Haematococcus pluvialis lysate, and preparation method and application thereof

Abstract

The present application relates to the field of cell agriculture and animal cell in vitro culture technology, and particularly relates to a Haematococcus pluvialis lysate and a preparation method and application thereof. Through mild wall breaking, controllable enzymolysis, clarification and impurity removal, two-stage tangential flow ultrafiltration molecular weight fractionation and other means, that is, separation is sequentially performed by using 10 kDa and 3 kDa membranes, and combined with stable delivery of lipid-soluble components and standardized compounding, an animal source-free culture supplement which can replace fetal bovine serum is obtained. The supplement can effectively promote fish cell proliferation and realize adipogenic differentiation, realize the integration of amplification and adipogenic compatibility in low serum / serum-free culture of cell cultured fish meat, and significantly reduce batch differences. The technical route can provide adhesion and proliferation support of serum in the proliferation stage, and play the roles of antioxidant, carrier protein-like buffer and lipid metabolism support in the adipogenic stage, and through a clear fractionation process and quality control index, quantifiable, predictable and repeatable production is realized.

Description

Technical Field

[0001] This invention relates to the fields of cell agriculture and in vitro culture of animal cells, specifically to a Haematococcus pluvialis lysate, its preparation method, and its application. Background Technology

[0002] The large-scale production of cell-cultured fish meat relies on high-density cell expansion and controlled differentiation. Fish myogenic cells determine tissue structure, protein deposition, and textural basis, while lipid-derived cells influence lipid droplet deposition, flavor precursor formation, and juicy texture. In existing processes, to maintain stable cell adhesion, proliferation, and metabolic homeostasis, fetal bovine serum is often added to the culture medium to provide adhesion-promoting factors, carrier proteins, lipids and hormone-like components, antioxidant buffers, and various complex nutrients, thereby reducing the sensitivity of the culture system to cell type and process disturbances to some extent.

[0003] However, fetal bovine serum (FBS) faces challenges such as limited availability, high price, highly complex composition, and significant batch-to-batch variations. This leads to batch-to-batch fluctuations in amplification rate, doubling time, and differentiation, making it difficult to meet the requirements for food-grade traceability and long-term process consistency. Furthermore, its animal-derived nature poses potential safety and compliance risks, hindering the industrialization of cell-cultured fish. Particularly during adipogenic differentiation, lipid metabolism-related factors and hormone-like substances in serum may introduce uncontrollable metabolic signals, causing fluctuations in lipid droplet formation rate, lipid composition, and microtissue maturity, thereby affecting the yield and quality of adipose microtissue.

[0004] Existing serum-free alternative strategies include recombinant protein and growth factor systems, plant hydrolysates, yeast extracts, and algal extracts. While some plant hydrolysates can provide amino acids and small peptides to support proliferation, they typically lack lipid-soluble antioxidants and membrane-protective components, making it difficult to balance rapid proliferation and efficient lipid formation. Recombinant protein systems have well-defined components but are costly and have complex formulations. Although algal raw materials have food-grade potential and a basis for large-scale production, directly using crude extracts or simple cell-wall-breaking solutions often faces problems such as pigment aggregation, impurity fluctuations, unstable osmotic pressure and ionic composition, and interference from small cytotoxic molecules. This results in a narrow effective concentration range, poor batch-to-batch consistency, and difficulty in stably replacing serum. Chinese patent CN115125195A discloses a combination of functional factors from single-celled green algae and its application in cultured meat, using these factors as a substitute for serum. Chinese patent CN115369084A discloses a culture medium for adipose-derived mesenchymal stem cells and its application, adding methyl lotusine and marine coccolithophores extract to the medium, eliminating the need for animal serum and effectively increasing the proliferation rate of adipose-derived mesenchymal stem cells. However, these algal extracts only achieve cell proliferation and cannot support myoblastic or adipogenic differentiation.

[0005] Haematococcus pluvialis is rich in carotenoids such as astaxanthin, proteins and peptides, soluble polysaccharides, and micronutrients, possessing potential for antioxidant, membrane protection, and nutritional supplementation. If intracellular components can be released gently and stably delivered through precise molecular weight fractionation and lipid-soluble component delivery, transforming it from crude lysate into a culture supplement with controllable composition, cell-friendly properties, and batch-standardizable formulation, it is hoped that stable proliferation of fish cells can be achieved under serum-free conditions, while simultaneously supporting adipogenic differentiation. This would meet the needs of the cell-cultured fish industry for scalable, traceable, and cost-controllable culture systems. Therefore, there is an urgent need to develop a serum alternative technology for cell-cultured fish. Summary of the Invention

[0006] The technical problem to be solved by this invention is that existing serum-free alternative strategies include recombinant protein and growth factor systems, plant hydrolysates, yeast extracts and algal extracts, etc. Although some plant hydrolysates can provide amino acids and small peptides to support proliferation, they usually lack lipid-soluble antioxidant and membrane protection components, making it difficult to achieve both rapid proliferation and efficient lipid formation.

