CN109055234A - The preparation method and its application method of a kind of expensive rotten bacterium dry powder of grape - Google Patents
The preparation method and its application method of a kind of expensive rotten bacterium dry powder of grape Download PDFInfo
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- CN109055234A CN109055234A CN201810896627.8A CN201810896627A CN109055234A CN 109055234 A CN109055234 A CN 109055234A CN 201810896627 A CN201810896627 A CN 201810896627A CN 109055234 A CN109055234 A CN 109055234A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The purpose of the present invention is to provide the preparation methods and its application method of a kind of expensive rotten bacterium dry powder of grape, a series of technological means that its overall process is accumulated and saved using spores for facilitating Botrytis cinerea, guarantee the mass propagation of Botrytis cinerea spore, final dry powder is able to maintain original activity, can successfully infect when dry powder activating is sprayed on grape and your corruption grape generated.The step of preparation method of the expensive rotten bacterium dry powder of grape of the present invention are as follows: (1) activation culture of Botrytis cinerea；(2) Liquid Culture of Botrytis cinerea, the expansion culture of (3) Botrytis cinerea；(4) the wheat bran culture of Botrytis cinerea；(5) vacuum freeze drying.The step of application method of the expensive rotten bacterium dry powder of grape of the present invention are as follows: (1) prepare emulsifier solution；(2) your corruption bacterium dry powder solution prepared；(3) your corruption bacterium dry powder clear liquid separated；(4) your corruption bacterium sprinkling is inoculated with；(5) your corruption grape maturity degree monitored.
Description
One, technical field:
The present invention relates to the preparation methods and its application method of a kind of expensive rotten bacterium dry powder of grape.
Two, background technique:
Botrytised wine is derived from a very precious sweet wine in Europe, because its grape material passes through a kind of grey mold
The infection effect of bacterium and generate.The natural conditions your corruption grape generates are harsher, it needs a kind of unique vineyard miniature
Weather could generate --- morning sombre rich water gas, afternoon illumination is hot, dry.Therefore the yield of wine is few, price, it is therefore named
" your corruption wine ".Your corruption effect can occur after infecting Botrytis cinerea close to the maturity period for grape, can as raw material using your corruption grape
To brew Botrytised wine.The process of the expensive corruption of grape includes the dehydration of complicated enzyme conversion and grape berry, is generated more highly concentrated
Sugar, acid, glycerol, minerals and the fragrance component of degree.Special fragrance include citrus fruit, orange peel or grape fruit, honey,
Caramel, crystallization fruit, walnut, fragrance and curry etc..Moreover, these fragrance components are typical, flavor complexity is pure.Grape it is natural
Your requirement of the corruption effect to weather conditions is very harsh, so that the yield of natural Botrytised wine is seldom, produces Botrytised wine
Risk it is also very big.The whole world possesses this unique geographical environment, brings up the vineyard producing region of such harsh weather condition with regard to that
It is several, as the Bordeaux producing region Su Dai, the producing region Hungary Tuo Kayi and German Rhine high yield area.China grape wine producing region from
Right condition is unsatisfactory for the formation of the expensive rotten grape of nature.Therefore, the raw material of Botrytised wine is not easy to produce, to cause the Portugal Gui Fu
The product and yield of grape wine are limited.
Three, summary of the invention
In view of this, of the invention provides the preparation method and its application method of a kind of expensive rotten bacterium dry powder of grape, in conjunction with
The production method of the culture and mould dry powder of Botrytis cinerea, overall process are accumulated using a series of spores for facilitating Botrytis cinerea
And the technological means saved, guarantee the mass propagation of Botrytis cinerea spore, final dry powder is able to maintain original activity, will
Dry powder activating can successfully infect your corruption grape generated when being sprayed on grape.
To achieve the above object, the technical solution adopted by the present invention are as follows: a kind of preparation method of the expensive rotten bacterium dry powder of grape,
It is characterized by: the step of described preparation method are as follows: 1) activation culture of Botrytis cinerea, the 2) Liquid Culture of Botrytis cinerea,
3) the expansion culture of Botrytis cinerea, 4) the wheat bran culture of Botrytis cinerea, 5) vacuum freeze drying.
1) activation culture of Botrytis cinerea: preferred B.byssoidea for masterplate is taken to be inoculated on slant medium, 25~28
It is cultivated 5 days in DEG C constant incubator；
2) Liquid Culture of Botrytis cinerea: the Botrytis cinerea after 1/6 step 1) activation culture of picking is together with PDA culture medium
It is inoculated into deep-layer liquid culture medium, is cultivated 5 days under the conditions of 180r/min, 25~28 DEG C of shaking table culture；
3) the expansion culture of Botrytis cinerea: the inoculum concentration aspiration step 2 by 1%) bacterium solution after Liquid Culture, and be inoculated with
Onto culture medium, shaking table culture 5 days under the conditions of 180r/min, 25~28 DEG C；
4) inoculation that the Botrytis cinerea bacteria suspension cultivated presses 2% the wheat bran culture of Botrytis cinerea: will be expanded by step 3)
Amount is inoculated into bran mass, and is sufficiently stirred, and inoculation is made uniformly to cultivate 8~10 in 25~28 DEG C of constant incubator
It, bran mass with a thickness of 1cm or so；
5) vacuum freeze drying: the Botrytis cinerea after step 4) wheat bran culture is taken out together with bran mass, vacuum
Freeze-drying for 24 hours, is made the expensive rotten bacterium dry powder of grape, is fitted into Fresco Bag and is vacuum-packed.
