NL2034548A

NL2034548A - A Preparation Method for Embedding n-3 Long Chain Polyunsaturated Fatty Acid Microcapsules

Info

Publication number: NL2034548A
Application number: NL2034548A
Authority: NL
Inventors: Xiang Xiaofeng; Xie Yuejie; Wang Kai; Wang Yihan; Wang Qiang; Gou Yao
Original assignee: Chongqing Univ Of Education Cque
Priority date: 2022-06-02
Filing date: 2023-04-12
Publication date: 2023-04-26
Also published as: CN119235013A; NL2034548B1

Abstract

The invention discloses a preparation method of fish oil microcapsules based on emulsification and aggregation of pea protein isolate, which comprises the following steps: 81. Preparation of PPI solution and SBP solution: PPl and SBP are dissolved respectively in phosphate buffer solution and stirred until fully dissolved to obtain PPl reserve solution and SBP reserve solution respectively; 82. Preparation of PPI stabilized oil-in-water emulsion of fish oil: PPl reserve solution was stirred and mixed with FO to obtain PPl oil-in- water emulsion of fish oil; 83. Preparation of FO-PPl-SBP complex condensates: PPl fish oil in water emulsion was mixed with SBP reserve solution, pH value was adjusted to 3.5- 4.5, and then standing for 24 hours to obtain FO-PPl-SBP complex condensates. 84. Preparation of fish oil microcapsules: the composite condensates of FO-PPl-SBP were sent into the micro spray dryer to form fish oil microcapsules through spray drying; By using FO- PPl-SBP composite condensate as the feed dispersion of fish oil microcapsules, this application greatly improves the powder yield, payload and encapsulation rate of fish oil microcapsules, and improves the denaturation temperature and network thermal stability of fish oil microcapsules

Description

Chongqing University of Education, CQUE 23/029 NL

A Preparation Method for Embedding n-3 Long Chain

Polyunsaturated Fatty Acid Microcapsules

Technical Field

The invention relates to the technical field of fish oil microcapsule preparation, in particular to a fish oil microcapsule based on emulsification and condensation of pea protein isolate and a preparation method thereof.

Background Technology

A large number of studies have shown that w-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) are one of the most effective bioactive lipids to improve human health, which can maintain infant visual and brain development, maintain cardiovascular health, and improve cardiovascular disease (CVD). To meet the demand for w-3 fatty acids in daily diets and communicate their benefits to consumers through traditional means, the food industry has worked to incorporate them into a variety of foods. Typically, w-3 fatty acids are found naturally in the form of triacylglycerol (TAG), such as Marine fish oil (also known as omega- 3 fish oil). As a result, they are extremely low in water solubility and generally do not disperse directly in water-based foods such as drinks, desserts, seasonings and sauces. Therefore, how to improve the dispersion of w-3 fatty acids in hydrophilic food substrates has been a major challenge for the food industry.

Summary of the Invention

In view of the above shortcomings of the prior art, the invention aims to provide a method for preparing fish oil microcapsules based on emulsification and condensation of pea protein isolate.

Further, the application provides a fish oil microcapsule based on emulsification and condensation of pea protein isolate.

To realize the above purposes, the technical means adopted by the invention are:

A method for preparing fish oil microcapsules based on emulsification and condensation of pea protein isolate comprises the following steps:

S1. Preparation of PPI solution and SBP solution: PPI and SBP were dissolved in phosphate buffer solution and stirred until fully dissolved to obtain PPI reserve solution and

SBP reserve solution respectively;

S2. Preparation of PPI stabilized oil-in-water emulsion for fish oil: PPI reserve solution prepared in Step S1 was mixed with FO at a mass ratio of 9:1 to obtain PPI oil-in-water emulsion for fish oil;

S3. Preparation of FO-PPI-SBP complex condensate: The PPI fish oil in water emulsion prepared in Step S2 and SBP reserve solution prepared in Step S1 were mixed according to the mass ratio of 5:1, the pH value was adjusted to 3.5~4.5, and then stood at 4°C for 24 hours to obtain FO-PPI-SBP complex condensate.

S4. Preparation of fish oil microcapsules: the FO-PPI-SBP composite condensates prepared in Step S3 were sent to the micro spray dryer at a feed rate of 0.15L /h and a suction flow rate of 40 L/h, and fish oil microcapsules were formed through spray drying.

