A poriferous microsphere material based on pachyman, a food preservative and a preparation method thereof

Abstract

The present application relates to food preservation material technical field, aiming at the technical problem of limited microsphere adsorption capacity of existing microspheres, and the microsphere preparation process does not meet the safety standard of food contact material, the present application provides a kind of poriferous microsphere material based on Pachyman, the poriferous microsphere material uses Pachyman and polyvinyl alcohol as matrix material, and is prepared by annealing technology and salting-out method coordination.This application adopts a kind of green preparation path of fusion annealing technology and salting-out method combination, and the physical pore-forming agent sodium chloride forms dispersed phase in the microsphere forming process, and is removed in subsequent purification process, so as to build hierarchical porous structure in the microsphere, and give its excellent specific surface area and material adsorption or slow-release capacity.

Description

Technical Field

[0001] This invention relates to the field of food preservation materials technology, and in particular to a poria cocos polysaccharide-based porous microsphere material, a food preservative, and a method for preparing the same. Background Technology

[0002] Food preservation is a core element in ensuring global food security, reducing post-harvest losses, and maintaining the nutritional value and sensory quality of food. Microbial contamination is one of the leading causes of food spoilage. The proliferation of microorganisms such as bacteria and molds not only damages the texture and flavor of food, but their metabolic products can also cause foodborne illnesses, seriously threatening public health. Traditionally, the food industry has relied on chemical preservatives (such as benzoates and nitrites) and physical barriers (such as plastic packaging and low-temperature refrigeration) to extend shelf life. However, the potential long-term health risks of chemical additives have raised widespread consumer concerns, while the widespread use of non-degradable plastic packaging has brought serious environmental problems. Therefore, developing "green" packaging materials that are naturally derived, biodegradable, and possess active preservation functions has become an important development direction in the field of food science and engineering.

[0003] Poria cocos polysaccharides (PCP) are a class of natural high-molecular-weight polysaccharides extracted from the dried sclerotia of the traditional medicinal fungus Poria cocos. Studies have shown that PCP possesses antioxidant activity, certain microbial inhibition potential, and recognized food safety, demonstrating great potential as a natural preservative or active packaging matrix. However, directly applying this material to food preservation systems faces fundamental challenges: its high molecular weight and strong interchain hydrogen bonding result in fatal defects such as extremely poor water solubility and insufficient dispersion stability; in addition, it is prone to agglomeration and inactivation during processing and is difficult to form a continuous film structure with good mechanical strength. These defects hinder the effective delivery and exercise of its active functions.

[0004] Microsphere carrier technology offers a promising path to address these challenges. Compared to traditional films, microsphere carriers exhibit several significant advantages: their porous internal structure endows the material with an extremely high specific surface area, significantly enhancing the loading capacity of active ingredients and strengthening the interaction interface between the carrier and the target factor; in terms of application, they can be independently packaged into small, breathable packets for internal use.

[0005] However, most existing microsphere carriers are solid or low-porosity structures, and their function mainly relies on surface adsorption mechanisms, which have limited adsorption capacity for gaseous putrefactive products and suspended microorganisms. Many efficient microsphere preparation processes rely on toxic chemical cross-linking agents or organic solvents, which do not meet the safety standards for food contact materials. Single polysaccharide microspheres often dissolve rapidly in aqueous media due to their strong hydrophilicity, leading to the destruction of their carrier structure and loss of function. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a poria cocos polysaccharide-based porous microsphere material, a food preservative, and its preparation method. It employs a green preparation path that combines annealing technology with salting-out method. Sodium chloride, a physical pore-forming agent, forms a dispersed phase during the microsphere forming process and is removed during subsequent purification, thereby constructing a hierarchical porous structure inside the microsphere, endowing it with excellent specific surface area and adsorption or sustained-release capabilities.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, this application discloses a poria cocos polysaccharide-based porous microsphere material, which is prepared by annealing technology and salting out using poria cocos polysaccharide and polyvinyl alcohol as matrix materials.

[0009] Furthermore, the porous microsphere material has a particle size of 3-5 mm and a porosity of 49-93%.

[0010] Furthermore, the annealing process is carried out at a temperature of 40°C for 48 hours; the salting-out process is carried out at a temperature of 100-110°C for 48 hours.

[0011] Secondly, this application discloses a poria cocos polysaccharide-based porous microsphere food preservative, which includes the above-mentioned poria cocos polysaccharide-based porous microsphere material and tannic acid loaded inside its porous structure.

[0012] Furthermore, the loading rate of the tannic acid is 3%.

[0013] Thirdly, this application discloses a method for preparing a poria cocos polysaccharide-based porous microsphere food preservative, comprising the following steps:

[0014] S1. Weigh out Poria cocos polysaccharide and polyvinyl alcohol in a mass ratio of 1:1 to 3, and dissolve Poria cocos polysaccharide and polyvinyl alcohol in dimethyl sulfoxide to obtain solution 1 and solution 2.

