Method for preparing sulfonated emulsifier based on cottonseed protein isolate

Abstract

The application discloses a method for preparing a sulfonated emulsifier based on cottonseed protein isolate, comprising: treating cottonseed meal to obtain cottonseed protein isolate; dissolving the cottonseed protein isolate in chlorosulfonic acid, fully stirring under nitrogen flow, then slowly adding an excess of an ice water mixture while stirring, stopping the reaction, and continuing to stir; removing residual acid and small molecule by-products through dialysis until the pH is constant; adjusting the pH to 7 using a sodium hydroxide solution, further dialyzing, freeze-drying, and obtaining sulfonated cottonseed protein hydrolysate, which is a sulfonated emulsifier based on cottonseed protein isolate. In the application, the sulfonation of CPI improves the structure, function and physical and chemical properties of CPI, increases flexibility and hydrophilicity, exposes hydrophobic groups, reduces pI, molecular weight and surface tension, which effectively improves the solubility and emulsifying capacity of CPI, and the prepared emulsion has excellent stability and uniformity under different pH, temperature and ionic strength.

Description

Technical Field

[0001] This invention relates to the field of emulsifier technology, and more specifically, to a method for preparing sulfonated emulsifiers based on cottonseed protein isolate. Background Technology

[0002] Cotton cultivation has traditionally been for fiber production, with cottonseed being a major byproduct. The chemical composition and functional properties of cottonseed make it an economically attractive agricultural-industrial waste with significant biotechnological applications. It contains storage proteins, primarily globulins, and non-storage proteins with substantial nutritional value, making it an excellent source of plant protein. Cottonseed is available in many temperate and tropical countries, but its use has been limited due to the toxicity of gossypol. However, in recent years, improvements in glandless cottonseed technology and techniques for safely recovering free gossypol from cottonseed protein, coupled with rapid global population growth and the increasing cost of traditional protein sources, have led to renewed interest in the utilization of cottonseed protein isolate (CPI). CPI exhibits broad bioactivity, good emulsifying properties, foaming properties, fluorescence intensity, oil / water absorption capacity, and surface hydrophobicity, suggesting potential applications in various fields. However, its sensitivity to environmental conditions limits its application.

[0003] Emulsions play a crucial role in the structural formation of many products and are being explored in numerous industries, including food, pharmaceuticals, and non-food sectors. However, emulsions are thermodynamically unstable, and as a result of one or more physicochemical mechanisms such as creaming, precipitation, aggregation, flocculation, Ostwald ripening, and phase inversion, they can decompose, thus reducing their shelf life. Therefore, surfactants with high mobility can rapidly adsorb onto interfaces and significantly reduce interfacial tension, thus stabilizing emulsions. Currently, the direct or indirect application of synthetic surfactants in the food / non-food industries carries potential toxicity and environmental inconvenience, increasing the importance of using natural substances and fostering interest in the application of proteins for emulsion stabilization. Plant proteins possess excellent emulsifying capabilities, are amphiphilic and surfactant molecules, contributing to emulsion stability, and are therefore commonly used in commercial food emulsions. However, plant proteins are structurally complex macromolecules that diffuse slowly to the oil-water interface, making them more difficult to manage in terms of emulsifying ability and emulsion stability compared to synthetic surfactants. Furthermore, natural protein-stabilized emulsions such as caseinate, cottonseed protein, whey protein, soy protein, and zein are sensitive to environmental factors such as pH, ionic strength, temperature, and mechanical stress, leading to aggregation / precipitation. Protein-stabilized emulsions flocculate at pH values ​​close to the protein's isoelectric point (pI). Since the isoelectric point of caseinate (CPI) is 5.1, it cannot function as an emulsifier under acidic conditions. This limits the industrial applicability of CPI, as many food, pharmaceutical, and non-food products are already in acidic environments. Therefore, various techniques are used to modify plant proteins to hydrolyze and improve their physicochemical and functional properties, overcoming their limitations and enabling multifunctional applications. Chemical modification of plant proteins by introducing different functional groups offers advantages such as low cost, low equipment requirements, and fast reaction time, making it superior to other methods (enzymatic, physical, and genetic methods).

[0004] Sulfonation is a common covalent chemical modification of biomolecules, involving the addition of sulfonic acid groups (SO3). -1 Sulfonation involves the transfer of sulfonation residues to hydroxyl or amino groups. It leads to conformational changes in both low and high molecular weight molecules, transforming them from lipophilic to amphiphilic molecules with excellent water solubility and lowering their pKa to around 1.5. Studies have found that hydrolysis during this reaction results in sulfonated molecules with lower molecular weights. Chlorosulfonic acid is chosen as both a solvent and sulfonating agent in sulfonation because it is inexpensive and readily available, and it can completely sulfonate serine, threonine, tyrosine, and tryptophan residues in a shorter time compared to other products. Despite the significant applications of sulfonation in biological systems and other polymers, few studies have focused on modifying and improving plant proteins. While residual chemicals after sulfonation are a concern, they can be easily removed by dialysis, and the modified protein can be recovered by drying the residue in the dialysis bag.

