Novel rebaudioside MX and application and biosynthesis method thereof

Abstract

The invention belongs to the technical field of biosynthesis, and particularly relates to novel rebaudioside MX as well as application and a biosynthesis method thereof. A coupled catalytic system of glycosyl transferase UGT94E13 and sucrose synthase AtSUS1 is constructed, and rebaudioside A is used as a substrate to realize directional glycosylation modification, so that the compound-rebaudioside MX with a novel structure is obtained, and the rebaudioside MX has high solubility and good dissolution stability, and has good application prospects in multiple fields of food, beverages, health care products, medicines and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a novel rebaudioside MX and an application and a biosynthesis method thereof. Background Art

[0002] Steviosides are a class of natural sweet compounds that are widely found in the leaves of the Stevia rebaudiana plant. As non-nutritive natural sweeteners, these compounds have attracted attention from the food industry and health field for their high sweetness and low calories. The sweetness of steviosides is usually tens to hundreds of times that of sucrose, and they have good safety, making them an important substance to replace traditional sugars.

[0003] Rebaudioside (Reb) is an important subclass of steviol glycosides, mainly including compounds with similar structures such as RebA, Reb D and Reb M. The molecular structures of these compounds are based on the same anthraquinone skeleton, but there are differences in the number of sugar groups and their connection positions, resulting in certain differences in their solubility, stability and sensory properties.

[0004] Although the properties of rebaudioside compounds are similar overall, they show significant differences in taste. Consumers' increasingly diverse sensory demands for sweeteners have driven research on new steviol glycoside compounds to further optimize their taste performance. In addition, existing rebaudioside compounds have certain differences in solubility and stability. Studies have shown that different rebaudioside compounds can synergistically improve solubility through reasonable compounding, thereby significantly improving compatibility with other food ingredients. These factors have jointly promoted the development of new compounds. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a novel rebaudioside MX compound, whose sweetness multiple is significantly higher than that of sucrose. The present invention also provides a sweetener composition, which comprises a rebaudioside compound and other ingredients, and can be used as a sweetener, a flavoring agent and / or a taste masking agent, so as to improve the applicability and taste of the product. At the same time, the present invention also discloses a process for preparing rebaudioside MX using an engineered bacterium and a biosynthetic method thereof.

[0006] In the first aspect, a novel rebaudioside MX is provided, and the chemical structural formula of the rebaudioside MX is as follows:

[0007]

[0008] In combination with the first aspect, the sweetness of rebaudioside MX is 166±20 times that of sucrose.

[0009] In combination with the first aspect, the aqueous solution of rebaudioside MX remains completely dissolved when the concentration is ≤70% (w / w), and no crystals are precipitated for 30 days under storage conditions of 25±2°C and 4±2°C.

[0010] In a second aspect, an engineered bacterium is provided, wherein the engineered bacterium expresses glycosyltransferase UGT94E13 and sucrose synthase AtSUS1.

[0011] In combination with the second aspect, in some embodiments of the second aspect, the amino acid sequence of the glycosyltransferase UGT94E13 is shown in SEQ ID NO:3.

[0012] In a third aspect, a method for biosynthesizing rebaudioside MX compounds is provided, using the engineered bacteria described in the second aspect to synthesize rebaudioside MX by biocatalytic reaction with rebaudioside A as a substrate.

[0013] In combination with the third aspect, in some embodiments of the third aspect, the substrate concentration of rebaudioside A is 40-100 g / L.

[0014] In a preferred embodiment of the third aspect, the substrate concentration of rebaudioside A is 60 g / L.

[0015] In combination with the third aspect, in some embodiments of the third aspect, the concentration of sucrose is 100-600 g / L.

[0016] In a preferred embodiment of the third aspect, the concentration of sucrose is 400 g / L.

[0017] In combination with the third aspect, in some embodiments of the third aspect, the conditions for the catalytic reaction are: 0.2 g / mL wet bacteria, 60 mM trisodium citrate, 100 mM sodium phosphate buffer at pH 8.0, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose and 60 g / L RA substrate, and the reaction temperature is 40°C.

[0018] In a fourth aspect, a sweetener composition is provided, comprising:

[0019] (a) Rebaudioside MX as described in the first aspect, and / or Rebaudioside MX obtained by the method described in the third aspect;

[0020] (b) optionally, carriers, excipients and / or auxiliary materials acceptable to food science, health care products science or daily chemical products science;

[0021] (c) optionally, other sweeteners or flavoring agents.

[0022] In combination with the fourth aspect, in some embodiments of the fourth aspect, the rebaudioside MX is in a crystalline form or an amorphous form.

[0023] In combination with the fourth aspect, in some embodiments of the fourth aspect, the composition is coated on a carrier, or contained in a carrier.

[0024] In the fifth aspect, there is provided the use of the compound described in the first aspect, or rebaudioside MX obtained by the method of the third aspect, or the sweetener composition of the fourth aspect, for use as a sweetener, flavoring agent and / or masking agent in a product.

