Glycosylated coconut protein and preparation method and application thereof

Abstract

The application belongs to the technical field of biochemistry, and discloses a glycosylated coconut protein as well as a preparation method and application thereof. The glycosylated coconut protein is prepared by using konjac glucomannan to modify the coconut protein. After the modification, the solubility, stability and emulsifying property of the coconut protein are simultaneously improved. The solubility of the glycosylated coconut protein under acidic conditions is significantly improved, the denaturation temperature is increased to 87.05 DEG C, which is increased by 26.84% compared with the coconut protein, the water holding capacity and oil holding capacity are increased by 42.79% and 30.58%, respectively, the emulsifying activity is increased to 3.63 m 2 / g, and the emulsion stability is increased to 84.77%. The preparation raw material of the glycosylated coconut protein is widely sourced and low in price. In the modification process, no toxic and harmful chemical reagents are added, and the glycosylated coconut protein has the advantages of low cost, high safety, strong process repeatability, easy industrialization and the like. The glycosylated coconut protein has important significance for promoting the high-value utilization and industrial application of the coconut protein.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology and relates to a glycosylated coconut protein, its preparation method and application. Background Technology

[0002] Coconut protein (CP) is a high-quality plant protein found in coconut processing byproducts such as coconut oil and coconut water. Its amino acid composition is balanced, rich in glutamic acid, arginine, and essential amino acids. The proportion of essential amino acids in coconut protein reaches 33%–38%, conforming to the FAO / WHO recommended model. Coconut protein also has the advantages of high digestibility and absorption, and lower allergenicity compared to soy protein. With a global annual coconut production exceeding 60 million tons, coconut protein, as a processing byproduct, is an abundant resource and a plant protein resource with significant development potential. However, in practical applications, coconut protein suffers from poor solubility, insufficient stability in acidic environments, inferior emulsifying properties compared to macromolecular emulsifiers, and a tendency to aggregate and denature during processing, which restricts its industrial application in beverages, emulsion systems, and other fields.

[0003] Existing methods for improving protein performance include physical modification (ultrasonic treatment, ultra-high pressure treatment), chemical modification (acylation, phosphorylation), and biological modification (enzymatic hydrolysis). While physical modification is simple to operate, it suffers from limited stability improvement, high equipment costs, and high energy consumption. Chemical modification is prone to reagent residues and numerous side reactions. Biological modification suffers from poor controllability, and excessive hydrolysis can actually reduce emulsifying properties. Furthermore, existing physical, chemical, and biological modification methods all have the limitation of only improving a single property. Therefore, developing a modification method that can simultaneously improve the solubility, stability, and emulsifying properties of coconut protein, while also being low-cost, safe, and controllable, is of great significance for promoting the high-value utilization and industrial application of coconut protein. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a glycosylated coconut protein, its preparation method, and its applications. This invention utilizes konjac glucomannan to glycosylate coconut protein. This polysaccharide modification simultaneously enhances the solubility, stability, and emulsifying properties of coconut protein. The glycosylated coconut protein exhibits significantly improved solubility under acidic conditions, with a denaturation temperature increased to 87.05℃, a 26.84% improvement compared to regular coconut protein. Water-holding capacity and oil-holding capacity are increased by 42.79% and 30.58%, respectively, and emulsifying activity is increased to 3.63 m. 2 / g, emulsification stability improved to 84.77%. The raw materials for the preparation of glycosylated coconut protein of this invention are widely available and inexpensive. No toxic or harmful chemical reagents need to be added during the modification process, which has the advantages of low cost and high safety. In addition, the process has strong repeatability and is easy to scale up industrially.

[0005] On one hand, the present invention seeks to protect a glycosylated coconut protein, which uses konjac glucomannan as a sugar source to glycosylate coconut protein.

[0006] On the other hand, the present invention seeks protection for a method for preparing the above-mentioned glycosylated coconut protein, which specifically includes the following steps:

[0007] S1: Solution preparation: Dissolve coconut protein and konjac glucomannan separately in PBS solution, mix the two and pre-stir at 750 rpm for 15 min to obtain a mixed solution.

[0008] Further, the mass ratio of the mixed coconut protein to konjac glucomannan is 1:3 to 5:1; preferably, the mass ratio of the coconut protein to konjac glucomannan is 1:1.

[0009] S2: Glycosylation reaction. The pH of the mixed solution is adjusted to 8.0, and the glycosylation reaction is carried out at 60~90℃ for 3~12 h to obtain glycosylated coconut protein.

[0010] Preferably, the glycosylation reaction is carried out at a temperature of 80°C for a reaction time of 9 h.

[0011] Specifically, this invention determined the optimal glycosylation reaction parameters for konjac glucomannan through single-factor experiments as follows: a coconut protein to konjac glucomannan mass ratio of 1:1, a reaction temperature of 80℃, a reaction time of 9 h, and a pH of 8.0. Under these conditions, the grafting degree, browning index, and lipid peroxidation resistance of the obtained product were all optimal.

[0012] On the other hand, the present invention seeks protection for the application of the above-described preparation method in improving the solubility, stability, and emulsifying properties of coconut protein.

[0013] Specifically, this invention tested the modification effects of different types of polysaccharides on coconut protein and found that the modification effects of different polysaccharides on coconut protein varied significantly, and it was often difficult to simultaneously improve its solubility, stability and emulsification properties. Only konjac glucomannan showed the best simultaneous improvement effect on the properties of coconut protein.

[0014] Furthermore, this invention seeks protection for the application of the above-mentioned glycosylated coconut protein in the preparation of fish oil microcapsules, wherein the glycosylated coconut protein is the wall material and the fish oil is the core material. The mass ratio of the core material to the wall material is 2:1 to 4:5; preferably, the mass ratio of the core material to the wall material is 4:5.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) The present invention tested the modification effects of different types of polysaccharides on coconut protein and found that the modification effects of different polysaccharides on coconut protein were significantly different. It was often difficult to simultaneously improve its solubility, stability and emulsification performance. Only konjac glucomannan showed the best effect in simultaneously improving the performance of coconut protein. For example, although chitosan can improve the stability of the coconut protein system and increase the denaturation temperature, the β-1,4 glycosidic bonds and a large number of intramolecular hydrogen bonds (between amino and hydroxyl groups) of chitosan result in strong molecular rigidity and poor chain flexibility. This leads to excessively high expansion modulus and insufficient flexibility at the oil-water interface, resulting in weak emulsification and spreading ability. Although the protonation of chitosan under acidic conditions can improve solubility, its solubilization effect on coconut protein is limited, and it cannot simultaneously achieve solubility, stability and emulsification performance. The direct thermal glycosylation efficiency of sodium alginate and coconut protein is limited: on the one hand, the reducing end reactivity of sodium alginate is low and the grafting degree is insufficient under conventional heating conditions; on the other hand, sodium alginate and positively charged proteins are prone to electrostatic phase separation. While the glycosylation modification of sodium alginate can be promoted by heating or microwave assistance, high temperatures accelerate browning and flavor deterioration. Furthermore, sodium alginate has reduced solubility under acidic conditions (it easily precipitates as alginic acid), making it difficult to simultaneously optimize the acidic solubility, stability, and emulsifying properties of coconut protein.

