CN106085792A - A kind of preparation method of tricholoma matsutake mycelium fermented type vinegar beverage - Google Patents
A kind of preparation method of tricholoma matsutake mycelium fermented type vinegar beverage Download PDFInfo
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Abstract
The preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage, the present invention relates to the preparation method of tricholoma matsutake mycelium fermented type vinegar beverage.The present invention is to solve in prior art all with Tricholoma matsutake (lto et lmai) Singer sporophore as material, utilize that form is single, cost is high and the problem of growth cycle length.The present invention is with tricholoma matsutake mycelium as raw material, obtain the former vinegar of mycelium by alcoholic fermentation and acetic fermentation, utilize response surface optimization analytic approach to analyze sweat, obtain optimal parameter, finally with fruit syrup and honey for allotment raw material, orthogonal test method is used to allocate.Determine alcoholic fermentation condition: fermentation temperature 28.5 DEG C, fermentation time 6d, pH 5, inoculum concentration 1.4%；Acetic fermentation condition: fermentation temperature 28.0 DEG C, shaking speed 146r/min, inoculum concentration 10%.Obtain and fill a prescription: the former vinegar of matsutake 10%, fruit syrup 8%, honey 5%, water 77%.The present invention is applied to mycelium health care new product development field.
Description
Technical field
The present invention relates to the preparation method of tricholoma matsutake mycelium fermented type vinegar beverage.
Background technology
Matsutake (Tricholoma matsutake) formal name used at school is Trichotoma matsutake, belongs to Basidiomycota, and Tricholomataceae belongs to, and is famous and precious
Wild edible and medical fungi, has the title of " king of edible mushroom ".It is rich in multiple human bodies such as rich in protein, amino acid, vitamins
Necessary nutritional labeling, matsutake also has radioresistance, antitumor, removing free radical, anti-ageing biological effectiveness of waiting for a long time simultaneously.Mesh
Before, the research and utilization to matsutake is all with fructification as material, matsutake be naturally born in autumn height above sea level 2000～4000 meters and more than
Free of contamination pine forest or theropencedrymion in, it has symbiosis with pine roots, needs again the shade of the broad-leaf forests such as robur
Cover condition.Mid-August to the early October in annual autumn is the collection season of matsutake.Owing to matsutake grows to environmental condition
Requiring strict, matsutake can't realize artificial cultivation at present, leans on wild collection completely, and therefore the yield of matsutake is very low and price is held high
Expensive.
Content of the invention
The present invention is to solve in prior art all with Tricholoma matsutake (lto et lmai) Singer sporophore as material, utilizes that form is single, cost is high, raw
Long period length and be difficult to the problem manually obtaining, and the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage proposing.
The preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage realizes according to the following steps:
Tricholoma after activation is inoculated into liquid by the inoculum concentration of 10% volume ratio by step one: carry out matsutake thalline activation
Cultivate on body culture medium, homogenate is carried out to the nutrient solution containing tricholoma matsutake mycelium after cultivation and filters, with sucrose by sugar content
The matsutake nutrient solution to 13.0%, after being allocated for the allotment；
Step 2: saccharomycete and acetic acid bacteria slant preservation bacterial classification are respectively connected to microzyme culture medium and acetic acid bacteria culture medium
In, it is enlarged cultivating；
Step 3: step 2 is expanded in the matsutake nutrient solution that the saccharomycete access step one after cultivating obtains and carry out alcohol
Fermentation single factor experiment；
Step 4: the alcoholic fermentation single factor experiment result according to step 3 carries out alcoholic fermentation Optimum Experiment, determines wine
Essence fermentation condition is: temperature is 28～29 DEG C, and fermentation time is 6～6.5 days, and pH is 5～6, and inoculum concentration is 1.4～1.5%；
Step 5: carry out fermentation under the fermentation condition that step 4 determines and obtain matsutake alcohol fermentation liquid, at matsutake alcohol
Zymotic fluid accesses the acetic acid bacteria after step 2 expands cultivation and carries out acetic fermentation single factor experiment；
Step 6: the acetic fermentation single factor experiment result according to step 5 carries out acetic fermentation Optimum Experiment, determines vinegar
Acid fermentation condition is: fermentation temperature is 28～29 DEG C, and shaking speed is 146～150r/min, and inoculum concentration is 10～11%；
Step 7: carry out fermentation under the fermentation condition that step 6 determines and obtain the former vinegar of matsutake, with the former vinegar of matsutake, high fructose corn
Slurry and honey be that influence factor carries out orthogonal test, determine the former vinegar of matsutake in tricholoma matsutake mycelium fermented type vinegar beverage, fruit syrup,
The mass percent of honey and water is 10:8:5:77.
Invention effect:
Acetic acid beverage belongs to the rising star of beverage industry, has the effects such as dispelling fatigue, hypotensive, beauty treatment, intestines and stomach health-care,
The present invention, will the currently study hotspot of edible mushroom industry and health beverage by combination creative to tricholoma matsutake mycelium and acetic acid beverage
The study hotspot of product has carried out organic fusion, is that matsutake is manually developed while providing new way also for health drink industry
Development new direction is provided.The present invention plants with the matsutake test tube strains of Laboratories Accession for test mother, plants, with mother, the liquid that spreads cultivation and is
Experiment material, formulated acquisition sweet and sour taste on the basis of to the optimization of process conditions of spread cultivation liquid alcoholic fermentation and acetic fermentation
Tricholoma matsutake mycelium fermented type vinegar beverage.
