CN109486731A - A kind of resistance to acetate ethanol bacterium and its application - Google Patents
A kind of resistance to acetate ethanol bacterium and its application Download PDFInfo
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- CN109486731A CN109486731A CN201910023909.1A CN201910023909A CN109486731A CN 109486731 A CN109486731 A CN 109486731A CN 201910023909 A CN201910023909 A CN 201910023909A CN 109486731 A CN109486731 A CN 109486731A
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- resistance
- fruit vinegar
- bacterium
- acetate ethanol
- acetobacter pasteurianus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/02—Acetobacter
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
- C12J1/04—Vinegar; Preparation or purification thereof from alcohol
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Abstract
The invention discloses a kind of resistance to acetate ethanol bacterium and its applications, the resistance to acetate ethanol bacterium is Acetobacter pasteurianus NF-171(Acetobacter pasteurianus), China typical culture collection center is preserved on November 26th, 2018, deposit number is CCTCC NO:M 2018824.The bacterium is to separate to obtain from Nanfeng orange many years ageing fruit vinegar magma, and ethanol tolerance ability with higher can be resistant to 12% concentration of alcohol.Tangerine orange fruit vinegar nutritional ingredient concentration using this plant of bacterium brewing is high, excellent in flavor.
Description
Technical field
The present invention relates to industrial microbial technology fields, more particularly, to a kind of resistance to acetate ethanol bacterium and its application.
Background technique
The not only rich content of organic acid in fruit vinegar, mineral matter element, amino acid and vitamin also rich in needed by human body；
Not only there is the function of traditional vinegar, also retains the original nutritive value of fruit.Fruit vinegar, which also possesses, adjusts human acid-base balance, promotees
It into digestion, whets the appetite, eliminates effect of fatigue, and Other Drinks can not be in contrast, therefore hair of the fruit vinegar in beverage industry
It is that there is very big potential in exhibition.
In the brewing process of fruit vinegar, first progress wine fermentation, further use on this basis acetic acid bacteria ferment for
Fruit vinegar, therefore strain Acetobacter xylinum is to influence one of production efficiency and the key factor of fruit vinegar quality.Currently, the acetic acid of tangerine orange fruit vinegar
Bacterium is mostly to adopt grain vinegar strain, such as 1.01 acetobacters are made in vinegar strain AS1.41 and Shanghai, and shortage is suitable for tangerine orange hair
The dedicated acetic acid bacteria strain of ferment, causes tangerine orange fruit vinegar flavor not protrude.
In addition, 8 ~ 14% currently are generally in the alcohol concentration that the wine fermentation stage obtains, and domestic manufacturer often uses AS1.41
1.01 production fruit vinegars are made with Shanghai, acetic fermentation could be continued by needing for fruit wine to be diluted 4% or less.On the one hand, it needs bigger
Fermentor ferments.On the other hand, the fruit vinegar magma nutritional ingredient concentration of acquisition of fermenting is diluted.Therefore, product quality
It cannot ensure, increased costs.
Summary of the invention
The object of the present invention is to provide a kind of resistance to acetate ethanol bacterium, are from spontaneous fermentation Nanfeng orange vinegar, Nanfeng County
It is dedicated that the fermentation of tangerine orange fruit vinegar is screened in tangerine orange orchard soil, in Nanfeng orange decayed fruit and in Nanfeng orange many years ageing fruit vinegar magma
Acetic acid bacteria strain, and it is finally obtained a kind of resistance to acetate ethanol bacterium, Acetobacter pasteurianus NF-171(Acetobacter
Pasteurianus).
In order to solve the above technical problems, the present invention adopts the following technical scheme:
A kind of resistance to acetate ethanol bacterium, the resistance to acetate ethanol bacterium are Acetobacter pasteurianus NF-171(Acetobacter
Pasteurianus), China typical culture collection center is preserved on November 26th, 2018, deposit number is CCTCC NO:M
2018824。
The present invention also provides application of the above-mentioned resistance to acetate ethanol bacterium in fruit vinegar fermentation.
Further, the fruit vinegar fermentation is that tangerine orange fruit vinegar ferments.
