CN104694371A - 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, be specifically related to a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare and preparation method thereof.
Background technology
Organge fruit vinegar is for raw material with fresh, ripe citrus fruits, through removing the peel, squeezing the juice, be separated, debitterize, the explained hereafter gained such as zymamsis, acetic fermentation, ageing, sterilizing, it contains abundant organic acid and VITAMIN, having relieves summer heat quenches one's thirst, Ginseng Extract, beautifying face and moistering lotion, improve a poor appetite, the health-care effect such as Antialcoholic liver-protecting, antisepsis and sterilization, be applicable to the dietotherapy of various diseases, especially having good treatment and prophylactic effect to cardiovascular disordeies such as hypertension, coronary heart disease, arteriosclerosis, is leavened food of new generation.Safety, produce that the smell of fruits is very sweet, the organge fruit vinegar of unique flavor is the key point that its industrialization can develop in a healthy way efficiently.Fruit vinegar needs to have unique local flavor and nutritive ingredient, this just to be suitable for brew organge fruit vinegar bacterial screening, raise and train work and production technique proposes new requirement.The bacterial classification that current China fruit vinegar produces family's use is mostly single yeast saccharomyces cerevisiae and vinegar bacterial classification, and the product special flavour produced, insufficient quality, yield rate is low, and facility investment is larger.About fruit vinegar production technology, there are liquid state fermentation and solid state fermentation two kinds.Solid state fermentation has the advantages such as local flavor is good, mouthfeel is good compared with liquid fermentation method, but also exists that fermentation period is long, labour intensity large, fermenting process is difficult to control, easily by shortcomings such as living contaminantses.And the advantage of liquid fermentation method is that fermentation period is short, labour intensity is little, fermenting process is easy to control, decrease the chance of living contaminants, but owing to being purebred cultivation, and fermentation time is limited, therefore the flavour substances of liquid fermentation method formation is few, and its local flavor and mouthfeel comparatively solid state fermentation are poor.At present, also the method for very not most people's will produces fruit vinegar, about improving the production technology of fruit vinegar, still in the exploratory stage.
Summary of the invention
For the technical problem existed in background technology, the object of the present invention is to provide a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare and preparation method thereof.With citrus and glutinous rice for raw material, mix through removing the peel, squeezing the juice, pull an oar, after saccharification, add composite yeast bacterial classification and compound acetic acid bacteria strain, through a zymamsis and an acetic fermentation, safety, produce that the smell of fruits is very sweet, unique flavor efficiently, be rich in the organge fruit vinegar of the nutritive ingredients such as amino acid, flavones, vitamins C.
The technical scheme adopted is as follows:
The organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare, is characterized in that its preparation method is as follows:
1) preparation of mulriple yeasts kind seed liquor: yeast activation medium liquid amount is 150mL/250mL, respectively get two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the thick bacterium of the abnormal mutation 1431 of Hansenula anomala move into nutrient solution respectively, be placed in shaking table in 28 DEG C, 180r/min cultivates 16h, obtain one-level nutrient solution, adjustment cell concentration is 10
7individual/mL; Four kinds of one-level nutrient solutions are linked in Fructus Hordei Germinatus substratum according to following volume percent: the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906,2% yeast saccharomyces cerevisiae 1306, then shaking table is placed in 28 DEG C, cultivate 16h under 180r/min, make thalline quantity reach 10
7-10
8individual/mL, is mulriple yeasts kind seed liquor.
2) preparation of compound acetic acid bacteria strain seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, respectively get two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai to 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, obtain one-level nutrient solution, adjustment cell concentration is 10
6individual/mL; It is in the Citrus Fruit Wine of 6-7% that four kinds of one-level nutrient solutions are linked into alcoholic strength according to following volume percent: 4% cider vinegar bacillus ZY.C4, and 3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518,2% Shanghai makes 1.01; Then be placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity and reaches 10
6-10
8individual/mL.
3) glutinous rice saccharified liquid preparation: rice rinses 2 times, soaks 2-4h; By weight water: rice=2.5:1 defibrination, size mixing to 4:1 ratio, add CaCl
2and MgCl
2, described CaCl
2and MgCl
2add-on be every 100mL slurries and add 1g, pH to 6.2-6.4 is adjusted with acetic acid and sodium carbonate, add α-amylase, the add-on of α-amylase is that 100mL slurries add 1g, be heated to the 90-95 DEG C of about 20-30min that liquefies, be cooled to 60 DEG C, tune pH is 4.5-5.0, obtain liquid A, liquid A obtains glutinous rice saccharified liquid through saccharification;
Its method for saccharifying is: add saccharifying enzyme and malt meal, and add-on is add 1g saccharifying enzyme and 10g malt meal in every 100mL liquid A, insulation saccharification 6-8h; Or add black song: the mixed starters of red colouring agent for food, also used as a Chinese medicine=3:1, the add-on of mixed starters is add 1g in 100mL liquid A, and saccharification time is 2-4h.