[0016] (2) The optimal glycosylation reaction parameters for konjac glucomannan were determined by single-factor experiments in this invention as follows: a mass ratio of coconut protein to konjac glucomannan of 1:1, a reaction temperature of 80℃, a reaction time of 9 h, and a pH of 8.0. Under these conditions, the grafting degree, browning index, and lipid peroxidation resistance of the obtained product were all optimal. FTIR spectroscopy analysis confirmed the successful preparation of the covalent complex. The glycosylation reaction led to the reconstruction of the protein's secondary structure, characterized by a decrease in the content of α-helices and β-sheets, while the proportion of random coils increased significantly. This structural disorder indicates that the protein molecule conformation is more relaxed, providing a structural basis for the improvement of functional properties. Functional property studies showed that compared with pure coconut protein (CP), the thermal stability, water-holding capacity, oil-holding capacity, emulsifying properties, and solubility of glycosylated coconut protein (CP-KGM) were significantly improved, and the heat denaturation temperature was increased by 26.84%, indicating that covalent grafting endowed the protein with excellent heat tolerance.

[0017] (3) This invention uses konjac glucomannan to glycosylate coconut protein, and the resulting protein-polysaccharide covalent complex has advantages such as low raw material cost, high food safety, and mild and controllable reaction process. Konjac glucomannan is widely available and inexpensive. As a natural non-digestible polysaccharide, it does not require the addition of toxic or harmful chemical reagents during the modification process. Covalent grafting can be achieved solely through a wet Maillard reaction, which meets the requirements of green food processing. At the same time, key parameters such as reaction temperature, time, and glycoprotein ratio are easy to adjust, the process is highly reproducible, and it is easy to scale up industrially.

[0018] (4) This invention further prepared coconut protein modified with konjac glucomannan into microcapsules, and further explored the influence of the core-to-wall ratio on the physicochemical properties, oxidative stability and flavor quality of the prepared fish oil microcapsules. The experiment showed that when the core-to-wall ratio was 4:5, the microcapsules exhibited the best comprehensive performance. The encapsulation rate was the highest and the surface oil content was the lowest, indicating that a complete and dense wall membrane structure was formed. Particle size analysis showed that the microcapsules had the lowest Span value (1.83) at this ratio, showing the best uniformity. The larger median diameter (D50 of 31.44 μm) and smaller specific surface area were conducive to the slow release and stable storage of the core material. Oxidative stability analysis showed that the induction time of the microcapsules was significantly prolonged at different acceleration temperatures at this ratio. The first-order reaction kinetic model predicted that its shelf life at 4℃ could reach 2688.58 hours, which was significantly better than other ratios. The microcapsule product had low moisture content (<4%) and high solubility (>83%), and had good storage stability and application characteristics. Using glycosylated coconut protein-fish gelatin as a wall material not only effectively masks the fishy smell components in fish oil, but also generates special pleasant flavor substances through Maillard reaction, forming a complex and harmonious aroma profile, thus achieving the masking of unpleasant flavors and the imparting of aroma. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The figure shows the effect of different glycosylation temperatures on the grafting degree, browning index, and lipid oxidation resistance of glycosylated coconut protein.

[0021] Figure 2 The figure shows the effect of different glycosylation times on the grafting degree, browning index, and lipid oxidation resistance of glycosylated coconut protein.

[0022] Figure 3 The figure shows the effect of different mixing ratios of coconut protein solution and konjac glucomannan solution on the degree of grafting of glycosylated coconut protein, browning index, and degree of lipid oxidation resistance.

[0023] Figure 4 Fourier transform infrared spectra of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein.

[0024] Figure 5Statistical graphs showing the solubility of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein at different pH values.

[0025] Figure 6 DSC thermograms of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein.

[0026] Figure 7 The results show the water-holding and oil-holding capacities of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein.

[0027] Figure 8 The figure shows the optimized pH results for the glycosylated coconut protein-fish gelatin solution. Figure 8 (A) in the figure is the Zeta potential diagram of glycosylated coconut protein solution, fish gelatin solution and glycosylated coconut protein-fish gelatin solution at different pH values; Figure 8 (B) in the image is an optical microscope image of the composite coagulation of glycosylated coconut protein-fish gelatin solution at different pH values; Figure 8 (C) in the figure represents the turbidity results of the glycosylated coconut protein-fish gelatin solution at different pH values; Figure 8 (D) in the figure represents the zeta potential diagram of the glycosylated coconut protein-fish gelatin solution at different pH values.

[0028] Figure 9 Optical micrographs of glycosylated coconut protein-fish gelatin emulsions, complex aggregates, and microcapsules with different core-wall ratios.

[0029] Figure 10 The yield of glycosylated coconut protein-fish gelatin complex aggregates with different core-wall ratios is shown in the figure.

[0030] Figure 11 A first-order reaction kinetic model for glycosylated coconut protein-fish gelatin microcapsules with different core-wall ratios. Detailed Implementation

[0031] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially. Unless otherwise specified, the percentages in the following embodiments refer to mass percentages.

[0032] Coconut protein was purchased from Yuexu Technology Co., Ltd.

[0033] Konjac glucomannan and fish gelatin were purchased from Sigma.

[0034] The tuna oil was purchased from Zhoushan Xin Nuojia Biotechnology Co., Ltd.

[0035] Lecithin was purchased from Aladdin Company.

[0036] Phthalate, sodium borate, SDS, β-mercaptoethanol, trichloroacetic acid, 2-thiobarbituric acid, and FeCl3 were all analytical grade and purchased from Zhanjiang Keming Co., Ltd.

[0037] The preparation method of glycosylated coconut protein specifically includes the following steps: S1: Solution preparation: Dissolve coconut protein and konjac glucomannan separately in PBS solution, mix the two and pre-stir at 750 rpm for 15 min to obtain a mixed solution.

[0038] Furthermore, the concentration of the coconut protein solution is 100 g / L, the concentration of the konjac glucomannan solution is 100 g / L, and the volume ratio of the mixture is 1:3 to 5:1.

[0039] S2: Glycosylation reaction. The pH of the mixed solution is adjusted to 8.0, and the glycosylation reaction is carried out at 60~90℃ for 3~12 h to obtain glycosylated coconut protein.

[0040] Example 1 This embodiment provides a single-factor experiment on the ratio of glycosylated coconut protein raw materials, reaction temperature, and reaction time.