[0025] Beneficial effects:

[0026] The present invention provides a novel steviol glycoside compound - rebaudioside MX, which has a sweetness multiple of up to 160 times that of sucrose. Its aqueous solution remains completely dissolved at a concentration of 70% (w / w), and no crystals are precipitated for more than 30 days under storage conditions of 25±2°C and 4±2°C, thereby solving the formula compatibility problem caused by the easy crystallization of traditional steviosides. It can not only meet the needs of high-concentration syrups and low-temperature foods, but also optimize formula design and reduce production costs, and greatly reduce the restrictions on application scenarios in multiple fields such as food, beverages, health products and medicines.

[0027] The present invention also provides a method for preparing rebaudioside MX, by constructing a coupled catalytic system of glycosyltransferase UGT94E13 and sucrose synthase AtSUS1, using rebaudioside A as a substrate to achieve directional glycosylation modification, and obtaining a novel compound-rebaudioside MX. In addition, the rebaudioside MX of the present invention also has low production cost, short production cycle, and does not require highly toxic organic solvents, and meets the green chemistry standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below in conjunction with the accompanying drawings. These drawings are only for illustrating the embodiments of the present invention, rather than for limiting the scope of the present invention.

[0029] Figure 1 This is a comparison chart of HPLC analysis of the biosynthetic reaction products of Example 3.

[0030] Figure 2 This is the negative ion mode result diagram of the mass spectrometry analysis of the reaction product of Example 4.

[0031] Figure 3 This is the result of the mass spectrometry analysis of the reaction product in Example 4 in positive ion mode.

[0032] Figure 4 This is the hydrogen spectrum of the nuclear magnetic resonance spectroscopy analysis of the reaction product of Example 4.

[0033] Figure 5 The carbon spectrum of the nuclear magnetic resonance spectroscopy analysis of the reaction product of Example 4.

[0034] Figure 6 The COSY spectrum of the nuclear magnetic resonance spectroscopy analysis of the reaction product of Example 4.

[0035] Figure 7 HSQC spectrum of the nuclear magnetic resonance spectroscopy analysis of the reaction product of Example 4.

[0036] Figure 8 The HMBC spectrum of the nuclear magnetic resonance spectroscopy analysis of the reaction product of Example 4 is shown in FIG.

[0037] Fig. 9 This is a radar chart for the sensory evaluation test of Rebaudioside MX.

[0038] Fig.10 This is a comparison chart of the dissolution of rebaudioside MX samples under room temperature for 30 days.

[0039] Fig.11 This is a comparison chart of the dissolution of rebaudioside MX samples under 4℃ conditions for 30 days.

[0040] Fig.12 This is a comparison chart of samples dissolved and precipitated from rebaudioside A at room temperature for one day.

[0041] Fig.13 This is a comparison chart of the dissolution of rebaudioside M samples under room temperature for one day. DETAILED DESCRIPTION

[0042] The structure of rebaudioside (Reb) is composed of an anthraquinone core skeleton connected to sugar groups of different numbers and arrangements. These compounds have the characteristics of natural sweetness, high sweetness and low calories, and are an ideal healthy sweetener.

[0043] The existing and more commonly used rebaudioside compounds are Rebaudioside A (Reb A), Rebaudioside D (Reb D) and Rebaudioside M (Reb M). Among them, Reb A is the most common type of rebaudioside, with a high sweetness (about 200 times that of sucrose), but it is often accompanied by an aftertaste or metallic taste, and its taste is relatively limited. Reb A has relatively low solubility in water and poor solubility stability. Crystals will precipitate in a short period of time when stored at room temperature, which limits its use in high-concentration application scenarios; Reb D's sweetness is closer to sucrose, the aftertaste is significantly reduced, and the sensory properties are better than Reb A, but its natural content is low, resulting in higher costs. Reb D has a better taste than Reb A and can be used in a wider range of foods and beverages, but its compatibility with high-sweetness formulas still has room for improvement; Reb M has a pure sweetness and sensory properties closest to sucrose, no obvious aftertaste, but a longer aftertaste. The natural content of Reb M in stevia leaves is extremely low and it is currently the most promising compound among rebaudioside.

[0044] The content of rebaudioside in stevia leaves is limited, and traditional plant extraction methods are difficult to mass produce. In particular, the content of Reb D and Reb M in stevia leaves is very low, the extraction efficiency is low, and the economy is poor. Therefore, large-scale production mainly relies on biosynthesis technology. For example, Reb A is used as a substrate, and Reb D or Reb M is prepared by catalyzing the glycosylation reaction of engineered bacteria (such as Escherichia coli, yeast or Pichia pastoris) expressing glycosyltransferase.

[0045] Although Reb A, Reb D and Reb M occupy an important position in the sweetener market, they still have defects such as taste differences, solubility limitations and insufficient development of combination potential. Therefore, the discovery of new rebaudioside compounds can effectively improve the utilization rate and product quality of these compounds by fully exploring the differences in the properties of different rebaudioside compounds and optimizing their sensory properties and solubility. In addition, the development of new compounds can further broaden the combination design space of sweeteners, achieve synergistic effects through reasonable compounding, thereby meeting the market's diverse needs for high-performance natural sweeteners, and promoting technological progress and sustainable development of the sweetener industry.

