Method for producing fermented plant-based milk liquid

Abstract

Provided is a method for producing a plant milk-fermented liquid having reduced plant smell. This method for producing a plant milk-fermented liquid comprises bringing, into contact with plant milk, a Lactobacillus strain including at least one selected from the group consisting of a Lactobacillus fermentum CP1299 strain, a Lactobacillus fermentum CP3024 strain, a Lactobacillus reuteri CP3017 strain, and a Lactobacillus reuteri CP3019 strain.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a fermented vegetable milk liquor. [Background technology]

[0002] Plant-based milk is made by breaking down and liquefying plant ingredients such as rice, wheat, and soybeans (see, for example, J. Food Sci. Technol (September 2016) 53(9):3408-3423). An example of plant-based milk is rice milk, which is made by saccharifying rice with enzymes, koji, etc., adding vegetable oil, etc., and seasoning it to form a milky consistency.

[0003] Generally, plant-based milk contains aldehydes as aroma components. Aldehydes can be desirable aroma components in beverages with a plant flavor. However, aldehydes are known to cause an unpleasant, undesirable plant odor in beverages with a fermented milk-like flavor. Methods for reducing the aldehydes contained in plant-based milk include, for example, the following methods.

[0004] For example, Japanese Patent Application Laid-Open No. 9-205999 describes a method for reducing medium-chain aldehydes to alcohols by contacting a food containing medium-chain aldehydes with the lactic acid bacteria Leuconostoc mesenteroides or Lactobacillus brevis without causing lactic acid bacteria growth. Japanese Patent Application Laid-Open No. 2020-17 also describes a lactic acid-fermented beverage obtained by lactic acid fermentation of rice-based plant milk using the lactic acid bacteria Lactobacillus sakei strain No. 7 or Lactobacillus sakei strain No. 16, and describes that the diacetyl and hexanal contents were reduced. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a method for producing a plant-based fermented milk liquid with a reduced plant odor. [Means for solving the problem]

[0006] The present invention provides a method for producing a plant-based milk fermented liquid, which comprises contacting plant-based milk with a lactic acid bacteria strain including at least one selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain, and Lactobacillus reuteri CP3019 strain.

[0007] In one embodiment, the plant-based milk in the production method may be derived from grains. In one embodiment, the plant-based milk in the production method may be derived from rice. In one embodiment, the plant-based milk in the production method may be saccharified plant-based milk. In one embodiment, contacting the lactic acid bacteria strain with the plant-based milk in the production method may involve fermentation of the plant-based milk by the lactic acid bacteria strain.

[0008] The present invention also provides a method for reducing aldehydes in plant-based milk, which comprises contacting plant-based milk containing aldehydes with a lactic acid bacteria strain including at least one selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain and Lactobacillus reuteri CP3019 strain.

[0009] In one embodiment, the plant-based milk in the reduction method may be derived from grains. In one embodiment, the plant-based milk in the reduction method may be derived from rice. In one embodiment, the plant-based milk in the reduction method may be saccharified plant-based milk. In one embodiment, the contact between the lactic acid bacteria strain and the plant-based milk in the reduction method may involve fermentation of the plant-based milk by the lactic acid bacteria strain.

[0010] Furthermore, the present invention provides the Lactobacillus fermentum CP3024 strain, accession number: NITE BP-3428, the Lactobacillus reuteri CP3017 strain, accession number: NITE BP-3426, and the Lactobacillus reuteri CP3019 strain, accession number: NITE BP-3427. [Effects of the Invention]

[0011] According to the present invention, a method for producing a plant-based fermented milk liquid with a reduced plant odor can be provided. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the ability of lactic acid bacteria to reduce low concentrations of acetaldehyde. [Figure 2] 1 is a graph showing the ability of lactic acid bacteria to reduce high concentrations of acetaldehyde. [Figure 3] 1 is a graph showing the ability of lactic acid bacteria to reduce low concentrations of hexanal. [Figure 4] 1 is a graph showing the ability of lactic acid bacteria to reduce high concentrations of hexanal. [Figure 5] 1 is a graph showing the ability of lactic acid bacteria to reduce low concentrations of benzaldehyde. [Figure 6] 1 is a graph showing the ability of lactic acid bacteria to reduce high concentrations of benzaldehyde. [Figure 7] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in plant-based milk derived from brown rice. [Figure 8] 1 is a graph showing the ability of lactic acid bacteria to reduce 2,4-decadienal contained in plant-based milk derived from brown rice. [Figure 9] 1 is a graph showing the ability of lactic acid bacteria to reduce 2-methylbutanal contained in plant-based milk derived from brown rice. [Figure 10] 1 is a graph showing the ability of lactic acid bacteria to reduce 2-methylpropanal contained in plant-based milk derived from brown rice. [Figure 11]1 is a graph showing the ability of lactic acid bacteria to reduce 3-methylbutanal contained in plant-based milk derived from brown rice. [Figure 12] 1 is a graph showing the ability of lactic acid bacteria to reduce hexanal contained in plant-based milk derived from brown rice. [Figure 13] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in plant-based milk derived from white rice. [Figure 14] 1 is a graph showing the ability of lactic acid bacteria to reduce hexanal contained in plant-based milk derived from white rice. [Figure 15] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in barley-derived plant-based milk. [Figure 16] 1 is a graph showing the reduction ability of lactic acid bacteria for 2,4-decadienal contained in barley-derived plant-based milk. DETAILED DESCRIPTION OF THE INVENTION

[0013] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below are merely examples of methods for producing plant-based milk fermented liquid in order to embody the technical concept of the present invention, and the present invention is not limited to the methods for producing plant-based milk fermented liquid described below.

