decreases the restraining effect of enzymolysis product concentration rising to enzyme digestion reaction, accelerates enzymolysis speed.
6. take off mycin in alcohol prozyme of the present invention for having the adsorbing alumino-silicate derivatives of strong selectivity, the multiple toxin such as energy active adsorption raw material (starchy material such as corn, wheat) middle aflatoxin, vomitoxin, zearalenone, T-2 toxin, and the nutritions such as amino acid, VITAMIN, trace element can not be adsorbed, can effectively prevent bulk toxin to the harm of saccharification in alcohol production process, fermented bacterium, promote its healthy propagation and fermentation, improve product enzyme, produce alcohol ability.
7. the science of alcohol prozyme antioxidant of the present invention is composite, prozyme enzyme molecular structure effectively can be prevented to be oxidized and to cause loss of enzyme activity, improve enzyme activity stability.12 months are stored under 0 DEG C and 40 DEG C of conditions, in prozyme, the loss alive of single enzyme enzyme is respectively 0.46% and 0.56%, 8.0% and 65% is reduced respectively than comparative example, effectively to prevent from links such as packaging, storage, transport, uses, because environment change, working method are improper and cause the inactivation of enzyme, especially can preventing the enzyme deactivation that high temperature causes.Antioxidant can consume the dissolved oxygen in alcohol production process fermented liquid simultaneously, slows down and prevents the miscellaneous bacterias such as acetic bacteria from oxidation of ethanol is caused alcohol losses.
8. in alcohol prozyme of the present invention, protective material science is composite, effectively slow down the moisture regain of compound enzymic preparation; Can strengthen prozyme simultaneously resistance toly to freeze, resistance toheat, keep identical enzyme activity, its heat resisting temperature can improve 20-30 DEG C, resistance to freezing temp can reduce 10-15 degree Celsius, effectively prevent the loss of prozyme enzyme activity in transport, preservation and use procedure, extend the quality guaranteed period of prozyme, reach same enzyme activity, can 2-3 be extended than the like product quality guaranteed period.
9. alcohol prozyme of the present invention adds inorganic salt as activator, create the top condition of enzyme catalysis, give full play to the vigor carrying enzyme and additional enzyme in Fructus Hordei Germinatus, the macromolecular substance such as starch, protein, hemicellulose, fat are thoroughly effectively decomposed in brewing materials, not only increase raw material availability, and add the nutritive substance of distiller's yeast and distiller's yeast, provide the necessary fragrance matter of white wine and precursor thereof simultaneously.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Prepared by embodiment 1 raw material
1. the preparation of plant milk extract: by Fructus Hordei Germinatus, wheat malt 6:4 Homogeneous phase mixing in mass ratio, be crushed to granularity 0.8mm, obtain pulverizing Fructus Hordei Germinatus, then by pawpaw, pineapple, Fructus Fici respectively at fill 0.4% sodium hydrogen carbonate solution Ultrasonic Cleaners at power 200W, ultrasonic cleaning 8min under frequency 30KHz condition, drain, granularity 0.8mm is crushed under room temperature, and 6:3:2 Homogeneous phase mixing in mass ratio, the pulverizing Fructus Hordei Germinatus adding mixture quality 3 times obtains raw mixture, add the water of raw mixture quality 2 times, be 3.5 by citric acid adjust ph, at power 200W, microwave extraction is carried out under frequency 2000Hz condition, wherein, each microwave exposure total time 70s, carry out compartment irradiation: irradiation 10s, interval 10s, control temperature 30 DEG C, irradiation like this 10 times, simultaneously at power 250W, ultrasonic-assisted extraction is carried out under frequency 35KHz condition, insulation 2h, then, carries out microwave extraction under power 300W, frequency 2000Hz condition, wherein, each microwave exposure total time 105s, carries out compartment irradiation: irradiation 15s, interval 10s, control temperature 50 DEG C, irradiation like this 10 times, simultaneously at power 400W, carry out ultrasonic-assisted extraction under frequency 45KHz condition, last Temperature fall to room temperature, in strength of electric field 30kV/cm, burst length 500 μ s, carries out pulsed electric field and extracts 18min under pulse-repetition 250Hz condition, extracting liquid filtering obtains the first filtrate, adds the water rinse of filter residue 2 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:1.5 Homogeneous phase mixing in mass ratio, namely mixed solution ultrafiltration and concentration, lyophilize, pulverize at low temperature obtain plant milk extract,
2. the preparation of aspartic protease
The preparation method of described aspartic protease comprises the steps:
(1) actication of culture