Slant medium in the step 1) is 20% potato, 2% glucose, 0.05% magnesium sulfate, 0.1% phosphorus
Acid dihydride potassium, 2% agar；
Deep-layer liquid culture medium in the step 2) be 3% glucose, 4% murphy juice, 0.1% potassium nitrate,
0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate；
Culture medium in the step 3) is 3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% sulfuric acid
Magnesium, 0.1% potassium dihydrogen phosphate；
Bran mass is 1% glucose solution in the step 4): wheat bran=1:1.1 ratio is prepared.
A kind of application method of the expensive rotten bacterium dry powder of grape, it is characterised in that: the method and step are as follows: 1) preparation emulsification
Agent solution, 2) your corruption bacterium prepare your corruption bacterium dry powder solution, 3) separate your corruption bacterium dry powder clear liquid, 4) sprinkling is inoculated with, 5) your corruption of monitoring
Grape maturity degree.
Step 1) prepares emulsifier solution: the emulsifier solution is soybean separation protein white water solution, first measures soybean
Protein isolate is sufficiently dissolved with 23~25 DEG C of warm clear water, makes its concentration 2%；
Your corruption bacterium dry powder solution is step 2) prepare: taking Botrytis cinerea dry powder, by the additive amount of 1g/L, is added to step 1)
It in obtained soybean separation protein white water solution, is sufficiently stirred under room temperature, ultrasonic wave or centrifugal method make spore be sufficiently disengaged from bran
Your corruption bacterium dry powder solution the skin surface of solids obtains；
Your corruption bacterium dry powder clear liquid is step 3) separate: the suspension that step 2) is obtained is separated by solid-liquid separation, and takes clear liquid standby
With；
Your corruption bacterium step 4) sprinkling is inoculated with: by 10L/ mus of dosage, continuous date of 3~5 day afternoon without rain is selected,
At dusk or early morning continuously sprays spores solution 3~7, and the specific number that sprays is depending on fruit ear infection conditions.
Your corruption grape maturity degree is step 5) monitor: after fruit ear infects botrytis cinerea, primary sugar, acid content are measured every five days,
Rationally determine picking time.
Compared with prior art, the invention has the advantages that and effect:
1, this invention simplifies operation cumbersome in your artificial corruption grape cultivating process, simplify Botrytis cinerea strain it is preferred,
Be inoculated with and spread cultivation and etc., the dry powder formulations of preferred B.byssoidea for masterplate are researched and developed, are inoculated with your corruption bacterium to grape by manually spraying
On, the colonization status of grape is controlled, your artificial corruption grape and brewing Botrytised wine have been produced.
2, the production method that the present invention combines the culture and mould dry powder of Botrytis cinerea, overall process are helped using a series of
In the spore accumulation of Botrytis cinerea and the technological means of preservation, including Liquid Culture, wheat bran culture, vacuum freeze drying, vacuum
Packaging etc. guarantees the mass propagation of Botrytis cinerea spore, and final dry powder is able to maintain original activity, and dry powder is living
Change can successfully infect your corruption grape generated when being sprayed on grape.
3, the production that can be carried out your artificial corruption grape using the technology of the present invention, has broken the region of Botrytised wine production
Limitation will promote the development of China's Botrytised wine industrial technology, form the distinguishing products of China's Botrytised wine.
Four, Detailed description of the invention:
Fig. 1 is operation of the present invention flow chart；
Fig. 2 is the culture of Botrytis cinerea: a.PDA plate culture；B. deep-layer liquid culture；C. wheat bran culture
The strain dry powder of Fig. 3 various concentration starch protection agent activity of beta-glucosidase in the case where simulating grape juice culture is surveyed
Fixed light absorption value；
Fig. 4 is the radar map of your artificial corruption wine and the late fragrance organoleptic analysis's result for adopting raw material sweet wine.
Five, specific embodiment
It is limited below with reference to specific embodiment technical solution of the present invention is further:
The present invention proposes on forefathers' Research foundation aiming at the problem that China's Botrytised wine raw material is difficult to self-assembling formation
Then a kind of production method of your artificial corruption grape, first screening B.byssoidea for masterplate carry out expansion training to optimizing bacterial strain
It supports, then bacteria suspension is sprayed on the grape fruit of normal growth, cause your corruption effect occurs on epidermis for grape berry.