[0007] To address the aforementioned issues, this invention provides a Haematococcus pluvialis lysate, its preparation method, and its applications. Through gentle cell disruption, controlled enzymatic hydrolysis, clarification and impurity removal, and two-stage tangential ultrafiltration molecular weight fractionation—specifically, separation using 10 kDa and 3 kDa membranes sequentially—and combined with stable delivery and standardized compounding of lipid-soluble components, an animal-free culture supplement that can replace fetal bovine serum is obtained. This supplement effectively promotes fish cell proliferation and adipogenic differentiation, achieving integrated low-serum / serum-free culture of fish cells that is compatible with both amplification and adipogenesis, while significantly reducing batch-to-batch variability. This technical approach provides both serum adhesion and proliferation support during the proliferation phase and antioxidant, carrier protein-like buffering, and lipid metabolism support during the adipogenic phase. Furthermore, through a defined fractionation process and quality control indicators, quantifiable, predictable, and reproducible production is achieved.

[0008] To achieve the above objectives, the technical solution of the present invention is: a method for preparing Haematococcus pluvialis lysate, comprising the following steps: (1) Pretreatment and low-temperature resuspension of Haematococcus pluvialis biomass: Weigh the dried Haematococcus pluvialis powder, add calcium- and magnesium-free PBS for resuspension, and obtain the algal suspension; place the suspension at 4°C for pre-cooling and use, so as to reduce the heat accumulation in the subsequent cell wall breaking and enzymatic hydrolysis process and reduce the oxidation of active components. (2) Low-temperature mechanical cell disruption: The pre-cooled algal suspension was subjected to high-pressure homogenization or bead milling at 0~15℃ to break the cell wall and release intracellular components. The temperature of the system was controlled between 0 and 15℃ during the cell disruption process to avoid thermal denaturation and oxidation of active components. After the cell disruption was completed, the sample was immediately placed at 4℃ for storage. (3) Controlled enzymatic hydrolysis: Neutral protease and / or compound flavor enzyme are added to the broken algae pulp obtained in step (2), wherein the amount of neutral protease added is 1.0-5.0 g / L, preferably 1.5-3.0 g / L; the amount of compound flavor enzyme added is 0.5-3.0 g / L, preferably 0.8-1.5 g / L; the pH of the system is adjusted to 6.5-7.5, preferably 6.8-7.2, more preferably 7.0; the enzymatic hydrolysis is carried out by stirring at 28-40℃ for 1-4 h, preferably 1.5-3 h, more preferably 2 h; after enzymatic hydrolysis, the enzyme is inactivated by high temperature, and then rapidly cooled to 4℃ in an ice bath to obtain the enzymatic hydrolysate. Enzymatic hydrolysis with protease yields a lysate containing small peptide fragments. The hydrolysis endpoint is controlled by the range of free amino nitrogen and the molecular weight distribution of peptide fragments, so that the molecular weight distribution of batch products is stably controlled within the target range of 3 to 10 kDa as the main component and less than 3 kDa as the auxiliary component. This balances cell uptake efficiency, osmotic pressure stability and cell compatibility, and provides a consistent basis for the subsequent stable achievement of low serum replacement capacity and FBS-free lipogenesis. (4) Clarification and pre-filtration: The enzymatic hydrolysate from step (3) is centrifuged at 8000-15000×g for 10-30 min, preferably at 9000-12000×g for 15-25 min, and more preferably at 10000×g for 20 min to remove cell wall residues and large insoluble particles, and the supernatant is collected; the supernatant is then filtered through a 0.45 μm pre-filter membrane and a 0.22 μm filter membrane in sequence to reduce the interference of particulate matter, aggregates and potential cytotoxic impurities on cell culture, and a clear lysate is obtained; (5) Molecular weight cutoff and fractionation: In the first stage, a 10 kDa MWCO ultrafiltration membrane was used to process the clarified lysate obtained in step (4-1), discarding the 10 kDa cutoff and collecting the 10 kDa permeate, which was denoted as F<10k; in the second stage, F<10k was further fractionated using a 3 kDa MWCO ultrafiltration membrane, collecting the 3 kDa cutoff as the 3~10 kDa enriched peptide fraction, which was denoted as F3~10k; collecting the 3 kDa permeate as the <3 kDa small molecule fraction, which was denoted as F<3k; F3~10k and F<3k are Haematococcus pluvialis lysate.

[0009] In the molecular weight fractionation step, this invention employs a fixed and industrially scalable two-stage tangential flow ultrafiltration process. In the first stage, the clarified lysate is treated with 10 kDa MWCO tangential flow ultrafiltration, discarding the 10 kDa cutoff, which contains components larger than 10 kDa, such as some large protein molecules, polysaccharide aggregates, and some pigment-protein complexes. The permeate smaller than 10 kDa is collected. In the second stage, the permeate smaller than 10 kDa is further fractionated using 3 kDa MWCO tangential flow ultrafiltration to obtain a 3-10 kDa peptide-enriched fraction and a smaller molecule fraction smaller than 3 kDa. The 3-10 kDa peptide-enriched fraction serves as the core functional fraction for promoting proliferation and maintaining cell state, while the smaller molecule fraction provides available small molecule nutrients, osmotic pressure buffering, and some metabolic support.

[0010] Furthermore, in step (1), the solid content of the algal suspension is 50-200 g / L, preferably 80-120 g / L, and more preferably 90-110 g / L.

[0011] Furthermore, in step (2), the high-pressure homogenization pressure is set to 60-120 MPa, preferably 70-100 MPa, and more preferably 80-90 MPa; the cycle is repeated 2-6 times, preferably 3-4 times; and the homogenization interval is cooled for 3-10 minutes under ice bath conditions, preferably 4-6 minutes.

