A microbial oil rich in Sn-2 DHA and its preparation method and application

Abstract

The present invention belongs to the technical field of oil and fat nutritional products, and relates to a microbial oil rich in Sn-2 DHA and its preparation method and application. The microbial oil is prepared by fermentation of a Schizochytrium strain, and the Schizochytrium strain is Schizochytrium sp. D2.0, and its deposit number is CCTCCNO: M20221481. The proportion of DHA at the Sn-2 position of triglycerides in the microbial oil prepared by the present invention is not less than 50%, and the content of triglyceride DHA is not less than 58%, and it has good stability and nutritional value.

Description

Technical Field

[0001] The present invention belongs to the technical field of oil and fat nutritional products, and in particular, relates to a microbial oil rich in Sn-2 DHA and a preparation method and application thereof. Background Art

[0002] In recent years, with the development of the demand for breast milk powder production, people have studied the fat composition of breast milk and found that breast milk contains 3% to 5% fat, of which glycerides account for more than 98%, saturated fatty acids account for 28.61% to 48.55%, and polyunsaturated fatty acids account for 15.47% to 27.31%. Most of the saturated fatty acids are palmitic acid, accounting for about 66%; the fat in the Sn-2 position is mostly palmitic acid, accounting for 62.01% to 77.27%. The study also found that the fatty acids in the Sn-2 position are more easily absorbed by infants and young children, meeting their production and development needs.

[0003] Docosahexaenoic acid (DHA) is an essential fatty acid that is indispensable for the production and development of infants. DHA algae oil usually exists in the form of triglycerides, and the absorption rate of the human body is about 50%. In order to improve the absorption rate of DHA oil in the human body, CN109247397 is enriched by distillation, enzyme method, and urea encapsulation method through hydrolysis (alkali method, enzyme method, acid method, high temperature and high pressure), and then purified by refining process, and Sn-2 DHA is obtained, and the content is 35% to 42%. The Sn-2 DHA obtained by this method solves the problem of easy oxidation of DHA, so that the bioavailability of DHA is greatly improved, but the process involved in this method is complicated. In addition, the new resource food announcement (Announcement No. 3 in 2010) stipulates that DHA algae oil must be a biological fermentation source, and the national standard of docosahexaenoic acid (DHA) oil also stipulates that its source should be a fermentation source, so the application of Sn-2 DHA prepared by enzyme method will be limited, causing it to be unable to be applied to infant formula.

[0004] CN110846346A discloses a method for preparing a microbial oil rich in Sn-2 DHA, which is prepared by inoculating Schizochytrium strain GDMCC No.60733 into a fermentation medium for fermentation. However, the proportion of DHA at the Sn-2 position of triglycerides in the microbial oil is about 43%, which needs to be further improved. Summary of the invention

[0005] In view of the deficiencies of the prior art and the requirements and restrictions of relevant regulations, the object of the present invention is to provide a DHA oil and a preparation method thereof that fully complies with regulatory requirements. The DHA oil prepared by the method is rich in Sn-2 DHA, wherein the proportion of DHA at the Sn-2 position of triglycerides is not less than 50%, and the content of triglyceride-type DHA is not less than 58%.

[0006] In order to achieve the above-mentioned technical purpose, the inventors conducted a large number of experimental studies and made unremitting efforts, and finally realized the industrial production of Sn-2 docosahexaenoic acid (DHA) oil through strain mutagenesis, fermentation culture and oil extraction, and obtained the following technical scheme: a microbial oil rich in Sn-2 DHA, the microbial oil is fermented by a Schizochytrium strain, the Schizochytrium strain is Schizochytrium sp. D2.0, which has been deposited in the China Center for Type Culture Collection (address: Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China) on September 23, 2022, and its preservation number is CCTCC NO: M20221481.

[0007] Secondly, the present invention also provides a method for preparing a microbial oil rich in Sn-2 DHA, which comprises preparing a microbial oil rich in Sn-2 DHA by seed culture and fermentation culture of a Schizochytrium strain, wherein the Schizochytrium strain is Schizochytrium sp. D2.0, and its preservation number is CCTCC NO: M20221481.

