Preparation method of high phospholipid content krill oil and application thereof

Abstract

This invention discloses a method for preparing krill oil with high phospholipid content, comprising the following steps: 1) mixing Antarctic krill oil with 90% ethanol, adding lipase B to react, enzymatically hydrolyzing the triglycerides in the Antarctic krill oil to release free fatty acids, which then combine in situ with ethanol to form fatty acid ethyl esters; 2) removing the solvent by rotary evaporation under reduced pressure; 3) removing the fatty acid ethyl esters by molecular distillation. Through this method, an Antarctic krill oil concentrate with a phospholipid content of over 90% is obtained. The entire process effectively avoids the damage to phospholipids and active ingredients caused by high temperature and oxidation. This invention does not use potentially harmful organic substances in the preparation process, ensuring the product's safety and reliability, and its applicability in food or pharmaceutical preparation. The preparation process of this invention is simple and suitable for large-scale industrial application.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a method for preparing Antarctic krill oil. Background Technology

[0002] Commercially available Antarctic krill oil typically contains around 40% phospholipids (purity), does not contain ethyl acetate, and its neutral lipids are mainly triglycerides. The DHA / EPA in Antarctic krill oil exists partly as triglycerides and partly as phospholipids.

[0003] Methods for purifying Antarctic krill oil include solvent extraction and chromatography. Chromatographic purification typically involves loading the lipid extract onto a silica gel column and eluting with a solvent of increased polarity to remove neutral lipids and unreacted phospholipids. After elution with methanol, lysophosphatidylcholine (LPC) containing approximately 75% EPA and DHA is obtained.

[0004] Patent application CN201910710461.0 discloses an EPA / DHA type lysophospholipid composition and its preparation method. Patent application CN202180026267.6 discloses an Antarctic krill oil composition rich in LPC-DHA and LPC-EPA.

[0005] Existing technologies remove byproducts such as ethyl esters from the reaction system through solvent extraction and chromatography. However, existing technologies do not provide methods to further increase the phospholipid content of Antarctic krill oil.

[0006] The testes continue to develop from birth to sexual maturity, significantly increasing in size and enhancing their ability to synthesize male hormones. Germ cells undergo active proliferation, division, and differentiation. The blood-testis barrier, a crucial channel for substance exchange within the testes, is composed of supporting cells and their tight junctions. It not only handles nutrient transport but also effectively prevents harmful substances from entering the seminiferous epithelium, thus maintaining the stable microenvironment required for spermatogenesis. This barrier gradually develops and matures with individual growth, becoming fully established in adulthood.

[0007] Studies have confirmed that dietary DHA supplementation can significantly increase DHA levels in the testes and sperm cells, and effectively improve sperm motility and function, highlighting the important role of DHA in the male reproductive system.

[0008] However, the effects of dietary Antarctic krill oil and dietary DHA supplementation on blood-testis barrier development have not been reported. Summary of the Invention

[0009] This invention discloses a method for the industrial-scale preparation of Antarctic krill oil with high phospholipid content.

[0010] This invention uses Antarctic krill oil as raw material and prepares Antarctic krill oil products with high phospholipid content (phospholipid content greater than 90%) through enzyme catalysis combined with molecular distillation technology.

[0011] This invention discloses a method for preparing Antarctic krill oil with high phospholipid content, comprising the following steps:

[0012] 1) Mix Antarctic krill oil with 90% ethanol, add lipase B to react. The triglycerides in Antarctic krill oil are hydrolyzed by lipase to release free fatty acids. The free fatty acids combine with ethanol in situ to form fatty acid ethyl esters.

[0013] 2) Solvent removal by rotary evaporation under reduced pressure;

[0014] 3) Molecular distillation removes fatty acid ethyl esters.

[0015] Furthermore, the lipase B in step 1) can be Candida antarcticis lipase B.

[0016] Furthermore, the reaction conditions for step 1) can be: reaction for 8 hours under nitrogen protection and constant temperature shaking at 45°C.

