Preparation method of functional plant beverage containing glycosides

Abstract

The application relates to the technical field of functional beverages and bioactive ingredient conversion utilization, and discloses a functional plant beverage containing glycosides and a preparation method thereof. The beverage comprises a plant-derived glycoside extract, a cyclodextrin glycosidase complex, a prebiotic, a stabilizer, an acidity regulator and water; the cyclodextrin glycosidase complex is a complex enzyme active unit formed by stabilizing cyclodextrin and glycosidase through a cross-linked immobilization network, and continuously promotes the conversion of glycosides into aglycones during storage. The preparation method comprises the steps of enzymatic conversion pretreatment, blending and homogenization, and ultrahigh pressure cold sterilization treatment. The application effectively improves the retention effect of the enzyme component in the beverage system, improves the suspended and dispersed state, and promotes the continuous conversion of glycosides during storage by cyclodextrin inclusion, glycosidase immobilization and the use of a composite stabilizer, so that the obtained functional plant beverage has good storage stability and active ingredient conversion effect.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and is further refined to the field of non-alcoholic beverage preparation, specifically a method for preparing a functional plant beverage containing glycosides. Background Technology

[0002] With the increasing application of plant-derived functional ingredients in the food and beverage industry, plant extracts containing glycosides are gradually being used to develop beverage products with nutritional supplementation and bioactivity regulation effects. Ginsenosides, steviol glycosides, geniposide, baicalin, and other plant-derived components are widely found in medicinal and edible raw materials or natural plant extracts, and their application prospects in beverage systems are quite broad. These components usually exist in glycoside form. In some cases, their corresponding aglycone form is more likely to exhibit the desired effects in terms of absorption, utilization, and efficacy. Therefore, how to achieve the rational conversion of glycoside components in beverage products has gradually become a focus of attention in related product development.

[0003] Chinese patent CN107410785B discloses a ginsenoside beverage that can quickly replenish energy. It adds a specific combination of ginsenoside monomers to the formula of conventional functional beverages, and determines a formulation with Rg2, Rh, Rb3 and Ro as the core, combined with some other monomer saponins, in order to improve the energy replenishment effect.

[0004] The published method discloses a method for preparing anthocyanin lemon slices, anthocyanin lemon vinegar, and their beverages. The method involves homogenizing steamed purple sweet potatoes with white vinegar and sweeteners, then soaking them with seeded lemon slices and white vinegar. This process allows the anthocyanins in the purple sweet potatoes to be fully dissolved and relatively stably retained in the beverage system. This technology utilizes steaming, acidic extraction, low-temperature soaking, and dehydrated sucrose coloring to improve the color, taste, and anthocyanin stability of the purple sweet potato and lemon compound beverage, thereby obtaining a lemon vinegar beverage that combines nutritional components with a long shelf life.

[0005] Therefore, existing technologies primarily focus on adding, retaining, or stabilizing existing active ingredients in beverages, without considering the introduction of a sustainable catalytic complex enzyme system into the finished beverage. They also fail to address the problems of enzyme components being easily inactivated, unstablely dispersed, and difficult to balance continuous conversion with storage stability in liquid beverage systems. Furthermore, they lack the technical means described in this application, which uses a cross-linked immobilization network and a compound stabilizing system to maintain the dispersion state of active enzyme components and the stability of the conversion process. Therefore, it remains necessary to provide a functional plant-based beverage and its preparation method that can maintain the stability of the beverage system while simultaneously preserving enzyme components and ensuring continuous glycoside conversion. Summary of the Invention

[0006] To achieve the above objectives, the present invention provides the following technical solution: a functional plant-based beverage containing glycosides, the beverage comprising the following components: The contents include 0.5–5 wt% plant-derived glycoside extract, 0.1–2 wt% cyclodextrin-glycosidase complex, 1–3 wt% prebiotics, 0.3–1 wt% stabilizer, 0.05–0.3 wt% acidity regulator, and the balance being deionized water. The cyclodextrin-glycosidase complex is composed of cyclodextrin and glycosidase forming an inclusion enzyme active unit, which is stabilized by a cross-linked immobilization network and used to continuously convert glycosides into aglycone forms during storage.

[0007] Furthermore, the plant-derived glycoside extract is selected from one or more of ginsenosides, steviol glycosides, geniposide, and baicalin.

