Selection method for lactic acid bacteria

Abstract

The objective is to provide lactic acid bacteria that are "sucrose non-assimilating" and "oligosaccharide assimilated." [Solution] A method for selecting lactic acid bacteria strains that are sucrose-non-assimilating and oligosaccharide-assimilating, comprising the step of subjecting a fermented product, prepared by adding a candidate lactic acid bacteria strain as a lactic acid bacteria starter to a fermentation raw material, to quantitative instrumental analysis.

Description

【Technical Field】 【0001】 The present invention relates to a method for selecting lactic acid bacteria that are, for example, "sucrose non-utilizing" and "oligosaccharide utilizing". 【Background Art】 【0002】 With the increase in the world population, environmental problems, and growing health consciousness in the future, the global demand for plant-based protein foods (Plant Based Food) using soybeans and the like has been expanding year by year. In Japan, in terms of production, the development of innovative technologies such as super-high-yield soybeans is being promoted, and at the same time, various processing technologies leading to their utilization are required. 【0003】 By the way, soybean processed products have unfavorable flavor characteristics such as beany odor, green odor, bitterness, and astringency, and in addition, there is a problem of "flatulence" caused by the oligosaccharides contained, which cause active gas production in the intestine, and it is expected to improve these by processing technologies. 【0004】 As one of the domestic soybean processing technologies, traditional fermentation processing technologies using Aspergillus oryzae and yeast, such as soy sauce and miso, are being inherited. In recent years, new protein fermentation foods such as soy milk yogurt using lactic acid bacteria have also emerged and are becoming established. Fermentation processing by lactic acid bacteria has been studied for a long time as one of the methods for improving the flavor of soy milk, and fermentation starter lactic acid bacteria with various characteristics have been proposed. However, there is still a large room for improvement in terms of flavor and physical properties in soybean lactic acid fermentation foods, and starter lactic acid bacteria with new characteristics are still required. 【0005】 For example, various fermentation starter lactic acid bacteria have been proposed for the purpose of producing yogurt-like fermented soy products and modifying their flavor. Patent Document 1 is a typical example, and it provides a "creamy soy milk fermented food with a smooth texture, no whey separation, and high viscosity" using lactic acid bacteria that have the ability to ferment soy milk. Such fermentation starter lactic acid bacteria carry out lactic acid fermentation using sugars in soy milk as a substrate, resulting in protein coagulation due to increased acidity and the production of taste-enhancing and aroma-enhancing metabolites. 【0006】 Furthermore, although taxonomically different from lactic acid bacteria (Lactobacillales), it is known from Non-Patent Document 1 that lactic acid fermented soy milk can also be produced using bifidobacteria (Bifidobacteriales). The bifidobacterial strain used in this document does not have the ability to assimilate sucrose (a disaccharide) which is the main component of soy milk, but it carries out fermentation by assimilating oligosaccharides such as stachyose (a tetrasaccharide) and raffinose (a trisaccharide) which are present in small amounts. 【0007】 Microorganisms that do not utilize the main sugars in fermentation raw materials are also used in techniques to prevent over-fermentation. Patent document 2 describes a technique for preventing over-fermentation during the storage period after fermentation in the production of fermented milk, using a mutant strain of lactic acid bacteria that does not utilize lactose, the main sugar in milk. 【0008】 Thus, various starter cultures have been developed for use in the fermentation of soy products. However, in the lactic acid fermentation of soy milk, sucrose, which has excellent sweetness intensity, is usually consumed preferentially, and lactic acid is produced instead. The remaining oligosaccharides exhibit only about 0.2 times the sweetness intensity of sucrose. Therefore, when conventional starter cultures are used, the natural sweetness of soy milk is greatly diminished by fermentation, and the sourness of the produced lactic acid becomes prominent, inevitably negatively impacting palatability. 