4) preparation of citrus juice: select the citrus without rotting, peeling, puts into juice extractor by citrus pulp, and adds the NaHSO of 0.01% (weight)
3solution, citrus pulp: NaHSO
3solution=1:1(weight ratio); Squeeze the juice, in juice, add pectinase enzymatic hydrolysis, the add-on of described polygalacturonase is add polygalacturonase 0.04g in every 100mL juice, hydrolysis temperature 60 DEG C, enzymolysis time 60min, and after enzymolysis, 4 layers of filtered through gauze, obtain citrus juice; As preferably, before enzymolysis, also add malt meal, add-on is add malt meal 10g in every 100mL juice.
5) fermented liquid preparation: citrus juice obtained above and glutinous rice saccharified liquid are mixed with the volume ratio of 3:1, sulfurous gas and ammonium sulfate is added in the ratio adding sulfurous gas 30mg and ammonium sulfate 50 mg in every 1L mixed solution respectively in mixed solution, mixing is adjust ph 4.48 also, regulate pol to 15-19 ° of Bx ', sterilization 15min in 85 DEG C of hot water, cool for subsequent use, obtain fermented liquid.
6) zymamsis: access mulriple yeasts kind seed liquor in the fermented liquid of step 5), inoculum size is 9.7-10% (volume percent), 28-28.5 DEG C of stirring, rotating speed is after 180r/min fermentation 1d, after airtight standing for fermentation 4-5d, sterilization, obtains Citrus Fruit Wine.As preferably, before fermentation, also add naringinase, the add-on of naringinase is add 0.04g in every 100mL fermented liquid.
7) acetic fermentation: Citrus Fruit Wine alcoholic strength is adjusted to 6-7%, access compound acetic acid bacteria strain seed liquor, inoculum size is 15.2-16% (volume percent), is 180r/min at rotating speed, and 31 DEG C are stirred fermentation 5-7d; Stop fermentation, pass into 80-100 DEG C of steam sterilizing 30min.
8) 7) in add salt in the liquid that obtains, the add-on of salt is add salt 20g in every 1L, normal temperature ageing 2-3 month, with diatomite filtration clarification, obtains organge fruit vinegar finished product.
By the organge fruit vinegar that above-mentioned technical process and control condition obtain, nutritious, this fruit vinegar tart flavour appropriateness, mouthfeel alcohol is good, fruital is pleasant, without bitter taste, is particularly rich in the amino acid required for HUMAN HEALTH and multiple antioxidant component, if carry out allotment and the dilution of appropriateness in addition again, popular drink can also be developed to.In addition, utilize composite bacteria brewing fruit vinegar, thalline reproduction speed is fast, and can produce the higher acidity of acid at the fermentation initial stage, acid production speed is fast simultaneously, within 5th day, maximum acidity is just reached what ferment, its fermentation time 3-4 days shorter in single culture fermentation time, therefore, utilizes this technique to reduce living contaminants, shorten the production cycle, stabilized product quality.
The present invention is owing to have employed cider vinegar bacillus ZY.C4, make the fruit vinegar flavor material such as the total acid of the standby organge fruit vinegar made, amino nitrogen, total ester, VC, flavones and nutritive ingredient bacterium higher than the fruit vinegar adopted containing the brew of AS1.41 acetic bacteria composite bacteria, and make the limonin substances decline 70-75% in organge fruit vinegar product, make its products taste soft, without bitter taste.
Embodiment
Below in conjunction with embodiment, explanation detailed is further done to the present invention.
Bacterial classification involved in embodiment and substratum as follows:
Yeast seeds: the abnormal mutation 1431 of yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, Hansenula anomala.Above bacterial classification is all purchased from Chinese industrial Microbiological Culture Collection administrative center.
Strain Acetobacter xylinum: Shanghai wine 1.01, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518, stench bacillus aceticus AS1.41, above bacterial classification is all purchased from Chinese industrial Microbiological Culture Collection administrative center; Cider vinegar bacillus (
acetobacter malorum) ZY.C4, be separated acquisition voluntarily, carry out culture presevation, depositary institution: CGMCC (is called for short in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preservation day: on 01 07th, 2014, preserving number: CGMCC NO: 8692.
Yeast activation medium: 5 ° of Be ' wort agars and the glutinous rice citrus nutrient solution 2:1 processed mix, and 121 DEG C of autoclaving 20min, cool for subsequent use.
