Method for preparing and use of whole fish-derived complex emulsified microspheres encapsulating phospholipase

Abstract

The application discloses a preparation method and application of full-fish-source composite emulsion microspheres coated with phospholipase, and the preparation method comprises the following steps: dissolving sturgeon skin collagen in a buffer solution to form a mixed solution; then uniformly mixing the mixed solution with sturgeon-source phospholipase A2, and adding sturgeon chondroitin sulfate solution dropwise, so as to obtain an inner water phase after reaction; sturgeon fish oil is used as an oil phase to be mixed and emulsified with the inner water phase, so as to obtain a water-in-oil type primary emulsion; sturgeon skin original collagen and sturgeon chondroitin gelatin are dissolved in deionized water to obtain an outer water phase, then the water-in-oil type primary emulsion is poured into the outer water phase, and secondary gentle dispersion is carried out, so as to form multiple emulsion droplets; the multiple emulsion droplets are continuously introduced into an ice water phase medium to be subjected to pure physical cold-induced solidification, and full-fish-source composite emulsion microspheres are obtained. The application can improve the caviar flavor quality of sturgeon and prolong the shelf life.

Description

Technical Field

[0001] This invention relates to the field of food technology, and in particular to a method for preparing and applying a whole-fish-derived composite emulsion microsphere encapsulating phospholipase. Background Technology

[0002] The flavor of sturgeon caviar primarily originates from the hydrolysis and lipid oxidation of phospholipids. Endogenous phospholipase A2 (PLA2) plays a crucial role in phospholipid metabolism and flavor formation. During storage and processing, the sn-2 position polyunsaturated fatty acids in sturgeon caviar can be hydrolyzed by endogenous phospholipase A2, releasing free fatty acids and lysophospholipids, which are further oxidized to generate volatile flavor compounds such as aldehydes, ketones, and alcohols. Experimental studies have shown that PLA2 promotes the formation of volatile flavor compounds in the early stages of sturgeon caviar processing and storage, thus contributing to its unique flavor. However, in the later stages of storage, the continued activity of PLA2 leads to excessive accumulation of acids and peroxidation products, resulting in increased rancidity and fermentation, intensified lipid oxidation, and overall quality deterioration. Therefore, overcoming the "early flavor formation—late quality deterioration" phenomenon in sturgeon caviar processing is a key challenge in traditional sturgeon caviar production.

[0003] The flavor deterioration of sturgeon caviar during storage is a complex biochemical process involving lipid oxidation, protein degradation, and microbial activity. At the sensory level, fresh caviar possesses a distinctive "grassy," "oily," and "salty" flavor, but these gradually diminish with prolonged storage, replaced by rancid and sour flavors dominated by lipid oxidation, as well as putrid odors produced by microbial metabolism. Nutritionally, the polyunsaturated fatty acids in sturgeon caviar are sensitive to oxidation, making it prone to peroxidation in later stages of storage, resulting in undesirable flavors and reduced quality.

[0004] Based on this, existing methods for flavor regulation of sturgeon caviar mainly rely on cold chain storage, high-salt conditions, or exogenous inhibitors to suppress PLA2 activity in the later stages. However, cold chain and high-salt conditions can only partially reduce enzyme activity and cannot achieve a dynamic balance between flavor generation and inhibition. On the other hand, PLA2 inhibitors have limited effects on regulating key subtypes in the food system and cannot achieve continuous and stable control over the release of free fatty acids and the generation of flavor substances. Furthermore, the addition of exogenous inhibitors can affect the natural flavor and compliance of sturgeon caviar, reducing the added value of the product.

[0005] Therefore, existing methods for flavor control of sturgeon caviar cannot promote flavor formation in the early stages, while also limiting excessive oxidation of substances in the sturgeon caviar in the later stages. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and applying whole fish-derived complex emulsion microspheres encapsulating phospholipase. This can improve the flavor and quality of sturgeon caviar and extend its shelf life.

[0007] The technical solution of the present invention: a method for preparing whole fish-derived composite emulsion microspheres encapsulating phospholipase, comprising the following steps:

[0008] A. Sturgeon skin collagen is extracted from sturgeon processing by-products. Then, the sturgeon skin collagen is dissolved in a buffer solution, and the pH of the buffer solution is controlled to be lower than the isoelectric point of the sturgeon skin collagen so that the surface of the sturgeon skin collagen molecules is positively charged, resulting in mixed solution a.

[0009] B. Mix the mixed solution a with sturgeon-derived phospholipase A2 at a mass ratio of 2:1 to form an enzyme mixed solution; then add a strongly negatively charged sturgeon chondroitin sulfate solution dropwise to the enzyme mixed solution, and the aqueous phase b is obtained after the reaction.

[0010] C. Sturgeon fish oil is used as the oil phase and mixed with the aqueous phase in b and emulsified to obtain c, a water-in-oil type primary emulsion;

[0011] D. Sturgeon skin collagen and sturgeon cartilage gelatin are dissolved together in deionized water to obtain an external aqueous phase. Then, the c water-in-oil type colostrum is poured into the external aqueous phase and gently dispersed again by low-speed shearing or high-frequency microporous vibration to form d multiple emulsion droplets.

[0012] E. Multiple emulsion droplets were continuously introduced into an ice-water phase medium at 0°C for pure physical cold-induced solidification, which physically thickened the outer sturgeon oil and promoted the triple helix restructuring and tightening of the internal collagen, and crosslinked with chondroitin sulfate to obtain whole fish-derived composite emulsion microspheres.

[0013] In the aforementioned method for preparing whole-fish-derived complex emulsion microspheres encapsulating phospholipase, the buffer solution in step A is a 0.1 mol / L citrate-sodium citrate buffer solution. Sturgeon skin collagen is dissolved in the citrate-sodium citrate buffer solution to form a 1.5 wt% collagen solution. The pH of the collagen solution is adjusted to 5.2-5.6 by the citrate-sodium citrate buffer solution, so that the surface of the sturgeon skin collagen molecules is positively charged.

[0014] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, step B, the preparation process of sturgeon-derived phospholipase A2 includes the following steps:

[0015] B11. Sturgeon caviar was added to Tris-HCl buffer at a material-to-liquid ratio of 1:4 w / v, and then homogenized at high speed under ice bath conditions to fully break the cells and release endogenous enzymes, resulting in B11 homogenate.

[0016] B12. Centrifuge the homogenized solution of b11, discard the solid precipitate at the bottom and collect the supernatant to obtain crude enzyme solution of b12;

[0017] B13. Under ice bath and continuous magnetic stirring conditions, add solid ammonium sulfate powder to the crude enzyme solution of b12 until the saturation of ammonium sulfate in the crude enzyme solution of b12 reaches 60%, to obtain crude enzyme solution of b13.

[0018] B14. The crude enzyme solution of b13 was subjected to continuous stirring in an ice bath and then allowed to stand for salting out to obtain the b14 salting out solution.

[0019] B15. Centrifuge the b14 salting-out solution, discard the supernatant and collect the protein precipitate at the bottom to obtain the b15 precipitate;

[0020] B16. Add Tris-HCl buffer in portions until the b15 precipitate is completely reconstituted. Then repeat steps B13-B15 to perform secondary fractionation salting out of the b15 precipitate to obtain the b16 protein precipitate.

[0021] B17. After fully reconstituted the b16 protein precipitate with Tris-HCl buffer, it was transferred to a hydrophilic dialysis bag. The dialysis bag was then immersed in ultrapure water at 4°C for continuous flow dialysis. After multiple cycles, the b17 desalting enzyme solution was obtained.

[0022] B18. The b17 desalting enzyme solution, along with the dialysis bag, was embedded in a solid polyethylene glycol powder matrix for external water absorption and concentration until the fluid volume inside the dialysis bag was reduced to 1 / 4 to 1 / 6 of the initial volume, thus obtaining sturgeon-derived phospholipase A2.

[0023] The preparation process of sturgeon chondroitin sulfate solution in step B includes the following steps:

[0024] B21. Sturgeon skull and trunk skeleton are used as raw materials and heated in a high-temperature water bath. After the raw materials are removed, the fish meat is removed and the skull and trunk skeleton are mechanically crushed into granules to obtain b21 fish bone granules.

[0025] B22. Place the b21 fish bone particles into a high-pressure reactor for high-pressure thermal liquefaction treatment to obtain b22 cartilage fluid;

[0026] B23. Adjust the pH of the b22 cartilage fluid to 6.8-7.2 for the complex protease, then add the complex neutral protease to the b22 cartilage fluid at an addition rate of 0.15-0.30% of the b22 cartilage fluid mass, and then enzymatically hydrolyze at a constant temperature of 52-58℃ for 8-12 hours to obtain the b23 cartilage hydrolysate.

[0027] B24. After the enzyme inactivation of the b23 cartilage hydrolysate at high temperature, the large molecular proteins and inorganic salt bone residues in the b23 cartilage hydrolysate are retained by a ceramic microfiltration membrane, and then the b23 cartilage hydrolysate is physically concentrated by a reverse osmosis membrane to obtain the b24 cartilage hydrolysate.

