Thermostable embedding method and application of alpha-amylase inhibitor

Abstract

The invention belongs to the technical field of food additives and new materials, and discloses a thermostable embedding method and application of an alpha-amylase inhibitor, and the method comprises the following steps: respectively adjusting the pH values of an alpha-AL stock solution and an iota-CG stock solution to 6, then mixing the stock solutions according to the mass ratio of alpha-AL to iota-CG of 2: 1, and carrying out heat preservation for 2-3 hours, so as to obtain the thermostable embedding method of the alpha-amylase inhibitor. According to the method disclosed by the invention, the stable ion compound is formed under the condition that the pH is equal to 6 by utilizing the electrostatic interaction and the hydrogen-bond interaction between the negatively charged sulfuric acid group in the iota-carrageenan molecule and the positively charged amino acid residue on the surface of the white kidney bean alpha-amylase inhibitor. According to the compound, stable combination among molecules is achieved by constructing a structured water molecular layer barrier, so that the tolerance of the white kidney bean alpha-amylase inhibitor to heat treatment is remarkably improved, and the defect that the heat stability of the white kidney bean alpha-amylase inhibitor is poor is overcome.

Description

Technical Field

[0001] This invention belongs to the field of food additives and new materials technology, and in particular to a heat-stable encapsulation method and application of an α-amylase inhibitor. Background Technology

[0002] The current technology for preparing α-amylase inhibitors has the following drawbacks:

[0003] 1. The peptides prepared by enzymatic method using white kidney beans are inactivated at 90℃ for 25 minutes, and the improvement in thermal stability is limited, still unable to meet the requirements of high-temperature processing such as baking.

[0004] 2. Using mannitol or maltitol to protect α-amylase inhibitors requires a high proportion of mannitol or maltitol (up to 1:3-8 w / w), which affects the flavor and texture of the product. In addition, the raw material cost is high in industrialization, making industrialization feasible.

[0005] 3. High-frequency, high-intensity ultrasonic processing technology is prone to damaging α-Al; excessively high ultrasonic intensity (6.5 W / cm²) can damage α-Al. 2 This leads to the destruction of glycoprotein structure, a sharp drop in inhibitory activity (only 97 U / g), severely impaired product function, difficulty in batch control, and poor repeatability.

[0006] 4. Microencapsulation requires strict temperature control, porous starch is prone to gelatinization, and the encapsulation structure is destroyed, posing operational risks in industrial applications.

[0007] The search revealed the following patent publications related to this invention's patent application:

[0008] 1. The "mannitol" technology used in the comparative document Xu Guomei, Huang Shouzheng, Xie Honglu. Protection of α-amylase inhibitor activity by mannitol [J]. Anhui Medical Journal, 2007, (07): 587-588. uses mannitol or maltitol to protect α-amylase inhibitors. The proportion of mannitol or maltitol used is relatively high (up to 1:3-8 w / w), which affects the flavor and texture of the product. Moreover, the raw material cost is high in industrialization, which poses a significant risk to the stability of the product.

[0009] 2. CN103159839B discloses a method for preparing a heat-stable α-amylase inhibitor and its application. This method involves mixing a mannitol or maltitol solution with dry α-amylase inhibitor powder to form a slurry, and then treating it with ultrasound at a specific temperature to protect its glycoprotein structure and improve its stability during heating. This technique utilizes a relatively high frequency (e.g., 2000 kHz) and a relatively high sound intensity (e.g., 6.5 W / cm²). 2 Under ultrasonic conditions, the inhibitory activity of amylase inhibitors decreased significantly, for example at 2000 kHz and 6.5 W / cm². 2The inhibitory activity after treatment was only 97 U / g. This technology is not ideal under some implementation conditions, and may even be counterproductive, leading to a decline in product quality.

