Interface limited calcium cross-linked glucose oxidase complex liposome for improving intestinal tract of pet

Abstract

The application discloses an interface limited calcium cross-linked glucose oxidase complex liposome for improving the intestinal tract of pets and belongs to the technical field of pet nutrition and functional food. 2+ The specific process of "preformed GOx liposome-LMP interface pre-adsorption-Ca 2+ After adding the limited cross-linking", the gastrointestinal stability of GOx is improved while the particle bridging and macroscopic gelation are inhibited, and the sustained available enzyme activity of GOx is maintained in the post-intestinal stage, so that a new food-grade delivery scheme is provided for pet intestinal environment regulation and fecal odor management.

Description

Technical Field

[0001] This invention relates to an interface-confined calcium-crosslinked glucose oxidase complex liposome for improving the gut health of pets, and the application of this complex liposome in regulating the gut environment of pets, belonging to the field of pet nutrition and functional food technology. Background Technology

[0002] Companion animals such as dogs and cats, due to their long-term intake of high-protein diets, may produce volatile metabolites containing nitrogen and sulfur during hindgut microbial fermentation. Therefore, regulating the intestinal fermentation environment, maintaining intestinal microecological stability, and reducing fecal odor in pets have practical application needs. Glucose oxidase (GOx) catalyzes the oxidation reaction of glucose with oxygen, producing gluconic acid while consuming oxygen. Therefore, it has the potential to regulate the intestinal microenvironment of companion animals, influence colonic fermentation processes, and reduce the production of some putrefactive metabolites. However, GOx is a water-soluble enzyme protein, and its enzyme activity is easily lost during food processing, storage, and under acidic gastrointestinal environments and proteolytic conditions. Therefore, improving the stability of GOx during oral delivery and ensuring its sufficient activity to reach the hindgut is a key technical problem limiting its application in the field of intestinal health.

[0003] Existing technologies have reported microencapsulation or liposome encapsulation of GOx. For example, CN101250515A uses calcium alginate and chitosan to form GOx microcapsules; previous studies have also used phospholipids and cholesterol to prepare GOx liposomes via thin-film evaporation-freeze-thaw or dehydration-rehydration methods to improve their encapsulation efficiency, storage stability, thermal stability, and proteolytic stability. However, none of the above technologies disclose the "pre-formed GOx liposomes, low-methoxyl pectin interface pre-adsorption, Ca2+" method used in this invention. 2+ A composite interface construction method of "post-confined cross-linking".

[0004] On the other hand, pectin-modified liposomes and low-methoxyl pectin-Ca 2+ Gelation technology already has applications. For example, CN106075414A discloses pectin-coated bromelain liposomes, but it mainly improves the stability of liposomes through pectin surface coating and does not further utilize Ca. 2+ Post-crosslinking of the pectin layer pre-adsorbed on the surface of liposomes; CN102553499A utilizes low-ester pectin and Ca... 2+ The integral microcapsule wall material is essentially a continuous pectin phase gelation, rather than a spatially confined ionic cross-linking at the pre-formed liposome interface.

[0005] Ensuring that GOx retains its effective activity after passing through the stomach and small intestine, and continues to be released and exert its effects in the hindgut, remains a technical challenge that needs to be addressed. Furthermore, the cat's gastrointestinal tract exhibits significant pH changes, being acidic in the stomach and rapidly transitioning to a neutral to slightly alkaline environment upon entering the small intestine.

[0006] Therefore, there is still a need for a GOx delivery technology that has both a novel interface embedding structure and preparation route, and can achieve a connection between protection of the pre-gastrointestinal tract and continuous action in the hindgut. Summary of the Invention

[0007] To address the aforementioned problems, this invention employs a method involving "pre-formed GOx liposomes—LMP interface pre-adsorption—Ca..." 2+ The specific process of "post-confined cross-linking" can improve the processing stability and gastrointestinal stability of GOx while inhibiting particle bridging and macroscopic gelation. It can also limit the premature release of GOx in the pre-gastrointestinal tract and maintain its continuous available enzyme activity in the hindgut stage, providing a new food-grade delivery solution for regulating the fermentation environment of the pet's hindgut, improving fecal condition, and managing fecal odor.

[0008] The first objective of this invention is to provide a method for interfacially confined calcium-crosslinked GOx complex liposomes, comprising the steps of: (1) Dissolve glucose oxidase and trehalose in water and disperse them evenly to obtain the aqueous phase of GOx; (2) Phospholipids were added to the aqueous phase of GOx, and the mixture was sheared, homogenized, and sonicated to obtain GOx pre-formed liposomes; (3) Low-methoxyl pectin was first added to the GOx preformed liposomes and stirred to allow the low-methoxyl pectin to complete the interfacial pre-adsorption; then CaCl2 solution was added to crosslink the liposomes to obtain crosslinked GOx liposomes. (4) Add a freeze-drying protectant to the cross-linked GOx liposomes, pre-freeze and freeze-dry to obtain GOx composite liposome powder.

[0009] In one embodiment, the low-methoxyl pectin is a pectin with an esterification degree of less than 50%.

[0010] In one embodiment, the phospholipid is selected from one or more of soybean lecithin, sunflower seed lecithin, egg yolk lecithin, and phosphatidylcholine.

[0011] In one embodiment, the concentration (mg / mL) ratio of glucose oxidase, phospholipids, and low-methoxyl pectin in the GOx complex liposome powder is 0.5~2:5~20:0.5~2.

[0012] In one embodiment, the concentration (mg / mL) ratio of glucose oxidase, phospholipids, and low-methoxyl pectin in the GOx complex liposome powder is 0.5~1.5:5~15:0.5~1.5.

[0013] In one embodiment, the GOx complex liposome powder contains Ca 2+ The molar ratio of carboxyl groups in low-methoxyl pectin is 0.02~0.15:1.

[0014] Optionally, in the GOx complex liposome powder, Ca 2+ The molar ratio of carboxyl groups in low-methoxyl pectin is 0.06~0.10:1.

[0015] In one embodiment, the mass concentration of glucose oxidase in the aqueous phase of GOx in step (1) is 0.5~2 mg / mL; the mass concentration of phospholipids in the GOx preformed liposome dispersion in step (2) is 5~20 mg / mL; and the mass concentration of low methoxy pectin in the cross-linked GOx liposomes in step (3) is 0.5~2 mg / mL.

[0016] Optionally, the mass concentration of glucose oxidase in the aqueous phase of GOx in step (1) is 0.5~1.5 mg / mL; the mass concentration of phospholipids in the GOx preformed liposome dispersion in step (2) is 5~15 mg / mL; and the mass concentration of low methoxy pectin in the cross-linked GOx liposomes in step (3) is 0.5~1.5 mg / mL.

[0017] In one embodiment, the dispersion temperature in step (1) is 4~10℃.

[0018] In one embodiment, the concentration ratio (mg / mL) of Gox and trehalose in step (1) is 1:3~8.

[0019] In one embodiment, the shearing, homogenization, and sonication in step (2) are performed at 4~10°C.

[0020] In one embodiment, the shearing in step (2) is performed at 7000~9000 rpm for 10~12 min.

[0021] In one embodiment, the homogenization in step (2) is a homogenization treatment at 20~40 MPa for 10~20 min.

[0022] In one embodiment, the dropping rate of the CaCl2 solution in step (3) is 0.05~0.50 mL / min.

[0023] In one embodiment, the dropping rate of the CaCl2 solution in step (3) is 0.1~0.3 mL / min.

[0024] In one embodiment, the CaCl2 solution in step (3) is added dropwise until the final concentration reaches 0.15~0.50 mM.

[0025] In one embodiment, the CaCl2 solution in step (3) is added dropwise until the final concentration reaches 0.2~0.40 mM.

[0026] In one embodiment, the stirring in step (3) allows for interfacial pre-adsorption of low-methoxyl pectin for 10 to 60 minutes.

[0027] In one embodiment, the crosslinking in step (3) is performed at 4~8°C for 20~30 min.

[0028] In one embodiment, the freeze-drying protectant is composed of trehalose and / or maltodextrin, wherein the mass ratio of trehalose to maltodextrin is 1 to 3:1.

[0029] In one embodiment, the concentration (mg / mL) ratio of the freeze-drying protectant to the phospholipid is 3~8:1.

[0030] A second objective of this invention is to provide interface-confined calcium-crosslinked GOx complex liposomes prepared by any of the methods described above.

[0031] A third objective of this invention is to provide the application of the above-described interface-confined calcium-crosslinked GOx complex liposomes in the food industry.

[0032] The fourth objective of this invention is to provide a pet food or pet nutritional supplement containing the above-mentioned interface-confined calcium crosslinked GOx complex liposomes, with an addition amount of 0.1%~1.0% w / w.

[0033] Optionally, the amount of the interface-confined calcium-crosslinked GOx complex liposome added to pet food is 0.5% (w / w).

[0034] In one embodiment, the pet is preferably a cat or a dog.

