WO2022092864A1 - Microorganisme présentant une capacité réduite de production de mucilage et produit de soja fermenté obtenu par utilisation de ce microorganisme - Google Patents

Microorganisme présentant une capacité réduite de production de mucilage et produit de soja fermenté obtenu par utilisation de ce microorganisme Download PDF

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WO2022092864A1
WO2022092864A1 PCT/KR2021/015335 KR2021015335W WO2022092864A1 WO 2022092864 A1 WO2022092864 A1 WO 2022092864A1 KR 2021015335 W KR2021015335 W KR 2021015335W WO 2022092864 A1 WO2022092864 A1 WO 2022092864A1
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soybean
microorganism
strain
amino acid
polypeptide
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PCT/KR2021/015335
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Korean (ko)
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최명현
박민주
박수영
한성욱
김형준
장경훈
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씨제이제일제당 (주)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/05—Mashed or comminuted pulses or legumes; Products made therefrom
    • A23L11/07—Soya beans, e.g. oil-extracted soya bean flakes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the present invention relates to a microorganism having reduced mucus-producing ability, a method for producing a soybean fermented product using the same, and a soybean fermented product and use thereof.
  • soybean meal In the feed market, defatted soybean meal (hereinafter referred to as “soybean meal”) accounts for the largest share of plant protein raw materials used as substitutes for animal protein such as fish meal, meat bone meal or plasma. Soybean meal refers to the residue left after squeezing soybean oil, and is also called soybean meal. Soybean meal contains 55-56% by weight of protein, 13-14% by weight of soluble carbohydrates, and 21-22% by weight of insoluble carbohydrates on a dry basis.
  • Patent Document 1 International Patent Publication No. WO2011-031020 (2011.03.17)
  • One object of the present application is to provide a polypeptide in which the 83th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
  • One object of the present application is to provide a polynucleotide encoding the polypeptide.
  • One object of the present application is to provide a recombinant vector comprising the polynucleotide.
  • Another object of the present application is to provide a soybean fermented product obtained by fermenting the soybean product with the microorganism.
  • Another object of the present application is to inoculate the soybean product with the microorganism; and culturing the microorganism.
  • Another object of the present application is to inoculate the soybean product with the microorganism; And to provide a method for reducing the production of mucilage during soybean fermentation comprising the step of culturing the microorganism.
  • Another object of the present application is to provide a feed composition comprising the fermented soybean.
  • Another object of the present application is to provide a use of the microorganism to produce a soybean fermented product and/or to ferment soybean product and/or to reduce mucilage production during soybean fermentation.
  • Another object of the present application is to provide a use of the microorganism in the production of a composition for producing a soybean fermented product and/or a composition for fermenting a soy product.
  • fermentation strains having improved properties useful for solid fermentation of soybean products, such as soybean meal, and uses thereof.
  • a genetic modification into a specific region of the wild-type strain, compared to the wild-type strain, the ability to produce mucus during the fermentation process is reduced, and the content of sticky mucilage in the fermented product is reduced, and there is little phenomenon of clumping into lumps.
  • a strain of Bacillus subtilis that is excellent in increasing aeration and/or productivity of the fermented product, as well as increasing proteolytic enzyme activity and protein.
  • soybean product such as soybean meal
  • soybean product is a generic term for by-products obtained during processing (eg, milking, etc.) of soybeans, and may be, for example, soybean meal, soybean protein concentrate, or a combination thereof.
  • soybean refers to a product obtained by inoculating and fermenting soybean products (soy protein concentrate and/or soybean meal) with a fermentation strain, for example, fermented soybean meal, fermented soybean protein concentrate, or these It may mean a combination of
  • soybean meal is also called soybean cake or oil cake, and is a product produced after milking from soybeans, and is the most used vegetable protein feed material.
  • soybean protein Concentrate refers to a concentration of protein content derived from soybeans by removing soluble non-protein substances from defatted soybeans.
  • the soybean protein concentrate may refer to a high-protein feed material in which the protein content is increased by using soybean meal, a by-product left after making edible oil from soybeans. It is classified as a representative vegetable high protein material along with fermented soybean meal and can be used as an alternative to fishmeal, which has been used as a major protein source in the past.
  • soy protein concentrate elutes defatted soybeans with water close to the isoelectric point (pH 4 to 5) or 20 to 80% ethanol, and removes water-soluble and non-protein substances such as carbohydrates to concentrate mainly globulinic proteins.
  • soy protein concentrate may be a soy protein containing protein in an amount of 50 to 90% by weight based on dry matter from which moisture is removed.