[0005] Ideal emulsifiers are typically unfolded amphiphiles, including those with sufficiently accessible hydrophobic groups, a good hydrophilic / hydrophobic ratio, charged groups, and relatively low molecular weight, exhibiting excellent solubility in a continuous aqueous phase. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0007] To achieve these objectives and other advantages of the present invention, a method for preparing a sulfonated emulsifier based on cottonseed protein isolate is provided, comprising the following steps:

[0008] Cottonseed protein isolate was dissolved in chlorosulfonic acid and stirred thoroughly for 1–12 hours at 20–35°C under a nitrogen atmosphere. Then, an excess of ice-water mixture was slowly added while stirring, the reaction was stopped, and stirring was continued for 20–40 minutes. Residual acid and small molecule byproducts were removed by dialysis until the pH was constant. The pH was adjusted to 7 using sodium hydroxide solution, and further dialysis and freeze-drying were performed to obtain sulfonated cottonseed protein hydrolysate, which is a sulfonated emulsifier based on cottonseed protein isolate.

[0009] Preferably, the preparation method of the cottonseed protein isolate is as follows: cottonseed meal is added to potassium hydroxide solution and stirred at 50-60°C for 30-60 min. The resulting mixture is centrifuged at 3-5°C and 2000-3000×g for 10-60 min. Then, the supernatant is adjusted to pH 5-5.5 with hydrogen chloride solution, centrifuged again, the precipitate is washed three times with deionized water, resuspended in water, the pH is adjusted to 7, and then freeze-dried to obtain cottonseed protein isolate.

[0010] Preferably, the mass-to-volume ratio of the cottonseed protein isolate to chlorosulfonic acid is 1g:20-30mL.

[0011] Preferably, the dialysis time is 42 to 54 hours, with the water changed every 6 hours.

[0012] Preferably, the concentration of the sodium hydroxide solution is 4–6 mol / L.

[0013] Preferably, the concentration of the potassium hydroxide solution is 0.05–0.15 mol / L.

[0014] Preferably, the mass-to-volume ratio of the cottonseed meal to the potassium hydroxide solution is 1g:10-20mL.

[0015] Preferably, the concentration of the hydrogen chloride solution is 0.05–0.15 mol / L.

[0016] The present invention also provides the application of the emulsifier prepared by the method for preparing sulfonated emulsifier as described above in the preparation of emulsions. The emulsifier is prepared into an emulsifier solution, and the emulsifier solution and MCT oil are homogenized at 10,000 to 13,000 rpm for 1 to 3 minutes to obtain a crude emulsion. The crude emulsion is treated with an ultrasonic homogenizer and the temperature is maintained in a plate cooler to obtain an emulsion.

[0017] Preferably, the concentration of the emulsifier solution is 5-15 mg / mL; the volume ratio of the emulsifier solution to MCT oil is 8-10:1; and the conditions for treating the crude emulsion with an ultrasonic homogenizer are: emulsification treatment at 750 W, 20 kHz and 40% amplitude for 15-20 min, using intermittent ultrasound, i.e., ultrasound for 3 seconds followed by a 5-second pause.

[0018] This invention offers at least the following beneficial effects: The sulfonated emulsifier prepared by this invention possesses advantages such as adjustable hydrophilicity / hydrophobicity, higher structural flexibility, lower isoelectric point, and lower molecular weight. Sulfonation of CPI improves its structure, function, and physicochemical properties, including unfolding, increased flexibility, increased hydrophilicity, exposure of hydrophobic groups, lower pI, lower molecular weight, and lower surface tension, which effectively enhances the solubility and emulsifying ability of CPI. Emulsions produced and stored under different pH, temperature, and ionic strength conditions are long-term stable and exhibit excellent flowability, indicating that sulfonated CPI is an excellent environmentally stable emulsifier, particularly performing well under acidic conditions. This invention contributes to understanding sulfonation as a feasible method for modifying and improving the properties of plant proteins, opening up new possibilities for their utilization and economic benefits.

[0019] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0020] Figure 1 The FTIR spectra of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention are shown below.

[0021] Figure 2 The molecular weight (Mw) and isoelectric point (pI) of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention are given.

[0022] Figure 3 The flexibility and turbidity of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0023] Figure 4 The surface hydrophobicity (H0) and carboxyl content of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention are used to determine their properties.

[0024] Figure 5 The emulsifying activity index (EAI) and emulsifying stability index (ESI) of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention are used.

