A method for preparing a cat grass plant stem cell extract, a feline health product and a method for preparing the same

Abstract

The present application belongs to the cross technical field of plant cell engineering and pet health food, and particularly relates to a preparation method of a cat grass plant stem cell extract, a feline health product and a preparation method thereof. The method is that callus of dactylis glomerata, wheat or oat is first proliferated in the dark, then is weak light photoconverted and is induced by methyl jasmonate and ultraviolet light, the harvested cells are pre-softened by cellulase, xylanase and beta-glucanase, and are homogenized by a two-stage pressure program of low pressure first and then high pressure, the endogenous membrane lipids in the cells are in-situ co-assembled into hybrid liposomes with a particle size of 50-200 nm in the presence of exogenous phosphatidylcholine and cholesterol, and the crushing and encapsulation are completed in the same field. The health product is composed of the extract dry powder, taurine, inulin, papain, fish meat flavor powder and malt dextrin, the extract has high encapsulation efficiency and gastric juice resistance, the preparation process is mild, the palatability is good, and the product is suitable for daily feeding of felines.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of plant cell engineering and pet health food, specifically relating to a method for preparing cat grass plant stem cell extract, the extract prepared by the method, a feline health product containing the extract, and a method for preparing the health product. Background Technology

[0002] Felines have an instinctive behavior of actively foraging for grasses in their natural state. The grasses they eat are generally called cat grass, and common species include orchardgrass (Dactylis glomerata), wheat (Triticum aestivum) seedlings, and oat (Avena sativa) seedlings. These young grasses are rich in chlorophyll, flavonoids, phenolic acids, and various digestive enzymes and minerals, providing felines with a natural pathway to obtain trace amounts of plant-derived bioactive components. However, fresh grass cultivation is constrained by season, location, and pests and diseases, resulting in significant fluctuations in bioactive component content between cultivation batches. Furthermore, home-grown fresh grass carries the risk of pesticide residues and mold contamination. From a physiological perspective, felines are obligate carnivores, with a very small proportion of plant-based components in their diets. Consuming cat grass is believed to be related to promoting hairball excretion through the digestive tract, supplementing trace amounts of bioactive components, and fulfilling behavioral needs. Because of this narrow window for plant-based intake, the efficiency of felines' uptake of plant-derived bioactive components is highly dependent on the concentration and form of the components, which places more stringent requirements on the extraction process than on human health supplements. Currently, commercially available cat grass products mainly consist of fresh potted plants, dried grass powder, and wheatgrass-based juice powders. In these products, chlorophyll and flavonoids exist in a free state and are significantly lost during processing and in the gastrointestinal environment. Using plant cell engineering techniques to culture and extract undifferentiated cells (callus) from cat grass plants can overcome the limitations of cultivation conditions and achieve standardized production of bioactive components, thus possessing clear industrial value. However, to truly translate the advantages of in vitro culture into bioactive intake that felines can utilize, it is also necessary to simultaneously address the issues of component preservation during extraction and the stability of oral delivery.

[0003] Several technologies have been reported regarding the extraction of plant in vitro cultures and their health applications. Chinese patent CN102960718B (Invention Title: Plant Stem Cell Health Products) discloses the application of plant extracts in the preparation of food or health products. It involves culturing rye and other grass cells in vitro, repeatedly freezing and thawing the cells to disrupt them, centrifuging to collect the supernatant, and then filtering it through a membrane with a molecular weight cutoff of 3000 Da to collect only small molecule components with a molecular weight below 3000 Da. This method has the following objective shortcomings: First, the freeze-thaw disruption is time-consuming and incomplete, making it difficult to adapt to large-scale continuous production; second, the 3000 Da cutoff filtration removes large molecules and amphiphilic active components such as chlorophyll-protein complexes, enzymes, polysaccharides, and cell membrane lipids as waste, resulting in low utilization of intracellular active substances; third, the resulting small molecule aqueous solution is not protected by any delivery system, and the active components are directly exposed to gastric acid and digestive enzymes after oral administration; fourth, this patent is aimed at human consumption and does not address the physiological characteristics and palatability requirements of felines.

[0004] US Patent 9155916B2 (Invention Title: Cosmetic preparation and method for preparing the same) discloses a cosmetic preparation containing whole-cell lysate of dedifferentiated apple (Malus domestica) cells. The preparation involves mixing apple callus suspension culture with lecithin and homogenizing it four times under high pressure at 1200 bar, encapsulating the cell lysate in liposomes with a particle size of approximately 50 nm, for topical application to human skin. The objective shortcomings of this patent are: firstly, the entire culture process is conducted in darkness, and the cells do not contain a thylakoid membrane system, limiting the types of endogenous membrane lipids available for vesicle assembly; secondly, the use of a single high-pressure constant-pressure multiple homogenization process, where cell wall disruption and vesicle formation occur in the same intensity field in one step, is unsuitable for the robust cell walls of grasses, and the cumulative temperature increase from multiple high-pressure cycles is detrimental to the heat-sensitive active ingredients; thirdly, the preparation is positioned as a topical cosmetic and does not address the stability of vesicles in an oral or gastrointestinal environment, nor does it involve applications in grasses or felines.

[0005] In the field of feline health products, US Patent 7368481B2 (Invention Title: Pet anti-aging wellness supplement for cats) discloses an oral health supplement composition for cats containing taurine, traditional plant extracts, and fatty acids. The plant extracts used are all derived from conventional solvent extraction of field-grown plants, without involving in vitro plant cultures. The batch-to-batch consistency of active ingredients is affected by raw material fluctuations; furthermore, the components are simply mixed in free form, lacking delivery protection for fat-soluble components, and their retention in the stomach is uncontrollable. US Patent 5820916A (Invention Title: Method for growing and preserving wheatgrass nutrients and products thereof) discloses a method for juicing and preserving field-grown wheatgrass, which is essentially still a fresh grass pressing route, not free from the constraints of cultivation conditions. Chlorophyll is easily degraded during pressing and storage, and the formula is not specifically designed for felines.

[0006] In summary, existing technologies have not yet solved the following core technical problems: The callus cells of grass-like catnip plants have tough cell walls, requiring high-intensity disruption to achieve complete release of intracellular active substances. However, the temperature rise associated with this high-intensity disruption damages heat-sensitive components such as chlorophyll, flavonoids, and enzymes. Furthermore, the extracted substances exist in a free form, making them difficult to tolerate in the gastrointestinal environment of felines after oral administration. Additionally, the cell membrane lipids are discarded as impurities in existing processes and are not utilized. It is particularly noteworthy that while conventional phosphatidylcholine liposomes are mature encapsulation carriers, their membrane stability in strong acid and bile salt environments is limited. Directly adopting encapsulation methods from human cosmetics or pharmaceuticals is insufficient for oral delivery in felines with their high gastric acidity and unique gastric emptying rhythm. Therefore, the field needs a method to completely disrupt grass callus cells under mild conditions, enabling the extraction of extracts to form gastrointestinal-resistant delivery vesicles in situ during disruption, and a health product based on the extract obtained using this method that is compatible with the physiological characteristics of felines. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, the first objective of this invention is to provide a method for preparing catnip plant stem cell extract, which involves completely disrupting callus cells of the Poaceae family under mild conditions, and allowing endogenous membrane lipids to assemble with exogenous phospholipids into hybrid liposomes during the disruption process, thereby achieving in-situ encapsulation of the active ingredients. The second objective of this invention is to provide catnip plant stem cell extract prepared by this method. The third objective of this invention is to provide a feline health product containing this extract and adapted to the digestive physiology of felines. The fourth objective of this invention is to provide a method for preparing this health product.

