Coconut milk product capable of improving emulsification stability and preparation process thereof
Through the interaction between coconut globulin and coffee polyphenols, a complex is formed, which solves the problem of insufficient stability of coconut milk and achieves the effect of improving the emulsification stability of coconut milk, enhancing nutritional value and taste.
Patent Information
- Application Number
- CN202410697376.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-02-07
AI Technical Summary
As an unstable emulsifier system, coconut milk has limitations on emulsifiers, which leads to its limited use in the food industry, and the added emulsifiers and thickeners will affect the taste and flavor of coconut milk.
By extracting coconut globulin and interacting with coffee polyphenols such as caffeic acid, chlorogenic acid and ferulic acid, it forms a complex to improve the emulsification performance of coconut globulin, thereby improving the stability of coconut milk.
This method not only improves the emulsification stability of coconut milk, but also enhances its nutritional value and taste, without affecting the original taste and flavor of coconut milk.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of coconut milk products, in particular to a coconut milk product with improved emulsification stability and a production process thereof. Background Art
[0002] Coconut milk contains proteins such as albumin, globulin, glutenin and gliadin. Coconut milk is an unstable emulsion system, which limits the development of the food industry. In coconut milk emulsions, protein is mainly used for stability. Globulin and albumin are used as emulsifiers to stabilize coconut milk emulsions. Coco globulin (CG) is the most abundant protein and the protein with the highest emulsification ability. At present, coconut milk is obtained by crushing, squeezing and extracting mature coconut meat. Coconut milk is nutritious and rich in fat, protein, minerals and vitamins. Coconut milk contains 31.0-35.0% fat and 3.5-4.0% protein. Coconut protein is an amphiphilic molecule that can adsorb, unfold and rearrange at the oil-water (O / W) interface to stabilize oil-in-water emulsions. Due to its high nutritional value and unique taste, it is widely used in beverages, ice cream, cooking, etc. However, like other emulsions, coconut milk represents a thermodynamically unstable system. Due to the limited emulsification of coconut protein, coconut milk droplets without emulsifiers tend to agglomerate and aggregate easily, and therefore tend to phase separate.
[0003] The most common way to improve the stability of coconut milk is to add emulsifiers, surfactants and thickeners, such as proteins, sorbitan esters, ethoxylated esters and sucrose esters. However, the amount of these additives used is limited for safety reasons. Previous research has shown that the stability of coconut milk can be improved by adding natural proteins (to improve emulsification) and starches (to increase viscosity). However, the addition of proteins and starches affects the taste and flavor of coconut milk, which is determined by the delicate balance of fat, protein and carbohydrates.
[0004] Coffee is one of the most popular beverages worldwide and is rich in polyphenols, such as caffeic acid (CA), chlorogenic acid (CHA), and ferulic acid (FA), which have potential antioxidant, hypoglycemic, antihypertensive, antimicrobial, and anti-inflammatory effects. In addition, certain studies have shown that coffee polyphenols (CPs), such as CA and CHA, can interact with proteins to enhance their functional properties. However, the mechanisms by which different CPs improve the emulsifying properties of proteins are still unclear. Summary of the invention
[0005] In view of this, the present invention provides a coconut milk product with improved emulsification stability and a production process thereof to solve the above problems.
[0006] The technical solution of the present invention is achieved in that a process for preparing a coconut milk product with improved emulsification stability comprises the following steps:
[0007] S1. Extraction of coconut globulin: using n-hexane to remove the oil in freeze-dried coconut meat powder, mixing 0.3-0.7 mol / L sodium chloride solution with coconut meat powder at a ratio of 1:3-6 (w / v) for extraction for 3-5 hours, and then freeze-drying the extract and storing it at 2-6°C to obtain coconut globulin CG;
[0008] S2, preparing globulin solution: the above-mentioned coconut globulin was dissolved in 0.005-0.015 mol / L phosphate buffer solution (PBS, pH 6.0), stirred for 1-3 h, and placed overnight to fully hydrate to obtain a globulin solution with a concentration of 1.5-2.5 mg / mL;
[0009] S3. Preparation of complex: respectively add caffeic acid CA, chlorogenic acid CHA and ferulic acid FA solution to the globulin solution, stir at low speed, adjust the pH value of all samples to 5.9-6.2, and obtain three solutions of coconut globulin-caffeic acid CG-CA, coconut globulin-chlorogenic acid CG-CHA, and coconut globulin-ferulic acid CG-FA, respectively. The solutions are marked as CG-CA, CG-CHA and CG-FA, and all samples are stored at 2-6°C for short-term storage;
[0010] S4. Preparation of emulsion: The above-mentioned coconut protein-caffeic acid CG-CA, coconut protein-chlorogenic acid CG-CHA, and coconut protein-ferulic acid CG-FA solutions are mixed with vegetable oil at a volume ratio of 7-10:1 to obtain a 9.0-12.5% oil phase mixture, and then sheared at a speed of 8000-12000 rpm for 1-3 min using a high-speed shearing machine, and then treated 1-3 times with a NanoGenzier high-pressure homogenizer (Genizer, USA) at 15-25 MPa to generate a microemulsion, and 0.01-0.03% of thimerosal by volume of the emulsion is added to the emulsion, and stored at 3-5° C. to obtain a coconut milk product.