[0028] B25. The enzymatic hydrolysate of B24 chondroitin was purified and refined by a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The retentate was then collected and dried to obtain B25 sturgeon chondroitin sulfate.

[0029] B26. Dissolve sturgeon chondroitin sulfate (b25) in 0.1 mol / L citrate-sodium citrate buffer solution to prepare a 0.5 wt% chondroitin sulfate solution, thus obtaining sturgeon chondroitin sulfate solution.

[0030] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres containing phospholipase, in step B, the mixed solution a is mixed with a quantitative amount of sturgeon-derived phospholipase A2, so that the enzyme activity concentration of phospholipase A2 in the enzyme mixed solution reaches 400-600 U / mL.

[0031] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, in step B, sturgeon chondroitin sulfate solution is added dropwise to the enzyme mixture at a dry weight ratio of sturgeon skin collagen to sturgeon chondroitin sulfate of 4:1, with a dropwise addition rate of 0.3–0.6 mL / min. During the dropwise addition, the enzyme mixture is continuously magnetically stirred in a constant temperature water bath at 4°C. After the dropwise addition is completed, the enzyme mixture is stirred for another 2–35 min in a constant temperature water bath at 4°C to obtain the aqueous phase in step b.

[0032] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, the preparation process of sturgeon oil in step C includes the following steps:

[0033] C1. Using fresh sturgeon fat as raw material, the fat raw material is crushed and added to deionized water at a material-to-liquid ratio of 1:2 w / v. The pH is then adjusted to 7.5-8.0 to obtain mixture C1.

[0034] C2. Add 1.0-1.5% (w / w) of alkaline protease to the mixture in c1, and then enzymatically hydrolyze it at 45-50℃ for 2.0 h. After hydrolysis, heat the solution to inactivate the enzyme, and obtain the mixture in c2.

[0035] C3. The mixture of C2 is centrifuged, and then the upper light phase crude fish oil is collected by separation to obtain C3 crude fish oil;

[0036] C4. After heating the C3 crude fish oil to 55-65℃, add hot distilled water at a mass of 2.0-3.0% of the C3 crude fish oil. Then, stir to allow the colloidal impurities to fully absorb water and coagulate. After the solution is allowed to stand and separate into layers, separate and remove the lower oil residue to obtain the C4 solution.

[0037] C5. Determine the acid value of solution C4, and add NaOH solution to solution C4 at an amount of 1.1 to 1.2 times the theoretical consumption of acid value. Neutralize and deacidify by stirring, and then collect the deacidified oil by separation to obtain deacidified oil C5.

[0038] C6. Add activated clay to C5 deacidified oil at an addition rate of 3.0-5.0% of the oil mass, and then place the oil under vacuum of ≤0.09MPa and temperature of 80-85℃ for continuous stirring and adsorption to remove pigments and free peroxides from the oil, thus obtaining C6 oil.

[0039] C7. C6 oil is pressure filtered through a microporous membrane to obtain C7 decolorized fish oil;

[0040] C8. Deodorize the c7 decolorized fish oil by depressurization and co-current flow under vacuum conditions of ≤0.095MPa and temperature of 110~120℃ to obtain sturgeon fish oil.

[0041] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, in step C, sturgeon fish oil and aqueous phase b are mixed at a mass ratio of 3:1 and then placed in a high-shear emulsifier with a 6°C constant-temperature circulating cooling jacket for continuous shear emulsification to obtain water-in-oil type primary emulsion c.

[0042] In the aforementioned method for preparing whole-fish-derived complex emulsion microspheres encapsulating phospholipase, the extraction method of sturgeon skin collagen in steps A and D includes the following steps:

[0043] D11. Using fresh sturgeon skin with lateral and ventral sacs as raw material, the sturgeon skin raw material is cut to obtain several fish skin pieces, resulting in d11 fish skin pieces.

[0044] D12. Place the d11 fish skin piece in boiling water and blanch for 30-60 seconds while stirring at a constant speed. Then remove the bone plates, scales and subcutaneous fat tissue of the d11 fish skin piece to obtain the d12 fish skin piece.

[0045] D13. The fish skin block d12 is rinsed and crushed in sequence to form square sturgeon skin fragments, resulting in fragment d13.

[0046] D14. Soak the d13 scraps in a 0.3-0.6 mol / L acetic acid solution at a material-to-liquid ratio of 1:25-30 w / v, and then add pepsin at a mass of 0.8-1.2% of the dry weight of the sturgeon skin raw material to obtain the d14 mixture.

[0047] D15. Stir the d14 mixture continuously at 4-8℃ for 20-28 hours, then filter to remove the undegraded residue in the mixture to obtain the d15 supernatant.

[0048] D16. Slowly add NaCl powder to the supernatant of d15 until the concentration is 0.9-1.2 mol / L, and then let the solution stand at 4℃ for 12-16 h to salt out, to obtain d16 salting-out solution;

[0049] D17. After centrifuging the salting-out solution of d16, collect the flocculent protein precipitate, and then redissolve the flocculent protein precipitate with 0.05-0.15 mol / L acetic acid solution to obtain solution d17.

[0050] D18. Transfer the d17 solution to a dialysis bag with a molecular weight cutoff of 50-100 kDa, dialyze continuously with deionized water at 4°C for 60-72 h, and then freeze-dry the dialysate under vacuum to obtain sturgeon skin collagen.

[0051] The preparation process of sturgeon cartilage gelatin in step D includes the following steps:

[0052] D21. Fresh sturgeon cartilage is crushed to obtain cartilage particles. Then, the cartilage particles are mixed with 0.05-0.15 mol / L NaHCO3 solution at a material-to-liquid ratio of 1:3-5 w / v and stirred continuously for 15-30 min to obtain d21 mixture.

[0053] D22. Wash the d21 mixture with distilled water in the reverse direction until the wash solution is neutral. Then, take out the cartilage particles and mix them with 0.05-0.15 mol / L citric acid solution at a material-to-liquid ratio of 1:3-5 w / v for 15-30 min. Wash with distilled water until neutral to obtain d22 cartilage particles.

[0054] D23. Mix d22 cartilage granules with distilled water at a material-to-liquid ratio of 1:3 to 5 w / v, and then extract by hot pressing at 115 to 125°C for 20 to 30 min to obtain d23 mixture;

[0055] D24. The mixture from d23 was centrifuged, and the supernatant rich in macromolecular degradation fragments was collected and freeze-dried under vacuum to obtain sturgeon cartilage gelatin.

[0056] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, in step D, the water-in-oil type primary emulsion (c) is pumped into the slowly stirred aqueous phase at a flow rate of 4-6 mL / min using a constant flow pump according to a mass ratio of c:water-in-oil type primary emulsion to external aqueous phase = 1:4-6. The stirring speed of the external aqueous phase is 300-400 rpm. After the water-in-oil type primary emulsion is pumped in, the external aqueous phase is continuously stirred and dispersed at 6°C for 8-12 min, so that the primary emulsion droplets are subjected to secondary mild shear cutting in the shear flow field of the external aqueous phase, thereby forming multiple emulsion droplets with a particle size of 30-80 μm, resulting in d multiple emulsion droplets.

[0057] In the aforementioned method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, step E specifically includes the following steps:

[0058] E1. Using an ice-water bath at 0℃ as the receiving liquid, the d-multiple emulsion droplets are continuously sprayed into the receiving liquid in the form of uniform and continuous micro-atomized droplets through a pressure spray device. After entering the receiving liquid, the d-multiple emulsion droplets are kept at a constant temperature for 40-45 minutes to obtain the e1 microsphere suspension.

[0059] E2. The E1 microsphere suspension is separated by gravity through a 350-400 mesh sieve, and the residue on the sieve is collected to obtain E2 solidified microsphere particles;

[0060] E3. The E2 solidified microspheres on the screen were rinsed multiple times with 4℃ deionized water, and then centrifuged and dehydrated at 4℃ to obtain whole fish-derived composite emulsion microspheres.

[0061] The aforementioned application of the whole fish-derived complex emulsion microspheres encapsulating phospholipase in the preparation of sturgeon caviar is as follows: the whole fish-derived complex emulsion microspheres are added to the surface of sturgeon eggs at an addition amount of 0.1 to 1.0 wt%, and then the mixture is stirred to ensure that the whole fish-derived complex emulsion microspheres are evenly attached to the surface of the sturgeon eggs.

