Use of strain having activity of converting fructose into allulose

Abstract

One example of the present invention provides a composition for producing allulose comprising one or more selected from the group consisting of a Microbacterium algeriense strain having activity for converting fructose into allulose, a culture of said strain, a lysate of said strain, and an allulose epimerizing enzyme produced by said strain. The Microbacterium algeriense strain according to the present invention exhibits optimal activity at a weakly acidic or neutral pH and has excellent activity for converting allulose and thermal stability. Therefore, when producing allulose from fructose using the Microbacterium algeriense strain according to the present invention, browning of sugars and the generation of by-products can be suppressed, the conversion reaction can be carried out for a long time without replacing the strain, and allulose productivity can be increased.

Description

Technology Field

[0001] The present invention relates to the use of a strain having the activity of converting fructose into allulose, and more specifically, to the use of a strain that exhibits optimal activity at a weakly acidic or neutral pH and has excellent thermal stability, so that browning of sugars and the formation of by-products can be suppressed when producing allulose from fructose and that it can be used for a long time. Background Technology

[0002] D-allulose is an epimer of the 3rd carbon of fructose and is also called D-psicose. D-allulose is a functional monosaccharide that can be applied as a sweetener in diet foods because it has a sweetness level of about 70% compared to sugar but contains almost no calories. In addition, D-allulose has the function of inhibiting glucose absorption and blood sugar levels, so it can be applied to foods for diabetic patients and foods for water consumption. Furthermore, since it can inhibit abdominal fat accumulation by inhibiting the activity of enzymes involved in lipid synthesis in the liver, it can be used in various functional foods, such as health foods.

[0003] Due to the characteristics mentioned above, allulose is a good source for replacing sugar, but because it belongs to the category of rare sugars—monosaccharides that exist extremely rarely in nature—an efficient method for producing allulose is required for its application in the food industry. The most efficient method for producing allulose for industrialization is to convert fructose into allulose using D-allulose 3-epimerase or microorganisms that have the activity to convert fructose into allulose.

[0004] Regarding the technology for producing allulose from fructose, Korean Registered Patent Publication No. 10-1944103 describes a fructose-containing substrate, Microbacterium polyorum ( Microbacterium foliorumA method for producing psychose by contacting a ) strain or a psychose-converting enzyme produced by said strain is disclosed. In addition, Korean Registered Patent Publication No. 10-1944104 discloses a fructose-containing substrate with Microbacterium oxidans ( Microbacterium oxydans ) and Microbacterium philosophera ( Microbacterium phyllosphaerae A method for producing psychose is disclosed by contacting a strain of the genus Microbacterium selected from the group consisting of ) or a psychose-converting enzyme. In addition, Korean Registered Patent Publication No. 10-2138862 discloses fructose with non-pathogenic Staphylococcus ( Staphylococcus A method for producing allulose by contacting it with microorganisms of the genus ) is disclosed. In addition, Korean Registered Patent Publication No. 10-1899061 describes fructose with caistia ( kaistia A method for producing allulose by contacting it with microorganisms of the genus is disclosed. The problem to be solved

[0005] The present invention is derived from the prior art background, and the objective of the present invention is to provide various uses for a strain that exhibits optimal activity at a weakly acidic or neutral pH, has excellent conversion activity of fructose to allulose, and has excellent thermal stability. means of solving the problem

[0006] When producing allulose from fructose using microorganisms that have the activity of converting fructose into allulose or the allulose-converting enzymes produced by them, it is desirable that the optimal pH be weakly acidic or neutral. When producing allulose from fructose under alkaline pH conditions, browning of the sugars or the formation of by-products can easily occur. In addition, if the thermal stability of the microorganisms that have the activity of converting fructose into allulose is poor, it becomes difficult to use the microbial cells for a long time when producing allulose from fructose, and the microbial cells must be replaced frequently, which leads to a problem of reduced allulose productivity.

