Method for purifying sweet protein thaumatin II
By employing steps such as high-speed centrifugation, D201 resin treatment, activated carbon removal, three-stage plate and frame filtration, and membrane encapsulation retention, the problem of impurities and pigments in the purification of thaumatin II was solved, resulting in high-purity thaumatin II protein suitable for use in food, brewing, and feed protein applications.
Patent Information
- Application Number
- CN202510778595.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-10-25
- Filing Date
- 2025-06-11
- Publication Date
- 2026-01-20
AI Technical Summary
How to efficiently purify the sweet protein Thaumatin II from Trichoderma reesei fermentation broth, remove impurities and pigments, and obtain high-purity Thaumatin II protein.
Thaumatin II protein was purified stepwise using a combination of high-speed centrifugation, D201 resin treatment, activated carbon removal, three-stage plate and frame filtration, 30kDa and 10kDa membrane encapsulation, and freeze-drying, along with pH adjustment and centrifugation steps.
It achieves efficient removal of pigments, inorganic salts, and impurities from fermentation broth, yielding high-purity Thaumatin II protein, suitable for the food, brewing, and feed protein industries.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a purification method of sweet protein Thaumatin II BACKGROUND
[0002] As a thermophilic saprophytic fungus, Trichoderma reesei is the main strain for producing cellulase and hemicellulase in industry. Compared with traditional expression host bacteria Escherichia coli and yeast, Trichoderma reesei has the following advantages: first, Trichoderma reesei has very strong protein secretion ability, and the yield of endogenous cellulase is as high as 100 g / L, which is incomparable to Escherichia coli and yeast; second, Trichoderma reesei has a similar glycosylation system to higher mammals, which can perform diverse post-translational modifications; third, Trichoderma reesei can secrete proteins outside the cell, which is beneficial for the separation and purification of target proteins; fourth, Trichoderma reesei is a food safety level industrial production strain certified by the US FDA, and the proteins produced by it can be used in food, brewing and feed protein industries; fifth, Trichoderma reesei is easy to cultivate and can use relatively inexpensive culture medium, which is very suitable for large-scale industrial fermentation. Based on the above advantages, Trichoderma reesei not only attracts many scientists, but also is favored by many biological enterprises, and is successfully applied to the expression and industrial production of many drugs, chemical reagents and enzyme reagents.
[0003] Sweet protein is a special bioactive molecule that can impart a remarkable sweet taste to food without increasing calorie and sugar content. This unique function makes it an important innovation in the modern food industry, providing an ideal alternative for consumers seeking healthy diets. Unlike traditional sugars, sweet protein acts on human taste buds, triggering a similar sweet response, but does not affect the rapid fluctuations in blood sugar levels, which is particularly important for diabetics and those who need to control sugar intake. Through precise biotechnological means, sweet protein is extracted and applied to various foods, including beverages, desserts, etc., achieving a perfect balance between flavor and health. With the continuous progress of food technology, sweet protein is expected to have broad application prospects in more fields, contributing to the healthy development of the food industry.
[0004] Thaumatin is a natural sweet protein derived from the native plants in the African region of Somalia. Its unique feature is that it can impart a surprising sweetness to food with extremely low calorie content, far exceeding conventional sugars. This natural sweet molecule acts on human taste buds, not only triggering a fresh and lasting sweetness, but also becoming an ideal choice for people pursuing a healthy lifestyle because it does not cause dramatic fluctuations in blood sugar levels.
[0005] Thaumatin I and Thaumatin II are two key natural sweet protein components derived from the fruit husk of a specific genus of canna (Thaumatococcus daniellii). Notably, Thaumatin II and Thaumatin I only differ in five positions in the amino acid sequence, which leads to subtle differences in physicochemical properties and sweetness characteristics. Despite the differences in chemical structure and content between Thaumatin I and Thaumatin II, they are both of great interest to the food industry due to their extremely high sweetness (up to thousands of times that of sucrose) and good processing stability, and are widely used in sugar-free foods, functional beverages, healthy snacks, and medical and health care products. Both of them exhibit great potential as natural and healthy sweeteners, not only meeting the dual needs of consumers for deliciousness and health, but also providing a new direction for innovation in the food industry.
