Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
  • A23L27/22—Synthetic spices, flavouring agents or condiments containing amino acids containing glutamic acids
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/88—Taste or flavour enhancing agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • 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
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01017—Lysozyme (3.2.1.17)
• • 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
• • 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
• • 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
• • 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
  • C12R2001/13—Brevibacterium
• • 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
  • C12R2001/15—Corynebacterium
• • 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
  • C12R2001/225—Lactobacillus
• • 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
  • C12R2001/225—Lactobacillus
  • C12R2001/24—Lactobacillus brevis

Definitions

• the present invention relates to a technology for improving the flavor of food.
• Patent Document 1 discloses a flavor base for enhancing taste obtained by fermentation of bacteria such as Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium casei, Corynebacterium efficiens, Brevibacterium lactofermentum, and Bacillus subtilis.
• Patent Document 2 discloses a flavor enhancer that contains yeast cells after the yeast contents have been removed, and gives examples of flavors such as spiciness.
• Patent Document 3 discloses a yeast extract that contains peptide and nucleic acid-based flavor components and can impart or enhance full-bodied flavor to foods by simultaneously imparting a long-lasting umami taste and a rich taste.
• Patent Document 4 discloses a food aroma improver that contains yeast-derived substances such as yeast extract, and gives examples of aromas such as spicy aromas.
• Patent Document 5 discloses a spice sensation enhancer that contains a product obtained by thermally reacting sugar and amino acid in the presence of alcohol.
• Patent Document 6 discloses a fermented seasoning composition that contains a fermented product produced by lactic acid bacteria and yeast and can be used to enhance the spiciness of spices.
• the objective of the present invention is to provide a technology that improves the flavor of food.
• the inventors discovered that the flavor of food can be significantly improved by adding lysozyme-treated cell walls of gram-positive bacteria (e.g., the cells of gram-positive bacteria), and thus completed the present invention.
• lysozyme-treated cell walls of gram-positive bacteria e.g., the cells of gram-positive bacteria
• a composition for improving the flavor of food comprising the following component (A): (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• a composition (specifically, the composition described in [1]) wherein the improvement in flavor is enhancement of spiciness and/or imparting full-bodied taste.
• the composition (specifically described in [1] or [2]) wherein the flavor improvement is the enhancement of the pungency and/or the addition of depth to the spices.
• the composition (specifically, any one of [1] to [3]) further contains a spice.
• a composition comprising the following component (A) and a spice: (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• the composition specifically, any one of [1] to [5]) wherein the component (A) is a lysozyme-treated product of the gram-positive bacterial body or a fragment thereof.
• the composition specifically, any one of [1] to [6]) wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria or Firmicutes.
• composition (specifically, any one of [1] to [7]) wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria.
• composition (specifically, any one of [1] to [8]) wherein the gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.
• composition (specifically, any one of [1] to [9]) wherein the Gram-positive bacterium is a bacterium of the genus Corynebacterium, a bacterium of the genus Brevibacterium, a bacterium of the genus Bifidobacterium, a bacterium of the genus Brachybacterium, a bacillus, a bacterium of the genus Enterococcus, or a bacterium of the genus Lactobacillus.
• the Gram-positive bacterium is a bacterium of the genus Corynebacterium, a bacterium of the genus Brevibacterium, a bacterium of the genus Bifidobacterium, a bacterium of the genus Brachybacterium, a bacillus, a bacterium of the genus Enterococcus, or a bacterium of the genus Lactobacillus.
• the gram-positive bacteria are selected from the group consisting of Corynebacterium casei, Corynebacterium flavescens, Corynebacterium ammoniagenes (Corynebacterium stationis), Corynebacterium glutamicum, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus
• the composition (specifically, any one of [1] to [10]) is selected from the group consisting of Lactobacillus mali, Lactobacillus hilgardii, and Lactobacillus Brevis.
• composition specifically, any one of [1] to [11] has a content of the component (A) of 0.1% (w/w) or more, calculated based on the dry weight of the original gram-positive bacterium cells.
• composition specifically, any one of [1] to [12] in which a fraction of the component (A) having a molecular weight cutoff of 10,000 Da or less contains bound diaminopimelic acid.
• the composition (specifically, the composition described in [13]) has a content of the bound diaminopimelic acid contained in a fraction of component (A) having a molecular weight cutoff of 10,000 Da or less per 100 g of dry weight of the composition (excluding the dry weight of the excipient if the composition contains an excipient) of 20.0 mg or more.
• the composition (specifically, any one of [1] to [14]) further contains the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the composition (specifically, the composition according to [15]) contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.
• the composition (specifically, the composition described in [16]) has a content of L-glutamic acid of 0.1 to 20 parts by weight per part by weight of the component (A), calculated as the dry weight of the original gram-positive bacterium cells.
• the composition (specifically, any one of [4] to [17]) has a content of the spice of 0.2 to 500 parts by weight per part by weight of the component (A) converted into the dry weight of the original gram-positive bacterium cells.
• the composition (specifically, any one of [4] to [18]) is characterized in that the spice is one or more spices selected from the group consisting of spices from the family Lauraceae, family Piperaceae, family Lamiaceae, family Umbelliferae, family Solanaceae, family Myrtaceae, family Liliaceae, family Myrtaceae, family Magnoliae, family Fabaceae, family Polygonaceae, family Brassicaceae, family Zingiberaceae, and family Rutaceae.
• the composition (specifically, any one of [4] to [19]) wherein the spice is a spice having a hot taste.
• composition (specifically, any one of [1] to [20]) which is a seasoning.
• composition specifically, any one of [1] to [21] wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 described below.
• the composition (specifically, any one of [1] to [22]) further comprises a culture of a gram-positive bacterium.
• a method for improving the flavor of a food product comprising: A method comprising the step of adding the following component (A) to a food ingredient: (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• a method for producing a food product having improved flavor comprising: A method comprising the steps of: adding the following component (A) to a food ingredient: (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium. [26] The method (specifically, the method described in [24] or [25]) in which the improvement in flavor is enhancement of spiciness and/or imparting full-bodied flavor. [27] The method (specifically, any one of [24] to [26]) wherein the improvement in flavor is enhancement of the pungency and/or addition of thickness to the spice.
• the Gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.
• the Gram-positive bacterium is a bacterium of the genus Corynebacterium, a bacterium of the genus Brevibacterium, a bacterium of the genus Bifidobacterium, a bacterium of the genus Brachybacterium, a bacillus, a bacterium of the genus Enterococcus, or a bacterium of the genus Lactobacillus.
• the gram-positive bacteria are selected from the group consisting of Corynebacterium casei, Corynebacterium flavescens, Corynebacterium ammoniagenes (Corynebacterium stationis), Corynebacterium glutamicum, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus The method (specifically, any one of [24] to [32]) described above, wherein the bacterium is Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus Brevis.
• the method (specifically, any one of [24] to [33]) described above, in which the component (A) is added so that its ingestible concentration is 0.005 to 2% (w/w) calculated as the dry weight of the original gram-positive bacterium cells.
• the method further comprises a step of adding the following component (B) to a food ingredient: (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the method (specifically, the method described in [35]) above, wherein at least the L-amino acid is added, and the L-amino acid is L-glutamic acid.
• the spice is one or more spices selected from the group consisting of spices from the family Lauraceae, family Piperaceae, family Lamiaceae, family Apiaceae, family Solanaceae, family Myristicae, family Liliaceae, family Myrtaceae, family Magnoliae, family Fabaceae, family Polygonaceae, family Brassicaceae, family Zingiberaceae, and family Rutaceae.
• the spice is a spice having a hot taste.
• the method (specifically, any one of [24] to [43]) wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.
• the method further comprises a step of adding a culture of a gram-positive bacterium to a food raw material.
• a seasoning comprising the following component (A): (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• the seasoning (specifically, as described in [46]) wherein the component (A) is a lysozyme-treated product of the gram-positive bacterium or a fragment thereof.
• the seasoning (specifically, the seasoning described in [46] or [47]) further contains the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• component (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the seasoning (specifically, as described in [48]) which contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.
• the seasoning (specifically, any one of [46] to [49]) further contains a spice.
• the seasoning (specifically, any one of [46] to [50]) wherein the Gram-positive bacterium is a bacterium capable of producing L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.
• the seasoning (specifically, any one of [46] to [51]) further contains a culture of a gram-positive bacterium.
• a method for producing a composition for improving flavor of food comprising the steps of:
• the composition comprises the following component (A): (A) a lysozyme-treated cell wall of a gram-positive bacterium, The method comprises a step of subjecting the cell wall of the gram-positive bacterium to lysozyme treatment to obtain the component (A).
• the method (specifically, the method described in [53]) above, wherein the improvement in flavor is enhancement of spiciness and/or imparting full-bodied flavor.
• the method (specifically described in [53] or [54]) wherein the improvement in flavor is enhancement of the pungency and/or addition of thickness to the spices.
• the method (specifically, the method described in [58]) in which the content of the bound diaminopimelic acid contained in a fraction of component (A) having a molecular weight cutoff of 10,000 Da or less per 100 g of dry weight of the composition (excluding the dry weight of the excipient if the composition contains an excipient) is 20.0 mg or more.
• the method (specifically, the method described in any one of [53] to [57]) above, wherein the composition further contains the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• a composition for improving the flavor of food comprising the following component (A): (A) Chitinase treatment of the cell wall of a Gram-positive bacterium. [66] The composition (specifically, the composition described in [65]) wherein the improvement in flavor is enhancement of spiciness and/or imparting full-bodied flavor.
• composition (specifically described in [65] or [66]) wherein the flavor improvement is enhancing the pungency and/or adding depth to the flavor of spices.
• the composition (specifically, any one of [65] to [67]) further contains a spice.
• a composition comprising the following component (A) and a spice: (A) Chitinase treatment of the cell wall of a Gram-positive bacterium.
• the composition (specifically, any one of [65] to [69]) wherein the component (A) is a chitinase-treated product of the gram-positive bacterium or a fragment thereof.
• composition (specifically, any one of [65] to [70]) wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria or Firmicutes.
• composition specifically, any one of [65] to [71] wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria.
• composition (specifically, any one of [65] to [72]) wherein the gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.
• the gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.
• composition (specifically, any one of [65] to [73]) wherein the Gram-positive bacterium is a bacterium of the genus Corynebacterium, a bacterium of the genus Brevibacterium, a bacterium of the genus Bifidobacterium, a bacterium of the genus Brachybacterium, a bacillus, a bacterium of the genus Enterococcus, or a bacterium of the genus Lactobacillus.
• the Gram-positive bacterium is a bacterium of the genus Corynebacterium, a bacterium of the genus Brevibacterium, a bacterium of the genus Bifidobacterium, a bacterium of the genus Brachybacterium, a bacillus, a bacterium of the genus Enterococcus, or a bacterium of the genus Lactobacillus.
• the gram-positive bacteria are selected from the group consisting of Corynebacterium casei, Corynebacterium flavescens, Corynebacterium ammoniagenes (Corynebacterium stationis), Corynebacterium glutamicum, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus
• the composition (specifically, any one of [65] to [74]) is selected from the group consisting of Lactobacillus mali, Lactobacillus hilgardii, and Lactobacillus Brevis.
• composition specifically, any one of [65] to [75] wherein the content of the component (A) is 0.1% (w/w) or more, calculated based on the dry weight of the original gram-positive bacterium cells.
• composition specifically, any one of [65] to [76] wherein a fraction having a molecular weight cutoff of 10,000 Da or less of the component (A) contains bound diaminopimelic acid.
• the composition (specifically, the composition described in [77]) has a content of the bound diaminopimelic acid contained in a fraction of component (A) having a molecular weight cutoff of 10,000 Da or less per 100 g of dry weight of the composition (excluding the dry weight of the excipient if the composition contains an excipient) of 20.0 mg or more.
• the composition (specifically, any one of [65] to [78]) further contains the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the composition (specifically, the composition according to [79]) contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.
• the composition (specifically, the composition described in [80]), wherein the content of L-glutamic acid is 0.1 to 20 parts by weight per 1 part by weight of the component (A), calculated as the dry weight of the original gram-positive bacterium cells.
• the composition (specifically, any one of [68] to [81]) has a content of the spice of 0.2 to 500 parts by weight per part by weight of the component (A) converted into the dry weight of the original gram-positive bacterium cells.
• the composition (specifically, any one of [68] to [82]) is characterized in that the spice is one or more spices selected from the group consisting of spices from the family Lauraceae, family Piperaceae, family Lamiaceae, family Umbelliferae, family Solanaceae, family Myristicae, family Liliaceae, family Myrtaceae, family Magnoliaceae, family Fabaceae, family Polygonaceae, family Brassicaceae, family Zingiberaceae, and family Rutaceae.
• the composition (specifically, any one of [68] to [83]) wherein the spice is a spice having a hot taste.
• composition which is a seasoning.
• composition which is a seasoning.
• the composition which is a seasoning.
• the composition wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.
• the composition (specifically, any one of [65] to [86]) further comprises a culture of a gram-positive bacterium.
• a method for improving the flavor of a food product comprising: A method comprising the steps of: adding the following component (A) to a food ingredient: (A) Chitinase treatment of the cell wall of a Gram-positive bacterium.
• a method for producing a food product having improved flavor comprising: A method comprising the steps of: adding the following component (A) to a food ingredient: (A) Chitinase treatment of the cell wall of a Gram-positive bacterium. [90] The method (specifically described in [88] or [89]) wherein the improvement in flavor is enhancement of spiciness and/or imparting kokumi (rich flavor). [91] The method (specifically, any one of [88] to [90]) wherein the improvement in flavor is enhancement of the pungency and/or addition of thickness to the spices.
• the Gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.
• the gram-positive bacteria are selected from the group consisting of Corynebacterium casei, Corynebacterium flavescens, Corynebacterium ammoniagenes (Corynebacterium stationis), Corynebacterium glutamicum, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus The method (specifically, any one of [88] to [96]) described above, wherein the bacterium is Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus Brevis.
• the method (specifically, any one of [88] to [97]) described above, wherein the component (A) is added so that the concentration of the component (A) consumed is 0.005 to 2% (w/w) calculated as the dry weight of the original gram-positive bacterium cells.
• the method further comprises a step of adding the following component (B) to a food ingredient: (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the method (specifically described in [99]) above, wherein at least the L-amino acid is added, and the L-amino acid is L-glutamic acid.
• the method (specifically, the method described in any one of [103] to [106]) wherein the spice is a spice having a hot taste.
• the method (specifically, any one of [88] to [107]) wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.
• the method (specifically, any one of [88] to [108]) further comprises the step of adding a culture of a gram-positive bacterium to a food ingredient.
• a seasoning comprising the following component (A): (A) Chitinase treatment of the cell wall of a Gram-positive bacterium.
• the seasoning (specifically, the seasoning described in [110]) in which the component (A) is a chitinase-treated product of the gram-positive bacterium or a fragment thereof.
• the seasoning (specifically, as described in [110] or [111]) further contains the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.
• the seasoning (specifically, the seasoning described in [112]) containing at least the L-amino acid, wherein the L-amino acid is L-glutamic acid.
• the seasoning (specifically, any one of [110] to [113]) further contains a spice.
• the seasoning (specifically, any one of [110] to [114]) wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 described below.
• the seasoning (specifically, any one of [110] to [115]) further contains a culture of a gram-positive bacterium.
• a method for producing a composition for improving flavor of food comprising the steps of:
• the composition comprises the following component (A): (A) a chitinase-treated product of the cell wall of a gram-positive bacterium,
• the method comprises a step of subjecting the cell wall of the gram-positive bacterium to chitinase treatment to obtain the component (A).
• the method (specifically, the method described in [117]) above, wherein the improvement in flavor is enhancement of spiciness and/or imparting full-bodied flavor.
• the method specifically described in [117] or [118] wherein the improvement in flavor is enhancement of the pungency and/or addition of thickness to the spices.
• the method (specifically, any one of [117] to [119]) wherein the composition further contains a spice.
• the method (specifically, the method described in any one of [117] to [120]) in which the cell wall of the gram-positive bacterium is subjected to the chitinase treatment in the form of a gram-positive bacterial body or a fragment thereof, and the component (A) is a chitinase-treated product of the gram-positive bacterial body or a fragment thereof.
• the method (specifically, any one of [117] to [121]) wherein a fraction having a molecular weight cutoff of 10,000 Da or less of the component (A) contains bound diaminopimelic acid.
• composition contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.
• the method (specifically, any one of [117] to [125]) wherein the Gram-positive bacterium is a bacterium having an ability to produce L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.
• the method (specifically, any one of [117] to [126]) wherein the composition further contains a culture of a gram-positive bacterium.
• the present invention can improve the flavor of food.
• Active Ingredient the following component (A) is used as an active ingredient: (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• the above ingredient (A) is also called the “active ingredient.”
• the flavor of food can be improved, that is, an effect of improving the flavor of food can be obtained.
• This effect is also called a "flavor improving effect.”
• the active ingredient has a function of improving the flavor of food.
• This function is also called a "flavor improving function.”
• the active ingredient may have a higher flavor improving function than the cell wall of a gram-positive bacterium.
• an improved flavor improving function may be obtained by treating the cell wall of a gram-positive bacterium with lysozyme.
