CN106167781A - Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes - Google Patents

Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes Download PDF

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CN106167781A
CN106167781A CN201610723314.3A CN201610723314A CN106167781A CN 106167781 A CN106167781 A CN 106167781A CN 201610723314 A CN201610723314 A CN 201610723314A CN 106167781 A CN106167781 A CN 106167781A
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pasteurianus
acetic acid
ethanol
vinegar
acetobacter
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CN106167781B (en
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胡勇
徐宁
李冬生
周梦舟
祁勇刚
高冰
汪超
石勇
徐梅
朱于鹏
龚元元
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Hubei University of Technology
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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
    • 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
    • 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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  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)

Abstract

The invention discloses the Acetobacter pasteurianus that a kind of temperature capacity is excellentAcetobacer pasteurianusDY4 and purposes, culture presevation place is China typical culture collection center (Wuhan, China Wuhan University), and preserving number is: CCTCC NO:M 2015462.Acetobacter pasteurianus of the present invention, under the conditions of 4% ethanol, can have more stable growth at 30 40 DEG C and produce acid amount (alcohol, acetic acid conversion ratio is equal > 90.6%), and 37 DEG C reach maximum conversion rate (97.5%), the OD when 43 DEG C of high temperature600Up to 0.72, producing acid is 40.0% up to 16.5g/L(maximum conversion rate)；37 DEG C, under the conditions of 8% ethanol, ethanol conversion reaches 95.6%.Therefore, strain of the present invention is suitable to brewing fruit vinegar in summer, and vinegar product special flavour is superior.

