CN104694371B - Citrus fruit vinegar prepared by composite strain mixed fermentation and preparation method thereof - Google Patents
Citrus fruit vinegar prepared by composite strain mixed fermentation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a citrus fruit vinegar prepared by composite strain mixed fermentation, belonging to the technical field of fruit vinegar brewage. The preparation method is characterized by comprising the following steps: 1) preparing a composite microzyme strain seed solution; 2) preparing a composite acetobacter strain seed solution; 3) preparing a glutinous rice saccharified solution; 4) preparing a citrus juice; 5) preparing a fermentation liquid; 6) carrying out alcohol fermentation; 7) carrying out acetic fermentation; and 8) aging at room temperature for 2-3 months, filtering with diatomite, and clarifying to obtain the citrus fruit vinegar finished product. By using the citrus and glutinous rice as the raw materials and adopting the fruit/grain co-brewage and composite strain mixed fermentation, the prepared citrus fruit vinegar has the advantages of abundant nutrition, favorable mouthfeel, pleasant fruital aroma and no bitterness.
Description
Technical field
The invention belongs to fruit vinegar brewing technical field, and in particular to citrus prepared by a kind of utilization composite bacteria mixed fermentation
Fruit vinegar and preparation method thereof.
Background technology
Organge fruit vinegar is that with fresh, ripe cedra fruits as raw material, Jing is removed the peel, squeezed the juice, separating, de- hardship, and alcohol is sent out
The technique productions such as ferment, acetic fermentation, ageing, sterilizing gained, it contains abundant organic acid and vitamin, quench one's thirst with relieving summer heat,
Dispelling fatigue, beautifying face and moistering lotion, improve a poor appetite, the health-care efficacy such as Antialcoholic liver-protecting, antisepsis and sterilization, it is adaptable to the dietotherapy of various diseases,
Especially there is good treatment and prevention effect to angiocardiopathies such as hypertension, coronary heart disease, artery sclerosis, be fermentation of new generation
Food.Produce that the smell of fruits is very sweet safe efficiently, the organge fruit vinegar of unique flavor is key that its industrialization can develop in a healthy way
It is located.Fruit vinegar needs the local flavor and nutrient content with uniqueness, and this is just to being suitable to brew the bacterial screening of organge fruit vinegar, raising and train work
Make and its production technology proposes new requirement.Current China's fruit vinegar produce the bacterial classification that family uses mostly be single saccharomyces cerevisiae and
Vinegar bacterial classification, the product special flavour produced, insufficient quality, yield rate is low, and equipment investment is larger.Skill is produced with regard to fruit vinegar
Art, there is two kinds of liquid state fermentation and solid state fermentation.Solid state fermentation is good, in good taste etc. excellent with local flavor compared with liquid fermentation method
Point, but exist fermentation period length, high labor intensive, sweat be difficult to control to, easily by living contaminants the shortcomings of.And liquid
The advantage of fermentation method is that fermentation period is short, labour intensity is little, sweat is easy to control, reduces the chance of living contaminants, but
That, due to being purebred culture, and fermentation time is limited, therefore the flavor substance that liquid fermentation method is formed is few, its local flavor and mouthfeel compared with
Solid state fermentation is poor.At present, the method production fruit vinegar also without very most people's will, the production technology about improving fruit vinegar still exists
Exploratory stage.
The content of the invention
For technical problem present in background technology, it is an object of the invention to provide one kind is mixed using composite bacteria
Organge fruit vinegar prepared by fermentation and preparation method thereof.With citrus and glutinous rice as raw material, mix Jing after removing the peel, squeeze the juice, be beaten, be saccharified
Close, add composite yeast bacterial classification and compound acetic acid bacteria strain, alcoholic fermentation of Jing and an acetic fermentation, it is safe efficiently raw
The smell of fruits is very sweet for output, unique flavor, the organge fruit vinegar rich in nutrient contents such as amino acid, flavones, vitamin Cs.
The technical scheme for being adopted is as follows：
Organge fruit vinegar prepared by a kind of utilization composite bacteria mixed fermentation, it is characterised in that its preparation method is as follows：
1）The preparation of mulriple yeasts kind seed liquor：Saccharomycete activation medium liquid amount is 150mL/250mL, respectively takes two
Bait saccharomycete 31906, saccharomyces cerevisiae 1306, the thick bacterium of saccharomyces cerevisiae 1425, Hansenula anomala exception mutation 1431 move into respectively
Nutrient solution, is placed in shaking table in 28 DEG C, and 180r/min culture 16h obtain one-level nutrient solution, and adjustment cell concentration is 107Individual/mL；
Four kinds of one-level nutrient solutions are linked in malt medium according to following percent by volume：The exception mutation of 4% Hansenula anomala
1431,3% saccharomyces cerevisiae 1425,3% saccharomyces cerevisiae 31906,2% saccharomyces cerevisiae 1306 is subsequently placed in shaking table in 28 DEG C, 180r/
16h is cultivated under min, makes thalline quantity reach 107-108Individual/mL, as mulriple yeasts kind seed liquor.
2）The preparation of compound acetic acid bacteria strain seed liquor：Acetic acid bacteria activation medium liquid amount is 30mL/150mL, respectively takes two
Ring apple acetobacter ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai and make 1.01 thick bacterium and move respectively
Enter nutrient solution, be placed in shaking table in 31 DEG C, 150r/min cultivates 48 hours, obtain one-level nutrient solution, adjustment cell concentration is 106
Individual/mL；Four kinds of one-level nutrient solutions are linked in the Citrus Fruit Wine that alcoholic strength is 6-7% according to following percent by volume：4% apple
Acetobacter ZY.C4,3% Pasteur acetobacter Luo Wang subspecies 7002,2% acetifies acidfast bacilli 22518, and 2% Shanghai makes 1.01；It is subsequently placed in and shakes
In 30 DEG C in bed, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity up to 106-108Individual/mL.
