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CN-120738011-B - Paracoccus martensii for biosynthesis of astaxanthin, pigment extract thereof and application thereof

CN120738011BCN 120738011 BCN120738011 BCN 120738011BCN-120738011-B

Abstract

The invention discloses a paracoccus equi and a pigment extract and application thereof for biosynthesis of astaxanthin. The Paracoccus martensii (Paracoccus marcusii) IHA069 is preserved in China general microbiological culture Collection center (CGMCC No. 25302) in the year 7 and 15 of 2022, and the 16s rDNA sequence is shown as SEQ ID No. 1. Culturing in fermentation medium for 48 hr, centrifuging, and extracting thallus with absolute ethanol to obtain pigment extract, wherein astaxanthin content in pigment extract is 40%. The marsupium paratungual pigment extract is used as a main material, corn starch, tween-80 and calcium chloride water are used as ingredients, and the nano particles with the antioxidation effect are obtained through blending and spray drying. Compared with IHA034, IHA069 strengthens the improvement of astaxanthin yield, is convenient for industrial production and improves the feasibility of use in different animals. The invention adopts the nanoparticle embedding technology to improve the retention rate of astaxanthin in the pigment extract, and improves the preservation temperature and the effective time.

Inventors

  • ZHANG YU
  • LOU BAO
  • HUANG FUYONG

Assignees

  • 浙江省农业科学院

Dates

Publication Date
20260505
Application Date
20250529

Claims (5)

  1. 1. A paracoccus equi of biosynthesis astaxanthin is characterized in that the paracoccus equi (Paracoccus marcusii) IHA069 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 25302 and a 16s rDNA sequence shown as SEQ ID No.1 in 7-15 of 2022.
  2. 2. A paracoccus equi pigment extract, which is characterized in that the paracoccus equi Paracoccus marcusii IHA069 is adopted to culture by a fermentation medium, and then the paracoccus equi pigment extract is obtained by centrifugation and extraction of thalli by absolute ethyl alcohol; the fermentation culture medium is prepared by sterilizing soybean powder, yeast powder, sucrose, calcium chloride, magnesium sulfate, ferrous sulfate, monopotassium phosphate and sodium citrate according to the mass percentages of 0.4%, 0.6%, 1.0%, 0.20%, 0.2%, 0.02%, 0.01% and 0.02% respectively and the balance of water; Inoculating the monoclonal strain of the Paracoccus marmoreus Paracoccus marcusii IHA and 069 into a fermentation culture medium, culturing for 48 hours at 28 ℃ under 200 r/min, centrifuging 6000 r/min to obtain a bacterial precipitate, adding absolute ethyl alcohol according to the mass ratio of volume=1:10, performing ultrasonic treatment for 10 minutes at 100: 100 Hz, centrifuging to obtain a supernatant, and treating at 60 ℃ until the ethyl alcohol is completely volatilized, thereby finally obtaining a pigment extract, wherein astaxanthin accounts for more than 50% of the total pigment.
  3. 3. The paracoccus equi pigment extract nano-particle is characterized in that the paracoccus equi pigment extract as defined in claim 2 is used as a main material, corn starch, tween-80 and calcium chloride water are used as ingredients, and the nano-particle with an antioxidant effect is obtained by blending and spray drying.
  4. 4. A method for preparing the paracoccus martensii pigment extract nano-particles as claimed in claim 3, which is characterized by weighing 20g of corn starch, weighing 200mL of water, heating the water to 80 ℃, adding starch, stirring, heating the temperature to 82-85 ℃, magnetically stirring 400-500 r/min for 20 minutes, cooling the starch matrix to 35-40 ℃, dissolving the pigment extract in 25 mL ethanol according to the concentration of 1 g/mL, preheating to 30 ℃, preparing high-speed dispersing equipment, starting 8000rpm high-speed dispersing, continuing to introduce nitrogen, slowly dropwise adding the solution according to the speed of 2 mL/min, emulsifying for 10-15 minutes, cooling to 30 ℃, stirring under reduced pressure, volatilizing for 15-20 minutes, preparing 2% calcium chloride solution 50 mL, slowly dropwise adding calcium chloride solution at room temperature, stirring according to 200-r/min, reacting 20-25 min, drying by a spray dryer, collecting 100-mesh screens, obtaining the nano-particles of 20-100 nm, and enabling the astaxanthin content in the nano-particles to reach 5.0%.
  5. 5. A feed capable of improving the activity of antioxidant enzymes of serum of yellow croaker and weever, which is characterized by comprising 0.2% of paracoccus equi extract nano-particles according to claim 3.

