Allulose-producing Staphylococcus microorganism and method for producing allulose using the same

Abstract

The present application relates to an allulose-producing microorganism and a method for producing allulose using the same.

Description

[Technical Field]

[0001] The present application relates to an allulose-producing microorganism and a method for producing allulose using the same. [Background technology]

[0002] D-Allulose is an epimer of the third carbon of D-fructose, and is a rare sugar found in nature in extremely small amounts. Its sweetness is about 70% of that of sugar, but it has almost no calories. Its ability to suppress blood sugar elevation and lipid synthesis has attracted much attention as a new food ingredient for use in functional foods.

[0003] Due to these characteristics, allulose is being considered for use in various foods as a sugar substitute sweetener, but because it exists in extremely small amounts in nature, there is a growing need for a method for efficiently producing allulose.

[0004] Conventional methods for producing allulose include chemical methods that use the catalytic action of molybdate ions or heat D-fructose with ethanol and triethylamine to produce allulose. However, these chemical methods have drawbacks such as low allulose production yields and high production costs.

[0005] To solve these problems, biological methods for producing allulose from fructose using microbial epimerization enzymes have been studied. In addition to techniques for producing allulose using genetically modified microorganisms carrying the D-allulose 3-epimerization gene derived from Agrobacterium tumefaciens, recent reports have also included methods for producing allulose from D-fructose by isolating microbial strains from the natural environment or food (Patent Documents 1, 2, 3, and 4). However, while biological techniques for producing allulose have been developed, the microorganisms used as biocatalysts have poor thermal stability and high production costs, so there is a pressing need to discover new microbial resources capable of producing allulose.

[0006] Against this background, the inventors conducted intensive research to develop a microorganism capable of producing allulose even in a high-temperature environment, and as a result, they confirmed that Staphylococcus microorganisms can produce allulose, leading to the completion of this application. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Korean Patent Publication No. 10-2011-0035805 [Patent Document 2] Korean Patent Registration No. 10-1804778 [Patent Document 3] Korean Patent Publication No. 10-2017-0067070 [Patent Document 4] Korean Patent Publication No. 10-2016-0081722 [Non-patent literature]

[0008] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-patent document 2] Rice et al., 2000, Trends Genet. 16: 276-277 Non-Patent Document 3 Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 Non-Patent Document 4 Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) Non-Patent Document 5 Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) Non-Patent Document 6 Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 Non-Patent Document 7 [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073 Non-Patent Document 8 https: / / www.ebi.ac.uk / Tools / msa / clustalo Non-Patent Document 9 Smith and Waterman, Adv. Appl. Math (1981) 2:482 Non-Patent Document 10 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) Non-Patent Document 11 Gribskov et al(1986) Nucl. Acids Res. 14: 6745 Summary of the Invention Problems to be Solved by the Invention

[0009] The present application provides a composition for producing allulose, comprising a Staphylococcus microorganism.

[0010] The present application provides a method for producing allulose using the composition. [Means for solving the problem]

[0011] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the following specific descriptions.

[0012] One aspect of the present application provides a composition for producing allulose, comprising a Staphylococcus microorganism or a culture of the Staphylococcus microorganism.

[0013] Specifically, the Staphylococcus microorganisms include Staphylococcus agnetis, Staphylococcus argensis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus condimentii, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus devriesei, Staphylococcus edaphicus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus faecalis, Staphylococcus felis felis, Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcus haemolyticushaemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus leei, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus lyticans, Staphylococcus massiliensis massiliensis, Staphylococcus microti, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pasteuri, Staphylococcus petrasii, Staphylococcus pettenkoferi, Staphylococcus piscifermentans, Staphylococcus pseudintermedius, Staphylococcus pseudorugdunensis pseudolugdunensis, Staphylococcus rostri, Staphylococcus saccharolyticus, Staphylococcus saprophyticussaprophyticus, Staphylococcus schleiferi, Staphylococcus schweitzeri, Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus stepanovicii, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri and Staphylococcus xylosus xylosus), but is not limited to these.