  • microorganism encompasses single-celled bacteria, and may be used interchangeably with “cell”, “strain”, and the like.
  • a polynucleotide which may be used interchangeably with “gene” or a polypeptide (which may be used interchangeably with “protein”) is “comprising or consisting of a specific nucleic acid sequence or amino acid sequence, or “expressed” may mean that the polynucleotide or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and the range that maintains the original function and/or the desired function of the polynucleotide or polypeptide It can be construed as including a "substantially equivalent sequence” in which nonsense mutations (deletions, substitutions, modifications, and/or additions) have been made to the specific nucleic acid sequence or amino acid sequence in (or not excluding the meaningless mutation). there is.
  • polypeptide variant in which a mutation is introduced in the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide variant may be a polypeptide in which the 83rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
  • a "polypeptide variant” or "polypeptide” in which a mutation is introduced in the amino acid sequence of SEQ ID NO: 1 means that a mutation is introduced in the amino acid sequence of SEQ ID NO: 1 as described herein. It may mean a polypeptide variant.
  • the 83rd amino acid (aspartic acid; D) from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is valine (V), asparagine (N), glutamic acid (E), glycine (G), alanine (A), Serine (S), Threonine (T), Cysteine (C), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), glutamine (Q), histidine (H), lysine (K), or arginine (R), such as valine.
  • the polypeptide is valine (V), asparagine (N), glutamic acid (E), glycine (G), alanine (A), serine (S) at position 83 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 , Threonine (T), Cysteine (C), Leucine (L), Isoleucine (I), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Glutamine (Q) , histidine (H), lysine (K), or arginine (R), such as a polypeptide having valine.
  • the polypeptide may include the amino acid sequence of SEQ ID NO: 3.
  • the variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 3, or may consist essentially of the amino acid sequence.
  • the amino acid corresponding to position 83 based on the amino acid sequence of SEQ ID NO: 1 is valine, and 70% or more, 75% or more, 80% or more, 85% of the amino acid sequence described in SEQ ID NO: 3 or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.9% or more, 99.1% or more, 99.3% or more, 99.6% or more, It comprises an amino acid sequence having 99.8% or more, or 99.9% or more homology or identity, or has 70% or more, 75% or more, 80% or more, 85% or more of homology or identity to the amino acid sequence set forth in SEQ ID NO: 3; 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.2% or more, 98.4% or more, 98.6% or more, 98.9% or
  • variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident
  • sequence additions or deletions naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.
  • conservative substitution means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.
  • variant means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties Refers to a polypeptide that is maintained.
  • variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation.
  • some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.
  • variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
  • variant is used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, if the term is used in a mutated sense, it is not limited thereto.
  • the variant may be a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 in which aspartic acid, an amino acid corresponding to position 83 of the amino acid sequence of SEQ ID NO: 1, is substituted with valine.
  • variants may include deletions or additions of amino acids having minimal effect on the properties and secondary structure of the polypeptide.
  • a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally.
  • the variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.
  • the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage.
  • the terms homology and identity can often be used interchangeably.
  • Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with a polynucleotide containing a common codon in a polynucleotide or a codon taking codon degeneracy into account.
  • a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
  • Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (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 end gaps.
  • the variant of the present application may have GTPase ObgE (NCBI Reference Sequence: WP_003229675.1) activity.
  • the mutant of the present application may have reduced mucin production compared to the wild-type polypeptide having GTPase ObgE activity.
  • the GTPase ObgE sequence can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having GTPase ObgE activity encoded by ObgE , but is not limited thereto.
  • corresponding to refers to an amino acid residue at a listed position in a polypeptide, or an amino acid residue that is similar, identical to, or homologous to a listed residue in a polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid of a sequence that refers to the specific sequence.
  • corresponding region generally refers to a similar or corresponding position in a related or reference protein.
  • any amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3 .
  • a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").
  • Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), but is not limited thereto, and a sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art may be appropriately used.
  • polynucleotide encoding the polypeptide.
  • the polynucleotide may include the nucleic acid sequence of SEQ ID NO: 4.
  • polynucleotide is a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are connected in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.
  • the polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3.
  • the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 4.
  • the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 4.
  • the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Variations can be made.
