Novel lactic acid bacteria strains and their applications

Abstract

The present invention aims to provide a lactic acid bacterium that is "sucrose non-assimilating" and "oligosaccharide assimilation capable," and a fermented product using said lactic acid bacterium, and specifically relates to the strain Leugolactobacillus coliniformis AL3G1, identified by accession number NITE BP-04155.

Description

Technical Field

[0001] The present invention relates to lactic acid bacteria that are, for example, "sucrose non-utilizing" and "oligosaccharide utilizing", and the production of fermented products using such lactic acid bacteria.

Background Art

[0002] Based on the future increase in the world's population, environmental problems, and the growing health consciousness, 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 concurrently, the development of various processing technologies leading to their utilization is required.

[0003] By the way, soybean processed products have unfavorable flavor characteristics such as beany odor, grassy 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 that utilize Aspergillus oryzae and yeast, such as soy sauce and miso, have been passed down. In recent years, new protein fermented 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 fermented starter lactic acid bacteria with various properties have been proposed. However, there is still a large room for improvement in terms of flavor and physical properties in soybean lactic acid fermented foods, and there is still a need for starter lactic acid bacteria with new properties.

[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 lactic acid bacterium that is "sucrose non-assimilating" and "oligosaccharide assimilated," which is useful as a fermentation starter. [Means for solving the problem]

[0014] As a result of diligent research to solve the above problems, we discovered the Loigolactobacillus coryniformis AL3G1 strain, a lactic acid bacterium that is "sucrose non-assimilating" and "oligosaccharide assimilation capable," which does not have the ability to assimilate sucrose but has the ability to assimilate oligosaccharides (e.g., stachyose, raffinose, melibiose), and thus completed the present invention.

[0015] In other words, the present invention encompasses the following: [1] The Loigolactobacillus coliniformis AL3G1 strain, identified by accession number NITE BP-04155. [2] A lactic acid bacteria starter containing the AL3G1 strain of *Lygolactobacillus coliniformis*, identified by accession number NITE BP-04155. [3] A fermented product obtained by adding a lactic acid bacteria starter to a fermentation raw material and fermenting it, wherein the lactic acid bacteria starter is the lactic acid bacteria strain AL3G1 identified by accession number NITE BP-04155. [4] The fermented product according to [3], wherein the fermentation raw material is a leguminous plant or a processed product thereof. [5] The fermented product described in [4], wherein the legume is soybean. [6] The fermented product described in [4], which is a processed product of a leguminous plant, such as soy milk. [7] A method for producing a fermented product, comprising the step of adding a lactic acid bacteria starter to a fermentation raw material and fermenting, wherein the lactic acid bacteria starter is the leugolactobacillus coliniformis AL3G1 strain identified by accession number NITE BP-04155. [8] The method according to [7], wherein the fermentation raw material is a leguminous plant or a processed product thereof. [9] The legume is soybean, [8] the method described.

[10] The method described in [8], wherein the processed product of a leguminous plant is soy milk.

[0016] This specification incorporates the disclosure of Japanese Patent Application No. 2024-210712, which is the basis of the priority of this application.

Advantages of the Invention

[0017] The lactic acid bacteria strain according to the present invention can be used for the fermentation processing of foods containing oligosaccharides such as stachyose, raffinose, and melibiose. 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 can be obtained. In addition, an effect of preventing flatulence caused by the intake of oligosaccharides can also be expected.

Brief Description of the Drawings

[0018] [Figure 1] Shows the NMR signal intensities of sucrose and oligosaccharides in the fermented soy milk supernatant in Example 1.

Modes for Carrying Out the Invention

[0019] Hereinafter, the present invention will be described in detail. The lactic acid bacteria strain according to the present invention is Lactobacillus colliniformis AL3G1 strain (hereinafter sometimes referred to as "AL3G1 strain") identified by the accession number NITE BP-04155. The AL3G1 strain is a lactic acid bacterium that is "sucrose non-assimilable" and "oligosaccharide assimilable", has no ability to assimilate sucrose, and has the ability to assimilate oligosaccharides (for example, stachyose, raffinose, melibiose).

[0020] When the AL3G1 strain is used as a lactic acid bacteria starter to ferment, for example, soy milk, it shows coagulation by lactic acid and a yogurt-like fermented product can be obtained. At this time, sucrose is not consumed, and only oligosaccharides are consumed. This is the same even when fermenting adjusted soy milk or the like to which sucrose is added as a sweetener, and as an effect, lactic acid fermentation can be carried out without impairing the sweetness of sucrose. In addition, this strain can be used for preventing over-fermentation as a sucrose non-assimilable lactic acid bacterium in soy milk, similar to the use of lactose non-assimilable lactic acid bacteria for preventing over-fermentation in the fermentation of milk.

[0021] Furthermore, the lactic acid bacteria species to which strain AL3G1 belongs is classified as a facultative heterozygous fermenting lactic acid bacterium. It performs homolactic fermentation in the presence of glucose, producing lactic acid as its main metabolite, and does not accumulate acetic acid, which produces a strong sour odor. In addition, unlike bifidobacteria, *L. coryniformis* is easy to culture. Therefore, strain AL3G1 exhibits excellent long-term subculturing in unsweetened soy milk alone as a culture medium, and also has excellent long-term survival in fermented soy milk, without requiring any special treatment. Thus, strain AL3G1 has characteristics that make it easy to use as a fermentation starter.