[0015] S2. After mixing solution 1 and solution 2 evenly, add the pore-forming agent, heat to dissolve, and obtain a mixed solution;

[0016] S3. The mixed solution was dropped into pure water to solidify and purify, resulting in poria cocos polysaccharide-based porous microspheres.

[0017] S4. Soak the poria polysaccharide-based porous microspheres in tannic acid solution for 24-48 hours, and then freeze-dry them to obtain the poria polysaccharide-based porous microsphere preservative.

[0018] Furthermore, in S2, the amount of pore-forming agent added is 0.25 times the total mass of Poria cocos polysaccharide and polyvinyl alcohol, and the heating and dissolution temperature is 100-110℃.

[0019] Furthermore, in S2, the pore-forming agent is sodium chloride.

[0020] Furthermore, in S3, the curing temperature is 40℃, the time is 30-60 min, and the purification time is 48 h.

[0021] Furthermore, in S4, the concentration of tannic acid solution is 10%, the freeze-drying time is 24-36 hours, and the freeze-drying temperature is -35℃ to -20℃.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention utilizes a green, synergistic preparation path combining physical pore formation and annealing gelation to construct porous microspheres based on Poria cocos polysaccharides, exhibiting both structural stability and high porosity. This fundamentally overcomes the technical shortcomings of traditional methods, such as closed pores and loose structures resulting from physical pore formation alone, and low porosity and insufficient specific surface area caused by annealing gelation alone. This preparation process completely eliminates the use of toxic chemical crosslinking agents and organic solvents, relying solely on sodium chloride for physical pore formation and temperature gradient-induced hydrogen bond self-assembly to achieve molding. This not only significantly reduces production difficulty and environmental burden but also ensures that the resulting microspheres meet the safety standards for food contact materials, providing reliable technical support for the large-scale application of green packaging materials.

[0024] Building upon this foundation, the present invention utilizes synergistic effects to achieve precise control over the microscopic morphology of the microspheres, resulting in a three-dimensional interconnected network structure with a particle size of 3-5 mm and a porosity of 49-93%. This unique structure not only endows the microspheres with excellent mechanical strength, effectively avoiding the risk of breakage during transportation and application, but also provides ample adsorption sites for the loading of functional factors. The high specific surface area and interconnected channels of the microspheres enable efficient physical adsorption of spoilage factors such as ethylene and odor molecules, and physical retention of microorganisms, thus constructing the first line of defense for preservation at the physical level.

[0025] Furthermore, this invention achieves long-term sustained release of tannic acid through a microsphere-stabilized network structure, constructing a dual preservation mechanism of "physical adsorption + chemical regulation." The dense network formed by annealing and gelation effectively immobilizes the tannic acid, extending its sustained release period to over 72 hours, continuously exerting its antioxidant and antibacterial functions. This synergistic effect of physical retention and chemical inhibition significantly improves preservation efficiency, solving the problems of excessively rapid release and poor persistence of traditional preservatives, and achieving quality protection for fresh products throughout their entire lifecycle. Attached Figure Description

[0026] Figure 1This is a photograph of the Poria cocos polysaccharide-based porous microsphere food preservative of the present invention;

[0027] Figure 2 The graph shows the spheroidization rate of the poria cocos polysaccharide-based porous microsphere food preservative of this invention.

[0028] Figure 3 This is a graph showing the sphericity results of the poria cocos polysaccharide-based porous microsphere food preservative of the present invention;

[0029] Figure 4 The swelling performance test results of the poria polysaccharide-based porous microsphere food preservative of this invention are shown in the figure.

[0030] Figure 5 The figure shows the porosity test results of the porosity of the poria polysaccharide-based porous microsphere food preservative of the present invention.

[0031] Figure 6 The graph shows the test results of the degradation performance of the poria cocos polysaccharide-based porous microsphere food preservative of this invention.

[0032] Figure 7 Infrared spectra of Poria cocos polysaccharide (PCP) and Poria cocos polysaccharide-based porous microspheres for food preservation.

[0033] Figure 8 This is a scanning electron microscope image of the poria cocos polysaccharide-based porous microsphere food preservative of the present invention;

[0034] Figure 9 This is a tannic acid release curve of the poria cocos polysaccharide-based porous microsphere food preservative of the present invention;

[0035] Figure 10 The figure shows the test results of the antibacterial performance of the Poria cocos polysaccharide-based porous microsphere food preservative of the present invention against Escherichia coli and Staphylococcus aureus.