[0035] In one embodiment, the interface-confined calcium-crosslinked GOx complex liposomes can be added to cat food by means of powder post-addition, oil coating carrying, roller coating, or premixing with palatability-enhancing powder.

[0036] In one embodiment, the pet food or pet nutritional supplement is used for non-therapeutic regulation of the pet's hindgut fermentation environment and reduction of fecal odor.

[0037] In one embodiment, the pet food or pet nutritional supplement enables glucose oxidase to maintain its activity in the simulated stomach and small intestine stages, continuously consume oxygen and reduce the pH of the system in the simulated hind intestine stage, and / or reduce the relative peak areas of methyl thiobutyrate, phenol, indole and 4-ethylphenyl isothiocyanate.

[0038] The fifth objective of this invention is to provide a method for improving the intestinal release rate of GOx liposomes, comprising the steps of preparing interfacially confined calcium-crosslinked GOx complex liposomes: (1) Dissolve glucose oxidase and trehalose in water and disperse them evenly to obtain the aqueous phase of GOx; (2) Phospholipids were added to the aqueous phase of GOx, and the mixture was sheared, homogenized, and sonicated to obtain GOx pre-formed liposomes; (3) Low-methoxyl pectin was first added to the GOx preformed liposomes and stirred to allow the low-methoxyl pectin to complete the interfacial pre-adsorption; then CaCl2 solution was added to cross-link the GOx liposomes. (4) Add a freeze-drying protectant to the cross-linked GOx liposomes, pre-freeze and freeze-dry to obtain GOx composite liposome powder.

[0039] In one embodiment, the low-methoxyl pectin is a pectin with an esterification degree of less than 50%.

[0040] In one embodiment, the concentration ratio (mg / mL) of Gox and trehalose in step (1) is 1:3~8.

[0041] In one embodiment, the phospholipid is selected from one or more of soybean lecithin, sunflower seed lecithin, egg yolk lecithin, and phosphatidylcholine.

[0042] In one embodiment, the concentration (mg / mL) ratio of glucose oxidase, phospholipids, and low-methoxyl pectin in the GOx complex liposome powder is 0.5~2:5~20:0.5~2.

[0043] In one embodiment, the concentration (mg / mL) ratio of glucose oxidase, phospholipids, and low-methoxyl pectin in the GOx complex liposome powder is 0.5~1.5:5~15:0.5~1.5.

[0044] In one embodiment, the GOx complex liposome powder contains Ca 2+ The molar ratio of carboxyl groups in low-methoxyl pectin is 0.02~0.15:1.

[0045] In one embodiment, the GOx complex liposome powder contains Ca 2+ The molar ratio of carboxyl groups in low-methoxyl pectin is 0.06~0.10:1.

[0046] In one embodiment, the mass concentration of glucose oxidase in the aqueous phase of GOx in step (1) is 0.5~2 mg / mL; the mass concentration of phospholipids in the GOx preformed liposome dispersion in step (2) is 5~20 mg / mL; and the mass concentration of low methoxy pectin in the cross-linked GOx liposomes in step (3) is 0.5~2 mg / mL.

[0047] Optionally, the mass concentration of glucose oxidase in the aqueous phase of GOx in step (1) is 0.5~1.5 mg / mL; the mass concentration of phospholipids in the GOx preformed liposome dispersion in step (2) is 5~15 mg / mL; and the mass concentration of low methoxy pectin in the cross-linked GOx liposomes in step (3) is 0.5~1.5 mg / mL.

[0048] In one embodiment, the dispersion temperature in step (1) is 4~10℃.

[0049] In one embodiment, the shearing, homogenization, and sonication in step (2) are performed at 4~10°C.

[0050] In one embodiment, the shearing in step (2) is performed at 7000~9000 rpm for 10~12 min.

[0051] In one embodiment, the homogenization in step (2) is a homogenization treatment at 20~40 MPa for 10~20 min.

[0052] In one embodiment, the dropping rate of the CaCl2 solution in step (3) is 0.05~0.50 mL / min.

[0053] In one embodiment, the dropping rate of the CaCl2 solution in step (3) is 0.1~0.3 mL / min.

[0054] In one embodiment, the CaCl2 solution in step (3) is added dropwise until the final concentration reaches 0.15~0.50 mM.

[0055] In one embodiment, the CaCl2 solution in step (3) is added dropwise until the final concentration reaches 0.2~0.40 mM.

[0056] In one embodiment, the stirring time in step (3) is 10 to 60 minutes.

[0057] In one embodiment, the crosslinking in step (3) is performed at 4~8°C for 20~30 min.

[0058] In one embodiment, the freeze-drying protectant is composed of trehalose and / or maltodextrin, wherein the mass ratio of trehalose to maltodextrin is 1 to 3:1.

[0059] In one embodiment, the concentration (mg / mL) ratio of the freeze-drying protectant to the phospholipid is 3~8:1.

[0060] In one embodiment, improving the intestinal release rate of Gox liposomes means that the glucose oxidase activity retention rate is not less than 90% after simulated gastric digestion and not less than 70% after simulated gastric-small intestine continuous digestion.

[0061] This invention also provides the application of the above-mentioned interface-confined calcium-crosslinked GOx complex liposomes in non-therapeutic regulation of the pet intestinal environment.

[0062] In one embodiment, the application includes feeding pet food or pet nutritional supplements containing the interface-confined calcium crosslinked GOx complex liposomes to regulate the hindgut fermentation environment, improve fecal condition, and / or reduce fecal odor.

[0063] In one embodiment, the application includes enhancing the activity retention of GOx during pet food processing, gastric digestion, and small intestinal digestion through the interface-confined calcium-crosslinked GOx complex liposomes, limiting the premature release of GOx in the pre-gastrointestinal tract, and enabling GOx to be gradually released and continuously exert its oxygen-consuming and acidifying effects in the hindgut stage.

[0064] Beneficial effects of the present invention The GOx composite liposomes formed in this invention exhibit staged particle characteristics during interfacial construction: the pre-formed GOx liposomes have an average particle size of 125 nm, a PDI of 0.18, and a zeta potential of -8 mV; after LMP pre-adsorption, the particle size is 165 nm, the PDI is 0.21, and the zeta potential is -39 mV; controlled Ca 2+ The particle size after addition was 220 nm, the PDI was 0.23, and the zeta potential was -32 mV.

[0065] Different interface construction order, Ca 2+ The comparison results of the pectin carboxyl molar ratio and LMP pre-adsorption time showed that pre-adsorption of LMP at the interface should be performed first, followed by the application of an appropriate amount of Ca. 2+ Post-crosslinking can maintain low particle size and PDI; Ca 2+ Excessive use or alteration of the assembly sequence leads to an increase in particle size and PDI, indicating that specific assembly sequences and controlled crosslinking conditions help suppress particle bridging and aggregation.

[0066] The encapsulation system prepared by this invention retains 95.00% GOx enzyme activity after simulated gastric digestion and 75.00% enzyme activity after simulated gastric-small intestine continuous digestion; after treatment at 75 °C for 10 min and at 85 °C for 5 min, it retains 98.90% and 82.10% enzyme activity, respectively.

[0067] During the simulated pet colon fermentation process, the GOx enzyme activity in the encapsulated group increased from 1385.991 U at 0 h to 2526.08 U at 48 h, while that in the unencapsulated group decreased from 692.99 U to 78.54 U. The dissolved oxygen in the encapsulated group decreased from 7.17 mg / L to 5.32 mg / L, accompanied by system acidification and a decrease in the relative peak areas of methyl thiobutyrate, phenol, indole, and 4-ethylphenyl isothiocyanate.

[0068] Furthermore, this invention employs a process of first preparing GOx pre-formed liposomes, then performing low-methoxyl pectin interfacial pre-adsorption, and finally adding Ca in a controlled manner. 2+ A specific construction sequence for confined crosslinking allows Ca to... 2+ It preferentially cross-links with low-methoxyl pectin enriched on the surface of liposomes, avoiding Ca2+ cross-linking. 2+ First, large-scale pectin gelation is induced in the continuous phase, and the bridging aggregation between liposome particles is reduced. This invention controls the amount of low-methoxyl pectin, the pre-adsorption time at the low-methoxyl pectin interface, and the Ca... 2+ The molar ratio of low-methoxy pectin carboxyl groups and the dropping rate of CaCl2 solution achieve a balance between interface enhancement, particle dispersion stability, gastrointestinal enzyme activity protection, and hindgut release. The interface-confined calcium-crosslinked GOx composite liposomes prepared by this invention have good particle dispersion stability, heat resistance stability and simulated gastrointestinal digestion stability, and can form a staged delivery characteristic of "low apparent release in the stomach stage - limited apparent release in the small intestine stage - gradually increasing apparent release ratio in the hindgut stage". The interface-confined calcium-crosslinked GOx complex liposomes prepared by this invention can maintain continuous and detectable GOx activity in a simulated pet colon fermentation system, accompanied by a decrease in dissolved oxygen, a decrease in pH, and a decrease in the relative peak area of ​​representative odor substances. They can be used for regulating the hindgut fermentation environment, improving fecal condition, and managing fecal odor in pets. Attached Figure Description

[0069] Figure 1 This is a schematic diagram of the process flow for the GOx composite liposome embedding method of the present invention.