[0006] Thaumatin is widely used in various fields, including sugar-free beverages, low-calorie desserts, medical and health care products, etc. It not only brings a pure sweet taste experience to food, but also meets the dual pursuit of deliciousness and health by consumers. As a pure natural sweetener, Thaumatin is leading the food and health care industry towards a greener and healthier future with its outstanding performance.
[0007] Due to the strong protein secretion ability of the fungus Trichoderma reesei, the Trichoderma reesei engineering bacteria secreting Thaumatin II protein will also secrete a large number of impurities and pigment molecules during the secretion and expression of Thaumatin II protein. Therefore, it is very important to develop a Thaumatin II protein purification method for removing impurities and pigments. SUMMARY
[0008] Therefore, the present application is proposed. To achieve the above-mentioned object, the present application provides a method for purifying sweet protein Thaumatin from Trichoderma reesei fermentation broth, which can effectively remove pigments, inorganic salts and impurities in the fermentation broth, and obtain high-purity Thaumatin II protein. The method comprises the following steps:
[0009] (1) solid-liquid separation is realized by high-speed centrifugation, and then Thaumatin II fermentation broth supernatant is obtained from Trichoderma reesei fermentation broth by removing the bacterial bodies;
[0010] (2) the pH of the fermentation broth supernatant obtained in step (1) is adjusted to 5-7, and D201 resin is used for treatment to remove part of the impurities and most of the pigments, and then activated carbon is used to remove the residual pigments in the fermentation broth;
[0011] (3) removing the residual activated carbon in the fermentation broth by high-speed centrifugation and three-stage plate-frame filtration to obtain a crude Thaumatin II protein solution;
[0012] (4) using a 30 kDa and a 10 kDa membrane package to cut off and concentrate the crude Thaumatin II protein solution to obtain a Thaumatin II protein concentrate;
[0013] (5) freeze-drying the Thaumatin II protein concentrate to obtain a Thaumatin II protein dry powder.
[0014] Further, the concentration of activated carbon used for removing the residual pigment in the fermentation broth in step (2) is 0.5-1.5%.
[0015] Further, the residual activated carbon in the fermentation broth is removed by three-stage plate-frame filtration in step (3). The first-stage filter membrane is a medium-speed qualitative filter paper, the second-stage filter membrane is a 1 μm filter membrane, and the third-stage filter membrane is a 0.22 μm filter membrane.
[0016] Further, the 30 kDa and above impurities in the crude Thaumatin II protein solution are cut off by using a 30 kDa membrane package in step (4) to obtain a permeate containing Thaumatin II protein.
[0017] Further, when the 30 kDa membrane package is used to cut off the impurities, the operating pressure is controlled at 0.2-0.3 MPa, and the operating temperature is room temperature.
[0018] Further, after the 30 kDa membrane package is used up, it is cleaned with a 0.3 M NaOH solution.
[0019] Further, the permeate containing Thaumatin II protein is concentrated by using a 10 kDa membrane package in step (4) to obtain a Thaumatin II protein concentrate.
[0020] Further, when the 10 kDa membrane package is used for concentration, the operating pressure is controlled at 0.2-0.3 MPa, and the operating temperature is room temperature.
[0021] Further, after the 10 kDa membrane package is used up, it is cleaned with a 0.3 M NaOH solution. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 SDS-PAGE verification diagram for 30 kDa membrane package cut-off effect;
[0023] Figure 2Figure 1 is a SDS-PAGE picture for 10 kDa membrane package retention effect verification;
[0024] Figure 3 Figure 4 is a picture of freeze-dried sample. DETAILED DESCRIPTION
[0025] The present application can be implemented in a number of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for describing the specific embodiments only and is not intended to be limiting of the application.
[0026] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.
