Lactobacillus mari strains and their uses

Abstract

This invention relates to a novel Lactobacillus mari strain exhibiting excellent glucose transfer activity and a method for producing glucose-transferable steviol glycosides using the same. The novel Lactobacillus mari CJST01 strain exhibits superior steviol glycoside transfer activity compared to Lactobacillus mari DSM20444 strain, which is known to produce glucose-transferable steviol glycosides. This results in higher productivity and a shorter enzymatic reaction time in the manufacturing process. The steviol glycosides produced using this strain have the advantage of superior taste quality and aroma compared to alpha-1,4 linked steviol glycosides and rebaudioside M. Furthermore, glucose-transferable rebaudioside A produced using Lactobacillus mari CJST01 strain exhibits reduced off-flavors and odors and superior solubility.

Description

Technical Field

[0001] [Cross - reference to Related Applications]

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10 - 2023 - 0066300, filed on May 23, 2023, and all the contents disclosed in the literature of the Korean patent application are incorporated herein by reference in their entirety.

[0003] The present invention relates to a novel Lactobacillus mali strain having excellent ability to produce glucose - transferred steviol glycoside and a method for producing glucose - transferred steviol glycoside using the same.

Background Art

[0004] Due to the risk of diseases (such as obesity) caused by sugar intake according to the World Health Organization (WHO)'s advice to reduce the daily intake, policies to reduce various sugar intakes are actively being discussed mainly in developed countries under the government's initiative. As a result, the market demand for various alternative sweetener materials is increasing, and alternative sweetener materials are being continuously developed and commonly used. Currently, synthetic high - intensity sweeteners (such as Saccharin, Aspartame, Sucralose, etc.) are mainly used as alternative sweeteners to replace sugar. However, as concerns about the safety of synthetic sweeteners continue to be raised, consumer demands for the development of natural alternative sweeteners are increasing.

[0005] Stevia, a recently spotlighted natural high - intensity sweetener, refers to a sweetener derived from the leaves of the plant Stevia rebaudiana Bertoni. Steviol glycoside, known as the main sweet component, has a sweetness about 200 - 400 times that of sugar for each component, and Rebaudioside A, Rebaudioside D, and Rebaudioside M are mainly used as sweeteners for food and beverages. However, Rebaudioside A has a peculiar bitter taste, and Rebaudioside D and Rebaudioside M have the disadvantages of low solubility, high price, and limited application, so their use is restricted.

[0006] To address these issues, glucose-transferred rebaudioside A produced using Lactobacillus malii has been studied. This rebaudioside A has a structure in which 1 to 4 glucose molecules are linked via alpha-1,6. It has been confirmed that it exhibits improved bitterness and superior sweetness preference compared to rebaudioside A, which is a steviol glycoside, thus demonstrating improved sensory characteristics.

[0007] Lactobacillus malii is a microorganism found in fermented foods such as cider and wine. The inventors completed this application by confirming that the newly discovered Lactobacillus malii strain CJST01, extracted from various fermented foods, is significantly superior to the Lactobacillus malii strain DSM20444, which is known to produce glucose-transferable steviol glycosides. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] One example of the present invention is the provision of the Lactobacillus mari CJST01 strain.

[0009] Another example of the present invention provides a method for producing glucose-transferable steviol glycosides using the aforementioned bacterial strain.

[0010] Another example of the present invention provides glucose-transferable steviol glycosides produced by the above-described method.

[0011] Another example of the present invention provides a sweetener containing the glucose-transferred steviol glycoside described above.

[0012] Another example of the present invention provides a composition for producing glucose-transferable steviol glycosides, comprising the aforementioned bacterial strain or its culture.

[0013] Another example of the present invention is the use of glucose-transferable steviol glycosides and / or glucose-transferable steviol glycosides of Lactobacillus malii CJST01 strain or its culture for the production of sweeteners.

[0014] Another example of the present invention is the use of compositions containing the aforementioned strain or culture thereof for the production of glucose-transferable steviol glycosides and / or sweeteners containing glucose-transferable steviol glycosides. [Means for solving the problem]

[0015] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not considered to be limited by the specific descriptions described later. Moreover, a person with ordinary skill in the art can recognize or confirm a number of equivalents to the particular aspects of this application described in this application using only ordinary experiments. Such equivalents are intended to be included in this application.