The slant strains of intact Aspergillus usamii CCTCCNO:M2013601 is inoculated in slant medium, cultivates 40h for 32 DEG C and carry out actication of culture, so activation 3 times;
(2) liquid seeds enlarged culturing
1. first order seed is cultivated: by step (1) activation back bevel bacterial classification with spore under aseptic washing, access in 500 ml shake flasks, liquid seed culture medium loading amount 100 milliliters, 32 DEG C, 100rpm shaking table cultivation 40h;
2. secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 8% inoculum size, liquid nutrient medium loading amount 1000 milliliters, 32 DEG C, 100rpm shaking table cultivation 40h;
4. seed tank culture: the first class seed pot being 150L with 8% inoculum size access cubic capacity by three grades of seeds, seed tank culture base loading amount 100L, control ph is 6, culture temperature 31 DEG C, stirring velocity 300rpm, ventilation (V/V) 1:1, incubation time 40h;
Described seeding tank fermented liquid cell concentration is 8.0x10
8individual/ml;
(3) ferment tank
By seeding tank liquid seeds in step (2) with 5% inoculum size access fermentation tank culture medium, culture temperature 32 DEG C, stirring velocity 300r/m, ventilation (V/V) 1:1.5, incubation time 11h; Then with 2 DEG C/h rate of temperature fall slow cooling to 28 DEG C, stirring velocity 500r/m, ventilation (V/V) 1:2, constant temperature culture 9h; Continue with 2 DEG C/h rate of temperature fall slow cooling to 24 DEG C, stirring velocity 600r/m, ventilation (V/V) 1:3, incubation time 27h, then, adds access fermentor tank by seeding tank liquid seeds in step (2) with 3% inoculum size, finally slowly be warming up to 32 DEG C with 2 DEG C/h temperature rise rate, stirring velocity 300r/m, ventilation (V/V) 1:1.5, constant temperature culture 11h; Then with 2 DEG C/h rate of temperature fall slow cooling to 24 DEG C, stirring velocity 600r/m, ventilation (V/V) 1:3, constant temperature culture 27h;
(4) fermentation liquor filter, concentrated, allotment, essence filter, dry solid acid proteolytic enzyme.
After the aspartic protease prepared through aforesaid method at room temperature preserves 12 months, enzyme loss of living is 1.8%, and fermented liquid aspartic protease enzyme activity can reach 22000U/mL.
Described slant medium consists of: glucose 20g, agar 20g, Chinese herbal medicine extract 7g, casein food grade 4g, dipotassium hydrogen phosphate 2g, magnesium chloride 0.6g, Repone K 0.8g, distilled water l000mL, pH value 5.8,121 DEG C of sterilizing 20min.
Described one-level, secondary, three grades of seed culture mediums consist of: wheat bran 70g, Semen Maydis powder 55g, soybean cake powder 38g, trehalose 12g, fish meal 8g, ammonium chloride 11g, calcium chloride 8g, casein food grade 8g, Chinese herbal medicine extract 8g, magnesium sulfate 3g, dipotassium hydrogen phosphate 2g, pure water l000mL, pH value 6,121 DEG C of sterilizing 20min;
Described seed tank culture base consists of: wheat bran 70g, Semen Maydis powder 55g, soybean cake powder 38g, trehalose 12g, Chinese herbal medicine extract 12g, fish meal 8g, ammonium chloride 11g, calcium chloride 8g, casein food grade 8g, magnesium sulfate 3g, dipotassium hydrogen phosphate 2g, pure water l000mL, pH value 6,121 DEG C of sterilizing 20min;
Described fermentation tank culture medium consists of: wheat bran 70g, Semen Maydis powder 55g, plant milk extract 50g, soybean cake powder 38g, Chinese herbal medicine extract 25g, trehalose 20g, fish meal 8g, ammonium chloride 11g, calcium chloride 8g, casein food grade 4g, magnesium sulfate 3g, dipotassium hydrogen phosphate 2g, saltpetre 2g, zinc sulfate 0.2g, pure water l000mL, pH value 6,121 DEG C of sterilizing 20min;
The preparation method of described Chinese herbal medicine extract is as follows: count by weight, takes the Radix Astragali 65 parts, Radix Angelicae Sinensis 55 parts, Radix Codonopsis 42 parts, 42 parts, Radix Glycyrrhizae, Herba Houttuyniae 40 parts, Divine Comedy 40 parts, radix bupleuri 12 parts, the root of large-flowered skullcap 12 parts; Said herbal medicine being crushed to particle diameter is less than 2 millimeters, then Homogeneous phase mixing add the water of 5 times of weight in container, control temperature 80 DEG C keeps 3h, is then cooled to 52 DEG C, the mixing enzyme preparation adding mixture gross weight 8% carries out enzymolysis, be 6.2 by lactic acid adjust ph, enzymolysis 3h, finally adds the mixture of mixture 2 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:1.5, control temperature to 70 DEG C keeps 4h, filters, obtains the first filtrate; Add the water of filter residue 2 times of weight, control temperature 90 DEG C keeps 2h, is then cooled to 30 DEG C, filters, obtains the second filtrate; First filtrate and the second filtrate are merged according to mass ratio 3:2, filter vacuum concentrates postlyophilization, pulverizes and obtains Chinese herbal medicine extract;
Described mixed enzyme is dextranase, zytase, pentosanase, polygalacturonase 5:3:1:0.5 Homogeneous phase mixing in mass ratio.