[0046] Based on this, the present invention provides a new type of rebaudioside, named as rebaudioside MX, which has no special meaning and is self-named. The molecular formula of the rebaudioside MX is C 56 H 90 O 33 , the chemical structure is as follows:

[0047]

[0048] In combination with the first aspect, the sweetness of rebaudioside MX is 166±20 times that of sucrose.

[0049] In combination with the first aspect, the aqueous solution of Rebaudioside MX remains completely dissolved when the concentration is ≤85% (w / w).

[0050] It remains completely dissolved at a concentration of ≤70% (w / w) and can be stored at 25±2°C and 4±2°C for 30 days without crystal precipitation.

[0051] The second aspect of the present invention provides an engineered bacterium, wherein the engineered bacterium expresses glycosyltransferase UGT94E13 and sucrose synthase AtSUS1.

[0052] Among them, the glycosyltransferase UGT94E13 is derived from Gardenia jasminoides. The amino acid sequence of the glycosyltransferase UGT94E13 is shown in SEQ ID NO: 3:

[0053] MKVLMLPWLAHGHISPFLELAKRLAKKNFHIYLCSTSVNLSSIKNKITGEYSDSIEPVELQLPCLPDLPPHYHTTNGLPPHLMTTLKTAYELSAPDFSNILTALHPDLVVY DFNQPWAAEIASSKNIPAVQFLPVGATMMAFSLHMLKYSGKEFPHPEIYIRDYEMLKGQSRNDQVNDVSDRERGLQALDLSCKILLVKSFKEIEEKFMNTLSVASGKKVVPV GPLVQDVNIDDIQDEEMEIIHWLDQKENASVVFVSFGSEYFLTKEERNEIARGLELSNVNFIWVIRFPLGGKITMEEALPEGFLERVGDRGKIVDGWAPQARILKHANTGAF LSHCGWSSMMESMKFGVPIIAMPMSVDQPVNARLIEAVGVGLEPLRDEKGNLQSAEIAKVIRKVLVDESGENVRRKAKELSEQMEMRGDEEEIDNLVEELLQLCRKNNGGC;

[0054] Sucrose synthase AtSUS1 is derived from Arabidopsis thaliana. The amino acid sequence of the sucrose synthase AtSUS1 is shown in SEQ ID NO: 4:

[0055] ;

[0056] In some embodiments of the second aspect, the host cell of the engineered bacteria is Escherichia coli; in a preferred embodiment, the Escherichia coli is BL21 (DE3) or JM109.

[0057] In some embodiments of the second aspect, the expression vector of the engineered bacteria is pET series, pCDFDuet, etc.; in a preferred embodiment, the expression vector is pETDuet-1.

[0058] The third aspect of the present invention provides a method for biosynthesizing rebaudioside compounds, using the engineered bacteria described in the second aspect to synthesize rebaudioside MX by biocatalytic reaction with rebaudioside A as a substrate.

[0059] In the third aspect, the engineered bacteria described in the second aspect are used, and the bacteria are resuspended and washed at least once with 100 mM sodium phosphate buffer at pH 8.0.

[0060] In combination with the third aspect, in some embodiments of the third aspect, in the biocatalytic reaction system, the concentration of wet bacteria is preferably 0.05-0.3 g / mL, for example 0.2 g / mL;

[0061] The concentration of trisodium citrate is preferably 40-200 mM, such as 60 mM;

[0062] Mg 2+ The concentration is preferably 0.01-0.5 mM, for example 0.1 mM;

[0063] The sucrose concentration is preferably 100-600 g / L, for example 400 g / L;

[0064] The RA substrate concentration is preferably 40-100 g / L, such as 60 g / L;

[0065] The xylene concentration is preferably 0.05%-5%, for example 1%;

[0066] The reaction temperature is preferably 30-60°C, for example 40°C;

[0067] The reaction pH is preferably 6.0-10.0, such as 8.0.

[0068] In a preferred embodiment of the third aspect, the conditions for the catalytic reaction are: 0.2 g / mL wet bacteria, 60 mM trisodium citrate, 100 mM sodium phosphate buffer at pH 8.0, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose and 60 g / L RA substrate, and the reaction temperature is 40°C.

[0069] In a preferred embodiment, the conditions for the catalytic reaction are: 0.2 g / mL wet bacteria, 60 mM trisodium citrate, 100 mM sodium phosphate buffer at pH 8.0, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose and 60 g / L RA substrate, and the reaction temperature is 40°C.

[0070] In combination with the third aspect, after the catalytic reaction is completed, an equal volume of methanol is added to quench, and the precipitate is removed by centrifugation at 12,000 rpm for 10 min, and the supernatant is taken to obtain a reaction product containing rebaudioside MX. The obtained reaction product can be further separated, dried, purified, etc. to obtain the desired rebaudioside MX.

[0071] The fourth aspect of the present invention provides a sweetener composition comprising:

[0072] (a) Rebaudioside MX according to claim 1, and / or Rebaudioside MX obtained by the method according to any one of claims 4 to 5;

[0073] (b) optionally, carriers, excipients and / or auxiliary materials acceptable to food science, health care products science or daily chemical products science;

[0074] (c) optionally, other sweeteners or flavoring agents.

