CN105018384B - One plant of acetic acid bacteria and its application in the apple vinegar that ferments - Google Patents
One plant of acetic acid bacteria and its application in the apple vinegar that ferments Download PDFInfo
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Abstract
Application the invention discloses one plant of acetic acid bacteria and its in the apple vinegar that ferments, belongs to industrial microorganism field.The acetic acid bacteria is one plant of Pasteur's acetobacter (Acetobacter pasteurianus) CYD159, and China typical culture collection center is preserved on June 30th, 2015, and preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2015418.The bacterium has higher acid producing ability, and acid producing ability reaches more than 110g/L, high using the apple vinegar acetate yield that ferments made from this plant of acetic acid bacteria, excellent flavor, is the acetic acid bacteria of one plant of suitable brewing apple vinegar.
Description
Technical field
Application the present invention relates to one plant of acetic acid bacteria and its in the apple vinegar that ferments, belongs to industrial microorganism field.
Background technology
Apple is one of " four big fruit of the world ", and apple tree is also that China's cultivated area is most wide, the most fallen leaves fruit of yield
Tree, apple are rich in minerals and vitamins, also containing a large amount of pectin, carbohydrate, fat, malic acid, citric acid, tannic acid and thin fibre
The indispensable substances of healths such as dimension have high nutritive value, are a kind of fruit of people's most often feeding.
Apple is largely processed to cider, applejack, apple vinegar, preserved apple, apple other than fresh food at present
Sauce, apple crisp slices etc., but apple vinegar type is few, poor quality.Commercially available apple beverage is mostly using allocating technology, product orientation category
In the inexpensive product scope that disappears soon, seldom focus on nutritional ingredient protection and the flavor characteristic of product.Existing apple vinegar generally mostly with
It is used instead of vinegar for cold and dressed with sauce seasoning, in mouthfeel based on bitterness, fermentation is highly seasoned, and irritation is strong, it is difficult to obtain consumers.
Minority fermentation apple vinegar is using natural apple juice fermentation, but the loss of its nutriment is larger, and yield is relatively low, and taste flavor is poor.
Fruit vinegar is using modern fermentation technique, using fruit or inspissated juice as primary raw material, by alcoholic fermentation and vinegar
Full of nutrition, the flavorful tart flavour drink of one kind that acid fermentation forms.The micro member such as potassium element, Zn-ef ficiency in general grain vinegar
It is plain insufficient, and apple vinegar can not only supplement these trace elements, but also containing compared with objects such as Multivitamin, polysaccharide, amino acid
Matter, nutrition and taste are better than making vinegar.In fruit vinegar production, strain plays a crucial role, and acetic acid bacteria is to determine
Determine acetate yield and the main bacterial strain of quality.Up to now 1.01 strains production liquid grain is often made with AS1.41 and Shanghai by domestic manufacturer
Vinegar, but with the increase of acetic acid concentration in zymotic fluid, Product inhibiton effect aggravation, the acid-producing activity of acetic acid bacteria reduces, this
Cost is virtually increased, while acetate yield and quality cannot also ensure.Therefore select that acid resistance is more excellent and flavor
The special acetic acid bacteria of excellent fruit vinegar is particularly significant for brewing high quality apple vinegar.
Invention content
The object of the present invention is to provide the characteristic acetic acid bacterial strains that one plant is suitable for fermentation apple vinegar, are one plant of Pasteur's acetobacters
(Acetobacter pasteurianus) CYD159 was preserved in China typical culture collection center on June 30th, 2015,
Preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2015418.
The present invention also provides the method that application Pasteur's acetobacter brews apple vinegar, by Pasteur's acetobacter activation culture
Afterwards, seed liquor is accessed in Acetic acid bacteria fermentation culture medium by 10% inoculum concentration, ventilation quantity 1vvm, 30 DEG C of temperature, speed of agitator
For 200-800rpm, dissolved oxygen 20%, rotating speed and dissolved oxygen coupling, stopping stream adding after per 1L, culture base flow adds 250g applejacks,
Ferment after 12h, stream plus applejack, and wine degree is controlled in culture medium to be no more than 3 ° (v/v) again, treat acidity do not continue to rise and
Stop fermentation when residual alcohol degree is 0.5 ° (v/v).
In one embodiment of the invention, the Acetic acid bacteria fermentation culture medium contains：Yeast extract 30g/L, Portugal
Grape sugar 20g/L, KH2PO41g/L, MgSO41g/L。
In one embodiment of the invention, the applejack is commercially available applejack.
