A bird's nest product based on bird's nest skeleton protein loaded with isomaltooligosaccharides, and a preparation method and application thereof

Abstract

This invention provides a bird's nest product based on isomaltooligosaccharide loaded with bird's nest skeletal proteins, its preparation method, and its application. The preparation method includes the following steps: cleaning and soaking the bird's nest raw materials to remove impurities; then, subjecting the soaked bird's nest to ultrasonic pretreatment; mixing it with an isomaltooligosaccharide solution and performing ultrasonic-assisted loading; and finally, stewing to obtain the bird's nest product. This invention improves the loading capacity and stability of isomaltooligosaccharide in bird's nest products through a three-step core process: "ultrasonic pretreatment protein structure modification – ultrasonic-assisted mass transfer and penetration – gentle stewing hydrogen bond stabilization." This constructs a sugar-protein interpenetrating network, significantly enhancing structural stability and resistance to water degradation under extreme environments. This allows ready-to-eat bird's nest to meet commercial aseptic production standards, combining the nourishing effects of bird's nest with the prebiotic function of isomaltooligosaccharide, while also possessing excellent antioxidant activity, thus expanding the application scenarios and market value of bird's nest products.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and in particular relates to a bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, its preparation method and application. Background Technology

[0002] Bird's nest is considered a top-grade tonic in traditional Chinese medicine. The *Compendium of Materia Medica Supplement* states: "Bird's nest is sweet, bland, and neutral in nature; it greatly nourishes lung yin, resolves phlegm, stops coughs, and is both nourishing and cleansing, making it a holy medicine for treating deficiency and fatigue." The protein in bird's nest mainly exists in the form of sialylated mucin (SiaMuc) glycoprotein, whose sugar chains are tightly bound to the protein, giving bird's nest its unique gelling properties and bioactivity.

[0003] Isomaltooligosaccharide (IMO) is a functional oligosaccharide produced from refined corn starch through enzymatic hydrolysis and transglycosylation. IMO possesses various physiological functions: as a prebiotic, it selectively promotes the proliferation of beneficial bacteria such as Bifidobacteria; it regulates the balance of the intestinal microecology; it promotes the absorption of minerals such as calcium and magnesium; it enhances immunity; it lowers serum cholesterol levels; it has low calorie properties (only 1 / 6 that of sucrose) and anti-caries effects.

[0004] Loading isomaltooligosaccharides into bird's nest can develop novel functional foods that combine the nourishing effects of bird's nest with the prebiotic functions of isomaltooligosaccharides. However, the existing technology has the following problems: (1) Ready-to-eat bird's nest is difficult to withstand standard commercial aseptic processing: In modern food industry, in order to achieve long shelf life and absolute microbial safety, a commercial aseptic sterilization process of 121°C is usually required. However, the original non-covalent bond network of bird's nest will undergo severe breakage and disintegration under the extreme thermal field of 121°C (i.e., "watering" phenomenon). In order to prevent watering, traditional processes are often forced to lower the sterilization temperature, which greatly limits the standardization and large-scale production of ready-to-eat bird's nest.

[0005] (2) Low loading efficiency of functional excipients: Conventional soaking or stewing methods are difficult to achieve efficient loading of functional excipients. The interaction mechanism between excipients and bird's nest skeleton proteins is unclear, and the loading process lacks scientific guidance. For example, Chinese invention patent with publication number CN110257459A discloses bird's nest small molecule peptides and their instant peptide powder, which add isomaltose as a sweetener to bird's nest products, but cannot achieve effective loading with bird's nest.

[0006] (3) Poor load selectivity: There are significant differences in the interaction between different functional excipients and bird's nest skeleton proteins. The lack of specific loading processes for different excipients leads to unstable loading effects.

[0007] (4) Insufficient optimization of process parameters: The influence of key parameters such as temperature and time on the load effect is unclear, making it difficult to achieve the best balance between load efficiency and nutrient retention.

[0008] Ultrasonic technology, as a non-thermal processing technique, has been widely used in the food processing industry. In recent years, research on the application of ultrasonic technology in bird's nest processing has gradually increased. Studies have shown that ultrasonic-assisted extraction can significantly improve the extraction rate of sialic acid in bird's nest, and ultrasonic-assisted enzymatic hydrolysis can improve the degree of hydrolysis and peptide yield of bird's nest protein. For example, Chinese invention patent CN115226886A discloses a method for preparing high-sialic acid bird's nest products by adding extract back. On the one hand, it uses the oscillation and microfluidics generated by ultrasound at the solid-liquid interface to change the pressure potential and osmotic potential of the bird's nest interface, increasing the penetration and diffusion of the external solution; on the other hand, it uses the cavitation effect formed inside the bird's nest by sound wave energy to promote the penetration of the external solution. However, research on applying ultrasonic technology to the loading of functional excipients in bird's nest has not been reported, and the mechanism of ultrasonic treatment on the interaction between bird's nest glycoprotein structure and isomaltooligosaccharide is still unclear.