[0010] 3. He Shaokai, Chi Yongnan, Zhong Yu, et al. Preparation and stability study of microencapsulated white kidney bean α-amylase inhibitor [J]. Food Science and Technology, 2022, 47(10):245-251. The technique of microencapsulation using porous starch as an adsorbent and resistant dextrin as a wall material is used. However, the optimal encapsulation temperature is 55℃. Excessively high temperatures (e.g., ≥65℃) can cause thermal denaturation of the active ingredient α-AI, significantly reducing its inhibitory activity. When the encapsulation temperature reaches 65-70℃, the porous starch may partially gelatinize, affecting its adsorption structure and leading to a decrease in encapsulation efficiency. In practical industrial applications, temperature control is a risk; improper control will directly result in a significant weakening of the product's functionality.

[0011] By comparison, the present invention patent application is fundamentally different from the aforementioned patent publications. Summary of the Invention

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide a heat-stable encapsulation method and application for α-amylase inhibitors.

[0013] The technical solution adopted by this invention to solve its technical problem is:

[0014] A heat-stable encapsulation method for an α-amylase inhibitor includes the following steps:

[0015] α-AL powder, an α-amylase inhibitor from white kidney beans, was dissolved in water and stirred at room temperature for 1.5-2.5 hours, followed by hydration overnight at 4°C to obtain a 2.5% (w / w) α-AL stock solution. ι-carrageenan (ι-CG) powder was dissolved in water preheated to 50-70°C and stirred in a water bath at 50-70°C for 1.5-2.5 hours, followed by stirring overnight at room temperature until fully hydrated to obtain a 1.25% (w / w) ι-CG stock solution.

[0016] The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6 respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 (α-AL:ι-CG) and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. The complex solution was stored at 4 °C.

[0017] Furthermore, the specific steps are as follows:

[0018] α-AL powder, an inhibitor of white kidney bean α-amylase, was dissolved in water and stirred at room temperature for 2 hours. Then, it was hydrated overnight at 4°C to obtain an α-AL stock solution with a mass concentration of 2.5%. ι-carrageenan, or ι-CG powder, was dissolved in water preheated to 60°C and stirred in a 60°C water bath for 2 hours. The solution was then stirred overnight at room temperature until completely hydrated to obtain an ι-CG stock solution with a mass concentration of 1.25%.

[0019] The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6, respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. A portion of the complex solution was stored at 4 °C. The remaining portion was frozen at -20 °C for at least 12 h and then freeze-dried for subsequent analysis.

[0020] Furthermore, the water is Milli-Q water.

[0021] Furthermore, the method also includes the following steps:

[0022] The complex solution was freeze-dried at -20°C for at least 12 hours.

[0023] The application of the heat-stable encapsulation method described above in the encapsulation of α-amylase inhibitors.

[0024] The advantages and positive effects of this invention are as follows:

[0025] 1. Enhances thermal stability, overcoming the bottleneck of heat inactivation during baking and processing:

[0026] This invention utilizes the negatively charged sulfate group (-OSO3) in the iota-carrageenan (ι-CG) molecule. - Electrostatic interactions between the α-AI and positively charged amino acid residues on the surface of the white kidney bean α-amylase inhibitor (α-AI) form an ionic complex at pH 6. This not only achieves stable intermolecular binding but also forms a structured water molecule layer barrier, significantly improving the heat tolerance of α-AI. Specific parameters are: α-AI concentration 1.25 (w / v), stirring speed 800 rpm. After treatment at 90℃ for 30 minutes, the inhibitory activity against α-amylase remained above 75%. This overcomes the limitations of traditional encapsulation methods that rely solely on physical barriers, achieving dual protection through an ionic layer and a hydration structure, exhibiting more stable thermal processing adaptability, and is particularly suitable for thermal processing scenarios such as baking, microwaving, and heat sterilization.