In one embodiment of the invention, the applejack is that concentrated apple juice is diluted to initial sugar with drinking water
Spend 250g/L, deployed to pour into sterilized fermentation tank, liquid amount 70%；By 2 ‰ inoculum concentration inoculating active dry ferments, in 25
It ferments at DEG C, until residual sugar amount stops fermentation after no longer changing.Residual yeast in wine liquid is removed in time after alcoholic fermentation
It goes, obtains applejack, stored at 4 DEG C spare.
Pasteur's acetobacter production acetic acid ability of the present invention is strong, and ethyl alcohol wear rate is up to 0.8g/Lh, fermentation gained apple
In vinegar, total acid (with Acetometer) is up to 120.12g/L, and acetic acid content is up to 114g/L, far above 50-70g/L in existing report
Acetate concentration.And in the volatile flavor substance in gained apple vinegar, acids and ester type compound relative amount are higher, are formed
Preferable flavor and taste.
Biomaterial preservation
One plant of Pasteur's acetobacter (Acetobacter pasteurianus) CYD159, was preserved on June 30th, 2015
China typical culture collection center, preservation address are Wuhan, China Wuhan University, and deposit number is CCTCC NO:M
2015418。
Description of the drawings
Fig. 1 Pasteur acetobacter CCTCC NO:2015418 seed growth curves of M
Concentration of alcohol change curve in Fig. 2 zymotic fluids
Acetic acid concentration change curve in Fig. 3 zymotic fluids
Specific embodiment
The formula of each culture medium is as follows in following embodiments：
Seed culture medium：Yeast extract 20g/L, peptone 20g/L, glucose 20g/L, natural ph, 115 DEG C,
20min high pressure sterilizations postcooling often adds in 3g absolute ethyl alcohols to room temperature in 100mL culture mediums.
Acetic acid bacteria fermentation culture medium：Yeast extract 30g/L, glucose 20g/L, KH2PO41g/L, MgSO41g/L.It is put in
In 7.5L U.S. NBS110 fermentation tanks, starting liquid amount is 1L, 115 DEG C of 15min that sterilize together with fermentation tank, and cooling is spare.
Experiment is purchased from Shaanxi Haisheng Fresh Fruit Juice Co., Ltd. with concentrated apple juice.
The efficient liquid phase chromatographic analysis of acetic acid and ethyl alcohol in apple vinegar zymotic fluid：(1) the 1mL bacterium solutions of sampling are put into
Centrifugation removal thalline, centrifugal condition 12000rpm, 1min in 1.5mL centrifuge tubes.(2) supernatant 0.75mL is taken to move into EP pipes,
The TCA solution of 0.75mL is added in, vibrates mixing, 4 DEG C of refrigerators is put into and stands 5h.(3) it is centrifuged after standing, centrifugal condition
12000rpm, 20min take supernatant 1mL to cross spare in 0.22 μm of water system film immigration liquid-phase inlet bottle.(4) using Shodex
SC1011 refractive index detection column (Showa electrician Co., Ltd, Tokyo, Japan) analysis measures ethyl alcohol and acetic acid content.At 50 DEG C,
Mobile phase is dilute H of 0.01mol/L2SO4Solution, with the flow velocity sample introduction of 1mL/min under the conditions of 50 DEG C.
Wine degree assay method refer to GB/T15038-2006, acidity assaying method refer to GB/T15038-2006, total reducing sugar with
Determination of Reducing Sugars refers to GB/T15038-2006.
1 acetic acid bacteria of embodiment isolates and purifies
1. the selection and breeding of starting strain
(1) spontaneous fermentation separation acetic acid bacteria
The sterile soil for weighing Mostviertel areas of the 50g from Alps northern foot, is ground with sterile mortar
After be put into 500mL conical flasks, add in 150mL sterile waters, gauze sealing, be placed in constant-temperature table and cultivated, condition of culture 30
℃、150rpm.Every taking zymotic fluid lmL for 24 hours, add in 9mL sterile waters and carry out gradient dilution to 10-7, take bacterium after 50 μ l dilutions
Liquid is put into the coating of YPD solid mediums, is put into 30 DEG C of incubators and cultivates 48h.