Acetic bacteria activation medium: glucose 1g, yeast extract paste 1g, alcohol (95%) 5mL, water 100mL, pH nature.Alcohol is added again after sterilizing.
embodiment 1: the preparation technology one of organge fruit vinegar
one,material preparation technology
(1) preparation of mulriple yeasts kind seed liquor: yeast activation medium liquid amount is 150mL/250mL, respectively get two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the thick bacterium of the abnormal mutation 1431 of Hansenula anomala move into nutrient solution respectively, be placed in shaking table in 28 DEG C, 180r/min cultivates 16h, obtain one-level nutrient solution, adjustment cell concentration is 10
7individual/mL; Four kinds of one-level nutrient solutions are linked in Fructus Hordei Germinatus substratum according to following volume percent: the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906,2% yeast saccharomyces cerevisiae 1306, then shaking table is placed in 28 DEG C, cultivate 16h under 180r/min, make thalline quantity reach 10
7-10
8individual/mL, is mulriple yeasts kind seed liquor.
(2) preparation of compound acetic acid bacteria strain seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, respectively get two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai to 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, obtain one-level nutrient solution, adjustment cell concentration is 10
6individual/mL; It is in the Citrus Fruit Wine of 6-7% that four kinds of one-level nutrient solutions are linked into alcoholic strength according to following volume percent: 4% cider vinegar bacillus ZY.C4, and 3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518,2% Shanghai makes 1.01; Then be placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity and reaches 10
6-10
8individual/mL.
(3) glutinous rice saccharified liquid preparation: rice rinses 2 times, soaks 2-4h; By weight water: rice=2.5:1 defibrination, size mixing to 4:1 ratio, add CaCl
2and MgCl
2, described CaCl
2and MgCl
2add-on be every 100mL slurries and add 1g, pH to 6.2-6.4 is adjusted with acetic acid and sodium carbonate, add α-amylase, the add-on of α-amylase is that 100mL slurries add 1g, is heated to the 90-95 DEG C of about 20-30min that liquefies, is cooled to 60 DEG C, tune pH is 4.5-5.0, add saccharifying enzyme and malt meal, add-on is add 1g saccharifying enzyme and 10g malt meal in every 100mL liquid, insulation saccharification 6-8h; Obtain glutinous rice saccharified liquid.
(4) preparation of citrus juice: select the citrus without rotting, peeling, puts into juice extractor by citrus pulp, and adds the NaHSO of 0.01% (weight)
3solution, citrus pulp: NaHSO
3solution=1:1(weight ratio); Squeeze the juice, add polygalacturonase and malt meal in juice, the add-on of polygalacturonase is add polygalacturonase 0.04g in every 100mL juice, and malt meal add-on is add malt meal 10g in every 100mL juice.Hydrolysis temperature 60 DEG C, enzymolysis time 60min, after enzymolysis, 4 layers of filtered through gauze, obtain citrus juice.
(5) fermented liquid preparation: citrus juice obtained above and glutinous rice saccharified liquid are mixed with the volume ratio of 3:1, sulfurous gas and ammonium sulfate is added in the ratio adding sulfurous gas 30mg and ammonium sulfate 50 mg in every 1L mixed solution respectively in mixed solution, mixing is adjust ph 4.48 also, regulate pol to 15-19 ° of Bx ', sterilization 15min in 85 DEG C of hot water, cool for subsequent use, obtain fermented liquid.
Two, zymamsis (the 1st fermentation)
9.7% mulriple yeasts kind seed liquor is added in above-mentioned fermented liquid (liquid amount is volume 4/5), after 28.5 DEG C of stirrings (rotating speed the is 180r/min) 1d that ferments, airtight standing for fermentation 4-5d, in detection fermented liquid, ethanol content is when 10%-12% V/V, stop fermentation, after filtration, pass into 80-100 DEG C of steam sterilizing 30min.
Three, acetic fermentation (the 2nd fermentation)
Above-mentioned fruit wine wine degree is adjusted to 6-7%, apparatus for acetic acid fermentation tank is loaded by 30% liquid amount, by 15.2% access compound strain Acetobacter xylinum seed liquor, add the naringinase (namely adding 0.04g in every 100mL Citrus Fruit Wine) of 0.04% again, in 31 DEG C of stirrings (rotating speed is 180r/min), ferment 5-7d, detect alcohol residue amount when 0.3%-0.5%, stop fermentation, pass into 80-100 DEG C of steam sterilizing 30min.
