CN102994409B

CN102994409B - New bacterial strain-proteus penneri CA8 for generating coenzyme Q10 and applications thereof

Info

Publication number: CN102994409B
Application number: CN201210260349.XA
Authority: CN
Inventors: 钟卫鸿; 孔卓怡; 柳华贵; 钟莉; 邱乐泉; 吴石金
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang Zhongke Long Life Medical Science And Technology Co Ltd
Priority date: 2012-07-20
Filing date: 2012-07-20
Publication date: 2014-09-03
Anticipated expiration: 2032-07-20
Also published as: CN102994409A

Abstract

The invention provides a new bacterial strain-proteus penneri CA8 for generating coenzyme Q10 and applications of the proteus penneri CA8 in preparation of the coenzyme Q10 by microbial fermentation. The bacterial strain is preserved in China Center for Type Culture Collection, the address is 430072, Wuhan University, Wuhan, China, the preservation number is CCTCC No: M2012208, and the preservation date is June 10, 2012. The new bacterial strain-proteus penneri CA8 for generating the coenzyme Q10 has the following main beneficial effects: the new coenzyme Q10 producing strain is provided, and the coenzyme Q10 is prepared by utilizing the microbial fermentation of the strain, so that yield is high, the subsequent extraction method is simple, and the industrial production is facilitated.

Description

Produce ubiquinone 10new bacterial strain P.penneri CA8 and application thereof

(1) technical field

The present invention relates to a strain and produce ubiquinone ₁₀new bacterial strain---P.penneri CA8 and prepare ubiquinone in microorganism fermentation ₁₀in application, belong to microbiological pharmacy field.

(2) background technology

Ubiquinone ₁₀(CoenzymeQ ₁₀, CoQ ₁₀) be extensively present in animal, plant and microbe, be hydrogen carrier important in biomass cells respiratory chain, be also a kind of good biochemical drug.It has oxidation-resistance, eliminates free radical, improves the functions such as immunity of organisms, has been widely used in the treatment of the diseases such as all kinds of heart troubles, diabetes, cancer, acute, chronic hepatitis, parkinsonism.Prepare ubiquinone ₁₀method mainly contain zooblast extraction method, tobacco leaf Salanesol semi-synthesis method, synthesis method, microbe fermentation method or culture plant cell method completely.Compare with additive method, microbe fermentation method has that its lytic activity is high, raw materials cost is low, easy to control and can realize the features such as scale operation.Obtaining good fermentation strain is Production by Microorganism Fermentation ubiquinone ₁₀one of key.The ubiquinone of having reported at present ₁₀fermentation strain mainly belongs to Pseudomonas, Saccharomycodes, photosynthetic bacterium genus and paracoccus etc.Fermentation thalline extracts ubiquinone ₁₀method mostly is alcohol alkali soap method for post extraction, and step is loaded down with trivial details, waste time and energy, and it is larger to extract solvent loss.

(3) summary of the invention

The object of the invention is in order to provide a strain to produce ubiquinone ₁₀output is new bacterial strain preferably, and utilizes this strain fermentating liquid to prepare ubiquinone ₁₀method.

The technical solution used in the present invention is:

One strain ubiquinone ₁₀bacterium---P.penneri (Proteus penneri) CA8, is preserved in Chinese Typical Representative culture collection center, address: China in generation, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M 2012208, preservation date: on June 10th, 2012.

Described P.penneri CA8 feature is as follows: bacterial strain on LB agar plate, cultivate after 1 day, be translucent moist, less, circle, smooth surface, easily provokes; It is shaft-like that thalline is, and size is (0.4～1.0) μ m * (0.8～1.8) μ m, and tool whole body flagellum, can move, and Gram-negative, without gemma; Amphimicrobian, phenylalanine test is positive, and oxydase reaction, gelatin hydrolysis test, indole test, IMViC test, V-P reaction are negative, and utilize glucose, lactose etc. to produce acid, and unfavorable with saligenin, optimum growth temperature is 37 ℃.

CA8 bacterial strain of the present invention, separated from the Ebolowa of Cameroon soil, through the Phylogenetic Analysis of morphology, Physiology and biochemistry, Biolog GEN Ш evaluation and 16S rRNA sequence, find that this bacterial strain belongs to P.penneri, called after Proteus penneri CA8.There is not yet at present relevant Bacillus proteus is both at home and abroad applied at ubiquinone ₁₀the research report of producing.

The present invention ferment thalline ultrasonication and extract ubiquinone ₁₀technique have no equally report.

The invention still further relates to described P.penneri CA8 and prepare ubiquinone in microorganism fermentation ₁₀in application.

