A low-sensitization natural sweet peptide compound preparation and a preparation method thereof

Abstract

The application belongs to the technical field of food sweeteners, and particularly relates to a low-sensitization natural sweet peptide compound preparation and a preparation method thereof. The preparation comprises 5-35 parts of low-sensitization natural sweet peptide, 20-60 parts of auxiliary natural sweetener, 25-55 parts of filling carrier, 0.5-5 parts of stabilizer and 0-2 parts of acidity regulator. The sweet peptide is prepared by controllable enzymolysis of brazzein, monellin or mabinlin by alkaline protease or papain, with an enzyme-substrate ratio of 1:20-1:100 and an enzymolysis time of 20-90 minutes. In the preparation, the sweet protein is first subjected to enzymolysis, and then subjected to ultrafiltration retention, concentration and drying to obtain peptide powder, which is then mixed with the remaining components or granulated. The application breaks the prejudice of sweet loss caused by enzymolysis, balances the desensitization and sweetness preservation by using the 'window effect', reduces the sensitization by 85%-92%, retains the sweet function domain, and has a compound sweetness of 150-250 times that of sucrose, a coordinated sweet feeling and synergistic effect, a simple process and easy industrialization.

Description

Technical Field

[0001] This invention belongs to the field of food sweetener technology, specifically relating to a low-allergenic natural sweet peptide compound preparation and its preparation method. Background Technology

[0002] Natural sweet proteins such as thaumatin (molecular weight approximately 22,000), monklin (molecular weight approximately 11,000), mabinlin (molecular weight approximately 12,400), and brazzein (molecular weight approximately 6,500) have advantages such as high sweetness, low calories, and natural origin, making them ideal substitutes for sucrose. However, these sweet proteins typically have relatively large molecular weights and complex spatial conformations, which may pose an allergenic risk to some individuals. Certain epitopes in their protein structure can be recognized by the body's immune system, triggering IgE-mediated allergic reactions. Studies have shown that thaumatin molecules contain multiple conformation-dependent IgE-binding epitopes, and its complete tertiary structure is the structural basis for allergenicity. This allergenicity issue severely limits its widespread application in infant formula, foods for special medical purposes, and general health foods.

[0003] To reduce protein allergenicity, existing technologies mainly employ the following methods: (1) Chemical modification, such as acetylation and succinylation, but these may introduce non-natural chemical groups, affecting the natural properties of the product, and their use in food is strictly limited; (2) Polysaccharide cross-linking, such as combining with dextran, but the cross-linking reaction conditions are not easy to control, which may result in a significant reduction in sweetness or uneven product; (3) Enzymatic hydrolysis, which reduces allergenicity by specifically cleaving peptide bonds with proteases and destroying allergenic epitopes.

[0004] Enzymatic hydrolysis for reducing protein allergenicity has been applied to various food proteins. For example, CN104782877A discloses a method for preparing low-allergenic soybean peptide powder by two-step enzymatic hydrolysis of soybean protein using a complex protease, resulting in peptides with molecular weights mainly between 1000 and 5000 Da accounting for over 90%, and reducing the content of allergenic proteins by over 90%. Similarly, hydrolysis desensitization techniques for whey protein, casein, and other proteins have also been extensively studied. However, these applications target common food proteins that do not rely on three-dimensional conformation to function; enzymatic hydrolysis only affects their allergenicity and does not lead to loss of function.

[0005] For natural sweet proteins, the application of enzymatic hydrolysis presents significant technical obstacles. A common technical bias exists in existing technologies: the belief that enzymatic hydrolysis of sweet proteins inevitably destroys their sweetness domains, as the sweetness of sweet proteins is highly dependent on their intact three-dimensional conformation. Specifically, the sweetness of sematrandine is closely related to the spatial arrangement of key residues such as the lysine-glutamate bridge in its molecule, while the sweetness of monellin depends on the special folding structure formed by its A and B chains; once peptide bonds break, leading to conformational disintegration, the sweetness will decrease significantly. This technical bias is prevalent in both academia and industry: on the one hand, structure-activity relationship studies of sweet proteins generally emphasize the necessity of intact tertiary structure for sweetness (e.g., sematrandine contains 8 pairs of disulfide bonds to maintain its conformation; destruction of any pair leads to loss of sweetness); on the other hand, existing technical routes for improving the properties of sweet proteins (such as WO2019233923A1 using PAD enzymes to modify arginine residues) deliberately avoid destroying the peptide bond backbone, instead modulating sweetness properties through side chain modification. Therefore, it is generally believed in the industry that enzymatic hydrolysis is difficult to achieve a balance between desensitization and sweetness retention. If the degree of hydrolysis is excessive, it will destroy the peptide structure necessary for the sweetness function, resulting in the loss of sweetness; if the hydrolysis is insufficient, the sensitization will not be reduced enough.