[0008] To achieve the above objectives, the technical concept of this invention is as follows: the culture procedure, cell membrane lipid composition, cell wall mechanical properties, and homogeneous fluid dynamics are designed as a unified causal chain. First, rapid proliferation of the cell population is achieved through light-protected heterotrophic culture. Then, a phototransformation phase is implemented, involving the removal of auxin, reduction of sucrose concentration, and provision of weak light, to promote the conversion of intracellular proplasts into chloroplasts. This directionally expands the thylakoid membrane lipid pool, primarily composed of monogalactosylglycerol and digalactosylglycerol. Simultaneously, methyl jasmonate and ultraviolet light are used to induce the phenylpropanoid metabolic pathway, leading to the accumulation of flavonoids and phenolic acids within the cells. After cell harvesting, a complex enzyme system consisting of cellulase, xylanase, and β-glucanase is used to treat the cells. The unique type II cell wall of this product undergoes controlled pre-softening, significantly reducing the mechanical energy required for subsequent disruption. High-pressure homogenization is then performed using a two-stage pressure program: low pressure followed by high pressure. The low-pressure stage completes cell disruption and release of contents, while the high-pressure stage, in the presence of exogenous phosphatidylcholine and cholesterol, reconstructs endogenous membrane lipids and exogenous lipids into nanoscale hybrid liposomes. This allows lipid-soluble active ingredients to be carried by their natural membrane environment, while water-soluble active ingredients are encapsulated in the vesicle water core, thus completing encapsulation simultaneously with extraction.

[0009] Specifically, the preparation method includes five stages: callus induction and dark proliferation, light conversion, enzyme softening, two-stage homogenization, and separation and purification. In the callus induction and dark proliferation stage, explants from at least one of the following grasses (Orchardgrass, wheat, and oats) are disinfected and inoculated into MS solid medium containing 1.0–2.0 mg / L 2,4-dichlorophenoxyacetic acid and 0.1–0.5 mg / L kinetin for induction in the dark. After subculture, the inoculum is transferred to liquid MS medium for suspension culture in the dark for 10–18 days. When the concentration of 2,4-dichlorophenoxyacetic acid is below 1.0 mg / L, dedifferentiation induction is incomplete; when it is above 2.0 mg / L, the callus is prone to browning. Kinetin works in conjunction with these concentrations to maintain cell division activity. During the photoconversion phase, dark-proliferating cells were transferred to liquid MS medium containing 10–15 g / L sucrose and free of 2,4-dichlorophenoxyacetic acid. The cells were cultured for 3–7 days under light intensities of 200–1000 lx and a 12 h:12 h light-dark cycle. During this period, methyl jasmonate at a final concentration of 50–200 μmol / L was added, and the cells were irradiated with ultraviolet light at a wavelength of 280–315 nm for 3–10 min. Chloroplast development was insufficient when light intensities were below 200 lx, and browning of the callus surface cells occurred when light intensities were above 1000 lx. Induction was insufficient when methyl jasmonate concentrations were below 50 μmol / L, and excessive oxidative damage occurred when concentrations were above 200 μmol / L. The purpose of reducing the sucrose concentration to 10–15 g / L was to relieve the inhibition of photoconversion-related metabolism by high sugar levels. During the enzyme softening stage, harvested cells are suspended in phosphate buffer, and cellulase, xylanase, and β-glucanase are added for limited enzymatic digestion, followed by cooling to 4–10°C. Cooling terminates the enzymatic reaction and prepares the cells for low-temperature homogenization. In the two homogenization stages, homogenization is first performed 2–3 times at 250–400 bar to complete cell disruption. Then, phosphatidylcholine and cholesterol are added, and homogenization is performed 2–3 times at 800–1200 bar to complete vesicle reconstruction. In the first stage, cell disruption is incomplete when the pressure is below 250 bar, and the temperature rise above 400 bar negates the purpose of low-temperature disruption. In the second stage, vesicle sizes are difficult to enter the 50–200 nm range when the pressure is below 800 bar, and the cumulative temperature rise above 1200 bar damages heat-sensitive components. In the separation and purification stage, cell wall residues are removed by centrifugation, and the supernatant is filtered through a 0.45μm filter membrane to obtain cat grass plant stem cell extract containing hybrid liposomes; if necessary, the extract powder can be prepared by spray drying or freeze drying under trehalose protection. During the dehydration process, trehalose fixes the vesicle membrane structure in a glassy matrix.

[0010] This invention also provides a cat grass plant stem cell extract prepared by the above-described method, and a feline health product. The health product is composed of a dried powder obtained from the extract, taurine, inulin, papain, fish flavor powder, and maltodextrin in defined mass proportions. The component design is adapted to the digestive physiology of felines: feline bile acids specifically bind with taurine to form taurocholate; the taurine in the formula ensures the supply of essential amino acids while providing sufficient bound bile acid substrate for the absorption of lipid-soluble components carried by hybrid liposomes via the mixed micelle pathway in the small intestine; inulin, as a fructan not digested by the small intestine, enters the hindgut for fermentation, producing short-chain fatty acids; papain is used to loosen the protein matrix of the hairball; fish flavor powder ensures palatability; and maltodextrin serves as an excipient. This invention also provides a method for preparing the health product, wherein after sieving, each component is mixed in equal increments at a temperature not exceeding 40°C until a specified degree of homogeneity is achieved, and then packaged.

[0011] Compared with existing technologies, the beneficial effects of this invention are reflected in the following aspects. First, the photoconversion stage enables callus cells to establish a thylakoid membrane system. Digalactosylglycerol enhances the compactness of the hybrid vesicle membrane through the hydrogen bond network between glycohead groups. The conical molecular geometry of monogalactosylglycerol adapts to the high curvature of small-diameter vesicles, increasing the retention rate of active ingredients in the extract in simulated gastric juice from 52.6% of pure phosphatidylcholine liposomes to 78.9% after 2 hours. Second, the pre-softening with compound enzymes enables cells to achieve a breakage rate of over 99% at a low pressure range of 250-400 bar. The discharge temperature throughout the process does not exceed 35°C, preserving heat-sensitive components such as total chlorophyll and enzymes. Simultaneously, xylanase releases ferulic acid bound to arabinoxylan in the cell wall into a free state, resulting in a free ferulic acid content of 0.3-0.8 mg / g in the dry powder. Third, the two-stage pressure process decouples vesicle fragmentation and reconstruction, allowing endogenous membrane lipids to be utilized in situ instead of being discarded. The total flavonoid encapsulation rate reaches over 79%, and the effective delivery rate is more than double that of the two-step process of extraction followed by encapsulation. Furthermore, the combined gain of photoconversion and in-situ hybridization significantly exceeds the sum of their individual gains, exhibiting a nonlinear synergistic effect. Fourth, callus culture is freed from seasonal and cultivation condition limitations, with stable content of active ingredients between batches. The total flavonoid accumulation in cells treated with inducers is significantly higher than that in uninduced cultures. Fifth, in the health supplement formulation, taurine and hybrid liposomes form a relay at the absorption level. Inulin and papain exert their effects through two pathways: intestinal fermentation and hairball matrix disintegration, respectively. Feeding trials show that the health supplement has good palatability and tolerability, and no abnormalities were observed in hematological and serum biochemical indicators. Attached Figure Description

[0012] Figure 1 This is a process flow diagram of the method for preparing cat grass plant stem cell extract according to the present invention.

[0013] Figure 2 This is a comparison chart of the total flavonoid encapsulation rate and simulated gastric fluid retention rate of the extracts obtained in each embodiment and comparative example.