[0041] Preparation of glycosylated coconut protein: 10 g coconut protein and 10 g konjac glucomannan were dissolved in 100 mL PBS solution to obtain a 100 g / L coconut protein solution and a 100 g / L konjac glucomannan solution. These two solutions were mixed at a 1:1 volume ratio and pre-stirred at 750 rpm for 15 min to obtain a mixed solution. The pH of the mixed solution was adjusted to 8.0, and the glycosylation reaction was carried out at 80℃ for 9 h to obtain glycosylated coconut protein, which was then lyophilized for later use.

[0042] Graft degree (DG) determination: The o-phthalaldehyde (OPA) method was used. 200 μL of the glycosylated sample was added to 4 mL of OPA solution and incubated at 35 °C for 2 min. The absorbance was measured at 340 nm using a UV-Vis spectrophotometer. The graft degree was calculated using the following formula. 200 μL of deionized water with 4 mL of OPA solution was used as a blank control. The OPA solution was prepared as follows: 40 mg of o-phthalaldehyde (OPA) was dissolved in 1 mL of methanol, then mixed with 2.5 mL of SDS (1%, w / v), 25 mL of sodium borate solution (0.1 mol / L), and 100 μL of β-mercaptoethanol, and finally diluted to 50 mL with ultrapure water.

[0043] DG(%)=(A0-A1) / A0×100%; In the formula: A0 is the absorbance value of the sample solution before incubation; A1 is the absorbance value of the sample solution after incubation.

[0044] Browning index detection: Pipette 1 mL of the sample solution (5 mg / mL) dissolved in water into a 10 mL test tube, and dilute it with 5 mL of SDS (0.1%, w / w). Using SDS as a blank control, measure the absorbance (A) of each sample solution at 420 nm using a UV-Vis spectrophotometer. 420 , with A 420 Characterizes the degree of browning.

[0045] Determination of anti-lipid oxidation capacity (I): Weigh 1 g of lecithin into a beaker and completely dissolve it in 100 mL of 0.01 mol / L pH 7.4 PBS solution to obtain a lecithin solution. Weigh 15 g of trichloroacetic acid and 0.37 g of 2-thiobarbituric acid, dissolve them, add 2 mL of concentrated HCl, and dilute to 100 mL with distilled water, labeling this as solution A. Take 1 mL of sample solution (lyophilized solid powder dissolved in distilled water), 1 mL of lecithin solution, and 1 mL of 0.4 mol / L FeCl3 solution, using distilled water as a blank control, mix thoroughly using a vortex mixer, incubate at 37℃ for 1 h, add 2 mL of solution A, incubate in a boiling water bath for 15 min, cool in cold water, centrifuge at 9000 r / min for 10 min, and measure the absorbance of the supernatant at 532 nm using a UV-Vis spectrophotometer. Calculate the anti-lipid oxidation capacity of glycosylated coconut protein according to the following formula.

[0046] I (%) = (A C -A S A C ×100%; In the formula, A c A represents the absorbance value of the blank control. S This indicates the absorbance value of the sample.

[0047] 1. Single-factor experiment on glycosylation temperature The glycosylation temperatures were 60℃, 70℃, 80℃, and 90℃. The grafting degree, browning index, and lipid peroxidation resistance of the glycosylated coconut proteins prepared at different glycosylation temperatures were measured. Figure 1 Each experiment was repeated three times, and results are expressed as mean ± standard deviation. One-way ANOVA and Tukey HSD multiple comparisons were performed using IBM SPSS Statistics 26 software, with differences determined at a confidence level of 95%. All plots in this paper were performed using Origin 2025 software.

[0048] Depend on Figure 1It can be seen that with increasing reaction temperature, the grafting degree, browning index, and lipid peroxidation resistance of the glycosylation reaction all show a trend of first increasing and then decreasing. When the glycosylation temperature is 80℃, the grafting degree and lipid peroxidation resistance reach their maximum values, which are 43.04±2.42% and 52.48%±0.62%, respectively, and the browning index (A... 420 nm The browning index was 0.452, which may be due to the fact that increased temperature causes the spatial structure of protein molecules to stretch, exposing the embedded active groups, while intensifying molecular thermal motion and increasing the probability of collisions, thus promoting the formation of glycosylation products. However, when the temperature reaches 90℃, both the grafting degree and the degree of lipid peroxidation resistance decrease significantly, and the browning index increases, indicating that coconut protein may undergo partial thermal denaturation and aggregation, leading to a decrease in grafting degree and glycosylation products. In addition, excessive reaction may cause cross-linking or degradation, destroying the antioxidant structure and slowing down or even reducing the growth of lipid peroxidation resistance. Excessive browning may also lead to the formation of undesirable flavors. Therefore, considering all factors, 80℃ was selected as the optimal temperature for the glycosylation reaction.

[0049] 2. Single-factor experiment on glycosylation time Glycosylation times were 3, 6, 9, and 12 h, and the grafting degree, browning index, and lipid peroxidation resistance of glycosylated coconut proteins prepared at different glycosylation temperatures were measured. Figure 2 Each experiment was repeated 3 times.

[0050] Depend on Figure 2 It can be seen that with the increase of glycosylation time, the grafting degree, browning index, and lipid peroxidation resistance of the glycosylation reaction also show a trend of first increasing and then decreasing. Within the glycosylation time range of 3–9 h, the grafting degree of glycosylated protein increases with prolonged heating time. This is because with prolonged heating time, coconut protein degrades, increasing the content of free amino acids and promoting the reaction probability of coconut protein with konjac glucomannan, thereby increasing the grafting degree, browning index, and lipid peroxidation resistance of glycosylated coconut protein. Within the glycosylation time range of 9–12 h, the grafting degree begins to decrease, indicating that excessive heating time leads to deamination and decarboxylation reactions of free amino acids, resulting in a decrease in the content of free amino acids and reducing the reaction probability of coconut protein with konjac glucomannan. The increased browning index and decreased lipid peroxidation resistance indicate that the increased reaction time produces more melanoidins. Therefore, considering all factors, 9 h was determined as the optimal time for the glycosylation reaction.

[0051] 3. Single-factor experiment on the mixing ratio of coconut protein solution and konjac glucomannan solution The volume ratios of coconut protein solution and konjac glucomannan solution were 5:1, 3:1, 1:1, and 1:3. The effects of different raw material ratios on the grafting degree, browning index, and lipid peroxidation resistance of glycosylated coconut protein were investigated. Each experiment was repeated three times. The results are shown below. Figure 3 As shown.