[0014] Specifically, the Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, Staphylococcus muscae, and the like. Staphylococcus muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteurii and Staphylococcus pasteuri, more specifically, Staphylococcus carnosus, Staphylococcus xylosus,Examples of bacteria that may be present include, but are not limited to, Staphylococcus xylosus or Staphylococcus vitulinus.

[0015] D-allulose (hereinafter referred to as allulose) in this application is an epimer of D-fructose and is produced from fructose (fruit sugar) using an epimerization enzyme. In this application, it is often referred to as psicose.

[0016] The fructose used as a substrate in the present application may be, but is not limited to, that obtained from sugar decomposed by a converting enzyme, that obtained from liquid fructose, or that is commercially available.

[0017] The Staphylococcus microorganism of the present application has the activity of converting D-fructose to D-allulose. The Staphylococcus microorganism produces D-allulose from D-fructose using its metabolic system, and the conversion reaction may occur intracellularly or may be secreted extracellularly. Any allulose production process may be used as long as the Staphylococcus microorganism is capable of producing allulose from fructose.

[0018] The Staphylococcus microorganisms of the present application include not only wild-type microorganisms but also mutant microorganisms that have naturally occurring or non-naturally occurring mutations. Specifically, non-naturally occurring mutations may be induced by irradiating wild-type or naturally occurring mutant microorganisms with UV rays or radiation (gamma rays, X-rays), or by using chemical mutagens. Even if they have genetic traits different from those of wild-type Staphylococcus microorganisms, they are included in the present application as long as they have the properties of Staphylococcus microorganisms that can produce allulose from fructose.

[0019] Furthermore, the Staphylococcus microorganism of the present application has the thermotolerance to produce allulose even in high temperature environments, for example, at temperatures of 50°C or higher, and therefore has the advantage of increasing the yield of allulose production.

[0020] The Staphylococcus microorganisms of the present application exhibit the activity of converting fructose to allulose, and are included in the present application as long as they exhibit an industrially applicable allulose conversion rate.

[0021] The conversion rate in the present application is expressed as the concentration of allulose produced after 12 hours of reaction divided by the initial fructose concentration (1 wt%), and the Staphylococcus microorganisms in the present application exhibit conversion rates of 0.1% or more, specifically 0.3% or more, 0.5% or more, 0.9% or more, 1.6% or more, 2.3% or more, 3.4% or more, 5.3% or more, 10.4% or more, and 24.5% or more, but are not limited to these.

[0022] The conversion rate in the present application can be measured by a method known in the art and is not limited to a specific method. For example, the conversion rate in the present application is measured as the result of a conversion reaction to allulose carried out for 12 hours under conditions of pH 7.5 and 55°C, but the reaction conditions (e.g., pH, temperature, time, etc.) can be appropriately selected by those skilled in the art to measure the conversion rate.

[0023] In particular, the Staphylococcus microorganisms are known to be non-pathogenic and have the advantage of being applicable to a variety of foods.

[0024] Therefore, the Staphylococcus microorganisms of the present application are non-pathogenic, but are not limited thereto. Specifically, Staphylococcus genus microorganisms known as non-pathogenic microorganisms include Staphylococcus argensis, Staphylococcus capitis, Staphylococcus devriesei, Staphylococcus faecalis, Staphylococcus sciuri, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus microti, and Staphylococcus piscifermentans. Staphylococcus piscifermentans, Staphylococcus schweitzeri, Staphylococcus simulans, Staphylococcus succinus, Staphylococcus arlettae, Staphylococcus auricularis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus delphini delphini, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarumgallinarum, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus muscae, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus vitulinus, Staphylococcus warneri, or Staphylococcus xylosus.