[0006] Furthermore, existing natural high-intensity sweeteners often suffer from drawbacks when used alone, such as impure sweetness, bitter aftertaste, and poor stability, requiring compound formulations for improvement. Compound formulations targeting low-allergenic sweet peptides also need to consider the compatibility of the carrier with the sweet peptides, stability during processing, and the physical properties of the product, placing high demands on both the formulation and the manufacturing process.

[0007] In conclusion, developing a sweet peptide compound formulation that can significantly reduce allergenicity through controlled enzymatic hydrolysis, maintain high sweetness and a balanced sweetness, and can be mass-produced using existing food processing equipment is of great practical significance. Summary of the Invention

[0008] The technical problem to be solved by this invention is to overcome the technical prejudice that "enzymatic hydrolysis of sweet proteins inevitably leads to loss of sweetness" and to provide a natural sweet peptide compound preparation that significantly reduces allergenicity through controllable enzymatic hydrolysis while maintaining high sweetness and a harmonious sweetness, as well as a preparation method that can be industrially produced.

[0009] Technical solution On one hand, the present invention provides a low-allergenic natural sweet peptide compound formulation, comprising the following components by weight: Low-allergenic natural sweet peptides: 5-35 parts Additional natural sweeteners: 20-60 parts Carrier: 25-55 parts Stabilizer: 0.5~5 parts Acidity regulator: 0-2 parts; The low-allergenic natural sweet peptides are obtained by controlled enzymatic hydrolysis of natural sweet proteins using alkaline protease or papain. The natural sweet proteins are selected from one or more of sematriol, monellin, and areca catechin. The enzyme-to-protein ratio for hydrolysis is 1:20 to 1:100 (w / w), and the hydrolysis time is 20 to 90 minutes. The proportion of peptides with a molecular weight less than 5000 Da in the hydrolysis products is 90 to 96%, and the proportion of small peptides with a molecular weight less than 1000 Da does not exceed 85%. The relative sweetness of the low-allergenic natural sweet peptides is 150 to 250 times that of sucrose.

[0010] Furthermore, the proportion of peptides with a molecular weight of less than 5000 Da in the hydrolysis product is 92-95%, and the proportion of small peptides with a molecular weight of less than 1000 Da is 60-70%.

[0011] Furthermore, the alkaline protease is Alcalase; the enzyme-to-protease ratio is 1:30 to 1:60 (w / w), and the hydrolysis time is 40 to 60 minutes.

[0012] Furthermore, the auxiliary natural sweetener is a combination of steviol glycosides and mogrosides, with a weight ratio of 1:0.5 to 1:2, to improve the sweetness profile.

[0013] Furthermore, the filling carrier is a combination of maltodextrin and erythritol in a weight ratio of 1:0.5 to 1:3, which can improve the dispersibility and moisture resistance of the formulation.

[0014] Furthermore, the stabilizer is one or more of sodium carboxymethyl cellulose, xanthan gum, gum arabic, and β-cyclodextrin.

[0015] Furthermore, the acidity regulator is citric acid, malic acid, tartaric acid, or sodium citrate.

[0016] Furthermore, the dosage form of the compound preparation is powder, granules, or instant tablets. Powder dosage forms can be obtained by dry mixing or spray drying; granule dosage forms can be obtained by wet granulation or fluidized bed granulation; tablets can be directly compressed after adding a glidant. The bulk density of the powder dosage form is 0.45~0.60 g / mL, and the moisture content is ≤4%.

[0017] The key discovery of this invention lies in the following: when a protease performs controlled enzymatic hydrolysis on natural sweet proteins, resulting in 90-96% of the hydrolysate being peptides with a molecular weight less than 5000 Da, the intact tertiary structure of the sweet protein is opened, the conformation-dependent IgE binding epitopes are destroyed, and the sensitization is significantly reduced (by 85%-92%). Furthermore, at this level of enzymatic hydrolysis, the key peptides of the sweetness functional domain are still retained as functional fragments, thus the sweetness is not significantly lost. This "window effect" is not revealed in existing technologies.