[0025] Figure 6 The dynamic interfacial tension of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0026] Figure 7 The average interfacial tension of SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0027] Figure 8 The percentage of protein absorbed (AP%) of the emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0028] Figure 9 Cream index (CI) of emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention after treatment at different pH values ​​(3.0, 4.0, 6.8, 8.0), different temperatures (40°C and 80°C), and different ionic strengths (100mM and 250mM);

[0029] Figure 10 Droplet size and ζ-potential of emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention after treatment at different pH values ​​(3.0, 4.0, 6.8, and 8.0);

[0030] Figure 11 Droplet size and ζ-potential of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after storage for 70 days at different pH values ​​(3.0, 4.0, 6.8, and 8.0);

[0031] Figure 12 Droplet size and ζ-potential of emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention after treatment at different temperatures (40°C and 80°C) and different ionic strengths (100 mM and 250 mM);

[0032] Figure 13 Droplet size and ζ-potential of emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention after being treated at different temperatures (40°C and 80°C) and different ionic strengths (100mM and 250mM) and stored for 70 days;

[0033] Figure 14Droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at pH 3.0;

[0034] Figure 15 Droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at pH 4.0;

[0035] Figure 16 Droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at pH 6.8;

[0036] Figure 17 Droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at pH 8.0;

[0037] Figure 18 The droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at 40°C;

[0038] Figure 19 Droplet size distribution of emulsions prepared for the present invention SCPH0, SCPH1, SCPH6, and SCPH12 after treatment at 80°C;

[0039] Figure 20 Droplet size distribution of emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention at a concentration of 100 mM NaCl;

[0040] Figure 21 Droplet size distribution of emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention at a concentration of 250 mM NaCl;

[0041] Figure 22 The relationship between apparent viscosity and shear rate of emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0042] Figure 23 The relationship between shear stress and shear rate of emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0043] Figure 24 Storage modulus of the emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0044] Figure 25The loss modulus of the emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention;

[0045] Figure 26 Storage modulus of emulsions prepared by SCPH0, SCPH1, SCPH6, and SCPH12 of this invention after 70 days of storage;

[0046] Figure 27 The emulsions prepared for SCPH0, SCPH1, SCPH6, and SCPH12 of this invention lost their modulus after 70 days of storage; Detailed Implementation

[0047] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0048] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0049] Example 1:

[0050] A method for preparing sulfonated emulsifiers based on cottonseed protein isolate includes the following steps:

[0051] Step 1: Add cottonseed meal to 0.1M potassium hydroxide solution (the mass-to-volume ratio of cottonseed meal to potassium hydroxide solution is 1g:12mL), stir at 55℃ for 40min, centrifuge the resulting mixture at 4℃ and 2470×g for 20min, then adjust the pH of the supernatant to 5.1 with 0.1M hydrogen chloride solution to precipitate the protein isolate, then centrifuge, wash the precipitate three times with deionized water, suspend it in water, adjust the pH to 7 with potassium hydroxide solution, and then freeze-dry to obtain cottonseed protein isolate (CPI);

[0052] Step 2: Dissolve 8g of cottonseed protein isolate obtained in Step 1 in 200mL of chlorosulfonic acid. Stir thoroughly for 1 hour at 25°C under a nitrogen atmosphere. Then, while stirring, slowly add an excess of ice-water mixture, stop the reaction, and continue stirring for 30 minutes. Dialyze for 48 hours, changing the water 8 times, to remove residual acid and small molecule byproducts until the pH is constant. Adjust the pH to 7 using 5M sodium hydroxide solution, dialyze further, and freeze-dry to obtain sulfonated cottonseed protein hydrolysate (SCPH1), which is a sulfonated emulsifier based on cottonseed protein isolate.

[0053] Example 2:

[0054] A method for preparing sulfonated emulsifiers based on cottonseed protein isolate includes the following steps:

[0055] Step 1: Add cottonseed meal to 0.1M potassium hydroxide solution (the mass-to-volume ratio of cottonseed meal to potassium hydroxide solution is 1g:12mL), stir at 55℃ for 40min, centrifuge the resulting mixture at 4℃ and 2470×g for 20min, then adjust the pH of the supernatant to 5.1 with 0.1M hydrogen chloride solution to precipitate the protein isolate, then centrifuge, wash the precipitate three times with deionized water, suspend it in water, adjust the pH to 7 with potassium hydroxide solution, and then freeze-dry to obtain cottonseed protein isolate (CPI);

[0056] Step 2: Dissolve 8g of cottonseed protein isolate obtained in Step 1 in 200mL of chlorosulfonic acid. Stir thoroughly for 6 hours at 25°C under a nitrogen atmosphere. Then, slowly add an excess of ice-water mixture while stirring, stop the reaction, and continue stirring for 30 minutes. Dialyze for 48 hours, changing the water 8 times, to remove residual acid and small molecule byproducts until the pH is constant. Adjust the pH to 7 using 5M sodium hydroxide solution, dialyze again, and freeze-dry to obtain sulfonated cottonseed protein hydrolysate (SCPH6), which is a sulfonated emulsifier based on cottonseed protein isolate.

[0057] Example 3:

[0058] A method for preparing sulfonated emulsifiers based on cottonseed protein isolate includes the following steps:

[0059] Step 1: Add cottonseed meal to 0.1M potassium hydroxide solution (the mass-to-volume ratio of cottonseed meal to potassium hydroxide solution is 1g:12mL), stir at 55℃ for 40min, centrifuge the resulting mixture at 4℃ and 2470×g for 20min, then adjust the pH of the supernatant to 5.1 with 0.1M hydrogen chloride solution to precipitate the protein isolate, then centrifuge, wash the precipitate three times with deionized water, suspend it in water, adjust the pH to 7 with potassium hydroxide solution, and then freeze-dry to obtain cottonseed protein isolate (CPI);

[0060] Step 2: Dissolve 8g of cottonseed protein isolate obtained in Step 1 in 200mL of chlorosulfonic acid. Stir thoroughly at 25°C under a nitrogen atmosphere for 12 hours. Then, while stirring, slowly add an excess of ice-water mixture, stop the reaction, and continue stirring for 30 minutes. Dialyze for 48 hours, changing the water 8 times to remove residual acid and small molecule byproducts until the pH is constant. Adjust the pH to 7 using 5M sodium hydroxide solution, dialyze further, and freeze-dry to obtain sulfonated cottonseed protein hydrolysate (SCPH12), which is a sulfonated emulsifier based on cottonseed protein isolate.

