Fermented composition, method for producing a fermented composition, method for improving the flavor of a fermented composition, method for determining aroma components involved in the aroma characteristics of fermented milk, method for evaluating the flavor of fermented milk

Abstract

To provide a fermentation composition with superior flavor. [Solution] A fermentation composition that satisfies at least one of the following conditions: (1a), (1b), (1c), and (1d). (1a) Maltol content is 8.8 μg / 100g or more. (1b) Cyclotene content is 0.07 μg / 100g or more. (1c) Sotolon content is 0.013 μg / 100g or more. (1d) Vanillin content is 0.016 μg / 100g or more.

Description

Technical Field

[0001] The present invention relates to a fermented composition, a method for producing the fermented composition, a method for improving the flavor of the fermented composition, a method for determining aroma components involved in the aroma characteristics of fermented milk, and a method for evaluating the flavor of fermented milk.

Background Art

[0002] Fermented milk is generally produced by fermenting milk such as cow milk with lactic acid bacteria. In recent years, consumer demand for fermented milk containing Bifidobacterium bacteria has been increasing. Bifidobacterium bacteria have a wide variety of physiological effects. Among Bifidobacterium bacteria, the Bifidobacterium bacteria having the property of living in the human intestinal tract, called HRB (Human Residential Bifidobacterium), have particularly many research reports on their physiological effects. Among HRB, Bifidobacterium longum subsp. longum and Bifidobacterium breve have been reported to have various physiological effects including intestinal regulatory function.

[0003] Bifidobacterium bacteria have a metabolic pathway different from that of lactic acid bacteria. A typical difference is acetic acid production by hetero-lactic fermentation. While acetic acid has physiological effects such as fatigue reduction and slimming function, its characteristic aroma has a great influence on flavor, and it has been one of the challenges in fermented milk where palatability is also emphasized. As described above, Bifidobacterium bacteria have flavor characteristics different from those of lactic acid bacteria, but there are not many studies on the flavor of Bifidobacterium bacteria compared to studies on the flavor of lactic acid bacteria. This is thought to be because in global laws and regulations (such as the CODEX of the EU), yogurt is stipulated to use lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus), and research on the flavor of lactic acid bacteria has naturally progressed.

[0004] On the other hand, controlling the flavor of beverages and the like by adding aroma components has been studied. Patent Document 1 discloses a composition for instant fermented tea beverages containing at least one selected from the group consisting of vanillin, ethyl vanillin, and heliotropin. Patent Document 2 discloses a non-fermented dark beer-flavored beverage containing one or more of pyrazines, ethyl maltol, and cyclotene. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2017 / 030187 [Patent Document 2] Japanese Patent Publication No. 2018-164463 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] As mentioned above, aroma components affect flavor. Therefore, in order to improve the flavor of fermented milk containing Bifidobacterium bacteria, it is considered effective to elucidate their characteristic aroma features and confirm the impact of these aroma features on the overall flavor of the fermented milk.

[0007] The present invention aims to provide a fermentation composition with excellent flavor and a method for producing the same. Another objective of this invention is to provide a method for improving the flavor of fermented compositions that is excellent in improving their flavor. Another objective of this invention is to provide a method for determining aroma components involved in the aroma characteristics of fermented milk containing Bifidobacterium bacteria, and to contribute to improving the flavor of fermented milk. Another objective of this invention is to provide an evaluation method that allows for the simple evaluation of the flavor of fermented milk containing Bifidobacterium bacteria. [Means for solving the problem]

[0008] The present invention has the following aspects. [1] A fermentation composition that satisfies at least one of the following conditions: (1a), (1b), (1c), and (1d). (1a) The maltol content is 8.8 μg / 100g or more. (1b) The cyclotene content is 0.07 μg / 100g or more. (1c) The sotolon content is 0.013 μg / 100g or more. (1d) The vanillin content is 0.016 μg / 100g or more. [2] A fermentation composition that satisfies at least one of the following (1e), (1f), and (1g). The content of (1e)trans-4,5-epoxy-(E)-decenal is 0.013 μg / 100g or less. (1f) The eugenol content is 0.06 μg / 100g or less. The content of (1g)(E)-2-nonenal is 0.006 μg / 100g or less. [3] The fermentation composition according to [1], further satisfying at least one of the following (1e), (1f), and (1g). The content of (1e)trans-4,5-epoxy-(E)-decenal is 0.013 μg / 100g or less. (1f) The eugenol content is 0.06 μg / 100g or less. The content of (1g)(E)-2-nonenal is 0.006 μg / 100g or less. [4] A fermented milk product, as described in any of [1] to [3]. [5] A fermentation composition according to any one of [1] to [4], comprising bacteria of the genus Bifidobacterium. [6] A method for producing a fermentation composition, comprising at least one of the following steps: (2a), (2b), (2c), and (2d). (2a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (2b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100g or more. (2c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (2d) A step to adjust the vanillin content in the fermentation composition to 0.016 μg / 100g or more. [7] A method for producing a fermentation composition, comprising at least one of the following steps: (2e), (2f), and (2g). (2e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100g or less. (2f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (2g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less. [8] A method for producing the fermentation composition according to [6], further comprising at least one of the following steps: (2e), (2f), and (2g). (2e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100g or less. (2f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (2g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less. [9] A method for improving the flavor of a fermented composition, comprising at least one of the following steps: (3a), (3b), (3c), and (3d). (3a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (3b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100g or more. (3c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (3d) A step to adjust the vanillin content in the fermentation composition to 0.016 μg / 100g or more.

[10] A method for improving the flavor of a fermented composition, comprising at least one of the following steps: (3e), (3f), and (3g). (3e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermented composition to 0.013 μg / 100 g or less. (3f) A step of adjusting the content of eugenol in the fermented composition to 0.06 μg / 100 g or less. (3g) A step of adjusting the content of (E)-2-nonenal in the fermented composition to 0.006 μg / 100 g or less.

[11] The method for improving the flavor of a fermented composition according to [9], further comprising at least one step selected from the following (3e), (3f) and (3g). (3e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermented composition to 0.013 μg / 100 g or less. (3f) A step of adjusting the content of eugenol in the fermented composition to 0.06 μg / 100 g or less. (3g) A step of adjusting the content of (E)-2-nonenal in the fermented composition to 0.006 μg / 100 g or less.

[12] A test sample which is a fermented milk containing a Bifidobacterium bacterium, and a control sample for comparing aroma characteristics are prepared, For each of the test sample and the control sample, an extract containing aroma components is obtained by solvent extraction, and the aroma components in the extract are analyzed by gas chromatography-mass spectrometry and gas chromatography-olfactometry, Sensory evaluation is performed on each of the test sample and the control sample, Correlation analysis is performed on the results of the analysis and the results of the sensory evaluation, and candidate components that may be involved in the predetermined aroma characteristic of the test sample are selected from among the aroma components, A determination method, wherein the candidate components are added to the test sample, sensory evaluation is performed again, and the aroma components involved in the predetermined aroma characteristic are determined.

[13] The determination method according to

[12] , wherein the predetermined aroma characteristic is sweet aroma / caramel odor, or yeast / animal / culture medium / heated odor.

[14] An evaluation method for fermented milk containing Bifidobacterium bacteria, comprising quantifying the aroma components determined to be involved in the predetermined aroma characteristics by the determination method described in

[12] or

[13] , and evaluating the flavor of the fermented milk based on the results of the quantification.