Four, ageing, filtration, clarification
The fruit vinegar completing acetic fermentation adds the salt of 2% (namely adding salt 20g in every 1L), deposits in clean airtight tank body, normal temperature ageing 2 months.Do filtration medium with diatomite and filtration clarification is carried out to vinegar unstrained spirits, obtain organge fruit vinegar finished product.
embodiment 2: the preparation technology two of organge fruit vinegar
one,material preparation technology
(1) preparation of mulriple yeasts kind seed liquor: yeast activation medium liquid amount is 150mL/250mL, respectively get two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the thick bacterium of the abnormal mutation 1431 of Hansenula anomala move into nutrient solution respectively, be placed in shaking table in 28 DEG C, 180r/min cultivates 16h, obtain one-level nutrient solution, adjustment cell concentration is 10
7individual/mL; Four kinds of one-level nutrient solutions are linked in Fructus Hordei Germinatus substratum according to following volume percent: the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906,2% yeast saccharomyces cerevisiae 1306, then shaking table is placed in 28 DEG C, cultivate 16h under 180r/min, make thalline quantity reach 10
7-10
8individual/mL, is mulriple yeasts kind seed liquor.
(2) preparation of compound acetic acid bacteria strain seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, respectively get two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai to 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, obtain one-level nutrient solution, adjustment cell concentration is 10
6individual/mL; It is in the Citrus Fruit Wine of 6-7% that four kinds of one-level nutrient solutions are linked into alcoholic strength according to following volume percent: 4% cider vinegar bacillus ZY.C4, and 3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518,2% Shanghai makes 1.01; Then be placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity and reaches 10
6-10
8individual/mL.
(3) glutinous rice saccharified liquid preparation: rice rinses 2 times, soaks 2-4h; By weight water: rice=2.5:1 defibrination, size mixing to 4:1 ratio, add CaCl
2and MgCl
2, described CaCl
2and MgCl
2add-on be every 100mL slurries and add 1g, pH to 6.2-6.4 is adjusted with acetic acid and sodium carbonate, add α-amylase, the add-on of α-amylase is that 100mL slurries add 1g, is heated to the 90-95 DEG C of about 20-30min that liquefies, is cooled to 60 DEG C, tune pH is 4.5-5.0, add black song: the mixed starters of red colouring agent for food, also used as a Chinese medicine=3:1, the add-on of mixed starters is add 1g in 100mL liquid, saccharification 2-4h; Obtain glutinous rice saccharified liquid.
(4) preparation of citrus juice: select the citrus without rotting, peeling, puts into juice extractor by citrus pulp, and adds the NaHSO of 0.01% (weight)
3solution, citrus pulp: NaHSO
3solution=1:1(weight ratio); Squeeze the juice, add polygalacturonase and malt meal in juice, the add-on of polygalacturonase is add polygalacturonase 0.04g in every 100mL juice, and malt meal add-on is add malt meal 10g in every 100mL juice.Hydrolysis temperature 60 DEG C, enzymolysis time 60min, after enzymolysis, 4 layers of filtered through gauze, obtain citrus juice.
(5) fermented liquid preparation: citrus juice obtained above and glutinous rice saccharified liquid are mixed with the volume ratio of 3:1, sulfurous gas and ammonium sulfate is added in the ratio adding sulfurous gas 30mg and ammonium sulfate 50 mg in every 1L mixed solution respectively in mixed solution, mixing is adjust ph 4.48 also, sugar addition to 15 ° Bx ', in 85 DEG C of hot water, sterilization kills wherein miscellaneous bacteria in 15 minutes, cools for subsequent use.
Two, zymamsis (the 1st fermentation)
10% mulriple yeasts kind seed liquor is added in above-mentioned fermented liquid (liquid amount is volume 4/5), after 28 DEG C of stirrings (rotating speed the is 180r/min) 1d that ferments, airtight standing for fermentation 6-7d, in detection fermented liquid, ethanol content is when 10%-12% V/V, stop fermentation, after filtration, pass into 80-100 DEG C of steam sterilizing 30min.
Three, acetic fermentation (the 2nd fermentation)
Above-mentioned fruit wine wine degree is adjusted to 6-7%, apparatus for acetic acid fermentation tank is loaded by 30% liquid amount, by 16% access compound strain Acetobacter xylinum seed liquor, in 32 DEG C of stirrings (rotating speed is 180r/min), ferment 5-7d, detect alcohol residue amount when 0.3%-0.5%, stop fermentation, pass into 80-100 DEG C of steam sterilizing 30min.
Four, ageing, filtration, clarification
The fruit vinegar completing acetic fermentation adds the salt of 2%, deposits in clean airtight tank body, normal temperature ageing 3 months.Do filtration medium with diatomite and filtration clarification is carried out to vinegar unstrained spirits, obtain organge fruit vinegar finished product.
embodiment 3: the isolation identification of cider vinegar bacillus ZY.C4
(1) the commercial citrus without decayed fruit is prepared in bacterium source, cleans, and making beating, carries out nature zymamsis and natural acetic fermentation.Every day measures its pol with saccharometer, when pol is down to 5 ° of BX, and no longer changes in a continuous week, starts to filter.With the 80 order gauze double medium filtrations boiled, after filtration, filtrate is kept in Stainless Steel Kettle, i.e. citrus wine.Hide pot face with gauze, carry out spontaneous fermentation at 32 DEG C, obtain the vinegar liquid of spontaneous fermentation.From vinegar liquid, get bacterium source after 5d, carry out screening and separating.