[0062] Compared with the prior art, the present invention has the following characteristics:

[0063] (1) By limiting the preparation method of the whole fish source composite emulsion microspheres, the prepared whole fish source composite emulsion microspheres can be fully dispersed in the form of soft particles and evenly attached to the surface of sturgeon eggs after adding sturgeon eggs in the early stage of salting, and can penetrate with the free water between sturgeon eggs; while the sturgeon source phospholipase A2 in the emulsion microspheres will be released to the outside in a moderate and slow manner, and hydrolyze the egg membrane phospholipids in a targeted manner; at the same time, the trace amount of polyunsaturated fatty acids contained in the sturgeon fish oil as the intermediate oil phase will be partially released, supplementing the flavor precursor substances, synergistically promoting the generation of the overall flavor precursors, thereby enhancing the fat aroma and mature flavor of caviar;

[0064] (2) When sturgeon eggs enter the dehydration stage and produce a large amount of brine during the salt mixing process, the positive and negative charged collagen inside the microspheres will undergo violent water loss and conformational contraction with the chondroitin sulfate network under hypertonic conditions, resulting in a surge in internal mass transfer resistance; at the same time, the sturgeon oil droplets on the periphery are redistributed under compression, which strongly blocks the outward diffusion channel of sturgeon-derived phospholipase A2, thus achieving the slow-release function.

[0065] (3) When the sturgeon caviar is being canned, the milky microspheres on the surface of the sturgeon caviar can also play a role in interface lubrication and physical buffering, thereby significantly reducing caviar damage caused by mechanical stress; at the same time, the microsphere system with high water holding capacity can also improve the brine balance in the can and improve the uniformity of ripening.

[0066] (4) When sturgeon caviar is stored at low temperature, the protein-polysaccharide charge network of the milky microspheres shrinks, the structural strength increases, and the viscosity of the sturgeon oil in the outer layer increases, forming a strong hydrophobic barrier, which greatly reduces the probability of PLA2 diffusion and substrate contact, thereby inhibiting the excessive hydrolysis of sturgeon caviar during long-term storage; at the same time, the sturgeon oil dispersed in the milky microspheres can also effectively adsorb and integrate the free separated fat components in the system, significantly reducing the typical oil separation phenomenon of sturgeon caviar during storage; and through the adsorption effect of the microsphere interface, the loss of volatile flavor substances is moderately reduced, lipid oxidation and rancidity are slowed down, and the quality is stabilized in the long term.

[0067] (5) With the above combination, the emulsion microspheres of the present invention can dynamically reconstruct the interface through physical barriers, temperature and osmotic pressure response during the salting, dehydration, pressing and cold storage maturation of sturgeon caviar, thereby achieving synergistic regulation of the early flavor promotion and late oxidation inhibition of sturgeon caviar; and since the emulsion microspheres are constructed entirely from endogenous substrates homologous to sturgeon caviar, there is no need to introduce complex chemical or mechanical elimination processes in the later stages of sturgeon caviar processing and maturation, and they can be directly retained in situ as natural lipids and nutrient matrix in the final product, thereby improving the application effect of the emulsion microspheres;

[0068] Therefore, this invention can improve the flavor and quality of sturgeon caviar and extend its shelf life. Attached Figure Description

[0069] Figure 1 This is a fatty acid accumulation diagram of the blank control group in Experiment Example 1;

[0070] Figure 2 This is a fatty acid packing diagram of the PLA2 treatment group in Experiment Example 1;

[0071] Figure 3 This is a fatty acid accumulation diagram of the inhibitor group in Experiment Example 1;

[0072] Figure 4 This is the volatile fingerprint spectrum of the blank control group in Experiment Example 1 compared with the group's volatile fingerprint spectrum;

[0073] Figure 5 This is the volatile fingerprint spectrum of the PLA2-treated group in Experiment Example 1 and the comparison spectrum of the other group;

[0074] Figure 6 This is the volatile fingerprint spectrum of the inhibitor group in Experiment Example 1 compared with the group's spectrum;

[0075] Figure 7 This is a line graph showing the total sensory evaluation scores of the four groups of samples in Experiment Example 2;

[0076] Figure 8 This is a radar chart of the sensory evaluation of the four groups of samples in Experiment Example 2;

[0077] Figure 9 These are the GC-IMS fingerprints and GC-IMS comparison spectra of Experiment 1 and Experiment 2 in Experiment Example 2;

[0078] Figure 10 These are the GC-IMS fingerprints and GC-IMS comparison spectra of control samples 1 and 2 in Experimental Example 2. Detailed Implementation

[0079] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0080] Example 1. A method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, comprising the following steps:

[0081] A. Sturgeon skin collagen is extracted from sturgeon processing by-products. Then, the sturgeon skin collagen is dissolved in a buffer solution, and the pH of the buffer solution is controlled to be lower than the isoelectric point of the sturgeon skin collagen so that the surface of the sturgeon skin collagen molecules is positively charged, resulting in mixed solution a.

[0082] B. Mix the mixed solution a with sturgeon-derived phospholipase A2 at a mass ratio of 2:1 to form an enzyme mixed solution; then add sturgeon chondroitin sulfate solution with a strong negative charge dropwise to the enzyme mixed solution, thereby utilizing the spatial electrostatic attraction of positive and negative charges to form an enzyme-containing complex protein network fluid with moderate toughness and weak viscoelasticity. After the reaction, the aqueous phase b is obtained.

[0083] C. Sturgeon fish oil is used as the oil phase and mixed with the aqueous phase in b and emulsified to obtain c, a water-in-oil type primary emulsion;

[0084] D. Sturgeon skin collagen and sturgeon cartilage gelatin are dissolved together in deionized water and prepared as an interfacial continuous phase stabilizer solution to obtain an outer aqueous phase. Then, using c-type water-in-oil colostrum as the dispersed phase and the outer aqueous phase as the continuous phase, c-type water-in-oil colostrum is poured into the outer aqueous phase. The natural amphiphilicity and interfacial affinity of sturgeon skin collagen are used as an outer continuous phase stabilizer. It is then gently dispersed again by low-speed shearing or high-frequency microporous vibration to form d-type multiple emulsion droplets.

[0085] E. Multiple emulsion droplets are continuously introduced into an ice-water phase medium at 0°C for pure physical cold-induced solidification, which physically thickens the outer sturgeon fish oil and promotes the triple helix restructuring and tightening of the internal collagen, crosslinking with chondroitin sulfate, and rapidly forming the cartilage gelatin in the outer aqueous phase. The entire mixture is rapidly solidified at the interface, thus obtaining discrete, smooth-surfaced, all-fish-derived composite emulsion microspheres.

[0086] The buffer solution in step A is a 0.1 mol / L citrate-sodium citrate buffer solution. After the sturgeon skin collagen is dissolved in the citrate-sodium citrate buffer solution, a 1.5 wt% collagen solution is formed. The pH of the collagen solution is adjusted to 5.4 by the citrate-sodium citrate buffer solution, so that the surface of the sturgeon skin collagen molecules is positively charged.

[0087] The preparation process of sturgeon-derived phospholipase A2 in step B includes the following steps:

[0088] B11. Sturgeon caviar was added to Tris-HCl buffer pre-cooled to 4°C at a material-to-liquid ratio of 1:4 w / v. The concentration of Tris-HCl buffer was 0.05 mol / L and the pH value of Tris-HCl buffer was 8.5. Then, the mixture was homogenized at high speed for 2 min under ice bath conditions to fully break the cells and release endogenous enzymes, resulting in B11 homogenate.

[0089] B12. Centrifuge the b11 homogenate at 4℃ and 10000r / min for 20min, discard the bottom solid precipitate and collect the supernatant rich in the target enzyme protein to obtain the b12 crude enzyme solution.

[0090] B13. Under ice bath and continuous magnetic stirring conditions, add solid ammonium sulfate powder to the crude enzyme solution of b12 until the saturation of ammonium sulfate in the crude enzyme solution of b12 reaches 60%, to obtain crude enzyme solution of b13.

[0091] B14. First, stir the crude enzyme solution of b3 continuously in an ice bath for 30 minutes, and then let it stand at 4°C for 2 hours to salt out, to obtain the b14 salting out solution;

[0092] B15. Centrifuge the b4 salting-out solution at 4℃ and 10000r / min for 15min, discard the supernatant and collect the bottom protein precipitate to obtain the b15 precipitate;

[0093] B16. Add Tris-HCl buffer in portions until the b15 precipitate is completely reconstituted. Then repeat steps B13-B15 to perform secondary fractional salting out of the b15 precipitate to obtain a recrystallized protein precipitate, thereby improving the purity of the target enzyme and obtaining the b16 protein precipitate.

[0094] B17. After fully reconstituted the b16 protein precipitate with Tris-HCl buffer, it was transferred to a hydrophilic dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis bag was then immersed in ultrapure water at 4°C for continuous flow dialysis. The external dialysis solution was replaced every 2 hours to completely remove residual ammonium sulfate ions from the solution through mass transfer of concentration gradient. After multiple cycles, the b17 desalting enzyme solution was obtained.

[0095] B18. The b17 desalting enzyme solution, along with the dialysis bag, was embedded in a solid polyethylene glycol powder matrix for external water absorption and concentration until the fluid volume inside the dialysis bag was reduced to 1 / 5 of the initial volume, thus obtaining sturgeon-derived phospholipase A2.

