CN105524901A - Baijiu-brewing compound enzyme containing acid protease and preparation method of Baijiu-brewing compound enzyme containing acid protease - Google Patents
Baijiu-brewing compound enzyme containing acid protease and preparation method of Baijiu-brewing compound enzyme containing acid protease Download PDFInfo
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Abstract
The invention discloses a Baijiu-brewing compound enzyme containing acid protease and a preparation method of the Baijiu-brewing compound enzyme containing the acid protease and belongs to the technical field of compound enzyme preparation. The Baijiu-brewing compound enzyme containing the acid protease and the preparation method have the advantages that on the basis of scientific compounding of food-grade enzyme preparations, high-activity acid protease and plant extracts containing plant enzymes and nutrient substances are compounded specially to enable an enzyme system of the compound enzyme to be more complete; activating agents enabling enzyme activity to be completely released and protective agents and antioxidants enabling enzyme activity to be more stable are added, and esterified red yeast and aroma-producing active dry yeast which make Baijiu more sufficient in aroma are compounded scientifically, so that starch, protein, fat, cellulose, hemicellulose, pectin and the like in grain can be hydrolyzed sufficiently and effectively, Baijiu yield can be increased by 24% to 29% remarkably, total ester content can be increased from 16.59% to 57.85%, and ethyl acetate content can be increased from 17.9% to 109%; taste of various types of Baijiu can be improved remarkably, and the compound enzyme can be suitable for brewing various types of Baijiu and addition of different Baijiu brewing methods.
Description
Technical field
The present invention relates to prozyme, be specifically related to a kind of brewed spirit prozyme containing aspartic protease and preparation method thereof.
Background technology
With the transformation of people's consumption idea, liquor consumption colony at present focuses on the mouthfeel of Liquor Products more.For multi-level, multi-class, the multifarious difference of liquor consumption demand, meet the consumers demand of constantly change, must to constantly bring forth new ideas product individuality and characteristic, simultaneously also must using the safety of white wine, health, health as primary demand, by quiet and tastefully laid out for fragrance exquisiteness, entrance soft sweet, enter that gorge circle profit is happy, after drink comfortable and side effect little of etc. main R&D direction.The main raw material of China's alcoholic is Chinese sorghum.Sorghum-brewed wine is the effect of the enzyme by Institute of Micro-biology's generation, makes the Starch Conversion in seed be the process that sugar produces alcohol then.If microbial host is by means of mould and yeast.The environment of whole wine brewing process and processing condition all should be applicable to the metabolic characteristic of microorganism used therefor, impel that they are energetic, effect large, starch is fully decomposed into sugar, then sugar is converted into alcohol.At present, mostly several famous brands of wine had won fame both at home and abroad are to cook major ingredient with Chinese sorghum or cook seasoning matter brew to form.In traditional fermentation liquor production process, often adopt cereal, potato class and plant block root class as raw material, normal Mierocrystalline cellulose, hemicellulose, beta-glucan and the pectin substance do not waited containing quantity in all kinds of raw material.And microorganism in distiller's yeast and raw material endogenous enzyme, enough capacity of decomposition are not had to these macromolecular substance, causes the reduction of raw material availability.Simultaneously because the aroma class material in raw material is wrapped up by these difficult macromole decomposed, fully can not discharge, make finished wine pungent having a surplus and mellow deficiency, cause wine quality to reduce or extend the latter stage of ripening of wine, increasing production cost.
The application on liquor production due to saccharifying enzyme and wine active dry yeast, replace traditional using wheat bran and certainly train the novel process that white wine produced by distiller's yeast, the simple the yield of liquor of technique is high, and liquor production cost reduces, and saves food.But white wine is alcoholic drink, not only require that also need a certain amount of fragrance matter, people develop again liquor production special composite enzyme for this reason containing a certain amount of ethanol.