The present invention on condition that artificial screening excellent B.byssoidea for masterplate Botrytis-C, China typical culture collection
Center deposit number: M2013661, depositary institution: China typical culture collection center, preservation address: Wuhan, China, preservation
Date: on December 13rd, 2013, classification naming: Botrytis cinerea Botrytis-C (Botvytis cinerea Botrytis-C).
Bacterial strain glycosidase activity with higher, and growth vigor is stronger, is the potentiality bacterial strain of your artificial corruption grape production.
The step of preparation method of the expensive rotten bacterium dry powder of grape of the present invention are as follows: (1) activation culture of Botrytis cinerea；(2) grey Portugal
The Liquid Culture of grape spore, the expansion culture of (3) Botrytis cinerea；(4) the wheat bran culture of Botrytis cinerea；(5) vacuum freeze drying.
The step of application method of the expensive rotten bacterium dry powder of grape of the present invention are as follows: (1) prepare emulsifier solution；(2) your corruption prepared
Bacterium dry powder solution；(3) your corruption bacterium dry powder clear liquid separated；(4) your corruption bacterium sprinkling is inoculated with；(5) your corruption grape maturity degree monitored.
Embodiment:
1. the preparation method (referring to Fig. 1) of your corruption bacterium dry powder:
(1) activation culture of Botrytis cinerea: preferred B.byssoidea for masterplate is taken to be inoculated in PDA slant medium (20% soil
Beans, 2% glucose, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 2% agar) on, in 25~28 DEG C of constant incubators
Culture 5 days；
(2) Liquid Culture of Botrytis cinerea: the Botrytis cinerea after 1/6 step of picking (1) activation culture is cultivated together with PDA
Base is inoculated into deep-layer liquid culture medium (3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% magnesium sulfate, 0.1% phosphorus
Acid dihydride potassium) in, it is cultivated 5 days under the conditions of 180r/min, 25~28 DEG C of shaking table culture；
(3) the expansion culture of Botrytis cinerea: the inoculum concentration aspiration step 2 by 1%) bacterium solution after Liquid Culture, and be inoculated with
To on culture medium (3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate),
180r/min, shaking table culture 5 days under the conditions of 25~28 DEG C.
(4) it the wheat bran culture of Botrytis cinerea: will be connect by the Botrytis cinerea bacteria suspension for expanding culture by 2% inoculum concentration
Kind is sufficiently stirred in bran mass (1% glucose solution: wheat bran=1:1.1 ratio is prepared), makes inoculation uniformly,
It is cultivated 8~10 days in 25~28 DEG C of constant incubator.Culture medium with a thickness of 1cm or so.
(5) vacuum freeze drying: the Botrytis cinerea after wheat bran culture is taken out together with bran mass, vacuum refrigeration
It is dry, dry powder is made, is fitted into Fresco Bag and is vacuum-packed.
Above culture medium is weight percentage.
2. the application method of dry powder:
Dry powder is applied to the production of your corruption grape, the specific steps are as follows:
(1) it prepares emulsifier solution: preparing soybean separation protein white water solution, concentration about 2% (i.e. 20g/L) first measures big
Beans protein isolate is sufficiently dissolved with a small amount of warm water, is then sufficiently dissolved in room temperature (23~25 DEG C) clear water；
(2) it prepares your corruption bacterium dry powder solution: taking Botrytis cinerea dry powder by the additive amount of 1g/L, be added to above-mentioned soybean point
Make spore be sufficiently disengaged from the wheat bran surface of solids (ultrasonic wave or centrifugation to can also be used from being sufficiently stirred under room temperature in protein solution
The methods of)；
(3) separate your corruption bacterium dry powder clear liquid: the suspension that (2) step is obtained is separated by solid-liquid separation, and takes clear liquid spare.
(4) your corruption bacterium sprinkling is inoculated with: by 10L/ mus of dosage, continuous date of 3~5 day afternoon without rain is selected, at dusk
Or early morning continuously sprays spores solution 3~7.Specific sprinkling number is depending on fruit ear infection conditions.
(5) monitor your corruption grape maturity degree: after fruit ear infects botrytis cinerea, the primary sugar of measurement, acid content, are closed every five days
It manages and determines picking time.
The present invention during productive manpower expensive rotten grape by Botrytis cinerea dry powder activating after be sprayed onto normal grape
On, so that your corruption grape obtained, reduce from bacterial screening, activation and the serial procedures for expanding culture, to simplify behaviour
Make, save the time, improves working efficiency.Experimental study shows to obtain after culture in bran mass 8~10 days a large amount of
Botrytis cinerea is then carried out vacuum freeze drying by botrytis cinerea spore together with bran mass, is not adding any protective agent
Under conditions of, the spore of the Botrytis cinerea after drying has higher survival rate and glycosidase activity.