[0017] Further, in step 1), Antarctic krill oil and 90% ethanol are mixed in a 1:1 ratio, and *Candida antarcticis* lipase B is added at one-fifth of the krill oil's mass. The mixture is reacted for 8 hours under nitrogen protection and constant temperature shaking at 45°C. Since *Candida antarcticis* lipase B preferentially hydrolyzes triglycerides, the triglycerides in the krill oil are specifically enzymatically hydrolyzed, releasing free fatty acids. In the ethanol reaction system, under the action of *Candida antarcticis* lipase B, during the 8-hour reaction under constant temperature shaking at 45°C, the free fatty acids combine in situ with ethanol to form fatty acid ethyl esters. During the enzymatic hydrolysis process, most of the phospholipids in the Antarctic krill oil are retained.

[0018] After the reaction is complete, most of the solvent is removed by rotary evaporation under reduced pressure in step 2) to obtain the concentrate.

[0019] Furthermore, the temperature for vacuum rotary evaporation in step 2) can be 40°C.

[0020] Furthermore, the conditions for molecular distillation in step 3) can be as follows: under a high vacuum of less than 10 Pa, first distill at 80–100 °C to remove the light components mainly composed of fatty acid ethyl esters, and then distill at 120–150 °C to collect the heavy components rich in phospholipids.

[0021] This invention also discloses the application of the above-mentioned method for preparing Antarctic krill oil with high phospholipid content in the industrial preparation of Antarctic krill oil.

[0022] The present invention also discloses Antarctic krill oil with high phospholipid content prepared by the above-mentioned method for preparing Antarctic krill oil with high phospholipid content.

[0023] Furthermore, the Antarctic krill oil prepared according to the method of the present invention has a phospholipid content of over 90%.

[0024] This invention also discloses the application of Antarctic krill oil in the preparation of products that promote the development of the blood-testis barrier. This invention reveals that Antarctic krill oil has a positive promoting effect on the development of the blood-testis barrier.

[0025] Furthermore, in the above applications, the intake of Antarctic krill oil is 1% of the food intake.

[0026] This invention also discloses the application of the above-mentioned high-phospholipid-content Antarctic krill oil in the preparation of products that promote the development of the blood-testis barrier. This invention reveals that the above-mentioned high-phospholipid-content Antarctic krill oil has a positive promoting effect on the development of the blood-testis barrier.

[0027] Furthermore, in the above applications, the intake of Antarctic krill oil with high phospholipid content is 1% of the food intake.

[0028] This invention first enzymatically hydrolyzes the triglycerides in Antarctic krill oil in ethanol, and then uses molecular distillation to remove light components, thereby obtaining Antarctic krill oil with high phospholipid content.

[0029] This invention does not use any potentially harmful organic substances in the preparation process, and the product is safe and reliable, and can be used in the preparation of food or medicine.

[0030] The preparation method of this invention yields Antarctic krill oil concentrate with a phospholipid content of over 90%, and the entire process effectively avoids the damage to phospholipids and other active ingredients caused by high temperature and oxidation.

[0031] The preparation process of Antarctic krill oil with high phospholipid content of the present invention is simple and suitable for large-scale industrial application. Attached Figure Description

[0032] Figure 1 This represents the sperm count detection result of one embodiment of the present invention.

[0033] Figure 2 This represents the relative transmittance detection result of Sulfo-NHS-LC-Biotin according to an embodiment of the present invention.

[0034] Figure 3 This represents the result of detecting the expression level of Mfsd2a in the testes according to an embodiment of the present invention.

[0035] Figure 4 This represents the serum testosterone level detection result of one embodiment of the present invention. Detailed Implementation

[0036] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0037] The Antarctic krill oil (phospholipid content 40%) used in this invention was purchased from Qingdao Kangjing Marine Biotechnology Co., Ltd.