[0007] The inventors of the present invention isolated a total of 341 strain colonies from various foods, soil, skin, etc., and first screened a total of 31 strains that showed an appropriate level of growth by culturing the isolated strain colonies in a minimal liquid medium containing allulose. Subsequently, the fructose-to-allulose conversion activity of the 31 strains screened in the first stage was evaluated, and a total of 3 strains were secondarily screened. Subsequently, the fructose-to-allulose conversion rate and thermal stability under various process conditions were evaluated for the 3 strains screened in the second stage, and Microbacterium alleriens (which exhibited excellent fructose-to-allulose conversion activity and thermal stability) Microbacterium algeriense The strain was selected as the final strain.

[0008] To solve the above problem, one example of the present invention is a Microbacterium algerian having the activity of converting fructose into allulose ( Microbacterium algeriense A composition for producing allulose is provided, comprising one or more selected from the group consisting of a strain, a culture of the strain, a lysate of the strain, and an allulose epimerizing enzyme produced by the strain.

[0009] To solve the above problem, one example of the present invention is a Microbacterium algerian having the activity of converting fructose into allulose ( Microbacterium algeriense A method for producing allulose is provided, comprising the step of converting fructose into allulose by reacting fructose with one or more selected from the group consisting of a strain, a culture of the strain, a lysate of the strain, and an allulose epimerizing enzyme produced by the strain. Effects of the invention

[0010] Microbacterium algerian according to the present invention ( Microbacterium algeriense The strain exhibits optimal activity at a weakly acidic or neutral pH and has excellent conversion activity to allulose and thermal stability. Therefore, the Microbacterium algerian according to the present invention ( Microbacterium algerienseWhen producing allulose from fructose using a strain, browning of sugars and the formation of by-products can be suppressed, the conversion reaction can be carried out for a long time without replacing the strain, and allulose productivity can be increased. Brief explanation of the drawing

[0011] FIG. 1 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the conversion activity of fructose to allulose by the C8A T4 strain at different reaction temperatures. FIG. 2 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the conversion activity of fructose to allulose by the C8A T4 strain according to reaction pH. FIG. 3 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the conversion activity of fructose to allulose by the C8A T4 strain according to the type of metal ion compound added to the reaction solution. FIG. 4 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the allulose productivity of the C8A T4 strain over time. Specific details for implementing the invention

[0012] The present invention will be described in detail below.

[0013] One aspect of the present invention relates to a composition for producing allulose used when producing allulose from fructose.

[0014] A composition for producing allulose according to one example of the present invention is a Microbacterium algerian having the activity of converting fructose into allulose ( Microbacterium algerienseIt comprises one or more selected from the group consisting of a microbial strain, a culture of said strain, a lysate of said strain, and an allulose epimerizing enzyme produced by said strain. The culture refers to a product obtained by culturing microorganisms in a known liquid or solid medium, and is a concept that includes the cultured microorganisms. Specifically, the culture may be a microbial culture solution or a solid form obtained by solidifying the microbial culture solution. The lysate refers to a lysate obtained by crushing the strain or a supernatant obtained by centrifuging said lysate, and includes an enzyme produced from said strain. Furthermore, the allulose production composition according to an example of the present invention may preferably further comprise one or more metal ion compounds selected from the group consisting of cobalt ion compounds, manganese ion compounds, and magnesium ion compounds. The metal ion compounds are compounds formed by the ionic bonding of a metal cation and a non-metal anion, and readily dissociate into metal cations and non-metal anions in a liquid medium. The Microbacterium algerian ( Microbacterium algeriense The isolate source of the strain is not significantly limited and can be selected from various natural environments, for example, human skin. The above Microbacterium algerian ( Microbacterium algeriense The conversion activity of fructose to allulose of the strain can be stable in a temperature range of 67 to 85°C, and preferably stable in 70 to 83°C. The above Microbacterium algerian ( Microbacterium algeriense The strain can more preferably exhibit maximum conversion activity of fructose to allulose at a temperature of approximately 80°C. The above-mentioned Microbacterium algerian ( Microbacterium algeriense The conversion activity of fructose to allulose of the strain can be stable in a pH range of 5.5 to 8, and preferably in a pH range of 5.6 to 7.5. The above Microbacterium algerian ( Microbacterium algerienseThe ) strain can more preferably exhibit maximum conversion activity of fructose to allulose at a pH of approximately 6.5. In addition, the above Microbacterium algerian ( Microbacterium algeriense The ) strain can increase the conversion activity of fructose to allulose by at least 1.8 times compared to the case in the absence of metal ions due to cobalt ions, manganese ions, or magnesium ions, and preferably by 1.9 to 3 times. In addition, the above Microbacterium algerian ( Microbacterium algeriense The AN01 strain (accession number: KCCM13558P) can maintain the conversion activity of fructose to allulose at over 90%, for example 91–95%, compared to the case without heat shock, even when subjected to heat shock for 48 hours at a temperature of 50°C. The above Microbacterium algerian ( Microbacterium algeriense The type of strain is not significantly limited as long as it has the activity of converting fructose into allulose, and preferably Microbacterium algerian( Microbacterium algeriense ) It is the AN01 strain (accession number: KCCM13558P).

