阅读说明：本技术 一种粘质沙雷氏菌itbb b5-1在生产灵菌红素中的应用 (Application of Serratia marcescens ITBB B5-1 in prodigiosin production ) 是由 谭德冠 张家明 付莉莉 马帅 孙雪飘 于 2020-07-27 设计创作，主要内容包括：本发明涉及微生物领域,特别涉及一种粘质沙雷氏菌ITBB B5-1在生产灵菌红素中的应用。该粘质沙雷氏菌的保藏编号为CGMCC No.7416。采用本发明所述菌株生产灵菌红素,其产量在每升培养基中达9.8～15.5g,显著高于所报道的菌株。本发明克服了粘质沙雷氏菌菌株低灵菌红素产量的制约,在医药市场中具有广阔的应用前景。(The invention relates to the field of microorganisms, and in particular relates to application of serratia marcescens ITBB B5-1 in prodigiosin production. The preservation number of the serratia marcescens is CGMCC No. 7416. The prodigiosin produced by the strain provided by the invention has the yield of 9.8-15.5 g in each liter of culture medium, and is obviously higher than that of the reported strain. The invention overcomes the restriction of low prodigiosin yield of the serratia marcescens strain and has wide application prospect in the medical market.)

1. The application of Serratia marcescens ITBB B5-1 in prodigiosin production is characterized in that the preservation number of the Serratia marcescens is CGMCC No. 7416.

2. A method for producing prodigiosin, which is characterized by comprising the following steps:

activating serratia marcescens with the preservation number of CGMCC No.7416, and then inoculating the serratia marcescens in a liquid culture medium for amplification culture to obtain seed liquid;

inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture, and purifying to obtain prodigiosin.

3. The method according to claim 2, wherein the medium used for the activation is LB medium.

4. The method according to claim 2 or 3, wherein the activation is carried out under dark conditions at 24-26 ℃ for 1-3 d.

5. The method of claim 2, wherein the liquid medium is formulated as: 15-25g/L tryptone, 10-15g/L yeast extract, 4-6 g/L sodium chloride and 6.8-7.2 of pH value.

6. The method according to claim 2 or 5, wherein the conditions of the expanded culture are: the inoculation amount is 1-2 CFU/100mL, and the shaking culture is carried out for 12-18h at 200-300 rpm under the dark condition at the temperature of 24-26 ℃.

7. The method of claim 2, wherein the fermentation medium is formulated as: 15-25g/L tryptone, 10-15g/L yeast extract, 4-6 g/L sodium chloride and 6.8-7.2 of pH value.

8. The method according to claim 2 or 7, wherein the conditions of the fermentation culture are: the inoculation ratio of the seed liquid to the fermentation medium is 3 vt-5 vt%, the liquid loading amount is 200-400 mL/1000mL, and the seed liquid is subjected to shake culture at 200-300 rpm for 48-96 h under the dark condition at the temperature of 22-28 ℃.

9. The method of claim 2, wherein the purification is: centrifuging the fermentation liquor to collect thalli, resuspending the thalli by using absolute ethyl alcohol, centrifuging after violent oscillation, and collecting an ethyl alcohol supernatant and a precipitate;

leaching the precipitate with ethyl acetate, and centrifuging to collect ethyl acetate supernatant;

concentrating and drying ethanol supernatant and ethyl acetate supernatant, and adding methanol for redissolving to obtain crude extract;

mixing the raw materials in parts by weight of acetone: ethyl acetate 95: and 5, taking the eluent as an eluent, carrying out chromatography on the crude extract by using a 200-mesh silica gel column, eluting the obtained red component by using methanol, and drying the eluent to obtain the purified prodigiosin.

10. The method according to claim 9, wherein the volume of the absolute ethanol is 4 to 6 times of the volume of the cells, the period of the vigorous shaking is 50 to 100s, the volume of the ethyl acetate is 4 to 6 times of the volume of the precipitate, and the rotation speed of the centrifugation is 10000 to 15000 g.

Technical Field

The invention relates to the field of microorganisms, and in particular relates to application of serratia marcescens ITBB B5-1 in prodigiosin production.