[0038] Example 1: Preparation of neutral lipids

[0039] The neutral lipids derived from Antarctic krill oil, used as a control in the experiment, were prepared by cold acetone extraction.

[0040] The specific method is as follows: Antarctic krill oil and -20℃ cold acetone are mixed evenly at a ratio of 1:5. After standing and separating into layers, the upper layer containing neutral lipids in cold acetone is taken, and the solvent is removed by rotary evaporation under reduced pressure to obtain neutral lipids derived from Antarctic krill oil.

[0041] Example 2: Preparation of enzymatically hydrolyzed Antarctic krill oil

[0042] First, Antarctic krill oil and 90% ethanol were mixed in a 1:1 ratio. Antarctic Candida lipase B (Novozymes 435 immobilized lipase) was added at one-fifth of the krill oil mass. The reaction was carried out under nitrogen protection and constant temperature shaking at 45°C for 8 hours. After the reaction, most of the solvent was removed by rotary evaporation under reduced pressure at 40°C using a RE-2000B rotary evaporator equipped with a circulating water vacuum pump (SHZ-III, Shanghai Yarong Biochemical Instrument Factory), yielding enzymatically hydrolyzed Antarctic krill oil.

[0043] Because Candida antarcticis lipase B preferentially hydrolyzes triglycerides, under the conditions of this embodiment, the triglycerides in Antarctic krill oil are specifically enzymatically hydrolyzed, and the released free fatty acids combine in situ with ethanol to form fatty acid ethyl esters, while most of the phospholipids in Antarctic krill oil are retained.

[0044] Example 3: Molecular distillation

[0045] The enzymatically hydrolyzed Antarctic krill oil obtained in Example 2 was added to a KD6 molecular distillation system (pilot-scale short-path distillation, molecular distillation) at UIC GmbH, Germany. Separation was carried out under high vacuum conditions below 10 Pa by precisely controlling the temperature gradient. First, the light components, mainly composed of fatty acid ethyl esters, were removed by distillation at 80–100 °C, and then the heavy components rich in phospholipids were collected by distillation at 120–150 °C.

[0046] The phospholipid content in the above-mentioned phospholipid-rich heavy components was determined by the molybdenum blue colorimetric method.

[0047] Weigh 100 mg of the recombinant lipid sample, add 0.5 ml of perchloric acid, and digest at 160 °C until colorless. Then add 3.5 ml of water and 1 ml of colorimetric reagent (2.5 g / ml ammonium molybdate: 10 g / ml vitamin C = 1:1), boil in a water bath for 7 min, cool, and measure the absorbance of each tube at 820 nm.

[0048] Using sodium dihydrogen phosphate as a standard, a standard curve was plotted with the phosphorus content corresponding to different concentrations of inorganic phosphorus standard solutions as the abscissa (X) and the absorbance value as the ordinate (Y). Linear regression was then performed to obtain the regression equation.

[0049] Based on the absorbance value measured from the sample, substitute it into the standard curve equation to calculate the inorganic phosphorus content in the sample reaction solution.

[0050] Based on the inorganic phosphorus content, the phospholipid content is calculated using the following formula:

[0051] (Equation 1)

[0052] In the formula, p represents the phosphorus content (mg) of the test solution in the standard curve; m represents the mass of the sample (mg); V1 represents the volume of the sample test solution (mL); V2 represents the volume of the sample test solution taken (mL); and 25 is the number of milligrams of phospholipids equivalent to each milligram of phosphorus.

[0053] The phospholipid content in the recombinant lipids was found to be 91.6%.

[0054] The phospholipid recovery rate is calculated using the following formula.

[0055] Phospholipid recovery rate (%) = Total mass of phospholipids in heavy components / Total mass of phospholipids in Antarctic krill oil × 100% (Equation 2)

[0056] The recovery rate of phospholipids in Antarctic krill oil was found to be 92.8%.