During application Botrytis cinerea dry powder obtains your artificial corruption grape, the work for carrying out your corruption bacterium dry powder is first had to
Change.Since there is waxy substance on grape fruit surface, therefore, it is necessary to add emulsifier (soybean protein isolate), to guarantee that sprinkling is lived
When Botrytis cinerea solution after change, spore can be attached on grape pomace, guarantee effectively infecting for your corruption bacterium, generate your corruption work
With.Continuous date of 3~5 day afternoon without rain is selected, sprays spores solution in the period in other evening to early morning, in order to avoid
Botrytis cinerea disseminates the dehiscent fruit after rain, and dip dyeing is caused excessively to cause disease.Continuous sprinkling 3~7 days, guarantee is disseminated successfully, often
Every the primary sugar of measurement on the five, acid content, picking time is rationally determined.Botrytised wine using dry powder brewing has citrus, coke
The special fragrance such as sugar, honey, milk, fragrance.
Experimental example:
The present invention is facilitated using preferred one plant of B.byssoidea for masterplate Botrytis C early period as material using a series of
The culture of Botrytis cinerea and store method, centrifugation is lyophilized and is freeze-dried two after wheat bran culture after research compares Liquid Culture
Kind method, the results showed that, after wheat bran culture, under the conditions of not adding protectant, carry out vacuum freeze drying, obtained ash
Grape spore dry powder survival rate is higher, reaches 83%, and activity of beta-glucosidase is up to 1.11U/mL.
1 purpose
Botrytised wine raw material aiming at the problem that being difficult to self-assembling formation, dry powder formulations are carried out to preferred Botrytis cinerea
Research and development, to reduce the bacterial screening in your artificial corruption wine production, optimization, expand and cultivate, by artificially controlling Portugal
Grape infect situation, convenient for the generation of your artificial corruption grape.
2 experiment contents
(1) by the pathogen of Botrytis cinerea of laboratory purification storage, activated culture, fermented and cultured obtain enough ashes
Botrytis liquid.
(2) Botrytis cinerea is cultivated using the methods of deep-layer liquid culture, wheat bran culture, obtains a large amount of spore.
(3) drying means and protection agent prescription of Botrytis cinerea, vigor and β-Portugal after keeping mould that dry powder is made are determined
Polyglycoside enzymatic activity guarantees the success of subsequent experimental in a higher level.
3 materials and instrument
3.1 strain material
B.byssoidea for masterplate: laboratory early period screening excellent B.byssoidea for masterplate Botrytis-C, the bacterial strain have compared with
High glycosidase activity, and growth vigor is stronger.
Grape material: the Vidal Blanc grape of Shangri-La, Yunnan Province chateau plantation and evening adopt Vidal Blanc grape material. 3.2
Instrument and reagent
MP-250B constant incubator (Shanghai laboratory equipment Co., Ltd)；NRY-2102C type shaking table Shanghai laboratory
Equipment Co., Ltd)；AIRTECH type aseptic operating platform (SuZhou Antai Air Tech Co., Ltd.)； LABCONCO77530-11
Type vacuum freeze drier (LABCONCO company of the U.S.)；(Japanese Thomas is public for TOMYSS-325 type full-automatic high-pressure autoclave
Department) RC-5CPLUS high speed freezing centrifuge (section of the U.S. high instrument (Hong Kong) Co., Ltd)；The light splitting of Cary60 type UV, visible light
Photometer (Anjelen Sci. & Tech. Inc)；GCMS-PQ2020 instrument (Shimadzu Corporation).
Glucose, magnesium sulfate, potassium dihydrogen phosphate, agar, yeast extract, malic acid, fructose, tannic acid, tartaric acid, chlorine
It is (raw to change ammonium, citric acid, dipotassium hydrogen phosphate, soluble starch, sucrose, glycerol (Xilong Chemical Co., Ltd), trehalose
Object reagent, MPBiomedicals), lactose (biological reagent, Beijing extensive and profound in meaning star biotechnology Co., Ltd), skimmed milk power
(Inner Mongolia Yili Industry Group Co., Ltd), 4- nitro-β-D glucoside (Sigma Co., USA).
3.3 culture medium
PDA culture medium formula: 20% potato, 2% glucose, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 2% fine jade
Rouge.
Deep-layer liquid culture medium prescription: 3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% magnesium sulfate,
0.1% potassium dihydrogen phosphate.
Bran mass formula: 1% glucose solution: wheat bran=1:1.1
Simulate grape juice formula: 11% glucose, 11% fructose, 0.3% tartaric acid, 0.03% malic acid, 0.05% chlorine
Change ammonium, 0.06% yeast extract, 0.2% tannic acid, 0.2% potassium dihydrogen phosphate, 0.02% magnesium sulfate.
4 test methods
The activation culture of 4.1 Botrytis cinereas
- 20 DEG C of presence of the preferred B.byssoidea for masterplate of going bail for is inoculated in inclined-plane PDA culture medium, in 25 DEG C of constant temperature incubations
It is cultivated 5 days in case.