• the active ingredient has a higher flavor improving function than the cell wall of a gram-positive bacterium may mean that the flavor improving effect obtained by using a certain amount of the active ingredient (which may be, for example, the amount of the active ingredient added in the method of the present invention described below) is greater than the flavor improving effect obtained by using the amount of the cell wall of a gram-positive bacterium used in preparing that amount of the active ingredient.
• the improvement of the flavor of food is also simply called “flavor improvement.” Specifically, by using the active ingredient, the flavor of food can be improved compared to when the active ingredient is not used. Thus, the active ingredient may be used to improve the flavor of food.
• the active ingredient may be utilized in the production of food (specifically, the production of food with improved flavor).
• Flavor includes taste and aroma. That is, the taste and/or aroma may be improved by using an active ingredient.
• “aroma” here may mean the aroma felt from the throat to the nasal cavity when eating food (i.e., retronasal aroma) and/or the aroma smelled directly from the nose (i.e., orthonasal aroma).
• “aroma” here may mean the aroma felt from the throat to the nasal cavity when eating food (i.e., retronasal aroma).
• Flavor improvements include enhancing the spiciness and imparting a full-bodied taste. Flavor improvements include, in particular, enhancing the spiciness.
• the active ingredient may have a high flavor improving function for one or more flavors compared to the cell wall of a gram-positive bacterium.
• the active ingredient may have a high flavor improving function for some or all of the flavors improved by using the active ingredient compared to the cell wall of a gram-positive bacterium.
• the active ingredient may have a high flavor improving function for some or all of the flavors improved by using the active ingredient compared to the cell wall of a gram-positive bacterium.
• the active ingredient may have a high flavor improving function for some or all of the flavors improved by using the active ingredient compared to the cell wall of a gram-positive bacterium.
• “Spicy” may mean a sensation felt due to the presence of spices. “Spicy” may also be called Spice Flavor, Spice Aroma, Pungency, Hotness, or Burningness. “Spicy” may specifically mean a sensation felt due to the presence of spices when eating a food containing spices. “Spicy” may be used interchangeably with “flavor of spices.” Examples of spice include the taste and aroma of spices. Examples of spice include the hotness of spices. “Enhancing spice” is not limited to enhancing the spice of a food that has a spice (e.g., a food that contains spices), but may also include imparting a spice to a food that does not have a spice (e.g., a food that does not contain spices). For example, by using an active ingredient in combination with a spice, a spice can be imparted to a food that does not have a spice (specifically, an enhanced spice compared to when the active ingredient is not used).
• “Kokumi” refers to a sensation that cannot be expressed by the five basic tastes: sweet, salty, sour, bitter, and umami, and more specifically, it can mean a taste that enhances not only the basic tastes, but also the marginal tastes and marginal flavors surrounding the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony.
• “Imparting kokumi” includes enhancing the basic tastes, and imparting or enhancing the accompanying marginal tastes surrounding the basic tastes, such as thickness, growth, continuity, and harmony.
• “Imparting full-bodied flavor” includes imparting or enhancing flavors such as complexity, a sense of maturity, richness, meatiness, milkiness, fruitiness, and full-bodiedness (such as a sugar-like full-bodiedness or a wine-like full-bodiedness). "Imparting full-bodied flavor” particularly includes imparting thickness.
• Flavors may be divided into, for example, initial taste, middle taste, and aftertaste.
• the terms "initial taste”, “middle taste”, and “aftertaste” refer to the flavors felt from 0 to 1 second, 1 to 3 seconds, and 3 to 5 seconds after ingestion (after the food is held in the mouth) in the case of a liquid (liquid food).
• the terms "initial taste”, “middle taste”, and “aftertaste” refer to the flavors felt from 0 to 4 seconds, 4 to 10 seconds, and 10 to 15 seconds after ingestion (after the food is held in the mouth) in the case of a solid (solid food).
• solid refers to a form other than a liquid, and includes pastes, gels, and the like.
• the initial taste, middle taste, aftertaste, or a combination thereof may be improved by utilizing an active ingredient. That is, by using the active ingredient, specifically, for example, the initial spiciness, the middle spiciness, the aftertaste spiciness, or a combination thereof may be enhanced. Also, by using the active ingredient, specifically, for example, the initial full-bodied taste, the middle full-bodied taste, the aftertaste full-bodied taste, or a combination thereof may be imparted. By using the active ingredient, in particular, the initial full-bodied taste (for example, the thickness of the initial taste) may be imparted. Also, the active ingredient may have the function of imparting a high initial full-bodied taste (for example, the thickness of the initial taste) compared to, for example, the cell wall of a gram-positive bacterium.
• Measurement and comparison of flavor can be performed, for example, by sensory evaluation by an expert panel.
• the active ingredient may be used to improve flavor or produce food products in the manner described in the method of the present invention below.
• the active ingredient can be produced by subjecting the cell walls of gram-positive bacteria to lysozyme treatment.
• this specification discloses a method for producing an active ingredient, which includes a step of subjecting the cell wall of a gram-positive bacterium to lysozyme treatment.
• This step is also referred to as a "lysozyme treatment step.”
• the lysozyme treatment step may be a step of subjecting the cell wall of a gram-positive bacterium to lysozyme treatment to obtain an active ingredient.
• the cell wall of the gram-positive bacteria may be subjected to the lysozyme treatment in any form.
• the cell wall of the gram-positive bacteria may be subjected to the lysozyme treatment in the form of, for example, the gram-positive bacterial cell or a fragment thereof. That is, the cell wall of the gram-positive bacteria subjected to the lysozyme treatment may specifically be the gram-positive bacterial cell or a fragment thereof.
• the cell wall of the gram-positive bacteria subjected to the lysozyme treatment may specifically be the cell wall constituting the gram-positive bacterial cell (i.e., contained in the cell) or the cell wall constituting a fragment of the gram-positive bacterial cell (i.e., contained in the fragment). That is, the active ingredient may specifically be a lysozyme-treated product of the gram-positive bacterial cell or a lysozyme-treated product of a fragment of the gram-positive bacterial cell.
• the active ingredient may specifically be a lysozyme-treated product of the cell wall constituting the gram-positive bacterial cell (i.e., contained in the cell) or the lysozyme-treated product of the cell wall constituting a fragment of the gram-positive bacterial cell (i.e., contained in the fragment).
• the cell walls of gram-positive bacteria that are subjected to lysozyme treatment include, in particular, the cells of gram-positive bacteria. That is, the active ingredient includes, in particular, the lysozyme-treated cells of gram-positive bacteria.
• Gram-positive bacteria are not particularly limited. Examples of gram-positive bacteria include bacteria belonging to the phylum Actinobacteria and Firmicutes. Examples of gram-positive bacteria include, in particular, bacteria belonging to the phylum Actinobacteria.
• Bacteria belonging to the Actinobacteria phylum include coryneform bacteria, bacteria belonging to the Bifidobacteriaceae family, and bacteria belonging to the Dermabacteraceae family. Bacteria belonging to the Actinobacteria phylum, in particular, include coryneform bacteria.
• coryneform bacteria examples include bacteria of the genera Corynebacterium, Brevibacterium, and Microbacterium.
• coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium casei Corynebacterium crenatum Corynebacterium flavescens Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium casei Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoni
• coryneform bacteria Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium casei JCM 12072 Corynebacterium crenatum AS1.542 Corynebacterium flavescens ATCC 10340 (NBRC 14136) Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium casei ATCC
• Coryneform bacteria include, in particular, bacteria of the genus Corynebacterium and bacteria of the genus Brevibacterium.
• Corynebacterium bacteria include, in particular, Corynebacterium casei, such as Corynebacterium casei JCM 12072, and Corynebacterium flavescens, such as Corynebacterium flavescens ATCC 10340.
• Brevibacterium bacteria include, in particular, Brevibacterium casei, such as Brevibacterium casei ATCC 35513.
• Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)).
• Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have now been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis, etc. (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).
• Examples of bacteria belonging to the Bifidobacteriaceae family include the genus Bifidobacterium.
• Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, and Bifidobacterium niger.
• Examples of the genus Bifidobacterium include Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum.
• Examples of the genus Bifidobacterium include Bifidobacterium longum.
• Examples of Bifidobacterium longum include ATCC 15697, ATCC 15707, ATCC 25962, ATCC 15702, ATCC 27533, BG7, DSM 24736, SBT 2928, NCC 490 (CNCM I-2170), and NCC 2705 (CNCM I-2618).
• Examples of Bifidobacterium breve include ATCC 15700, B632 (DSM 24706), Bb99 (DSM 13692), ATCC 15698, DSM 24732, UCC2003, YIT4010, YIT4064, BBG-001, BR-03, C50, and R0070.
• Examples of Bifidobacterium bifidum include ATCC 29521, OLB6378, and BF-1.
• Examples of Bifidobacterium adolescentis include ATCC 15703.
• Examples of Bifidobacterium dentium include DSM 20436.
• Examples of Bifidobacterium pseudocatenulatum include ATCC 27919.
• Examples of Bifidobacterium animalis include DSM 10140, Bb-12, DN-173 010, GCL2505, and CNCM I-3446.
• Examples of Bifidobacterium pseudolongum include JCM 5820 and ATCC 25526.
• Examples of Bifidobacterium thermophilum include ATCC 25525.
• Brachybacterium Bacteria belonging to the family Dermabacteraceae include the genus Brachybacterium.
• Brachybacterium include Brachybacterium alimentarium and Brachybacterium tyrofermentans.
• Brachybacterium include Brachybacterium alimentarium.
• a specific example of Brachybacterium alimentarium is ATCC 700067 (NBRC 16118).
• a specific example of Brachybacterium tyrofermentans is DSM 10673.
• Bacteria belonging to the phylum Firmicutes include bacteria belonging to the family Bacillaceae, bacteria belonging to the family Enterococcaceae, and bacteria belonging to the family Lactobacillaceae.
• Bacteria belonging to the family Bacillaceae include bacteria of the genus Bacillus.
• bacteria of the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus, and Bacillus velezensis.
• bacteria of the genus Bacillus include Bacillus subtilis in particular.
• Specific examples of Bacillus subtilis include 168 Marburg strain (ATCC 6051, JCM 1465) and PY79 strain (Plasmid, 1984, 12, 1-9).
• Specific examples of Bacillus amyloliquefaciens include T strain (ATCC 23842), N strain (ATCC 23845), AJ11708 strain (NITE BP-02609), and FZB42 strain (DSM 23117).
• Bacteria belonging to the family Enterococccaceae include bacteria of the genus Enterococcus.
• bacteria of the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium.
• bacteria of the genus Enterococcus include Enterococcus faecalis.
• a specific example of Enterococcus faecalis is ATCC 19433.
• a specific example of Enterococcus faecium is ATCC 19434.
• Lactobacillus bacteria include Lactobacillus mali, Lactobacillus hilgardii, Lactobacillus Brevis, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus gasseri, and Lactobacillus acidophilus.
• Lactobacillus bacteria include Lactobacillus mali, Lactobacillus hilgardii, and Lactobacillus Brevis.
• Lactobacillus mali include NBRC 102159 (ATCC 27053).
• Lactobacillus hilgardii include NBRC 15886 (ATCC 8290).
• Lactobacillus Brevis include JCM 1102 (ATCC 27305).
• strains can be obtained, for example, from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). That is, each strain is assigned a registration number, and can be obtained by using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where each strain was deposited.
• the gram-positive bacteria may be modified as appropriate. That is, examples of the gram-positive bacteria include modified strains derived from the strains exemplified above. Specific examples of such modified strains include modified strains derived from Corynebacterium casei JCM 12072.
• the purpose of the modification is not particularly limited. Examples of the modification include modifications for imparting or enhancing the ability to produce a target substance.
• the modified strain may be, for example, one bred by artificial modification. Examples of the artificial modification include modifications by genetic engineering techniques and modifications by mutation treatment.
• the modified strain may also be one that naturally occurs during the use of the gram-positive bacteria. Examples of such modified strains include mutant strains that naturally occur during the cultivation of the gram-positive bacteria.
• a modified strain may be constructed by one type of modification, or by a combination of two or more types of modifications.
• the gram-positive bacterium may have the ability to produce a target substance.
• “Ability to produce a target substance” means an ability to produce a target substance.
• "bacteria having ability to produce a target substance” means bacteria having the ability to produce a target substance.
• Bacteria having ability to produce a target substance may specifically mean bacteria having the ability to produce a target substance when cultured in a medium and accumulate the target substance in the medium and/or in the bacterial body to an extent that it can be recovered.
• the bacterium having ability to produce a target substance is a modified strain
• the bacterium having ability to produce a target substance may be a bacterium that can accumulate a larger amount of the target substance in the medium and/or in the bacterial body than a non-modified strain.
• non-modified strains include wild-type strains and parent strains.
• the bacterium having ability to produce a target substance may be a bacterium that can accumulate a target substance in a medium.
• the bacterium having ability to produce a target substance may be a bacterium that can accumulate a target substance in a medium, preferably at 0.5 g/L or more, more preferably at 1.0 g/L or more.
• the bacterium having ability to produce a target substance may have the ability to produce only one type of target substance, or may have the ability to produce two or more types of target substances.
• target substance there are no particular limitations on the target substance, so long as it can be produced by culturing gram-positive bacteria.
• target substances include ingredients that are used by being added to foods.
• Specific examples of target substances include L-amino acids, nucleic acids, and organic acids.
• L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline; aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and glycine; hydroxymonoaminocarboxylic acid amino acids such as L-threonine and L-serine; cyclic amino acids such as L-proline; aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan; sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine; acidic amino acids such as L-glutamic acid and L-aspartic acid; and amino acids with an amide group in the side chain such as L-glutamine and L-asparagine.
• L-amino acids in particular include L-glutamic acid.
• Nucleic acids include purine-based substances.
• Purine-based substances include purine nucleosides and purine nucleotides.
• Purine nucleosides include inosine, guanosine, xanthosine, and adenosine.
• Purine nucleotides include 5'-phosphate esters of purine nucleosides.
• Purine nucleoside 5'-phosphate esters include inosinic acid (inosine 5'-phosphate; IMP), guanosine 5'-phosphate (guanosine 5'-phosphate; GMP), xanthylic acid (xanthosine 5'-phosphate; XMP), and adenylic acid (adenosine 5'-phosphate; AMP).
• Purine-based substances include, in particular, inosine and guanosine.
• Purine-based substances further include, in particular, inosine.
• Examples of organic acids include carboxylic acids.
• Examples of carboxylic acids include monocarboxylic acids and dicarboxylic acids.
• Examples of monocarboxylic acids include monocarboxylic acids having 3 to 8 carbon atoms (C 3 -C 8 monocarboxylic acids).
• Specific examples of monocarboxylic acids include pyruvic acid.
• Examples of dicarboxylic acids include dicarboxylic acids having 3 to 8 carbon atoms (C 3 -C 8 dicarboxylic acids).
• dicarboxylic acids include ⁇ -ketoglutaric acid ( ⁇ -KG; also known as 2-oxoglutaric acid), malic acid, fumaric acid, succinic acid, itaconic acid, malonic acid, adipic acid, glutaric acid, pimelic acid, and suberic acid.
• ⁇ -KG also known as 2-oxoglutaric acid
• malic acid fumaric acid
• succinic acid succinic acid
• itaconic acid malonic acid
• adipic acid glutaric acid
• pimelic acid and suberic acid.
• the target substance When the target substance can form a salt, the target substance may be produced and/or used as a free form, as a salt, or as a combination thereof.
• the term “target substance” may mean the target substance in free form, its salt, or a combination thereof, unless otherwise specified.
• the target substance when the target substance can form a hydrate, the target substance may be produced and/or used as a non-hydrate, as a hydrate, or as a combination thereof.
• the term “target substance” (e.g., “target substance in free form” or “salt of target substance”) may include non-hydrate and hydrate, unless otherwise specified.
• the target substance may be in any form, such as an ion, when used.
• the amount of the target substance (e.g., content (concentration) or amount used) is calculated based on the value obtained by converting the mass of the salt or hydrate to the equimolar mass of the free form, when the target substance forms a salt or hydrate.
• Salts can be selected appropriately depending on various conditions such as the use of the target substance.
• a salt that can be ingested orally can be selected.
• Salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine.
• salts of basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid; salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid; and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
• inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid
• examples of salts of L-glutamic acid include sodium L-glutamate (e.g., monosodium L-glutamate; MSG) and ammonium L-glutamate (e.g., monoammonium L-glutamate). Note that one type of salt may be used, or two or more types of salts may be used in combination.
• Gram-positive bacteria may inherently have the ability to produce a target substance, or may be modified to have the ability to produce a target substance.
• Gram-positive bacteria having the ability to produce a target substance can be obtained, for example, by imparting the ability to produce a target substance to the above-mentioned gram-positive bacteria, or by enhancing the ability of the above-mentioned gram-positive bacteria to produce a target substance.
• the method for imparting or enhancing the ability to produce a target substance is not particularly limited.
• known methods can be used as the method for imparting or enhancing the ability to produce a target substance.
• the ability to produce a target substance can be imparted or enhanced, for example, by mutation or genetic engineering techniques.
• Methods for imparting or enhancing the ability to produce L-amino acids are disclosed, for example, in WO2006/070944, WO2015/060391, and WO2018/030507.
• Methods for imparting or enhancing the ability to produce nucleic acids are disclosed, for example, in WO2015/060391.
• the ability to produce L-glutamic acid can be imparted or enhanced by the procedure described in the Examples.
• Bacteria capable of producing L-glutamic acid include bacteria with "specific mutations.”