Enzyme activity unit defines: 1mL crude enzyme liquid, in 40 DEG C, under pH3.0 condition, the enzyme amount that 1min caseinhydrolysate produces 1 μ g tyrosine is 1 enzyme activity unit, represents with U/mL.
3. the preparation of Flos lupuli (Flos Humuli Lupuli) extract
The preparation method of described Flos lupuli (Flos Humuli Lupuli) extract is: put in the Ultrasonic Cleaners filling 0.7% sodium hydrogen carbonate solution fresh mature hops in 200W, 40KHz cleans 12min, drain, pulverize immediately after-20 DEG C of freezing 30min, freezing bed thickness 4cm, grinding particle size 2mm, add and pulverize the ethanol of hops weight 2 times and the mixture of propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 3:2, be 3.2 by lactic acid adjust ph, at power 200W, frequency 2000Hz, microwave extraction 25min is carried out under temperature 30 DEG C of conditions, simultaneously at power 250W, ultrasonic-assisted extraction is carried out under frequency 35KHz condition, insulation 2h, then, microwave extraction 18min is carried out under power 300W, frequency 2000Hz condition, under power 400W, frequency 45KHz condition, carry out ultrasonic-assisted extraction, last Temperature fall is to room temperature simultaneously, in strength of electric field 40kV/cm, burst length 500 μ s, carry out pulsed electric field (PEF) under pulse-repetition 250Hz condition and extract 25min, namely extracting solution concentrating under reduced pressure, vacuum lyophilization, pulverize at low temperature obtain Flos lupuli (Flos Humuli Lupuli) extract,
Described microwave extraction adopts intermittent type to extract, i.e. microwave exposure 20s stops 10s, so circulates;
Described alcohol concn is 80%.
Embodiment 2
Containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 25 parts, 20 parts, proteolytic enzyme, plant milk extract 12 parts, hemicellulase 11 parts, Flos lupuli (Flos Humuli Lupuli) extract 11 parts, polygalacturonase 9 parts, esterase 9 parts, high temperature resistant brewer's dried yeasts 9 parts, de-mycin 8 parts, phytase 6 parts, protective material 5 parts, activator 5 parts, antioxidant 0.9 part;
The zymin Homogeneous phase mixing that described amylase is made up of following mass percent forms: 70% saccharifying enzyme, 10% Pullulanase, 10% fungal alpha-amylase, 5% beta-amylase, 5% mesophilicα-diastase;
The zymin Homogeneous phase mixing that described proteolytic enzyme is made up of following mass percent forms: aspartic protease 80%, neutral protease 10%, 10% proline protein enzyme;
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms: 35% heatproof beta-glucan prozyme, 35% beta-glucan prozyme, 10% mannase, the zytase of 10%, the pentosanase of 10%;
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms: 80% lipase, 15% acid phosphatase, 5% phosphoesterase;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 20 parts, 12 parts, calcium chloride, 12 parts, sodium sulfate, 8 parts, magnesium chloride;
Described protective material is made up of the raw material of following parts by weight: ganoderan 25 parts, trehalose 25 parts, NaCl15 part, (NH
4)
2sO
412 parts, halfcystine 12 parts;
Described antioxidant is grape pip procyanidin, rosemary extract and apricot leaf extract 3:2:1.5 Homogeneous phase mixing in mass ratio;
Described plant milk extract, aspartic protease, Flos lupuli (Flos Humuli Lupuli) extract are embodiment 1 to be prepared.