[0016] One aspect of the present invention relates to a method for manufacturing allulose.

[0017] A method for producing allulose according to one example of the present invention comprises Microbacterium algerianse having the conversion activity of fructose to allulose ( Microbacterium algeriense The method comprises the step of converting fructose into allulose by reacting fructose with one or more selected from the group consisting of a strain, a culture of said strain, a lysate of said strain, and an allulose epimerizing enzyme produced by said strain. A method for producing allulose according to an example of the present invention may preferably further include the step of recovering the converted allulose. In a method for producing allulose according to an example of the present invention, the Microbacterium algerian ( Microbacterium algerienseThe strain, the culture of the strain, the lysate of the strain, or the allulose epimerizing enzyme produced by the strain may be provided in a form immobilized on a carrier. The type of carrier used for the immobilization is not significantly limited and may be selected from various known carriers. For example, the carrier may be alginate. The Microbacterium algerian ( Microbacterium algeriense The type of strain is not significantly limited as long as it has the activity of converting fructose into allulose, and preferably Microbacterium algerian( Microbacterium algeriense It is the AN01 (accession number: KCCM13558P) strain. In the method for producing allulose according to an example of the present invention, the fructose is preferably supplied in the form of a fructose-containing substrate. The fructose-containing substrate may preferably be a substrate solution containing fructose. The concentration of fructose in the fructose-containing substrate is not significantly limited and may, for example, be 1 to 600 g / L, and considering economic feasibility and workability, it is preferable to be 10 to 500 g / L. In the method for producing allulose according to an example of the present invention, the reaction temperature for converting fructose into allulose may be selected in the range of 50 to 85°C, and Microbacterium algerian ( Microbacterium algeriense Considering the thermal stability and conversion activity of the strain, it can preferably be selected in the range of 55 to 83°C. In the method for producing allulose according to one example of the present invention, the reaction pH for converting fructose to allulose can be selected in the range of 5.5 to 7.5, and Microbacterium algerian ( Microbacterium algerienseConsidering the conversion activity of the strain or the inhibition of side reactions, the pH may preferably be selected within a weakly acidic to neutral range of 6.0 to 7.5. In the method for producing allulose according to an example of the present invention, the reaction for converting fructose into allulose may preferably be carried out in the presence of one or more metal ions selected from the group consisting of cobalt ions, manganese ions, and magnesium ions, and more preferably in the presence of cobalt ions or manganese ions when considering the level of increase in conversion activity by the metal ions. The metal ions act as co-catalysts for the conversion reaction of fructose into allulose. The concentration of metal ions in the fructose-containing substrate is not significantly limited and may be, for example, 0.1 to 5 mM, and is preferably 0.5 to 2.5 mM. In the method for producing allulose according to one example of the present invention, the reaction time for converting fructose into allulose is not significantly limited and, for example, may be 6 hours or more, and may be selected from 10 to 40 hours when considering the equilibrium conversion rate and economic feasibility, and is preferably 15 to 30 hours, and more preferably 18 to 24 hours.