[0025] In this application, a non-pathogenic microorganism means a microorganism that does not cause disease symptoms in individuals, including humans, and refers to a safe strain that falls into risk group 1 of the internationally accepted biosafety level. Specifically, Staphylococcus agnetis, Staphylococcus argenteus, Staphylococcus aureus, Staphylococcus cornubiensis, Staphylococcus felis, Staphylococcus fleurettii, Staphylococcus hyicus, Staphylococcus lutrae, Staphylococcus massiliensis, Staphylococcus genus, Staphylococcus genus strains that fall into biosafety risk group 2 or higher are also included. This refers to all strains except for Staphylococcus massiliensis, Staphylococcus nepalensis, Staphylococcus petrasii, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus rostri, Staphylococcus schleiferi, Staphylococcus simiae, and Staphylococcus stepanovicii.

[0026] The non-pathogenic Staphylococcus microorganism of the present application is characterized by having the ability to produce allulose and not having any harmful effects on individuals, including humans.

[0027] It has been confirmed that various microorganisms belonging to the genus Staphylococcus in the present application, particularly non-pathogenic microorganisms, have the activity of converting fructose into allulose, and analysis of the 16S rRNA of the microorganisms has confirmed that they are genetically related to each other.

[0028] In this application, "16S rRNA" refers to 16S ribosomal RNA, which is the rRNA that constitutes the 30S subunit of the prokaryotic ribosome and is approximately 1,500 nucleotides in length. While the 16S rRNA sequence is largely highly conserved, there is high diversity in some sections of the base sequence, particularly between species, whereas diversity exists between different species. Therefore, this sequence is known as a commonly used sequence for identifying prokaryotes. That is, genetic relatedness can be determined by comparing the homology of 16S rRNA sequences, and the higher the homology (similarity) of 16S rRNA sequences, the more similar the genetic traits. In this application, "homology" or "identity" refers to the degree to which two given base sequences are related to each other, expressed as a percentage.

[0029] Homology and identity are often used interchangeably.

[0030] Homology or identity of conserved polynucleotide sequences can be determined by standard alignment algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences generally hybridize over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or full length under moderately or highly stringent conditions. This hybridization also takes into account polynucleotides containing degenerate codons in place of codons.

[0031] Whether any two polynucleotide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters, as described in, for example, Non-Patent Document 1. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using the BLAST, Clustal omega program (Non-Patent Document 8), or Clustal W from the National Center for Biotechnology Information.

[0032] Polynucleotide homology, similarity, or identity can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 9. Briefly, the GAP program defines homology as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application indicates the relevance between sequences.

[0033] The composition for producing allulose of the present application may contain one, two or more species of Staphylococcus microorganisms or cultures thereof, but is not limited thereto.

[0034] The composition for producing allulose of the present application can produce allulose by containing a Staphylococcus microorganism or a culture thereof that has the activity of converting fructose into allulose.

[0035] In this application, "culture" means growing microorganisms under artificially adjusted environmental conditions, and "culture" means a product obtained by culturing microorganisms, and includes all of the microorganisms and substances secreted by the microorganisms.

[0036] The method for culturing Staphylococcus microorganisms in the present application can be a method well known in the art, specifically, but not limited to, batch, continuous, or fed-batch culture using a batch process, fed-batch, or repeated fed-batch process.

[0037] The medium used for the culture must meet the requirements of the particular strain in a suitable manner, and the conditions for culturing Staphylococcus microorganisms are well known.

[0038] Specifically, sugar sources that can be used in the medium include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, fats and oils such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerin and ethanol, and organic acids such as acetic acid. These substances can be used alone or in mixtures, but are not limited to these.

[0039] Examples of carbon sources that can be used include raw sugar or glucose, and molasses containing a large amount of raw sugar. Specifically, purified glucose may be used, but the carbon sources are not limited to these, and a variety of other carbon sources can be used.

[0040] Nitrogen sources that can be used include, but are not limited to, peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources can be used alone or in mixtures.

[0041] Phosphorus sources that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts.

[0042] The culture medium may also contain metal salts necessary for growth, such as magnesium sulfate and iron sulfate. In addition to the above substances, essential growth substances, such as amino acids and vitamins, may also be used. Precursors suitable for the culture medium may also be used. The above-mentioned raw materials may be added to the culture in a suitable manner, either batchwise or continuously, during the culture process.

