Fish oil microcapsule based on pea protein isolate emulsification and coagulation and preparation method thereof

Abstract

The invention discloses a fish oil microcapsule based on pea protein isolate emulsification and coagulation and a preparation method thereof, and the method comprises the following steps: S1, preparing a PPI solution and an SBP solution: respectively dissolving PPI and SBP in a phosphate buffer solution, and stirring until the PPI and the SBP are fully dissolved to respectively obtain a PPI stock solution and an SBP stock solution; s2, preparing PPI stable fish oil oil-in-water emulsion: stirring and mixing the PPI stock solution and FO to obtain the PPI fish oil oil-in-water emulsion; s3, preparing an FO-PPI-SBP composite coagulator: stirring and mixing the PPI fish oil oil-in-water emulsion and the SBP stock solution, adjusting the pH value to 3.5-4.5, and then standing for 24 hours to obtain the FO-PPI-SBP composite coagulator; s4, preparing the fish oil microcapsules: feeding the FO-PPI-SBP composite condensate into a micro spray dryer, and performing spray drying to form the fish oil microcapsules; the FO-PPI-SBP composite coacervate is used as the feeding dispersion of the fish oil microcapsule, so that the powder production rate, the effective load and the encapsulation efficiency of the fish oil microcapsule are greatly improved, and the denaturation temperature and the network thermal stability of the fish oil microcapsule are improved.

Description

Technical Field

[0001] The invention relates to the technical field of fish oil microcapsule preparation, and in particular to fish oil microcapsule based on pea protein isolate emulsification and coagulation and a preparation method thereof. Background Art

[0002] Numerous studies have shown that ω-3 long-chain polyunsaturated fatty acids (n-3LC-PUFA) are one of the most effective bioactive lipids for improving human health. They can maintain infant visual and brain development, maintain cardiovascular health, improve cardiovascular disease (CVD), etc. In order to meet the demand for ω-3 fatty acids in the daily diet and convey their benefits to consumers through traditional means, the food industry has been working hard to incorporate them into various foods. Typically, ω-3 fatty acids exist naturally in the form of triacylglycerols (TAGs), such as marine fish oil (also known as ω-3 fish oil). Therefore, they have extremely low water solubility and generally cannot be directly dispersed in aqueous foods, such as beverages, desserts, seasonings and sauces. Therefore, how to improve the dispersibility of ω-3 fatty acids in hydrophilic food matrices has always been a major challenge facing the food industry. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for preparing fish oil microcapsules based on emulsification and coagulation of pea protein isolate.

[0004] Furthermore, the present application also provides a fish oil microcapsule based on the emulsification and coagulation of pea protein isolate.

[0005] To achieve the above purpose, the technical means adopted by the present invention are:

[0006] A method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation comprises the following steps:

[0007] S1. preparing PPI solution and SBP solution: dissolving PPI and SBP in phosphate buffer respectively and stirring until fully dissolved to obtain PPI stock solution and SBP stock solution respectively;

[0008] S2, preparing a PPI-stabilized fish oil oil-in-water emulsion: mixing the PPI stock solution prepared in step S1 with FO at a mass ratio of 9:1 to obtain a PPI fish oil oil-in-water emulsion;

[0009] S3, preparing FO-PPI-SBP complex coacervates: the PPI fish oil water-in-oil emulsion prepared in step S2 and the SBP stock solution prepared in step S1 were stirred and mixed at a mass ratio of 5:1, the pH value was adjusted to 3.5-4.5, and then allowed to stand at 4°C for 24 hours to obtain FO-PPI-SBP complex coacervates;

[0010] S4, preparing fish oil microcapsules: feeding the FO-PPI-SBP complex coacervate prepared in step S3 into a micro spray dryer at a feed rate of 0.15 L / h and a suction flow rate of 40 L / h, and forming fish oil microcapsules by spray drying.

[0011] Further, in step S1, the mass concentration of the PPI stock solution is 2.22wt%, and the pH is 7.0; the mass concentration of the SBP stock solution is 0.4wt%, and the pH is 7.0.

[0012] Further, the preparation of the PPI solution specifically includes the following steps:

[0013] S111, dissolving PPI in phosphate buffer and stirring with a mechanical stirrer at 500 rpm for 0.5 hour at room temperature to fully dissolve PPI in the phosphate buffer;

[0014] S112, adding NaOH solution to the solution prepared in step S111, adjusting its pH value to 9.5, and stirring for 2 hours to promote hydration, then adding HCl to adjust the solution pH to 7.0, and stirring for 10-12 hours to ensure that the solution is fully dissolved;

[0015] S113, centrifuging the solution prepared in step S112 at 6000 rpm and 4° C. for 15 minutes, and filtering to remove insoluble impurities therein; rechecking the pH value of the solution, and adjusting the pH value to 7.0 using NaOH or HCl to obtain a PPI stock solution with a mass concentration of 2.22 wt%.