Show that tricholoma matsutake mycelium and fructification nutritional labeling are close with content after deliberation, therefore tricholoma matsutake mycelium is matsutake removes son
What the outer another kind of entity was new utilizes form.The present invention, with tricholoma matsutake mycelium as primary raw material, not only greatly reduces cost of material,
And the Mycelium culture time is short；Tricholoma matsutake mycelium can be obtained by liquid fermentation and culture, is obtained by alcoholic fermentation, acetic fermentation
The former vinegar of mycelium, with fruit syrup and honey for allotment raw material, allocate.And extracellular many in tricholoma matsutake mycelium zymotic fluid
Sugar also can become the nutritional labeling in beverage.
Response surface optimization analytic approach is utilized to be analyzed sweat, to obtain optimal parameter.The optimal bar of alcoholic fermentation
Part is: fermentation temperature 28.5 DEG C, fermentation time 6d, pH 5, and during inoculum concentration 1.4%, alcoholic strength is 6.51%, parallel through three times
It is 6.50% that test obtains actual value, differs less with theoretical value, and fitness is preferable；Acetic fermentation optimal conditions is: fermentation temperature
Spending 28.0 DEG C, shaking speed 146r/min, during inoculum concentration 10%, total acidity is 34.25%, obtains reality through three parallel tests
Actual value is 34.1%, differs less with theoretical value, so its fitness is preferable.Whole sweat is carried out pre-by available the method
Survey and analyze.
Allotment use orthogonal test method, it is thus achieved that best flavor formula be: the former vinegar of matsutake 10%, fruit syrup 8%, honeybee
Honey 5%, water 77%.Finished product excellent in flavor and the tricholoma matsutake mycelium vinegar beverage with health-care efficacy, have certain market value.
Brief description
Fig. 1 is that fermentation temperature affects figure to alcoholic fermentation alcoholic strength；
Fig. 2 is that fermentation time affects figure to alcoholic fermentation alcoholic strength；
Fig. 3 is that pH affects figure to alcoholic fermentation alcoholic strength；
Fig. 4 is that inoculum concentration affects figure to alcoholic fermentation alcoholic strength；
Fig. 5 be fermentation temperature Dichlorodiphenyl Acetate fermentation total acidity affect figure；
Fig. 6 be inoculum concentration Dichlorodiphenyl Acetate fermentation total acidity affect figure；
Fig. 7 be shaking speed Dichlorodiphenyl Acetate fermentation total acidity affect figure；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Fig. 8 is Y=(A, B)；In figure the 6.1st, the 6.2nd,
6.3rd, 6.4 and 6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Fig. 9 is Y=(A, B)；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 10 is Y=(A, C)；In figure the 6.1st, the 6.2nd,
6.3rd, 6.4 and 6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 11 is Y=(A, C)；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 12 is Y=(A, D)；In figure the 6th, the 6.1st, the 6.2nd,
6.3rd, 6.4 and 6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 13 is Y=(A, D)；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 14 is Y=(B, C)；6.3rd, 6.4 and in figure
6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 15 is Y=(B, C)；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 16 is Y=(B, D)；In figure the 6.2nd, the 6.3rd,
6.4 and 6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 17 is Y=(B, D)；
The response surface figure of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 18 is Y=(C, D)；In figure the 6.2nd, the 6.3rd,
6.4 and 6.5 is alcoholic strength；
The contour map of tricholoma matsutake mycelium nutrient solution alcoholic fermentation test when Figure 19 is Y=(C, D)；
The response surface figure of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 20 is Y=(A, B)；28th, the 30th, 32 and in figure
34 is total acid；
The contour map of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 21 is Y=(A, B)；
The response surface figure of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 22 is Y=(B, C)；In figure the 28th, the 29th, 30/
31st, 32 and 33 is total acid；
The contour map of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 23 is Y=(B, C)；
The response surface figure of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 24 is Y=(A, C)；28th, the 30th, 32 and in figure
34 is total acid；
The contour map of tricholoma matsutake mycelium nutrient solution acetic fermentation test when Figure 25 is Y=(A, C)；
Figure 26 is flow chart of the present invention.
Detailed description of the invention
Detailed description of the invention one: as shown in figure 26, the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage include with
Lower step:
Tricholoma after activation is inoculated into liquid by the inoculum concentration of 10% volume ratio by step one: carry out matsutake thalline activation
Cultivate on body culture medium, homogenate is carried out to the nutrient solution containing tricholoma matsutake mycelium after cultivation and filters, with sucrose by sugar content
The matsutake nutrient solution to 13.0%, after being allocated for the allotment；
Step 2: saccharomycete and acetic acid bacteria slant preservation bacterial classification are respectively connected to microzyme culture medium and acetic acid bacteria culture medium
In, it is enlarged cultivating；
Step 3: step 2 is expanded in the matsutake nutrient solution that the saccharomycete access step one after cultivating obtains and carry out alcohol
Fermentation single factor experiment；
Step 4: the alcoholic fermentation single factor experiment result according to step 3 carries out alcoholic fermentation Optimum Experiment, determines wine
Essence fermentation condition is: temperature is 28～29 DEG C, and fermentation time is 6～6.5 days, and pH is 5～6, and inoculum concentration is 1.4～1.5%；
Step 5: carry out fermentation under the fermentation condition that step 4 determines and obtain matsutake alcohol fermentation liquid, at matsutake alcohol
Zymotic fluid accesses the acetic acid bacteria after step 2 expands cultivation and carries out acetic fermentation single factor experiment；
Step 6: the acetic fermentation single factor experiment result according to step 5 carries out acetic fermentation Optimum Experiment, determines vinegar
Acid fermentation condition is: fermentation temperature is 28～29 DEG C, and shaking speed is 146～150r/min, and inoculum concentration is 10～11%；
Step 7: carry out fermentation under the fermentation condition that step 6 determines and obtain the former vinegar of matsutake, with the former vinegar of matsutake, high fructose corn
Slurry and honey be that influence factor carries out orthogonal test, determine the former vinegar of matsutake in tricholoma matsutake mycelium fermented type vinegar beverage, fruit syrup,
The mass percent of honey and water is 10:8:5:77.
