CN107916191A - A kind of production method for not adding sulfur dioxide organic wine - Google Patents
A kind of production method for not adding sulfur dioxide organic wine Download PDFInfo
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- CN107916191A CN107916191A CN201711486293.9A CN201711486293A CN107916191A CN 107916191 A CN107916191 A CN 107916191A CN 201711486293 A CN201711486293 A CN 201711486293A CN 107916191 A CN107916191 A CN 107916191A
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- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G1/00—Preparation of wine or sparkling wine
- C12G1/02—Preparation of must from grapes; Must treatment and fermentation
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- C12G1/00—Preparation of wine or sparkling wine
- C12G1/02—Preparation of must from grapes; Must treatment and fermentation
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract
The present invention relates to a kind of production method for not adding sulfur dioxide organic wine, by the plants interplanting with expelling parasite insecticidal effect in orchard, manages orchard according to organic planting requirement, grape is made wine after harvesting ripe, up to organic wine.The advantages of present invention is made wine using Organic grape, has nutrition, health, safety, non agricultural chemical residuum.
Description
Technical field
The present invention relates to a kind of production method for not adding sulfur dioxide organic wine.
Background technology
Bread is the staff of life, strengthens food security work, the health and life security of relation China more than 1,300,000,000, it is necessary to
Grab tightly and tightly.These years, all departments have descended very big strength to grab food security, and food security situation constantly improves, but exist
The problem of it is still many, common people still have many expectations, it is necessary to redouble one's efforts, work is run business into particular one reality, it is ensured that the people
" safety on the tip of the tongue ".
Grape wine is with a long history, it is acted on aid digestion, beautifying face and moistering lotion and extra-nutrition, is favored by people, at present
The grape to make grape wine has a small amount of pesticide residue, and a poor taste more using common plantation, market need with non agricultural chemical residuum,
The grape wine of brewing grape in good taste.
The content of the invention
The technical problem to be solved by the invention is to provide a kind of producer for not adding sulfur dioxide organic wine
The advantages of method, the present invention are made wine using Organic grape, have nutrition, health, safety, non agricultural chemical residuum.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme:
A kind of production method for not adding sulfur dioxide organic wine, it is characterised in that follow the steps below:
By the plants interplanting with expelling parasite insecticidal effect in orchard, orchard is managed according to organic planting requirement, picks fruit
Ripe grape is made wine in garden, up to organic wine;
The plant with expelling parasite insecticidal effect includes garlic, lavender, fish pelargonium, common nepenthes, shichongcao and folium artemisiae argyi
In one kind or arbitrary proportion it is two or more;
The preparation of the grape wine follows the steps below:
A) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
B) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 160~
230g/L;
C) destemming crushes:The grape of harvesting is reduced into temperature to 5~20 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape
Slurry, is heated to 50~65 DEG C by grape slurry, keeps 20~60min, make the inactivating oxidase in grape slurry, be cooled to 15~25
DEG C, the grape slurry of cooling is added in the container sterilized, and 20~60mg/L of enzyme is added, container is filled with nitrogen before pan feeding
Or carbon dioxide, air-discharging is anti-oxidation, obtains grape mash;
D) cold soaking stain:Grape mash is reduced into temperature to 5~8 DEG C, impregnates 12~96h, daily Sprayer Circulation 1~2 time, every time
Spray the grape mash of 1/2~1/3 times of vessel volume amount;
E) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen or carbon dioxide removes
Oxygen;
F) low temperature clarification:Control temperature to add fining agent to 5~8 DEG C, staticly settle 24~48h in the turbid juice of grape, separate
Grape juice, 150~250NTU of turbidity, during which inflated with nitrogen or carbon dioxide remove oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 10~12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is not less than
106Cfu/ml, 10~12 DEG C of fermentation temperature, 24~72h of fermentation time;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 15~25 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is not low
In 106Cfu/ml, 16~20 DEG C of fermentation temperature, 24~72h of fermentation time;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 5 when monitoring alcoholic fermentation
~10 DEG C of separation wine feet, container are filled with nitrogen or carbon dioxide air-discharging before pan feeding;