[0016] Further, the preparation of the SBP solution specifically comprises the following steps:

[0017] S121, dispersing beet pectin in a phosphate buffer and stirring with a mechanical stirrer at 500 rpm for 5 hours at room temperature to fully dissolve the beet pectin in the phosphate buffer; then adding NaOH or HCl to adjust the pH value of the solution to 7.0;

[0018] S122, centrifuging the solution prepared in step S121 at 6000 rpm and 4°C for 15 minutes, filtering to remove insoluble impurities, rechecking the pH value of the solution, and adjusting the pH value to 7.0 using 0.1-2M NaOH or HCl if necessary, to obtain a SBP stock solution with a mass concentration of 0.4%.

[0019] Furthermore, the concentration of the phosphate buffer is 10 mM and the pH is 7.0; the concentrations of the NaOH and HCl are both 0.1-2 M.

[0020] Furthermore, the pH value of the FO-PPI-SBP complex coacervate is 3.5.

[0021] Furthermore, in step S4, the inlet and outlet air temperatures of the micro spray dryer are 180° C. and 105° C., respectively.

[0022] Furthermore, the present application also provides a fish oil microcapsule prepared by the above method.

[0023] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0025] Figure 1 a is the Zeta (ζ) potential diagram of PPI solution, SBP solution, PPI-stabilized fish oil-in-water emulsion, and FO–PPI–SBP complex coacervate;

[0026] Figure 1 b is the average particle diameter of PPI-stabilized fish oil-in-water emulsion and FO–PPI–SBP complex coacervate;

[0027] Figure 2 a is the phase diagram of PPI-stabilized FO emulsion and FO-PPI-SBP composite dispersion during acid titration;

[0028] Figure 2 b is a photograph showing the changes in the appearance of FO-PPI-SBP composite dispersion with changes in pH value;

[0029] Among them, □ represents a turbid solution; ▲ represents a precipitate and a turbid solution; ■ represents a precipitate and a clear solution;

[0030] Figure 3 a is the CLSM micrograph of the PPI stock solution at pH 7.0;

[0031] Figure 3 b, CLSM micrograph of SBP stock solution at pH 7.0;

[0032] Figure 3 c is the CLSM micrograph of the PPI fish oil-in-water emulsion at pH 7.0;

[0033] Figure 3 d is the CLSM micrograph of the PPI fish oil-in-water emulsion at pH 3.5;

[0034] Figure 3 e is the CLSM micrograph of FO-PPI-SBP complex coacervate at pH 7.0;

[0035] Figure 3 f is the CLSM micrograph of FO-PPI-SBP complex coacervate at pH 4.5;

[0036] Figure 3 g is the CLSM micrograph of FO-PPI-SBP complex coacervates at pH 4.0;

[0037] Figure 3 h is the CLSM micrograph of FO-PPI-SBP complex coacervate at pH 3.5;

[0038] Figure 4 a is the microstructure of the PPI fish oil in water emulsion with a pH value of 3.5;

[0039] Figure 4 b is the microstructure of FO-PPI-SBP complex coacervate at pH 4.5;

[0040] Figure 4 c is the microstructure of FO-PPI-SBP complex coacervate at pH 4.0;

[0041] Figure 4 d is the microstructure of FO-PPI-SBP complex coacervate at pH 3.5;

[0042] Figure 5 FTIR spectra of different wall materials;

[0043] Figure 6 DSC thermograms of fish oil microcapsules prepared from different dispersions;

[0044] Figure 7 a is the SEM micrograph of SBP;

[0045] Figure 7 b is the SEM micrograph of PPI;

[0046] Figure 7 c is a SEM micrograph of the PPI fish oil-in-water emulsion;

[0047] Figure 7 d is the SEM micrograph of FO-PPI-SBP complex coacervates at pH 4.5;

[0048] Figure 7 e is the SEM micrograph of FO-PPI-SBP complex coacervates at pH 4.0;

[0049] Figure 7 f is the SEM micrograph of FO-PPI-SBP complex coacervate at pH 3.5. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with specific embodiments:

[0051] Example

[0052] This embodiment provides a method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation, comprising the following steps:

[0053] S1. Prepare PPI solution and SBP solution: dissolve PPI (pea protein isolate) and SBP (beet pectin) in phosphate buffer respectively and stir until fully dissolved to obtain PPI stock solution and SBP stock solution respectively, wherein the mass concentration of the PPI stock solution is 2.22wt%, the pH is 7.0, and the mass concentration of the SBP stock solution is 0.4wt%, and the pH is 7.0.