[0011] Further, the preparation process of the coconut milk product with improved emulsion stability comprises the following steps:
[0012] S1. Coco globulin extraction: Use n-hexane to remove the oil in the freeze-dried coconut meat powder, extract it with 0.5 mol / L sodium chloride solution at a ratio of 1:5 (w / v) for 4 h, and then freeze-dry the extract and store it at 4°C to obtain coconut globulin CG;
[0013] S2, preparing a globulin solution: dissolving the above coconut globulin in 0.01 mol / L phosphate buffer solution, stirring for 2 h, and leaving overnight to fully hydrate to obtain a globulin solution with a concentration of 2.0 mg / mL;
[0014] S3. Preparation of complex: respectively add caffeic acid (CA), chlorogenic acid CHA and ferulic acid FA solution dropwise to the globulin solution, stir at low speed, adjust the pH value of all samples to 6.0, obtain three solutions of coconut globulin-caffeic acid CG-CA, coconut globulin-chlorogenic acid CG-CHA, coconut globulin-ferulic acid CG-FA, and store all samples at 4°C for short-term storage;
[0015] S4. Preparation of emulsion: The above solutions were mixed with soybean oil at a volume ratio of 9:1 to obtain a 10% oil phase mixture, the above solution was sheared at a speed of 10000 rpm for 1 min using a high-speed shearing machine, and then treated twice with a high-pressure homogenizer at 20 MPa to generate a microemulsion, 0.02% thimerosal by volume of the emulsion was added to the emulsion, and the mixture was stored at 4° C. to obtain a coconut milk product.
[0016] Furthermore, the volume mass mL / g ratio of n-hexane to freeze-dried coconut meat powder in S1 is 1:2-5.
[0017] Furthermore, the freeze-drying temperature in S1 is -40 to -60°C, and the freezing time is 23 to 28 hours.
[0018] Furthermore, in S3, caffeic acid CA, chlorogenic acid CHA and ferulic acid FA are respectively dissolved in 0.01 mol / L PBS (pH 6.0) to obtain caffeic acid CA, chlorogenic acid CHA and ferulic acid FA solutions with a concentration of 1.8-2.2 mg / mL.
[0019] Furthermore, the low-speed stirring rate in S3 is 40-100 rpm.
[0020] Furthermore, in S3, 0.1 mol / L HCl and 0.1 mol / L NaOH solutions were used to adjust the pH values of all samples to 5.9-6.2.
[0021] Furthermore, the vegetable oil in S4 includes, but is not limited to, soybean oil, peanut oil, linseed oil or olive oil, preferably soybean oil.
[0022] Furthermore, the temperature during the shearing process in S4 is maintained at 23-27°C.
[0023] Furthermore, the coconut milk product prepared by the production process has high emulsification stability, high nutritional value and excellent taste.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The present invention is based on the study of the interaction between coffee polyphenols and proteins to enhance their functional properties to explore the mechanism of different coffee polyphenols in improving the emulsification performance of proteins. CA, CHA and FA are selected as CP models to study the mechanism of different CPs improving the emulsification performance of CG. The present invention modifies coconut globulin, and the interaction between coconut globulin and coffee polyphenols can promote the adsorption of protein molecules at the O / W interface, thereby improving the emulsification stability, improving the emulsification performance, and thus enhancing the stability of coconut milk. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 Schematic diagram of the molecular docking of CG with CA, CHA and FA; in the figure, the crystal structure of CG and the structural formulas of CA, CHA and FA (a); the fully stimulated three-dimensional docking mode and interaction of the 2D docking amino acid residues of CG-CA (b), CG-CHA (c), and CG-FA (d).