The present invention also provides a kind of methods of resistance to acetate ethanol bacterium brewing fruit vinegar, which comprises the steps of:
Step 1: by Acetobacter pasteurianus NF-171(Acetobacter pasteurianus), it is inoculated into inclined-plane solid culture
It is carried out activation culture 12~24 hours on base, is then linked into sterilized seed culture medium with one ring strain of oese picking
In, it is placed in that temperature is 28~35 DEG C, revolving speed is in the shaking table of 120~160r/min, continuously cultivates 1~2 day, i.e., to pair of thallus
Number growth period；
Step 2: according to 1~10%(w/v) inoculum concentration above-mentioned cultured seed is seeded to containing 4%~14% ethyl alcohol or fruit wine
Fermentation medium in, fermentation and acid to content substantially constant.
Further, in step 1 seed culture medium ingredient and proportion are as follows: alcoholic strength 2% ~ 6%, glucose 5g~20g/
L, KH2PO41.2~3.2g/L, NH4NO30.5~1.0g/L of 2.0~5.0g/L, NaCl, 0.05~0.3g/L of yeast extract,
MgSO4·7H2O 0.25~0.5g/L, CaCl20.011~0.11g/L, pure water 1000mL；Adjust 5.0~7.0,121 DEG C of pH
Sterilize 30min under high steam.
Further, in step 2 fermentation medium ingredient and proportion are as follows: alcoholic strength 4% ~ 14%, KH2PO41.2~
3.2g/L, NaCl 0.5~1.0 g/L, MgSO4·7H2O 0.25~0.5g/L, CaCl20.011~0.11g/L, pure water
1000mL；Adjust the 30min that sterilizes under 5.0~7.0,121 DEG C of high steams of pH.
Further, the condition of culture in step 2 are as follows: 28~36 DEG C of temperature, 120~200r/min of revolving speed.
Compared with prior art, advantageous effects of the invention:
Fermentor service efficiency is improved using resistance to acetate ethanol bacterium fermenting and producing tangerine orange fruit vinegar of the invention, without diluting fruit
Wine saves cost, and low power consuming, the fruit vinegar magma activity component concentration obtained that ferments is high, excellent flavor.
Detailed description of the invention
The invention will be further described for explanation with reference to the accompanying drawing.
Fig. 1 be Acetobacter pasteurianus NF-171(Acetobacter pasteurianus of the present invention) colonial morphology；
Fig. 2 is bacterium colony shape under Acetobacter pasteurianus NF-171(Acetobacter pasteurianus of the present invention) optical microscopy
State；
Fig. 3 be Acetobacter pasteurianus NF171 ferment under different alcohol concentrations production acetic acid the case where；
Fig. 4 be using Acetobacter pasteurianus NF171 with Nanfeng orange fruit wine (12% ethyl alcohol) be fermenting raw materials production tangerine orange fruit vinegar.
Specific embodiment
A kind of resistance to acetate ethanol bacterium, the acetic acid bacteria are to isolate and purify acquisition from Nanfeng orange vinegar ageing magma,
Acetobacter pasteurianus NF-171(is named as through microbial taxonomy identificationAcetobacter pasteurianus), in 2018
Be preserved in China typical culture collection center on November 26, deposit number is CCTCC NO:M 2018824, the bacterium be resistant to compared with
High ethano concentration, can achieve 12%.