[0054] The preparation of the PPI solution specifically includes the following steps:

[0055] S111. Dissolve PPI in phosphate buffer and stir with a mechanical stirrer at 500 rpm for 0.5 hour at room temperature to fully dissolve PPI in the phosphate buffer; the concentration of the phosphate buffer is 10 mM, the pH is 7.0, and the temperature is maintained at 21-22°C.

[0056] S112. Add a 0.1-2M NaOH solution to the solution prepared in step S111, adjust the pH to pH 9.5, and stir for 2 hours to promote hydration, then add 0.1-2M HCl to adjust the pH of the solution to 7.0, and stir for 10-12 hours to ensure that the solution is fully dissolved.

[0057] S113, centrifuging the solution prepared in step S112 at 6000 rpm and 4° C. for 15 minutes, filtering to remove insoluble impurities, rechecking the pH value of the solution, and adjusting the pH value to 7.0 using 0.1-2 M NaOH or HCl if necessary, to obtain a PPI stock solution with a mass concentration of 2.22 wt%.

[0058] The preparation of SBP solution specifically comprises the following steps:

[0059] S121. Disperse beet pectin in phosphate buffer and stir with a mechanical stirrer at 500 rpm for 5 hours at room temperature to fully dissolve the beet pectin in the phosphate buffer, and then add NaOH or HCl to adjust the pH value of the solution to 7.0; the concentration of the phosphate buffer is 10 mM, the pH is 7.0, and the temperature is maintained at 21-22°C.

[0060] S122, centrifuging the solution prepared in step S121 at 6000 rpm and 4°C for 15 minutes, filtering to remove insoluble impurities, rechecking the pH value of the solution, and adjusting the pH value to 7.0 using 0.1-2M NaOH or HCl if necessary, to obtain a SBP stock solution with a mass concentration of 0.4%.

[0061] S2. Preparation of PPI-stabilized fish oil oil-in-water emulsion: The PPI stock solution prepared in step S1 was mixed with FO (fish oil) at a mass ratio of 9:1, and mixed with a high-speed stirrer at 10,000 rpm for 2 minutes to prepare a PPI fish oil oil-in-water emulsion.

[0062] In order to obtain fine-grained emulsion droplets, the prepared coarse-grained emulsion needs to be homogenized three times by a two-stage high-pressure valve homogenizer. The pressures of the first and second stages are 5000 and 500 psi, respectively. The final emulsion composition is fish oil with a mass concentration of 10% and PPI stock solution with a mass concentration of 2.22%.

[0063] S3. Preparation of FO-PPI-SBP complex coacervates: The PPI fish oil water-in-oil emulsion prepared in step S2 was mixed with the SBP stock solution prepared in step S1 at a mass ratio of 5:1. The mixture was magnetically stirred at 500 rpm for 10 minutes to allow the two solutions to be fully mixed. The pH value was adjusted from 7.0 to 3.5-4.5 by adding HCl (0.1-2.0 M). The use of different concentrations of HCl can reduce the dilution effect and conductivity change of the mixed solution. Finally, the FO-PPI-SBP aqueous phase mixture was allowed to stand at 4 ° C for 24 hours to reach phase equilibrium.

[0064] S4, preparing fish oil microcapsules: feeding the FO-PPI-SBP complex coacervate prepared in step S3 into a micro spray dryer at a feed rate of 0.15 L / h and a suction flow rate of 40 L / h to form fish oil microcapsules.

[0065] The inlet and outlet air temperatures were maintained at 180 °C and 105 °C, respectively. The above operating parameters were optimized through preliminary experiments. The total solid content of all prepared mixtures was about 6.0–6.2%. All the powders after spray drying were collected and stored in glass bottles at 4 °C before characterization.

[0066] In this embodiment, the EPA content in fish oil is greater than 9%, the DHA content is greater than 12.5%, and the total content of n-3PUFA in the form of triglycerides is greater than 30%; pea protein isolate (PPI) is extracted from commercial pea powder by alkaline extraction-isoelectric precipitation, and its composition is 79.50% protein, 5.28% water, 0.77% fat, 4.61% crude ash, and 9.84% carbohydrates; beet pectin (SBP). Sodium hydroxide (NaOH), hydrochloric acid (HCl) and other chemicals and reagents used in this application are of analytical grade. All solutions are prepared with ultrapure water.

[0067] Comparative Example

[0068] Different from the embodiment, in this comparative example, after the PPI-stabilized fish oil oil-in-water emulsion is prepared in step S2, the PPI-stabilized fish oil oil-in-water emulsion is directly fed into a Büchi micro spray dryer to prepare fish oil microcapsules.