[0016] This application provides a novel Lactobacillus maruli strain. The novel Lactobacillus maruli strain may be a strain that has the ability to produce glucose-transferable steviol glycosides.

[0017] In this application, as an example, in order to identify a strain with superior glucose-transferable steviol glycoside production ability, a crude enzyme solution was obtained from a culture medium cultured after homogenizing fermented food, and its stevioside glycosyltransferase activity was confirmed. As a result, a Lactobacillus mari strain with superior stevioside glycosyltransferase activity was selected and named Lactobacillus mari CJST01 strain. This strain was deposited with the Korean Collection for Type Cultures (KCTC), an international depositary organization under the Budapest Convention, on May 8, 2023, and was assigned accession number KCTC15433BP.

[0018] Therefore, the newly identified Lactobacillus mali strain may be the Lactobacillus mali CJST01 strain deposited under accession number KCTC15433BP.

[0019] The aforementioned Lactobacillus mari CJST01 strain possesses the 16S rRNA of Sequence ID No. 1.

[0020] In this application, the term "steviol glycoside" refers to a natural sweetener having a form in which glucose, rhamnose, xylose, etc., are bonded to the 13th and 19th-OH groups of chemical formula 1 (steviol).

[0021] [Chemical formula 1] [ka]

[0022] Generally, in chemical formula 1, R1 may be bonded to hydrogen (H) or to 1 to 3 glucose molecules via β-bonds, and R2 may be bonded to one glucose, xylose, or rhamnose molecule via β-bonds, with 0 to 2 glucose molecules further bonded via β-bonds, but is not limited to these.

[0023] α- / β-glycosidic bonds are distinguished by the anomeric position and the relative stereochemistry (R-type or S-type) of the stereocenter furthest from the first carbon atom of the monosaccharide. Generally, α-glycosidic bonds are formed when two carbon atoms have the same stereochemistry, while β-glycosidic bonds are formed when two carbon atoms have different stereochemistry.

[0024] The term "glucosyltransferase steviol glycoside" in the present application may refer to a form in which one to nine glucose molecules are added to the 19-OH position of steviol glycoside via an α-linkage using sugar and steviol glycoside as substrates by Lactobacillus mali strains. More specifically, it may refer to a form in which one to nine glucose molecules are added to the glucose molecule bound to the 19-OH position of steviol glycoside via an α-(1,6) linkage, but is not limited thereto.

[0025] In the present application, the steviol glycoside may be any one selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, dulcoside A, steviolbioside, and rubusoside, but is not limited thereto.

[0026] The Lactobacillus mali CJST01 strain may be excellent in the ability to produce glucosyltransferase steviol glycoside compared to known Lactobacillus mali strains.

[0027] The term "glucosyltransferase steviol glycoside production ability" in the present application means the ability to transfer glucose from a glucose donor to steviol glycoside to produce glucosyltransferase steviol glycoside, and may be used interchangeably with "glucosyltransferase steviol glycoside productivity" or "glucosyltransferase steviol glycoside conversion rate". The glucosyltransferase steviol glycoside production ability, productivity, or conversion rate can be confirmed by a reducing sugar quantification method (DNS method), HPLC analysis method, or LC-MS analysis method by reacting a crude enzyme solution obtained after culturing the strain with steviol glycoside and sugar, but is not limited thereto.

[0028] In an example of the present application, it was confirmed that the Lactobacillus mali CJST01 strain has a higher conversion rate from steviol glycoside to glucosyltransferase steviol glycoside than other microorganisms containing known enzymes under the same conditions.

[0029] The present application also provides a method for producing glucosyltransferase steviol glycoside using the Lactobacillus mali CJST01 strain.

[0030] The effects on the Lactobacillus malii CJST01 strain and glucose-transferable steviol glycosides are as described above.

[0031] The method for producing the glucose-transferred steviol glycoside may include a step of reacting sugar with a steviol glycoside in the presence of a Lactobacillus mari strain or its culture.