[0014] Figure 3 The diagram shows a comparison of the effective delivery rates and synergistic effects of each embodiment and comparative example. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise stated, the experimental methods used in the following embodiments are conventional methods, and the reagents, culture medium components, and enzyme preparations used are all commercially available. Specifically, the MS medium was prepared according to a conventional formula, with 7 g / L agar added to the solid culture medium, and the pH was adjusted to 5.8 before sterilization; the cellulase activity was 5 × 10⁻⁶. 4 U / g, xylanase activity is 1×10 5 U / g, β-glucanase activity is 4×10 4 U / g; phosphatidylcholine was derived from sunflower and had a purity of not less than 95%; high-pressure homogenization was performed using a high-pressure homogenizer with independently adjustable pressure in two stages, and the homogenization chamber was equipped with a circulating cooling jacket; phosphate buffer was 0.01 mol / L and pH 7.2 unless otherwise specified. The main instruments included a clean bench, a light incubator, an airlift bioreactor, a high-pressure homogenizer with independently adjustable pressure in two stages, a dynamic light scattering particle size analyzer, a high-performance liquid chromatograph, a UV-Vis spectrophotometer, a gas chromatograph, a spray dryer, and a freeze dryer, all of which were conventional commercial equipment. The detection methods in each example and comparative example are described in the detection methods section below, and the detection data are summarized in Tables 1 and 2.

[0016] Example 1 This embodiment uses orchardgrass (Dactylis glomerata) as the raw material plant, according to... Figure 1 The process shown is for preparing cat grass plant stem cell extract. The specific steps are as follows.

[0017] Callus induction and dark proliferation: Healthy, disease-free young leaves and stem tips of *Orchardgrass* were collected from June to July. After rinsing with running water, the tissues were treated with 70% ethanol (v / v) for 30 s in a clean bench, followed by treatment with 0.1% sodium hypochlorite solution (w / w) for 15 min. The tissues were rinsed three times with sterile water. Explants of 3-5 mm in length were cut with a sterile scalpel and inoculated onto MS solid medium containing 1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.2 mg / L kinetin, 30 g / L sucrose, 100 mg / L inositol, and 0.4 mg / L thiamine hydrochloride. The tissues were cultured at 26°C in the dark for 18 days. White to pale yellow, loosely textured callus tissues formed at the cut sites. Vigorously growing callus tissue was selected and subcultured every 24 days for a total of 4 subcultures to establish a stable cell line. The callus tissue was then transferred to liquid MS medium with the same hormone composition. After two subculture cycles in shake flasks, the cells were inoculated into a 20 L airlift bioreactor with a liquid volume of 14 L and an inoculum size of 1.2 g / L (based on cell dry weight). The cells were cultured in suspension at 25°C and an aeration rate of 0.12 vvm in the dark for 14 days until the cell dry weight reached 16.8 g / L, yielding dark-proliferating cells. Cell dry weight and cell viability were measured every 2 days during suspension proliferation. The growth curve showed a logarithmic growth phase from day 3 to day 10, stabilizing after day 12. The trypan blue rejection rate remained consistently above 95%. Based on this, the dark proliferation time was determined to be 14 days to balance biomass accumulation and cell viability.

[0018] Photoconversion: Dark-proliferating cells were collected through a 200-mesh filter and transferred to photoconversion medium at the original culture volume. The photoconversion medium was a liquid MS medium with a sucrose concentration of 12 g / L, free of 2,4-dichlorophenoxyacetic acid, and with the same components as the proliferation medium. On the day of transfer, i.e., day 1 of photoconversion, methyl jasmonate was added to a final concentration of 100 μmol / L. The cells were then cultured for another 5 days under conditions of 500 lx light intensity, a 12 h:12 h light-dark cycle, and 25°C. The culture medium color gradually changed from milky yellow to yellow-green. 24 hours before harvest, the cells were exposed to 305 nm ultraviolet light at an irradiance of 1.0 W / m². 2 Irradiated for 5 min. After culture, cells were collected by filtering through a 200-mesh filter cloth, washed three times with phosphate-buffered saline (pH 7.2), and drained to obtain 1.65 kg of wet cells. Measurements showed that the total chlorophyll content of the cells after photoconversion reached 3.2 mg / g (dry weight), while that of the same batch of dark-proliferating cells was only 0.21 mg / g. The total flavonoid content of the cells increased from 6.7 mg / g in the uninduced control to 12.4 mg / g (dry weight), indicating that the establishment of the thylakoid membrane system and flavonoid accumulation were completed simultaneously.

[0019] Enzymatic softening: Wet cells were suspended in phosphate buffer at a material-to-liquid ratio of 1:8 g / mL. The pH of the system was adjusted to 5.2 with dilute hydrochloric acid, and the temperature was raised to 38°C. Cellulase 0.3%, xylanase 0.2%, and β-glucanase 0.15% were added based on the wet weight of the cells. The mixture was stirred and hydrolyzed for 30 min. The system was then rapidly cooled to 6°C to terminate the enzymatic reaction, yielding enzyme-softened cytoplasm. The degree of hydrolysis was controlled under a microscope to the extent that the cell wall showed visible loosening while the overall cell outline was still maintained, avoiding premature rupture of protoplasts.

[0020] Two-stage homogenization: The enzyme-softened cell plasma was fed into a high-pressure homogenizer at a feed temperature of 6°C and homogenized twice at 300 bar. Between the two homogenizations, the material was cooled to 12°C through a jacket heat exchange. The cell disruption rate reached 99.2% under microscopic counting, resulting in a first-stage homogenized liquid. 2.5% of phosphatidylcholine and 0.8% cholesterol by mass were added to the first-stage homogenized liquid. After stirring and dispersing for 30 min, the mixture was homogenized three times at 1000 bar, with cooling between each homogenization. The final discharge temperature was 31°C, resulting in a second-stage homogenized liquid.

[0021] Separation, purification, and drying: The two homogenized solutions were centrifuged at 4000 r / min for 20 min. The supernatant was filtered through a 0.45 μm mixed cellulose ester membrane to remove residues and possible microorganisms, yielding a cat grass plant stem cell extract containing hybrid liposomes, which was a yellow-green translucent liquid. Dynamic light scattering analysis showed an average particle size of 132 nm and a polydispersity index of 0.19, within the defined range of 50–200 nm. 8% (by weight) of trehalose was added to the extract, and after thorough dissolution, it was spray-dried at an inlet air temperature of 130°C and an outlet air temperature of 55°C to obtain a yellow-green extract powder. The yield of the powder, based on cell dry weight, was 38.6%, and a total of 90.7 g of powder was obtained in this batch.

[0022] Characterization results: The total flavonoid content (calculated as rutin) of the dry powder in this embodiment was 8.6 mg / g, the total chlorophyll content was 1.24 mg / g, the free ferulic acid content was 0.42 mg / g, and the total flavonoid encapsulation rate was 82.4%. The total flavonoid retention rate after incubation in simulated gastric fluid for 2 h was 78.9%, and the cumulative release rate after incubation in simulated intestinal fluid for 6 h was 81.5%, resulting in an effective delivery rate of 53.0%. The DPPH free radical scavenging ability was determined using the same species of fresh Oryza sativa homogenate freeze-dried powder as a reference sample. The IC50 of the dry powder in this embodiment... 50 The concentration was 0.86 mg / mL, while the reference concentration was 2.35 mg / mL, indicating that the removal capacity per unit mass was significantly better than that of the traditional fresh grass route. The moisture content of the dried powder was 3.1%, and the total bacterial count did not exceed 1×10⁻⁶. 4 CFU / g, mold and yeast not exceeding 1×10 2The CFU / g levels of lead, arsenic, cadmium, and mercury meet the limits specified in GB 13078 Feed Hygiene Standard.

