Method for preparing sweetening agent from stevia rebaudiana and momordica grosvenori, sweetening agent and application of sweetening agent

Abstract

The invention discloses a method for preparing a sweetening agent from stevia rebaudiana and momordica grosvenori, the sweetening agent and application of the sweetening agent, and belongs to the technical field of compound sweetening agents.The method comprises the following steps that S1, stevia rebaudiana leaves and crushed momordica grosvenori are subjected to leaching and compound enzyme enzymolysis, and enzymatic hydrolysate is obtained; the compound enzyme is prepared from cellulase, pectinase and beta-glucosidase; s2, carrying out adsorption-elution on the enzymatic hydrolysate in macroporous adsorption resin, and collecting an eluent; and S3, carrying out nanofiltration, desalination and concentration on the eluent, and drying to obtain the sweetening agent. According to the technical scheme, stevia rebaudiana leaves and siraitia grosvenorii are subjected to buffer solution leaching and enzymolysis pretreatment, and the type of macroporous resin and the polarity of an elution solvent are optimized; finally, the prepared sweetening agent is high in sweetness, is stable in a pH range of 2-8, and is suitable for the fields of foods, beverages and medicines.

Description

Technical Field

[0001] This invention belongs to the field of compound sweetener technology, specifically a method for preparing a sweetener from stevia and monk fruit, and the sweetener and its application. Background Technology

[0002] With the popularization of healthy eating concepts, high-sweetness, low-calorie natural sweeteners have gradually become a research and development hotspot in the food, beverage, and pharmaceutical industries. Stevia and monk fruit, as common natural sweetener ingredients, contain highly effective sweetener components such as steviol glycosides and mogrosides, respectively. They are characterized by low calories and high safety, and have broad application prospects in the field of sweeteners.

[0003] Currently, technologies for preparing sweeteners using stevia or monk fruit have been reported, but sweeteners prepared from single raw materials suffer from problems such as poor taste and insufficient stability. Existing methods for preparing compound sweeteners generally suffer from low extraction rates of sweet components, low product purity, and flavor deficiencies. For example, traditional extraction methods struggle to fully release the sweet components in the raw materials, conventional enzymatic hydrolysis systems are inefficient, and impurities are not thoroughly removed during adsorption and separation, resulting in the final product's sweetness, stability, and palatability failing to meet market demands. Summary of the Invention

[0004] To overcome the aforementioned technical problems, this invention provides a method for preparing a sweetener from stevia and monk fruit, along with the sweetener and its applications. The technical solution of this invention involves pretreatment of stevia leaves and monk fruit through buffer extraction and enzymatic hydrolysis, as well as optimizing the type of macroporous resin and the polarity of the elution solvent. Ultimately, the resulting sweetener exhibits high sweetness, stability within the pH range of 2-8, and suitability for use in the food, beverage, and pharmaceutical industries.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention discloses a method for preparing a sweetener from stevia and monk fruit, comprising the following steps: S1. Stevia leaves and broken monk fruit are soaked and hydrolyzed with a compound enzyme to obtain an enzymatic hydrolysate; The complex enzyme contains cellulase, pectinase, and β -Glucosidase; S2. Adsorb and elute the enzymatic hydrolysate in a macroporous adsorption resin and collect the eluent. S3. The eluent is subjected to nanofiltration, desalting and concentration, and then dried to obtain a sweetener.

[0007] In S1, the ratio of the stevia leaf to the monk fruit is 3~30:10.

[0008] In S1, the immersion is carried out in a weakly acidic buffer solution with a pH of 5.0 to 7.0 at 30 to 50°C for 40 to 120 minutes; preferably, the immersion is carried out in a weakly acidic buffer solution with a pH of 5.5 to 6.5 at 35 to 45°C for 60 to 120 minutes.

[0009] In S1, the weakly acidic buffer solution is a citrate-disodium hydrogen phosphate buffer solution or an acetic acid-sodium acetate buffer solution.

[0010] In S1, the mass ratio of cellulase, pectinase and β-glucosidase in the complex enzyme is 3~5:2~3:1.

[0011] In S1, the total amount of the compound enzyme added is 0.5 to 1.5% of the total dry weight of the stevia leaves and the monk fruit.

[0012] In S1, the enzymatic hydrolysis temperature is 40~60℃, preferably 45~55℃.

[0013] In S1, the enzymatic hydrolysis time is 90~150 min.

[0014] In S1, the pH of the enzymatic hydrolysis maintenance system is between 4.5 and 6.0, preferably between 4.8 and 5.5.