[0014] Method for producing fermented plant-based milk liquid A method for producing a plant-based milk fermented liquid includes contacting plant-based milk with a lactic acid bacteria strain including at least one selected from the group consisting of Lactobacillus fermentum CP1299, Lactobacillus fermentum CP3024, Lactobacillus reuteri CP3017, and Lactobacillus reuteri CP3019.

[0015] By contacting a specific lactic acid bacteria strain with plant-based milk, the content of aldehydes contained in the plant-based milk is effectively reduced, the plant odor derived from the aldehydes is reduced, and a plant-based milk fermented liquid with an excellent yogurt-like flavor is produced. The produced plant-based milk fermented liquid has an excellent balance of sweetness and sourness and an improved mellow fermented milk flavor. This can be attributed to, for example, the specific lactic acid bacteria strain's excellent activity in reducing the aldehydes contained in plant-based milk.

[0016] In the method for producing a plant-based milk fermented liquid, the content of aldehydes contained in the plant-based milk is reduced by contacting specific lactic acid bacteria with the plant-based milk. That is, the method for producing a plant-based milk fermented liquid may be a method for reducing aldehydes in plant-based milk.

[0017] Lactic acid bacteria strains The method for producing a plant-based fermented milk liquor uses a specific lactic acid bacteria strain. The specific lactic acid bacteria strain includes at least one strain selected from the group consisting of Lactobacillus fermentum CP1299, Lactobacillus fermentum CP3024, Lactobacillus reuteri CP3017, and Lactobacillus reuteri CP3019. The Lactobacillus fermentum CP1299 strain is a type of lactic acid bacteria belonging to the Lactobacillus fermentum genus and was internationally deposited on January 18, 2013, with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), an international depository institution under the provisions of the Budapest Treaty, under the accession number NITE BP-1512. The Lactobacillus fermentum CP3024 strain is a species of lactic acid bacteria belonging to the Lactobacillus fermentum genus and was internationally deposited on March 1, 2021, under accession number NITE BP-03428. The Lactobacillus reuteri CP3017 strain is a species of lactic acid bacteria belonging to the Lactobacillus reuteri genus and was internationally deposited on March 1, 2021, under accession number NITE BP-03426. The Lactobacillus reuteri CP3019 strain is a species of lactic acid bacteria belonging to the Lactobacillus reuteri genus and was internationally deposited on March 1, 2021, under accession number NITE BP-03427.

[0018] In the method for producing a plant-based milk fermented liquid, in addition to the specific lactic acid bacteria, other lactic acid bacteria, bifidobacteria, etc. may be used in combination. or bifidobacteriaExamples of such strains include Lactobacillus fermentum other than the CP1299 and CP3024 strains (e.g., JCM1173 strain), Lactobacillus reuteri other than the CP3017 and CP3019 strains (e.g., JCM1112 strain), Lactobacillus gasseri, Lactobacillus oris, Lactobacillus mucosae, Lactobacillus fructivorans, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus spp. Lactobacillus species such as Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus jenseni, Lactobacillus johnsonii, Lactobacillus kefilgranum, Lactobacillus mali, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus tolerans; Bifidobacterium ·Adolescentis, Bifidobacterium · Angra Atam, Bifidobacterium Animalis, Bifidobacterium bifidum, Bifidobacterium Blebs, Bifidobacterium · Cateranatum, Bifidobacterium Dentium, Bifidobacterium Gallicum, Bifidobacterium Infantis, Bifidobacterium Longum, Bifidobacterium Pseudocatenulatum, Bifidobacterium Pseudolongum, Bifidobacterium ·Switzerland, Bifidobacterium Thermophilum etc. Bifidobacterium The genus Streptococcus includes Streptococcus thermophilus, Streptococcus lactis, Streptococcus cremoris, Streptococcus faecalis, and Streptococcus faecium; the genus Leuconostoc includes Leuconostoc mesenteroides, Leuconostoc dextrinicum, Leuconostoc cremoris, and Leuconostoc oenos; and the genus Pediococcus includes Pediococcus cerevisiae, Pediococcus acidilactici, and Pediococcus halophilus. do.

[0019] When the lactic acid bacteria used in the method for producing a plant-based fermented milk liquor contain at least one species of lactic acid bacteria and bifidobacteria other than specific lactic acid bacteria, the content thereof may be 50% or less, preferably 10% or less, or less than 5%, of the total lactic acid bacteria and bifidobacteria. The content of lactic acid bacteria and bifidobacteria may be determined from the number of bacteria of at least one species of lactic acid bacteria and bifidobacteria used, or when a preculture solution is used as at least one species of lactic acid bacteria and bifidobacteria, it may be determined based on the volume of the preculture solution.