Its ITS base sequence: CATGGGGGGCTGCTTACACATGCAAGTCGCACGAAGGTTTCGGCCTTAGTGG
CGGACGGGTGAGTAACGCGTAGGTATCTATCCATGGGTGGGGGATAACACTGGGAAACTGGTGCTAATACCGCATG
ACACCTGAGGGTCAAAGGCGCAAGTCGCCTGTGGAGGAGCCTGCGTTTGATTAGCTAGTTGGTGGGGTAAAGGCCT
ACCAAGGCGATGATCAATAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC
GGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGTCTT
CGGATTGTAAAGCACTTTCGACGGGGACGATGATGACGGTACCCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGC
AGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTGTAGGCGGTTTGTACAGT
CAGATGTGAAATCCCCGGGCTTAACCTGGGAGCTGCATTTGATACGTGCAGACTAGAGTGTGAGAGAGGGTTGTGG
AATTCCCAGTGTAGAGGTGAAATTCGTAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCAACCTGGCTCATTAC
TGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGC
TAGATGTTGGGTGACTTAGTCATTCAGTGTCGCAGTTAACGCGTTAAGCACACCGCCTGGGGAGTACGGCCGCAAG
GTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAA
CCTTACCAGGGCTTGAATGTAGAGGCTGCAGGCAGAGATGTCTGTTTCCCGCAAGGGACCTCTAACACAGGTGCTG
CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTAGTTGCC
ATCAGGTTGGGCTGGGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCA
TGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTAGGTGGTGACACCATGCTGA
TCTCTAAAAGCCGTCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGTGGAATCGCTAGTAATCGCGGA
TCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTGACCTTA
AGCCGGTGAGCGAACCGCAAGGACGCAGCCGACCACGTCGTTAGGGGT。
In solid medium (glucose 1%, yeast extract 1%, CaCO32%, dehydrated alcohol 6%vol, agar 2%, natural pH
Value) on colonial morphology it is rounded, surface is smooth, there is protrusion, and periphery of bacterial colonies has apparent calcium carbonate transparent circle.To strain NF-171
Gram's staining is carried out, it is negative.Under an optical microscope, no gemma is in rod-short, single or pairs of, chaining arrangement.
Embodiment 1: resistance to acetate ethanol bacterium (Acetobacter pasteurianus) NF-171 separation
(1) it enrichment culture: draws 1mL Nanfeng orange vinegar ageing magma and is added to the 250mL's containing 50mL enrichment culture liquid
In triangular flask, 3d is cultivated in 30 DEG C, the shaking table of 150r/min, the triangular flask that choosing has acetic acid to be carries out the strain point of next step
From.
(2) strain separating purifies: taking 1mL enrichment culture bacterium solution, is diluted to 10-6~10-8Concentration is coated on primary dcreening operation culture dish
In, place 30 DEG C of culture 3d in the incubator.The situation of observation culture daily, records transparent circle situation.Select transparent circle big
Bacterial strain.
(3) bacterial strain secondary screening: the bacterial strain obtained to primary dcreening operation carries out shaking flask Liquid Culture and (adds 10% anhydrous second in culture medium
Alcohol) secondary screening, 30 DEG C, 150r/min cultivates 3d.Bacterium solution is centrifuged, 10mL is taken to neutralize using NaOH, then 5% FeCl is added dropwise3, boil
5min is boiled, if red brown flocculent deposit occur tentatively is judged as acetic acid producing strains.By primary dcreening operation, secondary screening obtains a kind of energy production
Bacterial strain NF-171(such as Fig. 1, Fig. 2 of acetic acid).
The identification of 2 acetic acid bacteria of embodiment
(1) morphology is carried out to bacterial strain NF-171, carries out the physiological and biochemical test of identification acetic acid bacteria referring to Bai Jieshi Bacteria Identification
Handbook (the 8th edition).(table 1)
(2) 16Sr DNA is identified: (supplement DNA is extracted, PCR operation data)
Embodiment 3: resistance to ethyl alcohol experiment
The high acid acetic acid bacteria Acetobacter pasteurianus NF171 and Acetobacter pasteurianus that will be obtained by producing acid cut amount experiment screening
NF172 is in 32 DEG C, after 150r/min shaken cultivation (22 ± 2) h activation, be inoculated in respectively ethanol content be respectively 4%, 6%, 8%,
10%, 12%(v/v) production acid culture medium in, 32 DEG C, 150r/min shaken cultivation 72h, measurement produce acid amount.Under the same terms, survey
The production acid amount for determining bacterial strain is as shown in Figure 3.
Embodiment 4: Nanfeng orange vinegar fermentation process
Embodiment described above is only that preferred embodiment of the invention is described, and is not limited the scope of the present invention
Fixed, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements should all be fallen into the protection scope that claims of the present invention determines.
Claims (7)
1. a kind of resistance to acetate ethanol bacterium, which is characterized in that the resistance to acetate ethanol bacterium is Acetobacter pasteurianus NF-171
(Acetobacter pasteurianus) is preserved in China typical culture collection center, preservation on November 26th, 2018
Number be CCTCC NO:M 2018824.
2. application of the resistance to acetate ethanol bacterium according to claim 1 in fruit vinegar fermentation.