[0012] Furthermore, after enzymatic hydrolysis in step (3), the system is heated to 70°C and kept at that temperature for 15 min to inactivate the enzyme.

[0013] Furthermore, in step (5), two-stage molecular weight fractionation is performed using tangential flow ultrafiltration (TFF), with PES as the membrane material and the operating temperature controlled at 10°C.

[0014] A Haematococcus pluvialis lysate culture supplement comprising F3~10k, F<3k, and LF / HP-β-CD, wherein the preparation method of LF / HP-β-CD is as follows: (0-1) Pretreatment and low-temperature resuspension of Haematococcus pluvialis biomass: Weigh the dried Haematococcus pluvialis powder, add calcium- and magnesium-free PBS for resuspension, and prepare the algal suspension; place the suspension at 4℃ for pre-cooling and use. (0-2) Low-temperature mechanical cell disruption: The pre-cooled algal suspension was subjected to high-pressure homogenization or bead milling at 0~15℃; after the cell disruption was completed, the sample was immediately placed at 4℃ for storage. (0-3) Extraction of lipid-soluble active components: Take the cell-wall-broken algae slurry from step (0-2), add 95% food-grade ethanol, and extract with magnetic stirring under light-protected conditions; after extraction, centrifuge and collect the supernatant; repeat the ethanol extraction once, combine the supernatants from the two ethanol extractions, and remove the solvent by rotary evaporation under reduced pressure to obtain a lipid-soluble active component concentrate, denoted as LF. The LF mainly contains carotenoids, astaxanthin-related components, lipids, and other hydrophobic active ingredients; (0-4) Preparation of HP-β-CD encapsulated lipophilic fraction: Weigh hydroxypropyl-β-cyclodextrin (HP-β-CD) and dissolve it in sterile pure water, PBS, or basal culture medium to obtain an HP-β-CD solution of 10-200 mg / mL, preferably 50 mg / mL to 150 mg / mL, more preferably 80 mg / mL to 120 mg / mL; pre-disperse the above lipophilic enrichment LF with food-grade ethanol or sterile water-ethanol mixture, with the concentration of the lipophilic active component being 0.5 mg / mL to 20 mg / mL, more preferably 1 mg / mL to 10 mg / mL; then slowly add the lipophilic active component solution dropwise to the HP-β-CD solution, with the mass ratio of HP-β-CD to LF being 5:1 to 40:1, more preferably 10:1 to 25:1, and even more preferably 15:1 to 20:1; after the dropwise addition is complete, stir at 20℃ to 40℃ for 0.5 h to 6 h, preferably 1 h to 3 h. h, allowing it to fully form the encapsulated complex, and then ultrasonically dispersed for 1~20 min to improve the encapsulation efficiency and system uniformity; then filtered through 0.22 μm to remove unencapsulated particles, and obtained an aqueous dispersible HP-β-CD encapsulated lipophilic fraction, denoted as LF / HP-β-CD.

[0015] In the stabilization step of delivering lipophilic active components, this invention enriches the lipophilic active components in Haematococcus pluvialis lysate and encapsulates and mixes them with hydroxypropyl-β-cyclodextrin (HP-β-CD) to form an aqueously dispersible and stable delivery fraction. The lipophilic active components are preferably derived from lipophilic extracts obtained by centrifugation, alcohol extraction, or supercritical extraction of Haematococcus pluvialis lysate, and mainly include carotenoids, astaxanthin, lipids, and other hydrophobic active ingredients.

[0016] By quantitatively mixing and encapsulating the above-mentioned hydroxypropyl-β-cyclodextrin with the lipid-soluble active components in microalgal lysates, the self-aggregation of lipid-soluble pigments and hydrophobic components in the aqueous culture system can be significantly reduced, thereby reducing the local high-concentration stress on cells and improving the dispersion stability and delivery consistency of active components in different batches of supplements. This achieves both the maintenance of cell state during the low serum proliferation stage and the support for anti-oxidation, membrane protection and lipid accumulation during the FBS-free adipogenesis stage.

[0017] Further, in step (0-3), extraction of the lipid-soluble active components: Take the cell-wall-broken algae slurry from step (0-2), add 1-4 times the volume of 70%-95% (v / v) food-grade ethanol, preferably 1.5-3 times the volume of 85%-95% (v / v) food-grade ethanol, more preferably 2 times the volume of 95% (v / v) food-grade ethanol; extract magnetically at 20-40°C in the dark for 0.5-3 h, preferably at 25-35°C for 0.5-2 h, more preferably at 30°C for 1 h; after extraction, centrifuge at 6000-12000×g for 5-20 min, preferably at 7000-10000×g for 8-15 min. Centrifuge at 8000×g for 10 min, more preferably at 8000×g, and collect the supernatant; repeat the ethanol extraction 1 to 3 times, preferably 1 to 2 times, more preferably 1 time; combine the supernatants from each ethanol extraction, and remove the solvent by rotary evaporation under reduced pressure at 30 to 45°C, preferably at 35 to 42°C, more preferably at 40°C, to obtain the lipid-soluble active component enrichment LF.

[0018] Furthermore, the proportions of each component in the Haematococcus pluvialis lysate culture supplement are as follows: F3~10k accounts for 50%~80%, F<3k accounts for 10%~40%, and LF / HP-β-CD accounts for 1%~15%.