[0075] In (a) of the fourth aspect, rebaudioside MX, as the core component of the composition of the present invention, has a sweetness multiple significantly higher than that of sucrose, and can provide a strong sweetness perception at a low addition amount, which not only helps to reduce the amount of sugar used in the product, reduce calorie intake, but also effectively control production costs. At the same time, its superior sensory properties are manifested in pure sweetness, refreshing aftertaste, no bitterness or other undesirable aftertaste, and can bring a pure sweetness experience to consumers.

[0076] In (b) of the fourth aspect, the carrier may be a common food-grade carrier such as starch, dextrin, cyclodextrin, etc., which can provide a medium for the attachment and dispersion of rebaudioside MX and other ingredients, and help improve the stability and solubility of the composition; the excipient may be microcrystalline cellulose, lactose, mannitol, etc., which can adjust the shape and texture of the composition, making it easier to process and use; auxiliary materials such as acidity regulators such as citric acid and malic acid can be used to adjust the pH value of the composition, enhance the stability and flavor coordination of the product; antioxidants such as vitamin C, vitamin E, etc. can prevent the ingredients in the composition from oxidative deterioration during storage and use, and extend the shelf life of the product.

[0077] In (c) of the fourth aspect, the other sweeteners or flavoring agents are selected from functional additives such as natural sweeteners, synthetic sweeteners and flavoring agents, which give the composition more functional properties.

[0078] Natural sweeteners such as allulose, erythritol, glycyrrhizin, etc. are of natural origin and low in calories. When used in combination with rebaudioside MX, they can further enrich the layering and taste of sweetness while maintaining low calories. Synthetic sweeteners such as sucralose, acesulfame potassium, aspartame, etc. have the advantages of high sweetness and low cost. Adding them in appropriate amounts can synergize with rebaudioside MX to enhance the sweetness intensity and reduce the overall cost. Flavoring agents such as spices and acidulants improve the taste of the composition, reduce possible unpleasant tastes, and make the sweetness softer and more natural. The reasonable combination of these functional additives can enable the composition to meet the sweetness needs of different consumers while also having good taste and stability, expanding its application in food, health products, and daily chemical products.

[0079] In combination with the fourth aspect, in some embodiments of the fourth aspect, the rebaudioside MX is in a crystalline form or an amorphous form.

[0080] Rebaudioside MX has high purity and stability in crystalline form, which is convenient for storage and transportation, and its physical properties such as melting point and solubility can be accurately characterized. The amorphous form of Rebaudioside MX has a higher dissolution rate and better solubility, and is suitable for beverages or instant products with high solubility requirements. During the preparation process, the production of amorphous form can be achieved through technologies such as spray drying or freeze drying, which gives it good processing performance and application flexibility.

[0081] In combination with the fourth aspect, in some embodiments of the fourth aspect, the composition is coated on a carrier, or contained in a carrier.

[0082] In some preferred embodiments, rebaudioside MX can be uniformly coated on the surface of a carrier material, such as silicon dioxide, cyclodextrin or edible polymer, by microencapsulation or other coating techniques. This coating form can effectively improve the stability of the compound, prevent the external environment (such as humidity, light or temperature) from affecting its performance, and extend the shelf life;

[0083] In some preferred embodiments, rebaudioside MX can be contained in a carrier material by adsorption, embedding or embedding, etc. For example, by combining it with a soluble carrier (such as lactose or starch derivatives) through blending technology, its dispersibility and processing adaptability can be further improved, making it easier to use in powder formulations.

[0084] In the fifth aspect, there is provided the use of the compound described in the first aspect, or rebaudioside MX obtained by the method of the third aspect, or the sweetener composition of the fourth aspect, for use as a sweetener, flavoring agent and / or masking agent in a product.

[0085] In conjunction with the fifth aspect, the products include but are not limited to food, beverages, condiments, daily chemical products, pharmaceutical components, nutritional health products, and cosmetics.

[0086] In the food field, it can be used in various candies, cakes, dairy products, condiments and ready-to-eat foods. It not only provides pure sweetness, but also effectively reduces calorie intake and meets consumers' demand for healthy food.

[0087] In the beverage field, it can be widely used in carbonated beverages, fruit juice drinks, functional drinks and tea drinks. Its high sweetness and low calorie properties make it an ideal sugar substitute. It is suitable for beverage formulas that need to maintain a pure taste but control calories.

[0088] In the field of pharmaceutical ingredients, it can be used as a taste masking agent in pharmaceutical preparations, not only providing sweetness to mask the bitterness of the drug, but also improving the patient's taking experience. In addition, its natural origin and low toxicity make it suitable for strict drug safety standards.

[0089] In the field of nutritional health products, its natural sweetness can significantly improve the taste and consumer acceptance of the product. At the same time, it has good stability when combined with other nutritional ingredients and is suitable for use in various forms of health products such as functional beverages, tablets, powders and capsules.