[0015] In S1, the enzymatic hydrolysis is followed by an enzyme inactivation step, which involves inactivating the enzyme hydrolysate by maintaining it at 90-100°C for 8-15 minutes.

[0016] In S2, the macroporous adsorption resin is a methacrylate-type macroporous resin.

[0017] In S2, the adsorption flow rate is 0.5~3 BV / h, preferably 1~2 BV / h.

[0018] In step S2, the elution process involves first eluting with an ethanol aqueous solution of 15-25% (v / v) to remove impurities; then eluting with an ethanol aqueous solution of 45-55% (v / v) and collecting the eluent from this step.

[0019] In step S2, 0.05-0.2% L-arabinose (by mass of the total eluent) is added to the eluent, and the mixture is stirred at 60-75°C for 30-60 minutes under inert gas protection. The sweetness of steviol glycosides and mogrosides is primarily due to glycosidic bonds, which are easily broken by acidic or alkaline environments or oxidation. The hydroxyl groups of L-arabinose can form hydrogen bonds with glycosidic bonds, reducing their reactivity and decreasing the probability of hydrolysis and oxidative breakage. L-arabinose also possesses antioxidant properties, scavenging free radicals in the system and preventing them from attacking the molecular structure of sweet components, thus delaying oxidative degradation. This makes it more difficult for L-arabinose to undergo structural changes during subsequent nanofiltration, drying, and storage, thereby improving the overall stability of the product.

[0020] In S3, the molecular weight cutoff of the nanofiltration is 200~500 Da.

[0021] In S3, the operating pressure of the nanofiltration is 0.5~3.0MPa, preferably 1.0~2.5MPa.

[0022] In S3, the drying method is spray drying, with an inlet temperature of 160~185℃ and an outlet temperature of 75~85℃.

[0023] This invention discloses a sweetener prepared by the aforementioned method for preparing sweeteners from stevia and monk fruit.

[0024] According to some embodiments of the present invention, the sweetener is 200 to 380 times sweeter than sucrose, preferably 300 to 380 times sweeter.

[0025] According to some embodiments of the present invention, the sweetener contains ≥20wt% steviol glycosides, preferably 22-30% steviol glycosides.

[0026] According to some embodiments of the present invention, the sweetener contains ≥20wt% mogroside, preferably 24~32wt% mogroside.

[0027] According to some embodiments of the present invention, the particle size distribution D90 of the sweetener is ≤30μm.

[0028] The aforementioned sweeteners are used in food, beverages, or pharmaceuticals.

[0029] According to some embodiments of the present invention, the sweetener is stable in a pH range of 2 to 8.

[0030] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0031] Compared with the prior art, the beneficial effects of the present invention are: This invention involves immersing stevia and monk fruit in a weakly acidic buffer solution to avoid pH fluctuations affecting the stability of sweet components during the immersion process. The complex enzymes used include cellulase to break down the plant cell wall structure, pectinase to decompose pectin-like substances, and β-glucosidase to promote the release and conversion of sweet components such as steviol glycosides.

[0032] Adding L-arabinose can not only mask the slight bitterness that steviol glycosides may have, making the sweetness closer to that of sucrose, but also reduce the damage of glycosidic bonds caused by the external environment, thus improving the overall stability of the product.

[0033] This invention fundamentally solves the long-standing flavor defects of natural high-intensity sweeteners, especially stevia. The resulting sweetener product has a pure sweetness and a harmonious taste far superior to physically mixed products and most products on the market, possessing the core competitive advantage of the potential to replace or even surpass sucrose in taste.

[0034] The sweetener product obtained by this invention is stable within a pH range of 2-8, and can adapt to the acidic and alkaline environments of various fields such as food, beverages, and pharmaceuticals. Furthermore, the spray-dried product exhibits uniform particle size distribution, good flowability, and facilitates subsequent processing and application. Detailed Implementation

[0035] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0036] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0037] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be combined arbitrarily; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for specific parameters, it is understood that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this invention, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed herein; "0-5" is merely a shortened representation of these numerical combinations. Furthermore, when a parameter is described as an integer greater than or equal to 2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0038] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0039] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0040] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0041] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0042] Unless otherwise specified, the term "or" is inclusive in this invention. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0043] The raw material information used in the following examples is as follows: Cellulase ( Cellulase (CAS No.: 9012-54-8) was purchased from Taian Xindeli Biotechnology Co., Ltd., and its activity was 100,000 U / g; Pectinase ( Pectase (CAS No.: 9032-75-1) was purchased from Henan Zhengxing Food Additives Co., Ltd. It is obtained by fermentation and refining of Aspergillus niger and its activity is 50,000 U / g; β -glucosidase ( β-D-Glucosidase Purchased from Xi'an Darwen Biotechnology Co., Ltd. (CAS No.: 9001-45-0), its activity is 200,000 U / g; The macroporous adsorption resin is a methacrylate type macroporous resin purchased from Beijing Lvbaicao Technology Development Co., Ltd. HP2MGL. Its monomer and crosslinking agent are both methacrylic acid, it does not contain aromatic rings, and it is a medium polar adsorption resin. This includes, but is not limited to, the models from the above manufacturers.