[0070] Figure 2 This is a schematic diagram of the structure of the GOx complex liposome of the present invention.

[0071] Figure 3 The present invention is LMP, LMP+Ca 2+ GOx liposomes + LMP and GOx liposomes + LMP + Ca 2+ (Example 1) Fourier transform infrared spectrum, each group of spectra is displayed with longitudinal misalignment.

[0072] Figure 4This is a graph showing the trend of GOx enzyme activity changes in the encapsulated and unencapsulated groups during the simulated colonic fermentation process in pets.

[0073] Figure 5 This is a graph showing the trend of dissolved oxygen changes in the control group, the embedded group, and the unencapsulated group during the simulated colonic fermentation process in pets.

[0074] Figure 6 This is a pH change trend diagram for the control group, the embedded group, and the unencapsulated group during the simulated colonic fermentation process in pets.

[0075] Figure 7 A comparison of the relative peak areas of four representative odor substances in a simulated pet colon fermentation system. Detailed Implementation

[0076] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0077] The encapsulation process of this invention does not involve liposomes, pectin, and Ca. 2+ Instead of a simple combination, this method involves first directionally enriching low-methoxyl pectin (LMP) at the liposome interface based on pre-formed GOx liposomes, and then adding Ca in a controlled manner. 2+ This allows cross-linking to preferentially occur in the LMP layer on the surface of liposomes, and by controlling Ca 2+ The process controls the carboxyl ratio and crosslinking conditions of pectin to prevent overall gelation of the continuous phase and liposome bridging and aggregation. It also controls the assembly sequence, crosslinking location, and degree of crosslinking, forming a composite protective interface that differs from ordinary GOx liposomes, non-crosslinked pectin-coated liposomes, and monolithic pectin calcium microcapsules.

[0078] Existing GOx liposome studies mainly evaluate encapsulation efficiency, particle size, storage stability, thermal stability, and general proteolytic stability. However, no complete delivery validation has been found that simulates gastric digestion and gastrointestinal continuous digestion of this type of GOx complex liposomes, and further integrates a hindgut fermentation model to evaluate the continuous GOx activity, oxygen consumption, acidification, and changes in odor substances, as is the case in this invention.

[0079] The GOx complex liposome powder prepared by this invention exhibits a high degree of ionization of carboxyl groups in low-methoxyl pectin and a low Ca2+ content. 2+ The hydration, swelling, and permeability of cross-linked networks are all affected by environmental pH; pectin also has the characteristic of being utilized by intestinal microorganisms in the hindgut. This invention utilizes changes in gastrointestinal pH and the hindgut microbial environment to influence LMP-Ca... 2+ The stability and permeability of the interface are adjusted in stages, providing a further technical approach to achieve GOx pre-gastrointestinal protection and sustained release into the hindgut.

[0080] In one embodiment of the invention, a specific interface construction sequence is employed: GOx, phospholipids, LMP, and Ca are not combined in the same order. 2+ Instead of direct mixing, the process first forms complete pre-formed liposomes, then performs LMP interface pre-adsorption, and finally introduces Ca. 2+ .

[0081] In one embodiment of the invention, crosslinking space is controlled: LMP in Ca 2+ Before addition, the liposomes are enriched on their surface to facilitate the subsequent addition of Ca. 2+ It preferentially crosslinks with interfacial LMP rather than first forming a large-scale pectin calcium network in the continuous phase.

[0082] In one embodiment of the present invention, sub-macro gelation conditions are employed to balance interface enhancement and nano-dispersion, while avoiding excessive Ca. 2+ This leads to particle bridging.

[0083] In one embodiment of the present invention, the GOx composite liposome powder prepared by the present invention improves the retention of enzyme activity in the pre-gastrointestinal tract, continuously consumes oxygen and acidifies after entering the colon, and further reduces representative odor substances.

[0084] In one embodiment of the present invention, the GOx composite liposome powder prepared by the present invention can achieve staged enzyme release mediated by gastrointestinal environment differences: the LMP-Ca 2+ The complex interface exhibits staged hydration and release characteristics under varying gastrointestinal environments. In the acidic gastric environment, the LMP layer exhibits low swelling, which helps limit premature GOx release. Upon entering the neutral to slightly alkaline intestinal environment, as the carboxyl ionization of LMP increases, interfacial hydration and permeability improve, leading to gradual GOx release. In the hindgut stage, LMP further serves as a polysaccharide substrate usable by intestinal microorganisms, causing further erosion and relaxation of the interfacial layer, thus forming a delivery process that connects pre-gastrointestinal protection with sustained enzyme release in the hindgut.

[0085] In one embodiment of the present invention, the GOx composite liposome powder prepared by the present invention regulates GOx release through interfacial confined cross-linking and differences in the gastrointestinal environment, so that GOx maintains high activity in the stomach and small intestine stages, and is continuously released, oxygen-consuming and acidified in the colon stage, further reducing representative odor substances.

[0086] In one embodiment of the present invention, the interface-confined calcium-crosslinked GOx complex liposomes can enable glucose oxidase to maintain a high total recoverable GOx activity in simulated stomach and simulated small intestine stages, and limit the premature release of GOx in the pre-gastrointestinal tract.

[0087] In one embodiment of the present invention, the interface-confined calcium-crosslinked GOx complex liposomes have a low GOx apparent activity release ratio at the end of simulated gastric digestion, maintain a limited GOx apparent activity release ratio after simulated gastric-small intestine continuous digestion, and gradually increase the GOx apparent activity release ratio during simulated hindgut fermentation, thereby forming a delivery process that connects gastrointestinal protection and hindgut stage release.

[0088] In one embodiment of the present invention, low-methoxyl pectin-Ca 2+ The composite interface can regulate its hydration level, permeability, and structural integrity based on differences in the gastrointestinal environment. In the acidic environment of the stomach, the low-methoxyl pectin interface layer has a lower degree of swelling, which helps to limit the premature entry of GOx into the external phase. After entering the neutral to weakly alkaline environment of the small intestine, the hydration level and permeability of the composite interface increase with the increase of the carboxyl ionization of low-methoxyl pectin. After entering the hindgut fermentation environment, the interface layer undergoes further hydration, relaxation, or erosion, allowing the active GOx to be released gradually.

[0089] In one embodiment of the present invention, the interface-confined calcium-crosslinked GOx complex liposomes can maintain continuously detectable GOx activity during simulated hindgut fermentation, continuously consume oxygen in the system and reduce the pH of the system.

[0090] In one embodiment of the present invention, the interface-confined calcium-crosslinked GOx complex liposomes can reduce the relative peak areas of methyl thiobutyrate, phenol, indole, and 4-ethylphenyl isothiocyanate in a simulated pet colon fermentation system.

[0091] In one embodiment of the present invention, adding interface-confined calcium crosslinked GOx complex liposomes to pet staple food can regulate the hindgut fermentation environment of pets, improve fecal condition and / or reduce fecal odor.

[0092] In one embodiment of the present invention, interface-confined calcium crosslinked GOx complex liposomes were added to a complete cat diet at 0.5% of the cat diet mass and fed to healthy adult cats for 28 days. As a result, the total short-chain fatty acids and fecal sIgA in feces showed an increasing trend, while the rate of soft stools and the relative peak area of ​​fecal indole showed a decreasing trend.

[0093] In one embodiment of the present invention, the present invention employs "pre-formed GOx liposomes—low-methoxyl pectin interface pre-adsorption—Ca..." 2+ The controlled addition of Ca constructs the composite interface in a sequential manner, compared to adding Ca first. 2+ Then add low-methoxyl pectin, or combine low-methoxyl pectin with Ca 2+Simultaneous addition can achieve lower particle size and PDI, higher GOx activity retention rate after gastrointestinal digestion, more reasonable gastrointestinal staged apparent activity release characteristics, and better variation range of indicators for pet applications.

[0094] Raw materials used in the examples: Low-methoxyl pectin (LMP) was purchased from Anhui Yuning Biotechnology Co., Ltd., product model YN-1, with a measured degree of esterification of 35% and a molecular weight of 50,000 to 300,000. The food-grade phospholipid was soybean phospholipid PC70, purchased from Shandong Fengtai Biotechnology Co., Ltd., product model PC70, with a phosphatidylcholine content of ≥70%. GOx is derived from Aspergillus niger ( Aspergillus niger The fermentation product was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the enzyme activity was 12000 U / g.