J) malo-lactic fermentation:Be inoculated with the grape wine obtained to step i) Oneococcus onei and lactic acid bacteria 20~
60mg/L, 20~25 DEG C, 24~480h of fermentation time of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 10~15 DEG C of storages of temperature, period of storage 3~24 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon, U.S. pleasure, Cabernet Gernischt, Cabernet franc, hila, PINOT NOIR, Chardonnay, Italian Riesling and Riesling
In one kind or arbitrary proportion it is two or more;
The enzyme is two of one kind or arbitrary proportion in pectase, cellulase, papain, alginase and amylase
More than kind;
The fining agent is the two or more of one kind in gelatin, bentonite, PVPP and egg white or arbitrary proportion;
Fertilizer used in organic plantation is one in organic fertilizer, microbial manure, organic and inorganic fertilizer and biological organic fertilizer
Kind or arbitrary proportion are two or more;
Ecological seaweed biostimulant is added in the organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer;
The ecology seaweed biostimulant is prepared according to following steps:
A) seaweed fragment, is prepared:Seaweed after cleaning is crushed, obtains seaweed fragment;
B) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, under the conditions of 60~120 DEG C sterilize 5~
40min, is cooled to 27~45 DEG C of access enzymes, digests 30~60min, the mass ratio 5~30 of seaweed fragment, water and enzyme:70~
95:0.02~1, seaweed must be digested;
C) seaweed filtrate and seaweed slag, are prepared:Enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, residue obtained to be
Seaweed slag;
D) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and water content is adjusted to water as 50~70%, at 70~150 DEG C
Under the conditions of sterilize 5~40min, be cooled to 27~36 DEG C, obtain mixed culture medium, access rhodotorula mucilaginosa, rhodotorula mucilaginosa and mixing are trained
The mass ratio for supporting base is 0.1~2:98~99.9, it is 27~36 DEG C in temperature, oxygen concentration is 8~21% in fermentation tank, fermentation
6~144h, sterilize 20~40min under the conditions of being 70~150 DEG C in temperature, is cooled to 20~40 DEG C, obtains fermentation of seaweed thing;
E) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 95~99.8:0.2~5, be 25~35 DEG C in temperature, oxygen content for 1~
Under the conditions of 8mg/L, ferment 1~6 day, gained filtrate is ecological seaweed liquid biostimulant after press filtration;Filter residue is seaweed
Residue;
F) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The seaweed is that one kind in Enteromorpha, kelp, sargassum, opotism and kelp or arbitrary proportion are two or more.
Pesticide used in the orchard is lime sulfur, Bordeaux mixture, mineral oil, trichoderma harzianum, quinolinone suspending agent, second
Alcohol, soda, China tree alkali, rotenone, Dalmatian chrysanthemum missible oil and one kind in matrine or arbitrary proportion are two or more.
The preparation of thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improve cell permeability:Thawing method is at least repeated once, its step is:Step 1) is obtained
Expansion is further cultured for strain and is put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolves at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, 20.0~25.0g of agar;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition, wherein lysozyme according to following steps into
OK:
(A) add the water of 5 times of weight to egg white or whole egg liquid, after stirring evenly add homogenizer in, at least it is homogeneous once, it is even
Matter temperature is 30~60 DEG C, and homogeneous pressure is 2~40MPa, and the homogeneous time is 10~40min, is filtered after homogeneous, gained filtrate is
Dilution;
(B) the dilution molecular cut off for obtaining step (A) is the ultrafiltration membrane ultrafiltration of 15000~30000Da, is obtained
Thin liquid and dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
3~8, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.001~0.5mol/L,
Obtain eluent;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 2000~10000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained;
The resin is that the resin is cation exchange resin or macroporous absorbent resin;The model of cation exchange resin
For 001*7,732, AmberliteIR-120, Dowex-50, Lewatit-100, Diaion SK-1, AllassionCS,
One or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II,
One or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
Step c) the separation is included more than one or both of press filtration, filtering and centrifugation.
Step d) the mixed culture mediums to water content is 60%, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is 0.5:
99.5, it is 30 DEG C in temperature, oxygen concentration 12%, ferment 120h, and sterilize 20min under the conditions of being 120 DEG C in temperature.
The mass ratio of corynebacterium ammoniagenes prepared by step e) mixtures and thawing method is 98:2, it is 28 DEG C in temperature, it is oxygen-containing
Measure under the conditions of 3mg/L, to ferment 5 days.