[0019] The present invention will be explained in more detail below through examples. However, the following examples are intended only to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0021] 1. Screening of microbial strains with fructose-to-allulose conversion activity

[0022] (1) Isolation of microbial strain colonies from various natural environments

[0023] Microbial strain colonies were isolated by pre-treating isolation sources such as various foods (honey, soybean paste, red pepper paste, kimchi, figs, sourdough, etc.), soil (apple orchards, forests, flowerbeds, wheat fields, vineyards, areas near street trees, riversides, factories, etc.), and human skin, and culturing them on solid media.

[0024] Samples were obtained for each isolation source, the obtained samples were suspended in PBS solution, and the solids were precipitated to obtain the supernatant. Subsequently, the obtained supernatant was plated onto an allulose-containing minimal solid medium (components and concentrations: 3 g / L KH2PO4, 0.5 g / L NaCl, 6.78 g / L Na2HPO4, 1 g / L NH4Cl, 5 g / L allulose, 1 mM MgSO4, 0.1 mM CaCl2, 0.05 mM FeCl3, 0.1 mM citric acid, and 16 g / L agar) and incubated at 30°C for 48 hours, after which a total of 341 strain colonies were obtained.

[0026] (2) Primary screening of microbial strains

[0027] It was determined that strains growing to an appropriate level in a minimal medium containing allulose express allulose epimerase, which is involved in the conversion mechanism between allulose and fructose, and strains growing to an appropriate level in a minimal medium containing allulose were screened first.

[0028] A total of 341 microbial strain colonies isolated from various natural environments were each inoculated into LB (Luria-Bertani) liquid medium and incubated overnight at a temperature of 30°C and stirring at 200 rpm. Subsequently, 60 µl of the incubation solution of the microbial strain colonies was inoculated into 3 mL of allulose-containing minimal liquid medium (composition and concentration: 3 g / L KH2PO4, 0.5 g / L NaCl, 6.78 g / L Na2HPO4, 1 g / L NH4Cl, 5 g / L allulose, 1 mM MgSO4, 0.1 mM CaCl2, 0.05 mM FeCl3, and 0.1 mM citric acid) and incubated for 48 hours at a temperature of 30°C and stirring at 200 rpm. Subsequently, the optical density (OD) of the culture medium was measured at a wavelength of 600 nm using a spectrophotometer. If the OD value was 0.1 or higher, it was determined that the culture had grown to an optimal level, and the corresponding strain was selected as a primary candidate strain. PCR was performed on the primary candidate strains to determine the 16S rRNA sequence, and the species names were confirmed through identification processes such as phylogenetic tree construction and Blast using the Chunlab EzbioCloud program. As a result, it was confirmed that only a total of 31 microbial strains grew to an optimal level in the allulose-containing minimal liquid medium.

[0030] (3) Secondary screening of microbial strains

[0031] A total of 31 microbial strains selected as primary candidates were each introduced into a fructose-containing solution and the amount of allulose produced was measured after the conversion reaction, thereby screening strains with high fructose-to-allulose conversion activity in the second stage.

[0032] The strains selected as primary candidates were cultured sequentially in LB (Luria-Bertani) liquid medium and allulose-containing minimal liquid medium, identical to the primary screening process. Subsequently, the culture medium was centrifuged to remove the supernatant, and the bacterial cells were recovered. The recovered cells were then suspended in a 0.85% (w / v) NaCl solution, centrifuged to remove the supernatant, and the cells free from the medium components were obtained. Finally, 0.8 g (dcw: dry cell weight) / L of the cells, 10 g / L of fructose, and Mn were added to 50 mM Tris-HCl buffer (pH 7.0). 2+ A conversion reaction solution was prepared by adding ingredients to achieve a concentration of 1 mM, and the conversion reaction of fructose to allulose was carried out overnight at 55°C. Subsequently, a 10% (w / v) HCl solution was added to the conversion reaction solution to terminate the reaction. The reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC. The HPLC analysis conditions are as follows.

