Method for producing vegetable milk fermentation liquid

Abstract

To provide a method for producing a vegetable milk fermentation liquid reduced in benzaldehyde.SOLUTION: The method for producing the vegetable milk fermentation liquid includes bringing lactic acid bacteria containing at least one kind selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris and Lactobacillus mucosae into contact with vegetable milk.SELECTED DRAWING: Figure 3

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, Non-Patent Document 1). An example of plant-based milk is rice milk, which is a milky food product made by saccharifying rice with enzymes, koji, etc., adding vegetable oil, etc., and seasoning it.

[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 flavor. For example, benzaldehyde, an aromatic aldehyde, is known to have an almond-like, almond tofu-like aroma, and is a desirable aroma for beverages where an almond-like, almond tofu-like aroma is desired. However, benzaldehyde causes an off-flavor when a fermented milk-like flavor is desired. For example, the following methods can be used to reduce the aldehydes contained in plant-based milk.

[0004] For example, Patent Document 1 describes a method for reducing medium-chain aldehydes to alcohols by contacting a food containing the aldehyde with lactic acid bacteria Leuconostoc mesenteroides or Lactobacillus brevis without causing lactic acid bacteria growth. Patent Document 2 also describes a lactic acid fermented beverage obtained by lactic acid fermentation of rice-based plant milk using lactic acid bacteria Lactobacillus sakei strain No. 7 or Lactobacillus sakei strain No. 16, and describes that the diacetyl and hexanal contents were reduced. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 9-205999 [Patent Document 2] JP 2020-17 A [Non-patent literature]

[0006] [Non-Patent Document 1] J.Food Sci.Technol(September2016)53(9):3408-3423. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for producing a plant-based fermented milk liquid with reduced benzaldehyde content. [Means for solving the problem]

[0008] The present invention provides a method for producing a plant-based milk fermented liquid, which comprises contacting plant-based milk with lactic acid bacteria including at least one species selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris, and Lactobacillus mucosae.

[0009] 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 at least one species selected from the group consisting of rice and barley. In one embodiment, the rice from which the plant-based milk is derived in the production method may be brown rice or white rice. In one embodiment, the plant-based milk in the production method may be saccharified plant-based milk. In one embodiment, the lactic acid bacteria in the production method may include at least one species selected from the group consisting of Lactobacillus fermentum and Lactobacillus reuteri. In one embodiment, contacting the lactic acid bacteria with the plant-based milk in the production method may involve fermentation of the plant-based milk by the lactic acid bacteria.

[0010] The present invention also provides a method for reducing benzaldehyde in plant-based milk, which comprises contacting lactic acid bacteria including at least one species selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris, and Lactobacillus mucosae with plant-based milk containing benzaldehyde.

[0011] 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 at least one species selected from the group consisting of rice and barley. In one embodiment, the rice from which the plant-based milk in the reduction method is derived may be brown rice or white rice. In one embodiment, the plant-based milk in the reduction method may be saccharified plant-based milk. In one embodiment, the lactic acid bacteria in the reduction method may include at least one species selected from the group consisting of Lactobacillus fermentum and Lactobacillus reuteri. In one embodiment, contact between the lactic acid bacteria and the plant-based milk in the reduction method may involve fermentation of the plant-based milk by the lactic acid bacteria. [Effects of the Invention]

[0012] According to the present invention, a method for producing a plant-based fermented milk liquid with reduced benzaldehyde content can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a graph showing the ability of lactic acid bacteria to reduce low concentrations of benzaldehyde. [Figure 2] 1 is a graph showing the ability of lactic acid bacteria to reduce high concentrations of benzaldehyde. [Figure 3] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in plant-based milk derived from brown rice. [Figure 4] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in plant-based milk derived from white rice. [Figure 5] 1 is a graph showing the ability of lactic acid bacteria to reduce benzaldehyde contained in barley-derived plant-based milk. DETAILED DESCRIPTION OF THE INVENTION

[0014] 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.

[0015] Method for producing fermented plant-based milk liquid A method for producing a plant-based milk fermented liquid includes contacting plant-based milk with lactic acid bacteria including at least one species selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris, and Lactobacillus mucosae.

[0016] By contacting specific lactic acid bacteria with plant-based milk, the content of benzaldehyde contained in the plant-based milk is effectively reduced, the plant odor derived from benzaldehyde 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's excellent activity in reducing benzaldehyde contained in plant-based milk.

[0017] In the method for producing a plant-based milk fermented liquid, the content of benzaldehyde 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 the content of benzaldehyde in plant-based milk.