3）It is prepared by glutinous rice saccharified liquid：Rice is rinsed 2 times, soaks 2-4h；By weight water：Rice=2.5：1 defibrination, size mixing to
4:1 ratio, adds CaCl2And MgCl2, the CaCl2And MgCl2Addition be every 100mL slurries and add 1g, use acetic acid
PH to 6.2-6.4 is adjusted with sodium carbonate, α-amylase is added, the addition of α-amylase is that 100mL slurries add 1g, is heated to 90-95
DEG C liquefaction about 20-30min, is cooled to 60 DEG C, and tunes pH is 4.5-5.0, obtains liquid A, and the saccharification of liquid A Jing obtains glutinous rice and is saccharified
Liquid；
Its method for saccharifying is：Add carbohydrase and malt flour, addition be add in every 100mL liquid As 1g carbohydrase and
10g malt flours, insulation saccharification 6-8h；Or the black song of addition：Red yeast rice=3:1 mixed starters, the addition of mixed starters is 100mL liquid
Add 1g in body A, saccharificatinn period is 2-4h.
4) preparation of citrus juice：Select without rotten citrus, peeling, citrus pulp is put in juice extractor, and add
The NaHSO of 0.01% (weight)3Solution, citrus pulp：NaHSO3Solution=1:1（Weight ratio）；Squeeze the juice, pectin is added in juice
Enzyme is digested, and the addition of the pectase is addition pectase 0.04g, 60 DEG C of hydrolysis temperature, enzymolysis time in every 100mL juice
60min, 4 layers of filtered through gauze, obtains citrus juice after enzymolysis；Preferably, being additionally added malt flour before enzymolysis, addition is every
Malt flour 10g is added in 100mL juice.
5) prepared by zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 volume ratio mixing, in mixing
Sulfur dioxide and sulfuric acid are added in liquid in the ratio that sulfur dioxide 30mg and the mg of ammonium sulfate 50 are separately added in every 1L mixed liquors
Ammonium, mixes and adjusts pH value 4.48, adjusts pol to 15-19 ° of Bx, and sterilization 15min in 85 DEG C of hot water, cooling is standby, is sent out
Zymotic fluid.
6）Alcoholic fermentation：In step 5）Zymotic fluid in access mulriple yeasts kind seed liquor, inoculum concentration is 9.7-10%
(percent by volume), 28-28.5 DEG C of stirring, rotating speed is that after 180r/min fermentation 1d, after closed standing for fermentation 4-5d, sterilization is obtained
To Citrus Fruit Wine.Preferably, being additionally added naringinase before fermentation, the addition of naringinase is to add in every 100mL zymotic fluids
0.04g。
7）Acetic fermentation：Citrus Fruit Wine alcoholic strength is adjusted to 6-7%, compound acetic acid bacteria strain seed liquor is accessed, inoculum concentration is
15.2-16% (percent by volume), is 180r/min, 31 DEG C of stirring fermentation 5-7d in rotating speed；Terminate fermentation, be passed through 80-100 DEG C
Steam sterilizing 30min.
8）7）In add salt in the liquid that obtains, the addition of salt is addition salt 20g, normal temperature ageing in every 1L
2-3 month, filtered with diatomite and clarified, obtain organge fruit vinegar finished product.
The organge fruit vinegar as obtained in above-mentioned technological process and control condition, nutritious, the fruit vinegar tart flavour appropriateness, mouthfeel alcohol
Good, fruital is pleasant, without bitter taste, particularly rich in the amino acid required for health and various antioxidant contents, if in addition
Appropriate allotment and dilution is carried out again, can also develop into popular drink.Additionally, using composite bacteria brewing fruit vinegar, thalline is numerous
Grow speed soon, at the fermentation initial stage the higher acidity of acid can be produced, while acid production speed soon, maximum is just reached on the 5th day in fermentation
Acidity, its fermentation time is shorter than single culture fermentation time 3-4 days, therefore, can reduce living contaminants using the technique,
Shorten the production cycle, stabilized product quality.
The present invention is as a result of apple acetobacter ZY.C4 so that the total acid of the standby organge fruit vinegar of system, amino nitrogen, always
The fruit vinegar flavor such as ester, VC, flavones material and nutrient content bacterium are higher than using the fruit brewed containing AS1.41 acetic acid bacterias composite bacteria
Vinegar, and make limonin substances in organge fruit vinegar product decline 70-75% so as to which products taste is soft, without bitter taste.
Specific embodiment
Make further explanation in detail to the present invention with reference to embodiment.
Involved bacterial classification and culture medium are as follows in embodiment：
Yeast seeds：Saccharomycete 31906, saccharomyces cerevisiae 1306, the exception mutation of saccharomyces cerevisiae 1425, Hansenula anomala
1431.Above bacterial classification is purchased from Chinese industrial Microbiological Culture Collection administrative center.
Strain Acetobacter xylinum：Make 1.01, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518, stench acetic acid bar in Shanghai
Bacterium AS1.41, above bacterial classification is purchased from Chinese industrial Microbiological Culture Collection administrative center；Apple acetobacter ZY.C4, latin name
Acetobacter malorum, voluntarily separate and obtain, and culture presevation, depositary institution have been carried out：Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, postcode 100101), preservation day：On 01 07th, 2014, preserving number：CGMCC NO ：8692.