[0020] Plant-based milk Plant-based milk may be prepared by liquefying plant materials by physical crushing or saccharification with enzymes or koji, etc., or by other methods. Plant-based milk may be prepared by liquefying plant materials using known methods, or may be appropriately selected from commercially available products. Examples of plant materials include grains such as rice, barley, wheat, oats, rye, soybeans, and peanuts; nuts and seeds such as almonds, coconuts, cashews, macadamia nuts, and walnuts; and vegetables such as carrots, potatoes, sweet potatoes, cassava, and tomatoes. Among these, the plant material preferably contains grains because of its high sugar content, and more preferably contains at least one selected from the group consisting of rice and barley, and even more preferably contains at least rice or barley.

[0021] When rice is used as the plant raw material, it may be at least one type selected from the group consisting of unhulled rice, brown rice (including germinated brown rice), polished rice, red bran, medium bran, white bran, and upper-grade white bran. Polished rice includes, depending on the degree of polishing, 30-minute polished rice, 50-minute polished rice, 70-minute polished rice, and white rice, and the germ may remain. The rice used as the plant raw material is preferably at least one type selected from the group consisting of brown rice, polished rice, medium bran, and upper-grade white bran, more preferably at least one type selected from the group consisting of brown rice, polished rice, and upper-grade white bran, and even more preferably brown rice or white rice. The rice used as the plant raw material may be ground to a predetermined particle size using a known method, or may be in its intact state without being ground. It may also be saccharified by microbial or enzymatic treatment. Rice is classified into non-glutinous rice, glutinous rice, and other types depending on the starch it contains, and any of these may be used. When the plant raw material contains rice, it may also contain plant materials other than rice. In addition to rice, the plant material may contain other grains such as barley, wheat, oats, rye, soybeans, and peanuts, as well as nuts and seeds such as almonds, coconuts, cashews, macadamia nuts, and walnuts. The content of other grains contained in the rice-containing plant material may be, for example, 30% by mass or less, 20% by mass or less, or 10% by mass or less. The lower limit of the content of other grains contained in the rice-containing plant material may be, for example, 0.1% by mass or more, or 1% by mass or more.

[0022] When barley is used as the plant material, it may be at least one type selected from the group consisting of two-row barley, four-row barley, six-row barley, naked barley, and wild barley, or at least one type selected from the group consisting of two-row barley and six-row barley. Barley as the plant material may be ground to a predetermined particle size by a known method, or may be in its original state without being ground. It may also be saccharified by microbial or enzymatic treatment. When the plant material contains barley, it may further contain plant materials other than barley. The plant material may contain, for example, other grains such as rice, wheat, oats, rye, soybeans, and peanuts, as well as nuts and seeds such as almonds, coconuts, cashews, macadamia nuts, and walnuts, in addition to barley. The content of other grains, etc. contained in the plant material containing barley may be, for example, 30% by mass or less, 20% by mass or less, or 10% by mass or less. The lower limit of the content of other grains, etc. contained in the plant material containing barley may be, for example, 0.1% by mass or more, or 1% by mass or more.

[0023] Plant-based milk may be prepared, for example, by saccharification of starch contained in plant materials (preferably grains) using an appropriate enzyme to hydrolyze it. That is, plant-based milk may be saccharified plant-based milk. Plant-based milk made from rice is preferred because it contains a large amount of monosaccharides, and plant-based milk made from brown rice or polished rice is more preferred. As an enzyme for the saccharification, for example, α-amylase (EC3.2.1.1) can be used. The amount of enzyme added can be, for example, 0.01% to 1% based on the mass of the plant material. The conditions for the hydrolysis reaction can be appropriately selected depending on the enzyme used, the plant material, and the like. The reaction temperature can be, for example, 40°C to 100°C, and preferably 50°C to 70°C. The reaction time can be, for example, 1 hour to 24 hours, and preferably 2 hours to 12 hours.

[0024] In preparing plant-based milk by saccharification, the milk can be prepared by hydrolyzing the plant material with α-amylase and then saccharifying it with glucoamylase (EC 3.2.1.3) or the like. Alternatively, the plant-based milk may be obtained by saccharification treatment in which koji or the like is applied to plant raw materials.

[0025] Plant-based milk prepared by saccharification contains various sugars, such as monosaccharides such as glucose, disaccharides such as maltose, trisaccharides such as maltotriose, and tetrasaccharides or higher. The sugar composition of plant-based milk is preferably rich in glucose, since glucose is decomposed by lactic acid bacteria in the fermentation process described below and ultimately serves as the main source of sweetness. The content of monosaccharides such as glucose may be, for example, 30% by mass or more, preferably 50% by mass or more, and more preferably 70% by mass or more, of the total sugars contained in the plant-based milk.