Additional aspects and advantages of this application will be given in part in the description below, and in part will become apparent from the description below, or will become known through the practice of this application.

Brief Description of the Drawings

In order to more clearly illustrate the specific implementation methods of the invention or the technical scheme in the prior art, a brief introduction of the drawings required to be used in the description of the specific implementation methods or the prior art is presented below. In all drawings, similar elements or parts are generally identified by similar drawings markings. In the drawings, the components or parts are not necessarily drawn to the actual scale.

Figure 1a shows the Zeta (2) potential diagram of PPI solution, SBP solution, oil-in- water emulsion of PPI stabilized fish oil and FO-PPI-SBP composite condensate.

Figure 1b shows the average particle diameter of PPI stabilized oil-in-water emulsion and FO-PPI-SBP composite condensate.

Figure 2a shows the phase diagram of the PPI stable FO emulsion and FO-PPI-SBP complex dispersion during the acid titration process.

Figure 2b shows the appearance of FO-PPI-SBP composite dispersion changing with pH value.

Wherein, o represents the cloudy solution; A represents precipitation and turbid solution; = represents precipitation and clear solution;

Figure 3a shows the CLSM micrograph of PPI reserve solution with pH value of 7.0.

Figure 3b shows the CLSM micrograph of SBP reserve solution with pH value of 7.0.

Figure 3c shows the CLSM micrograph of PPI water-in-water fish oil emulsion with pH value of 7.0.

Figure 3d shows the CLSM micrograph of PPI fish oil in water emulsion with pH value of 3.5.

Figure 3e shows the CLSM micrograph of FO-PPI-SBP complex condensates with pH value of 7.0.

Figure 3f shows the CLSM micrograph of FO-PPI-SBP complex condensates with pH value of 4.5.

Figure 3g shows the CLSM micrograph of FO-PPI-SBP complex condensates with pH value of 4.0.

Figure 3h shows the CLSM micrograph of FO-PPI-SBP complex condensates with pH value of 3.5.

Figure 4a shows the microstructure of PPI water-in-water fish oil emulsion with pH value of 3.5.

Figure 4b shows the microstructure of FO-PPI-SBP complex condensates with pH value of 4.5.

Figure 4c shows the microstructure of FO-PPI-SBP composite condensates with pH value of 4.0.

Figure 4d shows the microstructure of FO-PPI-SBP complex condensates with pH value of 3.5.

Figure 5 shows FTIR spectra of different wall materials.

Figure 6 shows the DSC thermogram of fish oil microcapsules prepared by different dispersion systems.

Figure 7a shows the SEM micrograph of SBP.

Figure 7b shows the SEM micrograph of PPI.

Figure 7c shows the SEM micrograph of PPI water-in-water fish oil emulsion.

Figure 7d shows the SEM micrograph of FO-PPI-SBP composite condensate with pH value of 4.5.

Figure 7e shows the SEM micrograph of FO-PPI-SBP composite condensate with pH value of 4.0.

Figure 7f shows the SEM micrograph of FO-PPI-SBP complex condensate with pH value of 3.5.

Specific Implementation Methods

The invention is further described in combination with specific implementation methods as follows:

Implementation methods

This implementation method provides a method for preparing fish oil microcapsules based on emulsification and condensation of pea protein isolate, consisting of the following steps:

S1. Preparation of PPI solution and SBP solution: PPI (pea protein isolate) and SBP (beet pectin) were dissolved in phosphate buffer and stirred until fully dissolved, respectively, to obtain PPI reserve solution and SBP reserve solution, wherein the mass concentration of said PPI reserve solution is 2.22wt%, pH is 7.0, and the mass concentration of said SBP reserve solution is 0.4wt%. The pH is 7.0.

The preparation of PPI solution includes the following steps:

S111. The PPI was dissolved in the phosphate buffer and stirred with a mechanical agitator at 500rpm at room temperature for 0.5 h to make the PPI fully dissolved in the phosphate buffer; The concentration of phosphate buffer is 10mM, pH is 7.0, and the temperature is maintained at 21-22°C.

Add 0.1-2M NaOH solution into the solution prepared by Step S111, adjust its pH value to 9.5, stir for 2 hours to promote hydration, then add 0.1-2M HCI to adjust the pH of the solution to 7.0, stir for 10-12h to ensure that the solution is fully dissolved.

