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Acid-resistant, ethanol-resistant and acetic acid-resistant Acetobacer pasteurianus

A technology of acetic acid bacteria and ethanol resistance, applied in the direction of bacteria, microorganism-based methods, microorganisms, etc., can solve the problems of poor tolerance to acetic acid, high fermentation temperature, intolerance to ethanol and acetic acid, etc., and achieve a wide range of environmental tolerance Effect

Active Publication Date: 2016-11-30
HUBEI UNIV OF TECH
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Problems solved by technology

During acetic acid fermentation, due to the inefficient oxidative respiration of acetic acid bacteria, a large amount of fermentation heat will be generated, and the accumulation of these heat will inevitably make the fermentation temperature higher than 37 °C, resulting in slow cell growth and low acetic acid production (Adachi et al. 2003); therefore, breeding heat-resistant acetic acid bacteria can effectively reduce the cooling cost of acetic acid fermentation; in addition to high temperature, high concentration of alcohol also leads to weak cell viability and low fermentation efficiency (Ardhana et al. 2003), so it is used for acetic fermentation The initial ethanol concentration is generally not more than 8%, which has a certain relationship with the inoculation amount of acetic acid bacteria; in addition, acetic acid bacteria (AAB) have poor tolerance to acetic acid, and the tolerance of Acetobacter pasteuriani to acetic acid is only 6% so far.
[0003] In recent years, some progress has been made in the screening and breeding of acetic acid bacteria, but most of the research has focused on the screening of a certain characteristic of acetic acid bacteria, heat resistance or acetic acid resistance, such as the Chinese patent "High temperature resistant acetic acid bacteria and its fermentation to produce acetic acid Application No. 201310549668.7", the acetic acid bacteria in this patent grow and produce acid at 25-45 °C, but are not resistant to ethanol and acetic acid
At present, no research has shown that the existing acetic acid bacteria have good fermentation characteristics in a complex environment (high temperature, high-concentration ethanol, high-concentration acetic acid compound environment), but such a complex environment is inevitable in the acetic acid fermentation process, so , it is very necessary to obtain acetic acid bacteria that perform well in the harsh and complex fermentation environment of high temperature, high concentration of ethanol and high concentration of acetic acid

Method used

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  • Acid-resistant, ethanol-resistant and acetic acid-resistant Acetobacer pasteurianus
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  • Acid-resistant, ethanol-resistant and acetic acid-resistant Acetobacer pasteurianus

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Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Preliminary separation and purification of high-temperature-resistant high-alcohol acetic acid bacteria

[0024] (1) Selection of acetic acid bacteria isolation source: vinegar grains from Danyang Vinegar Factory;

[0025] (2) Preliminary separation and purification of high-temperature-resistant high-alcohol acetic acid bacteria

[0026] ① Enrichment culture of acetic acid bacteria:

[0027] Add 1g of vinegar unstrained spirits from a vinegar factory (multiple) with traditional solid-state fermentation technology to 100mL of water, culture at 30°C for 5-8 days, obtain 5mL of natural fermentation liquid, put it into a conical flask containing 100mL of enriched medium, 30°C, Cultivate at 180r / min for 2 days to obtain acetic acid bacteria enriched culture solution.

[0028] Enrichment medium composition: 2% glucose, 2% yeast powder, 0.01% MgSO 4 , and the rest is distilled water;

[0029] ②Qualitative experiment of acetic acid bacteria:

[0030] Take 20 mL o...

Embodiment 2

[0033] The identification of embodiment 2 acetic acid bacteria

[0034] (1) ZJ17 strain was identified morphologically, physiologically and biochemically according to the "Common Bacterial System Identification Manual" and "Bergey's Bacterial Identification Manual" eighth edition (Table 1).

[0035] Table 1 Physiological and biochemical characteristics of ZJ17 and AS1.41

[0036]

[0037] (2) 16Sr DNA identification:

[0038] Pick acetic acid bacteria ZJ17 and inoculate them in 10mL / 100mL Erlenmeyer flask liquid medium, shake and cultivate in a constant temperature shaking incubator at 30°C and 180r / min for 24h, take 2mL of the bacteria liquid in a centrifuge tube, centrifuge at 12000r / min for 2min, and remove the supernatant Genomic DNA of the bacterial strain was extracted according to the instructions of the SK1201-MNIQ-10 Column Bacterial Genomic DNA Extraction Kit from Shanghai Sangon Bioengineering Technology Service Co., Ltd. as a template. Select universal primers...

Embodiment 3

[0047] Example 3 High temperature resistance experiment

[0048] Take the logarithmic period Acetobacter pasteμ rianμ s 4mL of ZJ17 and AS1.41 bacteria solution were respectively added to 96mL liquid medium (2% glucose, 2% yeast powder, 0.01% MgSO 4 , 4% absolute ethanol) in a 500mL Erlenmeyer flask, shake culture at 30°C, 32°C, 35°C, 37°C, 39°C, 41°C, 42°C, shaker speed 180r / min, every day Measure the OD of bacterial growth 600 value and acid production until growth OD 600 value and acid production reached a stable level, draw a graph and compare the final growth and acid production ( Figure 3-A , Figure 3-B ).

[0049] in conclusion: Acetobacer pasteurianus ZJ17 has relatively stable growth and acid production at 30-42°C under the condition of 4% ethanol ( Figure 3-A ), and both perform better than AS1.41 ( Figure 3-A , Figure 3-B ); the optimum fermentation temperature of AS1.41 is 30°C, while the optimum fermentation temperature of ZJ17 is 37°C (the ethano...

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Abstract

The invention discloses acid-resistant, ethanol-resistant and acetic acid-resistant Acetobacer pasteurianus ZJ17, which has the preservation number of CCTCC M 2015463. According to the present invention, the Acetobacer pasteurianus ZJ17 retains the high ethanol-acetic acid conversion rates at temperatures of 37 DEG C and 42 DEG C under a 4% ethanol condition, wherein the ethanol-acetic acid conversion rates respectively are 96.2% and 62.5%; under a 30 DEG C condition, the ethanol-acetic acid conversion rates of the Acetobacer pasteurianus ZJ17 on 12% ethanol and 8% ethanol respectively are 34.2% and 72.7%; and the acetic acid resistance of the Acetobacer pasteurianus ZJ17 is significantly higher than the upper limit 6% of the acetic acid resistance of the Acetobacer pasteurianus reported in the prior art, such that the Acetobacer pasteurianus ZJ17 has the wide tolerance range to the environment, is suitable for brewing acetic acid under the conditions of high temperature, high concentration ethanol and high concentration acetic acid, and provides great application prospects in vinegar brewing in summer.

Description

technical field [0001] The invention belongs to the field of microorganism and fermentation industry, and in particular relates to a heat-resistant, ethanol-resistant and acetic acid-resistant Acetobacter pasteurii Acetobacer pasteurianus ZJ17. Background technique [0002] Acetic acid bacteria are obligate aerobic Gram-negative bacteria, which can convert ethanol into acetic acid through aerobic fermentation, and play an important role in the vinegar industry. Breeding excellent acetic acid bacteria is also one of the important contents of improving the acetic acid fermentation process one. At present, the breeding of acetic acid bacteria is almost all focused on improving heat resistance, ethanol resistance and acetic acid resistance. The optimum growth temperature of common acetic acid bacteria is usually 30°C, and these strains can generally grow at 37-40°C (Gullo et al.2008), but the growth and acid production become extremely slow at 37°C; Bacteria can achieve the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/02
CPCC12N1/205C12R2001/02
Inventor 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
Owner HUBEI UNIV OF TECH
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