[0027] 2. Achieving high loading and high activity retention under mild encapsulation conditions:

[0028] This invention uses Na + Ion-mediated double-helical aggregation of iota-carrageenan constructs a three-dimensional network gel, achieving efficient encapsulation of α-AI under mild conditions. This strategy addresses the narrow temperature control window limitation of existing microencapsulation techniques, using the following parameters: α-AI concentration 1.25% (w / v), ι-CG concentration 0.5% (w / v), and pH 6. Gel formation can be completed at moderate temperatures without relying on a cooling gradient, enabling continuous industrial operation.

[0029] 3. Enhanced synergistic degradation, empowering the development of low-GI foods:

[0030] This invention utilizes the hydrogen bonding between iota-carrageenan and the starch backbone to inhibit amylase binding to the substrate, while α-AI simultaneously inhibits enzyme activity, achieving a synergistic degradation inhibition mechanism. Compared to traditional degradation strategies relying solely on enzyme inhibitors, this method provides a structure-function synergistic regulatory pathway, resulting in a more stable reduction in the starch digestibility index (GI).

[0031] 4. Highly controllable process, adaptable to various food processing environments:

[0032] The entire system of this invention exhibits high controllability under physical parameters (such as pH 6, stirring speed 800 rpm, gelation temperature 50-60℃), strong adaptability to water-soluble systems, and is suitable for formulation systems of different forms such as solid powders and liquid slurries.

[0033] 5. Excellent raw material economics and high feasibility for industrialization:

[0034] The iota-carrageenan used in this invention is widely available and inexpensive (approximately 20-25% of the cost of mannitol), and its excellent thermal stability, water solubility, and protein compatibility give it superior processing performance. Overall cost control is significantly better than comparative methods, improving the feasibility of implementing the industrial chain.

[0035] 6. The sulfate group (-OSO3) of iota-carrageenan in this invention - The iota-carrageenan forms ionic bonds with the positively charged amino acid residues on the surface of the white kidney bean α-amylase inhibitor at pH 6.0, and the hydroxyl groups (-OH) in the iota-carrageenan molecular chain form a structured water molecule layer with the sulfate groups, further isolating the external environment. With a white kidney bean α-amylase inhibitor concentration of 1–1.5 w / v%, an iota-carrageenan concentration of 0.5–0.75 w / v in the complex, a pH of 6 in the complex system, and a stirring speed of 800 rpm / min, this method overcomes the limitations of traditional encapsulation materials that rely solely on physical encapsulation.

[0036] 7. This invention employs ion-mediated gel embedding technology, using Na... +By shielding the electrostatic repulsion between the iota-carrageenan double helices, a high-density counterionic layer is formed in the interhelical gap, generating a net attractive force that induces the aggregation of double helices to construct a three-dimensional network, forming a physical barrier. The concentrations of white kidney bean α-amylase inhibitor (1–1.5 w / v%), iota-carrageenan concentration in the complex (0.5–0.75 w / v%), gelation temperature (50–60 °C), and pH of the complex system (6) solve the problem of achieving high loading, high efficiency, and stable encapsulation without destroying the activity of the substances, providing a foundation for subsequent applications (such as functional foods).

[0037] 8. In this invention, white kidney bean α-amylase inhibitor and iota-carrageenan synergistically reduce starch digestibility. Utilizing the linear molecular structure of iota-carrageenan, its main chain interacts with starch via hydrogen bonds, protecting the starch binding sites to amylase and synergistically inhibiting α-amylase activity to reduce starch hydrolysis. The concentration of white kidney bean α-amylase inhibitor in the complex is 1–1.5 w / v%, the concentration of iota-carrageenan in the complex is 0.5–0.75 w / v%, and the pH of the complex system is 6. This solves the problem of limited or unstable effects of single starch degradation digestion strategies, achieving a more significant and stable reduction in the rate and extent of starch hydrolysis, and providing a new approach for developing more effective low-GI foods.