[0027] Figure 2 The solubility and hydrophobicity of the O / W interface of protein adsorption. In the figure, (a) is the solubility of protein adsorbed on the O / W interface, and (b) is the surface hydrophobicity;
[0028] Figure 3 is the particle size distribution diagram of emulsion droplets;
[0029] Figure 4 This is a stability spectrum analysis diagram of the emulsion, which shows the emulsification index (b) and droplet size (c) of the emulsions stabilized by CG, CG-CA, CG-CHA and CG-FA composite materials at different pH values; after adding different concentrations of NaCl, the emulsification index (d) and droplet size (e) of the emulsions stabilized by CG, CG-CA, CG-CHA and CG-FA. DETAILED DESCRIPTION
[0030] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.
[0031] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.
[0032] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.
[0033] The raw materials used in the examples of the present invention were obtained from the following sources: caffeic acid (≥99%, molecular weight 180.15 Da), chlorogenic acid (≥98%, molecular weight 354.21 Da) and ferulic acid (≥98%, molecular weight 194.18 Da) were purchased from Shanghai Deepak Biotechnology Co., Ltd. (Shanghai, China). Vegetable oil (food grade) was purchased from China National Cereals, Oils and Foodstuffs Co., Ltd. (Beijing, China). All other reagents were analytical grade reagents purchased from Aladdin Co., Ltd. (Shanghai, China); coconut was produced in Wenchang City, Hainan Province.
[0034] Example 1
[0035] A process for preparing a coconut milk product with improved emulsification stability comprises the following steps:
[0036] S1. Extraction of coconut globulin: using n-hexane to remove the oil in freeze-dried coconut meat powder, the volume mass mL / g ratio of n-hexane to freeze-dried coconut meat powder is 1:3, and 0.5 mol / L NaCl solution is mixed with coconut meat powder in a ratio of 1:5 (w / v) for 4 hours, and then the extract is freeze-dried at a temperature of -50°C for 25 hours and stored at 4°C to obtain coconut globulin CG;
[0037] S2, preparing globulin solution: the above-mentioned coconut globulin was dissolved in 0.01 mol / L phosphate buffer solution (PBS, pH 6.0), stirred for 1-3 h, and placed overnight to fully hydrate to obtain a globulin solution with a concentration of 2.0 mg / mL;
[0038] S3. Preparation of complexes: Caffeic acid CA, chlorogenic acid CHA and ferulic acid FA were dissolved in 0.01 mol / L PBS (pH 6.0) at a concentration of 2.0 mg / mL. CA, CHA and FA solutions were added dropwise to the globulin solution with low-speed stirring at 60 rpm. The pH values of all samples were adjusted to 6.0 to obtain three solutions, which were labeled as CG-CA, CG-CHA and CG-FA, respectively. CG solution (1 mg / mL) was used as a control, and all samples were stored at 4°C for short-term storage.
[0039] Example 2 - Fluorescence spectroscopy measurement
[0040] Fluorescence spectra were measured using a fluorescence spectrophotometer (F-7000, Hitachi, Japan). The CG solution was used in combination with CA, CHA and FA solutions to prepare samples with a CG concentration of 1 mg / mL in the final complex. The concentrations of CA, CHA and FA were 0, 20, 40, 60, 80, 100, 120 and 140 μmol / L, respectively. The interactions within the complex were analyzed at temperatures of 298, 303 and 308 K, using 0.01 mol / L PBS buffer (pH 6.0) as a blank. The intrinsic emission fluorescence spectra of the samples were recorded at an excitation wavelength of 280 nm, with wavelengths ranging from 290 to 550 nm. The scanning speed was 1200 nm / min, and the slit width was set to 2.5 nm.
[0041] The results showed that the fluorescence intensity of CG at an excitation wavelength of 280 nm decreased with the introduction of CPs. When polyphenols were introduced into a protein solution containing fluorophores, the fluorescent groups of the proteins were destroyed, resulting in a decrease in fluorescence intensity. Therefore, it can be concluded that the quenching of CG fluorescence intensity is the result of the interaction between CG and CP.
[0042] Example 3 - Molecular docking simulation
[0043] The molecular docking method was used to simulate the binding mode and affinity of polyphenols to CG. The CGID (5WPW) was entered into the RCSB database (https: / / www.rcsb.org / ) to obtain the CG molecular model. MOE2012 software was used for preprocessing, including adding hydrogen atoms, optimizing charges, and removing water molecules. The structure of polyphenols was obtained from PubChem (https: / / pubchem.ncbi.nlm.nih.gov). Since the entire protein represents a potential binding site, the conformation with the lowest binding energy was selected as the optimal conformation. The binding site and interaction force were analyzed.