[0033] * Column and column temperature: 87°C (BIO-RAD), 80°C

[0034] * Mobile phase and mobile phase flow rate: Water 100% (v / v), 0.6 ml / min

[0035] * Detector: Refractive Index Detector (Agilent 1260 TID)

[0036] Tables 1 and 2 below summarize the information on a total of 31 microbial strains selected during the first screening process and the amount of allulose produced during the second screening process.

[0037] Strain management number Separation source strain species name Allulose production amount (g / ℓ) C8A T4 human skin Microbacterium algeriense 2.60 CT8-12 Vineyard soil Ensifer adhaerens 2.69 CCD1-60 Apple orchard soil Agrobacterium fabrum 2.12 C66C MH2 human skin Microbacterium foliorum 2.61 C4C L1 human skin Microbacterium oxydans 2.46 C22A LB3 human skin Microbacterium paraoxydans 1.66 CT5-1 rice straw Pantoea dispersa 0.00 CT5 E1 rice straw Klebsiella michiganensis 0.00 CT3-2 E1 Forest soil Burkholderia pyrrocina 0.00 CT3-2 R1 Forest soil Paenibacillus taichungensis 0.00 CT6-5 Flower bed soil Pantoea hericii 0.00 CT6-12 Flower bed soil Pseudarthrobacter sulfonivorans 0.00 CT7-1M10 Wheat field soil Pantoea eucalypti 0.00 CT7-1M17 Wheat field soil Kosakonia sacchari 0.00 CT7-2M1 Wheat field soil Rhizobium graham 0.00 CT7-2M5 Wheat field soil Terrabacter tumescens 0.00

[0039] Strain management number Separation source strain species name Allulose production amount (g / ℓ) CT7-2HM10 Wheat field soil Peribacillus frigoritolerans 0.00 CT8-H9 Vineyard soil Metabacillus idriensis 0.00 CT8-E5 Vineyard soil Klebsiella quasipneumoniae 0.00 CT8-7 Vineyard soil Rhizobium altiplani 0.00 CT8-11 Vineyard soil Pseudomonas nitritireducens 0.00 CT8-14 Vineyard soil Pantoea rodasii 0.00 CT8-16 Vineyard soil Sphingobacterium prati 0.00 CT8-21 Vineyard soil Pseudomonas iranensis 0.00 CT8-20 Vineyard soil Pseudomonas nicosulfuronedens 0.00 CT8-H29 Vineyard soil Rhizobium cauense 0.00 CT10-18 Soil near street trees Flavobacterium acidificum 0.00 CT10H-11 Soil near street trees Mesorhizobium tamadayense 0.00 CT12-1M2 riverside soil Rahnella aceris 0.00 CT12-1 HM4 riverside soil Niallia circulans 0.00 CT12-1 HM15 riverside soil Rossellomorea marisflavi 0.00

[0040] Microbacterium algerianus as a secondary candidate strain based on the amount of allulose produced ( Microbacterium algeriense ) C8A T4 strain, Ensifer Adherence( Ensifer adhaerens) CT8-12 strain and Microbacterium polyorum ( Microbacterium foliorum ) The C66C MH2 strain was selected.

[0042] 2. Measurement of fructose-to-allulose conversion activity and thermal stability of secondary candidate strains

[0043] (1) Allulose productivity of secondary candidate strains according to reaction time

[0044] 400 g / L fructose and 1 mM Mn 2+ Cells of the second candidate strain were added to a 50 mM Tris-HCl buffer solution (pH 7.0) containing [substance] at a concentration of 20 g (dcw) / L, and the reaction was carried out at a temperature of 55°C while measuring the conversion rate of fructose to allulose over time. When the preset time was reached, a 10% (w / v) HCl solution was added to the reaction solution to terminate the reaction. Afterward, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the conversion rate of fructose to allulose.