[0009] Therefore, developing a method for preparing bird's nest products with high loading of isomaltooligosaccharides using ultrasonic processing technology is of great significance for improving the functional properties and nutritional value of bird's nest products. Summary of the Invention

[0010] In view of this, the present invention aims to propose a bird's nest product based on bird's nest skeleton protein loaded with isomaltooligosaccharide, its preparation method and application, and improve the penetration and adsorption efficiency of isomaltooligosaccharide by ultrasonic-assisted loading, and improve high temperature stability, so as to solve the problems of easy water dissolution during high temperature sterilization, low loading rate of functional excipients and poor retention of active ingredients in the existing process.

[0011] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a method for preparing a bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, the preparation method comprising the following steps: S1. After cleaning and removing impurities from the bird's nest raw materials, soak them in water. S2. Ultrasonic treatment of the soaked bird's nest; S3. The ultrasonically treated bird's nest is mixed with isomaltooligosaccharide solution and then ultrasonically treated to obtain a mixture. S4. Stew the mixture obtained in step S3 to obtain the bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide.

[0012] Further, in step S1, the temperature of the soaking treatment is 20~25℃, for example, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, and the soaking time is 4~6h, for example, 4h, 4.5h, 5h, 5.5h, 6h; Preferably, the mass ratio of bird's nest raw material to water used in the soaking process is 1:15~25, for example, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25.

[0013] Furthermore, the bird's nest raw materials comply with the GH / T 1092-2014 standard.

[0014] Further, in step S2, the ultrasonic processing power is 200~600W, for example, 200W, 250W, 300W, 350W, 400W, 450W, 500W, 550W, or 600W; the ultrasonic frequency is 20~40kHz, for example, 20kHz, 25kHz, 30kHz, 35kHz, or 40kHz; the processing temperature is 40~60℃, for example, 40℃, 45℃, 50℃, 55℃, or 60℃; and the processing time is 10~30min, for example, 10min, 15min, 16min, 17min, 18min, 19min, 20min, 25min, or 30min. Preferably, the ultrasonic processing power is 400W and the processing time is 15~20min.

[0015] Further, in step S3, the ultrasonic processing power is 200~400W, for example, 200W, 250W, 300W, 350W, or 400W; the ultrasonic frequency is 20~40kHz, for example, 20kHz, 25kHz, 30kHz, 35kHz, or 40kHz; the processing time is 15~20min, for example, 10min, 15min, or 20min; and the ultrasonic processing temperature is 25~40℃, for example, 25℃, 28℃, 30℃, 33℃, 35℃, 38℃, or 40℃. Preferably, the ultrasonic processing power is 300W and the processing time is 15min; Preferably, the mass concentration of the isomaltooligosaccharide solution is 3% to 10%, for example, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Preferably, the mass ratio of the isomaltooligosaccharide solution to the bird's nest raw material is 20-30:1, for example, it can be 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1. Preferably, the isomaltooligosaccharide is selected from one or more of isomaltose, isomalttriose, panose, isomaltetose, and isomaltpentose.

[0016] Furthermore, the isomaltooligosaccharide solution conforms to the GB / T 20881-2017 standard.

[0017] Further, in step S4, the stewing temperature is 85~100℃, for example, it can be 85℃, 90℃, 95℃, or 100℃, and the stewing time is 20~30min, for example, it can be 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, or 30min; Preferably, the stewing temperature is 95℃ and the stewing time is 25 minutes; Preferably, step S4 further includes the steps of cooling, filling, and sterilizing the stewed bird's nest.

[0018] In a second aspect, the present invention provides a bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, prepared according to the preparation method described in the first aspect.

[0019] Furthermore, the loading of isomaltooligosaccharide in the bird's nest product is ≥5%.

[0020] Furthermore, the water-holding weight ratio of the bird's nest product is ≥8, the water-holding expansion ratio is ≥6, and the solid content is ≥12%.

[0021] Furthermore, the bird's nest product has a sialic acid retention rate of ≥90% and a protein extraction rate of ≥85%.

[0022] Furthermore, after sterilization at 121°C for 20 minutes, the bird's nest product retains ≥80% of its drained solids.

[0023] Furthermore, after the bird's nest product is accelerated to age at 50℃ for 30 days, the retention rate of drained solids in the bird's nest product is ≥90%, and there is no obvious water separation or stratification phenomenon. Thirdly, the present invention provides the application of bird's nest products based on bird's nest skeletal protein loaded with isomaltooligosaccharide as described in the second aspect in the preparation of functional foods, health products or nutritional supplements.

[0024] Preferably, the functional food, health product, or nutritional supplement has the functions of regulating intestinal flora, promoting mineral absorption, enhancing immunity, or antioxidation.