• Specific mutations include those shown in Table 1.
• the mutations shown in Table 1 consist of 135 mutations, A-1 to A-135, and 92 mutations, B-1 to B-92.
• Mutations A-1 to A-135 are also referred to as “group A mutations.”
• Mutations B-1 to B-92 are also referred to as "group B mutations.”
• the "specific mutation” may be one or more mutations selected from the mutations shown in Table 1.
• the L-glutamic acid producing bacterium may have one or more mutations selected from the mutations shown in Table 1.
• the L-glutamic acid producing bacteria may have, for example, one or more mutations selected from the mutations of group A.
• the L-glutamic acid producing bacteria may have, for example, one or more mutations selected from the mutations of group B.
• the L-glutamic acid producing bacteria may have, for example, one or more mutations selected from the mutations of group A and one or more mutations selected from the mutations of group B.
• the L-glutamic acid producing bacteria may have, for example, one or more mutations selected from the mutations of group A, and may further have one or more mutations selected from the mutations of group B.
• the L-glutamic acid producing bacteria may have, for example, one or more mutations selected from the mutations of group B, and may further have one or more mutations selected from the mutations of group A.
• the "specific mutation” may be, for example, one or more mutations selected from the mutations of group A, one or more mutations selected from the mutations of group B, or a combination of one or more mutations selected from the mutations of group A and one or more mutations selected from the mutations of group B.
• the number of mutations selected from group A mutations possessed by the L-glutamic acid producing bacterium may be, for example, 1 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, or 130 or more, or 135 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less, or any compatible combination thereof.
• the number of mutations selected from the group A mutations possessed by the L-glutamic acid producing bacteria may be, for example, 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, or 130 to 135.
• the number of mutations selected from the group A mutations possessed by the L-glutamic acid producing bacteria may be, in particular, 1 or more, 50 or more, 100 or more, 120 or more, 130 or more, or 135.
• the number of mutations selected from group B mutations possessed by the L-glutamic acid producing bacterium may be, for example, 1 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more, or 92 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less, or any compatible combination thereof.
• the number of mutations selected from the group B mutations possessed by the L-glutamic acid producing bacteria may be, for example, 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 92.
• the number of mutations selected from the group B mutations possessed by the L-glutamic acid producing bacteria may be, for example, 1 or more, 30 or more, 60 or more, 70 or more, 80 or more, or 92.
• the L-glutamic acid producing bacteria may have, for example, 50 or more mutations selected from the mutations of group A and 30 or more mutations selected from group B.
• the L-glutamic acid producing bacteria may have, for example, 100 or more mutations selected from the mutations of group A and 60 or more mutations selected from group B.
• the L-glutamic acid producing bacteria may have, for example, 120 or more mutations selected from the mutations of group A and 80 or more mutations selected from group B.
• the L-glutamic acid producing bacteria may have, for example, 135 mutations of group A and 92 mutations of group B.
• the L-glutamic acid producing bacterium having a "specific mutation” may in particular be a coryneform bacterium.
• the L-glutamic acid producing bacterium having a "specific mutation” may more particularly be a bacterium of the genus Corynebacterium.
• the L-glutamic acid producing bacterium having a "specific mutation” may more particularly be Corynebacterium casei.
• the L-glutamic acid producing bacterium having a "specific mutation” may more particularly be a modified strain derived from Corynebacterium casei JCM 12072.
• L-glutamic acid producing bacteria having a "specific mutation” include Corynebacterium casei RUN5-2-96 strain (NITE BP-03688) and mutant strains derived therefrom.
• the RUN5-2-96 strain is also referred to as "AJ111891 strain.”
• mutant strains derived from the RUN5-2-96 strain include mutant strains obtained from the RUN5-2-96 strain in the Examples.
• the mutant strains obtained from the RUN5-2-96 strain in the Examples are also referred to as "RUN5-2-96-derived mutant strains of the Examples” or "AJ111891-derived mutant strains of the Examples.”
• the RUN5-2-96 strain is a modified strain derived from Corynebacterium casei JCM 12072, and has all 135 mutations of group A.
• the RUN5-2-96 strain was originally deposited as an international deposit at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NITE NPMD, Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on July 7, 2022, and has been assigned the accession number NITE BP-03688.
• the RUN5-2-96-derived mutant strain of the embodiment is a modified strain derived from Corynebacterium casei JCM 12072, and has all 135 mutations in group A and all 92 mutations in group B.
• the RUN5-2-96 strain can be obtained, for example, from NITE NPMD.
• NZ_CP004350.1 nucleotide sequence is the nucleotide sequence of the genome of Corynebacterium casei JCM 12072 (LMG S-19264) and has been made public with annotations.
• the NZ_CP004350.1 nucleotide sequence and annotations of each genomic location can be obtained, for example, from NCBI.
• Each mutation shown in Table 1 shall be interpreted as a mutation at a position in the genome of each bacterium that corresponds to the position of each mutation shown in Table 1.
• mutation A-1 shall be interpreted as a mutation at a position in the genome of each bacterium that corresponds to position 78,486 of the base sequence of NZ_CP004350.1.
• the positions of each mutation shown in Table 1 are described for the sake of convenience in identifying each mutation, and do not necessarily indicate the absolute position in the genome of each bacterium. In other words, the positions of each mutation shown in Table 1 indicate relative positions based on the base sequence of NZ_CP004350.1, and the absolute positions may vary due to deletion or insertion of nucleic acid residues, etc.
• the original position X becomes position X-1 or position X+1, respectively, and the mutation at the original position X is considered to be "a mutation at a position corresponding to position X in the base sequence of NZ_CP004350.1".
• the bases before the mutation shown in Table 1 are described for convenience in identifying each mutation, and do not need to be conserved in the genome of the bacteria before modification. In other words, if the genome of the bacteria before modification does not have the base sequence of NZ_CP004350.1, the bases before the mutation shown in Table 1 may not be conserved.
• introducing a mutation into a bacterium for each mutation shown in Table 1 means modifying the base at the position of each mutation shown in Table 1 in the genome of the bacteria before modification (this is any base other than the base after mutation) to the base after mutation shown in Table 1.
• introducing mutation A-1 into a bacterium means modifying the base at the position corresponding to position 78,486 of the base sequence of NZ_CP004350.1 in the genome of the bacterium before modification (which is C, G, or A) to T.
• the positions in the genome of each bacterium that correspond to the positions of each mutation shown in Table 1 can be determined by aligning the base sequence of the genome of each bacterium with the base sequence of NZ_CP004350.1. Alignment can be performed, for example, using known genetic analysis software. Examples of genetic analysis software include DNASIS made by Hitachi Solutions and GENETYX made by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37. 1987).
• the bacterium has a mutation" for each mutation shown in Table 1 means that the base at the position of the mutation in the genome of the bacterium is the mutated base shown in Table 1, and does not necessarily mean that the bacterium was obtained by introducing the mutation.
• the bacterium has a mutation" for each mutation shown in Table 1 may be a bacterium in which the base at the position of the mutation is originally the mutated base shown in Table 1, or may be a bacterium obtained by modifying a bacterium in which the base at the position of the mutation is not originally the mutated base shown in Table 1.
• the bacterium has mutation A-1 means that the base at the position of mutation A-1 in the genome of the bacterium (the position corresponding to position 78,486 in the base sequence of NZ_CP004350.1) is T, and does not necessarily mean that the bacterium was obtained by introducing mutation A-1.
• a "bacterium having mutation A-1” may be a bacterium that originally has a T base at the position of mutation A-1 in its genome (the position corresponding to position 78,486 in the base sequence of NZ_CP004350.1), or may be a bacterium obtained by modifying a bacterium that originally does not have a T base at the position of mutation A-1 in its genome.
• An L-glutamic acid producing bacterium having a "specific mutation” may in particular be one obtained by introducing part or all of the "specific mutation.”
• L-glutamic acid producing bacteria having a "specific mutation” can be obtained, for example, by introducing the "specific mutation” into bacteria that do not have the "specific mutation.” Also, L-glutamic acid producing bacteria having a "specific mutation” can be obtained, for example, by introducing the remainder of the "specific mutation” into bacteria that have a portion of the "specific mutation.”
• Mutations can be introduced by known techniques, for example.
• a desired mutation can be introduced at a desired position on the genome by site-specific mutagenesis.
• site-specific mutagenesis include a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and a method using phages (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)).
• Each mutation shown in Table 1 may be a mutation that improves the L-glutamic acid producing ability of the bacterium.
• Each mutation shown in Table 1 may be a mutation that improves the L-glutamic acid producing ability of the bacterium, in particular, compared to when the position of each mutation is the base before the mutation shown in Table 1.
• Each mutation shown in Table 1 may be, for example, a mutation in a gene (here meaning the coding region of a gene), a mutation in an expression regulatory region of a gene such as a promoter, or a mutation in an intergenic region.
• the region in which each mutation shown in Table 1 occurs e.g., a gene, an expression regulatory region of a gene, or an intergenic region
• the mutation may, for example, change (e.g., increase or decrease) the expression of the gene.
• the mutation may, for example, change (e.g., increase or decrease) the activity of a protein encoded by the gene.
• the mutation may, for example, cause a change (e.g., an increase or decrease) in the expression of the gene.
• a change in the expression of a gene may result in a change in the activity of a protein encoded by the gene (e.g., an increase or decrease).
• L-glutamic acid producing bacteria having a "specific mutation” may or may not have modifications other than the "specific mutation” as long as they have the ability to produce L-glutamic acid. Modifications other than the "specific mutation” include known modifications that confer or enhance L-glutamic acid producing ability. Modifications other than the "specific mutation” include mutations not selected as "specific mutations" among those shown in Table 1. L-glutamic acid producing bacteria having a "specific mutation” may have the ability to produce L-glutamic acid based on, for example, the "specific mutation", or may have the ability to produce L-glutamic acid based on a combination of the "specific mutation” and other modifications.
• L-glutamic acid producing bacteria include Corynebacterium casei A-013 (NITE BP-03806).
• the A-013 strain is also called the AJ120306 strain.
• the A-013 strain is a modified strain derived from Corynebacterium casei JCM 12072.
• the A-013 strain was originally deposited as an international deposit at the National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (NITE NPMD, Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on January 25, 2023, and has been assigned the accession number NITE BP-03806.
• the A-013 strain can be obtained, for example, from NITE NPMD.
• L-glutamic acid producing bacteria such as L-glutamic acid producing bacteria having a "specific mutation” may have, for example, a significantly higher L-glutamic acid producing ability than Corynebacterium casei JCM 12072.
• "Significantly higher L-glutamic acid producing ability than Corynebacterium casei JCM 12072” may mean, for example, the ability to produce and accumulate in a medium an amount of L-glutamic acid that is at least two times, at least three times, at least five times, or at least seven times that of JCM 12072 when cultured under appropriate culture conditions.
• L-glutamic acid producing bacteria such as L-glutamic acid producing bacteria having a "specific mutation” may have an L-glutamic acid producing ability equal to or greater than that of, for example, Corynebacterium casei RUN5-2-96 strain (NITE BP-03688), the RUN5-2-96-derived mutant strain of the examples, or the A-013 strain (NITE BP-03806).
• L-glutamic acid producing ability equivalent to or greater than that of Corynebacterium casei RUN5-2-96 strain (NITE BP-03688), the RUN5-2-96-derived mutant strain in the Examples, or A-013 strain (NITE BP-03806) may mean, for example, the ability to produce and accumulate in a medium an amount of L-glutamic acid that is 80% or more, 90% or more, 95% or more, or 100% or more of that of RUN5-2-96 strain, the RUN5-2-96-derived mutant strain in the Examples, or A-013 strain when cultured under appropriate culture conditions.
• Suitable culture conditions include the culture conditions for measuring the amount of L-glutamic acid produced described in Example 1 (1-2) below (i.e., the conditions of shaking culture at 30°C for 48 hours in 500 ⁇ L of evaluation medium (Table 4) placed in a 96-deep-well plate).
• the L-glutamic acid producing bacteria having a "specific mutation” may have a genomic base sequence that is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.9% or more, 99.95% or more, 99.97% or more, or 99.99% or more identical to that of, for example, Corynebacterium casei JCM 12072 strain, RUN5-2-96 strain (NITE BP-03688), the RUN5-2-96-derived mutant strain of the embodiment, or A-013 strain (NITE BP-03806).
• the cell wall of the gram-positive bacteria may be a commercially available product or may be obtained by appropriate production. There are no particular limitations on the method for producing the cell wall of the gram-positive bacteria.
• the cell wall of a gram-positive bacterium can be produced, for example, by culturing the gram-positive bacterium in a medium. That is, the method for producing an active ingredient may include, for example, a step of culturing the gram-positive bacterium in a medium prior to the lysozyme treatment step. This step is also referred to as a "cultivation step.” Specifically, the culturing step may be a step of culturing the gram-positive bacterium in a medium to obtain a culture.
• the medium to be used is not particularly limited as long as it allows the growth of gram-positive bacteria.
• a normal medium used for culturing bacteria such as coryneform bacteria can be used as the medium.
• the medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as necessary.
• the types and concentrations of the medium components may be appropriately set depending on various conditions such as the type of gram-positive bacteria used.
• the carbon source is not particularly limited as long as it can be assimilated by the gram-positive bacterium.
• Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids.
• one type of carbon source may be used, or two or more types of carbon sources may be used in combination.
• nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, hydrolyzed vegetable protein (HVP; for example, soy protein hydrolyzate, soy sauce, and pea sauce), ammonia, and urea.
• Ammonia gas and aqueous ammonia used for pH adjustment may also be used as nitrogen sources.
• the nitrogen source one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.
• phosphate sources include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate.
• phosphate source one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.
• sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.
• sulfur source one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.
• organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and hydrolyzed vegetable protein (HVP; for example, soy protein hydrolyzate, soy sauce, and pea sauce).
• Other organic and inorganic components include defoamers, osmotic pressure regulators for culture media, and osmotic pressure compensation substances.
• defoamers examples include silicone-based defoamers (oil type, solution type, oil compound type, emulsion type, self-emulsifying type, etc.), alcohol-based defoamers, oil-based defoamers, polyether-based defoamers, and vegetable oils (cottonseed oil, linseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.).
• Antifoaming agents can be used in any form, such as liquid, paste, solid, powder, emulsion, wax, etc.
• Osmotic pressure regulators for the medium include salts such as sodium chloride and potassium chloride, and polysaccharides that cannot be assimilated by microorganisms (sorbitol, dextrin, etc.).
• Osmotic pressure compensation substances include potassium ions, betaine (glycine betaine), glutamic acid, and trehalose. As these and other various organic components, one type of component may be used, or two or more types of components may be used in combination.
• Culture medium components also include food ingredients. That is, the medium may contain food ingredients. Cultivating gram-positive bacteria in a medium containing food ingredients is also referred to as "fermenting the food ingredients with gram-positive bacteria.” That is, the active ingredient may be, for example, a fermentation product of the food ingredients with gram-positive bacteria. Food ingredients may be used as medium components alone or in appropriate combination with other medium components.
• Food ingredients used as medium components include food ingredients described below.
• Food ingredients used as medium components can be selected independently of the food ingredients used in the method of the present invention.
• Food ingredients used as medium components may or may not be the same as the food ingredients used in the method of the present invention.
• Food ingredients used as medium components may be one type of ingredient, or two or more types of ingredients may be combined.
• Specific examples of food raw materials used as medium components include plants of the Solanaceae family.
• Solanaceae plants include plants of the genera Solanum and Capsicum.
• Solanaceae plants include tomatoes and eggplants.
• Capsicum plants include bell peppers, paprika, shishito peppers, and chili peppers.
• bell peppers include green and red bell peppers.
• Solanaceae plants include tomatoes.
• Solanaceae plants one type of plant may be used, or two or more types of plants may be used in combination.
• fresh produce specifically, fresh produce of edible parts such as fruits
• processing include cutting, crushing, straining, squeezing, fractionation, dilution, concentration, drying, and heating. These processes may be performed alone or in suitable combination.
• the skin and/or seeds may be removed by processing.
• processed products of solanaceae plants include juice, puree, and paste of solanaceae plants. That is, for example, processed products of tomatoes include tomato juice, tomato puree, and tomato paste.
• “Juice” of solanaceae plants such as tomato juice
• a “puree” of a solanaceous plant, such as tomato puree may mean, for example, a concentrate of juice of a solanaceous plant having a salt-free soluble solid content of 8% (w/w) or more and less than 24% (w/w).
• a "paste" of a solanaceous plant may mean, for example, a concentrate of juice of a solanaceous plant having a salt-free soluble solid content of 24% (w/w) or more.
• Processed products of solanaceous plants may or may not contain additives such as sodium chloride.
• Processed products of solanaceous plants may be concentrated and reduced to have the salt-free soluble solid content exemplified above.
• the culture conditions are not particularly limited as long as the gram-positive bacteria can grow.
• the culture can be performed under normal conditions used for culturing bacteria such as coryneform bacteria.
• the culture conditions can be set appropriately depending on various conditions such as the type of gram-positive bacteria used. For specific culture conditions, reference can be made to the culture conditions described in previous reports on the production of target substances by bacteria (WO2015/060391, WO2018/030507, WO2015/060391, etc.).
• the cultivation may be carried out, for example, in a liquid medium, under aerobic or microaerobic conditions.
• “Aerobic conditions” may mean conditions in which the dissolved oxygen concentration in the medium is 0.18 ppm or more, 0.33 ppm or more, or 1.5 ppm or more.