The Liquid Culture of 4.2 Botrytis cinereas
Botrytis cinerea after 1/6 activation culture of picking is inoculated into deep-layer liquid culture medium together with PDA culture medium,
180r/min is cultivated 5 days under the conditions of 25 DEG C of shaking table culture.
The expansion culture of 4.3 Botrytis cinereas
Bacterium solution after drawing Liquid Culture by 1% inoculum concentration, and be inoculated on deep-layer liquid culture medium, in 180r/
Min, shaking table culture 5 days under the conditions of 25 DEG C.
The wheat bran culture of 4.4 Botrytis cinereas
It will be inoculated into bran mass by the Botrytis cinerea bacteria suspension for expanding culture by 2% inoculum concentration, and sufficiently
Stirring keeps inoculation uniform.It is cultivated 8~10 days in 25 DEG C of constant incubator.Culture medium with a thickness of 1cm or so.
4.5 vacuum freeze drying
By through expansion culture after bacterium solution revolving speed be 8000r/min, centrifugation time be 20min under conditions of freeze
High speed centrifugation.Sediment addition trehalose, lactose, sucrose, skimmed milk power, glycerol after centrifugation carry out vacuum as protective agent
Freeze-drying, program parameter such as table 1.There are three different concentration (5%, 10%, 15%) for every kind of protective agent, in the ratio of 1:2
Protective agent, i.e. 1mL protective agent are added, the ratio of 2g thallus carries out experiment of single factor.It is vacuum-packed after freeze-drying with Fresco Bag.It sees
It examines and records.
1 vacuum freeze drying program of table
Botrytis cinerea after wheat bran culture is taken out together with bran mass, respectively add concentration be (0,10%,
20%) starch protection agent, and tile to be put into basin in vacuum freeze drier and carry out vacuum freeze drying for 24 hours, system
At dry powder, drying program parameter such as table 1.Dry complete to guarantee, the culture medium of tiling and the thickness of Botrytis cinerea are on the left side 5mm
It is right.It is vacuum-packed after freeze-drying with Fresco Bag.
4.6 survival rates and glycosidase activity measurement
Strain is inoculated into before freeze-drying after being cultivated 3 days on PDA plate culture medium, records clump count.With same after freeze-drying
Method culture and record.Survival rate after calculating freeze-drying.
Botrytis cinerea after freeze-drying is inoculated into simulation grape juice, 1d, 2d, 3d, 4d, 5d, β-Portugal of 6d are cultivated in measurement
Polyglycoside enzymatic activity.
The substrate of activity of beta-glucosidase measurement is 4- nitrobenzene-β-D-Glucose glycosides, and reaction system includes 100 μ L
Culture solution, 750 μ L citrate-phosphate hydrogen dipotassium buffers, 250 μ L (1mM) substrates, 37 DEG C reaction 30min after use 1M carbonic acid
Sodium solution terminates reaction.Light absorption value is measured at 400nm.Since light absorption value is directly proportional to glycosidase activity in a certain range,
Therefore glycosidase activity size can be indicated with light absorption value.
The application method of 4.7 dry powder
The use experiment that Botrytis cinerea dry powder is carried out on the chateau Vidal Blanc grape of Shangri-la, dry powder is pressed centainly
After method activation, the suitable time is selected to be sprayed on Vidal Blanc grape fruit, it is specific spray number view fruit ear infection conditions and
It is fixed, and sugar, acid content are measured, rationally determine picking time.
4.7.1 the brewing of your corruption wine:
The brewage process that Botrytised wine and evening adopt raw material sweet wine is carried out referring to the technique of sweet wine, main work
Steps are as follows for skill:
(1) raw material directly squeezes after crushing, takes juice with prestissimo, and the squeezing juice of last time is separated；
(2) immediately with the sulfur dioxide treatment of 40~70mg/L after squeezing；
(3) clarification for 24 hours, makes the turbidity of grape juice reach 500~600NTU (astigmatism turbidity unit) naturally；
(4) ammonium sulfate and VB1 that 100~150mg/L is added in fermentation liquid promote fermentation, and access 2% activation for 24 hours
Yeast juice, and by fermentation temperature control at 20~24 DEG C；
(5) when the potential wine degree of residual sugar and wine degree reach balance, closed separation is carried out, and with 200~350mg/L
Sulfur dioxide treatment, with bacteriostatic agent terminate ferment；
(6) content of a couple of days post analysis free sulur dioxide, and being separated, by free sulur dioxide content adjust to
60mg/L or so；
(7) conventional physical and chemical index measurement, organoleptic attribute analysis are carried out storage to May next year.
4.7.2 the measurement of grape wine routine physical and chemical index
The artificial Botrytised wine (NW) and Shangri-la evening that measurement is made using Botrytis cinerea dry powder respectively adopt Vidal Blanc
The conventional physical and chemical index of the naturally dense sweet wine (SW) of grape material brewing.Measuring method is as follows:
Residual glucose: fehling reagent measuring method；
Alcoholic strength: density bottle method；
Total acid and volatile acid: acid-base titration.