[0090] In the field of daily chemical products, it can be used in oral cleaning products (such as toothpaste and mouthwash), skin care products and other personal care products to enhance the comfort of product use by giving a light and sweet sensory experience.

[0091] Through these wide applications, the rebaudioside MX or sweetener composition of the present invention has important promotion value in the healthy, environmentally friendly and high-performance sweetener, flavor corrective and / or taste masking agent market, and provides a new solution for the development of related industries.

[0092] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0093] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the present technical field.

[0094] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0095] Material:

[0096] White sugar, purchased from COFCO Sugar Liaoning Co., Ltd.;

[0097] Rebaudioside A (RA) was purchased from Guilin Rhine Biotechnology Co., Ltd. with a purity of 97%;

[0098] Rebaudioside M (RM) is produced by Sichuan Yingjia Hesheng Technology Co., Ltd., batch number 20241203BM95D, with a purity of 95%.

[0099] Example 1 Construction of recombinant expression vector

[0100] 1. Cloning of glycosyltransferase and sucrose synthase genes

[0101] This example uses glycosyltransferase UGT94E13 and sucrose synthase AtSUS1, wherein the glycosyltransferase UGT94E13 is derived from Gardenia jasminoides. The amino acid sequence of the glycosyltransferase UGT94E13 is shown in SEQ ID NO: 3:

[0102] Sucrose synthase AtSUS1 is derived from Arabidopsis thaliana. The amino acid sequence of the sucrose synthase AtSUS1 is shown in SEQ ID NO: 4:

[0103] Glycosyltransferase UGT94E13 and sucrose synthase AtSUS1 were codon optimized for Escherichia coli, and the full-length DNA sequence was synthesized. The nucleotide sequence of glycosyltransferase UGT94E13 after codon optimization can be shown as SEQ ID NO: 1:

[0104]

[0105] The nucleotide sequence of the sucrose synthase AtSUS1 after codon optimization may be shown as SEQ ID NO: 2:

[0106]

[0107] 2. Construction of recombinant expression vector

[0108] Using pETDuet-1 as the expression vector, the In-Fusion strategy was used to construct a recombinant expression plasmid. PCR amplification primers containing homology arms were designed (see Table 1) to clone UGT94E13 and the linearized pETDuet-1 vector. According to the instructions of the kit, the DNA fragment and the linearized vector fragment were subjected to homologous recombination reaction. After reacting at 50 degrees for 1 hour, the competent cells of E. coli DH5α were transformed, and the Amp-resistant plates were coated and cultured at 37 degrees overnight. Positive clones were screened by colony PCR, and positive clones were picked in 5 mL of LB medium containing Amp for sequencing verification. The plasmid with correct sequencing was extracted to obtain the recombinant plasmid pETDuet-1-UGT94E13.

[0109] Using the same scheme as above, PCR amplification primers containing homology arms were designed (see Table 1) to clone AtSUS1 and the linearized pETDuet-1-UGT94E13 recombinant vector, and the recombinant plasmid pETDuet-1-UGT94E13-AtSUS1 was obtained using the same homologous recombination strategy as above.

[0110] Table 1. Primer sequence list

[0111]

[0112] Example 2 Construction of engineered bacteria

[0113] The recombinant plasmid pETDuet-1-UGT94E13-AtSUS1 constructed in Example 1 was transferred into E. coli BL21 (DE3) to obtain a recombinant strain, which was frozen in a -80°C refrigerator. The frozen strain was inoculated into 5-10 mL of LB medium containing Amp resistance and cultured at 37°C overnight to prepare a fermentation seed solution; the seed solution was inoculated into 50 mL of TB medium according to a 1% inoculation amount, and cultured at 200 rpm and 37°C to OD600-0.6-0.8, 0.1 mM IPTG was added, and expression was induced at 22°C for 24 hours, and then centrifuged at 12,000 rpm and 4°C to collect the bacterial cells to obtain the engineered bacteria, which were stored at -20°C for use.

[0114] Example 3 Biosynthesis of Rebaudioside MX

[0115] The engineered bacterial cells constructed in Example 2 were resuspended and washed twice with 100 mM sodium phosphate buffer at pH 8.0 and then used for whole-cell catalytic reaction.

[0116] The biotransformation reaction system was: 0.2 g / mL wet bacteria, 60 mM trisodium citrate, 100 mM sodium phosphate buffer at pH 8.0, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose and 60 g / L RA substrate, the reaction temperature was 40°C, and the reaction time was 24 h.

[0117] When the catalytic reaction is completed, an equal volume of methanol is added for quenching, and the mixture is centrifuged at 12,000 rpm for 10 min to remove the precipitate, and the supernatant is taken to obtain the reaction product containing rebaudioside MX.

[0118] HPLC confirmatory analysis

[0119] The supernatant was taken and filtered through a 0.22 μm filter membrane to obtain a sample for HPLC analysis. The HPLC detection conditions were: isocratic system, organic phase-water phase: 33% acetonitrile-67% phosphoric acid water (pH 3.0), UV detection wavelength of 210 nm, column temperature of 40°C.