[0008] Further preferably, in the method for preparing the microbial oil rich in Sn-2 DHA as described above, the seed culture is to activate the Schizochytrium strain to make a shake flask seed solution and inoculate it into a seed culture medium for culture; the culture medium for the seed culture is composed of: 28-32 g / L glucose, 14-16 g / L sodium glutamate, 4.5-5.5 g / L yeast powder, 2.5-3.5 g / L magnesium sulfate, 1.6-2.5 g / L potassium dihydrogen phosphate, and the pH is adjusted to 6.2-6.6; the seed culture is to inoculate the shake flask seed culture solution at an inoculation rate of 5-10%, a stirring speed of 80-120 r / min, and a ventilation volume of 0.2-0.6 m 3 / min, and the culture temperature was 25-28°C, and the first-level seed culture was carried out to make the Sn-2 DHA strain grow to the logarithmic phase to obtain the first-level seed liquid.

[0009] Further preferably, in the method for preparing the microbial oil rich in Sn-2 DHA as described above, the fermentation culture medium composition is: glucose 28-32 g / L / L, sodium glutamate 24-26 g / L, yeast powder 9-11 g / L, corn steep liquor powder 5-6 g / L, magnesium sulfate 4-5 g / L, potassium dihydrogen phosphate 1.5-2.3 g / L, and the pH is adjusted to 6.2-6.6.

[0010] Further preferably, in the method for preparing the microbial oil rich in Sn-2 DHA as described above, the fermentation culture conditions are as follows: the seeds are inoculated into a fermentation tank at a ratio of 5 to 10% for fermentation culture, the stirring speed is 50 to 70 r / min, the culture temperature is 25 to 28°C, and the ventilation volume is controlled at 180 to 260 m / s. 3 / h, the pH value of the fermentation liquid is controlled at 6.2-6.6 by intermittently adding ammonia water, the concentration of glucose is controlled at 25g / L-32g / L in the early stage of fermentation, and when the sugar concentration is lower than 25g / L, 50-60% glucose solution is used for sugar supplementation, and the flow addition of sugar is stopped in the late stage of fermentation until the glucose is completely consumed. The fermentation cycle is 135-140h.

[0011] Further preferably, the method for preparing the microbial oil rich in Sn-2 DHA as described above further comprises breaking the cell wall of the fermentation product to obtain the microbial oil.

[0012] Further preferably, in the method for preparing the microbial oil rich in Sn-2 DHA as described above, the method for cell wall separation is: after the fermentation culture is completed, the fermentation liquid is heated to 54-56°C, the pH is adjusted to 7.0-8.0, 2-5‰ alkaline protease is added, the mixture is kept warm and stirred for 4-8 hours, and microscopic examination is performed. After the cell wall is completely broken, the enzymatic hydrolysis is terminated, and the fermentation liquid is input into a three-phase centrifuge for oil separation to obtain a purely physically extracted Sn-2 DHA oil without solvent residue.

[0013] Further preferably, in the method for preparing the microbial oil rich in Sn-2 DHA as described above, the proportion of DHA at the Sn-2 position of triglycerides in the microbial oil is not less than 50%, and the content of triglyceride DHA is not less than 58%.

[0014] In addition, the present invention also provides a Schizochytrium sp. strain, which is Schizochytrium sp. D2.0. The strain is induced by EMS mutagenesis + ultraviolet irradiation. The specific method is as follows:

[0015] (1) Take OD 660 Add 0.8 mL of the prepared EMS stock solution to 20 mL of the 0.6-0.8 suspension of Schizochytrium, place in an incubator at 26-28°C and 120 rpm for mutagenesis for 40-60 minutes, take some of the mutagenesis solution and add it to the 5% sodium thiosulfate solution prepared in advance for 5 minutes, wash with PBS 2-3 times, dilute to an appropriate concentration, and apply to the selection medium.

[0016] (2) Select a 30W UV lamp and irradiate for 30 minutes in advance, and take the OD 66020 mL of bacterial suspension with a pH of 0.6 to 0.8 was placed in a flat-bottomed glass culture dish and stirred with a magnetic stirrer. The linear distance between the ultraviolet lamp and the culture dish was 30 cm. The bacterial solution irradiated for 10 to 30 seconds was appropriately diluted and spread on the inhibitory solid culture medium and cultured in the dark.