The rhodotorula mucilaginosa and corynebacterium ammoniagenes are purchased from Chinese industrial Microbiological Culture Collection administrative center, numbering difference
For CICC 31192 and CICC 10168.
Step A) described in it is homogeneous be homogeneous 2 times, homogeneous temperature be 40 DEG C, the homogeneous time be 15min, wherein first time it is even
Matter pressure is 25MPa, and second of homogeneous pressure is 4MPa.
The mode that ecological seaweed biostimulant is added in fertilizer is:With the interior addition manner of fertilizer material mixing granulation
With the outer addition manner for spraying to fertiliser granulates surface.
Invention has following advantageous effects:
1st, the present invention uses organic fruits and Organic grape as raw material, and the wine set that it is prepared is nutritious, health, peace
Entirely, the advantages of non agricultural chemical residuum.
2nd, grape base liquor technique of the present invention is unique, grape wine is had strong, pure and fresh fragrance, and mouthfeel is more compliant, and
Nutriment retains abundant;First using color and benefit materials in low temperature cold impregnation technology controlling extraction pericarp and fruit seed;
Then squeezing goes pericarp to carry out alcoholic fermentation using clarification grape juice, in the process order inoculation Non-Saccharomyces and wine brewing ferment
Mother fermentation generation aromatic substance and alcohol etc., increase the fragrance complexity of grape base liquor and strong degree, and pass through cold fermentation, storage
Hiding technique prevents the volatilization loss of aroma substance, increases the sophistication in wine;It is anti-by whole inflated with nitrogen or carbon dioxide etc.
Oxidation technology, increases the freshness and nutriment activity of grape base liquor to greatest extent;The present invention need not add preservative substance.
3rd, the present invention can reach expelling parasite and reduce pesticide by interplanting the plant with expelling parasite insecticidal effect in orchard
The purpose of usage amount.
4th, ecological seaweed biostimulant uses new fresh seaweed in the present invention, and life can largely be stimulated by containing in new fresh seaweed
Long stimulin and resistance material, activity is good, can achieve the purpose that to reduce Fertilizer application amount.
5th, ecological seaweed biostimulant is fermented under anaerobic using aerobic and anaerobism rhodotorula mucilaginosa in the present invention,
It can prevent the loss of seaweed nutritive component, and the alcohol that anaerobic fermentation produces is a supporting role seaweed extraction.
6th, ecological seaweed biostimulant is sent out under anoxic conditions using aerobic and anaerobism corynebacterium ammoniagenes in the present invention
Ferment, produces ATP, and ATP is a kind of high energy phosphate compound, and in cell, it can realize energy storage and put with mutually converting for ADP
Can, so as to ensure that the energy supply of cell items vital movement, promote plant growth.It is unreasonable due to long-term fertilization, make
Into soil acidification, corynebacterium ammoniagenes can reduce N03 -And urea, producing ammonia makes tunning be in alkalescent, can improve acidifying soil
Earth problem.
7th, ecological seaweed biostimulant has heavy metal certain fixation in the present invention.Contain in fermentate of the present invention
There are polypeptide, alginic acid and other protides, heavy metal can make albumen, alginic acid and polypeptide inactivation, while heavy metal also can
Fixed by albumen, alginic acid and polypeptide, so as to prevent crop from absorbing.
8th, the present invention is using aerobic and oxygen bacterium, and soil hardening is serious at present, and the microorganism applied in ground is often in anoxic shape
State, present soil environment is suitable for using aerobic and oxygen bacterium.
9th, ecological seaweed biostimulant improves the permeability of corynebacterium ammoniagenes cell by thawing method in the present invention, can be with
The ATP that corynebacterium ammoniagenes produce is set to be easier to penetrate into from cell membrane extracellularly, its product effects is significantly better than with without place
The product of the corynebacterium ammoniagenes of reason.
10th, the present invention is free of sulfur dioxide.
Embodiment
The present invention is further illustrated with reference to instantiation.