[0018] Lactic acid bacteria strains The method for producing a plant-based milk fermented liquor uses specific lactic acid bacteria. The specific lactic acid bacteria include at least one species selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris, and Lactobacillus mucosae. The Lactobacillus fermentum used in the production method may be any available strain, including the type strain Lactobacillus fermentum T. The Lactobacillus fermentum may also include other strains, such as the CP1299 strain and the CP3024 strain. The Lactobacillus reuteri may also be any available strain, including the type strain Lactobacillus reuteri T. The Lactobacillus reuteri may also include other strains, such as the CP3017 strain and the CP3019 strain. Lactobacillus oris may be any available strain, and may include the type strain Lactobacillus oris T, or may include strains other than the type strain. Lactobacillus mucosae may be any available strain, and may include the type strain Lactobacillus mucosae T, or may include strains other than the type strain.

[0019] The Lactobacillus fermentum CP1299 strain is a species of lactic acid bacteria belonging to the Lactobacillus fermentum genus and was internationally deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), an international depository institution under the provisions of the Budapest Treaty, under accession number NITE BP-1512 on January 18, 2013. The Lactobacillus fermentum CP3024 strain is a species of lactic acid bacteria belonging to the Lactobacillus fermentum genus and was internationally deposited with accession number NITE BP-03428 on March 1, 2021. The Lactobacillus reuteri CP3017 strain is a species of lactic acid bacterium belonging to the Lactobacillus reuteri genus and was deposited internationally on March 1, 2021, under accession number NITE BP-03426. The Lactobacillus reuteri CP3019 strain is a species of lactic acid bacterium belonging to the Lactobacillus reuteri genus and was deposited internationally on March 1, 2021, under accession number NITE BP-03427.

[0020] From the viewpoint of safety in terms of eating experience, the specific lactic acid bacteria preferably include at least one species selected from the group consisting of Lactobacillus fermentum and Lactobacillus reuteri, and more preferably include at least Lactobacillus fermentum.

[0021] In the method for producing a plant-based milk fermented liquid, in addition to specific lactic acid bacteria, other lactic acid bacteria, bifidobacteria, etc. may be used in combination. Examples of lactic acid bacteria or bifidobacteria other than specific lactic acid bacteria include Lactobacillus gasseri, Lactobacillus fructivorans, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, and Lactobacillus spp. Lactobacillus species such as Lactobacillus gallinarum, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefilgranum, Lactobacillus mali, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus tolerans; Bifidobacterium adolescentis, Bifidobacterium angraatum, Bifidobacterium animalis, Bifidobacterium bifidum, and Bifidobacterium Bifidobacterium species such as Bifidobacterium breve, Bifidobacterium cateranatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium suis, and Bifidobacterium thermophilum; Streptococcus thermophilus, Streptococcus These include the Streptococcus genus, such as Streptococcus lactis, Streptococcus cremoris, Streptococcus faecalis, and Streptococcus faecium; the Leuconostoc genus, such as Leuconostoc mesenteroides, Leuconostoc dextranicum, Leuconostoc cremoris, and Leuconostoc oenos; and the Pediococcus genus, such as Pediococcus cerevisiae, Pediococcus acidilactici, and Pediococcus halophilus.

[0022] 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.

[0023] Plant-based milk Plant-based milk may be prepared by liquefying plant materials by physical crushing or saccharification with enzymes or koji. Plant-based milk may be prepared by liquefying plant materials using a known method, or may be 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.

[0024] 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 by a known method, or may remain as is 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Aldehydes Plant-based milk contains benzaldehyde derived from plant materials. In addition to benzaldehyde, plant-based milk also contains other aldehydes other than benzaldehyde derived from plant materials. The other aldehydes may have, for example, a plant-like flavor. Examples of the other 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 anisaldehyde, cuminaldehyde, etc.

[0031] The benzaldehyde content in plant-based milk may be, for example, 10 ng / ml to 3000 ng / ml, preferably 10 ng / ml to 500 ng / ml, and more preferably 10 ng / ml to 200 ng / ml. The content of benzaldehyde and other aldehydes in plant-based milk can be measured, for example, by high-performance liquid chromatography-mass spectrometry (LC-MS / MS, etc.).

[0032] The method for producing a plant-based fermented milk liquor reduces the benzaldehyde content, but may also simultaneously reduce the content of other aldehydes. The other aldehydes whose contents are reduced 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, and 2,4-decadienal.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In the method for producing a plant-based fermented milk liquor, the benzaldehyde content in the plant-based milk is reduced by the fermentation process. The residual rate (%) obtained by dividing the benzaldehyde content in the resulting plant-based fermented milk liquor by the benzaldehyde 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. The lower limit of the residual rate (%) is not particularly limited, but may be, for example, 1% or more, preferably 5% or more.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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]

[0042] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0043] Reference example 1 The lactic acid bacteria strains listed in Table 1 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 1 and Figure 1.