[15] An evaluation method for fermented milk containing Bifidobacterium bacteria, comprising quantifying at least one of trans-4,5-epoxy-(E)-decenal, eugenol, (E)-2-nonenal, maltol, cyclotene, sotolon, and vanillin, and evaluating the flavor of the fermented milk based on the results of the quantification. [Effects of the Invention]

[0009] According to the present invention, a fermentation composition with excellent flavor and a method for producing the same can be provided. Furthermore, according to the present invention, a flavor improvement method that is excellent in improving the flavor of fermented compositions can be provided. Furthermore, according to the present invention, it is possible to determine the aroma components involved in the aroma characteristics of fermented milk containing Bifidobacterium bacteria, and to provide a determination method that contributes to improving the flavor of fermented milk. Furthermore, the present invention provides a simple evaluation method for assessing the flavor of fermented milk containing Bifidobacterium bacteria. [Modes for carrying out the invention]

[0010] In this specification, "fermented composition" means a composition containing a fermented product obtained by fermenting a fermentation substrate with fermenting bacteria. "Fermented milk" means a product obtained by fermenting milk or milk containing an equivalent or greater amount of non-fat milk solids with fermenting bacteria, resulting in a paste-like or liquid state. The milk may be of mammalian origin, and examples include milk from cows, sheep, or goats. Milk from cows (i.e., cow's milk) is preferred. In this specification, the symbol "~" used for numerical ranges means that the range includes both its lower and upper limits.

[0011] [Fermentation composition] A fermentation composition according to one embodiment (hereinafter also referred to as "fermentation composition (1)") is a fermentation composition that satisfies at least one of the following (1a), (1b), (1c), and (1d) (hereinafter collectively referred to as "(1a) to (1d)"). The fermentation composition (1) may also satisfy at least one of the following (1e), (1f), and (1g) (hereinafter, these will also be collectively referred to as "(1e) to (1g)"). Another fermentation composition according to one embodiment (hereinafter also referred to as "fermentation composition (2)") is a fermentation composition that satisfies at least one of (1e) to (1g). The content of each component in (1a) to (1d) and (1e) to (1g) is the mass of each component per 100g of the fermented composition. Hereafter, the term "fermented composition" will include both fermented compositions (1) and (2).

[0012] (1a) The maltol content is 8.8 μg / 100g or more. (1b) The cyclotene content is 0.07 μg / 100g or more. (1c) The sotolon content is 0.013 μg / 100g or more. (1d) The vanillin content is 0.016 μg / 100g or more.

[0013] The content of (1e)trans-4,5-epoxy-(E)-decenal is 0.013 μg / 100g or less. (1f) The eugenol content is 0.06 μg / 100g or less. The content of (1g)(E)-2-nonenal is 0.006 μg / 100g or less.

[0014] <(1a)~(1d)> The inventors have found, using the determination method described later, that maltol, cyclotene, sotolon, and vanillin are aroma components that contribute particularly to the sweet aroma (including caramel aroma) among the aroma characteristics of the fermented composition, and that the aromas from these aroma components contribute positively to the flavor. The flavor of the fermented composition is improved by satisfying at least one of (1a) to (1d).

[0015] The maltol content in (1a) is 8.8 μg / 100g or more, preferably 10.2 μg / 100g or more, more preferably 13.0 μg / 100g or more, and also preferably 34.8 μg / 100g or less, more preferably 28.0 μg / 100g or less, and more preferably 21.2 μg / 100g or less.

[0016] The cyclotene content in (1b) is 0.07 μg / 100g or more, preferably 0.11 μg / 100g or more, more preferably 0.19 μg / 100g or more, and also preferably 0.79 μg / 100g or less, more preferably 0.60 μg / 100g or less, and more preferably 0.41 μg / 100g or less.

[0017] The sotolon content in (1c) is 0.013 μg / 100g or more, preferably 0.03 μg / 100g or more, more preferably 0.05 μg / 100g or more, more preferably 0.13 μg / 100g or more, and also preferably 0.52 μg / 100g or less, more preferably 0.39 μg / 100g or less, and more preferably 0.26 μg / 100g or less.

[0018] The vanillin content in (1d) is 0.016 μg / 100g or more, preferably 0.02 μg / 100g or more, more preferably 0.03 μg / 100g or more, more preferably 0.06 μg / 100g or more, and also preferably 0.96 μg / 100g or less, more preferably 0.64 μg / 100g or less, and more preferably 0.32 μg / 100g or less.

[0019] The fermentation composition (1) only needs to satisfy at least one of (1a) to (1d), but in terms of flavor, it is preferable that it satisfies at least two of (1a) to (1d), more preferably at least three, and particularly preferable that it satisfies all of them.

[0020] The aroma components maltol, cyclotene, sotolon, and vanillin in the fermented composition may be produced by fermentation microorganisms during the production of the fermented composition, added to the raw materials or their fermented products, or both. If a fermentation composition contains Bifidobacterium bacteria, it is considered that at least some of the aroma components in the fermentation composition are produced by Bifidobacterium bacteria.

[0021] <(1e)~(1g)> The inventors have found, using the determination method described later, that trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal are aroma components that are particularly involved in the yeast odor, animal odor, culture medium odor, and heating odor of the fermentation composition, and that the aromas from these aroma components contribute negatively to the flavor. The flavor of the fermentation composition is improved by satisfying at least one of (1e) to (1g).

[0022] The trans-4,5-epoxy-(E)-decenal content in (1e) is 0.013 μg / 100g or less, preferably 0.012 μg / 100g or less, more preferably 0.010 μg / 100g or less, more preferably 0.09 μg / 100g or less, and more preferably 0.08 μg / 100g or less. From the standpoint of flavor, a lower trans-4,5-epoxy-(E)-decenal content is preferable, and there is no particular lower limit, but from the standpoint of ease of manufacture, 0.06 μg / 100g or more is preferable.

[0023] The eugenol content in (1f) is 0.06 μg / 100g or less, preferably 0.05 μg / 100g or less, more preferably 0.02 μg / 100g or less, and more preferably 0.01 μg / 100g or less. A lower eugenol content is preferable in terms of flavor, and there is no particular lower limit.

[0024] The (E)-2-nonenal content per (1g) is 0.006 μg / 100g or less, preferably 0.005 μg / 100g or less, more preferably 0.002 μg / 100g or less, and more preferably 0.001 μg / 100g or less. A lower (E)-2-nonenal content is preferable in terms of flavor, and there is no particular lower limit.

[0025] The fermented composition (2) only needs to satisfy at least one of (1e) to (1g), but in terms of flavor, it is preferable that it satisfies at least two of (1e) to (1g), and it is particularly preferable that it satisfies all of them. Similarly, fermentation composition (1) preferably satisfies at least two of (1e) to (1g), and particularly preferably satisfies all of them.

[0026] The aroma components trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal in the fermentation composition may be produced by fermenting bacteria during the production of the fermentation composition, added to the raw materials or their fermented products, or both. If the fermentation composition contains Bifidobacterium bacteria, it is considered that at least a portion of each aroma component in the fermentation composition is produced by Bifidobacterium bacteria.

[0027] The fermentation composition may be the same as a known fermentation composition, except that it satisfies at least one of (1a) to (1d) and / or at least one of (1e) to (1g).

[0028] The fermentation composition contains fermented products. If the fermented composition is fermented milk, it includes a fermented product obtained by fermenting a raw material composition containing milk raw materials with fermentation bacteria. As milk raw materials, those known in the production of fermented milk can be used, such as raw milk, cream, skimmed condensed milk, skimmed milk powder, and milk protein concentrate. One type of milk raw material may be used, or two or more types may be used in combination. If the fermented composition is not fermented milk, it includes a fermented product obtained by fermenting a raw material composition containing a fermentation substrate other than dairy ingredients with fermentation bacteria. Examples of fermentation substrates other than dairy include plant-based milk. Plant-based milk refers to a milky food obtained from plant ingredients. Examples of plant-based milk include soy milk, almond milk, rice milk, coconut milk, and oat milk. The fermentation substrate other than dairy may be one type or two or more types may be used in combination.