[0043] During the cultivation of the microorganism, the pH of the culture may be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. Furthermore, foam formation may be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture to maintain an aerobic state.

[0044] In addition, the composition for producing allulose of the present application may further contain, in addition to the Staphylococcus microorganism or a culture of the microorganism, the substrate fructose and / or an enzyme involved in allulose production, but is not limited to these.

[0045] Furthermore, the composition for allulose production of the present application may further contain any suitable excipient commonly used in compositions for allulose production, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0046] The composition for allulose production of the present application may further contain a metal ion or metal salt. By including the metal ion or metal salt in the composition, the composition for allulose production of the present application has the activity of converting fructose to allulose. The metal ion or metal salt is necessary for the process in which Staphylococcus microorganisms produce allulose from fructose, for example, but not limited to, those necessary for the action of enzymes that mediate the conversion process. The metal ion or metal salt contained in the composition for allulose production of the present application can be appropriately selected from metal ions or metal salts known to those skilled in the art, as long as the composition exhibits the activity of converting fructose to allulose. In one embodiment, the metal ion is a divalent cation, specifically at least one metal ion selected from the group consisting of Ni, Mg, Ni, Co, Mn, Fe, and Zn. More specifically, the composition for producing allulose of the present application further contains a metal salt, and even more specifically, the metal salt is at least one selected from the group consisting of NiSO4, MgSO4, MgCl2, NiCl2, CoSO4, CoCl2, MnCl2, MnSO4, FeSO4 and ZnSO4.

[0047] Another aspect of the present application provides the use of a Staphylococcus microorganism or a culture of said Staphylococcus microorganism in the production of allulose.

[0048] The "Staphylococcus microorganism" and the "culture of Staphylococcus microorganism" are as described above.

[0049] Yet another aspect of the present application provides a method for producing allulose, comprising the step of contacting fructose with the composition to convert the fructose into allulose.

[0050] Specifically, the Staphylococcus microorganisms include Staphylococcus agnetis, Staphylococcus argensis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus condimentii, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus devriesei, Staphylococcus edaphicus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus faecalis, Staphylococcus felis felis, Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcus haemolyticushaemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus leei, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus lyticans, Staphylococcus massiliensis massiliensis, Staphylococcus microti, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pasteuri, Staphylococcus petrasii, Staphylococcus pettenkoferi, Staphylococcus piscifermentans, Staphylococcus pseudintermedius, Staphylococcus pseudorugdunensis pseudolugdunensis, Staphylococcus rostri, Staphylococcus saccharolyticus, Staphylococcus saprophyticussaprophyticus, Staphylococcus schleiferi, Staphylococcus schweitzeri, Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus stepanovicii, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri and Staphylococcus xylosus xylosus), but is not limited to these.

[0051] More specifically, the Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, Staphylococcus muscae, and the like. Staphylococcus muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteurii and Staphylococcus pasteuri, more specifically, Staphylococcus carnosus, Staphylococcus xylosus,Examples of bacteria that may be present include, but are not limited to, Staphylococcus xylosus or Staphylococcus vitulinus.

[0052] The production method of the present application may further comprise, but is not limited to, obtaining fructose from a sugar, glucose, and methods for obtaining fructose from said sugar, glucose, particularly enzymatic production methods, are known in the art.

[0053] The production method of the present application has the advantages of being able to convert fructose into allulose even in a high-temperature environment, resulting in fewer contamination sources and a higher yield.

[0054] Specifically, the temperature in the step of converting fructose to allulose is 40°C to 70°C, more specifically 50°C to 70°C. The culture time is continued until the desired amount of allulose is produced, and is specifically, but not limited to, 5 to 120 hours, more specifically 10 to 30 hours. Furthermore, in the production method of the present application, the conversion of fructose to allulose is carried out at a pH of 5.0 to 9.0, specifically pH 6.0 to 8.0.