[0025] Fourthly, the present invention provides the application of the preparation method as described in the first aspect in improving the quality of bird's nest products.

[0026] Furthermore, the application includes at least one of the following: (1) Application in increasing the loading of isomaltooligosaccharides in bird's nest products; (2) Application in improving the protein extraction rate of bird's nest products; (3) Application in improving the sialic acid retention rate of bird's nest products; (4) Application in improving the integrity of the bird's nest gel network in bird's nest products; (5) Application in improving the water-holding capacity of bird's nest products; (6) Application in improving the water-holding and swelling ratio of bird's nest products; (7) Application in enhancing the prebiotic function of bird's nest products; (8) Application in enhancing the antioxidant activity of bird's nest products; (9) Application in improving the water resistance of bird's nest products; (10) Application in improving the high-temperature stability of bird's nest products.

[0027] Preferably, the prebiotic function includes at least one of the following: increasing beneficial intestinal bacteria, inhibiting the growth of harmful bacteria, and increasing the production of short-chain fatty acids.

[0028] Compared with existing technologies, the bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, its preparation method, and its application described in this invention have the following advantages: (1) This invention improves the loading of isomaltooligosaccharide in bird's nest products through a three-step core process of "ultrasonic pretreatment protein structure modification - ultrasonic-assisted mass transfer and penetration - gentle stewing hydrogen bond stabilization", constructs a sugar-protein interpenetrating network, significantly improves the structural stability and resistance to water degradation under extreme conditions, and enables ready-to-eat bird's nest to meet commercial aseptic production standards.

[0029] (2) This invention achieves a balance between high loading of isomaltooligosaccharide and high retention of bird's nest nutrition, breaking the vicious cycle of existing technology. It adopts a gentle stewing process to replace the traditional high-temperature treatment, and uses ultrasonic pretreatment to gently modify the protein structure, so that the sialic acid retention rate in the product is ≥90% and the protein extraction rate is ≥85%, which is more than 10% higher than the traditional process. While achieving high loading, the core nutritional value of bird's nest is fully preserved.

[0030] (3) This invention significantly improves the functional characteristics and quality of bird's nest products. The prepared bird's nest products maintain the integrity of the bird's nest gel network, with a water holding weight ratio ≥8, a water holding expansion ratio ≥6, a uniform texture, and a smooth taste. At the same time, it has the nourishing effects of bird's nest and the prebiotic function of isomaltooligosaccharide. In vitro experiments have verified that it can significantly increase the growth of beneficial bacteria in the intestines, inhibit the growth of harmful bacteria, and increase the production of short-chain fatty acids. It also has excellent antioxidant activity, expanding the application scenarios and market value of bird's nest products.

[0031] (4) The preparation process of the present invention is green and safe and suitable for industrial production. It adopts food-grade green non-thermal ultrasonic processing technology, without the addition of chemical reagents. The process parameters are controllable and the operation is simple. It can be directly adapted to the existing industrial production line of bird's nest food without large-scale equipment modification and has extremely high industrial application value. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a process flow diagram for bird's nest products based on bird's nest skeletal proteins loaded with isomaltooligosaccharides. Detailed Implementation

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0034] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0035] The main experimental materials and instruments used in the following examples are as follows: (1) Experimental materials: The bird's nest raw material was imported white bird's nest from Indonesia, which met the first-class standard of GH / T 1092-2014 "Quality Grade of Bird's Nest", with a sialic acid content of 11.2% and a protein content of 58.6% on a dry basis; the isomaltooligosaccharide was food grade, which met the standard of GB / T 20881-2017, with a total functional oligosaccharide content of 90%, and was purchased from Shandong Baolingbao Biotechnology Co., Ltd.; all reagents used in the experiment were analytical grade, and the experimental water was grade III water or higher that met the requirements of GB / T 6682.

[0036] (2) Experimental instruments: SB-5200DTDN ultrasonic cleaner (power adjustable from 0-600W, frequency 20-40kHz, Ningbo Xinzhi Biotechnology Co., Ltd.); HH-S constant temperature water bath (Jintan Medical Instrument Factory); Agilent 1260 high performance liquid chromatograph (Agilent Technologies); KDN-102F Kjeldahl nitrogen analyzer (Shanghai Fiber Inspection Instrument Co., Ltd.); TA-XT2i texture analyzer (Stable Micro Systems, UK); UV-2600 ultraviolet-visible spectrophotometer (Shimadzu Instruments Co., Ltd.).

[0037] The evaluation index measurement methods are as follows in the following examples: (1) Sample pretreatment (solid extraction): The prepared bird's nest product was placed in a centrifuge tube and centrifuged at 4℃ and 8000r / min for 15min to separate the supernatant from the bottom precipitate. The bottom precipitate was collected and washed twice with cold water at 4℃ to remove surface free sugars and prevent the gel network from releasing oligosaccharide molecules wrapped in deep pores due to thermal expansion and contraction. Then, it was pre-frozen at -80℃ for 12h and freeze-dried in a vacuum freeze dryer until constant weight. The freeze-dried solid was ground and passed through a 60-mesh sieve, sealed and dried to obtain the freeze-dried bird's nest solid sample, which was used as the sample for subsequent index determination.