[0008] Furthermore, the glycosidase in the cyclodextrin-glycosidase complex is selected from one or more of β-glucosidase, α-L-rhamnosidase, β-galactosidase, cellobiase, and amygdalinase. Furthermore, the cyclodextrin-glycosidase complex is a multi-component complex formed by cross-linking and immobilizing transglutaminase. The raw materials for preparing the cyclodextrin-glycosidase complex, by weight, include: 10-20 parts of hydroxypropyl-β-cyclodextrin, 5-18 parts of glycosidase, 12-30 parts of whey protein isolate, 8-20 parts of sodium caseinate, 3-12 parts of pectin, 2-10 parts of chitosan oligosaccharide, 2-8 parts of sodium alginate, 1-6 parts of trehalose, 0.5-4 parts of transglutaminase, 0.2-2 parts of calcium lactate, and 40-120 parts of buffer solution. The glycosidase is β-glucosidase, or a complex enzyme consisting of β-glucosidase and one or more of α-L-rhamnosidase and β-galactosidase. The transglutaminase is used to catalyze the cross-linking reaction between glutamine residues and lysine residues in the whey protein isolate and / or sodium caseinate, forming a protein cross-linking network. This allows the cyclodextrin inclusion, enzyme molecule immobilization, and protein-polysaccharide microgel embedding to synergistically constitute an immobilized enzyme complex structure. The amount of cyclodextrin binding to the glycosidase is 0.5–2.0 μmol / mg, based on the mass of the glycosidase.

[0009] Furthermore, the preparation method of the cyclodextrin-glycosidase complex includes the following steps: S11: Dissolve hydroxypropyl-β-cyclodextrin in phosphate buffer solution with pH 5.5 to 7.0, add the glycosidase, and stir at 4℃ to 15℃ for 0.5h to 3h to obtain cyclodextrin-glycosidase pre-encapsulation solution; S12: Prepare a protein dispersion by adding whey protein isolate and sodium caseinate to another buffer solution, adjust the pH to 6.0-7.5, and sonicate under ice bath conditions for 2-15 minutes. The sonication power is 150W-400W to induce the protein molecules to expand appropriately and expose reaction sites that can be cross-linked. S13: Pectin, chitosan oligosaccharide, sodium alginate and trehalose are added sequentially to the protein dispersion, and after stirring and mixing, the pre-encapsulation solution is added to it to obtain the composite precursor solution; S14: Add transglutaminase to the composite precursor solution and react at 10℃~35℃ for 1h~8h to form a protein cross-linking network of protein components and promote the embedding of glycosidase into the protein-polysaccharide network. S15: Add calcium lactate to the cross-linked system and continue the reaction for 10 min to 60 min to enhance the sodium alginate-related ion network structure and obtain a dual-network immobilization system. S16: After removing unimmobilized small molecules by centrifugation, ultrafiltration, membrane separation or dialysis, the obtained system is freeze-dried or spray-dried at low temperature to obtain cyclodextrin-glycosidase complex.

[0010] Furthermore, the prebiotic is selected from one or more of fructooligosaccharides, galactooligosaccharides, and inulin.

[0011] Furthermore, the stabilizer is a multi-component compound stabilizing system, comprising, by weight of the functional plant beverage, 0.05wt%–0.25wt% gellan gum, 0.08wt%–0.40wt% xanthan gum, 0.03wt%–0.20wt% pectin, and 0.01wt%–0.08wt% calcium citrate or calcium lactate; wherein, the calcium salt is used to adjust the network structure and viscoelasticity of the compound stabilizing system to improve the suspension stability, anti-sedimentation performance, and enzyme activity retention effect of the cyclodextrin-glycosidase complex in the beverage during storage.

[0012] This invention also provides a method for preparing a functional plant beverage containing glycosides as described above, comprising the following steps: 1) Mix the plant-derived glycoside compound extract with a portion of deionized water according to the specified ratio, add a portion of cyclodextrin-glycosidase complex, and react at 40–50°C for 1–2 hours to obtain a partial conversion solution; 2) Mix the partially converted liquid with the remaining cyclodextrin-glycosidase complex, prebiotics, stabilizers and acidity regulators, and add the remaining deionized water to the composition content of the functional plant beverage, and then homogenize to obtain the beverage base liquid. 3) After the beverage base liquid is filled, it is treated with ultra-high pressure cold sterilization technology at 4℃~10℃. The treatment pressure is 400MPa~600MPa and the holding time is 5min~10min to obtain the functional plant beverage.

[0013] Further, the amount of the cyclodextrin-glycosidase complex added in step 1) is 30-50% of the total amount of the complex, and the remaining part is added in step 2).

[0014] Furthermore, the homogenization process in step 2) employs high-pressure homogenization, with a homogenization pressure of 20MPa to 60MPa and a homogenization frequency of 1 to 3 times.

[0015] The present invention has the following beneficial effects: 1. This application introduces a cyclodextrin glycosidase complex into a beverage system, combining the inclusion effect of cyclodextrin with glycosidase immobilization treatment, so that the glycosidase no longer exists in a simple free state in the liquid system. This not only helps to slow down the inactivation of enzyme components during storage, but also improves their dispersion state in beverages, allowing glycosides to continue to transform into aglycone forms after preparation and during storage, thereby improving the continuity of the utilization process of active ingredients.

[0016] 2. This application constructs a cross-linked immobilized network comprising protein and polysaccharide components, enabling glycosidase inclusion units to be stably embedded within the microstructure. This structure helps maintain the enzyme component's state in the beverage system and improves the contact conditions between the substrate and the enzyme's active site, thereby making the conversion process of glycosides more stable. Compared to simple physical mixing methods, the complex formed in this application exhibits better retention during storage and is more suitable for functional plant-based beverage systems.