Saccharomycete activation medium：5 ° of Be ' wort agars and the glutinous rice citrus nutrient solution 2 for processing:1 mixing is equal
Even, 121 DEG C of autoclaving 20min are cooled down standby.
Acetic acid bacteria activation medium：Glucose 1g, yeast extract 1g, alcohol (95%) 5mL, water 100mL, pH natures.After sterilizing
Add alcohol.
Embodiment 1：The preparation technology one of organge fruit vinegar
First, material preparation technology
（1）The preparation of mulriple yeasts kind seed liquor：Saccharomycete activation medium liquid amount is 150mL/250mL, is respectively taken
Two bait saccharomycete 31906, saccharomyces cerevisiae 1306, the thick bacterium of saccharomyces cerevisiae 1425, Hansenula anomala exception mutation 1431 move respectively
Enter nutrient solution, be placed in shaking table in 28 DEG C, 180r/min culture 16h obtain one-level nutrient solution, and adjustment cell concentration is 107Individual/
mL；Four kinds of one-level nutrient solutions are linked in malt medium according to following percent by volume：4% Hansenula anomala becomes extremely
1431 are planted, 3% saccharomyces cerevisiae 1425,3% saccharomyces cerevisiae 31906,2% saccharomyces cerevisiae 1306 is subsequently placed in shaking table in 28 DEG C,
16h is cultivated under 180r/min, makes thalline quantity reach 107-108Individual/mL, as mulriple yeasts kind seed liquor.
（2）The preparation of compound acetic acid bacteria strain seed liquor：Acetic acid bacteria activation medium liquid amount is 30mL/150mL, respectively takes two
Ring apple acetobacter ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai and make 1.01 thick bacterium and move respectively
Enter nutrient solution, be placed in shaking table in 31 DEG C, 150r/min cultivates 48 hours, obtain one-level nutrient solution, adjustment cell concentration is 106
Individual/mL；Four kinds of one-level nutrient solutions are linked in the Citrus Fruit Wine that alcoholic strength is 6-7% according to following percent by volume：4% apple
Acetobacter ZY.C4,3% Pasteur acetobacter Luo Wang subspecies 7002,2% acetifies acidfast bacilli 22518, and 2% Shanghai makes 1.01；It is subsequently placed in and shakes
In 30 DEG C in bed, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity up to 106-108Individual/mL.
（3）It is prepared by glutinous rice saccharified liquid：Rice is rinsed 2 times, soaks 2-4h；By weight water：Rice=2.5：1 defibrination, sizes mixing
To 4:1 ratio, adds CaCl2And MgCl2, the CaCl2And MgCl2Addition be every 100mL slurries and add 1g, use vinegar
Acid and sodium carbonate adjust pH to 6.2-6.4, add α-amylase, and the addition of α-amylase is that 100mL slurries add 1g, is heated to 90-
95 DEG C of liquefaction about 20-30min, are cooled to 60 DEG C, and tune pH is 4.5-5.0, adds carbohydrase and malt flour, and addition is every
1g carbohydrase and 10g malt flours, insulation saccharification 6-8h are added in 100mL liquid；Obtain glutinous rice saccharified liquid.
（4）The preparation of citrus juice：Select without rotten citrus, peeling, citrus pulp is put in juice extractor, and add
The NaHSO of 0.01% (weight)3Solution, citrus pulp：NaHSO3Solution=1:1（Weight ratio）；Squeeze the juice, pectin is added in juice
Enzyme and malt flour, the addition of pectase is addition pectase 0.04g in every 100mL juice, and malt flour addition is every 100mL
Malt flour 10g is added in juice.60 DEG C of hydrolysis temperature, enzymolysis time 60min, 4 layers of filtered through gauze, obtains citrus juice after enzymolysis.
（5）It is prepared by zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 volume ratio mixing, in mixing
Sulfur dioxide and sulfuric acid are added in liquid in the ratio that sulfur dioxide 30mg and the mg of ammonium sulfate 50 are separately added in every 1L mixed liquors
Ammonium, mixes and adjusts pH value 4.48, adjusts pol to 15-19 ° of Bx, and sterilization 15min in 85 DEG C of hot water, cooling is standby, is sent out
Zymotic fluid.
2nd, alcoholic fermentation (the 1st fermentation)
9.7% mulriple yeasts kind seed liquor is added in above-mentioned zymotic fluid (liquid amount is volume 4/5), in 28.5 DEG C
After stirring (rotating speed is 180r/min) fermentation 1d, closed standing for fermentation 4-5d, alcohol content is 10% -12% in detection zymotic fluid
During V/V, terminate fermentation, 80-100 DEG C of steam sterilizing 30min is passed through after filtration.
3rd, acetic fermentation (the 2nd fermentation)
Above-mentioned fruit wine wine degree is adjusted to into 6-7%, by 30% liquid amount apparatus for acetic acid fermentation tank is loaded, accessed by 15.2% compound
Strain Acetobacter xylinum seed liquor, adds 0.04% naringinase（0.04g is added in i.e. per 100mL Citrus Fruit Wines）, in 31 DEG C of stirrings
(rotating speed is 180r/min) fermentation 5-7d, detection alcohol residue amount terminates fermentation, is passed through 80-100 DEG C of steaming in 0.3%-0.5%
Vapour sterilizing 30min.