[0018] Specifically, the mechanism of the present invention is as follows: (1) The IgE binding epitopes of natural sweet proteins are mainly conformation-dependent epitopes, which are concentrated in the structural domains with a molecular weight of >5000 Da. When enzymatic hydrolysis makes the proportion of <5000 Da peptides reach more than 90%, these macromolecular epitopes are effectively destroyed, and the sensitization is greatly reduced.

[0019] (2) Although the sweetness function of sweet proteins depends on their complete three-dimensional conformation, the key binding sites for their binding to sweet taste receptors (T1R2-T1R3) are distributed on specific amino acid sequence fragments. When the degree of enzymatic hydrolysis is controlled to a peptide ratio of 90-96% with a molecular weight of <5000 Da, the peptides containing these key binding sites (with a molecular weight usually in the range of 1000-5000 Da) are preserved and can still bind to sweet taste receptors to produce sweetness.

[0020] (3) When the proportion of peptides with a molecular weight <5000 Da is less than 90% (e.g., 42%), a large number of large peptides still retain intact epitopes, and the sensitization effect is reduced by only about 20%, which is insufficient. However, when the enzymatic hydrolysis is excessive and the proportion of small peptides with a molecular weight <1000 Da reaches 88%, the sweet functional domain is further cleaved into inactive small peptides, and the sweetness drops to less than 30 times that of sucrose and a bitter taste appears. Therefore, "the proportion of peptides with a molecular weight <5000 Da of 90~96% and the proportion of small peptides <1000 Da ≤85%" is a key technical threshold window, within which an unexpected balance between desensitization and sweetness preservation is achieved.

[0021] On the other hand, the present invention provides a method for preparing the above-mentioned low-allergenic natural sweet peptide compound preparation, comprising the following steps: (1) Dissolve natural sweet protein in water to prepare a solution with a mass concentration of 3-8%, adjust the pH to 6.0-9.0, and the temperature to 50-65℃. Add alkaline protease or papain and carry out enzymatic hydrolysis reaction for 20-90 minutes at an enzyme-to-protein ratio of 1:20-1:100 (w / w). Control the degree of enzymatic hydrolysis so that the proportion of peptides with a molecular weight of less than 5000 Da in the hydrolysate reaches 90-96%. After the reaction is completed, raise the temperature or adjust the pH to inactivate the enzyme and obtain the enzymatic hydrolysate. (2) The enzymatic hydrolysate is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3000~5000 Da, and the permeate with a molecular weight smaller than the cutoff is collected; the permeate is concentrated by nanofiltration or vacuum to a solid content of 10~25%, and then dried to obtain low-allergenic natural sweet peptide powder. (3) According to the formula amount, mix the low-allergenic natural sweet peptide powder obtained in step (2) with auxiliary natural sweetener, filler carrier, stabilizer and acidity regulator evenly, or use water or ethanol aqueous solution as wetting agent to wet granulate, dry and granulate to obtain the compound preparation.

[0022] Preferably, in step (1), the enzyme inactivation conditions are: maintaining at 90~100℃ for 10~20 minutes, or adjusting the pH to 2.0~3.0 and maintaining for 30 minutes.

[0023] Preferably, the drying method in step (2) is spray drying, freeze drying, or low-temperature belt drying. The inlet air temperature for spray drying is 170~190℃, and the outlet air temperature is 80~95℃; the vacuum degree for freeze drying is ≤20 Pa, and the temperature is ≤40℃.

[0024] Beneficial effects Compared with the prior art, the present invention has the following significant advantages: (1) Extremely low sensitization: Through controlled enzymatic hydrolysis, the sweet protein is degraded into peptides with a molecular weight of <5000 Da, and its intact allergen epitopes are destroyed. Indirect ELISA testing showed that the sensitization of the formulation of this invention (based on IgE binding capacity) was 85%–92% lower than that of the unhydrolyzed sweet protein stock solution, significantly improving safety. Human serum IgE binding assays further confirmed that the desensitizing effect was also effective against human IgE-mediated allergic reactions.

[0025] (2) Excellent sweetness quality: By precisely controlling the degree of enzymatic hydrolysis (90-96% of peptides with molecular weight <5000 Da and ≤85% of small peptides <1000 Da), the key binding peptides of the sweetness functional domain are preserved. At the same time, auxiliary natural sweeteners are added to eliminate the bitterness or metallic taste of single sweet peptides. The sweetness can reach 150-250 times that of sucrose, and the sweetness is pure and mellow, close to that of sucrose.