Embodiment 1
By garlic and lavender interplanting in vineyard, orchard is managed according to organic planting requirement, is picked ripe in orchard
Grape is made wine, up to organic wine;
The preparation of the grape wine follows the steps below:
A) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
B) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
C) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
D) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 1 time, 1/3 times of spray every time
The grape mash of vessel volume amount;
E) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
F) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 36h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 48h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is pectase, cellulase and papain according to mass ratio 3:1:1 composition;
The fining agent is PVPP and egg white according to mass ratio 1:1 composition.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate is dilution;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Embodiment 2
By garlic and lavender interplanting in the orchard of apple, pears, cherry and grape miscegenation, according to organic planting requirement pipe
Orchard is managed, ripe grape in orchard is picked and makes wine, up to organic wine;
The preparation of the grape wine follows the steps below:
A) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
B) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
C) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
D) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 1 time, 1/3 times of spray every time
The grape mash of vessel volume amount;
E) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
F) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 48h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 32h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is cellulase and papain according to mass ratio 1:1 composition;
The fining agent is PVPP.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 35 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate is dilution;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Embodiment 3
By garlic and lavender interplanting in vineyard, orchard is managed according to organic planting requirement, is picked ripe in orchard
Grape is made wine, up to organic wine;
The preparation of the grape wine follows the steps below:
A) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
B) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
C) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
D) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 2 times, 1/3 times of spray every time
The grape mash of vessel volume amount;
E) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
F) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 48h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 32h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is pectase, cellulase and papain according to mass ratio 2:1:1 composition;
The fining agent is PVPP.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 45 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate is dilution;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Embodiment 4
By garlic and lavender interplanting in the orchard of apple, pears, cherry and grape miscegenation, according to organic planting requirement pipe
Orchard is managed, ripe grape in orchard is picked and makes wine, up to organic wine;
The preparation of the grape wine follows the steps below:
A) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
B) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
C) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
D) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 1 time, 1/3 times of spray every time
The grape mash of vessel volume amount;
E) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
F) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 36h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 48h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is pectase, cellulase and papain according to mass ratio 3:1:1 composition;
The fining agent is PVPP.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 1 time, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 30min, and homogeneous pressure 25MPa, is filtered, gained filtrate is dilution after homogeneous;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Embodiment 5
By garlic and lavender interplanting in vineyard, orchard is managed according to organic planting requirement, is picked ripe in orchard
Grape is made wine, up to organic wine;
The preparation of the grape wine follows the steps below:
G) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
H) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
I) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
J) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 1 time, 1/3 times of spray every time
The grape mash of vessel volume amount;
K) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
L) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 36h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 48h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is pectase, cellulase and papain according to mass ratio 3:1:1 composition;
The fining agent is PVPP and egg white according to mass ratio 1:1 composition.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 3 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Third time homogeneous pressure is 4MPa, is filtered after homogeneous, gained filtrate is dilution;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Embodiment 6
By garlic and lavender interplanting in vineyard, orchard is managed according to organic planting requirement, is picked ripe in orchard
Grape is made wine, up to organic wine;
The preparation of the grape wine follows the steps below:
M) collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling,
When acidity reaches 6.0~6.5g/L, start to harvest;
N) harvest:The grape fruit of fresh, healthy, the disease-free decayed fruit of harvesting and raw green fruit in organic orchard, pol 180~
200g/L;
O) destemming crushes:The grape of harvesting is reduced into temperature to 6 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 60 DEG C, 30min is kept, makes the inactivating oxidase in grape slurry, be cooled to 18 DEG C, by the grape slurry of cooling
It is added in the container sterilized and adds enzyme 40mg/L, container is filled with nitrogen before pan feeding, and air-discharging is anti-oxidation, obtains grape
Wine with dregs;
P) cold soaking stain:Grape mash is reduced into temperature to 7 DEG C, impregnates 48h, daily Sprayer Circulation 1 time, 1/3 times of spray every time
The grape mash of vessel volume amount;
Q) extracting juice is pressed:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen and remove oxygen;
R) low temperature clarification:Control temperature to add fining agent to 7 DEG C in the turbid juice of grape, stand heavy 36h, separate grape juice,
Turbidity 200NTU, during which inflated with nitrogen removal oxygen;
G) the fragrant fermentation of production:Grape juice is risen again to 12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is 2*
106Cfu/ml, 12 DEG C of fermentation temperature, fermentation time 36h;
H) alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 20 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is 2*
106Cfu/ml, 20 DEG C of fermentation temperature, fermentation time 48h;
I) wine foot is separated:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 6 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen purge gas before pan feeding;
J) malo-lactic fermentation:Oneococcus onei and lactic acid bacteria 40mg/L are inoculated with the grape wine obtained to step i),
22 DEG C, fermentation time 120h of fermentation temperature, completes malo-lactic fermentation;
K) store:By the grape wine of malo-lactic fermentation in 12 DEG C of storages of temperature, period of storage 4 months;
L) clarify:The grape wine that step k) is obtained is clarified using fining agent, obtains grape base liquor, and grape base liquor uses
After 0.45 and 0.20 micron of film device is degerming, up to organic wine;
The grape is Cabernet Sauvignon and Mei Le according to mass ratio 1:1 composition;
The enzyme is pectase, cellulase and papain according to mass ratio 3:1:1 composition;
The fining agent is PVPP and egg white according to mass ratio 1:1 composition.