[0095] Test method: 1. GOx activity assay GOx activity was determined using a glucose oxidase-peroxidase coupled colorimetric method. Using β-D-glucose as a substrate, glucose oxidase catalyzes the oxidation of β-D-glucose to generate hydrogen peroxide. The generated hydrogen peroxide participates in the colorimetric reaction under the action of peroxidase, and GOx activity is calculated based on the change in absorbance per unit time. For samples without GOx encapsulation, GOx activity was measured directly. For the determination of total recoverable GOx activity in GOx liposomes and GOx complex liposomes, nonionic surfactants were used to assist in membrane rupture before detection, combined with vortex mixing and low-temperature sonication to fully release the GOx encapsulated in the liposomes before activity measurement. Three independent replicates were set up for each sample, and each independent replicate was measured in triplicate. The average of the three parallel measurements was used as the result of that independent replicate.

[0096] 2. GOx Encapsulation Efficiency Measurement GOx-containing liposomes and unencapsulated free GOx were separated by ultrafiltration centrifugation. A certain volume of GOx-containing liposome dispersion was added to an ultrafiltration centrifuge tube, centrifuged, and the filtrate was collected. The activity of free GOx in the filtrate was measured. Separately, a GOx-containing liposome dispersion from the same batch that had not undergone ultrafiltration was taken, and the total GOx activity of the system was measured after complete membrane rupture.

[0097] GOx encapsulation efficiency was calculated using the following formula: GOx encapsulation efficiency (%) = (Total GOx activity in the system - Free GOx activity in the filtrate) ÷ Total GOx activity in the system × 100%. Each sample was tested in triplicate, with each replicate measured three times.

[0098] 3. Particle size, PDI and ζ potential determination The average hydrodynamic particle size, PDI, and zeta potential of the composite liposomes were determined using a dynamic light scattering particle size and zeta potential analyzer. Samples were diluted 10-fold with ultrapure water as the dispersion medium and equilibrated at 25 °C before analysis. Dynamic light scattering was performed using a 173° backscattering mode. Three batches of samples were prepared independently for each process condition, and each batch was measured in triplicate. The average of the three parallel measurements for each batch was taken as the result for that batch, and the average of the results from the three independently prepared batches was then calculated. The particle size, PDI, and zeta potential values ​​in Tables 1 to 4 are the average values ​​of the three independently prepared batches and are presented as individual values. A PDI not exceeding 0.30 was considered a process criterion for maintaining good dispersion of the composite liposomes.

[0099] 4. Heat resistance stability test The GOx-containing liposome powders prepared in Example 1 and Comparative Examples 1-6 were reconstituted with ultrapure water, and adjusted to the same initial GOx activity concentration based on the measured GOx activity of each sample. Each group of samples was treated at 75 °C for 10 min or at 85 °C for 5 min. After heat treatment, the samples were immediately cooled to room temperature in an ice bath. Subsequently, the membranes were thoroughly perforated, and the total recoverable GOx activity in the system after heat treatment was measured.

[0100] Taking the total recoverable GOx activity of the untreated samples in the same group as 100%, the GOx activity retention rate after heat treatment was calculated according to the following formula: GOx activity retention rate after heat treatment (%) = Total recoverable GOx activity of the system after heat treatment ÷ Total recoverable GOx activity of the system before heat treatment × 100%. Each group was set up with 3 independent replicates, and each independent replicate was measured in parallel 3 times.

[0101] 5. Simulated gastrointestinal digestion and GOx activity retention rate determination Simulated digestion was performed using the INFOGEST static in vitro digestion method combined with the feline gastrointestinal environment. Samples prepared in Example 1 and Comparative Examples 1-6 were taken and adjusted to the same initial GOx activity equivalent based on the measured GOx activity of each sample. In the simulated stomach stage, the system was adjusted to pH 3.0, pepsin was added to achieve a final pepsin activity of 2000 U / mL, and digestion was carried out at 38 °C for 120 min. After the stomach stage, pepsin activity was terminated by adjusting the system pH. Simulated intestinal fluid was then added, the system was adjusted to pH 7.0, trypsin was added to achieve a final trypsin activity of 100 U / mL, and bile salts were added to a final concentration of 10 mmol / L. Digestion continued at 38 °C for 120 min. After the small intestine stage, the reaction was terminated using a protease inhibitor. Samples were taken at the end of the simulated stomach digestion and at the end of the simulated stomach-small intestine continuous digestion. Before detection, the samples were thoroughly perforated, and the total recoverable GOx activity in the corresponding digestion stage was measured.

[0102] The total recoverable GOx activity of the same group of samples before digestion was taken as 100%, and the GOx activity retention rate was calculated according to the following formula: GOx activity retention rate (%) = total recoverable GOx activity of the system after the corresponding digestion stage ÷ total recoverable GOx activity of the system before digestion × 100%. Each group was set up with 3 independent replicates, and each independent replicate was measured in parallel 3 times.

[0103] 6. Determination of the proportion of apparent GOx activity released at different gastrointestinal stages To evaluate the effects of different interface construction methods on the gastrointestinal staged release behavior of GOx complex liposomes, samples prepared in Example 1 and Comparative Examples 1-6 were subjected to simulated gastric digestion, simulated small intestinal digestion, and simulated feline colon fermentation. In the simulated gastric stage, the system was adjusted to pH 3.0 and treated at 38 °C for 2 h. After the gastric stage, the system was adjusted to pH 7.0 and simulated small intestinal digestion continued for 2 h, for a total continuous gastro-small intestinal digestion time of 4 h. After the continuous gastro-small intestinal digestion, the resulting samples were inoculated into a simulated colon fermentation system containing fecal microorganisms. The simulated colon fermentation system used a pre-reduced buffer medium with an initial pH of 6.8 and was cultured anaerobically at 38 °C for 24 h and 48 h. The colon fermentation time was calculated from the time the samples were inoculated into the fermentation system containing fecal microorganisms. Samples were taken at the end of simulated gastric digestion, the end of continuous gastro-small intestinal digestion, 24 h of feline colon fermentation, and 48 h of feline colon fermentation. Two parallel samples were set up at each independent replicate time point. One sample was separated from the carrier particles and the external phase by centrifugation or ultrafiltration, and the free GOx activity in the external phase was determined; another parallel sample was subjected to full membrane rupture treatment, and the total recoverable GOx activity in the system at the corresponding time points was determined.

[0104] The apparent GOx activity release ratio was calculated using the following formula: Apparent GOx activity release ratio (%) = Free GOx activity in the external phase at the corresponding time point ÷ Total recoverable GOx activity in the system at the corresponding time point × 100%. Each group had 3 independent replicates, and the GOx activity in each independent replicate was measured in triplicate.

[0105] 7. Preparation of cat fecal microbial inoculum and simulated colon fermentation system Fresh feces from healthy adult cats were collected naturally as the microbial source for the simulated colonic fermentation system. The cats maintained normal diet and routine feeding management before fecal collection. After collection, the feces were processed promptly under anaerobic or minimally oxygenated conditions. Fresh feces were added to a pre-reduced buffer solution, thoroughly homogenized, and filtered to remove larger food residues and insoluble particles, yielding a fecal microbial inoculum. All experimental groups used the same batch of fecal microbial inoculum and maintained the same inoculation ratio to minimize the impact of differences in the inoculated microbiota on the experimental results. The simulated colonic fermentation medium was pre-reduced, with the initial pH adjusted to 6.8. After adding the fecal microbial inoculum, the culture was incubated anaerobically at 38 °C, and samples were taken at 0, 6, 12, 24, and 48 h. Each group had three independent replicates.

[0106] 8. Simulated feline colonic fermentation and determination of relative apparent GOx activity To evaluate the effect of liposome encapsulation on the sustained activity of GOx in a simulated feline colonic environment, a blank control group, an unencapsulated GOx group, and an encapsulated GOx group were set up. The encapsulated and unencapsulated GOx groups were added to the simulated colonic fermentation system at the same theoretical initial GOx activity equivalent; the blank control group received no GOx. Each group was fermented anaerobically at 38 °C, and samples were taken at 0, 6, 12, 24, and 48 h. The absolute GOx activity at each time point was measured. Considering that the liposome encapsulation state might affect substrate entry and the apparent detectable GOx activity at 0 h, the measured GOx activity at 0 h for each group was normalized to 100%, and the relative apparent GOx activity was calculated using the following formula: Relative apparent GOx activity (%) = Measured GOx activity at fermentation time point t ÷ Measured GOx activity at 0 h for the same group × 100%. The GOx activity measured during fermentation is the apparent detectable activity, and its changes may be influenced by the gradual release of GOx, hydration of the complex interface, substrate accessibility, and changes in the fermentation system environment. Each group was set up with 3 independent replicates, and each independent replicate was measured in triplicate.

[0107] 9. Determination of dissolved oxygen and pH in a simulated feline colonic fermentation system In a simulated feline colon fermentation process, fermentation broths from the blank control group, the unencapsulated GOX group, and the GOX-encapsulated group were collected at 0, 6, 12, 24, and 48 h to measure dissolved oxygen and pH in the fermentation system. Dissolved oxygen was measured using a calibrated dissolved oxygen meter. Zero-point and air saturation calibrations were performed according to the instrument's operating requirements before measurement. During the measurement process, the time the samples were exposed to air was minimized to reduce the influence of external oxygen on the results. pH was measured using a calibrated pH meter. The instrument was calibrated using a standard buffer solution before measurement, and the temperature of samples in each group was kept consistent during the measurement process. Each group had three independent replicates, and each independent replicate was measured in triplicate. Dissolved oxygen test results correspond to... Figure 5 pH test results correspond Figure 6 .