[0036] Figure 11 This is a graph showing the test results of DPPH free radical scavenging rate of the poria cocos polysaccharide-based porous microsphere food preservative of the present invention;

[0037] Figure 12 The rheological test results of the poria cocos polysaccharide-based porous microsphere food preservative of the present invention are shown in the figure.

[0038] Figure 13 These are photographs showing the effect of the poria cocos polysaccharide-based porous microsphere food preservative of this invention on the preservation of grapes and cherry tomatoes;

[0039] Figure 14 These are photographs showing the effects of the poria cocos polysaccharide-based porous microsphere food preservative of this invention on the preservation of fresh-cut pears and fresh-cut cantaloupes.

[0040] Figure 15 The images show the liver, kidneys, pancreas, and blood routine results from animal experiments of this invention. Detailed Implementation

[0041] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0042] Example

[0043] This application discloses a poria cocos polysaccharide-based porous microsphere material, which is prepared by synergistic annealing and salting-out methods using poria cocos polysaccharide and polyvinyl alcohol as matrix materials.

[0044] Among them, the porous microsphere material has a particle size of 3-5 mm and a porosity of 49-93%.

[0045] The annealing process is carried out at a temperature of 40℃ for 48 hours; the salting-out process is carried out at a temperature of 100-110℃ for 48 hours.

[0046] This application also discloses a poria cocos polysaccharide-based porous microsphere food preservative, which includes the above-mentioned poria cocos polysaccharide-based porous microsphere material and tannic acid loaded inside its porous structure.

[0047] Furthermore, this application also discloses a method for preparing the above-mentioned Poria cocos polysaccharide-based porous microsphere food preservative, including the following steps:

[0048] S1. Dissolve Poria cocos polysaccharide (PCP) and polyvinyl alcohol (PVA) in dimethyl sulfoxide (DMSO) respectively to obtain solution 1 and solution 2;

[0049] The mass ratio of Poria cocos polysaccharide to polyvinyl alcohol is 1:1;

[0050] For example, weigh 0.6g of PCP and 0.6g of PVA and add them to 10mL of DMSO respectively, and mix them using a high-speed homogenizer to obtain solution 1 and solution 2.

[0051] It should be noted that the preparation method of Poria cocos polysaccharide in S1 includes:

[0052] S101. Disperse Poria cocos powder in a 0.9% NaCl solution, stir for 24 hours, and then centrifuge to obtain precipitate and supernatant.

[0053] Specifically, 20g of dried wild Poria cocos powder was accurately weighed and placed in a 250mL beaker. 120mL of 0.9% NaCl solution was added, and the mixture was placed on a magnetic stirrer and stirred at room temperature for 24 hours. After stirring, the mixture was poured into a centrifuge tube and centrifuged at 5000rpm for 20 minutes, retaining the precipitate.

[0054] S102. The precipitate was successively treated with pure water at high temperature, and then with NaOH solutions of concentrations of 0.4%, 0.8% and 5% for 24 hours. The supernatant of the last alkaline solution was collected, and the pH of the collected alkaline supernatant was adjusted to neutral using 36% glacial acetic acid solution to obtain a gel-like precipitate.

[0055] Specifically, 120 mL of purified water was added to the precipitate, and the mixture was transferred to an autoclave at 120°C for 25 minutes. After sterilization, the mixture was centrifuged at 5000 rpm for 20 minutes, and the precipitate was retained. 120 mL of a 0.4% NaOH solution was added to the precipitate, and the mixture was stirred for 24 hours. After stirring, the mixture was centrifuged at 5000 rpm for 20 minutes, and the precipitate was retained. 120 mL of a 0.8% NaOH solution was added to the precipitate, and the mixture was stirred for 24 hours. After stirring, the mixture was centrifuged at 5000 rpm for 20 minutes, and the precipitate was retained. 120 mL of a 5% NaOH solution was added to the precipitate, and the mixture was stirred for 24 hours. After stirring, the mixture was centrifuged at 5000 rpm for 20 minutes, and the supernatant was retained. The pH of the supernatant was adjusted to neutral (pH=7.0) using 36% glacial acetic acid, at which point the solution became a gel-like solid.

[0056] S103. The gel precipitate was washed with hot water, centrifuged, and freeze-dried to obtain Poria cocos polysaccharide.

[0057] Specifically, the gel-like solid was washed with hot water to remove residual acid, and then centrifuged at 5000 rpm for 20 minutes, retaining the gel precipitate. The gel precipitate was placed in a freeze dryer and freeze-dried for 48 hours to obtain a white, spongy solid of Poria cocos polysaccharide, which is Poria cocos polysaccharide (PCP).

[0058] S2. After mixing solution 1 and solution 2 evenly, add the pore-forming agent, heat to dissolve, and obtain a mixed solution;

[0059] In S2, the amount of sodium chloride, the pore-forming agent, added is 0.25 times the total mass of Poria cocos polysaccharide and polyvinyl alcohol, and the heating and dissolution temperature is 100-110℃.