[0026] (3) Significant synergistic effect of compound formulation: When low-allergenic sweet peptides are compounded with auxiliary natural sweeteners such as steviol glycosides and mogrosides, not only does the sweetness exhibit a synergistic enhancement effect (the actual sweetness after compounding is 15% to 45% higher than the sum of the theoretical sweetness of each component, depending on the compounding ratio), but the sweetness curve is also closer to that of sucrose, with a significant reduction in bitterness and metallic taste. Sensory evaluation shows that the sweetness purity score of the compound formulation is more than 35% higher than that of using sweet peptides alone.

[0027] (4) Mature production process, industrializable: The enzymatic hydrolysis tank, ultrafiltration membrane, spray drying tower, three-dimensional mixer, fluidized bed granulator and other equipment used are all conventional equipment in the food industry. The process parameters are clear and controllable, no special equipment is required, it is easy to scale up production, and the cost is controllable.

[0028] (5) Good product stability: Through the matching of filler carrier and stabilizer, the compound preparation powder has good flowability and low moisture absorption. The results of accelerated stability test (40℃ / 75%RH, 6 months) show that the sweetness retention rate of the preparation is ≥95%, there is no significant change in sensitization, and the bulk density and moisture content are within the control range. The shelf life can reach more than 18 months under room temperature and sealed conditions.

[0029] (6) Wide range of applications: The resulting formulations can be used in various food systems such as beverages, baking, candy, dairy products, and special diets, and can be flexibly made into powder, granules or tablets to meet different processing needs. Attached Figure Description

[0030] Figure 1 This is a process flow diagram for preparing the low-allergenic natural sweet peptide compound formulation of the present invention; Figure 2 The curves showing the effect of enzymatic hydrolysis time on the molecular weight distribution and sensitization (indicating relative IgE binding rate) of the hydrolysate in Example 1 are shown. Figure 3 The graph shows the results of accelerated stability testing of the compound formulation. Figure 4 This is a comparison chart of the sweetness curves of sweet peptides used alone and in combination formulations. Detailed Implementation

[0031] The present invention will be further described in detail below through examples, but the scope of protection of the present invention is not limited thereto. All parts in each example refer to parts by weight, and all raw materials used are commercially available food-grade products. Molecular weight distribution was determined by gel permeation chromatography (GPC), using polyethylene oxide as a standard, with a mobile phase of 0.1 mol / L phosphate buffer (pH 7.0), a flow rate of 0.8 mL / min, and a column temperature of 30°C.

[0032] Example 1 A low-allergenic natural sweet peptide compound preparation (powder type) has the following formula: Low-allergenic semathy hydrolyzed peptides: 20 servings Steviosides (95% Rebaudioside A content): 35 servings Maltodextrin (DE value 10~12): 43 parts Sodium carboxymethyl cellulose: 1.5 parts Citric acid: 0.5 parts The preparation of low-allergenic semathymidine hydrolysate peptides: Food-grade sematrandrol was dissolved in deionized water to prepare a 5% (w / w) solution. The pH was adjusted to 8.5 with 1 mol / L NaOH, and the solution was heated to 55°C. Alkaline protease (2.4 L, enzyme activity 2.4 AU / g) was added at an enzyme-to-peptide ratio of 1:50 (w / w), and the mixture was stirred for 45 minutes for enzymatic hydrolysis. After hydrolysis, the temperature was rapidly increased to 95°C and held for 15 minutes to inactivate the enzyme, then cooled to room temperature. The hydrolysate was passed through a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.2 MPa, and the permeate was collected. The permeate was concentrated using a nanofiltration membrane to a solids content of approximately 15%, and then spray-dried at an inlet air temperature of 185°C and an outlet air temperature of 90°C to obtain a low-allergenic sematrandrol hydrolyzed peptide powder. GPC analysis showed that peptides with a molecular weight less than 5000 Da accounted for 93.5%, and small peptides with a molecular weight less than 1000 Da accounted for 62.3%, with a relative sweetness approximately 200 times that of sucrose.

[0033] Preparation of compound preparations: The above-mentioned hydrolyzed peptide powder, steviol glycosides, maltodextrin, sodium carboxymethyl cellulose, and citric acid were added to a three-dimensional motion mixer according to the formula, and mixed at 25 rpm for 30 minutes. The mixture was then passed through an 80-mesh vibrating sieve to obtain a powdered, low-allergenic natural sweet peptide compound preparation. The product is a white to slightly yellow powder with a sweetness approximately 200 times that of sucrose (with sucrose as 1), a moisture content of 3.2%, and a bulk density of 0.52 g / mL.