[0017] 3. This application further incorporates a complex stabilizing system composed of multiple colloidal components into the beverage system, enabling the resulting beverage to maintain the conversion capacity of active ingredients while also ensuring a good suspension dispersion and uniform appearance. This complex stabilizing system helps to slow down the sedimentation and local aggregation of cyclodextrin glycosidase complexes in the beverage and improves the stratification of the beverage under static conditions, thereby resulting in better overall performance of the finished product during both storage and consumption.

[0018] 4. The preparation method employed in this application includes steps such as enzymatic conversion pretreatment, homogenization, and ultra-high pressure cold sterilization. This method considers both the initial conversion of glycosides and the requirements for activity retention and safety handling in the subsequent beverage system. By using part of the complex for pretreatment first and then adding the remaining complex to the finished product system, the beverage can function separately in the preparation and storage stages. This helps to balance the initial conversion effect with the need for continued conversion, thereby improving the application value of this type of functional plant beverage. Attached Figure Description

[0019] Figure 1 The image shown is a scanning electron microscope image of the cyclodextrin-glycosidase complex prepared in Example 1 of this application; Figure 2 The image shown is a scanning electron microscope image of the cyclodextrin-glycosidase complex prepared in Example 2 of this application; Figure 3 A scanning electron microscope image of the cyclodextrin-glycosidase complex prepared in Example 3 of this application is shown. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] All raw materials used in this application are commercially available and are food-grade. The instruments used in the corresponding preparation and testing methods can also be obtained and implemented using existing technologies.

[0022] Example 1 This embodiment provides a functional plant-based beverage containing glycosides. The functional plant-based beverage comprises the following components: 0.5 wt% plant-derived glycoside extract, 1.1 wt% cyclodextrin-glycosidase complex, 3.0 wt% prebiotic, 0.3 wt% stabilizer, 0.18 wt% acidity regulator, and the balance being deionized water. The plant-derived glycoside extract is geniposide extract, the prebiotic is fructooligosaccharide, and the acidity regulator is citric acid. The stabilizer is a multi-component compound stabilizing system, comprising, by weight of the functional plant-based beverage, 0.05 wt% gellan gum, 0.12 wt% xanthan gum, 0.08 wt% pectin, and 0.05 wt% calcium lactate.

[0023] The raw materials used in the preparation of the cyclodextrin-glycosidase complex in this embodiment are as follows: by weight, 10 parts of hydroxypropyl-β-cyclodextrin, 12 parts of glycosidase, 30 parts of whey protein isolate, 8 parts of sodium caseinate, 7 parts of pectin, 2 parts of chitosan oligosaccharide, 5 parts of sodium alginate, 6 parts of trehalose, 0.5 parts of transglutaminase, 1.0 part of calcium lactate, and 80 parts of phosphate buffer. The glycosidase is a complex enzyme of β-glucosidase and α-L-rhamnosidase, wherein the mass ratio of β-glucosidase to α-L-rhamnosidase is 4:1.

[0024] The cyclodextrin-glycosidase complex used in this embodiment was prepared by the following method: S11: Hydroxypropyl-β-cyclodextrin was added to phosphate buffer at pH 5.5 and stirred at 15°C to dissolve; then the glycosidase was added, and the mixture was stirred continuously at 15°C for 0.5 h to obtain a cyclodextrin-glycosidase pre-encapsulation solution. The amount of cyclodextrin bound to the glycosidase was controlled to be 0.5 μmol / mg based on the mass of the glycosidase.

[0025] S12: Add whey protein isolate and sodium caseinate to another buffer solution with pH 6.8 to prepare a protein dispersion, and sonicate in an ice bath for 2 minutes at a power of 150W.

[0026] S13: Subsequently, pectin, chitosan oligosaccharide, sodium alginate and trehalose are added to the protein dispersion in sequence. After stirring and mixing evenly, the cyclodextrin-glycosidase pre-encapsulation solution is added to obtain the composite precursor solution.

[0027] S14: Add transglutaminase to the composite precursor solution and cross-link at 10°C for 8 hours to cross-link glutamine residues and lysine residues in whey protein isolate and sodium caseinate to form a protein cross-linking network and embed the glycosidase into the protein-polysaccharide network.

[0028] S15: After cross-linking, calcium lactate was added, and the reaction continued for 35 minutes to enhance the sodium alginate-related ion network structure and form a dual-network immobilization system. Finally, the obtained system was centrifuged and ultrafiltered to remove unimmobilized small molecules, and then freeze-dried to obtain the cyclodextrin-glycosidase complex.

