A paracoccus marcusii for biosynthesis of astaxanthin, pigment extract and application thereof

Abstract

This invention discloses a *Paragonimus westermani* strain that biosynthesizes astaxanthin, its pigment extract, and its applications. This *Paragonimus westermani* (… Paracoccus marcusii IHA069 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 15, 2022, with accession number CGMCC No. 25302. Its 16S rDNA sequence is shown in SEQ ID No. 1. After culturing in fermentation medium for 48 hours, the bacterial cells were centrifuged and extracted with anhydrous ethanol to obtain a pigment extract, with astaxanthin accounting for 40% of the extract. Using *Paragonimus westermani* pigment extract as the main ingredient, and corn starch, Tween-80, and calcium chloride solution as auxiliary ingredients, the mixture was spray-dried to obtain nanoparticles with antioxidant effects. Compared to IHA034, IHA069 significantly increased astaxanthin yield, facilitating industrial production and improving its feasibility for use in different animals. This invention utilizes nanoparticle encapsulation technology to improve the retention rate of astaxanthin in the pigment extract, and to extend its shelf life and effective storage temperature.

Description

Technical Field

[0001] This invention relates to the fields of microbial engineering technology and aquaculture, specifically to a strain of Paracoccus martensii that biosynthesizes astaxanthin, its pigment extract, and its applications. Background Technology

[0002] Astaxanthin (AXT) is widely used in food, cosmetics, pharmaceuticals, and animal feed due to its anti-inflammatory, antioxidant, and anti-proliferative properties. It is known that algae such as Haematococcus pluvialis and Chlorella, yeasts such as Rhodotorula fafusense, and bacteria such as Paracoccus and Agrobacterium can synthesize natural astaxanthin. Among these, algal astaxanthin has a levorotatory-trans structure, which not only has strong antioxidant capacity but is also a safe source, making it the only astaxanthin approved by the U.S. Food and Drug Administration (FDA) for food use. However, due to long production cycles, low yields, contamination of algal biomass, and easy oxidation during storage, even astaxanthin is expensive in the feed industry. Compared to the Haematococcus pluvialis system (average 2.7 mg / L),... -1 d -1 ) and the Rhodotorula fafeii system (5.2 mg L -1 d -1 In Europe, America, and Japan, *Paragonimococcus carotenoides* Paracoccus carotinifaciens The astaxanthin yield can reach approximately 863 mg / L after 2 days of fermentation. -1 The relevant products have been approved for marketing in the European and American markets as highly efficient feed astaxanthin, but there are currently no related products for paracoccal feed additives in China.

[0003] The applicant previously developed a strain of *Paragonimella masculinii*, named IHA034, which was deposited on July 15, 2022, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC. 25301, and has been granted Chinese patent ZL 2023 1 0536738.9. However, compared with foreign products, IHA034 has a low astaxanthin yield, with the bacterial culture yielding only 50 mg / L and the bacterial powder containing 4.94% astaxanthin. To improve the astaxanthin production capacity of the strain, the inventors conducted targeted screening of the strain, optimized the fermentation formula, and developed dosage form preparation and application technologies to improve the product's biosafety and expand its application scope. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a *Paracoccus martensii* strain for biosynthesizing astaxanthin, its pigment extract, and its applications.

[0005] Paracoccus martensii, a biosynthetic astaxanthin-producing bacterium, Paracoccus marcusiiIHA069 was deposited at the China General Microbiological Culture Collection Center on July 15, 2022, with accession number CGMCC No. 25302. The 16S rDNA sequence is shown in SEQ ID No. 1.

[0006] A pigment extract of Paracoccus maseri, using the aforementioned Paracoccus maseri. Paracoccus marcusii IHA069 was obtained by culturing in fermentation medium for 48 hours, centrifuging, and extracting the cells with anhydrous ethanol. The proportion of astaxanthin in the pigment extract reached 40%.

[0007] The fermentation medium is prepared and sterilized by mixing soybean flour, yeast powder, sucrose, calcium chloride, magnesium sulfate, ferrous sulfate, potassium dihydrogen phosphate, and sodium citrate in the following mass percentages: 0.4%, 0.6%, 1.0%, 0.20%, 0.2%, 0.02%, 0.01%, and 0.02%, respectively, with the balance being water.

[0008] Paracoccus martensii Paracoccus marcusii After the IHA069 monoclonal strain was inoculated into the fermentation medium, it was cultured at 28℃ and 200 r / min for 48 hours. The bacterial precipitate was obtained by centrifugation at 6000 r / min. Anhydrous ethanol was added at a mass:volume ratio of 1:10, and the mixture was sonicated at 100 Hz for 10 minutes. The supernatant was collected by centrifugation and treated at 60℃ until the ethanol was completely evaporated, finally obtaining the pigment extract, in which astaxanthin accounted for more than 50% of the total pigment.