[0026] The plant-based milk may be, for example, a milk-like emulsion. Alternatively, the plant-based milk may be prepared by pulverizing and liquefying a plant material and then seasoning it with vegetable oil, animal oil, salt, sugar, amino acids, flavorings, thickeners, thickeners, emulsifiers, pH adjusters, etc.

[0027] Aldehydes Plant-based milk contains aldehydes derived from plant materials. The aldehydes may have, for example, a plant-like flavor. Examples of aldehydes include aliphatic aldehydes such as short-chain aliphatic aldehydes, medium-chain aliphatic aldehydes, and long-chain aliphatic aldehydes, as well as aromatic aldehydes. Short-chain aliphatic aldehydes have 5 or fewer carbon atoms, medium-chain aliphatic aldehydes have 6 to 12 carbon atoms, and long-chain aliphatic aldehydes have 13 or more carbon atoms. Specific examples of aliphatic aldehydes include acetaldehyde, malondialdehyde, butanal, 2-methylpropanal, pentanal, 2-methylbutanal, 3-methylbutanal, hexanal, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, 2,4-nonadienal, heptanal, octanal, nonanal, decanal, 2,4-decadienal, tetradecanal, etc. Specific examples of aromatic aldehydes include benzaldehyde, anisaldehyde, cuminaldehyde, etc.

[0028] The aldehydes whose content is reduced by the method for producing a plant-based milk fermented liquid may be an aliphatic aldehyde or an aromatic aldehyde, or may be at least one selected from the group consisting of short-chain aliphatic aldehydes, medium-chain aliphatic aldehydes, and aromatic aldehydes, or may be at least one selected from the group consisting of acetaldehyde, malondialdehyde, butanal, 2-methylpropanal, pentanal, 2-methylbutanal, 3-methylbutanal, hexanal, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, 2,4-nonadienal, heptanal, octanal, nonanal, decanal, 2,4-decadienal, and benzaldehyde.

[0029] The method for producing a plant-based milk fermented liquor includes a step of contacting specific lactic acid bacteria with plant-based milk. The step of contacting specific lactic acid bacteria with plant-based milk may be a fermentation step in which specific lactic acid bacteria are added to plant-based milk and lactic acid fermentation is carried out. Specifically, the desired plant-based milk fermented liquor can be obtained by maintaining the plant-based milk at a predetermined fermentation temperature, adding a predetermined amount of lactic acid bacteria culture solution, and carrying out lactic acid fermentation for a predetermined fermentation time.

[0030] The lactic acid bacteria used to contact the plant-based milk may be a pre-culture solution previously cultured in an appropriate medium, a frozen product obtained by mixing the pre-culture solution with a cryoprotectant and freezing it, or a powdered pre-culture solution obtained by freeze-drying it. The medium used to prepare the pre-culture solution may be appropriately selected from media commonly used for culturing lactic acid bacteria. Examples of the medium include MRS medium (manufactured by BD Japan). Culture conditions may be, for example, static or anaerobic conditions at 30°C to 40°C for 12 to 32 hours.

[0031] The amount of lactic acid bacteria preculture solution added to plant-based milk may be, for example, 0.1% by volume or more and 30% by volume or less, preferably 0.5% by volume or more and 20% by volume or less, and more preferably 1% by volume or more and 10% by volume or less, relative to the liquid volume of the plant-based milk.

[0032] The fermentation temperature and fermentation time may be appropriately selected depending on the culture conditions, the lactic acid bacteria strain to be added, etc. The fermentation temperature may be, for example, from 10° C. to 45° C., preferably from 20° C. to 40° C., and more preferably from 30° C. to 40° C. The fermentation time may be, for example, from 1 hour to 72 hours, preferably from 6 hours to 48 hours, and more preferably from 12 hours to 32 hours.

[0033] In the method for producing a plant-based fermented milk liquor, the aldehyde content in the plant-based milk is reduced by the fermentation process. The residual rate (%) obtained by dividing the aldehyde content in the resulting plant-based fermented milk liquor by the aldehyde content in plant-based milk obtained by the same process except without contact with lactic acid bacteria is, for example, 85% or less, preferably 70% or less, more preferably 60% or less, and even more preferably 50% or less, for aromatic aldehydes. The lower limit of the residual rate (%) is not particularly limited, but may be, for example, 1% or more, preferably 5% or more. For aliphatic aldehydes, the residual rate (%) of aldehydes is, for example, 55% or less, preferably 45% or less, more preferably 40% or less, even more preferably 35% or less, and even more preferably 30% or less, or 20% or less. The lower limit of the residual rate (%) is not particularly limited, but may be, for example, 0% or more, preferably 1% or more, or 2% or more.

[0034] The lactic acid bacteria contained in the plant-based milk fermented liquor obtained in the fermentation step may be left viable or may be killed by high-temperature treatment, etc. The high-temperature treatment may be carried out, for example, by treating the obtained plant-based milk fermented liquor at a high temperature of 63°C or higher for a predetermined period of time.