Detailed description of the invention two: present embodiment from unlike detailed description of the invention one: described step one carries out pine
The detailed process of fine and soft thalline activation is:
To preserve bacterial classification transfer needle and access in matsutake slant medium, and cultivate 7～8 days for 23～25 DEG C, described matsutake is oblique
Face culture medium is for by glucose 2g, peptone 0.3g, KH2PO4 0.1g、MgSO 40.05g and agar 2g adds potato liquor
In be settled to 100ml.
Other steps and parameter are identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment from unlike detailed description of the invention one or two: described step one connects
Planting the cultivation temperature cultivated on fluid nutrient medium is 20～22 DEG C, and shaking speed 150～160r/min cultivates 28～30
My god, described fluid nutrient medium is: by sucrose 3g, maltose 2g, dusty yeast 0.25g, wheat bran 0.25g, KH2PO40.025g and
MgSO4·7H2O 0.015g is added to the water and is settled to 100ml.
Other steps and parameter are identical with detailed description of the invention one or two.
Detailed description of the invention four: present embodiment from unlike one of detailed description of the invention one to three: described step 2
In be enlarged cultivate cultivation temperature 28～30 DEG C, shaking speed 170～180r/min, cultivate 22～24h.
Other steps and parameter are identical with one of detailed description of the invention one to three.
Detailed description of the invention five: present embodiment from unlike one of detailed description of the invention one to four: described step 3
The detailed process of middle alcoholic fermentation single factor experiment is:
Respectively with fermentation temperature, fermentation time, pH and inoculum concentration as influence factor, matsutake liquid medium is tried
Test, determine the impact on alcoholic fermentation alcoholic strength of fermentation temperature, fermentation time, pH and inoculum concentration；
Described fermentation temperature is respectively 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C and 32 DEG C, fermentation time be respectively 4 days, 5 days, 6
My god, 7 days and 8 days, pH is respectively the 3rd, the 4th, the 5th, 6 and 7, and inoculum concentration is respectively the 0.5%th, the 1%th, the 1.5%th, 2% and 2.5%.
Other steps and parameter are identical with one of detailed description of the invention one to four.
Detailed description of the invention six: present embodiment from unlike one of detailed description of the invention one to five: described step 4
In carry out the detailed process of alcoholic fermentation Optimum Experiment and be:
Alcoholic fermentation single factor experiment result according to step 3, respectively with fermentation time, fermentation temperature, inoculum concentration and pH
For influence factor, determine alcoholic fermentation condition by Responds Surface Methodology.
Other steps and parameter are identical with one of detailed description of the invention one to five.
Detailed description of the invention seven: present embodiment from unlike one of detailed description of the invention one to six: described step 5
The detailed process of middle acetic fermentation single factor experiment is:
Respectively with fermentation temperature, inoculum concentration and shaking speed as influence factor, the alcohol fermentation liquid obtaining step 3 enters
Row test, determines the impact on total acidity of fermentation temperature, inoculum concentration and shaking speed；
Described fermentation temperature is respectively 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C and 30 DEG C, and the 9%th, the 10%th, the 8%th, inoculum concentration be respectively
11% and 12%, shaking speed is respectively 130r/min, 140r/min, 150r/min, 160r/min and 170r/min.
Other steps and parameter are identical with one of detailed description of the invention one to six.
Detailed description of the invention eight: present embodiment from unlike one of detailed description of the invention one to seven: described step 6
The detailed process of middle acetic fermentation Optimum Experiment is:
Acetic fermentation single factor experiment result according to step 5, respectively with shaking speed, fermentation temperature, inoculum concentration as shadow
The factor of sound, determines acetic fermentation condition by Responds Surface Methodology.
Other steps and parameter are identical with one of detailed description of the invention one to seven.
Embodiment one:
1 materials and methods
1.1 materials, reagent and instrument
1.1.1 bacterial classification
Matsutake, saccharomycete, acetic acid bacteria are by the Food Science institute preservation of Agriculture and Animal Husbandry College of Tibet University and provide；Chemical reagent
It is domestic analysis pure；Fruit syrup, sucrose, honey are purchased in Fromlingzhi, tibet city.
1.1.2 culture medium
Matsutake slant medium: glucose the 2%th, peptone the 0.3%th, KH2PO40.1%th, MgSO40.05%th, agar
2%th, potato liquor constant volume 100ml.
Matsutake fluid nutrient medium: sucrose 3g, maltose 2g, dusty yeast 0.25g, wheat bran 0.25g, KH2PO40.025g,
MgSO4·7H2O 0.015g, water 100ml, pH value is natural；
Microzyme culture medium: glucose 2g, peptone 2g, dusty yeast 1g, be dissolved in 100ml water, 121 DEG C sterilizing 30min.
Acetic acid bacteria culture medium: glucose 1g, yeast extract 1g, CaCO30.5g, 121 DEG C sterilizing 30min, treat that temperature is down to 60
DEG C, 100ml water, 2.5% (volume) absolute ethyl alcohol.