Described being applied as: with P.penneri CA8, be inoculated in the liquid nutrient medium that is applicable to P.penneri, at 30 ~ 37 ℃, cultivate 48 ~ 60 h, the centrifugal collection wet thallus of gained fermented liquid, wet thallus extracts and obtains described ubiquinone through fragmentation ₁₀.

The described liquid nutrient medium that is applicable to P.penneri is the conventional liquid nutrient medium that is applicable to P.penneri, can prepare by following composition: carbon source 10 ~ 20 g, nitrogenous source 5 ~ 15 g, KH in the present invention ₂pO ₄0.5 g, Na ₂hPO ₄1.5 g, MgSO ₄0.5 g, water 1000 ml, pH6.0 ~ 8.0.

Described carbon source is the conventional carbon source for substratum preparation, as glucose, sucrose, maltose, lactose etc., is preferably lactose.

Described nitrogenous source is the conventional carbon source for substratum preparation, as yeast extract paste, ammonium sulfate etc., is preferably yeast extract paste.

More preferred, described in be applicable to P.penneri liquid nutrient medium by following composition, prepare: add lactose 40 g, yeast extract paste 40 g, KH ₂pO ₄0.5 g, Na ₂hPO ₄1.5 g, MgSO ₄0.5 g, water 1000 ml, pH8.0.

Preferably, described fragmentation is extracted as ultrasonication and extracts, and method is as follows: wet thallus is moved in centrifuge tube, add enough ethanol, mix, and after 300 ~ 500 W supersound process 8 ~ 10 min, centrifugal, filter, in filtrate, obtain described ubiquinone ₁₀.

Beneficial effect of the present invention is mainly reflected in: provide a strain new ubiquinone ₁₀produce bacterium, utilize this strain microorganism fermentation to prepare ubiquinone ₁₀, output is high, and subsequent extracted method is simple, is beneficial to suitability for industrialized production.

(4) accompanying drawing explanation

Fig. 1 is the Electronic Speculum figure (* 30000) of P.penneri (Proteus penneri) CA8;

Fig. 2 is that different carbon sources are to CA8 ubiquinone ₁₀the impact of output;

Fig. 3 is that different nitrogen sources is to CA8 ubiquinone ₁₀the impact of output;

Fig. 4 is that lactose concn is to CA8 ubiquinone ₁₀the impact of output;

Fig. 5 is that yeast extract paste concentration is to CA8 ubiquinone ₁₀the impact of output;

Fig. 6 is that temperature is to ubiquinone ₁₀the impact of output;

Fig. 7 is that liquid amount is to ubiquinone ₁₀the impact of output;

Fig. 8 is that initial pH value is to ubiquinone ₁₀the impact of output.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment: ubiquinone is produced in the separation of P.penneri (Proteus penneri) CA8, evaluation and fermentation thereof ₁₀characteristic

1, the separation of bacterial strain:

(1) collected specimens

Gather after the Ebolowa of Cameroon pedotheque, get 10 g, add sterilized water 90 ml, make Soil Slurry.Draw supernatant liquor and make 10 with sterilized water dilution ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6different extent of dilution.

(2) strains separation

Get bacteria suspension 1 ml, in the flat lining out of selectivity, selective medium was prepared by following composition: isoprene 0.5 ml, (NH ₄) ₂sO ₄1.0 g, KH ₂pO ₄4.5 g, Na ₂hPO ₄12H ₂o 21.6 g, agar 15～20 g, water 1000 ml, pH 7.0.

According to the ordinary method of microorganism Pure strain separation, above-mentioned separation and Culture is placed to 2～3 d based on 28 ℃.A plurality of single bacterium colonies of picking, are inoculated on slant medium, and numbering is preserved.In liquid medium within, carry out fermentable check, result obtains a strain ubiquinone again ₁₀the bacterial strain CA8 that output is higher.Substratum used was prepared by following composition: glucose 20 g, peptone 10 g, yeast extract paste 10 g, KH ₂pO ₄0.5 g, Na ₂hPO ₄1.5 g, MgSO ₄7H ₂o 0.5 g, water 1000 ml, pH7.0.(as being solid medium, adding again the agar of 20 g/L).

2, the evaluation of bacterial strain

(1) ubiquinone ₁₀produce thalline and the colony morphology characteristic of bacterium CA8

Ubiquinone ₁₀it is shaft-like producing bacterium CA8, and size is (0.4～1.0) μ m * (0.8～1.8) μ m, Gram-negative, and without gemma, tool whole body flagellum, can move; Bacterium colony is translucent moist, less, circle, and smooth surface, easily provokes.

(2) ubiquinone ₁₀produce the physiological and biochemical property of bacterium CA8

Physiological and biochemical property is: amphimicrobian, and phenylalanine test is positive, and oxydase reaction, gelatin hydrolysis test, indole test, IMViC test, V-P reaction are negative, and utilize glucose, lactose etc. to produce acid, the unfavorable saligenin of using.