The preparation method of the above-mentioned alcohol prozyme containing aspartic protease, comprises the steps:
First by described protective material, activator micronizing; immediately add de-mycin, Flos lupuli (Flos Humuli Lupuli) extract, plant milk extract Homogeneous phase mixing 20-30min successively; then amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, phytase Homogeneous phase mixing is added successively; finally add high temperature resistant brewer's dried yeasts and antioxidant successively, after mixing, pack and obtain alcohol prozyme.
Embodiment 3
Containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 20 part, 15 parts, proteolytic enzyme, plant milk extract 10 parts, hemicellulase 10 parts, Flos lupuli (Flos Humuli Lupuli) extract 10 parts, polygalacturonase 8 parts, esterase 8 parts, high temperature resistant brewer's dried yeasts 7 parts, de-mycin 5 parts, phytase 5 parts, protective material 4 parts, activator 4 parts, antioxidant 0.6 part;
Described amylase, proteolytic enzyme, hemicellulase, esterase quality composition is with embodiment 2;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 15 parts, 10 parts, calcium chloride, 10 parts, sodium sulfate, 5 parts, magnesium chloride;
Described protective material is made up of the raw material of following parts by weight: ganoderan 20 parts, trehalose 20 parts, NaCl10 part, (NH
4)
2sO
410 parts, halfcystine 10 parts;
Described antioxidant is grape pip procyanidin, rosemary extract and apricot leaf extract 2:1:1 Homogeneous phase mixing in mass ratio;
Described plant milk extract, aspartic protease, Flos lupuli (Flos Humuli Lupuli) extract are embodiment 1 to be prepared.
The preparation method preparation method of the above-mentioned alcohol prozyme containing aspartic protease is with embodiment 2.
Embodiment 4
Containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 30 parts, 25 parts, proteolytic enzyme, plant milk extract 15 parts, hemicellulase 12 parts, Flos lupuli (Flos Humuli Lupuli) extract 12 parts, polygalacturonase 10 parts, esterase 10 parts, high temperature resistant brewer's dried yeasts 10 parts, de-mycin 10 parts, phytase 7 parts, protective material 6 parts, activator 6 parts, antioxidant 1.2 parts;
Described amylase, proteolytic enzyme, hemicellulase, esterase quality composition is with embodiment 2;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 25 parts, 15 parts, calcium chloride, 15 parts, sodium sulfate, 10 parts, magnesium chloride;
Described protective material is made up of the raw material of following parts by weight: ganoderan 30 parts, trehalose 30 parts, NaCl20 part, (NH
4)
2sO
415 parts, halfcystine 15 parts;
Described antioxidant is grape pip procyanidin, rosemary extract and apricot leaf extract 4:3:2 Homogeneous phase mixing in mass ratio;
Described plant milk extract, aspartic protease, Flos lupuli (Flos Humuli Lupuli) extract are embodiment 1 to be prepared.
The preparation method preparation method of the above-mentioned alcohol prozyme containing aspartic protease is with embodiment 2.
Embodiment 5
Containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 25 parts, 20 parts, proteolytic enzyme, plant milk extract 12 parts, hemicellulase 11 parts, Flos lupuli (Flos Humuli Lupuli) extract 11 parts, polygalacturonase 9 parts, esterase 9 parts, high temperature resistant brewer's dried yeasts 9 parts, de-mycin 8 parts, phytase 6 parts, protective material 5 parts, activator 5 parts, grape pip procyanidin 0.9 part;
Described amylase, proteolytic enzyme, hemicellulase, esterase quality composition is with embodiment 2;
Described activator, protective material quality composition is with embodiment 4;
Described plant milk extract, aspartic protease, Flos lupuli (Flos Humuli Lupuli) extract are embodiment 1 to be prepared.
The preparation method preparation method of the above-mentioned alcohol prozyme containing aspartic protease is with embodiment 2.