The solution prepared by Step S112 was centrifuged at 6000rpm and 4°C for 15 min, and the insoluble impurities were removed by filtration. The pH value of the solution was checked again, and 0.1-2M NaOH or HCI was used to adjust the pH value to 7.0 if necessary to obtain a PPI reserve solution with a mass concentration of 2.22wt%.

The preparation of SBP solution includes the following steps:

S121. The beet pectin was dispersed in the phosphate buffer and stirred with a mechanical mixer at 500rpm at room temperature for 5 hours, so that the beet pectin was fully dissolved in the phosphate buffer. Then NaOH or HCI was added to adjust the pH value of the solution to 7.0; The concentration of phosphate buffer is 10mM, pH is 7.0, and the temperature is maintained at 21-22°C.

The solution prepared by Step S121 was centrifuged at 6000 rpm and 4°C for 15 min, and the insoluble impurities were removed by filtration. The pH value of the solution was checked again, and 0.1-2M NaOH or HCI was used to adjust the pH value to 7.0 if necessary to obtain the SBP reserve solution with a mass concentration of 0.4 %.

S2. Preparation of PPI stabilized oil-in-water emulsion for fish oil: PPI reserve solution prepared in Step S1 was mixed with FO (fish oil) in accordance with the mass ratio of 9:1, and a high-speed mixer was used to mix and stir at 10000 rpm for 2 minutes to prepare PPI oil-in-water emulsion for fish oil.

In order to obtain the fine emulsion drops, the coarse emulsion prepared was homogenized three times by a two-stage high-pressure valve homogenizer, with the pressure of 5000 psi in the first stage and 500 psi in the second stage, respectively. The final emulsion consisted of 10% fish oil and 2.22% PPI reserve solution.

S3. Preparation of FO-PPI-SBP complex condensates: PPI fish oil in water emulsion prepared in Step S2 and SBP reserve solution prepared in Step S1 were mixed according to the mass ratio of 5:1. Stir the mixture magnetically at 500 rpm for 10 minutes to mix the two solutions well. Adjust pH from 7.0 to 3.5 to 4.5 by adding HCI (0.1-2.0M). The dilution effect and conductivity change of the mixed solution can be reduced by using different concentrations of HCI. Finally, the aqueous mixture of FO-PPI-SBP was left to stand at 4°C for 24 hours to achieve phase equilibrium.

S4. Preparation of fish oil microcapsules: The FO-PPI-SBP composite condensates prepared in Step S3 were sent to the micro spray dryer at a feed rate of 0.15L /h and a 5 suction flow rate of 40 L/h to form fish oil microcapsules.

Inlet and outlet air temperatures should be maintained at 180°C and 105°C respectively.

The above operating parameters were optimized through preliminary experiments. The total solids content of all prepared mixtures is approximately 6.0-6.2 %. All the spray-dried powders were collected and stored in glass bottles at 4°C for characterization.

In this implementation method, EPA content in fish oil 29%, DHA content >12.5%, total n-3 PUFA content in triglyceride >30%; Pea protein isolate (PPI) is extracted from commercial pea flour by alkali extract-isoelectric precipitation method and its composition is 79.50% protein, 5.28% moisture, 0.77% fat, 4.61% crude ash and 9.84% carbohydrate. Beet pectin (SBP). The sodium hydroxide (NaOH), hydrochloric acid (HCI) and other chemicals and reagents used in this application are analytical grade. All solutions are prepared with ultra-pure water.

Case Contrast

Different from the implementation method, in this ratio, after preparing PPI stabilized fish oil in-water emulsion in Step S2, the PPI stabilized fish oil in-water emulsion is directly sent into Buchi micro spray dryer to prepare fish oil microcapsules.

In order to further characterize the structure and properties of PPI solution, SBP solution, PPI stabilized fish oil oil-in-water emulsion, FO-PPI-SBP composite condensate and fish oil microcapsules prepared in this application, the above solutions or products will be tested and analyzed as follows:

Test method: 1. Surface charge and particle size analysis of the feed dispersion

The surface charge and particle size of PPI stabilized emulsion and FO-PPI SBP complex dispersants were measured with pH(7.0-3.0) and reported as Z potential (, mV) and Z-mean diameter (nm), respectively. Before each measurement, the sample was diluted 20 times with the corresponding pH buffer and equilibrated at 25°C for 2 minutes. All measurements are taken in duplicate, with three readings recorded for each measurement. 2. Construction of state diagram