[0057] This method yields Antarctic krill oil concentrate with a phospholipid content of over 90%, resulting in high-phospholipid Antarctic krill oil. Furthermore, the entire preparation process effectively avoids the damage to phospholipids and active ingredients caused by high temperatures and oxidation.

[0058] Example 4: Mouse feeding experiment

[0059] Female and male ICR mice (6-8 weeks old) (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were housed under standard conditions (temperature 20±2℃, relative humidity 60%, 12 h / 12 ​​h light / dark cycle) with free access to food and water.

[0060] After one week of acclimatization, female mice were randomly divided into four groups according to their body weight: a control group fed with a basic diet; a neutral lipid group fed with a diet supplemented with 1% neutral lipids derived from Antarctic krill oil prepared in Example 1; a krill oil group fed with a diet supplemented with 1% Antarctic krill oil; and a high phospholipid krill oil group fed with a diet supplemented with 1% Antarctic krill oil with high phospholipid content prepared in Example 3.

[0061] The specific feed formulation is shown in Table 1. In Table 1, exogenous lipids indicate the content of supplemental lipids for that group.

[0062] Table 1. Experimental feed formulation (g / kg) Ingredients (g / kg) control group neutral lipid group Krill oil group High-phospholipid krill oil group casein 200.0 200.0 200.0 200.0 sucrose 100.0 100.0 100.0 100.0 corn starch 397.5 397.5 397.5 397.5 maltodextrin 132.0 132.0 132.0 132.0 Cellulose 50.0 50.0 50.0 50.0 mineral salt mixture 35.0 35.0 35.0 35.0 Vitamin Mixture 10.0 10.0 10.0 10.0 L-methionine 3.0 3.0 3.0 3.0 Choline bitartrate 2.5 2.5 2.5 2.5 TBHQ 0.02 0.02 0.02 0.02 Hydrogenated coconut oil 56.7 46.7 46.7 46.7 Exogenous lipids − 10.0 10.0 10.0 Safflower seed oil 13.3 13.3 13.3 13.3

[0063] Subsequently, mice were mated together at a female-to-male ratio of 2:1. After confirming pregnancy, the females were separated from the males. The females were fed their respective group diets throughout the gestation and lactation periods. After weaning, the F1 generation continued to be fed the same diet as their mothers until they reached 10 days, 20 days, 45 days, and 10 weeks of age, at which point subsequent experiments were conducted. Eight mice were in each group for each experiment at each time point.

[0064] Example 5: Sperm Count

[0065] At 10 days of age, the offspring were injected intravenously with sulfosuccinimide-6-(biotinamide)hexanoate (Sulfo-NHS-LC-Biotin, APExBIO Technology LLC, catalog number A8003), and the testicular tissue was removed 30 minutes after the injection.

[0066] Carefully separate the epididymal tissue, place the epididymal tail in preheated physiological saline at 37°C, cut it 3-5 times with ophthalmic scissors, and incubate it at 37°C for 20 minutes to allow the sperm to swim out fully. Then, filter it through two layers of lens paper to obtain a sperm suspension, and dilute it appropriately with physiological saline.

[0067] Sperm counts were performed according to the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (5th Edition) standards for sperm motility rating.

[0068] One-way ANOVA-LSD test was used to analyze the significance of differences between different groups. The criterion for statistical significance was p < 0.05. Different letters above the data bars in the figure indicate significant differences between groups.

[0069] Total sperm count results in one epididymis as follows Figure 1 As shown in the results, dietary supplementation with Antarctic krill oil and high-phospholipid Antarctic krill oil significantly increased sperm count, suggesting that intervention with Antarctic krill oil and high-phospholipid Antarctic krill oil may improve testicular function by promoting blood-testis barrier development. Furthermore, the high-phospholipid Antarctic krill oil was significantly more effective than Antarctic krill oil and neutral lipid Antarctic krill oil. There was no significant difference between the neutral lipid Antarctic krill oil group and the control group. These results indicate that phospholipids in Antarctic krill oil play an important role in promoting blood-testis barrier development.