(2) separation screening flow process is planted
---------------liquid triangular flask is cultivated, and------slant preservation bacterial strain---sieves---determining excellent acid-producing bacteria strain---slant preservation bacterial strain to slant culture to primary dcreening operation to diluting separation to multiplication culture again to determine primary dcreening operation bacterial strain in bacterium source
(3) prescreening method
Multiplication culture: the fermented vinegar liquid 10mL taking from right acetic acid, puts into the proliferated culture medium of 90mL respectively, and cultivates 2d in constant-temperature table (32 DEG C, 120r/min).
Calcium plate isolation is tested: gradient dilution multiplication culture liquid, for being coated with separation, in the thermostat container of 32 DEG C, cultivates 2d, observations, selects calcium dissolving circle large, moves in slant medium by the single bacterium colony of different colonial morphology, repeat 3-4 time, obtain purifying bacterial strain.
(4) multiple sieve
By bacterial strain picking two ring of calcium plate isolation, move in 20mL liquid basal medium (adding 6% dehydrated alcohol), cultivate 7d in constant-temperature table (32 DEG C, 120r/min), carry out acetimetry mensuration.The high strain excellent of acid amount is produced according to the screening of acid test result.
Produce the high strain excellent of acid amount through being accredited as cider vinegar bacillus (Acetobacter malorum) ZY.C4.
Above-mentioned separation and Culture culture medium prescription used is as follows:
Proliferated culture medium: yeast powder 1%, glucose 1%, the Viola crystallina 0.5% of 0.02%, nysfungin 2.5 × l0
6unit/L, pH5.5, adds 95% dehydrated alcohol of 3% (v/v).
Isolation medium: yeast powder l%, glucose l%, CaCO
31.5%, agar 2%, pH5.5, adds 95% dehydrated alcohol of 4% (v/v) and the CaCO of dry sterilization
3.
Slant medium: yeast powder 1%, glucose 1%, CaCO
31.5%, agar 2%, pH5.5, packing test tube, adds 95% dehydrated alcohol 4% (v/v).
Liquid basal medium: yeast powder 1%, glucose 1%, pH5.5.
embodiment 4: organge fruit vinegar nutritive ingredient and bitter substance comparision contents
by embodiment 1, embodiment 2 and contrast in organge fruit vinegar nutritive ingredient and
debitterize rate and mouthfeelcompare, in contrast, cider vinegar bacillus ZY.C4 adopts the muddy mutation of conventional fruit vinegar brewing bacterial classification AS1.41 stench bacillus aceticus to substitute, and all the other techniques are with embodiment 1.Concrete nutritive ingredient is more as shown in table 1.Adopt and prepare the fruit vinegar flavor material such as the total acid of organge fruit vinegar, amino nitrogen, total ester, VC, flavones and nutritive ingredient bacterium higher than the fruit vinegar adopted containing the brew of AS1.41 acetic bacteria composite bacteria containing the sub-liquid of cider vinegar bacillus ZY.C4 compound acetic acid bacteria strain, and make the limonin substances decline 70-75% in organge fruit vinegar product, make its products taste soft, without bitter taste.
Table 1 organge fruit vinegar nutritive ingredient compares
embodiment 5 mulriple yeasts bacterial classification ratio optimization
Saccharomycetic one-level enlarged culturing: yeast activation medium liquid amount is 150mL/250mL, each examination barms that supplies gets two baits thick bacterium immigration nutrient solution, is placed in shaking table (28 DEG C, 180r/min) and cultivates about 16 hours.
The preparation of fermented liquid: citrus juice obtained above and glutinous rice saccharified liquid are mixed with 3:1 ratio, sugar addition is 11 degree, regulates pH4.2 with citric acid, and then the sterilization in 85 DEG C of hot water of the fruit juice after adjustment cools for subsequent use to kill wherein miscellaneous bacteria in 15 minutes.
Mulriple yeasts bacterial classification ratio is preferred: adopt 4 factor 3 horizontal quadrature experiments, be optimized the inoculative proportion of yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the abnormal mutation of Hansenula anomala 1,431 four kinds of bacterial strains, its level of factor is as shown in table 2.The cell concentration of one-level scale-up medium is adjusted to 10
8four kinds of one-level nutrient solutions are accessed in new Fructus Hordei Germinatus substratum according to the ratio of orthogonal experiment scheme by individual/mL, are placed in shaking table (28 DEG C, 180r/min) and cultivate about 16 hours, make thalline quantity reach 10
7-10
8individual/mL.By the mixed strains access citrus glutinous rice mixed solution made, inoculum size is 10%, after 28 DEG C of stirrings (rotating speed the is 180r/min) 1d that ferments, and airtight standing for fermentation.Every day weighs to fermented liquid, when substantially no longer changing to weight, measures total reducing sugar.After total sugar content is stable at 4 ~ 5 ° of Be ', carry out the evaluation of every physical and chemical index mensuration such as ethanol content, amino-acid nitrogen content, product acid amount and Oranoleptic indicator thereof.