After standing for 24h, the state diagram of PPI stabilized emulsion and FO-PPI-SBP was obtained. After standing for 24 hours, state maps of the PPI stabilized emulsion and

FO-PPI-SBP complex dispersion were drawn based on observations of each phase equilibrium mixture. The phase is divided into five groups, marked with three symbols, namely: o,A.® represents turbidity solution, precipitation and turbidity solution and precipitation and clarification solution respectively. 3. Characterization of spray dried fish oil microcapsules 3.1 Encapsulation Efficiency (EE)

The surface oil content was measured by dispersing 2.5g microencapsulated powder in 10 mL hexane and rotating it for 2 min. The grout was then centrifuged at ambient temperature at 4000rpm for 10 min. Then wash the sediment twice and recover all the supernatant. The hexane was removed by nitrogen washing for 6h and the surface oil content was determined by gravimetry. The total oil content of the dry microcapsules (2.59) was determined by Soxhlet extractor. Then the solvent was completely removed by rotary evaporator, and the total oil content was determined by gravimetric method. The encapsulation efficiency (EE) of microcapsules was calculated using the following method.

EE=(Wt-Ws)/Wtx 100% where, Wt and Ws were the mass of total oil and surface oil of microcapsules, respectively (9). 3.2 Differential scanning calorimetry

The thermal properties of spray dried samples and control samples were studied by differential scanning calorimeter. Place a sample containing 10uL of distilled water (about 3.5 mg) into a 40 uL aluminum pot. Then, seal the pot and balance at room temperature overnight to distribute the water evenly. Samples were measured from 20 °C to 120 °C at a heating rate of 10 °C /min using a sealed empty aluminum disk as a reference. 3.3 Fourier Transform infrared spectrum

FTIR spectra of spray-dried samples and control samples were recorded at 25°C using a spectrophotometer equipped with a attenuated total reflection attachment, a Globar (MIR) source, a KBr beam separator, and a MCT detector. By accumulating 32 scans at a resolution of 4 cm-1, the sample was scanned in absorption mode from 4000 to 400 cm-1.

Sample background was collected before measurement. 3.4. The surface morphology was observed by scanning electron microscopy

The spray-dried powder adheres to a bonded carbon sheet on a cylindrical aluminum bracket, and the excess is blown off with a nitrogen stream. Then, the sample is sputtered and plated with gold to make it conductive. Finally, the same scanning electron microscope is used for detection. A micrograph (5000x) was selected as a representative of each sample. 5. Statistical analysis

The experimental data were processed in a completely randomized design (CRD) with two replicates. At least two measurements were made using the newly prepared sample, and the measurements were expressed as mean + standard deviation. Univariate analysis of variance and the F-test of SAS software were used to protect the least significant difference method for significant differences between the means (p< 0.05) analysis.

Test results and analysis 1. Effect of pH value on surface charge and particle size of fish oil dispersion

The formation of complex condensates between PPI and SBP is realized by electrostatic attraction between them. At a fixed biopolymer ratio (PPI/SBP=5:1), the environmental pH value is the main factor that determines the electrostatic interaction strength. The surface charge (Z- potential) and particle size (z-mean) of each dispersion were measured in order to determine the appropriate pH range for the formation of the fish

FO-PPI-SBP complex condensates. The effect of pH on the surface charge and particle size of PPI stabilized fish oil emulsions was also measured for comparison.

The measurement results are shown in Figure 1, where Figure 1a is the Zeta (0) potential diagram of PPI solution, SBP solution, PPI stabilized oil-in-water emulsion of fish oil and FO-PPI-SBP composite condensate, and Figure 1b is the average particle diameter of PPI stabilized oil-in-water emulsion of fish oil and FO-PPI-SBP composite condensate.