[0070] Example 6: Immunofluorescence Analysis

[0071] For mice aged 10, 20, and 45 days, interstitial injection of the testis was performed on the day of sacrifice. After anesthesia, the fat pad around the epididymis and testis was located using tissue forceps and gently pulled out to expose the testis. The left testis was operated on, and 40 μL of Sulfo-NHS-LC-Biotin solution was injected into the interstitial space via microinjection; the right testis was injected with 40 μL of physiological saline solution containing 1 mM CaCl2 as a control.

[0072] The skin incision was then sutured with surgical sutures, and the operated mouse was transferred to a heated pad. Thirty minutes after the injection, the mouse was euthanized according to the 8th edition of the Laboratory Animal Care and Use Guidelines, et al., published by the National Institute of Laboratory Animal Science, and the testicular tissue was collected.

[0073] Testicular tissue was placed in OCT embedding medium (Wuhan Saiweier Biotechnology Co., Ltd., catalog number G6059-110ML), flash-frozen in liquid nitrogen, and then stored at -80℃.

[0074] Testicular tissue was cut into 8 μm thick cross-sectional sections using a cryostat at -20°C and attached to glass slides for immunofluorescence analysis.

[0075] Frozen sections of testicular tissue were washed three times with PBS, then dried at 37°C in the dark for 30 minutes, followed by fixation with 4% paraformaldehyde for 30 minutes. After fixation, they were washed three times again with PBS. They were then blocked with 5% BSA solution at room temperature for 1 hour. Next, they were washed three times with PBS containing 0.1% Tween-20, and then subjected to ActivAb... TMFITC-labeled streptavidin (Beijing Solarbio Science & Technology Co., Ltd., catalog number SF068) was incubated overnight at 4°C. Afterwards, the slides were washed three times with 1×PBST wash buffer (ready-to-use) (Wuhan Saive Biotechnology Co., Ltd., catalog number G2157-1L), and counterstained with DAPI solution (ready-to-use) (Beijing Solarbio Science & Technology Co., Ltd., model C0065). The slides were then mounted and observed under a fluorescence microscope.

[0076] Immunofluorescence analysis was performed on the green fluorescence within the seminiferous tubules, with Ex = 492 nm / Em = 515 nm. Fluorescence intensity was calculated based on microscopic images; the ratio of intra-semiolecular fluorescence intensity to total intra-semiolecular fluorescence intensity represents the relative fluorescence intensity within the seminiferous tubules of each sample. The relative fluorescence intensity within the seminiferous tubules of the control group was normalized to 1 to obtain the inter-group relative fluorescence intensity within the seminiferous tubules.

[0077] Immunofluorescence analysis of the green fluorescence intensity in seminiferous tubules, such as Figure 2 As shown. Fluorescence intensity represents the permeation of Sulfo-NHS-LC-Biotin. At 10, 20, and 45 days of age, dietary supplementation with Antarctic krill oil neutral lipids, Antarctic krill oil, and high-phospholipid-content Antarctic krill oil significantly reduced the permeation of Sulfo-NHS-LC-Biotin in the seminiferous tubules. This indicates that dietary intervention using Antarctic krill oil neutral lipids, Antarctic krill oil, and high-phospholipid-content Antarctic krill oil promoted the development of the blood-testis barrier in developing mice, and the effect of high-phospholipid-content Antarctic krill oil was significantly better than that of Antarctic krill oil and Antarctic krill oil neutral lipids.

[0078] Example 7: Expression level of Mfsd2a

[0079] The blood-testis barrier function is primarily regulated by the bypass junctional complex between barrier cells and transcytosis. Sertoli cells transport nutrients required by spermatogenic cells during replication and division into the spermatogenic cell interior via transcytosis. Simultaneously, the migration of spermatocytes from the basal side of the seminiferous tubules to the lumen also depends on the dynamic reorganization and regulation of the junctional complex through endocytosis by Sertoli cells. Dietary phospholipids rich in DHA / EPA are dispersed into micro-clusters by the emulsification of bile salts, and subsequently converted into DHA / EPA-lysophospholipids under the action of phospholipases.