[0029] In the preparation of the cyclodextrin-glycosidase complex in this application, whey protein isolate itself contains glutamine and lysine residues, as does sodium caseinate. Tcase can promote the acyl transfer between the glutamine side chain and the ε-amino group of lysine in the protein cross-linking substrates (whey protein isolate and / or sodium caseinate), i.e., the formation of ε-(γ-glutamyl)lysine cross-linking bonds between protein-bound glutamine residues and lysine residues. Whey protein isolate and sodium caseinate, as the main substrates for transglutaminase activity, are used to construct a relatively stable protein cross-linking network, thereby providing a basic immobilization carrier for the cyclodextrin-glycosidase complex. Pectin, sodium alginate, and chitosan oligosaccharide jointly participate in forming an embedding network and play a regulatory role in the local mass transfer and diffusion processes in the system, helping to maintain the distribution of enzyme components in the composite structure. Trehalose is used to slow down the activity loss of enzyme components during preparation and storage, while calcium lactate is used to enhance the polysaccharide-related gel structure, making the resulting composite system more likely to maintain its intact microstructure. At the same time, the use of compound glycosidases can also enable the system to adapt to the conversion requirements of different types of glycosides.

[0030] The method for preparing a functional plant beverage using the above-mentioned cyclodextrin-glycosidase complex containing glycosides, as provided in this embodiment, includes the following steps: 1) First, mix the geniposide extract with some deionized water, then add a complex that accounts for 30% of the total amount of the cyclodextrin-glycosidase complex, and react at 40°C for 2 hours to obtain a partial conversion solution. 2) Then, the converted liquid is mixed with the remaining 70% of the cyclodextrin-glycosidase complex, fructooligosaccharide, the stabilizer and citric acid, and the remaining deionized water is added to the set total amount. The mixture is then homogenized at a pressure of 20 MPa for 3 times to obtain the beverage base liquid.

[0031] 3) After the beverage base liquid is filled, it is treated with ultra-high pressure cold sterilization technology at 10°C, with a treatment pressure of 400MPa and a holding time of 10min to obtain the functional plant beverage of this embodiment.

[0032] Example 2 This embodiment provides a functional plant-based beverage containing glycosides. The functional plant-based beverage comprises the following components: 3.2 wt% plant-derived glycoside extract, 0.1 wt% cyclodextrin-glycosidase complex, 1.0 wt% prebiotic, 0.75 wt% stabilizer, 0.05 wt% acidity regulator, and the balance being deionized water. The plant-derived glycoside extract is a mixture of baicalin extract and steviol glycoside extract, wherein the mass ratio of baicalin extract to steviol glycoside extract is 3:1. The prebiotic is galacto-oligosaccharide, and the acidity regulator is malic acid. The stabilizer is a multi-component compound stabilizing system, comprising, by weight of the functional plant-based beverage, 0.15 wt% gellan gum, 0.32 wt% xanthan gum, 0.20 wt% pectin, and 0.08 wt% calcium citrate.

[0033] The cyclodextrin-glycosidase complex used in this embodiment was prepared by the following method: 16 parts by weight of hydroxypropyl-β-cyclodextrin, 5 parts by weight of glycosidase, 18 parts by weight of whey protein isolate, 20 parts by weight of sodium caseinate, 3 parts by weight of pectin, 6 parts by weight of chitosan oligosaccharide, 2 parts by weight of sodium alginate, 3 parts by weight of trehalose, 2.5 parts by weight of transglutaminase, 0.2 parts by weight of calcium lactate, and 40 parts by weight of phosphate buffer. The glycosidase is β-glucosidase.

[0034] S11: Hydroxypropyl-β-cyclodextrin was added to phosphate buffer at pH 6.2 and stirred at 8°C to dissolve. Then, β-glucosidase was added, and the mixture was stirred continuously at 8°C for 2.0 h to obtain a cyclodextrin-glycosidase pre-encapsulation solution. Based on the mass of the glycosidase, the binding amount of cyclodextrin to the glycosidase was 1.2 μmol / mg. S12: Add whey protein isolate and sodium caseinate to another buffer solution with pH 6.0 to prepare a protein dispersion, and sonicate in an ice bath for 9 min at a power of 280W. S13: Subsequently, pectin, chitosan oligosaccharide, sodium alginate and trehalose are added to the protein dispersion in sequence, and after stirring and mixing evenly, the pre-encapsulation solution is added to it to obtain the composite precursor solution; S14: Add transglutaminase to the composite precursor solution and react at 25°C for 4 hours to form a protein cross-linking network and achieve immobilization of glycosidase in the protein-polysaccharide network. S15: After cross-linking is complete, calcium lactate is added, and the reaction continues for 10 minutes to enhance the ionic network structure. Finally, the resulting system is centrifuged, dialyzed, and then freeze-dried to obtain the cyclodextrin-glycosidase complex.