[0034] Example 2 A low-allergenic natural sweet peptide compound preparation (granule form), the formula is as follows: Low-allergenic mixed sweet peptides: 15 servings Monk fruit glycosides (monk fruit glycoside V content 60%): 25 servings Erythritol: 50 parts Gum arabic: 3 parts β-Cyclodextrin: 5 parts Malic acid: 2 parts Among them, the preparation of low-allergenic mixed sweet peptides: Monelin and areca catechin were mixed at a mass ratio of 2:1, dissolved in water to prepare a 4% solution, and the pH was adjusted to 6.5 with phosphate buffer. The solution was heated to 60°C, and papain (enzyme activity ≥ 1 million U / g) was added at an enzyme-to-protein ratio of 1:40 (w / w). Enzymatic hydrolysis was performed for 60 minutes. After hydrolysis, the temperature was raised to 90°C and maintained for 10 minutes to inactivate the enzyme. After cooling, the solution was centrifuged (4000 r / min, 10 minutes) to collect the supernatant. The supernatant was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The permeate was collected and concentrated by nanofiltration to 18% solids. Then, it was freeze-dried (pre-frozen at -40°C, sublimation stage vacuum 15 Pa, temperature -20~20°C) to obtain a mixed sweet peptide powder. GPC analysis showed that peptides with a molecular weight <5000 Da accounted for 95.2%, and small peptides <1000 Da accounted for 68.7%, with a relative sweetness approximately 180 times that of sucrose.

[0035] Preparation of compound granules: Mixed sweet peptide powder, mogrosides, erythritol, gum arabic, β-cyclodextrin, and malic acid are premixed in a high-efficiency mixer for 10 minutes, then transferred to a fluidized bed granulator. Purified water is used as a wetting agent, the spray rate is 50 mL / min, and the inlet air temperature is 70℃. After granulation, the mixture is boiled and dried at 50℃ until the moisture content is ≤3.5%. The granules are then sieved through an 18-mesh sieve to obtain the granular compound formulation. The product has uniform granules, good solubility, and a sweetness approximately 180 times that of sucrose.

[0036] Example 3 An instant tablet of a low-allergenic natural sweet peptide compound preparation, with the same formulation as in Example 1, plus magnesium stearate at 1.5% of the total mass of the above mixture as a lubricant.

[0037] The compound powder prepared in Example 1 was mixed evenly with magnesium stearate, and then directly compressed into tablets using a rotary tablet press at a pressure of 20 kN, a tablet diameter of 10 mm, and a tablet weight of 400 mg. The resulting tablets had a hardness of 7-9 kg, a disintegration time of <3 minutes, and were easy to use.

[0038] Example 4 A low-allergenic natural sweet peptide compound preparation (powder type), the formula is as follows: Low-allergenic monellin hydrolysate: 12 portions Steviosides (95% Rebaudioside A content): 18 portions Monk fruit glycosides (monk fruit glycoside V content 60%): 22 servings Maltodextrin (DE value 10~12): 35 parts Erythritol: 10 parts Xanthan gum: 1.5 parts Sodium citrate: 1.5 parts The preparation of low-allergenic monellin hydrolysate peptides: Food-grade monellin was dissolved in deionized water to prepare a 4% (w / w) solution. The pH was adjusted to 6.5 with phosphate buffer, and the solution was heated to 60°C. Papain (enzyme activity ≥ 1 million U / g) was added at an enzyme-to-peptide ratio of 1:40 (w / w), and the mixture was stirred for 50 minutes for enzymatic hydrolysis. After hydrolysis, the solution was rapidly heated to 95°C and held for 15 minutes to inactivate the enzyme, then cooled to room temperature. The hydrolysate was passed through a spiral-wound ultrafiltration membrane with a molecular weight cutoff of 5000 Da at an operating pressure of 0.2 MPa, and the permeate was collected. The permeate was concentrated by nanofiltration to a solids content of approximately 16%, and then spray-dried (inlet air temperature 180°C, outlet air temperature 88°C) to obtain low-allergenic monellin hydrolyzed peptide powder. GPC analysis showed that peptides with a molecular weight less than 5000 Da accounted for 91.8%, and small peptides < 1000 Da accounted for 65.1%, with a relative sweetness approximately 170 times that of sucrose.

[0039] The preparation of the compound formulation is the same as in Example 1. The product is approximately 170 times sweeter than sucrose, with a refreshing and mellow sweetness and a unique aftertaste characteristic of monk fruit.