[0060] Specifically, 0.15g NaCl was added as a pore-forming agent, and after homogenization and mixing, it was heated at 100℃ to dissolve and obtain a mixed solution.

[0061] S3. The mixed solution was dropped into pure water to solidify and purify, resulting in poria cocos polysaccharide-based porous microspheres.

[0062] In S3, the curing temperature is 40℃, the time is 30-60 min, and the purification time is 48 h.

[0063] Specifically, the obtained mixed solution was loaded into a syringe and added dropwise to pure water at 40°C using a dripping device (needle inner diameter 0.45 mm). After solidification for 5 minutes, preliminary microspheres were obtained. The preliminary microspheres were removed from the water and immersed in pure water for 48 hours for purification, with the water changed every 12 hours during this period to remove residual DMSO and NaCl, resulting in poria cocos polysaccharide-based porous microspheres.

[0064] S4. Soak the poria polysaccharide-based porous microspheres in tannic acid solution for 24-48 hours, and then freeze-dry them to obtain the poria polysaccharide-based porous microsphere preservative.

[0065] In S4, the concentration of tannic acid solution is 10%, the freeze-drying time is 24-36 hours, and the freeze-drying temperature is -35℃ to -20℃.

[0066] Specifically, after soaking and loading the poria polysaccharide-based porous microspheres in tannic acid solution for 24 hours, the poria polysaccharide-based porous microspheres were taken out and freeze-dried in a -20℃ refrigerator for 24 hours to obtain the poria polysaccharide-based porous microsphere preservative.

[0067] More specifically, for better comparative analysis, this application removed half of the poria cocos polysaccharide-based porous microspheres from S4 and placed them in a refrigerator for freezing, then directly freeze-dried (24h, -20℃) to obtain porous microspheres with added pore-forming agents but without tannic acid, labeled PCP-AM; porous microspheres without added pore-forming agents were labeled PCP-M. The other half of the poria cocos polysaccharide-based porous microspheres were placed in a 10% tannic acid (TA) solution and soaked at room temperature for 24h, then removed and frozen in a -10℃ refrigerator, and then freeze-dried (24h, -20℃) to obtain porous microspheres with tannic acid, labeled PCP-TA-M.

[0068] It should be noted that PCP-AM consists of porous microspheres with added pore-forming agents but without tannins; among them, PCP-AM4 consists of microspheres with 4% (w / w) concentration of Poria cocos polysaccharide; PCP-AM5 consists of microspheres with 5% (w / w) concentration of Poria cocos polysaccharide; and PCP-AM6 consists of microspheres with 6% (w / w) concentration of Poria cocos polysaccharide.

[0069] PCP-TA-M consists of porous microspheres loaded with tannic acid; among them, PCP-TA-M4 consists of microspheres with 4% (w / w) concentration of Poria cocos polysaccharide, which are then soaked and loaded with tannic acid; PCP-TA-M5 consists of microspheres with 5% (w / w) concentration of Poria cocos polysaccharide, which are then soaked and loaded with tannic acid; and PCP-TA-M6 consists of microspheres with 6% (w / w) concentration of Poria cocos polysaccharide, which are then soaked and loaded with tannic acid.

[0070] It should be noted that the 4%, 5%, and 6% PCP microspheres mentioned above are calculated using a weight-to-volume ratio, i.e., 0.4g PCP to 10ml DMSO. Additionally, it should be noted that the percentage concentrations here refer only to the initial dissolved concentration of PCP in DMSO. In the embodiments of this application, although an equal mass (e.g., 0.6g) of PVA was added when preparing solutions 1 and 2, the 4%, 5%, and 6% concentrations do not include the mass of PVA; they are merely indicators to distinguish different PCP addition gradients.

[0071] The calculation formula is: Mass concentration (%) = [Solute mass (g) ÷ Solvent volume (mL)] × 100%

[0072] In this application, the solute is PCP, the solvent is DMSO, and the solvent volume is fixed at 10 mL.

[0073] Taking 4% mass concentration microspheres as an example, the specific calculation process will be explained. The calculation method for other mass concentrations is the same, and will not be elaborated further here:

[0074] Weigh out 0.4g of PCP.

[0075] Add 10 mL of DMSO;

[0076] Calculate: (0.4g ÷ 10mL) × 100% = 4%.

[0077] The following describes the characterization of the structure and basic properties of the porous microspheres prepared in the examples and the study of their food preservation performance.