[0096] The preparation process of sturgeon chondroitin sulfate solution in step B includes the following steps:

[0097] B21. Sturgeon skull and trunk skeleton are used as raw materials and heated in a water bath at 95°C until the bone and flesh tissue of the raw materials are in a state that is easy to physically separate. Then, the raw materials are removed, the fish meat is removed, and the skull and trunk skeleton are mechanically crushed into granules to obtain b21 fish bone granules.

[0098] B22. The fish bone particles of b21 were placed in a high-pressure reactor for high-pressure thermal liquefaction treatment. The reaction temperature was 110℃ and the reaction pressure was 0.15MPa. After continuous treatment for 2 hours, cartilage fluid of b22 was obtained.

[0099] B23. After cooling the B22 cartilage fluid to 55°C, adjust the pH of the B22 cartilage fluid to 7.0. Then, add a compound neutral protease to the B22 cartilage fluid at an addition rate of 0.2% of the B22 cartilage fluid mass. The compound neutral protease is specifically the compound neutral protease from Nanning Dongheng Huadao Biotechnology Co., Ltd. Then, enzymatically hydrolyze the solution at 55°C for 10 hours to obtain the B23 cartilage hydrolysate.

[0100] B24. After heating the B23 cartilage enzymatic hydrolysate to 90℃, the enzyme was inactivated for 10 minutes. Then, the large molecular proteins and inorganic salt bone residues in the B23 cartilage enzymatic hydrolysate were retained through a ceramic microfiltration membrane. The B23 cartilage enzymatic hydrolysate was then physically concentrated through a reverse osmosis membrane to obtain the B24 cartilage enzymatic hydrolysate.

[0101] B25. The enzymatic hydrolysate of B24 chondroitin was purified and refined by a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The retentate was then collected and dried to obtain B25 sturgeon chondroitin sulfate with a high charge density.

[0102] B26. Dissolve sturgeon chondroitin sulfate (b25) in 0.1 mol / L citrate-sodium citrate buffer solution to prepare a 0.5 wt% chondroitin sulfate solution, thus obtaining sturgeon chondroitin sulfate solution.

[0103] In step B, the a mixed solution is mixed with a quantitative amount of sturgeon-derived phospholipase A2 to achieve an enzyme activity concentration of 500 U / mL in the enzyme mixed solution; after mixing, the two are stirred at 200 rpm for 15 min at 4℃ to ensure uniform dispersion of the enzyme in the mixed solution.

[0104] In step B, according to the dry weight ratio of sturgeon skin collagen (i.e., sturgeon skin collagen from step A) to sturgeon chondroitin sulfate (i.e., b25 sturgeon chondroitin sulfate from step B25) = 4:1, sturgeon chondroitin sulfate solution was added dropwise to the enzyme mixture solution at a rate of 0.45 mL / min. During the dropwise addition, the enzyme mixture solution was continuously magnetically stirred at 250 rpm in a constant temperature water bath at 4℃. After the dropwise addition was completed, the enzyme mixture solution was stirred at 65 rpm in a constant temperature water bath at 4℃ for 30 min until the solution became a uniform, slightly milky, weakly viscoelastic complex coagulated fluid without macroscopic precipitation, thus obtaining the aqueous phase in step b.

[0105] The preparation process of sturgeon fish oil in step C includes the following steps:

[0106] C1. Using fresh sturgeon fat as raw material, the fat raw material is crushed and added to deionized water at a material-to-liquid ratio of 1:2 w / v. The pH is then adjusted to 7.8 to obtain mixture C1.

[0107] C2. Add 1.2% by mass of alkaline protease to the mixture of c1, and then enzymatically hydrolyze it at 48℃ for 2.0 h. After enzymatic hydrolysis, heat the solution to 85℃ for 10 min to inactivate the enzyme, and obtain the mixture of c2.

[0108] C3. Centrifuge the c2 mixture at 4℃ and 8000r / min for 15min, then separate and collect the upper light phase crude fish oil to obtain c3 crude fish oil;

[0109] C4. After heating the C3 crude fish oil to 60°C, add hot distilled water at a mass of 2.5% of the C3 crude fish oil mass. Then, stir slowly at 200 r / min for 30 min to allow colloidal impurities to fully absorb water and coagulate. After the solution has settled and separated into layers, remove the lower oil residue to obtain the C4 solution.

[0110] C5. Determine the acid value of solution C4, and add NaOH solution to solution C4 at an amount of 1.15 times the theoretical consumption of acid value for neutralization and deacidification. The mass fraction of NaOH solution is 5wt%. After mixing solution C4 and NaOH solution, stir and react at 55℃ for 20 min, then let stand to discard the soap residue at the bottom and separate the deacidified oil to obtain deacidified oil C5.

[0111] C6. Add activated clay to C5 deacidified oil at an addition rate of 4.0% of the mass of C5 deacidified oil, and then place the oil solution under vacuum degree ≤0.09MPa and temperature 82℃ and continuously stir and adsorb for 30 minutes to remove pigments and free peroxides from the oil solution to obtain C6 oil solution.

[0112] C7. C6 oil was pressure filtered through a 0.45μm microporous membrane to obtain clear and transparent C7 decolorized fish oil;

[0113] C8. The decolorized fish oil from C7 is introduced into a vacuum deodorization device and deodorized under reduced pressure and co-current conditions at a vacuum degree ≤0.095MPa and a temperature of 115℃ for 1.5h to completely remove volatile fishy odor substances such as low molecular weight aldehydes and ketones, thus obtaining sturgeon fish oil.

[0114] In step C, sturgeon oil and the aqueous phase in step b are mixed at a mass ratio of 3:1 and then placed in a high-shear emulsifier with a constant temperature circulating cooling jacket at 6°C for continuous shear emulsification. During the emulsification process, the shearing speed of the high-shear emulsifier is 4000 rpm and the shear emulsification time is 5 min, resulting in water-in-oil primary emulsion c. The average particle size of the aqueous phase droplets in the water-in-oil primary emulsion c is in the range of 0.5 to 2.0 μm.

[0115] The extraction methods for sturgeon skin collagen in steps A and D include the following steps:

[0116] D11. Using fresh sturgeon skin as raw material, each sturgeon skin raw material has a row of lateral bony plates and abdominal bony plates. After washing the sturgeon skin raw material, it is cut into several rectangular fish skin pieces to obtain d11 fish skin pieces.

[0117] D12. Place the d11 fish skin piece in boiling water with 2.5 times its weight and blanch for 45 seconds while stirring at a constant speed. Then remove the bone plates, scales and subcutaneous fat tissue of the d11 fish skin piece to obtain the d12 fish skin piece.

[0118] D13. Rinse the d12 fish skin pieces three times in water at 25℃, then put them into a chopper and grind them until they form square sturgeon skin scraps with a side length of 0.8 to 1.2 cm, which is d13 scraps;

[0119] D14. Soak the d13 scraps in a 0.45mol / L acetic acid solution at a material-to-liquid ratio of 1:25w / v, and then add food-grade pepsin at a mass of 1.0% of the dry weight of the sturgeon skin raw material to obtain the d14 mixture.

[0120] D15. The mixture of d14 was continuously stirred at 6°C for 24 hours, and then filtered through a 200-mesh sieve to remove the undegraded residue in the mixture, to obtain the supernatant of d15.

[0121] D16. Slowly add NaCl powder to the supernatant of d15 until the concentration is 1.0 mol / L, and then let the solution stand at 4℃ for 14 h to salt out, to obtain d16 salting out solution;

[0122] D17. After centrifuging the salting-out solution of d16, collect the flocculent protein precipitate, and then redissolve the flocculent protein precipitate with 0.10 mol / L acetic acid solution to obtain solution d17.

[0123] D18. Transfer the d17 solution to a dialysis bag with a molecular weight cutoff of 100 kDa, and dialyze continuously with deionized water at 4°C for 72 h. Replace the external dialysate every 6 h to completely desalt the solution. Then freeze-dry the dialysate under vacuum to obtain sturgeon skin collagen.

[0124] The preparation process of sturgeon cartilage gelatin in step D includes the following steps:

[0125] D21. Fresh sturgeon cartilage was crushed using a chopper to obtain cartilage particles with an average particle size of 2-4 mm. The cartilage particles were then washed and mixed with 0.10 mol / L NaHCO3 solution at a material-to-liquid ratio of 1:4 w / v and stirred continuously for 20 min to obtain mixture d21.

[0126] D22. Wash the d21 mixture with distilled water in the reverse direction until the wash solution is neutral. Then, take out the cartilage particles and mix them with 0.10 mol / L citric acid solution at a material-liquid ratio of 1:4 w / v for 20 min. Wash with distilled water until neutral to obtain d22 cartilage particles.

[0127] D23. Mix d22 cartilage granules with distilled water at a material-to-liquid ratio of 1:4 w / v and transfer the mixture into a high-pressure steam sterilizer. Extract the mixture at 120°C for 25 min to obtain d23 mixture.