[0035] The method for preparing a functional plant beverage using the above-mentioned cyclodextrin-glycosidase complex containing glycosides, as provided in this embodiment, includes the following steps: 1) First, mix the plant-derived glycoside extract with some deionized water, then add a complex accounting for 40% of the total amount of cyclodextrin-glycosidase complex, and react at 45℃ for 1.5h to obtain a partial conversion solution; 2) Then, the converted liquid is mixed with the remaining 60% of the cyclodextrin-glycosidase complex, galactooligosaccharide, the stabilizer and malic acid, and the remaining deionized water is added to the set total amount. After high-pressure homogenization, the homogenization pressure is 40 MPa and the homogenization is performed twice to obtain the beverage base liquid. 3) After the beverage base liquid is filled, it is treated with ultra-high pressure cold sterilization technology at 7°C, with a treatment pressure of 500MPa and a holding time of 7min to obtain the functional plant beverage of this embodiment.

[0036] Example 3

[0037] This embodiment provides a functional plant-based beverage containing glycosides. The functional plant-based beverage comprises the following components: 5.0 wt% plant-derived glycoside extract, 2.0 wt% cyclodextrin-glycosidase complex, 2.2 wt% prebiotic, 1.0 wt% stabilizer, 0.30 wt% acidity regulator, and the balance being deionized water. The plant-derived glycoside extract is a mixture of ginsenoside extract, geniposide extract, and baicalin extract, wherein the mass ratio of the three is 2:2:1. The prebiotic is inulin, and the acidity regulator is a mixture of citric acid and malic acid, wherein the mass ratio of citric acid to malic acid is 1:1. The stabilizer is a multi-component compound stabilizing system, comprising, by weight of the functional plant-based beverage, 0.25 wt% gellan gum, 0.40 wt% xanthan gum, 0.20 wt% pectin, and 0.08 wt% calcium lactate.

[0038] The cyclodextrin-glycosidase complex used in this embodiment was prepared by the following method: 20 parts by weight of hydroxypropyl-β-cyclodextrin, 18 parts by weight of glycosidase, 12 parts by weight of whey protein isolate, 14 parts by weight of sodium caseinate, 12 parts by weight of pectin, 10 parts by weight of chitosan oligosaccharide, 8 parts by weight of sodium alginate, 1 part by weight of trehalose, 4 parts by weight of transglutaminase, 2 parts by weight of calcium lactate, and 120 parts by weight of phosphate buffer. The glycosidase is a complex enzyme of β-glucosidase, α-L-rhamnosidase, and β-galactosidase, wherein the mass ratio of the three is 5:2:1.

[0039] S11: Hydroxypropyl-β-cyclodextrin was added to phosphate buffer at pH 7.0 and stirred at 4°C to dissolve. Then, the compound glycosidase was added, and the mixture was stirred continuously at 4°C for 3.0 h to obtain a cyclodextrin-glycosidase pre-encapsulation solution. The amount of cyclodextrin bound to the glycosidase was 2.0 μmol / mg, based on the mass of the glycosidase. S12: Add whey protein isolate and sodium caseinate to another buffer solution with pH 7.5 to prepare a protein dispersion, and sonicate in an ice bath for 15 min at a power of 400W. S13: Subsequently, pectin, chitosan oligosaccharide, sodium alginate and trehalose are added to the protein dispersion in sequence, and after stirring and mixing evenly, the pre-encapsulation solution is added to it to obtain the composite precursor solution; S14: Add transglutaminase to the composite precursor solution and cross-link at 35°C for 1 hour to form a protein cross-linking network and immobilize the glycosidase in the protein-polysaccharide network. After cross-linking is complete, add calcium lactate and continue the reaction for 60 minutes to enhance the sodium alginate-related ion network structure. S15: Finally, after centrifugation and membrane separation to remove unimmobilized small molecules, the obtained system is spray-dried at low temperature to obtain the cyclodextrin-glycosidase complex.

[0040] The method for preparing a functional plant beverage using the above-mentioned cyclodextrin-glycosidase complex containing glycosides, as provided in this embodiment, includes the following steps: 1) First, mix the plant-derived glycoside extract with some deionized water, then add a complex accounting for 50% of the total amount of cyclodextrin-glycosidase complex, and react at 50℃ for 1.0 h to obtain a partial conversion solution; 2) Then, the converted liquid is mixed with the remaining 50% of the cyclodextrin-glycosidase complex, inulin, the stabilizer and acidity regulator, and the remaining deionized water is added to the set total amount. High-pressure homogenization is then performed at a pressure of 60 MPa and the number of homogenizations is 1 to obtain the beverage base liquid. 3) After the beverage base liquid is filled, it is treated with ultra-high pressure cold sterilization technology at 4°C, with a treatment pressure of 600MPa and a holding time of 5min to obtain the functional plant beverage of this embodiment.

[0041] Comparative Example 1 The difference between this comparative example and Example 2 is that the cyclodextrin-glucosidase complex used in Example 2 is a multi-component complex formed by cross-linking and immobilization of transglutaminase; in this comparative example, this complex is replaced by a physical mixture of free β-glucosidase and hydroxypropyl-β-cyclodextrin.

