A method for preparing and using hematin

Abstract

The application discloses a preparation method and application of hematin. The hematin is prepared from a fermentation culture of propionic acid bacteria (Corallicola sp.) SCSIO 13291 CGMCC No. 14538. The hematin is prepared from the propionic acid bacteria (Corallicola sp.) SCSIO 13291, and therefore can be applied to hematin-related drugs and food.

Description

Technical fields:

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing and applying heme. Background technology:

[0002] Heme is an iron-containing porphyrin derivative widely found in plants, animals, and microorganisms. Composed of protoporphyrin and a ferrous ion, it possesses various physiological functions, participating in vital life processes such as iron balance regulation, oxygen transport, and electron transfer. In the food industry, heme is commonly used as a safe and harmless colorant, replacing artificial pigments to maintain food color and enhance nutritional value. Based on this characteristic, heme has great potential in the production of artificial meat; adding exogenous heme can give artificial meat a more realistic meat color. Heme is also attracting widespread attention in the pharmaceutical industry. It can be used to produce drugs for treating hepatic hyperfunction, inflammation, and tumors; ferrous heme can be directly absorbed by the human body and used as a blood tonic. Currently, heme is mainly synthesized chemically or isolated from plant tissues and animal blood, but this method is costly, has low purity, and produces many byproducts. With the expansion of heme's applications, relying solely on chemical synthesis and extraction from plants and animals is clearly insufficient to meet market demand. Therefore, the production of heme using microorganisms is considered a highly feasible alternative method. Currently, the microbial synthesis of heme mainly utilizes Escherichia coli as a host bacterium through genetic engineering technology. Corynebacterium glutamicum, Aspergillus niger, and Bacillus subtilis can also serve as potential host bacteria for heme production. Summary of the Invention:

[0003] The first objective of this invention is to provide a method for preparing heme.

[0004] The method for preparing heme of the present invention is wherein the heme is prepared from the fermentation culture of Propionibacterium SCSIO 13291CGMCC No.14538.

[0005] The fermentation culture for preparing Propionibacterium SCSIO 13291 was obtained by fermentation culture using seawater tryptic soy peptone liquid medium as the fermentation medium.

[0006] Preferably, the seawater tryptone-soybean liquid culture medium contains 17g tryptone, 3.0g soybean peptone, 2.5g glucose, 5.0g sodium chloride, 2.5g K₂HPO₄, and the remainder is aged seawater, with a pH of 7.3. The seawater tryptone-soybean agar medium is prepared by adding agar to the seawater tryptone-soybean liquid culture medium, at a concentration of 15g / L.

[0007] Preferably, the fermentation culture is a bacterial cell.

[0008] Preferably, Propionibacterium SCSIO 13291 is activated by inoculating it onto seawater tryptic soy agar medium. Then, a single colony is picked and inoculated into an Erlenmeyer flask containing seawater tryptic soy liquid medium. The flask is placed on a shaker at 25°C and cultured at 160 rpm for 3 days to obtain the fermentation culture. The supernatant and bacterial cells in the fermentation culture are separated by centrifugation at 12,000 rpm at 4°C for 5 minutes. After removing the supernatant, the bacterial cells are resuspended in sterile deionized water and washed. After centrifugation at 12,000 rpm at 4°C for 5 minutes, the supernatant is discarded to obtain the bacterial cells.

[0009] A second objective of this invention is to provide the application of Propionibacterium SCSIO 13291 in the preparation of heme.

[0010] Preferably, the Propionibacterium SCSIO 13291 is a bacterial cell of Propionibacterium SCSIO 13291.

[0011] This invention prepares heme from Propionibacterium SCSIO 13291, and therefore it can be used in heme-related drugs and foods.

[0012] The present invention relates to *Corallicola* sp. SCSIO 13291, which was deposited on August 18, 2017, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 14538. This strain is disclosed in patent CN201711305261.4, entitled "A Propionibacterium and its Application in the Production of Folic Acid and Niacin". Attached image description:

[0013] Figure 1 It is a standard curve of heme concentration. Detailed implementation method:

[0014] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0015] Example 1:

[0016] Propionibacterium SCSIO 13291 was inoculated from 30% (w / v) glycerol cryopreservation tubes onto seawater tryptic soy agar medium (the medium formula is: 17g tryptic, 3.0g soy peptone, 2.5g glucose, 5.0g sodium chloride, 2.5g K2HPO4, 15g agar powder per liter, with the remainder being aged seawater filtered through a GF / F glass microfiber membrane; the preparation method is to mix all components evenly, adjust the pH to 7.3, and sterilize). After activation, single colonies were picked and inoculated into Erlenmeyer flasks, each containing 50 ml of seawater tryptic soy liquid medium (the medium formula is: 17g tryptic, 3.0g soy peptone, 2.5g glucose, 5.0g sodium chloride, 2.5g K2HPO4 per liter, with the remainder being aged seawater filtered through a GF / F glass microfiber membrane, pH 7.3). The culture was incubated on a shaker at 25°C for 3 days at 160 rpm to obtain the fermentation culture. The supernatant and bacterial cells were separated by centrifugation at 12,000 rpm for 5 minutes at 4°C. After removing the supernatant, the cells were resuspended in sterile deionized water and washed. The supernatant was discarded after centrifugation at 12,000 rpm for 5 minutes at 4°C, and the wet weight of the cells was weighed and recorded.