[0061] FTIR analysis was performed on unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 using the KBr method, with a wavenumber range of 4000–500 cm⁻¹. -1 The resolution is 4cm. -1 FTIR is used to characterize changes in the secondary structure of proteins after sulfonation. For example... Figure 1 As shown, three typical protein spectral characteristic bands were observed, namely amide I (~1655 cm⁻¹). -1 ), Amide II (~1540cm) -1 ) and amide III (~1392cm) -1 Following sulfonation, the intensity of the amide peak increased and became more pronounced with increasing hydrolysis time, but remained fairly constant after 6 hours. Furthermore, a peak of approximately 1223 cm⁻¹ was observed in the SCPHs. -1 The strong new peak, attributed to the stretching vibration of the sulfonic acid group, indicates that the sulfonic acid group was successfully introduced into CPI after sulfonation, leading to changes in protein structure. The spectrum clearly shows that the intensity of the new peak attributable to the sulfonic acid group becomes more prominent with increasing hydrolysis time, but stops changing significantly after 12 hours, indicating that the degree of CPI sulfonation increases significantly to its highest value at 6 hours, and then stops changing significantly with further increases in reaction time to 12 hours. Simultaneously, peaks can also be observed at 2900–3300 cm⁻¹. -1 The band intensity increases slightly from SCPH0 to SCPHs, which is related to the stretching vibrations of the -OH and NH groups of sulfonic acids. Additionally, after sulfonation, the band intensity at 1127 cm⁻¹... -1 The increase in peak values ​​is related to the CS bond, indicating that the sulfonation reaction leads to the formation of COS and CS bonds between CPI and chlorosulfonic acid.

[0062] The molecular weight (Mw) of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 was determined. The samples were dissolved in 0.1M NaNO3 containing 0.02% NaN3 as the mobile phase, filtered through a 0.22 μm filter membrane, and determined by gel permeation chromatography. The results are as follows: Figure 2 As shown, the molecular weight of CPI decreases significantly after sulfonation, and this decrease becomes more pronounced with increasing hydrolysis time.

[0063] The isoelectric points (pI) of unsulfonated cottonseed protein isolate (SCPH0) and the SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 were tested. The isoelectric points were determined by measuring the turbidity of 0.1 mg / mL SCPH at 600 nm using a UV-Vis spectrophotometer at different pH values ​​(5.8-1.5). The results are shown below. Figure 2 As shown, the pI decreases from 5.1 for SCPH0 to 1.7 for SCPH12, indicating that the sulfonate group is introduced into the protein molecule through sulfonation, which increases the electronegativity of the protein molecule surface and reduces the protein's pI. This broadens the applicable pH range compared to natural proteins, which is an ideal property for most food and non-food industry applications. This suggests that SCPH1, SCPH6, and SCPH12 have a wider range of applications.

[0064] The flexibility of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 were tested. 0.5 mg / mL SCPH was precisely prepared in 0.05 mol / L tris-HCl (pH 7.5), and trypsin (potency ≥2500 units / mg, 16:1, v / v) was added. After thorough mixing, the mixture was incubated at 38°C for 10 minutes, then centrifuged at 5000 × g for 30 minutes. The absorbance of the supernatant was measured at 280 nm using a UV spectrophotometer. This value quantitatively represents the flexibility of SCPH. The results are as follows: Figure 3 As shown, SCPH1, SCPH6, and SCPH12 exhibit higher conformational flexibility compared to the unsulfonated SCPH0. The flexibility increases from 0.186 for SCPH0 to 0.913 for SCPH12, and this increase in flexibility is correlated with the sulfonation reaction time. This confirms that the protein unfolds during this process, and the acidic conditions employed denature the protein, leading to the unfolding of the protein molecule.

[0065] The turbidity of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 was tested, and the results are as follows: Figure 3 As shown. Turbidity indicates the size and quantity of suspended matter in an aqueous solution and can be used to estimate the degree of protein aggregation; higher turbidity indicates greater protein aggregation. It can be seen that SCPH0 has the highest turbidity at 1.13, decreasing with increasing sulfonation time, while SCPH12 has the lowest turbidity at 0.03. When more and larger protein aggregates appear, protein turbidity increases, which alters light scattering and hinders light transmission. Sulfonation reduces the molecular weight of proteins, increasing the surface area available for light scattering, thus leading to a decrease in turbidity.