Description

Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes
Technical field
The invention belongs to microorganism and fermentation industry field, be specifically related to Pasteur's acetic acid bar that a kind of temperature capacity is excellent BacteriumAcetobacer pasteurianusDY4 and purposes.
Background technology
Acetic acid bacteria (AAB) is obligate aerobic gram negative bacteria, has the ability of oxidation of ethanol acetic acid Deppenmeier et al. 2010).AAB generally separates (Wu et al. 2010) from natural habitat and raw vinegar.? In sweat, acetic acid bacteria can by various stress, such as high temperature.The optimum growth temperature of AAB is 25-
30 DEG C, although acetic acid bacteria also can be at 37-40 DEG C of growth (Gullo et al.2008), but poor growth.Acetic acid bacteria ferments During accumulation fermentation fever necessarily make fermentation temperature be higher than 37 DEG C, cause the low (Adachi of cell poor growth, acetate yield Et al.2003), this situation can become more serious in summer.
Fruit vinegar receives the welcome in market in recent years, such as apple vinegar, grape vinegar, Fructus Myricae rubrae vinegar, Fructus Kaki vinegar, pears vinegar etc..Summer, temperature Spending higher, conventional acetic acid bacteria AS1.41, Shanghai wine 1.01 etc. is difficult to tolerate high temperature, and acetate yield is the highest, makes above fruit vinegar needs Higher cooling cost, fruit price is higher the most in addition so that final finished fruit vinegar is too expensive.For improving under hot conditions Acetic fermentation level, the improvement of the heat-resisting function of acetic acid bacteria has become study hotspot, as Application No. CN201310549668, The Chinese patent of " high temperature resistant acetic acid bacteria and the application of fermenting and producing acetic acid thereof ", the acetic acid bacteria in this patent can be 45 DEG C of growths (high temperature resistant)), the Ethanol-Acetic Acid conversion ratio at 37 DEG C and 7% ethanol condition is 88.8%, at 37 DEG C and applejack (7% ethanol) bar The Ethanol-Acetic Acid conversion ratio of part is 93%, there is presently no the acetic acid bacteria strain that report resistance to elevated temperatures is more superior.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the Acetobacter pasteurianus that a kind of temperature capacity is excellentAcetobacer pasteurianusDY4, and utilize this bacterium to carry out Citrullus vulgaris vinegar brew in summer, this strain can effectively reduce cold But cost, and the Citrullus vulgaris vinegar excellent in flavor obtained.
In order to realize the purpose of the present invention, the present inventor, by lot of experiments research and unremitting exploration, is finally obtained Following technical scheme:
The present invention separates a strain acetic acid bacteria DY4 from raw vinegar, through taxonomic identification, is defined as Acetobacter pasteurianusAcetobacer pasteurianusDY4, culture presevation place is China typical culture collection center (Wuhan, China Wuhan University), preservation Number it is: CCTCC NO:M 2015462, preservation date is on July 23rd, 2015.
The present invention coupleAcetobacer pasteurianusDY4 has done morphology, physiological and biochemical property, homology and has sent out Ferment feature is studied, and concrete outcome is as follows:
Morphological feature: colonial morphology is neat in edge, smooth surface, milky, protruding circular colonies (Fig. 1)；The individual bodily form State is elongated rod shape, in list, to or catenation (Fig. 2).
Physiological and biochemical property: bacterial strain DY4, under the conditions of 1% acetic acid, can form the peroxidating (table 1) of Dichlorodiphenyl Acetate.
Homology features: with round pcr, the 16SrDNA sequence of amplification 16SrDNA, DY4 and Pasteur's acetic acid in Genbank Bacillus MSU10 similarity is the highest, reaches 99.98%.Comprehensive morphological, Physiology and biochemistry, homology, classify as bacterial strainAcetobacer pasteurianus(Fig. 3)。
Fermenting characteristic:Acetobacer pasteurianusDY4, under the conditions of 4% ethanol, can have relatively at 30-40 DEG C Stable growth and produce acid amount (Ethanol-Acetic Acid conversion ratio is equal > 90.6%), the suitableeest fermentation temperature is 37 DEG C, and 37 DEG C reach maximum turning Rate (97.5%, i.e. 40.2g/L acetic acid)；The OD when 43 DEG C of high temperature600Up to 0.72, produce acid up to 16.5g/L(maximum conversion rate It is 40.0%) (Fig. 4,5).
Compared with prior art, advantages of the present invention is: the Acetobacter pasteurianus of the present inventionAcetobacer pasteurianusDY4 is wide to the tolerance range of temperature, is suitable under ambient temperature higher strip part (such as summer) and makes vinegar Acid, has the biggest application prospect in summer in terms of brewing fruit vinegar.Compared with conventional AS1.41, cooling power consumption reduces；Make with DY4 Citrullus vulgaris vinegar processed, it is thus achieved that excellent Citrullus vulgaris vinegar product, its color and luster is orange limpid, and its fruital and tart flavour are denseer, and its mouthfeel is felt well sweet easypro Suitable, have a well filled-out figure.
Accompanying drawing explanation
Fig. 1 isAcetobacer pasteurianusDY4 is high temperature resistant plating medium transparent circle (43 DEG C) and bacterium colony Morphological characteristic figure.
Fig. 2 isAcetobacer pasteurianusDY4 is strain aspect graph under high power lens (40 x 10).
Fig. 3 isAcetobacer pasteurianusDY4 cladogram.
Fig. 4 is under different temperaturesAcetobacer pasteurianusThe growing state of DY4.
Fig. 5 is under different temperaturesAcetobacer pasteurianusThe product acid amount of DY4.
Detailed description of the invention
The initial gross separation purification of embodiment 1 is high temperature resistant high alcohol acetic acid bacteria
For the technical characterstic of the present invention program can be clearly described, below in conjunction with specific embodiment, the present invention is illustrated.But Protection scope of the present invention is not limited to these embodiments.Every change without departing substantially from present inventive concept or equivalent substitute and all include Within protection scope of the present invention.
The selection of acetic acid bacteria separation source:
(1) raw vinegar is from Danyang vinegar factory；
(2) the initial gross separation purification of high temperature resistant high alcohol acetic acid bacteria
1. the enrichment culture of acetic acid bacteria:
Vinegar factory (multiple) the raw vinegar 1g of conventional solid-state fermentation technology, adds in 100mL water, cultivates 5-8 days at 30 DEG C, obtain nature Fermentation liquid 5mL, loads in the triangular flask containing 100mL enrichment medium, and 30 DEG C, 180r/min cultivates 2 days, i.e. can get acetic acid Bacterium enrichment culture liquid.
Enrichment medium forms: 2% glucose, 2% yeast powder, 0.01% MgSO4, remainder is distilled water；
2. the qualitative experiment of acetic acid bacteria:
Take the centrifugation 3min with 10000r/min of the enrichment culture liquid 20mL containing acetic acid bacteria, be centrifuged off acetic acid bacteria fermentation Thalline in liquid, takes supernatant 5mL and is neutralized to PH 7.0 with 2.5mol/L NaOH solution, boil, and adds mass concentration 5% Ferric chloride solution 4-6 drips, and as occurred, reddish-brown precipitation then strain creates acetic acid really；