[0052] Depend on Figure 3 It was found that when the ratio of coconut protein to konjac glucomannan was 1:1, the glycosylation grafting degree, browning degree, and lipid oxidation resistance all reached their maximum values, with a grafting degree of 52.16% ± 0.61%, a browning degree of 0.444, and an lipid oxidation resistance of 55.18% ± 0.58%. As the proportion of konjac glucomannan increased, the grafting degree actually decreased. This may be because increasing the polysaccharide ratio leads to increased solution viscosity and decreased molecular fluidity. Simultaneously, excessive KGM chains can cause steric hindrance and electrostatic repulsion, hindering effective collision and binding between individual KGM molecules and protein active sites, thus reducing the grafting degree. When the proportion of coconut protein increased, the total number of amino functional groups in the protein far exceeded the total number of reducing carbonyl functional groups provided by KGM. A large number of protein molecules failed to be glycosylated due to their inability to access glycoligands, resulting in a decrease in the grafting degree. It is evident that an appropriate ratio of protein to polysaccharide can not only improve the reaction rate and degree, but also effectively reduce the formation of harmful substances. Therefore, considering all factors, the optimal mass ratio of coconut protein to konjac glucomannan is determined to be 1:1.

[0053] Example 2 This embodiment provides performance determination of coconut protein (CP), coconut protein-konjac glucomannan mixture (CP / KGM-MIX), and glycosylated coconut protein (CP-KGM).

[0054] 1. Determination of Fourier Transform Infrared Spectroscopy (FTIR) Coconut protein, a mixture of coconut protein and konjac glucomannan, and glycosylated coconut protein were each mixed with KBr at a mass ratio of 1:100, ground, and then pressed into thin sheets. Fourier transform infrared spectroscopy was performed from 4000 to 400 cm⁻¹. -1 The range is used to obtain the FTIR plot as shown below. Figure 4 As shown.

[0055] Depend on Figure 4It can be seen that, comparing the infrared spectra of coconut protein (CP), the physical mixture of coconut protein and konjac glucomannan (CP / KGM-MIX), and glycosylated coconut protein (CP-KGM), the absorption intensities of CP / KGM-MIX and CP-KGM in the amide-I and amide-II bands are significantly lower than those of CP. This phenomenon indicates that a glycosylation reaction occurred between coconut protein and konjac glucomannan. During the reaction, the free amino groups in coconut protein undergo carbonyl-amine condensation with the reducing carbonyl group of konjac glucomannan to generate a Schiff base, which then undergoes Amadori rearrangement to form a stable glycosylated product. Because the free amino and carbonyl groups are consumed in the reaction, the C=O bond (corresponding to 1650 cm⁻¹) is weakened. -1 (at) and NH bending vibration (1530 cm) -1 The signal at the amide-I and amide-II bands weakens, resulting in a significant decrease in the infrared absorption intensity of these bands. Furthermore, the infrared absorption intensity at 1400–1000 cm⁻¹ is also reduced. -1 Within the range, the number and intensity of absorption peaks of the conjugate both increased, further confirming that coconut protein and konjac glucomannan form a glycosylated complex through covalent binding, which causes changes in protein conformation.

[0056] 2. Determination of protein secondary structure The secondary structures of samples (coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein) were determined at room temperature using circular dichroism spectroscopy. Samples were dissolved in ultrapure water to prepare protein solutions of 0.5 mg / mL. Circular dichroism spectra were scanned and recorded in the far-ultraviolet region (190–250 nm) using quartz cuvettes with a 10 mm path length. The secondary structures were interpreted through spectral visualization and the computer program Circular Dichroism Deconvolution Version 2.1. The relative contents of four secondary structures (α-helix, β-sheet, β-turn, and random coil) were calculated (Table 1). α-helix is ​​a dense, ordered structure closely related to protein structural stability; β-sheet and β-turn are relatively extended ordered structures; while random coil is a disordered structure related to protein emulsification stability.

[0057] Table 1. Relative contents of coconut protein, coconut protein-konjac glucomannan mixture, and secondary structures of glycosylated coconut protein.

[0058] Table 1 shows that the secondary structure of coconut protein (CP), coconut protein-konjac glucomannan mixture (CP / KGM-MIX), and glycosylated coconut protein (CP-KGM) is mainly composed of β-sheets and random coils. Compared with pure CP, the α-helix and β-sheet contents of CP / KGM-MIX and CP-KGM are reduced, while the proportions of β-turns and random coils are significantly increased. The structural changes in CP-KGM are more pronounced than those in the CP / KGM-MIX physical mixture. The decrease in α-helix content may be related to the prolonged high-temperature environment of coconut protein during the glycosylation reaction. This condition leads to the disruption of the protein's spatial conformation, the unfolding of the internal structure, and consequently, the unfolding of α-helices, resulting in a decrease in orderliness and a change in the molecular structure from dense to loose. In addition, the reduction of α-helices also indicates that konjac glucomannan was successfully grafted onto the coconut protein molecule. The significant reduction of β-sheet structure after glycosylation indicates that lysine residues participated in the Maillard reaction, and their side chain ε-amino groups were covalently modified. The increased proportion of random coils indicates protein unfolding, exposing more internal hydrophobic regions and thus increasing surface hydrophobicity. This structural change helps improve the protein's emulsifying properties. These changes in secondary structure are consistent with a significant decrease in the absorption intensity of the amide I and amide II bands in the infrared spectrum. The weakening of the amide band signal confirms a carbonylamine condensation reaction between the free amino group and the carbonyl group, followed by Amadori rearrangement to generate a covalently bound glycosylated product. Successful glycosylation between coconut protein and konjac glucomannan not only formed a stable covalent complex but also caused a shift in the protein's secondary structure from ordered to disordered, providing a structural basis for the alteration of its functional properties.

[0059] 3. Determination of solubility Dissolve 0.1 g of sample (coconut protein, coconut protein-konjac glucomannan mixture, glycosylated coconut protein) in 100 mL of pure water, stir well, adjust pH to 3.0-11.0 with 1 mol / L HCl or NaOH solution, centrifuge at 10000 r / min for 15 min, and collect the supernatant. Determine the soluble protein content using the biuret method: add 4.0 mL of biuret reagent, let stand at room temperature for 30 min, and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Plot a standard curve using bovine serum albumin (BSA) as the standard, calculate the protein concentration in the supernatant, and calculate the protein solubility using the following formula. The results are as follows: Figure 5 As shown.

[0060] Solubility (%) = C1 / C0 × 100%; In the formula: C0 is the protein concentration in the sample solution before centrifugation, mg / mL; C1 is the protein concentration in the sample supernatant after centrifugation, mg / mL.