[0032] In this application, the term "culture" means a culture solution containing microbial cells or a crude enzyme solution from which microbial cells have been removed. The "culture" includes various substances released into the culture medium by the microorganisms during their growth, along with the culture medium components composed for microbial culture, and specifically includes glucose transferases having sugar hydrolysis activity. The enzyme having sugar hydrolysis activity may, but is not limited to, those that decompose sugar into glucose or α-link 1 to 9 glucose molecules to the 19-OH position of steviol glycosides via α-(1,6) bonds.

[0033] The culture medium used must meet the requirements of the specific strain in an appropriate manner. Culture media for Lactobacillus mari strains are publicly known. For example, the microorganism of this application can be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, etc., while controlling the temperature, pH, etc. In this case, the carbon source may include carbohydrates such as glucose, fructose, and sucrose, and amino acids such as glutamic acid and cysteine. Specifically, natural organic nutrient sources such as starch hydrolysates and molasses can be used, preferably carbohydrates such as glucose, fructose, and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars), and a variety of other appropriate amounts of carbon sources can be used without limitation, but are not limited thereto. As for nitrogen sources, inorganic nitrogen sources such as ammonia; amino acids such as glutamic acid and cysteine, and peptones, meat extracts, yeast extracts, etc. may be used as organic nitrogen sources. These nitrogen sources may be used alone or in combination, but are not limited thereto. The culture medium may, but is not limited to, phosphoric acid, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate or a corresponding sodium-containing salt as a phosphorus source. Inorganic compounds such as magnesium sulfate, iron sulfate, manganese sulfate, and calcium chloride may be used, and other amino acids, vitamins, and appropriate precursors may also be included. These culture media or precursors may, but are not limited to, be added to the culture in a batch or continuous manner.

[0034] During cultivation, the pH of the culture can be adjusted by adding compounds such as potassium hydroxide, ammonia, and phosphoric acid in an appropriate manner. Furthermore, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation during cultivation. Oxygen or oxygen-containing gas can also be injected into the culture to maintain an aerobic state. The culture temperature is 27°C to 37°C, specifically 30°C to 33°C. The cultivation period can continue until the desired amount of useful substance is achieved, specifically 20 to 120 hours.

[0035] The method for producing the glucose-transferred steviol glycoside of this application may include the steps of preparing a composition comprising the Lactobacillus mari strain or its culture, sugar, and the steviol glycoside; and mixing the compositions.

[0036] As one specific example, the manufacturing method may include a step of administering an additive to a composition containing the Lactobacillus mari strain or its culture.

[0037] As another specific example, the additive may be a cryoprotectant or an excipient, and the process may further include a drying step after the additive administration step. Cryoprotectants and excipients are as described later.

[0038] The steviol glycoside may be any one selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, dulcoside A, steviolbioside, and rubusoside, but is not limited thereto.

[0039] Furthermore, this application provides a glucose-transferable steviol glycoside produced by the method for producing the glucose-transferable steviol glycoside, and a sweetener containing the glucose-transferable steviol glycoside.

[0040] The glucose-transferable steviol glycoside produced and the sweetener containing the same may have reduced off-flavors and off-odors and improved sweetness quality.

[0041] Furthermore, this application provides a composition for producing glucose-transferable steviol glycosides, comprising the Lactobacillus malii CJST01 strain or its culture.

[0042] The effects on the Lactobacillus malii CJST01 strain and glucose-transferable steviol glycosides are as described above.

[0043] The composition may further contain a cryoprotectant or an excipient. The cryoprotectant or excipient may be, but is not limited to, a non-naturally occurring substance or a naturally occurring substance. As one specific example, the cryoprotectant or excipient may be, but is not limited to, a substance that Lactobacillus malii CJST01 strain does not naturally come into contact with or is not naturally present with the strain. As another specific example, the composition may further contain one or more cryoprotectants selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder, and starch, and / or one or more excipients selected from the group consisting of glucose, dextrin, and skim milk. The cryoprotectant of this application may be present in the composition in an amount of 0.01% to 20% by weight or 0.01% to 10% by weight relative to the total weight of the composition. Specifically, the composition may contain 5% to 20% by weight of glycerol, 2% to 10% by weight of trehalose, 2% to 10% by weight of maltodextrin, 0.5% to 2% by weight of skim milk powder, and 0.1% to 1% by weight of starch. Furthermore, the excipient may be present in the composition in an amount of 75% to 95% by weight or 85% to 95% by weight relative to the total weight of the composition.