[0038] 9. The method of this invention utilizes the electrostatic and hydrogen bonding interactions between the negatively charged sulfate groups in the ι-carrageenan molecule and the positively charged amino acid residues on the surface of the white kidney bean α-amylase inhibitor to form a stable ionic complex at pH=6. This complex achieves stable intermolecular binding by constructing a structured water molecule layer barrier, thereby significantly improving the heat treatment tolerance of the white kidney bean α-amylase inhibitor and overcoming its poor thermal stability. Attached Figure Description

[0039] Figure 1 This is a graph showing the effect of mass ratio and pH on the amino acid concentration and grafting degree of the α-AL / ι-CG complex in this invention;

[0040] Figure 2 This is a graph showing the effect of mass ratio and pH on the inhibitory activity of the α-AL / ι-CG complex on α-amylase in this invention;

[0041] Figure 3 This is a scanning electron microscope image of the α-AL / ι-CG complex in this invention;

[0042] Figure 4 The fluorescence spectra of the α-AL / ι-CG complex before and after heat treatment in this invention are shown.

[0043] Figure 5 The Fourier transform infrared and Raman spectra of the α-AL / ι-CG complex in this invention are shown. Detailed Implementation

[0044] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0045] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0046] A heat-stable encapsulation method for an α-amylase inhibitor includes the following steps:

[0047] α-AL powder, an α-amylase inhibitor from white kidney beans, was dissolved in water and stirred at room temperature for 1.5-2.5 hours, followed by hydration overnight at 4°C to obtain a 2.5% (w / w) α-AL stock solution. ι-carrageenan (ι-CG) powder was dissolved in water preheated to 50-70°C and stirred in a water bath at 50-70°C for 1.5-2.5 hours, followed by stirring overnight at room temperature until fully hydrated to obtain a 1.25% (w / w) ι-CG stock solution.

[0048] The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6 respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 (α-AL:ι-CG) and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. The complex solution was stored at 4 °C.

[0049] Preferably, the specific steps are as follows:

[0050] α-AL powder, an inhibitor of white kidney bean α-amylase, was dissolved in water and stirred at room temperature for 2 hours. Then, it was hydrated overnight at 4°C to obtain an α-AL stock solution with a mass concentration of 2.5%. ι-carrageenan, or ι-CG powder, was dissolved in water preheated to 60°C and stirred in a 60°C water bath for 2 hours. The solution was then stirred overnight at room temperature until completely hydrated to obtain an ι-CG stock solution with a mass concentration of 1.25%.

[0051] The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6, respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. A portion of the complex solution was stored at 4 °C. The remaining portion was frozen at -20 °C for at least 12 h and then freeze-dried for subsequent analysis.

[0052] Preferably, the water is Milli-Q water.

[0053] Preferably, the method further includes the following steps:

[0054] The complex solution was freeze-dried at -20°C for at least 12 hours.

[0055] The application of the heat-stable encapsulation method described above in the encapsulation of α-amylase inhibitors.

[0056] Specifically, the relevant preparation and testing methods are as follows:

[0057] Example 1

[0058] A heat-stable encapsulation method for an α-amylase inhibitor includes the following steps:

[0059] α-AL powder, an α-amylase inhibitor from white kidney beans, was dissolved in Milli-Q water and stirred at room temperature for 2 hours, followed by hydration overnight at 4°C to prepare an α-AL stock solution (2.5% w / v, mass concentration). ι-carrageenan powder, ι-CG powder, was dissolved in Milli-Q water preheated to 60°C and stirred in a 60°C water bath for 2 hours. The solution was then stirred overnight at room temperature until completely hydrated to prepare an ι-CG stock solution (1.25% w / v, mass concentration).

[0060] Adjust the pH values ​​of the α-AL stock solution and the ι-CG stock solution to 3, 4, 5, 6, 7, 8 and 9 respectively, and then mix the α-AL stock solution and the ι-CG stock solution at a mass ratio of 2:1 of α-AL:ι-CG; or, adjust the pH values ​​of the α-AL and ι-CG stock solutions to 6 respectively, and then mix the α-AL stock solution and the ι-CG stock solution at a mass ratio of 1:1, 2:1, 3:1, 4:1 and 5:1 of α-AL:ι-CG.

