An acetylated soluble soybean polysaccharide-linalool nanoparticle, and a preparation method and application thereof

Abstract

An acetylated soluble soybean polysaccharide-linalool nanoparticle, a preparation method and application thereof, the acetylated soluble soybean polysaccharide-linalool nanoparticle is acetylated soluble soybean polysaccharide as a carrier, and loaded with linalool. The acetylated soluble soybean polysaccharide is used as the carrier of linalool, and has good combination stability; efficient loading and stable wrapping of linalool are realized; the core-shell structure formed by the acetylated soluble soybean polysaccharide can block oxygen and light, inhibit oxidation and volatilization of linalool, delay the release rate, realize long-acting slow release of linalool, and meet the long-term action requirement.

Description

Technical Field

[0001] This invention relates to a sustained-release linalool, its preparation method and application, specifically to an acetylated soluble soybean polysaccharide-linalool, its preparation method and application. Background Technology

[0002] Linalool is a monoterpene alcohol mainly found in plants such as coriander, Sichuan pepper, and prickly ash. It is a volatile secondary plant metabolite and has attracted much attention not only for its excellent gas-phase bactericidal properties but also for its antioxidant, anti-inflammatory, and anti-tumor effects. Numerous studies have confirmed that linalool can effectively inhibit foodborne pathogens by reducing bacterial intracellular membrane potential, causing leakage of intracellular macromolecules (DNA, RNA, and proteins), inhibiting energy synthesis, leading to metabolic dysfunction, and inhibiting the activity of key enzymes, thereby causing abnormal bacterial cell structure. Linalool exhibits strong inhibitory effects against a variety of pathogens: it shows good antibacterial activity against Escherichia coli, Bacillus cereus, Staphylococcus aureus, white cocci, and Aspergillus; linalool can effectively inhibit the activity of Pseudomonas aeruginosa and Pseudomonas berries by disrupting bacterial structure. Although linalool exhibits considerable antibacterial activity and is classified as a safe substance, its hydrophobicity, volatility, and susceptibility to oxidation limit its application.

[0003] To address the problems associated with free linalool, previous studies have attempted to optimize it through encapsulation and loading, but the results have generally been unsatisfactory. For example, the technology of loading linalool onto single polysaccharide-based nanocarriers uses a single natural polysaccharide (such as chitosan, β-glucan, or gum arabic) as a nanocarrier, encapsulating linalool through physical adsorption and hydrogen bonding. Utilizing the biocompatibility and film-forming properties of polysaccharides, this approach aims to improve the stability and water dispersibility of linalool. The core principle is to encapsulate linalool using the spatial conformation of the polysaccharide molecular chains, reducing the impact of the external environment. However, this method generally results in low encapsulation rates, loose encapsulation structures that cannot effectively block oxygen, light, and other factors, and are unsuitable for long-term storage; the sustained-release effect is also poor: the encapsulated structure easily disintegrates rapidly in an aqueous environment, with linalool typically released completely within 60 hours, making it difficult to achieve a long-lasting effect.

[0004] To further improve upon the single polysaccharide nanocarrier technology for loading linalool, a binary / ternary biopolymer nanocarrier technology for loading linalool has been developed. This type of technology uses two or three biopolymers as carriers (such as gum arabic / maltodextrin, chitosan / alginate, casein phosphopeptide / chitosan / gallic acid), and enhances the encapsulation effect of linalool through the synergistic effects between different polymers (such as the superposition of ionic bonds, hydrogen bonds, and hydrophobic interactions). However, this technology has a complex preparation process: it requires precise control of the ratio, compounding order, and reaction conditions of multiple polymers, making the steps cumbersome and difficult to scale up; the procurement of multi-component raw materials and the process control during compounding increase production costs, making it unsuitable for low-cost applications; the binary / ternary system relies on the synergistic effects between multiple components, and the significant differences in the structural characteristics (such as charge properties and hydrophilicity / hydrophobicity) of each component lead to compatibility issues, and some compounded systems are prone to stratification and aggregation, affecting product stability; these problems limit its industrial application. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an acetylated soluble soybean polysaccharide-linalool nanoparticle with simple preparation method, high encapsulation rate and good sustained release effect, as well as its preparation method and application.

[0006] The technical solution adopted by this invention to solve its technical problem is as follows: an acetylated soluble soybean polysaccharide-linalool nanoparticle, using acetylated soluble soybean polysaccharide as a carrier and loading linalool; the loading amount is 2%~20%;

[0007] The degree of acetylation of the acetylated soluble soybean polysaccharide is 0.6~1.2;

[0008] The molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0009] Preferably, the preparation method of the acetylated soluble soybean polysaccharide is as follows: soluble soybean polysaccharide is prepared into a solution and reacted with acetic anhydride; the temperature during the reaction is 35℃~45℃, and the pH value is controlled at 8.0~10.0; after the target product is generated, the reaction is terminated, and after removing impurities, acetylated soluble soybean polysaccharide is obtained.

[0010] Preferably, the concentration of the solution prepared from soluble soybean polysaccharides is 0.01 g / mL to 0.04 g / mL.

[0011] Preferably, the ratio of soluble soybean polysaccharide to acetic anhydride is 1g:12~18mL.

[0012] Preferably, the reaction is terminated by adjusting the pH value to below 7.0.

[0013] Preferably, the reaction time between soluble soybean polysaccharide and acetic anhydride is 1 h to 2 h.

[0014] Based on the same inventive concept, this invention also provides a method for preparing the acetylated soluble soybean polysaccharide-linalool nanoparticles, comprising:

[0015] Acetylated soluble soybean polysaccharide was dispersed in PBS buffer as a carrier solution, linalool solution was added and stirred; centrifuged at 3000rpm~5000rpm and 0℃~4℃ for 10~20 minutes, unloaded small molecules were removed by ultrafiltration, the retentate solution was collected and freeze-dried to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0016] Preferably, the pH value of the PBS buffer is 7.0 to 7.4.

[0017] Preferably, the concentration of the PBS buffer is 5mM to 20mM.

[0018] Preferably, the concentration of acetylated soluble soybean polysaccharide in the carrier solution is 1.5 mg / mL to 2.5 mg / mL.

[0019] Preferably, the concentration of the linalool solution is 0.8 mg / mL to 1.6 mg / mL.

[0020] Preferably, the process of adding linalool solution and stirring is carried out under light-protected conditions.

[0021] Based on the same inventive concept, the present invention also provides the application of one or more of the acetylated soluble soybean polysaccharide-linalool nanoparticles or the acetylated soluble soybean polysaccharide-linalool nanoparticles prepared by the method thereof in the fields of preservation, antibacterial, pharmaceutical, and bioactive substance delivery.

