Caproic acid bacterium proliferation culture medium and caproic acid fermentation and caproic acid bacterium screening method

Abstract

The invention belongs to the technical field of bacterial fermentation and functional bacteria screening, and discloses a caproic acid bacterium proliferation culture medium and a caproic acid fermentation and caproic acid bacterium screening method. The proliferation culture medium comprises 10.0 g / L of peptone, 10.0 g / L of beef powder, 3.0 g / L of yeast powder, 1.0-5.0 g / L of glucose, 1.0 g / L ofsoluble starch, 5.0 g / L of sodium chloride, 3.0-6.0 g / L of sodium acetate, 0.5 g / L of cysteine hydrochloride, 0.3-1.0 g / L of ascorbic acid, 0.5-2.0 g / L of agar and distilled water composition. Nutrients such as 0.005-0.01 g / L of hemin are selectively added, so that the growth and division of some extreme bacteria are facilitated, the cell density is increased, and the screening range of caproic acid bacteria is expanded. Ethanol and sodium butyrate are added midway to promote the synthesis of hexanoic acid. A fermentation container is transparent, and fermentation liquor is preliminarily screened according to the conditions of gas production and acid production in the fermentation liquor, so that the screening efficiency is improved.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation and screening and identification of functional bacteria, and in particular relates to a caproic acid bacteria proliferation medium, caproic acid fermentation and caproic acid bacteria screening methods. Background technique [0002] Currently, the closest prior art: [0003] Baijiu (baijiu) is a unique distilled liquor in China. Luzhou-flavor liquor has the main aroma of "mainly ethyl caproate, mixed with a small amount of ethyl acetate, ethyl butyrate and ethyl lactate" due to the "solid-state fermentation in mud pit" process. As the precursor of ethyl caproate, caproic acid mainly comes from caproic acid bacteria in pit mud. Caproic acid bacteria, without strict taxonomic significance, refer to a type of bacteria that can produce caproic acid. The reason why many wineries can produce high-grade Luzhou-flavored puree wine mostly depends on the use of old cellars with a long hist...

AI Technical Summary

Problems solved by technology

When detecting hexanoic acid in the fermentation broth, although 2-ethylbutyric acid was also used as an internal standard, the ethanol solution (including the fermentation broth) was not directly measured by gas phase, but a fermentation broth, internal standard The step of fully mixing liquid and ether, and finally taking the upper ether layer for detection

The defect is: ether gas is harmful to the human body and is not conducive to safe operation; the pretreatment process of the fermented liquid is not simple enough, which consumes manpower and reagents; the extraction process of ethyl ether to hexanoic acid will be affected by the acidity in the fermented liquid, and there is no Obvious buffer reagents lead to differences in the extraction yields of hexanoic acid in fermentation broths with different acidities, and the quantitative detection results are unstable

The defect is: the reaction is disturbed by the pH of the solution (easy to produce copper hydroxide when alkaline) and the fat-soluble substances in the yeast extract (easy to appear in the ether layer), and in the process of making the standard curve, the hexanoic acid standard must be Dissolved in a diluent with a certain concentration of ethanol, which has a negative effect on color rendering (light absorption), resulting in poor fidelity

The defect is: since the system belongs to the natural culture medium, which itself contains caproic acid and other substances, it is impossible to distinguish whether the caproic acid in the fermentation broth comes from the initial culture medium or the synthetic metabolism of the bacteria to be tested; the patent includes a variety of bacteria, even Yeast, the composition of the fermentation liquid is more complex, which is the result of the joint action of multiple strains, which is not conducive to the analysis of the metabolic profile of a single pure strain

Its defect is: the energy substance of EAM liquid culture medium is less, it is difficult to reach higher cell density; Fermentation process does not adopt anaerobic means such as deoxygenation and isolation; Fermentation liquid is directly (not prepared into ethanol solution) into gas chromatographic detection , it is easy to be contaminated with miscellaneous bacteria in the sample transportation process; the preliminary screening uses the copper sulfate ether method, which is time-consuming and easy to pollute (not as good as the fermented liquid gas production blackening and the acid indicator primary screening method in the present invention, which is intuitive and convenient); use test tubes Fermentation, it is not convenient to prolong the fermentation of the slow-growing bacteria to be tested. Due to the evaporation and loss of water in the fermentation broth, there will be certain errors in the quantitative detection of caproic acid concentration

Although the prepared cellar protection solution contains high caproic acid, how to quickly screen caproic acid bacteria to prove that a certain strain is caproic acid bacteria is not suitable for this method;

[0017] (2) The culture medium component used in the prior art is complicated, is unfavorable for the analysis and screening of purebred bacterial metabolism

In the process of sample preparation, the pretreatment operation is complicated, and harmful gases such as ether are used, which is not conducive to a large number of screening work;

[0018] (3) The detection method of hexanoic acid in the prior art has relatively general sensitivity, accuracy and stability; the copper sulfate colorimetric method is cumbersome and has large errors; the fermentation vessel is large and occupies a large area, making it difficult to detect many different High-throughput screening of bacteria (strains)

[0019] (4) In the screening process of caproic acid bacteria in the prior art, there are defects in some caproic acid fermentation conditions, causing some caproic acid bacteria to only produce butyric acid, valeric acid, isobutyric acid (intermediate metabolite) or isocaproic acid, etc., while Unable to complete terminal caproic acid synthesis, which will cause the leakage of caproic acid bacteria to a certain extent

However, some extreme bacteria that are demanding on culture conditions are still difficult to isolate, and even if they are isolated, they are not successfully identified as caproic acid bacteria in time, which seriously hinders the speed of application and transformation of this type of caproic acid bacteria

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