[0023] Storage stability: The dry powder of this embodiment was sealed in an aluminum foil bag and stored at 25°C in the dark for 6 months. Samples were taken every 2 months for rehydration characterization. After 6 months, the average particle size after rehydration changed from 132 nm to 139 nm, the polydispersity index changed from 0.19 to 0.22, the total flavonoid retention rate was 96.8%, the total chlorophyll retention rate was 94.2%, and the retention rate in simulated gastric juice after 2 hours was 77.1%. Compared with the initial values, there was no significant deterioration, indicating that the trehalose glassy matrix is ​​effective in fixing the hybrid liposome membrane structure under normal storage conditions. In addition, the dry powder was placed at 40°C and 75% relative humidity for 3 months. The total flavonoid retention rate was 91.5%, and the average particle size after rehydration was 151 nm, which was still within the limited range of 50-200 nm.

[0024] Example 2 This embodiment uses wheat (Triticum aestivum) seedlings as the raw material to investigate the implementation effect when the lower limit values ​​of each process parameter are taken. Young leaf bases and root tip meristems of sterile wheat seedlings that have germinated for 7 days are used as explants. The sterilization and cutting methods are the same as in Example 1. They are inoculated into MS solid medium containing 1.0 mg / L 2,4-dichlorophenoxyacetic acid and 0.1 mg / L kinetin, and cultured at 24°C in the dark for 14 days to induce callus tissue. Subculture is performed every 21 days for a total of 3 subcultures before being transferred to liquid culture. Suspension culture is carried out in a 5 L airlift bioreactor with a liquid volume of 3.5 L, a temperature of 24°C, an aeration rate of 0.10 vvm, and incubation in the dark for 10 days, until the cell dry weight reaches 14.2 g / L. During the photoconversion stage, cells were transferred to liquid MS medium containing 10 g / L sucrose and free of 2,4-dichlorophenoxyacetic acid. Methyl jasmonate was added to a final concentration of 50 μmol / L on day 1 of photoconversion. Cells were cultured for 3 days under a light intensity of 200 lx and a light-dark cycle of 12 h:12 h. Twelve hours before harvest, cells were cultured at a wavelength of 305 nm and an irradiance of 0.5 W / m². 2The cells were irradiated with ultraviolet light for 3 min, filtered, and washed to obtain 348 g of wet cells. Enzyme softening was performed by resuspending the cells at a material-to-liquid ratio of 1:5 g / mL, pH 5.0, and temperature 35°C. Cellulase 0.2%, xylanase 0.1%, and β-glucanase 0.1% were added based on the wet weight of the cells. Enzymatic hydrolysis was carried out for 20 min, followed by cooling to 4°C. Two homogenization stages were performed. The first stage was homogenized twice at 250 bar, achieving a 98.6% cell disruption rate. After adding 2% phosphatidylcholine and 0.5% cholesterol from the first stage's disruption solution, the second stage was homogenized twice at 800 bar, with the final discharge temperature at 29°C. Centrifugation was performed at 3500 r / min for 25 min. The supernatant was filtered through a 0.45 μm filter membrane, and 5% (by weight) of trehalose (by mass of the extract) was added. The mixture was then freeze-dried to obtain a dry powder. The freeze-drying process involved pre-freezing at -40°C for 4 h, drying at -20°C for 24 h, and then desorption drying at 25°C for 8 h. The resulting dry powder had a moisture content of 2.6%. The extract obtained in this example had an average particle size of 147 nm and a polydispersity index of 0.23. The total flavonoid content of the dry powder was 7.9 mg / g, the total chlorophyll content was 1.02 mg / g, the free ferulic acid content was 0.51 mg / g, the total flavonoid encapsulation rate was 79.6%, the retention rate in simulated gastric juice after 2 h was 76.4%, the cumulative release rate in simulated intestinal juice after 6 h was 79.8%, and the effective delivery rate was 48.5%. These results indicate that the method can still be stably implemented when all parameters are taken at their lower limits. The wheat cell wall arabinoxylan content is high, and its free ferulic acid content is higher than that in Example 1.

[0025] Example 3 This embodiment uses oat (Avena sativa) seedlings as the raw material to investigate the implementation effect and scale-up feasibility of each process parameter at its upper limit. Explant collection and disinfection were the same as in Example 1. The induction medium contained 2.0 mg / L 2,4-dichlorophenoxyacetic acid and 0.5 mg / L kinetin, and the cells were cultured at 27°C in the dark for 21 days, subcultured every 28 days for a total of 6 subcultures. Suspension culture was carried out in a 100 L airlift bioreactor with a liquid volume of 70 L, a temperature of 26°C, an aeration rate of 0.15 vvm, and in the dark for 18 days, until the cell dry weight reached 15.1 g / L. During the photoconversion stage, the sucrose concentration in the medium was 15 g / L, the final concentration of methyl jasmonate was 200 μmol / L, the light intensity was 1000 lx, and the culture was carried out for 7 days. 24 h before harvest, the cells were cultured at a wavelength of 305 nm and an irradiance of 1.5 W / m². 2The sample was irradiated with ultraviolet light for 10 min. Enzymatic softening was performed at a material-to-liquid ratio of 1:10 g / mL, pH 5.5, and temperature of 40°C. Cellulase 0.5%, xylanase 0.3%, and β-glucanase 0.3% were added based on the wet weight of the cells. After enzymatic hydrolysis for 40 min, the sample was cooled to 10°C. The first homogenization was performed three times at 400 bar, achieving a 99.5% breakage rate. After adding 3% phosphatidylcholine and 1% cholesterol (by weight of the first homogenate), the second homogenization was performed three times at 1200 bar, with the final discharge temperature at 33°C. The sample was centrifuged at 4500 r / min for 15 min, filtered, and then trehalose (by weight of the extract) was added. The sample was then spray-dried at an inlet air temperature of 140°C and an outlet air temperature of 60°C. The extract obtained in this embodiment had an average particle size of 118 nm and a polydispersity index of 0.20; the total flavonoid content of the dry powder was 7.2 mg / g, the total chlorophyll content was 1.31 mg / g, the free ferulic acid content was 0.38 mg / g, the total flavonoid encapsulation rate was 80.8%, the retention rate in simulated gastric juice after 2 h was 77.5%, the cumulative release rate in simulated intestinal juice after 6 h was 80.2%, and the effective delivery rate was 50.2%. These results indicate that the method operates stably at a scale of 100 L, and the product quality is comparable to that of the small-scale test when all parameters are taken at their upper limits. A horizontal comparison of the three embodiments also shows that cells from the three catnip plants exhibited consistent process behavior under this process. The differences between species are mainly reflected in the relative proportions of the active ingredient profiles rather than differences in process adaptability, indicating that the parallel limitation of species has a common implementation basis.

[0026] Comparative Example 1 This comparative study investigated the effect of omitting the photoconversion stage. Except for replacing the photoconversion stage of Example 1 with continued light-protected culture in the original proliferation medium for 5 days, adding methyl jasmonate at a final concentration of 100 μmol / L on day 1 of this stage, and omitting light and UV irradiation, all other materials and operations were the same as in Example 1. The resulting cells had a total chlorophyll content of only 0.09 mg / g on a dry weight basis, indicating that the thylakoid membrane system was not established. The final dry powder had a total flavonoid content of 7.1 mg / g, a total chlorophyll content of 0.09 mg / g, a total flavonoid encapsulation rate reduced to 61.3%, a 2-hour retention rate in simulated gastric juice reduced to 58.2%, and an effective delivery rate of only 28.2%. The results indicate that, in the absence of thylakoid galactosides, the vesicles obtained by two-stage homogenization are mainly composed of plasma membrane and endomembrane system phospholipids and exogenous phosphatidylcholine. Their membrane resistance to gastric juice is significantly lower than that of hybrid liposomes containing galactosides. In other words, the photoconversion stage is the material basis for hybrid liposomes to obtain oral stability.