Description

Paracoccus martensii for biosynthesis of astaxanthin, pigment extract thereof and application thereof Technical Field The invention relates to the technical field of microbial engineering and the field of aquaculture, in particular to a paracoccus malassezia for biosynthesis of astaxanthin, a pigment extract thereof and application thereof. Background Astaxanthin (Astaxanthin, AXT) is widely used in the fields of food, cosmeceuticals, medicine and animal feeds due to its anti-inflammatory, antioxidant and antiproliferative properties. It is known that natural astaxanthin can be synthesized by algae such as rhodococcus pluvialis and chlorella, yeasts such as rhodotorula freudenreichii and bacteria such as Paracoccus, agrobacterium and the like. Among them, astaxanthin from algae has a L-trans structure, is not only strong in antioxidant ability but also safe in origin, and thus is only approved by the U.S. Food and Drug Administration (FDA) as astaxanthin for food. But astaxanthin is expensive even in the feed field due to problems of long production cycle, low yield, pollution of algal biomass, easy oxidation during storage, etc. Compared to haematococcus pluvialis system (average 2.7 mg L -1d-1) and rhodotorula freudenreichii system (5.2 mg L -1 d-1). In European and Japanese areas, the yield of astaxanthin fermented by the paracoccus carotovorus Paracoccus carotinifaciens for 2 days can reach about 863 mg L -1, related products are approved to be marketed in European and Japanese markets and used as high-efficiency feed astaxanthin, but no paracoccus feed additive related products exist in China at present. The applicant developed a strain of Paracoccus malae earlier, named IHA034, which was deposited in China general microbiological culture Collection center (address: north Star Xway No. 1, 3 of the Korean area North Star of Beijing) on day 7 and 15 of 2022, with a deposit number of CGMCC.25301, and obtained a Chinese patent ZL 2023 1 05360338.9. But compared with foreign products, the IHA034 astaxanthin yield is lower, the astaxanthin yield of the bacterial liquid is only 50 mg/L, and the astaxanthin content in the bacterial powder is 4.94 percent. In order to improve the astaxanthin production capacity of the strain, the inventor performs directional screening and fermentation formula optimization on the strain, and develops preparation and application technologies of the dosage form so as to improve the biological safety of the product and expand the application range. Disclosure of Invention In order to overcome the defects of the prior art, the invention aims to provide paracoccus equi, pigment extract and application thereof for biosynthesis of astaxanthin. A Paracoccus marmoreus for biosynthesis of astaxanthin, paracoccus marmoreus (Paracoccus marcusii) IHA069 is preserved in China general microbiological culture Collection center (CGMCC No. 25302) in the year 2022 and the preservation number is shown as SEQ ID No. 1. The paracoccus malaise pigment extract is obtained by culturing paracoccus malaise Paracoccus marcusii IHA069,069 in a fermentation medium for 48 hours, centrifuging and extracting thalli by using absolute ethyl alcohol, wherein the proportion of astaxanthin in the pigment extract is up to 40 percent. The fermentation medium is prepared by sterilizing soybean powder, yeast powder, sucrose, calcium chloride, magnesium sulfate, ferrous sulfate, monopotassium phosphate and sodium citrate according to the mass percentages of 0.4%, 0.6%, 1.0%, 0.20%, 0.2%, 0.02%, 0.01% and 0.02% respectively and the balance of water. Inoculating the monoclonal strain of the Paracoccus marmoreus Paracoccus marcusii IHA and 069 into a fermentation culture medium, culturing for 48 hours at 28 ℃ under 200 r/min, centrifuging 6000 r/min to obtain a bacterial precipitate, adding absolute ethyl alcohol according to the mass ratio of volume=1:10, performing ultrasonic treatment for 10 minutes at 100: 100 Hz, centrifuging to obtain a supernatant, and treating at 60 ℃ until the ethyl alcohol is completely volatilized, thereby finally obtaining a pigment extract, wherein astaxanthin accounts for more than 50% of the total pigment. The nanometer particles of the martensii subspecies pigment extract take the martensii subspecies pigment extract as a main material, corn starch, tween-80 and calcium chloride water as ingredients, and are prepared by blending and spray drying to obtain the nanometer particles with the antioxidation effect. A preparation method of the marshy paratunguiculata pigment extract nanoparticle comprises the steps of weighing 20g of corn starch, weighing 200 mL g of water, heating the water to 80 ℃, adding starch, stirring, heating to 82-85 ℃, magnetically stirring 400-500 r/min for 20 minutes, cooling a starch substrate to 35-40 ℃, dissolving pigment extract in 25mL ethanol according to the concentration of 1 g/mL, preheating to 30 ℃, preparing high-speed dispersing equipment, starting 8000rpm f