  • the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% homology or identity to the sequence of SEQ ID NO: 4 or more, 98% or more, 98.68% or more, 98.76% or more, 98.83% or more, 98.91% or more, 98.99% or more, 99.07% or more, 99.15% or more, 99.22% or more, 99.3% or more, 99.38% or more, 99.46% or more, It has or contains a nucleotide sequence having homology or identity of 99.53% or more, 99.61% or more, 99.69% or more, 99.77% or more, 99.84% or more, or 99.92% or more, or homology or identity with the sequence of SEQ ID NO: 4 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more
  • the polynucleotide of the present application may be a probe that can be prepared from a known gene sequence, for example, a sequence capable of hybridizing under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application, without limitation. may be included.
  • the “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).
  • polynucleotides with high homology or identity 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.68% or more, 98.76% or more, 98.83% or more, 98.91% or more, 98.99% or more, 99.07% or more, 99.15% or more, 99.22% or more, 99.3% or more, 99.38% or more, 99.46% or more, 99.53% or more, 99.61% Conditions under which polynucleotides having homology or identity of at least 99.69%, at least 99.77%, at least 99.84%, or at least 99.92% do not hybridize with each other, and polynucleotides with lower homology or identity do not hybridize, or conventional Equivalent to the washing conditions of southern hybridization at 60°C, 1 ⁇ SSC, 0.1% SDS, specifically 60°C, 0.1
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
  • the term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.
  • a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using the hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions.
  • the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
  • the appropriate stringency for hybridizing the polynucleotide depends on the length of the polynucleotide and the degree of complementarity, and the parameters are well known in the art (eg, J. Sambrook et al., supra).
  • the recombinant vector may be for inserting the polynucleotide into the genome of a host cell or replacing a corresponding gene in the genome of a host cell.
  • the vector of the present application is a DNA preparation comprising a nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.
  • a suitable expression control region or expression control sequence
  • the expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the vector After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
  • the vector used in the present application is not particularly limited, and any vector known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used.
  • pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used.
  • pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pTop33ori vectors and the like can be used.
  • a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
  • the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
  • the selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression Markers that give ? may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
  • the term "transformation” refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. .
  • the transformed polynucleotide can be expressed in the host cell, it may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome.
  • the polynucleotide includes DNA and/or RNA encoding a target polypeptide.
  • the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
  • the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.
  • microorganism comprising the polypeptide, a polynucleotide encoding the polypeptide, or a recombinant vector comprising the polynucleotide.
  • the microorganism may be a fermentation strain having the ability to produce starch, protein, and cellulosic enzyme. More specifically, the microorganism may be a fermented strain of soybean product.
  • the microorganism may be homogeneous belonging to the genus Bacillus.
  • the bacteria belonging to the genus Bacillus may be at least one selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus megaterium or Bacillus clausii, and the like, for example, Bacillus subtilis strain.
  • the microorganism may be a Bacillus subtilis CR01-0016 strain of accession number KCCM12814P.
  • strain or microorganism
  • strain includes both wild-type microorganisms or microorganisms in which genetic modification has occurred naturally or artificially. As a result of which a specific mechanism is weakened or enhanced, it may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.
  • the strain of the present application includes a strain comprising any one or more of the variant of the present application, the polynucleotide of the present application, and a vector including the polynucleotide of the present application; a strain modified to express a variant of the present application or a polynucleotide of the present application; a variant of the present application, or a strain expressing the polynucleotide of the present application (eg, a recombinant strain); Or it may be a strain having the mutant activity of the present application (eg, a recombinant strain), but is not limited thereto.
  • the term "unmodified microorganism” does not exclude strains containing mutations that can occur naturally in microorganisms, and is either a wild-type strain or a natural-type strain itself, or a genetic mutation caused by natural or artificial factors. It may mean the strain before being changed.
  • the unmodified microorganism may refer to a strain in which the GTPase ObgE variant described herein is not introduced or before it is introduced.
  • the "unmodified microorganism” may be used interchangeably with "strain before modification", “microbe before modification”, “unmodified strain”, “unmodified strain”, "unmodified microorganism” or "reference microorganism”.
  • the microorganism may include the polynucleotide by expressing the polypeptide in place of the wild-type ObgE protein, and/or replacing the wild-type ObgE gene.
  • the microorganism is a microorganism belonging to the genus Bacillus, such as a strain of Bacillus subtilis
  • the endogenous ObgE gene eg, SEQ ID NO: 2
  • a wild-type ObgE protein eg, SEQ ID NO: 1 in the microorganism (eg, SEQ ID NO: 2)
  • the 83rd amino acid of SEQ ID NO: 1 may be mutated to encode a mutated polypeptide (eg, SEQ ID NO: 3) (eg, to have the nucleic acid sequence of SEQ ID NO: 4).