[0061] The mixture was stirred at room temperature for 1 hour to form the α-AL / ι-CG complex. The final concentration of α-AL in all complex solutions was 12.5 mg / mL. A portion of the complex solution was stored at 4 °C; the remaining portion was frozen at -20 °C for at least 12 hours and then freeze-dried for subsequent analysis.

[0062] The relevant tests are as follows:

[0063] 1. Free amino content

[0064] A heat-stable embedding method for an α-amylase inhibitor was used, identical to that in Example 1. The free amino content of the α-AL / ι-CG complex at different mass ratios or pH values ​​was determined using the OPA method. Sodium tetraborate decahydrate (7.620 g) and SDS (200 mg) were dissolved in 150 mL of Milli-Q water, and OPA (160 mg) was dissolved in 4 mL of anhydrous ethanol. After complete dissolution, the two solutions were mixed, and DTT (176 mg) was added. Finally, the volume was adjusted to 200 mL with Milli-Q water, mixed thoroughly, and stored in a brown bottle. 600 μL of the test sample was mixed with 4.5 mL of OPA reagent in a dark tube. After a precise reaction time of 2 min, the absorbance was immediately measured at 340 nm using a UV-Vis spectrophotometer. A standard curve was constructed using serine to calculate the free amino content in the sample. The grafting degree can be calculated based on the change in the free amino content on the protein surface in the α-AL / ι-CG complex. The calculation formula is as follows (where Aα-AL, Aα-AL / ι-CG, and AOPA represent the absorbance values ​​at 340 nm of the α-AL and OPA reagent mixture, the α-AL / ι-CG and OPA reagent mixture, and the OPA reagent, respectively):

[0065]

[0066] The degree of complexation between white kidney bean α-amylase inhibitor and ι-carrageenan was assessed by measuring the content of residual free amino groups on α-AL after binding interaction. Results are as follows: Figure 1 The results showed that as the ι-CG mass ratio increased, the free amino concentration decreased, while the grafting degree increased accordingly. The free amino concentration increased from 0.89 mmol / L. -1 Decreased to 0.53 mmol / L -1The grafting degree increased from 33.71% to 60.99%. This is mainly attributed to the increased number of effective binding sites for negatively charged sulfate groups in the system, which promoted the interaction with the α-AL surface. Furthermore, the highest grafting degree between α-AL and ι-CG was found at pH 6.0, reaching 57.88 ± 0.36%. This phenomenon can be attributed to the fact that extremely acidic environments disrupt hydrophobic interactions, while extremely alkaline environments disrupt hydrogen bonds and electrostatic interactions. Moreover, this pH value is close to the isoelectric point of α-AL, the net charge of α-AL approaches zero, the molecular conformation expands, exposing more hydrophobic regions. Simultaneously, the semi-rigid helical structure of ι-CG significantly enhances the exposure of hydrophobic regions, binding with the hydrophobic domains of α-AL. The synergistic effect of electrostatic attraction, hydrophobic interaction, and non-covalent interactions such as hydrogen bonding between the two reaches its optimum at pH 6.0 and an α-AL:ι-CG ratio of 2:1. It can also be seen that the two conditions of pH 6.0 and α-AL:ι-CG mass ratio of 2:1 in the method of the present invention have a synergistic effect, which can synergistically improve the relevant properties of the prepared product.