[0044] Molecular docking technology can provide a deeper understanding of the interaction between CP and CG by visualizing the binding sites and interaction forces between receptor and ligand molecules. Figure 1It shows the three-dimensional docking mode of the docking simulation and the interactions between the two-dimensional docking amino acid residues. The order of the molecular docking energies of CGs and CPs is CG-CHA (-6.01 kJ / mol) < CG-FA (-5.34 kJ / mol) < CG-CA (-4.36 kJ / mol), indicating that the CG-CHA complex has the most stable molecular conformation. This finding is consistent with the results of fluorescence fitting. The hydrogen bond interactions between CG and CA are mediated by Lys218, His195, and Arg57. CG and CHA interact through four hydrogen bonds involving amino acids His156, Lys293, Arg295, and Asn300. Hydrogen bond interactions were found between CG and FA through Lys319. These results suggest that the main driving force for the formation of CG-CPs complexes is mainly hydrogen bonds.
[0045] In summary, all CPs can interact robustly with CG. Due to the limited number of –OH and –COOH groups, the interaction between CG and FA is mainly hydrophobic, followed by hydrogen bonds. CA has two –OH groups and one –COOH group, ensuring that its non-covalent interaction with the CG molecule is mainly hydrogen bonds, followed by hydrophobic interactions. CHA consists of an ester formed by CA and quinic acid, indicating that CHA can easily form non-covalent interactions with proteins. CHA forms a stable complex with CG through strong hydrogen bonds and hydrophobic interactions.
[0046] Example 4 - Determination of the Structures of CG, CG-CA, CG-CHA, and CG-FA
[0047] SDS-PAGE was performed using a precast Biosharp PAGE gel (Tris-Gly, 4%–20%, 10×). Samples were prepared by mixing a 1 mg / mL sample solution with loading buffer (SDS-PAGE loading buffer, 5×) at a volume ratio of 4:1 and then denaturing the mixture by heating in a water bath at 100 °C for 3–5 minutes. A volume of 10 μL of each sample solution was loaded into each well, and the voltage was adjusted to 90 V (for stacking gel) / 120 V (for separating gel). The gel was stained with Coomassie Brilliant Blue R250 and then decolorized with a decolorizing solution.
[0048] CD spectra were obtained using a Bio-Logic MOS-500 circular dichroism spectrometer (Isère, France) in the range of 190–250 nm. The sample solution was diluted to 0.5 mg / mL, and deionized water, CA, CHA, and FA solution without CG were used as controls. The spectra were averaged over three scans.
[0049] The FTIR spectra of the samples were measured using an FTIR spectrometer (TENSOR27, Bruker, Germany). The freeze-dried powder samples (10 mg) were mixed with KBr at a ratio of 1:100 (w / w) and pressed into tablets. The spectra were recorded in the wavenumber range of 4000–400 cm-1 with air as the background. Each FTIR spectrum consisted of an average of 32 scans.
[0050] Free -SH and SS single bond content of the sample: The lyophilized sample was dissolved in Tris-glycine urea buffer (0.086mol / LTris, 0.090mol / L glycine, 8mol / L urea, 0.004mol / L ethylenediaminetetraacetic acid, pH8.0) to prepare a 6mg / mL solution. The supernatant was centrifuged, and then 80μL (4mg / mL) of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was added to 2mL of the supernatant and reacted at 25°C for 5 minutes. The -SH content was calculated by measuring the absorbance at 412nm. In order to determine the SS single bond content, 2mL of the above supernatant was reacted with β-mercaptoethanol (4mg) at 25°C for 2 hours. Then, 12% trichloroacetic acid solution was added and centrifuged for 1h, and the precipitate was washed repeatedly. After adding 80 μL of DTNB solution, the absorbance was measured at 412 nm, and the total S-S content was calculated according to the following formulas (1) and (2):
[0051]
[0052] Where A412 is the absorbance value at 412 nm, D is the dilution ratio, and C is the protein concentration in the sample.
[0053] result:
[0054] The results of SDS-PAGE experiments of CG, CG-CA, CG-CHA, and CG-FA showed that in their simplified forms, CG and CG-CP were observed between 17 and 55 kDa. CG and CG-CPs mainly showed a band of 55 kDa, corresponding to 11S globulin (cocos nuciferol), which plays a vital role in maintaining the stability of coconut milk. In its native state, 11S globulin exists as a hexamer, mainly composed of two acidic polypeptides (32 kDa) and two basic polypeptides (22 kDa), which are connected by disulfide bonds. In addition, 7S globulin is another key protein, which consists of bands of 19, 29, and 33 kDa. The bands of the CG-CPs complex reflect those of CG, and no new bands are obvious, indicating that the CG-CPs complex is formed by non-covalent interactions rather than forming a new substance through covalent bonds. The 55 kDa band is slightly darker due to the incorporation of CA, CHA, and FA. At the same time, the bands corresponding to the low molecular weight components of CG-CPs gradually faded. These observations suggest that aggregated macromolecules are formed due to the interaction between CG and CP.