1.1.3 instrument
HZQ-F160A horizontal constant temperature oscillator Shanghai Yiheng Scientific Instruments Co., Ltd；On BPH-9162 constant incubator
Hai Yiheng scientific instrument Co., Ltd；Hong Ke instrument plant of Jintan City of JJ-2 high-speed tissue mashing machine；UV-2102PCS type ultraviolet-can
See the permanent magnetic electronic Science and Technology Ltd. in spectrophotometer Shanghai；Hand-held digital display refractometer Zhejiang Li Chen instrument Science and Technology Ltd.；
2XZ-2 type rotary-vane vaccum pump Linhai City Yong Hao vacuum equipment Co., Ltd；On YX-280A portable stainless steel steam disinfecting apparatus
Hai Sanshen Medical Devices Co., Ltd..
1.2 method
1.2.1 technology path
1.2.2 the expansion of bacterial classification is cultivated
The activation of matsutake thalline: bacterial classification transfer needle will be preserved and access in matsutake slant medium, and cultivate 7d for 24 DEG C.
The Liquid Culture of tricholoma matsutake mycelium: the inoculation shovel of the Tricholoma after activation is accessed by the inoculum concentration of 10% volume ratio
On fluid nutrient medium, rotating speed 160r/min, cultivates 30d under 22 DEG C of cultivation temperature；Afterwards the homogenate of matsutake zymotic fluid is filtered, use
Sucrose loads 500mL triangular flask after sugar content allotment to 13.0%, and sample-loading amount is 200mL, 121 DEG C of sterilizing 30min, cooling,
Standby.
It is respectively connected to saccharomycete, acetic acid bacteria slant preservation kind in saccharomycete and acetic acid bacteria mother culture media, temperature 30 DEG C,
Rotating speed 180r/min, cultivates 24h, standby.
1.2.3 single factor experiment
1.2.3.1 alcoholic fermentation single factor experiment
In matsutake liquid medium, respectively with fermentation temperature (the 24th, the 26th, the 28th, the 30th, 32 DEG C), fermentation time (the 4th, the 5th, the 6th, the 7th,
8h), pH (the 3rd, the 4th, the 5th, the 6th, 7), inoculum concentration (the 0.5th, the 1st, the 1.5th, the 2nd, 2.5%) are influence factor, are probed into wine by single factor experiment
The impact of precision, inoculum concentration refers to move into the ratio of nutrient solution volume after the volume of seed liquor and inoculation.
1.2.3.2 acetic fermentation single factor experiment
In gained alcohol fermentation liquid, respectively with fermentation temperature (the 22nd, the 24th, the 26th, the 28th, 30 DEG C), inoculum concentration (the 8th, the 9th, the 10th, the 11st,
12%), shaking speed (the 130th, the 140th, the 150th, the 160th, 170r/min) is influence factor, is probed into total acidity by single factor experiment
Impact.
1.2.4 process optimization test
1.2.4.1 technology of alcohol Optimum Experiment
According to single factor experiment result, design response surface optimization test influence factor, respectively with fermentation time, fermentation temperature
Degree, inoculum concentration, pH are influence factor, obtain optimum extraction process parameter by Responds Surface Methodology.
1.2.4.2 acetic fermentation process Optimum Experiment
According to single factor experiment result, design response surface optimization test influence factor, respectively with shaking speed, fermentation temperature
Degree, inoculum concentration are influence factor, obtain optimum extraction process parameter by Responds Surface Methodology.
1.2.5 tricholoma matsutake mycelium fermented type vinegar beverage formula mixture experiment design Optimum Experiment
Matsutake stoste through alcoholic fermentation and acetic fermentation is allocated by a certain percentage with fruit syrup, honey,
Test according to substantial amounts of the groping of early stage, obtain being closer to the formula of optimal mouthfeel, carry out orthogonal test, determine optimum formula
Ratio, orthogonal test factor level table is shown in Table 1.
Table 1 orthogonal test factor level table
1.2.5 assay method
According to GB/T 10345-2007 " Liquor Analysis Methods ", alcohol meter method is used to measure alcoholic strength；According to GB/T
The 12456-2008 mensuration of total acid " in the food ", uses determination of acid-basetitration total acid content；Total sugar content uses phenolsulfuric acid
Method is measured；Soluble solid content uses compound microcapsule to measure；Content of reducing sugar uses Fehling Regent titration measuring；
PH value uses digital display pH meter to measure；The mensuration of volatile acid uses Direct Determination, i.e. first isolates total volatile acid, with mark after cooling
Quasi-alkali-titration.
Tricholoma matsutake mycelium fermented type vinegar beverage subjective appreciation standard
Table 2 subjective appreciation standards of grading
2 results and analysis
2.1 single factor experiment
2.1.1 alcoholic fermentation single factor experiment
2.1.1.1 the impact on alcoholic fermentation alcoholic strength for the fermentation temperature
Inoculate 1% saccharomycete in 250mL conical flask, pH 5, fermentation temperature respectively with the 24th, the 26th, the 28th, the 30th, 32 DEG C carry out
Fermentation 5d, probes into the impact on alcoholic fermentation for the fermentation temperature.
As seen from Figure 1, fermentation temperature be less than 28 DEG C, alcoholic strength increases with the rising of fermentation temperature, 28 DEG C with
After reduce with the rising of fermentation temperature, reach peak 6.29% when 28 DEG C.Main cause is that saccharomycete most preferably ferments
Temperature is about 28 DEG C, and extremes of temperature all can affect saccharomycetic fermentation, and alcohol angle value changes also with it, it is also possible to
Being the increase due to temperature, causing the volatilization of alcohol, alcoholic strength reduces.
2.1.1.2 the impact on alcoholic fermentation alcoholic strength for the fermentation time
Inoculating 1% saccharomycete in 250mL conical flask, pH 5, fermentation temperature 26 DEG C, the 5th, the 6th, the 7th, the 4th, fermentation time be respectively
8d, probes into the impact on alcoholic fermentation for the fermentation time.