[0029] The raw material composition may further contain known components in fermentation compositions, such as water, sugars, vegetable fats, stabilizers, flavorings, pH adjusters, fruit juice / pulp, sweeteners, yeast extract, vitamins, minerals, and dietary fiber. Examples of stabilizers include agar, gelatin, pectin, and starch. Examples of sweeteners include sucralose, acesulfame potassium, licorice, and stevia. Yeast extract is a nutrient source obtained by moderately breaking down yeast using autodigestive enzymes. Yeast extract may be in liquid, powder, or other forms. Commercially available yeast extract can be used. The solid content of yeast extract in the raw material composition is preferably 0.005% by mass or more, more preferably 0.01% by mass or more, even more preferably 0.02% by mass or more, and preferably 0.10% by mass or less, and more preferably 0.07% by mass or less, relative to the total mass of the raw material composition. When the yeast extract content is above the lower limit, the effects of the yeast extract are sufficiently obtained. When the yeast extract content is below the upper limit, the flavor tends to be better.

[0030] Examples of fermentation microorganisms include Bifidobacterium bacteria, lactic acid bacteria, and yeast. One type of fermentation microorganism may be used, or two or more may be used in combination. A well-known lactic acid bacteria starter may also be used as the fermentation microorganism. The fermentation bacteria preferably include Bifidobacterium bacteria, as this enhances the flavor by satisfying at least one of (1a) to (1d) and / or at least one of (1e) to (1g). From the standpoint of fermentation efficiency, it is preferable that the fermentation bacteria include lactic acid bacteria.

[0031] The Bifidobacterium species (hereinafter also referred to as Bifidobacteria) is not particularly limited, and any Bifidobacteria known in fields such as fermentation can be used. For example, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium breve, Bifidobacterium longum subsp. suis, Bifidobacterium animalis subsp. longum, Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. Examples include Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. Note that Bifidobacterium longum subspecies longum is sometimes simply abbreviated as Bifidobacterium longum.Furthermore, Bifidobacterium longum subspecies infantis is sometimes simply abbreviated as Bifidobacterium infantis. One type of Bifidobacterium may be used, or two or more types may be used in combination.

[0032] The bifidobacteria may be HRB (Human Residential Bifidobacterium), or other bifidobacteria (non-HRB), and these may be used in combination. As for HRBs, since a variety of physiological effects, including intestinal regulation, have been reported, at least one selected from the group consisting of Bifidobacterium longum subspecies longum, Bifidobacterium longum subspecies infantis, and Bifidobacterium breve is preferred. As a non-HRB, Bifidobacterium animalis subspecies lactis is preferred because it is the most commonly used Bifidobacterium bacterium in the food industry.

[0033] Examples of Bifidobacterium longum subspecies longum include NITE BP-02621, ATCC 15707, ATCC 25962, DSM 20219, and JCM 1217. Among these, NITE BP-02621 is preferred. Bifidobacterium longum subspecies longum may be used individually or in combination of two or more strains. The bacterium assigned accession number NITE BP-02621 was internationally deposited on January 26, 2018, under the Budapest Convention, with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan). This bacterium is identical to Bifidobacterium longum subspecies longum BB536.

[0034] Examples of Bifidobacterium longum subspecies infantis include NITE BP-02623, ATCC 15697, ATCC 15702, DSM 20088, and JCM 1222. Among these, NITE BP-02623 is preferred. Bifidobacterium longum subspecies infantis may be used individually or in combination of two or more strains. The bacterium assigned accession number NITE BP-02623 was internationally deposited on January 26, 2018, under the Budapest Convention, with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan). This bacterium is identical to Bifidobacterium longum subspecies infantis M-63.

[0035] Examples of Bifidobacterium breve include NITE BP-02622, FERM BP-11175, ATCC 15700, ATCC 15698, DSM 20213, DSM 24706, DSM 13692, DSM 24732, DSM 24736, DSM16604, JCM 1192, NCC 2705, NCC 490, YIT 4010, YIT 4064, SBT 2928, UCC 2003, BBG-001, C50, R0070, and BG7. Among these, NITE BP-02622 and FERM BP-11175 are preferred. Bifidobacterium breve may be used individually or in combination of two or more strains. The bacteria assigned accession number NITE BP-02622 were internationally deposited on January 26, 2018, under the Budapest Convention, with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) using accession number NITE BP-02622. The bacterium assigned accession number FERM BP-11175 was internationally deposited on August 25, 2009, under the Budapest Convention, with the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (now the Patent Organism Depository Center of the National Institute of Technology and Evaluation, Room 120, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan). This bacterium is identical to Bifidobacterium breve MCC1274.

[0036] The lactic acid bacteria used are not particularly limited, and any lactic acid bacteria known in the field of fermented foods can be used. Examples include bacteria of the genera Lactobacillus, Streptococcus, Lactococcus, Enterococcus, and Leuconostoc. One type of lactic acid bacteria may be used, or two or more types may be used in combination. In this invention, Bifidobacteria are not considered lactic acid bacteria.

[0037] Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus derubruekii subsp. bulgaricus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus helveticus, Lacticaseibacillus paracasei, Lacticaseibacillus casei, Lacticaseibacillus rhamunosus, Lactobacillus delbrueckii, and Lactiplantibacillus plantarum. Lactobacillus delbrukii subspecies bulgaricus is sometimes simply abbreviated as Lactobacillus bulgaricus.

[0038] An example of a bacterium belonging to the genus Streptococcus is Streptococcus thermophilus.

[0039] Examples of bacteria belonging to the genus Lactococcus include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus plantarum. Lactococcus lactis subsp. lactis is sometimes abbreviated simply as Lactococcus lactis, and Lactococcus lactis subsp. cremoris is sometimes abbreviated simply as Lactococcus cremoris.

[0040] Lactic acid bacteria belonging to the Enterococciaceae family include Enterococcus species such as Enterococcus faecalis and Enterococcus faecium.

[0041] Lactic acid bacteria belonging to the Leuconostoc family include Leuconostoc mesenteroides and Leuconostoc mesenteroides subsp. cremoris, which are all genus Leuconostoc.

[0042] From the viewpoint of fermentation efficiency, the lactic acid bacteria preferably include Lactobacillus delbrookii subspecies bulgaricus and Streptococcus thermophilus. In addition to Lactobacillus delbrookii subspecies bulgaricus and Streptococcus thermophilus, other lactic acid bacteria may also be further included. As for other lactic acid bacteria, Lactococcus lactis subspecies lactis is preferred from the viewpoint of improving the survival rate of bifidobacteria.

[0043] The fermentation composition may consist solely of fermented products, or it may further contain other raw materials besides fermented products. Other ingredients include, for example, animal or vegetable oils and fats, dietary fiber, emulsifiers, stabilizers, thickeners, sugars, sugar alcohols, polysaccharides, pH adjusters, fatty acid esters, flavoring and odor-correcting agents, flavorings, sweeteners, colorings, excipients, minerals, vitamins, etc.

[0044] The fermented composition preferably contains Bifidobacterium. Fermented compositions containing Bifidobacterium have a characteristic aroma of Bifidobacterium, and tend to have a greater difference in flavor compared to fermented compositions fermented with lactic acid bacteria alone, depending on whether at least one of (1a) to (1d) and / or at least one of (1e) to (1g) is satisfied or not. The Bifidobacteria in the fermentation composition may be in either a live or dead state. From the viewpoint of probiotic effect, it is preferable to include live Bifidobacteria. The amount of live Bifidobacterium is not particularly limited, but for example, 10 × 10⁶ live bacteria per 100g of fermentation composition is acceptable. 8 cfu / 100g or more, and even 10x10 9 cfu / 100g or more, and even 10x10 10 It is cfu / 100g or more, and also, for example, 10 x 10 13 cfu / 100g or less, and even 10x10 12 cfu / 100g or less, and even 5x10 11 It is less than cfu / 100g. cfu stands for colony forming unit.