It seems from existing research, aspartic protease role in brewed spirit be mainly manifested in following several in: 1. accelerate growth of microorganism: the growth of microorganism needs protein provides nitrogenous source and energy for it.The acidity in the acidity of fermentation grain and fermentation initial stage cellar for storing things when entering to store, equal meta-acid, the aspartic protease now in distiller's yeast, can strengthen breaks down proteins is amino acid, amino acid whose content during rich liquor is cruel, promotion microbial growth.During the fermentation, when fermentation proceeds to certain phase, just there will be fermenting speed slack-off, the phenomenon that yeast activity reduces, this is because yeast also exists a tolerance problem to alcohol.Will suppress saccharomycetic activity after the accumulation of alcohol reaches certain limit, thus fermenting speed reduces.If but keep high-caliber FAN to utilize for Yeast Growth, improve the gentle growth of synthetic water of Yeast cell, it just can be made to keep vigorous vigor.2. improve raw-material utilization ratio: during the fermentation, can be more and more low to the utilization ratio of raw material, fermenting speed slows down, and attenuation degree is more and more incomplete, and this is caused by saccharomycetic activity reduces.During fermentation growth form yeast cell to the utilization ratio of sugar far away higher than non-growth form cell.The use of proteolytic enzyme, not only can promote growth and the propagation of yeast cell, simultaneously can by the proteolysis of parcel starch granules, destroy feed particles mesenchymal cell wall construction, make starch release, increase utilization of carbon source, thus can effectively improve raw-material utilization ratio.3. provide raw fragrant precursor substance and flavour substances: aspartic protease is in the sour environment of wine brewing, material protein is hydrolyzed into amino acid, multiple amino acids is the precursor substance generating liquor flavor composition, through the metabolism of different microorganisms and enzyme, generates multiple fragrance material.Such as, the katabolism such as glycine, L-Ala, Threonine, Serine and half moon bright propylhomoserin can change pyruvic acid into, and pyruvic acid is except changing ethanol into, also can be converted into the organic acids such as acetic acid, butyric acid, caproic acid, lactic acid, L-Ala and Threonine can metabolic transformation be propyl alcohol, pick propylhomoserin and are converted into isopropylcarbinol, Isoleucine is converted into isovaleric acid, phenylalanine changes phenylethyl alcohol etc. into, and these materials, as multiple bouquet principles matter precursor, can be converted into multiple vinegar and other fragrance matters.4. relevant to fragrant liquor: the proportionlity that in wine, the content of various trace ingredients is how many and suitable, be the important factor forming various famous liquor style odor type.There are some researches show, the aspartic protease content of aromatic type Daqu higher than nearly one times of fragrant yeast, and giving off a strong fragrance Daqu than delicate fragrance Daqu and Xifeng Daqu content high, illustrate that aspartic protease may have certain dependency with different flavor.5. improve clarity and the quality of white spirit stability of beer: in brewage, skin class material is the important sources forming the muddy precursor substance of beer, utilizes aspartic protease to their Decomposition, can improve the clarity of beer.In addition, add proteolytic enzyme, can eliminate " solidifying mist phenomenon " because many skin one polyphenol complexes are formed.
To sum up, liquor production is multiple-microorganism ferment altogether, aspartic protease adds in the material unstrained spirits of liquor production, the protein decomposed in material unstrained spirits is little peptide or amino acid, promote the growth of liquor production flora, promote the ability of producing wine and producing fragrance matter, therefore aspartic protease is the indispensable a kind of enzyme of liquor production.Current most of distilled liquor factory produces white wine and changes the production technique adding saccharifying enzyme and active dry yeast into novel process with wine prozyme, and not only the simple the yield of liquor of production technique is high, and the raciness of wine.Wine prozyme by saccharifying enzyme, aspartic protease, head mold is bent, aroma yeast is bent, wine uses yeast fungus is bent etc., and multiple biological products form by different ratios, can select the optimum proportioning of these biotechnological formulations by local flavor according to demand.