  • the microorganism When the microorganism is used for fermenting soybean products, it may be to reduce mucus production or to prevent mucilage from being produced.
  • the microorganism may have a reduced ability to produce mucus compared to a wild-type strain (eg, a wild-type Bacillus subtilis strain). More specifically, the microorganism expresses a wild-type ObgE protein (eg, ObgE protein of a wild-type Bacillus subtilis strain; SEQ ID NO: 1), and/or a wild-type ObgE gene (eg, an ObgE gene of a wild-type Bacillus subtilis strain; Compared to the microorganism comprising SEQ ID NO: 2), the mucus production ability may be reduced.
  • a wild-type StragE protein eg, ObgE protein of a wild-type Bacillus subtilis strain; SEQ ID NO: 1
  • a wild-type ObgE gene eg, an ObgE gene of a wild-
  • Another example provides a composition for producing a soybean fermented product or a composition for fermenting soybean product comprising the microorganism.
  • Another example provides a soybean fermented product obtained by fermenting the soybean product with the microorganism.
  • the fermented soybean product may have a lower mucilage content compared to a fermented product obtained by fermenting the soybean product with a microorganism comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the polypeptide.
  • mucilage refers to a substance that increases the viscosity (viscosity) of fermented soybeans, and levan form fructan and polyglutamate derived from soybean sugar, protein, or a combination thereof. ), but is not limited thereto.
  • the fermented soybean has a moisture content, by weight, of 30 to 60%, 30 to 55%, 30 to 50%, 30 to 45%, 30 to 40%, 30 to 38%, 32 to 60%, 32 to 55%, 32 to 50%, 32 to 45%, 32 to 40%, 32 to 38%, 35 to 60%, 35 to 55%, 35 to 50%, 35 to 45%, 35 to 40%, or 35 to 38%; Total protein content (based on dry matter weight) of at least 40%, at least 45%, or at least 50%, such as 40 to 70%, 40 to 65%, 40 to 60%, 45 to 70%, 45 to 65%, 45 to 60%, 50 to 70%, 50 to 65%, or 50 to 60%; the pH may be at least 7, at least 7.2, at least 7.5, at least 7.7, or at least 8 (the upper limit may be pH 9 or 8.5); The number of viable cells may be 1x10 5-12 CFU/g, 1x10 6-12 CFU/g, 1x10 7-12 CFU/g, or 1x10 8-12 CFU/g, but is not limited
  • Another example includes inoculating the microorganism into soybean products; and culturing the microorganism.
  • the soybean fermented product thus prepared may have a reduced mucilage content in the fermented product as compared to the case of using a wild-type microorganism.
  • Another example includes inoculating the microorganism into soybean products; and culturing the microorganism. It provides a method of reducing mucilage production during soybean fermentation (or a method of reducing the mucilage content in soybean fermentation product).
  • the method may include the following steps: (a) adding moisture to the soybean product and heat-treating; (b) inoculating the fermented bacteria after cooling the heat-treated soybean product; (c) obtaining a soybean fermented product by solid-culturing the inoculated bacteria in the soybean product.
  • the fermenting bacteria refers to the microorganisms described above.
  • a process of hydrolyzing the raw material soybean product before heat treatment may be necessary.
  • the raw soybean product may be directly sprayed or mixed with an appropriate amount of water before solid fermentation to adjust the moisture content and then heat-treated for a certain period of time.
  • the purpose of the heat treatment may be to provide a chemical composition in which a target microorganism can actively grow by killing and/or destroying soybean cell walls and denaturing proteins in the raw soybean product.
  • the moisture content of the soybean product added in step (a) is 30 to 80% (v/w), 30 to 70% (v/w), or 40 to 60% (v/w) ) can be
  • the moisture content range is a preferable range in terms of preventing a delay in the fermentation rate due to low moisture, improving the costly problems in the transport and drying process of soybean products after fermentation, and in terms of thermal efficiency.
  • the water-added soybean product may be heat-treated.
  • the heat treatment process may use a variety of methods known in the art, for example, may be heat treatment using steam (steam) or superheated steam (superheated steam), but is not limited thereto.
  • the heat treatment of step (a) may be a short-time heat treatment for several seconds to several minutes with steam at a temperature of 70 to 130° C. for 10 to 60 minutes or with superheated steam at a temperature of 200 to 300° C. Steam at °C steam for 10 to 30 minutes, or steam at 80 to 121.1 °C steam for 10 to 30 minutes.