[0022] The present invention has the following beneficial effects: Using acetylated soluble soybean polysaccharide as a carrier to load linalool, the acetylated soluble soybean polysaccharide, as a carrier of linalool, can spontaneously form a core-shell structure of "hydrophobic core-hydrophilic shell." This structure efficiently binds linalool through hydrophobic interactions while enhancing water dispersibility and biocompatibility through the hydrophilic shell. Simultaneously, the retained hydroxyl groups can form hydrogen bonds with linalool, strengthening binding stability. This achieves efficient loading and stable encapsulation of linalool. The core-shell structure can block oxygen and light, inhibiting the oxidation and volatilization of linalool. Linalool, encapsulated within the hydrophobic core, needs to gradually diffuse through the hydrophilic shell and polysaccharide molecular chain network, thus slowing the release rate and achieving long-term sustained release, meeting the requirements for long-term action.

[0023] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0024] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0025] Figure 1 This is a release curve of acetylated soluble soybean polysaccharide-linalool nanoparticles and free linalool in Example 1 of the present invention.

[0026] Figure 2 These are DPPH free radical scavenging activity curves of different concentrations of acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 2 of the present invention and the control group after storage for a period of time. Figure 2 A: On day 0; Figure 2 B: On the 10th day; Figure 2 C: On the 20th day; Figure 2 D: On the 30th day;

[0027] Figure 3 These are ABTS free radical scavenging activity curves of different concentrations of acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 2 of the present invention and the control group after storage for a period of time. Figure 3 A: On day 0; Figure 3 B: On the 10th day; Figure 3 C: On the 20th day; Figure 3 D: On the 30th day;

[0028] Figure 4 The cytotoxicity of different concentrations of acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 2 of this invention and the control group ( Figure 4 A) and anti-tumor cell proliferation activity ( Figure 4 B);

[0029] Figure 5 The total bacterial count of acetylated soluble soybean polysaccharide-linalool nanoparticles in Example 3 of this invention and the control group when used for the preservation of fresh chicken is ( Figure 5 A) and TVB-N value ( Figure 5 B) Curve graph showing the change over time;

[0030] Figure 6 The total bacterial count of acetylated soluble soybean polysaccharide-linalool nanoparticles in Example 4 of this invention and the control group when used for preserving dried bean curd is ( Figure 6 A) and TBARS value ( Figure 6 B) Curve graph showing the change over time. Detailed Implementation

[0031] To make the objectives, solutions, and beneficial technologies of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be noted that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0032] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0033] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, "multiple" in "one or more" means two or more, and "more than" in "one or more" means two or more.

[0034] The soluble soybean polysaccharides (SSPS, >98% purity) in the following cases were purchased from Tianjin Buer Protein Co., Ltd.; the molecular weight is 5kDa~50kDa.

[0035] An embodiment of the present invention provides acetylated soluble soybean polysaccharide-linalool nanoparticles, using acetylated soluble soybean polysaccharide as a carrier and loading linalool; the loading amount is 2%~20%;

[0036] The degree of acetylation of the acetylated soluble soybean polysaccharide is 0.6~1.2;

[0037] The molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0038] In some embodiments of the present invention, the linalool loading of the acetylated soluble soybean polysaccharide-linalool nanoparticles is 12% to 15%.

[0039] In some embodiments of the present invention, the degree of acetylation of the acetylated soluble soybean polysaccharide is 0.8 to 1.2.

[0040] Unmodified soluble soybean polysaccharides exhibit low encapsulation efficiency and low loading capacity for linalool, limiting their application scenarios. Due to their low loading capacity and poor stability, they are only suitable for short-term applications where the retention of active substances is less critical, failing to meet the needs of long-term food preservation and long-term delivery of bioactive substances. However, research has shown that acetylation modification of soluble soybean polysaccharides significantly improves their encapsulation effect. As a carrier for linalool, they can spontaneously form a core-shell structure of "hydrophobic core-hydrophilic shell," efficiently binding linalool through hydrophobic interactions while enhancing water dispersibility and biocompatibility through the hydrophilic shell. Simultaneously, the retained hydroxyl groups can form hydrogen bonds with linalool, strengthening binding stability. This achieves efficient loading and stable encapsulation of linalool. The core-shell structure blocks oxygen and light, inhibiting the oxidation and volatilization of linalool. Linalool, encapsulated within the hydrophobic core, needs to gradually diffuse through the hydrophilic shell and polysaccharide molecular chain network, slowing the release rate and achieving long-term sustained release, meeting the requirements for long-term action.

[0041] This invention achieves efficient loading through acetylation modification of a single soluble soybean polysaccharide, without the need for multi-component compounding. The method is simple, the raw material cost is low, and it is easy to scale up production, overcoming the shortcomings of complex and costly binary / ternary carrier processes.

[0042] Further experiments revealed that the acetylated soluble soybean polysaccharide-linalool nanoparticles of this invention have multiple functions, including highly efficient antibacterial (inhibiting Escherichia coli, Listeria monocytogenes, Aspergillus flavus, etc.), antioxidant, and antitumor (inhibiting HepG2 cell proliferation) activities, and have no obvious toxicity to normal cells, making them suitable for a wide range of applications.

[0043] In an embodiment of the present invention, the preparation method of the acetylated soluble soybean polysaccharide is as follows: soluble soybean polysaccharide is prepared into a solution and reacted with acetic anhydride; the temperature during the reaction is 35℃~45℃, and the pH value is controlled at 8.0~10.0; after the target product is generated, the reaction is terminated, and after removing impurities, acetylated soluble soybean polysaccharide is obtained.

[0044] This method for preparing acetylated soluble soybean polysaccharides uses acetic anhydride as the acetylation reagent to introduce acetyl groups by substituting the hydroxyl groups of the polysaccharide. The method is simple and easy to operate. The reaction needs to be carried out under alkaline conditions to activate the hydroxyl groups of the polysaccharide and ensure smooth acetylation. Common pH adjusters such as sodium hydroxide (alkaline) and hydrochloric acid (acidic) can be used to adjust the pH, as long as no other side reactions occur. The reaction has a suitable rate at a temperature of 35℃~45℃.

[0045] In embodiments of the present invention, the concentration of the solution prepared from soluble soybean polysaccharides is 0.01 g / mL to 0.04 g / mL.

[0046] In embodiments of the present invention, the ratio of soluble soybean polysaccharide to acetic anhydride is 1 g: 12-18 mL. Within this range, the resulting acetylated soluble soybean polysaccharide exhibits a suitable degree of acetylation.