[0027] Comparative Example 2 This comparative study investigated the effect of omitting the enzyme softening step. Except for the elimination of the enzymatic hydrolysis and the direct entry of wet cell suspension into the two-stage homogenization process, all other raw materials and operations were the same as in Example 1. Under the same 300 bar first-stage and 1000 bar second-stage programs, the cell breakage rate was only 78.5%, the dry powder yield (based on cell dry weight) decreased to 25.1%, the total flavonoid content in the dry powder was 6.8 mg / g, the free ferulic acid content was only 0.07 mg / g, the total flavonoid encapsulation rate was 70.2%, and the effective delivery rate was 42.4%. If the second-stage pressure was increased to 1500 bar and the number of homogenization cycles increased to 5 to achieve a breakage rate comparable to Example 1, the breakage rate could reach 97.8%, but the final discharge temperature rose to 48°C, and the total chlorophyll content in the dry powder decreased to 0.69 mg / g, showing significant demagnesiation and browning. These results indicate that pre-softening of the type II cell wall by the complex enzyme is a prerequisite for achieving complete low-pressure disruption and preservation of heat-sensitive components, and that the release of bound ferulic acid by xylanase is a unique concomitant contribution of this method.

[0028] Comparative Example 3 This comparative study investigated the effect of replacing a two-stage pressure program with a single-stage constant-pressure homogenization. Except for replacing the two-stage homogenization with direct homogenization at 1200 bar for four cycles and adding phosphatidylcholine and cholesterol all at once before homogenization, all other raw materials and operations were the same as in Example 1. This homogenization method is comparable to the process conditions disclosed in US9155916B2. The resulting cell disruption rate was 93.4%, the final discharge temperature was 41°C, the average particle size of the extract was 156 nm, the polydispersity index increased to 0.31, the total flavonoid encapsulation rate was 65.8%, the retention rate in simulated gastric juice after 2 hours was 64.5%, and the effective delivery rate was 32.8%. These results indicate that when disruption and vesicle reconstruction are completed in one step within the same high-intensity field, large cell wall fragments compete with lipids for shear energy, resulting in a wider vesicle size distribution. Furthermore, the absence of inter-stage low-pressure buffering leads to cumulative temperature rise, validating the necessity of the decoupled design of the two-stage pressure program.

[0029] Comparative Example 4 This comparative study investigated the effect of replacing in-situ hybrid encapsulation with a two-step extraction-encapsulation method. Callus culture, photoconversion, and enzyme softening were the same as in Example 1. No lipids were added during homogenization; the mixture was homogenized twice at 300 bar and three times at 1000 bar, then centrifuged and filtered to obtain the free extract. Blank liposomes were prepared using the same amount of phosphatidylcholine and cholesterol as in Example 1 via thin-film hydration. The free extract was then mixed with the blank liposomes, and encapsulated by ultrasonic treatment for 20 min. The resulting liposomes had an average particle size of 168 nm, a polydispersity index of 0.28, a total flavonoid encapsulation efficiency of 58.7%, a 2-hour retention rate in simulated gastric juice of 52.6%, and an effective delivery rate of 23.2%. The results indicate that in the two-step method, endogenous membrane lipids are discarded with the centrifugation residue, the encapsulation membrane is a pure phosphatidylcholine membrane, lacking the hydrogen bond densification contribution of digalactosylglycerol, and the post-encapsulation has low encapsulation efficiency for the already free active substances, verifying the core role of in-situ hybrid encapsulation, i.e., the simultaneous completion of fragmentation and encapsulation.

[0030] Comparative Example 5 This comparative study investigated the impact of key parameters exceeding the specified range, setting up two exceedance groups. Exceedance group one increased the second-stage homogenization pressure of Example 1 to 1500 bar, while maintaining the same parameters as Example 1. The final discharge temperature was increased to 52°C, resulting in a decrease in total flavonoid encapsulation efficiency to 66.1%, a decrease in total chlorophyll content in the dry powder to 0.71 mg / g, a 2-hour retention rate in simulated gastric juice of 63.0%, and an effective delivery rate of 32.0%. This indicates that when the second-stage pressure exceeded 1200 bar, the cumulative temperature rise damaged both the vesicle membrane and the heat-sensitive active ingredient. In the second group, the final concentration of methyl jasmonate during the photoconversion stage was increased to 400 μmol / L, while the rest remained the same as in Example 1. The cells underwent significant browning, with a browning rate of 38%, and the cell dry weight decreased to 9.8 g / L. The total flavonoid content in the dry powder actually decreased to 4.9 mg / g, and the effective delivery rate was 49.3%. This indicates that excessive methyl jasmonate induced excessive oxidative stress, and phenols were oxidized and polymerized, verifying the critical significance of the upper limit of the 50~200 μmol / L range.

[0031] Comparative Example 6 This comparative example is a double-deleted baseline group used for the quantitative decomposition of the synergistic effect. The photoconversion stage was omitted (processing method same as Comparative Example 1), and a two-step encapsulation method was used (processing method same as Comparative Example 4), otherwise the same as in Example 1. The resulting liposomes had an average particle size of 172 nm, a polydispersity index of 0.30, a total flavonoid encapsulation efficiency of 49.5%, a 2-hour retention rate in simulated gastric juice of 47.1%, and an effective delivery rate of 17.3%. This comparative example, together with Comparative Example 1, Comparative Example 4, and Example 1, constitutes a 2×2 factorial design, and its data are used for the quantitative demonstration of the nonlinear synergistic effect described later.

[0032] The comparative examples and embodiments above together demonstrate that the technical features of the present invention are not isolated parameter selections, but rather a chain configuration around the same core technical problem: the photoconversion stage determines the composition of the membrane lipid pool, enzyme softening determines the energy threshold required for disruption, the two-stage pressure program determines whether disruption and vesicle reconstruction can be decoupled, and in-situ hybridization determines whether endogenous membrane lipids can be utilized. If any of these four links is replaced with conventional practices in the prior art, the effective delivery rate will decrease significantly, and this decrease cannot be compensated for by strengthening the other links.