[0061] Depend on Figure 5 It was found that among the three groups of samples, the coconut protein-konjac glucomannan mixture (CP / KGM-MIX) consistently exhibited the highest solubility, while pure coconut protein (CP) showed the lowest solubility. This phenomenon is mainly attributed to the extremely strong hydrophilicity and hydration capacity of konjac glucomannan (KGM). KGM molecules can rapidly bind water molecules to form a high-viscosity colloidal solution, constructing a continuous hydration network in the CP / KGM-MIX system. This effectively disperses and stabilizes protein particles through non-covalent interactions such as hydrogen bonds, significantly improving the overall solubility of the system. Compared to CP / KGM-MIX, the decreased solubility of CP-KGM may be due to the continuous heat treatment during the reaction process, which causes partial denaturation and aggregation of the protein, reducing its solubility. Furthermore, the isoelectric point of coconut protein is between pH 4.0 and 5.0. All samples exhibited low solubility near this region, consistent with the theoretical expectation that proteins have zero net charge at their isoelectric point and are prone to molecular aggregation and precipitation. However, CP / KGM-MIX and CP-KGM, due to the introduction of KGM, effectively delay the sedimentation of protein aggregates by means of its hydration and steric hindrance effects, thus exhibiting higher solubility than CP.

[0062] 4. Determination of thermal stability The thermal behavior of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein was measured using differential scanning calorimetry (DSC). 5.0 mg of sample was weighed into a solid aluminum pot, sealed with an aluminum lid, and heated to 20–200 °C at a rate of 10 °C / min using an empty sample pan as a reference. Nitrogen flow was applied at a rate of 50 mL / min, and DSC thermograms were recorded. Figure 6 ).

[0063] Depend on Figure 6It is known that the denaturation temperature of coconut protein (CP) is 68.63℃. After physical mixing with konjac glucomannan (CP / KGM-MIX), its denaturation temperature increases to 80.96℃, an increase of 17.97%. The denaturation temperature of glycosylated coconut protein (CP-KGM) prepared by glycosylation further increases to 87.05℃, an increase of 26.84% compared to CP. This shows that the addition of konjac glucomannan (KGM) can significantly improve the thermal stability of CP, and the thermal stability is further enhanced after glycosylation. In the physical mixture (CP / KGM-MIX), the improvement in thermal stability mainly stems from the excellent thermal stability of KGM itself and the non-covalent interactions (such as hydrogen bonds, hydrophobic interactions, and van der Waals forces) formed between KGM and protein molecules. These interactions effectively inhibit the molecular chain movement of proteins during heating, thereby delaying their thermal denaturation and aggregation process, and thus increasing the denaturation temperature. The glycosylated covalent complex (CP-KGM) inhibits heat-induced protein unfolding through steric hindrance and enhances the overall conformational rigidity of the molecule through the coating effect of the long KGM chain, thereby significantly improving the thermal stability of the complex. In summary, glycosylation covalent modification shows significant advantages in improving protein thermal stability.

[0064] 5. Determination of water-holding and oil-holding capacity Weigh 0.1 g of the sample, denoted as M0. Place the sample into a centrifuge tube, denoted as M1. Add 10 mL of deionized water to the centrifuge tube, vortex for 60 s, centrifuge at 3500 g for 20 min, and remove residual water with absorbent paper. Record the total mass of the centrifuge tube and the precipitate after water absorption as M2. Calculate the water-holding capacity (WAC) using the following formula. The results are as follows: Figure 7 As shown.

[0065] Weigh 0.2 g of the sample, denoted as M3. Place the sample into a centrifuge tube, denoted as M4. Add 10 mL of soybean oil to the centrifuge tube, vortex for 60 s, centrifuge at 3500 g for 20 min, and remove residual oil with absorbent paper. Record the total mass of the centrifuge tube and the precipitate after oil removal as M5. Calculate the oil holding capacity (OAC) using the following formula. The results are as follows: Figure 7 As shown.

[0066] WAC = (M2) M1) / M0×100%; OAC=(M5) M4) / M3×100%.

[0067] Depend on Figure 7It is evident that CP-KGM outperforms CP and CP / KGM-MIX in both water-holding and oil-holding properties, and CP / KGM-MIX is generally superior to pure CP. CP's water-holding and oil-holding capacities are 2.01 g / g and 2.78 g / g, respectively; CP-KGM exhibits the highest water-holding and oil-holding capacities, at 2.87 g / g and 3.63 g / g, respectively, representing increases of 42.79% and 30.58%, a significant improvement. The increased water-holding capacity may be due to the covalent introduction of the hydrophilic polysaccharide konjac glucomannan during glycosylation, whose molecular chain contains numerous hydroxyl groups that can effectively bind and lock water molecules through hydrogen bonds. In physical mixtures, KGM can also improve the hydrophilic environment of the system through physical processes, but its effect is not as significant as the formation of covalent complexes. The improved oil-holding capacity may be due to the increased content of random coils in the protein, resulting in more hydrophobic groups exposed on the molecular surface, enhancing their hydrophobic interaction with oil molecules. The protein-polysaccharide covalent complexes generated by glycosylation have larger molecular weights and more extended three-dimensional network structures. This physical structure can more effectively adsorb and encapsulate lipids.

[0068] 6. Determination of emulsifying activity (EAI) and emulsifying stability (ESI) Weigh 9 g of sample and dissolve it in 0.1 mol / L sodium chloride solution. Pre-emulsify the sample for 5 min at 1500 rpm using a digital overhead stirrer. Emulsify the pre-emulsified sample for 10 min at 20000 rpm using a digital display homogenizer to obtain an emulsion. Take 0.5 mL of the emulsion and dilute it 100 times with 0.1% sodium dodecyl sulfate solution. Measure the absorbance using sodium dodecyl sulfate solution as a blank solution. Measure the absorbance at 500 nm using a UV-Vis spectrophotometer and calculate the emulsifying activity (EAI). The calculation results are shown in Table 2. After the diluted mixture is allowed to stand at room temperature for 12 h, the absorbance is measured again to calculate the emulsifying stability (ESI). The calculation results are shown in Table 2.

[0069] Table 2. Emulsifying activity and emulsifying stability results of coconut protein, coconut protein-konjac glucomannan mixture, and glycosylated coconut protein emulsion.

[0070] Table 2 shows that different treatment methods significantly affected the emulsifying properties of coconut protein emulsions (P < 0.05). The EAI of the CP emulsion was 3.23 ± 0.09 m. 2 / g, ESI was 68.73±0.39%, and EAI of CP / KGM-MIX emulsion was 3.32±0.06m. 2The concentration of konjac glucomannan (KGM) was not significantly different from that of the CP group (P > 0.05), indicating that under physical mixing conditions, KGM mainly improves emulsion stability by increasing the viscosity of the continuous phase and slowing down the droplet migration rate, but it failed to change the interfacial adsorption behavior of coconut protein. Therefore, it did not significantly improve the EAI, but the ESI increased to 78.40 ± 0.09%, which was significantly higher than that of the CP group (P < 0.05). The CP-KGM emulsion showed the best emulsifying performance, with an EAI of 3.63 ± 0.08 g / g. 2 The emulsifying activity (EAI) and ESI (84.77±0.10%) of coconut protein were significantly higher than those of the other two groups (P<0.05). This may be because the polysaccharide chains increase the steric hindrance and hydrophilicity of the protein molecules, promoting their rapid adsorption and directional alignment at the oil-water interface, thereby improving the EAI. In addition, the protein-polysaccharide complex can construct a thicker and more elastic interfacial film on the oil droplet surface, and the stability of the emulsion is also enhanced. In summary, the glycosylation modification of coconut protein with konjac glucomannan significantly enhances the emulsifying activity and stability of coconut protein.