[0044] Example 1 The method for preparing a sweetener from stevia and monk fruit in this embodiment is as follows: S1. Raw material pretreatment, leaching and enzymatic hydrolysis Weigh the dried stevia leaves and crushed monk fruit at a dry weight ratio of 1:1 and mix them evenly to obtain the mixed raw materials. The mixed raw materials were added to an acetate-sodium acetate buffer solution with a pH of 6.0, and the material-to-liquid ratio was controlled at 1:10 (g / mL). The mixture was then slowly stirred at 40℃ for 60 min for extraction. Add a compound enzyme to the above-mentioned leaching system. The ratio of the compound enzyme is cellulase: pectinase: β-glucosidase = 4:2.5:1 (mass ratio); the total amount of compound enzyme added is 1.0% of the dry weight of the mixed raw materials. Maintain the pH of the enzymatic hydrolysis system at 5.3-5.5 and the temperature at 50℃. Under these conditions, carry out the enzymatic hydrolysis reaction for 120 minutes with continuous stirring. After enzymatic hydrolysis, the hydrolysate is rapidly heated to 95°C and maintained at this temperature for 10 minutes to completely inactivate the enzyme. The inactivated hydrolysate is then cooled to 25°C for later use.

[0045] S2. Adsorption-elution The cooled enzymatic hydrolysate was loaded into an adsorption column packed with macroporous adsorption resin, and the adsorption flow rate was controlled at 1.5 BV / h. After adsorption saturation, elute with a 20% (v / v) ethanol aqueous solution for about 2-3 column volumes until the eluent is colorless, thus removing most of the impurities; then switch to a 50% (v / v) ethanol aqueous solution for elution and collect the eluent rich in the target sweet component at this stage. Add 0.1% L-arabinose by total mass to the eluent, place the reaction system in a sealed container, and purge with nitrogen to replace the air for protection; then stir in a water bath at 65°C for 45 min.

[0046] S3. Nanofiltration, concentration, and drying The eluent after L-arabinose treatment was subjected to nanofiltration with a molecular weight cutoff of 300 Da and an operating pressure of 1.8 MPa for desalting and concentration to obtain a concentrate with a solid content of 28%. The concentrate was spray-dried with an inlet temperature of 175°C and an outlet temperature of 80°C. The powder product was collected with a particle size distribution D90 of 22 μm.

[0047] Example 2 The difference between this embodiment and Embodiment 1 is as follows: Stevia leaves: Monk fruit = 1:2 (dry weight); The other raw materials, steps and parameters are the same as in Example 1.

[0048] Example 3 The difference between this embodiment and Embodiment 1 is as follows: In S1, the enzymatic hydrolysis pH was maintained at 4.8~5.0, and the temperature was maintained at 58℃; The other raw materials, steps and parameters are the same as in Example 1.

[0049] Example 4 The difference between this embodiment and Embodiment 1 is as follows: Cellulase: Pectinase: β The mass ratio of glucosidase is 5:3:1; The other raw materials, steps and parameters are the same as in Example 1.

[0050] Example 5 The difference between this embodiment and Embodiment 1 is as follows: Add 0.1% L-arabinose by total mass to the eluent and stir in a water bath at 75°C for 40 min; The other raw materials, steps and parameters are the same as in Example 1.

[0051] Example 6 The difference between this embodiment and Embodiment 1 is as follows: In this embodiment S1, the leaching was carried out using hot water at 45°C for 1 hour, and the leaching was not carried out using a weak acid buffer solution of acetic acid-sodium acetate. The other raw materials, steps and parameters are the same as in Example 1.

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is as follows: In this comparative example, the compound enzyme contained only cellulase and pectinase, with a cellulase:pectinase mass ratio of 2:1. The total amount of compound enzyme added was 1.5% of the dry weight of the mixed raw materials. The other raw materials, steps and parameters are the same as in Example 1.

[0053] Test case The sweeteners prepared in the above examples and comparative examples were subjected to the following tests, and the test results are shown in Table 1.