【0009】 When sucrose-non-assimilating bifidobacteria are used in soy milk fermentation, lactic acid can be produced by consuming oligosaccharides instead of sucrose. However, bifidobacteria are generally more difficult to cultivate and maintain than lactic acid bacteria. In the example in Non-Patent Document 1, the bifidobacteria were not pre-cultured in milk or soy milk, and the culture medium was directly inoculated into the soy milk. Furthermore, since bifidobacteria produce acetic acid as their main metabolite, an undesirable sour odor is imparted to the fermented product. 【0010】 In lactic acid bacteria, sugar assimilation patterns differ at the strain level, and it is not impossible to search for strains with patterns suited to specific purposes. However, sugar assimilation patterns like those of Bifidobacterium are special cases that arose as a survival strategy against other microorganisms. In lactic acid bacteria, being "sucrose non-assimilated" and "oligosaccharide assimilated" is an unusual phenotype, and therefore there are no examples of it being realized as a fermentation starter. [Prior art documents] [Patent Documents] 【0011】 [Patent Document 1] Japanese Patent Publication No. 2007-014303 [Patent Document 2] Japanese Patent Publication No. 2023-102164 [Non-patent literature] 【0012】 [Non-Patent Document 1] Fermentability and sugar utilization of soy milk by Bifidobacterium (1992), Matsuyama et al., Journal of the Japan Society for Food Science and Technology 39(10): 887-893 [Overview of the project] [Problems that the invention aims to solve] 【0013】 In view of the above circumstances, the present invention aims to provide a method for selecting lactic acid bacteria that are "sucrose non-assimilating" and "oligosaccharide assimilation-enabled" and useful as fermentation starters. [Means for solving the problem] 【0014】 As a result of diligent research to solve the above problems, we have discovered that by using quantitative instrumental analysis methods such as a highly sensitive and high-resolution nuclear magnetic resonance (NMR) spectrometer capable of simultaneous quantitative analysis of multiple components, it is possible to select lactic acid bacteria strains that are "sucrose non-assimilating" and "oligosaccharide assimilationable" while discriminating even slight sugar consumption, thus completing the present invention. 【0015】 In other words, the present invention encompasses the following: [1] A method for selecting sucrose-non-assimilating and oligosaccharide-assimilating lactic acid bacteria strains, comprising the step of subjecting a fermented product, obtained by adding a candidate lactic acid bacteria strain as a lactic acid bacteria starter to a fermentation raw material, to quantitative instrumental analysis. [2] When the signal intensity of sucrose is the same as that of an unfermented sample, and the signal intensity of oligosaccharides is lower; Compared to lactic acid bacteria strains that utilize sucrose but not oligosaccharides, is it the case that the signal intensity for sucrose is high and the signal intensity for oligosaccharides is low? Compared to lactic acid bacteria strains that utilize both sucrose and oligosaccharides, the signal intensity for sucrose is higher and the signal intensity for oligosaccharides is the same; or When the signal intensity for sucrose is the same as that for lactic acid bacteria strains that do not assimilate for sucrose but do as oligosaccharides, and the signal intensity for oligosaccharides is the same as that for lactic acid bacteria strains that do not assimilate for sucrose but do as oligosaccharides, The aforementioned candidate lactic acid bacteria strain is identified as a lactic acid bacteria strain that does not assimilate with sucrose and assimilates with oligosaccharides. [1] The method described. [3] The method according to [1] or [2], wherein quantitative instrumental analysis is selected from the group consisting of nuclear magnetic resonance analysis, high-performance liquid chromatography analysis, high-performance anion exchange chromatography analysis by pulsed amperometric detection, and liquid chromatography-mass spectrometry. [4] The method described in [3], wherein the quantitative instrumental analysis is nuclear magnetic resonance analysis. [5] The method according to any one of [1] to [4], wherein the fermentation raw material is a leguminous plant or a processed product thereof. [6] The method according to [5], wherein the leguminous plant is soybean. [7] The method according to [5], wherein the processed product of the leguminous plant is soy milk. [8] The method according to any one of [1] to [7], wherein the oligosaccharide is stachyose and / or raffinose. [Advantages of the Invention] 【0016】 According to the method for selecting lactic acid bacteria of the present invention, lactic acid bacteria strains that cannot assimilate sucrose and can assimilate oligosaccharides can be efficiently selected by a quantitative simultaneous analysis method. 