[0045] If the fermentation composition contains Bifidobacteria, the pH of the fermentation composition at 10°C is preferably 3.7 to 5.0, and more preferably 4.0 to 4.8.

[0046] The fermentation composition may contain both bifidobacteria and lactic acid bacteria. When the fermentation composition contains bifidobacteria and lactic acid bacteria, the ratio of lactic acid bacteria content (unit: cfu / 100g) to bifidobacteria content (unit: cfu / 100g) in the fermentation composition, "lactic acid bacteria / bifidobacteria," is preferably 0.001 to 2.0, more preferably 0.002 to 1.0, even more preferably 0.005 to 0.7, and particularly preferably 0.01 to 0.5.

[0047] The solid content of the fermentation composition is, for example, 3 to 30% by mass, more specifically, 5 to 25% by mass, or more specifically, 8 to 20% by mass, relative to the total mass of the fermentation composition.

[0048] The solid content is calculated using the formula: "Solid content (unit: mass%) = 100 - Moisture (unit: mass%)". The moisture content is measured using the atmospheric pressure heating and drying method. Specifically, the amount of weight lost when the sample is dried in a 99°C incubator for 4 hours is measured, and the moisture content is calculated using the following formula. Moisture content (unit: mass%) = (Mass of sample before drying (unit: g) - Mass of sample after drying (unit: g)) / Mass of sample before drying (unit: g) × 100

[0049] If the fermented composition is fermented milk, the non-fat milk solids content of the fermented composition is, for example, 3 to 23% by mass, more specifically, 5 to 18% by mass, or more specifically, 8 to 14% by mass, relative to the total mass of the fermented composition.

[0050] Non-fat milk solids are the value obtained by subtracting the fat content (also called milk fat) from the solids derived from milk (also called milk solids). The fat content is measured by the Reese-Gottlieb method. Specifically, 3 g of the sample is placed in a Majornie tube, 7 mL of water, 1 drop of phenolphthalein, and 2 mL of aqueous ammonia are added, and the tube is gently shaken. Then, 10 mL of ethanol, 25 mL of ethyl ether, and 25 mL of petroleum ether are added, stopping the tube after each addition and shaking 2 or 3 times. After centrifugation of the Majornie tube, the solvent layer is transferred to a dish and the solvent is allowed to evaporate. This residue is the fat. The residue is weighed to obtain the fat mass. The ratio of the fat mass to 3 g of sample is determined and expressed as the fat content (unit: mass%).

[0051] The fermentation composition may be in any form, such as liquid, semi-solid, or solid. Specific examples of fermented compositions include, but are not limited to, yogurts, cheeses, creams, dairy beverages, lactic acid bacteria beverages, and supplements. The fermentation composition is preferably a fermented food or beverage, and more preferably fermented milk.

[0052] [Method for producing fermented composition] A method for producing a fermented composition according to one embodiment (hereinafter also referred to as "production method (1)") includes at least one of the following steps: (2a), (2b), (2c), and (2d) (hereinafter collectively referred to as "(2a) to (2d)"). Manufacturing method (1) may further include at least one of the following steps: (2e), (2f), and (2g) (hereinafter, these will also be collectively referred to as "(2e) to (2g)"). A method for producing a fermented composition according to another embodiment (hereinafter also referred to as "production method (2)") includes at least one of the steps (2e) to (2g). The content of each component in (2a) to (2d) and (2e) to (2g) is the mass of each component per 100g of the fermented composition to be manufactured. According to manufacturing method (1), a fermentation composition (1) is obtained. According to manufacturing method (2), a fermentation composition (2) is obtained.

[0053] (2a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (2b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100g or more. (2c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (2d) A step to adjust the vanillin content in the fermentation composition to 0.016 μg / 100g or more.

[0054] (2e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100g or less. (2f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (2g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less.

[0055] <(2a)~(2d)> In each of the steps (2a) to (2d), the content of each component, maltol, cyclotene, sotolon, and vanillin, can be adjusted, for example, by adjusting the composition of the raw material composition or fermentation conditions in the production of the fermented product, by adding each component to the fermented product, or by a combination thereof. The process of producing fermented products will be explained in more detail later. Regarding the composition of the raw material, for example, it is known that the amount of bifidobacteria can be increased by increasing the amount of yeast extract, and this may increase the content of these substances. Regarding fermentation conditions, for example, it has been found that the amount of Bifidobacteria can be increased by suppressing the dissolved oxygen concentration during fermentation, which may increase the amount of these substances. The preferred contents of maltol, cyclotene, sotolon, and vanillin in (2a) to (2d) are the same as the preferred contents of maltol, cyclotene, sotolon, and vanillin in (1a) to (1d).

[0056] <(2e)~(2g)> In each step from (2e) to (2g), the content of each component, trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal, can be adjusted, for example, by adjusting the composition of the raw material composition, sterilization conditions, and fermentation conditions (e.g., fermentation temperature, atmospheric composition during fermentation, and dissolved oxygen concentration) in the production of the fermented product, by genetically modifying fermenting bacteria such as Bifidobacterium, or by a combination of these methods. Furthermore, it is expected that the flavor can be improved by incorporating masking agents to make the negative flavor characteristics of these substances less noticeable. The preferred contents of trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal in (2e) to (2g) are the same as the preferred contents of maltol, cyclotene, sotolon, and vanillin in (1e) to (1g).

[0057] <Production of fermented products> Fermented products are produced by preparing a raw material composition containing fermentation bacteria and fermenting it. The composition of the raw material composition is preferably designed according to the composition of the fermented product to be obtained. The total mass of the raw material composition is the same as the total mass of the fermented product at the end of fermentation.

[0058] In the preparation process of the raw material composition, raw materials other than fermentation bacteria are mixed, preferably subjected to homogenization, heat sterilization, and after cooling, fermentation bacteria are added (also called inoculation) to obtain the raw material composition. Homogenization and heat sterilization can be carried out by conventional methods. Homogenization can be carried out by conventional methods. For example, one method is to use a homogenizer and homogenize the material at a temperature of 65-80°C and a pressure of 5-25 MPa, but this method is not limited to these. Heat sterilization can be carried out by conventional methods. Heat sterilization can be carried out using heat sterilization equipment such as plate sterilizers, tubular sterilizers, direct heating sterilizers, and jacketed tanks. Heat sterilization can be carried out by heating to, for example, 80-150°C or 80-120°C. The cooling temperature should be any temperature at which the fermentation bacteria can survive, for example, 30-40°C.

[0059] In the fermentation process, the raw material composition containing fermentation bacteria is maintained at a predetermined fermentation temperature for fermentation. The fermentation temperature should be within the range in which the fermentation bacteria can efficiently grow; for example, 35°C to 43°C is preferred, and 37°C to 41°C is more preferred. As fermentation progresses, the pH of the raw material composition decreases. Once a predetermined endpoint pH is reached, the mixture is cooled to obtain the fermented product. The endpoint pH is preferably between 4.0 and 5.0, and more preferably between 4.2 and 4.8. The temperature after cooling is preferably between 3°C and 15°C, and more preferably between 5°C and 10°C. The fermentation process can be carried out by known methods.

[0060] The fermented product obtained after the fermentation process may be used as is as a fermented composition, or it may be processed by adding other raw materials to form a fermented composition.