[0066] The surface hydrophobicity (H0) of unsulfonated cottonseed isolate protein (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 was tested. Surface hydrophobicity (H0) was determined using 1-aniline-8-naphthalenesulfonate (ANS). SCPH0 / SCPHs solutions (0.1–1.0 mg / mL) in phosphate buffer (100 mM, pH 7.0) were centrifuged at 10000 × g for 20 minutes, and the protein content in the supernatant was determined using the Bradford method. Then, 30 μL of ANS (8.0 mM) was added to 6 mL of the sample solution, and the fluorescence intensity was measured using a multi-functional microplate reader with an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The H0 value is expressed as the initial slope of the fluorescence intensity versus protein concentration function graph. The results are shown below. Figure 4 As shown, H0 increases from 125.3 in SCPH0 to 939.4 in SCPH12. This means that more hydrophobic groups that were originally located inside the protein are exposed. The sulfonation reaction changes the tertiary conformation of the protein, increases its flexibility, causes the protein molecule to unfold, and increases the proportion of surface hydrophobic regions.

[0067] The carboxyl content of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 was tested. Carboxyl content (Mol) COOH / g pepedie The determination was performed using conductivity titration, and the calculations are as follows:

[0068]

[0069] Figure 4 The image shows the carboxyl content of the peptides formed after sulfonation for 1, 6, and 12 hours. The carboxyl content increases with increasing reaction time, indicating that the peptides become more hydrophilic. Chlorosulfonic acid acts as a protein deamidizing agent, causing deamidation during the reaction and converting side-chain amino acids (such as glutamine and asparagine) in the protein into glutamic acid and aspartic acid, respectively, leading to an increase in carboxyl content. This is also confirmed by FTIR spectroscopy at approximately 1540 cm⁻¹. -1 The nearby peak also represents carboxyl groups, and this peak increases with increasing reaction time.

[0070] The emulsifying activity index (EAI) and emulsifying stability index (ESI) of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 were tested. 3 mL of MCT oil was added to 9 mL of sample solution and homogenized at 10000 rpm for 1 minute. Then, 50 μL of the emulsion was removed from the bottom at 0 and 10 minutes, immediately diluted and mixed with 5 mL of 0.1% SDS solution. The absorbance was then read at 500 nm. The EAI and ESI were calculated as follows:

[0071]

[0072]

[0073] The emulsifying ability of emulsifiers is evaluated using EAI and ESI; higher EAI and ESI values ​​indicate stronger emulsifying ability. Figure 5 Sulfonation significantly increased the EAI and ESI of CPI, but longer reaction times had little effect on the EAI value. The quaternary conformational flexibility of proteins can be expressed via EAI, while their tertiary conformational flexibility can be expressed via ESI. Proteins with higher EAI exhibit greater flexibility in their tertiary structure by influencing the structural reorganization of proteins absorbed at the interface. EAI follows the same pattern as the increase in protein flexibility, and the observed phenomenon can also be attributed to enhanced hydrophilicity and increased exposure of hydrophobic sites, which promotes interfacial interactions between the protein and nearby oil droplets.

[0074] The dynamic and mean interfacial tensions of unsulfonated cottonseed protein isolate (SCPH0) and SCPHs (SCPH1, SCPH6, SCPH12) prepared in Examples 1-3 were tested. The interfacial tension between MCT oil and a 5 mg / mL SCPH0 / SCPHs dispersion was analyzed using a K100 tensiometer and the Wilhelmy plate method at 25°C. Tests were performed for 60 seconds between the aqueous and oil phases, with each test repeated at least three times. Data are expressed as mean and standard deviation. Interfacial tension plays a crucial role in the adsorption behavior and structural modification of proteins at the oil-water interface during emulsion formation. Figure 6 The dynamic interfacial tension (DIFT) of the MCT oil / water interface for SCPH0, 1, 6 and 12 at a concentration of 5 mg / mL and pH 7 is shown. The interfacial tension value decreases significantly with increasing reaction time, indicating that the protein undergoes flexible stretching and sustained adsorption at the oil-water interface. Figure 7The mean interfacial tension (mean SFT) decreased from 14.77 mN / m (SCPH0) to 11.84 mN / m (SCPH12). The interfacial tension values ​​of SCPHs are lower than those of SCPH0, meaning they are more suitable for absorption at oil / water interfaces. Shortly after the experiment began, the DIFT values ​​of SCPHs approached equilibrium. Although not fully reached equilibrium by the end of the experiment, they exhibited a gradual and marginal decrease, which can be attributed to the sulfonation modification increasing the hydrophobic / hydrophilic ratio and flexibility of the peptides. SCPH0 exhibited typical globular protein behavior at oil-water interfaces, decreasing rapidly immediately after interface formation, followed by a slower decrease. The interfacial properties of SCPH0 can be attributed to their tertiary and secondary structures, which require more time to unfold compared to the more flexible SCPHs.

[0075] Example 4:

[0076] An application of a sulfonated emulsifier prepared by the method described in Example 1 in the preparation of an emulsion: The emulsifier is prepared into an emulsifier solution (10 mg / mL), and 27 mL of the emulsifier solution and 3 mL of MCT oil are homogenized at 12000 rpm for 2 min to obtain a crude emulsion; the crude emulsion is treated with an ultrasonic homogenizer and the temperature is maintained in a plate cooler to obtain an emulsion; the conditions for treating the crude emulsion with the ultrasonic homogenizer are: emulsification treatment at 750 W, 20 kHz and 40% amplitude for 18 min, and intermittent ultrasonication is used, i.e., ultrasonication for 3 seconds and pause for 5 seconds.