[0017] (3) Strain selection: The larger strains on the inhibition plate are selected as candidate strains for fermentation culture (the fermentation container can be a microtiter plate or a conical flask, and the fermentation volume can be minimized to improve the mutagenesis efficiency).

[0018] (4) Standard curve for determining oil content using vanillin phosphate: Take an appropriate amount of fermentation liquid (calculate the oil content in advance), dilute it several times (to keep the oil content within 0-10 g / L), and measure the OD 515 .

[0019] (5) Vanillin phosphate reaction method for rapid determination of bacterial lipid content: Take 0.5 mL of uniform fermentation liquid, add 1.5 mL of sterile water to wash, centrifuge at 8000 r / min for 5 min, and repeat twice. After dilution with 2 mL of sterile water, shake evenly, take 0.1 mL of bacterial suspension and place it in a 25 mL stoppered test tube (distilled water is the control sample), add 2 mL of 98% H2SO4, boil in boiling water for 10 min, and then bath in room temperature water for 5 min. Take out the stoppered test tube and add 5 mL of vanillin phosphate solution, shake evenly, bath in 37°C water for 15 min, and then bath in room temperature water for 10 min. Measure the OD value at 515 nm, and set the OD 515 The strains with larger values ​​and the strains with the highest DHA content at the Sn-2 position among the strains analyzed by DHA structure were preserved as high-yield strains with the preservation number of CCTCC M20221481.

[0020] Finally, the present invention provides the use of the above-mentioned microbial oil in food, and the food includes: infant formula food, special medical food, special diet, and health food.

[0021] Compared with the prior art, the microbial oil rich in Sn-2 DHA and the preparation method thereof provided by the present invention have the following advantages and significant progress:

[0022] (1) The microbial oil produced by fermentation of Schizochytrium sp. D2.0 obtained by mutation of the present invention has a ratio of DHA at the Sn-2 position of triglyceride of not less than 50% and a content of triglyceride-type DHA of not less than 58%, and has good stability and nutritional value.

[0023] (2) The present invention adopts a solvent-free extraction method for production, has higher food safety, and is suitable for the production and application of infant formula food, special medical food, special diet, health food, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The colony morphology of Schizochytrium schizochytrium;

[0025] Figure 2 The morphology of Schizochytrium under a microscope;

[0026] Figure 3 Gas chromatogram of the fatty acid composition of Sn-2 DHA oil. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below through specific embodiments. If no specific technical operation steps or conditions are specified in the examples, they are all carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0028] In addition, the test method for the DHA content at the Sn-2 position in the present invention is as follows:

[0029] 1) Weigh 0.1g of the sample into a 10mL centrifuge tube, add about 30mg of lipase (porcine pancreatic lipase) and 2mL of 1mol / L tris(hydroxymethyl)aminomethane buffer solution, shake evenly, then add 0.5mL of 1‰ sodium cholate solution and 0.2mL of calcium chloride solution, cover the stopper and shake carefully, immediately put the centrifuge tube into a 40℃ water bath, and keep shaking for 1min. Take out the centrifuge tube from the water bath, shake vigorously at 40℃ with an oscillator for 2min, immediately add 1ml of hydrochloric acid and 1ml of dichloromethane, shake vigorously with an oscillator, centrifuge, transfer the organic layer with a syringe, and filter it with a 0.45um filter membrane into the liquid chromatography sample for use.

[0030] 2) Liquid chromatography separation: mobile phase is acetonitrile (containing 0.1% glacial acetic acid): dichloromethane = 50:50; chromatographic column is amino column 4.6mm×250mm, 5um; column temperature 40℃; flow rate 1ml / min; evaporative light scattering detector, offset tube 70℃, nebulizing gas pressure 0.3Mpa.

[0031] 3) Separation of monoglycerides by chromatographic column: Inject 40ul of monoglyceride standard into the liquid chromatograph to determine the retention time of monoglyceride, then inject 40ul of sample into the liquid chromatograph and collect the monoglyceride fractions according to the retention time of monoglyceride.