• “Microaerobic conditions” may mean conditions in which the dissolved oxygen concentration in the medium is less than 0.33 ppm, or less than 0.18 ppm.
• the dissolved oxygen concentration in the medium under microaerobic conditions may be 0.30 ppm or less, 0.25 ppm or less, 0.20 ppm or less, 0.15 ppm or less, 0.10 ppm or less, or 0.05 ppm or less.
• the dissolved oxygen concentration can be measured, for example, using a sensor such as a PL electrode or a DO electrode.
• the cultivation may be, for example, aerated or shaken.
• the pH of the medium may be, for example, pH 3-10, or pH 4.0-9.5. During the culture, the pH of the medium can be adjusted as necessary.
• the pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide.
• the culture temperature can be, for example, 20 to 45°C, or 25°C to 37°C.
• the culture period can be, for example, 10 hours to 120 hours.
• the culture can be continued, for example, until the carbon source in the medium is consumed or the activity of the gram-positive bacteria is lost.
• a culture of gram-positive bacteria (specifically, a culture containing gram-positive bacterial cells) is obtained.
• the bacterial cells may be subjected to the lysozyme treatment while still contained in the culture (specifically, the medium), or may be recovered from the culture (specifically, the medium) and subjected to the lysozyme treatment. That is, the method for producing an active ingredient may include, for example, a step of recovering the bacterial cells from the culture (specifically, the medium) before the lysozyme treatment step.
• the recovery of the bacterial cells can be carried out, for example, by a known method. Such methods include natural sedimentation, centrifugation, and filtration.
• the bacterial cells may also be subjected to an appropriate treatment before the lysozyme treatment.
• the method for producing an active ingredient may include, for example, a step of subjecting the bacterial cells to a treatment other than the lysozyme treatment before the lysozyme treatment step.
• the treatment other than the lysozyme treatment include concentration, drying, and heating. Concentration, drying, and heating can all be carried out, for example, by a known method. Known drying methods include spray drying and freeze drying. The heating temperature may be, for example, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 110°C or higher, or 120°C or higher.
• the cell wall of the gram-positive bacteria (specifically, the bacterial body of the gram-positive bacteria) subjected to the lysozyme treatment may be a culture of the gram-positive bacteria, a bacterial body recovered from the culture, or a processed product thereof. That is, the bacterial body may be subjected to the lysozyme treatment in the form of, for example, a culture of the gram-positive bacteria, a bacterial body recovered from the culture, a processed product thereof, or a combination thereof.
• the cell wall of the gram-positive bacteria (specifically, the bacterial body of the gram-positive bacteria) subjected to the lysozyme treatment may be a bacterial body contained in a culture of the gram-positive bacteria, a bacterial body recovered from the culture, or a bacterial body contained in a processed product thereof. That is, the bacterial cells may be subjected to the lysozyme treatment in the form of, for example, bacterial cells contained in a culture of a gram-positive bacterium, bacterial cells recovered from the culture, bacterial cells contained in a processed product thereof, or a combination thereof.
• the processed product may be bacterial cells (for example, bacterial cells contained in a culture or bacterial cells recovered from a culture) that have been subjected to the treatment.
• the bacterial cells may be live bacterial cells, dead bacterial cells, or a combination thereof.
• the bacterial cells of a gram-positive bacterium contain the cell wall of the gram-positive bacterium.
• bacterial fragments can be prepared from the bacterial cells.
• bacterial disruption containing bacterial fragments can be prepared by disrupting the bacterial cells. That is, the method for producing the active ingredient may include, for example, a step of disrupting the bacterial cells before the lysozyme treatment step.
• the disruption of the bacterial cells can be carried out, for example, by a known method. Such a method includes ultrasonic disruption.
• the bacterial fragments may be subjected to lysozyme treatment while still contained in the bacterial disruption, or may be recovered from the bacterial disruption and subjected to lysozyme treatment.
• the method for producing the active ingredient may include, for example, a step of recovering the bacterial fragments from the bacterial disruption before the lysozyme treatment step.
• the recovery of the bacterial fragments can be carried out, for example, by a known method.
• a known method includes natural sedimentation, centrifugation, and filtration.
• the bacterial fragments may be subjected to appropriate treatment before the lysozyme treatment.
• the method for producing the active ingredient may include, for example, a step of treating the bacterial fragments before the lysozyme treatment step.
• the treatment include concentration, drying, and heating. Concentration, drying, and heating can all be performed by, for example, known techniques. Examples of known drying techniques include spray drying and freeze drying.
• the heating temperature may be, for example, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 110°C or higher, or 120°C or higher. That is, the cell wall of the gram-positive bacteria (specifically, the fragments of the gram-positive bacteria cells) subjected to the lysozyme treatment may include the disrupted gram-positive bacteria cells, the fragments of the gram-positive bacteria cells recovered from the disrupted cells, and the processed products thereof.
• the fragments of the bacteria cells may be subjected to the lysozyme treatment in the form of, for example, the disrupted gram-positive bacteria cells, the fragments of the gram-positive bacteria cells recovered from the disrupted cells, the processed products thereof, or a combination thereof.
• the cell wall of the gram-positive bacteria specifically, the cell fragments of the gram-positive bacteria
• the cell fragments contained in the processed products thereof includes the cell fragments contained in the disrupted product of the gram-positive bacteria, the cell fragments recovered from the disrupted product, and the cell fragments contained in the processed products thereof.
• the cell fragments may be subjected to the lysozyme treatment in the form of, for example, the cell fragments contained in the disrupted product of the gram-positive bacteria, the cell fragments recovered from the disrupted product, the cell fragments contained in the processed products thereof, or a combination thereof.
• the processed product includes the cell fragments (for example, the cell fragments contained in the disrupted product or the cell fragments recovered from the disrupted product) subjected to the treatment.
• the cell fragments of the gram-positive bacteria (for example, the disrupted product of the gram-positive bacteria, the cell fragments recovered from the disrupted product, or the processed products thereof) contain the cell wall of the gram-positive bacteria.
• the gram-positive bacterial cells or fragments thereof may be treated by heating or the like. That is, the cell walls of the gram-positive bacteria to be subjected to the lysozyme treatment may be treated by heating or the like. That is, the method for producing an active ingredient may include, for example, a step of subjecting the cell walls of the gram-positive bacteria, such as the gram-positive bacterial cells or fragments thereof, to a treatment such as heating before the lysozyme treatment step. The cell walls of the gram-positive bacteria to the lysozyme treatment may be particularly heat-treated.
• Subjecting the cell walls of the gram-positive bacteria to treatment includes cases where any form of the cell walls of the gram-positive bacteria are subjected to treatment, unless otherwise specified, in other words, cases where the cell walls of the gram-positive bacteria themselves or any fractions containing the cell walls are subjected to treatment.
• heat-treating the cells of the gram-positive bacteria includes cases where any fractions containing the cells of the gram-positive bacteria, such as a culture of the gram-positive bacteria, cells recovered from the culture, or processed products thereof, are subjected to heat treatment, unless otherwise specified.
• the method for producing an active ingredient may include, for example, a step of subjecting a culture of a gram-positive bacterium to a treatment such as heating before the lysozyme treatment step.
• a treatment such as heating
• "subjecting a culture to a treatment” includes any form of culture to the treatment, in other words, the culture itself or any fraction containing the cell wall of a gram-positive bacterium prepared therefrom to the treatment.
• a treatment such as heating
• a cell wall of a gram-positive bacterium that has been subjected to a treatment such as heating may be obtained.
• the target substance may be produced by culturing the gram-positive bacteria.
• the culture of the gram-positive bacteria may contain the target substance.
• the target substance may be appropriately recovered from the culture.
• the target substance may be recovered as a suitable fraction containing the target substance.
• An example of such a fraction is the culture supernatant.
• the target substance may also be further separated and purified from the above-mentioned fraction.
• the cells of the gram-positive bacteria may be separated from the culture to obtain a culture supernatant, and the target substance may be recovered from the culture supernatant.
• “recovery of the target substance” may include removal of impurities from a fraction containing the target substance.
• the target substance may be recovered, for example, by a known method used for separating and purifying compounds.
• the method for recovering the target substance may be appropriately selected depending on various conditions such as the type of target substance. These methods may be used alone or in combination.
• the recovered target substance may contain other components other than the target substance, such as the cells of the gram-positive bacteria, medium components, water, and metabolic by-products of the gram-positive bacteria.
• the purity of the recovered target substance may be, for example, 30% (w/w) or more, 50% (w/w) or more, 70% (w/w) or more, 80% (w/w) or more, 90% (w/w) or more, or 95% (w/w) or more.
• the recovered target substance may be used separately from the active ingredient, or may be used in combination with the active ingredient.
• the target substance may be prepared as a fermented seasoning containing the target substance, and used in combination with the active ingredient.
• the recovered target substance may also be combined with the cell wall of a gram-positive bacterium and subjected to lysozyme treatment.
• the target substance may be prepared, for example, as a fermented seasoning containing the target substance, and combined with the cell wall of a gram-positive bacterium and subjected to lysozyme treatment.
• a fraction containing both the target substance and the cell walls of the gram-positive bacteria may be subjected to lysozyme treatment.
• a culture containing both the target substance and the cells of the gram-positive bacteria may be subjected to lysozyme treatment as is or after appropriate drying.
• the conditions for the lysozyme treatment are not particularly limited as long as a treated product having a flavor-improving function is obtained.
• the conditions for the lysozyme treatment may be set, for example, so as to obtain a treated product having a higher flavor-improving function than the cell wall of a gram-positive bacterium.
• “Lysozyme” may mean a protein (EC 3.2.1.17) that has the activity of catalyzing the hydrolysis of peptidoglycan. This activity is also called “lysozyme activity.”
• lysozyme may be derived from any of microorganisms, animals, plants, etc.
• egg white-derived lysozyme is used as lysozyme.
• Egg white is hen's egg white.
• known lysozyme homologues may be used as lysozyme.
• known lysozymes or artificially modified versions of their homologues may be used as lysozyme.
• Such homologues or artificially modified versions are not particularly limited as long as they have lysozyme activity.
• lysozyme may be obtained by heterologous expression (i.e., recombinant enzyme).
• lysozyme may be used, or lysozyme obtained by appropriate production may be used.
• Commercially available lysozyme is Lysozyme BIO (manufactured by Japan Biocon, derived from egg white).
• Lysozyme can be produced, for example, by isolating (e.g., extracting) from agricultural, marine, and livestock products (e.g., egg white) that contain lysozyme.
• Lysozyme can be produced, for example, by culturing host cells that produce lysozyme.
• the host cells that produce lysozyme may be ones that inherently produce lysozyme, or may be ones that have been modified to produce lysozyme.
• the host cells that produce lysozyme can be obtained, for example, by introducing a gene that codes for lysozyme into the host cells so that it can be expressed.
• the culture conditions for the host cells that produce lysozyme are not particularly limited, so long as lysozyme is produced.
• the host cells that produce lysozyme can be cultured, for example, under normal conditions for culturing host cells.
• Lysozyme may or may not contain components other than lysozyme.
• purified lysozyme may be used as the lysozyme, or a material containing lysozyme may be used. Lysozyme may be purified to a desired degree.
• lysozyme one type of lysozyme may be used, or two or more types of lysozyme may be used in combination.
• Lysozyme treatment can be carried out by contacting lysozyme with the cell wall of gram-positive bacteria. Subjecting the cell wall of gram-positive bacteria to lysozyme treatment is also referred to as "acting lysozyme on the cell wall of gram-positive bacteria.” Lysozyme treatment may be carried out, for example, in a liquid. Examples of liquids include aqueous media such as water and aqueous buffer solutions. For example, a suspension containing the cell wall of gram-positive bacteria may be prepared, and lysozyme may be added thereto. Alternatively, for example, a treatment liquid containing lysozyme may be prepared, and the cell wall of gram-positive bacteria may be added thereto. Alternatively, for example, a suspension containing the cell wall of gram-positive bacteria and a treatment liquid containing lysozyme may be prepared, and then they may be mixed.
• aqueous media such as water and aqueous buffer solutions.
• the lysozyme treatment may or may not be performed under heating.
• the temperature of the lysozyme treatment may be, for example, a temperature at which lysozyme is active.
• the temperature of the lysozyme treatment may be, for example, 10°C or higher, 20°C or higher, 30°C or higher, 40°C or higher, or 50°C or higher, or 60°C or lower, 50°C or lower, 40°C or lower, 30°C or lower, or 20°C or lower, or any compatible combination thereof.
• the temperature of the lysozyme treatment may be, for example, 10-20°C, 20-30°C, 30-40°C, 40-50°C, or 50-60°C.
• the temperature of the lysozyme treatment may be, for example, 20-60°C, 30-60°C, or 40-60°C.
• the pH of the lysozyme treatment may be, for example, a pH at which lysozyme is active.
• the pH of the lysozyme treatment may be, for example, 3-9.
• the time of lysozyme treatment may be, for example, a time that provides a flavor improving function that is improved to a desired degree.
• the time of lysozyme treatment may be, for example, 0.5 hours or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, or 18 hours or more, or 24 hours or less, 18 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less, or a non-contradictory combination thereof.
• the time of lysozyme treatment may specifically be, for example, 0.5 to 1 hour, 1 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 8 hours, 8 to 10 hours, 10 to 12 hours, 12 to 15 hours, 15 to 18 hours, or 18 to 24 hours.
• the time of lysozyme treatment may specifically be, for example, 0.5 to 24 hours, 1 to 18 hours, or 2 to 12 hours.
• the amount of lysozyme used is, for example, 0.00005 g or more, 0.0001 g or more, 0.0002 g or more, 0.0005 g or more, 0.001 g or more, 0.002 g or more, 0.005 g or more, 0.01 g or more, 0.02 g or more, 0.05 g or more, 0.1 g or more, 0.2 g or more, or 0.5 g per 1 g of dried gram-positive bacteria.
• g or more may be 1 g or less, 0.5 g or less, 0.2 g or less, 0.1 g or less, 0.05 g or less, 0.02 g or less, 0.01 g or less, 0.005 g or less, 0.002 g or less, 0.001 g or less, 0.0005 g or less, 0.0002 g or less, or 0.0001 g or less, or any non-consistent combination thereof.
• the amount of lysozyme used may be, for example, 0.00005-0.0001 g, 0.0001-0.0002 g, 0.0002-0.0005 g, 0.0005-0.001 g, 0.001-0.002 g, 0.002-0.005 g, 0.005-0.01 g, 0.01-0.02 g, 0.02-0.05 g, 0.05-0.1 g, 0.1-0.2 g, 0.2-0.5 g, or 0.5-1 g per 1 g of dried gram-positive bacterial cells.
• the amount of lysozyme used may be, for example, 0.00005 to 1 g, 0.00005 to 0.2 g, 0.0001 to 0.1 g, or 0.0002 to 0.05 g per 1 g of dried gram-positive bacterial cells.
• the amount of lysozyme used may be, for example, 1,950 FIP units or more, 3,900 FIP units or more, 7,800 FIP units or more, 19,500 FIP units or more, 39,000 FIP units or more, 78,000 FIP units or more, 195,000 FIP units or more, 390,000 FIP units or more, 780,000 FIP units or more, 1,950,000 FIP units or more, 3,900,000 FIP units or more, 7,800,000 FIP units or more, or 19,500,000 FIP units or more per gram of dried gram-positive bacteria.
• FIP units may be not more than 39,000,000 FIP units, not more than 19,500,000 FIP units, not more than 7,800,000 FIP units, not more than 3,900,000 FIP units, not more than 1,950,000 FIP units, not more than 780,000 FIP units, not more than 390,000 FIP units, not more than 195,000 FIP units, not more than 78,000 FIP units, not more than 39,000 FIP units, not more than 19,500 FIP units, not more than 7,800 FIP units, or not more than 3,900 FIP units, or any combination thereof that is not inconsistent.
• the specific amounts of lysozyme used per gram of dried gram-positive bacteria are, for example, 1,950-3,900 FIP units, 3,900-7,800 FIP units, 7,800-19,500 FIP units, 19,500-39,000 FIP units, 39,000-78,000 FIP units, 78,000-195,000 FIP units, 195,000-390,000 FIP units, and FIP units, 390,000 to 780,000 FIP units, 780,000 to 1,950,000 FIP units, 1,950,000 to 3,900,000 FIP units, 3,900,000 to 7,800,000 FIP units, 7,800,000 to 19,500,000 FIP units, or 19,500,000 to 39,000,000 FIP units.
• the amount of lysozyme used may be, for example, 1,950 to 39,000,000 FIP units, 1,950 to 7,800,000 FIP units, 3,900 to 3,900,000 FIP units, or 7,800 to 1,950,000 FIP units per gram of dried gram-positive bacterial cells.
• the dry weight of the cell walls of gram-positive bacteria may be converted to the equivalent dry bacterial body weight of gram-positive bacteria.
• "per 1 g of dry gram-positive bacterial bodies” may be read as, for example, "per cell wall prepared from 1 g of gram-positive bacterial bodies by dry weight.”
• Lysozyme activity can be measured by the following procedure.
• 1 FIP unit is defined as the amount of enzyme that reduces the absorbance at 450 nm by 0.001 per minute at pH 7.0 and 25°C when an enzyme reaction is carried out using a suspension of Micrococcus lysodeikticus cells as the substrate (Shugar D, Biochimica et Biophysica Acta, Vol. 8, 302-309 (1952)).