[0080] Major Facilitator Superfamily Domaincontaining 2a (Mfsd2a) is a highly efficient transporter of DHA / EPA-lysophospholipids. Antarctic krill oil with high phospholipid content may be transported to the testes via Mfsd2a and regulate caveolin-mediated transcytosis to maintain barrier integrity.

[0081] This embodiment detected the expression level of Mfsd2a in testicular tissue.

[0082] RNA was extracted from the testes using the Trizol method. Testicular tissue was removed from a -80°C cryogenic freezer and added to Invitrogen TRIzol reagent (Thermo Fisher Scientific (China) Co., Ltd., catalog number 15596-026) at a mass-to-volume ratio of 1:10. The tissue was homogenized in a KZ-III-F high-speed cryogenic tissue homogenizer (Wuhan Saive Biotechnology Co., Ltd.) at 4°C and 60Hz for 30 seconds, followed by standing at room temperature for 5 minutes. After centrifugation, the supernatant was collected, and chloroform was added followed by vortexing. After centrifugation, isopropanol was added to the supernatant, and the mixture was vortexed again. The precipitate was then resuspended in pre-cooled 75% ethanol (-20°C), centrifuged, and the supernatant was collected. The precipitate was dissolved in DEPC water to obtain RNA.

[0083] Take a certain amount of RNA and perform reverse transcription to obtain cDNA according to the instructions of the All-In-One 5X RT MasterMix reverse transcription kit (abm, Canada, catalog number G592).

[0084] The Mfsd2a primers were synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are as follows.

[0085] Forward primer: 5'-AGAAGCAGCAACTGTCCATTT-3' Seq_1

[0086] Reverse primer: 5'-CTCGGCCCACAAAAAGGATAAT-3' Seq_2

[0087] qPCR amplification was performed on a BioRad iQ5 real-time PCR system (Bio-Rad Laboratories, INC.) according to the instructions of the universal qPCR kit BlasTaqTm 2X qPCR MasterMix (ABM, Canada, catalog number G891). The qPCR reaction program was as follows: pre-denaturation at 95℃ for 3 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 60 s. β-actin was used as an internal control to correct for the mRNA expression level of the target gene.

[0088] The expression level of Mfsd2a in testicular tissue is as follows: Figure 3 As shown in the figure. The results showed that dietary supplementation with Antarctic krill oil and high-phospholipid Antarctic krill oil significantly upregulated the expression of Mfsd2a in the testes, suggesting that intervention with Antarctic krill oil and high-phospholipid Antarctic krill oil may promote the development of the blood-testis barrier by regulating Mfsd2a expression, and the effect of high-phospholipid Antarctic krill oil was significantly better than that of Antarctic krill oil and Antarctic krill oil neutral lipids.

[0089] Example 8: The effect of Antarctic krill oil with high phospholipid content on blood-testis barrier development

[0090] Blood samples were collected from mice at 10 weeks of age and centrifuged at 4°C and 3000 g for 15 minutes to obtain serum. The serum sample was placed in a 10 mL centrifuge tube, and 4 mL of ethyl acetate-n-hexane mixture (3:2, v / v) was added. The mixture was vortexed for 3 minutes. The organic layer was transferred to a new tube, and 1 mL of 0.1 mol / L NaOH solution was added. The mixture was vortexed for 3 minutes. The organic layer was transferred again to a new tube, and the mixture was concentrated under reduced pressure to approximately 1 mL. This concentrated solution was then transferred to a 1.5 mL centrifuge tube and concentrated until dry. The residue was reconstituted with 100 μL of water-methanol mixture (7:3, v / v), vortexed for 2 minutes, sonicated for 5 minutes, and then centrifuged at 4°C and 13000 rpm for 15 minutes.