[0067] 2. α-Amylase inhibitory activity

[0068] A heat-stable embedding method for an α-amylase inhibitor was used, identical to that in Example 1. The inhibitory activity of α-AL and α-AL / ι-CG complexes at different mass ratios or pH levels before and after heat treatment was determined using the DNS method. 0.25 mL of 0.05 mg / mL α-amylase solution and 0.25 mL of the test sample were added to 0.5 mL of phosphate-buffered saline (PBS, 0.1 M) and incubated at 37°C for 10 min. Then, maintaining the temperature in a 37°C water bath, 0.5 mL of 1% soluble starch solution was added, and the reaction was allowed to proceed precisely for 5 min. Finally, 1 mL of DNS reagent was added to terminate the reaction. The reaction solution was heated in a boiling water bath for 10 min, then rapidly cooled to room temperature in an ice bath. 5 mL of Lmilli-Q water was added, and after thorough mixing, the absorbance was measured at 540 nm. A standard curve was established using maltose standards to calculate the concentration of maltose produced in the reaction. Four groups were set up in the experiment: blank tubes (containing α-amylase solution and starch solution but no sample), blank control tubes (containing starch solution but no α-amylase solution or sample), inhibition tubes (containing sample, α-amylase solution and starch solution), and inhibition control tubes (containing starch solution and sample but no α-amylase solution). All unadded components were replaced with an equal volume of PBS. The formula for calculating the α-amylase activity φ in the blank group is:

[0069] A1 and A2 represent the absorbance values ​​of the blank tube and blank control tube at the corresponding maltose concentrations, respectively; f(A) represents the maltose concentration, mg / mL. The formula for calculating the α-amylase inhibitory activity θ in the sample is as follows (where V and m represent the volume and mass of the sample to be tested, respectively; φ represents the α-amylase activity added to the reaction system; A1, A2, A3, and A4 represent the absorbance values ​​of the blank tube, blank control tube, inhibition tube, and inhibition control tube at the corresponding maltose concentrations, respectively; f(A) represents the maltose concentration, mg / mL):

[0070]

[0071] The inhibitory activity of α-amylase was investigated by measuring the α-AL / ι-CG complex formed before and after heat treatment, as well as under different mass ratios and pH conditions. The results are as follows: Figure 2 As shown, the inhibitory activity of free α-AL significantly decreased after heat treatment. This is because high temperature induces conformational changes in the protein, leading to the destruction of the tertiary structure of the active site, thereby weakening the specific binding ability of α-AL to α-amylase. Compared with free α-AL, the inhibitory activity of the α-AL / ι-CG complex against α-amylase increased. This phenomenon is attributed to the synergistic effect of ι-CG, whose aggregated network can physically block the contact between starch and α-amylase through steric hindrance.

[0072] However, at a mass ratio of 1:1, the excess ι-CG leads to the shielding of key active sites on the α-AL molecule surface involved in α-amylase inhibition, thereby weakening its inhibitory function. Under acidic conditions (pH 3-4), the inhibitory activity of the α-AL / CG complex significantly decreases after heat treatment. This is due to the decreased solubility of ι-CG and protonation of the sulfate ester groups in the acidic environment (pH 3-4), reducing the accessibility of binding sites. When the pH is greater than 4, the loss rate of α-amylase inhibitory activity of the α-AL / CG complex after heat treatment is significantly lower. Therefore, the complex with a mass ratio of 2:1 and a pH of 6 exhibits the highest retention rate and greatest inhibitory activity against α-amylase after heat treatment, decreasing only from 7864.19±239.90U to 6094.88±88.27U. It can also be seen that the pH 6.0 and the α-AL:ι-CG mass ratio of 2:1 in the method of this invention have a synergistic effect, which can synergistically improve the relevant properties of the prepared product.

[0073] 3. Scanning electron microscope (SEM)

[0074] The microstructure of the single components (α-AL, ι-CG) and their α-AL / ι-CG complex (prepared under the conditions of pH 6.0 and a mass ratio of α-AL:ι-CG of 2:1 in Example 1) was characterized by scanning electron microscopy. α-AL exhibited a lamellar or fibrous network structure with globular protein aggregates distributed on its surface. ι-CG, due to the strong hydrophilicity of its polysaccharide chains, aggregated into a continuous large lamellar structure. When the two were combined, scanning electron microscopy analysis showed that the α-AL / ι-CG complex retained the lamellar framework structure of ι-CG, but irregular filamentous and fragmented protrusions appeared on its surface, indicating cross-linking between α-AL and ι-CG molecules. Figure 3 As shown.