[0061] The product form of the fermentation composition is not particularly limited. For example, in the production of fermented milk, static fermented milk can be obtained by fermenting the raw material composition while it is contained in a product container such as a cup. Alternatively, a stirred-type fermented milk or a drinkable type of fermented milk can be obtained by fermenting the raw material composition in a tank or the like, stirring and crushing the resulting fermented product (also called curd), and filling it into a product container. In particular, when producing drinkable fermented milk, it is preferable to smooth the fermented product (also called curd). Smoothing refers to the process of crushing the fermented product to make it smooth. The crushing means can be any known crushing means used in the production of fermented milk. Specific examples include homogenizers, back pressure valves, filters, pumps, etc.

[0062] [Method for improving the flavor of fermented compositions] A method for improving the flavor of a fermented composition according to one embodiment (hereinafter also referred to as "flavor improvement method (1)") includes at least one of the following steps: (3a), (3b), (3c), and (3d) (hereinafter collectively referred to as "(3a) to (3d)"). The flavor improvement method (1) may further include at least one of the following steps: (3e), (3f), and (3g) (hereinafter, these will also be collectively referred to as "(3e) to (3g)"). A method for improving the flavor of a fermented composition according to another embodiment (hereinafter also referred to as "flavor improvement method (2)") includes at least one of the steps (3e) to (3g). The content of each component in (3a) to (3d) and (3e) to (3g) is the mass of each component per 100g of the fermented composition after flavor improvement.

[0063] (3a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (3b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100g or more. (3c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (3d) A step to adjust the vanillin content in the fermentation composition to 0.016 μg / 100g or more.

[0064] (3e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100g or less. (3f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (3g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less.

[0065] Steps (3a) to (3d) are the same as those described above in (2a) to (2d). Steps (3e) to (3g) are the same as those (2e) to (2g) described above.

[0066] [Method for determining aroma components involved in the aroma characteristics of fermented milk] The determination method according to one embodiment is: (1) Prepare a sample of fermented milk containing Bifidobacterium and a control sample to compare their aroma characteristics. (2) For each of the above-mentioned sample and the control sample, an extract containing aroma components is obtained by solvent extraction, and the aroma components in the extract are analyzed by gas chromatography-mass spectrometry (hereinafter also referred to as "GC-MS") and gas chromatography-olfactometry (hereinafter also referred to as "GC-O"). (3) Sensory evaluation is performed on each of the above-mentioned sample and the control sample. (4) A correlation analysis is performed on the results of the analysis and the sensory evaluation, and candidate components are selected from the aroma components that may be involved in the predetermined aroma characteristics of the sample. (5) Add the candidate components to the sample and perform another sensory evaluation to determine the aroma components involved in the predetermined aroma characteristics. There are no particular restrictions on the order in which steps (2) and (3) are performed. Either one may be performed first, or both steps may be performed in parallel.

[0067] The sample is not particularly limited as long as it is fermented milk containing Bifidobacterium, and it may be manufactured by a known method or be commercially available. You may prepare one or more types of samples. For example, you may prepare two or more samples that contain different types or amounts of Bifidobacterium.

[0068] The control sample can be any sample with different aroma characteristics from the target sample, and can be appropriately selected according to the "specified aroma characteristics" being targeted. When determining the aroma components that contribute to the distinct aroma characteristics of fermented milk containing Bifidobacterium compared to fermented milk fermented solely with Lactobacillus, it is preferable to use fermented milk fermented solely with Lactobacillus as a control sample.

[0069] The aroma components extracted by solvent extraction (hereinafter also referred to as "endogenous aromas") include aroma components that are involved in the characteristic aroma features of Bifidobacterium. Solvent extraction can be carried out by conventional methods. For example, a sample or control sample, water, solvent, and internal standard substance are mixed, stirred, and then allowed to stand to separate into an aqueous phase and a solvent phase. The solvent phase is then separated and recovered, and concentrated as necessary to obtain an extract containing aroma components and the internal standard substance. Examples of solvents include dichloromethane, but the solvent is not limited to this. Internal standards are used to quantify aroma components in extracts. In GC-MS and GC-O, the content of aroma components is determined as an approximate content calculated from the peak area ratio with respect to the internal standard, with a response factor of 1. Examples of internal standards include, but are not limited to, 4-octanol.

[0070] GC-MS is used to identify and quantify the aroma components contained in the extract. In GC-O, the extract is diluted stepwise, and both the extract and its dilution are injected into a gas chromatograph (GC). Panelists then smell the aroma gas emitted from the GC and evaluate its odor. Details of GC-MS and GC-O are described in the examples below.

[0071] Traditionally, the most common method for analyzing aroma components involves placing the sample in a container and analyzing the aroma in the headspace within the container. Solid-phase microextraction (SPME) is a widely used method for analyzing headspace aromas. In the SPME method, volatile components are concentrated in an SPME fiber and then introduced into a GC or GC-MS. This method is suitable for analyzing highly volatile components, mainly top-note flavors, but it proved unsuitable for our purpose. This is likely because the sensory evaluation terms used to describe the characteristic odor of Bifidobacterium in the examples described later were aroma descriptions characteristic of the middle to base notes.

[0072] In sensory evaluation, predetermined aroma characteristics are assessed for both the sample and the control sample. For example, trained panelists taste both the sample and the control sample and evaluate the degree of intensity (or weakness) of the predetermined aroma characteristics. The predetermined aroma characteristics can be set arbitrarily. Examples of predetermined aroma characteristics include the following. One or more aroma characteristics may be evaluated. "Green smell (pickle smell, tomato smell)" "Stuffy odor (sweat odor, pungent odor)" "Butter cream vanilla scent" "Rancid smell (cheese)" "Citrus scent (lemon / citrus)" "Grain / bread smell" "Yeast, animal, culture medium, heating odor" "Vaccinium odor" "Sweet aroma, caramel scent" "Early tobacco smell" "Chemical smell" "Soy sauce smell" "A characteristic scent of bifidobacteria" "Deliciousness" Note that "yeast, animal, culture medium, and heating odor" refers to one or more of the following odors: yeast odor, animal odor, culture medium odor, and heating odor. The same applies to "sweet aroma / caramel odor," "butter / cream / vanilla odor," "grain / bread odor," and "earthy / tobacco odor." Regarding the "aroma characteristic of bifidobacteria," there was no well-established sensory evaluation index for the aroma characteristic of fermented milk containing bifidobacteria. However, the results of the examples described later revealed that there are aroma characteristics such as yeast, animal, culture medium, heating odor, acetic acid odor, sweet aroma, caramel odor, earthy, tobacco odor, and medicinal odor. These complex aromas were designated as a single evaluation term for the aroma characteristic of bifidobacteria, and the overall odor unique to fermented milk containing bifidobacteria, which is not present in fermented milk fermented with lactic acid bacteria alone, was evaluated.

[0073] According to the inventors' research, among the above aroma characteristics, the sweet aroma / caramel odor and the yeast / animal / culture medium / heat odor are characteristic of Bifidobacterium and have a significant impact on flavor. The sweet aroma / caramel odor positively contributes to the deliciousness of fermented milk containing Bifidobacterium. The yeast / animal / culture medium / heat odor negatively contributes to the deliciousness of fermented milk containing Bifidobacterium. Therefore, suitable aroma characteristics for sensory evaluation include sweet aromas, caramel odors, or yeast, animal, culture medium, and heating odors.

[0074] Correlation analysis, for example, uses the JMP14 software package (SAS Institute Inc., Cary, North Carolina, USA) to perform analysis of variance (ANOVA) and Tukey-Kramer's honestly significant difference test (TK HSD) on GC-MS and GC-O data. By performing correlation analysis, it is possible to select candidate components that may be involved in a given aroma characteristic.