[0035] The plant-based milk fermented liquor obtained by this production method may be used as a lactic acid fermented beverage as is, or may be subjected to a predetermined post-processing step to produce a lactic acid fermented beverage or lactic acid fermented food in a desired form. Examples of post-processing steps include a concentration step to form a syrup, a step of adding desired additives (flavorings, etc.), a step of mixing with a carbonated beverage, a fruit juice beverage, an alcoholic beverage, etc., and a food processing step.

[0036] Examples of additives used in the post-treatment process include sugar alcohols such as sorbitol, erythritol, maltitol, and xylitol; high-intensity sweeteners such as aspartame, stevioside, sucralose, and acesulfame K; organic acids such as citric acid, tartaric acid, malic acid, succinic acid, and lactic acid; vitamins such as L-ascorbic acid, dl-α-tocopherol, B vitamins, nicotinamide, and calcium pantothenate; surfactants such as glycerin fatty acid esters, polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, and propylene glycol fatty acid esters; thickeners such as gum arabic, carrageenan, pectin, and agar; stabilizers such as casein and gelatin; amino acids, minerals such as calcium salts; additives such as sodium ascorbate, sodium erythorbate, glycerin, and propylene glycol, colorants, flavorings, and preservatives.

[0037] Examples of lactic acid fermented beverages obtained by the post-treatment step include carbonated beverages, fruit juice beverages, alcoholic beverages, syrups, fruit-flavored beverages, etc. The lactic acid fermented beverage can also be mixed with other beverages, such as soy milk, to the extent that the effects of the present invention are not impaired. Examples of lactic acid fermented foods obtained by the post-treatment step include frozen desserts such as jelly, yogurt, pudding, and ice cream, candy, soft candy, gum, and jam. [Example]

[0038] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0039] Reference example 1 The lactic acid bacteria strains listed in Table 1 were prepared. All strains were type strains. Lactic acid bacteria were cultured for 16 hours at 37°C under anaerobic conditions in MRS medium (BD Japan) supplemented with 0.05% cysteine ​​hydrochloride by volume to prepare a preculture solution for each lactic acid bacterium. Next, 0.05% cysteine ​​hydrochloride by volume and 0.05% acetaldehyde were added to MRS medium (BD Japan), followed by the addition of 5% of the lactic acid bacteria preculture solution. Cultures were then centrifuged to remove the bacterial cells, yielding culture supernatants. The acetaldehyde concentration in the culture supernatants was quantified using F-kit Acetaldehyde (JK International). The residual acetaldehyde rate (%) was calculated by dividing the acetaldehyde concentration in each culture supernatant by the average acetaldehyde concentration in uninoculated culture media and multiplying the result by 100. The number of data points (n) for each sample was 3. The results are shown in Table 1 and Figure 1.

[0040] [Table 1]

[0041] It is clear that the ability to reduce acetaldehyde differs depending on the species of lactic acid bacteria.

[0042] Reference example 2 The acetaldehyde reduction ability of each lactic acid bacterium was evaluated in the same manner as in Reference Example 1, except that the strains shown in Table 2 were used and the concentration of acetaldehyde added was 0.2% by volume. The results are shown in Table 2 and Figure 2. Statistical analysis was performed using Student's t-test, comparing the residual rate with that of Lactobacillus brevis T. A P<0.05 level was considered to indicate a significant difference. The number of data points per sample (n) was 3.

[0043] [Table 2]

[0044] It was confirmed that under high-concentration conditions, certain bacterial species exhibited a higher acetaldehyde-reducing ability than Lactobacillus brevis T. It was confirmed that Lactobacillus fructovorans T exhibited a high acetaldehyde-reducing ability similar to certain bacterial species under low-concentration conditions, but under high-concentration conditions, its ability was equivalent to that of Lactobacillus brevis T. This shows that the acetaldehyde-reducing ability differs depending on the bacterial species and strain.

[0045] Reference example 3 The lactic acid bacteria strains listed in Table 3 were prepared. All strains prepared were type strains. Lactic acid bacteria were cultured for 16 hours at 37°C under anaerobic conditions using MRS medium (manufactured by BD Japan) supplemented with 0.05% cysteine ​​hydrochloride by mass to prepare a preculture solution for each lactic acid bacterium. Next, 0.05% cysteine ​​hydrochloride by mass and 0.05% hexanal by volume were added to MRS medium (manufactured by BD Japan), and then 5% of the lactic acid bacteria preculture solution was added. The culture was then centrifuged to remove the bacterial cells, yielding a culture supernatant. The hexanal concentration in the culture supernatant was quantified using a modified LC-MS / MS method described in Drug Test. Analysis, 8, 458-464 (2016). Details of the quantification method are described below. The residual rate (%) of hexanal was calculated by dividing the hexanal concentration in each culture supernatant by the average hexanal concentration in the uninoculated medium and multiplying the result by 100. The number of data points per sample (n number) was 3. The results are shown in Table 3 and Figure 3.

[0046] [Table 3]

[0047] It was confirmed that the ability to reduce hexanal differs depending on the species of lactic acid bacteria.