4th, ageing, filtration, clarification
The fruit vinegar for completing acetic fermentation adds 2% salt (adding salt 20g in i.e. per 1L), deposits in clean closed
In tank body, normal temperature ageing 2 months.Filter medium is made of diatomite carries out filtration clarification to vinegar unstrained spirits, obtains organge fruit vinegar finished product.
Embodiment 2：The preparation technology two of organge fruit vinegar
First, material preparation technology
（1）The preparation of mulriple yeasts kind seed liquor：Saccharomycete activation medium liquid amount is 150mL/250mL, is respectively taken
Two bait saccharomycete 31906, saccharomyces cerevisiae 1306, the thick bacterium of saccharomyces cerevisiae 1425, Hansenula anomala exception mutation 1431 move respectively
Enter nutrient solution, be placed in shaking table in 28 DEG C, 180r/min culture 16h obtain one-level nutrient solution, and adjustment cell concentration is 107Individual/
mL；Four kinds of one-level nutrient solutions are linked in malt medium according to following percent by volume：4% Hansenula anomala becomes extremely
1431 are planted, 3% saccharomyces cerevisiae 1425,3% saccharomyces cerevisiae 31906,2% saccharomyces cerevisiae 1306 is subsequently placed in shaking table in 28 DEG C,
16h is cultivated under 180r/min, makes thalline quantity reach 107-108Individual/mL, as mulriple yeasts kind seed liquor.
（2）The preparation of compound acetic acid bacteria strain seed liquor：Acetic acid bacteria activation medium liquid amount is 30mL/150mL, respectively takes two
Ring apple acetobacter ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai and make 1.01 thick bacterium and move respectively
Enter nutrient solution, be placed in shaking table in 31 DEG C, 150r/min cultivates 48 hours, obtain one-level nutrient solution, adjustment cell concentration is 106
Individual/mL；Four kinds of one-level nutrient solutions are linked in the Citrus Fruit Wine that alcoholic strength is 6-7% according to following percent by volume：4% apple
Acetobacter ZY.C4,3% Pasteur acetobacter Luo Wang subspecies 7002,2% acetifies acidfast bacilli 22518, and 2% Shanghai makes 1.01；It is subsequently placed in and shakes
In 30 DEG C in bed, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity up to 106-108Individual/mL.
（3）It is prepared by glutinous rice saccharified liquid：Rice is rinsed 2 times, soaks 2-4h；By weight water：Rice=2.5：1 defibrination, sizes mixing
To 4:1 ratio, adds CaCl2And MgCl2, the CaCl2And MgCl2Addition be every 100mL slurries and add 1g, use vinegar
Acid and sodium carbonate adjust pH to 6.2-6.4, add α-amylase, and the addition of α-amylase is that 100mL slurries add 1g, is heated to 90-
95 DEG C of liquefaction about 20-30min, are cooled to 60 DEG C, and tune pH is 4.5-5.0, adds black song：Red yeast rice=3:1 mixed starters, mixed starters
Addition be 100mL liquid in plus 1g, be saccharified 2-4h；Obtain glutinous rice saccharified liquid.
（4）The preparation of citrus juice：Select without rotten citrus, peeling, citrus pulp is put in juice extractor, and add
The NaHSO of 0.01% (weight)3Solution, citrus pulp：NaHSO3Solution=1:1（Weight ratio）；Squeeze the juice, pectin is added in juice
Enzyme and malt flour, the addition of pectase is addition pectase 0.04g in every 100mL juice, and malt flour addition is every 100mL
Malt flour 10g is added in juice.60 DEG C of hydrolysis temperature, enzymolysis time 60min, 4 layers of filtered through gauze, obtains citrus juice after enzymolysis.
（5）It is prepared by zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 volume ratio mixing, in mixing
Sulfur dioxide and sulfuric acid are added in liquid in the ratio that sulfur dioxide 30mg and the mg of ammonium sulfate 50 are separately added in every 1L mixed liquors
Ammonium, mixes and adjusts pH value 4.48, and sugar addition to 15 ° of Bx kills wherein miscellaneous bacteria, cooling in sterilized 15 minutes in 85 DEG C of hot water
It is standby.
2nd, alcoholic fermentation (the 1st fermentation)
10% mulriple yeasts kind seed liquor is added in above-mentioned zymotic fluid (liquid amount is volume 4/5), in 28 DEG C of stirrings
After (rotating speed is 180r/min) fermentation 1d, closed standing for fermentation 6-7d, alcohol content is in 10% -12% V/V in detection zymotic fluid
When, terminate fermentation, 80-100 DEG C of steam sterilizing 30min is passed through after filtration.
3rd, acetic fermentation (the 2nd fermentation)
Above-mentioned fruit wine wine degree is adjusted to into 6-7%, by 30% liquid amount apparatus for acetic acid fermentation tank is loaded, by 16% Composite vinegar is accessed
Sour bacterium bacterial classification seed liquor, in 32 DEG C of stirring (rotating speed is 180r/min) fermentation 5-7d, detection alcohol residue amount is in 0.3%-0.5%
When, terminate fermentation, it is passed through 80-100 DEG C of steam sterilizing 30min.
4th, ageing, filtration, clarification
The fruit vinegar for completing acetic fermentation adds 2% salt, in depositing in clean closed tank body, normal temperature ageing 3 months.
Filter medium is made of diatomite carries out filtration clarification to vinegar unstrained spirits, obtains organge fruit vinegar finished product.
Embodiment 3：The separation identification of apple acetobacter ZY.C4
（1）Bacterium source prepares the commercial citrus without decayed fruit, cleans, beating, carries out nature alcoholic fermentation and natural acetic fermentation.