[0055] The interaction between CPs and CG may actually lead to the rearrangement of the intermolecular forces that maintain the secondary structure of CG, resulting in conformational changes in CG. Far-UV CD analysis is widely used to study such changes in the secondary structure after binding with CP. CPs have the potential to interact with the side chains of various amino acid residues in CG as well as the carbonyl and amino groups in the peptide bonds, leading to the formation of hydrogen bonds. This interaction can change the hydrogen bond structure and loosen the peptide chains in native proteins, leading to protein denaturation. Therefore, the interaction between CG and CPs destroys the secondary structure of CG molecules and transforms them from a loosely structured, ordered, dominant form to a partially disordered structure.
[0056] Example 5 - Particle size, zeta potential and turbidity measurements
[0057] The sample solution (5 mL) was diluted with deionized water to a concentration of 0.5 mg / mL, and the particle size and zeta potential were measured three times using a Malvern ZS90 (Malvern Instruments, Malvern, Worcestershire, UK). During the measurement, the temperature was maintained at 25° C. The sample was fully diluted to 0.5 mg / mL, and the absorbance was measured at 600 nm using a UV spectrophotometer (TU-1901, Universal Instruments, Beijing).
[0058] The results showed that with the addition of CPs, the particle sizes of CG-CA, CG-CHA and CG-FA increased significantly to 247, 245 and 249 nm, respectively (p<0.05), indicating that CPs caused CG aggregation. TEM images further confirmed that the interaction between CG and CP led to the increase in CG particle size. In addition, the absolute potential values of CG, CG-CA and CG-CHA complexes were higher than that of CG (-12.07 mV). These data indicate that CPs promote CG to expose more negatively charged amino acid residues, thereby enhancing the electrostatic repulsion between complex molecules and inhibiting their aggregation. Compared with CG, the turbidity values of CG-CA, CG-CHA and CG-FA all increased, among which CG-CHA increased significantly (p<0.05). This increase can be attributed to the interaction between CG and CA / CHA / FA.
[0059] In summary, the interaction between CGs and CPs resulted in an increase in both particle size and absolute potential value, while the PDI of CG-CPs decreased. These changes indicate that the stability of CG-CPs complexes is enhanced, reflecting the key role of CPs in regulating the physical properties of the system.
[0060] Example 6 - Determination of properties of CG, CG-CA, CG-CHA and CG-FA
[0061] Protein solubility was modified by Bradford method. Prepare bovine serum albumin (BSA) standard solution and record standard curve. Centrifuge the sample solution (1 mg / mL) at 8000 r / min for 20 min, collect 1 mL of supernatant and measure protein concentration. Protein solubility was expressed as protein concentration in supernatant relative to protein concentration before centrifugation.
[0062] Surface hydrophobicity was determined using the bromophenol blue method. The prepared protein solution (1 mg / mL, 0.1 mL) was diluted to an appropriate concentration, mixed with a 1 mg / mL bromophenol blue solution (20 μL), and centrifuged. The absorbance of the supernatant was measured at 595 nm. The surface hydrophobicity of the protein is represented by the amount of bromophenol blue bound.
[0063] The dynamic interfacial tension was measured at 25 °C using a drop profile tensiometer (OT100, Ningbo NB Scientific Instruments, China). Due to the technical requirements of the surface tensiometer, the sample was fully diluted and the volume of each drop was kept at 10 μL while the soybean oil was loaded into the syringe. The dynamic interfacial tension was measured for 7200 s until no further changes in the interfacial tension were observed.
[0064] result: Figure 2a shows that the presence of CPs leads to a decrease in the solubility of CG. This is because the –OH and –COOH groups in CA, CHA, and FA molecules act as hydrogen bond donors and acceptors, forming strong hydrogen bond interactions with CG molecules to form an interconnected network structure, leading to precipitation. At the same time, CPs expand the CG structure, exposing the internal hydrophobic groups, hindering the interaction between the hydrophilic groups on the surface of the protein and water molecules, leading to a decrease in solubility.