[0055] The production method of the present application may further include, but is not limited to, a step of recovering allulose converted by the Staphylococcus microorganism.

[0056] Specifically, the method may involve crushing Staphylococcus microorganisms to recover allulose, or may involve separating allulose from a culture of Staphylococcus microorganisms. As long as it is possible to recover allulose converted by Staphylococcus microorganisms, the method is not limited to a specific method.

[0057] Allulose can be separated by conventional methods known in the art. Separation methods include centrifugation, filtration, ion exchange chromatography, crystallization, etc. For example, the culture can be centrifuged at low speed to remove the biomass, and the resulting supernatant can be separated by ion exchange chromatography, but this is not limiting.

[0058] Furthermore, the production method of the present application may further include, but is not limited to, a step of purifying allulose. Commonly used methods can be used for the purification. Examples include, but are not limited to, dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography, and fractional crystallization. The purification may involve a single method or two or more methods in combination. For example, the allulose-producing reaction product may be purified by chromatography. The separation of sugars by chromatography can be achieved by utilizing the small difference in binding strength between the sugars to be separated and the metal ions attached to the ion resin.

[0059] In addition, the present application may further include a step of decolorizing and / or desalting before or after the purification step of the present application. By performing the decolorizing and / or desalting, a more purified allulose reactant without impurities can be obtained. [Effects of the Invention]

[0060] The Staphylococcus microorganism of the present application has the effect of being able to produce allulose even in a high-temperature environment. [Brief explanation of the drawings]

[0061] [Figure 1] Figure 1 shows allulose production in Staphylococcus delphini KCTC3592. [Figure 2] FIG. 1 shows a phylogenetic tree of non-pathogenic Staphylococcus strains. [Figure 3] FIG. 1 shows a phylogenetic tree of 20 non-pathogenic staphylococcal strains. DETAILED DESCRIPTION OF THE INVENTION

[0062] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, the scope of this application is not limited to the following specific description.

[0063] To achieve the above object, the present application provides a composition for producing allulose, which comprises a Staphylococcus microorganism or a culture of the Staphylococcus microorganism. [Example]

[0064] The present application will be described in detail below with reference to examples. However, these examples are merely illustrative of the present application and are not intended to limit the scope of the present application. [Example]

[0065] Confirmation of allulose production by Staphylococcus aureus microorganisms To confirm the allulose-producing ability of Staphylococcus microorganisms, 32 non-pathogenic Staphylococcus microorganisms were selected, and 20 of these Staphylococcus microorganisms were examined to determine whether they could produce allulose from D-fructose.

[0066] Specifically, two species of Staphylococcus carnosus (KCTC3580, KACC13250), two species of Staphylococcus xylosus (KCTC3342, KACC16180), four species of Staphylococcus vitulinus (KACC15803, KACC15804, KACC15805, KACC13211), Staphylococcus delphini (KCTC3592), Staphylococcus aequorum (KCTC3589), Staphylococcus epidermidis (KCTC1917), Staphylococcus cohnii (KCTC3574), Staphylococcus chromogenes (KCTC3579), Staphylococcus caprae (KCTC3583), and Staphylococcus difficile (KCTC3584). Staphylococcus warneri (KCTC3340), Staphylococcus lentus (KCTC3577), Staphylococcus muscae (KCTC3576), Staphylococcus saprophyticus (KCTC3345), Staphylococcus pasteuri (KCTC13167), Staphylococcus intermedius (KCTC3344), Staphylococcus arlette (KCTC3588), Staphylococcus krusii (KCTC3590), Staphylococcus haemolyticus (KCTC3341), Staphylococcus gallinarum (KCTC3585), and Staphylococcus auricularis (KCTC3584) were collected from the Korean Biological Resource Center (KCTC3340). Collection for Type Cultures (KCTC) and the Korean Agricultural Culture Collection (KACC).