• the lysozyme-treated product may be used as an active ingredient as is or after being subjected to an appropriate treatment. That is, the method for producing the active ingredient may include, for example, a step of subjecting the lysozyme-treated product to additional treatment. Examples of the additional treatment include concentration, drying, heating, and fractionation. All of these treatments can be carried out, for example, by known techniques. Examples of known drying techniques include spray drying and freeze drying. Heating may be carried out, for example, so as to inactivate the lysozyme. The heating temperature may be, for example, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 110°C or higher, or 120°C or higher.
• Fractionation may also include separation (for example, purification) of a specific component. Fractionation may be carried out so as to obtain a fraction or component having a flavor improving function. Fractions or components having flavor-improving properties include the supernatant of a lysozyme-treated product, the low-molecular-weight fraction of a lysozyme-treated product, and components contained therein. The supernatant can be obtained, for example, by centrifugation or filtration. Filtration can be performed, for example, using a microfilter (e.g., a membrane with a pore size of 0.1 to 0.5 ⁇ m), filter paper, or filter cloth.
• a microfilter e.g., a membrane with a pore size of 0.1 to 0.5 ⁇ m
• the "low-molecular-weight fraction” may mean a fraction obtained as a permeate by filtering a target substance (e.g., a supernatant) through an ultrafiltration membrane with a nominal molecular weight cutoff of 30,000 or less, 10,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less.
• a target substance e.g., a supernatant
• Components contained in the supernatant or low-molecular-weight fraction of a lysozyme-treated product include components characteristic of peptidoglycan.
• Components characteristic of peptidoglycan include N-acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, D-isoglutamine, D-alanine, and compounds containing one or more of these as components. That is, the active ingredient can be the lysozyme-treated product itself or one that has been subjected to additional processing.
• the active ingredient may be used in combination with other ingredients (i.e., ingredients other than the active ingredient).
• ingredients other than the active ingredient include the target substance and a culture of gram-positive bacteria.
• the flavor improving effect may be enhanced compared to when the active ingredient is used alone.
• the active ingredient in combination with L-glutamic acid and/or a culture of gram-positive bacteria the flavor improving effect may be enhanced compared to when the active ingredient is used alone.
• the umami of the food may be enhanced compared to when the active ingredient is used alone.
• using the active ingredient in combination with L-glutamic acid includes cases where the active ingredient is used in combination with any form of L-glutamic acid, in other words, cases where the active ingredient is used in combination with L-glutamic acid itself or any fraction containing it (for example, a culture containing L-glutamic acid).
• the target substance is as described above.
• the target substance may be a commercially available product or may be obtained by appropriate production.
• the target substance can be produced, for example, by chemical synthesis, enzyme reaction, fermentation, extraction, or a combination thereof.
• the target substance can be produced, for example, by a fermentation method using a microorganism capable of producing the target substance.
• the microorganism capable of producing the target substance may be the same as the gram-positive bacterium from which the active ingredient is derived, or it may not be.
• the target substance may be, for example, one produced together with the production of the active ingredient, or one produced separately from the active ingredient.
• the target substance may be, or may not be, purified to a desired degree.
• the target substance may be a purified product, or a material containing the target substance.
• material containing the target substance refers to a material having a target substance content of 0.1% (w/w) or more.
• Specific examples of materials containing the target substance include fermentation products such as cultures, bacterial cells, and culture supernatants obtained by culturing microorganisms capable of producing the target substance, and processed products thereof.
• processed products include those obtained by subjecting materials such as the above-mentioned fermentation products to treatments such as heating, concentration, dilution, drying, and fractionation.
• the content of the target substance in the material containing the target substance may be, for example, 1% (w/w) or more, 3% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, 30% (w/w) or more, 50% (w/w) or more, 70% (w/w) or more, 90% (w/w) or more, or 95% (w/w) or more.
• “using an active ingredient and a target substance in combination” is not limited to the case where an active ingredient and a target substance obtained individually are combined and used, but also includes the case where an active ingredient and a target substance obtained together are used.
• an active ingredient and a target substance obtained together are used is a case where a culture containing both the target substance and the cells of a gram-positive bacterium is subjected to lysozyme treatment to prepare and use the active ingredient.
• the target substance may be one that has been subjected to lysozyme treatment.
• the culture of gram-positive bacteria as the other component can be obtained by culturing gram-positive bacteria.
• the culturing of gram-positive bacteria is as described above.
• the gram-positive bacteria from which the culture as the other component is derived may or may not be the same as the gram-positive bacteria from which the active ingredient is derived.
• the culture as the other component may be, for example, one produced together with the production of the active ingredient, or one produced separately from the active ingredient.
• the culture as the other component may be used as it is, or after being subjected to a treatment such as fractionation (separation of bacterial cells or separation of the target substance), concentration, drying, or heating, as an additional component.
• fractionation for example, 90% or more, 95% or more, 97% or more, or 99% or more of the total number of bacterial cells may be separated (i.e., removed from the culture).
• the culture as the other component may or may not contain gram-positive bacterial cells.
• the culture as the other component may or may not contain the target substance (e.g., L-glutamic acid).
• target substance e.g., L-glutamic acid
• An example of using an active ingredient and a culture obtained together is when a culture containing gram-positive bacterial cells is treated with lysozyme to prepare and use the active ingredient.
• the culture as the other ingredient may be one that has been treated with lysozyme.
• composition of the present invention is a composition containing an active ingredient.
• composition of the present invention is a composition containing the following component (A): (A) Lysozyme treatment of the cell wall of a Gram-positive bacterium.
• the flavor of food can be improved, i.e., a flavor improving effect can be obtained.
• the composition of the present invention may be used to improve the flavor of food. That is, the composition of the present invention may be, for example, a composition for improving the flavor of food.
• the improvement of flavor may be, for example, an enhancement of the spiciness and/or the addition of fullness.
• the improvement of flavor may in particular be an enhancement of the pungency of spices and/or the addition of depth.
• the composition of the present invention may be utilized in the production of food (specifically, the production of food with improved flavor). That is, the composition of the present invention may be, for example, a composition for use in the production of food (specifically, the production of food with improved flavor).
• composition of the present invention may be, for example, a seasoning.
• composition of the present invention may be, for example, a seasoning for improving the flavor of food, or a seasoning for use in the production of food (specifically, the production of food with improved flavor).
• composition of the present invention may or may not have an improved flavor by itself.
• the composition of the present invention may have an enhanced spiciness by itself (specifically, an enhanced spiciness compared to when the active ingredient is not contained).
• composition of the present invention may be used to improve flavor or produce food products in the manner described in the method of the present invention below.
• composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient.
• the composition of the present invention may exclude a composition consisting of an active ingredient.
• the ingredient other than the active ingredient one type of ingredient may be used, or two or more types of ingredients may be used in combination.
• the active ingredient contained in the composition of the present invention may be produced by the method for producing the active ingredient described above.
• this specification discloses a method for producing the composition of the present invention, which includes a step of producing an active ingredient by the above-mentioned method for producing an active ingredient.
• the step of producing an active ingredient in the method for producing the composition of the present invention may be a step of subjecting the cell wall of a gram-positive bacterium to lysozyme treatment to obtain the active ingredient.
• Ingredients other than the active ingredient are not particularly limited, so long as they do not lose the flavor improving effect (i.e., the flavor improving effect of the active ingredient can be obtained).
• Ingredients other than the active ingredient can be appropriately selected depending on various conditions such as the type of food. Examples of ingredients other than the active ingredient include ingredients that are blended into foods or medicines.
• ingredients other than the active ingredient include ingredients that are effective in the production of food.
• ingredients that are effective in the production of food include the food ingredients described below.
• ingredients other than the active ingredient include, in particular, a culture of gram-positive bacteria, a target substance, and spices.
• the composition of the present invention may be, for example, a composition containing an active ingredient and a culture of gram-positive bacteria (e.g., a seasoning), a composition containing an active ingredient and a target substance (e.g., a seasoning), a composition containing an active ingredient and a spice (e.g., a seasoning), a composition containing an active ingredient, a culture of gram-positive bacteria, and a spice (e.g., a seasoning), a composition containing an active ingredient, a target substance, and a spice (e.g., a seasoning), or a composition containing an active ingredient, a culture of gram-positive bacteria, a target substance, and a spice (e.g., a seasoning).
• a composition containing an active ingredient and a culture of gram-positive bacteria e.g., a seasoning
• a target substance e.g., a seasoning
• a spice e.g., a seasoning
• a composition containing an active ingredient and a target substance is not limited to cases where the active ingredient and target substance obtained separately are contained in the composition, but also includes cases where the active ingredient and target substance obtained collectively are contained in the composition.
• An example of a case where the active ingredient and target substance obtained collectively are contained in the composition is a case where a culture containing both the target substance and Gram-positive bacterial cells is treated with lysozyme to prepare an active ingredient, which is then contained in the composition.
• the culture of gram-positive bacteria as an ingredient other than the active ingredient is as described above.
• the composition contains an active ingredient and a culture of gram-positive bacteria is not limited to the case where the active ingredient and culture obtained separately are contained in the composition, but also includes the case where the active ingredient and culture obtained collectively are contained in the composition.
• An example of the case where the active ingredient and culture obtained collectively are contained in the composition is the case where a culture containing the cells of gram-positive bacteria is treated with lysozyme to prepare an active ingredient and then contained in the composition.
• “Spices” may mean the leaves, stems, bark, roots, flowers, buds, seeds, fruits, or peels of plants that are used for the purpose of giving a special flavor to foods. “Spices” may also include so-called “herbs”. Spices include spices from the Lauraceae family (laurel, cinnamon, etc.), spices from the Piperaceae family (black pepper, white pepper, etc.), spices from the Lamiaceae family (thyme, sage, basil, oregano, marjoram, rosemary, mint, etc.), spices from the Umbelliferae family (fennel, caraway, cumin, coriander, parsley, Italian parsley, celery, celery seed, dill, etc.), spices from the Solanaceae family (chili pepper, cayenne pepper, etc.), and spices from the Myristicae family.
• Lauraceae family laurel, cinnamon, etc.
• spices from the Piperaceae family black pepper, white pepper, etc.
• Spices include spices from the family Onionaceae (nutmeg, mace, etc.), spices from the family Myrtaceae (garlic powder, onion powder, etc.), spices from the family Myrtaceae (allspice, cloves, etc.), spices from the family Melastomataceae (star anise, etc.), spices from the family Fabaceae (fenugreek, etc.), spices from the family Polygonaceae (polygonum, etc.), spices from the family Brassicaceae (garden cress, wasabi, Japanese mustard, etc.), spices from the family Zingiberaceae (cardamom, turmeric, ginger, etc.), and spices from the family Rutaceae (Japanese pepper, etc.).
• Spices include, in particular, spices from the family Piperaceae, Solanaceae, Brassicaceae, and Rutaceae that have a hot taste.
• garlic and onion which are used in cooking in a raw state as fresh vegetables according to common practice, may be excluded from the spices.
• one type of spice may be used, or two or more types of spices may be used in combination.
• composition of the present invention can be produced, for example, by appropriately combining (e.g., mixing) the active ingredient and, optionally, other ingredients. Some or all of the other ingredients may be mixed with the cell wall of the gram-positive bacteria before the lysozyme treatment and then subjected to the lysozyme treatment. That is, some or all of the other ingredients may have been subjected to the lysozyme treatment.
• composition of the present invention may be formulated as appropriate, for example.
• additives may be used as appropriate.
• additives include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, diluents, surfactants, and solvents.
• Additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.
• the shape of the composition of the present invention is not particularly limited.
• the composition of the present invention may be in any shape, such as a powder, flakes, tablet, paste, liquid, etc.
• the content and content ratio of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention are not particularly limited as long as a flavor improving effect is obtained.
• the content and content ratio of each component in the composition of the present invention can be appropriately set according to various conditions such as the mode of use of the composition of the present invention.
• the content of the active ingredient in the composition of the present invention is greater than 0% (w/w) and less than 100% (w/w).
• the content of the active ingredient in the composition of the present invention may be, for example, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.5% (w/w) or more, 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, 20% (w/w) or more, 30% (w/w) or more, 50% (w/w) or more, or 70% (w/w) or more, and may be greater than 100%.
• the content of the active ingredient in the composition of the present invention may be, for example, 0.1% (w/w) to 1% (w/w), 1% (w/w) to 10% (w/w), 10% (w/w) to 20% (w/w), 20% (w/w) to 30% (w/w), 30% (w/w) to 50% (w/w), 50% (w/w) to 70% (w/w), or 70% (w/w) to 100% (w/w).
• the content of the active ingredient in the composition of the present invention may be, for example, 0.1 to 100% (w/w), 0.2 to 70% (w/w), or 0.5 to 50% (w/w).
• the content of the active ingredient in the composition of the present invention is, for example, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.5% (w/w) or more, 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, 20% (w/w) or more, 50% (w/w) or more, 100% (w/w) or more, 200% (w/w) or more, 500% (w/w) or more, 1000% (w/w) or more, 2000% (w/w) or more, or 5000% (w/w).
• % (w/w) or more may be 10,000% (w/w) or less, 5,000% (w/w) or less, 2,000% (w/w) or less, 1,000% (w/w) or less, 500% (w/w) or less, 200% (w/w) or less, 100% (w/w) or less, 50% (w/w) or less, 20% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, 2% (w/w) or less, 1% (w/w) or less, 0.5% (w/w) or less, or 0.2% (w/w) or less, or any non-consistent combination thereof.
• the content of the active ingredient in the composition of the present invention may be, specifically, for example, 0.1 to 0.2% (w/w), 0.2 to 0.5% (w/w), 0.5 to 1% (w/w), 1 to 2% (w/w), 2 to 5% (w/w), 5 to 10% (w/w), 10 to 20% (w/w), 20 to 50% (w/w), 50 to 100% (w/w), 100 to 200% (w/w), 200 to 500% (w/w), 500 to 1000% (w/w), 1000 to 2000% (w/w), 2000 to 5000% (w/w), or 5000 to 10000% (w/w) calculated based on the dry weight of the original Gram-positive bacterial cells.
• the content of the active ingredient in the composition of the present invention may be, for example, 0.1 to 10,000% (w/w), 0.2 to 5,000% (w/w), or 0.5 to 2,000% (w/w) in terms of the dry weight of the original Gram-positive bacterial cells.
• the "dry weight of the original Gram-positive bacterial cells” means the dry weight of the Gram-positive bacterial cells from which the active ingredient is derived.
• the dry weight of the bacterial cells is also referred to as the "dry cell weight (DCW)."
• DCW dry cell weight
• the dry weight of the cell wall of the Gram-positive bacterial cell may be converted into the dry bacterial weight of the corresponding Gram-positive bacterial cell.
• the content of the active ingredient in the composition of the present invention is 1% (w/w) in terms of the dry weight of the original Gram-positive bacterial cells.
• a content of more than 100% (w/w) calculated based on the dry weight of the original gram-positive bacterial cells means that the active ingredient is concentrated and contained in the composition of the present invention. That is, for example, a content of 200% (w/w) means that the active ingredient is concentrated twice and contained in the composition of the present invention.
• the content of the active ingredient in the composition of the present invention is 200% (w/w) calculated based on the dry weight of the original gram-positive bacterial cells.
• the composition of the present invention may, for example, satisfy one or more (e.g., one, two, three, or all four) conditions selected from the following conditions (1) to (4). That is, the content of the active ingredient in the composition of the present invention may be, for example, an amount such that the composition of the present invention satisfies one or more (e.g., one, two, three, or all four) conditions selected from the following conditions (1) to (4).
• All of the following conditions (1) to (4) may be applied, for example, when the composition of the present invention is present in a form containing a liquid fraction such as a liquid or paste, or when the composition of the present invention is dispersed (e.g., dissolved or suspended) in an aqueous medium such as water or an aqueous buffer solution.
• Condition (1) The content of components characteristic of peptidoglycan in the clarified fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention is within a specified range.
• “Clarified fraction” may mean a liquid fraction obtained by subjecting the composition of the present invention to centrifugation or filtration.
• “Medium-low molecular weight fraction” may mean a fraction obtained as a permeate by filtering the target substance (e.g., clarified fraction) through an ultrafiltration membrane with a nominal molecular weight cutoff of 30,000 or less, 10,000 or less, 5,000 or less, 3,000 or less, or 1,000 or less.
• Components characteristic of peptidoglycan include N-acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, D-isoglutamine, and D-alanine.
• the predetermined range in condition (1) may be, for example, a molar ratio of the content of each component in the clear fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention to the content of the same component in the composition of the present invention, which may be 0.025 or more, preferably 0.05 or more, more preferably 0.1 or more, more preferably 0.15 or more, more preferably 0.2 or more, and even more preferably 0.25 or more.
• the content of each component in condition (1) means the total content of the same component present in any form (e.g., a form in which the component exists as a single molecule, or a form in which the component forms a compound with other components, etc.).
• the content of N-acetylglucosamine in the composition of the present invention in condition (1) means the total content of N-acetylglucosamine present in any form (e.g., a form in which the component exists as a single molecule, or a form in which the component forms a peptidoglycan, etc.).
• the total content of each component, including the form in which the component forms a peptidoglycan, can be measured after decomposing the peptidoglycan by a treatment such as hydrolysis using an acid or alkali.
• a heating reaction is carried out in 6 molar hydrochloric acid at 120°C for 24 hours under reduced pressure.