[0065] Figure 2 b shows that in its natural state, the surface hydrophobicity of CG is weak. However, the addition of CPs can promote the unfolding of the CG molecular structure, exposing the internal hydrophobic groups, thereby enhancing the surface hydrophobicity of CG. The bromophenol blue method can be used to measure the surface hydrophobicity of proteins.
[0066] During the formation of the O / W interfacial film, the hydrophobic groups of the protein face the oil phase and the hydrophilic groups face the water phase, which helps to reduce the interfacial tension. The hydrophobic groups of the CG molecules gradually adsorb on the surface of the oil droplets and rearrange to form an interfacial film. Compared with the equilibrium interfacial tension of water, the interfacial tension of CG and its complex is significantly reduced. As a globular protein, CG maintains its original conformation at the O / W interface and exhibits a low rearrangement rate.
[0067] During the formation of the O / W interfacial film, CG formed a thin interfacial film at the interface after adsorption and rearrangement. Polyphenols CA, CHA, and FA promoted the unfolding of CG, exposing more hydrophobic groups and enhancing the protein-oil interaction. The –COOH and –OH groups of CA formed hydrogen bonds with the amino acid residues of CG, promoting the formation of a dense and thick interfacial film at the O / W interface. The presence of quinic acid in CHA introduced additional hydrogen bonding groups, making it easier for CG-CHA molecules to form a strong interfacial film. The hydrogen bonds between CG and CP promoted molecular cross-linking and produced a dense interfacial film. CPs promoted the formation of SsinglebondS and improved the stability and viscoelasticity of the interfacial film.
[0068] The interaction between CP and CG promotes the unfolding of the CG structure, which masks the surface hydrophilic groups, reduces the interaction with water molecules, and reduces the solubility. At the same time, this interaction exposes the internal hydrophobic groups, resulting in increased surface hydrophobicity. The affinity of CG-CPs to the O / W interface is enhanced, among which the strong hydrogen bond between CHA and CG is particularly important, which is conducive to the formation of a dense and thick interfacial film.
[0069] Example 7 - Emulsion Preparation
[0070] CG and CG-CPs solutions were mixed with soybean oil in a volume ratio of 9:1 to obtain a 10% oil phase mixture with a final CG concentration of 10 mg / mL. The above solution was sheared at a speed of 10000 rpm for 1 min using a high-speed shearing machine. Then, a NanoGenzier high-pressure homogenizer (Genizer, USA) was used to treat twice at 20 MPa to generate a microemulsion. The temperature was maintained at 25°C during the shearing process to produce an oil-in-water emulsion stabilized by CG, CG-CA, CG-CHA and CG-FA. Subsequently, 0.02% thimerosal by emulsion volume was added to each emulsion to inhibit microbial growth. All emulsions were stored at 4°C.
[0071] Example 8 - Emulsifying activity
[0072] The droplet size distribution and average droplet size of the emulsion were measured using a Malvern Master Sizer 2000 instrument (Malvern Instruments, Malvern Hills, UK). The experiment used a general spherical analysis model, with the refractive indices of oil droplets and dispersed water being 1.46 and 1.33, respectively, and the obscuration values ranging from 5% to 15%.
[0073] The viscosity measurements were performed using a HaakeMars40 rheometer (ThermoFisher, USA). The shear rate was gradually increased from 1s to 100s-1. During the measurement, the temperature of the sample (1mL) was maintained at 25°C. By comparing the fluidity of different samples, the effect of adding CPs on the fluidity of CG-stabilized emulsions was determined. All data provided are the average of three replicates.
[0074] Results: The droplet sizes of emulsions stabilized by CG, CG-CA, CG-CHA, and CG-FA showed that droplet aggregation in CG-CPs-stabilized emulsions was effectively suppressed. This suppression may be due to the increase in electrostatic repulsion and steric hindrance between droplets, especially for CHA. The droplet crosslinking promoted by the interaction between CG and CP led to an increase in apparent viscosity. This phenomenon suggests that the interaction between CG and CP restricts the movement of molecules. The strong hydrogen bonding interaction between CG and CHA leads to extensive crosslinking and a denser structure, which in turn leads to an increase in viscosity. CG-CPs exhibited greater resistance to deformation at the O / W interface, probably due to the formation of a dense and thick interfacial film.
[0075] Example 9 - Emulsion stability under different conditions
[0076] (1) Storage stability: The droplet morphology of the emulsion after storage for 0, 7, 14, 21 and 28 days was directly observed using an inverted microscope (Leica Microsystems CMS GmbH, Germany).