[0069] In order to further characterize the structure and performance of the PPI solution, SBP solution, PPI-stabilized fish oil oil-in-water emulsion, FO-PPI-SBP complex coacervate and fish oil microcapsule prepared in this application, the above solutions or products are tested and analyzed as follows:

[0070] Test method:

[0071] 1. Surface charge and particle size analysis of feed dispersion

[0072] The surface charge and particle size of PPI-stabilized emulsions and FO-PPI-SBP composite dispersants were measured as a function of pH (7.0-3.0) and reported as zeta potential (ζ, mV) and z-average diameter (nm), respectively. Before each measurement, the samples were diluted 20 times with buffer of the corresponding pH and equilibrated at 25°C for 2 minutes. All measurements were performed in duplicate, and three readings were recorded for each measurement.

[0073] 2. Construction of state diagram

[0074] After standing for 24 hours, the state diagrams of PPI-stabilized emulsions and FO-PPI-SBP were obtained. After standing for 24 hours, the state diagrams of PPI-stabilized emulsions and FO-PPI-SBP composite dispersions were drawn based on the observations of each phase equilibrium mixture. The phase separation was divided into five groups, marked with three symbols, namely: □, ▲, ■ represent turbid solution, precipitation and turbid solution, and precipitation and clear solution, respectively.

[0075] 3. Characterization of spray-dried fish oil microcapsules

[0076] 3.1 Encapsulation Efficiency (EE)

[0077] The surface oil content was measured by dispersing 2.5 g of microcapsule powder in 10 mL of hexane and rotating for 2 min, then centrifuging the slurry at 4000 rpm for 10 min at ambient temperature. The precipitate was washed twice and all supernatant was recovered. The hexane was removed by flushing with nitrogen for 6 h, and the surface oil content was determined gravimetrically. The total oil content of the dried microcapsules (2.5 g) was determined using a Soxhlet oil extractor, and the solvent was completely removed using a rotary evaporator, flushed with nitrogen, and finally the total oil content was determined gravimetrically. The encapsulation efficiency (EE) of the microcapsules was calculated as follows.

[0078] EE=(Wt-Ws) / Wt×100%

[0079] Wherein, Wt and Ws are the mass of total lipid and surface lipid of microcapsules, respectively (g).

[0080] 3.2 Differential Scanning Calorimetry

[0081] The thermal properties of the spray-dried samples and control samples were studied using a differential scanning calorimeter. The sample (about 3.5 mg) containing 10 μL of distilled water was placed in a 40 μL aluminum pan. The pan was then sealed and equilibrated at room temperature overnight to allow the water to be evenly distributed. The sample was measured from 20°C to 120°C at a heating rate of 10°C / min using a sealed empty aluminum pan as a reference.

[0082] 3.3 Fourier Transform Infrared Spectroscopy

[0083] FTIR spectra of the spray-dried samples and controls were recorded at 25 °C using a spectrophotometer equipped with an attenuated total reflectance accessory, a Globar (MIR) source, a KBr beam splitter, and an MCT detector. -1 32 scans were accumulated in absorbance mode from 4000 to 400 cm -1 Scan the sample. Collect background before measuring the sample.

[0084] 3.4 Scanning electron microscopy observation of surface morphology

[0085] The spray-dried powder adheres to the bonded carbon sheet on the columnar aluminum support, and the excess is blown off with a nitrogen stream. The samples are then sputter-coated with gold to make them conductive and finally examined using the same scanning electron microscope. The selected micrographs (5000×) are representative of each sample.

[0086] 5. Statistical analysis

[0087] The experimental data were processed in a completely randomized design (CRD) with two replicates. At least two measurements were performed using freshly prepared samples, and the measured values ​​were expressed as mean ± standard deviation. One-way ANOVA and SAS software F test protection of the least significant difference method were used to analyze the significant differences (p < 0.05) between the means.

[0088] Test results and analysis

[0089] 1. Effect of pH on surface charge and particle size of fish oil dispersions

[0090] The formation of complex coacervates between PPI and SBP is achieved through the electrostatic attraction between them. At a fixed biopolymer ratio (PPI / SBP = 5:1), the environmental pH is the main factor determining the strength of the electrostatic interaction. In order to determine the appropriate pH range required for the formation of fish oil-PPI-SBP complex coacervates, the surface charge (ζ-potential) and particle size (z-average) of each dispersion were measured experimentally. For comparison, the effect of pH on the surface charge and particle size of PPI-stabilized fish oil emulsions was also measured.

[0091] The measurement results are as follows Figure 1 As shown, Figure 1 a is the Zeta potential diagram of PPI solution, SBP solution, PPI-stabilized fish oil-in-water emulsion and FO-PPI-SBP complex coacervate. Figure 1 b Average particle diameter of PPI-stabilized fish oil-in-water emulsion and FO–PPI–SBP complex coacervate.