[0045] Table 3 below summarizes the results of measuring the conversion rate of fructose to allulose according to reaction time of the second candidate strain.

[0046] Second candidate strain Conversion rate (%) of fructose to allulose by reaction time 0 hr 2 hr 3.5 hr 16 hr 24 hr Microbacterium algeriense C8A T4 0 10.4 15.1 27 28.2 Ensifer adhaerens CT8-12 0 5 8 21.2 24.3 Microbacterium foliorum ) C66C MH2 0 17.5 22.8 28.6 28.4

[0047] As shown in Table 3 above, Microbacterium algerian ( Microbacterium algeriense ) The C8A T4 strain is Ensifer Adherence ( Ensifer adhaerens It showed higher allulose productivity compared to the CT8-12 strain.

[0049] (2) Thermal stability of the second candidate strain

[0050] Cells of the second candidate strain were suspended in 50 mM Tris-HCl buffer (pH 7.0) and subjected to heat shock while stored at 50°C for a preset time. Subsequently, 400 g / L fructose and 1 mM Mn 2+Cells of the secondary candidate strain subjected to heat shock were added to a 50 mM Tris-HCl buffer solution (pH 7.0) containing [a specific substance] at a concentration of 5 g (dcw) / L and reacted for 1 hour at a temperature of 55°C, after which a 10% (w / v) HCl solution was added to terminate the reaction. Subsequently, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the residual activity of the secondary candidate strain for the conversion of fructose to allulose according to the time of heat shock.

[0051] The results of measuring the thermal stability of the second candidate strain are summarized in Table 4 below.

[0052] Second candidate strain Residual activity (%) by heat shock duration 0 hr 8 hr 12 hr 16 hr 24 hr 36 hr 48 hr Microbacterium algeriense C8A T4 100.0 96.3 96.1 95.6 95.1 94.4 93.9 Ensifer adhaerens CT8-12 100.0 91.9 88.9 82.8 81.1 71.1 64.4 Microbacterium foliorum ) C66C MH2 100.0 92.4 91.0 79.7 52.1 17.6 14.1

[0053] As shown in Table 4 above, Microbacterium algerian ( Microbacterium algeriense The C8A T4 strain maintained its fructose-to-allulose conversion activity at approximately 94% of the level observed in the non-heat shock case, even when subjected to heat shock for 48 hours at a temperature of 50°C. Microbacterium algerians ( Microbacterium algeriense ) The C8A T4 strain is Ensifer Adherence ( Ensifer adhaerens ) CT8-12 strain and Microbacterium polyorum ( Microbacterium foliorum It has very high thermal stability compared to the C66C MH2 strain, so it is expected that the cells can be used for a long time.

[0055] (3) Selection of final strain

[0056] Considering the fructose-to-allulose conversion rate, fructose-to-allulose conversion rate, and thermal stability, Microbacterium algerianus was selected as the final strain ( Microbacterium algeriense ) The C8A T4 strain was selected. Microbacterium algerianus ( Microbacterium algeriense The 16S rDNA of the C8A T4 strain consists of the nucleotide sequence of SEQ ID NO. 1, and is a Microbacterium algerian ( disclosed in the NCBI database) Microbacterium algerienseIt was confirmed to have 99.86% homology with the 16S rDNA of the G1 strain.

[0058] 3. Microbacterium algerians ( Microbacterium algeriense Establishment of optimal conditions for the conversion of fructose to allulose by the C8A T4 strain

[0059] (1) Microbacterium algerian according to reaction temperature ( Microbacterium algeriense ) Transformation activity of C8A T4 strain

[0060] Fructose at a concentration of 10 g / L and Mn at a concentration of 1 mM 2+ Microbacterium algerian in a 50 mM Tris-HCl buffer solution (pH 7.0) containing Microbacterium algeriense Cells of the C8A T4 strain were added at a concentration of 0.8 g (dcw) / L and reacted for 1 hour at a temperature range of 40–90°C, after which a 10% (w / v) HCl solution was added to terminate the reaction. Subsequently, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the conversion rate of fructose to allulose.