[0019] The aqueous dispersible lipophilic fraction is not used alone as a culture supplement, but is further standardized and compounded with the aforementioned 3-10 kDa peptide-enriched fraction and the smaller molecule fraction (less than 3 kDa). In other words, this invention first obtains three types of functional fractions: ① 3-10 kDa peptide-enriched fraction, ② smaller molecule fraction (less than 3 kDa), and ③ hydroxypropyl-β-cyclodextrin-encapsulated lipophilic fraction. These three types of functional fractions are then compounded in a predetermined ratio to form the final Haematococcus pluvialis lysate culture supplement. The aqueous fraction includes the 3-10 kDa peptide-enriched fraction and the smaller molecule fraction (less than 3 kDa). Preferably, based on the total solids of the supplement, the 3 to 10 kDa fraction accounts for 50% to 80%, more preferably 55% to 70%; the fraction less than 3 kDa accounts for 10% to 40%, more preferably 15% to 30%; and the hydroxypropyl-β-cyclodextrin-encapsulated lipophilic fraction accounts for 1% to 15%, more preferably 3% to 10%. In a more preferred embodiment, based on the total solids of the supplement, the 3 to 10 kDa fraction accounts for 65%, the fraction less than 3 kDa accounts for 25%, and the hydroxypropyl-β-cyclodextrin-encapsulated lipophilic fraction accounts for 10%.

[0020] This invention further provides a standardized quality control method to ensure batch consistency and reproducibility of supplements. Key quality control indicators include at least the total protein and total peptide concentration range, the proportion of peptides from 3 to 10 kDa as a core release indicator, the proportion of peptides less than 3 kDa, the total sugar and soluble polysaccharide range, the content of fat-soluble active components expressed as carotenoid or astaxanthin equivalents, pH and osmotic pressure, microbial limits and endotoxins, and rapid cell compatibility screening indicators, such as 24 to 72 h viability, doubling time, or CCK-8 metabolic activity. The above quality control targets the final formulated cultured supplement, and process control can also be performed separately on the three functional fractions to ensure the consistency of the final formulated product.

[0021] Application of the above-mentioned Haematococcus pluvialis lysate culture supplement in fish cell proliferation culture.

[0022] Furthermore, the supplement of this invention promotes cell adhesion and proliferation in low-serum systems, thereby replacing the additional serum required in conventional 10% FBS systems. The amount of the supplement added to the culture medium can be defined by volume fraction or equivalent total peptide concentration, preferably 0.5%~5% (v / v), 1%~2% (v / v), and 0.01~2.0 g / L (v / L), preferably 0.05~1.0 g / L (v / L). At the application level, the supplement is a single finished product resulting from the combination of the above three functional fractions, rather than three independent substitutes added separately to the culture medium.

[0023] Application of the above-mentioned Haematococcus pluvialis lysate culture supplement in adipogenic differentiation of cells.

[0024] Furthermore, the supplement of this invention is added to the basal culture system under FBS-free conditions to support lipid droplet formation and lipid accumulation in adipose-derived stem cells. The amount of the supplement added during the adipogenesis phase is maintained at a volume fraction of 1%–5% or a total peptide concentration of 0.2–1.0 g / L. Simultaneously, the rate of lipid droplet formation and the level of lipid accumulation can be controllably regulated by adjusting the ratio of 3–10 kDa peptide fractions, fractions smaller than 3 kDa, and lipophilic fractions encapsulated with cyclodextrin. Its innovation lies in using the standardized aqueous peptide fractions to provide nutrition and support for cell state under low serum or serum-free conditions, and the stable delivery of the lipophilic active component, cyclodextrin, to provide antioxidant and membrane protection functions, thereby achieving stable adipogenesis in an FBS-free environment.

[0025] Application of the above-mentioned Haematococcus pluvialis lysate culture supplement in cell-cultured fish meat.

[0026] In the fish cell expansion stage, a 3% FBS supplement system with the present invention is used for large-scale proliferation. In the adipose microtissue construction stage, an FBS-free system with the present invention is used to achieve lipid droplet formation and lipid accumulation. It can also be coupled with microcarrier culture technology to form cell-carrying microtissues enriched with adipocytes on microcarriers, which are used for composite assembly with myogenic microtissues, thereby obtaining cell culture fish meat structural units with adjustable muscle fat ratio.

[0027] Furthermore, when the supplement is used for cell culture, the final concentration of the hydroxypropyl-β-cyclodextrin-encapsulated lipophilic fraction in the culture system is 0.001%–0.10% (w / v), preferably 0.005%–0.08% (w / v), more preferably 0.01%–0.075% (w / v); the corresponding final concentration of the lipophilic active component is 0.5–60 μg / mL, preferably 2–50 μg / mL, more preferably 5–45 μg / mL.

[0028] During the low serum proliferation phase, the final concentration of the encapsulated lipophilic fraction is preferably controlled at 0.005% to 0.03% (w / v), more preferably 0.008% to 0.02% (w / v); the corresponding final concentration of the lipophilic active component is preferably 2 to 15 μg / mL, more preferably 5 to 10 μg / mL, in order to maintain antioxidant and membrane protection effects and avoid excessive hydrophobic components from interfering with cell proliferation.