[0054] Sweetness test: A 1% sucrose standard solution was prepared as a reference solution. Sample solutions of sweeteners with different concentrations were prepared and subjected to sensory evaluation by 5 professional sensory evaluators under neutral conditions at 25°C. By diluting or concentrating the sample solutions, a sample concentration with a sweetness equivalent to that of the sucrose standard solution was found, and the relative sweetness was calculated according to the formula.

[0055] The content of steviol glycosides and mogrosides in the sweetener was detected by high performance liquid chromatography (HPLC).

[0056]

[0057] pH stability test: The sweeteners prepared in each example and comparative example were dissolved in buffer solutions with different pH values, i.e., buffer solutions with pH values ​​of 2, 4, 6 and 8 were prepared respectively. The test was conducted at 40°C for 1 month. Within the pH range of 2 to 8, there was no significant change in sweetness and component content, which showed high stability. Specific data are shown in Table 2.

[0058]

[0059] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this invention. It should be understood that the above descriptions are merely specific embodiments of this invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method of preparing a sweetener from stevia and monk fruit, characterized by, The method comprises the following steps: S1. Stevia leaves are immersed and complex enzyme hydrolysis is performed with broken monk fruit to obtain an enzyme hydrolysis solution; The complex enzyme comprises a cellulase, a pectolytic enzyme and β - a glucosidase; S2. The enzyme hydrolysis solution is subjected to adsorption-elution in a macroporous adsorption resin, and the eluate is collected; S3. The eluate is subjected to nanofiltration and concentration, and the sweetener is obtained after drying.

2. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, At least one of the following conditions a-c is met: a. The mass ratio of the Stevia leaves to the monk fruit is 3-30:10; b. The immersion is performed in a weak acid buffer solution with a pH value of 5.0-7.0 at 30-50°C for 40-120 min; preferably, the immersion is performed in a weak acid buffer solution with a pH value of 5.5-6.5 at 35-45°C for 60-120 min; c. The weak acid buffer solution is a citric acid-disodium hydrogen phosphate buffer solution or an acetic acid-sodium acetate buffer solution.

3. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, At least one of the following conditions a-b is met: a. The mass ratio of cellulase, pectinase and β-glucosidase in the complex enzyme is 3-5:2-3:1; b. The total amount of the complex enzyme added is 0.5-1.5% of the total dry weight of the Stevia leaves and the monk fruit.

4. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, At least one of the following conditions a-d is met: a. The temperature of the enzyme hydrolysis is 40-60°C, preferably 45-55°C; b. The time of the enzyme hydrolysis is 90-150 min; c. The pH of the enzyme hydrolysis maintaining system is 4.5-6.0, preferably 4.8-5.5; d. The enzyme hydrolysis further comprises a step of enzyme inactivation, i.e. the enzyme hydrolysis solution is maintained at 90-100°C for 8-15 min for enzyme inactivation.

5. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, The macroporous adsorption resin is a methacrylate macroporous resin.

6. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, At least one of the following conditions a-c is met: a. The flow rate of the adsorption is 0.5-3 BV / h, preferably 1-2 BV / h; b. The elution is first performed with an ethanol aqueous solution with a volume concentration of 15-25% to remove impurities; then an ethanol aqueous solution with a volume concentration of 45-55% is used for elution and the eluate collected in this section is collected; c. L-arabinose accounting for 0.05-0.2% of the total mass of the eluate is added to the eluate, and stirring is performed at 60-75°C for 30-60 min under the protection of inert gas.

7. The method of preparing a sweetener from Stevia rebaudiana and Momordica grosvenori of claim 1, wherein, At least one of the following conditions a-c is met: a. The molecular weight cut-off of the nanofiltration is 200-500 Da; b. The operating pressure of the nanofiltration is 0.5-3.0 MPa, preferably 1.0-2.5 MPa; c. The drying method is spray drying, the inlet temperature is 160-185°C, and the outlet temperature is 75-85°C.

8. A sweetener, characterized by, The sweetener is prepared by the method of any one of claims 1-7.

9. The sweetener of claim 8, wherein, At least one of the following conditions a-d is met: a. The sweetness of the sweetener is 200-380 times that of sucrose; b. The particle size distribution D90 of the sweetener is ≤30 μm; c. The sweetener contains ≥20 wt% of steviol glycosides; d. The sweetener contains ≥20 wt% of mogrosides.

10. Use of a sweetener according to any one of claims 8 to 9 in a food, beverage or pharmaceutical product, characterized in that, The sweetener is stable in the pH value range of 2-8.

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