Chinese patent CN101481682B discloses a kind of complex enzyme special for white spirit, and it is made up of cellulase, hemicellulase, beta-glucanase, saccharifying enzyme, polygalacturonase and proteolytic enzyme.Complex enzyme special for white spirit of the present invention has synergy, effectively can transform the compositions such as starch, fat and the protein in brewing materials and promote fermentation; In fermenting process while reservation white wine self odor type feature, increase considerably the content of flavour ingredient, accelerated the molecular balance between alcohol, aldehyde, acid, ester, improve the quality of white wine, shorten fermentation period, decrease period of storage.Chinese patent CN102108324B discloses a kind of drinks high-efficient flavor intensifier, and it is produced or application in liquid phase process brewing wine with raw materials and the white wine utilizing this sweetener to manufacture or liquor at white spirit by solid state method.This sweetener is combined by a certain percentage by prozyme of making wine, esterified red yeast and aroma-producing active dry yeast, and be applicable to solid-state, semi-solid state and liquid phase process wine-making technology, its consumption is the 0.1-5% of charging capacity.Use this sweetener, make solid-state scent type distilled liquor total ester that ferments to reach 260-270mg/100ml, ethyl acetate reaches 150-160mg/100ml.This high-efficient flavor intensifier has the advantages such as consumption is few, flavouring Be very effective, wine body flavor taste is good, purposes is wide.Described wine brewing prozyme comprises zytase, lipase and cellulase.There is following defect in above-mentioned disclosed prozyme involved by patent: 1) enzyme class is composite incomplete, can not solve the correspondence of additional enzyme preparation and material composition comprehensively.2) do not have the composite zymin vigor that makes to give full play to and prevent enzyme activity loss, the composition such as anti-oxidant, prozyme effect and vigor unstable.3) not open or not concrete openly proteolytic enzyme enzymatic property.
Therefore, the prozyme preparing the applicable brewed spirit containing aspartic protease that a kind of enzyme system is comprehensive, enzyme activity is stablized, enzyme effect is high is still the long-sought of industry technician.
Summary of the invention
Technical problem solved by the invention overcomes that proteinase activity power in existing brewed spirit prozyme is low, prozyme enzyme system is incomplete, the defects such as prozyme resistance of oxidation is low, enzyme activity loss is large, with food-grade enzyme preparations such as high vigor aspartic proteases for main raw material, science compound botanical extract, antioxidant, esterified red yeast, aroma-producing active dry yeast, protective material, activator etc.; Prepare the prozyme of the applicable brewed spirit containing aspartic protease that a kind of enzyme system is comprehensive, enzyme activity is stablized, enzyme effect is high.
In order to achieve the above object, the present invention takes following technical scheme:
Containing a brewed spirit prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 10-20 part, proteolytic enzyme 10-15 part, plant milk extract 10-15 part, hemicellulase 10-12 part, esterified red yeast 10-12 part, aroma-producing active dry yeast 8-10 part, polygalacturonase 5-8 part, esterase 5-8 part, protective material 4-6 part, activator 4-6 part, antioxidant 0.2-0.5 part;
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, 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 (PEF) 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,
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 75%, neutral protease 15%, 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 amylase is made up of following mass percent forms:
60% saccharifying enzyme, 20% Pullulanase, 10% fungal alpha-amylase, 5% beta-amylase, 5% mesophilicα-diastase;
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms:
30% heatproof beta-glucan prozyme, 25% beta-glucan prozyme, 15% mannase, the zytase of 15%, the pentosanase of 15%.
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms:
70% lipase, 25% acid phosphatase, 5% phosphoesterase.
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component:
Zinc chloride 20-30 part, calcium chloride 10-20 part, sodium sulfate 10-20 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 brewed spirit prozyme containing aspartic protease, comprises the steps:
First by described protective material, activator micronizing; immediately add amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, plant milk extract Homogeneous phase mixing successively; finally add esterified red yeast, aroma-producing active dry yeast and antioxidant successively, after mixing, pack and obtain brewed spirit prozyme.
Another object of the present invention is the application of brewed spirit prozyme in brewed spirit 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 45 DEG C.
2. using method: mix with distiller's yeast powder when 1) using and execute koji fermentation, also can add in material moistening process and carry out raw materials pretreatment; 2) by prozyme and 45 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: 0.05-0.3% when executing koji fermentation being raw grain dry weight; Be the 0.01-0.05% of raw grain dry weight during raw materials pretreatment.