[0038] (2) Determination of isomaltooligosaccharide loading: Accurately weigh the above-mentioned freeze-dried bird's nest solid sample, and perform high performance liquid chromatography (HPLC) according to Appendix A of GB / T20881-2017. Use a Sugar-Pak I calcium-type sugar analytical column (6.5 mm × 300 mm), with ultrapure water as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 80 °C, and a differential refractive index detector. The loading is calculated on a dry basis, and the formula is as follows: Loading weight (%) = (mass of isomaltooligosaccharide in solid sample / dry weight of bird's nest) × 100%.

[0039] (3) Determination of sialic acid content and retention rate: Accurately weigh the above-mentioned freeze-dried bird's nest solid sample, and determine it by high performance liquid chromatography according to GB31614.1-2023 "National Food Safety Standard - Determination of Sialic Acid in Food"; the formula for calculating the sialic acid retention rate is as follows: Retention rate (%) = (Total mass of sialic acid in solid sample / Total mass of sialic acid in bird's nest raw material) × 100%.

[0040] (4) Protein content and extraction rate determination: Accurately weigh the above-mentioned freeze-dried bird's nest solid sample and determine the protein content using the Kjeldahl method, Method II, according to GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food"; the protein extraction rate is calculated using the following formula: Extraction rate (%) = (mass of soluble protein in solid sample / total mass of protein in bird's nest raw material) × 100%.

[0041] (5) Water-holding capacity test: Water-holding weight ratio: Accurately weigh a certain mass of freeze-dried solid sample as the initial dry weight (W1), completely immerse it in purified water, and let it stand at 25℃ for 30 minutes to fully absorb water and swell; take out the rehydrated gel, gently blot the surface free water with filter paper, and weigh the wet weight after water absorption (W2). The calculation formula is: Water-holding weight ratio = W2 / W1. Water-holding swelling ratio: Place the above freeze-dried solid sample in a graduated cylinder, gently tap and compact it, and record the initial dry volume (V1). Add an appropriate amount of purified water and let it stand at 25℃ for 30 minutes to fully rehydrate and swell. Record the volume of the swollen gel (V2). The calculation formula is: Water-holding swelling ratio = V2 / V1.

[0042] (6) Determination of soluble solids content: Refer to the determination method of soluble solids in GB / T 10786-2022 "Test Methods for Canned Foods" and use the refractive method for determination.

[0043] (7) Evaluation of prebiotic function: An in vitro anaerobic fermentation model using fecal microbiota from healthy adults was employed. A basic fermentation medium (containing tryptone, yeast extract, inorganic salts, etc.) was prepared and the pH was adjusted to 6.8. Freeze-dried bird's nest solids samples were added to the medium at a final concentration of 1.0% (w / v). Fresh feces were collected from three healthy volunteers and a 10% fecal suspension was prepared using 0.1 mol / L PBS buffer. This suspension was then inoculated into the aforementioned medium containing the samples at a 10% (v / v) inoculation rate. Fermentation was carried out at 37℃ under anaerobic conditions for 24 h. Changes in pH, short-chain fatty acid content, and gut microbiota structure were measured. Three replicates were set up for each group.

[0044] (8) Evaluation of antioxidant activity: The scavenging ability of the sample against DPPH and ABTS free radicals was determined. Different concentrations (1-10 mg / mL) of bird's nest solid extract were taken: (8-1) DPPH measurement method: 2 mL of sample solution was thoroughly mixed with 2 mL of 0.1 mmol / L anhydrous DPPH ethanol solution, reacted at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm; (8-2) ABTS measurement method: 0.5 mL of sample solution was mixed with 4 mL of prepared ABTS working solution, reacted at room temperature in the dark for 6 min, and the absorbance was measured at 734 nm. The half-maximum scavenging concentration (IC50) was calculated using a nonlinear regression curve. A lower IC50 value indicates stronger antioxidant activity. Example 1

[0045] like Figure 1As shown, the preparation method of bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide in this embodiment includes the following steps: (1) Pretreatment of bird's nest: Take 10g of bird's nest raw material (dry weight), clean it to remove impurities such as down feathers and impurities, add 200mL of purified water (20 times dry weight), soak it at room temperature of 25℃ for 5 hours, and drain it after soaking. (2) Ultrasonic pretreatment: Place the soaked bird's nest along with the soaking liquid in an ultrasonic cleaner, set the ultrasonic power to 400W, the ultrasonic frequency to 40kHz, the system temperature to 50℃, and ultrasonically treat for 15min. (3) Ultrasonic assisted loading: Prepare 300 mL of 5% isomaltooligosaccharide solution (30:1 mass ratio with dry weight of bird’s nest), add it to the bird’s nest system after ultrasonic pretreatment, set ultrasonic power to 300 W, ultrasonic frequency to 40 kHz, system temperature to 30 °C, and ultrasonic treatment for 15 min. (4) Gentle stewing: Transfer the mixture obtained in step (3) to a sealed stewing pot and stew in a 95°C constant temperature water bath for 25 minutes; (5) Post-processing: Cool the stewed product to room temperature under normal pressure to obtain bird's nest product based on bird's nest skeleton protein loaded with isomaltooligosaccharide.