As seen from Figure 2, fermentation time is before 5d, and alcoholic strength increases with the increase of fermentation time, after 5d
As the increase of fermentation time reduces, when fermentation time is 5d, alcoholic strength reaches maximum 6.14%.Main cause may
After being fermentation time 5d, owing to alcoholic strength reaches certain value, saccharomycetes to make fermentation is affected, so alcohol angle value gradually drops
Low.
2.1.1.3 the impact on alcoholic fermentation alcoholic strength for the pH
Inoculating 1% saccharomycete in 250mL conical flask, fermentation temperature 26 DEG C, the 4th, the 5th, the 6th, the 3rd, fermentation time 5d, pH are respectively
7, probe into the impact on alcoholic fermentation for the pH.
Being drawn by Fig. 3, before pH value is 5, alcoholic strength must increase with pH value and increase, after reaching 5, with pH value
Increasing, alcoholic strength decreases, and when pH is 5, alcoholic strength reaches maximum 6.02%.Reason is probably yeasting pH value pair
Saccharomycete obtains growth and breeding and has a great impact, and after pH value is more than 5, Yeast Growth is suppressed, and alcoholic strength is relatively
Reduce.
2.1.1.4 the impact on alcoholic fermentation alcoholic strength for the inoculum concentration
Respectively so that the 0.5th, the 1st, the 1.5th, the 2nd, 2.5% inoculation yeast bacterium is in 250mL conical flask, pH 5, fermentation temperature 26 DEG C enters
Row fermentation 5d, probes into the impact on alcoholic fermentation for the inoculum concentration.
As shown in Figure 4, when inoculum concentration is less than 1.5%, alcoholic strength increases with the increase of inoculum concentration, inoculum concentration
After 1.5%, alcoholic strength reduces with the increase of inoculum concentration, and when inoculum concentration 1.5%, alcoholic strength reaches maximum
6.06%%.Reason be probably inoculum concentration less when, in saccharomycetic growing environment, nutrient is more, and saccharomycete can fast-growing soon
Long, after inoculum concentration reaches certain value, owing to saccharomycetic quantity is increasing, and its growing environment is not changed in, and fights for nutrition
Material, nutriment major part is used for the breeding of saccharomycete own growth, so its total acid content reduces.
2.1.2 acetic fermentation single factor experiment
2.1.2.1 the impact of fermentation temperature Dichlorodiphenyl Acetate fermentation total acidity
Inoculate 10% acetic acid bacteria in 250mL conical flask, fermentation temperature respectively with the 22nd, the 24th, the 26th, the 28th, 30 DEG C, shaking speed
150r/min ferments, and probes into the impact of fermentation temperature Dichlorodiphenyl Acetate fermentation.
As shown in Figure 5, fermentation temperature less than 28 DEG C when, total acid increases with the rising of fermentation temperature, 28 DEG C with
After, total acid reduces with the increase of fermentation temperature, and when fermentation temperature is 28 DEG C, total acid reaches maximum 34%.Reason is vinegar
The optimum growh environment temperature of acid bacterium is about 28 DEG C, and higher or lower temperature all the breeding of Dichlorodiphenyl Acetate bacteria growing can produce shadow
Ring.
2.1.2.2 the impact of inoculum concentration Dichlorodiphenyl Acetate fermentation total acidity
Respectively by the 8th, the 9th, the 10th, the 11st, 12% inoculation acetic acid bacteria in 250mL conical flask, fermentation temperature 26 DEG C, shaking speed
150r/min ferments, and probes into the impact of inoculum concentration Dichlorodiphenyl Acetate fermentation.
It will be appreciated from fig. 6 that when inoculum concentration is less than 10%, at long last as the increase of inoculum concentration increases, be higher than in inoculum concentration
After 10%, total acid decreases with the increase of inoculum concentration, and when inoculum concentration is 10%, total acid reaches maximum 34%.
Reason be probably inoculum concentration less when, in the growing environment of acetic acid bacteria, nutrient is more, acetic acid bacteria can fast-growth, conversion produce
Thing, after inoculum concentration reaches certain value, owing to the quantity of acetic acid bacteria is increasing, and its growing environment is not changed in, and fights for nutrition
Material, nutriment major part is used for the breeding of acetic acid bacteria own growth, so its total acid content reduces.
2.1.2.3 the impact of shaking speed Dichlorodiphenyl Acetate fermentation total acidity
Inoculating 10% acetic acid bacteria in 250mL conical flask, fermentation temperature 26 DEG C, the 140th, the 150th, the 130th, shaking speed be respectively
160th, 170r/min ferments, and probes into the impact of shaking speed Dichlorodiphenyl Acetate fermentation.
As shown in Figure 7, shaking speed less than 150r/min when, total acid increases with the increase of shaking speed, when shaking
Bed rotating speed is higher than 150r/min, and total acid tends towards stability substantially with the increase of shaking speed, is 150r/min in shaking speed
When, total acid reaches maximum 34%.It is big that reason is probably rotating speed, and dissolved oxygen amount is high, beneficially acetic acid bacteria fermentation.
2.2 tricholoma matsutake mycelium response surface optimization tests
2.2.1 alcoholic fermentation response surface optimization is tested
After matsutake liquid medium is filtered, in filtrate, add sucrose so that it is (use after soluble solid to 13 ° of Bx
Saccharometer is measured, and contains exocellular polysaccharide in the nutrient solution after filtration), access saccharomycete and carry out alcoholic fermentation test, by response
The process conditions determining tricholoma matsutake mycelium nutrient solution alcoholic fermentation are tested in interview, and experimental factor water-glass is shown in Table 3.