[0040] Example 5 A low-allergenic natural sweet peptide compound preparation (powder type), the formula is as follows: Low-allergenic semathymidine-monelin mixed hydrolyzed peptides: 18 servings Steviosides (95% Rebaudioside A content): 20 servings Monk fruit glycosides (monk fruit glycoside V content 60%): 20 servings Maltodextrin (DE value 10~12): 33 parts Erythritol: 6 parts β-Cyclodextrin: 2 parts Citric acid: 1 part The preparation of low-allergenic semathymidine-monelin mixed hydrolyzed peptides: Food-grade semaphore and monellin were mixed at a mass ratio of 3:1 and dissolved in deionized water to prepare a 5% solution. The pH was adjusted to 8.0 with 1 mol / L NaOH, and the solution was heated to 55°C. Alcalase (enzyme activity 2.4 AU / g) was added at an enzyme-to-protein ratio of 1:50 (w / w), and enzymatic hydrolysis was performed for 50 minutes. Enzyme inactivation conditions were the same as in Example 1. The hydrolysate was passed through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, and the permeate was collected. The permeate was concentrated to 15% solids by nanofiltration and spray-dried (inlet air temperature 180°C, outlet air temperature 88°C) to obtain a mixed hydrolyzed peptide powder. GPC analysis showed that peptides with a molecular weight <5000 Da accounted for 94.1%, and small peptides <1000 Da accounted for 64.5%, with a relative sweetness approximately 210 times that of sucrose.

[0041] The preparation of the compound formulation is the same as in Example 1. The product is approximately 210 times sweeter than sucrose, with a rich and pure sweetness that combines the persistence of sematriol with the refreshing sensation of monetarine, and has no aftertaste.

[0042] Example 6 The compound preparation obtained in Example 1 was applied to a lemon-flavored carbonated beverage at an addition amount of 0.025% (w / v) to replace the sucrose in the original formula (the original formula contained 10% sugar).

[0043] The product was evaluated by a 10-person sensory panel. The sweetness was not significantly different from the original formula (containing 10% sucrose) (p>0.05), exhibiting a pure sweetness without any aftertaste of bitterness. After storage at 25℃ for 3 months, the sweetness retention rate was 97.8%, with no precipitation or flocculation observed, demonstrating good product stability. The control group (using unhydrolyzed sucrose instead of sucrose) showed slight turbidity under the same storage conditions.

[0044] Example 7 The compound preparation obtained in Example 1 was applied to a low-sugar sponge cake, with an addition amount of 0.04% of the flour mass, replacing 50% of the sucrose in the original formula.

[0045] After baking at 200℃ for 25 minutes, the cake's sweetness was comparable to the full-sugar control group, and its texture was soft. Testing showed that the sweetness retention rate of the compound preparation after baking was 92.3%, indicating that the preparation has good thermal stability. ELISA testing for allergenicity showed no significant difference in IgE binding rate after baking compared to before baking, indicating that high-temperature baking does not increase allergenicity.

[0046] Comparative Example 1 The compound preparation was prepared according to the method and formulation of Example 1, except that the low-allergenic semathy hydrolyzed peptide was replaced with an equal amount of unhydrolyzed semathy protease powder, while the other components and steps were the same.

[0047] Tests showed that the sensitization of this comparative example (in vitro IgE binding assay) was 12.6 times that of the product in Example 1, and its sweetness was about 1600 times that of sucrose (because the sweet protein activity was not preserved due to the lack of enzymatic hydrolysis). However, the sensitization was too high and did not meet the requirements for low sensitization.

[0048] Comparative Example 2 Referring to Example 1, extending the enzymatic hydrolysis time to 150 minutes resulted in 88% of the hydrolysate being peptides with a molecular weight less than 1000 Da, and 98.5% being peptides with a molecular weight less than 5000 Da. Most of the sweetness was lost, with a relative sweetness of only 30 times that of sucrose, and a noticeable bitterness appeared, failing to meet the requirements of an ideal sweetener. This comparative example illustrates that excessive enzymatic hydrolysis exceeded the upper limit of the "window effect," leading to the complete destruction of the sweetness functional domain.

[0049] Comparative Example 3 Referring to Example 1, the enzymatic hydrolysis time was shortened to 15 minutes, with peptides with a molecular weight <5000 Da accounting for only 42% and small peptides <1000 Da accounting for only 15%. The sensitization rate was only reduced by about 20% compared to the original protein, indicating an insufficient desensitization effect. This comparative example demonstrates that insufficient enzymatic hydrolysis did not reach the lower limit of the "window effect," and the large molecular weight allergen epitopes were not effectively destroyed.