[0108] 10. Detection of representative odor substances in a simulated feline colon fermentation system Volatile odor compounds in a simulated feline colon fermentation system were detected using headspace solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). Fermentation broth samples were collected from the blank control group, the unencapsulated GOX group, and the GOX-encapsulated group at 48 h of simulated feline colon fermentation. Equal volumes of fermentation broth were placed in headspace vials and sealed. After headspace equilibration at a constant temperature, volatile compounds in the headspace were extracted using a solid-phase microextraction fiber. After extraction, the extraction fiber was inserted into the gas chromatograph inlet for thermal desorption and GC-MS detection. Target compounds were qualitatively identified using chromatographic retention time and the NIST mass spectrometry database, and relative quantification was performed using peak area. Representative target compounds detected included methyl thiobutyrate, phenol, indole, and 4-ethylphenyl isothiocyanate.

[0109] The average peak area of ​​the corresponding compound in the blank control group was defined as 100%. The relative peak areas of the target compounds in the unencapsulated GOx group and the encapsulated GOx group were calculated according to the following formula: Relative peak area of ​​target compound (%) = Peak area of ​​target compound in treatment group ÷ Average peak area of ​​corresponding compound in blank control group × 100%. Each group was set up with 3 independent replicates, and each independent replicate was measured in triplicate.

[0110] 11. Observation on the application of non-invasive staple food for healthy adult domestic cats Ten healthy adult domestic cats of any breed and sex were selected, all in good general health and capable of normal eating and defecation. Before the experiment, the selected cats showed no persistent vomiting, diarrhea, or other abnormalities that might affect normal eating, fecal condition, or intestinal indicators. A 7-day acclimatization period was implemented. During this period, the cats were fed the same basic complete cat food, and their eating habits, mental state, frequency of defecation, and fecal condition were recorded. After the acclimatization period, the cats were stratified and randomly assigned to three groups based on weight, sex, and fecal condition during the acclimatization period: Example 1, Comparative Example 1, and Comparative Example 2. Each group consisted of three cats: Example 1 (4 cats), Comparative Example 1 (3 cats), and Comparative Example 2 (3 cats).

[0111] Example 1 group was fed a complete cat diet supplemented with the GOx complex liposome powder prepared in Example 1; Comparative Example 1 group was fed a complete cat diet supplemented with the GOx complex liposome powder prepared in Comparative Example 1; Comparative Example 2 group was fed a complete cat diet supplemented with the GOx complex liposome powder prepared in Comparative Example 2. All three groups used the same basic complete cat diet. Samples from each group were standardized based on measured GOx activity to ensure consistent actual GOx activity levels across the three groups. The total amount of complex liposome powder and the same excipients added to each group was 0.5% of the cat diet mass. If the required mass of complex liposome powder to achieve the same GOx activity level differed, the same lyophilized protectant or excipient without GOx was used to supplement the total amount.

[0112] Feeding continued for 28 days. Except for the test samples, the basal diet, daily feeding method, and routine husbandry conditions remained consistent throughout the experiment. Before the application observation began, the animal owners were informed of the purpose of the observation, the observation period, the method of dietary addition, the content of fecal samples collected from natural defecation, and possible dietary adaptation changes, and their written informed consent was obtained. This application observation only involved dietary feeding and the collection of naturally excreted fecal samples; no blood collection, anesthesia, surgery, gavage, or other invasive procedures were performed. During the observation period, the cats' eating status, mental state, defecation frequency, fecal condition, and other abnormalities were recorded. Fresh feces were collected from natural excretions before the intervention and after 28 days of continuous feeding. The feces were immediately mixed and aliquoted after collection; one portion was used for fecal dry matter and related indicator testing, and the other portion was stored at low temperature for the detection of total short-chain fatty acids, sIgA, and indole.

[0113] 12. Determination of total short-chain fatty acids in feces Fresh feces were collected from cats in each group before and 28 days after the intervention. One portion of the samples was used to determine the fecal dry matter content, while the other portion was aliquoted and processed... Store at 80 °C for short-chain fatty acid detection. Take 0.5 g of fecal sample, add 4.5 mL of ultrapure water, vortex thoroughly and homogenize. Centrifuge at 4 °C and collect the supernatant. After acidification, re-centrifugation and filtration, the supernatant is used to determine acetic acid, propionic acid, butyric acid and other target short-chain fatty acids by gas chromatography. Quantification is performed based on standard curves established using the standards for each short-chain fatty acid. Total short-chain fatty acids are the sum of the measured target short-chain fatty acid contents, converted according to the fecal dry matter content, and the results are expressed as mmol / kg DM. Each cat is considered an independent biological replicate, and each fecal sample is measured in triplicate.

[0114] 13. Fecal sIgA determination Fresh feces were collected from cats in each group before and 28 days after the intervention. 0.2 g of fecal sample was added to 2.0 mL of pre-cooled phosphate buffer, vortexed and homogenized thoroughly, and then centrifuged at 4 ℃. The supernatant was collected for analysis. The sIgA content in the fecal extract was determined using a feline secretory immunoglobulin A (sIgA) enzyme-linked immunosorbent assay (ELISA) kit. Samples and standards were added, incubated, washed, developed, and the reaction terminated according to the kit instructions. Absorbance was measured using an ELISA reader, and the sIgA content was calculated based on the standard curve. Results were converted from fecal extract volume and fecal dry matter content and expressed as mg / g DM. Each cat was considered an independent biological replicate, and each sample was measured in triplicate.

[0115] 14. Fecal Stool Score and Soft Stool Rate Calculation A 5-point scoring system for fecal formation was used to evaluate each natural defecation session based on factors such as water content, consistency, structural integrity, and collectability. A higher score indicates better fecal formation. Specific criteria are as follows: 1 point: Watery or liquid stool, without a fixed shape, and cannot be picked up; 2 points: Unformed soft stool, high water content, in a clump or pasty state, difficult to pick up intact; 3 points: The stool is basically formed but the texture is soft. The fecal structure is relatively loose and a lot of residue can be left after picking it up. 4 points: Well-formed, moderate texture, relatively complete structure, easy to pick up, leaving only a small amount of residue; 5 points: Excellent fecal formation, intact fecal structure, appropriate softness and hardness, relatively dry surface, can be picked up completely and leaves almost no residue.

[0116] Each natural defecation was scored by uniformly trained observers who were unfamiliar with the experimental group information. If the stool condition varied across different parts of the same defecation, the lower score corresponding to the less formed portion was used as the final score for that defecation. Defecation records with scores of 1 or 2 were considered soft stools. A score of 3 indicated that the stool was mostly formed but soft and was not counted in the soft stool count. The soft stool rate before intervention was calculated based on defecation records during the 7-day adaptation period; the soft stool rate after intervention was calculated based on defecation records during the last 7 days of the formal intervention period. The soft stool rate was first calculated for each cat during the corresponding observation period, and then the average soft stool rate for the same group of animals was calculated. The soft stool rate was calculated using the following formula: Soft stool rate (%) = Number of defecations with a score ≤ 2 during the corresponding observation period ÷ Total number of defecations during the corresponding observation period × 100%.

[0117] 15. Determination of the relative peak area of ​​indole in feces Fresh feces were collected from cats in Example 1, Comparative Example 1, and Comparative Example 2 before and 28 days after intervention. Indole in the feces was detected using SPME-GC-MS. Equal masses of fecal samples were placed in headspace vials and sealed. After headspace equilibration at a constant temperature, volatile components in the headspace were extracted using solid-phase microextraction fiber, followed by GC-MS analysis. Indole was qualitatively analyzed using chromatographic retention time and the NIST mass spectrometry database, and relative quantification was performed using peak area. The average peak area of ​​indole before intervention in each group was normalized to 100%. The relative peak area of ​​indole after intervention was calculated using the following formula: Relative peak area of ​​indole (%) = Peak area of ​​indole in fecal sample after intervention ÷ Average peak area of ​​indole in fecal sample before intervention in the same group × 100%. Each cat was considered an independent biological replicate, and each sample was measured in triplicate.

[0118] 16. Fourier transform infrared spectroscopy determination Take low-methoxyl pectin (LMP), LMP and Ca 2+ Fourier transform infrared spectroscopy was performed on the cross-linked products, the GOx liposomes and LMP complex, and the interface-confined calcium-crosslinked GOx composite liposomes prepared in Example 1.

[0119] After freeze-drying, each sample was analyzed using a Fourier transform infrared spectroscopy (FTIR) instrument in [ATR mode / potassium bromide pellet method], with a wavenumber range of 4000–600 cm⁻¹. -1 The resolution is [4 cm] -1

[32] scans were performed cumulatively. Spectral signals are expressed as transmittance. For ease of comparison, each group of spectra is displayed in a vertically offset manner, and the absolute transmittance after the offset is not used for quantitative comparison between groups.