[0075] The selected candidate components are added to the sample, and sensory evaluation is performed again. Sensory evaluation may be performed on multiple samples with different amounts of the candidate components added. If the predetermined aroma characteristics of the sample to which the candidate component has been added are stronger than the predetermined aroma characteristics of the sample before the candidate component was added, it is determined that the candidate component is an aroma component involved in the predetermined aroma characteristics. If the characteristics are not stronger, it is determined that the candidate component is not an aroma component involved in the predetermined aroma characteristics.

[0076] [Method for evaluating the flavor of fermented milk] In one embodiment of the evaluation method, the aroma components determined to be involved in a predetermined aroma characteristic by the above-described determination method are quantified in fermented milk containing Bifidobacterium, and the flavor of the fermented milk is evaluated based on the results of the quantification.

[0077] The aforementioned determination method allows for the identification of aroma components involved in any particular aroma characteristic of fermented milk containing Bifidobacterium. Since the strength (or weakness) of an aroma characteristic correlates with the content of the aroma components involved in that characteristic, the content of aroma characteristics that affect the flavor of fermented milk containing Bifidobacterium serves as an indicator for evaluating the flavor of that fermented milk.

[0078] According to the inventors' research, in fermented milk containing Bifidobacterium, in addition to acetic acid, aroma components that contribute to the characteristic aroma of Bifidobacterium include maltol, cyclotene, sotolon, vanillin, trans-4,5-epoxy-(E)-2-decenal, eugenol, δ-undecalactone, furaneol, butyric acid, (E)-2-nonenal, etc. At least one of these may be used as the target for quantitative analysis.

[0079] According to the inventors' research, the aroma characteristics that particularly affect the flavor of fermented milk containing Bifidobacterium are yeast, animal, culture medium, and heating odors, as well as sweet and caramel odors. Aroma components involved in yeast, animal, culture medium, and heating odors include, for example, trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal. Aroma components involved in sweet and caramel odors include maltol, cyclotene, sotolon, and vanillin. At least one of these may be used as the target for quantitative analysis. [Examples]

[0080] The present invention will be described in more detail below using examples. However, the present invention is not limited to these examples.

[0081] ● Sample preparation Using Bifidobacterium longum subspecies longum and Bifidobacterium breve, both HRBs; Bifidobacterium animalis subspecies lactis, the most commonly used non-HRB; and Lactobacillus delbryuki subspecies bulgaricus and Streptococcus thermophilus, the most commonly used lactic acid bacteria, six types of yogurt samples (BL, BB, SL, 3BL, 3BB, and 3BA) were prepared using the following procedure.

[0082] First, the following ingredients were mixed in the following proportions to make a total of 1 kg. • Low-heat skim milk powder (manufactured by Morinaga Milk Industry Co., Ltd.) 10% by mass • Yeast extract 21-NYP (Fuji Foods Industry Co., Ltd.) 0.025% by mass ·RO water 89.975% by mass RO water is water that has been filtered through a reverse osmosis membrane (RO membrane).

[0083] Next, the mixed raw materials were filled into 1L glass bottles (manufactured by Durand GmbH, Germany) and autoclaved at 90°C for 10 minutes. Afterward, the temperature was adjusted to 37°C, and the appropriate starter culture (marked with a circle in Table 1) was added. Fermentation was then allowed to reach pH 4.7. Immediately after reaching pH 4.7, the mixture was cooled to 5°C and refrigerated. All samples were prepared by repeating the process three times.

[0084] [Table 1]

[0085] ● Sensory evaluation of the sample • Evaluation method A sensory evaluation test of samples was conducted with 10 trained male and female panelists aged 20 to 40. In the sensory evaluation test, the panelists tasted the samples one day after the start of refrigeration without being able to see them, and evaluated each evaluation item shown in Table 2 on a 7-point scale (1: not perceived, 2: slightly perceived, 3: slightly perceived, 4: perceived, 5: strongly perceived, 6: quite strongly perceived, 7: very strongly perceived). Sensory evaluation was performed each time for three samples that were prepared. The arithmetic mean of the results from the 10 panelists was used as the evaluation result for each item.

[0086] ·result The evaluation results are shown in Table 2. As shown in Table 2, there were significant differences between the sample containing only lactic acid bacteria commonly used in yogurt (SL) and the samples containing bifidobacteria (BL, BB, 3BL, 3BB, 3BA) in evaluation terms such as acetic acid odor, sweet aroma, caramel aroma, and aroma characteristic of bifidobacteria. Furthermore, significant differences were observed between the sample containing only lactic acid bacteria commonly used in yogurt (SL) and the sample containing non-HRB (3BA) in evaluation terms such as yeast, animal, culture medium, and heating odor, and grain and bread odor. The Tukey-Kramer honestly significant difference test (TK HSD) using SPSS ver28 (IBM, USA) was used to test for statistical significance.

[0087] [Table 2]

[0088] ● Gas chromatography-mass spectrometry (GC-MS) pretreatment • Capture of headspace aromas using SPME As described above, 1 g of the prepared sample was weighed into a 20 mL vial and equilibrated at 38°C for 1 minute. Headspace aroma was then collected for 30 minutes using an SPME fiber (PDMS / DVB). Headspace aroma was collected for each of the three prepared samples. The SPME fibers from which headspace aroma was collected from each sample were subjected to GC-MS analysis.

[0089] • Extraction of endogenous aromas by solvent extraction As described above, 200 g of the prepared sample and 400 mL of deionized water were transferred to a 2 L conical flask, and 4-octanol (100 μg / mL methanol, 2.5 mL) was added as an internal standard. 600 mL of dichloromethane (hereinafter sometimes referred to as DCM) was slowly added, and the mixture was gently stirred at room temperature for 1 hour. The separated DCM phase was then dried on anhydrous sodium sulfate. The extract was concentrated in a rotary evaporator to obtain a final volume of extract of approximately 1 mL. Endogenous aroma extraction was performed on each of the three prepared samples. The extracts from each sample were subjected to GC-MS analysis.

[0090] ●GC-MS analysis GC-MS analysis was performed three times repeatedly using a 5975C mass-selective detector (Agilent Technologies) equipped with a fused silica capillary column DB-WAX (60 m × 0.25 mm, film thickness 0.25 μm; Agilent Technologies). The helium carrier gas flow rate was 1.6 mL / min. The oven temperature was programmed at 50°C for 2 minutes from the start, then increased to 220°C at 3°C / min and held at 220°C for 75 minutes. The injection port was maintained at 250°C. In SPME, the SPME fiber containing headspace aromas was heated to 250°C, and the entire volume of the desorbed aroma extract was injected through the injection port. In solvent extraction, the operation was performed in split mode (split rate 10:1), and 3 μL of the extract was injected through the injection port. Semi-quantification of volatile compounds was performed using relative peak area and internal standards. Volatile compounds were identified by comparing their mass spectra or odor characteristics with those of standard compounds, and by comparing their linear retention indices using n-alkanes with 6 to 32 carbon atoms with those of standard compounds. Standard compounds for identifying volatile compounds were purchased from Tokyo Chemical Industry Co., Ltd., Sigma-Aldrich Japan Co., Ltd., NARD Institute, LTD., Ambeed, Inc., or Soda Aromatic Co., Ltd. When standard compounds were unavailable, compounds were provisionally identified by matching them with mass spectral data using the Wiley Registry 11th Edition / NIST 2017 Mass Spectral Library (John Wiley and Sons, Inc. Hoboken, NJ, USA).