Background

Prodigiosin-like pigments (Prodigiosins) are natural haematochrome with a methoxypyrrole skeleton structure having 3 pyrrole rings, including Prodigiosin, cycloalkylprodigiosin, UndecylprodigiosinMesoproducin (Metacylioprodigiosin), streptorubicin B (Streptoryubin B) and the like (Yu, Wang Yu Jie, Sun Shi Qing, Liu Xiao Xia, the research progress of prodigiosin production by a microbial fermentation method. the biological engineering journal, 2016, 32 (10): 1332-1347). Wherein Prodigiosin (Prodigiosin) has huge potential application value in the medical field. Prodigiosin is an efficient and low-toxic anticancer drug. The related studies showed that prodigiosin is at the mean semi-Inhibitory Concentration (IC)₅₀) The antibody has stronger resistance to 60 cancer cell lines when the antibody is 2.1umol/L, and does not generate toxicity to normal cells. Prodigiosin has an immunosuppressive function and can be used as an immunosuppressant to be applied to clinical organ transplantation. In addition, it can be used as antimalarial medicine with good effect.

Prodigiosin is a microbial secondary metabolite mainly derived from Serratia marcescens (Serratiamarcescens) or actinomycetes. However, the yield of prodigiosin from the currently reported serratia marcescens strain capable of producing prodigiosin is low. The prodigiosin yield after fermenting for 2 days by serratia marcescens with the preservation number of CGMCC No.2593 in the publication number CN 101392227A is 7.85 g/L; the yield of prodigiosin after fermentation of Serratia marcescens with the preservation number of CCTCC NO of M2010347 for 2.8 days in the publication number CN 102277323A is 3-5 g/L; the maximum concentration of prodigiosin at the end of 48h fermentation of serratia marcescens with the preservation number of CGMCC No.12714 in the publication number CN 1059697029 is 5.29 g/L. The low prodigiosin yield characteristic of the serratia marcescens strain restricts the production of prodigiosin and the application thereof in the medical market.

Disclosure of Invention

In view of the above, the invention provides an application of Serratia marcescens ITBB B5-1 in prodigiosin production. The prodigiosin yield can reach 9.8-15.5 g/L and is obviously higher than that of the reported strains when the strains are used for producing prodigiosin.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of serratia marcescens ITBB B5-1 in prodigiosin production, wherein the preservation number of the serratia marcescens is CGMCC No. 7416.

The serratia marcescens ITBB B5-1 disclosed by the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2013, 04 and 07, and the address is No. 3 of the West Lu No.1 of the Beijing city, Chaoyang district, and the preservation number is CGMCC No.7416 at the institute of microorganisms of the Chinese academy of sciences. And the strain is disclosed in a patent with a publication number of CN103333810A, and the strain is separated from latex of rubber trees and belongs to Serratia marcescens through morphological and molecular biological identification.

The invention also provides a method for producing prodigiosin, which comprises the following steps:

activating serratia marcescens with the preservation number of CGMCC No.7416, and then inoculating the serratia marcescens in a liquid culture medium for amplification culture to obtain seed liquid;

inoculating the obtained seed liquid into a fermentation culture medium for fermentation culture, and purifying to obtain prodigiosin.

Preferably, the medium used for activation is LB medium.

Preferably, the activation is carried out for 1-3 days under the dark condition at 24-26 ℃.

Preferably, the activation is 2d under dark conditions at 25 ℃.

Preferably, the formula of the liquid culture medium is as follows: 15-25g/L tryptone, 10-15g/L yeast extract, 4-6 g/L sodium chloride and 6.8-7.2 of pH value.

Preferably, the liquid medium has the formula: 15-25g/L of tryptone, 10-15g/L of yeast extract, 5g/L of sodium chloride and 7.0 of pH value.

Preferably, the conditions for the scale-up culture are: the inoculation amount is 1-2 CFU/100mL, and the shaking culture is carried out for 12-18h at 200-300 rpm under the dark condition at the temperature of 24-26 ℃.

Preferably, the conditions of the scale-up culture are: the inoculation amount is 1CFU/100mL, and the shaking culture is carried out for 12-18h at the temperature of 25 ℃ and the rpm under the dark condition.

Preferably, the formula of the fermentation medium is as follows: 15-25g/L tryptone, 10-15g/L yeast extract, 4-6 g/L sodium chloride and 6.8-7.2 of pH value.

Preferably, the formulation of the fermentation medium is: 15-25g/L of tryptone, 10-15g/L of yeast extract, 5g/L of sodium chloride and 7.0 of pH value.

Preferably, the conditions of the fermentation culture are: the inoculation ratio of the seed liquid to the fermentation medium is 3 vt-5 vt%, the liquid loading amount is 200-400 mL/1000mL, and the seed liquid is subjected to shake culture at 200-300 rpm for 48-96 h under the dark condition at the temperature of 22-28 ℃.

Preferably, the conditions of the fermentation culture are: the inoculation ratio of the seed solution to the fermentation medium is 3 vt-5 vt%, the liquid loading amount is 300mL/1000mL, and the seed solution is subjected to shake culture at 250rpm under the dark condition at the temperature of 22-28 ℃ for 48-96 h.