Daily its pol is determined with saccharometer, when pol is down to 5 ° of BX, and a continuous week no longer changes, start to filter.With boiling
The 80 mesh gauze double medium filtrations crossed, are stored in filtrate in stainless-steel pan, i.e. citrus wine after filtration.Pot face is covered with gauze, 32
Spontaneous fermentation is carried out at DEG C, the vinegar liquid of spontaneous fermentation is obtained final product.Bacterium source is taken from vinegar liquid after 5d, screening separation is carried out.
（2）Plant separation screening flow process
Bacterium source --- --- --- train diluting separation Multiplying culture by primary dcreening operation --- inclined-plane culture --- liquid triangular flask
Support and --- determine primary dcreening operation bacterial strain --- slant preservation bacterial strain --- secondary screening --- determining excellent acid-producing bacteria strain --- slant preservation bacterium
Strain
（3）Prescreening method
Multiplying culture：Fermentation vinegar liquid 10mL of right acetic acid is taken from, in being respectively put into the proliferated culture medium of 90mL, and in constant temperature
Shaking table（32 DEG C, 120r/min）Culture 2d.
Calcium plate isolation is tested：Gradient dilution Multiplying culture liquid, separates for being coated with, and cultivates in 32 DEG C of insulating box
2d, observes result, selects calcium dissolving and encloses big, and the single bacterium colony of different colonial morphologies is moved in slant medium, repeats 3-4
It is secondary, obtain purifying bacterial strain.
（4）Secondary screening
By the ring of bacterial strain picking two of calcium plate isolation, in moving into 20mL liquid basal mediums (plus 6% absolute ethyl alcohol),
In (32 DEG C, 120r/min) culture 7d of constant-temperature table, acetimetry measure is carried out.Acid is produced according to the screening of acidity assaying result
The high strain excellent of amount.
Produce the high strain excellent of acid amount and be identified as apple acetobacter (Acetobacter malorum) ZY.C4.
It is above-mentioned that to be separately cultured culture medium prescription used as follows：
Proliferated culture medium：Dusty yeast 1%, glucose 1%, 0.02% crystal violet 0.5%, 2.5 × l0 of Nysfungin6Unit/L,
PH5.5, adds 95% absolute ethyl alcohol of 3% (v/v).
Isolation medium：Dusty yeast l%, glucose l%, CaCO31.5%, agar 2%, pH5.5 add 4% (v/v's)
The CaCO of 95% absolute ethyl alcohol and hot air sterilization3。
Slant medium：Dusty yeast 1%, glucose 1%, CaCO31.5%, agar 2%, pH5.5 dispense test tube, add 95%
Absolute ethyl alcohol 4% (v/v).
Liquid basal medium：Dusty yeast 1%, glucose 1%, pH5.5.
Embodiment 4：Organge fruit vinegar nutrient content and bitter substance comparision contents
Organge fruit vinegar nutrient content in embodiment 1, embodiment 2 and control and de- hardship rate and mouthfeel are compared, it is right
Substituted using conventional fruit vinegar brewing bacterial classification AS1.41 stenches acetobacter muddiness mutation according to middle apple acetobacter ZY.C4, remaining work
Skill is with embodiment 1.Concrete nutrient content is more as shown in table 1.Using containing the compound acetic acid bacteria seed liquors of apple acetobacter ZY.C4
Prepare the fruit vinegar flavor materials such as total acid, amino nitrogen, total ester, VC, the flavones of organge fruit vinegar and nutrient content bacterium to contain higher than adopting
There is the fruit vinegar that AS1.41 acetic acid bacterias composite bacteria is brewed, and make the limonin substances in organge fruit vinegar product decline 70-
75% so as to which products taste is soft, without bitter taste.
The organge fruit vinegar nutrient content of table 1 compares
The mulriple yeasts bacterial classification ratio optimization of embodiment 5
Saccharomycetic one-level Amplification Culture：Saccharomycete activation medium liquid amount is 150mL/250mL, each for examination saccharomycete
Plant and take two baits thickness bacterium immigration nutrient solution, be placed in shaking table（28 DEG C, 180r/min）About 16 hours of culture.
The preparation of zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 ratio mixes, and sugar addition is 11
Degree, with citric acid pH4.2 is adjusted, the fruit juice after adjustment in 85 DEG C of hot water sterilized 15 minutes killing wherein miscellaneous bacteria, Ran Houleng
It is standby.
Mulriple yeasts bacterial classification ratio is preferred：Tested using the horizontal quadrature of 4 factor 3, to saccharomycete 31906, saccharomyces cerevisiae
1306th, the inoculative proportion of saccharomyces cerevisiae 1425, Hansenula anomala exception 1,431 4 kinds of bacterial strains of mutation is optimized, its factor water
It is flat as shown in table 2.The cell concentration of one-level scale-up medium is adjusted to into 108Individual/mL, by four kinds of one-level nutrient solutions according to orthogonal
The ratio of experimental program is accessed in new malt medium, is placed in shaking table about 16 hours of (28 DEG C, 180r/min) cultures,
Thalline quantity is set to reach 107-108Individual/mL.Mixed bacteria accesses citrus glutinous rice mixed liquor by made by, and inoculum concentration is 10%, in 28
DEG C stirring (rotating speed is 180r/min) fermentation 1d after, closed standing for fermentation.Zymotic fluid is weighed daily, to weight substantially not
When changing again, total reducing sugar is determined.Total sugar content is stable at after 4 ~ 5 ° of Be ', is carried out alcohol content, amino-acid nitrogen content, is produced acid amount etc.