[0047] In embodiments of the invention, the reaction is terminated by adjusting the pH value to below 7.0. Adjusting the pH value to neutral or acidic will terminate the reaction. Conventional pH adjusters, such as sodium hydroxide (alkaline) and hydrochloric acid (acidic), can be used to adjust the pH value, as long as they do not cause other side reactions.

[0048] In an embodiment of the present invention, the reaction time of soluble soybean polysaccharide with acetic anhydride is 1h to 2h.

[0049] In embodiments of the present invention, impurity removal can be carried out by various methods such as dialysis, filtration, precipitation, washing, and drying, and finally acetylated soluble soybean polysaccharides with the required molecular weight are separated.

[0050] The preparation method of acetylated soluble soybean polysaccharide-linalool nanoparticles according to the embodiments of the present invention includes:

[0051] Acetylated soluble soybean polysaccharides were dispersed in PBS buffer as a carrier solution, and linalool solution was added and stirred. The mixture was centrifuged at 3000-5000 rpm and 0-4℃ for 10-20 minutes. Unloaded small molecules were removed by ultrafiltration, and the retentate was collected and freeze-dried to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder. Stirring and centrifugation allow linalool to be loaded onto acetylated soluble soybean polysaccharides to form nanoparticles. Ultrafiltration allows small molecules to pass through, while larger nanoparticles are retained in the retentate. Ultrafiltration can be performed using an ultrafiltration tube.

[0052] In an embodiment of the present invention, the pH value of the PBS buffer is 7.0 to 7.4.

[0053] In embodiments of the present invention, the concentration of the PBS buffer is 5mM to 20mM.

[0054] In an embodiment of the present invention, the concentration of acetylated soluble soybean polysaccharide in the carrier solution is 1.5 mg / mL to 2.5 mg / mL.

[0055] In embodiments of the present invention, the concentration of the linalool solution is 0.8 mg / mL to 1.6 mg / mL. Linalool solutions within this concentration range exhibit excellent encapsulation efficiency.

[0056] In embodiments of the present invention, the process of adding linalool solution and stirring is carried out under light-protected conditions. Light-protected conditions reduce the oxidation of linalool.

[0057] The application of one or more of the acetylated soluble soybean polysaccharide-linalool nanoparticles or the acetylated soluble soybean polysaccharide-linalool nanoparticles prepared by the method of the present invention in the fields of preservation, antibacterial, pharmaceutical, and bioactive substance delivery.

[0058] Acetylated soluble soybean polysaccharide-linalool nanoparticles possess antioxidant and antibacterial activities, exhibit low toxicity to normal cells while inhibiting tumor cell proliferation, and have a food preservation effect, making them suitable for a wide range of applications.

[0059] Example

[0060] The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight. Unless otherwise stated, all reagents used in the examples are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, all instruments used in the examples are available commercially.

[0061] Example 1

[0062] In this embodiment, the acetylated soluble soybean polysaccharide-linalool nanoparticles use acetylated soluble soybean polysaccharide as a carrier and load linalool; wherein the degree of acetylation of the acetylated soluble soybean polysaccharide is 0.8~1.2; and the molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0063] The preparation method of acetylated soluble soybean polysaccharides is as follows:

[0064] (1) Weigh 1.00g of soluble soybean polysaccharide and disperse it in 50mL of deionized water; stir with a magnetic stirrer at 500rpm for 1 hour, and then sonicate for 15 minutes to obtain a uniform soybean polysaccharide solution to avoid raw material clumping and affecting subsequent reactions.

[0065] (2) Place the above homogeneous solution in a constant temperature water bath, heat it to 40°C and keep it constant, and stir continuously; slowly adjust the pH of the solution to 8.0~9.0 with 0.1mol / L sodium hydroxide solution. This alkaline environment can promote the activation of hydroxyl groups in soybean polysaccharide molecules and create conditions for the incorporation of acetyl groups.

[0066] (3) Keep the system temperature at 40℃ and the stirring speed at 500rpm, and add acetic anhydride and 0.1mol / L sodium hydroxide solution dropwise alternately. Acetic anhydride is added at a ratio of 1:15 (g:mL) (i.e., 15mL of acetic anhydride is added for every 1.00g of raw material). During the dropwise addition, strictly control the pH of the system to be stable at 8.0~10.0 to prevent the reaction from failing due to excessively high or low pH. After the dropwise addition is completed, continue to keep the temperature and stir for 1.5 hours to complete the acetylation modification.

[0067] (4) After the target product is generated, the pH of the system is slowly adjusted to 7.0 with 0.1 mol / L hydrochloric acid solution to terminate the acetylation reaction; then the reaction solution is poured into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with distilled water for 48 hours to remove unreacted small molecule impurities in the system.

[0068] (5) Take out the dialysis solution, add 4 times the volume of anhydrous ethanol, and place it in an environment of 4℃ for alcohol precipitation overnight to allow the acetylated soybean polysaccharide to be fully separated; then centrifuge at 8000g speed for 10 minutes at 4℃ and collect the bottom precipitate; wash the precipitate repeatedly with anhydrous ethanol 3 times to remove residual acetic anhydride and other impurities.

[0069] (6) The washed precipitate was redispersed in a small amount of deionized water and dialyzed again for 12 hours for further purification. Finally, the dialyzed solution was freeze-dried to obtain acetylated soybean polysaccharide powder.

[0070] A method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles includes:

[0071] 20 mg of acetylated soluble soybean polysaccharide was dissolved in 10 mL of PBS (10 mM, pH 7.0) and stirred at 700 rpm for 1 hour to obtain an acetylated soluble soybean polysaccharide solution. Different concentrations of linalool solution (0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL) were added to the prepared acetylated soluble soybean polysaccharide solution and stirred for 2 hours under light-protected conditions. After centrifugation at 4000 rpm and 4℃ for 15 minutes, the solution was separated and purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The solution in the ultrafiltration tube was collected and lyophilized to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0072] Example 2

[0073] In this embodiment, the acetylated soluble soybean polysaccharide-linalool nanoparticles use acetylated soluble soybean polysaccharide as a carrier and load linalool; wherein the degree of acetylation of the acetylated soluble soybean polysaccharide is 0.8~1.2; and the molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0074] The preparation method of acetylated soluble soybean polysaccharides is as follows:

[0075] (1) Weigh 1.00g of soluble soybean polysaccharide and disperse it in 60mL of deionized water; stir with a magnetic stirrer at 600rpm for 1.5 hours, and then sonicate for 20 minutes to obtain a uniform soybean polysaccharide solution.

[0076] (2) Place the above homogeneous solution in a constant temperature water bath, heat it to 37°C and keep it constant, and stir continuously; slowly adjust the pH of the solution to 8.0~9.0 with 0.1mol / L sodium hydroxide solution;

[0077] (3) Keep the system temperature at 37℃ and the stirring speed at 600rpm, and add acetic anhydride and 0.1mol / L sodium hydroxide solution dropwise to the solution alternately; the acetic anhydride is added at a ratio of 1:12 (g:mL) and the pH of the system is strictly controlled to be stable at 8.0~10.0 during the dropwise addition; after the dropwise addition is completed, continue to keep the temperature and stir the reaction for 2 hours.

[0078] (4) After the target product is generated, the pH of the system is slowly adjusted to 7.0 with 0.1 mol / L hydrochloric acid solution; then the reaction solution is poured into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with distilled water for 48 hours.

[0079] (5) Take out the dialysis solution, add 4 times the volume of anhydrous ethanol, and place it in an environment of 4℃ for alcohol precipitation overnight; then centrifuge at 8000g for 10 minutes at 4℃ and collect the bottom precipitate; wash the precipitate repeatedly with anhydrous ethanol 3 times.

[0080] (6) The washed precipitate was redispersed in a small amount of deionized water and dialyzed again for 12 hours for further purification. Finally, the dialyzed solution was freeze-dried to obtain acetylated soybean polysaccharide powder.

[0081] A method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles includes:

[0082] 20 mg of acetylated soluble soybean polysaccharide was dissolved in 10 mL of PBS (15 mM, pH 7.0) and stirred at 700 rpm for 1 hour to obtain an acetylated soluble soybean polysaccharide solution. Linalool solution (1.2 mg / mL) was added to the prepared acetylated soluble soybean polysaccharide solution and stirred for 1 hour in the dark. After centrifugation at 3000 rpm and 2 °C for 15 minutes, the solution was separated and purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The solution in the ultrafiltration tube was collected and lyophilized to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0083] Example 3

[0084] In this embodiment, the acetylated soluble soybean polysaccharide-linalool nanoparticles use acetylated soluble soybean polysaccharide as a carrier and load linalool; wherein the degree of acetylation of the acetylated soluble soybean polysaccharide is 0.6~1.2; and the molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0085] The preparation method of acetylated soluble soybean polysaccharides is as follows:

[0086] (1) Weigh 1.00g of soluble soybean polysaccharide and disperse it in 50mL of deionized water; stir with a magnetic stirrer at 500rpm for 2 hours, and then sonicate for 15 minutes to obtain a uniform soybean polysaccharide solution.

[0087] (2) Place the above homogeneous solution in a constant temperature water bath, heat it to 43°C and keep it constant, and stir continuously; slowly adjust the pH of the solution to 8.0~9.0 with 0.1mol / L sodium hydroxide solution;

[0088] (3) Keep the system temperature at 43℃ and the stirring speed at 500rpm, and add acetic anhydride and 0.1mol / L sodium hydroxide solution dropwise alternately. Acetic anhydride is added at a ratio of 1:18 (g:mL) and the pH of the system is strictly controlled to be stable at 8.0~10.0 during the dropwise addition. After the dropwise addition is completed, continue to keep the temperature and stir the reaction for 1.5 hours.

[0089] (4) After the target product is generated, the pH of the system is slowly adjusted to 7.0 with 0.1 mol / L hydrochloric acid solution; then the reaction solution is poured into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with distilled water for 48 hours.

[0090] (5) Take out the dialysis solution, add 4 times the volume of anhydrous ethanol, and place it in an environment of 4℃ for alcohol precipitation overnight; then centrifuge at 8000g for 10 minutes at 4℃ and collect the bottom precipitate; wash the precipitate repeatedly with anhydrous ethanol 3 times.

[0091] (6) The washed precipitate was redispersed in a small amount of deionized water and dialyzed again for 12 hours for further purification. Finally, the dialyzed solution was freeze-dried to obtain acetylated soybean polysaccharide powder.

[0092] A method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles includes:

[0093] 15 mg of acetylated soluble soybean polysaccharide was dissolved in 10 mL of PBS (10 mM, pH 7.2) and stirred at 700 rpm for 1 hour to obtain an acetylated soluble soybean polysaccharide solution. Linalool solution (1.2 mg / mL) was added to the prepared acetylated soluble soybean polysaccharide solution and stirred for 1 hour in the dark. After centrifugation at 4500 rpm and 4℃ for 15 minutes, the solution was separated and purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The solution in the ultrafiltration tube was collected and lyophilized to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0094] Example 4

[0095] In this embodiment, the acetylated soluble soybean polysaccharide-linalool nanoparticles use acetylated soluble soybean polysaccharide as a carrier and load linalool; wherein the degree of acetylation of the acetylated soluble soybean polysaccharide is 0.8~1.2; and the molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

[0096] The preparation method of acetylated soluble soybean polysaccharides is as follows:

[0097] (1) Weigh 1.00g of soluble soybean polysaccharide and disperse it in 40mL of deionized water; stir with a magnetic stirrer at 600rpm for 1 hour, and then sonicate for 10 minutes to obtain a uniform soybean polysaccharide solution.

[0098] (2) Place the above homogeneous solution in a constant temperature water bath, heat it to 40°C and keep it constant, and stir continuously; slowly adjust the pH of the solution to 8.0~9.0 with 0.1mol / L sodium hydroxide solution;

[0099] (3) Keep the system temperature at 40℃ and the stirring speed at 600rpm, and add acetic anhydride and 0.1mol / L sodium hydroxide solution dropwise to the solution alternately; the acetic anhydride is added at a ratio of 1:16 (g:mL) and the pH of the system is strictly controlled to be stable at 8.0~10.0 during the dropwise addition; after the dropwise addition is completed, continue to keep the temperature and stir the reaction for 1 hour.

[0100] (4) After the target product is generated, the pH of the system is slowly adjusted to 7.0 with 0.1 mol / L hydrochloric acid solution; then the reaction solution is poured into a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with distilled water for 48 hours.

[0101] (5) Take out the dialysis solution, add 4 times the volume of anhydrous ethanol, and place it in an environment of 4℃ for alcohol precipitation overnight; then centrifuge at 8000g for 10 minutes at 4℃ and collect the bottom precipitate; wash the precipitate repeatedly with anhydrous ethanol 3 times.

[0102] (6) The washed precipitate was redispersed in a small amount of deionized water and dialyzed again for 12 hours for further purification. Finally, the dialyzed solution was freeze-dried to obtain acetylated soybean polysaccharide powder.