[0078] I. Structure and Basic Properties

[0079] 1. Appearance

[0080] After dialysis of the microspheres prepared by combining annealing and salting out, irregular and agglomerated microspheres are removed, while regular microspheres are retained and freeze-dried to obtain the microspheres. Figure 1 These are physical images of the PCP-AM4, PCP-AM5, PCP-AM6, PCP-TA-M4, PCP-TA-M5, and PCP-TA-M6 microspheres of the present invention. Figure 1 It can be seen that the microspheres without the active substance tannic acid are white spheres (such as PCP-AM4, PCP-AM5, and PCP-AM6) and have intact spherical shape; the microspheres with the active substance tannic acid are uniformly light yellow in color (such as PCP-TA-M4, PCP-TA-M5, and PCP-TA-M6) and still maintain a good spherical shape without collapse or adhesion, indicating that the loading of tannic acid did not destroy the basic molding structure of the microspheres.

[0081] 2. Ball formation rate

[0082] The sphericity of six groups of microspheres was statistically analyzed using a counting method. All microspheres after curing were collected, and the number of intact, non-adhesive, and unbroken microspheres was counted. Each sample was tested in triplicate, and the average value was taken. The results are shown below. Figure 2 The results showed that the sphericity of the PCP-AM series microspheres gradually increased with formulation optimization, with PCP-AM6 exhibiting the highest sphericity, significantly higher than PCP-AM5 and PCP-AM4. Among the PCP-TA-M series microspheres, PCP-TA-M6 showed the best sphericity among its peers, and compared to its corresponding matrix, PCP-AM6 showed no significant decrease in sphericity after loading with active substances. In contrast, PCP-TA-M5 and PCP-TA-M4 both experienced varying degrees of decrease in sphericity after loading with active substances. These results indicate that the optimal formulation of the Poria cocos polysaccharide-PVA composite system in this invention possesses the best sphericity performance, and the loading of tannic acid has no negative impact on the molding effect, thus solving the technical problems of unstable sphericity formation with single polysaccharides and a significant decrease in molding rate after loading with active substances.

[0083] 3. Swelling properties

[0084] The swelling properties of six groups of microspheres were tested using a gravimetric method. After being freeze-dried, the microspheres were precisely weighed and immersed in phosphate buffered saline (PBS, pH=7.4) at 37℃. At preset time points, the microspheres were removed, surface liquid was removed, and they were precisely weighed again. The changes in swelling rate at different time points were statistically analyzed. Each sample was tested in triplicate. The results are shown in the table below. Figure 4 The results showed that all six groups of microspheres exhibited a swelling pattern of rapid initial water absorption followed by a stable swelling in PBS solution. PCP-AM6 showed the fastest water absorption rate and the highest equilibrium swelling ratio among the PCP-AM series. Within the PCP-TA-M series, PCP-TA-M6 had the best equilibrium swelling ratio, although slightly lower than PCP-AM6 with the same matrix, it was still higher than PCP-TA-M5 and PCP-TA-M4. The swelling ratios of the other two groups of microspheres loaded with active substances decreased significantly compared to their corresponding unloaded groups. Only PCP-TA-M6 maintained excellent water absorption and swelling performance after being loaded with tannic acid, demonstrating structural stability in high-humidity food packaging environments. Simultaneously, it reduced free water within the packaging through water absorption, meeting the application requirements for food preservation.

[0085] 4. Swelling properties

[0086] The porosity of PCP-TA-M series microspheres was tested using the ethanol impregnation method. The lyophilized microspheres were accurately weighed, immersed in anhydrous ethanol until adsorption saturation, and after removing free ethanol from the surface, they were accurately weighed again. The porosity of each group of microspheres was calculated. Each group of samples was tested in triplicate. The results are shown in the table below. Figure 5The results showed that among the PCP-TA-M series microspheres, PCP-TA-M6 had the highest porosity and the best internal pore connectivity; the porosity of PCP-TA-M5 and PCP-TA-M4 decreased sequentially, and both exhibited a large number of closed pore structures. These results indicate that microspheres prepared under the optimal formulation can construct a interconnected porous structure, providing ample adsorption sites for spoilage factors such as ethylene and odor molecules, as well as sufficient channels for the loading and sustained release of tannic acid, thus overcoming the technical shortcomings of existing microspheres, such as low porosity and limited adsorption capacity.

[0087] 5. Degradation performance

[0088] Six groups of microspheres were placed in PBS (pH=7.4) solution at 37℃ for in vitro degradation experiments. An equal amount of lyophilized microspheres from each group were immersed in PBS solution. Samples were removed at preset time points, freeze-dried to constant weight, and accurately weighed. Changes in degradation rate were statistically analyzed. Each group of samples was tested in triplicate. Results are shown below. Figure 6 The results showed that PCP-AM4 and PCP-AM5 in the PCP-TA-M series had relatively slow degradation rates, exhibiting significant mass loss and structural breakage in the early stages of degradation, and were unable to maintain structural stability during the shelf life of food products.