Table 3 Box-Behnken experimental design factor level table
Each influence factor when produced alcoholic strength is optimal during for obtaining alcoholic fermentation under each factor common acts on
Technological parameter.With fermentation temperature, fermentation time, pH value, inoculum concentration as factor of influence, with alcoholic strength as reference index, ring
Answer face experimental design, experimental design and result as shown in table 4.
Table 4 Box-Behnken experimental design and result
Utilize Design-Expert 8.06 software to carry out data analysis, obtain quadratic regression equation: alcoholic strength=6.50+
0.11A+0.020B+0.008333C-0.055D-0.025AB-0.057AC+0.063AD+0.052BC+0.012BD-
0.050CD-0.20A2-0.093B2-0.13C2-0.17D2.The results of analysis of variance is as shown in table 5.
Table 5 variance analysis
" * * " represents that difference is extremely notable, P < 0.01；" * " represents significant difference, 0.01 < P < 0.05；"-" represents that difference does not shows
Write, P > 0.05；R2=0.9790；R2Adj=0.9581.
Carry out variance analysis to it, as shown in table 5, the P value of its model is less than 0.01, and it loses intends item P=0.9957 >
0.05, difference is not notable, its R2Value is 0.9790, so this model can analog reslt well.Can come with this model
Certain prediction and analysis are carried out to test process.Being learnt by table 5 data, fermentation time and inoculum concentration difference are extremely notable, to wine
The impact of precision is relatively big, is ranked up according to the size of F value, and obtaining its main influence factor size is: fermentation temperature > inoculation
Amount > fermentation time > pH.
From Fig. 8 and Fig. 9, fermentation temperature is when fermentation time is relatively low, little on alcoholic strength impact, but when fermentation
Between be continuously increased, alcoholic strength gradually rises, until fermentation time is to 6d, alcoholic strength reaches maximum 6.5%, afterwards alcoholic strength
As the increase of fermentation time has declined on the contrary, entirety is rendered as parabolic shape.Main cause be probably fermentation time 5d it
After, owing to alcoholic strength reaches certain value, saccharomycetes to make fermentation is affected, so alcohol angle value is gradually lowered.
From Figure 10 and Figure 11, fermentation temperature is when pH is relatively low, little on alcoholic strength impact, but with the continuous increasing of pH
Adding, alcoholic strength gradually rises, until pH to 5.2, alcoholic strength reaches maximum 6.5%, afterwards alcoholic strength with the increase of pH anti-
And declined, entirety is rendered as parabolic shape.Reason is probably yeasting pH value and obtains growth and breeding to saccharomycete and have very big
Impact, when pH value more than 5 after, Yeast Growth is suppressed, and alcoholic strength decreases relatively.
From Figure 12 and Figure 13, fermentation temperature is when inoculum concentration is relatively low, little on alcoholic strength impact, but with inoculum concentration
Be continuously increased, alcoholic strength gradually rises, until inoculum concentration is to 1.4%, alcoholic strength reaches maximum 6.5%, afterwards alcoholic strength
As the increase of inoculum concentration has declined on the contrary, entirety is rendered as parabolic shape.Reason be probably inoculum concentration less when, saccharomycete
Growing environment in nutrient more, saccharomycete can fast-growth, after inoculum concentration reaches certain value, due to saccharomycetic quantity
Increasing, and its growing environment being not changed in, and fights for nutriment, it is numerous that nutriment major part is used for saccharomycete own growth
Grow, so its total acid content reduces.
From Figure 14 and Figure 15, fermentation time is when pH is relatively low, little on alcoholic strength impact, but with the continuous increasing of pH
Adding, alcoholic strength gradually rises, until pH to 5, alcoholic strength reaches maximum 6.5%, afterwards alcoholic strength with pH increase on the contrary
Having declined, entirety is rendered as parabolic shape.Reason is probably yeasting pH value and obtains growth and breeding to saccharomycete and have very big
Impact, after pH value is more than 5, Yeast Growth is suppressed, and alcoholic strength decreases relatively.
From Figure 16 and Figure 17, fermentation time is when inoculum concentration is relatively low, little on alcoholic strength impact, but with inoculum concentration
Be continuously increased, alcoholic strength gradually rises, until inoculum concentration is to 1.4%, alcoholic strength reaches maximum 6.5%, afterwards alcoholic strength
As the increase of inoculum concentration has declined on the contrary, entirety is rendered as parabolic shape.Reason be probably inoculum concentration less when, saccharomycete
Growing environment in nutrient more, saccharomycete can fast-growth, after inoculum concentration reaches certain value, due to saccharomycetic quantity
Increasing, and its growing environment being not changed in, and fights for nutriment, it is numerous that nutriment major part is used for saccharomycete own growth
Grow, so its total acid content reduces.
From Figure 18 and Figure 19, pH is when inoculum concentration is relatively low, little on alcoholic strength impact, but continuous with inoculum concentration
Increasing, alcoholic strength gradually rises, until inoculum concentration is to 1.4%, alcoholic strength reaches maximum 6.5%, and alcoholic strength is with connecing afterwards
The increase planting amount has declined on the contrary, and entirety is rendered as parabolic shape.Reason be probably inoculum concentration less when, saccharomycetic growth
In environment, nutrient is more, saccharomycete can fast-growth, after inoculum concentration reaches certain value, due to saccharomycetic quantity increase
Adding, and its growing environment being not changed in, fight for nutriment, nutriment major part is used for the breeding of saccharomycete own growth,
So its total acid content reduces.