[0120] The HPLC results showed Figure 1 As shown, Figure 1 In the figure, B is the control sample - rebaudioside A, and the arrow points to the newly generated product. Figure 1 Comparative analysis with the rebaudioside A standard showed that obvious new products were generated in the reaction system.

[0121] Example 4 Structural Identification of Novel Rebaudioside MX

[0122] 1. Preparation and purification of reaction products

[0123] The whole-cell catalytic reaction was carried out in the same manner as in Example 3 to prepare a novel rebaudioside compound. After the catalytic reaction was completed, the reaction solution was heated at 95° C. for 10 min to quench the reaction, an equal volume of methanol was added to precipitate the protein, and the precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane and purified using a semi-preparative high performance liquid chromatography system to prepare a purified reaction product.

[0124] HPLC conditions were: isocratic system, organic phase-aqueous phase: 33% acetonitrile-67% phosphoric acid water (pH 3.0), UV detection wavelength at 210 nm, column temperature at 40°C.

[0125] 2. High-resolution mass spectrometry

[0126] The purified reaction product was analyzed by high resolution mass spectrometry (HRMS). Figure 2 and Figure 3 As shown, Figure 2 The results are for negative ion mode. Figure 3 The results are in positive ion mode.

[0127] The negative ion mode results showed: [MH] at m / z 1290.52713, 1289.52389 - The peak of the ion and its isotope peak; the positive ion mode results show: [M+Na] at m / z1314.59715,1313.59354 - The peak of the ion and its isotope peak, corresponding to the molecular formula C 56 H 90 O 33 , indicating that the product is a disaccharide derivative of rebaudioside A.

[0128] 3. Nuclear Magnetic Resonance Analysis

[0129] The purified reaction product was dissolved in deuterated pyridine and purified by 1D ( f1 H and 13 C) and 2D NMR (COSY, HSQC and HMBC) spectroscopy were used to analyze the complete structure of the product.

[0130] Data were collected using a Bruker Avance III 400 MHz spectrometer. 1 The H spectrum detection frequency is 400MHz. 13 The C spectrum is 151MHz. 1 H-NMR has typical signal characteristics of steviol glycosides. The peak with chemical shift less than 2.8ppm comes from terpene aglycones, and the peak with chemical shift from 3.6-6.0ppm comes from sugar rings. 1 H and 1 H- 13 C HSQC spectrum results show that δ H 5.88(δ C 95.72),δ H 5.45(δ C 104.84),δ H 5.14(δ C 104.41),δ H 5.00(δ C 98.40),δ H 4.96(δ C 104.03),δ H 4.81(δ C 103.82) has 6 abnormal protons, confirming that there are 6 sugar units in the structure of the product. H 5.88(8.2Hz),δ H 5.45(7.8Hz),δ H 5.14(7.8Hz),δ H5.00(8.2Hz),δ H 4.96(7.6Hz),δ H The high J dipole moment observed at 4.81 (7.6 Hz) indicates that all six glucose residues are in β configuration. The chemical shifts of H and C of the new derivatives were assigned in detail by 1D and 2D NMR. The results are shown in Figure 4-Figure 8 As shown, and summarizes its 1 H and 13 The C chemical shift assignments are shown in Table 2.

[0131] The new product was determined to be the core diterpene structure of rebaudioside A, with a glucose group each connected to the glucose groups at C13 and C19 via a β-1,6-bond, and was named rebaudioside MX.

[0132] Table 2. 1H and 13C chemical shift assignments (pyridine-d5)

[0133]

[0134]

[0135] Example 5 Determination of the sweetness multiple of Rebaudioside MX

[0136] In this example, the sample rebaudioside MX is the purified reaction product prepared in Example 4, and the purity is 95%.

[0137] Take the Rebaudioside MX samples and prepare them into aqueous solutions with concentrations of 0.0176%, 0.0188%, 0.02%, 0.0214%, and 0.0231%, respectively. Ask the sensory testers to compare the sweetness of the Rebaudioside MX solutions of each concentration with the 3% sucrose solution (control sample) and select the solution that they think is sweeter (only compare the sweet peak). Count the results of the sweetness comparison between different Rebaudioside MX concentrations and the control sample, record it as a, and the number of sensory testers is recorded as b, and calculate the ratio A of the sweetness of different Rebaudioside MX concentrations that is stronger than the sweetness of the control sample.

[0138]

[0139] A sweetness intensity ratio scatter plot is drawn with the x-axis as the concentration of Rebaudioside MX and the y-axis as A, and a fitting equation is obtained. A=50% (i.e., y=50%) is substituted into the fitting equation to obtain LD50, i.e., the concentration of the Rebaudioside MX solution when 50% of the personnel cannot correctly distinguish the sweetness intensity of a certain concentration of Rebaudioside MX solution from that of the control sample. It is generally believed that the sweetness of the Rebaudioside MX solution at this concentration is consistent with that of the control sample. The sweetness multiple of Rebaudioside MX is calculated according to the following formula.

[0140]

[0141] The fitting equation is the linear regression equation y=4621.7x-0.3327, the LD50 is 0.0180%, and the sweetness multiple of rebaudioside MX is converted to 166 times.