[0089] 6. Infrared spectroscopy characterization

[0090] The structure of six groups of microspheres was characterized using Fourier transform infrared spectroscopy, with a scanning range of [missing information]. The results are shown Figure 7 The results showed that all three groups of PCP-AM series microspheres exhibited characteristic absorption peaks of Poria cocos polysaccharide and polyvinyl alcohol, with no new chemical cross-linking characteristic peaks appearing. This proves that the present invention achieved the gelation and molding of microspheres through physical annealing without introducing chemical cross-linking agents, meeting the safety requirements for food contact materials. Among them, PCP-AM6 showed the highest degree of broadening of the hydroxyl characteristic peak, indicating that the hydrogen bonding between Poria cocos polysaccharide and PVA molecules was the strongest, and the internal network structure was the most stable. All three groups of PCP-TA-M series microspheres exhibited characteristic absorption peaks of tannic acid, proving that tannic acid was successfully loaded into the microspheres. Among them, PCP-TA-M6 showed the most significant tannic acid characteristic peak, with the strongest hydrogen bonding with the polysaccharide matrix, and the best tannic acid loading effect. In contrast, the tannic acid characteristic peaks of PCP-TA-M5 and PCP-TA-M4 were weaker, indicating relatively poor loading effects.

[0091] 7. Microscopic morphology characterization

[0092] The surface and cross-sectional micromorphology of the seven groups of microspheres were characterized using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 8The results showed that PCP-AM6 exhibited a uniformly rough porous morphology, without a dense skin layer or cracks. The cross-section revealed an interconnected three-dimensional porous network structure with uniform pore size distribution and no closed pores. PCP-AM5 and PCP-AM4, however, exhibited uneven pore size distribution, partial pore closure, and thickened pore walls. Among the PCP-TA-M series, PCP-TA-M6 maintained an excellent interconnected porous structure, without pore blockage or collapse, providing a good channel for the sustained release of tannic acid.

[0093] II. Verification of the functional activity and preservation performance of microspheres

[0094] This experiment uses six groups of microspheres—PCP-AM4, PCP-AM5, PCP-AM6, PCP-TA-M4, PCP-TA-M5, and PCP-TA-M6—as research subjects. It systematically compares the release behavior of active ingredients, antioxidant properties, antibacterial properties, biosafety, and actual food preservation effects of each group of microspheres, comprehensively verifying the functional differences of microspheres with different formulations and clarifying the optimal implementation plan.

[0095] 1. Tannic acid release

[0096] The tannic acid release performance of three groups of PCP-TA-M4, PCP-TA-M5, and PCP-TA-M6 microspheres carrying active substances was tested using dialysis. A release environment was simulated in PBS solution at 37℃ and pH 7.4. Samples were taken at preset time points to detect the cumulative tannic acid release rate. Each group of samples was tested in triplicate. The results are shown below. Figure 9 The results showed that among the three groups of microspheres carrying active substances, PCP-TA-M6 exhibited the best tannic acid release behavior, displaying a biphasic release characteristic of "rapid onset in the early stage and long-term sustained release in the later stage," with no burst release phenomenon, the highest cumulative release rate, and the longest sustained release period. PCP-TA-M5 and PCP-TA-M4, on the other hand, showed significant burst release in the early stage and incomplete release in the later stage, failing to maintain a stable preservation function throughout the entire shelf life of food. These results indicate that the optimal interconnected porous structure and hydrogen-bonded binding system of PCP-TA-M6 can achieve controllable sustained release of tannic acid, perfectly meeting the application requirements for long-term food preservation.

[0097] 2. Antioxidant properties

[0098] The antioxidant properties of six groups of microspheres were tested using the DPPH free radical scavenging method. Microsphere sample solutions of different concentrations were prepared, mixed with DPPH ethanol solution, and allowed to stand in the dark. The absorbance was measured, and the free radical scavenging rate was calculated. Vitamin C was used as a positive control. Each sample group was tested in triplicate. The results are shown in the table below. Figure 11The results showed that all three groups of PCP-TA-M series microspheres exhibited significant antioxidant activity. Among them, PCP-TA-M6 showed the highest DPPH free radical scavenging rate. At the same test concentration, its scavenging effect was not significantly different from the positive control vitamin C, but was much higher than PCP-TA-M5 and PCP-TA-M4. These results indicate that the microsphere carrier prepared with the optimal formulation can maximize the retention of tannic acid's antioxidant activity. Through the continuous release of tannic acid, it can efficiently scavenge free radicals in food systems, delaying the deterioration processes such as oil oxidation and browning of fruits and vegetables.

[0099] 3. Antibacterial properties

[0100] The antibacterial properties of two groups of microspheres, PCP-AM6 and PCP-TA-M6, against common food spoilage pathogens, Escherichia coli and Staphylococcus aureus, were tested using a co-culture method combined with optical density (OD) measurement. Each group of samples was tested in triplicate. Results are shown below. Figure 10 .