[0077] Example 5:

[0078] An application of a sulfonated emulsifier prepared by the method described in Example 2 in the preparation of an emulsion: The emulsifier is prepared into an emulsifier solution (10 mg / mL), and 27 mL of the emulsifier solution and 3 mL of MCT oil are homogenized at 12000 rpm for 2 min to obtain a crude emulsion; the crude emulsion is treated with an ultrasonic homogenizer and the temperature is maintained in a plate cooler to obtain an emulsion; the conditions for treating the crude emulsion with the ultrasonic homogenizer are: emulsification treatment at 750 W, 20 kHz and 40% amplitude for 18 min, and intermittent ultrasonication is used, that is, ultrasonication for 3 seconds and pause for 5 seconds.

[0079] Example 6:

[0080] An application of a sulfonated emulsifier prepared by the method described in Example 3 in the preparation of an emulsion: The emulsifier is prepared into an emulsifier solution (10 mg / mL), and 27 mL of the emulsifier solution and 3 mL of MCT oil are homogenized at 12000 rpm for 2 min to obtain a crude emulsion; the crude emulsion is treated with an ultrasonic homogenizer and the temperature is maintained in a plate cooler to obtain an emulsion; the conditions for treating the crude emulsion with the ultrasonic homogenizer are: emulsification treatment at 750 W, 20 kHz and 40% amplitude for 18 min, and intermittent ultrasonication is used, that is, ultrasonication for 3 seconds and pause for 5 seconds.

[0081] The percentage of absorbed protein (AP%) of the prepared emulsion was measured: The freshly prepared emulsion was centrifuged at 13000×g for 15 minutes to separate the emulsion into an aqueous phase and a cream layer. The aqueous phase was removed using a syringe, and the emulsion was filtered through a 0.22 μm filter. The peptide concentration (Cp) of the filtered aqueous phase was determined using the Bradford method. f The initial SCPH solution used was centrifuged, and the peptide concentration in the supernatant was measured (C0). s AP% is calculated as follows:

[0082]

[0083] Where C0 represents the initial protein concentration of SCPH.

[0084] The cream stability of the emulsion was analyzed using the cream index (CI). Freshly prepared emulsions were transferred to glass bottles, stored, and the layering of the emulsion was observed and recorded. The CI was calculated as follows:

[0085]

[0086] Where H s Indicates the height of the serum layer, H t This indicates the total height of the emulsion.

[0087] The particle size, distribution, and zeta potential of SCPH were measured using a Zetasizer Nano ZS90 at 25°C and a scattering angle of 90°. The samples were diluted 100-fold with the same aqueous solution used for dispersion to avoid multiple scattering effects.

[0088] Environmental stability of the emulsion: 5 mL of the emulsion was centrifuged at 13000 × g for 15 min. The effect of pH on emulsion stability was assessed; the pH of the freshly prepared emulsion was 6.8, and 30 mL of SCPH emulsion was adjusted to pH 3.0–8.0 using 1 M HCl / NaOH solution. The effect of temperature on emulsion stability was assessed by incubating 30 mL of SCPH emulsion in a water bath at different temperatures (40 and 80 °C) for 30 min. The effect of ionic strength on the SCPH emulsion was assessed by treating 30 mL of freshly prepared emulsion with stock NaCl to produce final concentrations of 100 and 250 mM. The treated emulsions were poured into capped glass tubes, and droplet size and zeta potential were measured for all treated samples after treatment and 70 days of storage.

[0089] like Figure 8 As shown, sulfonation significantly increased the percentage of protein absorbed in the emulsion from 16.58% (SCPH0) to 60.63% (SCPH12). This was due to increased flexibility and hydrophobic / hydrophilic ratio, and a decrease in Mw, resulting in more protein adsorbed at the oil droplet interface. This is also reflected in the CI results, such as... Figure 9 As shown, the CI of SCPH0 emulsion is significantly higher than that of SCPHs emulsion. Droplets of SCPH0 emulsion are flocculated and aggregated together, while droplets of SCPHs emulsion are uniformly dispersed in small size. When more proteins are absorbed on the surface of the droplets, the electrostatic repulsion between the droplets becomes stronger, which essentially hinders emulsion aggregation and phase separation, proving that the sulfonation reaction significantly improves the stability of the emulsion.