[0029] In the FBS-free lipogenesis stage, the final concentration of the encapsulated lipophilic fraction is preferably increased to 0.03%–0.10% (w / v), more preferably 0.05%–0.08% (w / v); the corresponding final concentration of the lipophilic active component is preferably 15–60 μg / mL, more preferably 20–50 μg / mL, to enhance lipid droplet formation, lipid accumulation and cell membrane stability.

[0030] The beneficial effects of this invention are as follows: Compared with existing technologies, this invention has at least the following beneficial effects and innovations. First, this invention establishes a well-defined, scalable, and feasible molecular weight fractionation process using two-stage tangential flow ultrafiltration from 10 kDa to 3 kDa, transforming Haematococcus pluvialis lysate from an unstable crude extract into a standardized culture supplement, significantly reducing batch-to-batch variability and impurity interference. Second, this invention constructs a compatible culture strategy for low-serum amplification and FBS-free lipogenesis. During the proliferation stage, a supplement can be added to the low-serum system to replace the extra serum used in the conventional 10% FBS system. During the lipogenesis stage, the supplement is added under FBS-free conditions to promote lipid droplet formation and triglyceride accumulation, thereby reducing serum dependence and improving the stability of adipose microstructure. Third, through the proportional design and quality control system of peptide fractionation and lipophilic delivery fractionation, a quantifiable, predictable, and reproducible process is achieved, meeting the requirements of food-grade cell agriculture for clearly defined components, traceability, and cost control. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 : The number of cells cultured in the culture medium prepared in each embodiment of the present invention for 72 hours for the large yellow croaker muscle satellite cells; Figure 2 : Cell viability of large yellow croaker muscle satellite cells after culturing in the culture medium prepared in each embodiment of the present invention for 72 hours; Figure 3 : CCK-8 values ​​detected after culturing the large yellow croaker muscle satellite cells in the culture medium prepared in each embodiment of the present invention for 72 hours; Figure 4 Images of lipid fluorescence staining of cells cultured for 72 hours in the culture medium prepared according to various embodiments of the present invention for stem cells derived from fat in large yellow croaker; Figure 5 The triglyceride content of cells cultured for 72 hours in the culture medium prepared according to various embodiments of the present invention for stem cells derived from fat in large yellow croaker was detected. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to embodiments. The Haematococcus pluvialis lysate culture supplement of the present invention is used to reduce the amount of fetal bovine serum and achieve partial replacement of serum. It is suitable for fish cells, including myosal cells, adipose-derived stem cells, fibroblasts, etc., for proliferation and adipogenic differentiation in planar culture, microcarrier suspension culture, and cell-borne microtissue culture. Unless otherwise stated, "serum replacement" in this specification specifically refers to: under conventional 10% (v / v) FBS culture conditions, the supplement of the present invention is used to construct a low-serum culture system; in this specific embodiment, 1% (v / v) FBS + the supplement of the present invention is used as the low-serum proliferation medium during the cell proliferation stage, and FBS-free + the supplement of the present invention is used as the adipogenic differentiation medium during the adipogenic differentiation stage.

[0034] To verify the efficacy of this invention, proliferation performance was characterized by cell doubling time, viable cell percentage, EdU positivity rate, and CCK-8 metabolic activity, and compared with a 10% FBS conventional system and a low-serum control system to evaluate the proliferative support capacity of the supplement under low-serum conditions. Lipogenic performance was evaluated under FBS-free conditions, characterized by the positive area of ​​BODIPY or Oil Red O lipid droplet staining and triglyceride expression levels, and the difference in lipid accumulation between the control group without supplementation and the group with supplementation was compared. Simultaneously, cell adhesion rate, cell yield per unit volume, and batch consistency under microcarrier suspension culture conditions needed to be assessed.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available or can be prepared by existing methods.

[0036] Example 1: Preparation of Haematococcus pluvialis lysate culture supplement. Raw materials and pretreatment: Weigh 100g of dried Haematococcus pluvialis powder and resuspend it in 1000mL of calcium- and magnesium-free PBS to obtain an algal suspension with a solid content of approximately 100 g / L. Pre-cool the suspension at 4℃ for 45min to reduce heat accumulation during subsequent cell disruption and enzymatic hydrolysis processes and minimize oxidation of active components.

[0037] Low-temperature cell wall disruption: The pre-cooled algal suspension was subjected to high-pressure homogenization at 4–10℃, with a homogenization pressure of 80 MPa, for three consecutive cycles; each homogenization interval was followed by 5 min cooling in an ice bath. Immediately after disruption, the samples were stored at 4℃ for later use. Microscopic observation revealed that most algal cell walls had ruptured, and intracellular components were fully released.

[0038] Controlled enzymatic hydrolysis: Add 2.0 g of neutral protease and 1.0 g of compound flavor enzyme to the above-mentioned broken algal slurry, adjust the pH of the system to 7.0, and stir and hydrolyze at 32℃ for 2 h. After the enzymatic hydrolysis is completed, heat the system to 70℃ and hold for 15 min to inactivate the enzyme, and then quickly cool it to 4℃ in an ice bath.

[0039] Clarification and pre-filtration: The enzyme hydrolysate was centrifuged at 10000×g for 20 min to remove cell wall residues and large insoluble particles, and the supernatant was collected. The supernatant was then filtered sequentially through a 0.45 μm pre-filter membrane and a 0.22 μm filter membrane to obtain a clear lysate.