  • step (a) before or after step (b)), it may further include the step of pre-culturing the fermented bacteria in a conventional manner.
  • the fermenting bacteria may be the microorganisms described above.
  • the final number of viable cells of the seed culture medium thus obtained may be in the range of (1-5)x10 9 cfu/mL, but is not limited thereto.
  • the soybean product heat-treated in step (a) is cooled to a temperature capable of solid fermentation, and then inoculated with fermented bacteria.
  • the cooling may proceed naturally after the steaming is finished, and may proceed through a transfer process using a conveyor type cooler to prevent overheating and uniformly cool the cooling rate by increasing the cooling rate, but is not limited thereto.
  • the temperature of the soybean product cooled in this step may be 30 to 50 °C, 35 to 45 °C, or 37 °C.
  • the inoculation of the fermented bacteria may be performed as uniformly as possible by diluting the pre-culture solution of the fermented bacteria in the prepared soybean product medium as it is or by appropriately diluting it with sterile water.
  • the amount of the inoculated fermenting bacteria may be an important factor influencing the solid fermentation of soybean products.
  • the inoculation amount of the fermented bacteria may be an amount such that the number of bacteria immediately after inoculation becomes 10 5 to 10 9 CFU/g.
  • the culture in this step may be a solid culture.
  • solid fermentation means culturing microorganisms using defatted soybean meal and/or soy protein concentrate remaining after separating fat (soybean oil) from soybeans, and using the extract of soybean meal " a method distinct from “liquid culture or liquid fermentation”.
  • the fermentation may proceed in a conventional manner, for example, may proceed in a packed-bed fermentor, but is not limited thereto.
  • packed bed fermenters such as batch aeration culture equipment, closed culture equipment, and continuous aeration culture equipment, and suitable equipment can be selected and used without limitation depending on the production scale.
  • the fermentation may be performed at a temperature of 20 to 50 °C for 12 to 72 hours, at a temperature of 30 to 45 °C for 12 to 48 hours, or at 35 to 40 °C for 12 to 24 hours.
  • step (d) drying and/or pulverizing the soybean fermented product at low temperature and low humidity may be additionally included.
  • the soybean product when the soybean product is fermented, partially weakly agglomerated lumps may be formed.
  • the fermented soybean product can be pulverized to a uniform particle size after drying.
  • the drying and grinding may be carried out by various methods known in the art. For example, it can be dried at a low temperature that does not kill live cells, and can be dried with hot air at low temperature and low humidity.
  • the soybean fermented product may be pulverized to various sizes depending on the intended purpose, and a hammer mill may be used as the pulverizing method, but is not limited thereto.
  • soybean products By fermenting soybean products according to the above method, various anti-nutritional factors contained in soybean products are reduced, digestion and absorption rate are improved by hydrolysis of protein, and absolute value as feed is improved by increasing protein content, so it can replace animal protein.
  • a fermented soybean useful as a high-quality protein feedstock can be obtained.
  • the fermented soybean contains Bacillus, which has strong viability even during distribution, and thus has the advantage of having a function of helping the intestinal tract of animals that have consumed the feed.
  • Another example provides a feed composition comprising the fermented soybean.
  • the feed composition is used to encompass all of the feed, feed raw materials for use in feed production, and feed additives.
  • the feed may be for application to mammals, poultry, fish and/or crustaceans.
  • the mammals include pigs, cattle, horses, deer, goats, dogs, cats, and/or rabbits
  • the poultry includes chickens, ducks, geese, and/or turkeys
  • the fish and crustaceans include trout, salmon , and/or shrimp.
  • the feed composition may include, in addition to the fermented soybean, organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid; phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate; vitamin; mineral; Polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, may additionally include one or more selected from the group consisting of natural antioxidants such as tannic acid, anti-influenza agents, buffers, and / Or other conventional additives such as a bacteriostatic agent may be additionally included.
  • organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid
  • phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate
  • vitamin mineral
  • Polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan may additionally include one or more selected from the group consisting of natural antioxidants such as tannic acid, anti-in
  • diluents, dispersants, surfactants, binders, and/or lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., or an appropriate form such as capsules, granules or tablets.
  • the feed composition may include the fermented soybean in an amount of about 10 to 500 g, or 10 to 100 g per 1 kg by dry weight, but is not limited thereto.