[0032] 4) The collected monoglycerides were blown dry with a nitrogen blower, 1 ml of n-hexane was added to dissolve, 0.2 ml of potassium hydroxide methanol (2 mol / L) solution was added, and the mixture was shaken vigorously for 2 min. The mixture was allowed to stand for stratification, and the n-hexane layer was transferred to a gas chromatography sample bottle for later use.

[0033] 5) Determine fatty acids according to GB5009.168 method.

[0034] Example 1 Screening of strains for Sn-2 DHA oil production

[0035] The samples recovered from the beach humus in Bohai Bay were diluted and spread on solid culture medium (glucose 20.0 g / L, yeast extract 10.0 g / L, peptone 20.0 g / L, agar 20.0 g / L, sea crystal 5 g / L), and cultured at 27°C for 48 h to obtain multiple single colonies; then the colonies were picked into liquid culture medium (glucose 30 g / L, sodium glutamate 15 g / L, yeast powder 5 g / L, magnesium sulfate 3 g / L, potassium dihydrogen phosphate 2 g / L) for shake flask culture, the culture temperature was controlled at 27±0.5°C, the rotation speed was 150 r / min, the culture was carried out for 96 h, the fermentation was terminated, and the strain morphology (such as Figure 1 ), and the cell morphology observed under a microscope is that of a binary fission cell (e.g. Figure 2 ), and the peak time of Sn-2 DHA was consistent with the standard peak time (such as Figure 3 ). It was identified as Schizochytrium sp. by gene sequencing.

[0036] Example 2 Mutation of Sn-2 DHA oil production strains

[0037] (1) Take OD 660 20mL of 0.7 bacterial suspension, add 0.8mL of prepared EMS stock solution, put in an incubator at 26-28℃, 120rmp for mutagenesis, respectively for 40-60min, take some of the mutagenesis bacterial solution and add it to the 5% sodium thiosulfate solution prepared in advance for 5 minutes, wash with PBS 2-3 times. After diluting to an appropriate concentration, apply it to the selection medium.

[0038] (2) Select a 30W UV lamp and irradiate for 30 minutes in advance, and take the OD 660 20 mL of bacterial suspension with a concentration of 0.7 was placed in a flat-bottomed glass culture dish and stirred with a magnetic stirrer. The linear distance between the UV lamp and the culture dish was 30 cm. The bacterial suspension irradiated for 10 to 30 seconds was appropriately diluted and spread on the inhibitory solid culture medium and cultured in the dark.

[0039] (3) Strain selection: The larger strains on the inhibition plate are selected as candidate strains for fermentation culture (the fermentation container can be a microtiter plate or a conical flask, and the fermentation volume can be minimized to improve the mutagenesis efficiency).

[0040] (4) Standard curve for determination of oil content by vanillin phosphate: The fermentation broth was released and diluted several times (to keep the oil content within 0-10 g / L) to measure the OD 515 .

[0041] (5) Vanillin phosphate reaction method for rapid determination of bacterial lipid content: Take 0.5 mL of uniform fermentation liquid, add 1.5 mL of sterile water to wash, centrifuge at 8000 r / min for 5 min, and repeat twice. After dilution with 2 mL of sterile water, shake evenly, take 0.1 mL of bacterial suspension and place it in a 25 mL stoppered test tube (distilled water is the control sample), add 2 mL of 98% H2SO4, boil in boiling water for 10 min, and then bath in room temperature water for 5 min. Take out the stoppered test tube and add 5 mL of vanillin phosphate solution, shake evenly, bath in 37°C water for 15 min, and then bath in room temperature water for 10 min. Measure the OD value at 515 nm, and set the OD 515 The strains with larger values ​​and the strains with high DHA content at the Sn-2 position in the DHA structure analysis were preserved as high-yield strains with the preservation number: CCTCC M20221481.