[0009] A nanoparticle containing a Marsupialia paraglobin extract is prepared by mixing and spray drying with the Marsupialia paraglobin extract as the main ingredient, corn starch, Tween-80, and calcium chloride solution as auxiliary ingredients to obtain nanoparticles with antioxidant effects.

[0010] A method for preparing the aforementioned Marsepia spheroid pigment extract nanoparticles involves weighing 20g of corn starch and measuring 200mL of water; heating the water to 80℃, adding the starch, and stirring; raising the temperature to 82-85℃, and magnetically stirring at 400-500 r / min for 20 minutes; cooling the starch matrix to 35-40℃, dissolving the pigment extract in 25mL of ethanol at a concentration of 1 g / mL, and preheating to 30℃; preparing a high-speed dispersion device; starting the high-speed dispersion at 8000rpm, and continuously introducing nitrogen gas; slowly adding the solution dropwise at a rate of 2 mL / min, emulsifying for 10-15 minutes; cooling to 30℃, and stirring under reduced pressure; evaporating for 15-20 minutes; preparing 50mL of 2% calcium chloride solution, cooling the system temperature to room temperature, slowly adding the calcium chloride solution dropwise, stirring at 200 r / min, and reacting for 20-25 minutes; drying in a spray dryer, collecting and passing through a 100-mesh sieve to obtain nanoparticles of 20-100 nm; the astaxanthin content in the nanoparticles reaches 5.0%.

[0011] A feed that can enhance the activity of antioxidant enzymes in the serum of small yellow croaker and sea bass contains 0.2% by weight of the Paracoccus masculinii extract nanoparticles.

[0012] Beneficial effects of this invention:

[0013] 1. Compared to IHA034, IHA069 enhances astaxanthin production and facilitates industrial production.

[0014] 2. The pigment extracts are mainly astaxanthin and lycopene, which can activate the antioxidant capacity of intestinal cells in aquatic animals.

[0015] 3. The pigment extract isolates beneficial components, improving its feasibility for use in different farmed animals.

[0016] 4. To address the issue of astaxanthin's susceptibility to oxidation in exposed environments, this invention employs nanoparticle encapsulation technology to improve the retention rate of astaxanthin, extend the storage temperature and effective time range, and facilitate its use. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Comparison of astaxanthin production between Paracoccus martensii strains IHA069 and IHA034

[0019] In this embodiment of the invention, two strains of *Paragonimella maseri* were used: IHA034 and IHA069. The culture medium used was a first-generation fermentation medium, namely: 4 g peptone, 6 g yeast extract, 10 g glucose, 2 g calcium chloride, 0.72 g magnesium sulfate, 0.05 g ferrous sulfate, and 0.1 g potassium hydrogen phosphate were added to 1 L of water. After sterilization at 120℃ for 30 min, a single clone was inoculated and cultured at 28℃ for 72 hours. After centrifugation at 6000 rpm for 25 minutes, the precipitate was washed with distilled water and then freeze-dried at -40℃ to obtain freeze-dried powder 2.

[0020] Table 1 shows the results obtained after weighing and testing the astaxanthin content of two types of lyophilized bacterial powders, IHA034 and IHA069.

[0021] Table 1 Comparison of biomass and astaxanthin content in freeze-dried bacterial powder

[0022]

[0023] The results showed that, compared with IHA034, IHA069 not only had a 3-fold increase in dry weight after fermentation, but also a 64% increase in astaxanthin content in the freeze-dried powder.

[0024] Example 2: Effect of optimized fermentation medium on astaxanthin content in IHA069

[0025] This invention compares the effects of two culture media on the astaxanthin yield of IHA069. The freshwater culture medium consisted of 4 g peptone, 6 g yeast extract, 10 g glucose, 2 g calcium chloride, 0.72 g magnesium sulfate, 0.05 g ferrous sulfate, and 0.1 g potassium hydrogen phosphate in 1 L of water. The second-generation culture medium consisted of 4.0 g soybean flour, 6.0 g yeast extract, 10 g sucrose, 2.0 g calcium chloride, 2.0 g magnesium sulfate, 0.2 g ferrous sulfate, 0.1 g potassium dihydrogen phosphate, and 0.2 g trisodium citrate in 1 L of water. After inoculating the monoclonal strain into the fermentation medium, the cultures were incubated at 26–28 °C and 200 r / min for 48 hours. The precipitate obtained by centrifugation at 6000 r / min was frozen and freeze-dried at -40 °C to obtain freeze-dried powder. The astaxanthin content of the two freeze-dried powders, IHA034 and IHA069, was weighed and measured, as shown in Table 2.

[0026] Table 2 Comparison of bacterial cell lyophilized powder biomass and astaxanthin content after culture medium optimization.