[0042] Specifically, in this comparative example, whey protein isolate, sodium caseinate, pectin, chitosan oligosaccharide, sodium alginate, trehalose, transglutaminase, and calcium lactate were not used to prepare the immobilized complex; only β-glucosidase and hydroxypropyl-β-cyclodextrin were added to deionized water at a mass ratio of 1:4 and stirred at 25°C for 1 h to obtain a physical mixture system of free glycosidase and hydroxypropyl-β-cyclodextrin.

[0043] In preparing functional plant beverages, the physical mixing system described above is used to replace the cyclodextrin-glycosidase complex in Example 2, while the other functional plant beverage components and preparation steps are the same as in Example 2.

[0044] Comparative Example 2 The difference between this comparative example and Example 2 is that the cyclodextrin-glycosidase complex used in Example 2 is a complex formed by transglutaminase catalysis to form a protein-polysaccharide cross-linked immobilization network; in this comparative example, only a simple premixed inclusion system of hydroxypropyl-β-cyclodextrin and β-glucosidase is used, without any protein components, polysaccharide network components, transglutaminase cross-linking immobilization steps, or calcium lactate densification treatment.

[0045] Specifically, in this comparative example, only 16 parts of hydroxypropyl-β-cyclodextrin, 5 parts of β-glucosidase, and 40 parts of phosphate buffer were weighed. The hydroxypropyl-β-cyclodextrin was added to phosphate buffer at pH 6.2 and stirred at 8°C to dissolve. Then, β-glucosidase was added, and the mixture was stirred continuously at 8°C for 2.0 h to obtain the cyclodextrin-glucosidase premixed inclusion system. During the preparation of this system, whey protein isolate, sodium caseinate, pectin, chitosan oligosaccharide, sodium alginate, trehalose, transglutaminase, and calcium lactate were not added, nor were ultrasonic preactivation, enzymatic cross-linking immobilization, ion densification, or freeze-drying steps performed.

[0046] In preparing functional plant beverages, the cyclodextrin-glycosidase premixed inclusion system described above is used to replace the cyclodextrin-glycosidase complex in Example 2. The other functional plant beverage components and preparation steps are the same as in Example 2.

[0047] Comparative Example 3 The difference between this comparative example and Example 2 is that the stabilizer used in Example 2 is a multi-component compound stabilizing system; in this comparative example, the stabilizing system is replaced with a single sodium carboxymethyl cellulose stabilizing system.

[0048] Specifically, in this comparative example, sodium carboxymethyl cellulose 0.75wt% was used to replace gellan gum 0.15wt%, xanthan gum 0.32wt%, pectin 0.20wt%, and calcium citrate 0.08wt% in Example 2, and the complex network stabilization system composed of gellan gum, xanthan gum, pectin, and calcium salt was no longer used.

[0049] In preparing functional plant beverages, the preparation method of cyclodextrin-glycosidase complex, plant-derived glycoside extract, prebiotics, acidity regulators and other preparation step parameters are the same as in Example 2, except that the stabilizer added in step 2) is replaced with 0.75wt% sodium carboxymethyl cellulose.

[0050] To observe the microstructure of the cyclodextrin glycosidase complexes with protein cross-linking networks prepared in Examples 1 to 3, scanning electron microscopy (SEM) was performed on the lyophilized complex samples. Specifically, the complex samples obtained in each example were first lyophilized, ground into powder, and a small amount was evenly spread on the surface of a conductive sample stage. During the test, the accelerating voltage was controlled at 5000V, the working distance at 8mm-10mm, and surface morphology images of each sample were acquired under the same magnification to ensure the comparability of image results between different examples. The obtained images were mainly used to characterize the construction of the protein cross-linking network in the complex, the pore distribution, and the binding morphology of the embedded phase within the network. The results are as follows: Figures 1-3 As shown.

[0051] from Figures 1-3 As can be seen, the complexes obtained in Examples 1 to 3 are quite similar in overall morphology, all exhibiting a continuously distributed network framework structure, accompanied by a certain degree of granular or clustered regions. This indicates that under different formulation compositions and process conditions, the cyclodextrin inclusion and proteoglycan cross-linking immobilization methods used in this application can all form a relatively stable microscopic support structure. No obvious large-area collapse was observed in any of the samples, and the overall network remained relatively intact, indicating that the obtained complexes still possess a certain spatial structure after drying.

[0052] Further comparison reveals that the pores in Example 1 are more pronounced, the network distribution in Example 2 is more uniform, and the local framework in Example 3 is slightly thicker. However, the differences between the three are mainly in the details of local areas, with no significant difference in overall morphology. This indicates that the cyclodextrin glycosidase complex prepared in this application can form a continuous cross-linked network under different implementation conditions, playing a role in carrying and dispersing the enzyme active components, thus providing a foundation for its stable existence and continuous transformation in beverage systems.

[0053] The cyclodextrin glycosidase complex provided in this application is not a simple physical mixture of particles, but rather an embedded structure with spatial support formed on the basis of a protein cross-linking network. This type of structure helps maintain the dispersion state of the enzyme components in the system and creates better conditions for substrate contact, local mass transfer, and continuous enzymatic transformation during storage. Therefore, it helps glycosides in functional plant beverages to continuously convert to aglycone forms during storage.