[0050] Synergistic effect comparison test To verify the synergistic effect of low-allergenic sweet peptides and co-natural sweeteners, the following comparative experiment was designed: Group A: The low-allergenic semathy hydrolyzed peptide powder prepared in Example 1 was used alone at an addition rate of 0.05% (w / v). Group B: Steviosides were used alone, at an addition rate of 0.10% (w / v); Group C: A simple mixture of low-allergenic semathymide hydrolysate (0.05%) and steviol glycosides (0.10%); Group D: The compound preparation of Example 1 (containing 0.05% hydrolyzed peptides + 0.10% steviol glycosides + carrier and stabilizer) was added according to the formula ratio.

[0051] All the above groups were dissolved in pure water, with a 10% sucrose solution as a control. A sensory panel of 10 people conducted quantitative assessments and descriptive analyses. The results are shown in the table below:

[0052] The results showed that the relative sweetness of group D (compound formulation) (approximately 240 times) was significantly higher than the theoretical sum of the expected values ​​of group A (150 times) and group B (180 times) (theoretical sum approximately 165 times), with a synergistic enhancement of sweetness of approximately 45%. Simultaneously, group D had the highest sweetness purity score (8.8 points), and the bitterness and metallic taste were eliminated, indicating that the compound formulation not only achieved synergistic sweetness but also significantly improved sweetness quality. Under different compound ratios and raw material combinations, the synergistic enhancement ranged from 15% to 45%, with the synergistic effect being most significant at the ratio shown in Example 1.

[0053] Accelerated stability test The compound formulation powder prepared in Example 1 was sealed and packaged, and then subjected to accelerated stability testing in a 40℃ / 75%RH constant temperature and humidity chamber. Samples were taken and tested at 0, 1, 2, 3, and 6 months. The results are shown in the table below:

[0054] The results showed that after 6 months under accelerated conditions, the sweetness retention rate of the formulation was ≥96.8%, the moisture content was ≤4%, the bulk density showed no significant change, and the sensitization (relative IgE binding rate) did not increase significantly. Based on ICH guidelines, the shelf life at room temperature could reach over 18 months.

[0055] Allergenicity and sweetness evaluation The allergenicity of the samples was tested using an indirect competitive ELISA method: a samaram-specific rabbit polyclonal antibody was used as the primary antibody, and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody. The binding ability of each sample to the antibody was detected. With undigested samaram as the 100% binding rate, the relative binding rate of the sample in Example 1 was 8.5%, representing a 91.5% reduction in sensitization.

[0056] To further verify the desensitization effect, serum samples were collected from five patients sensitized by sematrandextrose for human serum IgE binding assay. The results showed that the relative binding rate of human IgE in the sample of Example 1 was 9.2% (with unhydrolyzed sematrandextrose as 100%), and the sensitization was reduced by 90.8%, consistent with the results of the rabbit polyclonal antibody assay, confirming that the desensitization effect of the present invention is also effective for human IgE-mediated allergic reactions.

[0057] The molecular weight distribution and sweetness of the hydrolyzed peptide powders prepared in each example were tested, and the results are summarized as follows: Example 1 (Symatran, Alcalase hydrolysis for 45 min): <5000 Da accounted for 93.5%, <1000 Da accounted for 62.3%, and the sweetness was 200 times; Example 2 (Monelin + Areca catechin, Papain hydrolysis for 60 min): <5000 Da accounted for 95.2%, <1000 Da accounted for 68.7%, and the sweetness was 180 times; Example 4 (Monelin, Papain hydrolysis for 50 min): <5000 Da accounted for 91.8%, <1000 Da accounted for 65.1%, and the sweetness was 170 times; Example 5 (Symatran + Monelin, Alcalase hydrolysis for 50 min): <5000 Da accounted for 94.1%, <1000 Da accounted for 64.5%, and the sweetness was 210 times. All embodiments are within the "window effect" range (<5000 Da accounted for 90~96%, <1000 Da accounted for ≤85%), achieving a balance between desensitization and sweetness preservation.

[0058] Sweetness assessment was conducted by a trained sensory panel of 10 people, using a 10% (w / v) sucrose solution as a control. The sweetness multiple was determined using a quantitative assessment method, and the sweetness characteristics were described. Example 1: The formulation had a pure sweetness with no aftertaste of bitterness; Example 2: It had a refreshing sweetness upon entry with a licorice-like aftertaste; Example 4: The sweetness was rounded with a pronounced monk fruit aftertaste; Example 5: The sweetness was rich and mellow, possessing both persistence and a refreshing quality.