The thermal stability analysis of embodiment 6 aspartic protease
At embodiment 1 acidic protein enzymic fermentation crude enzyme liquid is placed in 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C respectively, live every 10 minutes sampling and measuring enzymes.At 40 DEG C, 45 DEG C, 50 DEG C, 60 minutes enzymes are lived and are not declined.At 55 DEG C and 60 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 90% in 95%, 60 minutes.At 65 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes.At 70 DEG C, what within 30 minutes, drop to constitutive enzyme work drops to 75% of constitutive enzyme work for 80%, 60 minutes.Illustrate that acidic protein enzyme heat stability is stronger.
The pH stability analysis of embodiment 7 aspartic protease
Embodiment 1 acidic protein enzymic fermentation crude enzyme liquid is placed in pH value 2.0,2.5,3.5,4.5,5.5,6.0 times respectively, lives every 10 minutes sampling and measuring enzymes.Crude enzyme liquid pH value 2.0,2.5,3.5 times, 60 minutes enzymes are lived and are not declined.PH value 4.5 times, what within 30 minutes, drop to constitutive enzyme work drops to 90% in 95%, 60 minutes.PH value 5.5 times, what within 30 minutes, drop to constitutive enzyme work drops to 85% in 90%, 60 minutes.PH value 6.0 times, what within 30 minutes, drop to constitutive enzyme work drops to 80% in 85%, 60 minutes, illustrates that aspartic protease a wider range of resistance to pH is general.
Embodiment 8 plant milk extract is on the impact of aspartic protease enzyme activity
Adopt the preparation method of aspartic protease in the embodiment of the present invention 1, the fermentation conditions such as other processing condition, processing parameter, substratum are identical, unique difference is that fermention medium does not add plant milk extract, form comparative example, ferment complete, adopt same detection method to measure fermented liquid acid protease activity, detected result is as table 1:
Table 1 fermented liquid acid protease activity
| Project | Embodiment 1 | Comparative example | Difference | Amplification |
| Fermented liquid acid protease activity | 22000U/mL | 14000U/mL | 8000U/mL | 57.14％ |
Above result shows: plant milk extract prepared by the present invention contains higher acid protease activity, plant milk extract is added in fermention medium, fermented liquid acid protease activity can be increased to 22000U/mL from 14000U/mL, aspartic protease enzyme activity improves 57.14%, also show: science compound botanical extract in alcohol prozyme of the present invention, can provide abundant, comprehensive plant enzyme system simultaneously.
Embodiment 9 antioxidant is on the impact of alcohol prozyme enzyme activity
Adopt the alcohol prozyme containing aspartic protease prepared by the embodiment of the present invention 2, other raw material, material component, preparation method are identical, unique difference is that feed composition is not containing antioxidant, form comparative example, 12 months are stored respectively under 0 DEG C and 40 DEG C of conditions, detection method in " GB8726-2006 foodstuff additive Glucoamylase preperation " is adopted to measure the enzyme activity of saccharifying enzyme, calculate enzyme rate of loss alive, enzyme rate of loss alive refers to that the enzyme activity of actual detection and the difference of product annotation enzyme activity account for the percentage marking enzyme activity, and result is as table 2
Glucoamylase enzyme vigor rate of loss in table 2 shelf lives prozyme
Above result shows, 12 months are stored under 0 DEG C and 40 DEG C of conditions, saccharifying enzyme in embodiment 2 is lived in losing than the glucoamylase enzyme in comparative example and is reduced by 8.0% and 65% respectively, illustrate that the science of antioxidant is composite, significantly improve the vigor of each component enzymes in prozyme, enzyme is lived in losing and is significantly reduced, and effect is more remarkable especially under the high temperature conditions.
Embodiment 10 alcohol prozyme of the present invention is on the impact of different material thick mash fermentation
One, test site: a certain grain distillery in Hunan brewing workshop.
Two, test period: on May 18 ,-2014 years on the 18th November in 2013, last 180 days.