Preferably, the purification is: centrifuging the fermentation liquor to collect thalli, resuspending the thalli by using absolute ethyl alcohol, centrifuging after violent oscillation, and collecting an ethyl alcohol supernatant and a precipitate;

leaching the precipitate with ethyl acetate, centrifuging and collecting ethyl acetate supernatant;

concentrating and drying ethanol supernatant and ethyl acetate supernatant, and adding methanol for redissolving to obtain crude extract;

mixing the raw materials in parts by weight of acetone: ethyl acetate 95: and 5, taking the eluent as an eluent, carrying out chromatography on the crude extract by using a 200-mesh silica gel column, eluting the obtained red component by using methanol, and drying the eluent to obtain the purified prodigiosin.

Preferably, the volume of the absolute ethyl alcohol is 4-6 times of the volume of the bacteria, the time of violent oscillation is 50-100 s, the volume of the ethyl acetate is 4-6 times of the volume of the sediment, and the rotating speed of centrifugation is 10000-15000 g.

Preferably, the volume of the absolute ethanol is 5 times of the cell volume, the vigorous shaking time is 60s, the volume of the ethyl acetate is 5 times of the precipitation volume, and the rotation speed of the centrifuge is 12000 g.

The invention provides an application of serratia marcescens ITBB B5-1 in prodigiosin production, wherein the preservation number of the serratia marcescens is CGMCC No. 7416. The invention has the technical effects that:

the prodigiosin produced by the strain provided by the invention has the yield of 9.8-15.5 g in each liter of culture medium, and is obviously higher than that of the reported strain. The invention overcomes the restriction of low prodigiosin yield of the serratia marcescens strain and has wide application prospect in the medical market.

Detailed Description

The invention discloses an application of serratia marcescens ITBB B5-1 in prodigiosin production, and a person skilled in the art can use the contents to refer to the text and appropriately improve process parameters to realize the production. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The method for efficiently producing prodigiosin by using serratia marcescens ITBB B5-1 comprises the following steps:

1. preparing a seed solution: activating Serratia marcescens ITBB B5-1 in LB culture solid medium in the dark at 25 deg.C for 2d, inoculating single colony in liquid culture medium containing 100mL tryptone 15-25g/L, yeast extract 10-15g/L, sodium chloride 5g/L, pH value 7.0 at 25 deg.C under the dark at 250rpm, shaking for culturing for 12-18h to obtain bacterial liquid OD₆₀₀0.8-1.0 as seed liquid.

2. Fermentation conditions are as follows: inoculating the seed liquid into a culture medium containing tryptone 15-25g/L, yeast extract 10-15g/L and sodium chloride 5g/L, pH with the value of 7.0 according to the proportion of 3-5%, wherein the liquid loading amount is 300mL/1000mL, the shake flask culture is carried out at 250rpm under the dark condition, the fermentation temperature is 22-28 ℃, and the fermentation time is 48-96 h.

3. And (3) detecting the yield of prodigiosin of fermentation liquor: centrifuging 1mL of fermentation liquid in 12000g of a 1.5mL centrifuge tube for 5min, discarding the supernatant, adding 1mL of acidified ethanol (4% (v/v)1M hydrochloric acid in 95% ethanol) to resuspend the thallus precipitate to extract thallus prodigiosin, shaking vigorously for 30s, centrifuging 12000g for 5min, collecting the supernatant in a new centrifuge tube, and measuring the light absorption value at the wavelength of 536 nm. Preparing a standard solution from a prodigiosin standard substance, diluting the standard solution into different concentration gradients, and measuring a light absorption value at a wavelength of 536nm to prepare a standard curve. And calculating the yield of prodigiosin in the fermentation liquor through a standard curve.

Centrifuging fermentation liquor at 12000g for 5min to collect thalli, adding 5 times volume of absolute ethyl alcohol to resuspend the thalli, violently shaking for 60s, centrifuging at 12000g to collect supernatant, adding 5 times volume of ethyl acetate to precipitate for leaching, and centrifuging at 12000g to collect supernatant; respectively drying the supernatants, adding methanol for redissolution, adding acetone: ethyl acetate 95: and 5, performing chromatography by using a 200-mesh silica gel column as an eluent to obtain a red component, collecting the component, eluting by using methanol, and drying to finally obtain the purified prodigiosin. The purified Prodigiosin was analyzed by LC-ESI-MS/MS and Q-TOF MS and found to be purified Prodigiosin (Prodigiosin).

The reagent or the instrument used in the application of the serratia marcescens ITBB B5-1 in the prodigiosin production can be purchased from the market.

The invention is further illustrated by the following examples: