Aastaxanthin-containing pickering emulsion, its preparation method and application in beef

Abstract

The application provides a kind of astaxanthin Pickering emulsion and its preparation method and application in beef, belongs to food processing technical field.The application provides astaxanthin Pickering emulsion and its preparation method, through the precise self-assembly of bovine serum albumin and xanthan gum, the microstructure and interface property of composite nanoparticles are regulated, and the effect of stable loading astaxanthin is realized.The application further provides the application of astaxanthin Pickering emulsion in beef coloring, which can be used as a partial substitute for nitrite, can effectively maintain the redness of beef during storage, significantly inhibit lipid oxidation, and help to stabilize myoglobin state, while reducing the safety risk that may be brought by traditional nitrite use, providing a new way for developing natural and efficient beef coloring scheme.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a Pickering emulsion containing astaxanthin, its preparation method, and its application in beef. Background Technology

[0002] Beef, rich in high-quality protein, minerals, and B vitamins, is a popular and important meat product. However, beef is highly susceptible to quality deterioration during post-slaughter storage, transportation, and sales, primarily manifested in: 1) Color deterioration: Under the influence of oxygen, enzymes, and light, myoglobin in the muscle transforms from bright red oxymyoglobin to brown methemoglobin, leading to a decrease in commercial value; 2) Lipid oxidation: Unsaturated fatty acids undergo oxidative rancidity, producing volatile undesirable substances such as aldehydes and ketones, resulting in a rancid taste; 3) Microbial contamination: This leads to spoilage and food safety issues. To inhibit these deteriorations, extend shelf life, and impart a stable pink color to the product, nitrites have long been widely added to cured meat products and some processed meat products. Nitrites have multiple functions: inhibiting the growth of pathogenic bacteria such as Clostridium botulinum; reacting with myoglobin to form stable nitrosomyoglobin, resulting in a lasting pink color; and possessing certain antioxidant properties. However, the potential safety issues associated with nitrites cannot be ignored. Under acidic conditions or during high-temperature cooking, nitrites may react with amine compounds in meat to form nitrosamines, which are recognized as potent carcinogens. Therefore, developing safe and effective natural alternatives to nitrites has become a research hotspot and challenge in the field of meat science.

[0003] Astaxanthin is a naturally occurring ketocarotenoid widely found in organisms such as Haematococcus pluvialis, Antarctic krill, and salmon. Numerous studies have confirmed that astaxanthin possesses superior antioxidant activity, with its free radical scavenging ability far exceeding that of other antioxidants such as vitamin E and beta-carotene. Furthermore, astaxanthin itself is a deep pink color, possessing natural coloring potential. Therefore, astaxanthin is considered one of the ideal natural candidates to replace nitrites for antioxidant and color-enhancing purposes. However, astaxanthin is a highly unsaturated, lipid-soluble molecule, almost insoluble in aqueous solutions, and extremely sensitive to light, heat, and oxygen, readily undergoing oxidative degradation and exhibiting low bioavailability. These characteristics severely limit its direct and effective application in aquatic food systems (such as meat products). Therefore, constructing a carrier system that can effectively encapsulate and protect astaxanthin and stably deliver it into meat tissue is crucial for realizing its coloring applications.

[0004] Pickering emulsions are emulsion systems stabilized by micron- or nano-sized solid particles (rather than traditional surfactants) adsorbed at the oil-water interface, preventing droplet aggregation. Traditional pickering emulsions often use single protein or polysaccharide particles as stabilizers, making it difficult to simultaneously meet core requirements such as three-phase contact angle matching, nano-scale monodispersion, sufficient interfacial activity, and tolerance to food environments. Astaxanthin is a highly fat-soluble and easily oxidized and degraded active substance. Conventional emulsion systems struggle to effectively encapsulate, protect, and deliver it. When applied to meat product systems with high salt, high protein, and dynamically changing pH, conventional emulsions are prone to stratification, demulsification, active substance leakage, and degradation, failing to achieve integrated coloring, color protection, and antioxidant functions. Therefore, developing an astaxanthin pickering emulsion and using it as a natural colorant for meat products has significant theoretical and practical value. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide an astaxanthin-containing Pickering emulsion and its preparation method, wherein the astaxanthin-containing Pickering emulsion exhibits high stability and effectively protects the activity of astaxanthin.

[0006] A second objective of this invention is to provide an application of the above-mentioned astaxanthin-containing Pickering emulsion, which can be used as a coloring agent for beef and partially replace nitrites.

[0007] A third objective of this invention is to provide a beef coloring agent.

[0008] A fourth objective of this invention is to provide a method for coloring beef and the resulting colored beef product.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing an astaxanthin-containing Pickering emulsion, comprising the following steps: (1) Bovine serum albumin and xanthan gum were dispersed in water at a mass ratio of 10:1 to 1:8. The pH was adjusted, the mixture was stirred, and allowed to stand at low temperature to hydrate, thus obtaining a dispersion, which was used as the aqueous phase. (2) Astaxanthin was dissolved in edible oil to obtain an astaxanthin oil solution with an astaxanthin concentration of 0.2 mg / mL, which was used as the oil phase; (3) Mix the oil phase and water phase, disperse them at high speed and emulsify them according to the oil phase volume ratio of 20%~60% to obtain a Pickering emulsion containing astaxanthin.

[0010] Preferably, the concentration of bovine serum albumin in the dispersion is 0.1~5 mg / mL.

[0011] Preferably, the pH value of the dispersion is 3.5~8.0; the stirring time is 0.5~2h; and the low-temperature static hydration condition is standing at 4℃ for 8~12h.

[0012] Preferably, the edible oil is soybean oil.

[0013] Preferably, the conditions for high-speed dispersion are: rotation speed 11000 rpm and time 1 min.

[0014] The present invention also provides a Pickering emulsion containing astaxanthin prepared by the above preparation method.

[0015] The present invention also provides the application of the above-mentioned astaxanthin-containing Pickering emulsion in any of the following: (1) As a coloring agent for beef; (2) Preparation of beef coloring agent; (3) To partially or completely replace nitrites for coloring beef; (4) It is used in combination with nitrite for coloring beef.

[0016] The present invention also provides a beef coloring agent comprising the above-mentioned astaxanthin-containing Pickering emulsion.

[0017] The present invention also provides a method for coloring beef, comprising the following steps: applying the above-mentioned astaxanthin-containing Pickering emulsion to the interior of beef.

[0018] The present invention also provides beef products prepared by the above method.

[0019] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention overcomes the technical limitation that natural macromolecules are difficult to use as efficient pickering stabilizers by precisely self-assembling bovine serum albumin and xanthan gum to regulate the microstructure and interfacial properties of composite nanoparticles. This allows for the stable encapsulation of astaxanthin and its successful application in beef preservation and coloring. When this emulsion is used for beef coloring, it can serve as a partial (or complete) substitute for nitrites, effectively maintaining the redness of beef during storage. It significantly inhibits lipid oxidation (manifested as a decrease in TBARS value) and helps stabilize myoglobin status, while effectively reducing the safety risks that may be caused by the use of traditional nitrites, providing a new approach for developing natural and efficient beef coloring solutions.

[0020] The astaxanthin-containing Pickering emulsion of this invention uses safe raw materials, has a simple process, and is easy to industrialize and promote: the BSA, xanthan gum, vegetable oil and natural astaxanthin used are all GRAS-recognized safe food ingredients or natural extracts, which are in line with the clean label trend; and the preparation process does not require complicated equipment, the conditions are mild, and it is suitable for large-scale production in food factories, with good market conversion prospects. Attached Figure Description

[0021] Figure 1 : Curves showing the average particle size and zeta potential variation of composite nanoparticles prepared at different BSA to XG mass ratios.

[0022] Figure 2 Microscopic photograph of astaxanthin Pickering emulsion.

[0023] Figure 3 Comparison of macroscopic states of astaxanthin Pickering emulsion after 1 day and 15 days of static storage at 4°C.

[0024] Figure 4 Average particle size and particle size distribution of astaxanthin Pickering emulsion.

[0025] Figure 5 Curves showing the change in internal redness value of beef samples from different treatment groups over time during storage at 4℃.

[0026] Figure 6 : Curves showing the change of thiobarbituric acid reactant values ​​over time in beef samples from different treatment groups during storage at 4℃. Detailed Implementation

[0027] This invention provides a method for preparing an astaxanthin-containing Pickering emulsion, comprising the following steps: (1) Bovine serum albumin and xanthan gum were dispersed in water at a mass ratio of 10:1 to 1:8. The pH was adjusted, the mixture was stirred, and allowed to stand at low temperature to hydrate, thus obtaining a dispersion, which was used as the aqueous phase. (2) Astaxanthin was dissolved in edible oil to obtain an astaxanthin oil solution with an astaxanthin concentration of 0.2 mg / mL, which was used as the oil phase; (3) Mix the oil phase and water phase, disperse them at high speed and emulsify them according to the oil phase volume ratio of 20%~60% to obtain a Pickering emulsion containing astaxanthin.

[0028] The present invention describes step (1) for preparing a bovine serum albumin-xanthan gum composite nanoparticle dispersion. In this invention, bovine serum albumin solution and xanthan gum solution are mixed in a specific ratio, the pH value of the system is adjusted, and the mixture is stirred to ensure full interaction. Then, the mixture is allowed to stand at low temperature for hydration to obtain a uniform nanoparticle dispersion. As an optional implementation, the present invention prepares bovine serum albumin aqueous solution and xanthan gum aqueous solution separately (both are stirred at 500 rpm during preparation, with bovine serum albumin stirred at room temperature for 0.5 h and xanthan gum stirred at 40℃ for 1 h). Under stirring conditions, the bovine serum albumin aqueous solution is slowly added dropwise to the xanthan gum aqueous solution, and the composite process is completed by constant stirring at 600 rpm. Then, the pH of the mixture is adjusted to the target value using 0.1 mol / L dilute acid (such as HCl) or dilute alkali (such as NaOH), and stirring is continued to ensure a complete reaction. The mixture is then allowed to stand overnight in a 4℃ refrigerator to ensure full hydration and equilibrium, resulting in a uniform, semi-transparent nanoparticle dispersion.

[0029] Preferably, in the dispersion of the present invention, the concentration of bovine serum albumin is 0.1~5 mg / mL, more preferably 0.4 mg / mL, 0.625 mg / mL, 1.25 mg / mL, or 2.5 mg / mL; the mass ratio of bovine serum albumin to xanthan gum is more preferably 1:1 or 1:6; the pH value of the dispersion is 3.5~8.0, more preferably 6.0 or 7.0; the stirring time is 0.5~2 h, more preferably 1 h~1.5 h; and the low-temperature static hydration conditions are 4°C for 8~12 h, more preferably 9 h, 10 h, or 11 h.

[0030] This invention uses composite nanoparticles formed by the self-assembly of bovine serum albumin and xanthan gum through intermolecular forces such as hydrogen bonding and hydrophobic interactions as a solid particle stabilizer. The mass ratio of bovine serum albumin to xanthan gum is optimized and a suitable pH is maintained. The resulting nanoparticles are small in size, uniformly distributed, and have good stability in the dispersion. The nanoparticle dispersion constitutes the aqueous phase of an astaxanthin-containing Pickering emulsion.

[0031] Step (2) of this invention involves preparing the astaxanthin oil phase. This invention dissolves astaxanthin in edible oil to obtain a homogeneous astaxanthin oil solution. As an optional embodiment, this invention adds precisely weighed astaxanthin powder to edible soybean oil, maintains a constant temperature magnetic stirrer at 35°C, sets the rotation speed to 400 r / min, and continuously stirs for 10 min to completely dissolve the astaxanthin, until a clear astaxanthin oil solution is obtained. Preferably, the edible oil in this invention is soybean oil. The concentration of the astaxanthin oil solution in this invention is 0.2 mg / mL.

[0032] Step (3) of this invention constructs a Pickering emulsion. As an optional embodiment, this invention mixes the astaxanthin oil phase obtained in step (2) with the nanoparticle dispersion obtained in step (1) in a specific ratio, and emulsifies under high-speed dispersion conditions to obtain an astaxanthin-containing Pickering emulsion. The oil phase volume ratio of this invention is 20%~60%, that is, the oil phase and water phase are mixed at a volume ratio of 20:80 to 60:40, and the more preferred volume ratio of the oil phase and water phase is 30:70. The high-speed dispersion conditions of this invention are: rotation speed 11000 rpm, time 1 min. Under these conditions, after high-speed dispersion and emulsification, an O / W (oil-in-water) type Pickering emulsion with uniform droplet size, a strong interface, and excellent storage stability can be formed.

[0033] This invention also provides an astaxanthin-containing Pickering emulsion prepared by the above method, which is an orange-colored astaxanthin Pickering emulsion. The efficient astaxanthin delivery and protection system constructed in this invention utilizes bovine serum albumin and xanthan gum, two common food ingredients, to construct composite nanoparticles as Pickering stabilizers. These nanoparticles form a robust interfacial film at the oil-water interface through physical adsorption, greatly improving the physical stability of the emulsion (resisting aggregation and stratification) and effectively isolating it from external factors such as oxygen and light, significantly enhancing the chemical stability of astaxanthin during processing and storage.

[0034] The present invention also provides the application of the above-mentioned astaxanthin-containing Pickering emulsion in any of the following: (1) as a beef coloring agent; (2) preparation of a beef coloring agent; (3) partial or complete replacement of nitrite for beef coloring; (4) compounding with nitrite for beef coloring.

[0035] The present invention also provides a beef coloring agent comprising the above-mentioned astaxanthin-containing Pickering emulsion.

[0036] This invention applies astaxanthin Pickering emulsion to beef coloring or the preparation of beef coloring agents, and demonstrates that it can simultaneously play the following key roles: (a) natural coloring and color protection: the red color of astaxanthin itself can directly contribute to the meat color, while its strong antioxidant capacity may indirectly stabilize the bright red color of beef (maintaining a high a) by reducing methemoglobin or protecting myoglobin from oxidation. (a) Highly effective antioxidant: Astaxanthin in the emulsion can effectively penetrate and act on beef tissue, quench free radicals in the lipid oxidation chain reaction, significantly inhibit the rise of TBARS value during storage, and delay rancidity; (b) Potential auxiliary antibacterial: Astaxanthin has certain antibacterial activity, and its emulsion system may inhibit some spoilage bacteria and pathogenic bacteria on the surface of beef, which, in synergy with the physical barrier effect, helps to extend shelf life.

[0037] This invention applies astaxanthin Pickering emulsion to partially or completely replace nitrite, providing a reliable pathway for reducing nitrite content and even achieving nitrification-free coloring. Specifically, based on the coloring effect of the maximum permitted addition amount of nitrite (e.g., 150 mg / kg) according to national standards, astaxanthin Pickering emulsion is used to replace 20%, 40%, 60%, 80%, and 100% of the "functional share." This includes both the combined use of astaxanthin emulsion and low-dose nitrite, as well as the use of astaxanthin emulsion entirely. It has been demonstrated that using the astaxanthin Pickering emulsion of this invention to replace 40%~60% of nitrite effectively maintains the color of beef (a...). While achieving an antioxidant effect that is not inferior to or even superior to the full nitrite group (TBARS is close to the nitrite group), it also obtains a better (far superior to the latter) antioxidant effect, thereby improving the overall coloring effect while significantly reducing the risk of nitrite residue and nitrosamine formation in the final product.

[0038] This invention also provides a method for coloring beef, comprising the following steps: applying the above-mentioned astaxanthin-containing Pickering emulsion to the interior of beef. This invention can treat beef raw materials or products with the above-mentioned astaxanthin-containing Pickering emulsion by injection, tumbling, soaking, or spraying. Preferably, the application amount of the astaxanthin-containing Pickering emulsion, calculated as astaxanthin, in beef at a final concentration of 0.02~0.10 mg / g (i.e., 20~100 mg / kg), preferably 0.06 mg / g (60 mg / kg). The astaxanthin-containing Pickering emulsion of this invention can replace 20%~100% of the color-maintaining and antioxidant functions of nitrite in coloring.

[0039] This invention also provides a beef product prepared by the above method, wherein the redness value (a) of the beef product after storage at 4°C for 5 days is... The levels showed almost no decrease, and the TBARS value was below 1.0 mg MDA / kg.

[0040] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1 1. Preparation, optimization and characterization of bovine serum albumin (BSA)-xanthan gum (XG) composite nanoparticles.