The orchard uses the organic fertilizer for adding ecological seaweed biostimulant;
The ecology seaweed biostimulant is prepared according to following steps:
1) seaweed fragment, is prepared:Fresh Laminaria Japonica after cleaning is dried, dry kelp is crushed using pulverizer, is obtained
Seaweed fragment;
2) enzymolysis seaweed, is prepared:Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition;
3) seaweed filtrate and seaweed slag, are prepared:The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag;
4) fermentation of seaweed thing, is prepared:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and is adjusted to water content as 60% with water, goes out under the conditions of 120 DEG C
Bacterium 20min, is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of being 120 DEG C in temperature
20min, is cooled to 35 DEG C, obtains fermentation of seaweed thing;
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared:The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed thing that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant;Filter residue is seaweed residue;
6) ecological seaweed biostimulant, is prepared:Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant;
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below:
I) expansion of strain is further cultured for:Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain;
II) thawing method improves the permeability of cell:In triplicate, its step is thawing method:The expansion that step 1) is obtained
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, it is cold
Freeze 4h, take out, dissolve, be placed again into -20 DEG C of refrigerator at room temperature, freeze 4h, take out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams of immersions
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, added after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate is dilution;
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:
5, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained.
Beneficial effects of the present invention are further illustrated with reference to experimental data:
Experiment one
1st, material to be tested is tested
1 materials and methods:
1.1 test sites and experimental subjects:Yantai university chemistry analysis inspection center.
1.2 experiment detections:Survey the activity and the rate of recovery of lysozyme.
1.3 material to be tested:It is (even without homogenizer after the preparation method of lysozyme is except stirring evenly for common process
Matter, other production methods are consistent with embodiment 1), embodiment 1, embodiment 2, embodiment 3, prepare in embodiment 4 and embodiment 5
Lysozyme do effect and compare.
1.4 experimental design:Using high performance liquid chromatography to sample treatment.
This experiment is consistent with other operations in addition to processing difference except testing.
2 results and analysis
Common process (homogeneous without homogenizer after stirring evenly, other production methods are consistent with embodiment 1), implement
Bacteriolyze enzymatic treatment prepared by example 1, embodiment 2, embodiment 3, embodiment 4 and embodiment 5 is compared, and data are shown in Table 1
Table 1
The rate of recovery (%) | Enzymatic activity (U/mg) | |
Common process | 72.7 | 14567 |
Embodiment 1 | 91.7 | 21101 |
Embodiment 2 | 84.7 | 19831 |
Embodiment 3 | 88.3 | 20790 |
Embodiment 4 | 91.7 | 20903 |
Embodiment 5 | 89.3 | 20737 |
Compared by 1 embodiment 1 of table with common process, the present invention is either in terms of the lysozyme rate of recovery, or enzymatic activity
All it is significantly better than common process;Compared by embodiment 1 to embodiment 3 it can be seen that the rate of recovery and work of the homogeneous temperature to lysozyme
Property still have certain influence, wherein embodiment 1 is best using 40 DEG C of general effects;Compared by embodiment 1, embodiment 4 and embodiment 5
Relatively it can be seen that homogeneous pressure and number also have certain influence to the rate of recovery and activity of lysozyme, and non-pressure is bigger, the time
It is more long better, 1 best results of Integrated comparative embodiment.