4.7.3 organoleptic analysis
The organoleptic analysis of wine sample, with reference to (2013) such as Peng, it is special to major in grape wine by 15 using sense organ quantization trial test method
The graduate and undergraduate composition taste panel of industry carries out sensory evaluation to NW and two wine sample of SW.5~8s of static news perfume shakes
Dynamic wineglass hears 5~10s of perfume, and difference hears 1~2min of fragrant operating interval, then drinks grape wine, wine liquid covers lingual surface, gently inhales one
Implication stirs the tip of the tongue, feels 5~6s, discharge wine liquid, nasal cavity 3~4s of feedback feeling, to 5 scales of organoleptic feature of perception
Method is quantified: 1- is very weak, and 2- is weak, and in 3-, 4- is strong, and 5- is very strong.The final quantization intensity value (MF%) of a certain odor characteristic is comprehensive
Trial test group is closed to the frequency of use (F%) of this odor characteristic vocabulary and the information of average strength (I%), quantify to use with
Lower formula calculates:
5 results and analysis
The culture of 5.1 Botrytis cinereas and the collection of spore
Go bail for -20 DEG C of presence artificial screening excellent B.byssoidea for masterplate Botrytis-C, be inoculated in PDA culture medium
On, it is cultivated 5 days in 25 DEG C of constant incubators.The breeding of Botrytis cinerea first grows the mycelia of white, and then mycelia range is gradually
Expand.Culture started to produce spore after three days, and the color of bacterium colony starts to be changed into grey.It observes under the microscope, sporophore end is swollen
Greatly, have branch, there is kick, generate from end and much attend to anything else spores, be in grape spike, spore of attending to anything else is unit cell, in spherical or
Elliposoidal.
It is observed that white millet is granular after Botrytis cinerea after taking activation culture carries out deep-layer liquid culture culture 3 days
Hypha body, at this time the color of culture solution be it is light yellow.Due to the generation of spore, culture solution becomes celadon after 5 days, with
Incubation time increases, and cenobium becomes larger.The phenomenon that observing when expanding culture is similar to Liquid Culture.
There is white hypha generation at wheat bran culture 3 days, 5 days whens have a small amount of spore to generate, and spore is concentrated mainly on wheat bran training
Support the contact surface of primary surface and culture medium and conical flask.
The culture of Botrytis cinerea such as Fig. 1.
5.2 vacuum freeze-drying methods and protectant determination
Liquid Culture-centrifugation freeze-drying
Will be enlarged by culture after bacterium solution revolving speed be 8000r/min, under conditions of be centrifuged 20min.By the precipitating after centrifugation
Object (thallus) trehalose (5%, 10%, 15%), lactose (5%, 10%, 15%), sucrose (5%, 10%, 15%), degreasing
Milk powder (5%, 10%, 15%), glycerol (5%, 10%, 15%) are lyophilized as protective agent.It is added in the ratio of 1:2, i.e.,
The ratio of 1mL protective agent, 2g thallus carries out experiment of single factor.
Protective agent is trehalose, humidity is big after the freeze-drying of lactose, sucrose, skimmed milk power, shape such as dough, color
It is very sticky after protective agent is the freeze-drying of the bacterium of glycerol and distilled water for canescence, shape is not fixed, color is in grayish green
Color.It is not ideal dry powder.
Bacterium after freeze-drying is inoculated into deep-layer liquid culture medium and is cultivated.The different protectant strains of addition are in culture
Different material can be generated, causes the color of culture solution and fragrance different.The color of the bigger culture solution of protective agent additive amount is more
Deep, protective agent has the peat-reek of nail polish and fruit respectively after the culture for sucrose and skimmed milk power.
Wheat bran culture-freeze-drying
Botrytis cinerea after wheat bran culture is taken out together with bran mass, respectively add concentration be (0,10%,
20%) starch protection agent, and tile to be put into basin in vacuum freeze drier and carry out vacuum freeze drying, it is made
Dry powder.
Survival rate after vacuum freeze drying
As shown in Table 2, having higher survival rate when not adding any protective agent is 83.34% ± 2.54%, adds starch
The survival rate of Botrytis cinerea reduces instead after protective agent, and survival rate is 80.81% ± 0.00% when adding 10% starch, adds
Survival rate is 53.04% ± 2.53% when adding 20% starch.The survival rate highest of starch protection agent, the shallow lake of addition are not added
The amplitude that the fewer survival rate of powder content reduces is smaller.The agent of different content starch protection is added after measuring wheat bran culture-freeze-drying
Survival rate after freeze-drying is as shown in table 2.
The survival rate of Botrytis cinerea after 2 wheat bran culture of table-freeze-drying
Note: lowercase indicates the otherness of every row, α=0.05
Activity of beta-glucosidase under the starch of Different adding amount
It will be inoculated into after dry powder activating in simulation grape juice, 1d, 2d, 3d, 4d, 5d, the beta-glucosidase of 6d are cultivated in measurement
Enzymatic activity measures situation such as table 3, daily light absorption value such as Fig. 3.