[0101] During the test, single colonies of *Escherichia coli* and *Staphylococcus aureus* were inoculated into sterile LB broth and cultured at 37°C with shaking until the logarithmic growth phase. The bacterial concentration was then adjusted to a suitable range using fresh sterile LB broth. Equal volumes of irradiated sterilized PCP-AM6 and PCP-TA-M6 microspheres were added to the bacterial culture, with an equal volume of culture without microspheres serving as a blank control. All groups were co-cultured at 37°C with shaking. Samples were taken at 4h, 8h, 12h, and 24h, and the OD value of the bacterial culture at 600nm was measured using a UV spectrophotometer. Changes in OD value characterized bacterial proliferation. After culture, colony counting was performed on each group using the plate spread method to evaluate the antibacterial effect. Simultaneously, the minimum inhibitory concentration (MIC) of the two microspheres against the two pathogenic bacteria was determined using a micro-broth dilution method combined with OD value measurement.

[0102] The co-culture experiment results showed that the OD value of the bacterial culture in the PCP-AM6 group at each culture time point was not significantly different from that in the blank control group. After 24 hours of culture, the total number of colonies was close to that in the blank control group. It did not have a significant inhibitory effect on the proliferation of Escherichia coli and Staphylococcus aureus, and the inhibition rate was less than 10%, indicating that the PCP-AM6 microspheres without tannic acid loading do not have effective antibacterial ability.

[0103] 4. Rheological properties

[0104] The rheological behavior of six groups of microspheres was characterized using a rotational rheometer. The test temperature was 25℃, and the angular frequency scan range was 0.1-100 rad / s. The storage modulus and loss modulus of the microspheres were tested as a function of angular frequency. The results are shown in [Figure number missing]. Figure 12The results showed that throughout the entire test angular frequency range, the storage modulus of all six microsphere groups was consistently higher than their loss modulus, exhibiting typical elastic gel behavior. Among them, PCP-AM6 had the highest storage modulus, the best mechanical strength, and the best structural stability in the PCP-AM series, while the moduli of PCP-AM5 and PCP-AM4 decreased sequentially. In the PCP-TA-M series, PCP-TA-M6 had the highest storage modulus and the best structural stability, significantly higher than PCP-TA-M5 and PCP-TA-M4. This demonstrates that under the optimal formulation, loading tannic acid can further enhance the stability of the microsphere network structure through hydrogen bonding, allowing the microspheres to maintain structural integrity and resist breakage during transportation, storage, and application.

[0105] 5. Performance in food preservation applications

[0106] Complete Fruit Preservation Application

[0107] Fresh grapes and cherry tomatoes were selected for preservation. Fruits with uniform ripeness, no mechanical damage, and no pests or diseases were randomly divided into a blank control group, a PCP-AM6 group, and a PCP-TA-M6 group, with three replicates in each group. The corresponding microspheres were encapsulated in breathable non-woven bags and placed together with the fruit in a PE preservation box. The bags were stored at 25℃ and 60% relative humidity. Changes in fruit appearance were observed, and the rate of decay and weight loss were recorded. Results after 15 days of storage were observed... Figure 13 The results showed that after 15 days of storage, the fruits in the blank control group showed signs of rotting, mold, dehydration, and shrinkage. Among PCP-AM6 and PCP-TA-M6, PCP-TA-M6 had the most outstanding preservation effect. The grapes and cherry tomatoes maintained their plump appearance and bright color, without mold or rotting. The rotting rate and weight loss rate were the lowest among all groups, far superior to PCP-AM6, and could significantly extend the shelf life of whole fruits.

[0108] 6. Applications in preserving fresh-cut fruits

[0109] Fresh pears and cantaloupes were used for preservation. After washing, peeling, and cutting, uniformly sized fresh-cut fruit pieces were obtained and randomly divided into a blank control group, a PCP-AM6 group, and a PCP-TA-M6 group, with three replicates in each group. The corresponding microspheres were encapsulated in breathable non-woven bags and placed together with the fresh-cut fruit pieces in a sterile preservation box, stored at 4°C. Browning, softening, and microbial growth of the fruit pieces were observed. Results after 1-2 days of storage were observed... Figure 14The results showed that after 10 days of storage, the blank control group exhibited severe browning of the cut fruit pieces, softening of the flesh, oozing of juice, and mold growth, with the total bacterial count exceeding the food hygiene standard limit. In contrast, the PCP-TA-M6 group showed no obvious browning of the cut fruit pieces, the flesh was crisp and tender, with no oozing of juice or mold growth, and the total bacterial count remained within the safe limit. Its preservation effect far exceeded that of PCP-TA-M5 and PCP-TA-M4, demonstrating excellent application results in the cold storage and preservation of fresh-cut fruits.