[0092] like Figure 1 As shown in a, the ζ potential of the PPI solution is -24.17 mV at pH = 7.0, +27.18 mV at pH = 3.0, and zero net charge appears at around pH = 4.7. The ζ potential of the SBP solution is negative over the entire pH range, which is a common surface charge pattern for anionic polysaccharides. When the pH value decreases from 7.0 to 5.0, the ζ potential of the SBP solution decreases in a gradient. A further decrease in pH causes a sharp drop in the net value of the ζ potential. This is caused by the protonation of the carboxyl functional group (pKa≈3.5) on the galacturonic acid unit of SBP. When the pH value is 7.0-3.0, the ζ potential of fish oil is between monomeric PPI and monomeric SBP, and a net zero charge appears at pH 3.5. In general, complex coacervation driven by electrostatic attraction requires that the biopolymers have opposite charges. According to the ζ-potential results, when the pH is less than 4.5, an overall positively charged PPI (-NH 4+) can generate electrostatic attraction with anionic SBP (-COO-) to form complex coacervation. For PPI-stabilized fish oil in water emulsions, the ζ-potential of fish oil droplets is very similar to that of PPI solution, and the net charge increases when the pH value is far from the isoelectric point (IEP≈4.7).

[0093] The pH dependence of z-average diameter of fish oil emulsion and fish oil-PPI-SBP composite dispersion is shown in Figure 1 As shown in Figure 2b. At different pH values, the particle size of the PPI-stabilized fish oil emulsion changes in a bell-shaped pattern, which is a typical protein-stabilized lipid droplet morphology. At pH 7.0 or 3.0, due to the high net charge (>20 mV) of the PPI-stabilized fish oil emulsion, there is a strong electrostatic repulsion between them, which hinders aggregation. When the pH value is 4.0-6.0, the particles are widely aggregated (>10 μm) because the particles carry little or no net charge. In contrast, the fish oil-PPI-SBP composite dispersion has the smallest particle size at pH 7.0 and 6.5, which means that the interaction between PPI and SBP is weak or non-existent. With the decrease of pH, the particle size increases significantly. The appearance of larger particles may be due to the enhanced protonation of the amino side group of PPI, which triggers the electrical attraction with the negatively charged carboxyl group of SBP. In the medium pH range, the particle size of the fish oil-PPI-SBP composite dispersion is even smaller than that of the emulsion. This may be due to the fact that the electrostatic repulsion is not strong enough to counteract the attractive interactions (such as van der Waals forces or hydrophobic interactions).

[0094] 2. Effect of pH on the phase behavior and microstructure of fish oil dispersions

[0095] To better visualize the effect of pH on the appearance of fish oil in two different feed dispersions, we constructed a state diagram ( Figure 2 a) to show the phase behavior of PPI-stabilized fish oil emulsions and FO–PPI–SBP composite dispersions as a function of pH (7.0–3.0) after 24 h at 25 °C. The phase behavior of PPI-stabilized fish oil emulsions is very classical ( Figure 2 a), a single-phase solution can be observed at pH (7.0, 6.5, 2.0). However, as the pH value decreases, it changes to precipitation and cloudy forms (▲), and becomes a condensed precipitate (■) at pH 5.0. For the fish oil-PPI-SBP composite dispersion system, three distinguishable phase behaviors are shown in the state diagram. At neutral to acidic pH values ​​(7.0, 6.5, 6.0), a homogeneous phase (□) is observed in the mixture, which is the result of the repulsion of the same charges of PPI and SBP ( Figure 2b). When the pH is lowered to acidic conditions (5.5-2.0), two insoluble complexes (condensation or precipitation) with different phase behaviors appear, with different structural properties. Since the pH value (5.5-4.0) is close to the IEP of PPI, the condensation shows precipitation and cloudy appearance (▲). This phase behavior is due to the mutual attraction between the negative charge of the polyanion SBP and the opposite color spots on the surface of PPI. But at higher pH values ​​(5.5, 5.0, 4.5), the repulsive force within the system is strong, preventing drastic phase separation, while obvious phase separation occurs at pH 4.0. At pH 3.5 and 3.0, phase behaviors of precipitation and transparent solution (■) are formed.