As shown in Table 3, grape juice culture first three days are simulated, the glycosidase activity variation of three kinds of situations is consistent, culture first
Glycosidase activity is minimum after it, is increased within second day, and it is high that third day is decreased slightly as low rear increase.Starch protection agent is not added
Sharply increase in culture the when glycosidase activity, glycosidase activity reaches maximum value.10% starch additive amount at the 6th day
When glycosidase activity reach 1U/mL.Variation and the place of no starch additive amount of the processing of 10% starch additive amount at the 4th~6 day
Reason is identical in 3~5 days variation tendencies, and 20% variation is delayed.
It can be obtained by Fig. 2 analysis, the light absorption value without starch addition after culture 5 days increases and light absorption value maximum at this time.
The strain of 10% starch additive amount is increased when cultivating 6 days, and light absorption value at this time is maximum.Starch is not added since the 4th day
Protectant light absorption value rises most fast, is secondly the strain that starch additive amount is 10%, on the maximum light absorption value of starch additive amount
It rises most slow.And the light absorption value without adding starch protection agent reaches maximum value at first.Thus starch known to analysis is to grey grape
The growth of spore has inhibiting effect, and the intensity inhibited is related with the content of starch.
The strain dry powder of the addition various concentration starch protection agent of table 3 beta-glucosidase enzyme activity in the case where simulating grape juice culture
The measurement result of property
Note: lowercase indicates the otherness of every row, α=0.05
The quality analysis of Botrytised wine
It is the artificial Botrytised wine of your the corruption bacterium dry powder gained grape material brewing made using the present invention shown in table 4
(NW) the conventional physical and chemical index analysis result of the dense sweet wine (SW) of raw material brewing is adopted with the grape evening.Two sweet wine
Little compared to total difference, each index is all normal, and specific value is slightly different.The wine degree of NW wine sample is that 8.79 (%vol) are high
In SW wine sample 0.9 (%vol) left and right；Volatile acid is all slightly above 1.1g/L, is lower than 1.2g/L；Total acid NW wine sample lacks SW wine sample 4g/
L；19g/L more than residual sugar content NW ratio SW.
Your the artificial corruption wine of table 4 and evening adopt the conventional physical and chemical index analysis result of raw material sweet wine
Note: lowercase indicates the otherness of every row, α=0.05
It is the radar map of your artificial corruption wine and the late fragrance organoleptic analysis's result for adopting raw material sweet wine shown in Fig. 4, is related to fragrance
Type has 10 kinds, and for the characteristic perfume such as caramel, honey, coffee, orange peel, fragrance etc. of your corruption wine, this two wine, which has, is in
It is existing.The tart fruit class of NW wine sample, hesperidium class, fragrance of a flower class, the MF value of small berries and citrus are greater than 50%, and SW wine sample only has acid
Fruit, hesperidium class, fragrance of a flower class, four kinds of small berries be greater than 50%.The fragrance power trend that two wine is presented is much the same, but NW
More coordinate compared with SW, not particularly pertinent fragrance.
6 conclusions
Experiment facilitates ash using a series of using preferred one plant of B.byssoidea for masterplate Botrytis C early period as material
The culture of grape spore and store method, research compare centrifugation freeze-drying and two kinds of freeze-drying after wheat bran culture after Liquid Culture
Your corruption bacterium dry powder is method prepare.
It is not as a result ideal thalli dry powder state after Liquid Culture-centrifugation freeze-drying, and dry with wheat bran culture freezing
The step for dry method is compared, increases centrifugation, therefore more demand to centrifugation apparatus.In comparison, wheat bran is trained
It is easy to operate support-to be freeze-dried this method, reduces device requirement, and wheat bran is cheap, is easy to get.Therefore it uses
Wheat bran culture-freeze-drying is not only easy to operate but also at low cost.
It is freeze-dried this method using wheat bran culture-, in the case where not adding protectant situation, vacuum freeze drying pair
The injury of spore is small, and the survival rate of spore is high.It is added to the dry powder of starch protection agent, β-Portugal when with simulation grape juice culture
Dry powder of the polyglycoside enzymatic activity lower than without addition starch protection agent.In culture to bacterium solution it has been observed that starch is to grey grape
The activity of beta-glucosidase of spore has inhibiting effect, and the starch protection agent concentration of addition is higher, and glycosidase activity is lower.Therefore
Addition protective agent is not needed when preparing Botrytis cinerea dry powder with wheat bran culture-freeze-drying method.After comparative liquid culture from
Two methods are freeze-dried after heart freeze-drying and wheat bran culture, the results showed that, it is any protectant not adding after wheat bran culture
Under the conditions of, wheat bran is mixed with strain and carries out vacuum freeze drying, obtains survival rate high (83.34%), beta-glucosidase enzyme activity
Property big (1.11U/mL) your corruption bacterium dry powder.