[0103] A method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles includes:

[0104] 25 mg of acetylated soluble soybean polysaccharide was dissolved in 10 mL of PBS (8 mM, pH 7.0) and stirred at 700 rpm for 1 hour to obtain an acetylated soluble soybean polysaccharide solution. Linalool solution (1.2 mg / mL) was added to the prepared acetylated soluble soybean polysaccharide solution and stirred for 1 hour in the dark. After centrifugation at 4000 rpm and 4℃ for 10 minutes, the solution was separated and purified using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The solution in the ultrafiltration tube was collected and lyophilized to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0105] Comparative Example

[0106] In this comparative example, linalool was directly loaded onto soluble soybean polysaccharides to obtain soluble soybean polysaccharide-linalool nanoparticles. The preparation method was basically the same as in Example 1, except that acetylated soluble soybean polysaccharides were replaced with soluble soybean polysaccharides. The specific method is as follows:

[0107] (1) Take commercially available soluble soybean polysaccharides, select the portion with a molecular weight of 3.5kDa~30kDa by dialysis and freeze-dry to obtain soluble soybean polysaccharide freeze-dried products;

[0108] (2) Take 20 mg of soluble soybean polysaccharide freeze-dried product and dissolve it in 10 mL of PBS (10 mM, pH 7.0). Stir at 700 rpm for 1 hour to obtain acetylated soluble soybean polysaccharide solution. Add linalool solutions of different concentrations (0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL) to the prepared acetylated soluble soybean polysaccharide solution and stir for 2 hours under light-protected conditions. Centrifuge at 4000 rpm and 4℃ for 15 minutes, and then separate and purify using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Collect the solution in the ultrafiltration tube and freeze-dry it to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

[0109] Detection and Analysis

[0110] (1) Evaluation of linalool loading rate and release effect

[0111] (1.1) Determination of loading capacity and encapsulation efficiency

[0112] The nanoparticles from Example 1 and the comparative example were centrifuged at 4000 rpm for 15 minutes. The supernatant was then placed in an ultrafiltration tube with a molecular cutoff of 3 kDa and centrifuged at 4000 rpm for 15 minutes. After appropriate dilution, the absorbance was measured at 278 nm using a UV-Vis spectrophotometer to plot the linalool standard curve (Y = 0.0281x + 0.0432, R0). 2 =0.9996), the linalool encapsulation efficiency and loading in the nanoparticles were calculated using the following formulas: Encapsulation efficiency (%) = (Total linalool - Free linalool content) / Total linalool; Loading (%) = (Total linalool - Free linalool content) / Total nanoparticles. The results are shown in Table 1.

[0113] Table 1 Encapsulation efficiency and loading of nanoparticles with different linalool concentrations

[0114]

[0115] Table 1 shows that the encapsulation efficiency (77.97%~87.53%) and loading capacity (3.90%~13.34%) of acetylated soluble soybean polysaccharides for linalool are significantly higher than those for soluble soybean polysaccharides. When linalool is loaded onto acetylated soluble soybean polysaccharides, the loading capacity increases gradually with increasing linalool concentration, exhibiting a relatively ideal encapsulation efficiency over a wide concentration range. This invention's acetylation modification introduces acetyl groups onto the soybean polysaccharide molecular chain, achieving efficient loading of linalool by altering the molecular hydrophilicity-hydrophobicity balance, spatial conformation, and interaction type.

[0116] (1.2) Assessment of linalool release rate

[0117] Release profiles of bioactive substances / drugs on nanocarriers are crucial for evaluating the suitability of the encapsulation matrix as a controlled-release carrier. The linalool release assay method is as follows:

[0118] Free linalool (3 mg / mL) and acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 1 (22.85 mg / mL, equivalent to 3 mg / mL linalool) were dissolved in PBS (pH 7.0). 15 mL of this solution was injected into a dialysis bag (molecular weight cutoff 10 kDa). The dialysis bag was placed in a flask containing 165 mL of PBS (pH 7.0) to achieve a final linalool concentration of 0.4 mg / mL. The flask was sealed with plastic film and placed in an air bath shaker at 25°C and 140 rpm. At predetermined time intervals, 3 mL of PBS was removed and fresh buffer was added to maintain a constant total volume. The concentration of free linalool generated from the nanoparticles in the buffer was measured.

[0119] Figure 1Release curves of free linalool and acetylated soluble soybean polysaccharide-linalool nanoparticles are presented. The linalool released from the acetylated soluble soybean polysaccharide-linalool nanoparticles exhibits a two-stage characteristic: a rapid release stage and a slow, sustained release stage. In the first 100 hours, approximately 39.14% of the released linalool is adsorbed onto the surface of the acetylated soluble soybean polysaccharide. Over the next 400 hours, linalool within the nanoparticles is gradually released. This can be attributed to the longer time required for linalool within the nanoparticles to detach from the carrier, indicating that the sustained-release characteristics of the acetylated soluble soybean polysaccharide-linalool nanoparticles endow them with long-lasting antioxidant and antibacterial activity. Free linalool, however, is almost completely released within the first 60 hours and is susceptible to environmental stress, leading to a decrease or even loss of biological activity.

[0120] (2) Evaluation of antioxidant, cytotoxic and antibacterial activities

[0121] The following tests were conducted using the acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 2, and the acetylated soluble soybean polysaccharide and linalool raw materials and soluble soybean polysaccharide raw materials obtained in the preparation process of Example 2 were used as control groups when necessary.

[0122] (2.1) Antioxidant properties

[0123] The antioxidant capacity of acetylated soluble soybean polysaccharide-linalool nanoparticles relative to free linalool was evaluated using DPPH and ABTS free radical scavenging assays. The test methods are as follows:

[0124] DPPH free radical scavenging activity assay: Different concentrations of acetylated soluble soybean polysaccharide, linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticle solutions were prepared as test samples and placed at room temperature (25℃). Then, on the day of sample preparation and on days 10, 20, and 30 thereafter, 0.5 mL of each sample was taken for testing, and 2.5 mL of 0.1 mM DPPH ethanol solution was added. After thorough stirring, the mixture was placed in the dark at 25℃ for 30 minutes, and the absorbance value was finally measured at a wavelength of 517 nm, which is As. Simultaneously, anhydrous ethanol was used instead of the DPPH solution, and the absorbance value was measured, which is Ab. Anhydrous ethanol was used instead of the sample solution, and the absorbance value was measured, which is Ac.

[0125] The DPPH radical scavenging rate is calculated using the following formula: DPPH radical scavenging rate (%) = [1-(As-Ab) / Ac]×100.