3. application according to claim 2, which is characterized in that the fruit vinegar fermentation is that tangerine orange fruit vinegar ferments.
4. the method for resistance to acetate ethanol bacterium brewing fruit vinegar according to claim 1, which comprises the steps of:
Step 1: by Acetobacter pasteurianus NF-171(Acetobacter pasteurianus), it is inoculated into inclined-plane solid culture
It is carried out activation culture 12~24 hours on base, is then linked into sterilized seed culture medium with one ring strain of oese picking
In, being placed in temperature is that 28~35 DEG C of revolving speeds are in the shaking table of 120~160r/min, is continuously cultivated 1~2 day, i.e., to pair of thallus
Number growth period；
Step 2: according to 1~10%(w/v) inoculum concentration above-mentioned cultured seed is seeded to containing 4%~14% ethyl alcohol or fruit wine
Fermentation medium in, fermentation and acid to content substantially constant.
5. the method for resistance to acetate ethanol bacterium brewing fruit vinegar according to claim 4, which is characterized in that seed is trained in step 1
Support the ingredient and proportion of base are as follows: alcoholic strength 2% ~ 6%, glucose 5g~20g/L, KH2PO41.2~3.2g/L, NH4NO32.0~
0.5~1.0g/L of 5.0g/L, NaCl, yeast extract 0.05~0.3g/L, MgSO4·7H2O 0.25~0.5g/L, CaCl2
0.011~0.11g/L, pure water 1000mL；Adjust the 30min that sterilizes under 5.0~7.0,121 DEG C of high steams of pH.
6. the method for resistance to acetate ethanol bacterium brewing fruit vinegar according to claim 4, which is characterized in that training of fermenting in step 2
Support the ingredient and proportion of base are as follows: alcoholic strength 4% ~ 14%, KH2PO41.2~3.2g/L, NaCl 0.5~1.0 g/L, MgSO4·
7H2O 0.25~0.5g/L, CaCl20.011~0.11g/L, pure water 1000mL；5.0~7.0,121 DEG C of high pressures of pH are adjusted to steam
Sterilize 30min under vapour.
7. the method for resistance to acetate ethanol bacterium brewing fruit vinegar according to claim 4, which is characterized in that cultivated in step 2
Condition are as follows: 28~36 DEG C of temperature, 120~200r/min of revolving speed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748491A (en) * | 2020-06-15 | 2020-10-09 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN113969245A (en) * | 2021-10-25 | 2022-01-25 | 爱康食品(青岛)有限公司 | Sushi vinegar acetic acid bacteria, method for fermenting sushi vinegar and application of sushi vinegar to sushi |
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| CN102433268A (en) * | 2011-08-08 | 2012-05-02 | 丽水市鱼跃酿造食品有限公司 | Acetobacter pasteurium and its application method in producing citrus vinegar |
| CN104611269A (en) * | 2015-02-06 | 2015-05-13 | 丽水学院 | Acetobacter malorum and application thereof |
| CN106167780A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | A kind of ethanol heat-resisting, resistance to, the acetic acid bacteria of resistance to acetic acid |
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes |
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2019
- 2019-01-10 CN CN201910023909.1A patent/CN109486731A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433268A (en) * | 2011-08-08 | 2012-05-02 | 丽水市鱼跃酿造食品有限公司 | Acetobacter pasteurium and its application method in producing citrus vinegar |
| CN104611269A (en) * | 2015-02-06 | 2015-05-13 | 丽水学院 | Acetobacter malorum and application thereof |
| CN106167780A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | A kind of ethanol heat-resisting, resistance to, the acetic acid bacteria of resistance to acetic acid |
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748491A (en) * | 2020-06-15 | 2020-10-09 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN111748491B (en) * | 2020-06-15 | 2022-12-16 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN113969245A (en) * | 2021-10-25 | 2022-01-25 | 爱康食品(青岛)有限公司 | Sushi vinegar acetic acid bacteria, method for fermenting sushi vinegar and application of sushi vinegar to sushi |
| CN113969245B (en) * | 2021-10-25 | 2022-07-29 | 爱康食品(青岛)有限公司 | Sushi vinegar acetic acid bacteria, method for fermenting sushi vinegar and application of sushi vinegar to sushi |
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