Experiment two
1st, material to be tested is tested
1 materials and methods:
1.1 test site:Laboratory.
1.2 material to be tested:The water in fruit and grape and the organic orchard of the present invention in common orchard (non-organic orchard)
Fruit and grape, compare.
This experiment is consistent with other management in addition to processing difference except testing.
1.3 experimental design:The fruit in common orchard (non-organic orchard) and grape and the organic fruit of the present invention are picked respectively
Fruit and grape in garden make grape wine according to the method for embodiment 2, detect the NO in grape wine2 -And pesticide residue.
2 results and analysis
NO2 -Content (mg/kg) | SO2Content (mk/L) | Pesticide residue (mg/kg) | |
Common orchard grape wine | 0.7 | 102 | 0.8 |
Grape wine of the present invention | 0 | 0 | 0 |
As can be seen from the above data, the grape wine made of the present invention and vin ordinaire are more excellent in terms of food health
Gesture.
Experiment three
1st, material to be tested is tested
1 materials and methods:
1.1 test site:Yantai Qixia City Su Jiadian towns vineyard.
1.2 material to be tested:Common orchard (in addition to not there is no the plant of expelling parasite insecticidal effect, other production methods and this hair
It is bright consistent), contrast 1 is (when preparing ecological seaweed liquid biostimulant, except corynebacterium ammoniagenes are without thawing method processing directly use
Outside, other production methods are consistent with the present invention) and embodiment 1, compare.
This experiment is consistent with other management in addition to processing difference except testing.
1.3 experimental design:The consistent vineyard of 6 mu of age of trees is selected, 2 is parallel, and 1 mu is 1 processing unit.For common orchard
(in addition to the plant with expelling parasite insecticidal effect is not interplanted, other production methods are consistent with the present invention), contrast 1 (prepare ecological sea
During algae solution body biostimulant, except corynebacterium ammoniagenes are without the processing of thawing method directly in addition to use, other production methods with this hair
It is bright consistent) and embodiment 1 contrast, observe orchard insect pest situation, statistics orchard vintage.
2 results and analysis
Grape average product (kg) | Orchard insect pest situation | |
Common orchard | 2028.6 | Insect pest is serious |
Contrast 1 | 2371.4 | Substantially without worm |
The present invention | 2611.5 | Substantially without worm |
Compared by common orchard with the present invention, it can be seen that the present invention has obvious anthelminthic effect, using the teaching of the invention it is possible to provide grape
Yield;Being compared by embodiment 1 and contrast 1 can show that corynebacterium ammoniagenes through thawing method high treating effect, can improve vintage.
" managing orchard according to organic planting requirement " of the present invention refers to according to GB/T19630《Organic products》Mark
Quasi- management orchard.
Claims (10)
1. a kind of production method for not adding sulfur dioxide organic wine, it is characterised in that follow the steps below:
By the plants interplanting with expelling parasite insecticidal effect in orchard, orchard is managed according to organic planting requirement, is picked in orchard
Ripe grape is made wine, up to organic wine;
The plant with expelling parasite insecticidal effect is included in garlic, lavender, fish pelargonium, common nepenthes, shichongcao and folium artemisiae argyi
A kind of or arbitrary proportion is two or more;
The preparation of the grape wine follows the steps below:
a)Collecting period determines:After grape fruit annesl, the change of the pol, acidity index of grape fruit is measured by sampling, works as acid
Degree reaches 6.0~6.5g/L, starts to harvest;
b)Harvesting:The grape fruit of fresh, healthy, disease-free decayed fruit and raw green fruit, 160~230g/ of pol are harvested in organic orchard
L;
c)Destemming crushes:The grape of harvesting is reduced into temperature to 5~20 DEG C, flexible removing carpopodium is carried out, crushes, obtain grape slurry,
Grape slurry is heated to 50~65 DEG C, 20~60min is kept, makes the inactivating oxidase in grape slurry, is cooled to 15~25 DEG C, will
The grape slurry of cooling is added in the container sterilized, and adds 20~60mg/L of enzyme, container be filled with before pan feeding nitrogen or
Carbon dioxide, air-discharging is anti-oxidation, obtains grape mash;
d)Cold soaking stain:Grape mash is reduced into temperature to 5~8 DEG C, impregnates 12~96h, daily Sprayer Circulation 1~2 time, spray every time