[0044] (1) Solution preparation: Accurately weigh 0.5 g of BSA and XG respectively, and prepare stock solutions with a mass concentration of 5 mg / mL using ultrapure water. Stirring was performed at 500 rpm for both preparations. Bovine serum albumin was stirred at room temperature for 0.5 h, and xanthan gum was stirred at 40℃ for 1 h until completely dissolved.

[0045] (2) Nanoparticle preparation: BSA and XG were prepared at mass ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, and 1:8, respectively. The BSA solution was added dropwise to an equal volume of XG solution under magnetic stirring at 600 rpm. After mixing, the pH of the mixture was adjusted to 7.0 with 0.1 mol / L HCl or NaOH solution. Stirring was continued for 1 h to ensure sufficient interaction. The resulting mixture was then transferred to a 4°C refrigerator and allowed to stand overnight (12 h) for hydration, yielding a BSA-XG composite nanoparticle dispersion.

[0046] (3) Characterization and stability testing: a. Particle size and Zeta potential: Dilute the above samples 10 times with ultrapure water and measure their hydrodynamic average particle size (Z-average), polydispersity index (PDI), and Zeta potential at 25°C using a particle size potentiometer. Each sample was measured three times.

[0047] The results are as follows Figure 1 As shown, when the BSA:XG ratio is 1:1, the average particle size of the composite nanoparticles is the smallest (about 607 nm), and the PDI is 0.98, indicating that the particle size distribution is uniform; the Zeta potential is about -29 mV, and the high absolute value indicates that the system has a good electrostatic stabilizing effect.

[0048] b. Storage stability: The freshly prepared dispersion (pH 7.0) was stored at 4°C, and samples were taken on days 0, 1, 3, 7, and 10. The absorbance (OD) was measured at 600 nm using a UV spectrophotometer. 600 The physical stability of a dispersion is evaluated by the change in absorbance (turbidity change). The smaller the turbidity change, the more stable the system is, and the less likely it is to aggregate or precipitate.

[0049] Table 1. Storage stability test results (OD) 600 value)

[0050] The results showed that in the 10-day storage stability test, when the BSA:XG ratio was 1:6, the OD of the sample was [missing value]. 600 The value variation was the smallest, confirming that it had the best long-term storage stability. The next best was a BSA:XG ratio of 1:1.

[0051] c. pH stability: The pH of freshly prepared BSA:XG dispersions at ratios of 1:1 and 1:6 was adjusted to 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, and 8.0 (7 gradients) using 0.1 mol / L HCl and 0.1 mol / L NaOH, and allowed to stand for equilibration for 30 min. The hydrodynamic mean particle size (Z-average), polydispersity index (PDI), and zeta potential were measured at 25 °C using a particle size potentiometer. Each sample was measured three times.

[0052] Table 2. Particle size, PDI, and Zeta potential of BSA:XG composite nanoparticles at different pH values ​​(1:1 and 1:6).

[0053] When adjusting the pH, the nanoparticles with a BSA:XG ratio of 1:1 at pH 3.5, 4.0, 4.5, and 5.0 showed significant agglomeration and were therefore discarded. Comparing the two ratios, the BSA:XG=1:1 ratio showed smaller particle size and better data repeatability at pH 7.0 and 8.0, with significant differences compared to the other pH groups and the 1:6 group (P < 0.05). While the BSA:XG=1:6 ratio remained relatively stable within the pH range of 6–8, its overall particle size was significantly larger than the 1:1 group (P < 0.05), and agglomeration was more intense under acidic conditions.

[0054] Therefore, bovine serum albumin-xanthan gum composite nanoparticles with a BSA to XG mass ratio of 1:1 were subsequently selected for the preparation of astaxanthin Pickering emulsion.

[0055] 2. Construction and determination of basic properties of astaxanthin Pickering emulsion (1) Emulsion preparation: Astaxanthin (96% content, Shanghai Ron Reagent Co., Ltd.) was accurately weighed and added to soybean oil at 35℃, fixing the concentration of astaxanthin in the oil phase at 0.2 mg / mL. Nanoparticle dispersions and astaxanthin-soybean oil stock solutions were measured into sample tubes according to oil phase volume fractions of 20% (E20), 30% (E30), 40% (E40), 50% (E50), and 60% (E60), respectively. A series of Pickering emulsions were prepared by high-speed dispersion at 11000 rpm for 1 minute using a high-speed disperser.