  • Bacillus subtilis C27 strain (KCCM12814P) according to the present application exhibits reduced mucilage production ability during the fermentation process and has excellent protein and cellulolytic enzyme activity. It is possible to manufacture high-quality fermented soybean meal with improved digestion and absorption rate and feed efficiency due to low molecular weight and increased crude protein content due to protein hydrolysis, and decreased content of anti-nutritive factors such as non-digestible polysaccharides.
  • 1 is a graph showing the viscosity of fermented soybean meal obtained using strain C27 according to an embodiment compared with that of fermented soybean meal obtained using the parent strain (CJ2042 strain).
  • FIG. 2 is a photograph of fermented soybean meal obtained using strain C27 according to an embodiment and fermented soybean meal obtained using the parent strain (CJ2042 strain).
  • FIG 3 is a graph showing the viscosity of fermented soybean meal obtained using the BS_obgE strain according to an embodiment compared with that of the fermented soybean meal obtained using the parent strain (BS3135 strain).
  • FIG. 4 is a photograph of a fermented soybean meal obtained using the BS_obgE strain according to an embodiment and a fermented soybean meal obtained using the parent strain (BS3135 strain).
  • the screening of starch, protein, and cellulolytic enzyme high activity strain was 1% (w/v) soluble starch (Difco, USA), 2% (w/v) skim milk (Difco, USA), or 1%, respectively.
  • CMC carboxymethyl cellulose
  • YM agar medium yeast extract 3.0 g, malt extract 3.0 g, peptone 10.0 g, agar 20.0 g
  • the selected starch, protein and cellulolytic enzyme high-producing strains were each prepared in TSB medium (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, final pH: After inoculation at 7.3 ⁇ 0.2 at 25 °C), cultured at 37 °C and 200 rpm for 12 hours, and the culture medium was added dropwise by 1.0 ⁇ l to YM agar medium containing soluble starch, skim milk, or CMC. After culturing the agar medium at 37° C. for 16 hours, the diameter of the transparent ring formed on the medium was measured, and the results are shown in Table 1.
  • the eight strains shown in Table 1 were selected as high-producing strains of starch, protein, and cellulase. Among them, the CJ2042 strain having excellent starch, protein, and cellulase activity was finally selected.
  • the CJ2042 strain selected in Example 1 exhibits high activity of starch, protein and cellulase, but contains mucilage due to the characteristics of the CJ2042 strain.
  • the generation of such mucilage makes it difficult to stir during solid culture later, causing problems in controlling dissolved oxygen, temperature, etc. in the fermented product, making it difficult to transport.
  • UV irradiation was performed on the CJ2042 strain as follows. mutation was induced.
  • strain CJ2042 on TSB agar medium (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, agar 15.0 g, final pH: 7.3 ⁇ 0.2 at 25 °C ) and incubated at 37° C. for 12 hours to activate the strain.
  • the main culture is a seed suspension in a previously prepared TSB medium (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, final pH: 7.3 ⁇ 0.2 at 25°C) 1% inoculation was carried out by shaking culture at 37 °C at 180 rpm. After incubation, the culture medium was centrifuged at 25° C. at 8000 rpm for 10 minutes to separate the cells and the supernatant, and only the cells were taken and washed with 0.8% NaCl sterilizing solution. After washing, the recovered cells were irradiated with UV light at a wavelength of 254 nm using a UV lamp (VIBER LOURMAT, 115 V, 60 Hz) to induce mutation.
  • TSB medium enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g,
  • UV-irradiated mixture was added to TSA plate medium (tryptic soy agar, enzymatic digest of casein 15 g, enzymatic digest of soybean meal 5 g, NaCl 5 g, agar 15 g, final pH 7.3 ⁇ 0.2 at 25°C) ), incubated at 37° C. for 12 hours, and compared with the parent strain CJ2042 strain, UV-irradiated mutant strains that visually formed colonies not containing mucilage were isolated.
  • TSA plate medium tryptic soy agar, enzymatic digest of casein 15 g, enzymatic digest of soybean meal 5 g, NaCl 5 g, agar 15 g, final pH 7.3 ⁇ 0.2 at 25°C
  • Example 2 The enzyme ability of each mutant strain isolated from Example 2 was confirmed.
  • Bacillus subtilis CJ2042 strain selected in Example 1 was used as a control.
  • each of the mutant strains was prepared in TSB medium (medium composition: enzymatic digest of casein 17.0g, enzymatic digest of soybean meal 3.0g, NaCl 5.0g, dipotassium phosphate 2.5g, dextrose 2.5g, final pH 7.3 ⁇ 0.2 at 25 °C) at 37 °C, 200 rpm for 12 h.