[0110] III. Safety Testing

[0111] SD mice were selected as experimental animals. After 7 days of acclimatization, they were randomly divided into a blank control group, a PCP-AM6 experimental group, and a PCP-TA-M6 experimental group. The number of mice and the male-to-female ratio were the same in each group, and there was no significant difference in initial body weight. Mice in the blank control group were fed a basal maintenance diet, while the two experimental groups were fed experimental diets mixed with the corresponding microspheres. The dosage of microspheres added to both groups was the same. All groups had free access to food and water for 7 consecutive days.

[0112] During the experiment, the mice's mental state, eating habits, and activity were observed daily, and their weight changes were recorded weekly. After the feeding cycle ended, whole blood was collected from the mice for routine blood tests. Simultaneously, the liver, kidneys, and pancreas were completely dissected, fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE) for histopathological observation. The results are shown below. Figure 15 .

[0113] General observation results showed that throughout the feeding period, neither the PCP-AM6 group nor the PCP-TA-M6 group mice exhibited abnormal behavior, signs of poisoning, or death. Their mental state and dietary activity were normal, and their weight gain trend was not significantly different from that of the blank control group.

[0114] Histopathological observations showed that the liver, kidneys, and pancreas of the control group mice had normal morphology and structure, with no pathological changes such as inflammatory cell infiltration, cell degeneration, or necrosis. Although there was no obvious pathological damage in the organs of the PCP-AM6 group mice, very slight inflammatory cell infiltration was observed in some areas. In contrast, the liver, kidneys, and pancreas of the PCP-TA-M6 group mice had intact morphology and clear structure, with no visible differences from the control group, and no pathological changes related to the test substance, such as inflammatory cell infiltration, cell degeneration, or necrosis. The organ tissue safety of the PCP-TA-M6 group was significantly better than that of the PCP-AM6 group.

[0115] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A porous microsphere material based on Poria cocos polysaccharide, characterized in that, The porous microsphere material is prepared by synergistic annealing and salting-out methods using Poria cocos polysaccharide and polyvinyl alcohol as matrix materials.

2. The poria cocos polysaccharide-based porous microsphere material according to claim 1, characterized in that, The porous microsphere material has a particle size of 3-5 mm and a porosity of 49-93%.

3. The poria cocos polysaccharide-based porous microsphere material according to claim 1, characterized in that, The annealing process is carried out at a temperature of 40°C for 48 hours; the salting-out process is carried out at a temperature of 100-110°C for 48 hours.

4. A poria cocos polysaccharide-based porous microsphere food preservative, characterized in that, The poria cocos polysaccharide-based porous microsphere preservative includes the poria cocos polysaccharide-based porous microsphere material as described in any one of claims 1-3, and tannic acid loaded inside its porous structure.

5. The poria cocos polysaccharide-based porous microsphere food preservative according to claim 4, characterized in that, The tannic acid loading rate was 3%.

6. The preparation method of the Poria cocos polysaccharide-based porous microsphere food preservative according to claim 4, characterized in that, Includes the following steps: S1. Weigh out Poria cocos polysaccharide and polyvinyl alcohol in a mass ratio of 1:1 to 3, and dissolve Poria cocos polysaccharide and polyvinyl alcohol in dimethyl sulfoxide to obtain solution 1 and solution 2. S2. After mixing solution 1 and solution 2 evenly, add the pore-forming agent, heat to dissolve, and obtain a mixed solution; S3. The mixed solution was dropped into pure water to solidify and purify, resulting in poria cocos polysaccharide-based porous microspheres. S4. Soak the poria polysaccharide-based porous microspheres in tannic acid solution for 24-48 hours, and then freeze-dry them to obtain the poria polysaccharide-based porous microsphere preservative.

7. The method for preparing a poria cocos polysaccharide-based porous microsphere food preservative according to claim 6, characterized in that, In S2, the amount of pore-forming agent added is 0.25 times the total mass of Poria cocos polysaccharide and polyvinyl alcohol, and the heating and dissolution temperature is 100-110℃.

8. The method for preparing a poria cocos polysaccharide-based porous microsphere food preservative according to claim 7, characterized in that, In S2, the pore-forming agent is sodium chloride.

9. The method for preparing a poria cocos polysaccharide-based porous microsphere food preservative according to claim 6, characterized in that, In S3, the curing temperature is 40℃, the time is 30-60 min, and the purification time is 48 h.

10. The method for preparing a poria cocos polysaccharide-based porous microsphere food preservative according to claim 6, characterized in that, In S4, the concentration of tannic acid solution is 10%, the freeze-drying time is 24-36 hours, and the freeze-drying temperature is -35℃ to -20℃.

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