[0046] The bird's nest product prepared in Example 1 was tested for the following indicators: isomaltooligosaccharide loading 5.0% (dry basis), water holding weight ratio 8.8, water swelling ratio 7.2, soluble solids content 14.2%, sialic acid retention rate 91.8%, and protein extraction rate 86.5%. Examples 2-4

[0047] The only difference between Examples 2-4 and Example 1 is that the mass concentration of the isomaltooligosaccharide solution in step (3) is 3%, 7%, and 10%, respectively; the other steps are the same as in Example 1. The bird's nest products obtained in Examples 2-4 were tested for their indicators, and the results are shown in Table 1.

[0048] Table 1. Results of index measurement in Examples 2-4 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 1, the loading capacity increases significantly with the increase of isomaltooligosaccharide solution concentration; when the concentration is ≥5%, the loading capacity can be ≥5%; however, when the concentration exceeds 10%, the sialic acid retention rate and protein extraction rate decrease significantly. Therefore, this invention limits the mass concentration of isomaltooligosaccharide solution to 3-10% to balance loading capacity and nutrient retention. Examples 5-7

[0049] The only difference between Examples 5-7 and Example 1 is that the ultrasonic pretreatment power in step (2) is 200W, 600W, and 500W respectively; the other steps are the same as in Example 1. The bird's nest products obtained in Examples 5-7 were tested for their indicators, and the results are shown in Table 2.

[0050] Table 2 Results of index measurement in Examples 5-7 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 2, when the ultrasonic pretreatment power is too low, the protein structure is not fully unfolded, the binding sites are not sufficiently exposed, and the loading efficiency is low. When the ultrasonic pretreatment power is too high, the protein undergoes excessive denaturation and aggregation, the number of binding sites decreases, the loading capacity decreases, and the retention rate of active ingredients also decreases. The loading capacity reaches its peak when the ultrasonic pretreatment power is 400W. Therefore, this invention limits the ultrasonic pretreatment power to 200-600W, and preferably to 400W. Examples 8-10

[0051] The only difference between Examples 8-10 and Example 1 is that the stewing temperatures in step (4) are 85℃, 100℃, and 115℃, respectively. The other steps are the same as in Example 1. The indicators of the bird's nest products obtained in Examples 8-10 were measured, and the results are shown in Table 3.

[0052] Table 3 Results of index measurement in Examples 8-10 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 3, when the stewing temperature is too low (<85℃), the bird's nest gel network is not fully constructed, resulting in poor load stability. When the stewing temperature exceeds 100℃, the non-covalent bonds of the bird's nest will break violently because the gel network is not yet stable, and the oligosaccharides have not yet completed multi-point anchoring, leading to irreversible "water-disintegration" of the system. When the stewing temperature is 95℃, the load reaches its peak, and the retention rate of active ingredients is at a relatively high level. Therefore, this invention limits the stewing temperature to 85-100℃, and preferably to 95℃. Only after the network reconstruction is completed under this mild condition can the product have the mechanical support to withstand the subsequent extreme commercial sterilization at 121℃. Examples 11-12

[0053] The only difference between Examples 11-12 and Example 1 is that the ultrasonic treatment power in step (3) is 200W and 400W respectively, while the other steps are the same as in Example 1. The bird's nest products obtained in Examples 11-12 were tested for their indicators, and the results are shown in Table 4.

[0054] Table 4. Results of index measurement in Examples 11-12 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 4, when the ultrasonic power is too high, the strong cavitation mechanical shear force causes the already formed hydrogen bonds to break, leading to the desorption of oligosaccharides and the destruction of some proteins. When the ultrasonic power is too low, the mass transfer and permeation effects decrease, resulting in a reduction in the loading of isomaltooligosaccharides. The mass transfer enhancement effect is optimal at a degradation power of 300W, with the highest collision and binding rate between polyhydroxyl groups and polar groups, and the loading reaches its peak. Therefore, this invention limits the ultrasonic power to 200-400W. Examples 13-14

[0055] The only difference between Examples 13-14 and Example 1 is that the ultrasonic treatment temperature in step (3) is 25℃ and 40℃ respectively, while the other steps are the same as in Example 1. The bird's nest products obtained in Examples 13-14 were tested for their indicators, and the results are shown in Table 5.