[0067] The microorganisms were inoculated into tryptic soy broth (peptone 17 g / L, soytone 3 g / L, psicose 10 g / L, NaCl 5 g / L, KHPO 2.5 g / L, agar 15 g / L) supplemented with 1% allulose and cultured for 18 hours at 30 or 37° C. The cultured cells were then collected, washed with 0.85% (w / v) NaCl, and used to carry out a pre-cell conversion reaction.

[0068] The cells were suspended in 50 mM potassium phosphate buffer (pH 7.5) containing 1% (w / w) D-fructose at a cell concentration of 20% (w / w), and the conversion reaction was carried out at 55°C for 12 hours.

[0069] The supernatant from the conversion reaction was analyzed by HPLC to confirm allulose production. HPLC analysis was performed using an HPLC (Agilent, USA) Refractive Index Detector (Agilent 1260 RID) equipped with an Aminex HPX-87C column (BI0-RAD). The mobile phase solvent was water, the temperature was 80°C, and the flow rate was 0.6 mL / min. The allulose conversion rate was calculated as the ratio of the weight of allulose produced after the reaction to the weight of the substrate (D-fructose) before the reaction (allulose concentration (12 h reaction) / initial fructose concentration (1 wt%)).

[0070] As a result, it was confirmed that all 20 species of Staphylococcus microorganisms produced allulose from D-fructose (Table 1 and Figure 1).

[0071] [Table 1] [Example]

[0072] Confirmation of 16S rRNA similarity among Staphylococcus species Example 2-1. Confirmation of 16S rRNA similarity of non-pathogenic Staphylococcus microorganisms In this example, microorganisms classified as non-pathogenic (biosafety level risk group 1) Staphylococcus microorganisms are Staphylococcus argensis, Staphylococcus capitis, Staphylococcus devriesei, Staphylococcus faecalis, Staphylococcus sciuri, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus microti, Staphylococcus piscifermentans, Staphylococcus schweitzeri, Staphylococcus simulans, Staphylococcus succinus, Staphylococcus arlettae, Staphylococcus auricularis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus muscae, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus vitulinus, Staphylococcus warneri, and Staphylococcus xylosus.

[0073] Among the nonpathogenic staphylococci, the 16s rRNA sequences of 31 species were analyzed for interstrain sequence homology using the Clustal omega program (Non-Patent Document 8), excluding one species, Staphylococcus faecalis, for which the 16s rRNA sequence could not be identified. Representative 16s rRNA sequences for each strain were identified in the NCBI database.

[0074] As a result, it was confirmed that the homology of the 16s rRNA sequences between strains of Staphylococcus species, which are known to be non-pathogenic, was 92.78% or higher (Figure 2). Most of the 31 species of Staphylococcus species showed homology of 95% or higher, with Staphylococcus argensis showing homology of 92.78% to 95.21%.

[0075] This result confirms that all 31 species of Staphylococcus classified as non-pathogenic share a high degree of genetic relatedness.

[0076] Example 2-2. Confirmation of 16S rRNA similarity among 20 non-pathogenic Staphylococcus species The 16s rRNA sequences of the 20 Staphylococcus species whose allulose conversion ability was confirmed in Example 1 were analyzed for sequence homology using the Clustal omega program (Non-Patent Document 8). Representative 16s rRNA sequences of each strain were confirmed in the NCBI database.

[0077] As a result, it was confirmed that the homology of the 16s rRNA sequences of the 20 Staphylococcus species that were confirmed to have allulose-producing ability was 95.97% or more.

[0078] From the above examples, it was confirmed that all non-pathogenic Staphylococcus microorganisms have high genetic relatedness between species, and that these highly genetically related Staphylococcus microorganisms have the activity to produce allulose from fructose.