Every physical and chemical index measure and its evaluation of organoleptic indicator.
The composite yeast bacterial classification ratio optimization orthogonal test factor level table of table 2
Composite yeast bacterial classification ratio preferred result：Hansenula anomala exception mutation 1431, saccharomyces cerevisiae 1425, wine brewing ferment
Female 31906, the inoculum concentration of saccharomyces cerevisiae 1306 is respectively 4%, 3%, 3%, when 2%, the alcohol content, amino-acid nitrogen content after fermentation
It is higher with total ester content, and aroma is strong, and have the fragrance of fruit.
Embodiment 6：Composite yeast bacterial classification technology of alcohol optimizes
It is prepared by the seed liquor of composite yeast bacterial classification：Saccharomycete activation medium liquid amount is 150mL/250mL, takes two bait ferment
Four kinds of different thick bacterium such as female bacterium 31906, saccharomyces cerevisiae 1306, the exception mutation 1431 of saccharomyces cerevisiae 1425, Hansenula anomala
Nutrient solution is moved into respectively, after being placed in (28 DEG C, 180r/min) of shaking table about 16 hours of culture, adjustment cell concentration is 108/
mL；By four kinds of one-level nutrient solutions according to the exception mutation 1431 of 4% Hansenula anomala, 3% saccharomyces cerevisiae 1425,3% saccharomyces cerevisiae
31906, the ratio of 2% saccharomyces cerevisiae 1306 is accessed in new malt medium, is placed in shaking table (28 DEG C, 180r/min) trainings
About 16 hours are supported, makes thalline quantity reach 107-108Individual/mL.
Technology of alcohol optimizes：First to ammonium sulfate addition, sulfur dioxide addition, composite bacteria inoculum concentration, fermentation
Each factors such as time, fermentation temperature, initial pol and initial pH carry out single factor experiment, with select more excellent level carry out it is excellent
Change.Composite yeast is accessed in citrus glutinous rice mixed liquor, alcohol content, amino-acid nitrogen content are determined Jing after alcoholic fermentation, gone forward side by side
Row subjective appreciation.Each factorial experiments level is as follows：Sulfur dioxide addition is：0、30、60、90 mg/L；Ammonium sulfate addition
For：0、25、50、75 、100 mg/L；Mixed bacteria inoculum concentration is 6%, 8%, 10%, 12%, 14%；Fermentation time be 1,2,3,4,
5、6、7、8d；Fermentation temperature is 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C；Initial pol be 13 ° of Bx ', 15 ° of Bx ', 17 ° of Bx ',
19 ° of Bx ', 21 ° of Bx ', 5 pol gradients；Initial pH is 2.8,3.5,4.5,5.5,6.5.In the base of above single factor experiment result
On plinth, initial pol, initial pH value, inoculum concentration, time, the value of temperature major influence factors in alcoholic fermentation process is determined
Scope, carries out the quadratic orthogonal rotating composite test (half implement, have 36 combinations) of the level of 5 factor 5, using alcohol content as
Evaluation index, the result to testing is analyzed, and integrated sensory's evaluation score verifies that the alcohol for obtaining optimizing is sent out to result
Ferment process conditions.Its technology of alcohol experimental factor level code table is as shown in table 3.
The technology of alcohol experimental factor level code value table of table 3
The most suitable process conditions of composite yeast bacterial classification alcoholic fermentation：Add 30 in citrus juice and glutinous rice saccharified liquid mixed liquor
Mg/L sulfur dioxide, 50 mg/L ammonium sulfate, adjust initial pol for 19 ° of Bx ', initial pH values 4.48, wherein access 9.7
% composite yeast bacterial classifications, at 28.5 DEG C, after 28.5 DEG C of stirring (rotating speed is 180r/min) fermentation 1d, closed standing for fermentation 4-5d
Fermentation, gained Citrus Fruit Wine alcohol content is up to 11.7 %, and aroma is strong, and has fruit aroma.
Embodiment 7：Compound acetic acid bacteria strain ratio is preferred
The one-level Amplification Culture of acetic acid bacteria：Saccharomycete activation medium liquid amount is 30mL/150mL, takes four kinds of two ring not
Same thick bacterium moves into respectively nutrient solution, is placed in shaking table about 48 hours of (30 DEG C, 150r/min) cultures.
The preparation of zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 ratio mixes, in citrus juice and glutinous
Add 30 mg/L sulfur dioxide, 50 mg/L ammonium sulfate in rice saccharified liquid mixed liquor, adjust initial pol and be 19 ° of Bx ', use lemon
Then acid for adjusting pH 4.48, the sterilization in 85 DEG C of hot water of the fruit juice after adjustment is cooled down standby for 15 minutes with killing wherein miscellaneous bacteria.
Citrus Fruit Wine ferments：Add 9.7% mulriple yeasts a variety of in above-mentioned zymotic fluid (liquid amount is volume 4/5)
Sub- liquid, after 28.5 DEG C of stirring (rotating speed is 180r/min) fermentation 1d, closed standing for fermentation 4-5d, alcohol contains in detection zymotic fluid
Amount terminates fermentation in 10% -12% V/V, and 80-100 DEG C of steam sterilizing 30min is passed through after filtration.