【0017】 In addition, the lactic acid bacteria strains obtained by the method for selecting lactic acid bacteria of the present invention can be used for the fermentation processing of foods containing oligosaccharides such as stachyose and raffinose. Mainly, beans are suitable, and when used in soy milk, which is a processed food of soybeans, a yogurt-like fermented product with a good flavor that does not impair the sweetness of sucrose can be obtained. In addition, an effect of preventing flatulence caused by the intake of oligosaccharides can also be expected. [[ID=十七]][Brief Description of the Drawings] 【0018】 [Figure 1] An example of rapid analysis of the saccharide-degrading ability of lactic acid bacteria strains by 1H NMR spectrum analysis of a soy milk fermented product in Example 1 is shown. [Figure 2] An example of rapid analysis of the saccharide-degrading ability of lactic acid bacteria strains by 1H NMR spectrum analysis of a soy milk fermented product in Example 1 is shown. [Modes for Carrying Out the Invention] 【0019】 Hereinafter, the present invention will be described in detail. 【0020】 The method for selecting lactic acid bacteria strains that do not assimilate sucrose and assimilate oligosaccharides according to the present invention (hereinafter referred to as "this method") includes a step of subjecting a fermented product prepared by adding a candidate lactic acid bacteria strain as a lactic acid bacteria starter to quantitative instrumental analysis. For example, in this method, a candidate lactic acid bacteria strain is identified as a lactic acid bacteria strain that does not assimilate sucrose and assimilates oligosaccharides if, compared to an unfermented sample, the signal intensity of sucrose is the same and the signal intensity of oligosaccharides is low; compared to a lactic acid bacteria strain that assimilates sucrose and does not assimilate oligosaccharides, the signal intensity of sucrose is high and the signal intensity of oligosaccharides is low; compared to a lactic acid bacteria strain that assimilates sucrose and assimilates oligosaccharides, the signal intensity of sucrose is high and the signal intensity of oligosaccharides is the same; or compared to a lactic acid bacteria strain that does not assimilate sucrose and assimilates oligosaccharides, the signal intensity of sucrose is the same and the signal intensity of oligosaccharides is the same. 【0021】 In this method, quantitative instrumental analysis refers to an analysis that quantitatively detects sucrose and oligosaccharides as signal intensity using analytical instruments (apparatus). Examples of such quantitative instrumental analysis include nuclear magnetic resonance (NMR) analysis, high-performance liquid chromatography (HPLC) analysis, high-performance anion exchange chromatography (HPAEC-PAD) analysis using pulsed amperometric detection, and liquid chromatography-mass (LC-MS) analysis, with NMR analysis being preferred. 【0022】 In this method, the candidate lactic acid bacteria strains are not particularly limited as long as they belong to the order Lactobacillales, but examples include Loigolactobacillus coryniformis, Latilactobacillus sakei, Lactiplantibacillus plantarum, Leuconostoc mesenteroides, Lentilactobacillus buchneri, Lacticaseibacillus paracasei, Lactococcus lactis, Levilactobacillus brevis, and Limosilactobacillus reuteri. Lactobacillus reuteri, Lactobacillus delbrueckii, Enterococcus faecalis, Streptococcus thermophilus, Ligilactobacillus ruminis, Pediococcus pentosaceus, Weissella cibaria, Fructilactobacillus fructivorans, Companilactobacillus alimentarius, Paucilactobacillus vaccinostercus), Liquorilactobacillus nagelii, Secundilactobacillus shirageiExamples include *Fructobacillus silagei*, *Schleiferilactobacillus perolens*, *Tetragenococcus halophilus*, *Furfurilactobacillus rossiae*, *Lapidilactobacillus concavus*, *Carnobacterium divergens*, *Aerococcus vaginalis*, *Oenococcus oeni*, and *Fructobacillus tropaeoli*. 【0023】 Furthermore, the fermentation raw materials are not particularly limited as long as they contain sucrose and oligosaccharides (e.g., stachyose, raffinose, melibiose, etc.), and examples include crushed, ground, soaked, boiled beans, boiling liquid, residue (okara) of leguminous plants such as soybeans, kidney beans, broad beans, peanuts, chickpeas, kudzu, red clover, licorice, and apios (preferably soybeans), or processed products of these leguminous plants (preferably soy milk), or vegetables such as sugar beets. As soy milk, it may also be adjusted soy milk to which sucrose has been added as a sweetener. 【0024】 In this method, first, a candidate strain of lactic acid bacteria is added to the fermentation raw material as a lactic acid bacteria starter (fermentation starter) to prepare the fermented product. 