[0033] The detection method is as follows: Cell disruption rate was determined by microscopic counting. Samples before and after homogenization were stained with trypan blue, and intact cells were counted in a hemocytometer. The disruption rate was expressed as the ratio of the difference in the number of intact cells before and after homogenization to the number of intact cells before homogenization. Average particle size and polydispersity index were determined using dynamic light scattering at 25°C. Samples were diluted 100-fold with phosphate buffer. Total flavonoid content was determined using the sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method at 510 nm, with rutin as the standard for quantification. Total chlorophyll content was determined according to the Arnon method, after extraction with 80% acetone in the dark, and absorbance was measured and calculated at 645 nm and 663 nm. Free ferulic acid content was determined by high-performance liquid chromatography (HPLC) using a C18 column at a detection wavelength of 320 nm, with gradient elution of methanol and 0.1% phosphate aqueous solution. The total flavonoid encapsulation efficiency was determined by separating free flavonoids from vesicle-bound flavonoids using dextran gel column chromatography. The encapsulation efficiency was the percentage of vesicle-bound flavonoids in the total amount. Simulated gastric juice was prepared according to current pharmacopoeia methods as a pH 1.2 hydrochloric acid solution containing pepsin. After incubation at 37°C for 2 h, the remaining proportion of total flavonoids in the vesicles was measured and recorded as the simulated gastric juice 2-h retention rate. Simulated intestinal juice was a pH 6.8 phosphate solution containing pancreatin and bile salts. After incubation at 37°C for 6 h, the proportion of cumulatively released total flavonoids in the vesicles was measured and recorded as the simulated intestinal juice 6-h cumulative release rate. The effective delivery rate was the product of the total flavonoid encapsulation efficiency, the simulated gastric juice 2-h retention rate, and the simulated intestinal juice 6-h cumulative release rate, divided by 10000, expressed as a percentage. This rate characterizes the proportion of active ingredients that can be absorbed by the small intestine after oral ingestion. DPPH free radical scavenging capacity was expressed as IC50. 50The results showed that after serial dilution, the samples were reacted with DPPH ethanol solution in the dark for 30 min, and measured at a wavelength of 517 nm. Fecal short-chain fatty acids were determined by gas chromatography with a flame ionization detector. Serum total antioxidant capacity was determined using the ABTS method kit. The coefficient of variation for mixing uniformity was determined using taurine as a tracer component. At the mixing endpoint, 10 samples were taken from different parts of the material layer, and the taurine content of each sample was determined by high-performance liquid chromatography, and the coefficient of variation was calculated. The moisture content of the dried powder was determined using the 105°C constant weight method. Fecal hair excretion was determined by soaking all feces collected from the long-haired subgroup within 14 days in warm water, dispersing the hair, and then passing it through a 40-mesh sieve. The hair remaining on the sieve was washed, dried at 60°C to constant weight, and then weighed. Fecal chlorophyll recovery rate was the percentage of total chlorophyll recovered from feces relative to the total chlorophyll ingested during the same period. All data were analyzed using SPSS 26.0 software with one-way ANOVA and Duncan multiple comparisons; P < 0.05 was considered statistically significant.

[0034] Example 4 This embodiment describes the formulation of a feline health supplement and its feeding validation. The complete formula group C1 was prepared according to the following mass proportions: 15 parts of the extract powder obtained in Example 1, 6 parts of taurine, 22 parts of inulin, 2 parts of papain, 4 parts of fish flavor powder, and 51 parts of maltodextrin. Each component was passed through an 80-mesh sieve and then mixed at room temperature (26°C) using an equal-incremental mixing method. The measured coefficient of variation for the mixing uniformity at the mixing endpoint was 2.8%, meeting the requirement of not exceeding 5%. The mixture was then packaged in 2g portions into aluminum foil bags to obtain the powdered feed supplement. To differentiate the contributions of the subgroups, two missing groups, C2 and C3, were established: C2 did not contain taurine, and C3 did not contain inulin. The missing portions were supplemented with maltodextrin to the same total mass proportion, with the remaining operations identical to C1. The control group C4 was a commercially available cat nutritional supplement powder, whose labeled ingredients included taurine, fish oil, and field-grown wheatgrass powder.

[0035] Thirty-two healthy adult domestic cats, aged 2-6 years and weighing 3.5-5.5 kg, were selected for the feeding trial. Sixteen long-haired and sixteen short-haired cats were randomly divided into four groups of eight each, with a balanced distribution of long / short hair and weight among the groups. After a 7-day acclimatization period, the cats entered a 28-day trial period. Each group was fed 2 g of the corresponding sample daily, evenly mixed into their basal diet, which was a commercially available complete cat food from the same batch. Palatability was determined using the two-bowl method. Each cat underwent eight pairing tests with sample C4 during the trial period, and the initial food choice was recorded. Daily food intake was recorded throughout the trial period. On days 0 and 28, fasting blood samples were collected in the morning to measure hematological and serum biochemical indicators and serum total antioxidant capacity. For the long-haired subgroup, all feces were collected on the last 14 days of the trial. The feces were washed, separated, and the hair excreted in the feces was weighed. Simultaneously, fresh fecal samples were taken to determine the short-chain fatty acid content and chlorophyll recovery rate.

[0036] The feeding results are summarized in Table 3. The preferred feeding rate of the two-bowl method (C4) in group C1 was 78.1%, and the average daily feed intake increased by 6.2% compared to the adaptation period, indicating good palatability of the hybrid liposome extract combined with fish flavor powder. The fecal hair excretion in the long-haired subgroup was 1.86 g in group C1, significantly higher than the 1.12 g in group C4, suggesting that the loosening of the hairball matrix by papain and the propulsion of intestinal contents by inulin fermentation products promoted the physiological excretion of hair through feces. The fecal acetic acid, propionic acid, and butyric acid contents in group C1 were 58.2 μmol / g, 24.6 μmol / g, and 12.8 μmol / g, respectively, all significantly higher than those in inulin-deficient groups C3 and C4, while those in group C2 (taurine-deficient) were similar to those in group C1, consistent with the expectation that inulin was the only source of incremental short-chain fatty acids. The total antioxidant capacity of serum in group C1 increased by 21.4% compared to day 0, while that in group C2 increased by only 12.6%. Furthermore, the fecal chlorophyll recovery rate in group C2 was 63.5%, significantly higher than the 41.2% in group C1. This indicates that the supply of conjugated bile acids is limited when taurine is lacking, leading to reduced absorption of lipid-soluble active ingredients carried by hybrid liposomes via the mixed micelle pathway and increased excretion in feces. This verifies the relay relationship between taurine and hybrid liposomes in the absorption process. The total antioxidant capacity of serum in group C3 increased by 19.8%, similar to that of group C1, while its fecal short-chain fatty acid level was significantly lower than that of group C1. This suggests that the inulin pathway and the antioxidant absorption pathway are independent and do not compensate for each other. Juxtaposing the data from groups C2 and C3 shows that when each pathway is individually interrupted, their respective indicator groups decline independently. However, the complete formulation C1 causes both groups of indicators to be at their highest levels simultaneously. This orthogonal division of labor among components is the structural feature that distinguishes the composition of this invention from simple mixed commercially available products. During the trial period, all groups exhibited good mental and coat condition, with no signs of intolerance such as vomiting or diarrhea. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine levels were all within the reference range, with no statistically significant differences between groups. Body weight remained stable, indicating good tolerability and safety of the supplement in felines. Fecal characteristics were recorded daily using a 5-point scale. The median score for each group was 3 points (formed stool), with no statistically significant differences in distribution between groups. In the later stages of the trial, the residue rate in the mixed feed in group C1 was less than 3%, and no anorexia was observed, suggesting good compliance with the formula during 28 days of continuous feeding.

[0037] The preparation process indicators of each embodiment and comparative example are summarized in Table 1, and the active ingredients and delivery performance indicators are summarized in Table 2.