[0044] The composition may, but is not limited to, be used in liquid, granular, or powder form.

[0045] Furthermore, this application provides a use for producing glucose-transferable steviol glycosides and / or sweeteners containing glucose-transferable steviol glycosides derived from the Lactobacillus malii CJST01 strain or its culture.

[0046] Furthermore, this application provides a use for a composition containing the Lactobacillus malii CJST01 strain or its culture for the production of glucose-transferable steviol glycosides and / or sweeteners containing glucose-transferable steviol glycosides.

[0047] The effects on the Lactobacillus malii CJST01 strain and glucose-transferable steviol glycosides are as described above. [Effects of the Invention]

[0048] This invention relates to a novel Lactobacillus mari strain and a method for producing glucose-transferable steviol glycosides using the same. The novel Lactobacillus mari CJST01 strain exhibits superior steviol glycoside transglycosylation activity compared to the Lactobacillus mari DSM20444 strain, which is known to produce glucose-transferable steviol glycosides. This results in higher productivity and a shorter enzymatic reaction time during the manufacturing process. The steviol glycosides produced using this strain have the advantage of superior taste quality and aroma compared to alpha-1,4 linked steviol glycosides and rebaudioside M. Furthermore, glucose-transferable rebaudioside A produced using Lactobacillus mari CJST01 strain exhibits reduced off-flavors and odors and superior solubility. [Brief explanation of the drawing]

[0049] [Figure 1] This figure shows the results of measuring the sugar hydrolysis activity of Lactobacillus malii strain CJST01 using the DNS method, compared to the control group, Lactobacillus malii strain DSM20444. [Figure 2] This figure shows the results of comparing the conversion rates from rebaudioside A to glucose-transferase rebaudioside A at different amounts of crude enzyme solution added to Lactobacillus malii strain CJST01 and the control group Lactobacillus malii strain DSM20444. [Figure 3] This figure shows a chromatogram obtained by HPLC analysis of the reaction mixture after adding 15% crude enzyme solution of Lactobacillus malii strain CJST01 and the control group Lactobacillus malii strain DSM20444. [Figure 4] This figure shows the results of HPLC analysis of the reaction solution components after adding a 5-fold concentrated 10% crude enzyme solution of Lactobacillus mari CJST01 to 10% of a mixture of 10% rebaudioside A and 10% sugar. [Figure 5]This figure shows the results of LC-MS analysis of the reaction solution components after adding 10% rebaudioside A and 10% sugar to a 10% concentration of Lactobacillus mari CJST01 crude enzyme solution, which had been concentrated five times. [Figure 6] This figure shows the results of LC-MS analysis of the reaction solution components after adding a 5-fold concentrated 10% crude enzyme solution of Lactobacillus mari CJST01 to 10% of a mixture of 10% rebaudioside A and 10% sugar. [Figure 7] This figure shows chromatograms comparing the reaction of different steviol glycosides in the crude enzyme solution of Lactobacillus malii CJST01. [Figure 8] This is the result of an intensity analysis of detailed flavor attributes of glucose-transferable rebaudioside A, prepared using a crude enzyme solution of Lactobacillus malii CJST01. [Modes for carrying out the invention]

[0050] The present invention will be described in more detail below with reference to the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

[0051] Example 1. Selection of novel bacterial strains possessing glucose transfer activity to stevioside. Microbial library stocks, isolated from samples collected from traditional fermented foods such as kimchi, soy sauce, vinegar, and fermented alcoholic beverages, as well as from their manufacturing processes, were spread on MRS agar medium and cultured at 30°C for 24 hours. Of these, strains with rapid growth rates were selected visually, and the colonies were inoculated into 3 mL of MRS broth and cultured at 30°C for 24 hours. Each culture solution was centrifuged at 8000 rpm for 10 minutes to separate the cells from the supernatant, and the supernatant (crude enzyme solution) was collected. The sugar hydrolysis ability of each crude enzyme solution was confirmed by the DNS method, and the stevioside glycosyltransferase ability was confirmed by reacting it with 5% each of sugar and stevioside, as shown in Table 1.