[0044] [Table 1]

[0045] It was confirmed that the ability to reduce benzaldehyde differed depending on the species of lactic acid bacteria.

[0046] Reference example 2 The benzaldehyde 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 added benzaldehyde 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 significant. Lactobacillus mucosae strain A and Lactobacillus mucosae strain B, as well as Lactobacillus oris strain C and Lactobacillus oris strain D, are lactic acid bacteria strains of human origin. The number of data points per sample (n) was 3.

[0047] [Table 2]

[0048] 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.

[0049] Example 1 The lactic acid bacteria strains listed in Table 3 were prepared. Each lactic acid bacteria strain was cultured for 16 hours under anaerobic conditions at 37°C in MRS medium (manufactured by BD Japan) supplemented with 0.05% cysteine ​​hydrochloride by mass at 37°C to prepare a preculture solution for each lactic acid bacteria strain. Concentrated rice milk (manufactured by Kikkoman Corporation, raw material: processed brown rice) was diluted 1.67-fold with purified 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 cultured statically at 37°C for 16 hours to obtain a plant-based milk fermentation broth. The concentration of benzaldehyde in the resulting culture solution 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 medium by the average benzaldehyde concentration in the uninoculated medium and multiplying the result by 100. The results are shown in Table 3 and Figure 3. 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.

[0050] [Table 3]

[0051] It was confirmed that benzaldehyde was significantly reduced in certain bacterial species compared to the control, Lactobacillus gasseri T.

[0052] Example 2 The strains of lactic acid bacteria shown in Table 4 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 rice flour for confectionery (Tomizawa Shoten Co., Ltd., raw material: non-glutinous rice) and pure water were mixed in a weight ratio of 3:7, and 0.03% by volume of amylase (Novozymes, BAN480L) was added. The mixture was then incubated at 60°C for 6 hours. After the reaction, the mixture was stirred and sterilized at 95°C for 15 minutes to prepare a white rice rice milk medium. A 5% vol. lactic acid bacteria preculture was added to the white rice rice milk medium and incubated at 37°C for 16 hours to obtain a plant-based milk fermentation broth. The benzaldehyde concentration in the resulting culture broth 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 benzaldehyde rate (%) was calculated by dividing the benzaldehyde concentration in each culture broth by the average benzaldehyde concentration in the uninoculated medium and multiplying by 100. The results are shown in Table 4 and Figure 4. 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.

[0053] [Table 4]

[0054] It was confirmed that benzaldehyde was significantly reduced in certain bacterial species compared to the control, Lactobacillus gasseri T.

[0055] Example 3 The lactic acid bacteria strains shown in Table 5 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. 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 lactic acid bacteria preculture solution 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 concentration of benzaldehyde in the resulting culture medium 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 medium by the average benzaldehyde concentration in the uninoculated medium and multiplying the result by 100. The results are shown in Table 5 and Figure 5. 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.

[0056] [Table 5]

[0057] It was confirmed that benzaldehyde was significantly reduced in certain bacterial species compared to the control, Lactobacillus gasseri T.

[0058] Quantitative method for benzaldehyde 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 quantifying benzaldehyde. 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).

[0059] 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.

[0060] 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 6 below, and Table 7 below.

[0061] [Table 6]

[0062] [Table 7]

[0063] 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, benzaldehyde contained in the culture medium or culture supernatant was derivatized under the above conditions, and the concentrations of benzaldehyde contained in the culture supernatant (Reference Examples 1 and 2) and the culture medium (Examples 1 to 3) were quantified.

[0064] The disclosure of Japanese Patent Application No. 2021-052283 (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]

[0065] NITE BP-1512 NITE BP-03426 NITE BP-03427 NITE BP-03428

Claims

1. A method for producing a plant-based milk fermented liquid, comprising contacting plant-based milk with lactic acid bacteria including at least one species selected from the group consisting of Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus oris, and Lactobacillus mucosae.

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

3. A method for producing a plant-based milk fermented liquid according to claim 1 or 2, wherein the plant-based milk is derived from at least one species selected from the group consisting of rice and barley.

4. The method for producing a plant-based milk fermented liquid according to claim 3, wherein the rice is brown rice or white rice.

5. The method for producing a plant-based milk fermented liquid according to any one of claims 1 to 4, wherein the plant-based milk is saccharified plant-based milk.

6. The method for producing a plant-based milk fermented liquid according to any one of claims 1 to 5, wherein the lactic acid bacteria include at least one species selected from the group consisting of Lactobacillus fermentum and Lactobacillus reuteri.

7. A method for producing a plant-based milk fermented liquid according to any one of claims 1 to 6, wherein the contact between the lactic acid bacteria and the plant-based milk involves fermentation of the plant-based milk by the lactic acid bacteria.

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