The grape mash of 1/2~1/3 times of vessel volume amount;
e)Press extracting juice:The grape mash of cold soaking stain is pressed into extracting juice, the turbid juice of grape is obtained, during which rushes nitrogen or carbon dioxide deoxygenation
Gas;
f)Low temperature clarification:Control temperature to add fining agent to 5~8 DEG C in the turbid juice of grape, staticly settle 24~48h, separate grape
Juice, 150~250NTU of turbidity, during which inflated with nitrogen or carbon dioxide remove oxygen;
g)The fragrant fermentation of production:Grape juice is risen again to 10~12 DEG C, the fragrant Non-Saccharomyces of inoculation production, yeast quantity is not less than
106Cfu/ml, 10~12 DEG C of fermentation temperature, 24~72h of fermentation time;
h)Alcoholic fermentation:The grape juice for producing fragrant fermentation is risen again to 15~25 DEG C, is inoculated with saccharomyces cerevisiae, yeast quantity is not less than
106Cfu/ml, 16~20 DEG C of fermentation temperature, 24~72h of fermentation time;
i)Separate wine foot:The proportion and temperature of grape mash, when proportion is down to 0.993, are cooled to 5~10 when monitoring alcoholic fermentation
DEG C separation wine foot, container is filled with nitrogen or carbon dioxide air-discharging before pan feeding;
j)Malo-lactic fermentation:To step i)20~60mg/L of Oneococcus onei and lactic acid bacteria is inoculated with obtained grape wine,
20~25 DEG C, 24~480h of fermentation time of fermentation temperature, completes malo-lactic fermentation;
k)Storage:By the grape wine of malo-lactic fermentation in 10~15 DEG C of storages of temperature, period of storage 3~24 months;
l)Clarification:By step k)Obtained grape wine is clarified using fining agent, obtains grape base liquor, and grape base liquor uses 0.45
With 0.20 micron of film device it is degerming after, up to organic wine;
The grape is in Cabernet Sauvignon, U.S. pleasure, Cabernet Gernischt, Cabernet franc, hila, PINOT NOIR, Chardonnay, Italian Riesling and Riesling
It is a kind of or arbitrary proportion two or more;
The enzyme be two kinds of one kind or arbitrary proportion in pectase, cellulase, papain, alginase and amylase with
On;
The fining agent is the two or more of one kind in gelatin, bentonite, PVPP and egg white or arbitrary proportion;
Fertilizer used in organic plantation be organic fertilizer, microbial manure, organic and inorganic fertilizer and biological organic fertilizer in one kind or
Arbitrary proportion is two or more;
Ecological seaweed biostimulant is added in the organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer;
The ecology seaweed biostimulant is prepared according to following steps:
a), prepare seaweed fragment:Seaweed after cleaning is crushed, obtains seaweed fragment;
b), prepare enzymolysis seaweed:Seaweed fragment and water are fitted into agitator tank, under the conditions of 60~120 DEG C sterilize 5~
40min, is cooled to 27~45 DEG C of access enzymes, digests 30~60min, the mass ratio 5~30 of seaweed fragment, water and enzyme:70~
95:0.02~1, seaweed must be digested;
c), prepare seaweed filtrate and seaweed slag:Enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and residue obtained is seaweed
Slag;
d), prepare fermentation of seaweed thing:By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to quality
Than 95.5:1:0.5:1:1:1 is added in fermentation tank and mixes, and water content is adjusted to water as 50~70%, in 70~150 DEG C of conditions
5~40min of lower sterilizing, is cooled to 27~36 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, rhodotorula mucilaginosa and mixed culture medium
Mass ratio be 0.1~2:98~99.9, it is 27~36 DEG C in temperature, oxygen concentration is 8~21% in fermentation tank, fermentation 6~
144h, sterilize 20~40min under the conditions of being 70~150 DEG C in temperature, is cooled to 20~40 DEG C, obtains fermentation of seaweed thing;
e), prepare ecological seaweed liquid biostimulant and seaweed residue:By step 3)Obtained seaweed filtrate is added to step
4)Stirred and evenly mixed in obtained fermentation of seaweed thing, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and thawing
The mass ratio of corynebacterium ammoniagenes prepared by method is 95~99.8:0.2~5, it is 25~35 DEG C in temperature, oxygen content is 1~8mg/L
Under the conditions of, ferment 1~6 day, gained filtrate is ecological seaweed liquid biostimulant after press filtration;Filter residue is seaweed residue;
f), prepare ecological seaweed biostimulant:Ecological seaweed liquid biostimulant is concentrated with after spray drying, gives birth to obtain the final product
State seaweed bio stimulin;
The seaweed is that one kind in Enteromorpha, kelp, sargassum, opotism and kelp or arbitrary proportion are two or more.