Compound acetic acid bacteria strain ratio is preferred：Tested using the horizontal quadrature of 4 factor 3,1.01, apple acetobacter is made to Shanghai
ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, the inoculative proportion for acetifying 22,518 4 kinds of bacterial classifications of acidfast bacilli are optimized, its because
Plain level is as shown in table 4.The cell concentration of the one-level nutrient solution of four bacterial classifications is adjusted to into 107Individual/mL, according to orthogonal experiment side
It is in 6-7% Citrus Fruit Wines, to be placed in shaking table about 24 hours of (30 DEG C, 150r/min) cultures that the ratio of case moves into wine degree, makes bacterium
Body quantity reaches thalline quantity up to 106~108Individual/mL, obtains mixing acetic acid bacteria strain.
Acetic fermentation：It is 6-7% that the mixed bacteria by made by accesses wine degree, and in 31 DEG C (rotating speed is 180r/min) is stirred
Fermentation, detection alcohol residue amount terminates fermentation in 0.3%-0.5%, determines it and produces acid amount, total ester content and amino acid peptide nitrogen
Content, and subjective appreciation is carried out to its local flavor.With reference to every physical and chemical index and results of sensory evaluation, suitable Ponkan fruit vinegar is filtered out
The acetic acid bacteria inoculum concentration optimal proportion of fermentation.
Table 4 is combined the preferred orthogonal test factor level table of strain Acetobacter xylinum ratio
Compound strain Acetobacter xylinum ratio preferred result：When 4 kinds of strain Acetobacter xylinum ratios be 4% apple acetobacter ZY.C4,3%
Pasteur acetobacter Luo Wang subspecies 7002,2% Shanghai makes 1.01, and 2% when acetifying acidfast bacilli 22518, measures acidity, amino acid peptide nitrogen content
Maximum is reached with total ester content, and obtained fruit vinegar tart flavour is soft, good smell has the fragrance of fruit vinegar and grain vinegar concurrently.
Embodiment 8 is combined the optimization of strain Acetobacter xylinum acetic fermentation process
It is prepared by compound strain Acetobacter xylinum seed liquor：Acetic acid bacteria activation medium liquid amount is 30mL/150mL, takes two ring apples
Fruit vinegar bacillus ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai and make 1.01 etc. four kinds of different thickness
Bacterium moves into respectively nutrient solution, and after being placed in shaking table about 48 hours of (30 DEG C, 150r/min) cultures, adjustment cell concentration is 107
Individual/mL；By four kinds of one-level nutrient solutions according to 4% apple acetobacter ZY.C4,3% Pasteur acetobacter Luo Wang subspecies 7002,2% acetifies acid
Bacillus 22518, it is in 6-7% Citrus Fruit Wines, to be placed in shaking table (30 DEG C, 150r/min) that 2% Shanghai is made 1.01 ratio and moves into wine degree
About 24 hours of culture, make thalline quantity reach thalline quantity up to 106~108Individual/mL.
Acetic fermentation process optimizes：First to fermentation time, initial pH, liquid amount, inoculum concentration, initial wine degree and fermentation temperature
Experiment of single factor is carried out etc. each factor, compound acetic acid bacteria is accessed in Citrus Fruit Wine, acetic acid content, ammonia are determined Jing after acetic fermentation
Ground state nitrogen content, total ester content, and subjective appreciation is carried out, to filter out the optimum level of each factor.Each factorial experiments level is such as
Under：Fermentation time be 1,2,3,4,5,6,7,8d；The initial pH of fermentating wine is 2.8,3.5,4.5,5.5,6.5；Liquid amount is
10%th, 20%, 30%, 40%, 50%；The inoculum concentration of compound acetic acid bacteria is 5%, 10%, 15%, 20%；Initial wine degree is 5%, 6%, 7%, 8%,
9%；Fermentation temperature is 25 DEG C, 28 DEG C, 31 DEG C, 34 DEG C, 37 DEG C.On the basis of above single factor experiment, using the level of 5 factor 5
Quadratic orthogonal rotating composite test (half implements, totally 36 combinations), investigates time, initial pH, liquid amount, initial wine degree and optimal
The impact of the factor Dichlorodiphenyl Acetate ferment effect such as inoculum concentration of the mixed bacteria of combination, is contained with zymotic fluid total acid content, amino acid peptide nitrogen
The content of amount and total ester optimizes Ponkan fruit vinegar acetic fermentation process conditional parameter as evaluation index with this.Its acetic fermentation work
Skill experimental factor level code table is as shown in table 5.
The most suitable process conditions of compound strain Acetobacter xylinum acetic fermentation：Fermentation time 5.3 days, initial wine degree 6.4%, initially
PH3.51, inoculum concentration 15.2%, liquid amount 30%, with this understanding, producing acid amount up to 66.4g/L, amino-acid nitrogen content is
0.712g/L, total ester content are 0.962g/L, can greatly improve the rate of producing acid of orange fruit vinegar using the technique and produce acid amount, and
The fruit vinegar tart flavour appropriateness, mouthfeel alcohol is good, and fruital is pleasant.