[0059] In summary, by controlling the degree of enzymatic hydrolysis within a critical window range of 90-96% for peptides with a molecular weight of <5000 Da and ≤85% for small peptides with a molecular weight of <1000 Da, and by using appropriate auxiliary sweeteners and carriers, this invention achieves an ideal balance between low allergenicity and high sweetness quality. Moreover, the preparation process relies entirely on conventional equipment, making it highly applicable to the industry.

[0060] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A low-allergenic natural sweet peptide compound preparation and its preparation method, characterized in that: By weight, it includes the following components: Low-allergenic natural sweet peptides: 5-35 parts; Additional natural sweeteners: 20-60 parts; Carrier: 25-55 parts; Stabilizer: 0.5~5 parts; Acidity regulator: 0-2 parts; The low-allergenic natural sweet peptides are obtained by controlled enzymatic hydrolysis of natural sweet proteins using alkaline protease or papain. The natural sweet proteins are selected from one or more of sematriol, monellin, and areca catechin. The enzyme-to-protein ratio for hydrolysis is 1:20 to 1:100 (w / w), and the hydrolysis time is 20 to 90 minutes. The proportion of peptides with a molecular weight less than 5000 Da in the hydrolysis products is 90 to 96%, and the proportion of small peptides with a molecular weight less than 1000 Da does not exceed 85%. The relative sweetness of the low-allergenic natural sweet peptides is 150 to 250 times that of sucrose.

2. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The proportion of peptides with a molecular weight of less than 5000 Da in the hydrolysis products is 92-95%, and the proportion of small peptides with a molecular weight of less than 1000 Da is 60-70%.

3. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The alkaline protease is Alcalase; the enzyme-to-protein ratio is 1:30 to 1:60 (w / w), and the hydrolysis time is 40 to 60 minutes.

4. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The auxiliary natural sweetener is a combination of steviol glycosides and mogrosides, with a weight ratio of 1:0.5 to 1:

2.

5. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The filling carrier is a combination of maltodextrin and erythritol, with a weight ratio of 1:0.5 to 1:

3.

6. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The stabilizer is one or more of sodium carboxymethyl cellulose, xanthan gum, gum arabic, and β-cyclodextrin.

7. The low-allergenic natural sweet peptide compound preparation according to claim 1, characterized in that: The acidity regulator is citric acid, malic acid, tartaric acid, or sodium citrate.

8. The low-allergenic natural sweet peptide compound preparation according to any one of claims 1 to 7, characterized in that: The compound preparation is in the form of powder, granules or instant tablets; the bulk density of the powder dosage form is 0.45~0.60 g / mL and the moisture content is ≤4%.

9. A method for preparing a low-allergenic natural sweet peptide compound formulation as described in claim 1, comprising the following steps: (1) Dissolve natural sweet protein in water to prepare a solution with a mass concentration of 3-8%, adjust the pH to 6.0-9.0, and the temperature to 50-65℃. Add alkaline protease or papain and carry out enzymatic hydrolysis reaction for 20-90 minutes at an enzyme-to-protein ratio of 1:20-1:100 (w / w). Control the degree of enzymatic hydrolysis so that the proportion of peptides with a molecular weight of less than 5000 Da in the hydrolysate reaches 90-96%. After the reaction is completed, inactivate the enzyme to obtain the enzymatic hydrolysate. (2) The enzymatic hydrolysate is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3000~5000 Da, and the permeate is collected; the permeate is concentrated to a solid content of 10~25%, and then dried to obtain low-allergenic natural sweet peptide powder. (3) According to the formula amount, mix the low-allergenic natural sweet peptide powder obtained in step (2) with auxiliary natural sweetener, filler carrier, stabilizer and acidity regulator evenly, or granulate by wet method, dry and granulate to obtain the low-allergenic natural sweet peptide compound preparation.

10. The preparation method according to claim 9, characterized in that: In step (1), the enzyme inactivation conditions are: maintaining at 90~100℃ for 10~20 minutes, or adjusting the pH to 2.0~3.0 and maintaining for 30 minutes; in step (2), the drying is spray drying, freeze drying or low temperature belt drying; the inlet air temperature of the spray drying is 170~190℃, and the outlet air temperature is 80~95℃; in step (3), the mixing time is 20~40 minutes, and the mixture is passed through a 60~100 mesh sieve.

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