[0052] [Table 1]

[0053] As shown in Table 1, a total of six strains of Bifidobacterium bifidum, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus salivarius, and Lactobacillus mari were selected. Sugar hydrolysis activity and stevioside transferability activity were examined. Of the six strains, five exhibited sugar hydrolysis activity, but only the Lactobacillus mari strain was found to possess stevioside transferability activity.

[0054] Example 2. Identification of 16S rRNA sequences of selected Lactobacillus maritima strains. Upon examining the 16S rRNA sequence of the Lactobacillus mali strain selected in Example 1, it was confirmed that no other strain had the same sequence, and that it differed from the nucleotide sequence of known Lactobacillus mali strains. Therefore, the Lactobacillus mali strain selected in Example 1 was confirmed to be a novel strain, and was named Lactobacillus mali CJST01. The 16S rRNA sequence was registered in NCBIGenBank.

[0055] Furthermore, the Lactobacillus mari CJST01 strain was deposited with the Korean Collection for Type Cultures (KCTC), an international depositary under the Budapest Convention, on May 8, 2023, and was assigned accession number KCTC15433BP.

[0056] Example 3. Confirmation of sugar hydrolysis activity of Lactobacillus malii CJST01 strain. To confirm that the novel Lactobacillus malii strain CJST01 has superior sugar hydrolysis activity, it was compared with the sugar hydrolysis activity of Lactobacillus malii strain DSM20444, which is known to have sugar hydrolysis activity.

[0057] Specifically, two strains of Lactobacillus malii, CJST01 and DSM20444, were cultured in MRS broth with added sugar. The cultures were then centrifuged at 8000 rpm for 10 minutes to separate the cells from the supernatant, and only the supernatant (crude enzyme solution) was collected. The obtained crude enzyme solution was reacted with sugar, and the sugar hydrolysis activity was measured using the reducing sugar quantification method (DNS method), as shown in Figure 1. 50 mM sodium acetate buffer was mixed with a 200 mM sugar aqueous solution and each of the aforementioned fractions in a 1:1 ratio. The mixture was then reacted in a 40°C water bath for 10 minutes, followed by deactivation at 100°C. DNS reagent was added in a 1:3 ratio and mixed, and the reaction was carried out at 100°C for 5 minutes. The DNS reaction was then immediately stopped with ice. Subsequently, the absorbance (OD) of the DNS reagent was measured at 575 nm, and the activity was confirmed by substituting it into the prepared fructose standard curve equation. In this context, 1 Unit is defined as the amount (mL) of enzyme that increases reducing sugar by 1 umole per minute.

[0058] As a result, as shown in Figure 1, it was confirmed that the novel Lactobacillus mari CJST01 strain exhibited approximately 1.7 times higher sugar hydrolysis activity compared to the Lactobacillus mari DSM20444 strain.

[0059] Example 4. Evaluation of conversion from rebaudioside A to glucose-transferase rebaudioside A. The conversion rates from rebaudioside A to glucose-transferose rebaudioside A of the crude enzyme solutions of Lactobacillus malii CJST01 strain and Lactobacillus malii DSM20444 strain, prepared in Example 3, were compared as follows.

[0060] Specifically, 10% each of rebaudioside A (Ohira) and sugar (CJ Daiichi Sugar) were added to 50 μM sodium acetate buffer (pH 5.0). Then, 5%, 15%, or 25% of the crude enzyme solution of the two Lactobacillus mari strains prepared in Example 3 were added, and the mixture was reacted at 40°C for 24 hours. After the reaction, the mixture was inactivated at 100°C, and the conversion rate to glucose-transferable rebaudioside A was confirmed by HPLC, as shown in Figure 2. Furthermore, after the reaction with 15% crude enzyme solution added, the chromatograms of each reaction solution were compared and shown in Figure 3. The HPLC analysis conditions are shown in Table 2 below.

[0061] [Table 2]

[0062] A comparison of the conversion rates to rebaudioside A at different amounts of crude enzyme solutions from two Lactobacillus mali strains was shown in Figure 2. When 5%, 15%, and 25% of the crude enzyme solution was added, the conversion rate of the Lactobacillus mali CJST01 strain crude enzyme solution was approximately 1.3 to 1.5 times higher than that of the Lactobacillus mali DSM20444 strain crude enzyme solution. This indicates that using the Lactobacillus mali CJST01 strain, compared to the Lactobacillus mali DSM20444 strain, requires less Lactobacillus crude enzyme solution to produce glucose-transferable rebaudioside A at the same conversion rate, thus offering an advantage in the glucose-transferable rebaudioside A production process.