[0071] Example 3 This embodiment demonstrates the effects of different types of polysaccharides on the properties of glycosylated coconut protein.

[0072] Coconut protein was glycosylated with konjac glucomannan, chitosan, and sodium alginate, respectively. The thermal stability, solubility (pH=9), and emulsifying properties of the glycosylated coconut protein were tested using the same methods as in Example 2. The test results are shown in Table 3.

[0073] Table 3. Performance of glycosylated coconut protein prepared from different types of polysaccharides

[0074] Table 3 shows that although chitosan can improve the stability of coconut protein systems and increase the denaturation temperature, its high molecular rigidity and poor chain flexibility due to the β-1,4 glycosidic bonds and numerous intramolecular hydrogen bonds (between amino and hydroxyl groups) result in excessively high expansion modulus and insufficient flexibility at the oil-water interface, leading to weak emulsification and spreading ability. While protonation of chitosan under acidic conditions can improve solubility, its solubilizing effect on coconut protein is limited, failing to simultaneously achieve solubility, stability, and emulsification performance. The direct thermal glycosylation efficiency of sodium alginate with coconut protein is limited: on the one hand, sodium alginate has low reactivity at the reducing end, resulting in insufficient grafting under conventional heating conditions; on the other hand, sodium alginate easily undergoes electrostatic phase separation with positively charged proteins. Although the glycosylation modification of sodium alginate can be promoted by heating or microwave assistance, high temperatures accelerate browning and flavor deterioration. Furthermore, the solubility of sodium alginate decreases under acidic conditions (easily precipitating as alginic acid), making it difficult to simultaneously optimize the acidic solubility, stability, and emulsification performance of coconut protein. In contrast, the konjac glucomannan of the present invention has good chain flexibility and a special branched structure. After glycosylation and grafting, it can improve stability and solubility by providing strong steric hindrance through long chains, without hindering interfacial adsorption and spreading. It can simultaneously improve the three major properties of solubility, environmental stability and emulsification.

[0075] Example 4 This embodiment provides the application of glycosylated coconut protein in the preparation of microcapsules.

[0076] Fish oil microcapsules were prepared using a glycosylated coconut protein-fish gelatin complex as the wall material and fish oil as the core material. Previous experiments showed that dissolving the raw materials in sodium chloride solution yielded microcapsules with smaller particle sizes. 9 g of glycosylated coconut protein and 1 g of fish gelatin were dissolved in 200 mL of 0.1 mol / mL sodium chloride solution. The pH of the solution was adjusted to 3.75 by dropwise addition of 1% (v / v) phosphoric acid solution to obtain a composite solution. Tuna oil was then added, resulting in microcapsules with a core-to-wall ratio of 4:5. The initial temperature for microencapsulation was maintained at 50℃ for 1 h, followed by cooling to 5℃ at a rate of 12℃ / h. After holding the sample at 5℃ for 30 min, 50 mL of 10% (w / w) transglutaminase solution was added to induce cross-linking of the microcapsule shell. The solution temperature was then increased to 25℃ at a heating rate of 5℃ / h and held at 25℃ for 5 h to complete cross-linking. The stirring speed was maintained at 700 r / min during microencapsulation. The resulting liquid microcapsules were freeze-dried and ready for use.

[0077] 1. Optimization of pH of composite solution 1g of glycosylated coconut protein, 1g of fish gelatin, and 0.9g of glycosylated coconut protein and 0.1g of fish gelatin were dissolved in 100mL of ultrapure water (ultrapure water is sufficient for parameter optimization that does not involve microcapsule particle size). The pH of the resulting solutions was adjusted to 2.5, 3.5, 4.5, 5.5, and 6.5 respectively using 1M HCl and NaOH. The zeta potential of the solutions was measured using a nanopotential meter (Zeta sizer Nano ZS90). The results are as follows. Figure 8 As shown in (A) above. 0.9 g of glycosylated coconut protein and 0.1 g of fish gelatin were dissolved in 100 mL of ultrapure water. Then, 1% (v / v) phosphoric acid solution was added dropwise to adjust the pH of the solution to 5.64 and to 3.5–4.2. The morphology of the complex aggregates in the solution was observed using an electron microscope. The results are shown below. Figure 8 As shown in (B) above. 0.9 g of glycosylated coconut protein and 0.1 g of fish gelatin were dissolved in 100 mL of ultrapure water and placed in a 50°C constant temperature water bath. The solution was stirred at 500 rpm, and the pH was adjusted to 3.5–4.2 using phosphoric acid solution. The turbidity of the solution at 600 nm was measured using a UV spectrophotometer, and the Zeta potential was detected. The results are shown below. Figure 8 As shown in (C) and (D) in the diagram.

[0078] Depend on Figure 8 As shown in (A), the zeta potential of glycosylated coconut protein is negative within the pH range of 2.5 to 6.5, while fish gelatin exhibits a positive potential within the same pH range. The composite system shows a zeta potential close to zero within the pH range of 4.0 to 4.2, indicating that the negative charge of the glycosylated coconut protein and the positive charge of the fish gelatin reach electrostatic equilibrium, achieving complete neutralization. Figure 8 As shown in (B), complex coagulation occurs when pH ≤ 4.0. From... Figure 8 As shown in (C) and (D), the system reaches its maximum turbidity between pH 3.7 and 3.8. Furthermore, the Zeta potential of the composite condensate drops to 0 mV near pH 3.75. In conclusion, pH approximately 3.75 is the optimal condition for studying the condensation behavior of this composite system.

[0079] 2. Optimization of core-to-wall ratio (1) Effect of different core-to-wall ratios on composite aggregates Electron microscopy was used to observe the morphology of glycosylated coconut protein-fish gelatin complex aggregates at different core-to-wall ratios (2:1, 5:4, 1:1, and 4:5) at initial pH and pH 3.75, as well as the morphology of microcapsules at different core-to-wall ratios. The results are as follows: Figure 9 As shown, the yield (CY) of the composite aggregate was calculated using the following formula, and the results are as follows. Figure 10 As shown.

[0080] CY (%) = m i / m0×100% In the formula, m i The mass (g) of the composite coagulant dried to constant weight is given, and m0 is the total mass (g) of the glycosylated coconut protein-fish gelatin and fish oil used to prepare the composite coagulant.