2. do not add the production method of sulfur dioxide organic wine as claimed in claim 1, it is characterised in that:The orchard institute
It is lime sulfur, Bordeaux mixture, mineral oil, trichoderma harzianum, quinolinone suspending agent, ethanol, soda, China tree alkali, fish with pesticide
Rattan ketone, Dalmatian chrysanthemum missible oil and one kind in matrine or arbitrary proportion are two or more.
3. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:Thawing method is produced
The preparation of ammonia bar bacterium follows the steps below:
ⅰ)The expansion of strain is further cultured for:Using the thalline in exponential phase as seed, liquid is inoculated in 10% inoculum concentration
In culture medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for strain;
ⅱ)Thawing method improves the permeability of cell:Thawing method is at least repeated once, its step is:By step 1)Obtained expansion
It is further cultured for strain to be put into -20 DEG C of refrigerator, freezes 4h, takes out, dissolve at room temperature;
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio:Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, 20.0~25.0g of agar;
The preparation method of wherein beef infusion broth follows the steps below:Lean beef is cleaned, and chopping, weighs 550 grams and be soaked in
In 1375ml water, soak a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
4. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:The enzyme is
Lysozyme and pectase are according to mass ratio 5:1 composition, wherein lysozyme follow the steps below:
(A) add the water of 5 times of weight to egg white or whole egg liquid, after stirring evenly add homogenizer in, at least it is homogeneous once, homogeneous temperature
Spend for 30~60 DEG C, homogeneous pressure is 2~40MPa, and the homogeneous time is 10~40min, is filtered after homogeneous, and gained filtrate is dilution
Liquid;
(B) the dilution molecular cut off for obtaining step (A) is the ultrafiltration membrane ultrafiltration of 15000~30000Da, obtains thin liquid
And dope;
(C) add resin in the thin liquid that step (B) obtains to be adsorbed, the part by weight of the thin liquid and resin is 1:3~
8, the resin after being adsorbed;
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.001~0.5mol/L, is obtained
Eluent;
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention molecule of the ultrafiltration membrane are obtained
Measure as 2000~10000Da;
(F) after the ultrafiltrate drying obtained step (E), that is, lysozyme is obtained;
The resin is that the resin is cation exchange resin or macroporous absorbent resin;The model of cation exchange resin
001*7、732、AmberliteIR-120、Dowex-50、Lewatit-100、Diaion SK-1、AllassionCS、
One or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II,
One or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
5. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:Step c)Institute
Stating separation is included more than one or both of press filtration, filtering and centrifugation.
6. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:Step d)Institute
It is 60% to state mixed culture medium to water content, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is 0.5:99.5, it is 30 in temperature
DEG C, oxygen concentration 12%, ferment 120h, and sterilize 20min under the conditions of being 120 DEG C in temperature.
7. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:Step e)It is mixed
The mass ratio of corynebacterium ammoniagenes prepared by compound and thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L,
Fermentation 5 days.
8. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:The glue is red
Yeast and corynebacterium ammoniagenes are purchased from Chinese industrial Microbiological Culture Collection administrative center, and numbering is respectively 31192 Hes of CICC
CICC 10168。
9. the production method as claimed in claim 4 for not adding sulfur dioxide organic wine, it is characterised in that:Step A)In
It is described it is homogeneous be homogeneous 2 times, homogeneous temperature is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, the
Secondary homogeneous pressure is 4MPa.
10. the production method as claimed in claim 1 for not adding sulfur dioxide organic wine, it is characterised in that:Ecological sea
The mode that algae biostimulant is added in fertilizer is:With the interior addition manner of fertilizer material mixing granulation and spraying to fertilizer
The outer addition manner on grain surface.
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