[0063] Furthermore, by comparing the chromatograms obtained by HPLC analysis of the reaction solutions after adding 15% crude enzyme solutions of two Lactobacillus malii strains, as shown in Figure 3, it was confirmed that when crude enzyme solutions of the two strains were added, the peak of rebaudioside A, the reactant, decreased, and the peak of glucose-transferable rebaudioside A increased. In addition, it was confirmed that the rebaudioside A peak decreased more significantly in the reaction solution with crude enzyme solution of Lactobacillus malii strain CJST01 compared to the reaction solution with crude enzyme solution of Lactobacillus malii strain DSM20444.

[0064] Example 5. Evaluation of glucose transfer rebaudioside A conversion rate under different reaction conditions. The conversion rates from rebaudioside A to glucose-transferose rebaudioside A in crude enzyme solutions of Lactobacillus malii strains CJST01 and DSM20444 were evaluated under different reaction conditions.

[0065] Specifically, the rebaudioside A conversion rate was analyzed by HPLC using the same method as in Example 4, under the conditions of adding 5%, 10%, 15%, or 25% of the crude enzyme solution of Lactobacillus malai CJST01 strain and Lactobacillus malai DSM20444 strain prepared in Example 3, with substrate (rebaudioside A) concentrations of 5%, 7%, or 10%, and reaction times of 6 hours, 24 hours, or 48 hours. The results are shown in Tables 3 and 4 below.

[0066] [Table 3]

[0067] [Table 4]

[0068] The conversion rates of rebaudioside A of crude enzyme solutions from two Lactobacillus malii strains were examined under different reaction conditions. As shown in Tables 3 and 4, when the substrate concentration was 10%, the crude enzyme solution added was 5%, 10%, or 15%, and the reaction time was 6–48 hours, the crude enzyme solution from Lactobacillus malii DSM20444 strain showed a conversion rate of 8–80%, while the crude enzyme solution from Lactobacillus malii CJST01 strain showed a conversion rate of 13–90%. These results indicate that when using Lactobacillus malii CJST01 strain, a higher conversion rate is observed at the same substrate concentration or crude enzyme solution addition amount.

[0069] Furthermore, the reaction solution to which the crude enzyme solution of Lactobacillus mari DSM20444 strain was added showed a rebaudioside A conversion rate of 90% under conditions of substrate concentration of 7%, crude enzyme solution addition amount of 15%, and reaction time of 48 hours. The reaction solution to which the crude enzyme solution of Lactobacillus mari CJST01 strain was added also showed a rebaudioside A conversion rate of 90% under conditions of substrate concentration of 10%, crude enzyme solution addition amount of 15%, and reaction time of 48 hours. This indicates that when using the crude enzyme solution of Lactobacillus mari CJST01 strain, a reaction solution with the same conversion rate can be obtained at a high substrate concentration.

[0070] Example 6. Analysis of glucose-transferred rebaudioside A To determine how many glucose molecules were bound to the glucose-transferable rebaudioside A converted by adding a crude enzyme solution of Lactobacillus maritima strain, the following analysis was performed.

[0071] Specifically, 10% each of rebaudioside A (Ohira) and sugar (CJ Daiichi Sugar) were added to 50uM sodium acetate buffer (pH 5.0). The crude enzyme solution of Lactobacillus mari CJST01 strain prepared in Example 3 was concentrated fivefold and added to the buffer at a concentration of 10%, and the mixture was reacted at 40°C for 24 hours. After the reaction, the mixture was inactivated at 100°C, and the results of HPLC analysis and LC-MS analysis under the conditions shown in Table 5 below are shown in Figures 4-6.

[0072] [Table 5]

[0073] As a result, as shown in Figures 4-6, it was confirmed that rebaudioside A in the reaction solution prepared using the crude enzyme solution of Lactobacillus malii CJST01 strain had 1 to 9 glucose molecules transferred to it.