Table 2 composite yeast bacterial classification ratio optimization orthogonal test level of factor table
Composite yeast bacterial classification ratio preferred result: the inoculum size of the abnormal mutation 1431 of Hansenula anomala, yeast saccharomyces cerevisiae 1425, yeast saccharomyces cerevisiae 31906, yeast saccharomyces cerevisiae 1306 is respectively 4%, 3%, 3%, when 2%, ethanol content after fermentation, amino-acid nitrogen content and total ester content are all higher, and aroma is strong, and there is the fragrance of fruit.
embodiment 6:
composite yeast bacterial classification technology of alcohol is optimized
The seed liquor preparation of composite yeast bacterial classification: yeast activation medium liquid amount is 150mL/250mL, the thick bacterium of getting two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the abnormal mutation 1431 of Hansenula anomala etc. four kinds different moves into nutrient solution respectively, be placed in shaking table (28 DEG C, 180r/min) cultivate about after 16 hours, adjustment cell concentration is 108/mL; By four kinds of one-level nutrient solutions according to the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906, the ratio of 2% yeast saccharomyces cerevisiae 1306 accesses in new Fructus Hordei Germinatus substratum, be placed in shaking table (28 DEG C, 180r/min) cultivate about 16 hours, make thalline quantity reach 10
7-10
8individual/mL.
Technology of alcohol is optimized: carry out single factor experiment first to each factors such as ammonium sulfate addition, sulfurous gas addition, composite bacteria inoculum size, fermentation time, leavening temperature, initial pol and initial pH, is optimized to select more excellent level.In citrus glutinous rice mixed solution, access composite yeast, after zymamsis, measure ethanol content, amino-acid nitrogen content, and carry out subjective appreciation.Each factorial experiments level is as follows: sulfurous gas addition is: 0,30,60,90 mg/L; Ammonium sulfate addition is: 0,25,50,75,100 mg/L; Mixed strains inoculum size is 6%, 8%, 10%, 12%, 14%; Fermentation time is 1,2,3,4,5,6,7,8d; Leavening temperature is 24 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C; Initial pol is 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.On the basis of above single factor experiment result, determine the span of the initial pol in alcoholic fermentation process, initial pH value, inoculum size, time, temperature major influence factors, carry out quadratic orthogonal rotating composite test (half enforcement of 5 factor 5 levels, have 36 combinations), using ethanol content as evaluation index, analyze the result of test, integrated sensory evaluates score and verifies result, the technology of alcohol condition be optimized.Its technology of alcohol experimental factor level code table is as shown in table 3.
Table 3 technology of alcohol experimental factor level code value table
The suitableeest processing condition of composite yeast bacterial classification zymamsis: add 30 mg/L sulfurous gas, 50 mg/L ammonium sulfate in citrus juice and glutinous rice saccharified liquid mixed solution, adjusting initial pol is 19 ° of Bx ', initial pH value 4.48, access wherein 9.7 % composite yeast bacterial classification, at 28.5 DEG C, after 28.5 DEG C of stirrings (rotating speed the is 180r/min) 1d that ferments, airtight standing for fermentation 4-5d fermentation, gained Citrus Fruit Wine ethanol content can reach 11.7 %, and aroma is strong, and there is fruit aroma.
embodiment 7: compound acetic acid bacteria strain ratio is preferred
The one-level enlarged culturing of acetic bacteria: yeast activation medium liquid amount is 30mL/150mL, the thick bacterium of getting four kinds, two ring different moves into nutrient solution respectively, is placed in shaking table (30 DEG C, 150r/min) and cultivates about 48 hours.
The preparation of fermented liquid: citrus juice obtained above and glutinous rice saccharified liquid are mixed with 3:1 ratio, 30 mg/L sulfurous gas, 50 mg/L ammonium sulfate are added in citrus juice and glutinous rice saccharified liquid mixed solution, adjust initial pol to be 19 ° of Bx ', to regulate pH4.48 with citric acid, then the sterilization in 85 DEG C of hot water of fruit juice after adjustment cools for subsequent use to kill wherein miscellaneous bacteria in 15 minutes.