[0120] 17. Data Presentation and Statistical Analysis All in vitro experiments were conducted with three independent replicates, and each independent replicate was performed in triplicate. The mean of the results from the triplicate replicates was calculated first, and then used as the result for the corresponding independent replicate. For experiments involving independently prepared samples, three batches of independently prepared samples were used for testing. Animal application observations used each cat as an independent biological replicate. Original data from each batch, replicate, parallel assay, and individual animal were preserved. In vitro experimental data were analyzed using one-way ANOVA combined with Tukey multiple comparisons, or Kruskal-Wallis nonparametric tests, based on the data distribution characteristics. In animal application observations, paired analysis was performed on individual data for each cat before and after intervention. Data conforming to a normal distribution were analyzed using paired t-tests, and data not conforming to a normal distribution were analyzed using Wilcoxon signed-rank tests. Intergroup comparisons were analyzed using changes in the amount of stool before and after intervention for each animal, and one-way ANOVA combined with Tukey multiple comparisons or Kruskal-Wallis nonparametric tests were used based on the data distribution. The soft stool rate was calculated as the proportion of soft stools to total defecation frequency for each cat during the corresponding observation period, and the analysis was performed using the individual animal as the statistical unit.

[0121] Example 1 1. Preparation of GOx complex liposome powder A method for preparing interfacially confined calcium-crosslinked GOx complex liposomes, the process flow is as follows: Figure 1 As shown, the steps include: (1) Dissolve glucose oxidase (GOx) and trehalose (as a GOx protectant) in water and disperse them evenly at 4°C to obtain an aqueous phase containing GOx; the mass concentration of GOx in the aqueous phase containing GOx is 1.0 mg / mL and the mass concentration of trehalose is 5 mg / mL. (2) Food-grade phospholipids (soybean phospholipid PC70) were added to the aqueous phase of GOx, sheared at 8000 rpm for 10 min at 4℃, homogenized at 30 MPa for 10 min, and sonicated for 15 min to obtain GOx pre-formed liposomes; wherein the mass concentration of food-grade phospholipids in the GOx pre-formed liposome dispersion was 10 mg / mL. (3) Low-methoxyl pectin (LMP) was added to the GOx pre-formed liposomes to a final concentration of 1.0 mg / mL, and stirring was continued for 30 min to allow the LMP to fully contact the liposomes and complete the interfacial pre-adsorption. 10 mM CaCl2 solution was added to the system at a dropping rate of 0.20 mL / min to increase the Ca concentration in the system. 2+ The final concentration reached 0.30 mM, and Ca was controlled. 2+ The molar ratio of the carboxyl group to the low-methoxyl pectin was 0.08:1. After the addition was completed, cross-linking was continued at 8 °C for 20 min to obtain cross-linked GOx liposomes. (4) A freeze-drying protectant (trehalose and maltodextrin mixed at a mass ratio of 2:1) was added to the cross-linked GOx liposomes to a final concentration of 50 mg / mL. The mixture was pre-frozen at low temperature (-40℃, 4 h) and freeze-dried (48 h) to obtain GOx composite liposome powder with the structure shown in Figure 1. Figure 2 As shown.

[0122] 2. Particle characteristics at different stages Liposomes prepared at different stages in Example 1 were analyzed for particle size, with excessive cross-linking (Ca) as the determining factor. 2+ As a control, a low-methoxyl pectin carboxyl molar ratio of 0.20:1 was used, with the remaining steps kept consistent. The results are shown in Table 1.

[0123] Table 1 Liposome Particle Size

[0124] The results showed that with the pre-adsorption of low-methoxyl pectin at the interface and Ca 2+ During confined cross-linking, the liposome particle size increased from 125 nm to 220 nm, while the PDI remained below 0.23, indicating that low-methoxyl pectin and Ca... 2+ Crosslinking forms an interfacial protective layer, but does not cause significant aggregation; compared to Ca 2+ The particle size of the excess control group increased to 520 nm, indicating that the controlled Ca... 2+ The addition helps maintain the dispersion stability of the complex liposomes.

[0125] 3. Fourier transform infrared spectroscopy characterization LMP and LMP+Ca were tested respectively. 2+ GOx liposomes + LMP and GOx liposomes + LMP + Ca 2+ (The sample prepared in Example 1) underwent Fourier transform infrared spectroscopy analysis, and the results are as follows: Figure 3 As shown.

[0126] LMP and LMP+Ca 2+ The overall spectral profiles are similar, but the carboxylate-related absorption bands change, with COO... - The asymmetric stretching absorption peak is from 1605 cm⁻¹ -1 Moved to 1618 cm -1 COO - The symmetric stretching absorption peak is from 1413 cm⁻¹ -1 Moved to 1423 cm -1 This indicates that Ca 2+ The addition alters the chemical environment of the LMP carboxyl group, supporting the interaction between LMP and Ca. 2+ Coordination and cross-linking effects are formed between them.

[0127] GOx liposomes + LMP group at approximately 2922 cm -1 and 2852 cm -1 The absorption exhibits lipid hydrocarbon chain CH2 stretching vibration-related absorption, while retaining the characteristic absorption of LMP; with the addition of Ca... 2+ Afterwards, GOx liposomes + LMP + Ca 2+ COO of the group - The asymmetric stretching absorption peak is from 1604 cm⁻¹ -1 Moved to 1616 cm - ¹, COO - The symmetric stretching absorption peak is from 1412 cm⁻¹ -1 Moved to 1422 cm -1 Its direction of change is related to the addition of Ca to LMP. 2+ The subsequent changes were consistent, approximately 2922 cm. -1 2852 cm -1 1737 cm -1 1240 cm -1 and 1081 cm -1 Nearby lipid-related absorption persists; simultaneously, the cross-linked system did not exhibit significant gelation, precipitation, or abnormally increased particle size / PDI, indicating that the Ca... 2+ Crosslinking did not manifest as overall solution gelation or nonspecific aggregation, indicating that Ca... 2+ Cross-linking mainly occurs in the adsorption layer on the surface of liposomes, forming a complex protective interface on the surface of liposomes.

[0128] The above results indicate that adding Ca to the composite system of liposomes and LMP... 2+ Subsequently, the LMP carboxyl group participated in coordination crosslinking, while liposome-related structural features were preserved. This result is consistent with the staged changes in particle size, PDI, and zeta potential, supporting the LMP-Ca... 2+ The formation of composite interfaces.

[0129] Example 2 Based on Example 1, change Ca in step (3) 2+ The pectin carboxyl molar ratio was 0.04:1 and 0.20:1, while the other steps remained the same, to prepare GOx complex liposome powder.

[0130] Table 2 Ca 2+ Effect of pectin carboxyl molar ratio on particle characteristics

[0131] Detecting Ca 2+The effect of the pectin carboxyl molar ratio on particle characteristics is shown in Table 2. The results indicate that the system maintains low particle size and PDI under the conditions of 0.04:1 and 0.08:1; when Ca 2+ When the carboxyl molar ratio of pectin was increased to 0.20:1, the particle size increased to 520 nm, the PDI increased to 0.43, and the absolute value of the zeta potential decreased, indicating that excess Ca... 2+ It can promote particle bridging and aggregation.

[0132] Example 3 Based on Example 1, the stirring time (i.e., pre-adsorption time) after adding low methoxy pectin in step (3) was changed to 0 min and 60 min respectively, while the other steps remained the same, and GOx composite liposome powder was prepared.

[0133] The effect of pre-adsorption time on particle characteristics was investigated, and the results are shown in Table 3. The results indicate that adding Ca without pre-adsorption... 2+ At the initial stage, the particle size and PDI were relatively high; after 30 min of pre-adsorption, the system reached a lower particle size and PDI. Extending the time to 60 min did not bring significant further improvement, indicating that 30 min can be regarded as the preferred time to balance interface stability and production efficiency.

[0134] Table 3 Effect of pre-adsorption time on particle characteristics

[0135] Comparative Example 1 Based on Example 1, the order of addition in step (3) was changed to: first add CaCl2 solution, then add low methoxy pectin for cross-linking, and the remaining steps were consistent with Example 1, so as to prepare GOx composite liposome powder.

[0136] Comparative Example 2 Based on Example 1, the order of addition in step (3) was changed to: low methoxy pectin and CaCl2 solution were added together to GOx liposomes, and the remaining steps were kept the same as in Example 1 to prepare GOx composite liposome powder.

[0137] Table 4. Influence of different interface construction sequences on particle characteristics

[0138] The effect of the preparation order on particle characteristics was investigated, and the results are shown in Table 4. The results indicate that simply changing the order of addition can lead to systematic changes in particle size, PDI, and zeta potential; the example group maintained a narrower particle size distribution, while the group with Ca added first... 2+ The increased particle bridging and aggregation when added simultaneously indicates that LMP pre-adsorption is a necessary step in forming a stable interfacial cross-linked layer.