[0126] ABTS radical scavenging activity assay: Different concentrations of acetylated soluble soybean polysaccharide, linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticle solutions were prepared as test samples and stored at room temperature (25℃). A 7mM ABTS aqueous solution and a 2.5mM potassium persulfate aqueous solution were mixed in a brown flask and reacted at 25℃ in the dark for 16 hours. The ABTS+ solution was diluted with 10mM PBS (pH 7.4) to a wavelength absorbance of 0.70 ± 0.02 at 734 nm to obtain the ABTS+ radical working solution, which was stored at room temperature (25℃). On the day of sample preparation and on days 10, 20, and 30 thereafter, 0.8 mL of ABTS+ was added to 200 μL of the sample solution. The ABTS+ free radical working solution was reacted at 25°C for 6 minutes; then 200 μL of the sample was transferred to a 96-well plate; the absorbance value was measured at 734 nm using a microplate reader, which is As; at the same time, the absorbance value was measured by replacing the sample solution with distilled water, which is Ab.

[0127] The ABTS radical scavenging rate is calculated using the following formula: ABTS radical scavenging rate (%) = [(Ab-As) / Ab]×100.

[0128] Figure 2 and Figure 3 The results of the free radical scavenging tests of DPPH and ABTS were summarized separately.

[0129] like Figure 2 As shown, on day 0 ( Figure 2 A) Linalool exhibited slightly better DPPH radical scavenging ability than acetylated soluble soybean polysaccharide-linalool nanoparticles, while acetylated soluble soybean polysaccharide showed virtually no antioxidant activity. From day 10 onwards, the DPPH radical scavenging ability of acetylated soluble soybean polysaccharide-linalool nanoparticles gradually increased compared to free linalool. At all concentrations (10, 20, 30, 40, 50 μg / mL) on day 10, the DPPH radical scavenging ability of acetylated soluble soybean polysaccharide-linalool nanoparticles was significantly higher than that of the control group. Figure 2 B). By day 30, the DPPH radical scavenging rate of acetylated soluble soybean polysaccharide-linalool nanoparticles still reached 82.88%, while that of free linalool was only 29.76%. Figure 2 D). This indicates that acetylated soluble soybean polysaccharide-linalool nanoparticles have good protective and sustained-release effects on linalool. On day 0, due to the above two effects, the DPPH free radical scavenging ability of acetylated soluble soybean polysaccharide-linalool nanoparticles is lower than that of free linalool. However, with the extension of storage time, free linalool is easily oxidized by environmental oxygen due to the lack of protection from acetylated soluble soybean polysaccharide, resulting in the loss of some DPPH free radical scavenging ability.

[0130] like Figure 3 As shown, the ABTS radical scavenging activity and DPPH radical scavenging capacity of acetylated soluble soybean polysaccharide-linalool nanoparticles at different storage times were similar. Overall, the antioxidant activity of the samples increased with increasing sample concentration and decreased with prolonged storage time; the antioxidant activity of acetylated soluble soybean polysaccharide-linalool nanoparticles decreased only slightly with increasing storage time, while the antioxidant activity of free linalool decreased significantly with prolonged storage time. By day 30, acetylated soluble soybean polysaccharide-linalool nanoparticles still maintained 85.38% of the ABTS radical scavenging capacity, while the ABTS radical scavenging activity of free linalool was only 36.03% (…). Figure 3 D). These results indicate that acetylated soluble soybean polysaccharide-linalool nanoparticles possess long-lasting antioxidant capabilities.

[0131] (2.2) Cytotoxicity

[0132] Using free linalool, soluble soybean polysaccharide, and acetylated soluble soybean polysaccharide as control groups, the cytotoxicity of acetylated soluble soybean polysaccharide-linalool nanoparticles to normal cells was tested. The specific method was as follows:

[0133] Cell viability of normal cells (human umbilical vein endothelial cells, HUVECs) and tumor cell line (HepG2) under different sample interventions was detected, and cytotoxicity of each sample was evaluated. Each sample was tested at four concentrations (10, 25, 50, and 100 μg / mL), and cell viability was determined using the MTT assay.

[0134] Cytotoxicity to HUVEC cell lines: see [link to relevant documentation] Figure 4 A, cytotoxicity to HepG2, see [link / reference]. Figure 4 B.

[0135] like Figure 4 As shown in Figure A, soluble soybean polysaccharides, acetylated soluble soybean polysaccharides, and acetylated soluble soybean polysaccharide-linalool nanoparticles showed no significant toxicity to HUVEC cell lines, while free linalool exhibited a concentration-dependent inhibitory effect on HUVEC cells, but with low cytotoxicity. Even at a free linalool concentration of 100 mg / mL, the survival rate of the HUVEC cell line remained at 83.12%. Notably, when free linalool was combined with acetylated soluble soybean polysaccharides to form nanoparticles, its cytotoxicity was significantly reduced, indicating that acetylated soluble soybean polysaccharides can effectively encapsulate linalool and mitigate the cytotoxicity of free linalool.

[0136] Figure 4The in vitro antiproliferative activity results of each sample against HepG2 tumor cells showed that HepG2 exhibited a conventional dose-dependent antiproliferative effect, and the antiproliferative effect of acetylated soluble soybean polysaccharide-linalool nanoparticles on HepG2 tumor cells was better than that of free linalool, indicating that the nano-linalool of the present invention can more effectively inhibit the proliferation of tumor cells.

[0137] (2.3) Antibacterial activity

[0138] The antibacterial and antifungal activities of acetylated soluble soybean polysaccharide, free linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticles were determined by agar diffusion method. The specific methods are as follows: the antibacterial activity against Listeria monocytogenes, Escherichia coli, Aspergillus flavus, and Aspergillus parasiticus was determined; the sample solution concentration was 1%; the sample petri dishes were incubated at 30℃ for 24 h and 72 h, respectively; and the diameter of the inhibition zone was measured using vernier calipers.

[0139] The results are shown in Table 2. After 24 hours of incubation following inoculation, acetylated soluble soybean polysaccharides exhibited certain antibacterial activity, indicating that acetylated soluble soybean polysaccharides possess antibacterial properties. Free linalool showed significant antibacterial activity, while the antibacterial activity of acetylated soluble soybean polysaccharide-linalool nanoparticles was slightly higher than that of acetylated soluble soybean polysaccharides. After 72 hours of incubation following inoculation, the diameter of the inhibition zones of both acetylated soluble soybean polysaccharides and free linalool did not increase significantly, while the diameter of the inhibition zones of acetylated soluble soybean polysaccharide-linalool nanoparticles against *Escherichia coli*, *Listeria monocytogenes*, *Aspergillus flavus*, and *Aspergillus parasiticus* increased significantly, demonstrating the protective and controlled release effect of the nanoparticles on linalool.

[0140] Table 2. Diameter of inhibition zones (mm) for each sample against bacteria and fungi.