As shown in Figure 1a, the Z potential of PPI solution values is -24.17mV when it at pH=7.0, it is +27.18mV when it at pH=3.0, and zero net charge occurs at pH=4.7. The ( potential of SBP solution is negative throughout the pH range, which is a common surface charge pattern for anionic polysaccharides. The Z potential of SBP solution decreased gradiently when the pH value decreased from 7.0 to 5.0. The net value of the Z potential drops sharply with a further drop in pH value. This is because the SBP of galacturonic acid carboxyl functional groups on the unit (pKa= 3.5) caused by the protonation between. The Z potential of fish oil is between monomer PPI and monomer SBP at pH 7.0-3.0, and net zero charge occurs at pH 3.5. In general, composite condensation driven by electrostatic attraction requires that biopolymers have opposite electric charges. According to the results of {- potential, when pH is less than 4.5, the positive charge PPI(-NH4+) can generate electrostatic attraction with the anion SBP(-coo-) to form the complex condensation. For PPI and stable water bag fish oil emulsion, fish oil droplets of zeta potential and PPI solution is very similar, when pH value away from the isoelectric point, net charge increase (the ieps require curricular modules = 4.7).

The dependence between the z mean diameter and pH value of fish oil emulsion and fish FO-PPI-SBP composite dispersion is shown in Figure 1b. At different pH values, the particle size of PPI stable fish oil emulsions showed a bell shape change, which is a typical protein stable lipid droplet morphology. At pH 7.0 or 3.0, fish oil emulsions with PPI stability have a higher net charge (>20mV), so there is a strong electrostatic repulsion between them, which prevents polymerization. When pH value is 4.0~6.0, the particles have little or no net charge, resulting in extensive aggregation of particles (>10um). In contrast, the fish FO-PPI-

SBP complex dispersions have the smallest particle size at pH 7.0 and 6.5, suggesting that the interaction between PPI and SBP is weak or non-existent. The particle size increases significantly with the decrease of pH. 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 value. In the medium pH range, the particle size of the fish FO-PPI-SBP complex dispersion is even smaller than that of the emulsion. This may be caused by electrostatic repulsion being insufficient to counteract attractive interactions (such as van der Waals forces or hydrophobic interactions). 2. The effect of pH value on the phase behavior and microstructure of fish oil dispersion

In order to better visualize the effect of pH on the appearance of fish oil in two different feed dispersions, we constructed a state diagram (Figure 2a) to show that the phase behavior of the PPI stable fish oil emulsion and the FO-PPI-SBP complex dispersions after 24h at 25°C is a function of pH (7.0-3.0). PPI stabilizes the phase behavior of fish oil emulsions very classically (Figure 2a), and single-phase solutions are observed at pH(7.0,6.5,2.0). But as pH decreases, it changes to a precipitation and cloudy form (A), and atpH 5.0 to a condensed sediment (m). For the fish FO-PPI-SBP complex dispersion system, the state diagram shows three distinguishable phase behaviors. At neutral to acidic pH values (7.0,6.5,6.0), a homogeneity (0) was observed in the mixture as a result of the same charge repulsion of PPI and SBP (Figure 2b). When the pH is reduced to acidic conditions (5.5-2.0), two insoluble complexes (condensation or precipitation) with different phase behavior appear, with different structural properties. Since the pH (5.5-4.0) is close to the

PPI IEP, condensation shows a precipitation and cloudy appearance (4). This phase behavior is due to the mutual attraction between the negative charge of the polyanion SBP and the opposite stain on the PPI surface. However, at higher pH values (5.5, 5.0, 4.5), the repulsive force within the system is strong, preventing violent phase separation, while at pH 4.0, significant phase separation occurs. At pH 3.5 and 3.0, the phase behavior of precipitated and transparent solutions (=) is formed.

CLSM (confocal laser scanning microscope) was then applied to evaluate the effect of pH on the in-situ morphological properties of PPI stabilized fish oil emulsion and fish FO-

PPI-SBP composite dispersions (Figure 3). The PPI is labeled by the FITC and appears as green dots in the CLSM micrograph (Figure 3a), while the fish oil is stained with Nile red.

Compared with CLSM micrographs of PPI and SBP solutions (Figure 3 a and b}, no signs of flocculation of emulsified droplets were observed at pH 7.0 (Figure 3c), which may be attributed to the high Z potential of the droplets. When the emulsion was acidified to a pH of 3.5, locally clustered large particle sizes appeared (white arrow in Figure 3d), which was consistent with particle size measurements. The increased droplet flocculation is due to the added acid lowering the pH from 7.0 to 3.5. This allows the pH value to be absorbed through the PPIl's IEP, thus bringing the droplets close to each other. For the fish FO-PPI-SBP composite dispersion (Figure 3e), the microstructure is similar to that of the emulsified 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 PPI IEP), only a small amount of flocculant mixed with green particles (Figure 3f and 39) was observed, and the microstructure was similar to that of emulsion with a pH of 3.5. The observed size of the condensate is also consistent with the results in Figure 1b. Under this pH composite condensed the number and size of low may be due to dispersion of relatively strong electrostatic repulsion ([Z/>20). As a rule, a slight decrease in pH from 4.0 to 3.5 significantly promotes the formation of complex, irregularly shaped condensates (Figure 3h).