(2) purifying culture
To having cultivated the acetic acid bacteria strain of 48h, the macroscopic bacterium colony grown in picking culture medium, difference in incubator
It moves on YPD solid mediums, after 30 DEG C of constant temperature incubations, purifying are until obtain single bacterium colony, are forwarded to slant tube conduct and set out
Bacterial strain obtains 58 plants of bacterium.
2. acetic acid bacterial strain primary dcreening operation
Picking single bacterium is fallen in 100mLYPD fluid nutrient mediums, constant-temperature table culture, 30 DEG C of condition of culture, 200r/min.
The bacterium solution of activation is accessed in seed culture medium, inoculum concentration 10%, 30 DEG C, carry out acetic acid hair under the conditions of the shaking table of 200r/min
Ferment, sampling progress qualitative determination after fermentation 72 hours.
It takes bacterium solution 6000r/min, centrifuge 20min at 4 DEG C, take supernatant for use.5mL is taken to remove the supernatant of thalline, with
1mol/L sodium hydroxides are neutralized to pH7.0, and 5% FeCl is added in after boiling3Solution 5-7 drops, forming reddish-brown precipitation, person is production
Acetic acid bacteria obtains 5 plants of bacterium CYD13, CYD36, CYD79, CYD127, CYD159.
3. acetic acid bacteria preservation
The a large amount of bacteria suspension 1mL in bottom in above-mentioned 5 plants of bacterium biological seed culture solution is taken in 2mL glycerol stocks pipes, to add in
The glycerine of 1mL30% is positioned over preservation in -80 DEG C of ultra low temperature freezers.
2 acetic acid bacteria seed growth curve of embodiment
1. actication of culture：It is drawn with 1 ring of thalline in oese picking glycerol stocks pipe in YPD solid mediums upper flat plate
Line.Liquid Culture：Single bacterium is fallen in seed culture medium on picking tablet, constant-temperature table culture for 24 hours, 30 DEG C of condition of culture,
150rpm。
2. in acetic fermentation inoculation of medium seed culture fluid, inoculum concentration 10% is sealed with gauze, is placed in constant-temperature table
It is cultivated, condition of culture：30 DEG C, 150rpm is sealed with gauze.
3. 0.5mL bacterium solutions are taken respectively in 0h, 7h, 9.5h, 12.5h, 15.5h, 20.5h, 23.5h, 34.5h, 39.5h,
Suitable multiple is diluted, absorbance is measured under the conditions of λ=600.
Experimental result is as shown in Figure 1, it can be seen from the figure that the cell concentration of acetic acid bacteria has been up to OD600=6,
Middle CYD159 has higher cell concentration, and CYD36 takes second place, and the growth ability of acetic acid bacteria CYD13, CYD79, CYD127 are weaker.
3 Brewing Process of Apple Vinegar of embodiment
Concentrated apple juice is diluted to initial pol 250g/L with pure water, it is deployed to pour into sterilized fermentation tank,
Liquid amount 70%.It by 2 ‰ inoculating active dry ferments of fermentation medium, ferments at 25 DEG C, until residual sugar amount no longer changes
Stop fermentation afterwards.Residual yeast in wine liquid is removed in time after alcoholic fermentation, obtains applejack, is stored at 4 DEG C spare.
A ring acetic acid bacteria is taken to be inoculated into YPD solid mediums with oese glycerol stocks strain, 30 DEG C of constant temperature incubations
24h.The preferable single bacterium colony of growing way is seeded in seed culture medium in picking tablet, shaking table constant temperature incubation for 24 hours, condition of culture：30
DEG C, 150rpm.Seed liquor is accessed in Acetic acid bacteria fermentation culture medium by 10% inoculum concentration, per 1L, culture base flow adds 250g applejacks
Stop flowing afterwards adding, after the 12h that ferments, stream plus applejack control 1-3 ° of wine degree (v/v) in fermentation tank again.Fermentating controling condition：pH
Natural trend, ventilation quantity 1vvm, dissolved oxygen 20%, 30 DEG C, speed of agitator 200-800rpm of temperature, rotating speed are even with dissolved oxygen
Connection.Treat that residual alcohol degree for 0.5 ° (v/v), stops fermentation when acidity does not continue to rise.