Three, plan design: 1. test as single-factor designs, test group and control group are set, the raw material of control group and test group, technique, equipment, operator, environment and way to manage are all identical, difference is: test group adds the alcohol prozyme of the embodiment of the present invention 2 preparation at saccharifying, and control group adds the alcohol special composite enzyme of commercially available corresponding raw material (corn, cassava and potato).2. test group and control group all add alcohol prozyme by raw material dry weight 0.01-0.15%.3. same raw material test group and control group respectively feed intake 10 batches, calculating mean value.4. pair different material thick mash fermentation process raw material availability, ethanol production index of correlation carry out detected result as table 3
Table 3 alcohol prozyme is on the impact of different material thick mash fermentation
Above result shows: compared with commercially available corn alcohol special composite enzyme, cassava alcohol special composite enzyme and potato alcohol special composite enzyme, alcohol prozyme of the present invention can effectively improve liquor ratio of raw material and ethanol concn, compared with control group, alcoholic strength improves 13.63% (2.23%), 36.25% (4.76%) and 17.44% (2.72%) respectively; Liquor ratio of raw material improves 17.41% (5.91%), 38.81% (14.33%) and 20.04% (7.59%) respectively, significantly reduces Alcohol Production cost.Also show that alcohol prozyme of the present invention is applicable to adopting thick mash fermentation explained hereafter alcohol with various ethanol production materials simultaneously.
Claims (10)
1., containing an alcohol prozyme for aspartic protease, be made up of the raw material of following parts by weight: amylase 2 0-30 part, proteolytic enzyme 15-25 part, plant milk extract 10-15 part, hemicellulase 10-12 part, Flos lupuli (Flos Humuli Lupuli) extract 10-12 part, polygalacturonase 8-10 part, esterase 8-10 part, high temperature resistant brewer's dried yeasts 7-10 part, de-mycin 5-10 part, phytase 5-7 part, protective material 4-6 part, activator 4-6 part, antioxidant 0.6-1.2 part;
The zymin Homogeneous phase mixing that described amylase is made up of following mass percent forms: 70% saccharifying enzyme, 10% Pullulanase, 10% fungal alpha-amylase, 5% beta-amylase, 5% mesophilicα-diastase;
The zymin Homogeneous phase mixing that described proteolytic enzyme is made up of following mass percent forms: aspartic protease 80%, neutral protease 10%, 10% proline protein enzyme;
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms: 35% heatproof beta-glucan prozyme, 35% beta-glucan prozyme, 10% mannase, the zytase of 10%, the pentosanase of 10%;
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms: 80% lipase, 15% acid phosphatase, 5% phosphoesterase;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 15-25 part, calcium chloride 10-15 part, sodium sulfate 10-15 part, magnesium chloride 5-10 part;
Described protective material is made up of the raw material of following parts by weight: ganoderan 20-30 part, trehalose 20-30 part, NaCl10-20 part, (NH
4)
2sO
410-15 part, halfcystine 10-15 part.
2. a kind of alcohol prozyme containing aspartic protease as claimed in claim 1, it is characterized in that, the preparation method of described plant milk extract is: by Fructus Hordei Germinatus and wheat malt 5-7:3-5 Homogeneous phase mixing in mass ratio, be crushed to granularity 0.5-1mm, obtain pulverizing Fructus Hordei Germinatus, then by pawpaw, pineapple, Fructus Fici respectively in Ultrasonic Cleaners at power 200W, ultrasonic cleaning 5-10min under frequency 30KHz condition, drain, granularity 0.5-1mm is crushed under room temperature, and 5-7:2-4:1-3 Homogeneous phase mixing in mass ratio, add mixture quality 1.5-3 pulverizing Fructus Hordei Germinatus doubly and obtain raw mixture, add raw mixture quality 1-3 water doubly, be 3-4 by citric acid adjust ph, at power 150-300W, microwave extraction is carried out under frequency 2000Hz condition, wherein, each microwave exposure total time 60-80s, carry out compartment irradiation: irradiation 10s, interval 10s, control temperature 20-35 DEG C, irradiation like this 10 times, simultaneously at power 200-300W, ultrasonic-assisted extraction is carried out under frequency 30-40KHz condition, insulation 1-3h, then, microwave extraction is carried out under power 200-400W, frequency 2000Hz condition, wherein, each microwave exposure total time 90-105s, carry out compartment irradiation: irradiation 15s, interval 10s, control temperature 40-60 DEG C, irradiation like this 10 times, simultaneously at power 300-500W, carry out ultrasonic-assisted extraction under frequency 40-50KHz condition, last Temperature fall to room temperature, in strength of electric field 25-35kV/cm, burst length 400-600 μ s, carries out pulsed electric field and extracts 15-20min under pulse-repetition 200-300Hz condition, extracting liquid filtering obtains the first filtrate, adds the water rinse of filter residue 2 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:1-2 Homogeneous phase mixing in mass ratio, namely mixed solution ultrafiltration and concentration, lyophilize, pulverize at low temperature obtain plant milk extract.