This trend reflects the progressive charge neutralization of the system, in which biopolymer interactions reach electrical equivalent points while allowing attractive forces such as van der Waals forces and hydrophobic forces to dominate the interactions.

According to scanning electron microscopy (SEM), at different pH values, the microstructure of the freeze-dried fish oil emulsion and FO-PPI-SBP composite condensates is different without polishing (Figure 4a), and their shapes are flacy-like, with smooth surfaces and no fragments, which may be caused by the aggregation of emulsion droplets during drying. Similar morphology was observed in the FO-PPi-SBP complex condensates formed at pH 4.5, except that the microstructure became less condensed as it extended to one dimension (Figure 4b). Again, this indicates that no composite condensates will form due to the strong repulsion between biopolymers. Notably, the spongy porous structure appears as pH decreases. A multi-dimensional network with multiple patterned apertures is formed when the pH of the condensate is 4.0 (Figure 4c). Further lowering the pH value to 3.5 causes the network to become more concentrated (Figure 4d). This morphological change in lyophilized composite condensates confirms that the strength of electrostatic interactions varies at different pH values, which may significantly affect biopolymer interactions. Combined with the results of the state diagram, CLSM and SEM, it can be confirmed that the FO-PPI-SBP condensate exhibits a relatively dense network structure at pH 3.5, which is mainly due to charge neutralization. 3. Microcapsule analysis of fish oil 3.1 Encapsulation efficiency

Table 1 shows the powder yield (PY), encapsulation efficiency (EE), encapsulation yield (EY) and payload (PL) of spray-dried microcapsule powders prepared by emulsification and composite condensation. Powder yield is the key parameter to determine the recovery rate of solid substance after spray drying. All microencapsulated powders had very similar

PY values, except that the composite condensates formed at pH 3.5 produced powders with the lowest PY values (42.0%). Of the two different dispersion preparation methods, the microcapsule powder prepared by PPI stabilized emulsion had the lowest EE (p< 0.05), indicating improved EE of the latter method and higher packaging efficiency in the pH range of 3.5-4.5. The EE of the three fish oil microcapsules prepared by composite condensation is the same, which is independent of the pH value at the time of formation. Two factors can lead to low EE values. In this study, fewer wall materials were used, which had a direct impact on EE values. In addition, a more rigorous and powerful solvent washing regimen was used to try to completely remove the surface oil, rather than determine the content of the easily extracted ail in it. Compared with the control study without SBP, the encapsulation efficiency (EE), encapsulation rate (EY) and payload (PL) of fish oil microcapsules with SBP can achieve similar or better effects under wider pH conditions. Fish oil microcapsule powder dried by PPI stabilization emulsion and fish FO-PPI -- SBP composite condensate spray showed similar PY, EY, and PL.

Table 1

Powder yield (PY), package efficiency (EE), package yield (EY}, and payload (PL) * of PPI stabilized emulsion or PPI-SBP Composite condensate spray dried FO microcapsules. * Values in a column with different superscript letters indicate that in p< 0.05, there was a statistically significant difference (FO: fish oil; PPI: pea protein isolate; SBP: sugar beet pectin)

The major chemical functional groups of SBP are identified in spectra with wave number range 1000-2000cm™. It includes 1734cm (C=O stretching vibration), 1622cm™’ (tensile carboxylate ions) and 1441cm™ (asymmetric stretch-CH3). In the position of 3012cm™, we can observe fish oils olefin double bond (-HC=CH-) absorption peak.