[0048] Reference example 4 The hexanal reduction ability of each lactic acid bacterium was evaluated in the same manner as in Reference Example 3, except that the strains shown in Table 4 were used and the concentration of hexanal added was 0.2% by volume. The results are shown in Table 4 and Figure 4. Statistical analysis was performed using Student's t-test, and the results were compared with the residual rate in the case of Lactobacillus brevis T. A P<0.05 level was considered to indicate a significant difference. The number of data points per sample (n) was 3.

[0049] [Table 4]

[0050] It was confirmed that under high-concentration conditions, certain bacterial species exhibited a higher hexanal reduction ability than Lactobacillus brevis T. Under low-concentration conditions, Lactobacillus fructovorans T exhibited a high hexanal reduction ability similar to certain bacterial species, but under high-concentration conditions, its hexanal reduction ability was equivalent to that of Lactobacillus brevis T. This indicates that hexanal reduction ability differs depending on the bacterial species and strain.

[0051] Reference example 5 The lactic acid bacteria strains listed in Table 5 were prepared. All strains prepared were type strains. Lactic acid bacteria were cultured for 16 hours at 37°C under anaerobic conditions using MRS medium (manufactured by BD Japan) supplemented with 0.05% cysteine ​​hydrochloride by mass to prepare a preculture solution for each lactic acid bacterium. Next, 0.05% cysteine ​​hydrochloride by mass and 0.05% benzaldehyde by volume were added to MRS medium (manufactured by BD Japan), and then 5% of the lactic acid bacteria preculture solution was added. The culture was then centrifuged to remove the bacterial cells, yielding a culture supernatant. The benzaldehyde concentration in the culture supernatant was quantified using a modified LC-MS / MS method described in Drug Test. Analysis, 8, 458-464 (2016). Details of the quantification method are described below. The residual rate (%) of benzaldehyde was calculated by dividing the benzaldehyde concentration in each culture supernatant by the average benzaldehyde concentration in the uninoculated medium and multiplying the result by 100. The number of data points per sample (n) was 3. The results are shown in Table 5 and Figure 5.

[0052] [Table 5]

[0053] It was confirmed that the ability to reduce benzaldehyde differed depending on the species of lactic acid bacteria.

[0054] Reference example 6 The benzaldehyde reduction ability of each lactic acid bacterium was evaluated in the same manner as in Reference Example 5, except that the strains shown in Table 6 were used and the concentration of added benzaldehyde was 0.2% by volume. The results are shown in Table 6 and Figure 6. Statistical analysis was performed using Student's t-test, and the results were compared with the residual rate in the case of Lactobacillus brevis T. A P<0.05 level was considered to indicate a significant difference. The number of data points per sample (n) was 3.

[0055] [Table 6]

[0056] It was confirmed that under high-concentration conditions, certain bacterial species exhibited a higher benzaldehyde reduction ability than Lactobacillus brevis T. Under low-concentration conditions, Lactobacillus fructovorans T exhibited a high benzaldehyde reduction ability similar to certain bacterial species, but under high-concentration conditions, its benzaldehyde reduction ability was equivalent to that of Lactobacillus brevis T. This indicates that benzaldehyde reduction ability differs depending on the bacterial species and strain.

[0057] Example 1 The lactic acid bacteria strains shown in Tables 7 to 12 were prepared. Each lactic acid bacteria was cultured for 16 hours under anaerobic conditions at 37°C using MRS medium (manufactured by BD Japan) supplemented with 0.05% by mass of cysteine ​​hydrochloride, to prepare a preculture solution for each lactic acid bacteria strain. Rich rice milk (manufactured by Kikkoman Corporation, raw material: processed brown rice) was diluted 1.67-fold with pure water and sterilized at 95°C for 15 minutes to prepare a brown rice rice milk medium. Five volume percent of the lactic acid bacteria preculture solution was added to the brown rice rice milk medium, and the mixture was cultured stationary at 37°C for 16 hours to obtain a culture solution as a plant-based milk fermentation liquid. The concentrations of benzaldehyde, 2,4-decadienal, 2-methylbutanal, 2-methylpropanal, 3-methylbutanal, and hexanal contained in the resulting culture medium were quantified using a modified LC-MS / MS method described in Drug Test. Analysis, 8, 458-464 (2016). Details of the quantification method are described below. The residual rate (%) of each aldehyde was calculated by dividing the concentration of each aldehyde in each culture medium by the average concentration of each aldehyde in the uninoculated medium and multiplying the result by 100. The results are shown in Tables 7 to 12 and Figures 7 to 12. Statistical analysis was performed using Student's t-test, comparing the residual rates with those of Lactobacillus gasseri T. The number of data points per sample (n) was 3. A P<0.05 level was considered significant.

[0058] [Table 7]

[0059] [Table 8]

[0060] [Table 9]

[0061] [Table 10]

[0062] [Table 11]

[0063] [Table 12]

[0064] It was confirmed that the levels of various aldehydes were significantly reduced in certain strains compared to the control strain, Lactobacillus gasseri T.