[0054] To evaluate the overall performance of the functional plant beverages prepared in this application, the aglycone conversion rate, enzyme activity retention rate after 30 days of storage, centrifugal sedimentation rate, average particle size, and sensory stability score were tested for Examples 1-3 and Comparative Examples 1-3, respectively. The results are shown in Table 1. As can be seen from Table 1, Examples 1-3 showed significantly better performance than Comparative Examples 1-3 in terms of aglycone conversion rate and enzyme activity retention rate. Simultaneously, they exhibited lower centrifugal sedimentation rates, smaller average particle sizes, and higher sensory stability scores. This indicates that the synergistic construction of the compound stabilization system through cyclodextrin inclusion, protein cross-linking immobilization, and compound stabilization helps improve the retention effect, suspension dispersion state, and continuous conversion performance of enzyme components in functional plant beverages during storage. The test results are shown in Table 1. The specific test methods are as follows: 1) Aglycone conversion rate test Take appropriate amounts of each sample, centrifuge to remove insoluble matter, and then use high-performance liquid chromatography (HPLC) to determine the content of glycosides and their corresponding aglycones in the supernatant. The aglycone conversion rate is characterized by the percentage increase in aglycone content relative to the initial glycoside content, which is used to evaluate the conversion of glycosides to aglycones during beverage preparation and storage.

[0055] 2) Enzyme activity retention test after 30 days of storage The initial enzyme activity of each sample was measured after preparation, and the enzyme activity was measured again after storing the samples at 4°C in the dark for 30 days. The enzyme activity retention rate was calculated by comparing the latter with the former, which is used to characterize the enzyme activity preservation of the beverage during storage.

[0056] 3) Centrifugal sedimentation rate test Take 10 mL of each sample and place it in a centrifuge tube. Centrifuge at 4000 rpm for 10 min, discard the supernatant, and weigh the sediment. The percentage of sediment mass to the initial total mass of the sample is used as the centrifugal sedimentation rate to characterize the dispersion stability and anti-sedimentation ability of the beverage system.

[0057] 4) Average particle size test Take appropriate amounts of each sample, dilute with deionized water to a suitable test concentration, and disperse by gentle shaking. The average particle size of the sample is then determined using dynamic light scattering. This indicator is used to characterize the dispersion state and particle size distribution of composite particles in a beverage system.

[0058] 5) Sensory stability score After each sample was filled, it was left to stand at room temperature for 24 hours. Then, 10 basic-trained evaluators conducted sensory evaluations. The evaluation included the uniformity of appearance, the presence of layering or sedimentation, the harmony of color, and the consistency of taste. Each item was scored out of 10, and the average value was taken as the sensory stability score, which reflects the beverage's appearance retention and drinking quality when left to stand.

[0059] Table 1 Performance test results of Examples 1-3 and Comparative Examples 1-3

[0060] As shown in Table 1, the overall performance of Examples 1 to 3 was superior to that of Comparative Examples 1 to 3. Examples 2 and 3 showed better performance in terms of aglycone conversion rate and enzyme activity retention after 30 days of storage. For example, the aglycone conversion rate of Example 3 reached 79.3%, significantly higher than the 41.7% of Comparative Example 1, indicating that the cyclodextrin inclusion and cross-linking immobilization complex system constructed in this application is more conducive to the continuous conversion of glycosides to aglycone form. Meanwhile, the centrifugal sedimentation rate of the Example groups was generally lower, and the particle size distribution was more concentrated, indicating that the complex dispersion in the beverage system was more stable and less prone to significant aggregation or sedimentation. In contrast, Comparative Examples 1 to 3 showed varying degrees of decline in enzyme activity retention, sedimentation control, and sensory stability, indicating that simple physical mixing, simple premixing inclusion, or single stabilizer systems are difficult to simultaneously achieve continuous enzymatic conversion and beverage storage stability.

[0061] The accompanying drawings of the embodiments disclosed in this invention only involve the structures involved in the embodiments of this disclosure. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of this invention can be combined with each other. The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A functional plant-based beverage containing glycosides, characterized in that, The beverage comprises the following components: The contents include 0.5–5 wt% plant-derived glycoside extract, 0.1–2 wt% cyclodextrin-glycosidase complex, 1–3 wt% prebiotics, 0.3–1 wt% stabilizer, 0.05–0.3 wt% acidity regulator, and the balance being deionized water. The cyclodextrin-glycosidase complex is composed of cyclodextrin and glycosidase forming an inclusion enzyme active unit, which is stabilized by a cross-linked immobilization network and used to continuously convert glycosides into aglycone forms during storage.

2. The functional plant-based beverage according to claim 1, characterized in that, The plant-derived glycoside extracts are selected from one or more of ginsenosides, steviol glycosides, geniposide, and baicalin.