[0056] Table 5. Results of index measurement in Examples 13-14 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 5, excessively high ultrasonic temperatures lead to thermal breakage of the newly formed hydrogen bond network; excessively low ultrasonic temperatures reduce the kinetic energy of molecular thermal motion, resulting in decreased mass transfer efficiency; and the bird's nest product exhibits the best overall performance when the ultrasonic temperature is 30℃. Therefore, this invention limits the ultrasonic temperature to 25-40℃. Examples 15-16

[0057] The only difference between Examples 15-16 and Example 1 is that the ultrasonic treatment time in step (3) is 10 min and 20 min respectively, while the other steps are the same as in Example 1. The bird's nest products obtained in Examples 15-16 were tested for indicators, and the results are shown in Table 6.

[0058] Table 6 Results of index measurement in Examples 15-16 (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 6, when the ultrasonic treatment time is too long, it not only fails to significantly increase the loading of isomaltooligosaccharides, but also leads to a decrease in the extraction rate of sialic acid and protein due to the continuous thermal and mechanical effects. When the ultrasonic treatment time is too short, the mass transfer and permeation do not reach equilibrium, resulting in insufficient loading of isomaltooligosaccharides. When the ultrasonic treatment time is 15 min, the mass transfer and permeation reach dynamic equilibrium, and the resulting bird's nest product has the best overall performance. Therefore, this invention limits the ultrasonic treatment time to 15-20 min.

[0059] Comparative Example 1 This comparative example uses a conventional stewing process to prepare bird's nest products containing isomaltooligosaccharides. The preparation method includes the following steps: (1) Pretreatment of bird's nest: Take 10g of bird's nest raw material (dry weight), clean it to remove impurities such as down feathers and impurities, add 200mL of purified water (20 times dry weight), soak at room temperature of 25℃ for 5 hours, and drain it after soaking. (2) Soaking treatment: Prepare 300 mL of 5% isomaltooligosaccharide solution, add the soaked bird's nest, and soak at room temperature for 30 min; (3) Stewing treatment: Transfer the mixture obtained in step (2) to a sealed stewing pot and stew in a boiling water bath at 100°C for 30 minutes; (4) Post-processing: Cool the stewed product to room temperature under normal pressure, fill and sterilize to obtain bird's nest products containing isomaltooligosaccharides.

[0060] The bird's nest product prepared in Comparative Example 1 was tested for the following indicators: isomaltooligosaccharide loading 1.6% (dry basis), water holding weight ratio 7.5, water swelling ratio 5.8, soluble solids content 10.5%, sialic acid retention rate 82.5%, and protein extraction rate 75.2%.

[0061] Compared with Example 1, the isomaltooligosaccharide loading of Comparative Example 1 prepared by conventional stewing process was only 32% of that of the present invention. The sialic acid retention rate, protein extraction rate and water holding capacity were significantly lower than those of the present invention. This confirms that the process of the present invention, which adopts "ultrasonic pretreatment protein structure modification - ultrasonic-assisted mass transfer and penetration - gentle stewing hydrogen bond stabilization", can simultaneously achieve the core objectives of high isomaltooligosaccharide loading and high nutritional retention of bird's nest.

[0062] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the ultrasonic pretreatment in step (2) was not performed; the other steps are the same as in Example 1.

[0063] The bird's nest product prepared in Comparative Example 2 was tested for the following indicators: isomaltooligosaccharide loading 2.9% (dry basis), sialic acid retention rate 92.1%, protein extraction rate 85.8%, and water holding capacity 8.1. Compared with the product prepared in Example 1, the ultrasonic pretreatment used in this invention promotes the appropriate unfolding of the bird's nest glycoprotein structure through ultrasonic cavitation effect, exposing polar groups to provide hydrogen bonding sites, which can increase the loading by more than 72%.

[0064] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that ultrasound was not performed during the loading process in step (3): 300 mL of a 5% isomaltooligosaccharide solution (mass ratio of 30:1 to the dry weight of the bird's nest) was prepared and added to the bird's nest system after ultrasound pretreatment, and the mixture was left to soak at room temperature for 15 min. The remaining steps were the same as in Example 1.

[0065] The bird's nest product prepared in Comparative Example 3 was tested for the following indicators: isomaltooligosaccharide loading 3.2% (dry basis), sialic acid retention rate 91.5%, protein extraction rate 86.2%, and water holding capacity 8.3. Compared with the product prepared in Example 1, it can be seen that the ultrasonic-assisted loading method of this invention can significantly improve the penetration and binding efficiency of isomaltooligosaccharide through mass transfer enhancement, which is a key process for achieving high loading.