[0027]

[0028] The results showed that after the culture medium was optimized, the bacterial dry weight yield after 48 hours was more than twice that of the bacterial dry weight yield after 72 hours in the first-generation culture medium, and the astaxanthin content reached 7.82%.

[0029] Example 3: Component Analysis of Pigment Extract from Paracoccus martensii IHA069

[0030] In this invention, the carotenoid content of the pigment product of IHA069 cultured in second-generation medium for 48 hours was detected. 4.0 g soybean powder, 6.0 g yeast powder, 10 g sucrose, 2.0 g calcium chloride, 2.0 g magnesium sulfate, 0.2 g ferrous sulfate, 0.1 g potassium dihydrogen phosphate, and 0.2 g trisodium citrate were added to 1 L of water. After inoculating the monoclonal strain into the fermentation medium, it was cultured at 28°C and 200 r / min for 48 hours, and the precipitate was obtained by centrifugation at 6000 r / min. Anhydrous ethanol was added at a mass:volume ratio of 1:10, and the mixture was sonicated at 100 Hz for 10 minutes. The supernatant was collected by centrifugation and treated with a rotary evaporator at 60℃ until the ethanol was completely evaporated to obtain the pigment extract. This extract was then dissolved in anhydrous ethanol at a mass:volume ratio of 1.0 mg:1.0 ml. 1 mL of the resulting solution was lyophilized, diluted 1000 times, and extracted with a hexane / acetone / ethanol mixture containing 0.01% dibutylhydroxytoluene (volume ratio 1:1:1). The mixture was vortexed at room temperature for 20 min, centrifuged at 12000 r / min for 5 min at 4℃, and the supernatant was discarded. The extraction was repeated once, and the supernatants were combined by centrifugation to obtain the concentrated extract. This concentrated extract was redissolved in 100 μL of dichloromethane, filtered through a 0.22 μm filter membrane, and analyzed by liquid chromatography-tandem mass spectrometry. The results are shown in Table 3.

[0031] Table 3. Carotenoid content in extracts

[0032]

[0033] The results showed that the main pigment components in the extract were astaxanthin (content of 4.08 μg / mg) and phytoene (content of 3.07 μg / mg), with astaxanthin accounting for >50%.

[0034] Example 4: Effect of incubation of pigment extract on the antioxidant capacity of intestinal cells of small yellow croaker

[0035] Using isolated intestinal epithelial cells of small yellow croaker as the research object, the pigment extract was dissolved in dimethyl sulfoxide at a concentration of 4 mg / mL, and co-incubated with the intestinal epithelial cells at a final concentration of 7.5 μg / mL for 4 hours. After that, the cell culture medium was changed, and hydrogen peroxide was added to a concentration of 700 μM / mL. The results are shown in Table 4.

[0036] Table 4. Effects of pigment extracts on the antioxidant capacity of intestinal cells of small yellow croaker

[0037]

[0038] The results showed that hydrogen peroxide treatment reduced the activity of cellular antioxidant enzymes and increased intracellular malondialdehyde content, leading to cell growth inhibition. In contrast, cells incubated with pigment extracts exhibited stronger cellular antioxidant enzyme activity, thus resisting the damage caused by hydrogen peroxide.

[0039] Example 5: Storage stability and DPPH free radical scavenging ability test of pigment extract nanoparticles

[0040] The prepared pigment extract nanoparticles were dissolved in distilled water at a concentration of 5.0 mg / mL and stored at 4℃, 25℃, and 37℃. Astaxanthin retention and DPPH free radical scavenging ability were tested on days 5, 10, and 15. Retention rate test: 1 mL of nanoparticle solution was added to 5 mL of anhydrous ethanol, vortexed for 60 s, and then ultrasonically extracted from the particles using a frequency of 50 Hz. The mixture was centrifuged at 1000 r / min for 10 min, and the supernatant was collected. This process was repeated until the supernatant was colorless, and the absorbance of astaxanthin was measured at 476 nm. DPPH free radical scavenging ability test: 200 μL of astaxanthin nanoparticles were mixed with 800 μL of 0.2 mmol / L DPPH solution, reacted in the dark for 30 min, and the absorbance was measured at 517 nm. The results are shown in Table 5.

[0041] Table 5. Astaxanthin retention rate and DPPH free radical scavenging ability of pigment extracts at different times

[0042]

[0043] It is evident that after encapsulation with nanoparticles, the astaxanthin retention rate is better at 4℃, and even after storage at 25℃ for 15 days, the astaxanthin retention rate is still 70% and the DPPH free radical scavenging rate is still over 70%.