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 (Aspergillususamil) 801-2.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:
Brewed spirit 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 simultaneously, and science composite can make liquor flavor more sufficient esterified red yeast and aroma-producing active dry yeast, 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, Chinese distillate spirits can be significantly improved, the output of aromatic Chinese spirit and fen-flavor type white spirit, white wine output 24% is improved respectively compared with control group, 28% and 29%.Can fully discharge aroma class material and precursor thereof, outstanding wine product style, improve wine quality, can significantly improve the total ester of Main Fragrance and the ethyl acetate content of white wine, compared with control group, Chinese distillate spirits improves 57.85% and 66.29% respectively simultaneously; Rich fragrance wine improves 35.86% and 109% respectively; Scent type wine improves 16.59% and 17.9% respectively, is particularly suitable for brewageing of giving off a strong fragrance and Chinese distillate spirits.Simultaneously as can be seen from the comprehensive mouth feel score result of white wine, test group significantly can promote the mouthfeel of all kinds white wine, to sum up, and the use of prozyme, mouthfeel and the quality of white wine can be significantly improved, be applicable to the interpolation of brewageing various types of white wine and different liquor brewing method.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 brewed spirit, thoroughly can decompose the high molecular weight protein in raw material, improve white wine non-biostability.
4. the science of brewed spirit 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 0.50% and 0.72%, 7.41% and 60.0% 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.
5. in brewed spirit 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.
6. brewed spirit 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.
Embodiment 2
Containing a brewed spirit prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 15 parts, 12 parts, proteolytic enzyme, plant milk extract 12 parts, hemicellulase 11 parts, esterified red yeast 11 parts, aroma-producing active dry yeast 9 parts, polygalacturonase 7 parts, esterase 6 parts, protective material 5 parts, activator 5 parts, antioxidant 0.4 part;
The zymin Homogeneous phase mixing that described proteolytic enzyme is made up of following mass percent forms: aspartic protease 75%, neutral protease 15%, 10% proline protein enzyme;
The zymin Homogeneous phase mixing that described amylase is made up of following mass percent forms: 60% saccharifying enzyme, 20% Pullulanase, 10% fungal alpha-amylase, 5% beta-amylase, 5% mesophilicα-diastase;
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms: 30% heatproof beta-glucan prozyme, 25% beta-glucan prozyme, 15% mannase, the zytase of 15%, the pentosanase of 15%;
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms: 70% lipase, 25% acid phosphatase, 5% phosphoesterase;
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, 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 are embodiment 1 to be prepared;
The preparation method of the above-mentioned brewed spirit prozyme containing aspartic protease:
First by protective material, activator micronizing; immediately add amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, plant milk extract Homogeneous phase mixing successively; finally add esterified red yeast, aroma-producing active dry yeast and antioxidant successively, after mixing, pack and obtain brewed spirit prozyme.
Embodiment 3
Containing a brewed spirit prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 10 parts, 10 parts, proteolytic enzyme, plant milk extract 10 parts, hemicellulase 10 parts, esterified red yeast 10 parts, aroma-producing active dry yeast 8 parts, polygalacturonase 5 parts, esterase 5 parts, protective material 4 parts, activator 4 parts, antioxidant 0.2 part;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 20 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 proteolytic enzyme, amylase, hemicellulase, esterase mass percent composition is with embodiment 2;
Described plant milk extract, aspartic protease are embodiment 1 to be prepared;
Containing the preparation method of the brewed spirit prozyme of aspartic protease with embodiment 2.
Embodiment 4
Containing a brewed spirit 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 15 parts, hemicellulase 12 parts, esterified red yeast 12 parts, aroma-producing active dry yeast 10 parts, polygalacturonase 8 parts, esterase 8 parts, protective material 6 parts, activator 6 parts, antioxidant 0.5 part;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 30 parts, 20 parts, calcium chloride, 20 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 proteolytic enzyme, amylase, hemicellulase, esterase mass percent composition is with embodiment 2;
Described plant milk extract, aspartic protease are embodiment 1 to be prepared;
Containing the preparation method of the brewed spirit prozyme of aspartic protease with embodiment 2.