【0025】 The candidate lactic acid bacteria strain may be a culture obtained by appropriately culturing the lactic acid bacteria strain, or it may be a suspension obtained by washing the strain with a solution such as physiological saline after culturing, and then suspending the washed strain in a solution such as physiological saline. Examples of culturing (pre-culturing) of the lactic acid bacteria strain include culturing in Lactobacillus MRS liquid medium (manufactured by BD) at 30-37°C for 24 hours. 【0026】 Next, the candidate lactic acid bacteria strain is added to the fermentation raw material as a lactic acid bacteria starter, and fermentation is carried out. The amount of the candidate lactic acid bacteria strain added to the fermentation raw material is not particularly limited, but if soy milk is the fermentation raw material, for example, 10 5 The bacterial count can be expressed as cfu / g. The fermentation temperature can be, for example, 30-37°C, and the fermentation time can be as long as sufficient time is required for the pH to decrease (acidity to increase). For example, if soy milk is the raw material for fermentation, this can be 24-48 hours. 【0027】 The fermented products obtained in this way can take various forms, such as a yogurt-like fermented product (fermented food) when soy milk is fermented, as well as processed products resembling fresh cheese or cream cheese obtained using the fermented product. 【0028】 In this method, the fermented product is subjected to quantitative instrumental analysis such as nuclear magnetic resonance (NMR) analysis. Specifically, for example, the supernatant obtained by centrifuging the fermented product is subjected to nuclear magnetic resonance (NMR) analysis. The centrifugation supernatant is mixed with NMR measurement buffer (125 mM deuterium aqueous solution potassium phosphate buffer pH 7.0, containing 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt and maleic acid as internal standards) to prepare the NMR measurement sample. 1 For the 1H NMR spectrum, the signal intensity is normalized using the internal standard maleic acid, and the signal intensities of sucrose and oligosaccharides (mainly stachyose and / or raffinose) are detected. 【0029】 Examples of unfermented samples for comparison include samples in which fermentation has not been carried out with the candidate lactic acid bacteria strain, such as the supernatant obtained by centrifugation of a coagulated product obtained by adding a small amount of lactic acid (e.g., 1% final concentration) to a fermentation raw material such as soy milk after warming, and a mixture of the candidate lactic acid bacteria strain and the fermentation raw material before fermentation. 【0030】 Furthermore, as a negative control, for example, the supernatant obtained by centrifugation of a fermented product using a conventional lactic acid bacteria strain (a lactic acid bacteria strain that utilizes sucrose but does not utilize oligosaccharides) can be used. An example of such a conventional lactic acid bacteria strain is Lactobacillus paracasei subsp. paracasei NCFB 206. 【0031】 Alternatively, a negative control could be the supernatant obtained by centrifugation of a fermented product using a conventional lactic acid bacteria strain (a lactic acid bacteria strain that utilizes both sucrose and oligosaccharides). An example of such a conventional lactic acid bacteria strain is Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293. T These are some examples. 【0032】 Furthermore, as a positive control, a lactic acid bacterium strain that does not assimilate sucrose and assimilates oligosaccharides, for example, the supernatant obtained by centrifugation of a fermented product using the Lougolactobacillus coryniformis AL3G1 strain (hereinafter sometimes referred to as "AL3G1 strain"), which was identified by the inventors under accession number NITE P-04155, can be used. The AL3G1 strain is a lactic acid bacterium that is "sucrose-non-assimilate" and "oligosaccharide-assimilate," lacking the ability to assimilate sucrose but possessing the ability to assimilate oligosaccharides (e.g., stachyose, raffinose, melibiose). In addition, the lactic acid bacterium species to which the AL3G1 strain belongs is classified as a facultative heterozygous fermenting lactic acid bacterium, and since it performs homolactic acid fermentation in the presence of glucose, it produces lactic acid as its main metabolite and does not accumulate acetic acid, which produces a strong sour odor. Moreover, unlike Bifidobacteria, Lougolactobacillus coryniformis is easy to culture. Therefore, the AL3G1 strain exhibits excellent long-term subculturing properties in unsweetened soy milk alone as a culture medium, and also shows superior long-term survival in fermented soy milk, even without special treatment. Thus, the AL3G1 strain possesses characteristics that make it easy to use as a fermentation starter. 【0033】 The AL3G1 strain was deposited with the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) on September 12, 2024, under accession number NITE P-04155. 