[0075] 4. Fluorescence spectrum

[0076] A heat-stable encapsulation method for an α-amylase inhibitor was prepared under the following conditions: α-AL:ι-CG ratio of 2:1, pH 6, and the remaining preparation steps were the same as in Example 1. The intrinsic fluorescence and surface hydrophobicity changes of α-AL (6.25 mg / mL, pH 6) and α-AL / ι-CG (mass ratio 2:1, α-AL concentration 6.25 mg / mL, pH 6) before and after heat treatment were measured using a fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at 25°C. The excitation wavelength was 280 nm, the slit width was 5 nm, and the scan rate was 240 nm / min, scanning the emission spectrum in the range of 300-420 nm.

[0077] In addition, changes in the surface hydrophobicity of the samples were determined using an ANS fluorescent probe. The sample was vortexed with an ANS-PBS solution (8 mM) at a volume ratio of 50:1 and incubated at room temperature in the dark for 30 min. Fluorescence intensity was measured at an excitation wavelength of 380 nm, and emission spectra were collected in the 400-600 nm range.

[0078] After heat treatment, the maximum fluorescence emission wavelength of free α-AL redshifted from 344 nm to 347 nm, and its intrinsic fluorescence intensity significantly increased from 2006 a.u. to 2552 a.u., indicating that the unfolding of the internal structure of α-AL led to an increase in the polarity of the Trp residue microenvironment. In contrast, the α-AL / ι-CG complex showed only a slight redshift after heat treatment, and the fluorescence intensity did not change significantly, only from 2257 a.u. to 2235 a.u., indicating that the complexation of -AL and -CG reduced the intramolecular quenching of Trp residues and preserved more of the native secondary structure.

[0079] Compared to free α-AL, the addition of ι-CG significantly reduced the peak intensity of α-AL, indicating that the complexation masked the hydrophobic residues on the α-AL surface. The fluorescence intensity of free α-AL increased significantly after heat treatment, from 126 a.u. to 641 a.u., while the fluorescence intensity of the α-AL / ι-CG complex remained essentially unchanged before and after heat treatment. This suggests that the formation of the α-AL / ι-CG complex effectively reduced the exposure of hydrophobic groups in α-AL, thereby reducing the aggregation of α-AL during heat treatment. Figure 4 As shown.

[0080] 5. Fourier transform infrared and Raman spectra

[0081] A thermostable encapsulation method for an α-amylase inhibitor was prepared under the following conditions: α-AL:ι-CG ratio of 2:1, pH 6, and the remaining preparation steps were the same as in Example 1. Fourier transform infrared spectroscopy (Thermo Fisher Scientific, Waltham, MA, USA) and Raman spectroscopy (HORIBA Scientific, Palaiseau, France) were used to obtain information on the functional groups and interactions in α-AL, ι-CG, and the α-AL / ι-CG complex. For FTIR, the sample was lyophilized for 48 h, then mixed with KBr powder at a ratio of 1:100 (w / w, mass ratio), ground, and pressed into tablets. The tablets were then heated at 4000-400 cm⁻¹. -1 Within the wavenumber range, at 4cm -1 The spectrum was acquired through 64 high-resolution scans.

[0082] For Ram, the lyophilized sample was ground into a fine powder and filled into a quartz sample cell. An excitation wavelength of 785 nm was used, and the sample was analyzed at 400-2000 cm⁻¹. -1 Within 1cm -1 Spectra were acquired at a resolution of 50 μm, a slit width of 5 s, and an integration time of 5 s. The obtained spectra were baseline corrected using OMNIC software, and characteristic functional group peaks were identified.