[0074] Example 7. Evaluation of glucose transfer activity by crude enzyme solution of Lactobacillus mari strains for each steviol glycoside. To evaluate the glucose transfer activity of different steviol glycosides using crude enzyme solutions from Lactobacillus mari strains, the following experiment was conducted.

[0075] Specifically, 10% each of 12 steviol glycosides (stevioside, rebaudioside A, B, C, D, E, F, M, N, dulcoside A, steviol bioside, rubusoside) and sugar (CJ First Sugar) were added as substrates to 50 μM sodium acetate buffer (pH 5.0). Crude enzyme solution of two Lactobacillus malii strains prepared in Example 3 was added, and the mixture was reacted at 40°C for 24 hours. After the reaction, the mixture was inactivated at 100°C, and the production of glucose-transferable steviol glycosides was confirmed by HPLC, as shown in Table 6 and Figure 7 below.

[0076] [Table 6]

[0077] As a result, as shown in Table 6 and Figure 7, the crude enzyme solution of Lactobacillus mari CJST01 strain demonstrated glucose transfer activity against stevioside, rebaudioside A, C, and F, dulcoside A, steviolbioside, and rubusoside, among 12 types of steviol glycosides. Rebaudioside E showed activity only with the crude enzyme solution of Lactobacillus mari DSM20444, while steviolbioside showed activity only with the crude enzyme solution of Lactobacillus mari CJST01.

[0078] Example 8. Functional description analysis of aqueous solution of glucose-transferred rebaudioside A The sensory differences between CJST01 glucose-transferred rebaudioside A (CJ), produced by the method for producing the glucose-transferred steviol glycoside described above, and the comparative samples rebaudioside M (RM) and alpha-1,4 glucose-transferred rebaudioside A (A14) were analyzed.

[0079] Specifically, this evaluation was conducted using descriptive analysis with a recruited expert sensory panel. Prior to the evaluation, concentrations corresponding to the same level of sweetness were calculated from 100 female consumers aged 19 to 40 who are sensitive to sweetness. The sensory evaluation method used was a paired comparison test, and the final calculated concentrations are shown in Table 7 below. In this process, the CJ and A14 sample concentrations were graded into quintiles based on the RM concentration, and consumers were asked to select the closest concentration. The manufacturing method involved dissolving the substance in a drinkable solution at a constant concentration (w / w). The consumer panel was provided with 10 mL samples in clear plastic cups, and two samples were randomly assigned. They were asked to select the sample that they perceived as sweeter. Based on this, the samples that showed statistical equivalence among the overall differences between samples were calculated as the final descriptive analysis sample concentrations.

[0080] [Table 7]

[0081] Sensory evaluation by descriptive analysis was performed as follows, using the sample concentrations for descriptive analysis derived above. The purpose of the sensory evaluation was to clarify the overall sensory profile of each sweetener aqueous solution when the sweetness level was the same. The sensory evaluation was repeated twice with a panel of six trained experts.

[0082] Specifically, samples were provided in 10 mL portions in clear plastic cups at room temperature, and the panel was given three cups of each sample to describe its sensory characteristics. Evaluation was carried out on randomly provided samples, with a 2-3 minute pause between samples to eliminate the influence of sensory differences between samples while investigating detailed flavor attributes. Evaluation items perceived by the panel were derived through pre-sample evaluation, and the following standard samples were provided for detailed flavor / texture characteristics. The general details regarding this are shown in Table 8 below.

[0083] [Table 8]

[0084] As shown in Table 8, the sensory characteristics of the five sweeteners were derived through sensory evaluation using descriptive analysis, consisting of "sweetness, bitterness, five aroma characteristics, and two texture characteristics." For bitterness, standard substances were provided at different intensities (2 points: anhydrous caffeine 0.055%, 4 points: anhydrous caffeine 0.067%).

[0085] The results of the intensity analysis for detailed flavor attributes are shown in Table 9 below, and the results visualized using a Spider Map are shown in Figure 8.

[0086] [Table 9]

[0087] 1) Means of two replicates.Data were scored on a 15 point category scale, where 1week intensity of the attribute and 15=strong intensity of the attribute.Values ​​within a row not sharing a superscript letter are significantly different(p<0.0 5Duncan's multiple range test).