[0081] Depend on Figure 9 It was found that the initial pH of the emulsions formed by adding different masses of fish oil was around 5.65. Larger oil droplets were observed in the composite condensates with core-to-wall ratios of 2:1 and 5:4, while the oil droplets were relatively smaller at core-to-wall ratios of 1:1 and 4:5, indicating that the wall material more effectively covered the oil droplet surface, forming a better condensed phase. The morphology of the four microcapsules showed no significant difference, all exhibiting elongated shapes. Figure 10 It can be seen that the yield of composite aggregates in all groups is greater than 70%, with the highest yield (79.17±0.51%) when the core-to-wall ratio is 1:1. This indicates that the interaction between the wall material and the core material is in a better state under this condition, which promotes the formation of stable complexes and yields more products.

[0082] (2) Effect of different core-to-wall ratios on the moisture content and solubility of microcapsules The mass loss of the sample during the drying process was determined by direct drying at 101.3 kPa and 105℃. After the sample cooled to room temperature, the data were recorded, and the moisture content of the microcapsules was calculated. The results are shown in Table 4. 1 g of microcapsule (m0) sample was dissolved in 30 mL of water, stirred (1000 r / min, 10 min), centrifuged (3000 r / min, 5 min), and the supernatant was transferred to a culture dish of known weight. The sample was dried at 105℃ to constant weight. The formula for calculating the solubility of the microcapsules is as follows, and the calculation results are shown in Table 4.

[0083] Solubility (%) = [m1 / (m0×(1-w)] c ))]×100% In the formula, m1 is the weight of the dried microcapsules (g) after subtracting the weight of the container, and w c The moisture content (%) of the microcapsules.

[0084] Table 4. Results of moisture content and solubility tests for glycosylated coconut protein-fish gelatin-fish oil microcapsules with different core wall ratios.

[0085] Table 4 shows that as the proportion of wall material increases, the moisture content of the microcapsules exhibits a slow upward trend, rising from 2.18±0.05% to 2.56±0.09%. Lower moisture content indicates lower hygroscopicity, reducing powder clumping, minimizing microbial growth, and improving storage stability. Although the moisture content of the microcapsules increased, it remained below 4%, meeting the quality requirements for powdered food materials and thus demonstrating potential for application in the food industry. The solubility of all groups was above 83%, with no significant differences between groups. This may be because the surface structure and density of the microcapsules are relatively similar, thus having little impact on solubility. However, higher solubility is beneficial for their use in food processing.

[0086] (3) Determination of microcapsule encapsulation characteristics with different core-to-wall ratios Surface oil (W) s Determination: 2 g of microcapsule powder (W) was added. m Dissolve in 30 mL of n-hexane and vortex for 60 s. Centrifuge at 10,000 rpm for 30 min at 4 °C, remove the n-hexane fraction, and remove the solvent by rotary evaporation at 50 °C. Then place at 90 °C to remove residual solvent and water and dry to constant weight. After cooling to room temperature, calculate the surface oil content by gravimetric method.

[0087] Total oil (W) t Determination: Dissolve 2 g of microcapsules in 20 mL of 4 M hydrochloric acid, vortex for 60 s, then add 20 mL of n-hexane, vortex for 60 seconds. Mix the mixture at room temperature for 14 h to ensure the oil is fully dissolved in the hexane. Centrifuge the mixture at 10,000 rpm for 30 min at 4 °C, then remove the n-hexane fraction and remove the solvent by rotary evaporation at 50 °C. Then, place the mixture at 90 °C to remove residual solvent and water, and dry to constant weight. After cooling to room temperature, determine the total oil content by gravimetric method.

[0088] Encapsulation efficiency (EE), payload (PL), and packaging efficiency (EY) were calculated using the following formulas, and the statistical results are shown in Table 5.

[0089] Surface oil content (%) = W s / W m ×100% EE (%) = (W t -W s ) / W t ×100% PL (%) = W t / W m ×100% EY (%) = W t / W i ×100% In the formula, Wi The mass (g) of the oil used to form microcapsules.

[0090] Table 5. Detection results of microcapsule encapsulation characteristics of glycosylated coconut protein-fish gelatin-fish oil microcapsules with different core-wall ratios.

[0091] Table 5 shows that the encapsulation efficiencies of microcapsules with different core-to-wall ratios were 95.53%, 96.32%, 96.37%, and 96.49%, respectively. This indicates that increasing the amount of wall material helps form a more complete and dense wall film structure, effectively inhibiting oil droplet leakage during emulsification and drying. The surface oil content decreased from 2.64±0.17% to 1.45±0.20%, which also indicates a more thorough encapsulation process, enhanced stability of the emulsion system, improved coverage and protection of the core material by the wall material, and reduced leakage. This result has a positive impact on the storage stability of the final product. Combined with indicators such as effective load and encapsulation efficiency, it can be seen that glycosylated coconut protein as a wall material has good load-bearing capacity for fish oil, and the preparation process is stable, allowing for relatively complete and efficient encapsulation and utilization of the core material.

[0092] (4) Determination of microcapsule particle size with different core-wall ratios After diluting the microcapsule powder by a certain factor to ensure dispersion, the average size and size distribution of the microcapsules were measured using a laser particle size analyzer. The distribution index (Span) was calculated according to the following formula, and the results are shown in Table 6.

[0093] Span=(D90-D10) / D50 In the formula, D90, D50, and D10 are the equivalent diameters (average particle sizes) of the largest particles when the cumulative distribution in the distribution curve is 90%, 50%, and 10%, respectively.

[0094] Table 6. Particle size determination results of glycosylated coconut protein-fish gelatin-fish oil microcapsules with different core-wall ratios.

[0095] Table 6 shows that as the core-to-wall ratio increased from 2:1 to 4:5, the median diameter (D50) of the microcapsules significantly increased from 10.54±0.13 μm to 31.44±0.43 μm (P<0.05), indicating that increasing the wall material content leads to an increase in microcapsule size. The volume average diameter (D[4,3]) showed the same increasing trend as D50, and D[4,3] was greater than D50, indicating the presence of a small number of coarse particles that contribute significantly to the volume. The Span value is a key indicator for measuring the width of the particle size distribution. The smaller the Span value, the more concentrated the particle size distribution and the more uniform the sample. The Span value decreased significantly from 3.45±0.08 to 1.83±0.02 (P<0.05), indicating that the microcapsule particle size distribution was more concentrated and uniform. Furthermore, the average surface area diameter (D[3,2]) significantly increased from 5.26±0.06 μm to 8.75±0.26 μm, indicating a decrease in the specific surface area of ​​the microcapsules. This facilitates a slower and more sustained release, reducing the degree of oxidation. Therefore, while increasing the core-to-wall ratio increases the microcapsule size, it also improves the uniformity of their distribution, thus slowing down the oxidation process.