[0066] Performance Test Example 1: Evaluation of Prebiotic Function The products prepared in Example 1 and Comparative Example 1 were used to conduct in vitro intestinal flora fermentation experiments. Ordinary bird's nest products (without isomaltooligosaccharide, and the rest of the process is the same as in Example 1) were used as the control group to evaluate their prebiotic function. The results are shown in Table 7.

[0067] Table 7 Comparison of Prebiotic Functions (Note: Different lowercase letters after the data in the same column indicate significant differences, p<0.05) As shown in Table 7, the product prepared by this invention has a significant advantage over the product prepared in Comparative Example 1 and the product without isomaltooligosaccharide loading in terms of producing short-chain fatty acids, promoting the growth of beneficial bacteria such as Bifidobacteria, and inhibiting harmful bacteria (p<0.05). This strongly demonstrates that the "high loading amount (≥5%)" achieved by the ultrasound-assisted targeted loading technology of this invention can stimulate the prebiotic function of the product, breaking through the technical bottleneck of "functional ineffectiveness due to extremely low loading amount" in traditional processes.

[0068] Performance Test Example 2: Evaluation of Antioxidant Activity The antioxidant activity of the products prepared in Example 1 and Comparative Example 1 was determined, with ordinary bird's nest products (without isomaltooligosaccharide, and the rest of the process was the same as in Example 1) serving as the control group. The results are shown in Table 8.

[0069] Table 8 Comparison of antioxidant activities (Note: A smaller IC50 value indicates stronger antioxidant capacity. Different lowercase letters after the data from the same group indicate significant differences between the two groups, p<0.05) As shown in Table 8, the product prepared in Comparative Example 1, due to its low isomaltooligosaccharide loading, exhibited slightly better antioxidant capacity than ordinary bird's nest, but the improvement was limited. In contrast, the product prepared in this invention not only had a high concentration of isomaltooligosaccharide loading, but also, during the preparation process (mild ultrasonic modification and mild simmering at 95°C), the original antioxidant active proteins of the bird's nest were largely preserved. The synergistic effect of these two processes resulted in a breakthrough improvement in its ability to scavenge DPPH and ABTS free radicals.

[0070] Performance Test Example 3: Water Stability Test under Commercial Sterilization Conditions The products prepared in Example 1 and Comparative Example 1 were subjected to water-resistant stability tests. Pure water was used as the control group (the difference from Example 1 is that the isomaltooligosaccharide solution in step (3) was replaced with an equal volume of pure water, and the other steps were the same as in Example 1). The three groups of products were sealed and subjected to a destructive thermal stress test at 121°C for 20 min (commercial sterilization simulation). After cooling, the retention rate of drained solids was measured. The results are shown in Table 9.

[0071] Table 9 Comparison of water resistance stability under commercial sterilization conditions (Note: A smaller IC50 value indicates stronger antioxidant capacity. Different lowercase letters after the data from the same group indicate significant differences between the two groups, p<0.05) As shown in Table 9, the bird's nest without ultrasonic loading protection (pure water control group) underwent severe structural depolymerization under an extreme thermal field of 121℃, with the solid content retention rate dropping to 56.5%. In the traditional process group (Comparative Example 1), isomaltooligosaccharide (IMO) was only physically free in the broth, resulting in a low loading amount on the protein. This not only failed to protect the bird's nest skeleton, but its extremely strong hydration ability also triggered a "competitive solvation effect," accelerating the stretching and dissolution of loosened proteins at high temperatures, leading to a further drop in the solid content retention rate to 52.8%. In contrast, the bird's nest product prepared by this invention still maintained a solid content retention rate as high as 85.4% after sterilization at 121℃. This demonstrates that the present invention, through ultrasonic pretreatment and ultrasonic-assisted loading, forces IMO molecules into the deep pores of the protein, acting as high-density "molecular rivets." When the original non-covalent bonds break due to high temperature, the polyhydroxy IMO molecules can quickly form a dense "glycoprotein cogel" hydrogen bond network with free protein fragments, effectively resisting extreme thermal degradation and completely breaking through the industrial bottleneck that ready-to-eat bird's nest cannot withstand commercial sterilization at 121℃.

[0072] Performance Test Example 4: Accelerated Aging Stability Evaluation The products prepared in Example 1 and Comparative Example 1 were subjected to accelerated aging stability tests. Pure water was used as the control group (the difference from Example 1 is that the isomaltooligosaccharide solution in step (3) was replaced with an equal volume of pure water, and the other steps were the same as in Example 1). The products were placed in a 50°C constant temperature incubator for 30 days of accelerated aging test to simulate the structural degradation of ready-to-eat bird's nest in the whole life cycle. After cooling, the retention rate of drained solids was measured. The results are shown in Table 10.