• a heating reaction is carried out in 4 molar hydrochloric acid at 105°C for 12 hours under reduced pressure.
• N-acetylglucosamine is converted to glucosamine
• N-acetylmuramic acid is converted to muramic acid, so the contents of N-acetylglucosamine and N-acetylmuramic acid can be measured as the contents of the respective converted substances (glucosamine and muramic acid).
• each component constituting peptidoglycan can be determined by measuring the content of each component (amount in free form) under conditions where peptidoglycan hydrolysis is not performed, and subtracting it from the content of each component (total amount in free form and those constituting peptidoglycan) under conditions where peptidoglycan hydrolysis is performed.
• the predetermined range in condition (1) regarding the content of diaminopimelic acid may be, for example, 20.0 mg or more, preferably 25.0 mg or more, more preferably 30.0 mg or more, even more preferably 35.0 mg or more, even more preferably 37.5 mg or more, and even more preferably 40.0 mg or more as the content of bound diaminopimelic acid contained in a fraction having a molecular weight cutoff of 10,000 Da or less per 100 g of the dry weight of the composition of the present invention (excluding the weight of the excipient if the composition of the present invention contains the excipient).
• Condition (2) The ratio of the content of reducing sugar ends to the content of total sugars in the clarified fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention is within a specified range.
• the specified range in condition (2) may be, for example, a molar ratio of 0.001 or more, preferably 0.005 or more, more preferably 0.01 or more, more preferably 0.015 or more, and even more preferably 0.02 or more.
• the clear fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention has the function of enhancing the response of the TRPV1 receptor to a spicy component (e.g., capsaicin).
• a spicy component e.g., capsaicin
• the clear fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention may have the function of enhancing the response of the TRPV1 receptor to a spicy component (e.g., capsaicin) by 1.1 times or more, 1.3 times or more, 1.5 times or more, or 2 times or more.
• a spicy component e.g., capsaicin
• the molecular weight or a value reflecting the molecular weight of the glycopeptide or glycan derived from peptidoglycan contained in the clear fraction (particularly the medium-low molecular weight fraction) of the composition of the present invention is within a predetermined range.
• the molecular weight can be measured, for example, by mass spectrometry, gel filtration chromatography, gel exclusion chromatography, or light scattering.
• An example of a value reflecting the molecular weight is the diffusion coefficient determined by two-dimensional NMR.
• the content of the spice in the composition of the present invention may be, for example, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.5% (w/w) or more, 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, 20% (w/w) or more, 30% (w/w) or more, 50% (w/w) or more, or 70% (w/w) or more, or 90% (w/w) or less, 70% (w/w) or less, 50% (w/w) or less, 30% (w/w) or less, 20% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, 2% (w/w) or less, or 1% (w/w) or less, or any compatible combination thereof.
• the content of the spice in the composition of the present invention may be, for example, 0.1% (w/w) to 1% (w/w), 1% (w/w) to 10% (w/w), 10% (w/w) to 20% (w/w), 20% (w/w) to 30% (w/w), 30% (w/w) to 50% (w/w), 50% (w/w) to 70% (w/w), or 70% (w/w) to 90% (w/w).
• the content of the spice in the composition of the present invention may be, for example, 0.2 to 90% (w/w), 0.5 to 50% (w/w), or 1 to 20% (w/w).
• the content of the spice in the composition of the present invention may be, for example, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 50 parts by weight or more, or 100 parts by weight or more, or 1000 parts by weight or less, 500 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 2 parts by weight or less, or any compatible combination thereof, per 1 part by weight of active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacterial cells).
• the content of the spice in the composition of the present invention may be, for example, 0.2 to 500 parts by weight, 1 to 500 parts by weight, 5 to 500 parts by weight, 50 to 500 parts by weight, 0.2 to 100 parts by weight, 1 to 100 parts by weight, 5 to 100 parts by weight, 50 to 100 parts by weight, 0.2 to 100 parts by weight, 0.5 to 50 parts by weight, or 1 to 20 parts by weight, per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacteria).
• the content of the spice in the composition of the present invention may be, in particular, 0.2 to 500 parts by weight, or 50 to 500 parts by weight, per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacteria).
• the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.5% (w/w) or more, 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, or 20% (w/w) or more, or 90% (w/w) or less, 50% (w/w) or less, 20% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, 2% (w/w) or less, or 1% (w/w) or less, or any compatible combination thereof.
• a target substance e.g., L-glutamic acid
• the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, 0.2-90% (w/w), 0.5-50% (w/w), 1-50% (w/w), 1-20% (w/w), 5-50% (w/w), 10-50% (w/w), or 20-50% (w/w).
• the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, or 5 parts by weight or more per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacterial cells), or 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 2 parts by weight or less, or 1 part by weight or less, or a compatible combination thereof.
• 0.1 parts by weight or more 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, or 5 parts by weight or more per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacterial cells), or 50 parts by
• the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, specifically, 0.1 to 20 parts by weight per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of the original gram-positive bacterial cells).
• the content of the culture of gram-positive bacteria in the composition of the present invention may be, for example, 0.01% (w/w) or more, 0.1% (w/w) or more, 1% (w/w) or more, 5% (w/w) or more, or 10% (w/w) or more, calculated based on the amount of the original culture, and may be 10,000% (w/w) or less, 5,000% (w/w) or less, or 10 ...
• the content may be 1000% (w/w) or less, 500% (w/w) or less, 300% (w/w) or less, 200% (w/w) or less, 150% (w/w) or less, 100% (w/w) or less, 70% (w/w) or less, 50% (w/w) or less, 30% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, or 1% (w/w) or less, or a combination thereof that is compatible with the above.
• the "original culture” refers to a culture in which no change in concentration, such as concentration or dilution, has occurred after culture, and specifically, may be a culture immediately after culture.
• a content of more than 100% (w/w) in terms of the amount of the original culture means that the culture is concentrated and contained in the target (here, the composition of the present invention). That is, for example, a content of 200% (w/w) means that the culture is concentrated twice and contained in the target.
• each component i.e., the active ingredient and optionally other ingredients
• the content of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention can be set, for example, so as to obtain the amount of each component added in the method of the present invention described below.
• composition of the present invention may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination.
• the composition of the present invention may be provided as a set of components each packaged separately. In such a case, the components contained in the set can be used together as appropriate when used.
• the method of the present invention is a method including a step of utilizing an active ingredient.
• the method of the present invention is a method comprising a step of utilizing the following component (A): (A) Lysozyme treatment of a fraction containing cell walls of Gram-positive bacteria.
• the method of the present invention can improve the flavor of food, i.e., a flavor improving effect can be obtained.
• the method of the present invention may be carried out to improve the flavor of food. That is, the method of the present invention may be, for example, a method for improving the flavor of food. This method is also referred to as the "flavor improving method of the present invention.”
• the improvement in flavor may be, for example, an enhancement of the spiciness and/or the addition of a fuller body.
• the improvement in flavor may, in particular, be an enhancement of the pungency of the spices and/or the addition of depth.
• the method of the present invention can produce food with improved flavor.
• the method of the present invention may be carried out for the production of food (specifically, the production of food with improved flavor). That is, the method of the present invention may be, for example, a method for producing food (specifically, the production of food with improved flavor). This method is also referred to as the "food production method of the present invention.”
• the active ingredient can be added to food ingredients during food production to improve flavor or to be used in food production. That is, the use of an active ingredient can be, for example, adding the active ingredient to food ingredients. That is, the method of the present invention can specifically be, for example, a method of improving food flavor, which includes adding the active ingredient to food ingredients. Also, the method of the present invention can specifically be, for example, a method of producing food (specifically, producing food with improved flavor), which includes adding the active ingredient to food ingredients. "Adding" can also be called "blending.”
• the active ingredient may be utilized in the method of the present invention, for example, in the form of a composition of the present invention. That is, "utilization of an active ingredient” also includes utilization of a composition of the present invention. For example, “addition of an active ingredient” also includes addition of a composition of the present invention.
• the food obtained by the method of the present invention is also called the "food of the present invention.”
• the food of the present invention is a food with improved flavor.
• the food of the present invention is a food to which an active ingredient has been added.
• Flavor improvement or food production may be carried out in the same manner as normal food production, for example, except that an active ingredient is used.
• flavor improvement or food production may be carried out using the same ingredients and under the same production conditions as normal foods, for example, except that an active ingredient is used.
• both the food ingredients and production conditions may be appropriately modified for use in flavor improvement or food production.
• Foods include beverages. Foods also include seasonings. Foods may be, for example, liquid or solid. Specific examples of foods include beverages such as milk, soft drinks, alcoholic drinks, and soups; processed meat foods such as ham, sausages, dumplings, shumai, hamburger steak, fried chicken, and pork cutlets; processed seafood foods such as salmon flakes, spicy cod roe, salted cod roe, grilled fish, dried fish, salted fish, fish sausage, kamaboko, boiled fish, tsukudani, and canned goods; sweets such as potato chips, potato snacks, corn snacks, wheat snacks, cinnamon cookies, rice crackers, and arare; noodle soups such as udon soup, soba soup, somen soup, ramen soup, champon soup, and pasta sauce; cooked rice dishes such as rice balls, pilaf, fried rice, mixed rice, porridge, and ochazuke; stews such as curry, stew,
• ingredients include dishes; roux such as stew roux and curry roux; processed vegetable products such as kimchi and pickles; other processed foods such as bread, noodles, gratin, croquettes, and mashed potatoes; sauces such as Chinese sauce, oyster sauce, cheese sauce, tomato sauce, white sauce, demi-glace sauce, curry sauce, Genoa sauce, chili sauce, and Tabasco sauce; seasoning oils such as chili oil; basic seasonings such as soy sauce and miso; flavor seasonings such as bonito flavor, chicken flavor, pork flavor, and beef flavor; spicy seasonings such as shichimi pepper, doubanjiang, and gochujang; menu seasonings (special seasonings tailored to the menu being cooked); and other seasonings such as dressings, miso, mayonnaise, tomato ketchup, and consommé.
• sauces such as Chinese sauce, oyster sauce, cheese sauce, tomato sauce, white sauce, demi-glace sauce, curry sauce, Genoa sauce, chili sauce, and Tabasco sauce
• seasoning oils such as chili oil
• Soft drinks may mean non-alcoholic beverages (drinks with an alcohol concentration of less than 1%) excluding milk and dairy products.
• Specific examples of soft drinks include water, fruit juice, vegetable juice, tea (chai, cinnamon tea, etc.), coffee drinks (coffee, milk drinks with coffee, etc.), carbonated drinks (ginger ale, lemon carbonated drinks, etc.), and sports drinks.
• Specific examples of soups include dal soup, tom yum goong, soup with eggs, soup with seaweed, soup with shark fin, Chinese-style soup, consommé soup, curry-flavored soup, clear soup, miso soup, and potage soup.
• foods include foods containing spices (for example, the above-mentioned foods containing spices). Examples of foods include spices themselves.
• Foods may be provided in a form that can be eaten as is, or in a form that requires preparation before or at the time of eating, such as concentrated or dried products.
• foods are not limited to general foods, and also include so-called health foods or medical foods, such as nutritional supplements, nutritional functional foods, and foods for specified health uses. That is, for example, the foods exemplified above may be provided as general foods, or as health foods or medical foods.
• Food ingredients refers to food materials used to manufacture food. There are no particular limitations on the food ingredients, so long as they can be used to manufacture food. Food ingredients can be selected appropriately depending on various conditions, such as the type of food. Food ingredients include ingredients that can be commonly used in the manufacture of foods, such as those exemplified above. Specific examples of food ingredients include ingredients such as grains, vegetables, meat, seafood, and eggs; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; dairy products such as milk and cheese; spices; flavorings; oils and fats; and alcohol. Examples of organic acids, nucleic acids, and amino acids include those exemplified as target substances.
• the active ingredient may be added to the food ingredients at any stage of the food manufacturing process, so long as the flavor improving effect is obtained.
• the "food ingredients” to which the active ingredient is added may be at any stage of the food manufacturing process.
• the "food ingredients” to which the active ingredient is added may include finished foods before the active ingredient is added.
• the active ingredient may be added to the food ingredients as is, or after being appropriately prepared into a desired form such as a solution. "Addition of an active ingredient” may collectively refer to the operation of causing the active ingredient to coexist with the food ingredients.
• Ingredients other than the active ingredient may also be added to the food ingredients as appropriate.
• the method of the present invention may further include adding ingredients other than the active ingredient to the food ingredients.
• ingredients other than the active ingredient there are no particular limitations on the ingredients other than the active ingredient, so long as the flavor improving effect is not lost (i.e., the flavor improving effect of the active ingredient is obtained).
• the ingredients other than the active ingredient may be appropriately selected depending on various conditions such as the type of food. Examples of ingredients other than the active ingredient include cultures of gram-positive bacteria, target substances, and spices. The descriptions regarding the addition of active ingredients also apply mutatis mutandis to the addition of ingredients other than the active ingredient.
• the ingredients i.e., the active ingredient and optionally other ingredients
• the amount and ratio of each component (i.e., active ingredient and optional other ingredients) added in the method of the present invention are not particularly limited as long as a flavor improving effect is obtained.
• the amount and ratio of each component added in the method of the present invention can be appropriately set according to various conditions such as the type of food raw material and the type of food.
• the active ingredient may be added to the food ingredients so that the ingestible concentration of the active ingredient is within a desired range (e.g., the ingestible concentration range of the active ingredient described below).
• the ingested concentration of the active ingredient may be, for example, 0.005% (w/w) or more, 0.01% (w/w) or more, 0.02% (w/w) or more, 0.05% (w/w) or more, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.5% (w/w) or more, 1% (w/w) or more, 2% (w/w) or more, 5% (w/w) or more, 10% (w/w) or more, or 20% (w/w) or more, 3
• the content of the active ingredient in the composition of the present invention may be, for example, 0.005-0.01% (w/w), 0.01-0.05% (w/w), 0.05-0.1% (w/w), 0.1-0.5% (w/w), 0.5-1% (w/w), 1-5% (w/w), or 5-30% (w/w).
• the content of the active ingredient in the composition of the present invention may be, for example, 0.005 to 30% (w
• the ingested concentration of the active ingredient is, for example, 0.005% (w/w) or more, 0.007% (w/w) or more, 0.01% (w/w) or more, 0.02% (w/w) or more, 0.03% (w/w) or more, 0.04% (w/w) or more, 0.05% (w/w) or more, 0.07% (w/w) or more, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.3% (w/w) or more, 0.4% (w/w) or more, 0.5% (w/w) or more, 0.7% (w/w) or more, or 1% (w % (w/w) or more, or may be 5% (w/w) or less, 2% (w/w) or less, 1% (w/w) or less, 0.7% (w/w) or less, 0.5% (w/w) or less, 0.4% (w/w/w/
• the ingested concentration of the active ingredient may be, for example, 0.005-0.01% (w/w), 0.01-0.05% (w/w), 0.05-0.1% (w/w), 0.1-0.5% (w/w), 0.5-1% (w/w), or 1-5% (w/w) in terms of the dry weight of the original gram-positive bacterial cells.
• the ingested concentration of the active ingredient may be, for example, 0.005-2% (w/w), 0.005-1% (w/w), 0.005-0.5% (w/w), 0.01-2% (w/w), 0.02-1% (w/w), or 0.03-0.5% (w/w) in terms of the dry weight of the original gram-positive bacterial cells.
• composition of the present invention can be added so as to obtain the amount of the active ingredient exemplified above.
• the food of the present invention may contain spices in addition to the active ingredient. That is, the food of the present invention may be manufactured so as to contain spices. That is, the method of the present invention may further include adding spices to the raw materials of the food.
• the food containing spices may be manufactured, for example, by adding the spices themselves, or by adding a spice-containing material such as a seasoning containing spices.
• the spices may be added by adding the composition of the present invention.
• the addition of spices can be carried out in the same manner as the addition of active ingredients.
• the spices may be added to the raw materials of the food, for example, so that the content of the spices in the food of the present invention is within a desired range (for example, the content range described below).
• the raw materials of the food may originally contain spices.
• the content of the spices in the food of the present invention may be, for example, in terms of consumption concentration, 0.01% (w/w) or more, 0.02% (w/w) or more, 0.03% (w/w) or more, 0.04% (w/w) or more, 0.05% (w/w) or more, 0.07% (w/w) or more, 0.1% (w/w) or more, 0.2% (w/w) or more, 0.3% (w/w) or more, 0.4% (w/w) or more, 0.5% (w/w) or more,
• the spice content in the food product of the present invention may be 0.7% (w/w) or more, or 1% (w/w) or more, or 5% (w/w) or less, 2% (w/w) or less, 1% (w/w) or less, 0.7% (w/w) or less, 0.5% (w/w) or less, 0.4% (w/w) or less, 0.3% (w/w) or less,
• the content of the spice in the food product of the present invention may be, for example, 0.01 to 0.05% (w/w), 0.05 to 0.1% (w/w), 0.1 to 0.5% (w/w), 0.5 to 1% (w/w), or 1 to 5% (w/w) in terms of the concentration to be consumed.
• the spice content in the food of the present invention may be, for example, 0.01 to 2% (w/w), 0.02 to 1% (w/w), or 0.03 to 0.5% (w/w) as an eating concentration.