[0128] D24. The mixture from d23 was continuously centrifuged at 6000 r / min for 20 min, and then the supernatant rich in macromolecular degradation fragments was collected and freeze-dried under vacuum to obtain sturgeon cartilage gelatin.

[0129] In step D, the water-in-oil type primary emulsion (c) and the external aqueous phase are pumped into the external aqueous phase at a flow rate of 5 mL / min using a constant flow pump. The stirring speed of the external aqueous phase is 350 rpm. After the water-in-oil type primary emulsion (c) is pumped in, the external aqueous phase is continuously stirred and dispersed at 6°C for 10 min. This allows the primary emulsion droplets to undergo secondary mild shear cutting in the shear flow field of the external aqueous phase, thereby forming multiple emulsion droplets with a particle size of 30–80 μm, resulting in multiple emulsion droplets (d).

[0130] Step E specifically includes the following steps:

[0131] E1. Using an ice-water bath at 0℃ as the receiving liquid, droplets of the d-multiple emulsion were introduced into a pressure spray device equipped with a 200μm microporous nozzle. The d-multiple emulsion droplets were continuously sprayed into the receiving liquid in the form of uniform and continuous micro-atomized droplets through the pressure spray device. After entering the receiving liquid, the d-multiple emulsion droplets were kept at a constant temperature for 42 minutes to obtain the e1 microsphere suspension.

[0132] E2. The E1 microsphere suspension is separated by gravity through a 350-mesh stainless steel microporous sieve, and the residue on the sieve is collected to obtain E2 solidified microsphere particles.

[0133] E3. The e2 solidified microspheres on the screen were rinsed multiple times with deionized water at 4℃ to completely remove the free sturgeon-derived phospholipase A2 and unadsorbed external aqueous phase components remaining on the outer surface of the microspheres. Then, the e2 solidified microspheres were centrifuged at 4000 rpm for 5 min at 4℃ and dehydrated to obtain off-white, uniform, discrete, and non-adhesive whole fish-derived composite emulsion microspheres.

[0134] This embodiment, by defining the extraction method of sturgeon skin collagen, can retain the surface charge activity of sturgeon skin collagen and remove impurities, enzymes, and fat components that may cause caviar off-flavors and unsaturated fatty acid oxidation. By defining the preparation method of sturgeon fish oil and sturgeon chondroitin sulfate, the thermal oxidative degradation of sensitive highly unsaturated fatty acids such as EPA and DHA in sturgeon oil can be avoided, while free fatty acids, peroxides, and volatile impurities that may damage the PLA2 enzyme activity in the microspheres and cause caviar off-flavors can be removed.

[0135] By adjusting the pH of the system in steps A and B, the electrostatic attraction between the dissociated collagen (positively charged amino groups) and chondroitin sulfate (negatively charged sulfate groups) can reach an equilibrium threshold, effectively inhibiting excessive aggregation and precipitation or non-reaction. Under a static low-shear flow field, the two macromolecules undergo precise charge self-assembly, constructing a dense physical network. This network relies on strong Coulomb forces to physically confine the water-soluble PLA2 molecules within the spatial grid, significantly reducing the free movement of enzyme molecules, thereby raising the thermal denaturation temperature of natural fish collagen and synergistically improving the thermal stability of the core enzyme structure.

[0136] By utilizing medium-to-high shear forces in steps C and D to drive the highly miniaturized aqueous droplets, the encapsulation rate is ensured. Simultaneously, the shear heat is dissipated at a low temperature of 6°C, mitigating the destructive effects of thermal effects on endogenous enzyme activity. Strictly limiting the shear rate during the re-emulsion dispersion stage reduces the impact of fluid shear stress on the multi-layered structure, preventing primary emulsion droplet rupture and premature enzyme leakage. Furthermore, the collagen component in the external aqueous phase spontaneously adsorbs at the fluid interface due to its amphiphilic nature, stabilizing the droplets through steric hindrance and effectively preventing spontaneous aggregation and demulsification of microspheres during large-scale production.

[0137] By defining the preparation method of the whole-fish-derived composite emulsion microspheres in step E, the lipid components in the external phase sturgeon oil can be physically thickened when the d-multiplex emulsion droplets come into contact with an ice-water bath, forming a protective shell. On the other hand, the collagen adsorbed inside and at the interface of the d-multiplex emulsion droplets undergoes a spatial conformational change, and its hydrogen bonds reassemble at low temperature, physically cross-linking with chondroitin sulfate segments. This synergistic network, combined with the cold gelation properties of chondroitin gelatin in the external aqueous phase, endows the emulsion microspheres with excellent shear and compressive mechanical strength. Subsequent micropore washing removes residual free enzymes from the microsphere surface, avoiding uncontrollable direct catalysis before salt mixing and ensuring the precision of enzyme activity timing control.

[0138] Example 2. Application of whole fish-derived complex emulsion microspheres encapsulated with phospholipase in the preparation of sturgeon caviar. Specifically, the whole fish-derived complex emulsion microspheres are the same as those in Example 1. The application method is as follows: after the sturgeon eggs are washed, rubbed, rinsed, drained, and graded, the whole fish-derived complex emulsion microspheres are added to the surface of the sturgeon eggs at a dosage of 0.5 wt%. Then, the whole fish-derived complex emulsion microspheres are stirred and mixed to ensure that the whole fish-derived complex emulsion microspheres are evenly attached to the surface of the sturgeon eggs. The sturgeon eggs are then placed in a constant temperature workshop at 10°C for 4 hours to allow the whole fish-derived complex emulsion microspheres encapsulated with PLA2 to fully exert their effects, resulting in the finished sturgeon caviar.

[0139] When sturgeon caviar is salted, 3.7% w / w salt is added for curing. This allows the whole-fish-derived complex emulsion microspheres to adhere tightly to the roe due to their excellent interfacial adhesion, and to benefit from the abundant free water permeation between the roe. The physical pressure difference between the inside and outside of the whole-fish-derived complex emulsion microspheres drives the moderate release of PLA2, which directionally catalyzes the degradation of lecithin in the roe membrane to release free fatty acids. At the same time, trace amounts of polyunsaturated fatty acids from the fish oil are released synergistically, supplementing flavor precursors and giving caviar its characteristic mature fatty and nutty aromas.

[0140] During the canning and pressing process of sturgeon caviar, the hypertonic environment created by salting drives dehydration within the microspheres through a chemical potential difference, causing the internal protein-polysaccharide charge network to contract tightly and the micropore size to decrease. This compact physical structure increases the diffusion resistance of the macromolecular phospholipase A2, thereby moderately blocking the mass transfer channels from the enzyme molecules to the surrounding substrate. Simultaneously, the all-fish-derived composite emulsion microspheres between the sturgeon eggs can effectively absorb stress through their own deformation, preventing localized concentration of mechanical stress on the surface of the sturgeon eggs and thus reducing the breakage rate. The high water-holding capacity of the all-fish-derived composite emulsion microspheres also helps to smooth out local brine concentration gradients, improving the uniformity of the sturgeon caviar's ripening process.

[0141] After being sealed in pressurized cans, sturgeon caviar is stored long-term in a cold storage facility at -2℃ to 2℃ to allow its quality to mature. During the cold storage process, the self-assembled network inside the whole fish-derived composite emulsion microspheres continuously shrinks, and the viscosity of the outer sturgeon oil increases, forming a strong physical hydrophobic barrier. This significantly reduces the probability of PLA2 substrate contact, inhibits excessive lipid oxidation caused by PLA2 in the later stages of processing, and greatly mitigates excessive hydrolysis and rancidity. Simultaneously, the microsphere interface can physically adsorb the free oil that spontaneously separates during the later stages of sturgeon caviar storage, thus locking in flavor to some extent. This also improves the typical technical problems of oil separation and bitterness / rancidity in sturgeon caviar, contributing to extended shelf life and stabilizing and improving the quality of the sturgeon caviar.

[0142] Experimental Example 1: Three sets of test samples were prepared according to the following steps:

[0143] S1. Weigh 3.0 g of freeze-dried sturgeon caviar powder into centrifuge tubes, add 20 mL of chloroform-methanol mixture (2:1, V / V), vortex for 2 min, and then extract with ultrasonic assistance for 20 min. Centrifuge at 4℃ and 8000 r / min for 10 min, and collect the lower organic phase. Repeat the extraction twice, then combine all organic phases. Transfer the combined organic phase to a rotary evaporator and evaporate under reduced pressure at 40℃ until it no longer boils, obtaining the total lipid extract of sturgeon caviar. Place the above total lipid extract into centrifuge tubes, slowly add 5 times the volume of pre-cooled acetone, mix thoroughly, and then place in a -20℃ freezer for 4 h to allow the phospholipids to fully precipitate. After removing the extract, centrifuge at 4℃ and 10000 r / min for 15 min, discard the supernatant, and collect the yellow precipitate at the bottom, which is the crude phospholipid. Finally, the collected phospholipid precipitate was fully dissolved in a small amount of chloroform-methanol mixture (2:1, V / V), and the solvent was slowly dried under a nitrogen blower at 40℃ to obtain purified sturgeon caviar phospholipids.