[0077] (2) Centrifugal stability: The centrifugal stability of the emulsion (0.4 mL) was further analyzed using a LUMiSizer stability analyzer (LUMiSizer, LUMGmbH, Germany). The light wavelength was set to 870 nm, the optical factor was 1.00, the contour line was set to 300, the time interval was set to 10 s, and the required time was set to 50 min.
[0078] (3) pH stability: Fresh emulsion samples (10 mL) were adjusted to pH 4.0, 5.0, 6.0, 7.0 and 8.0 using 0.1 mol / L HCl and 0.1 mol / L NaOH solutions. The droplet size of the emulsion was measured. The emulsification index (CI) of the emulsion was determined using the following formula (3):
[0079]
[0080] where HE and HS represent the total height of the emulsion and emulsion-serum layers, respectively.
[0081] (4) Salt stability: NaCl solutions of different concentrations (0.05-0.3 mol / L) were added to the emulsion (10 mL). The emulsion index and droplet size were measured after 3 h.
[0082] The results are as follows:
[0083] Storage stability: By recording the changes in the microstructure of the emulsion droplets within 28 days ( Figure 3 ), which demonstrated that the storage stability of CG-CPs-stabilized emulsions was relatively higher, especially that of CG-CHA-stabilized emulsions. In CG-stabilized emulsions, droplet merging and aggregation were more prominent, while in fresh emulsions stabilized by CG-CPs, droplets were small and uniform in size, and also changed slightly after 28 days of storage. This instability may be due to the aggregation and coalescence of emulsion droplets.
[0084] Centrifugal stability: Using emulsions as demonstration materials, the spatiotemporal changes in the transmitted liquid during centrifugation were recorded. After centrifugation, the emulsions underwent unstable changes, with the heavier aqueous phase settling to the bottom and the lighter oil droplets migrating to the top. The movement of the emulsion droplets can be visualized and the unstable processes therein quantified by the change in the intensity of the transmitted light over time. Changes in the transmittance of CG and CG-CPs-stabilized emulsions during centrifugation. The transmittance of the CG-stabilized emulsion changed rapidly, with the transmittance of most emulsions increasing from less than 10% to more than 40%, indicating that it quickly became unstable during centrifugation. In contrast, the centrifugal stability of the CG-CPs-stabilized emulsions increased, with the transmittance gradually increasing. It is particularly noteworthy that after centrifugation of the CG-CHA-stabilized emulsion, the transmittance of most emulsion layers remained below 20%, indicating that it has higher stability.
[0085] pH stability: The effect of pH on the appearance, CI and droplet size of the prepared emulsions was investigated. Figure 4 bc showed that between pH 6.0 and 8.0, the emulsions stabilized by CGs and CG-CPs remained relatively stable with little change in droplet size. However, when the pH dropped to 4.0, the droplet size of the emulsions stabilized by CG and CG-CPs increased to about 16 μm. This indicates that the droplets aggregated into larger units near the isoelectric point. CG is an acidic protein that easily aggregates in an acidic environment, resulting in reduced emulsion stability. At pH 4.0 and 5.0, the emulsions stabilized by CG, CG-CA, and CG-FA separated into a transparent serum layer at the bottom of the container, while the emulsion stabilized by CG-CHA showed no detectable phase separation. Under neutral or alkaline conditions, CA, CHA, and FA are susceptible to oxidation, especially CA and CHA, which have catechol structures that can react with nucleophilic groups in CG and covalently couple to form yellow polymers. In addition, the covalent coupling of CG with CA / CHA / FA can promote further protein development and exposure of hydrophobic groups, thereby enhancing the emulsification properties.
[0086] Figure 4 de showed that different salt ion concentrations had a significant effect on the stability of CG and CG-CPs stabilized emulsions. At low Na concentrations, salt dissolution improved the emulsification stability of the emulsion. However, at higher NaCl concentrations, salt ions masked the electrostatic repulsion between droplets, leading to droplet aggregation and coalescence. Compared with the CG-stabilized emulsion, the droplet size and CI of the CG-CPs-stabilized emulsion were smaller. This indicates that the CG-CPs complex effectively enhanced the salt ion stability of the CPs-modified emulsion. In summary, the effect of salt ion concentration on emulsion stability needs to take into account the characteristics of the components in the emulsion.