[0142] Through statistical analysis of the data, it was found that the sweetness multiple of Rebaudioside MX was 166±20 times.

[0143] Example 6 Sensory Evaluation Test of Rebaudioside MX

[0144] The sensory panel for this evaluation consisted of 12 people (4 men and 8 women), and the sensory panel members were screened, tested, trained and evaluated. The screening and selection process included physical health status, eating habits, basic taste recognition, basic smell recognition, taste matching, gradient recognition, difference detection and numerical estimation, etc. In addition, after completing the descriptive training, the panel members were able to use professional descriptive vocabulary to describe the stevia samples relatively accurately.

[0145] The test sample rebaudioside MX used in the sensory evaluation experiment was the purified reaction product prepared in Example 4; and a control was set, and the control was rebaudioside M (RM).

[0146] Using purified water, the test samples and controls were prepared into samples with a concentration of 400 ppm. 10 mL of sample solution was taken and provided to the panel members in turn, and randomly coded with three-digit numbers. The order of presentation of the samples was randomized to avoid bias caused by the presentation order. Clean water and unsalted crackers were provided to cleanse the taste buds.

[0147] Panelists were asked to rate different attributes of the sample solutions, including sweetness onset, sweetness peak, bitterness, astringency, sweetness aftertaste and bitterness aftertaste, and overall taste. The experiment used a linear scale as the scoring scale, with a scoring range of 0-10, that is, a 10cm line segment was marked to represent the position that represents the intensity of the taster's perception of each attribute, with 0 at the far left of the line segment, representing "no perception", and 10 at the far right of the line segment representing "strongest", where 0 represents very delayed sweetness onset, no intensity, no sweetness aftertaste, and low bitterness, while 10 represents fast sweetness onset, high sweetness peak, long sweetness aftertaste and strong bitterness.

[0148] In the evaluation, the sweetness is based on an 8% sucrose aqueous solution by mass fraction. The sweetness is equal to 8% sucrose, and 10 points, and no sweetness is detected at all, 0 points. The comprehensive evaluation is given 0-10 points based on the overall taste, 10 points represents the taste of 8% sucrose, and bitterness, astringency and other miscellaneous tastes are deducted points. Miscellaneous tastes refer to other unpleasant tastes besides sweetness, bitterness and astringency, such as alcohol, plastic, metal, licorice, chemical and other unpleasant tastes. When tasting, ensure that the sweetness of each sample is basically the same, and compare their other tastes besides sweetness, such as bitterness, miscellaneous tastes, etc.

[0149] The evaluation results are as follows Fig. 9 As shown, combined Fig. 9 From the analysis results, it can be seen that the new Rebaudioside MX compound has a relatively excellent taste (Rebaudioside M has the sensory properties closest to sucrose, without obvious aftertaste, and the new Rebaudioside MX of the present invention has a hook close to Rebaudioside M), which provides important information for the development of new stevioside natural sweeteners.

[0150] Example 7 Solubility Test of Rebaudioside MX

[0151] In this example, the test sample rebaudioside MX adopts the purified reaction product prepared in Example 4.

[0152] At room temperature, pure water was used to prepare solutions of rebaudioside MX with mass concentrations of 70%, 50%, 25%, 10%, and 5%, respectively, and the solubility stability tests were carried out at room temperature and 4°C, respectively. The results are shown in Table 3 and Fig.10 , 11 shown.

[0153] Table 3 Dissolution of MX under room temperature and refrigeration conditions for 30 days

[0154] Serial number concentration% Crystal precipitation at room temperature Crystal precipitation at 4°C 1 70 No crystals precipitated after 30 days No crystals precipitated after 30 days 2 50 No crystals precipitated after 30 days No crystals precipitated after 30 days 3 25 No crystals precipitated after 30 days No crystals precipitated after 30 days 4 10 No crystals precipitated after 30 days No crystals precipitated after 30 days 5 5 No crystals precipitated after 30 days No crystals precipitated after 30 days

[0155] And set up control examples, control example 1 is rebaudioside A (RA), and control example 2 is rebaudioside M (RM).

[0156] At room temperature, pure water was used to prepare solutions of 0.1%, 0.3%, 0.5%, 0.7%, 1%, 2%, 3%, 4%, and 5% of the control example 1-Rebaudioside A (RA), and the solubility stability test was carried out at room temperature. The results are as follows Fig.12 shown.

[0157] At room temperature, pure water was used to prepare solutions of 0.06%, 0.12%, 0.15%, 0.2%, 0.3%, 0.4%, and 0.5% mass concentration of control example 2-Rebaudioside M (RM), and the solubility stability test was carried out at room temperature. The results are as follows Fig.13 shown.

[0158] From Example 7 and Comparative Examples 1 and 2, it can be seen that the solubility of the new rebaudioside MX is significantly better than that of the commonly used rebaudioside A and rebaudioside M. The solubility of rebaudioside A at room temperature is only 1%, and the solubility of rebaudioside M is even lower, only 0.15%, while rebaudioside MX can remain clear and transparent at a concentration of up to 70%, and no crystals are precipitated after storage at room temperature and 4°C for 30 days. This feature brings many advantages to the application of rebaudioside MX, especially in greatly reducing the restrictions on application scenarios.