[0083] Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy provided crucial evidence for elucidating the functional groups and molecular interactions involved in the binding of α-AL to ι-CG. The results are as follows: Figure 5 As shown, for the natural α-AL sample, amide A and amide B are located at 3203 cm⁻¹. -1 and 2932cm -1 The amide II and III regions are located at 1585 cm⁻¹, respectively. -1 and 1393cm -1 At 1043cm -1Characteristic absorptions similar to those observed in ι-CG were observed, which are related to the stretching vibrations of the OH / CC glycosyl groups in the protein. After the formation of the α-AL / ι-CG complex (i.e., the one prepared in Example 1), the peak intensity of the α-AL amide region decreased and shifted to varying degrees, indicating that the anionic groups of ι-CG (-COO- / -OSO3)... - ) and the protonated amino group (-NH3) of α-AL + The complex is bound by electrostatic interactions, and the broadening of its absorption peak at 3400 cm⁻¹, along with a slight shift in the COH vibration in ι-CG, indicates a hydrogen bond between α-AL and ι-CG. Similarly, Ram's results show that α-AL at 1315 cm⁻¹... -1 The spectral band is the protein amide III band (CN / -NH coupling vibration). Compared to the single component, the addition of ι-CG significantly weakens the characteristic Raman signal intensity of the α-AL amide band, and the 856 cm⁻¹ band in ι-CG is also significantly weakened. -1 COS vibration of sulfated galactose, 1074 cm⁻¹ -1 CO / CC stretching vibration and 1260cm -1 Significant changes in the antisymmetric stretching vibrations of the sulfate ester group are consistent with the electrostatic interaction and hydrogen bond-dominated binding mechanism.

[0084] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A heat-stable encapsulation method for an α-amylase inhibitor, characterized in that: Includes the following steps: α-AL powder, an inhibitor of white kidney bean α-amylase, was dissolved in water and stirred at room temperature for 1.5-2.5 h, followed by hydration at 4 °C overnight to obtain an α-AL stock solution with a mass concentration of 2.5%. ι-carrageenan, or ι-CG powder, was dissolved in water preheated to 50 °C-70 °C and stirred in a water bath at 50 °C-70 °C for 1.5-2.5 h. The solution was then stirred at room temperature overnight until completely hydrated to obtain an ι-CG stock solution with a mass concentration of 1.25%. The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6 respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 (α-AL:ι-CG) and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. The complex solution was stored at 4 °C.

2. The thermally stable embedding method according to claim 1, characterized in that: The specific steps are as follows: α-AL powder, an inhibitor of white kidney bean α-amylase, was dissolved in water and stirred at room temperature for 2 hours. Then, it was hydrated overnight at 4°C to obtain an α-AL stock solution with a mass concentration of 2.5%. ι-carrageenan, or ι-CG powder, was dissolved in water preheated to 60°C and stirred in a 60°C water bath for 2 hours. The solution was then stirred overnight at room temperature until completely hydrated to obtain an ι-CG stock solution with a mass concentration of 1.25%. The pH values ​​of the α-AL and ι-CG stock solutions were adjusted to 6, respectively. Then, the α-AL and ι-CG stock solutions were mixed at a mass ratio of 2:1 and stirred at room temperature for 1 h to form an α-AL / ι-CG complex. The final concentration of α-AL in the complex solution was 12.5 mg / mL. A portion of the complex solution was stored at 4 °C. The remaining portion was frozen at -20 °C for at least 12 h and then freeze-dried for subsequent analysis.

3. The thermally stable embedding method according to claim 1 or 2, characterized in that: The water is Milli-Q water.

4. The thermally stable embedding method according to claim 1, characterized in that: The method further includes the following steps: The complex solution was freeze-dried at -20°C for at least 12 hours.

5. The application of the heat-stable encapsulation method as described in any one of claims 1 to 4 in the encapsulation of α-amylase inhibitors.

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