[0144] S2. Weigh 5.00 g of standard sturgeon caviar and add 20 mL of pre-cooled 0.05 mol / L Tris-HCl buffer (pH 8.5). Homogenize for 2 min on ice. Then centrifuge at 10,000 rpm for 20 min at 4 °C, discard the precipitate, and collect the supernatant as the crude enzyme solution. Slowly add ammonium sulfate powder to the crude enzyme solution until 60% saturation, stir on ice for 30 min, and let stand at 4 °C for 2 h. Then centrifuge at 10,000 rpm for 15 min at 4 °C, discard the supernatant, and collect the protein precipitate. Dissolve the precipitate thoroughly with a small amount of Tris-HCl buffer, add ammonium sulfate again for a second salting-out, let stand, centrifuge, and collect the precipitate. Redissolve the precipitate and transfer it to a dialysis bag. Dialyze in ultrapure water at 4 °C, changing the dialysis solution every 2 h for a total of 4 times to completely remove the ammonium sulfate. After dialysis, the dialysis bag was placed in solid PEG20000 for water absorption and concentration until the enzyme solution volume was significantly reduced, resulting in concentrated and purified PLA2 enzyme solution.

[0145] S3. A simulated system was established by combining the extracted purified sturgeon caviar phospholipids and PLA2 enzyme solution. The system containing only phospholipids served as the blank control group, the system with added PLA2 enzyme solution served as the PLA2 treatment group, and the system with further addition of PLA2 inhibitor served as the inhibitor group. Samples from each group were stored at 25℃ and collected at 0, 3, 5, 7, and 9 days to obtain test samples for the three groups.

[0146] (1) Fatty acid determination: Accurately weigh 10 mg of each of the three groups of test samples, add 2% H2SO4-methanol solution and mix thoroughly. After purging with nitrogen and sealing, react in a water bath at 80℃ for 1 h. After cooling to room temperature, add 1 mL of deionized water and 1 mL of n-hexane respectively, vortex to extract, allow to stand for layering, collect the upper organic phase, filter through a 0.22 μm organic phase filter membrane and then analyze. The fatty acid composition of sturgeon caviar phospholipids was determined by gas chromatography-mass spectrometry. The chromatographic conditions were as follows: HP-5ms capillary column; injection port temperature 250℃; carrier gas was high-purity helium, flow rate 1.5 mL / min; split ratio 10:1; injection volume 1.0 μL.

[0147] The 13 fatty acids (C14:0-C22:6n3) in the three groups of samples were quantitatively analyzed during storage using the above method. The dynamic changes of different treatment groups during storage days 0, 3, 5, 7, and 9 were also investigated. The results are as follows: Figures 1-3 As shown in the figure, on day 3 of storage, the content of all free fatty acids in the PLA2 treatment group increased significantly compared with the baseline on day 0. Among them, the increase of n-3 long-chain polyunsaturated fatty acids was the most significant. The content of DHA (C22:6n3) increased from 565.75 mg / kg to 1420.54 mg / kg (+151.1%), and the content of EPA (C20:5n3) increased from 194.85 mg / kg to 435.88 mg / kg (+123.7%). In addition, the increases of C20:2 (+114.8%), C18:2n6c (+112.7%) and C16:0 (+109.9%) all exceeded 100%. The total fatty acid content increased from 2617.5 mg / kg to 5652.1 mg / kg (+116.0%).

[0148] In contrast, the inhibitor group showed effective inhibition on day 3, with all fatty acid contents below or close to baseline levels. The most significant decreases were observed in C24:1 (-23.4%), C20:4n6 (-22.5%), and C18:0 (-19.4%), with total fatty acids at only 2311.3 mg / kg (-11.7%). The blank control group showed a natural hydrolysis trend on day 3, with increases in each fatty acid ranging from 26.0% (C24:1) to 106.2% (C22:6n3), reaching a total fatty acid content of 4630.4 mg / kg (+76.9%). Compared to the FFA release produced by the blank control group on day 3 of storage, the PLA2 treatment group, through the external addition of PLA2, significantly accelerated the hydrolysis of phospholipid substrates, causing a sharp increase in total FFA content to 5652.1 mg / kg, an increase of approximately 22% compared to the blank control group. This indicates that phospholipase A2 can hydrolyze phospholipids to generate free fatty acids. Compared with the blank control group, the addition of PLA2 significantly accelerated the hydrolysis of phospholipids and the release of fatty acids. The fluctuation in fatty acid content caused by the addition of the inhibitor was due to the synergistic effect of phospholipid auto-oxidation and residual PLA2.

[0149] (2) Gas chromatography-ion mobility spectrometry (GC-IMS) analysis: Accurately weigh 1 mg of each of the three groups of test samples and transfer them to 20 mL headspace vials. Incubate at 60 °C and 500 r / min for 20 min. The injection needle temperature was 85 °C, and the carrier gas was 99.999% pure nitrogen. Inject 500 μL of sample in splitless mode, with a cleaning time of 30 s. Use a DB-5 column at 60 °C for 25 min, and an IMS temperature of 45 °C for 25 min.

[0150] Test results as follows Figures 4-6 As shown, the blank control group contained 16 volatile substances, mainly including 2,3-pentanedione, 3-methyl-2-butenal, trans-2-butenal, propional, and 3-pentanone. The content of these substances varied at different storage times. During storage, the contents of octanal, hexanal, and 2,3-butanedione significantly increased. These aldehydes and ketones typically impart floral, creamy, and fruity aromas. Furthermore, butyraldehyde was only detected in the early stages of storage, suggesting that butyraldehyde may be a volatile component unique to fresh phospholipids.

[0151] Compared to the blank control group, the changes in volatile compound content in the PLA2-treated group were more pronounced. This is because PLA2 acts on phospholipids, causing them to release fatty acids such as ARA, DHA, and EPA. These fatty acids oxidize over time, producing aldehydes and ketones, which contribute to aroma. Furthermore, octanal, propanol, 3-methyl-2-butenal, 2,3-2-butanedione, 1,3-hexadiene, methyl 2-methyl-2-acrylate, and ethyl formate were relatively high in the middle of storage. As oxidation continued, volatile compounds with unpleasant, pungent odors, such as propanal and hexanal, were produced, reducing the edibility of the food. This indicates that the hydrolysis of PLA2 in the early stages provided the substrate, lipid oxidation in the middle of storage led to an increase in aldehydes and ketones, and microbial metabolism in the later stages promoted the accumulation of esters and alcohols. Overall, this signifies that the flavor quality and phospholipid structure of the sturgeon caviar had begun to deteriorate irreversibly.

[0152] The fingerprint chromatograms of the inhibitor group showed very low levels of several volatile compounds, including nonanal, butyraldehyde, hexanal, propionaldehyde, 2-butanone, 3-methyl-2-butenal, 1-propanone, 3-pentanone, 1-hexene, and 2-methyl-2-butene. This is because the inhibitors blocked the catalytic action of PLA2, preventing the degradation of phospholipids to produce precursors for these volatile substances. However, trace amounts of PLA2, which were not completely inhibited, continued to promote the breakdown of phospholipids into fatty acids, resulting in the presence or slight detection of these substances. Several characteristic volatile compounds were detected in the inhibitor group, including octanal, 2,3-pentanedione, 2,3-butanedione, and 3-pentanol. Among these, short-chain diketones such as 2,3-pentanedione and 2,3-butanedione mainly originate from the β-oxidation or non-enzymatic degradation of free fatty acids and generally provide a rich creamy and sweet aroma.

[0153] The above experimental results demonstrate that PLA2 has a significant effect on regulating the quality of sturgeon caviar. This invention uses whole fish-derived composite emulsion microspheres containing PLA2 to encapsulate sturgeon caviar during the later stages of production, thereby achieving synergistic regulation of early-stage flavor enhancement and late-stage oxidation inhibition, thus improving the flavor and storage quality of sturgeon caviar.

[0154] Experimental Example 2: This experimental example sets up Experimental Sample 1, Experimental Sample 2, and Comparative Sample 1 and Comparative Sample 2 respectively. Experimental Sample 1 is made by attaching the whole fish-derived composite emulsion microspheres prepared in Example 1 to the surface of sturgeon eggs according to the application method in Example 2. Then, the sturgeon eggs are subjected to salting, canning and storage treatment in sequence according to the standard sturgeon caviar processing procedure.

[0155] Experiment 2, based on the process of Example 1, removed the step of adding sturgeon-derived phospholipase A2 in step B, while the rest of the process was the same as in Example 1, thus preparing PLA2-free emulsion microspheres. These emulsion microspheres were then attached to the surface of sturgeon eggs according to the application method of Example 2, and the sturgeon eggs were then subjected to salting, canning, and storage treatments according to the standard sturgeon caviar processing procedure.