On the basis of said process, utilize Design-Expert 8.0.6 software to be analyzed this model, obtain
Excellent parameter is: fermentation temperature 28.46 DEG C, fermentation time 6.07d, pH 5.02, inoculum concentration 1.44%, obtains alcoholic strength
6.51636%.For ease of operation, experimental condition is set as: fermentation temperature 28.5 DEG C, fermentation time 6d, pH 5, inoculum concentration
1.4%, with this theoretical value condition, carry out 3 groups of parallel tests, averaged, obtaining alcoholic strength is: 6.53%, this result
Close to theoretical value, illustrate that models fitting is preferable.
2.2.2 alcohol fermentation liquid acetic fermentation response surface optimization test
Add acetic acid bacteria to carry out acetic fermentation in above-mentioned alcohol fermentation liquid, determine tricholoma matsutake mycelium by response surface experiments
The process conditions of nutrient solution acetic fermentation, experimental factor water-glass is shown in Table 6.
Table 6 Box-Behnken experimental design factor level table
For studying under different factor effect, tricholoma matsutake mycelium alcohol fermentation liquid acetic fermentation optimal processing parameter.With fermentation
Temperature, shaking speed, inoculum concentration are influence factor, and total acidity is inspection target, carry out response surface optimization experimental design, and test sets
Meter and result are as shown in table 7.
Table 7 Box-Behnken experimental design and result
Utilize Design-Expert 8.06 software to carry out data analysis, obtain quadratic regression equation: total acid=33.40+
1.38A+0.75B+1.13C-1.75AB-1.00AC-0.25BC-0.70A2-1.45B2-2.70C2.The results of analysis of variance such as table 8
Shown in.
Table 8 variance analysis
" * * " represents that difference is extremely notable, P < 0.01；" * " represents significant difference, 0.01 < P < 0.05；"-" represents that difference does not shows
Write, P > 0.05；R2=0.9340；R2 Adj=0.8492.
Carry out variance analysis to it, as shown in table 8, the P value of its model is less than 0.01, and it loses intends item P=0.3762 >
0.05, difference is not notable, its R2Value is 0.9340, so this model can analog reslt well.Therefore this model can be used
Describe test, certain prediction and analysis are carried out to its process.Being learnt by its table 8 data, fermentation temperature difference is extremely notable, therefore
It is relatively big on the impact of total acidity, is ranked up according to the size of F value, and obtaining its main influence factor size is: fermentation temperature
Degree > inoculum concentration > shaking speed.
From Figure 20 and 21, fermentation temperature is when shaking speed is relatively low, little on total acidity impact, but as shaking table turns
Being continuously increased of speed, total acidity gradually rises, until shaking speed reaches 150r/min, total acidity reaches maximum 34%,
Total acidity has declined on the contrary with the increase of shaking speed after this, but it is very obvious to decline degree, and entirety is rendered as throwing
Thing wire.It is big that reason is probably rotating speed, and dissolved oxygen amount is high, beneficially acetic acid bacteria fermentation.
From Figure 22 and 23, shaking speed is when inoculum concentration is relatively low, little on total acidity impact, but with inoculum concentration
Being continuously increased, total acidity gradually rises, until inoculum concentration reaches 10.25%, total acidity reaches maximum 33%, after this always
Acidity has declined on the contrary with the increase of inoculum concentration, and entirety is rendered as parabolic shape.Reason be probably inoculum concentration less when, vinegar
Acid bacterium growing environment in nutrient more, acetic acid bacteria can fast-growth, converted product, after inoculum concentration reaches certain value, by
Increasing in the quantity of acetic acid bacteria, and its growing environment is not changed in, and fights for nutriment, and nutriment major part is used for vinegar
Acid bacterium own growth breeding, so its total acid content reduces.
From Figure 24 and 25, fermentation temperature is when inoculum concentration is relatively low, little on total acidity impact, but with inoculum concentration
Being continuously increased, total acidity gradually rises, until inoculum concentration reaches 10.10%, total acidity reaches maximum 34%, after this always
Acidity has declined on the contrary with the increase of inoculum concentration, and entirety is rendered as parabolic shape.Reason be probably inoculum concentration less when, vinegar
Acid bacterium growing environment in nutrient more, acetic acid bacteria can fast-growth, converted product, after inoculum concentration reaches certain value, by
Increasing in the quantity of acetic acid bacteria, and its growing environment is not changed in, and fights for nutriment, and nutriment major part is used for vinegar
Acid bacterium own growth breeding, so its total acid content reduces.
On the basis of said process, utilize Design-Expert 8.0.6 software to be analyzed this model, obtain
Excellent result is: fermentation temperature 28.00 DEG C, shaking speed 146.53r/min, inoculum concentration 10.04%, total acid 34.2516%.For just
In test operation, experimental condition is set as: fermentation temperature 28.0 DEG C, shaking speed 146r/min, inoculum concentration 10%, theoretical with this
Value is condition, has carried out 3 parallel tests, has averaged, and obtaining total acidity is: 34.1%, and this result is close to theoretical value, explanation
Models fitting is preferable.
2.3 tricholoma matsutake mycelium fermented type vinegar beverage formula mixture experiment design Optimum Experiments
For obtaining best flavor beverage, carry out orthogonal test with the former vinegar of matsutake, fruit syrup, honey for factor of influence, choosing
Take 120 undergraduates of Food Science institute of Agriculture and Animal Husbandry College of Tibet University and carry out subjective appreciation, orthogonal experiment data and range analysis
The results are shown in Table 9.
Table 9 orthogonal experiment data and range analysis result
Table 10 orthogonal test variance analysis
Being found by table 10, result of the test is had a significant impact by the former vinegar of matsutake, therefore optimum combination is: A2B1C2.I.e. optimum proportioning
For: the former vinegar of matsutake 10%, fruit syrup 8%, honey 5%, water 77%.