[0090] Depending on storage conditions such as pH, ionic strength, and temperature, emulsions may become unstable due to flocculation, aggregation, Ostwald ripening, and gravity separation. Therefore, the environmental stability of emulsions stable with SCPH0 and SC PHs (SCPH1, SCPH6, SCPH12) was investigated, and the results are as follows: Figures 9-21 As shown. From Figure 10 It can be seen that all samples had the lowest droplet size at pH 6.8, with SC PHs being significantly smaller than SCPH0, and decreasing to the minimum with increasing sulfonation time. SC PHs-stabilized emulsions remained stable under pH fluctuations, while SCPH0-stabilized emulsions showed the greatest droplet size change under pH fluctuations. However, at pH 3, except for SCPH12-stabilized emulsions, all freshly prepared emulsions showed flocculation and whey layers shortly after emulsification. Furthermore, from... Figures 14-17It can be seen that the SCPH12 emulsion, stable at all pH levels, exhibits a single-peak droplet size distribution with similar shape, narrow distribution, and small droplets, reflecting the efficiency of SCPH12 in generating uniformly sized oil droplets during homogenization. In contrast, the droplet size distribution (DSD) of SCPH1 and SCPH6 is bimodal at pH 6.8 and single-peaked at pH 4 and pH 8. SCPH0 shows a bimodal DSD with a wide distribution and larger droplet size at all pH levels, exhibiting a main peak near 500 nm at pH 8, shifting towards 1 μm, while the small peak near 5 μm becomes larger as the pH becomes more acidic. Furthermore, the droplet size of the SCPH emulsions is significantly smaller than that of the SCPH0 emulsion, which can be attributed to the increased flexibility and hydrophilic / hydrophobic ratio, as well as the decrease in Mw. This effect facilitates faster protein adsorption at the oil-water interface, significantly reduces interfacial tension, and ultimately promotes droplet breakup during emulsification. Figure 10 As shown, SCPH emulsions exhibit a high negative charge at pH 8, which decreases slightly as the pH moves to pH 4. The zeta potential of SCPH emulsions is significantly lower than that of SCPH0 emulsions. This phenomenon can be explained by the lowering effect of sulfonation on the isoelectric point of CPI, leading to the introduction of negatively charged sulfonic acid groups onto the protein surface. All SCPH emulsions have a zeta potential below -50 mV. Since emulsions with a zeta potential below -30 mV are widely considered to have the ability to prevent droplet aggregation through electrostatic mechanisms, this indicates that SCPH emulsions possess high stability. Figure 10 and 11 As shown, after 70 days of storage, the SCPH emulsions stored at pH 4.0 remained homogeneous, and their droplet size and zeta potential were almost identical to those of the freshly prepared emulsions, indicating that SCPHs have good emulsifying and stabilizing properties at pH 4.0. Similar trends were observed at pH 6.8 and 8.0, which means that SCPHs can act as emulsifiers in acidic, neutral, and alkaline environments.

[0091] from Figure 12 It can be seen that the droplet size of the SCPHs emulsion increased after heat treatment, but remained lower than that of the SCPH0 emulsion, indicating that the sulfonated emulsifier is beneficial to the thermal stability of the emulsion. Figure 13 As shown, after a 70-day storage period, compared to the stable emulsion of SCPH0, the droplet size and ζ-potential of the SCPHs emulsion increased slightly, and there was no emulsification, demonstrating the good thermal stability of the SCPHs emulsion. Furthermore, as... Figure 18 , Figure 19As shown, they exhibit a single-peaked DSD across all temperature variations, reflecting uniform and adequate coverage of oil droplets during homogenization. SCPHs interact more favorably with emulsified oil droplets during homogenization, which helps break down large aggregates into smaller particles. SCPH0 emulsions are unstable; proteins adsorbed at the interface denature upon heat treatment, exposing hydrophobic residues in SCPH0. Increased temperature leads to increased hydrophobic attraction and collision frequency of droplets, making them more prone to aggregation and agglomeration, thus destabilizing the emulsion. Furthermore, heating the emulsion may cause conformational changes in adsorbed proteins, which facilitates rearrangement of their interfacial structure. In summary, SCPH emulsions exhibit excellent thermal stability.

[0092] Since food and pharmaceutical products typically require exposure to environments with varying ionic strengths, the stability of the emulsion was examined at different NaCl concentrations (100 mM, 250 mM), and the results are as follows: Figure 12 , Figure 13 , Figure 20 and Figure 21 As shown, the average droplet size and ζ-potential of the emulsions change very little under different ionic intensities, and the droplet size and ζ-potential of the SCPHs emulsion are significantly smaller than those of the SCPH0 emulsion. Furthermore, from... Figure 20 and Figure 21 It can be seen that they also exhibit a single-peaked DSD with similar shape, narrow distribution, and containing small droplets, indicating that SCPHs have excellent stability under different ionic intensities. This can be attributed to the shielding effect of the introduced ions at the droplet interface, providing a thicker spatial barrier interface and preventing emulsion droplet aggregation. Furthermore, as... Figure 9 and Figure 13 As shown, during the storage of SCPH0 stable emulsion, an emulsion layer forms, and droplets aggregate at all NaCl concentrations. This is due to the electrostatic shielding of droplet charge caused by high ionic strength, which reduces the repulsive force between droplets, promotes aggregation and agglomeration, and thus leads to demulsification.