[0042] Example 3 Sn-2 DHA oil production

[0043] 1. Preparation of seed culture medium: glucose 30g / L, sodium glutamate 15g / L, yeast powder 5g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 2g / L;

[0044] 2. Preparation of fermentation medium: glucose 30g / L, sodium glutamate 25g / L, yeast powder 10g / L, corn syrup powder 6g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 2g / L

[0045] 3. Seed culture: Add 10% of the shake flask seed culture solution into the primary fermentation seed tank, stir at a speed of 100 r / min, and a ventilation volume of 0.4 m 3 / min, and the culture temperature was 26.5°C, and the first-level seed culture was carried out to make the Sn-2 DHA strain grow to the logarithmic phase, or to obtain the first-level seed liquid.

[0046] 4. Fermentation culture: Inoculate the first-grade seed liquid into the fermentation tank at a ratio of 10% for fermentation culture. During the culture process, the stirring speed is 70r / min, the culture temperature is 27.5℃, and the ventilation volume is controlled at 200m 3 / h, the pH of the fermentation liquid is controlled at 6.5 by intermittently adding ammonia water. The glucose concentration is controlled at 25g / L-32g / L in the early stage of fermentation. When the sugar concentration is lower than 25g / L, 50-60% glucose solution is used for sugar supplement. After 90h of fermentation, the flow addition of sugar is stopped until the glucose is completely consumed. The fermentation cycle is 135h.

[0047] 5. The oil extraction: After the fermentation culture is completed, the fermentation liquid is heated to 55°C, the pH is adjusted to 7.5, 5‰ alkaline protease is added, and the mixture is stirred for 8 hours under a microscope. After the cell wall is completely broken, the enzymolysis is terminated. The fermentation liquid is input into a three-phase centrifuge for oil separation to obtain a pure physically extracted Sn-2 DHA oil without solvent residue. The fermentation results (Sn-2DHA content, DHA content, total oil) are shown in Table 1.

[0048] Comparative Example 1 Fermentation production of Sn-2 DHA by different strains

[0049] 1. Another strain of Schizochytrium (strain collection number: CCTCC M2013459; patent publication number: CN103602591A) independently developed by our company is used as the strain to produce Sn-2 DHA.

[0050] 2. Preparation of seed culture medium: glucose 30g / L, sodium glutamate 15g / L, yeast powder 5g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 2g / L

[0051] 3. Preparation of fermentation medium: glucose 100g / L, sodium glutamate 25g / L, yeast powder 10g / L, corn syrup powder 6g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 2g / L

[0052] 4. Seed culture: Add 10% of the shake flask seed culture solution into the primary fermentation seed tank, stir at a speed of 100 r / min, aeration of 0.4 m3 / min, and culture at a temperature of 26.5°C to carry out primary seed culture until the Sn-2 DHA strain grows to the logarithmic phase, or obtain the primary seed solution.

[0053] 5. Fermentation culture: Inoculate the first-grade seed liquid into the fermentation tank at a ratio of 10% for fermentation culture. During the culture process, the stirring speed is 70r / min, the culture temperature is 27.5℃, and the ventilation volume is controlled at 200m 3 / h, the pH of the fermentation liquid is controlled at 6.5 by intermittently adding ammonia water. The glucose concentration is controlled at 30g / L in the early stage of the fermentation process. When the sugar concentration is too low, 50-60% glucose solution is used for sugar supplement. After 90h of fermentation, the flow addition of sugar is stopped until the glucose is completely consumed. The fermentation cycle is 135h.

[0054] 6. The oil extraction: After the fermentation culture is completed, the fermentation liquid is heated to 55°C, the pH is adjusted to 7.5, 5‰ alkaline protease is added, and the mixture is stirred for 8 hours under a microscope. After the cell wall is completely broken, the enzymolysis is terminated. The fermentation liquid is input into a three-phase centrifuge for oil separation to obtain a pure physically extracted Sn-2 DHA oil without solvent residue. The fermentation results (Sn-2DHA content, DHA content, total oil) are shown in Table 1.