3 quality index
3.1 physical and chemical index
Total sugar content 10.5%；Total acid content (with acetometer) 4.0g/L；Soluble solid content 10%；Arsenic must not be examined
Go out；Lead < 5 × 10-4g/kg。
3.2 sanitary index
Total number of bacteria≤50mL-1；Escherichia coli≤30L-1；Pathogenic bacteria must not detect.
Claims (8)
1. the preparation method of a tricholoma matsutake mycelium fermented type vinegar beverage, it is characterised in that the detailed process of described preparation method
For:
Tricholoma after activation is inoculated into liquid training by the inoculum concentration of 10% volume ratio by step one: carry out matsutake thalline activation
Support and cultivate on base, homogenate is carried out to the nutrient solution containing tricholoma matsutake mycelium after cultivation and filters, by sucrose, sugar content is allocated
Matsutake nutrient solution to 13.0%, after being allocated；
Step 2: be respectively connected to saccharomycete and acetic acid bacteria slant preservation bacterial classification in microzyme culture medium and acetic acid bacteria culture medium,
It is enlarged cultivating；
Step 3: step 2 is expanded in the matsutake nutrient solution that the saccharomycete access step one after cultivating obtains and carry out alcoholic fermentation
Single factor experiment；
Step 4: the alcoholic fermentation single factor experiment result according to step 3 carries out alcoholic fermentation Optimum Experiment, determines that alcohol is sent out
Ferment condition is: temperature is 28～29 DEG C, and fermentation time is 6～6.5 days, and pH is 5～6, and inoculum concentration is 1.4～1.5%；
Step 5: carry out fermentation under the fermentation condition that step 4 determines and obtain matsutake alcohol fermentation liquid, in matsutake alcoholic fermentation
Liquid accesses the acetic acid bacteria after step 2 expands cultivation and carries out acetic fermentation single factor experiment；
Step 6: the acetic fermentation single factor experiment result according to step 5 carries out acetic fermentation Optimum Experiment, determines that acetic acid is sent out
Ferment condition is: fermentation temperature is 28～29 DEG C, and shaking speed is 146～150r/min, and inoculum concentration is 10～11%；
Step 7: carry out fermentation and obtain the former vinegar of matsutake under the fermentation condition that step 6 determines, with the former vinegar of matsutake, HFCS and
Honey is that influence factor carries out orthogonal test, determines the former vinegar of matsutake, fruit syrup, honey in tricholoma matsutake mycelium fermented type vinegar beverage
Mass percent with water is 10:8:5:77.
2. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 1, it is characterised in that described
The detailed process carrying out matsutake thalline activation in step one is:
To preserve bacterial classification transfer needle and access in matsutake slant medium, and cultivate 7～8 days for 23～25 DEG C, described matsutake inclined-plane is trained
Foster base is: by glucose 2g, peptone 0.3g, KH2PO4 0.1g、MgSO 4It is fixed that 0.05g and agar 2g adds in potato liquor
Hold to 100ml.
3. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 2, it is characterised in that described
Being inoculated into the cultivation temperature cultivated on fluid nutrient medium in step one is 20～22 DEG C, shaking speed 150～160r/min,
Cultivating 28～30 days, described fluid nutrient medium is: by sucrose 3g, maltose 2g, dusty yeast 0.25g, wheat bran 0.25g, KH2PO4
0.025g and MgSO4·7H2O 0.015g is added to the water and is settled to 100ml.
4. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 3, it is characterised in that described
Step 2 is enlarged the cultivation temperature 28～30 DEG C cultivated, shaking speed 170～180r/min, cultivates 22～24h.
5. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 4, it is characterised in that described
In step 3, the detailed process of alcoholic fermentation single factor experiment is:
Respectively with fermentation temperature, fermentation time, pH and inoculum concentration as influence factor, matsutake liquid medium is tested, really
Determine the impact on alcoholic fermentation alcoholic strength of fermentation temperature, fermentation time, pH and inoculum concentration；
Described fermentation temperature is respectively 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C and 32 DEG C, and fermentation time is respectively 4 days, 5 days, 6 days, 7 days
With 8 days, pH is respectively the 3rd, the 4th, the 5th, 6 and 7, and inoculum concentration is respectively the 0.5%th, the 1%th, the 1.5%th, 2% and 2.5%.
6. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 5, it is characterised in that described
The detailed process carrying out alcoholic fermentation Optimum Experiment in step 4 is:
Alcoholic fermentation single factor experiment result according to step 3, respectively with fermentation time, fermentation temperature, inoculum concentration and pH as shadow
The factor of sound, determines alcoholic fermentation condition by Responds Surface Methodology.
7. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 6, it is characterised in that described
In step 5, the detailed process of acetic fermentation single factor experiment is:
Respectively with fermentation temperature, inoculum concentration and shaking speed as influence factor, the alcohol fermentation liquid obtaining step 3 tries
Test, determine the impact on total acidity of fermentation temperature, inoculum concentration and shaking speed；
Described fermentation temperature is respectively 22 DEG C, 24 DEG C, 26 DEG C, 28 DEG C and 30 DEG C, and inoculum concentration is respectively the 8%th, the 9%th, the 10%th, 11%
With 12%, shaking speed is respectively 130r/min, 140r/min, 150r/min, 160r/min and 170r/min.
8. the preparation method of a kind of tricholoma matsutake mycelium fermented type vinegar beverage according to claim 7, it is characterised in that described
In step 6, the detailed process of acetic fermentation Optimum Experiment is:
Acetic fermentation single factor experiment result according to step 5, respectively with shaking speed, fermentation temperature, inoculum concentration for affect because of
Element, determines acetic fermentation condition by Responds Surface Methodology.
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