【0034】 Furthermore, the AL3G1 strain possesses the following bacteriological characteristics: it is a Gram-positive rod-shaped bacillus, lacks spore-forming ability, is facultative anaerobic and catalase-negative, negative for gas production in lactic acid fermentation from glucose, and primarily produces D-lactic acid. It also grows in MRS medium for lactobacilli at 15°C and shows tolerance to 4% salt, but does not grow at 45°C. 【0035】 Compared to the unfermented sample, the signal intensity of sucrose (for example) 1 The 5.40 ppm doublet peak in the 1H NMR spectrum is the same, and the oligosaccharides (for example, stachyose and raffinose) are the same. 1 A candidate lactic acid bacteria strain can be identified as a lactic acid bacteria strain that does not assimilate sucrose and assimilates oligosaccharides if: the signal intensity of the 5.42 ppm doublet peak in the 1H NMR spectrum is low; the signal intensity of sucrose is high and the signal intensity of oligosaccharides is low compared to a lactic acid bacteria strain that assimilates sucrose and does not assimilate oligosaccharides; the signal intensity of sucrose is high and the signal intensity of oligosaccharides is the same compared to a lactic acid bacteria strain that assimilates sucrose and does not assimilate oligosaccharides; or the signal intensity of sucrose is the same and the signal intensity of oligosaccharides is the same compared to a lactic acid bacteria strain that does not assimilate sucrose and does not assimilate oligosaccharides. Here, "the signal intensity of sucrose is the same" means, for example, that the ratio of signal area values ​​based on signal intensity is 1.00 ± 0.10, and "the signal intensity of sucrose is high" means, for example, that the ratio of signal area values ​​is higher than 1.10. Furthermore, "low signal intensity of oligosaccharides" means, for example, that the ratio of signal area values ​​is less than 0.9, preferably less than 0.5, and most preferably less than 0.3, while "same signal intensity of oligosaccharides" means, for example, that the ratio of signal area values ​​is 1.00 ± 0.10. [Examples] 【0036】 The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples. 【0037】 [Example 1] As an example of selecting lactic acid bacteria strains with distinctive metabolic capabilities, we searched for lactic acid bacteria strains with sugar assimilation patterns suitable for fermentation processing of soy products, using unsweetened soy milk as the culture medium. 【0038】 The test strains used were lactic acid bacteria strains that were previously determined to be negative for sucrose assimilation and positive for raffinose assimilation (Figure 1), and lactic acid bacteria strains that were determined to be positive for sucrose assimilation and positive / negative for raffinose assimilation (Figure 2), using a medium prepared by replacing the glucose in the composition of MRS liquid medium with various sugars and adding a pH indicator (BCP: 1-bromo-3-chloropropane). In this medium, the determination of negative or positive results was made by observing the color development of the indicator due to the decrease in pH when the lactic acid bacteria strain consumed the added sugars and produced organic acids. 【0039】 Each test strain was pre-cultured in Lactobacillus MRS liquid medium (BD) at 30°C for 24 hours, and 3 μL of the culture solution was aseptically inoculated into 5 mL of unsweetened soy milk. After fermentation at 30°C for 48 hours, the supernatant was centrifuged and subjected to simultaneous quantitative analysis of multiple components by nuclear magnetic resonance (NMR). 130 μL of the centrifugation supernatant was mixed with 520 μL of NMR measurement buffer (heavy water 125 mM potassium phosphate buffer pH 7.0, containing 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt and maleic acid as internal standards) to prepare the NMR sample. For unfermented samples (uninoculated samples), a small amount of lactic acid was added to the incubated soy milk to a final concentration of 1% to induce coagulation, and the supernatant was subjected to NMR analysis as control data. 【0040】 Each sample 1For the 1H NMR spectra, the signal intensity was standardized using the internal standard maleic acid, and the signal intensities of detected sucrose and oligosaccharides (mainly stachyose and raffinose) were compared. 