[0040] Molecular weight cutoff and fractionation: Two-stage molecular weight fractionation was performed using tangential flow ultrafiltration (TFF) with PES membrane material and operating temperature controlled at 10℃. In the first stage, a 10 kDa MWCO ultrafiltration membrane was used to treat the clarified lysis buffer. The 10 kDa cutoff was discarded, and the 10 kDa permeate was collected, denoted as F<10k. In the second stage, F<10k was further fractionated using a 3 kDa MWCO ultrafiltration membrane. The 3 kDa cutoff was collected as the 3–10 kDa enriched peptide fraction, denoted as F3–10k; the 3 kDa permeate was collected as the <3 kDa small molecule fraction, denoted as F<3k. After lyophilization, the solids content of F3–10k was 12.6 g, and the solids content of F<3k was 6.8 g.

[0041] Extraction of lipid-soluble active components: 200 mL of the above-mentioned broken-cell algal slurry was added to 400 mL of 95% food-grade ethanol, and extracted with magnetic stirring at 30℃ in the dark for 1 h. After extraction, the mixture was centrifuged at 8000×g for 10 min, and the supernatant was collected. The ethanol extraction was repeated once, and the supernatants from the two extractions were combined. The solvent was removed by rotary evaporation under reduced pressure at 40℃ to obtain 1.60 g of lipid-soluble active component enrichment, denoted as LF. The LF mainly contains carotenoids, astaxanthin-related components, lipids, and other hydrophobic active ingredients.

[0042] Preparation of HP-β-CD encapsulated lipophilic fraction: 24.0 g of hydroxypropyl-β-cyclodextrin was weighed and dissolved in 240 mL of sterile pure water to obtain a 100 mg / mL HP-β-CD solution. 1.60 g of the above lipophilic concentrate LF was pre-dispersed in 16 mL of 20% (v / v) ethanol aqueous solution, and then slowly added dropwise to the HP-β-CD solution to achieve a LF to HP-β-CD mass ratio of 1:15. After the addition was complete, the mixture was stirred at 30 °C for 2 h and then ultrasonically dispersed for 10 min. Unencapsulated particles were then removed by 0.22 μm filtration to obtain an aqueously dispersible HP-β-CD encapsulated lipophilic fraction, denoted as LF / HP-β-CD.

[0043] Standardized compounding: F3–10k, F<3k and LF / HP-β-CD three functional fractions are compounded according to the solid mass ratio to obtain the final Haematococcus pluvialis lysate culture supplement.

[0044] Example 2: This embodiment evaluates the supportive effect of the supplement of the present invention on fish cell proliferation under low serum conditions. Large yellow croaker muscle satellite cells were used and seeded in 24-well plates at an initial seeding density of 50,000 cells / well. The basal medium was DMEM / F12, and the culture temperature was 28°C.

[0045] The supplement for low serum proliferation of this invention employs a proliferation-optimized compound ratio, namely, a compound of three functional fractions—F3–10k, F<3k, and LF / HP-β-CD—at a solids mass ratio of 75:20:5. In this ratio, the proportion of F3–10k is increased to enhance cell proliferation and maintain cell state; the proportion of LF / HP-β-CD is decreased to reduce interference from excessive hydrophobic components on the proliferation phase. The final compound supplement is prepared as a sterile stock solution and added to the basal culture medium at a final concentration of 2 mg / mL. Based on this total concentration, the final concentration of F3–10k in the culture medium is 1.50 mg / mL, the final concentration of F<3k is 0.40 mg / mL, and the final concentration of LF / HP-β-CD is 0.10 mg / mL.

[0046] The experimental groups are set as follows: (1) Positive control group: DMEM / F12 + 10% (v / v) FBS; (2) Low serum control group: DMEM / F12 + 1% (v / v) FBS; (3) The supplement group of the present invention: DMEM / F12 + 1% (v / v) FBS + the supplement of the present invention (total solids final concentration 2 mg / mL, F3–10k:F<3k:LF / HP-β-CD=75:20:5); (4) Single-stage control group A: DMEM / F12 + 1% (v / v) FBS + F3–10k (final concentration 1.50 mg / mL). (5) Two-stage control group B: DMEM / F12 + 1% (v / v) FBS + F3–10k + F<3k (total final concentration 1.90 mg / mL, mass ratio 75:20, without LF / HP-β-CD).

[0047] Cells were cultured for 72 h before analysis. Viability was determined using trypan blue staining; cell doubling time was calculated from cell counts at 0 h and 72 h; metabolic activity was measured using the CCK-8 assay kit, specifically the OD450 value.

[0048] See results Figure 1-3 The results showed that, under 1% FBS low serum conditions, the final supplement formed by the combination of three functional fractions significantly improved cell viability, doubling time, and CCK-8 metabolic activity compared to the 1% FBS low serum control group, and approached those of the 10% FBS positive control group. Furthermore, it was superior to the control groups that only added F3–10k or only added F3–10k + F<3k, indicating that a combination strategy of high F3–10k and low LF / HP-β-CD during the low serum proliferation phase is more conducive to achieving proliferation performance close to that of a conventional serum system.