As shown in Figure 5, in the spectra of fish oil microcapsules prepared with PPI stabilization emulsion at pH 3.5, the intensity of absorption peaks at 3012, 2926, 2852 and 1146cm™ gradually increased, indicating the presence of fish oil. In addition, when the fish oil was encased, PPI initial peak of 1633, 1529, 1450cm™, the blue shift magnitude 8, 4, and 4 cm, respectively or so. These results suggest that the hydrophobic regions of fish oil and

PPI may interact with each other during spray drying. A similar FTIR spectrum was found in spray-dried microcapsules, where low water soluble chlorophyll was able to bind to tryptophan or tyrosine residues of whey protein, causing blue shift. Although the spectra of fish oil microcapsules formed by composite condensation are very similar to those of fish ail microcapsules formed by emulsification, some significant differences have been found. The spectra of fish oil microcapsules formed by composite condensation showed a new peak at 1016cmrt, indicating that SBP had been successfully introduced into the composite condensate. In addition, the peak value of 1392cm™ in PPI and emulsified fish oil micro- capsules was not found in all complex condensed microcapsules. Because this peak was assigned to -COO- of the amino acid, its disappearance confirmed that SPB and PPI formed complex condensates through electrostatic interaction. In addition, the spectra of the fish oil microcapsules based on the composite agglomerations show a wide band (about 1020-1080cm™), which may be attributed to the superposition of the spectra of PPI (peak 1063 cm} and SBP (peak 1012cm), mainly due to their electrostatic interaction during the formation of the composite agglomerations. 3.3. Thermal analysis

The thermal properties of PPI in different fish oil microcapsules were studied by DSC in this application (Figure 6).

The DSC maps of both PPI and SBP showed endothermic peaks corresponding to the melting of the network structure of the two biopolymers. PPI raw material melting point of about 86.66°C. The melting point of PPI may be that water evaporation from SBP leads to the destruction and breaking of non-covalent bonds and hydrogen interactions between polar and non-polar groups, thus destroying the secondary and tertiary structure of PPI.

When PPI was used as emulsifier to cover the surface of FO droplets, the melting point of microcapsules decreased to 76.83°C, indicating that the synergic structure of PPI was poor after spray drying. This is reasonable because the hydrophobic interaction between FO and the high-pressure homotropic PPI is weaker than the interaction between the hydrophobic amino acid patch in the raw PPI. On the contrary, the FO microcapsules prepared by the

FO-PPI-SBP composite coacervation system showed a small and narrow melting peak, which was attributed to the formation of a strong new network structure. This structure inhibits denaturation of the PPI and therefore maintains a melting point similar to that of the original PPI. A similar phenomenon is consistently observed in microcapsules made from complex condensates, such as soy protein isolate and chitosan, as this structure generally increases the protein's stability against thermal denaturation. The pH value affects the thermal performance of microcapsules, with larger and wider endothermic peaks forming complexes at higher pH (4.5) 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 zeta - potentials (Figure 1a). 3.4 Morphology of fish oil microcapsules

Scanning electron microscopy (SEM) can be used to analyze the morphologies of fish oil microcapsules prepared by different feeding dispersants (Figure 7). The microstructure of SBP(Figure 7a) and PPI(Figure 7b) showed irregular and plate morphology, which were difficult to identify, respectively. All of the fish oil microcapsules have some similarities in their morphology, for example, they all have no cracks or holes in their surface, which ensures the capsule's low permeability and provides protection against the fish oil. However, different feed dispersions have different effects on its morphology. For fish oil microcapsules prepared from a PPI stabilized emulsion with a pH of 3.5, the particles were observed to be spherical with a certain roughness (Figure 7c). The size of microcapsules varied greatly from 0.5um to 7.5um. Notably, some oil spots can also be seen on the surface of the microcapsule (red arrow in Figure 7c). The size of fish oil microcapsules obtained from composite agglomerates is generally larger than that obtained from emulsification, because pectin is produced during agglomerate formation. Compared with emulsified microcapsules, there was almost no oil spot, which indicated that the encapsulation effect of composite coagulant had been improved. The pH value affects the size of fish oil microcapsules prepared from complex condensates. Especially when the pH value is 4.5, the size of the capsule changes dramatically (Figure 7d); The maximum particle size appeared at pH 3.5 (Figure 7f). The uniform size of the microcapsules (Figure 7e) was produced at an intermediate pH value (4.0). This observation is consistent with the results in Figure 4, further demonstrating that the composite condensate formed at pH3.5 is the optimal condition for coating fish oil before spray drying.