The most high temperature resistant acetic acid bacteria screens:
Take the enrichment culture liquid 1mL containing acetic acid bacteria, be diluted to 10 with normal saline successively-6, take 200 μ L 10 respectively-5With 10-6 Diluent coat on calcium carbonate flat board, cultivate under the conditions of 43 DEG C and produce transparent circle to bacterium colony, select transparent circle maximum Single bacterium colony, named DY4(Fig. 1).
4. the microscopic morphology of acetic acid bacteria observes (Fig. 2).
The qualification of embodiment 2 acetic acid bacteria DY4
(1) basis " common bacteria system identification handbook " and " primary Jie Shi Bacteria Identification handbook " the 8th edition carries out form to bacterial strain Learning, Physiology and biochemistry has carried out identifying (table 1).
The physiological and biochemical property of table 1 DY4 Yu AS1.41
(2) acetic acid bacteria 16Sr DNA identifies
Picking acetic acid bacteria is inoculated in 10mL/100mL triangular flask fluid medium, 30 DEG C, 180r/min constant-temperature shaking culture Shaken cultivation 24h in case.Taking 2mL bacterium solution in centrifuge tube, 12000r/min is centrifuged 2min, removes supernatant, raw according to Shanghai The SK1201-Μ NIQ-10 pillar bacterial genomes DNA extraction agent box of work biotechnology Services Co., Ltd is said Bright book extracts the genomic DNA of bacterial strain, as template.Select universal primer to carry out 16S rDNA amplification to test:
Primer 1 sequence 7f(5'-CAGAGTTTGATCCTGGCT-3'),
Primer 2 sequence 1540r(5'-AGGAGGTGATCCAGCCGCA-3').
PCR amplification system: Template genome 1 μ L, Primer μ p (10 μMs) 0.5 μ L, Primer down (10 μM) 0.5 μ L, dNTP mix (10Mm each) 0.5 μ L, 10 x Taq reaction B μ ffer 2.5 μ L, Taq (5 μ/μ L) 0.2 μ L, H2O 24.8 μL。
PCR amplification program: the condition of denaturation is 94 DEG C of 5min；The condition of degeneration is 94 DEG C of 30s, and the condition of annealing is 55 DEG C of 30s, the condition of extension is 72 DEG C of 1min, from degeneration to extending 30 circulations；72 DEG C extend 8min, are incubated 5min；Fall Take out PCR primer to 16 DEG C, use 1% agarose to carry out gel electrophoresis, after EB dyeing, gel imaging system detect.
(3) amplified production order-checking and sequence analysis
16S rDNA amplified production, after purity detecting, entrusts raw work gene sequencing center, Shanghai to check order.The sequence recorded Compare and similarity analysis with known array in NCBI data base.
(4) acetic acid bacteria qualification result:
Compare with the 16S rDNA sequence reported in NCBI data base.Acetic acid bacteria DY4 andAcetobacter paste μ rianμ s MSU10 evolution position is closest, so bacterial strain DY4 isAcetobacter pasteμ rianμ s(Fig. 3).
Experiment that embodiment 3 DY4 acetic acid bacteria is high temperature resistant
Take the logarithm the phaseAcetobacter pasteμ rianμ s DY4、AS1.41 bacterium solution 4mL joins to be trained containing 96mL liquid Support base (2% glucose, 2% yeast powder, 0.01% MgSO4, 4% dehydrated alcohol) 500mL conical flask in, respectively at 30 DEG C, 33 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, the condition of 44 DEG C is little shakes training, shaking speed 180r/min, measures every day Increment OD of strain600Value and product acid amount, until increment OD600Till value and product acid amount reach to stablize, draw, compare final Increment and product acid amount, draw the limiting temperature (Fig. 4, Fig. 5) that strain tolerates.
Conclusion: bacterium DY4 can 30-40 DEG C have more stable growth and produce acid amount (Ethanol-Acetic Acid conversion ratio is equal > 90.6%), the suitableeest fermentation temperature is 37 DEG C, and 37 DEG C reach maximum conversion rate (97.5%, i.e. 40.2g/L acetic acid)；When 43 DEG C of high temperature OD600Up to 0.72, producing acid is 40.0% up to 16.5g/L(maximum conversion rate) (Fig. 4,5).The suitableeest fermentation temperature of AS1.41 is 33 DEG C, the conversion ratio ＜ 90% during more than 35 DEG C, 40 DEG C of later growths and produce acid drastically decline, conversion ratio when 41 DEG C only have 0.6%, when temperature i.e. stops producing acid (Fig. 4,5) more than 41 DEG C.
Embodiment 4 utilizes acetic acid bacteria DY4 to prepare Citrullus vulgaris vinegar
(1) pretreatment of raw material: select non-rot, color is natural, and melon skin is flexible, taps and has clear and melodious sound, ripe west Melon, and clean above earth with clear water and impurity is standby；
(2) prepare Pulp Citrulli juice: cut the Citrullus vulgaris cleaned, scoop out flesh of Pulp Citrulli with spoon, squeeze out Pulp Citrulli juice with six layers with juice extractor Filtered through gauze, the Pulp Citrulli juice leached is poured in Porcelain Jar or aluminum pot, is heated to 70 DEG C, keeps 20 minutes, treats Pulp Citrulli juice cooling clarification After, with suction pipe sucking-off supernatant liquid, put into the Porcelain Jar through disinfection or porcelain altar.First measure Citrullus vulgaris with hand-held saccharometer Sugar content in juice, adds pure white sugar and melon juice sugar content is adjusted to 20%；
(3) saccharomyces cerevisiae and the preparation of ester-producing yeast seed liquor: the saccharomyces cerevisiae CICC1027 taking slant preservation is seeded to 500mL In Pulp Citrulli juice, cultivate 22h, obtain saccharomyces cerevisiae seed liquor for 28 DEG C；
The Hansenula anomala CICC1650 taking slant preservation is seeded in 500mL Pulp Citrulli juice, cultivates 22h for 26 DEG C, obtains producing ester Yeast starter liquid；
(4) alcohol fermentation: with 3%(v/v) inoculum concentration by saccharomyces cerevisiae seed liquor with 1.5%(v/v) inoculum concentration will be abnormal Hansenula yeast seed liquor accesses in step (2) in the Pulp Citrulli juice of sugar content 20%, and for preventing from becoming sour, every 100 kilograms of Pulp Citrulli juice add Enter sodium sulfate 10g, after deployed Pulp Citrulli juice stirs, sealed fermenting, 26 DEG C of fermentations, survey the ethanol of watermelon wine every day Content, reaches 8%(v/v until alcohol content) time, stop alcohol fermentation；
(5) acetic fermentation: the acetic acid bacteria DY4 taking slant preservation is seeded in 500mL Pulp Citrulli juice, cultivates 22h, obtains vinegar for 28 DEG C Acetic acid bacteria DY4 seed liquor is joined in the watermelon wine containing 8% ethanol by the inoculum concentration of 6% by acid bacterium DY4 seed liquor, 37 DEG C of fermentations 15d；
(6) preparation of Citrullus vulgaris vinegar: be centrifuged the fermentation liquid fermented, filters, sterilizing, and packaging i.e. can get Citrullus vulgaris vinegar.
Conclusion: 8% ethanol, the Ethanol-Acetic Acid conversion ratio of 37 DEG C reaches 95.6%.
Embodiment 5 utilizes acetic acid bacteria AS1.41 to prepare Citrullus vulgaris vinegar
The same as in Example 4, except substituting DY4 with AS1.41.Conclusion: 8% ethanol, the Ethanol-Acetic Acid conversion ratio of 37 DEG C reaches 62.5%。
The quality evaluation of embodiment 4,5 products obtained therefrom is shown in Table 2.
Table 2 DY4 Yu AS1.41 brewages the comparison of Citrullus vulgaris vinegar
。