[0049] Example 3: This embodiment evaluates the promoting effect of the supplement of the present invention on adipogenic differentiation of fish adipose-derived stem cells under FBS-free conditions. The cells used were fish adipose-derived stem cells, the basal culture medium was DMEM / F12, and the culture temperature was 28°C. The supplement of the present invention used for adipogenic differentiation employed an optimized adipogenic compound ratio, namely, a compound of three functional fractions—F3–10k, F<3k, and LF / HP-β-CD—at a solids mass ratio of 60:25:15. Compared with Example 2, the LF / HP-β-CD ratio was increased in this embodiment to enhance antioxidant, membrane protection, and lipid accumulation support effects. The final compounded supplement was prepared as a sterile stock solution and added to the culture system at a final concentration of 5 mg / mL. Based on this total concentration, the final concentration of F3–10k in the culture medium was 3.00 mg / mL, the final concentration of F<3k was 1.25 mg / mL, and the final concentration of LF / HP-β-CD was 0.75 mg / mL.

[0050] The experimental groups are set as follows: (1) Control group without FBS: DMEM / F12; (2) The supplement group of the present invention: DMEM / F12 + the supplement of the present invention (total solids final concentration 5 mg / mL, F3–10k:F<3k:LF / HP-β-CD=60:25:15); (3) Single-stage control group A: DMEM / F12 + F3–10k (final concentration 3.00 mg / mL); (4) Two-stage control group B: DMEM / F12 + F3–10k + F<3k (total final concentration 4.25 mg / mL, mass ratio 60:25, without LF / HP-β-CD).

[0051] Cells were cultured for 7 days before analysis. Lipid droplet formation was detected using BODIPY staining; triglyceride (TG) levels were determined using a triglyceride (TG) assay kit.

[0052] See results Figure 4-5The results showed that, under FBS-free conditions, the final supplement formed by the combination of the three functional fractions significantly promoted lipid droplet formation, triglyceride accumulation, and the expression of adipogenic markers, which was superior to the FBS-free blank control group and also superior to the comparison group containing only F3–10k or only F3–10k+F<3k. This indicates that the combination strategy of increasing the LF / HP-β-CD ratio is more conducive to lipid accumulation and adipogenic differentiation in the FBS-free adipogenic stage.

[0053] Comparative Example 1: Pretreatment, low-temperature cell disruption, controlled enzymatic hydrolysis, centrifugation, and 0.45 μm / 0.22 μm filtration were performed according to Example 1 to obtain a clear lysate, but without 10 kDa and 3 kDa TFF fractionation. Subsequently, LF / HP-β-CD was prepared using the lipophilic extraction and embedding method described in Example 1, and the clear lysate was directly compounded with LF / HP-β-CD as a comparative supplement. To maintain comparability with Examples 2 and 3, Comparative Example 1 was added at a final total solids concentration of 2 mg / mL in the proliferation experiment and at a final total solids concentration of 5 mg / mL in the lipogenesis experiment.

[0054] Figure 1-5 The results showed that the crude lysis buffer compound system exhibited greater batch fluctuations under low serum proliferation and FBS-free lipogenesis conditions, and the cell proliferation efficiency, lipid droplet formation rate and TG accumulation were all lower than those of the supplement of this invention at the same addition amount, indicating that the clear two-level molecular weight fractionation of 3 kDa-10 kDa is the key step to obtain stable culture results.

[0055] Comparative Example 2: F3–10k and F<3k were obtained according to Example 1, but LF / HP-β-CD was not added to the final supplement. In the proliferation experiment, an aqueous phase combination of F3–10k:F<3k=75:20 was used, with a final total solids concentration of 2 mg / mL; in the lipogenesis experiment, an aqueous phase combination of F3–10k:F<3k=60:25 was used, with a final total solids concentration of 5 mg / mL. The remaining experimental conditions were the same as in Examples 2 and 3.

[0056] Figure 1-5 The results showed that, under the condition of no LF / HP-β-CD, low serum proliferation could be improved to some extent, but not as much as the complete three-component compound system; in the FBS-free lipogenesis stage, the lipid droplet formation rate and TG accumulation were significantly weaker than the complete supplement containing LF / HP-β-CD, indicating that the stable delivery of lipophilic active components has an important beneficial effect on the FBS-free lipogenesis stage.

[0057] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing Haematococcus pluvialis lysate, characterized in that... Includes the following steps: (1) Pretreatment and low-temperature resuspension of Haematococcus pluvialis biomass: Weigh the dried Haematococcus pluvialis powder, add calcium- and magnesium-free PBS for resuspension, and prepare the algal suspension; place the suspension at 4°C for pre-cooling. (2) Low-temperature mechanical cell disruption: The pre-cooled algal suspension was subjected to high-pressure homogenization or bead milling at 0~15℃; after the cell disruption was completed, the sample was immediately placed at 4℃ for storage. (3) Controlled enzymatic hydrolysis: Add neutral protease and / or compound flavor enzyme to the broken algae pulp obtained in step (2), wherein the amount of neutral protease added is 1.0 to 5.0 g / L; the amount of compound flavor enzyme added is 0.5 to 3.0 g / L; adjust the pH of the system to 6.5 to 7.5; stir and hydrolyze for 1 to 4 h at 28 to 40 °C; after enzymatic hydrolysis, inactivate the enzyme at high temperature, and then quickly cool down to 4 °C in an ice bath to obtain the enzymatic hydrolysate; (4) Clarification and pre-filtration: Centrifuge the enzymatic hydrolysate from step (3) at 8000–15000×g for 10–30 min and collect the supernatant; filter the supernatant through a 0.45 μm pre-filter membrane and a 0.22 μm filter membrane in sequence to obtain a clear lysate; (5) Molecular weight cutoff and fractionation: In the first stage, a 10 kDa MWCO ultrafiltration membrane was used to process the clarified lysate obtained in step (4-1), discarding the 10 kDa cutoff and collecting the 10 kDa permeate, which was denoted as F<10k; in the second stage, F<10k was further fractionated using a 3 kDa MWCO ultrafiltration membrane, collecting the 3 kDa cutoff as the 3~10 kDa enriched peptide fraction, which was denoted as F3~10k; collecting the 3 kDa permeate as the <3 kDa small molecule fraction, which was denoted as F<3k; F3~10k and F<3k are Haematococcus pluvialis lysate.