3. a kind of alcohol prozyme containing aspartic protease as claimed in claim 1, it is characterized in that, the preparation method of described Flos lupuli (Flos Humuli Lupuli) extract is: put by hops in Ultrasonic Cleaners in 200W, 40KHz cleans 10-15min, drain, pulverize immediately after-18--22 DEG C of freezing 20-40min, freezing bed thickness 3-5cm, grinding particle size 0.5-3mm, add and pulverize hops weight 1-3 ethanol doubly and the mixture of propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 2-4:1-3, be 3.0-3.5 by lactic acid adjust ph, at power 150-300W, frequency 2000Hz, microwave extraction 20-30min is carried out under temperature 20-35 DEG C of condition, simultaneously at power 200-300W, ultrasonic-assisted extraction is carried out under frequency 30-40KHz condition, insulation 1-3h, then, microwave extraction 15-20min is carried out under power 200-400W, frequency 2000Hz condition, under power 300-500W, frequency 40-50KHz condition, carry out ultrasonic-assisted extraction, last Temperature fall is to room temperature simultaneously, in strength of electric field 35-45kV/cm, burst length 400-600 μ s, carry out pulsed electric field under pulse-repetition 200-300Hz condition and extract 20-30min, namely extracting solution concentrating under reduced pressure, vacuum lyophilization, pulverize at low temperature obtain Flos lupuli (Flos Humuli Lupuli) extract.
4. a kind of alcohol prozyme containing aspartic protease as claimed in claim 3, it is characterized in that, described hops is fresh mature hops, compression film clips or compressing grains hops.
5. a kind of alcohol prozyme containing aspartic protease as claimed in claim 1, it is characterized in that, the preparation method of described aspartic protease comprises the following steps: the bacterial classification of intact Aspergillus usamii CCTCCNO:M2013601 through slant strains activation, one-level, secondary, three grades of liquid seeds enlarged culturing to seeding tank, by seeding tank liquid seeds with 5% inoculum size access fermentation tank culture medium, culture temperature 31-32 DEG C, stirring velocity 200-400r/m, ventilation 1:1-2, incubation time 10-12h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 26-30 DEG C, stirring velocity 400-600r/m, ventilation 1:1-3, constant temperature culture 8-10h; Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 23-25 DEG C, stirring velocity 500-700r/m, ventilation 1:2-4, incubation time 22-31h, then, adds access fermentor tank by seeding tank liquid seeds with 3% inoculum size, finally slowly be warming up to 31-32 DEG C with 1-2 DEG C/h temperature rise rate, stirring velocity 200-400r/m, ventilation 1:1-2, constant temperature culture 10-12h; Then with 1-2 DEG C/h rate of temperature fall slow cooling to 23-25 DEG C, stirring velocity 500-700r/m, ventilation 1:2-4, constant temperature culture 22-31h; Fermentation liquor is filtered, concentrated, allotment, essence filter, dry solid acid proteolytic enzyme.
6. a kind of alcohol prozyme containing aspartic protease as claimed in claim 1, is characterized in that, described antioxidant is any or several combination in grape pip procyanidin, rosemary extract and apricot leaf extract.
7. the preparation method of a kind of alcohol prozyme containing aspartic protease as claimed in claim 1; it is characterized in that; comprise the steps: first by described protective material, activator micronizing; immediately add de-mycin, Flos lupuli (Flos Humuli Lupuli) extract, plant milk extract Homogeneous phase mixing 20-30min successively; then amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, phytase Homogeneous phase mixing is added successively; finally add high temperature resistant brewer's dried yeasts and antioxidant successively, after mixing, pack and obtain alcohol prozyme.
8. the preparation method of a kind of alcohol prozyme containing aspartic protease as claimed in claim 7, it is characterized in that, time prepared by described Flos lupuli (Flos Humuli Lupuli) extract, microwave extraction adopts intermittent type to extract, i.e. microwave exposure 20s stops 10s.
9. the preparation method of a kind of alcohol prozyme containing aspartic protease as claimed in claim 7, it is characterized in that, described antioxidant is grape pip procyanidin, rosemary extract and apricot leaf extract 2-4:1-3:1-2 Homogeneous phase mixing in mass ratio.
10. a kind of application of alcohol prozyme in Alcohol Production containing aspartic protease as claimed in claim 1.
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Application publication date: 20160427 |