The application of your corruption bacterium dry powder the result shows that, the expensive rotten grape material of gained can successfully brew Botrytised wine.Often
Physical and chemical index analysis result and fragrance organoleptic analysis are advised the results show that every conventional index of artificial Botrytised wine is in sweet tea Portugal
In the ideal range of grape wine, odor characteristic is more coordinated, the typicalness with Botrytised wine.
Claims (5)
1. a kind of preparation method of the expensive rotten bacterium dry powder of grape, it is characterised in that: the step of the described preparation method are as follows: 1) grey grape
The activation culture of spore, 2) Liquid Culture of Botrytis cinerea, 3) the expansion culture of Botrytis cinerea, 4) the wheat bran culture of Botrytis cinerea,
5) vacuum freeze drying.
2. a kind of preparation method of the expensive rotten bacterium dry powder of grape according to claim 1, it is characterised in that:
1) activation culture of Botrytis cinerea: taking preferred B.byssoidea for masterplate to be inoculated on slant medium, in 25~28 DEG C of perseverances
It is cultivated 5 days in warm incubator；
2) Liquid Culture of Botrytis cinerea: the Botrytis cinerea after 1/6 step 1) activation culture of picking is inoculated into together with PDA culture medium
In deep-layer liquid culture medium, cultivated 5 days under the conditions of 180r/min, 25~28 DEG C of shaking table culture；
3) the expansion culture of Botrytis cinerea: the inoculum concentration aspiration step 2 by 1%) bacterium solution after Liquid Culture, and it is inoculated into culture
On base, shaking table culture 5 days under the conditions of 180r/min, 25~28 DEG C；
4) the wheat bran culture of Botrytis cinerea: the Botrytis cinerea bacteria suspension for expanding culture by step 3) is connect by 2% inoculum concentration
Kind is sufficiently stirred into bran mass, cultivates inoculation uniformly 8~10 days in 25~28 DEG C of constant incubator, bran
Skin culture medium with a thickness of 1cm or so；
5) vacuum freeze drying: the Botrytis cinerea after step 4) wheat bran culture is taken out together with bran mass, vacuum refrigeration
Drying for 24 hours, is made the expensive rotten bacterium dry powder of grape, is fitted into Fresco Bag and is vacuum-packed.
3. a kind of preparation method of the expensive rotten bacterium dry powder of grape according to claim 1, it is characterised in that:
Slant medium in the step 1) is 20% potato, 2% glucose, 0.05% magnesium sulfate, 0.1% biphosphate
Potassium, 2% agar；
Deep-layer liquid culture medium in the step 2) is 3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% sulphur
Sour magnesium, 0.1% potassium dihydrogen phosphate；
Culture medium in the step 3) be 3% glucose, 4% murphy juice, 0.1% potassium nitrate, 0.05% magnesium sulfate,
0.1% potassium dihydrogen phosphate；
Bran mass is 1% glucose solution in the step 4): wheat bran=1:1.1 ratio is prepared.
4. a kind of application method of the expensive rotten bacterium dry powder of grape as described in claim 1, it is characterised in that: the method and step
Are as follows: 1) prepare emulsifier solution, 2) prepare your corruption bacterium dry powder solution, 3) separate your corruption bacterium dry powder clear liquid, 4) sprinkling inoculation your corruption
Bacterium, 5) your corruption grape maturity degree monitored.
5. a kind of application method of the expensive rotten bacterium dry powder of grape according to claim 4, it is characterised in that:
Step 1) prepares emulsifier solution: the emulsifier solution is soybean separation protein white water solution, first measures soybean separation
Albumen is sufficiently dissolved with 23~25 DEG C of warm clear water, makes its concentration 2%；
Your corruption bacterium dry powder solution is step 2) prepare: taking Botrytis cinerea dry powder, by the additive amount of 1g/L, is added to what step 1) obtained
It in soybean separation protein white water solution, is sufficiently stirred under room temperature, ultrasonic wave or centrifugal method make spore be sufficiently disengaged from wheat bran solid
Your corruption bacterium dry powder solution surface obtains；
Your corruption bacterium dry powder clear liquid is step 3) separate: the suspension that step 2) is obtained is separated by solid-liquid separation, and takes clear liquid spare；
Your corruption bacterium step 4) sprinkling is inoculated with: by 10L/ mu of dosage, selection continuous date of 3~5 day afternoon without rain, in the dusk or
Early morning continuously sprays spores solution 3~7, and the specific number that sprays is depending on fruit ear infection conditions.
Your corruption grape maturity degree is step 5) monitor: after fruit ear infects botrytis cinerea, measuring primary sugar, acid content every five days, rationally
Determine picking time.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104255384A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Method for cultivating botrytised grapes |
| CN104263659A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Preparation method of botrytis cinerea agent |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104255384A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Method for cultivating botrytised grapes |
| CN104263659A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Preparation method of botrytis cinerea agent |
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| 陶永胜等: "人工贵腐葡萄酒香气的仪器分析与感官评价", 《农业机械学报》 * |
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