[0044] Example 6 Effect of feeding nanoparticles on serum antioxidant enzyme activity in small yellow croaker

[0045] Juvenile yellow croaker weighing approximately 30g were used as the subjects for feeding. Three experimental groups (control group, experimental group 1, and experimental group 2) were designed, with three replicates per group and 50 fish per replicate. The control group was fed a basal diet, experimental group 1 was fed 2g / kg of Haematococcus pluvialis additive (10% astaxanthin content), and experimental group 2 was fed 4g / kg of pigment extract nanoparticles. The feeding period was 60 days. At the end of the experiment, the serum antioxidant enzyme activity of the yellow croaker was measured, and the results are shown in Table 6.

[0046] Table 6. Effects of feeding extract nanoparticles on serum antioxidant enzyme activity in small yellow croaker.

[0047]

[0048] The results showed that feeding astaxanthin freeze-dried powder could increase the activity of antioxidant enzymes in the serum of small yellow croaker.

[0049] Example 7: Effect of feeding nanoparticles on the antioxidant capacity of juvenile bass serum

[0050] Juvenile largemouth bass weighing approximately 14 g were used as the feeding subjects. Feeding groups were designed with 0.0 g / kg, 1.0 g / kg, 2.0 g / kg, and 4.0 g / kg nanoparticles, with three replicates per group and 10 fish per replicate. The feeding period was 60 days. At the end of the experiment, the activity of antioxidant enzymes in the bass serum was measured. The results are shown in Table 7.

[0051] Table 7. Effects of feeding extract nanoparticles on serum antioxidant enzyme activity in juvenile largemouth bass

[0052]

[0053] The results of serum antioxidant enzyme activity are shown in Table 4. It can be seen that the nanoparticles of Paracoccus martensii pigment extract effectively increased the activity of three antioxidant enzymes in the serum of juvenile sea bass.

[0054] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the design concept of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalent technical solutions.

Claims

1. A strain of *Paracoccus martensii* that biosynthesizes astaxanthin, characterized in that: Paracoccus martensii ( Paracoccus marcusii IHA069 was deposited at the China General Microbiological Culture Collection Center on July 15, 2022, with accession number CGMCC No. 25302. The 16S rDNA sequence is shown in SEQ ID No.

1.

2. A pigment extract of Paracoccus masculinus, characterized in that: Using Paracoccus maseri as described in claim 1 Paracoccus marcusii IHA069 was obtained by culturing in fermentation medium, centrifuging, and extracting the cells with anhydrous ethanol. The fermentation medium is prepared and sterilized by mixing soybean flour, yeast powder, sucrose, calcium chloride, magnesium sulfate, ferrous sulfate, potassium dihydrogen phosphate, and sodium citrate in the following mass percentages: 0.4%, 0.6%, 1.0%, 0.20%, 0.2%, 0.02%, 0.01%, and 0.02%, respectively, with the balance being water. Paracoccus martensii Paracoccus marcusii After the IHA069 monoclonal strain was inoculated into the fermentation medium, it was cultured at 28℃ and 200 r / min for 48 hours. The bacterial precipitate was obtained by centrifugation at 6000 r / min. Anhydrous ethanol was added at a mass:volume ratio of 1:10, and the mixture was sonicated at 100 Hz for 10 minutes. The supernatant was collected by centrifugation and treated at 60℃ until the ethanol was completely evaporated, finally obtaining the pigment extract, in which astaxanthin accounted for more than 50% of the total pigment.

3. A nanoparticle containing a pigment extract from Paracoccus masculinii, characterized in that: Using the *Paragonimus maseri* pigment extract as the main ingredient, and corn starch, Tween-80, and calcium chloride solution as auxiliary ingredients, the mixture is spray-dried to obtain nanoparticles with antioxidant effects.

4. A method for preparing Paracoccus maseri pigment extract nanoparticles as described in claim 3, characterized in that: Weigh 20g of corn starch and measure 200mL of water; heat the water to 80℃, add the starch, and stir; raise the temperature to 82-85℃, and magnetically stir at 400-500 r / min for 20 minutes; cool the starch matrix to 35-40℃, dissolve the pigment extract in 25mL of ethanol at a concentration of 1 g / mL, and preheat to 30℃; prepare a high-speed dispersion device; turn on the high-speed dispersion at 8000rpm, and continuously purge with nitrogen; slowly add the solution dropwise at a rate of 2 mL / min, and emulsify for 10-15 minutes; cool to 30℃ and stir under reduced pressure; The mixture was allowed to evaporate for 15-20 minutes; 50 mL of 2% calcium chloride solution was prepared, the system temperature was lowered to room temperature, and the calcium chloride solution was slowly added dropwise while stirring at 200 r / min for 20-25 min; the mixture was dried in a spray dryer and collected through a 100-mesh sieve to obtain nanoparticles of 20-100 nm; the content of astaxanthin in the nanoparticles reached 5.0%.

5. A feed that can increase the activity of serum antioxidant enzymes in small yellow croaker and sea bass, characterized in that: It contains 0.2% by weight of the Paracoccus masculinii extract nanoparticles as described in claim 3.

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