[0091] Samples were analyzed using an Agilent 1260 Infinity HPLC-MS system (Agilent 6410B, Agilent Technologies, USA), with an injection volume of 10 μL. Chromatographic separation was performed using an Agilent Porshell 120EC-C18 UPLC column (2.1 × 150 mm, 2.7 μm) (Agilent Technologies, catalog number 693775-902), at a column temperature of 50 °C. Mobile phases: A was a 0.1% formic acid aqueous solution, and B was a methanol solution containing 0.1% formic acid; gradient elution program: 0 min, 60% A; 5 min, 30% A; 5.1 min, 2% A; 7.0 min, 2% A; 7.1 min, 60% A. Mass spectrometry conditions: positive ion electrospray ionization (ESI) source was used. + The source temperature was 350℃, the atomization pressure was 40 psi, the drying gas flow rate was 10 L / min, and the capillary voltage was ±4000 V. Multiple reaction monitoring (MRM) was used to detect testosterone (m / z 289.2→109.1, fragmentation voltage 125 V, impact energy 27 eV) and dihydrotestosterone (DHT) (m / z 291.2→255.2, fragmentation voltage 125 V, impact energy 25 eV).

[0092] Serum testosterone level analysis results as follows Figure 4 As shown in the results, dietary supplementation with Antarctic krill oil neutral lipids, Antarctic krill oil, and Antarctic krill oil with high phospholipid content significantly increased serum testosterone levels in mice. The effect of Antarctic krill oil with high phospholipid content was significantly better than that of Antarctic krill oil alone, and the effects of Antarctic krill oil with high phospholipid content and Antarctic krill oil were significantly better than those of Antarctic krill oil neutral lipids.

Claims

1. A method for preparing Antarctic krill oil with high phospholipid content, comprising the following steps: 1) Mix Antarctic krill oil with 90% ethanol, add lipase B to react, the triglycerides in Antarctic krill oil are enzymatically hydrolyzed to release free fatty acids, and the free fatty acids combine with ethanol in situ to form fatty acid ethyl esters. 2) Solvent removal by rotary evaporation under reduced pressure; 3) Molecular distillation removes fatty acid ethyl esters.

2. The method for preparing Antarctic krill oil with high phospholipid content according to claim 1, characterized in that: The lipase B in step 1) is Candida antarcticis lipase B.

3. The method for preparing Antarctic krill oil with high phospholipid content according to claim 2, characterized in that: The reaction conditions for step 1) are: reaction for 8 hours under nitrogen protection and constant temperature shaking at 45°C.

4. The method for preparing Antarctic krill oil with high phospholipid content according to claim 1, characterized in that: In step 2), the temperature for vacuum rotary evaporation is 40°C.

5. The method for preparing Antarctic krill oil with high phospholipid content according to any one of claims 1 to 4, characterized in that: In step 3), molecular distillation specifically involves distilling at 80–100°C under a high vacuum condition below 10 Pa to remove the light components, which are mainly composed of fatty acid ethyl esters, and then distilling at 120–150°C to collect the heavy components rich in phospholipids.

6. The application of the method for preparing Antarctic krill oil with high phospholipid content as described in any one of claims 1 to 5 in the industrial preparation of Antarctic krill oil.

7. Antarctic krill oil with high phospholipid content prepared by the method for preparing high phospholipid content Antarctic krill oil according to any one of claims 1 to 5.

8. The Antarctic krill oil with high phospholipid content according to claim 7, characterized in that: Antarctic krill oil contains over 90% phospholipids.

9. The use of the high phospholipid content Antarctic krill oil according to claim 7 or 8 in the preparation of products that promote the development of the blood-testis barrier.

10. The application according to claim 9, characterized in that: The intake of Antarctic krill oil, which is high in phospholipids, should be 1% of the total food intake.

Images