• the content of the spice in the food of the present invention may, for example, be 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 50 parts by weight or more, or 100 parts by weight or more, or 1000 parts by weight or less, 500 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 2 parts by weight or less, or any compatible combination thereof, per 1 part by weight of active ingredient contained in the food of the present invention (converted to the dry weight of gram-positive bacterial cells).
• the content of the spice in the food of the present invention may be, for example, 0.2 to 500 parts by weight, 1 to 500 parts by weight, 5 to 500 parts by weight, 50 to 500 parts by weight, 0.2 to 100 parts by weight, 1 to 100 parts by weight, 5 to 100 parts by weight, 50 to 100 parts by weight, 0.2 to 100 parts by weight, 0.5 to 50 parts by weight, or 1 to 20 parts by weight, per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of the gram-positive bacterial cells).
• the content of the spice in the food of the present invention may be, in particular, 0.2 to 500 parts by weight, or 50 to 500 parts by weight, per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of the gram-positive bacterial cells).
• the food of the present invention may contain a target substance in addition to the active ingredient. That is, the food of the present invention may be manufactured to contain the target substance. That is, the method of the present invention may further include adding the target substance to the raw material of the food.
• the target substance is as described above.
• the food containing the target substance may be manufactured, for example, by adding the target substance itself, or by adding a material containing the target substance, such as a seasoning containing the target substance. Note that "adding the active ingredient and the target substance” is not limited to the case where the active ingredient and the target substance obtained individually are added, but also includes the case where the active ingredient and the target substance obtained together are added.
• the active ingredient and the target substance obtained together are added is a case where a culture containing both the target substance and the cells of a gram-positive bacterium is subjected to a lysozyme treatment to prepare and add the active ingredient.
• the target substance may be added by adding the composition of the present invention.
• the addition of the target substance can be carried out in the same manner as the addition of the active ingredient.
• the target substance may be added to the food ingredients, for example, so that the content of the target substance in the food of the present invention falls within a desired range (for example, the content range described below).
• the food ingredients may originally contain the target substance.
• the content of the target substance (e.g., L-glutamic acid) in the food of the present invention is, for example, 0.01% (w/w) or more, 0.02% (w/w) or more, 0.03% (w/w) or more, 0.04% (w/w) or more, 0.05% (w/w) or more, 0.07% (w/w) or more, 0.1% (w/w) or more, 0.2% (w/w) or more, as an intake concentration.
• the content of the target substance (e.g., L-glutamic acid) in the food of the present invention may be, specifically, for example, 0.01 to 2% (w/w), 0.02 to 1% (w/w), or 0.03 to 0.5% (w/w) as an ingestible concentration.
• the content of the target substance (e.g., L-glutamic acid) in the food of the present invention may be, for example, 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, or 5 parts by weight or more per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of the gram-positive bacterial cells), or 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 2 parts by weight or less, or 1 part by weight or less, or any combination thereof that is not contradictory.
• 0.1 parts by weight or more 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, or 5 parts by weight or more per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of the gram-positive bacterial cells), or 50
• the content of the target substance (e.g., L-glutamic acid) in the food of the present invention may be, for example, specifically, 0.1 to 20 parts by weight per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of the gram-positive bacterial cells).
• the food of the present invention may contain a culture of gram-positive bacteria in addition to the active ingredient. That is, the food of the present invention may be manufactured to contain a culture of gram-positive bacteria. That is, the method of the present invention may further include adding a culture of gram-positive bacteria to the raw material of the food.
• the culture of gram-positive bacteria as an ingredient other than the active ingredient is as described above.
• the food containing a culture of gram-positive bacteria may be manufactured, for example, by adding a culture of gram-positive bacteria itself, or by adding a material containing a culture of gram-positive bacteria.
• “adding an active ingredient and a culture of gram-positive bacteria” is not limited to the case where an active ingredient and a culture obtained individually are added, but also includes the case where an active ingredient and a culture obtained collectively are added.
• An example of the case where an active ingredient and a culture obtained collectively are added is a case where a culture containing cells of gram-positive bacteria is subjected to lysozyme treatment to prepare and add an active ingredient.
• the composition of the present invention contains a culture of gram-positive bacteria
• the culture of gram-positive bacteria may be added by adding the composition of the present invention.
• the addition of the culture of gram-positive bacteria can be carried out in the same manner as the addition of the active ingredient.
• the gram-positive bacterial culture may be added to the food raw material, for example, so that the content of the gram-positive bacterial culture in the food of the present invention is within a desired range (for example, the content range described below).
• the food raw material may originally contain the gram-positive bacterial culture.
• the content of the culture of gram-positive bacteria in the food of the present invention may be, for example, 0.01% (w/w) or more, 0.1% (w/w) or more, 1% (w/w) or more, 5% (w/w) or more, or 10% (w/w) or more, converted into the amount of the original culture, and may be 10,000% (w/w) or less, 5,000% (w/w) or less, ), 1000% (w/w) or less, 500% (w/w) or less, 300% (w/w) or less, 200% (w/w) or less, 150% (w/w) or less, 100% (w/w) or less, 70% (w/w) or less, 50% (w/w) or less, 30% (w/w) or less, 10% (w/w) or less, 5% (w/w) or less, or 1% (w/w) or less, or any compatible
• the present specification also discloses active ingredients for use in the applications exemplified above. That is, the present specification discloses, for example, active ingredients for use in improving flavor or producing food products, and active ingredients for use in producing compositions for improving flavor or producing food products.
• this specification discloses the use of the active ingredient for combination with other ingredients (e.g., spices).Also, this specification discloses the active ingredient for combination with other ingredients (e.g., spices).The active ingredient may be combined with other ingredients (e.g., spices) for the uses exemplified above.
• L-Glu or "Glu” refers to L-glutamic acid.
• Example 1 Acquisition of a strain highly producing L-glutamic acid from Corynebacterium casei JCM 12072 (1-1) Preparation of a mutant library of C. casei JCM 12072 strain C. casei JCM 12072 strain (wild type strain) was inoculated into a Sakaguchi flask containing 30 mL of the medium described in Table 2 (A), and cultured at 30°C for 1 day with shaking, and then the cells were collected. The cells were suspended in a solution containing 0.1 M potassium phosphate buffer (pH 7.0), 6.0% dimethyl sulfoxide, and 0.1 mg/mL N-methyl-N-nitrosoguanidine (NTG), and allowed to stand at room temperature for 50 minutes.
• 0.1 M potassium phosphate buffer pH 7.0
• NTG N-methyl-N-nitrosoguanidine
• the cells were collected and washed three times with 0.1 M potassium phosphate buffer (pH 7.0).
• the cells were cultured at 30°C for 2 hours in a Sakaguchi flask containing 30 mL of the recovery culture medium described in Table 2 (B), and then the cells were collected.
• the cells were suspended in 20% glycerol and stored at ⁇ 80° C. This was used as a mutant strain library.
• the 26 candidate strains were cultured in 500 ⁇ L of evaluation medium (Table 4) placed in a 96-deep-well plate with shaking at 30°C for 48 hours, and the amount of L-Glu accumulated in the medium was measured using a Biotech Analyzer AS210 (Sakura SI Co., Ltd.).
• the RUN5-2-96 strain (NITE BP-03688), which had the highest L-Glu concentration, was selected.
• the RUN5-2-96 strain is also referred to as the "AJ111891 strain.”
• the RUN5-2-96 strain (AJ111891 strain) has all 135 mutations in group A (i.e., all of the mutations A-1 to A-135).
• Example 2 Preparation of dried cells of C. casei JCM 12072 strain and AJ111891-derived mutant strain
• the culture of C. casei JCM 12072 strain and AJ111891-derived mutant strain was carried out by jar culture using the medium shown in Table 5.
• a fermenter with a volume of 1 L was used for jar culture.
• the culture temperature was controlled at a constant 30°C
• the culture pH was controlled to 6.8 with ammonia gas
• the dissolved oxygen concentration was controlled by stirring control to be 23% or more as a relative value with the saturated dissolved oxygen concentration being 100%.
• the obtained culture solution was sterilized at 80°C for 20 minutes, and the medium components were removed by centrifugation to obtain a cell pellet.
• the cells were washed by adding water in an amount equal to the medium, and the supernatant was removed by centrifugation, which was repeated twice to wash the cells.
• the washed bacterial pellet was frozen at -80°C and then the water was removed using a freeze dryer to obtain dry bacterial cells of C. casei JCM 12072 strain and the AJ111891-derived mutant strain.
• Example 3 Preparation of fermented seasoning using C. casei AJ111891 strain and AJ111891-derived mutant strain (3-1) L-Glu fermentation was carried out using C. casei AJ111891 and its derived mutant strains as follows.
• seed culture of the AJ111891 strain and the AJ111891-derived mutant strain was carried out by jar culture using the seed medium shown in Table 6.
• the culture temperature was controlled at a constant 30°C, and the culture pH was controlled to 6.8 with ammonia gas.
• main culture of the AJ111891 strain and the AJ111891-derived mutant strain was carried out by jar culture using the medium shown in Table 7.
• the culture pH was controlled to 6.8 with ammonia gas, as in the seed culture.
• OD620 reached 40 the culture temperature was controlled at 30°C and the dissolved oxygen concentration was 23% or more.
• the temperature was raised to 35°C, and the culture was continued until glucose was depleted.
• the fine powder of activated carbon that had leaked into the filtrate was removed by dispensing the filtrate into a centrifuge tube (Himac500PA 330437A), centrifuging the filtrate at 7,000 rpm (9,400 ⁇ G) at 25°C for 10 minutes using a centrifuge (Hitachi CR20 GIII, PRP9-2 rotor), and collecting the supernatant by decantation.
• the C. casei AJ111891 fermentation broth/cell-containing seasoning contains 6% by weight of the cells of the C. casei AJ111891 strain.
• the fermentation broth and cell-containing seasoning of the C. casei AJ111891-derived mutant strain contained 6% by weight of the C. casei AJ111891-derived mutant strain cells.
• Example 4 Effect of enzyme treatment of Corynebacterium casei cells on the hotness enhancing effect 0.2 g of dried cells of the mutant strain derived from Corynebacterium casei AJ111891 obtained in Example 2 was mixed with 19.0 g of distilled water to obtain a suspension.
• lysozyme BIO manufactured by Japan Biocon Co., Ltd., derived from egg white; 39000 FPI units/mg
• papain manufactured by Amano Enzyme Co., Ltd.
• Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g.
• the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• 10 g of the resulting reaction solution was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a negative control (NC) was made by mixing 10 g of distilled water with 90 g of curry sauce.
• a positive control (PC) was made by mixing 10 g of a 1% suspension of untreated bacteria with 90 g of curry sauce.
• Sensory evaluation was performed by a rating method based on the consensus of two expert panelists.
• the spiciness intensity of the negative control was 0 points, and the spiciness intensity when untreated bacteria was added was 100 points. It was also confirmed that when 0.1% of untreated bacteria was added to mashed potatoes containing 0.2% by weight of chili pepper, it showed the same level of spiciness as mashed potatoes containing 0.4% by weight of chili pepper.
• Example 5 Effect of enzyme treatment of Corynebacterium casei bacteria on thickness enhancement
• the enzyme reaction solution of the bacteria obtained in Example 4 was used to examine the thickness enhancement effect in a cheese sauce system. 10 g of the resulting reaction solution was mixed with 90 g of cheese sauce ("Ragout for Cheese" manufactured by Mizkan Co., Ltd.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of cheese sauce.
• a positive control (PC) was prepared by mixing 10 g of a 1% suspension of untreated bacteria with 90 g of cheese sauce. Sensory evaluation was performed by a scoring method based on the consensus of two expert panelists. The thickness intensity of the negative control was 0 points, and the thickness intensity when untreated bacteria was added was 100 points.
• Example 6 Effect of enzyme treatment of Corynebacterium casei cells (wild strain) on the hotness enhancement effect 0.2 g of dried cells of Corynebacterium casei JCM 12072 strain (wild strain) obtained in Example 2 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in amounts of 1/100 and 1/100 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution was 20 g. After the addition of the enzyme solution, the reaction was carried out at 50°C for 6 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• 10 g of the obtained reaction solution was mixed with 90 g of curry sauce (17.2 g of curry roux produced by House Co., Ltd. mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a mixture of 10 g of distilled water and 90 g of curry sauce was used as a negative control (NC).
• a mixture of 10g of a 1% suspension of untreated bacteria and 90g of curry sauce was used as a positive control (PC).
• Sensory evaluation was performed by a panel of two experts using a scoring system. The spiciness of the negative control was scored as 0 points, and the spiciness of the addition of untreated bacteria was scored as 100 points. Separately, it was confirmed that the addition of 0.1% untreated bacteria to mashed potatoes containing 0.2% by weight of chili peppers produced the same level of spiciness as mashed potatoes containing 0.4% by weight of chili peppers.
• Example 7 Effect of enzyme treatment of Corynebacterium casei cells (wild strain) on thickness enhancement
• the enzyme reaction solution of the cells obtained in Example 6 was used to examine the thickness enhancement effect in cheese sauce.
• 10 g of the resulting reaction solution was mixed with 90 g of cheese sauce ("Ragout for Cheese" manufactured by Mizkan Co., Ltd.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of cheese sauce.
• a positive control (PC) was prepared by mixing 10 g of a 1% suspension of untreated cells with 90 g of cheese sauce.
• Sensory evaluation was performed by a scoring method based on the consensus of two expert panelists. The thickness intensity of the negative control was 0 points, and the thickness intensity when untreated cells were added was 100 points.
• Example 8 Effect of enzyme treatment of seasoning containing Corynebacterium casei fermentation broth and bacteria on the hotness enhancement effect 0.4 g of the seasoning containing C. casei AJ111891 strain fermentation broth and bacteria (bacterial content 6.0%) obtained in Example 3 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in an amount of 1/10,000 to 1/10 of the weight of the bacteria. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• the reaction was carried out at 50°C for 6 hours, and after the reaction, the mixture was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution for the seasoning.
• 10 g of the obtained reaction solution was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a negative control (NC) was made by mixing 10g of distilled water with 90g of curry sauce.
• a positive control (PC) was made by mixing 10g of a 2% suspension of the fermentation broth and cell-containing seasoning of untreated C. casei AJ111891 strain with 90g of curry sauce.
• Sensory evaluation was performed by a joint evaluation by two expert panelists. The spiciness intensity of the negative control was 0 points, and the spiciness intensity when untreated seasoning was added was 100 points.
• Example 9 Effect of enzyme treatment of seasoning containing Corynebacterium casei fermentation broth and bacteria on the thickness enhancement effect
• the enzyme reaction solution of the seasoning obtained in Example 8 was used to examine the thickness enhancement effect in a cheese sauce system. 10 g of the resulting reaction solution was mixed with 90 g of cheese sauce ("Ragout for Cheese" manufactured by Mizkan Co., Ltd.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of cheese sauce.
• a positive control (PC) was prepared by mixing 10 g of a 2% suspension of seasoning containing fermentation broth and bacteria of C. casei AJ111891 strain that had not been treated with enzymes with 90 g of cheese sauce.
• the sensory evaluation was performed by a scoring method based on the consensus of two expert panelists. The thickness intensity of the negative control was 0 points, and the thickness intensity when the seasoning not treated with enzymes was added was 100 points.
• Example 10 Effect of lysozyme treatment of Corynebacterium casei fermentation broth/cell-containing seasoning on the hotness enhancement effect 0.4 g of the fermentation broth/cell-containing seasoning (cell content 6.0%) derived from C. casei AJ111891 mutant obtained in Example 3 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in an amount of 1/10,000 to 1/10 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• the reaction was carried out at 50°C for 6 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution for the seasoning.
• 10 g of the obtained reaction solution was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a negative control (NC) was made by mixing 10g of distilled water with 90g of curry sauce.
• a positive control (PC) was made by mixing 10g of a 2% suspension of fermentation broth and cell-containing seasoning of untreated C. casei AJ111891 mutant with 90g of curry sauce.
• Sensory evaluation was performed by a joint rating system consisting of two expert panelists. The spiciness intensity of the negative control was 0 points, and the spiciness intensity when untreated seasoning was added was 100 points.
• Example 11 Effect of enzyme treatment of Corynebacterium casei fermentation broth/cell-containing seasoning on thickness enhancement effect
• the enzyme reaction solution of the seasoning obtained in Example 10 was used to examine the thickness enhancement effect in cheese sauce systems. 10 g of the resulting reaction solution was mixed with 90 g of cheese sauce ("Ragout for Cheese" manufactured by Mizkan Co., Ltd.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of cheese sauce.
• a positive control (PC) was prepared by mixing 10 g of a 2% suspension of a fermentation broth/cell-containing seasoning containing a mutant strain of C. casei AJ111891 that had not been treated with enzymes with 90 g of cheese sauce.
• the sensory evaluation was performed by a scoring method based on the consensus of two expert panelists. The thickness intensity of the negative control was 0 points, and the thickness intensity when the seasoning not treated with enzymes was added was 100 points.
• Example 12 Effect of enzyme treatment of Corynebacterium casei fermentation broth/cell-containing seasoning on the quality of Atsumi (cheese sauce type)
• the enzyme reaction solution of the seasoning obtained in Example 10 by adding 1/100 of the bacterial weight of lysozyme was used to evaluate the effect of enhancing the thickness felt from the beginning to the aftertaste in a cheese sauce system.
• 10 g of the resulting reaction solution was mixed with 90 g of cheese sauce (Heinz Classic Alfred) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of cheese sauce.