[0096] (5) Detection of oxidative stability of microcapsules with different core-wall ratios Oxidation time assay: Accelerated oxidation tests were performed using a Rancimat accelerated oxidation assay instrument to determine the oxidative stability of various microcapsules. In short, 2 g of microcapsules were heated in purified air at a flow rate of 20 L / h at 80°C, 90°C, 100°C, 110°C, and 120°C. The induction time of the samples was recorded as the oxidation stability index (OSI), and the results are shown in Table 7.

[0097] Table 7 Accelerated oxidation time of glycosylated coconut protein-fish gelatin fish oil microcapsules with different core-wall ratios at different temperatures.

[0098] Table 7 shows that the core-to-wall ratio has a significant impact on the antioxidant properties of microcapsules. With increasing wall material ratio, the oxidation time of microcapsules at all test temperatures (80–120 °C) is significantly prolonged, indicating a significant enhancement in oxidative stability. This trend is consistent with the results of the encapsulation characteristic analysis, suggesting that a more complete and dense wall membrane structure effectively reduces free oil content, thus providing a superior barrier protection for the core material and greatly delaying the lipid oxidation process. In conclusion, a core-to-wall ratio of 4:5 provides optimal oxidative stability and is the best choice for extending the shelf life of microcapsule products.

[0099] (6) Oxidation kinetics analysis of microcapsules with different core-to-wall ratios By establishing a first-order reaction kinetic model of temperature versus oxidation induction time ( Figure 11The kinetic parameters are shown in Table 8. The oxidative degradation behavior of microcapsules with different core-to-wall ratios was analyzed.

[0100] Table 8. First-order reaction kinetic parameters of glycosylated coconut protein-fish gelatin-fish oil microcapsules with different core-wall ratios.

[0101] Depend on Figure 11 As shown in Table 8, the regression equations for microcapsules with different core-to-wall ratios all exhibit extremely high regression coefficients (R²). 2 The value >0.99 indicates that the oxidation process follows good first-order reaction kinetics, demonstrating strong model reliability. As the wall material ratio increases (from 2:1 to 4:5), the intercept of the regression equation significantly increases, indicating that the oxidation induction time is prolonged and thermal stability is enhanced at the same temperature. Based on this model, it is predicted that under normal storage conditions at 25℃, the shelf life of the 4:5 formulation (approximately 2688.58 hours) is significantly longer than that of the 2:1 formulation (approximately 1054.54 hours), with a stability improvement of more than 2.5 times. In summary, increasing the wall material ratio can effectively delay core material oxidation and significantly extend product shelf life, with 4:5 being the optimal formulation.

[0102] (7) Analysis of volatile substances in fish oil, fish gelatin fish oil microcapsules, and glycosylated coconut protein-fish gelatin fish oil microcapsules 2 g of sample was weighed into a 20 mL headspace vial. Volatile flavor compounds were determined using gas chromatography-ion mobility spectrometry (GC-IMS) (FlavourSpec), and GC-IMS chromatograms and fingerprints were obtained. Injection conditions: Incubation was performed at 80 °C and 500 r / min for 15 min. 500 μL of headspace sample was injected into the autosampler in splitless mode using a 1 mL gas-tight heated syringe at 80 °C. GC conditions: MTX-5 column, column temperature 60 °C, carrier gas / drift gas nitrogen (purity > 99.999%). Carrier gas flow program: 2 mL / min for 0–2 min, linearly increased to 100 mL / min for 2–25 min, and held for 30 min. IMS conditions: IMS was run at 45 °C and a flow rate of 150 mL / min.

[0103] According to the GC-IMS comparison, fish oil microencapsulation significantly increases the amount of flavor compounds. Dividing the fingerprint spectrum into five regions (A, B, C, D, and E) allows for effective differentiation of the flavor characteristics of the three types of microcapsules. A total of 86 volatile compounds were detected in regions A through E. Region A (20 compounds) is shared by all three types; Region B (35 compounds) is shared by both types of microcapsules; Region C (21 compounds) is unique to glycosylated coconut protein-fish gelatin fish oil microcapsules; while Region D (6 compounds) and Region E (4 compounds) are unique to fish gelatin fish oil microcapsules and fish oil, respectively. Fish oil microencapsulation reduces substances in Region E such as 4-methyl-5-vinylthiazole and 1-methyl-2-pyrrolidone, which are prone to producing unpleasant odors, indicating that fish oil microencapsulation helps mask fishy odors. Compared to fish gelatin-fish oil microcapsules, glycosylated coconut protein-fish gelatin-fish oil microcapsules contain more furans formed through the dehydration or cyclization of reducing sugars, resulting in a roasted flavor. This may be related to the presence of polysaccharides in the wall material. The formation of furans not only enhances flavor complexity but also synergizes with the fruity and floral aromas of esters, further masking unpleasant odors and thus improving the overall flavor. Therefore, glycosylated coconut protein, compared to fish gelatin, has a more significant advantage in masking fishy odors as a microcapsule wall material, resulting in a superior overall flavor for the microcapsules.

[0104] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A glycosylated coconut protein, characterized in that, Coconut protein was glycosylated using konjac glucomannan as a sugar source.

2. The method for preparing glycosylated coconut protein according to claim 1, characterized in that, include: After dissolving and mixing coconut protein and konjac glucomannan, and pre-stirring, a mixed solution is obtained; The mixed solution was heated to carry out a glycosylation reaction to obtain the glycosylated coconut protein.

3. The method for preparing glycosylated coconut protein according to claim 2, characterized in that, The mass ratio of the mixed coconut protein to konjac glucomannan is 1:3 to 5:

1. The solution used for dissolution is PBS solution.

4. The method for preparing glycosylated coconut protein according to claim 3, characterized in that, The mass ratio of the mixed coconut protein to konjac glucomannan is 1:

1.

5. The method for preparing glycosylated coconut protein according to claim 2, characterized in that, The pre-stirring was carried out at 750 rpm for 15 min.

6. The method for preparing glycosylated coconut protein according to claim 2, characterized in that, The glycosylation reaction is carried out at a temperature of 60-90°C for 3-12 hours. The pH of the mixed solution was adjusted to 8.0 before the glycosylation reaction.

7. The method for preparing glycosylated coconut protein according to claim 6, characterized in that, The glycosylation reaction was carried out at a temperature of 80°C for 9 hours.

8. The application of the method for preparing glycosylated coconut protein according to any one of claims 2 to 7 in improving the solubility, stability, and emulsifying properties of coconut protein.

9. The application of the glycosylated coconut protein according to claim 1 in the preparation of fish oil microcapsules, characterized in that, The glycosylated coconut protein is used as the wall material, and fish oil is used as the core material; The mass ratio of the core material to the wall material is 2:1 to 4:

5.

10. The application according to claim 9, characterized in that, The mass ratio of the core material to the wall material is 4:5.

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