[0017] Resuspend 0.33 ± 0.03 g of bacterial cells in 500 μL of 20 mmol / L oxalic acid solution, then transfer to a 1.5 mL amber centrifuge tube, gently mix, and incubate at 4 °C for 16 hours. Next, add 500 μL of 2 mmol / L oxalic acid solution to the centrifuge tube and mix well. Transfer another 500 μL sample to another 1.5 mL amber centrifuge tube and heat in a 95 °C water bath for 30 minutes for the reaction, used for the detection of total fluorescence values ​​of porphyrin and heme. The remaining 500 μL sample is kept at room temperature for the detection of porphyrin concentration fluorescence values.

[0018] After the sample cooled to room temperature, it was centrifuged at 12,000 rpm for 5 minutes. 200 μL of the supernatant was then transferred to a 96-well plate with a black transparent bottom for fluorescence measurement. The detection conditions were an excitation wavelength of 400 nm and an emission wavelength of 620 nm. The difference between the two sets of fluorescence values ​​was calculated; this difference represents the fluorescence value corresponding to the heme concentration being measured.

[0019] Different concentrations of ferric chloride heme standard samples were prepared using 0.25% (w / v) Na₂CO₃ solution. The standard samples of different concentrations were mixed with 2 mol / L oxalic acid solution in a 1:1 ratio, and then heated in a 95°C water bath for 30 minutes. After the standard samples cooled to room temperature, 200 μL of the supernatant was transferred to a 96-well plate with a black transparent bottom for fluorescence measurement. The detection conditions were excitation wavelength 400 nm and emission wavelength 620 nm. A standard curve was plotted based on the fluorescence value and heme concentration, with fluorescence value on the ordinate and heme concentration on the abscissa. Figure 1 .

[0020] The difference in fluorescence values ​​obtained from the above experimental samples was used as the y-value. Substituting this value into the formula y = 1643.9x + 2229, the x-value was calculated, which is the heme concentration in the experimental sample. Then, based on the wet weight of the bacterial cells used for heme extraction, the heme content per gram of wet bacterial cells was calculated, resulting in 8.46 ± 1.43 micrograms / gram of wet bacterial cells.

Claims

1. A method for preparing heme, characterized in that, The heme mentioned is from Propionibacterium acnes ( Corallicola The fermentation culture of Propionibacterium SCSIO 13291 (sp.) was prepared by fermenting Propionibacterium SCSIO 13291 using seawater tryptone-soybean broth as the fermentation medium. The seawater tryptone-soybean broth contained 17 g tryptone, 3.0 g soybean peptone, 2.5 g glucose, 5.0 g sodium chloride, 2.5 g K2HPO4, and the remainder was aged seawater, with a pH of 7.

3. The fermentation culture consisted of bacterial cells, and the preservation number of Propionibacterium SCSIO 13291 was CGMCC No. 14538.

2. The preparation method according to claim 1, characterized in that, Propionibacterium SCSIO 13291 was activated by inoculating it onto seawater tryptic soy agar medium. Single colonies were then picked and inoculated into Erlenmeyer flasks containing seawater tryptic soy liquid medium. The flasks were placed on a shaker at 25°C and cultured at 160 rpm for 3 days to obtain the fermentation culture. The supernatant and bacterial cells were separated by centrifugation at 12,000 rpm at 4°C for 5 minutes. After removing the supernatant, the bacterial cells were resuspended in sterile deionized water and washed. The supernatant was then discarded after centrifugation at 12,000 rpm at 4°C for 5 minutes to obtain the bacterial cells. The seawater tryptic soy agar medium is seawater tryptic soy liquid medium with agar added.

3. The application of Propionibacterium SCSIO 13291 in the preparation of heme is to prepare heme using the preparation method described in claim 1, wherein the preservation number of Propionibacterium SCSIO 13291 is: CGMCC No.14538.

4. The application according to claim 3, characterized in that, The aforementioned Propionibacterium SCSIO 13291 is the bacterial cell of Propionibacterium SCSIO 13291.

Images