[0056] (2) Emulsion characterization and stability evaluation: a. Liquefaction Index (CI): Freshly prepared emulsions were placed into graduated centrifuge tubes and heated in a 100°C water bath for 30 minutes. The emulsion separation index, total column height (H0), and lower supernatant height (H1) were then recorded. The emulsion separation index was calculated using the formula CI = (H1 / H0) × 100%. A lower CI value indicates better emulsion anti-stratification stability.

[0057] Table 3. Liquefaction Index (CI, %)

[0058] b. Light transmittance: Freshly prepared emulsions from group E30 were placed into centrifuge tubes, sealed, and stored at 4°C for 15 days. The macroscopic morphology of the emulsions was observed on day 1 (C0) and day 15 (C1), and any unstable phenomena such as turbidity or layering were recorded. The absorbance of the emulsions was measured at 600 nm using a UV spectrophotometer, and the results were recorded according to... If the transmittance is greater than 90%, it indicates that the emulsion has good storage stability within a 15-day storage period.

[0059] Table 4. Light transmittance (%)

[0060] c. Observation of microstructure and macroscopic state: Take a small amount of freshly prepared emulsion from group E30, dilute it appropriately with deionized water, and drop it onto a glass slide. Cover with a coverslip and observe and photograph the morphology of the emulsion and the distribution of droplet size under an optical microscope. The results are as follows: Figure 2 As shown.

[0061] Comparison photos of the macroscopic state of E30 group astaxanthin Pickering emulsion after standing and storing at 4℃ for 1 day and 15 days are shown below. Figure 3 show.

[0062] d. Particle size distribution: The average particle size and particle size distribution of the astaxanthin Pickering emulsion from group E30 were analyzed, and the results are as follows: Figure 4 As shown.

[0063] The results show that the emulsion prepared under 11000 rpm shear conditions has a particle size mainly distributed between 0 and 200 nm, and small particles are visible adhering to the droplet surface, consistent with Pickering emulsion characteristics. Therefore, the emulsion prepared with the following process parameters—BSA to XG mass ratio of 1:6, oil phase volume fraction of 30%, and shear conditions of 11000 rpm for 1 min—performs the best emulsion.

[0064] Example 2 Preparation of astaxanthin Pickering emulsion: (1) Preparation of bovine serum albumin (BSA)-xanthan gum (XG) composite nanoparticle dispersion: A composite nanoparticle dispersion was prepared with a bovine serum albumin (BSA) to xanthan gum (XG) mass ratio of 1:1 and a bovine serum albumin concentration of 2.5 mg / mL. A certain concentration of BSA and XG aqueous solutions were prepared separately. Under magnetic stirring at 600 rpm, the BSA solution was added dropwise to an equal volume of XG solution. After mixing, the pH of the mixture was adjusted to 7.0 with 0.1 mol / L HCl / NaOH solution, and stirring was continued for 1 hour. The resulting mixture was then transferred to a 4°C refrigerator and allowed to stand for 12 hours to obtain the BSA-XG composite nanoparticle dispersion.

[0065] (2) Preparation of astaxanthin oil phase: Accurately weighed astaxanthin (content 96%, Shanghai Ron Reagent Co., Ltd.) was added to edible soybean oil at 35℃. The temperature of the constant temperature magnetic stirrer was kept at 35℃, the speed was set to 400r / min, and the stirring was continued for 10min to completely dissolve the astaxanthin and obtain an astaxanthin oil solution with a concentration of 0.2mg / mL.

[0066] (3) Constructing the Pickering emulsion: Measure the astaxanthin oil phase from step (2) and the composite nanoparticle dispersion from step (1) at a volume fraction of 20% for the oil phase, and place them in a container. Use a high-speed disperser to shear at a predetermined speed (11000 rpm) for 1 min to obtain an orange astaxanthin Pickering emulsion.