2. The preparation method according to claim 1, characterized in that: In step (1), the solid content of the algal suspension is 50-200 g / L; in step (2), the high-pressure homogenization pressure is set to 60-120 MPa and continuously cycled 2-6 times; each homogenization interval is cooled for 3-10 min under ice bath conditions; in step (3), after enzymatic hydrolysis, the system is heated to 70℃ and kept for 15 min to inactivate the enzyme; in step (5), tangential flow ultrafiltration (TFF) is used for two-stage molecular weight fractionation, the membrane material is PES, and the operating temperature is controlled at 10℃.

3. A Haematococcus pluvialis lysate culture supplement comprising Haematococcus pluvialis lysate, characterized in that: This includes F3~10k, F<3k, and LF / HP-β-CD, with the preparation method of LF / HP-β-CD as follows: (0-1) Pretreatment and low-temperature resuspension of Haematococcus pluvialis biomass: Weigh the dried Haematococcus pluvialis powder, add calcium- and magnesium-free PBS for resuspension, and prepare the algal suspension; place the suspension at 4℃ for pre-cooling and use. (0-2) Low-temperature mechanical cell disruption: The pre-cooled algal suspension was subjected to high-pressure homogenization or bead milling at 0~15℃; after the cell disruption was completed, the sample was immediately placed at 4℃ for storage. (0-3) Extraction of lipid-soluble active components: Take the cell wall broken algae pulp from step (0-2), add 95% food-grade ethanol, and extract with magnetic stirring under light-protected conditions; after extraction, centrifuge and collect the supernatant; repeat the ethanol extraction once, combine the supernatants from the two ethanol extractions, remove the solvent by rotary evaporation under reduced pressure, and obtain the lipid-soluble active component enrichment, denoted as LF; (0-4) Preparation of HP-β-CD-encapsulated lipophilic fraction: Weigh hydroxypropyl-β-cyclodextrin (HP-β-CD) and dissolve it in sterile pure water, PBS, or basal culture medium to obtain an HP-β-CD solution of 10-200 mg / mL; pre-disperse the above-mentioned lipophilic active component enrichment LF with food-grade ethanol or sterile water-ethanol mixture, with an added concentration of 0.5 mg / mL to 20 mg / mL; then slowly add the lipophilic active component enrichment solution dropwise to the HP-β-CD solution, with a mass ratio of HP-β-CD to LF of 5:1 to 40:1; after the addition is complete, stir at 20℃ to 40℃ for 0.5 h to 6 h, and then perform ultrasonic dispersion for 1 to 20 min; subsequently, filter through 0.22 μm to obtain an aqueous dispersible HP-β-CD-encapsulated lipophilic fraction, denoted as LF / HP-β-CD.

4. The Haematococcus pluvialis lysate culture supplement as described in claim 3, characterized in that: Step (0-3) Extraction of lipid-soluble active components: Take the cell wall-broken algae slurry from step (0-2), add 1-4 times the volume of the cell wall-broken algae slurry in 70%-95% (v / v) food-grade ethanol, and extract with magnetic stirring at 20-40℃ in the dark for 0.5-3 h; after extraction, centrifuge at 6000-12000×g for 5-20 min and collect the supernatant; repeat the ethanol extraction 1-3 times, combine the supernatants from each ethanol extraction, and remove the solvent by rotary evaporation under reduced pressure at 30-45℃ to obtain the lipid-soluble active component enrichment LF.

5. The Haematococcus pluvialis lysate culture supplement as described in claim 3, characterized in that: The proportions of each component in the Haematococcus pluvialis lysate culture supplement are as follows: F3~10k accounts for 50%~80%, F<3k accounts for 10%~40%, and LF / HP-β-CD accounts for 1%~15%.

6. The application of the Haematococcus pluvialis lysate culture supplement as described in claim 3 in fish cell proliferation culture.

7. The application as described in claim 6, characterized in that: The supplement is added to the culture medium at a volume fraction of 0.5% to 5% and a total peptide concentration of 0.01 to 2.0 g / L.

8. The application of the Haematococcus pluvialis lysate culture supplement as described in claim 3 in adipogenic differentiation of cells.

9. The application as described in claim 8, characterized in that: The amount of the supplement added during the lipogenesis phase is maintained in the range of 1% to 5% by volume or 0.2 to 1.0 g / L of total peptides.

10. The application of the Haematococcus pluvialis lysate culture supplement as described in claim 3 in cell-cultured fish meat.

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