[0088] As shown in Table 8 and Figure 8, RM was characterized by a strong "watery, fishy flavor" and strong metallic properties, while the "bitter and astringent sensation" was expressed more weakly compared to the A14 sample. The A14 sample showed relatively strong "bitter / clove flavor / astringent sensation." In the case of the CJ sample, the metallic intensity was weaker than that of the RM sample, and the bitter / astringent sensation was evaluated at a similar level. Compared to the A14 sample, the bitter / astringent sensation attribute was expressed more weakly, while the remaining attribute intensity was found to be equivalent.

[0089] In other words, the results above confirm that the CJ sample has superior applicability in terms of the sensory performance of the sweetener compared to the comparative sample (fewer off-notes). Since sweeteners aim to replace sugar, other properties besides "sweetness" are not necessary, and considering their application to a variety of foods, the mentioned properties are extremely important for universal use. Therefore, taking the findings of this descriptive analysis into account, it can be concluded that glucose-transferable rebaudioside A according to the present invention has demonstrated its excellence as a food sweetener.

[0090] Example 9. Evaluation of the solubility of glucose-transferred rebaudioside A To evaluate the solubility of steviol glycosides and glucose-transferred rebaudioside A, aqueous solutions prepared by adding 10 ml of distilled water to 2 g each of rebaudioside A (RebA), rebaudioside D (RebD), rebaudioside M (RebM), and stevioside (STV) were mixed with an aqueous solution prepared by adding 10 ml of distilled water to 8 g of glucose-transferred rebaudioside A (RebA-Glu). These solutions were dissolved in a sonicator at 50°C for 60 minutes. These solutions were incubated at 10, 25, 40, and 55°C for 4 days. Each aqueous solution was centrifuged at 12000 rpm for 10 minutes, and 1 ml of the supernatant was dried in a dry oven at 105°C. The solubility was measured and is shown in Table 10. In this case, glucose-transferred rebaudioside A was prepared in the same manner as the method used for glucose-transferred rebaudioside A in Example 7.

[0091] [Table 10]

[0092] As a result, as shown in Table 10, glucose-transferred rebaudioside A showed a significant increase in solubility compared to the reaction substrate rebaudioside A, and its solubility was far improved compared to other steviol glycosides, rebaudioside D, rebaudioside M, and stevioside.

[0093] From the above description, a person with ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the embodiments described above should be understood to be illustrative and not limiting in all respects. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts.

[0094] [Accession Number] Depository name: Korea Institute of Biotechnology, Bioresource Center (KCTC) Accession number: KCTC15433BP Date of acceptance: 20230508

[0095] [Table 11]

Claims

1. Lactobacillus mari CJST01 strain deposited under accession number KCTC15433BP.

2. The strain according to claim 1, wherein the strain has the 16S rRNA sequence of Sequence ID No.

1.

3. The strain according to claim 1, wherein the steviol glycoside is one selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, dulcoside A, steviolbioside, and rubusoside.

4. A method for producing glucose-transferable steviol glycosides using the bacterial strain described in claim 1.

5. The method for producing glucose-transferred steviol glycoside according to claim 4, comprising the step of reacting sugar with a steviol glycoside in the presence of the Lactobacillus mari strain or a culture thereof.

6. The method for producing a glucose-transferable steviol glycoside according to claim 4, wherein the steviol glycoside is selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, dulcoside A, steviolbioside, and rubusoside.

7. The method for producing a glucose-transferable steviol glycoside according to claim 6, wherein the steviol glycoside is a steviol glycoside in which glucose is attached via an α-(1,6) bond through a glucose bond at the 19-OH position of the steviol glycoside.

8. The method for producing a glucose-transferred steviol glycoside according to claim 7, wherein the steviol glycoside contains 1 to 9 glucose molecules.

9. A glucose-transferred steviol glycoside produced by the method described in any one of claims 4 to 8.

10. The glucose-transferable steviol glycoside according to claim 9, wherein the steviol glycoside is selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, dulcoside A, steviolbioside, and rubusoside.

11. A sweetener comprising the glucose-transferable steviol glycoside described in claim 9.

12. A composition for producing glucose-transferable steviol glycosides, comprising the bacterial strain or culture thereof described in claim 1.

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