The acetic fermentation process factor level coding schedule of table 5
Claims (6)
1. a kind of method that utilization composite bacteria mixed fermentation prepares organge fruit vinegar, it is characterised in that the preparation method is as follows：
1）The preparation of mulriple yeasts kind seed liquor：Saccharomycete activation medium liquid amount is 150mL/250mL, respectively takes two bait ferment
Female bacterium 31906, saccharomyces cerevisiae 1306, the thick bacterium of saccharomyces cerevisiae 1425, Hansenula anomala exception mutation 1431 move into respectively culture
Liquid, is placed in shaking table in 28 DEG C, and 180r/min culture 16h obtain one-level nutrient solution, and adjustment cell concentration is 107Individual/mL；By four
Plant one-level nutrient solution to be linked in malt medium according to following percent by volume：The exception mutation 1431 of 4% Hansenula anomala,
3% saccharomyces cerevisiae 1425,3% saccharomyces cerevisiae 31906,2% saccharomyces cerevisiae 1306 is subsequently placed in shaking table in 28 DEG C, under 180r/min
Culture 16h, makes thalline quantity reach 107-108Individual/mL, as mulriple yeasts kind seed liquor；
2）The preparation of compound acetic acid bacteria strain seed liquor：Acetic acid bacteria activation medium liquid amount is 30mL/150mL, respectively takes two ring apples
Fruit vinegar bacillus Acetobacter malorum ZY.C4, Pasteur acetobacter Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai
Make 1.01 thick bacterium and move into nutrient solution respectively, be placed in shaking table in 31 DEG C, 150r/min cultivates 48 hours, obtains one-level nutrient solution,
Adjustment cell concentration is 106Individual/mL；Four kinds of one-level nutrient solutions are linked into into alcoholic strength for 6-7%'s according to following percent by volume
In Citrus Fruit Wine：4% apple acetobacter Acetobacter malorum ZY.C4,3% Pasteur acetobacter Luo Wang subspecies 7002,2%
Acidfast bacilli 22518 is acetified, 2% Shanghai makes 1.01；It is subsequently placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline
Quantity reaches thalline quantity up to 106-108Individual/mL；
Described apple acetobacter Acetobacter malorum ZY.C4 are obtained voluntarily to separate, and deposit number is CGMCC
NO ：8692；
3）It is prepared by glutinous rice saccharified liquid：Rice is rinsed 2 times, soaks 2-4h；By weight water：Rice=2.5：1 defibrination, sizes mixing to 4:1
Ratio, adds CaCl2And MgCl2, the CaCl2And MgCl2Addition be every 100mL slurries and add 1g, with acetic acid and carbon
Sour sodium adjusts pH to 6.2-6.4, adds α-amylase, and the addition of α-amylase is that 100mL slurries add 1g, is heated to 90-95 DEG C of liquid
Change 20-30min, be cooled to 60 DEG C, tune pH is 4.5-5.0, obtain liquid A, the saccharification of liquid A Jing obtains glutinous rice saccharified liquid；
4) preparation of citrus juice：Select without rotten citrus, peeling, citrus pulp is put in juice extractor, and add weight hundred
Point concentration is 0.01% NaHSO3Solution, weight ratio is citrus pulp：NaHSO3Solution=1:1；Squeeze the juice, fruit is added in juice
Glue enzyme is digested, and the addition of the pectase is addition pectase 0.04g, 60 DEG C of hydrolysis temperature, during enzymolysis in every 100mL juice
Between 60min, 4 layers of filtered through gauze, obtains citrus juice after enzymolysis；
5) prepared by zymotic fluid：By citrus juice produced above and glutinous rice saccharified liquid with 3:1 volume ratio mixing, in mixed liquor
Ratio in sulfur dioxide 30mg and the mg of ammonium sulfate 50 is separately added in every 1L mixed liquors adds sulfur dioxide and ammonium sulfate, mixes
It is even and adjust pH value 4.48, pol is adjusted to 15-19 ° of Bx, sterilization 15min in 85 DEG C of hot water, cooling is standby, obtains zymotic fluid；
6）Alcoholic fermentation：In step 5）Zymotic fluid in access mulriple yeasts kind seed liquor, inoculum concentration is percent by volume
9.7-10%, 28-28.5 DEG C of stirring, rotating speed is that after 180r/min fermentation 1d, after closed standing for fermentation 4-5d, sterilization obtains citrus
Fruit wine；
7）Acetic fermentation：Citrus Fruit Wine alcoholic strength is adjusted to 6-7%, compound acetic acid bacteria strain seed liquor is accessed, inoculum concentration is volume
Percentage 15.2-16%, is 180r/min, 31 DEG C of stirring fermentation 5-7d in rotating speed；Terminate fermentation, be passed through 80-100 DEG C of steam and go out
Bacterium 30min；
8）7）In add salt in the liquid that obtains, the addition of salt is addition salt 20g, normal temperature ageing 2-3 in every 1L
Individual month, filtered with diatomite and clarified, obtain organge fruit vinegar finished product.
2. the method that a kind of utilization composite bacteria mixed fermentation according to claim 1 prepares organge fruit vinegar, its feature exists
In：The step 3）Method for saccharifying be：Carbohydrase and malt flour are added, addition is that 1g saccharification is added in every 100mL liquid As
Enzyme and 10g malt flours, insulation saccharification 6-8h.
3. the method that a kind of utilization composite bacteria mixed fermentation according to claim 1 prepares organge fruit vinegar, its feature exists
In：The step 3）Method for saccharifying be：Add black song：Red yeast rice=3:1 mixed starters, the addition of mixed starters is 100mL liquid
Add 1g in A, saccharificatinn period is 2-4h.
4. the method that a kind of utilization composite bacteria mixed fermentation according to claim 1 prepares organge fruit vinegar, its feature exists
In：The step 3）Malt flour is additionally added before enzymolysis, addition is addition malt flour 10g in every 100mL juice.
5. the method that a kind of utilization composite bacteria mixed fermentation according to claim 1 prepares organge fruit vinegar, its feature exists
In：The step 7）Naringinase is additionally added before fermentation, the addition of naringinase is to add 0.04g in every 100mL Citrus Fruit Wines.
6. according to prepared by the method that any one in claim 1-5 prepares organge fruit vinegar using composite bacteria mixed fermentation
Organge fruit vinegar.
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