4 acetic acid bacteria of embodiment fermentation apple vinegar during ethyl alcohol, acetic acid content measure
1mL bacterium are taken respectively in acetic fermentation 0h, 7h, 9.5h, 12.5h, 15.5h, 20.5h, 23.5h, 34.5h, 39.5h
Liquid carries out efficient liquid phase chromatographic analysis to apple vinegar zymotic fluid
1. ethyl alcohol variation tendency is as shown in Figure 2
The ethyl alcohol consumption abilities of five plants of acetic acid bacterias as shown in Fig. 2, the wear rate of ethyl alcohol is consistent with the growth rate of thalline,
Bacterial strain CYD159 have higher ethyl alcohol wear rate, ferment 73h when concentration of alcohol fallen below from initial 61.1g/L
1.825g/L, ethyl alcohol wear rate are up to 0.812g/Lh, and CYD36 takes second place, the ethyl alcohol of acetic acid bacteria CYD13, CYD79, CYD127
Wear rate is most slow, has just consumed the ethyl alcohol in zymotic fluid after the 121h that ferments.
2. acetic acid variation tendency is as shown in Figure 3
The sour higher and rate of producing acid of acetic acid bacteria CYD159 productions is very fast as can be seen from Figure 3, ferment 73h when acetic acid concentration
Reached 81.935g/L, ferment 121h when acetic acid product reached maximum concentration 100g/L, and acetic acid bacteria CYD36, CYD13,
CYD79, CYD127 most high acid amount only have 50g/L or so.Therefore acetic acid bacteria CYD159 is one plant and is suitble to make the high-quality of fruit vinegar
Acetic acid bacteria chooses this plant of acetic acid bacteria and carries out follow-up test.During bacterial strain screening it has also been found that after ethyl alcohol has consumed thalline acetic acid
Concentration can be reduced slightly, it is therefore desirable to ethyl alcohol and acetic acid concentration in the real time measure zymotic fluid, when acetic acid concentration reaches highest
Stop fermentation immediately.
Each index variation in 5 acetic acid bacteria fermentation process of embodiment
Each index variation is as shown in table 1 in apple vinegar brewing process, passes through the method for stream plus applejack so that in fermentation tank
Wine degree is no more than 3 ° (v/v).Cell concentration rises smaller during the fermentation, but acetic acid concentration persistent accumulation, fermentation process
In total acid concentration be up to 120.36g/L.Highest acetic acid is dense during the fermentation for most of fruit vinegars in domestic existing report
Degree only has 50-70g/L, and the acetic acid concentration in apple vinegar can be significantly improved with acetic acid bacteria CYD159 brewing apple vinegars, right
Fruit vinegar production plays an important roll.
Each index variation in 1 apple vinegar fermentation process of table
The measure of flavor substance and organic acid in 6 apple vinegar of embodiment
Volatile flavor substance and organic acid in the apple vinegar of acetic acid bacteria CYD159 brewings are measured using GC-MS and HPLC
Content obtains result such as table 2 and table 3.The main volatile flavor substance of apple vinegar have acid, alcohol, aldoketones, esters, hydro carbons and
Other a small amount of compounds.Wherein acids and ester type compound relative amount be higher, they have cooperatively formed apple vinegar
Flavour.The higher compound of component content has 2- hydroxy-propionic acids methyl esters (16.077%), acetic acid (12.405%), benzene first
Sour (9.461%).
Volatile flavor substance content in 2 apple vinegar of table
| Compound name | Content/% |
| 3,3- dimethoxy-2-butanones | 0.216 |
| 2- oxo-propionic acid methyl esters | 0.513 |
| Methyl chloroacetate | 0.08 |
| 3- hydroxy-2-butanones | 0.745 |
| 2- hydroxy-propionic acid methyl esters | 16.077 |
| Methyl dichloroacetate | 0.143 |
| Hydroxy methyl acetate | 0.07 |
| Trimethoxy-ethane | 1.222 |
| Dimethyl oxalate | 0.312 |
| Ethyl acetate | 2.12 |
| Furfural | 0.207 |
| Acetic acid | 12.405 |
| 2- hydroxy-3-methyls-methyl valerate | 0.151 |
| Dimethyl malenate | 0.539 |
| 2- hydroxy-4-methyls-methyl valerate | 0.649 |
| Dimethyl succinate | 5.986 |
| Methyl benzoate | 6.81 |
| Dimethyl glutarate | 0.12 |
| Malonic methyl ester nitrile | 0.896 |
| Methyl phenylacetate | 0.869 |
| 2- methyl -2- apple dimethyl phthalates | 0.584 |
| Apple dimethyl phthalate | 4.022 |
| Trimethyl -1,2,3- tricarballylic acid esters | 0.557 |
| Alpha-hydroxy-methyl phenylpropionate | 2.712 |
| One methyl esters of succinic acid | 0.73 |
| Trimethyl citrate | 0.218 |
| Benzoic acid | 9.461 |
| N- acetyl group-dl- aspartic acid dimethyl esters | 0.153 |
| N- acetyl group-Pidolidone dimethyl ester | 0.266 |
| N- acetyl group-L-phenylalanine methyl esters | 0.266 |
| Palmitic acid | 0.08 |
Kinds of organic acids and content are as shown in table 3 in apple vinegar, and sample mesotartaric acid, lactic acid, succinic acid content are less, second
Acid content is up to 114.443g/L, is most important organic acid in apple vinegar.The type of organic acid determines acid with content composition
Tart flavour quality, as shown in table 3, relatively abundant and organic acid the relative amount of the type of organic acid highlights apple vinegar acetic acid hair
The characteristics of ferment.Organic acid in fruit vinegar can effectively maintain the acid-base balance in human body, and nutriment in food is promoted to be absorbed,
Stomach strengthening and digestion promoting has good health-care efficacy.