[0065] Example 2 The lactic acid bacteria strains shown in Tables 13 and 14 were prepared. Each lactic acid bacteria strain was cultured for 16 hours under anaerobic conditions at 37°C using MRS medium (manufactured by BD Japan) supplemented with 0.05% by mass of cysteine ​​hydrochloride to prepare a preculture solution for each lactic acid bacteria strain. Commercially available rice flour for confectionery (Tomizawa Shoten Co., Ltd., raw material: non-glutinous rice) was mixed with purified water at a weight ratio of 3:7, and 0.03% by volume of amylase (Novozymes, BAN480L) was added. The mixture was then reacted at 60°C for 6 hours, stirred, and sterilized at 95°C for 15 minutes to prepare a white rice rice milk medium. Five percent by volume of the lactic acid bacteria preculture solution was added to the white rice rice milk medium and cultured at 37°C for 16 hours to obtain a plant-based milk fermentation liquid. The concentrations of benzaldehyde and hexanal contained in the resulting culture medium were quantified using a modified LC-MS / MS method described in Drug Test. Analysis, 8, 458-464 (2016). Details of the quantification method are described below. The residual rate (%) of each aldehyde was calculated by dividing the concentration of each aldehyde in each culture medium by the average concentration of each aldehyde in the uninoculated medium and multiplying the result by 100. The results are shown in Tables 13 and 14 and Figures 13 and 14. Statistical analysis was performed using Student's t-test, comparing the residual rate with that of Lactobacillus gasseri T. The number of data points per sample (n) was 3. A P<0.05 level was considered significant.

[0066] [Table 13]

[0067] [Table 14]

[0068] It was confirmed that the levels of various aldehydes were significantly reduced in certain strains compared to the control strain, Lactobacillus gasseri T.

[0069] Example 3 The strains of lactic acid bacteria shown in Tables 15 and 16 were prepared. Each lactic acid bacterium was cultured for 16 hours under anaerobic conditions at 37°C using MRS medium (manufactured by BD Japan) supplemented with 0.05% by mass of cysteine ​​hydrochloride, to prepare a preculture solution for each lactic acid bacterium strain. Commercially available barley flour (Omugi Club Co., Ltd., raw material: whole wheat flour) and purified water were mixed in a weight ratio of 2:8, and 0.02% by volume of amylase (Novozymes, BAN480L) was added. The mixture was then reacted at 60°C for 6 hours with stirring, and then sterilized at 95°C for 15 minutes to prepare a barley milk medium. Five percent by volume of the preculture solution of lactic acid bacteria was added to the barley milk medium, and the mixture was cultured stationary at 37°C for 16 hours to obtain a plant-based milk fermentation liquid. The concentrations of benzaldehyde and 2,4-decadienal contained in the resulting culture medium were quantified using a modified LC-MS / MS method described in Drug Test. Analysis, 8, 458-464 (2016). Details of the quantification method are described below. The residual rate (%) of each aldehyde was calculated by dividing the concentration of each aldehyde in each culture medium by the average aldehyde concentration in the uninoculated medium and multiplying the result by 100. The results are shown in Tables 15 and 16 and Figures 15 and 16. Statistical analysis was performed using Student's t-test, comparing the residual rate with that of Lactobacillus gasseri T. The number of data points per sample (n) was 3. A P<0.05 level was considered significant.

[0070] [Table 15]

[0071] [Table 16]

[0072] It was confirmed that the levels of various aldehydes were significantly reduced in certain strains compared to the control strain, Lactobacillus gasseri T.

[0073] Quantitative method for aldehydes The internal standard was hexanal d-12 (Sigma-Aldrich), which was dissolved in 50% ethanol at a concentration of 25 ng / μl to prepare an internal standard solution. 40 μl of this internal standard solution was mixed with 80 μl of a solution of dibutylhydroxytoluene (Tokyo Chemical Industry Co., Ltd.) dissolved in 100% ethanol at a concentration of 1 mg / ml, 440 μl of 100% ethanol, and 440 μl of the culture supernatant or culture solution for which aldehydes were to be quantified. The mixture was centrifuged at 3700 g for 10 minutes, and 750 μl of the supernatant was collected. 325 μl of this supernatant was mixed with 325 μl of a solution of 2,4-dinitrophenylhydrazine hydrochloride (Tokyo Chemical Industry Co., Ltd.) dissolved at a concentration of 0.05 mol / L in a mixed solvent (acetonitrile:acetic acid:pure water, volume ratio = 80:10:10) and incubated at 60°C for 2 hours. This mixture was then mixed with 1 ml of pure water and subjected to a total of four liquid-liquid extractions using 500 μl of hexane. The hexane solution obtained by the liquid-liquid extraction was evaporated to dryness using a centrifugal evaporator, and this dried product was dissolved in 1 ml of a mixed solvent (0.03% acetic acid aqueous solution:acetonitrile, volume ratio = 60:40). The resulting solution was used as a derivatized sample for high-performance liquid chromatography (HPLC).