[0067] Example 3 Preparation of astaxanthin Pickering emulsion: (1) Preparation of bovine serum albumin (BSA)-xanthan gum (XG) composite nanoparticle dispersion: A composite nanoparticle dispersion was prepared with a bovine serum albumin (BSA) to xanthan gum (XG) mass ratio of 1:6 and a BSA concentration of 0.625 mg / mL. A certain concentrations of BSA and XG aqueous solutions were prepared separately. Under magnetic stirring at 600 rpm, the BSA solution was added dropwise to an equal volume of XG solution. After mixing, the pH of the mixture was adjusted to 7.0 with 0.1 mol / L HCl / NaOH solution, and stirring was continued for 1 hour. The resulting mixture was then transferred to a 4°C refrigerator and allowed to stand for 8 hours to obtain the BSA-XG composite nanoparticle dispersion.

[0068] (2) Preparation of astaxanthin oil phase: Accurately weighed astaxanthin (content 96%, Shanghai Ron Reagent Co., Ltd.) was added to edible soybean oil at 35℃. The temperature of the constant temperature magnetic stirrer was kept at 35℃, the speed was set to 400r / min, and the stirring was continued for 10min to completely dissolve the astaxanthin and obtain an astaxanthin oil solution with a concentration of 0.2mg / mL.

[0069] (3) Constructing the Pickering emulsion: Measure the astaxanthin oil phase from step (2) and the composite nanoparticle dispersion from step (1) with an oil phase volume fraction of 30% and place them in a container. Use a high-speed disperser to shear at a predetermined speed (11000 rpm) for 1 min to obtain an orange astaxanthin Pickering emulsion.

[0070] Example 4 The astaxanthin Pickering emulsion prepared in Example 3 was used to verify its application and effect evaluation in beef coloring.

[0071] 1. Materials and Grouping Ingredients: Fresh beef hind leg (within 24 hours after slaughter), with visible fat and connective tissue removed, cut into pieces of approximately 50±5g.

[0072] Experimental Design: Four treatment groups were set up, with at least five replicate meat samples in each group. The amount of compound marinade added was uniformly 25% of the meat weight, and the amount of salt added was uniformly 3% of the meat weight, as detailed below: CK group (blank control group): No coloring agent was added, and the treatment was carried out with an equal amount of sterile water and salt.

[0073] Group AN (total nitrite group): 150 mg / kg of sodium nitrite (refer to the maximum allowable amount for pre-processed meat products in GB 2760-2024) + salt were added.

[0074] AS60 group: Astaxanthin emulsion replaces 60% of the nitrite function, that is, add 60 mg / kg sodium nitrite + corresponding astaxanthin emulsion (calculated based on astaxanthin content of 60 mg / kg) 300 mL / kg + salt.

[0075] AS100 group: Astaxanthin emulsion replaces 100% of the nitrite function, that is, no sodium nitrite is added, only astaxanthin emulsion is used for coloring (calculated based on astaxanthin content of 100mg / kg) 500mL / kg + salt.

[0076] 2. Sample processing Sodium nitrite (food grade) was dissolved in a small amount of sterile water, and the astaxanthin emulsion was used directly. All treatment solutions were injected evenly into the beef chunks using a multi-point, multi-directional injection method, followed by brief tumbling to ensure uniform distribution. All treated meat samples were placed on sterile trays, covered with a colored film, and stored in a cold storage at 4±1℃. Samples were taken on days 0, 1, 2, 3, 4, and 5 of storage for index determination.

[0077] 3. Measurement Indicators and Result Analysis (1) Color: Using an intelligent spectrophotometer with a D65 light source and a 10° observation angle, the surface of the meat sample was measured, and the redness value (a) was recorded. Each meat sample was measured at three different locations, and the average value was taken.

[0078] Color changes such as Figure 5 As shown: During the entire 5-day storage period, group CK a The value decreased the fastest, and by day 4 it had already turned a distinct brown. The AN group (nitrite) maintained its red color better. The value decreased gradually. (AS60 group a) The value retention effect was not statistically significantly different from the AN group (P>0.05), and was even slightly higher than the AN group in the later stage of storage. The AS100 group (full replacement) had a... Although the value was slightly lower than that of group AN, it was significantly higher than that of group CK, indicating that the astaxanthin emulsion itself has a certain color-protecting ability. This proves that astaxanthin Pickering emulsion can replace 60% of nitrite without excessively sacrificing the color-protecting effect.