[0061] FIG. 1 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the fructose-to-allulose conversion activity of the C8A T4 strain at different reaction temperatures. As shown in Figure 1, Microbacterium algerian ( Microbacterium algeriense The C8A T4 strain was found to have maximum conversion activity at approximately 80°C.

[0063] (2) Microbacterium algerian depending on reaction pH ( Microbacterium algeriense ) Transformation activity of C8A T4 strain

[0064] Fructose, MnSO4, and Microbacterium algerianus in each of the McIlvaine buffer solutions having a pH range of 5–9 ( Microbacterium algeriense ) By adding cells of the C8A T4 strain, 10 g / L fructose, 1 mM Mn 2+Reaction solutions containing cells at a concentration of 0.8 g (dcw) / L and having various pH conditions were prepared. Subsequently, the reaction solutions were reacted at a temperature of 80°C for 10 minutes, and then a 10% (w / v) HCl solution was added to terminate the reaction. Afterward, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the conversion rate of fructose to allulose.

[0065] FIG. 2 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the fructose-to-allulose conversion activity of the C8A T4 strain according to reaction pH. As shown in Figure 2, Microbacterium algerian ( Microbacterium algeriense The C8A T4 strain was confirmed to have maximum conversion activity at a pH of approximately 6–7, preferably 6.5. Microbacterium algerianus ( Microbacterium algeriense Since the C8A T4 strain exhibits maximum conversion activity under weakly acidic to neutral pH conditions, browning of sugars and the formation of by-products can be inhibited when converting fructose to allulose.

[0067] (3) Microbacterium algerian depending on metal ions ( Microbacterium algeriense ) Transformation activity of C8A T4 strain

[0068] Fructose, metal ion compounds (CaCl2, CoCl2, CuSO4, FeSO4, MnSO4, NiSO4, ZnSO4) and Microbacterium algerianus in McIlvaine buffer solution at pH 6.5 ( Microbacterium algerienseA reaction solution containing various types of metal ions was prepared by adding cells of the C8A T4 strain, 10 g / L of fructose, 1 mM of metal ions, and 0.8 g (dcw) / L of cells. Subsequently, the reaction solution was reacted at a temperature of 80°C for 10 minutes, after which a 10% (w / v) HCl solution was added to terminate the reaction. Afterward, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the conversion rate of fructose to allulose.

[0069] FIG. 3 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the fructose-to-allulose conversion activity of the C8A T4 strain according to the type of metal ion compound added to the reaction solution. As shown in Figure 3, Microbacterium algerian ( Microbacterium algeriense The C8A T4 strain showed enhanced conversion activity of fructose to allulose by cobalt (Co), manganese (Mn), magnesium (Mg), iron (Fe), and nickel (Ni) ions compared to a control group (None) in the absence of metal ions. In particular, cobalt, manganese, and magnesium ions, respectively, Microbacterium algerians ( Microbacterium algeriense ) Increased the conversion activity of fructose to allulose of the C8A T4 strain by about 3 times, about 2.2 times, and about 2 times.

[0071] (4) Microbacterium algerian according to reaction time ( Microbacterium algeriense ) Allulose productivity of C8A T4 strain

[0072] 400 g / L fructose and 1 mM Mn 2+ Microbacterium algerian in a 50 mM Tris-HCl buffer solution (pH 7.0) containing Microbacterium algerienseCells of the C8A T4 strain were added at a concentration of 20 g (dcw) / L, and the reaction was carried out at a temperature of 55°C for 55 hours, during which the conversion rate of fructose to allulose was measured at each reaction time. When the preset time was reached, a 10% (w / v) HCl solution was added to the reaction solution to terminate the reaction. Afterward, the reaction solution was centrifuged to recover the supernatant, and the amount of allulose produced was analyzed by HPLC to calculate the conversion rate of fructose to allulose.