• a positive control was prepared by mixing 10 g of a 2% suspension of a fermentation broth/cell-containing seasoning of a mutant strain derived from C. casei AJ111891 that had not been treated with enzymes with 90 g of cheese sauce.
• the sensory evaluation was performed by a scoring method based on the consensus of five expert panelists.
• the thickness of the negative control was 0 points, and the thickness of the seasoning when the bacterial cells were not treated with enzymes was 100 points.
• the results are shown in Table 16.
• the reaction solution obtained by adding lysozyme at 1/100th the weight of the bacteria showed a strong effect of enhancing thickness compared to the untreated seasoning.
• the reaction solution obtained by adding lysozyme at 1/100th the weight of the bacteria showed a particularly remarkable effect of enhancing the thickness felt earlier.
• Example 13 Effect of enzyme treatment of Corynebacterium casei fermentation broth/cell-containing seasoning on the enhancement of the flavor felt from the beginning to the end (beef consommé type)
• the enzyme reaction solution of the seasoning obtained in Example 10 by adding and reacting lysozyme at a weight of 1/100 of the weight of the bacteria was used to evaluate the effect of enhancing the thickness felt from the beginning to the aftertaste in beef consommé.
• 10 g of the resulting reaction solution was mixed with 90 g of beef consommé (prepared with direction chicken from "Maggi additive-free beef consommé” manufactured by Nestle) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 10 g of distilled water with 90 g of beef consommé.
• a positive control was prepared by mixing 10 g of 2% suspension of fermentation broth and bacteria-containing seasoning of untreated C. casei AJ111891 mutant strain with 90 g of cheese sauce. The sensory evaluation was performed by a scoring method based on the consensus of five expert panelists. The thickness of the negative control was 0 points, and the thickness of the bacteria when untreated with enzymes was 100 points.
• the results are shown in Table 17.
• the reaction solution obtained by adding lysozyme at 1/100th the weight of the bacteria showed a strong effect of enhancing thickness compared to the untreated seasoning.
• the sample obtained by adding lysozyme at 1/100th the weight of the bacteria showed a particularly remarkable effect of enhancing the thickness felt earlier.
• Example 14 Effect of various enzyme treatments on the hotness enhancing effect of seasonings containing Corynebacterium casei fermentation broth and cells
• a suspension was obtained by mixing 0.2 g of the seasoning containing fermentation broth and cells of the mutant strain C. casei AJ111891 (cell content 6.0%) obtained in Example 3 with 9.5 g of distilled water.
• lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• papain produced by Amano Enzyme Co., Ltd.
• cellulase produced by Amano Enzyme Co., Ltd.
• hemicellulase produced by Amano Enzyme Co., Ltd.
• nuclease produced by Amano Enzyme Co., Ltd.
• lipase produced by Amano Enzyme Co., Ltd.
• a negative control was prepared by mixing 5 g of distilled water with 45 g of curry sauce.
• a positive control was prepared by mixing 5 g of a 2% suspension of a fermentation broth and cell-containing seasoning derived from an enzyme-untreated C. casei AJ111891 mutant strain with 45 g of curry sauce.
• the sensory evaluation was carried out by a scoring method based on the consensus of two expert panelists. The spiciness intensity of the negative control was 0 points, and the spiciness intensity when the enzyme-untreated seasoning was added was 100 points.
• Example 15 Effect of various enzyme treatments of Corynebacterium casei fermentation broth/cell-containing seasoning on thickness enhancement effect
• the enzyme reaction solution of the seasoning obtained in Example 14 was used to examine the thickness enhancement effect in cheese sauce systems.
• 2 g of the resulting reaction solution was mixed with 18 g of cheese sauce ("Alfred” manufactured by Kraft Heinz Co.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 2 g of distilled water with 18 g of cheese sauce.
• a positive control (PC) was prepared by mixing 2 g of a 2% suspension of a fermentation broth/cell-containing seasoning containing a mutant strain of C. casei AJ111891 that had not been treated with enzymes with 18 g of cheese sauce.
• the sensory evaluation was performed by a scoring method based on the consensus of two expert panels.
• the thickness intensity of the negative control was 0 points, and the thickness intensity when the seasoning was added with no enzyme treatment was 100 points.
• the results are shown in Table 19.
• the reaction solution obtained by adding lysozyme at a weight equal to one-tenth of the bacterial weight showed a stronger thickness-enhancing effect than the untreated seasoning.
• the reaction solutions obtained by adding papain, cellulase, hemicellulase, nuclease, or lipase treatment all showed thickness-enhancing effects equivalent to those of the untreated seasoning.
• Example 16 Effect of solubilization A suspension was obtained by mixing 0.2 g of dried cells of the mutant strain derived from Corynebacterium casei AJ111891 obtained in Example 2 with 19.0 g of distilled water. To this suspension, lysozyme (produced by Japan Biocon, derived from egg white) or papain (produced by Amano Enzyme) was added in an amount of 1/1000 to 1/10 of the weight of the cells. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g.
• lysozyme produced by Japan Biocon, derived from egg white
• papain produced by Amano Enzyme
• the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes, cooled on ice, and centrifuged at 2500 rpm for 15 minutes to obtain a supernatant fraction.
• 10 g of the obtained supernatant fraction was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a supernatant fraction was obtained from the suspension of the cells before the enzyme treatment in the same manner, and 10 g of the resulting supernatant fraction was mixed with curry sauce to form e-NC.
• a negative control was prepared by mixing 10 g of distilled water with 90 g of curry sauce.
• a positive control was prepared by mixing 10 g of a 1% suspension of untreated cells with 90 g of curry sauce. Sensory evaluation was performed by a joint rating system consisting of two expert panelists. The spiciness intensity of the negative control was 0 points, and the spiciness intensity when untreated cells were added was 100 points. It was also confirmed that when 0.1% untreated cells were added to mashed potatoes containing 0.2% by weight of chili pepper, the spiciness was the same as that of mashed potatoes containing 0.4% by weight of chili pepper.
• Example 17 Effect of chitinase treatment of Corynebacterium casei fermentation broth/cell-containing seasoning on the hotness enhancement effect 0.2 g of the fermentation broth/cell-containing seasoning (cell content 6.0%) derived from C. casei AJ111891 mutant obtained in Example 3 and 9.5 g of distilled water were mixed to obtain a suspension. Chitinase (Nagase & Co., Ltd.) was added to this suspension in an amount equivalent to 1/10 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution was 10 g.
• the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the seasoning.
• 5 g of the obtained reaction solution was mixed with 45 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a mixture of 5 g of distilled water and 45 g of curry sauce was used as a negative control (NC).
• 5g of 2% suspension of untreated seasoning was mixed with 45g of curry sauce to serve as a positive control (PC).
• Sensory evaluation was performed by two expert panelists using a scoring method. The spiciness intensity of the negative control was 0 points, and the spiciness intensity when untreated seasoning was added was 100 points.
• Example 18 Effect of chitinase treatment of seasoning containing Corynebacterium casei fermentation broth and bacteria on the thickness-enhancing effect
• the enzyme reaction solution of the seasoning obtained in Example 14 was used to examine the thickness-enhancing effect in a cheese sauce system. 2 g of the resulting reaction solution was mixed with 18 g of cheese sauce ("Alfred” manufactured by Kraft Heinz Co.) to obtain a sample for sensory evaluation.
• a negative control (NC) was prepared by mixing 2 g of distilled water with 18 g of cheese sauce.
• a positive control (PC) was prepared by mixing 2 g of a 2% suspension of the seasoning that was not enzyme-treated with 18 g of cheese sauce.
• the sensory evaluation was performed by a rating method by two expert panelists. The thickness intensity of the negative control was 0 points, and the thickness intensity when the seasoning that was not enzyme-treated was added was 100 points.
• Example 19 Preparation of dried cells of Corynebacterium flavescens Corynebacterium flavescens NBRC 14136 strain was inoculated into a 300 mL volume oblique pleated Erlenmeyer flask containing 100 mL of the medium described in Table 23, and cultured at 30 ° C for 1 day with shaking. The obtained culture solution was sterilized at 120 ° C for 20 minutes, and the medium components were removed by centrifugation to obtain a cell pellet. Furthermore, the cells were washed by adding the same amount of water as the medium and removing the supernatant by centrifugation twice, thereby washing the cells. The washed cell pellet was frozen at -80 ° C, and then the moisture was removed using a freeze dryer to obtain dried cells of Corynebacterium flavescens NBRC 14136 strain.
• Example 20 Effect of lysozyme treatment of Corynebacterium flavescens cells on the hotness enhancing effect 0.2 g of dried cells of C. flavescens NBRC 14136 strain obtained in Example 19 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in amounts of 1/100 and 1/100 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution was 20 g. After the addition of the enzyme solution, the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• 10 g of the obtained reaction solution was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a mixture of 10 g of distilled water and 90 g of curry sauce was used as a negative control (NC).
• a mixture of 10 g of a 1% suspension of enzyme-untreated C. casei AJ111891 mutant cells obtained in Example 2 and 90 g of curry sauce was used as a positive control (PC).
• PC positive control
• a mixture of 10 g of a 1% suspension of enzyme-untreated C. flavescens NBRC 14136 cells and 90 g of curry sauce was also subjected to sensory evaluation.
• the sensory evaluation was performed by a panel of three experts using a scoring method.
• the spiciness intensity of the negative control was rated at 0 points, and the spiciness intensity of the positive control was rated at 100 points.
• Example 21 Preparation of dried cells of Corynebacterium ammoniagenes
• the Corynebacterium ammoniagenes ATCC21280 strain was cultured by jar culture using the medium shown in Table 25.
• a fermenter with a volume of 1L was used for jar culture.
• the culture temperature was controlled at a constant 34°C
• the culture pH was controlled to 6.8 with ammonia gas
• the dissolved oxygen concentration was controlled by stirring control so that it was 23% or more relative to the saturated dissolved oxygen concentration of 100%.
• the obtained culture solution was sterilized at 70°C for 10 minutes, and the medium components were removed by centrifugation to obtain a cell pellet.
• the cells were washed by adding the same amount of water as the medium and removing the supernatant by centrifugation twice, thereby washing the cells.
• the washed cell pellet was frozen at -80°C, and then the moisture was removed using a freeze dryer to obtain dried cells of Corynebacterium ammoniagenes ATCC21280 strain.
• the obtained dried cells were heated at 105° C. for 4 hours, and the loss on drying was measured to determine the solid content in the dried cells.
• Example 22 Effect of lysozyme treatment of Corynebacterium ammnoniageses cells on the hotness enhancing effect 0.2 g (solid content) of dried cells of C. ammoniagenes ATCC21280 strain obtained in Example 21 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in an amount of 1/100 and 1/1000 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• 10 g of the obtained reaction solution was mixed with 90 g of curry sauce (17.2 g of House curry roux mixed with 400 g of hot water) to obtain a sample for sensory evaluation.
• a mixture of 10 g of distilled water and 90 g of curry sauce was used as a negative control (NC).
• a mixture of 10 g of a 1% suspension of enzyme-untreated C. casei AJ111891 mutant strain cells obtained in Example 2 and 90 g of curry sauce was used as a positive control (PC).
• a mixture of 10 g of a 1% suspension of enzyme-untreated C. ammoniagenes ATCC21280 strain cells and 90 g of curry sauce was also subjected to sensory evaluation.
• the sensory evaluation was performed by a rating method by three expert panelists.
• the spiciness intensity of the negative control was 0 points, and the spiciness intensity of the positive control was 100 points.
• Example 23 Preparation of dried cells of Corynebacterium glutamicum Corynebacterium glutamicum ATCC13869 strain was cultured by jar culture using the medium shown in Table 25.
• a fermenter with a volume of 1 L was used for jar culture.
• the culture temperature was controlled at a constant 31.5°C
• the culture pH was controlled to 6.8 with ammonia gas
• the dissolved oxygen concentration was controlled by stirring control to be 23% or more relative to the saturated dissolved oxygen concentration of 100%.
• the obtained culture solution was sterilized at 70°C for 10 minutes, and the medium components were removed by centrifugation to obtain a cell pellet.
• the cells were washed by adding the same amount of water as the medium and removing the supernatant by centrifugation twice, thereby washing the cells.
• the washed cell pellet was frozen at -80°C, and the moisture was removed by a freeze dryer to obtain dried cells of Corynebacterium glutamicum ATCC13869 strain.
• the obtained dried cells were heated at 105° C. for 4 hours, and the loss on drying was measured to determine the solid content in the dried cells.
• Example 24 Effect of lysozyme treatment of Corynebacterium glutamicum cells on the hotness enhancing effect 0.2 g (solid content) of dried cells of C. glutamicum ATCC13869 strain obtained in Example 23 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in an amount equivalent to 1/100 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g. After the addition of the enzyme solution, the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction solution was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• a mixture of 1 g of a 1% suspension of enzyme-untreated Corynebacterium glutamicum cells, 0.2 g of the fermented umami seasoning, and 99 g of curry sauce was also subjected to sensory evaluation.
• the sensory evaluation was performed by a rating method by three expert panelists.
• the spiciness intensity of the negative control was 0 points, and the spiciness intensity of the positive control was 100 points.
• Example 25 Preparation of dried cells of Corynebacterium stationis
• the Corynebacterium stationis ATCC6872 strain was cultured by jar culture using the medium shown in Table 28.
• a fermenter with a volume of 1L was used for jar culture.
• the culture temperature was controlled at a constant 34°C
• the culture pH was controlled to 6.8 with ammonia gas
• the dissolved oxygen concentration was controlled by stirring control to be 23% or more relative to the saturated dissolved oxygen concentration of 100%.
• the obtained culture solution was sterilized at 80°C for 10 minutes, and the medium components were removed by centrifugation to obtain a cell pellet.
• the cells were washed by adding the same amount of water as the medium and removing the supernatant by centrifugation twice, thereby washing the cells.
• the washed cell pellet was frozen at -80°C, and then the moisture was removed by a freeze dryer to obtain dried cells of Corynebacterium stationis ATCC6872 strain.
• the obtained dried cells were heated at 105° C. for 4 hours, and the loss on drying was measured to determine the solid content in the dried cells.
• Example 26 Effect of lysozyme treatment of Corynebacterium stationis cells on the hotness enhancing effect 0.2 g (solid content) of dried cells of C. stationis ATCC6872 strain obtained in Example 25 was mixed with 19.0 g of distilled water to obtain a suspension. Lysozyme (produced by Japan Biocon Co., Ltd., derived from egg white) was added to this suspension in an amount equivalent to 1/100 of the cell weight. Distilled water was added appropriately so that the total weight of the enzyme reaction solution became 20 g. After the addition of the enzyme solution, the reaction was carried out at 45°C for 4 hours, and after the reaction, the reaction solution was heated at 100°C for 10 minutes and cooled on ice to obtain an enzyme reaction solution of the cells.
• Lysozyme produced by Japan Biocon Co., Ltd., derived from egg white
• Example 27 Relationship between the content of bound diaminopimelic acid and the effect of lysozyme treatment of Corynebacterium casei cells on the hotness enhancement effect. Using the enzyme reaction solution of the seasoning obtained in Examples 8 and 10 by adding and reacting with lysozyme in an amount of 1/10,000 to 1/10 of the cell weight, the diaminopimelic acid content in the fraction with a molecular weight of 10,000 Da or less was measured.
• the positive control obtained in Example 10 the reaction solution obtained by adding lysozyme to the enzyme reaction solution of the seasoning obtained in Example 10 so that the amount was 1/10000, 1/1000, and 1/10 of the bacterial weight, and the reaction solution obtained by adding lysozyme to the enzyme reaction solution of the seasoning obtained in Example 8 so that the amount was 1/10000, 1/1000, and 1/10 of the bacterial weight were filtered using a microfiltration filter with a pore size of 0.45 ⁇ m, and then centrifuged at 2800 rpm for 15 minutes with a Vivaspin (registered trademark) 20 (manufactured by Sartorius), which is a centrifugal filter membrane with a MWCO of 10000 Da, to obtain a fraction with a molecular weight of 10000 Da.
• Vivaspin registered trademark
• the free diaminopimelic acid content was calculated by measuring the diaminopimelic acid content in the obtained fraction.
• concentrated hydrochloric acid was added to the obtained fraction so that the hydrochloric acid concentration was 6 M, and then hydrochloric acid hydrolysis was performed by heating at 120 ° C. under reduced pressure for 24 hours. Thereafter, hydrochloric acid was removed by distillation using a vacuum concentrator, and the solution was diluted to a predetermined volume to obtain an analytical sample.
• the diaminopimelic acid content in the analytical sample was measured using LC-MS (high performance liquid chromatography-mass spectrometry).
• the bound diaminopimelic acid content was calculated by subtracting the free diaminopimelic acid content from the measured diaminopimelic acid content in the analytical sample.
• Table 30 The results are shown in Table 30.
• the presence or absence of the "improvement effect on enhancing pungency” in Table 30 is determined based on the results of the sensory evaluation in Examples 8 and 10.
• a pungency intensity of 110 points or more was determined to have an "improvement effect on enhancing pungency”
• a pungency intensity of less than 110 points was determined to have no "improvement effect on enhancing pungency”.
• the content of bound diaminopimelic acid in the fraction with a molecular weight of 10,000 Da in Table 30 is calculated by converting the content of bound diaminopimelic acid calculated above into the content per 100 g of dry weight of the fermentation broth/cell-containing seasoning of the C.

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