[0079] (2) Degree of lipid oxidation (TBARS value): Refer to GB5009.181—2016 "National Food Safety Standard - Determination of malondialdehyde in Food". Weigh 3.00 g of minced meat sample, add 30 mL of 7.5% trichloroacetic acid (TCA) solution containing 0.1% EDTA, homogenize and filter. Accurately transfer 5 mL of the above filtrate and 5 mL of the standard series solutions into 25 mL stoppered colorimetric tubes respectively. Take another 5 mL of trichloroacetic acid mixture as a sample blank, add 5 mL of thiobarbituric acid aqueous solution to each, stopper, mix well, and react in a 90 ℃ water bath for 30 min. Remove and cool to room temperature. Adjust the zero point with the sample blank, and measure the absorbance values ​​of the sample solution and the standard series solutions at 532 nm with a 1 cm optical path. Plot a standard curve with the mass concentration of the standard series solutions as the abscissa and the absorbance value as the ordinate.

[0080] Lipid oxidation inhibition, such as Figure 6 As shown, the MDA content in all groups increased with prolonged storage time. The CK group showed the fastest increase and the highest content. The AS60 group demonstrated the best performance in inhibiting MDA accumulation, with its TBARS value remaining very close to that of the AN group (nitrite) throughout the storage period, significantly better than the CK and AS100 groups. This indicates that at the AS60 concentration, the powerful antioxidant capacity of astaxanthin was effectively utilized in the emulsion system and successfully delivered into the beef, greatly inhibiting oxidative rancidity of the fat. This demonstrates that the AS60 group's astaxanthin emulsion exhibited the best antioxidant effect.

[0081] In summary, the astaxanthin Pickering emulsion prepared by this invention exhibits excellent stability and effectively encapsulates and protects astaxanthin. When applied to beef coloring, replacing 40%–60% of nitrite (e.g., the AS60 group), it not only achieves color retention comparable to the full-volume nitrite group (AN group), but also provides significantly superior antioxidant protection (significantly lower TBARS values). This enables quality improvement under "nitrite reduction" or even "nitrite-free" conditions, representing a highly promising composite natural coloring solution.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Pickering emulsion containing astaxanthin, characterized in that, Includes the following steps: (1) Bovine serum albumin and xanthan gum were dispersed in water at a mass ratio of 10:1 to 1:

8. The pH was adjusted, the mixture was stirred, and allowed to stand at low temperature to hydrate, thus obtaining a dispersion, which was used as the aqueous phase. (2) Astaxanthin was dissolved in edible oil to obtain an astaxanthin oil solution with an astaxanthin concentration of 0.2 mg / mL, which was used as the oil phase; (3) Mix the oil phase and water phase, disperse them at high speed and emulsify them according to the oil phase volume ratio of 20%~60% to obtain a Pickering emulsion containing astaxanthin.

2. The preparation method according to claim 1, characterized in that, The concentration of bovine serum albumin in the dispersion is 0.1~5 mg / mL.

3. The preparation method according to claim 1, characterized in that, The pH value of the dispersion is 3.5~8.0; the stirring time is 0.5~2h; the low-temperature static hydration condition is 4℃ standing for 8~12h.

4. The preparation method according to claim 1, characterized in that, The edible oil is soybean oil.

5. The preparation method according to claim 1, characterized in that, The conditions for high-speed dispersion are: rotation speed 11000 rpm, time 1 min.

6. The astaxanthin-containing Pickering emulsion prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the astaxanthin-containing Pickering emulsion of claim 6 in any of the following, characterized in that, (1) As a coloring agent for beef; (2) Preparation of beef coloring agent; (3) To partially or completely replace nitrites for coloring beef; (4) It is used in combination with nitrite for coloring beef.

8. A beef coloring agent, characterized in that, The beef coloring agent includes the astaxanthin-containing Pickering emulsion as described in claim 6.

9. A method for coloring beef, characterized in that, The procedure includes the following steps: applying the astaxanthin-containing Pickering emulsion of claim 6 to the interior of beef.

10. The beef product prepared by the method of claim 9.

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