[0073] FIG. 4 shows Microbacterium algerian (in an embodiment of the present invention) Microbacterium algeriense This is the result of measuring the allulose productivity of the C8A T4 strain according to reaction time. As shown in Figure 4, Microbacterium algerian ( Microbacterium algeriense When fructose was converted to allulose at a temperature of 55°C using the C8A T4 strain, the maximum conversion rate was nearly reached after about 20 hours of reaction time.

[0075] 4. Microbacterium algerians ( Microbacterium algeriense ) Renaming and Deposit of C8A T4 Strain

[0076] The inventor of the present invention, for the convenience of management, Microbacterium algerian ( Microbacterium algeriense The name of the C8A T4 strain is Microbacterium algerianse( Microbacterium algeriense ) changed to AN01 and on April 2, 2025, proceeded with an international patent deposit pursuant to the Treaty of Budapest at the accredited depositary, the Korean Culture Center of Microorganisms (KCCM; Address: 3F, Yurim Bldg., 45 Hongjenae 2-ga-gil, Seodaemun-gu, Seoul, Republic of Korea), and was assigned accession number KCCM13558BP. The above Microbacterium algerians ( Microbacterium algeriense ) AN01 (Entrustment No.: KCCM13558P) may be distributed to a third party within the procedures prescribed by the Treaty of Budapest.

[0078] Although the present invention has been described above through the embodiments, the invention is not necessarily limited thereto, and it is understood that various modifications are possible within the scope and spirit of the invention. Accordingly, the scope of protection of the present invention should be interpreted to include all embodiments falling within the scope of the claims attached to the present invention.

[0080] Depository Name: Korean Culture Collection Center (KCCM) Trustee Number: KCCM13558P Date of Deposit: 2025-04-02

Claims

Claim 1 Microbacterium algerianus having the activity of converting fructose to allulose ( Microbacterium algeriense A composition for producing allulose comprising one or more selected from the group consisting of a strain, a culture of said strain, a lysate of said strain, and an allulose epimerizing enzyme produced by said strain. Claim 2 A composition for producing allulose according to claim 1, further comprising one or more metal ion compounds selected from the group consisting of cobalt ion compounds, manganese ion compounds and magnesium ion compounds. Claim 3 In paragraph 1 or 2, the above-mentioned Microbacterium algerian ( Microbacterium algeriense A composition for producing allulose, wherein the conversion activity of fructose to allulose by the strain is stable in a temperature range of 67 to 85°C and a pH range of 5.5 to 8. Claim 4 In paragraph 1 or 2, the above-mentioned Microbacterium algerian ( Microbacterium algeriense A composition for producing allulose, wherein the strain increases the conversion activity of fructose to allulose by more than 1.8 times compared to the case in the absence of metal ions by cobalt ions, manganese ions, or magnesium ions. Claim 5 Microbacterium algerianus having the activity of converting fructose to allulose ( Microbacterium algeriense A method for producing allulose, comprising the step of converting fructose into allulose by reacting fructose with one or more selected from the group consisting of a strain, a culture of the strain, a lysate of the strain, and an allulose epimerizing enzyme produced by the strain. Claim 6 A method for producing allulose according to claim 5, wherein the reaction temperature for converting fructose into allulose is selected in the range of 50 to 85°C. Claim 7 A method for producing allulose according to claim 5, wherein the reaction pH for converting fructose into allulose is selected from the range of 5.5 to 7.

5. Claim 8 A method for producing allulose according to claim 5, wherein the reaction for converting fructose into allulose is carried out in the presence of one or more metal ions selected from the group consisting of cobalt ions, manganese ions, and magnesium ions. Claim 9 A method for producing allulose according to claim 5, wherein the reaction time for converting fructose into allulose is selected in the range of 10 to 40 hours.

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