[0055] Table 1 Fermentation results (Sn-2DHA content, DHA content, total oil)

[0056]

[0057] It can be seen from the above data that the content of Sn-2DHA produced by the induced strains of the present invention is significantly higher than that of other strains. Example 4 In vitro simulated gastrointestinal digestion test of Sn-2 DHA

[0058] 2g of DHA oil produced in Example 3 and DHA oil produced in Comparative Example 1 were mixed with 50ml of simulated gastric juice and placed in a 250ml enzyme reactor, the pH was adjusted to 6.0, kept at 37°C for 10min, and then 16mg of pepsin and 50mg of Rhizopus oryzae lipase were added, mixed evenly and reacted for 1h, during which time 0.2mol / L NaOH was used to titrate the system to keep the pH at 6. 25ml of the gastric juice reacted above was mixed with 25ml of simulated intestinal juice and the pH was adjusted to 7.5, 250mg of bile salt and 80mg of pancreatin were added, reacted at 37°C for 2h, titrated with 0.2mol / L NaOH to keep the pH of the system at 7.5, and samples were taken every 20min to detect the metabolic absorption rate of fatty acids. The specific test data are shown in Table 2.

[0059] Table 2 Metabolic absorption rate of different DHA in gastrointestinal digestion

[0060]

[0061] The above is only a preferred specific implementation method of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes within the technical scope disclosed by the present invention according to the technical scheme and inventive concept of the invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a microbial oil rich in Sn-2 DHA, characterized in that: The method comprises the steps of: preparing a microbial oil rich in Sn-2 DHA by seed culture and fermentation culture of a Schizochytrium strain, wherein the Schizochytrium strain is Schizochytrium ( Schizochytrium sp. )D2.0, and its deposit number is CCTCC NO: M20221481.

2. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 1, characterized in that: The seed culture is to make a shake flask seed solution after activating the Schizochytrium strain and inoculate it into a seed culture medium for culture; the culture medium composition of the seed culture is: 28-32 g / L glucose, 14-16 g / L sodium glutamate, 4.5-5.5 g / L yeast powder, 2.5-3.5 g / L magnesium sulfate, 1.6-2.5 g / L potassium dihydrogen phosphate, and the pH is adjusted to 6.2-6.6; the culture conditions of the seed culture are: stirring speed 80-120 r / min, ventilation volume 0.2-0.6 m 3 / min, temperature 25~28℃.

3. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 1, characterized in that: The culture medium for the fermentation culture is composed of 28-32 g / L / L glucose, 24-26 g / L sodium glutamate, 9-11 g / L yeast powder, 5-6 g / L corn steep liquor powder, 4-5 g / L magnesium sulfate, and 1.5-2.3 g / L potassium dihydrogen phosphate, and the pH is adjusted to 6.2-6.

6.

4. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 1, characterized in that: The fermentation culture conditions are as follows: stirring speed 50-70 r / min, culture temperature 25-28°C, ventilation volume 180-260 m 3 / h, fermentation cycle 135~140h, the pH of the fermentation liquid is controlled at 6.2~6.6 by intermittent addition of ammonia water, the glucose concentration is controlled at 25g / L~32g / L in the early stage of fermentation, and when the sugar concentration is lower than 25g / L, 50~60% glucose solution is used for sugar supplementation. In the late stage of fermentation, the flow addition of sugar is stopped until the glucose is completely consumed.

5. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 1, characterized in that: The method also includes breaking the cell wall of the fermented product to obtain microbial oil.

6. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 5, characterized in that: The cell wall breaking and separation method is: heating the fermentation liquid to 54-56°C, adjusting the pH to 7.0-8.0, adding 2-5‰ alkaline protease, keeping warm and stirring for 4-8 hours, stopping the enzymatic hydrolysis after the cell wall is completely broken, and inputting the fermentation liquid into a three-phase centrifuge for oil separation to obtain microbial oil rich in Sn-2 DHA.

7. The method for preparing the microbial oil rich in Sn-2 DHA according to claim 1, characterized in that: The proportion of DHA at the Sn-2 position of triglycerides in the microbial oil is not less than 50%, and the content of triglyceride-type DHA is not less than 58%.

8. A Schizochytrium strain, which is Schizochytrium ( Schizochytrium sp. )D2.0, the deposit number is CCTCCNO: M20221481.

Images