Embodiment 5
Containing a brewed spirit prozyme for aspartic protease, be made up of the raw material of following parts by weight:
Amylase 15 parts, 12 parts, proteolytic enzyme, plant milk extract 12 parts, hemicellulase 11 parts, esterified red yeast 11 parts, aroma-producing active dry yeast 9 parts, polygalacturonase 7 parts, esterase 6 parts, protective material 5 parts, activator 5 parts, grape pip procyanidin 0.4 part;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 20 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 proteolytic enzyme, amylase, hemicellulase, esterase mass percent composition is with embodiment 2;
Described plant milk extract, aspartic protease are embodiment 1 to be prepared;
Containing the preparation method of the brewed spirit prozyme of aspartic protease 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 42.86%, also show: science compound botanical extract in brewed spirit prozyme of the present invention, can provide abundant, comprehensive plant enzyme system simultaneously.
Embodiment 9 antioxidant is on the impact of brewed spirit prozyme enzyme activity
Adopt the brewed spirit prozyme containing aspartic protease prepared by the embodiment of the present invention 2, other raw material, feed composition, 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 7.41% and 60% 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 prozyme is on the impact of brewing spirit seed output and quality
One, test site: a certain distillery in Hunan brewing workshop.
Two, test period: on June 8 ,-2014 years on the 8th December 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 brewed spirit prozyme prepared of embodiment of the present invention 2-5 executing bent process, and control group does not add.2. accurately take brewed spirit prozyme prepared by the embodiment of the present invention (2-5) by raw material dry weight 0.05-0.3%, by itself and 45 DEG C of water 1:10 Homogeneous phase mixing in mass ratio, adjusted to ph is 4, activation 40min, even application, to distiller's yeast powder surface, executes koji fermentation after stirring.3. in the base wine output of pair dissimilar brewing spirit and finished product white wine, flavour ingredient detects.4. in test group and control group, often kind of prozyme (embodiment 2-5) feeds intake 10 batches, calculating mean value.
Four, detected result
1. prozyme is on the impact of white wine output
1) Chinese distillate spirits wine-making: white spirit return-enclosure yeast phase 18 days, prozyme addition 0.25%.
Table 3 prozyme affects unit to Chinese distillate spirits output: kg
| Prozyme | Test group | Control group | Difference |
| Embodiment 2 | 1028 | 822 | 25％ |
| Embodiment 3 | 1017 | 834 | 22％ |
| Embodiment 4 | 1030 | 817 | 26％ |
| Embodiment 5 | 1025 | 833 | 23％ |
| On average | 1025 | 827 | 24％ |
2) aromatic Chinese spirit wine-making: giving off a strong fragrance return-enclosure yeast phase 34 days, prozyme addition 0.1%.
Table 4 prozyme affects unit to aromatic Chinese spirit output: kg
| Prozyme | Test group | Control group | Difference |
| Embodiment 2 | 1865 | 1435 | 30％ |
| Embodiment 3 | 1859 | 1452 | 28％ |
| Embodiment 4 | 1855 | 1449 | 28％ |
| Embodiment 5 | 1850 | 1457 | 27％ |
| On average | 1857 | 1448 | 28％ |
3) fen-flavor type white spirit wine-making: adopt tradition " steamed secondary is clear " method to brewage, first time fermentation 28 days, second time fermentation 35 days, prozyme addition 0.2%, first time executes 65% of Qu Tianjia prozyme total amount, second time adds remaining 35%, output Yi Tou Cha wine with Er Cha wine mixing total amount.