[0139] Comparative Example 3 Based on Example 1, high-methoxyl pectin was used to replace low-methoxyl pectin, while keeping the other steps the same, to prepare GOx complex liposome powder.

[0140] Comparative Example 4 Based on Example 1, sodium alginate was used to replace low-methoxyl pectin, while keeping the other steps the same, to prepare GOx complex liposome powder.

[0141] Comparative Example 5 Based on Example 1, the amount of low-methoxyl pectin was changed to 6 mg / mL, while the other steps remained the same, to prepare GOx complex liposome powder.

[0142] Comparative Example 6 Based on Example 1, the dropping rate of the CaCl2 solution was changed to 1 mL / min, while the other steps remained the same, to prepare GOx composite liposome powder.

[0143] Example 4 The properties of the GOx composite liposome powders prepared in Example 1 and Comparative Examples 1-6 were tested, as follows: (1) Heat resistance and stability in simulated gastrointestinal digestion The heat resistance and simulated gastrointestinal digestion stability of the obtained GOx composite liposome-encapsulated powder were evaluated. The GOx activity retention rate was used to evaluate the change in total recoverable GOx activity in the system before and after treatment. Before the test, the encapsulation system was fully ruptured to release the GOx and avoid the influence of the encapsulation state on the accessibility of the substrate.

[0144] Table 5. Heat resistance of the encapsulation system and retention rate of GOX activity after simulated gastrointestinal digestion.

[0145] The results are shown in Table 5. The interface-confined calcium-crosslinked GOx composite liposomes prepared in Example 1 showed high GOx enzyme activity retention rates after simulated gastric digestion, simulated gastrointestinal continuous digestion, and heat treatment. The enzyme activity retention rates after simulated gastric digestion and simulated gastrointestinal continuous digestion in Example 1 were 95.00% and 75.00%, respectively, both higher than those of the control groups.

[0146] Comparative Examples 1 and 2 changed the relationship between LMP and Ca 2+ The order of addition of LMP and Ca resulted in lower enzyme activity retention rates compared to Example 1, with LMP and Ca... 2+ The decrease was even more pronounced in Comparative Example 2, indicating that the addition of Ca after LMP pre-adsorption was more significant. 2+It is beneficial for forming a uniform and stable interfacial cross-linked layer. After high-methoxyl pectin replaced LMP, the retention rate of enzyme activity after gastrointestinal digestion further decreased, indicating that the Ca of low-methoxyl pectin... 2+ Responsive crosslinking properties are an important basis for the formation of composite protective interfaces.

[0147] Although the sodium alginate substitution group showed some protective effect, the overall enzyme activity retention rate was still lower than that in Example 1. Increasing the final LMP concentration to 6.0 mg / mL or increasing the CaCl2 solution dropping rate to 1.00 mL / min both resulted in a decrease in enzyme activity retention, indicating that excessively high LMP dosage and excessively rapid CaCl2 solution dropping rates were detrimental. 2+ The addition rate is detrimental to simultaneously achieving interface enhancement, particle dispersion, and enzyme activity protection. The above results indicate that the LMP concentration, interfacial pre-adsorption sequence, and Ca2+ concentration used in Example 1 are optimal. 2+ Controlled dripping conditions can achieve better processing stability and better retention of pre-gastrointestinal enzyme activity.

[0148] (2) Staged release characteristics of GOx under different gastrointestinal environments To evaluate the release behavior of GOx complex liposomes at different gastrointestinal stages, the resulting encapsulation system was subjected to simulated gastric digestion, simulated small intestinal digestion, and simulated feline colonic fermentation.

[0149] In the simulated stomach stage, the system was adjusted to pH 3.0 and treated at 38 °C for 2 h. After the stomach stage, the system was adjusted to pH 7.0 and simulated small intestinal digestion was continued for 2 h. The total time for continuous stomach-small intestinal digestion was 4 h.

[0150] After continuous digestion in the stomach and small intestine, the resulting samples were inoculated into a simulated colon fermentation system containing cat fecal microorganisms. The simulated colon fermentation system was cultured in a pre-reducing buffer medium with an initial pH of 6.8 under anaerobic conditions at 38 °C for 24 h and 48 h, and the pH changes during the fermentation process were recorded.

[0151] At the end of each stage, two parallel samples were prepared. One sample was subjected to centrifugation or ultrafiltration to separate the carrier particles from the external phase, and the free GOx activity in the external phase was measured. The other parallel sample underwent complete membrane rupture treatment, and the total recoverable GOx activity in the system at the corresponding time point was measured. The apparent GOx activity release ratio at the end of each stage was calculated using the following formula: GOx apparent activity release rate (%) = Free GOx activity in the external phase at the corresponding time point ÷ Total recoverable GOx activity in the system at the corresponding time point × 100%.

[0152] Table 6. Apparent activity release ratio of GOx at different gastrointestinal stages

[0153] The results are shown in Table 6. The GOx composite liposomes prepared in Example 1 exhibited a relatively ideal gastrointestinal staged apparent release characteristic. After 2 hours of simulated gastric digestion, the free GOx activity in the external phase of Example 1 accounted for 8% of the total recoverable GOx activity at the corresponding time point; after 4 hours of continuous digestion in the stomach and small intestine, the GOx apparent activity release ratio was 25%, indicating that the constructed LMP-Ca 2+ The composite interface can limit the premature release of GOx in the stomach and small intestine.

[0154] After entering the feline colonic fermentation system, the apparent GOx activity release rate of Example 1 significantly increased, reaching 60% and 80% at 24 h and 48 h of fermentation, respectively. The results indicate that with prolonged colonic fermentation time, the proportion of active GOx in the external phase of the total recoverable GOx activity gradually increases at the corresponding time points. The microbial fermentation environment of fecal matter may promote further hydration, relaxation, or erosion of the complex interface, thereby gradually releasing the active GOx in the encapsulated system during the colonic stage.

[0155] Comparative Examples 1 and 2 changed the relationship between LMP and Ca 2+ The order of addition of Ca resulted in GOx apparent activity release rates of 39% and 47% at the end of continuous gastrointestinal digestion, respectively, both higher than the 25% in Example 1, indicating that adding Ca first... 2+ Or combine LMP with Ca 2+ Simultaneous addition easily leads to the formation of a heterogeneous cross-linked structure, allowing a higher proportion of recoverable active GOx to enter the external phase in the pre-gastrointestinal tract. After entering the cat colon fermentation system, the apparent GOx activity release rates of Comparative Examples 1 and 2 at 48 h were 68% and 65%, respectively, both lower than the 80% in Example 1, indicating that changing the interface construction order is not conducive to simultaneously achieving restricted release in the pre-gastrointestinal tract and active GOx release in the colon.

[0156] Comparative Example 3, which used high-methoxyl pectin instead of low-methoxyl pectin, achieved a 54% apparent GOx activity release rate at the end of continuous gastrointestinal digestion, the highest among all groups. This indicates that high-methoxyl pectin is unlikely to form stable Ca under the conditions of this invention. 2+ The responsive cross-linking interface results in a higher proportion of recoverable active GOx entering the external phase in the pre-gastrointestinal tract. Its apparent GOx activity release rate after 48 h of fermentation in the cat colon was 70%, still lower than the 80% in Example 1, indicating that its gastrointestinal staged release characteristics are inferior to those of Example 1.

[0157] Comparative Examples 4 and 5 showed apparent GOx activity release rates of 18% and 15% respectively at the end of gastrointestinal continuous digestion, lower than the 25% in Example 1. However, after 48 hours of fermentation in the cat colon, their apparent GOx activity release rates were only 58% and 52%, also lower than the 80% in Example 1. These results indicate that the cross-linked structure formed by sodium alginate or excessive amounts of low-methoxyl pectin may make the interfacial layer too dense. While this can further restrict GOx from entering the external phase in the pre-gastrointestinal tract, it also hinders the release of active GOx in the colon. Therefore, a lower apparent release rate in the pre-gastrointestinal tract does not necessarily represent better delivery; responsive release in the colon must also be considered.

[0158] Comparative Example 6, with the CaCl2 solution drop rate increased to 1.00 mL / min, showed a GOx apparent activity release rate of 36% at the end of continuous gastric-small intestine digestion, higher than the 25% in Example 1; while the GOx apparent activity release rate at 48 h of feline colon fermentation was 66%, lower than the 80% in Example 1. These results indicate that rapid CaCl2 addition may cause localized Ca2+ accumulation. 2+ The instantaneous increase in concentration and uneven cross-linking increase particle bridging, resulting in an increased proportion of active phase in the pre-gastrointestinal tract and insufficient apparent release in the colon.

[0159] In summary, Example 1 employs "LMP interface pre-adsorption—Ca 2+ By using a controlled, post-construction sequence and controlling the appropriate amount of LMP and the CaCl2 drop rate, a better balance can be achieved between the pre-gastric active protection, the restriction of external phase release, and the release of active GOx in the colonic stage, forming a phased delivery pattern of "low apparent release in the stomach stage - limited apparent release in the small intestine stage - gradually increasing apparent release ratio in the colonic stage".