[0093] The rheological properties of the newly prepared SCPH emulsion were characterized using a controlled stress rheometer. Measurements were taken at 25 °C using a serrated plate-flat geometry (60 mm in diameter, 1° angle), and all measurements were repeated at least twice. The results were obtained within the range of 0.01–200 s. -1The shear rate was adjusted within a certain range, and flow curves were recorded and analyzed using a steady-rate flow mode. To determine the relationship between the structure and viscoelastic properties of the SCPH stabilized emulsion, dynamic oscillation measurements were performed. First, strain scans were measured at a fixed angular frequency of 6.283 rad / s within the range of 0.1–100% to determine the limits of the linear viscoelastic region. Frequency scans of the samples were then measured within the angular frequency range of 0.01–10 Hz to determine the storage modulus (G′) and loss modulus (G″). Within the linear viscoelastic range, a strain of 1% was applied, and the results are as follows: Figures 22-27 As shown. From Figure 22 and 23 It can be seen that although the apparent viscosity of all emulsions decreases with increasing shear rate, the shear stress increases proportionally with the shear rate. The shear stress decreases with increasing degree of sulfonation, which is due to the unfolding and increased flexibility of proteins during the reaction, as well as the introduction of sulfonate groups, which prevents protein aggregation. The apparent viscosity decreases with increasing sulfonation reaction time, because more proteins are adsorbed on the surface of the emulsion droplets, further increasing electrostatic and steric repulsion. This indicates that SCPHs-stabilized emulsions have better flowability.

[0094] from Figures 24-25 It can be seen that both the storage modulus and loss modulus increase steadily with increasing oscillation frequency, and the dependence is very small. Furthermore, the G' value is greater than the G” value, indicating that the emulsion exhibits a typical weak gel structure. Both G' and G” decrease with increasing sulfonation time, meaning that the emulsion exhibits liquid-like behavior consistent with its apparent viscosity. This is due to increased flexibility, decreased molecular weight, and reduced turbidity, resulting in less protein aggregation and enhanced protein adsorption at the oil droplet interface, leading to a smaller oil droplet size after homogenization. This makes SCPHs-stabilized emulsions suitable for industries requiring easy mixing and flow through pipelines and packaging. Figures 26-27 The gel structure of these emulsions changed during long-term storage. After 70 days of storage, the gel network was strengthened and became similar to that of freshly prepared SCPH0, which is due to the flocculation process that had begun in the emulsion system.

[0095] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing sulfonated emulsifiers based on cottonseed protein isolate, characterized in that, Includes the following steps: Cottonseed protein isolate was dissolved in chlorosulfonic acid and stirred thoroughly for 1-12 hours at 20-35°C under a nitrogen atmosphere. Then, an excess of ice-water mixture was slowly added while stirring, the reaction was stopped, and stirring was continued for 20-40 minutes. Residual acid and small molecule byproducts were removed by dialysis until the pH was constant. The pH was adjusted to 7 using sodium hydroxide solution, and further dialysis and freeze-drying were performed to obtain sulfonated cottonseed protein hydrolysate, i.e., a sulfonated emulsifier based on cottonseed protein isolate. The mass-to-volume ratio of cottonseed protein isolate to chlorosulfonic acid was 1 g: 20-30 mL.

2. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 1, characterized in that, The method for preparing cottonseed protein isolate is as follows: cottonseed meal is added to potassium hydroxide solution and stirred at 50-60℃ for 30-60 min. The resulting mixture is centrifuged at 3-5℃ and 2000-3000×g for 10-60 min. The supernatant is then adjusted to pH 5-5.5 with hydrogen chloride solution, centrifuged again, and the precipitate is washed three times with deionized water. The precipitate is then suspended in water, the pH is adjusted to 7, and then freeze-dried to obtain cottonseed protein isolate.

3. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 1, characterized in that, The dialysis time is 42-54 hours, with the water changed every 6 hours.

4. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 1, characterized in that, The concentration of the sodium hydroxide solution is 4~6 mol / L.

5. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 2, characterized in that, The concentration of the potassium hydroxide solution is 0.05~0.15 mol / L.

6. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 2, characterized in that, The mass-to-volume ratio of cottonseed meal to potassium hydroxide solution is 1g:10~20mL.

7. The method for preparing sulfonated emulsifier based on cottonseed protein isolate as described in claim 2, characterized in that, The concentration of the hydrogen chloride solution is 0.05~0.15 mol / L.

8. The use of an emulsifier prepared by the method for preparing a sulfonated emulsifier according to any one of claims 1-7 in the preparation of an emulsion, characterized in that, The emulsifier was prepared into an emulsifier solution, and the emulsifier solution and MCT oil were homogenized at 10,000~13,000 rpm for 1~3 min to obtain a crude emulsion. The crude emulsion was treated with an ultrasonic homogenizer and the temperature was maintained in a plate cooler to obtain an emulsion.

9. The application of an emulsifier prepared by the method for preparing a sulfonated emulsifier as described in claim 8 in the preparation of an emulsion, characterized in that, The concentration of the emulsifier solution is 5~15 mg / mL; the volume ratio of the emulsifier solution to MCT oil is 8~10:1; the conditions for treating the crude emulsion with an ultrasonic homogenizer are: emulsification treatment at 750W, 20kHz and 40% amplitude for 15~20 min, and intermittent ultrasound, i.e., ultrasound for 3 seconds and pause for 5 seconds.

Images