[0027] Example 1
[0028] The removal of pigment residues in the fermentation broth is carried out according to the following steps:
[0029] (1) The solid-liquid separation is achieved by high-speed centrifugation, and then the Thaumatin II protein fermentation broth supernatant containing Thaumatin II protein is obtained from the Trichoderma reesei fermentation broth, and the Thaumatin II protein fermentation broth supernatant is collected;
[0030] (2) The pH of the collected fermentation broth supernatant is adjusted to 5-7, and the D201 resin is used for treatment to remove part of the impurities and most of the pigments, and then activated carbon is used to remove the residual pigments in the fermentation broth;
[0031] (3) The residual activated carbon in the fermentation broth is removed by high-speed centrifugation and three-stage plate and frame filtration to obtain the Thaumatin II protein crude extract; wherein the first filter membrane is a medium-speed qualitative filter paper, the second filter membrane is a 1 μm filter membrane, and the third filter membrane is a 0.22 μm filter membrane;
[0032] Example 2
[0033] The removal of impurities in the fermentation broth and the obtaining of freeze-dried powder are carried out according to the following steps:
[0034] (1) using 30kDa membrane package to protein crude extract of 30kDa above impurities for intercept. Operating pressure control at 0.2-0.3MPa, operating temperature is room temperature, get containing Thaumatin II protein permeate. 30kDa membrane package using after, with 0.3M NaOH solution is cleaned. By Figure 1 It can be seen that the protein in 30kDa membrane package permeate is mainly Thaumatin II protein, no obvious detection of 25kDa above impurities, indicating that 25kDa above impurities are successfully intercepted by 30kDa membrane package.
[0035] (2) using 10kDa membrane package to contain Thaumatin II protein permeate is concentrated. Operating pressure control at 0.2-0.3MPa, operating temperature is room temperature, get Thaumatin II protein concentrate. 10kDa membrane package using after, with 0.3M NaOH solution is cleaned. By Figure 2 It can be seen that the intercept liquid contains a large number of Thaumatin II protein, and the purity is very high, indicating that Thaumatin II protein is successfully intercepted by 10kDa membrane package.
[0036] (3) Thaumatin II protein concentrate is freeze-dried, get Thaumatin II protein dry powder, Figure 3 The display is the Thaumatin II protein sample after freeze-drying.
[0037] The above only for the embodiment of the present application, not therefore limit the patent range of the present application, any equivalent structure or equivalent process transformation, or directly or indirectly used in other related technical field, all the same reason includes in the patent protection scope of the present application.
Claims
1. A method for purifying the sweet protein Thaumatin II, characterized in that, The method includes the following steps: (1) Solid-liquid separation was achieved by high-speed centrifugation, and the supernatant of the fermentation broth containing the sweet protein Thaumatin II, which has been depleted of the mycelium, was obtained from the fermentation broth of Trichoderma reesei. (2) Adjust the pH of the supernatant of the fermentation broth obtained in step (1) to 5-7, treat it with D201 resin to remove some impurities and most of the pigments, and then use activated carbon to remove the pigments remaining in the fermentation broth. (3) The residual activated carbon in the fermentation broth was removed by high-speed centrifugation and three-stage plate and frame filtration to obtain crude extract of Thaumatin II protein. (4) The crude thomatin II protein solution was retained and concentrated using 30kDa and 10kDa membranes to obtain thomatin II protein concentrate. (5) Thaumatin II protein concentrate was freeze-dried to obtain Thaumatin II protein powder.
2. The method according to claim 1, characterized in that, The concentration of activated carbon used in step (2) to remove residual pigments from the fermentation broth is 0.5-1.5%.
3. The method according to claim 1, characterized in that, In step (3), residual activated carbon in the fermentation broth is removed by a three-stage plate and frame filter. The primary filter membrane is a medium-speed qualitative filter paper, the secondary filter membrane is a 1μm filter membrane, and the tertiary filter membrane is a 0.22μm filter membrane.
4. The method according to claim 1, characterized in that, In step (4), a 30kDa membrane is used to retain impurities with a value greater than 30kDa in the crude extract of Thaumatin II protein, resulting in a permeate containing Thaumatin II protein.
5. The method according to any one of claims 1-5, characterized in that, When using a 30kDa membrane to retain impurities, the operating pressure should be controlled at 0.2-0.3MPa and the operating temperature at room temperature.
6. The method according to any one of claims 1-6, characterized in that, After use, the 30kDa membrane pack was cleaned with 0.3M NaOH solution.
7. The method according to claim 1, characterized in that, In step (4), a 10 kDa membrane is used to concentrate the permeate containing Thaumatin II protein to obtain Thaumatin II protein concentrate.
8. The method according to any one of claims 1-8, characterized in that, When using a 10kDa membrane pack for concentration, the operating pressure should be controlled at 0.2-0.3MPa and the operating temperature at room temperature.
9. The method according to any one of claims 1-9, characterized in that, After use, the 10kDa membrane pack was cleaned with 0.3M NaOH solution.