[0096] CLSM (confocal laser scanning microscopy) was then used to evaluate the effect of pH on the in situ morphological properties of PPI-stabilized fish oil emulsions and fish oil–PPI–SBP composite dispersions. Figure 3 ). PPI is labeled with FITC, as shown in the CLSM micrograph ( Figure 3 a) is shown as green dots, while fish oil is stained with Nile red. CLSM micrographs of PPI and SBP solutions ( Figure 3 a and b), at pH 7.0 ( Figure 3 c), no signs of flocculation of the emulsion droplets were found, which may be attributed to the high zeta potential of the droplets. When the emulsion was acidified to pH 3.5, localized aggregation of large particles ( Figure 3 d white arrow), which is consistent with the particle size measurement results. The increased droplet flocculation is due to the addition of acid to lower the pH from 7.0 to 3.5. This changes the pH by allowing the IEP of the adsorbed PPI to move the droplets closer to each other. For the fish oil–PPI–SBP composite dispersion ( Figure 3 e), whose microstructure was similar to that of the emulsion droplets at pH 7.0. When the pH of the mixture was reduced from 7.0 to 4.5 or 4.0 (close to the IEP of PPI), only a small amount of floccules mixed with green particles were observed ( Figure 3 f and 3g), the microstructure is similar to that of the emulsion with a pH value of 3.5. The size of the observed aggregates is also similar to that of the emulsion with a pH value of 3.5. Figure 1 b. The lower number and size of complex coacervates at this pH may be due to the relatively strong electrostatic repulsion of the dispersion (|ζ|>20 mV). As a rule, a slight decrease in pH from 4.0 to 3.5 significantly promotes the formation of complex irregularly shaped coacervates ( Figure 3 h). This trend reflects the progressive charge neutralization of the system, where the biopolymer interactions reach a point of electrical equivalence while allowing attractive forces such as van der Waals and hydrophobic forces to dominate the interactions.

[0097] From the scanning electron microscopy (SEM), it can be seen that the microstructures of freeze-dried fish oil emulsion and FO-PPI-SBP complex coacervate at different pH values ​​are different without polishing ( Figure 4 a), which exhibited a flake-like shape with a smooth surface and no debris, which may be due to the aggregation of emulsion droplets during the drying process. A similar morphology was observed in the FO–PPI–SBP complex coacervates formed at pH 4.5, except that the microstructure became less condensed as it extended in one dimension ( Figure 4 b). This again indicates that complex coacervates are not formed due to the strong repulsion between the biopolymers. Notably, sponge-like porous structures emerge with decreasing pH. At pH 4.0 of the coacervates, a multidimensional network with multiple patterned pore sizes is formed ( Figure 4 c). Further lowering the pH to 3.5 resulted in an even more concentrated network ( Figure 4 d). This morphological change of the freeze-dried complex coacervates confirms that the strength of electrostatic interactions is different at different pH values, which may significantly affect the interactions of biopolymers. Combining the results of the state map, CLSM, and SEM, it can be confirmed that the FO-PPI-SBP coacervates exhibit a relatively dense network structure at pH 3.5, which is mainly derived from charge neutralization.

[0098] 3. Analysis of fish oil microcapsules

[0099] 3.1 Packaging efficiency

[0100] Table 1 shows the powder yield (PY), encapsulation efficiency (EE), encapsulation yield (EY), and payload (PL) of the spray-dried microcapsule powders prepared by emulsification and complex coacervation. Powder yield is a key parameter that determines the recovery of solid matter after spray drying. The PY of all microcapsule powders was very similar, except that the complex coacervate formed at pH 3.5 produced the lowest PY value (42.0%). The microcapsule powder prepared by PPI-stabilized emulsion had the lowest EE (p < 0.05) among the two different dispersion preparation methods, indicating that the latter method has improved EE and higher encapsulation efficiency in the pH range of 3.5-4.5. The EE of the three fish oil microcapsules prepared by complex coacervation was the same, regardless of the pH at formation. Two factors may have contributed to the lower EE values. In this study, less wall material was used, which had a direct impact on the EE value. In addition, a more stringent and aggressive solvent washing protocol was used in an attempt to completely remove the surface oil instead of measuring the amount of easily extractable oil in it. Compared with the control group without SBP, the encapsulation efficiency (EE), encapsulation rate (EY) and payload (PL) of fish oil microcapsules after SBP addition were able to achieve similar or better results under a wider pH condition. The fish oil microcapsule powders spray-dried from PPI-stabilized emulsions and fish oil-PPI-SBP complex coacervates showed similar PY, EY and PL.

[0101] Table 1

[0102] Powder yield (PY), encapsulation efficiency (EE), encapsulation yield (EY), and payload (PL) of spray-dried FO microcapsules of PPI-stabilized emulsions or PPI-SBP complex coacervates*

[0103]

[0104] * Values ​​with different superscript letters in a column indicate statistically significant differences at p < 0.05 (FO: fish oil; PPI: pea protein isolate; SBP: beet pectin)

[0105] 3.2 Interaction of wall materials

[0106] The main chemical functional groups of SBP are in the wave number range of 1000-2000 cm -1 The spectrum of -1 (C=O stretching vibration), 1622 cm -1 (carboxylate ion stretching) and 1441 cm -1 (Asymmetric stretching – CH 3 ). At 3012cm -1 The absorption peak of olefin double bond (–HC=CH–) in fish oil can be observed.