[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A process for producing a coconut milk product having improved emulsion stability, characterized in that: The following steps are involved: S1. Extraction of coconut protein: using n-hexane to remove the oil in freeze-dried coconut meat powder, mixing 0.3-0.7 mol / L sodium chloride solution with coconut meat powder in a ratio of 1:3-6 (w / v) for 3-5 hours, and then freeze-drying the extract and storing it at 2-6°C to obtain coconut protein; S2, preparing a globulin solution: dissolving the above coconut globulin in a 0.005-0.015 mol / L phosphate buffer solution, stirring for 1-3 h, and leaving overnight to fully hydrate to obtain a globulin solution with a concentration of 1.5-2.5 mg / mL; S3. Preparation of complex: respectively add caffeic acid CA, chlorogenic acid CHA and ferulic acid FA solution to the globulin solution, stir at low speed, adjust the pH value of all samples to 5.9-6.2, and obtain three solutions of coconut globulin-caffeic acid CG-CA, coconut globulin-chlorogenic acid CG-CHA, and coconut globulin-ferulic acid CG-FA, and store all samples at 2-6°C for short-term storage; S4, emulsion preparation: the above solutions are mixed with vegetable oil at a volume ratio of 7-10:1 to obtain an oil phase mixture of 9.0-12.5%, and then a high-speed shearing machine is used to shear at a speed of 8000-12000 rpm for 1-3 minutes, and then a high-pressure homogenizer is used to treat 1-3 times at 15-25 MPa to generate a microemulsion, and 0.01-0.03% of thimerosal by volume of the emulsion is added to the emulsion, and the mixture is stored at 3-5° C. to obtain a coconut milk product.
2. A process for producing a coconut milk product having improved emulsion stability as claimed in claim 1, characterized in that: The following steps are involved: S1. Coco globulin extraction: using n-hexane to remove the oil in the freeze-dried coconut meat powder, extracting it with 0.5 mol / L sodium chloride solution at a ratio of 1:5 (w / v) for 4 h, and then freeze-drying the extract and storing it at 4°C to obtain coconut globulin; S2, preparing a globulin solution: dissolving the above coconut globulin in 0.01 mol / L phosphate buffer solution, stirring for 2 h, and leaving overnight to fully hydrate to obtain a globulin solution with a concentration of 2.0 mg / mL; S3. Preparation of complex: respectively add caffeic acid (CA), chlorogenic acid CHA and ferulic acid FA solution dropwise to the globulin solution, stir at low speed, adjust the pH value of all samples to 6.0, obtain three solutions of coconut globulin-caffeic acid CG-CA, coconut globulin-chlorogenic acid CG-CHA, coconut globulin-ferulic acid CG-FA, and store all samples at 4°C for short-term storage; S4. Preparation of emulsion: The above solutions were mixed with soybean oil at a volume ratio of 9:1 to obtain a 10% oil phase mixture, the above solution was sheared at a speed of 10000 rpm for 1 min using a high-speed shearing machine, and then treated twice with a high-pressure homogenizer at 20 MPa to generate a microemulsion, 0.02% thimerosal by volume of the emulsion was added to the emulsion, and the mixture was stored at 4° C. to obtain a coconut milk product.
3. A process for producing a coconut milk product having improved emulsion stability as claimed in claim 1, characterized in that: The volume mass mL / g ratio of n-hexane to freeze-dried coconut meat powder in S1 is 1:2-5.
4. A process for producing a coconut milk product having improved emulsion stability as claimed in claim 1, characterized in that: The freeze drying temperature in S1 is -40 to -60°C, and the freezing time is 23 to 28 hours.
5. A process for producing a coconut milk product with improved emulsion stability as claimed in claim 1, characterized in that: In the S3, caffeic acid CA, chlorogenic acid CHA and ferulic acid FA are respectively dissolved in 0.01 mol / L phosphate buffer solution to obtain caffeic acid CA, chlorogenic acid CHA and ferulic acid FA solutions with a concentration of 1.8-2.2 mg / mL.
6. A process for producing a coconut milk product with improved emulsion stability as claimed in claim 1, characterized in that: The low speed stirring rate of S3 is 40-100 rpm.
7. A process for producing a coconut milk product with improved emulsion stability as claimed in claim 1, characterized in that: The pH values of all samples were adjusted to 5.9-6.2 using 0.1 mol / L HCl and 0.1 mol / L NaOH solutions in S3.
8. A process for producing a coconut milk product with improved emulsion stability as claimed in claim 1, characterized in that: The vegetable oil in S4 includes but is not limited to soybean oil, peanut oil, linseed oil or olive oil.
9. A process for producing a coconut milk product with improved emulsion stability as claimed in claim 1, characterized in that: The temperature during the shearing process in S4 was maintained at 23-27°C.
10. A coconut milk product prepared by the production process according to any one of claims 1 to 9, which has high emulsification stability, high nutritional value and excellent taste.