Claims (3)

1. the Acetobacter pasteurianus that a temperature capacity is excellent, it is characterised in that: this Acetobacter pasteurianus isAcetobacer pasteurianusDY4, is preserved in China typical culture collection center on July 23rd, 2015, and deposit number is: CCTCC NO:M 2015462.
The purposes of the Acetobacter pasteurianus that a kind of temperature capacity the most as claimed in claim 1 is excellent, it is characterised in that: this Pasteur AcetobacterAcetobacer pasteurianusDY4 can brew vinegar under the high temperature conditions.
The purposes of the Acetobacter pasteurianus that a kind of temperature capacity the most as claimed in claim 2 is excellent, it is characterised in that: this Pasteur AcetobacterAcetobacer pasteurianusDY4 can under the conditions of high temperature 43 DEG C brew vinegar, under the conditions of 43 DEG C produce acid can Reach 16.5g/L, Ethanol-Acetic Acid conversion ratio 40.0%.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486731A (en) * 2019-01-10 2019-03-19 江西科技师范大学 A kind of resistance to acetate ethanol bacterium and its application
CN110195025A (en) * 2019-01-10 2019-09-03 江西科技师范大学 A kind of heat-resisting acetic acid bacteria and its application
CN116286559A (en) * 2023-05-10 2023-06-23 阳西美味鲜食品有限公司 High-yield high-temperature-resistant acetobacter pasteurii M112 and application thereof

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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 pasteurianus and application method thereof in production of citrus vinegar
CN103614318A (en) * 2013-11-08 2014-03-05 天地壹号饮料股份有限公司 High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation

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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 pasteurianus and application method thereof in production of citrus vinegar
CN103614318A (en) * 2013-11-08 2014-03-05 天地壹号饮料股份有限公司 High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486731A (en) * 2019-01-10 2019-03-19 江西科技师范大学 A kind of resistance to acetate ethanol bacterium and its application
CN110195025A (en) * 2019-01-10 2019-09-03 江西科技师范大学 A kind of heat-resisting acetic acid bacteria and its application
CN116286559A (en) * 2023-05-10 2023-06-23 阳西美味鲜食品有限公司 High-yield high-temperature-resistant acetobacter pasteurii M112 and application thereof
CN116286559B (en) * 2023-05-10 2023-09-05 阳西美味鲜食品有限公司 High-yield high-temperature-resistant acetobacter pasteurii M112 and application thereof

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