【0041】 The results of spectral analysis are shown in Figures 1 and 2. Among the signals for sucrose and oligosaccharides in the soy milk sample, independent signals for anomeric protons were detected around 5.40 ppm and 5.42 ppm in the NMR spectrum, respectively. Regarding the oligosaccharide signals, samples from each strain that had been previously determined to be sucrose (Suc) negative and raffinose (Raf) positive showed a clear decrease in oligosaccharide signal intensity compared to the unfermented sample, reflecting the assimilation of raffinose. However, regarding the sucrose signal, even in strains that had been previously determined to be sucrose-negative, a slight decrease in sucrose signal intensity was observed, except for the AL3G1 strain. No decrease in sucrose was detected in fermentation by the AL3G1 strain. Instead, oligosaccharides (mainly stachyose and raffinose) decreased significantly, indicating that these were used as substrates for lactic acid fermentation and for the production of organic acids. Furthermore, samples from each strain that had been previously determined to be sucrose-positive showed a significantly reduced sucrose signal intensity compared to unfermented samples, regardless of whether or not they were able to assimilate raffinose, reflecting the ability to assimilate sucrose. These strains indicated that sucrose, either alone or in combination with oligosaccharides, was used as a substrate for lactic acid fermentation. 【0042】 Thus, by using a quantitative, simultaneous analysis method of multiple components, it was possible to select characteristic lactic acid bacteria strains while discriminating even slight sugar consumption. It is possible that strains previously determined to be negative for sucrose assimilation using conventional methods actually consumed small amounts of sucrose in soy milk, which could not be detected with the accuracy of non-quantitative discrimination methods. Furthermore, there are significant differences in the growth environment for lactic acid bacteria between the culture medium used in conventional methods and the soy milk used as the actual fermentation raw material, including the coexistence of multiple types of sugars, differences in nutrient composition, and the presence of growth inhibitors. Due to these environmental factors, the sugar assimilation ability determined by conventional methods may not be reflected in soy milk. This selection method, which uses a simple and quantitative simultaneous analysis of multiple components, can efficiently select characteristic lactic acid bacteria strains suitable for soy milk fermentation by using quantitative changes in not only sugars but also organic acids, amino acids, nucleic acids, and their metabolites as indicators. [Accession Number] 【0043】 NITE P-04155

Claims

[Claim 1] A method for selecting lactic acid bacteria strains that are non-sucrose-assimilating and oligosaccharide-assimilating, comprising the step of subjecting a fermented product, prepared by adding a candidate lactic acid bacteria strain as a lactic acid bacteria starter to fermentation raw materials, to quantitative instrumental analysis. [Claim 2] Compared to the unfermented sample, is it the case that the signal intensity of sucrose is the same, and the signal intensity of oligosaccharides is lower? Compared to a lactic acid bacteria strain that assimilates sucrose but does not assimilate oligosaccharides, is it the case where the signal intensity for sucrose is high and the signal intensity for oligosaccharides is low? Compared to lactic acid bacteria strains that utilize both sucrose and oligosaccharides, the signal intensity for sucrose is higher and the signal intensity for oligosaccharides is the same; or When the signal intensity for sucrose is the same as that for lactic acid bacteria strains that do not assimilate for sucrose but do as oligosaccharides, and the signal intensity for oligosaccharides is the same as that for lactic acid bacteria strains that do not assimilate for sucrose but do as oligosaccharides, The aforementioned candidate lactic acid bacteria strain is identified as a lactic acid bacteria strain that does not assimilate with sucrose and assimilates with oligosaccharides. The method according to claim 1. [Claim 3] The method according to claim 1 or 2, wherein the quantitative instrumental analysis is selected from the group consisting of nuclear magnetic resonance analysis, high-performance liquid chromatography analysis, high-performance anion exchange chromatography analysis by pulsed amperometric detection, and liquid chromatography-mass spectrometry. [Claim 4] The method according to claim 3, wherein the quantitative instrumental analysis is nuclear magnetic resonance analysis. [Claim 5] The method according to claim 1 or 2, wherein the fermentation raw material is a leguminous plant or a processed product thereof. [Claim 6] The method according to claim 5, wherein the leguminous plant is soybean. [Claim 7] The method according to claim 5, wherein the processed product of a leguminous plant is soy milk. [Claim 8] The method according to claim 1 or 2, wherein the oligosaccharide is stachyose and / or raffinose.

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