[0079] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features of the present application. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents. The present invention includes the following aspects. [1] A composition for producing allulose comprising a Staphylococcus microorganism or a culture of the Staphylococcus microorganism. [2] The Staphylococcus microorganisms include Staphylococcus agnetis, Staphylococcus argensis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, and Staphylococcus chromogenes. chromogenes, Staphylococcus cohnii, Staphylococcus condimentii, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus devriesei, Staphylococcus edaphicus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus faecalis, Staphylococcus felis felis, Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcus haemolyticushaemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus leei, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus lyticans, Staphylococcus massiliensis massiliensis, Staphylococcus microti, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pasteuri, Staphylococcus petrasii, Staphylococcus pettenkoferi, Staphylococcus piscifermentans, Staphylococcus pseudintermedius, Staphylococcus pseudorugdunensis pseudolugdunensis, Staphylococcus rostri, Staphylococcus saccharolyticus, Staphylococcus saprophyticussaprophyticus, Staphylococcus schleiferi, Staphylococcus schweitzeri, Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus stepanovicii, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri and Staphylococcus xylosus Item 1, wherein the composition is any one selected from the group consisting of: [3] The Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, and Staphylococcus muscae. Staphylococcus muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteurii Item 1. The composition according to item 1, which is any one selected from the group consisting of: [4] The composition described in item 1 above, wherein the Staphylococcus microorganism is non-pathogenic. [5] A method for producing allulose, comprising the step of contacting fructose with a Staphylococcus microorganism or a culture of the Staphylococcus microorganism to convert the fructose into allulose. [6] The Staphylococcus microorganisms include Staphylococcus agnetis, Staphylococcus argensis, Staphylococcus argenteus, Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, and Staphylococcus chromogenes. chromogenes, Staphylococcus cohnii, Staphylococcus condimentii, Staphylococcus cornubiensis, Staphylococcus delphini, Staphylococcus devriesei, Staphylococcus edaphicus, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus faecalis, Staphylococcus felis felis, Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcus haemolyticushaemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus leei, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus lyticans, Staphylococcus massiliensis massiliensis, Staphylococcus microti, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pasteuri, Staphylococcus petrasii, Staphylococcus pettenkoferi, Staphylococcus piscifermentans, Staphylococcus pseudintermedius, Staphylococcus pseudorugdunensis pseudolugdunensis, Staphylococcus rostri, Staphylococcus saccharolyticus, Staphylococcus saprophyticussaprophyticus, Staphylococcus schleiferi, Staphylococcus schweitzeri, Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus simulans, Staphylococcus stepanovicii, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri and Staphylococcus xylosus Item 6. The method of producing according to item 5, wherein the genus is any one selected from the group consisting of: [7] The Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, and Staphylococcus muscae. Staphylococcus muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteurii Item 6. The method of producing according to item 5, wherein the fermented food is any one selected from the group consisting of: [8] The method according to item 5, further comprising recovering the converted allulose. [9] The method according to item 5, wherein the step of converting fructose into allulose is carried out at a temperature of 40°C to 70°C.

[10] Use of a Staphylococcus microorganism or a culture of the Staphylococcus microorganism in the production of allulose.

Claims

1. A composition for producing allulose comprising a Staphylococcus microorganism or a culture of the Staphylococcus microorganism, The Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, Staphylococcus muscae, and the like. muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteuri.

2. A method for producing a Staphylococcus microorganism, comprising culturing the Staphylococcus microorganism in a broth containing allulose before contacting the microorganism with fructose; A step of contacting a Staphylococcus microorganism or a culture of the Staphylococcus microorganism with fructose to convert the fructose into allulose. A method for producing allulose, comprising: The Staphylococcus microorganisms include Staphylococcus carnosus, Staphylococcus xylosus, Staphylococcus vitulinus, Staphylococcus epidermidis, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus cohnii, Staphylococcus muscae, and the like. muscae, Staphylococcus lentus, Staphylococcus chromogenes, Staphylococcus caprae, Staphylococcus auricularis, Staphylococcus gallinarum, Staphylococcus arlettae, Staphylococcus equorum, Staphylococcus kloosii, Staphylococcus delphini, and Staphylococcus pasteuri.

3. The method of claim 2, further comprising recovering the converted allulose.

4. The method according to claim 2 or 3, wherein the step of converting fructose into allulose is carried out at a temperature of 40°C to 70°C.

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