[0141]

[0142] (3) Application test of fresh chicken preservation

[0143] The following tests were conducted using the acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 3, and the acetylated soluble soybean polysaccharide and linalool raw materials obtained during the preparation process of Example 3 were used as a control group.

[0144] The following are methods for preserving chicken:

[0145] Fresh chicken meat was rinsed with 5% saline solution to remove mucus and some microorganisms. The fresh chicken was then cut into uniform pieces (approximately 2cm × 4cm × 2cm) and randomly divided into 10 groups. Each group was coated with either pure water (blank control group) or different preservatives (i.e., a solution of acetylated soluble soybean polysaccharide, linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticles, 0.5wt%). The meat pieces were then drained. The chicken pieces were placed in sterile bags and refrigerated at 4°C. On the day of sample preparation and on days 5, 10, 15, 20, 25, and 30 thereafter, three samples were randomly selected from each group for total bacterial count and total volatile basic nitrogen (TVB-N) testing.

[0146] Method for determining total bacterial count: The determination of total bacterial count shall be performed in accordance with GB 4789.2—2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count"; 10g of chicken sample shall be added to 90mL of 0.9% sodium chloride solution to prepare 10... -1 Diluent, shake well for 30 minutes; then prepare 10 by a tenfold incremental dilution method. -2 10 -3 and 10 -4 Diluents: Take 100 μL of each diluent and spread it on the surface of an agar plate. After incubation at 37°C for 1 day, count the colonies.

[0147] TVB-N value determination method: Take 10g of chicken sample and add 90mL of 6% perchloric acid, homogenize for 1 minute. Then filter the sample, and repeatedly distill the filtrate with 20% sodium hydroxide solution. Titrate the distillate with 0.01mol / L hydrochloric acid solution.

[0148] Total bacterial count is a key indicator for evaluating the storage quality of fresh chicken products. Figure 5A shows the changes in bacterial colony count of fresh chicken stored at 4°C for 0 to 30 days; as shown in the figure, the inhibition efficiency of the three samples is in the order of acetylated soluble soybean polysaccharide-linalool nanoparticles > free linalool > acetylated soluble soybean polysaccharide, and this effect is concentration-dependent. According to industry standards, products with a total bacterial count exceeding 6.0 lg CFU / g are not edible. On day 15 of storage, the bacterial count in the blank control group was 6.22 lg CFU / g. After coating with acetylated soluble soybean polysaccharides, the total bacterial count of the chicken remained above this level on days 20 (6.29 lg CFU / g), 25 (6.68 lg CFU / g), and 30 (7.29 lg CFU / g). When coated with free linalool, it showed better antibacterial effect, with the total bacterial count of the chicken exceeding 6.37 lg CFU / g on day 30 of storage. Notably, the group treated with acetylated soluble soybean polysaccharide-linalool nanoparticle coating maintained a total bacterial count within the range of 3.32~5.52 lg CFU / g after 30 days of storage, exhibiting the best preservation effect and extending the shelf life by more than 15 days compared to the blank control group.

[0149] TVB-N is often used as one of the main indicators for assessing chicken freshness. It refers to important nitrogenous substances such as amines and ammonia produced after protein is broken down by endogenous enzymes and microorganisms. Figure 5 As shown in B, the TVB-N value of the blank control group was significantly higher than that of the treatment group, reaching 27.68 mg / 100g after 20 days of storage and 54.23 mg / 100g after 30 days of storage. The TVB-N values ​​of the groups treated with acetylated soluble soybean polysaccharide, free linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticles remained in the ranges of 10.36 mg / 100g~27.88 mg / 100g, 9.82 mg / 100g~23.64 mg / 100g, and 10.21 mg / 100g~18.06 mg / 100g, respectively. According to national standards for TVB-N content in fresh meat, Grade 1 fresh meat <15mg / 100g, Grade 2 fresh meat <20mg / 100g, and spoiled meat >25mg / 100g. Chicken in the blank control group and the acetylated soluble soybean polysaccharide group were inedible after 20 and 25 days of storage, respectively, while the free linalool treatment group and the acetylated soluble soybean polysaccharide-linalool nanoparticle treatment group remained within the edible range. Notably, the acetylated soluble soybean polysaccharide-linalool nanoparticle treatment group had the lowest TVB-N value after 30 days of storage; this result shows a similar trend to the total bacterial count test results.

[0150] (4) Application test of dried bean curd preservation

[0151] The following tests were conducted using the acetylated soluble soybean polysaccharide-linalool nanoparticles from Example 4, and the acetylated soluble soybean polysaccharide and linalool raw materials obtained during the preparation process of Example 4 were used as a control group.

[0152] The following are methods for preserving dried tofu:

[0153] A 0.5 wt% solution of acetylated soluble soybean polysaccharide, linalool, and acetylated soluble soybean polysaccharide-linalool nanoparticles was used as a preservative, with a blank control group using sterile water instead of preservatives. Dried bean curd was soaked in the preservatives, air-dried at 20℃ for 5 min, and then individually packaged in vacuum bags, vacuum-sealed, and sterilized under pressure (80℃, 0.1 kPa, 30 min). The samples were then stored at 25℃. On the day of sample preparation and on days 5, 10, 15, 20, 25, and 30 thereafter, three samples were randomly selected from each group for total bacterial count and thiobarbituric acid reactive substances (TBARS) values.

[0154] Method for determining total bacterial count: The determination of total bacterial count shall be performed in accordance with GB 4789.2—2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count". Take 10g of dried bean curd sample from each group and add it to 90mL of 0.9% sodium chloride solution to prepare 10... -1 Diluent, shake well for 30 minutes; then prepare 10 by a tenfold incremental dilution method. -2 10 -3 and 10 -4 Diluents: Take 100 μL of each diluent and spread it on the surface of an agar plate. After incubation at 37°C for 1 day, count the colonies.

[0155] TBARS value determination method: Take 5g of dried bean curd sample and add 15mL of 7.5% trichloroacetic acid [containing 0.1% butylated hydroxyanisole (BHA) and 0.1% ethylenediaminetetraacetic acid (EDTA)], then homogenize using a high-speed shear dispersion device for 30 seconds. Filter the homogenate and collect the filtrate. Take 2.5mL of the filtrate and add 2.5mL of 0.02mol / L 2-thiobarbituric acid, heat in a boiling water bath for 40 minutes, and immediately cool in an ice bath. Then add 5mL of chloroform and mix thoroughly, centrifuge at 2℃ and 2000rpm for 10 minutes. Collect the supernatant and measure its absorbance at 532nm wavelength.