[0160] Example 5 The application effects of the GOx complex liposomes prepared in Example 1 in a simulated pet colonic fermentation system and pet food were tested. This example includes a simulated colonic fermentation experiment containing cat fecal microorganisms, and an observation of its application in a non-invasive staple food for healthy adult domestic cats, as detailed below.

[0161] (1) Simulating continuous enzyme activity, oxygen consumption and acidification in pet colon fermentation To evaluate the effect of encapsulation on the sustained action of GOx in the colon, a blank control group, an unencapsulated GOx group, and an encapsulated GOx group were set up. Both the encapsulated and unencapsulated GOx groups were added to a simulated colonic fermentation system at the same theoretical initial GOx activity equivalent, with three independent replicates for each group. Fermentation was carried out under anaerobic conditions at 38 °C, and samples were taken at 0, 6, 12, 24, and 48 h to measure GOx activity, dissolved oxygen, and pH.

[0162] Considering that the liposome encapsulation state may affect substrate entry and the apparent detectable activity of GOx at 0 h, in addition to reporting the absolute enzyme activity at each time point, the measured GOx activity at 0 h for each group was normalized to 100%, and the relative apparent GOx activity was calculated according to the following formula: Relative apparent GOx activity (%) = A t / A0h ×100 Among them, A t At represents the GOx activity measured at fermentation time point t, and A0h represents the GOx activity measured at 0 h in the same group.

[0163] The results are as follows Figure 4 As shown, the apparent enzyme activity in the encapsulated group gradually increased from 1385.991 U at 0 h to 2526.08 U at 48 h, while that in the unencapsulated group decreased from 692.99 U to 78.54 U. This indicates that the encapsulation system helps GOx maintain detectable activity and continue to function in a simulated pet colon environment. The increase in apparent enzyme activity may be related to the gradual hydration of the encapsulation structure, GOx release, and improved substrate accessibility, and does not indicate an increase in the number of enzyme molecules.

[0164] Dissolved oxygen and pH were measured simultaneously. In the encapsulated group, dissolved oxygen decreased from 7.17 mg / L to 5.32 mg / L at 48 h, a decrease greater than that in the control and unencapsulated groups. Results are shown in [Figure number missing]. Figure 5 The pH of the embedded group decreased from 6.85 to 5.18 at 24 h and remained at 5.32 at 48 h. The results are shown in [see attached table]. Figure 6 .

[0165] (2) Representative odor substances in a simulated pet colon fermentation system To further evaluate the effect of GOx complex liposomes on odor-related metabolites during simulated pet colon fermentation, representative volatile odor substances in the simulated colon fermentation system were detected.

[0166] Using the average peak area of ​​the corresponding compound in the blank control group as 100%, the relative peak areas of the target compounds in the unencapsulated GOx group and the encapsulated GOx group were calculated. The results are as follows: Figure 7 As shown, the relative peak areas of methyl thiobutyrate, phenol, indole, and 4-ethylphenyl isothiocyanate in the GOx-encapsulated group were 45.6%, 16.4%, 23.0%, and 5.5%, respectively, all lower than those in the corresponding blank control group and the unencapsulated GOx group. These results indicate that GOx-encapsulated liposomes can continuously exert oxygen-consuming and acidifying effects during simulated pet colonic fermentation, accompanied by a decrease in the relative peak areas of representative odor substances.

[0167] (3) Observation on the application of feline intestinal health To evaluate the effect of different interface construction sequences on the application efficacy of GOx complex liposomes in pets, healthy adult cats were randomly divided into three groups: Example 1, Comparative Example 1, and Comparative Example 2. The three groups were fed a complete cat diet supplemented with GOx complex liposomes prepared in Example 1, Comparative Example 1, or Comparative Example 2, respectively. All groups used the same basal diet, the same actual GOx activity dose, and the same total supplementation amount. The GOx complex liposomes were added at 0.5% of the cat diet mass, and the feeding was continuous for 28 days. Fresh feces were collected before the intervention and after 28 days of intervention to detect total short-chain fatty acids, fecal sIgA, soft stool rate, and indole relative peak area. The results are shown in Table 7.

[0168] Table 7. Effects of GOx complex liposomes with different interface construction sequences on feline gut health and odor-related indicators.

[0169] The results are shown in Table 7. After 28 days of intervention, the total SCFA and fecal sIgA in Example 1 group increased by 58.1% and 41.2% respectively compared with before the intervention, while the soft stool rate and the relative peak area of ​​indole decreased by 65.0% and 56.0% respectively. The overall change was better than that of Comparative Example 1 and Comparative Example 2.

[0170] In Comparative Example 1, total SCFA and fecal sIgA increased by 33.4% and 18.5%, respectively, while the soft stool rate and indole relative peak area decreased by 35.0% and 32.0%, respectively. In Comparative Example 2, the changes in the corresponding indicators were further reduced. These results indicate that pre-adsorption at the LMP interface followed by controlled addition of Ca... 2+ The resulting complex interface helps GOx maintain its activity in the anterior gastrointestinal tract and be continuously released in the hindgut, thereby achieving better regulation of the intestinal fermentation environment and improvement of odor-related indicators.

[0171] Change LMP and Ca 2+ After the addition of certain components, the particle dispersion stability, gastrointestinal enzyme activity retention, and hindgut release capacity of the system all decreased, and its animal application effect was correspondingly weakened, further illustrating that the specific interface construction sequence is an important condition for obtaining the above application effects.

[0172] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing interface-confined calcium-crosslinked GOx complex liposomes, characterized in that, Including the following steps: (1) Dissolve glucose oxidase and trehalose in water and disperse them evenly to obtain the aqueous phase of GOx; wherein the concentration of glucose oxidase in the aqueous phase of GOx is 0.5~2 mg / mL; (2) Phospholipids were added to the aqueous phase of GOx, and the mixture was sheared, homogenized, and sonicated to obtain GOx pre-formed liposomes; wherein the concentration of phospholipids in the GOx pre-formed liposome dispersion was 5~20 mg / mL. (3) Low-methoxyl pectin was first added to the preformed GOx liposomes and stirred; then CaCl2 solution was added to crosslink the liposomes, resulting in crosslinked GOx liposomes; wherein the concentration of low-methoxyl pectin in the crosslinked GOx liposomes was 0.5~2 mg / mL, and CaCl2 solution was added to obtain crosslinked GOx liposomes. 2+ The molar ratio of carboxyl groups in low-methoxyl pectin to low-methoxyl pectin is 0.02~0.15:1; (4) Add a freeze-drying protectant to the cross-linked GOx liposomes, pre-freeze and freeze-dry to obtain GOx composite liposome powder.

2. The method according to claim 1, characterized in that, The dispersion temperature in step (1) is 4~10℃.

3. The method according to claim 1, characterized in that, In step (2), the shearing is performed at 7000~9000 rpm for 10~12 min, and the homogenization is performed at 20~40 MPa for 10~20 min.

4. The method according to claim 1, characterized in that, In step (3), the dropping rate of the CaCl2 solution is 0.05~0.50 mL / min.

5. The method according to claim 1, characterized in that, In step (3), the interfacial pre-adsorption stirring time for low methoxyl pectin is 10~60 min.

6. The interface-confined calcium-crosslinked GOx complex liposomes prepared by the method according to any one of claims 1 to 5.

7. The application of the interface-confined calcium crosslinked GOx complex liposomes according to claim 6 in the field of pet food.

8. A pet food or pet nutritional supplement, characterized in that, The pet food or pet nutritional supplement contains the interface-confined calcium crosslinked GOx complex liposomes as described in claim 6, with an addition amount of 0.1%~1.0% w / w.

9. A method for improving the intestinal release rate of GOx liposomes, characterized in that, Preparation of interfacially confined calcium-crosslinked GOx complex liposomes includes the following steps: (1) Dissolve glucose oxidase and trehalose in water and disperse them evenly to obtain the aqueous phase of GOx; wherein the concentration of glucose oxidase in the aqueous phase of GOx is 0.5~2 mg / mL; (2) Phospholipids were added to the aqueous phase of GOx, and the mixture was sheared, homogenized, and sonicated to obtain GOx pre-formed liposomes; wherein the concentration of phospholipids in the GOx pre-formed liposome dispersion was 5~20 mg / mL. (3) Low-methoxyl pectin was first added to the preformed GOx liposomes and stirred; then CaCl2 solution was added to crosslink the liposomes, resulting in crosslinked GOx liposomes; wherein the concentration of low-methoxyl pectin in the crosslinked GOx liposomes was 0.5~2 mg / mL, and CaCl2 solution was added to obtain crosslinked GOx liposomes. 2+ The molar ratio of carboxyl groups in low-methoxyl pectin to low-methoxyl pectin is 0.02~0.15:1; (4) Add a freeze-drying protectant to the cross-linked GOx liposomes, pre-freeze and freeze-dry to obtain GOx composite liposome powder.

Images