[0038] Table 1. Preparation process indicators of each embodiment and comparative example Example 1 16.8 99.2 31 132 0.19 38.6 Example 2 14.2 98.6 29 147 0.23 36.2 Example 3 15.1 99.5 33 118 0.20 37.4 Comparative Example 1 16.5 99.0 31 121 0.22 37.9 Comparative Example 2 16.8 78.5 33 139 0.26 25.1 Comparative Example 3 16.8 93.4 41 156 0.31 33.5 Comparative Example 4 16.8 99.0 32 168 0.28 30.8 Comparative Example 5, Group 1 (crossing the boundary) 16.8 99.4 52 106 0.24 34.0 Comparative Example 5, Group 2 (crossing the boundary) 9.8 99.1 31 135 0.21 28.7 Comparative Example 6 16.4 99.0 32 172 0.30 29.5 Table 2. Active ingredients and delivery performance of the dry powders in each example and comparative example. Example 1 8.6 1.24 0.42 82.4 78.9 81.5 53.0 0.86 Example 2 7.9 1.02 0.51 79.6 76.4 79.8 48.5 0.94 Example 3 7.2 1.31 0.38 80.8 77.5 80.2 50.2 0.98 Comparative Example 1 7.1 0.09 0.40 61.3 58.2 79.0 28.2 1.21 Comparative Example 2 6.8 1.15 0.07 70.2 74.9 80.6 42.4 1.05 Comparative Example 3 8.1 0.98 0.39 65.8 64.5 77.2 32.8 0.92 Comparative Example 4 8.3 1.18 0.41 58.7 52.6 75.2 23.2 0.90 Comparative Example 5, Group 1 (crossing the boundary) 7.4 0.71 0.36 66.1 63.0 76.8 32.0 1.02 Comparative Example 5, Group 2 (crossing the boundary) 4.9 1.20 0.35 78.9 77.2 80.9 49.3 1.42 Comparative Example 6 7.0 0.08 0.39 49.5 47.1 74.0 17.3 1.26 Table 3 Results of the health supplement feeding trial (28 days) The preferred rate (%) for the C4 two-bowl method 78.1 75.0 76.6 — Change in average daily feed intake (%) +6.2 +5.8 +5.5 +1.2 Fecal hair excretion (g / 14 d) 1.86 1.79 1.52 1.12 Fecal acetic acid (μmol / g) 58.2 55.9 41.5 43.8 Fecal propionic acid (μmol / g) 24.6 23.8 16.2 17.5 Fecal butyric acid (μmol / g) 12.8 12.1 7.4 8.2 Increase in serum total antioxidant capacity (%) +21.4 +12.6 +19.8 +8.9 Fecal chlorophyll recovery rate (%) 41.2 63.5 43.0 — As can be seen from Tables 1 and 2, Examples 1 to 3 all achieved a breakage rate of no less than 98.6%, a discharge temperature of no more than 35°C throughout the process, a vesicle particle size of 50~200 nm, and a total flavonoid encapsulation rate of more than 79% under the combination of the median, lower limit, and upper limit of the parameter window. Moreover, the performance of Example 3 on a 100 L scale is comparable to that of Example 1 on a 20 L scale, proving that the method can be stably implemented and has the feasibility of scale-up within the specified parameter range. Figure 2 The total flavonoid encapsulation rate and the 2-hour retention rate of simulated gastric juice were compared in the form of a bar chart for each group. Figure 2 As shown, both metrics of the three embodiments are significantly higher than those of all comparative examples. The difference with Comparative Example 4 directly reflects the advantage of in-situ hybrid encapsulation over the traditional two-step method. Further examination of each group reveals several details with mechanistic implications. The average particle size of the vesicles obtained in Comparative Example 1 is 121 nm, smaller than 132 nm in Example 1, but its encapsulation efficiency and gastric fluid retention rate are significantly lower, indicating that vesicle size itself is not the dominant factor determining oral stability; the lipid composition of the membrane is the key variable. The smallest particle size in Comparative Example 5 (out-of-bounds group 1) is 106 nm, but the discharge temperature of 52°C impairs membrane density, resulting in a decrease in encapsulation efficiency instead of an increase. This suggests that sacrificing temperature control for particle size refinement is not worthwhile in this system. Although the performance of Comparative Example 5 (Group 2) at the vesicle level was close to that of Example 1, the browning and biomass loss at the cell level significantly reduced the yield of active ingredients per unit culture volume. The total flavonoid content of its dry powder was only 4.9 mg / g, which fell outside the specified quality index range. This confirmed the necessity of the upper limit of methyl jasmonate concentration from the raw material perspective.

[0039] The quantitative demonstration of the nonlinear synergistic effect is as follows. Example 1, Comparative Example 1, Comparative Example 4, and Comparative Example 6 constitute a 2×2 factorial design with the photoconversion stage and in-situ hybrid encapsulation as two factors. The effective delivery rates of the four groups are 53.0%, 28.2%, 23.2%, and 17.3%, respectively. Taking the double-deficient Comparative Example 6 as the baseline, the gain from recovering only the photoconversion stage is the difference between Comparative Example 4 and Comparative Example 6, i.e., 5.9 percentage points; the gain from recovering only the in-situ hybrid encapsulation is the difference between Comparative Example 1 and Comparative Example 6, i.e., 10.9 percentage points. If the effects of the two methods are independent, the expected effective delivery rate, based on linear summation, should be 17.3% + 5.9 percentage points + 10.9 percentage points, i.e., 34.1%. However, the measured value of Example 1, which possesses both methods simultaneously, is 53.0%, exceeding the expected linear summation by 18.9 percentage points, a relative excess of 55%. Figure 3As shown, the super-addition and gain demonstrate a clear interdependence between the two methods: the value of in-situ hybrid encapsulation can only be fully realized when the photoconversion stage provides a thylakoid membrane lipid library dominated by digalactosylglycerol and monogalactosylglycerol. Conversely, if the galactosylglycerol accumulated during photoconversion is discarded with centrifugation residue in the two-step process, it cannot participate in vesicle assembly. The two are upstream and downstream links in the same causal chain, rather than separate parallel methods; their combination produces a technical effect greater than the sum of their parts. Furthermore, Comparative Examples 2 and 3 show that when enzyme softening and the two pressure programs are absent, the effective delivery rate drops to 42.4% and 32.8%, respectively, accompanied by a decrease in yield or damage from temperature rise, indicating that each step of this method is interconnected and jointly supports the final performance. From an industrial implementation perspective, all steps of this method utilize common equipment used in the food and feed industries, eliminating the need for high-cost units such as organic solvent extraction and chromatographic purification. Enzyme preparations, phosphatidylcholine, cholesterol, and trehalose are all readily available commercial raw materials. The two homogenization processes can be achieved by switching programs on the same high-pressure homogenizer. The key control points during scale-up are only the inter-stage cooling capacity and the uniformity of illumination during the photoconversion stage. The 100 L scale data in Example 3 shows that the above control points can be stably met in engineering. This method is ready for direct transfer to pilot-scale and production.