  • the culture solution of each mutant strain was spotted by 1.0 ⁇ l on YM agar medium containing soluble starch, skim milk, or CMC. After culturing the agar medium at 37° C. for 16 hours, the diameter of the transparent ring formed on the medium was measured, and the results are shown in Table 2.
  • mutant strain C27 strain has superior starch, protein and cellulase ability than the parent strain Bacillus subtilis CJ2042 strain and Bacillus subtilis TP6 strain.
  • Soybean meal fermentability of the mutant strain C27 strain selected in Example 3 was confirmed.
  • the parent strain CJ2042 strain mentioned in Example 2 and the control Bacillus subtilis TP6 (KCCM11438P) were used.
  • GYP medium glucose 10g/L, yeast extract 8g/L, soy pepton 2g/L
  • 1% of the culture solution obtained through the pre-culture is inoculated again in GYP medium to A660nm 6 or higher incubated until
  • Soybean meal was prepared, the moisture content was adjusted to about 43%, and heat-treated at 100° C. for 30 minutes, followed by cooling.
  • the culture solution of the C27 strain was inoculated into the pre-treated soybean meal in an amount of 10% by weight based on the weight of the soybean meal, and the moisture content was adjusted to about 46%.
  • the soybean meal inoculated with the C27 strain was fermented for 16 hours in a thermo-hygrostat maintained at 37° C. and 95% humidity. Before fermentation, the pH of the soybean meal was about 6.6, and each strain was inoculated at an amount of about 10 ⁇ 7 CFU/g.
  • the fermented product of each strain contained a moisture content of about 35% to 38%, the pH was 8 or more, and the number of viable cells was about 10 ⁇ 9CFU/g or more. .
  • the protein content increased by about 4-6% or more compared to the protein content of the raw material.
  • the fermented product obtained in Example 4 was measured for viscosity (6 kPa) using Anton Paar RheoCompass, and the results are shown in FIGS. 1 and 2 .
  • the maximum viscosity measurement was 8.8387 kPa, and it was confirmed that the viscosity of the fermented product was lower than that of the parent strain CJ2042 strain (10.238 kPa).
  • Example 5 For identification of the C27 strain mutated in Example 5, the 16S rRNA gene nucleic acid sequence of the strain was analyzed.
  • Nucleic acid sequence was confirmed by sequencing using BigDye® Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems Inc., USA), and analyzed with ABI 3730XL DNA Analyzer (Applied Biosystems, 3.850 Lincoln Center Drive Foster City, CA 94404 USA) did The analyzed nucleic acid sequence was compared with the nucleic acid sequence registered from the EzTaxon server (http://eztaxon-e.ezbiocloud.net/) and GenBank/EMBL/DDBJ to confirm the similarity with other gene nucleic acid sequences. After performing multiple sequence alignment for the nucleic acid sequence, a phylogenic tree was prepared using the MEGA 6 program and the taxonomic position was analyzed.
  • the mutant strain C27 strain was named Bacillus subtilis CR01-0016, and was deposited under the Budapest Treaty with the Korean Culture of Microorganisms (KCCM) as accession number KCCM12814P on October 26, 2020.
  • KCCM Korean Culture of Microorganisms
  • Example 7 Full-length analysis of the genome of a mutant with reduced mucilage production capacity
  • Example 8 Mutation introduction for reducing mucilage production ability and confirmation of mucilage production amount of the mutation-introduced strain
  • a strain in which the ObgE gene is mutated in a wild-type Bacillus subtilis strain was prepared.
  • pTop33ori prepared from pTOP Blunt V2 plasmid vector (Enzynomics) was used as the basic plasmid vector for gene replacement, and was inserted at the BamHI restriction site of the plasmid.
  • the gene fragment for constructing the substitution vector was obtained through PCR using the genomic DNA of C27 mutated with ObgE as a template.
  • the PCR conditions for constructing the ObgE gene replacement vector are as follows: After denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 50°C for 15 seconds, and polymerization at 68°C for 1 minute after repeating 30 times, 68°C polymerization was carried out for 5 minutes at Primer sequence information used to construct the vector is shown in Table 5 below.
  • the PCR product of about 2 kb thus amplified was mixed with pTop33ori treated with BamHI restriction enzyme, and a pTOP33ori_obgE substitution vector was prepared using an infusion enzyme.
  • Example 8-2 Confirmation of production of BS_obgE strain introduced with mutation and reduction of mucilage production ability
  • the obgE gene replacement vector, pTOP33ori_obgE, prepared in Example 8-1 was introduced into the Bacillus subtilis KCTC 3135 T strain to prepare a mutated strain BS_obgE.