The Physiological-biochemical Characters of the P.penneri that above-mentioned mycology feature and document (uncle Jie Shi Bacteria Identification handbook) are edited and recorded matches.In conjunction with 16S rRNA sequence homology analysis and the Biolog systems analysis result of bacterial strain, this bacterium pearl is accredited as the product ubiquinone that belongs to P.penneri ₁₀new bacterium pearl.

3, produce ubiquinone ₁₀performance

P.penneri (Proteus penneri) CA8, with 5 % inoculum sizes, is inoculated in to 500 ml 100 ml liquid nutrient mediums (20 g carbon sources, 10 g peptones, 10 g yeast extract pastes, KH are housed ₂pO ₄0.5 g, Na ₂hPO ₄1.5 g, MgSO ₄0.5 g, water complements to 1000 ml, pH 7.0) shaking flask in, using these several conventional carbon sources of glucose, sucrose, maltose and lactose as the carbon source of fermentation, investigate its cell growth total amount and ubiquinone ₁₀the impact of total amount, carries out single factor research of carbon source.

Result as shown in Figure 2, shows that the utilization of carbon source is to bacteria growing amount and ubiquinone ₁₀yield effect is more inhomogeneous.From bacteria growing amount and ubiquinone ₁₀proportionlity between output, lactose is optimum carbon source.

Carry out on this basis the impact research of lactose concn interpolation, result as shown in Figure 4, ubiquinone when Inclusion of Lactose is 40 g/L ₁₀output is higher.

P.penneri (Proteus penneri) CA8, with 5 % inoculum sizes, is inoculated in to liquid nutrient medium (40 g lactose, 10 g nitrogenous sources, KH ₂pO ₄0.5 g, Na ₂hPO ₄1.5 g, MgSO ₄0.5 g, water complements to 1000 ml, pH 7.0), as shown in Figure 3, result shows the result of nitrogenous source: in organic nitrogen source, take yeast extract paste as best, itself and inorganic nitrogen-sourced ammonium sulfate comparison, the inorganic nitrogen-sourced thalli growth that is unfavorable for.Carry out on this basis the impact research of yeast extract paste concentration interpolation, as shown in Figure 5, result shows result: ubiquinone when yeast extract paste addition is 40 g/L ₁₀output is higher.

Investigate respectively differing temps, liquid amount, initial pH value cell growth total amount and ubiquinone ₁₀the impact of total amount.Result, as shown in Fig. 6, Fig. 7, Fig. 8, shows when temperature, liquid amount, initial pH are divided into 30 ℃, 80 ml, fermentation preparation of cozymase Q 8.0 time ₁₀best.

With this understanding, by bacterial strain P.penneri (Proteus penneri) CA8 fermentation, produce ubiquinone ₁₀output the highest, reached 105.1 mg/L, than before optimizing, improved 500%.

Claims

1. a strain ubiquinone ₁₀bacterium---P.penneri (Proteus penneri) CA8, is preserved in Chinese Typical Representative culture collection center, address: China in generation, Wuhan, Wuhan University, 430072, deposit number: CCTCC No:M2012208, preservation date: on June 10th, 2012.

2. P.penneri CA8 as claimed in claim 1 prepares ubiquinone in microorganism fermentation ₁₀in application.

3. application as claimed in claim 2, described in it is characterized in that, be applied as: with P.penneri CA8, be inoculated in the liquid nutrient medium that is applicable to P.penneri, at 30～37 ℃, cultivate 48～60h, the centrifugal collection wet thallus of gained fermented liquid, wet thallus extracts and obtains described ubiquinone through fragmentation ₁₀.

4. application as claimed in claim 3, the liquid nutrient medium that is applicable to P.penneri described in it is characterized in that is prepared by following composition: carbon source 10～20g, nitrogenous source 5～15g, KH ₂pO ₄0.5g, Na ₂hPO ₄1.5g, MgSO ₄0.5g, water 1000ml, pH6.0～8.0.

5. application as claimed in claim 4, is characterized in that described carbon source is lactose.

6. application as claimed in claim 4, is characterized in that described nitrogenous source is yeast extract paste.

7. application as claimed in claim 3, the liquid nutrient medium that is applicable to P.penneri described in it is characterized in that is prepared by following composition: add lactose 40g, yeast extract paste 40g, KH ₂pO ₄0.5g, Na ₂hPO ₄1.5g, MgSO ₄0.5g, water 1000ml, pH8.0.

8. application as claimed in claim 3, is characterized in that described fragmentation is extracted as ultrasonication and extracts, and method is as follows: wet thallus is moved in centrifuge tube, add enough ethanol, mix, after 300～500W supersound process, 8～10min, centrifugal, filter, in filtrate, obtain described ubiquinone ₁₀.