[0156] The comparison product directly processes sturgeon roe according to the standard sturgeon caviar processing procedure, including draining, grading, salting, canning, and storage.

[0157] Comparative product 2 involved processing sturgeon eggs according to the standard sturgeon caviar processing procedure, including draining, grading, salting, canning, and storage. During the draining, grading, and salting processes, the sturgeon-derived phospholipase A2 from Example 1 was evenly sprayed onto the surface of the sturgeon eggs at an addition rate of 800U of free enzyme activity per 1kg of sturgeon eggs and thoroughly mixed in a container to ensure even distribution.

[0158] All four groups of samples were stored in a container at 0℃ for one week. The samples were then removed and stored at 4℃ for another week. Samples were taken and measured at 0, 3, 5, 7 and 9 days.

[0159] A professional evaluation team comprehensively assessed four groups of sturgeon caviar samples based on indicators such as color, taste, and aroma, thus directly reflecting the impact of different processing and storage times on the quality of sturgeon caviar. Specifically, a team of 10 trained sensory evaluators conducted sensory evaluations of the different sturgeon caviar samples. Before evaluation, all samples were equilibrated to the same temperature at 25°C and then randomly numbered, and the evaluation was conducted blindly. Sensory evaluation indicators included appearance, color, gloss, flavor characteristics, and texture characteristics, each scored on a 20-point scale, as shown in Table 1. Each group of samples was evaluated three times, and the average value was used as the final sensory score.

[0160] Table 1 Sensory Evaluation Scores of Sturgeon Caviar

[0161]

[0162] Sensory evaluation comprehensive score results as follows Figure 7As shown, the overall scores of different samples showed a continuous downward trend during storage, indicating that the quality of sturgeon caviar gradually deteriorated with prolonged storage. However, the rate of decline varied significantly among the samples, reflecting the regulatory effect of PLA2 and the whole-fish-derived complex emulsion microspheres containing PLA2 on the quality changes of sturgeon caviar. The differences between groups were small from 0 to 3 days, indicating that the quality changes were not significant in the early stages of storage. From 3 to 7 days of storage, the scores of the groups began to differentiate significantly, with the control sample 2 showing a faster decline, while the experimental sample 1 showed a relatively slower decline. From 7 to 9 days of storage, control sample 2 had the lowest score, indicating the most severe quality deterioration, while experimental sample 1 maintained a relatively high level, suggesting that the whole-fish-derived complex emulsion microspheres containing PLA2 slowed down the quality decline process.

[0163] Sensory evaluation radar chart as follows Figure 8 As shown, the decline in odor and taste was most pronounced with prolonged storage. Comparative product 2 showed the most significant decrease in odor indicators in the later stages, characterized by increased sourness and off-flavors, which is closely related to lipid oxidation and the accumulation of acidic substances. Simultaneously, its taste and gloss declined rapidly, indicating accelerated lipid structure damage and changes in moisture content. In contrast, experimental product 1 showed a smaller overall decline in all indicators, particularly maintaining good odor and appearance. This suggests that the all-fish-derived complex emulsion microspheres reduce fatty acid release and subsequent oxidation reactions, thereby delaying the formation of undesirable flavor compounds. Comparative product 1 showed a moderate level of change, indicating that its quality deterioration was mainly controlled by spontaneous oxidation. Experimental product 2 had a lower overall score, indicating that the all-fish-derived complex emulsion microspheres without PLA2 could not achieve the flavor regulation effect of initial enhancement followed by subsequent suppression. Overall, by synergistically introducing the processing strategy of all-fish-derived complex emulsion microspheres, including phospholipase A2, into the standard process flow of sturgeon caviar enterprises, the flavor and overall sensory quality of sturgeon caviar can be effectively improved, and the quality stability during storage can be enhanced.

[0164] The differences in volatile compounds among the four groups of sturgeon caviar samples were analyzed by GC-IMS, and the results are as follows: Figures 9-10As shown in the figure, fingerprint spectroscopy allows for a relatively intuitive comparison of differences in volatile substances and identification of their dynamic changes. Each row in the spectroscopy represents a sample, and each column represents a signal peak of a volatile substance. Using the NIST database, 46 volatile compounds were screened from four groups of sturgeon caviar, with 45 compounds in Comparative Standard 1, 41 in Comparative Standard 2, 46 in Experimental Standard 1, and 40 in Experimental Standard 2. Some compounds may exhibit multiple signal values ​​or response points due to differences in concentration. Hexanal, heptanal, nonanal, decanal, 2-hexenal, and 2-octenal were observed in different treatment groups; these volatile substances are typical characteristic products of the autoxidation of unsaturated fatty acids. (E,E)-2,4-heptadienal, (E,E)-2,4-octadienal, and 1-penten-3-ol were observed only in Comparative Sample 1, while 3-nonanone, 2-pentylfuran, and (E,E)-2,4-decadienal were detected only in Comparative Sample 2. The production of these substances indicates an increased degree of lipid oxidation, contributing fruity and oily flavors to the sturgeon caviar. Notably, Sample 1 contained more volatile compounds than the other groups. This suggests that when the sturgeon caviar is encapsulated in PLA2 whole-fish-derived complex emulsion microspheres, the metabolic pathways that would normally be induced by PLA2 during the later stages of storage—such as the hydrolysis of phospholipids like PC and PE to produce fatty acids or further metabolism to form various volatile compounds—are inhibited. Instead, non-enzymatic oxidation or other hydrolytic reactions occur during storage, resulting in a more diverse range of secondary metabolites. The experimental results above show that when the whole fish-derived composite emulsion microspheres of the present invention are applied to sturgeon caviar, they can achieve synergistic regulation of the early flavor enhancement and late oxidation inhibition of sturgeon caviar, thereby improving the flavor and storage quality of sturgeon caviar.

Claims

1. A method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase, characterized in that, Includes the following steps: A. Sturgeon skin collagen is extracted from sturgeon processing by-products. Then, the sturgeon skin collagen is dissolved in a buffer solution, and the pH of the buffer solution is controlled to be lower than the isoelectric point of the sturgeon skin collagen so that the surface of the sturgeon skin collagen molecules is positively charged, resulting in mixed solution a. B. Mix the mixed solution a with sturgeon-derived phospholipase A2 at a mass ratio of 2:1 to form an enzyme mixed solution; then add a strongly negatively charged sturgeon chondroitin sulfate solution dropwise to the enzyme mixed solution, and the aqueous phase b is obtained after the reaction. C. Sturgeon fish oil is used as the oil phase and mixed with the aqueous phase in b and emulsified to obtain c, a water-in-oil type primary emulsion; D. Sturgeon skin collagen and sturgeon cartilage gelatin are dissolved together in deionized water to obtain an external aqueous phase. Then, the c water-in-oil type colostrum is poured into the external aqueous phase and gently dispersed again by low-speed shearing or high-frequency microporous vibration to form d multiple emulsion droplets. E. Multiple emulsion droplets were continuously introduced into an ice-water phase medium at 0°C for pure physical cold-induced solidification, which physically thickened the outer sturgeon oil and promoted the triple helix restructuring and tightening of the internal collagen, and crosslinked with chondroitin sulfate to obtain whole fish-derived composite emulsion microspheres.

2. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1, characterized in that: The buffer solution in step A is a 0.1 mol / L citrate-sodium citrate buffer solution. After the sturgeon skin collagen is dissolved in the citrate-sodium citrate buffer solution, a 1.5 wt% collagen solution is formed. The pH value of the collagen solution is adjusted to 5.2-5.6 by the citrate-sodium citrate buffer solution, so that the surface of the sturgeon skin collagen molecules is positively charged.

3. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1, characterized in that, The preparation process of sturgeon-derived phospholipase A2 in step B includes the following steps: B11. Sturgeon caviar was added to Tris-HCl buffer at a material-to-liquid ratio of 1:4 w / v, and then homogenized at high speed under ice bath conditions to fully break the cells and release endogenous enzymes, resulting in B11 homogenate. B12. Centrifuge the homogenized solution of b11, discard the solid precipitate at the bottom and collect the supernatant to obtain crude enzyme solution of b12; B13. Under ice bath and continuous magnetic stirring conditions, add solid ammonium sulfate powder to the crude enzyme solution of b12 until the saturation of ammonium sulfate in the crude enzyme solution of b12 reaches 60%, to obtain crude enzyme solution of b13. B14. The crude enzyme solution of b13 was subjected to continuous stirring in an ice bath and then allowed to stand for salting out to obtain the b14 salting out solution. B15. Centrifuge the b14 salting-out solution, discard the supernatant and collect the bottom protein precipitate to obtain the b15 precipitate; B16. Add Tris-HCl buffer in portions until the b15 precipitate is completely reconstituted. Then repeat steps B13-B15 to perform secondary fractionation salting out of the b15 precipitate to obtain the b16 protein precipitate. B17. After fully reconstituted the b16 protein precipitate with Tris-HCl buffer, it was transferred to a hydrophilic dialysis bag. The dialysis bag was then immersed in ultrapure water at 4°C for continuous flow dialysis. After multiple cycles, the b17 desalting enzyme solution was obtained. B18. The b17 desalting enzyme solution, along with the dialysis bag, was embedded in a solid polyethylene glycol powder matrix for external water absorption and concentration until the fluid volume inside the dialysis bag was reduced to 1 / 4 to 1 / 6 of the initial volume, thus obtaining sturgeon-derived phospholipase A2. The preparation process of sturgeon chondroitin sulfate solution in step B includes the following steps: B21. Sturgeon skull and trunk skeleton are used as raw materials and heated in a high-temperature water bath. After the raw materials are removed, the fish meat is removed and the skull and trunk skeleton are mechanically crushed into granules to obtain b21 fish bone granules. B22. Place the b21 fish bone particles into a high-pressure reactor for high-pressure thermal liquefaction treatment to obtain b22 cartilage fluid; B23. Adjust the pH of the b22 cartilage fluid to 6.8-7.2, then add a compound neutral protease to the b22 cartilage fluid at an addition rate of 0.15-0.30% of the b22 cartilage fluid mass, and then enzymatically hydrolyze at a constant temperature of 52-58℃ for 8-12 hours to obtain the b23 cartilage hydrolysate. B24. After the enzyme inactivation of the b23 cartilage hydrolysate at high temperature, the large molecular proteins and inorganic salt bone residues in the b23 cartilage hydrolysate are retained by a ceramic microfiltration membrane, and then the b23 cartilage hydrolysate is physically concentrated by a reverse osmosis membrane to obtain the b24 cartilage hydrolysate. B25. The enzymatic hydrolysate of B24 chondroitin was purified and refined by a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The retentate was then collected and dried to obtain B25 sturgeon chondroitin sulfate. B26. Dissolve sturgeon chondroitin sulfate (b25) in 0.1 mol / L citrate-sodium citrate buffer solution to prepare a 0.5 wt% chondroitin sulfate solution, thus obtaining sturgeon chondroitin sulfate solution.

4. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1 or 3, characterized in that, In step B, the mixed solution a is mixed with a quantitative amount of sturgeon-derived phospholipase A2 to achieve an enzyme activity concentration of 400–600 U / mL in the enzyme mixture.

5. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1 or 3, characterized in that: In step B, according to the dry weight ratio of sturgeon skin collagen to sturgeon chondroitin sulfate = 4:1, sturgeon chondroitin sulfate solution is added dropwise to the enzyme mixture at a rate of 0.3-0.6 mL / min. During the dropwise addition, the enzyme mixture is continuously magnetically stirred in a constant temperature water bath at 4℃. After the dropwise addition is completed, the enzyme mixture is stirred in a constant temperature water bath at 4℃ for another 2-35 min to obtain the aqueous phase in step b.

6. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1, characterized in that, The preparation process of sturgeon fish oil in step C includes the following steps: C1. Using fresh sturgeon fat as raw material, the fat raw material is crushed and added to deionized water at a material-to-liquid ratio of 1:2 w / v. The pH is then adjusted to 7.5-8.0 to obtain mixture C1. C2. Add 1.0-1.5% (w / w) of alkaline protease to the mixture in c1, and then enzymatically hydrolyze it at 45-50℃ for 2.0 h. After hydrolysis, heat the solution to inactivate the enzyme, and obtain the mixture in c2. C3. The mixture of C2 is centrifuged, and then the upper light phase crude fish oil is collected by separation to obtain C3 crude fish oil; C4. After heating the C3 crude fish oil to 55-65℃, add hot distilled water at a mass of 2.0-3.0% of the C3 crude fish oil. Then, stir to allow the colloidal impurities to fully absorb water and coagulate. After the solution is allowed to stand and separate into layers, separate and remove the lower oil residue to obtain the C4 solution. C5. Determine the acid value of solution C4, and add NaOH solution to solution C4 at an amount of 1.1 to 1.2 times the theoretical consumption of acid value. Neutralize and deacidify by stirring, and then collect the deacidified oil by separation to obtain deacidified oil C5. C6. Add activated clay to C5 deacidified oil at an addition rate of 3.0-5.0% of the oil mass, and then place the oil under vacuum of ≤0.09MPa and temperature of 80-85℃ for continuous stirring and adsorption to remove pigments and free peroxides from the oil, thus obtaining C6 oil. C7. C6 oil is pressure filtered through a microporous membrane to obtain C7 decolorized fish oil; C8. Deodorize the c7 decolorized fish oil by depressurization and co-current flow under vacuum conditions of ≤0.095MPa and temperature of 110~120℃ to obtain sturgeon fish oil.

7. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1 or 6, characterized in that: In step C, sturgeon oil and aqueous phase b are mixed at a mass ratio of 3:1 and then placed in a high-shear emulsifier with a 6°C constant temperature circulating cooling jacket for continuous shear emulsification to obtain water-in-oil primary emulsion c.

8. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1, characterized in that, The extraction methods for sturgeon skin collagen in steps A and D include the following steps: D11. Using fresh sturgeon skin with lateral and ventral sacs as raw material, the sturgeon skin raw material is cut to obtain several fish skin pieces, resulting in d11 fish skin pieces. D12. Place the d11 fish skin piece in boiling water and blanch for 30-60 seconds while stirring at a constant speed. Then remove the bone plates, scales and subcutaneous fat tissue of the d11 fish skin piece to obtain the d12 fish skin piece. D13. The fish skin block d12 is rinsed and crushed in sequence to form square sturgeon skin fragments, resulting in fragment d13. D14. Soak the d13 scraps in a 0.3-0.6 mol / L acetic acid solution at a material-to-liquid ratio of 1:25-30 w / v, and then add pepsin at a mass of 0.8-1.2% of the dry weight of the sturgeon skin raw material to obtain the d14 mixture. D15. Stir the d14 mixture continuously at 4-8℃ for 20-28 hours, then filter to remove the undegraded residue in the mixture to obtain the d15 supernatant. D16. Slowly add NaCl powder to the supernatant of d15 until the concentration is 0.9-1.2 mol / L, and then let the solution stand at 4℃ for 12-16 h to salt out, to obtain d16 salting-out solution; D17. After centrifuging the salting-out solution of d16, collect the flocculent protein precipitate, and then redissolve the flocculent protein precipitate with 0.05-0.15 mol / L acetic acid solution to obtain solution d17. D18. Transfer the d17 solution to a dialysis bag with a molecular weight cutoff of 50-100 kDa, dialyze continuously with deionized water at 4°C for 60-72 h, and then freeze-dry the dialysate under vacuum to obtain sturgeon skin collagen. The preparation process of sturgeon cartilage gelatin in step D includes the following steps: D21. Fresh sturgeon cartilage is crushed to obtain cartilage particles. Then, the cartilage particles are mixed with 0.05-0.15 mol / L NaHCO3 solution at a material-to-liquid ratio of 1:3-5 w / v and stirred continuously for 15-30 min to obtain d21 mixture. D22. Wash the d21 mixture with distilled water in the reverse direction until the wash solution is neutral. Then, take out the cartilage particles and mix them with 0.05-0.15 mol / L citric acid solution at a material-to-liquid ratio of 1:3-5 w / v for 15-30 min. Wash with distilled water until neutral to obtain d22 cartilage particles. D23. Mix d22 cartilage granules with distilled water at a material-to-liquid ratio of 1:3 to 5 w / v, and then extract by hot pressing at 115 to 125°C for 20 to 30 min to obtain d23 mixture; D24. The mixture from d23 was centrifuged, and the supernatant rich in macromolecular degradation fragments was collected and freeze-dried under vacuum to obtain sturgeon cartilage gelatin.

9. The method for preparing whole-fish-derived composite emulsion microspheres encapsulating phospholipase according to claim 1, characterized in that, Step E specifically includes the following steps: E1. Using an ice-water bath at 0℃ as the receiving liquid, the d-multiple emulsion droplets are continuously sprayed into the receiving liquid in the form of uniform and continuous micro-atomized droplets through a pressure spray device. After entering the receiving liquid, the d-multiple emulsion droplets are kept at a constant temperature for 40-45 minutes to obtain the e1 microsphere suspension. E2. The E1 microsphere suspension is separated by gravity through a 350-400 mesh sieve, and the residue on the sieve is collected to obtain E2 solidified microsphere particles; E3. The E2 solidified microspheres on the screen were rinsed multiple times with 4℃ deionized water, and then centrifuged and dehydrated at 4℃ to obtain whole fish-derived composite emulsion microspheres.

10. The application of the whole fish-derived complex emulsion microspheres encapsulating phospholipase as described in any one of claims 1-9 in the preparation of sturgeon caviar, characterized in that: After adding the whole fish-derived composite emulsion microspheres to the surface of sturgeon eggs at an addition rate of 0.1–1.0 wt%, the whole fish-derived composite emulsion microspheres are then mixed evenly to ensure uniform adhesion to the surface of the sturgeon eggs.

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