[0107] like Figure 5 As shown, in the spectrum of fish oil microcapsules prepared from PPI-stabilized emulsion at pH 3.5, the peaks at 3012, 2926, 2852, and 1146 cm -1 The absorption peak intensity at 1633, 1529, and 1450 cm-1 gradually increased, indicating the presence of fish oil. In addition, when fish oil was embedded, the initial peaks of PPI were 1633, 1529, and 1450 cm-1. -1 , and their blue shift amplitudes are 8, 4, and 4 cm respectively -1 About. These results indicate that the hydrophobic regions of fish oil and PPI may have interacted with each other during spray drying. Similar FTIR spectra were also found in spray-dried microcapsules, in which low water-soluble chlorophyll was able to bind to the tryptophan or tyrosine residues of whey protein, resulting in a blue shift. Although the spectrum of fish oil microcapsules formed by complex coacervation is very similar to that of fish oil microcapsules formed by emulsification, some significant differences were also found. A new peak appeared in the spectrum of fish oil microcapsules formed by complex coacervation at 1016cm-1, indicating that SBP has been successfully introduced into the complex coacervate. In addition, the peak at 1392cm in PPI and emulsified fish oil microcapsules -1 The peak of did not appear in all complex coacervation microcapsules. Because this peak is assigned to -COO- of amino acids, its disappearance confirms that SPB and PPI form complex coacervates through electrostatic interaction. In addition, the spectrum of fish oil microcapsules based on complex coacervation showed a broadband (about 1020–1080 cm-1), which may be attributed to PPI (peak 1063 cm -1 ) and SBP (peak 1012cm -1 ) is mainly caused by their electrostatic interaction during the formation of complex condensates.

[0108] 3.3 Thermal Analysis

[0109] The present application studies the thermal properties of PPI in different fish oil microcapsules by DSC (e.g. Figure 6 ).

[0110] The DSC thermograms of both PPI and SBP showed endothermic peaks corresponding to the melting of the network structures of the two biopolymers. The average melting point of the raw PPI was about 86.66 °C. The melting point of PPI may be due to the evaporation of water from SBP, which led to the destruction and scission of non-covalent bonds and hydrogen interactions between polar and non-polar groups, thus destroying the secondary and tertiary structures of PPI. When PPI was used as an emulsifier to cover the surface of FO droplets, the melting point of the microcapsules decreased to 76.83 °C, indicating that the synergistic structure of PPI after spray drying was poor. This is reasonable because the hydrophobic interaction between FO and high-pressure homogenization denatured PPI is weaker than the interaction between the hydrophobic amino acid patches in the raw PPI. In contrast, the FO microcapsules prepared from the feed FO–PPI–SBP complex coacervates showed a small and narrow melting peak, which was attributed to the formation of a strong new network structure. This structure can inhibit the denaturation of PPI, thus maintaining a melting point similar to that of the original PPI. Similar phenomena are constantly observed in microcapsules made from complex coacervates such as soy protein isolate and chitosan, as this structure generally increases the stability of proteins against thermal denaturation. The pH value affects the thermal properties of the microcapsules, and the endothermic peaks of the complexes formed at higher pH (4.5) are larger and broader than those formed at pH 4.0 and 3.5. This may also be caused by the strong electrostatic interaction between PPI and SBP, as evidenced by their ζ-potential ( Figure 1 a).

[0111] 3.4 Morphology of fish oil microcapsules

[0112] The morphology of fish oil microcapsules prepared with different feed dispersants can be analyzed using scanning electron microscopy (SEM). Figure 7 ). SBP( Figure 7 a) and PPI ( Figure 7 The microstructures of the microcapsules of fish oil and fish oil are shown as irregular and plate-like morphologies that are difficult to identify, respectively. The morphologies of all fish oil microcapsules have some similarities, for example, there are no cracks or holes on their surfaces, which ensures the low permeability of the capsules and protects the fish oil. However, different feed dispersions have different effects on their morphology. For the fish oil microcapsules prepared from the PPI-stabilized emulsion at pH 3.5, it can be observed that the particles are spherical with a certain degree of roughness ( Figure 7 c). The size of the microcapsules varies greatly between 0.5 μm and 7.5 μm. It is worth noting that some oil spots can be seen on the surface of the microcapsules ( Figure 7c). The size of the fish oil microcapsules obtained from the complex coacervates was generally larger than that obtained from emulsification because pectin was produced during the formation of the coacervates. Compared with the emulsified microcapsules, it showed almost no oil spots, indicating that the encapsulation effect of the complex coacervate was improved. The pH value affected the size of the fish oil microcapsules prepared from the complex coacervates. In particular, when the pH value was 4.5, the size of the capsules changed dramatically ( Figure 7 d); the maximum particle size appears at pH 3.5 ( Figure 7 f). Uniform size of microcapsules ( Figure 7 e) is produced at an intermediate pH value (4.0). This observation is consistent with Figure 4 This is consistent with the results of the previous study, further indicating that the complex coacervates formed at a pH of 3.5 are the optimal conditions for coating fish oil before spray drying.