  • the KCTC 3135 T strain was used after receiving from KCTC (Korea Research Institute of Bioscience and Biotechnology Biological Resources Center).
  • pTOP33ori_obgE a substitution vector
  • strains into which the vector was inserted into the chromosome by recombination of homologous sequences were selected on an agar nutrient medium containing chloramphenicol 7.5 mg/L.
  • the selected primary strain was again cultured with shaking in LB medium without chloramphenicol antibiotics for 16 hours, and after dilution and smearing on LB agar plate medium, strains susceptible to chloramphenicol antibiotics were individually selected from the colonies that grew up, and through nucleotide sequence confirmation, mutant strains was obtained. Whether the final transformed strain was mutated was confirmed through sequencing after PCR was performed using the primer pair of SEQ ID NOs: 5 and 6.
  • the mutant (BS_obgE strain) colonies were subcultured in a nutrient medium, and then in GYP medium (glucose 10g/L, yeast extract 8g/L, soy pepton 2g/L). After pre-culturing, the culture solution obtained through the pre-culture was again inoculated into GYP medium in an amount of 1% and cultured until A660nm 6 or higher to prepare the culture of the BS_obgE strain.
  • GYP medium glucose 10g/L, yeast extract 8g/L, soy pepton 2g/L
  • Soybean meal was prepared, the moisture content was adjusted to about 43%, and heat-treated at 100° C. for 30 minutes, followed by cooling.
  • the prepared culture solution of the BS_obgE strain was inoculated into the pretreated soybean meal in an amount of 10% by weight based on the weight of the soybean meal, and the moisture content was adjusted to about 46%.
  • the soybean meal inoculated with the BS_obgE strain was fermented for 12 hours in a thermo-hygrostat maintained at 37° C. and 95% humidity.
  • the obtained fermented product was measured for viscosity using Anton Paar Rheo Compass. The measurement was performed at 6 Kpa.

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Abstract

La présente invention concerne des microorganismes présentant une capacité réduite de production de mucilage, un procédé de production d'un produit de soja fermenté l'utilisant, le produit de soja fermenté ainsi produit et leurs utilisations.
PCT/KR2021/015335 2020-10-29 2021-10-28 Microorganisme présentant une capacité réduite de production de mucilage et produit de soja fermenté obtenu par utilisation de ce microorganisme WO2022092864A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006025785A (ja) * 2004-06-15 2006-02-02 Mitsukan Group Honsha:Kk 糸引性低下納豆菌、該納豆菌を用いて製造された糸引性低下納豆
KR20110027535A (ko) * 2009-09-09 2011-03-16 씨제이제일제당 (주) 바실러스균을 이용한 발효 대두박의 제조방법
KR101517326B1 (ko) * 2014-01-28 2015-05-04 씨제이제일제당 (주) 발효 대두박 생산능이 향상된 바실러스 속 균주 및 이를 이용하여 발효 대두박을 제조하는 방법
JP2015208319A (ja) * 2014-04-24 2015-11-24 茨城県 糸引性低下納豆菌株及び該納豆菌株による納豆の製造方法と納豆
WO2019209576A1 (fr) * 2018-04-24 2019-10-31 Danisco Us Inc Souches fongiques filamenteuses comprenant des phénotypes à viscosité réduite

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* Cited by examiner, † Cited by third party
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WO2011031020A2 (fr) 2009-09-09 2011-03-17 Cj Cheiljedang Corporation Procédé de préparation d'une farine de soja fermentée au moyen de souches de bacillus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006025785A (ja) * 2004-06-15 2006-02-02 Mitsukan Group Honsha:Kk 糸引性低下納豆菌、該納豆菌を用いて製造された糸引性低下納豆
KR20110027535A (ko) * 2009-09-09 2011-03-16 씨제이제일제당 (주) 바실러스균을 이용한 발효 대두박의 제조방법
KR101517326B1 (ko) * 2014-01-28 2015-05-04 씨제이제일제당 (주) 발효 대두박 생산능이 향상된 바실러스 속 균주 및 이를 이용하여 발효 대두박을 제조하는 방법
JP2015208319A (ja) * 2014-04-24 2015-11-24 茨城県 糸引性低下納豆菌株及び該納豆菌株による納豆の製造方法と納豆
WO2019209576A1 (fr) * 2018-04-24 2019-10-31 Danisco Us Inc Souches fongiques filamenteuses comprenant des phénotypes à viscosité réduite

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