Kinds of organic acids and content in 3 apple vinegar of table
| Type | Oxalic acid | Tartaric acid | Malic acid | Lactic acid | Acetic acid | Citric acid | Succinic acid |
| Content (g/L) | 0.556 | 0.485 | 3.536 | 0.305 | 114.443 | 1.195 | 0.263 |
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Claims (7)
1. one plant of acetic acid bacteria, which is characterized in that the acetic acid bacteria is one plant of Pasteur's acetobacter
(Acetobacterpasteurianus) CYD159 was preserved in China typical culture collection center on June 30th, 2015,
Preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M 2015418.
2. the method for application one plant of acetic acid bacteria brewing apple vinegar described in claim 1.
3. according to the method described in claim 2, it is characterized in that, the apple vinegar is fermented type apple vinegar.
4. according to the method described in claim 2, it is characterized in that, by after Pasteur's acetobacter activation culture, by 10% inoculum concentration
Seed liquor is accessed in Acetic acid bacteria fermentation culture medium, ventilation quantity 1vvm, 30 DEG C of temperature, speed of agitator 200-800rpm is molten
Solution oxygen 20%, rotating speed and dissolved oxygen coupling, stopping stream adding after per 1L, culture base flow adds 250g applejacks, ferments after 12h, flows again
Add applejack so that wine degree is no more than 3 ° in fermentation tank, stops when acidity does not continue to rise and residual alcohol degree is 0.5 °
Fermentation.
5. according to the method described in claim 4, it is characterized in that, the Acetic acid bacteria fermentation culture medium contains：Yeast extract
30g/L, glucose 20g/L, KH2PO41g/L, MgSO4 1g/L。
6. according to the method described in claim 4, it is characterized in that, the applejack is commercially available applejack.
7. according to the method described in claim 4, it is characterized in that, the preparation of the applejack is by concentrated apple with drinking water
Juice is diluted to initial pol 250g/L, deployed to pour into sterilized fermentation tank, liquid amount 70%, by fermentation medium
2 ‰ inoculating active dry ferments, ferment at 25 DEG C, until residual sugar amount stops fermentation after no longer changing；After alcoholic fermentation
Residual yeast in wine liquid is removed in time, obtains applejack.
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| CN106011023B (en) * | 2016-07-11 | 2019-04-05 | 山西省农业科学院农作物品种资源研究所 | One plant of acetic acid bacteria for being isolated from traditional fermented food acid congee and its application |
| MX2020008397A (en) | 2018-02-09 | 2022-05-26 | Haldor Topsoe As | Process of producing alpha-hydroxy compounds and uses thereof. |
| CN109234207B (en) * | 2018-11-12 | 2022-04-01 | 江苏恒顺醋业股份有限公司 | Acetobacter strain and application thereof |
| CN109735464B (en) * | 2019-01-25 | 2020-09-04 | 华中农业大学 | Acetobacter pasteurianus for converting ethanol into acetic acid and application of acetobacter pasteurianus in fruit vinegar brewing |
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| CN110923270A (en) * | 2019-12-06 | 2020-03-27 | 鹤山市东古调味食品有限公司 | Method for producing acetic acid by utilizing chemical ethanol fermentation |
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