[0113] The present application uses FO-PPI-SBP complex coacervates as feed dispersions for fish oil microcapsules, thereby greatly improving the powder yield, effective load and encapsulation efficiency of fish oil microcapsules; due to the interaction between PPI and SBP, the prepared fish oil microcapsules have improved denaturation temperature and network thermal stability; since the droplet size, zeta potential, phase behavior and microstructure of the feed dispersion are all affected by pH, the present application adjusts the FO-PPI-SBP complex coacervates at a suitable pH value to generate fish oil microcapsules with larger particle size and denser network structure. Experiments have shown that the FO-PPI-SBP complex coacervates formed at a pH of 3.5 have larger particle size and denser network structure than those generated at higher pH (4.5 and 4.0).

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the technical solution. Although the applicant has described the present invention in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that those modifications or equivalent substitutions of the technical solution of the present invention without departing from the purpose and scope of the technical solution should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation, characterized in that: The steps include: S1. preparing PPI solution and SBP solution: dissolving PPI and SBP in phosphate buffer respectively and stirring until fully dissolved to obtain PPI stock solution and SBP stock solution respectively; S2, preparing a PPI-stabilized fish oil oil-in-water emulsion: mixing the PPI stock solution prepared in step S1 with FO at a mass ratio of 9:1 to obtain a PPI fish oil oil-in-water emulsion; S3, preparing FO-PPI-SBP complex coacervates: the PPI fish oil water-in-oil emulsion prepared in step S2 and the SBP stock solution prepared in step S1 were stirred and mixed at a mass ratio of 5:1, the pH value was adjusted to 3.5-4.5, and then allowed to stand at 4°C for 24 hours to obtain FO-PPI-SBP complex coacervates; S4, preparing fish oil microcapsules: feeding the FO-PPI-SBP complex coacervate prepared in step S3 into a micro spray dryer at a feed rate of 0.15 L / h and a suction flow rate of 40 L / h, and forming fish oil microcapsules by spray drying.

2. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 1, characterized in that: In step S1, the mass concentration of the PPI stock solution is 2.22wt%, and the pH is 7.0; the mass concentration of the SBP stock solution is 0.4wt%, and the pH is 7.

0.

3. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 2, characterized in that: The preparation of the PPI solution specifically comprises the following steps: S111, dissolving PPI in phosphate buffer and stirring with a mechanical stirrer at 500 rpm for 0.5 hour at room temperature to fully dissolve PPI in the phosphate buffer; S112, adding NaOH solution to the solution prepared in step S111, adjusting its pH value to 9.5, and stirring for 2 hours to promote hydration, then adding HCl to adjust the solution pH to 7.0, and stirring for 10-12 hours to ensure that the solution is fully dissolved; S113, centrifuging the solution prepared in step S112 at 6000 rpm and 4° C. for 15 minutes, and filtering to remove insoluble impurities therein; rechecking the pH value of the solution, and adjusting the pH value to 7.0 using NaOH or HCl to obtain a PPI stock solution with a mass concentration of 2.22 wt%.

4. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 2, characterized in that: The preparation of the SBP solution specifically comprises the following steps: S121, dispersing beet pectin in a phosphate buffer and stirring with a mechanical stirrer at 500 rpm for 5 hours at room temperature to fully dissolve the beet pectin in the phosphate buffer; then adding NaOH or HCl to adjust the pH value of the solution to 7.0; S122, centrifuging the solution prepared in step S121 at 6000 rpm and 4°C for 15 minutes, filtering to remove insoluble impurities, rechecking the pH value of the solution, and adjusting the pH value to 7.0 using 0.1-2M NaOH or HCl if necessary, to obtain a SBP stock solution with a mass concentration of 0.4%.

5. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 3 or 4, characterized in that: The concentration of the phosphate buffer is 10 mM and the pH is 7.0; the concentrations of the NaOH and HCl are both 0.1-2 M.

6. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 3 or 4, characterized in that: The pH value of the FO–PPI–SBP complex coacervate was 3.

5.

7. The method for preparing fish oil microcapsules based on pea protein isolate emulsification and coagulation according to claim 3 or 4, characterized in that: In step S4, the inlet and outlet air temperatures of the micro spray dryer are 180° C. and 105° C. respectively.

8. A fish oil microcapsule, characterized in that: Prepared by the method according to any one of claims 1 to 7.

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