[0091] • Additional solvent extraction GC-MS testing GC-O analysis, described later, suggested that certain substances (sotolon, δ-undecalactone, eugenol, (E)-2-nonenal, vanillin) are highly likely to be involved in the characteristic aroma of Bifidobacterium. To semi-quantify these substances, large-scale extraction was performed to increase their concentration in the extract. 1600g of the sample was transferred to four 5L conical flasks, each containing 400g. 1200mL of deionized water and 4-octanol (1mg / mL methanol, 0.5mL) as an internal standard were added to each flask. 1600mL of DCM was slowly added to each flask, and the mixture was gently stirred at room temperature for 1 hour before the DCM was separated. The four resulting DCM phases were combined and dried on anhydrous sodium sulfate. The extract was concentrated using a rotary evaporator to obtain a final volume of approximately 1mL. This extract was analyzed by GC-MS and quantified in SIM mode. This additional test was conducted only on 3BL (samples fermented with *Streptococcus thermophilus*, *Lactobacillus bulgaricus*, and strain BB536).

[0092] ● Gas chromatography-olfactometry (GC-O) analysis As will be discussed later, solvent extraction was effective in extracting the characteristic aroma of Bifidobacterium. Therefore, GC-O analysis was performed only when solvent extraction was used as the pretreatment. GC-O analysis samples were prepared by mixing three batches of equal volumes of the extracts obtained by solvent extraction as described above, and then sequentially diluting them three-fold with DCM (from 1:3 to 1:81). GC-O analysis of the extracts and their DCM dilutions was repeated three times using CharmAnalysis® and an Agilent 6890GC (modified by DATU, Inc., Geneva, New York, USA) (Acree, TE, Barnard, J., and Cunningham, DG (1984). A procedure for the sensory analysis of gas chromatographic effluents. Food Chem., 14, 273-286). A DB-WAX fused silica capillary column (15 m × 0.32 mm, film thickness 0.25 μm; Agilent Technologies) was used, with a helium carrier gas flow rate of 3.2 mL / min. The oven temperature was programmed to start at 40°C, increased to 230°C at 6°C / min, and then held at 230°C for 30 minutes. The injection port and detector were maintained at 225°C, and 1 μL of sample was injected in splitless mode. The GC exhaust gases from each sample were identified by panelists with over five years of experience in humidified air. The number of volatile compounds and radioisotopes detected by these panelists were confirmed with panelists with over 20 years of experience.

[0093] The odor activity of volatile compounds obtained by GC-O dilution analysis was expressed using charm value (CMV) and aroma descriptors (Acree, TE, Barnard, J., and Cunningham, DG (1984). A procedure for the sensory analysis of gas chromatographic effluents. Food Chem., 14, 273-286). CMV is an index of odor intensity and is calculated by integrating the time duration and dilution using the following formula. In the following formula, F is the dilution factor, n is the number of dilutions, and di is the time duration.

[0094]

number

[0095] The relative intensity of volatile compounds is expressed as an odor spectral value (OSV). OSV is a normalized charm value modified by an approximate Stevens law exponent (0.5 example) for the odor component with the strongest detected odor intensity (Acree, TE (1997), GC / olfactometry, Anal. Chem. News Features, 69, 170A-175A). OSV is calculated using the following formula and is used to approximate the relative intensity of odors by considering the exponential properties of olfactory psychophysics. OSV = {(Charm Value) / (Charm Value) max} 1 / 2 × 100

[0096] ●Statistical analysis We performed analysis of variance (ANOVA) and the Tukey-Kramer honestly significant difference test (TK HSD) on GC-MS and GC-O data using the JMP14 software package (SAS Institute Inc., Cary, North Carolina, USA).

[0097] ·SPME GC-MS analysis results Tables 3-8 below show the results of SPME GC-MS analysis. 36 compounds were identified from 6 samples. The mean concentration and relative standard deviation (RSD) of each compound, as well as the results of multiple comparison studies (TK HSD) after one-way ANOVA for all samples, are shown in each table.

[0098] [Table 3]

[0099] [Table 4]

[0100] [Table 5]

[0101] [Table 6]

[0102] [Table 7]

[0103] [Table 8]

[0104] • Solvent extraction GC-MS analysis results Tables 9-14 below show the results of solvent extraction GC-MS analysis. 68 compounds were identified from 6 samples. The mean concentration and relative standard deviation (RSD) of each compound, as well as the results of multiple comparison studies (TK HSD) after one-way ANOVA for all samples, are shown in each table.

[0105] [Table 9A]

[0106] Table 9B

[0107] Table 10A

[0108] Table 10B

[0109] Table 11A

[0110] Table 11B

[0111] Table 12A

[0112] Table 12B

[0113] Table 13A

[0114] Table 13B

[0115] Table 14A

[0116] Table 14B

[0117] ·Analysis results In the table, "Concentration" is a semi-quantitative value calculated from the relative peak area of ​​each compound and the internal standard. "RSD" is the relative standard deviation of three measurements. "TK HSD" indicates the results of the Tukey-Kramer HSD test, where there was a significant difference (p<0.05) between samples showing different letters.

[0118] The volatile compounds detected by GC-MS as described above do not necessarily correspond to the odor intensity of the sample. Therefore, GC-O (CharmAnalysis®) was performed to clarify the characteristic aroma components of Bifidobacterium and their odor intensity. The analysis results are shown in Table 15 below. "Charm value" represents the average value of three measurements.

[0119] [Table 15]

[0120] SPME GC-MS Based on the aroma characteristics of the components that showed a significant difference, we concluded that no correlation was found with other sensory evaluation results. While methods for collecting headspace aromas, such as SPME, are suitable for analyzing highly volatile components, mainly top note flavors, and are the most frequently used method for analyzing yogurt due to their ease of use, they proved unsuitable for our purpose. This is likely because the sensory evaluation terms cited as characteristic of the Bifidobacterium odor were aroma descriptions characteristic of the middle to base notes.

[0121] • Solvent extraction GC-MS and GC-O Since the target aroma components could not be captured by SPME, we decided to perform solvent extraction as a pretreatment, followed by GC-MS and GC-O analysis. The column used in this study releases aroma components over approximately 30 minutes. Therefore, it is possible to estimate each characteristic aroma component of Bifidobacterium that was perceived in the sensory evaluation. GC-O analysis revealed several components with aroma characteristics that were perceived as different in sensory evaluation. The identified candidate components are maltol, sotolon, cyclotene, norfuraneol, 4-vinylguaiacol, trans-4,5-epoxy-(E)-2-decenal, octanoic acid, geraniol, furaneol, γ-dodecalactone, butyric acid, eugenol, δ-undecalactone, (E)-2-nonenal, and vanillin.

[0122] ●Drilling test To identify the selected substances as characteristic aroma components of Bifidobacterium, we decided to conduct an injection test, in which each substance is injected into a sample and then evaluated by sensory evaluation.

[0123] • Preparation of target samples Each substance was added to 95% ethanol to prepare a standard for injection testing. The concentration of each substance in the standard was adjusted so that when the standard was injected into the sample at a concentration of 0.01% by volume per 100% of the sample, the concentration of each substance in SL (a sample fermented only with Bulgarian bacteria and Thermophilus bacteria) was equal to the difference between the quantitative values ​​of each substance in 3BL (a sample fermented with Bulgarian bacteria, Thermophilus bacteria, and BB536).

[0124] • Testing The prepared sample was injected into SL at an additive concentration of 0.01% by volume. The same sensory evaluation as described above was performed on the samples injected with the sample.

[0125] The results of the inoculation test revealed that the components thought to be involved in the characteristic aroma of Bifidobacterium (selection criteria: components in which some sensory evaluation index increased or decreased by 0.2 points or more compared to the SL sample) are as shown in Table 16 below.