[0159] For example, the high solubility of Rebaudioside MX enables it to be easily used in high-concentration syrups and concentrated beverages without worrying about crystallization or precipitation. Traditional sweeteners (such as Rebaudioside A and M) tend to precipitate crystals at high concentrations, affecting the appearance and taste of the product, while Rebaudioside MX remains stable at 70% concentration, greatly expanding its application in concentrated syrups, functional beverages and energy drinks.

[0160] Rebaudioside MX has no crystals after being stored at 4°C for 30 days, indicating that it has excellent stability in low temperature environments. This property makes it very suitable for use in refrigerated or frozen foods, while traditional sweeteners are prone to crystallization problems at low temperatures. These advantages make Rebaudioside MX expected to be widely used in more innovative products.

[0161] Example 8 Sweetener Composition and Application

[0162] The composition of the present invention can be used to prepare various foods, beverages, condiments, daily chemical products, pharmaceutical components, nutritional health products, and cosmetics.

[0163] 1. High concentration syrup

[0164] Rebaudioside MX: 0.1%-0.5%

[0165] Water: 70%-80%

[0166] Glycerin: 5%-10%

[0167] Citric acid: 0.1%-0.3%

[0168] Natural flavor - caramel: appropriate amount.

[0169] The high-concentration syrup is used as a flavoring syrup for beverages such as coffee, milk tea, and cocktails.

[0170] 2. Sweeteners for ice cream

[0171] Rebaudioside MX: 50%-70%

[0172] Erythritol: 20%-40%

[0173] Maltodextrin: 5%-10%

[0174] Natural vanilla extract: 0.1%-0.5%.

[0175] The ice cream sweetener is used for frozen desserts such as ice cream and frozen yogurt, and is added at 0.1%-0.5% of the total weight of the ice cream to provide sweetness and improve the taste. The sweetener has high solubility and low-temperature stability and is suitable for frozen foods.

[0176] 3. Sweeteners for functional beverages

[0177] Rebaudioside MX: 40%-60%

[0178] Sucralose: 10%-20%

[0179] Sodium citrate: 5%-10%

[0180] Vitamin C: 1%-5%.

[0181] The functional beverage sweetener is added at 0.05%-0.2% of the total weight of the beverage to provide sweetness and enhance functionality.

[0182] 4. Sweeteners for daily chemical products

[0183] Rebaudioside MX: 50%-70%

[0184] Sorbitol: 20%-30%

[0185] Mint flavor: 1%-5%

[0186] Silicon dioxide: 1%-5%.

[0187] The sweetener for daily chemical products is added at 0.05%-0.1% of the total weight of the daily chemical products, is suitable for products such as toothpaste and lip balm, provides sweetness and improves user experience.

[0188] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A novel rebaudioside MX, characterized in that: The chemical structural formula of rebaudioside MX is as follows:

2. A novel rebaudioside MX according to claim 1, characterized in that: The aqueous solution of rebaudioside MX remains completely dissolved when the concentration is ≤70% (w / w), and no crystals are precipitated for 30 days under storage conditions of 25±2° C. and 4±2° C.

3. An engineered bacterium for synthesizing rebaudioside MX, characterized in that: The engineered bacteria express glycosyltransferase UGT94E13 and sucrose synthase AtSUS1.

4. An engineered bacterium for synthesizing rebaudioside MX, characterized in that: The amino acid sequence of the glycosyltransferase UGT94E13 is shown in SEQ ID NO:

3.

5. A method for biosynthesizing rebaudioside MX, characterized in that: Rebaudioside MX is synthesized by biocatalytic reaction using the engineered bacteria described in any one of claims 3 to 4, sucrose as a glycosyl donor and rebaudioside A as a substrate.

6. A method for biosynthesizing rebaudioside MX according to claim 5, characterized in that: The conditions for the catalytic reaction were: 0.2 g / mL wet bacteria, 60 mM trisodium citrate, 100 mM sodium phosphate buffer at pH 8.0, 0.1 mM MgCl 2, 1% (v / v) xylene, 400 g / L sucrose and 60 g / L RA substrate, and the reaction temperature was 40°C.

7. A sweetener composition, characterized in that contain: (a) Rebaudioside MX according to claim 1, and / or Rebaudioside MX obtained by the method according to any one of claims 5 to 6; (b) optionally, carriers, excipients and / or auxiliary materials acceptable to food science, health care products science or daily chemical products science; (c) optionally, other sweeteners or flavoring agents.

8. The sweetener composition according to claim 7, characterized in that The rebaudioside MX is in a crystalline form or an amorphous form.

9. The sweetener composition according to claim 7, characterized in that The composition is coated on a carrier or contained in a carrier.

10. Use of the compound according to claim 1, or rebaudioside MX obtained by the method of any one of claims 5-6, or the sweetener composition according to claims 7-9 as a sweetener, flavoring agent and / or taste masking agent in a product.

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