[0074] The HPLC column and guard column used were a Waters Atlantis T3 C18 column (3 μm, 2.1 × 150 mm) and a Waters Atlantis guard column T3 C18 (3 μm, 2.1 × 10 mm) as described in Drug Test. Analysis, 8, 458-464 (2016). The modified LC-MS / MS equipment included a Shimadzu Nextera HPLC system (Communication Bus Module: CBM-20A; Pump: LC-30AD; Autosampler: SIL-30AC; Degasser: DGU-20A5R; Column Oven: CTO-20AC) and an AB SCIEX Sciex Triple Quad 6500+ tandem mass spectrometry (MS / MS) system.

[0075] The modified LC-MS / MS conditions were as follows: mobile phase A was 0.03% acetic acid (volume) aqueous solution, and mobile phase B was 100% acetonitrile (volume). The initial concentration was 60:40 (mobile phase A:B volume ratio). After 2 minutes, the mobile phase A:B volume ratio was changed to 52:48 (mobile phase A:B volume ratio). This was maintained for 37 minutes, and then after 4 minutes, the mobile phase A:B volume ratio was changed to 0:100 (mobile phase A:B volume ratio). This was maintained for 8 minutes. The sample injection volume into the HPLC was 1 μL. The compound-specific modification conditions are shown in Table 17 below, and Table 18 below.

[0076] [Table 17]

[0077] [Table 18]

[0078] Based on the above conditions, a quantitative method was established in multiple reaction monitoring mode using hexanal d-12 as an internal standard. Benzaldehyde (Tokyo Chemical Industry Co., Ltd.), hexanal (Tokyo Chemical Industry Co., Ltd.), 2-methylpropanal (Tokyo Chemical Industry Co., Ltd.), 3-methylbutanal (Tokyo Chemical Industry Co., Ltd.), 2,4-decadienal (Tokyo Chemical Industry Co., Ltd.), 2-methylbutanal (Tokyo Chemical Industry Co., Ltd.), and 2,4-nonadienal (Tokyo Chemical Industry Co., Ltd.) were derivatized under the above conditions, and calibration curves for each aldehyde were prepared. Subsequently, under the above conditions, aldehydes contained in the culture medium or culture supernatant were derivatized, and the concentrations of aldehydes contained in the culture supernatant (Reference Examples 3 to 6) and the culture medium (Examples 1 to 3) were quantified.

[0079] The disclosure of Japanese Patent Application No. 2021-052277 (filing date: March 25, 2021) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference. [Accession number]

[0080] NITE BP-1512 NITE BP-03426 NITE BP-03427 NITE BP-03428

Claims

1. The method comprises contacting plant-based milk with at least one lactic acid bacteria strain selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain, and Lactobacillus reuteri CP3019 strain; A method for producing a plant-based milk fermented liquid, wherein the plant-based milk is derived from at least one plant material selected from the group consisting of rice and barley.

2. A method for producing a plant-based milk product, comprising contacting a lactic acid bacteria strain containing at least one selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain, and Lactobacillus reuteri CP3019 strain with plant-based milk; A method for producing a plant-based milk fermentation liquid, wherein the plant-based milk is plant-based milk that has been saccharified.

3. The method for producing a plant-based milk fermented liquid according to claim 1 or 2, wherein the plant-based milk is derived from rice.

4. A method for producing a plant-based milk fermentation liquid described in any one of claims 1 to 3, wherein the plant-based milk is obtained by breaking down the plant raw material into smaller pieces by physical crushing or saccharification and liquefying it.

5. A method for producing a plant-based milk fermented liquid according to any one of claims 1 to 4, wherein the contact of the lactic acid bacteria strain with the plant-based milk involves fermentation of the plant-based milk by the lactic acid bacteria strain.

6. The method comprises contacting a lactic acid bacteria strain comprising at least one selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain and Lactobacillus reuteri CP3019 strain with plant-based milk containing aldehydes; A method for reducing aldehydes in plant-based milk, wherein the plant-based milk is derived from at least one plant material selected from the group consisting of rice and barley.

7. A method for producing a lactic acid bacteria strain comprising at least one selected from the group consisting of Lactobacillus fermentum CP1299 strain, Lactobacillus fermentum CP3024 strain, Lactobacillus reuteri CP3017 strain and Lactobacillus reuteri CP3019 strain, and contacting the strain with plant-based milk containing aldehydes; A method for reducing aldehydes in plant-based milk, wherein the plant-based milk is plant-based milk that has been saccharified.

8. The method for reducing aldehydes in plant-based milk according to claim 6 or 7, wherein the plant-based milk is derived from rice.

9. A method for reducing aldehydes in plant-based milk described in any one of claims 6 to 8, wherein the plant-based milk is obtained by breaking down the plant raw material into smaller pieces by physical crushing or saccharification and liquefying it.

10. 10. A method for reducing aldehydes in plant-based milk according to any one of claims 6 to 9, wherein the contact of the lactic acid bacteria strain with the plant-based milk involves fermentation of the plant-based milk by the lactic acid bacteria strain.

11. Lactobacillus fermentum strain CP3024, accession number: NITE BP-3428.

12. Lactobacillus reuteri strain CP3017, accession number: NITE BP-3426.

13. Lactobacillus reuteri CP3019 strain with accession number NITE BP-3427.

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