[0126] [Table 16]

[0127] Maltol, cyclotene, sotolon, vanillin, trans-4,5-epoxy-(E)-2-decenal, eugenol, δ-undecalactone, furaneol, butyric acid, and (E)-2-nonenal were identified as being involved in the characteristic aroma of Bifidobacterium. These results demonstrate the effectiveness of this analytical method, which combines sensory evaluation with GC-MS and GC-O analysis using solvent extraction to perform a detailed analysis of the middle to base notes in fermented milk. At the very least, it is considered an optimal method for analyzing the characteristic aroma components of Bifidobacterium in fermented milk containing Bifidobacterium.

[0128] A sweet aroma was identified as having a desirable effect on taste, and it was determined that maltol, cyclotene, sotolon, and vanillin are involved in this aroma. If the content of these aromatic components can be increased, it is believed that it will be possible to develop fermented milk containing bifidobacteria with a more desirable flavor.

[0129] Yeast odor, animal odor, culture medium odor, and heating odor were identified as aromas that undesirably affect the taste, and it was also identified that trans-4,5-epoxy-(E)-2-decenal, eugenol, and (E)-2-nonenal are involved in these aromas. If the content of these aroma components can be reduced, it is thought that it will be possible to develop fermented milk containing bifidobacteria with a more desirable flavor. Possible methods for reducing the content of these aroma components include, for example, genetic modification of bifidobacteria, adjustment of the raw materials for the milk preparation, adjustment of the sterilization conditions of the milk preparation, adjustment of the fermentation temperature, atmospheric composition during fermentation, and dissolved oxygen concentration, as well as the development of masking agents.

[0130] • Additional exam 1 To determine the optimal content of maltol, cyclotene, sotolon, and vanillin—aroma components responsible for sweet and caramel-like scents—similar injection tests were conducted, varying the injection amounts by 0.5 times, 2 times, 5 times, or 10 times the quantitative difference. Furthermore, similar tests were conducted with all four components (maltol, cyclotene, sotolon, and vanillin) injected. The results are shown in Tables 17-19 below.

[0131] [Table 17]

[0132] [Table 18]

[0133] [Table 19]

[0134] From the above results, it was revealed that the optimal dosage ranges are 8.85–12.95 μg / mL for maltol, 0.07–0.19 μg / mL for cyclotene, 0.01–0.26 μg / mL for sotolon, and 0.02–0.32 μg / mL for vanillin. Furthermore, it was found that when all four of these substances are present simultaneously, the caramel-like aroma characteristic of Bifidobacterium is more easily reproduced and has a favorable effect on taste when maltol is present at 8.85-12.95 μg / mL, cyclotene at 0.07-0.19 μg / mL, sotolon at 0.01-0.05 μg / mL, and vanillin at 0.02-0.06 μg / mL.

[0135] • Additional exam 2 To determine the optimal content of trans-4,5-epoxy-(E)-decenal, eugenol, and (E)-2-nonenal, which are aroma components involved in yeast, animal, culture medium, and heating odors, the same injection tests as above were conducted, but with the injection amount varied by 0.2 times, 0.5 times, or 0.8 times the quantitative difference. The results are shown in Table 20 below.

[0136] [Table 20]

[0137] From the above results, it became clear that the optimal ranges for trans-4,5-epoxy-(E)-decenal are 0.012 μg / mL or less, for eugenol 0.05 μg / mL or less, and for (E)-2-nonenal 0.005 μg / mL or less.

Claims

1. A fermentation composition that satisfies at least one of the following conditions: (1a), (1b), (1c), and (1d). (1a) The maltol content is 8.8 μg / 100g or more. (1b) The cyclotene content is 0.07 μg / 100g or more. (1c) The sotolon content is 0.013 μg / 100g or more. (1d) The vanillin content is 0.016 μg / 100g or more.

2. A fermentation composition that satisfies at least one of the following (1e), (1f), and (1g). (1e) The content of trans-4,5-epoxy-(E)-decenal is 0.013 μg / 100g or less. (1f) The eugenol content is 0.06 μg / 100g or less. The (1g) (E)-2-nonenal content is 0.006 μg / 100g or less.

3. The fermentation composition according to claim 1, further satisfying at least one of the following (1e), (1f), and (1g). (1e) The content of trans-4,5-epoxy-(E)-decenal is 0.013 μg / 100g or less. (1f) The eugenol content is 0.06 μg / 100g or less. The (1g) (E)-2-nonenal content is 0.006 μg / 100g or less.

4. A fermented milk composition according to any one of claims 1 to 3.

5. A fermentation composition according to any one of claims 1 to 3, comprising bacteria of the genus Bifidobacterium.

6. A method for producing a fermentation composition, comprising at least one of the following steps: (2a), (2b), (2c), and (2d). (2a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (2b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100 g or more. (2c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (2d) A step of adjusting the vanillin content in the fermentation composition to 0.016 μg / 100g or more.

7. A method for producing a fermentation composition, comprising at least one of the following steps: (2e), (2f), and (2g). (2e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100 g or less. (2f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (2g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less.

8. A method for producing a fermented composition according to claim 6, further comprising at least one of the following steps: (2e), (2f), and (2g). (2e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100 g or less. (2f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (2g) A step to adjust the content of (E)-2-nonenal in the fermentation composition to 0.006 μg / 100g or less.

9. A method for improving the flavor of a fermented composition, comprising at least one of the following steps: (3a), (3b), (3c), and (3d). (3a) A step of adjusting the maltol content in the fermentation composition to 8.8 μg / 100g or more. (3b) A step of adjusting the cyclotene content in the fermentation composition to 0.07 μg / 100 g or more. (3c) A step of adjusting the sotolon content in the fermentation composition to 0.013 μg / 100g or more. (3d) A step of adjusting the vanillin content in the fermentation composition to 0.016 μg / 100g or more.

10. A method for improving the flavor of a fermented composition, comprising at least one of the following steps: (3e), (3f), and (3g). (3e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100 g or less. (3f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (3g) A step to adjust the content of (E)-2-nonenal in the fermented composition to 0.006 μg / 100g or less.

11. A method for improving the flavor of a fermented composition according to claim 9, further comprising at least one of the following steps: (3e), (3f), and (3g). (3e) A step of adjusting the content of trans-4,5-epoxy-(E)-decenal in the fermentation composition to 0.013 μg / 100 g or less. (3f) A step to adjust the eugenol content in the fermentation composition to 0.06 μg / 100g or less. (3g) A step to adjust the content of (E)-2-nonenal in the fermented composition to 0.006 μg / 100g or less.

12. A sample of fermented milk containing Bifidobacterium bacteria and a control sample for comparing aroma characteristics were prepared. For each of the aforementioned sample and the aforementioned control sample, an extract containing aroma components was obtained by solvent extraction, and the aroma components in the extract were analyzed by gas chromatography-mass spectrometry and gas chromatography-olfactometry. Sensory evaluation was performed on each of the aforementioned sample and the aforementioned control sample. A correlation analysis is performed on the results of the above analysis and the results of the above sensory evaluation to select candidate components from among the aroma components that may be involved in the predetermined aroma characteristics of the sample. A method for determining the aroma components involved in the predetermined aroma characteristics by adding the candidate components to the sample and performing another sensory evaluation.

13. The determination method according to claim 12, wherein the predetermined aroma characteristic is a sweet aroma, a caramel smell, or a yeast, animal, culture medium, or heating smell.

14. An evaluation method for fermented milk containing Bifidobacterium bacteria, comprising quantifying the aroma components determined to be involved in the predetermined aroma characteristics by the determination method described in claim 12 or 13, and evaluating the flavor of the fermented milk based on the results of the quantification.

15. An evaluation method for fermented milk containing Bifidobacterium bacteria, comprising quantifying at least one of the following: trans-4,5-epoxy-(E)-decenal, eugenol, (E)-2-nonenal, maltol, cyclotene, sotolon, and vanillin, and evaluating the flavor of the fermented milk based on the results of the quantification.

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