[0073] Table 10 Comparison of water resistance stability under commercial sterilization conditions (Note: Different lowercase letters after the data in the same row indicate significant differences between the two groups, p<0.05) As shown in Table 10, in the accelerated aging test under continuous thermal stress at 50℃, the solids retention rates of the pure water control group and the traditional process group (Comparative Example 1) showed a significant decreasing trend over time, dropping to 68.5% and 71.4% respectively by day 30. Macroscopically, this exhibited obvious colloidal dehydration shrinkage, water separation, and marginal water phenomena. This indicates that free sugars in traditional physical mixing cannot inhibit the long-term thermodynamic structural degradation of bird's nest. In contrast, the high-load bird's nest product of this invention, with its deeply embedded IMO molecules assisted by ultrasound, constructs an extremely stable interpenetrating cross-linked network within the framework. This network effectively locks in the moisture within the system and significantly restricts the secondary aggregation and slow depolymerization of bird's nest protein chains from a kinetic perspective, maintaining an extremely high retention rate of 91.6% even at the end of the 30-day test. This demonstrates that the ultrasonic pretreatment and ultrasonic-assisted IMO loading process of this invention can endow the product with excellent physical stability throughout its entire shelf life (resistance to water separation / water degradation).

[0074] As can be seen from the above embodiments and comparative examples, the present invention achieves highly stable loading of isomaltooligosaccharides in bird's nest through an ultrasound-mediated targeted loading process, constructs a glycoprotein interpenetrating network, significantly improves structural stability and resistance to water degradation under extreme environments, enables ready-to-eat bird's nest to meet commercial aseptic production standards, solves the core defects of existing technologies, and at the same time retains the core active ingredients of bird's nest to the greatest extent. The product has excellent gel quality, prebiotic function and antioxidant activity, expands the application scenarios of bird's nest products, and has extremely high industrial application value.

[0075] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A method for preparing a bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, characterized in that, The preparation method includes the following steps: S1. After cleaning and removing impurities from the bird's nest raw materials, soak them in water. S2. Ultrasonic treatment of the soaked bird's nest; S3. The ultrasonically treated bird's nest is mixed with isomaltooligosaccharide solution and then ultrasonically treated to obtain a mixture. S4. Stew the mixture obtained in step S3 to obtain the bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide.

2. The preparation method according to claim 1, characterized in that, In step S1, the soaking temperature is 20~25℃ and the soaking time is 4~6h; Preferably, the mass ratio of bird's nest raw material to water used in the soaking process is 1:15~25.

3. The preparation method according to claim 1, characterized in that, In step S2, the ultrasonic processing power is 200~600W, the ultrasonic frequency is 20~40kHz, the processing temperature is 40~60℃, and the processing time is 10~30min. Preferably, the ultrasonic processing power is 400W and the processing time is 15~20min.

4. The preparation method according to claim 1, characterized in that, In step S3, the ultrasonic processing power is 200~400W, the ultrasonic frequency is 20~40kHz, the processing time is 15~20min, and the processing temperature is 25~40℃. Preferably, the ultrasonic processing power is 300W and the processing time is 15min; Preferably, the mass concentration of the isomaltooligosaccharide solution is 3% to 10%. Preferably, the mass ratio of the isomaltooligosaccharide solution to the bird's nest raw material is 20-30:1; Preferably, the isomaltooligosaccharide is selected from one or more of isomaltose, isomalttriose, panose, isomaltetose, and isomaltpentose.

5. The preparation method according to claim 1, characterized in that, In step S4, the stewing temperature is 85~100℃ and the stewing time is 20~30min; Preferably, the stewing time is 25 minutes.

6. The bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide, prepared by any one of the preparation methods according to claims 1-5.

7. The bird's nest product according to claim 6, characterized in that: The loading of isomaltooligosaccharide in the bird's nest product is ≥5%; And / or, the water-holding weight ratio of the bird's nest product is ≥8, the water-holding expansion ratio is ≥6, and the solid content is ≥12%; And / or, the sialic acid retention rate of the bird's nest product is ≥90%, and the protein extraction rate is ≥85%.

8. The application of the bird's nest product based on bird's nest skeletal protein loaded with isomaltooligosaccharide as described in claim 6 or 7 in the preparation of functional foods, health products or nutritional supplements.

9. The application of the preparation method as described in any one of claims 1-5 in improving the quality of bird's nest products.

10. The application according to claim 9, characterized in that, The application includes at least one of the following: (1) Application in increasing the loading of isomaltooligosaccharides in bird's nest products; (2) Application in improving the protein extraction rate of bird's nest products; (3) Application in improving the sialic acid retention rate of bird's nest products; (4) Application in improving the integrity of the bird's nest gel network in bird's nest products; (5) Application in improving the water-holding capacity of bird's nest products; (6) Application in improving the water-holding and swelling ratio of bird's nest products; (7) Application in enhancing the prebiotic function of bird's nest products; (8) Application in enhancing the antioxidant activity of bird's nest products; (9) Application in improving the water resistance of bird's nest products; (10) Application in improving the high-temperature stability of bird's nest products.

Images