Citrus Fruit Wine ferments: in above-mentioned fermented liquid (liquid amount is volume 4/5), add 9.7% mulriple yeasts kind seed liquor, after 28.5 DEG C of stirrings (rotating speed the is 180r/min) 1d that ferments, airtight standing for fermentation 4-5d, in detection fermented liquid, ethanol content is when 10%-12% V/V, stop fermentation, after filtration, pass into 80-100 DEG C of steam sterilizing 30min.
Compound acetic acid bacteria strain ratio is preferred: adopt 4 factor 3 horizontal quadrature experiments, be optimized the inoculative proportion of Shanghai wine 1.01, cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22,518 four kinds of bacterial classifications, its level of factor is as shown in table 4.The cell concentration of the one-level nutrient solution of four bacterial classifications is adjusted to 10
7individual/mL, moving into wine degree according to the ratio of orthogonal experiment scheme is in 6-7% Citrus Fruit Wine, is placed in shaking table (30 DEG C, 150r/min) and cultivates about 24 hours, make thalline quantity reach thalline quantity and reach 10
6 ~10
8individual/mL, obtains mixing acetic acid bacteria strain.
Acetic fermentation: it is 6-7%'s that the mixed strains made is accessed wine degree, ferment in 31 DEG C of stirrings (rotating speed is 180r/min), detect alcohol residue amount when 0.3%-0.5%, stop fermentation, measure the content that it produces acid amount, total ester content and amino acid peptide nitrogen, and subjective appreciation is carried out to its local flavor.In conjunction with every physical and chemical index and results of sensory evaluation, filter out the acetic bacteria inoculum size optimum proportion of applicable Pon hesperidium aurantium vinegar fermentation.
Table 4 compound strain Acetobacter xylinum ratio preferred orthogonal test level of factor table
Compound strain Acetobacter xylinum ratio preferred result: when 4 kinds of strain Acetobacter xylinum ratios are 4% cider vinegar bacillus ZY.C4,3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% Shanghai makes 1.01, during 2% acetify acidfast bacilli 22518, record acidity, amino acid peptide nitrogen content and total ester content and reach maximum, and obtained fruit vinegar tart flavour is soft, good smell, has the fragrance of fruit vinegar and grain vinegar concurrently.
embodiment 8 compound strain Acetobacter xylinum acetic fermentation process is optimized
Prepared by compound strain Acetobacter xylinum seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, the thick bacterium of getting four kinds, two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai wine 1.01 etc. different moves into nutrient solution respectively, be placed in shaking table (30 DEG C, 150r/min) cultivate about after 48 hours, adjustment cell concentration is 10
7individual/mL; By four kinds of one-level nutrient solutions according to 4% cider vinegar bacillus ZY.C4,3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518, it is in 6-7% Citrus Fruit Wine that the ratio of 2% Shanghai wine 1.01 moves into wine degree, be placed in shaking table (30 DEG C, 150r/min) cultivate about 24 hours, make thalline quantity reach thalline quantity and reach 10
6 ~10
8individual/mL.
Acetic fermentation process is optimized: first carry out experiment of single factor to fermentation time, initial pH, liquid amount, inoculum size, initial each factor such as wine degree and leavening temperature, compound acetic bacteria is accessed in Citrus Fruit Wine, acetic acid content, amino-acid nitrogen content, total ester content is measured after acetic fermentation, and carry out subjective appreciation, to filter out the optimum level of each factor.Each factorial experiments level is as follows: fermentation time is 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%, 20%, 30%, 40%, 50%; The inoculum size of compound acetic bacteria is 5%, 10%, 15%, 20%; Initial wine degree is 5%, 6%, 7%, 8%, 9%; Leavening temperature is 25 DEG C, 28 DEG C, 31 DEG C, 34 DEG C, 37 DEG C.On above single factor experiment basis, adopt quadratic orthogonal rotating composite test (half enforcement of 5 factor 5 levels, totally 36 combinations), the impact of the factor Dichlorodiphenyl Acetate ferment effects such as the inoculum size of investigation time, initial pH, liquid amount, the initially mixed strains of wine degree and best of breed, using the content of fermented liquid total acid content, amino acid peptide nitrogen content and total ester as evaluation index, optimize Pon hesperidium aurantium vinegar acetic fermentation process conditional parameter with this.Its acetic fermentation process experimental factor level code table is as shown in table 5.
The suitableeest processing condition of compound strain Acetobacter xylinum acetic fermentation: fermentation time 5.3 days, initial wine degree 6.4%, initial pH3.51, inoculum size 15.2%, liquid amount 30%, with this understanding, produce acid amount up to 66.4g/L, amino-acid nitrogen content be 0.712g/L, total ester content is 0.962g/L, utilizes this technique greatly can improve the rate of producing acid of orange fruit vinegar and produce acid amount, and this fruit vinegar tart flavour appropriateness, mouthfeel alcohol is good, and fruital is pleasant.