[0156] The changes in bacterial count of dried tofu stored at 25℃ for 30 days are as follows: Figure 6As shown in Figure A, the inhibition efficiency of the three samples was in the order of acetylated soluble soybean polysaccharide-linalool nanoparticles > free linalool > acetylated soluble soybean polysaccharide, and this effect was concentration-dependent. According to industry standards, products with a total bacterial count exceeding 5.0 lg CFU / g are not edible. On day 20 of storage, the bacterial count in the blank control group was 5.07 lg CFU / g. After coating with acetylated soluble soybean polysaccharides, the total bacterial count of dried soybeans remained above this level on day 25 (5.21 lg CFU / g) and day 30 (6.65 lg CFU / g). After 30 days of storage, the total bacterial count of the groups treated with free linalool and acetylated soluble soybean polysaccharide-linalool nanoparticle coatings was 4.86 lg CFU / g and 4.12 lg CFU / g, respectively. The acetylated soluble soybean polysaccharide-linalool nanoparticles showed better antibacterial effect and extended the shelf life by more than 10 days compared with the blank control group.

[0157] The TBARS value is an important indicator for judging the degree of lipid oxidation. When the TBARS value is greater than 2.0 mg / kg, the lipids are prone to oxidation and deterioration, producing an unpleasant odor. Figure 6 As shown in Figure B, the TBARS value of the blank group of dried bean curd was 2.12 mg / kg after 20 days, indicating that the dried bean curd had undergone significant oxidative spoilage at this time. The TBARS values ​​of the bean curd treated with acetylated soluble soybean polysaccharide and free linalool exceeded the threshold at 20 and 25 days, respectively, reaching 2.07 mg / kg and 2.08 mg / kg. The TBARS value of the group treated with acetylated soluble soybean polysaccharide-linalool nanoparticles was the lowest, with a value of 1.76 mg / kg after 30 days of storage. The results indicate that acetylated soluble soybean polysaccharide alone cannot effectively inhibit lipid oxidation; linalool has an inhibitory effect on lipid oxidation in the short term, but the long-term effect is poor; while acetylated soluble soybean polysaccharide-linalool nanoparticles can inhibit lipid oxidation in the long term.

[0158] This invention utilizes acetylated soluble soybean polysaccharides as a delivery carrier for linalool. Compared to free linalool, acetylated soluble soybean polysaccharide-based nanoparticles impart sustained antioxidant and antibacterial activity to linalool. This conclusion is verified through preservation experiments on chicken and dried tofu. The inhibitory effect of acetylated soluble soybean polysaccharide-linalool nanoparticles on total bacterial count is related to their sustained antibacterial activity; the inhibitory effect on lipid peroxidation (TBARS) is related to their sustained antioxidant activity; and the inhibition of nitrogenous substance (TVB-N) formation is related to their sustained antibacterial and antioxidant effects. The antibacterial and antioxidant capabilities of the nanoparticles prepared in this invention meet or exceed the levels of previous research. Based on this, the nanoparticles were also applied to preservation experiments on real foods (chicken and dried tofu), verifying their actual efficacy in food systems.

[0159] In summary, this invention significantly improves the loading capacity of soluble soybean polysaccharides for linalool through acetylation treatment. In the prepared acetylated soluble soybean polysaccharide-linalool nanoparticles, the protective and sustained-release effects of acetylated soluble soybean polysaccharides on linalool significantly enhance the long-term antibacterial and antioxidant capabilities of linalool. Through synergistic effects such as protective effects, sustained-release effects, and alteration of intermolecular hydrophilicity-hydrophobicity balance, the acetylated soluble soybean polysaccharide-linalool nanoparticles exhibit good anti-proliferative effects against tumor cells. These results suggest that acetylated soluble soybean polysaccharide-linalool nanoparticles have potential applications in food preservation and drug / bioactive substance delivery.

Claims

1. An acetylated soluble soybean polysaccharide-linalool nanoparticle, characterized in that, Acetylated soluble soybean polysaccharide was used as a carrier to load linalool; the loading amount was 2%~20%. The degree of acetylation of the acetylated soluble soybean polysaccharide is 0.6~1.2; The molecular weight of the acetylated soluble soybean polysaccharide is 3.5kDa~30kDa.

2. The acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 1, characterized in that, The preparation method of the acetylated soluble soybean polysaccharide is as follows: soluble soybean polysaccharide is prepared into a solution and reacted with acetic anhydride; the temperature during the reaction is 35℃~45℃ and the pH value is controlled at 8.0~10.0; after the target product is generated, the reaction is terminated and impurities are removed to obtain acetylated soluble soybean polysaccharide.

3. The acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 2, characterized in that, The concentration of the solution prepared with soluble soybean polysaccharide is 0.01 g / mL to 0.04 g / mL; the ratio of soluble soybean polysaccharide to acetic anhydride is 1 g: 12 to 18 mL.

4. The acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 2, characterized in that, The reaction was terminated by adjusting the pH value below 7.

0.

5. The acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 2, characterized in that, The reaction time between soluble soybean polysaccharides and acetic anhydride is 1 to 2 hours.

6. The method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles according to any one of claims 1 to 5, characterized in that, include: Acetylated soluble soybean polysaccharide was dispersed in PBS buffer as a carrier solution, linalool solution was added and stirred; the mixture was centrifuged at 3000 rpm to 5000 rpm and 0℃ to 4℃ for 10 to 20 minutes, and unloaded small molecules were removed by ultrafiltration. The retentate solution was collected and freeze-dried to obtain acetylated soluble soybean polysaccharide-linalool nanoparticle powder.

7. The method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 6, characterized in that, The pH value of the PBS buffer is 7.0~7.4; the concentration of the PBS buffer is 5mM~20mM.

8. The method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles according to claim 6, characterized in that, The concentration of acetylated soluble soybean polysaccharide in the carrier solution is 1.5 mg / mL to 2.5 mg / mL; the concentration of linalool solution is 0.8 mg / mL to 1.6 mg / mL.

9. The method for preparing acetylated soluble soybean polysaccharide-linalool nanoparticles according to any one of claims 6 to 8, characterized in that, The process of adding linalool solution and stirring is carried out under light-protected conditions.

10. The application of acetylated soluble soybean polysaccharide-linalool nanoparticles obtained by any one of claims 1 to 5 or any one of claims 6 to 9 in the preservation of food.

11. The use of acetylated soluble soybean polysaccharide-linalool nanoparticles obtained by any one of claims 1 to 5 or any one of claims 6 to 9 in the preparation of antibacterial agents.

12. The use of acetylated soluble soybean polysaccharide-linalool nanoparticles obtained by any one of claims 1 to 5 or any one of claims 6 to 9 in the preparation of antitumor drugs.

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