Table 5 prozyme affects unit to fen-flavor type white spirit output: kg
| Prozyme | Test group | Control group | Difference |
| Embodiment 2 | 2764 | 2110 | 31％ |
| Embodiment 3 | 2751 | 2132 | 29％ |
| Embodiment 4 | 2717 | 2156 | 26％ |
| Embodiment 5 | 2749 | 2131 | 29％ |
| On average | 2745 | 2132 | 29％ |
Above result shows: brewed spirit prozyme of the present invention can significantly improve the output of Chinese distillate spirits, aromatic Chinese spirit and fen-flavor type white spirit, improve white wine output 24%, 28% and 29% respectively compared with control group, also illustrate that brewed spirit prozyme of the present invention is better to the output results improving fen-flavor type white spirit simultaneously.
2. prozyme is on the primary quality measure of the impact of quality of white spirit to the finished wine of above-mentioned test group and the dissimilar white wine of control group: total acid, total ester, ethyl acetate detect, engage simultaneously 10 veteran professional white wine comment the comprehensive mouthfeel of wine teacher to dissimilar white wine carry out 100 score value scorings (average, test group-A; Control group B; ), result is as table 6
Table 6 prozyme is on the impact of quality of white spirit
Above result shows: the total ester of Main Fragrance that brewed spirit prozyme of the present invention can significantly improve white wine and ethyl acetate content, and compared with control group, Chinese distillate spirits improves 57.85% and 66.29% respectively; Rich fragrance wine improves 35.86% and 109% respectively; Scent type wine improves 16.59% and 17.9% respectively, is particularly suitable for brewageing of giving off a strong fragrance and Chinese distillate spirits.Simultaneously as can be seen from the comprehensive mouth feel score result of white wine, test group significantly can promote the mouthfeel of all kinds white wine, and to sum up, the use of prozyme, can significantly improve mouthfeel and the quality of white wine.
Claims (9)
1. the brewed spirit prozyme containing aspartic protease, be made up of the raw material of following parts by weight: amylase 10-20 part, proteolytic enzyme 10-15 part, plant milk extract 10-15 part, hemicellulase 10-12 part, esterified red yeast 10-12 part, aroma-producing active dry yeast 8-10 part, polygalacturonase 5-8 part, esterase 5-8 part, protective material 4-6 part, activator 4-6 part, antioxidant 0.2-0.5 part;
The zymin Homogeneous phase mixing that described amylase is made up of following mass percent forms: 60% saccharifying enzyme, 20% 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 75%, neutral protease 15%, 10% proline protein enzyme;
The zymin Homogeneous phase mixing that described hemicellulase is made up of following mass percent forms: 30% heatproof beta-glucan prozyme, 25% beta-glucan prozyme, 15% mannase, the zytase of 15%, the pentosanase of 15%;
The zymin Homogeneous phase mixing that described esterase is made up of following mass percent forms: 70% lipase, 25% acid phosphatase, 5% phosphoesterase;
Described activator is formed by the inorganic salt Homogeneous phase mixing of following quality component: zinc chloride 20-30 part, calcium chloride 10-20 part, sodium sulfate 10-20 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. the brewed spirit 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. the brewed spirit prozyme containing aspartic protease as claimed in claim 2, is characterized in that, fill the sodium hydrogen carbonate solution of 0.3-0.5% in described Ultrasonic Cleaners.
4. the brewed spirit 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.
5. the brewed spirit prozyme containing aspartic protease as claimed in claim 4, it is characterized in that, 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.
6. the brewed spirit 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. as claimed in claim 1 containing the preparation method of the brewed spirit prozyme of aspartic protease; it is characterized in that; comprise the steps: first by described protective material, activator micronizing; immediately add amylase, proteolytic enzyme, hemicellulase, polygalacturonase, esterase, plant milk extract Homogeneous phase mixing successively; finally add esterified red yeast, aroma-producing active dry yeast and antioxidant successively, after mixing, pack and obtain brewed spirit prozyme.
8., as claimed in claim 7 containing the preparation method of the brewed spirit prozyme of aspartic protease, 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.
9. as claimed in claim 7 containing the preparation method of the brewed spirit prozyme of aspartic protease, it is characterized in that, the preparation method of Chinese herbal medicine extract time prepared by described aspartic protease in substratum is as follows: count by weight, takes 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.10. the application of brewed spirit prozyme in brewed spirit containing aspartic protease as claimed in claim 1.
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