3. The functional plant-based beverage according to claim 1, characterized in that, The glycosidase in the cyclodextrin-glycosidase complex is selected from one or more of β-glucosidase, α-L-rhamnosidase, β-galactosidase, cellobiase, and amygdalinase.

4. The functional plant-based beverage according to claim 1, characterized in that, The cyclodextrin-glycosidase complex is a multi-component complex formed by cross-linking and immobilizing transglutaminase. The raw materials for preparing the cyclodextrin-glycosidase complex, by weight, include: 10-20 parts of hydroxypropyl-β-cyclodextrin, 5-18 parts of glycosidase, 12-30 parts of whey protein isolate, 8-20 parts of sodium caseinate, 3-12 parts of pectin, 2-10 parts of chitosan oligosaccharide, 2-8 parts of sodium alginate, 1-6 parts of trehalose, 0.5-4 parts of transglutaminase, 0.2-2 parts of calcium lactate, and 40-120 parts of buffer solution. The glycosidase is β-glucosidase, or a complex enzyme consisting of β-glucosidase and one or more of α-L-rhamnosidase and β-galactosidase. The transglutaminase is used to catalyze the cross-linking reaction between glutamine residues and lysine residues in the whey protein isolate and / or sodium caseinate to form a protein cross-linking network; the amount of cyclodextrin bound to the glycosidase is 0.5–2.0 μmol / mg based on the mass of the glycosidase.

5. The functional plant-based beverage according to claim 4, characterized in that, The preparation method of the cyclodextrin-glycosidase complex includes the following steps: S11: Dissolve hydroxypropyl-β-cyclodextrin in phosphate buffer solution with pH 5.5 to 7.0, add the glycosidase, and stir at 4℃ to 15℃ for 0.5h to 3h to obtain cyclodextrin-glycosidase pre-encapsulation solution; S12: Prepare a protein dispersion by adding whey protein isolate and sodium caseinate to another buffer solution, adjust the pH to 6.0-7.5, and sonicate under ice bath conditions for 2-15 minutes. The sonication power is 150W-400W to induce the protein molecules to expand appropriately and expose reaction sites that can be cross-linked. S13: Pectin, chitosan oligosaccharide, sodium alginate and trehalose are added sequentially to the protein dispersion, and after stirring and mixing, the pre-encapsulation solution is added to it to obtain the composite precursor solution; S14: Add transglutaminase to the composite precursor solution and react at 10℃~35℃ for 1h~8h to form a protein cross-linking network of protein components and promote the embedding of glycosidase into the protein-polysaccharide network. S15: Add calcium lactate to the cross-linked system and continue the reaction for 10 min to 60 min to enhance the sodium alginate-related ion network structure and obtain a dual-network immobilization system. S16: After removing unimmobilized small molecules by centrifugation, ultrafiltration, membrane separation or dialysis, the obtained system is freeze-dried or spray-dried at low temperature to obtain cyclodextrin-glycosidase complex.

6. The functional plant-based beverage according to claim 1, characterized in that, The prebiotics are selected from one or more of fructooligosaccharides, galactooligosaccharides, and inulin.

7. The functional plant-based beverage according to claim 1, characterized in that, The stabilizer is a multi-component compound stabilizing system, comprising, by weight of the functional plant beverage, 0.05wt%–0.25wt% gellan gum, 0.08wt%–0.40wt% xanthan gum, 0.03wt%–0.20wt% pectin, and 0.01wt%–0.08wt% calcium citrate or calcium lactate. The calcium salt is used to adjust the network structure and viscoelasticity of the compound stabilizing system to improve the suspension stability, anti-sedimentation properties, and enzyme activity retention during storage of the cyclodextrin-glycosidase complex in the beverage.

8. A method for preparing a functional plant-based beverage containing glycoside compounds as described in any one of claims 1-7, characterized in that, Includes the following steps: 1) Mix plant-derived glycoside extract with a portion of deionized water according to the content ratio described in claim 1, add a portion of cyclodextrin-glycosidase complex, and react at 40–50°C for 1–2 hours to obtain a portion of the conversion solution; 2) Mix the partially converted liquid with the remaining cyclodextrin-glycosidase complex, prebiotics, stabilizers and acidity regulators, and add the remaining deionized water to the composition content of the functional plant beverage as described in claim 1, and then perform homogenization to obtain the beverage base liquid; 3) After the beverage base liquid is filled, it is treated with ultra-high pressure cold sterilization technology at 4℃~10℃. The treatment pressure is 400MPa~600MPa and the holding time is 5min~10min to obtain the functional plant beverage.

9. The preparation method according to claim 8, characterized in that, The amount of the cyclodextrin-glycosidase complex added in step 1) is 30-50% of the total amount of the complex, and the remaining part is added in step 2).

10. The preparation method according to claim 8, characterized in that, The homogenization process in step 2) uses high-pressure homogenization, with a homogenization pressure of 20MPa to 60MPa and a homogenization frequency of 1 to 3 times.

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