In this application, FO-PPI-SBP composite condensate is used as the feed dispersion of fish oil microcapsules, which greatly improves the powder yield, payload and encapsulation rate of fish oil microcapsules. Due to the interaction between PPI and SBP,

the denaturation temperature and network thermal stability of the prepared fish oil microcapsules were improved. Since the droplet size, { potential, phase behavior and microstructure of the feed dispersion are all affected by pH, this application is designed to generate fish oil microcapsules with larger particle size and denser network structure by adjusting the FO-PPI-SBP composite condensate at appropriate pH value. The FO-PPI-SBP composite condensates formed at pH3.5 have larger particle size and denser network structure than those formed at higher pH4.5 and pH4.0.

Finally, it shall be noted that the above implementation methods are used only to illustrate the technical scheme of the invention and not to limit the technical scheme.

Notwithstanding the detailed description of the invention by the applicant with reference to the better implementation methods, ordinary technicians in the field shall understand that those technical schemes of the invention modified or substituted without deviating from the purpose and scope of the technical scheme. Shall be covered by the claims of the invention.

Claims

Conclusions

A method for preparing fish oil microcapsules based on the emulsification and condensation of pea protein isolates, characterized in that the method comprises the following steps:

S1. Preparation of PPl solution and SBP solution: Dissolve PPI and SBP in phosphate buffer solution and stir until completely dissolved to obtain PPI stock solution and SBP stock solution, respectively;

S2. Preparation of a PPI stabilized oil-in-water emulsion for fish oil: mix the PPI stock solution prepared in Step S1 with FO in a mass ratio of 9:1 to obtain a PPI oil-in-water emulsion for fish oil;

S3. Preparation of a FO-PPI-SBP complex condensate: mix the PPl-fish oil-in-water emulsion prepared in Step S2 and the SBP stock solution prepared in Step S1 in a mass ratio of 5:1, adjust the pH value to 3.5-4.5 and leave the mixture at 4°C for 24 hours, yielding a FO-PPI-SBP complex condensate.

S4. Preparation of fish oil microcapsules: send the FO-PPI-SBP complex condensates prepared in Step S3 to the microspray dryer at a feed rate of 0.15 L/h and a suction rate of 40 L/h, forming fish oil microcapsules by spray drying.

A process for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 1, characterized in that the mass concentration of the PPI stock solution is 2.22% by weight and the pH value is 7. is 0, and the mass concentration of the SBP stock solution is 0.4 wt% and the pH value is 7.0.

A method for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 2, characterized in that the preparation of the PPI1 solution comprises the following steps: S111. Dissolve the PPI in the phosphate buffer and stir with a mechanical agitator at 500 rpm for half an hour at room temperature to completely dissolve the PPI in the phosphate buffer; Add NaOH solution to the solution prepared in Step S111, adjust the pH to 9.5 and stir for 2 hours to promote hydration, then add HCl to adjust the pH of the solution to 7.0 and stir for 10-12 hours to ensure that the solution is completely dissolved; S113. Centrifuge the solution prepared in Step S112 at 6000 rpm for 15 min and 4°C, and remove the insoluble impurities by filtration;

Recheck the pH of the solution and adjust the pH to 7.0 using NaOH or HCl to obtain a PPI stock solution with a mass concentration of 2.22% by weight.

A method for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 2, characterized in that the preparation of the SBP solution specifically comprises the following steps: S121. Disperse the beet pectin in the phosphate buffer and stir with a mechanical mixer at 500 rpm for 5 hours at room temperature so that the beet pectin is completely dissolved in the phosphate buffer; then add NaOH or HCl to bring the pH of the solution to 7.0; Centrifuge the solution prepared in Step S121 at 6000 rpm for 15 min at 4°C and remove the insoluble impurities by filtration; Recheck the pH of the solution and if necessary use 0.1-2M NaOH or HCl to bring the pH back to 7.0, yielding an SBP stock solution with a mass concentration of 0.4%.

A method for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 3 or claim 4, characterized in that the concentration of the phosphate buffer solution is 10mM and the pH is 7.0, and the concentration of NaOH and HCl is 0.1-2M.

Process for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 3 or claim 4, characterized in that the pH value of the condensate of the FO-PPI-SBP complex is 3 .5.

A method for preparing fish oil microcapsules based on emulsifying and condensing pea protein isolates according to claim 3 or claim 4, characterized in that the temperature of the incoming and outgoing air of the micro spray dryer in Step S4 is 180 °C, resp. is 105°C.

Fish oil microcapsule, characterized in that it has been prepared according to the method of any one of claims 1-7.