[0040] The mechanism by which the technical effects of this invention are achieved can be summarized as a causal chain connecting the three levels of culture, disruption, and delivery. At the culture level, the removal of 2,4-dichlorophenoxyacetic acid and the reduction of sucrose concentration relieved the inhibition of plastid development by hormones and high sugar. Weak light of 200-1000 lx was sufficient to initiate the transformation of proplasts into chloroplasts without causing photodamage to the surface cells, and the thylakoid membrane was subsequently established, with its membrane lipids mainly consisting of monogalactosylglycerol and digalactosylglycerol. Simultaneously applied methyl jasmonate and short-term ultraviolet irradiation activated the phenylpropane metabolic pathway, causing flavonoids and phenolic acid products to accumulate in the cells. That is, the photoconversion stage simultaneously completed the dual enrichment of the encapsulation material library and the encapsulated active substance library. At the fragmentation level, the cell walls of grasses belong to type II cell walls, with cellulose microfibrils and cross-linked arabinoxylan and mixed-bond glucan forming the backbone. The combined action of cellulase, xylanase and β-glucanase hydrolyzes this cross-linked network to a limited extent, reducing the mechanical strength of the cell wall. The fragmentation can be completed in a low-pressure range of 250~400 bar, and the temperature rise is controlled throughout the process. When xylanase hydrolyzes arabinoxylan, it releases ferulic acid linked by ester bonds into a free state, which in turn increases the phenolic acid level of the extract. At the delivery level, the two homogeneous high-shear fields cause endogenous membrane lipid fragments to co-assemble with exogenous phosphatidylcholine and cholesterol. The cone-shaped molecular geometry of monogalactosylglycerol buffers the high curvature stress of small vesicles, and the digalactosylglycerol head group forms a hydrogen bond network on the vesicle surface. Together with the membrane sequence regulation effect of cholesterol, these factors improve the membrane compactness and acid resistance, allowing the vesicles to remain intact in the gastric juice environment. After entering the intestinal juice, the contents are released under the action of bile salts and pancreatic enzymes. During spray drying or freeze drying, trehalose replaces water molecules as a glassy matrix to maintain the hydrogen bond coordination of the polar head of the membrane lipids, and the vesicle structure is preserved in the dry powder state. It is worth noting that cholesterol, with its rigid steroidal ring inserted into the acyl chain region in the hybrid membrane, compresses the free volume within the membrane, reducing the permeation of protons and pepsin into the membrane. Simultaneously, it mitigates membrane phase separation near the phase transition temperature, maintaining the hybrid membrane in a homogeneous state within the storage, transport, and body temperature range. The average particle size of the hybrid vesicles is smaller than that of the pure phosphatidylcholine liposomes in Comparative Example 4. This is because the cone-shaped molecular geometry of monogalactosylglycerol diglyceride reduces the membrane bending elasticity at small radii of curvature, causing the system to tend towards a smaller stable particle size under the same homogeneous energy input. At the animal level, feline bile acids specifically bind to taurine. The taurine provided by the formulation ensures taurolate levels, enabling the lipid-soluble active ingredients released from the vesicles to efficiently enter the intestinal epithelium via a mixed micelle pathway. Inulin ferments in the hindgut to produce short-chain fatty acids, and papain loosens the protein matrix of the hair bulb. Both of these, along with the antioxidant components of the extract, form a complementary and non-overlapping synergistic effect. The above-mentioned steps are interconnected, and the absence of any step will manifest as a quantifiable performance loss in the comparative example, with the mechanism and data mutually corroborating each other.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications, equivalent substitutions, and improvements that can be made by those skilled in the art based on the disclosed technical solutions of the present invention without inventive effort should be considered to fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing cat grass plant stem cell extract, characterized in that, Includes the following steps: S1. Explants from *Corydalis yanhusuo* (a type of grass) were disinfected and inoculated into MS solid medium containing 1.0–2.0 mg / L 2,4-dichlorophenoxyacetic acid and 0.1–0.5 mg / L kinetin. Callus formation was induced in the dark, and the cells were subcultured. Then, they were transferred to liquid MS medium and cultured in suspension in the dark for 10–18 days to obtain dark-proliferating cells. The *Corydalis yanhusuo* species is at least one of *Orchardgrass*, wheat, and oats. S2. The dark-proliferating cells were transferred to photoconversion medium and cultured for 3–7 days at a light intensity of 200–1000 lx and a light-dark cycle of 12 h:12 h. During culture, methyl jasmonate at a final concentration of 50–200 μmol / L was added, and the cells were irradiated with ultraviolet light at a wavelength of 280–315 nm for 3–10 min. The cells were then harvested. The photoconversion medium was liquid MS medium with a sucrose concentration of 10–15 g / L and without 2,4-dichlorophenoxyacetic acid. S3. The cells harvested in S2 are suspended in phosphate buffer, and cellulase, xylanase and β-glucanase are added for enzymatic hydrolysis. After enzymatic hydrolysis, the cells are cooled to 4-10°C to obtain enzyme-softened cytoplasm. S4. The enzyme-softened cytoplasm is homogenized 2-3 times at 250-400 bar to obtain a fragment of lysate. Phosphatidylcholine and cholesterol are added to the first-stage homogenate, and homogenized 2-3 times at 800-1200 bar to obtain a second-stage homogenate; S5, the second-stage homogenate is centrifuged, and the supernatant is filtered through a 0.45 μm filter membrane to obtain cat grass plant stem cell extract containing hybrid liposomes; the hybrid liposomes are formed by the assembly of endogenous membrane lipids of cells harvested in S2 and phosphatidylcholine and cholesterol added in S4, with a particle size of 50-200 nm.

2. The preparation method according to claim 1, characterized in that, In S1, the explants are young leaves, stem tips, or root tips with a length of 3-5 mm; the disinfection is performed by treating with 70% ethanol for 30 s, followed by treatment with 0.1% sodium hypochlorite solution for 15 min, and rinsing three times with sterile water; the induction is carried out at 24-27°C for 14-21 days; the subculture is performed every 21-28 days, for a total of 3-6 subcultures; the suspension culture is carried out in an airlift bioreactor at a temperature of 24-26°C and an aeration rate of 0.1-0.15 vvm.

3. The preparation method according to claim 1, characterized in that, In S2, methyl jasmonate is added on day 1 after transfer to the light conversion medium; the ultraviolet irradiance is 0.5~1.5 W / m². 2 Irradiation was carried out 12-24 hours before harvest; at harvest, cells were collected by filtering with a 200-mesh filter cloth and washed 2-3 times with phosphate buffer solution at pH 7.0-7.

4.

4. The preparation method according to claim 1, characterized in that, In S3, the ratio of cell wet weight to phosphate buffer was 1:5 to 1:10 g / mL; the pH of the enzymatic hydrolysis system was 5.0 to 5.5, the hydrolysis temperature was 35 to 40°C, and the hydrolysis time was 20 to 40 min; based on cell wet weight, the amount of cellulase added was 0.2% to 0.5%, the amount of xylanase added was 0.1% to 0.3%, and the amount of β-glucanase added was 0.1% to 0.3%, all by mass fraction.

5. The preparation method according to claim 1, characterized in that, In S4, the feed temperature of the first homogenization stage is 4~10°C, and the material is cooled to no higher than 15°C between adjacent homogenization stages; the amount of phosphatidylcholine added is 2%~3% of the mass of the first homogenization liquid, and the amount of cholesterol added is 0.5%~1% of the mass of the first homogenization liquid; the discharge temperature of the second homogenization stage is no higher than 35°C.

6. The preparation method according to claim 1, characterized in that, In step S5, the centrifugation speed is 3500~4500 r / min and the centrifugation time is 15~25 min; after filtration, trehalose is added to the obtained extract, the amount of trehalose added is 5%~10% of the extract mass, and the extract is prepared into dry powder by spray drying or freeze drying; the inlet air temperature of the spray drying is 120~140°C and the outlet air temperature is 50~60°C.

7. Catgrass plant stem cell extract prepared by the preparation method according to any one of claims 1 to 6.

8. A feline health product, characterized in that, It is composed of the following components in parts by weight: 10-20 parts of dried powder obtained by drying the cat grass plant stem cell extract of claim 7, 4-8 parts of taurine, 15-30 parts of inulin, 1-3 parts of papain, 3-6 parts of fish flavor powder and 40-60 parts of maltodextrin.

9. The feline health product according to claim 8, characterized in that, The dry powder contains 5.0-12.0 mg / g of total flavonoids (calculated as rutin), 0.5-2.0 mg / g of total chlorophyll, and 0.3-0.8 mg / g of free ferulic acid.

10. The method for preparing the feline health product according to claim 8 or 9, characterized in that, The process includes the following steps: passing the dry powder, taurine, inulin, papain, fish flavor powder, and maltodextrin described in claim 8 through an 80-mesh sieve, mixing them according to the mass ratio described in claim 8 using an equal-incrementing method, with a mixing temperature not exceeding 40°C, mixing until the coefficient of variation of the mixing uniformity is not greater than 5%, and then packaging them to obtain the final product.

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