Table 5 acetic fermentation process level of factor coding schedule
Claims (6)
1. the organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare, is characterized in that its preparation method is as follows:
1) preparation of mulriple yeasts kind seed liquor: yeast activation medium liquid amount is 150mL/250mL, respectively get two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the thick bacterium of the abnormal mutation 1431 of Hansenula anomala move into nutrient solution respectively, be placed in shaking table in 28 DEG C, 180r/min cultivates 16h, obtain one-level nutrient solution, adjustment cell concentration is 10
7individual/mL; Four kinds of one-level nutrient solutions are linked in Fructus Hordei Germinatus substratum according to following volume percent: the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906,2% yeast saccharomyces cerevisiae 1306, then shaking table is placed in 28 DEG C, cultivate 16h under 180r/min, make thalline quantity reach 10
7-10
8individual/mL, is mulriple yeasts kind seed liquor;
2) preparation of compound acetic acid bacteria strain seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, respectively get two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai to 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, obtain one-level nutrient solution, adjustment cell concentration is 10
6individual/mL; It is in the Citrus Fruit Wine of 6-7% that four kinds of one-level nutrient solutions are linked into alcoholic strength according to following volume percent: 4% cider vinegar bacillus ZY.C4, and 3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518,2% Shanghai makes 1.01; Then be placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity and reaches 10
6-10
8individual/mL;
3) glutinous rice saccharified liquid preparation: rice rinses 2 times, soaks 2-4h; By weight water: rice=2.5:1 defibrination, size mixing to 4:1 ratio, add CaCl
2and MgCl
2, described CaCl
2and MgCl
2add-on be every 100mL slurries and add 1g, pH to 6.2-6.4 is adjusted with acetic acid and sodium carbonate, add α-amylase, the add-on of α-amylase is that 100mL slurries add 1g, be heated to the 90-95 DEG C of about 20-30min that liquefies, be cooled to 60 DEG C, tune pH is 4.5-5.0, obtain liquid A, liquid A obtains glutinous rice saccharified liquid through saccharification;
4) preparation of citrus juice: select the citrus without rotting, peeling, puts into juice extractor by citrus pulp, and adds the NaHSO of 0.01% (weight)
3solution, citrus pulp: NaHSO
3solution=1:1(weight ratio); Squeeze the juice, in juice, add pectinase enzymatic hydrolysis, the add-on of described polygalacturonase is add polygalacturonase 0.04g in every 100mL juice, hydrolysis temperature 60 DEG C, enzymolysis time 60min, and after enzymolysis, 4 layers of filtered through gauze, obtain citrus juice;
5) fermented liquid preparation: citrus juice obtained above and glutinous rice saccharified liquid are mixed with the volume ratio of 3:1, sulfurous gas and ammonium sulfate is added in the ratio adding sulfurous gas 30mg and ammonium sulfate 50 mg in every 1L mixed solution respectively in mixed solution, mixing is adjust ph 4.48 also, regulate pol to 15-19 ° of Bx ', sterilization 15min in 85 DEG C of hot water, cool for subsequent use, obtain fermented liquid;
6) zymamsis: access mulriple yeasts kind seed liquor in the fermented liquid of step 5), inoculum size is 9.7-10% (volume percent), 28-28.5 DEG C of stirring, rotating speed is after 180r/min fermentation 1d, after airtight standing for fermentation 4-5d, sterilization, obtains Citrus Fruit Wine;
7) acetic fermentation: Citrus Fruit Wine alcoholic strength is adjusted to 6-7%, access compound acetic acid bacteria strain seed liquor, inoculum size is 15.2-16% (volume percent), is 180r/min at rotating speed, and 31 DEG C are stirred fermentation 5-7d; Stop fermentation, pass into 80-100 DEG C of steam sterilizing 30min;
8) 7) in add salt in the liquid that obtains, the add-on of salt is add salt 20g in every 1L, normal temperature ageing 2-3 month, with diatomite filtration clarification, obtains organge fruit vinegar finished product.
2. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, it is characterized in that: the method for saccharifying of described step 3) is: add saccharifying enzyme and malt meal, add-on is add 1g saccharifying enzyme and 10g malt meal in every 100mL liquid A, insulation saccharification 6-8h.
3. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, it is characterized in that: the method for saccharifying of described step 3) is: add black song: the mixed starters of red colouring agent for food, also used as a Chinese medicine=3:1, the add-on of mixed starters is add 1g in 100mL liquid A, and saccharification time is 2-4h.
4. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, is characterized in that: described step 3) also adds malt meal before enzymolysis, and add-on is add malt meal 10g in every 100mL juice.
5. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, is characterized in that: described step 6) also adds naringinase before fermentation, and the add-on of naringinase is add 0.04g in every 100mL Citrus Fruit Wine.
6. the preparation method of the organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to any one in claim 1-5.
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