[0022] The AL3G1 strain was deposited with the Patent Microbial 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. Furthermore, the AL3G1 strain was transferred to international depositary status at the Patent Microbial 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 November 11, 2025, under accession number NITE BP-04155.

[0023] 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.

[0024] A lactic acid bacteria starter containing the AL3G1 strain may be a culture obtained by appropriately culturing the AL3G1 strain, or it may be a suspension obtained by washing the cultured strain with a solution such as physiological saline and suspending the washed strain in a solution such as physiological saline.

[0025] The AL3G1 strain can be cultured in the same manner as general lactic acid bacteria. Suitable culture media include, for example, Lactobacillus MRS liquid medium (BD) and GYP medium. In particular, for culture intended for food applications, it can be cultured in soy milk, vegetable or fruit juices, or in culture media prepared with food-grade culture medium components.

[0026] Furthermore, as shown in Table 4 below, suitable culture temperatures include, for example, a temperature range higher than 15°C and lower than 45°C, with the optimal temperature being around 30-37°C.

[0027] Furthermore, regarding the culture period, for example, when culturing in MRS medium, it is approximately 24 hours, which is when the growth reaches its peak.

[0028] Furthermore, the present invention relates to a method for producing a fermented product (e.g., a fermented food), which includes a step of adding the AL3G1 strain as a lactic acid bacteria starter to a fermentation raw material and fermenting it, as well as a fermented product produced by said method.

[0029] Here, 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.

[0030] In the method for producing fermented products, the AL3G1 strain of lactic acid bacteria is added to the fermentation raw materials, and fermentation is carried out.

[0031] The amount of AL3G1 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 load can be expressed as cfu / g.

[0032] The temperature during fermentation should be, for example, 30 to 37°C, and the fermentation time should be long enough for the pH to decrease (acidity to increase) sufficiently. When soy milk is the raw material for fermentation, for example, 20 to 96 hours, preferably 20 to 30 hours.

[0033] Alternatively, the fermented product obtained once may be added to new fermentation raw materials, and the subgeneration process may be repeated, for example, 1 to 20 times.

[0034] The fermented products obtained in this way contain a higher amount of sucrose and a lower amount of oligosaccharides compared to fermented products obtained by conventional lactic acid fermentation. In terms of form, for example, when soy milk is fermented, it becomes a yogurt-like fermented product (fermented food), and processed products such as fresh cheese-like and cream cheese-like products obtained using the fermented product are also included. [Examples]

[0035] 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.

[0036] [Example 1] Each lactic acid bacteria strain, including strain AL3G1, was pre-cultured in 5 mL of Lactobacillus MRS liquid medium (BD) at 30°C for 24 hours. After collection, the cells were washed with physiological saline and resuspended in the same volume of physiological saline.

[0037] Aseptically dispensed unsweetened soy milk (manufactured by Company A) was inoculated with this resuspension at a volume of 0.1%, and fermentation was carried out at 30°C for 24 hours. After that, coagulation was observed and the resulting pH was measured.

[0038] Furthermore, the supernatant obtained by centrifugation was subjected to component analysis by nuclear magnetic resonance (NMR). For uninoculated samples, a small amount of lactic acid was added to the soy milk after warming to a final concentration of 1%, and the supernatant obtained by centrifugation was subjected to NMR analysis and used as control data. 1For the 1H NMR spectra, the signal intensity was standardized using 1 mM maleic acid added as an internal standard, and the signal intensities of sucrose and oligosaccharides (mainly stachyose and raffinose) detected in the NMR spectra of each sample were compared.

[0039] The results of spectral analysis are shown in Figure 1. In strains that did not coagulate soy milk, sucrose was not consumed and the pH did not decrease compared to the uninoculated control. Conversely, in all strains that did coagulate soy milk, sucrose was consumed, and a decrease in pH due to the production of lactic acid was observed, but no consumption of oligosaccharides was observed. In the AL3G1 strain, despite the decrease in pH and coagulation, sucrose was not consumed, but oligosaccharides were.

[0040] [Example 2] Regarding the sugar assimilation pattern of the AL3G1 strain, the reference strain is L. coryniformis subsp. coryniformis JCM 1164. T Stock and L. coryniformis subsp. torquens JCM 1166 T A comparison was made with the strain. The assimilation pattern was analyzed using the Api50 CH kit and Api50 CHL medium (manufactured by bioMérieux Japan). For stachyose assimilation, a 10% stachyose solution was filtered and sterilized, then added to Api50 CHL medium to a final concentration of 1% (w / v). Assimilation was determined after incubation at 30°C for 2 days.

[0041] Table 1 shows the results of a comparison of sugar assimilation patterns. While the L. coryniformis reference strain showed assimilation or weak assimilation of sucrose, the AL3G1 strain showed no assimilation. Furthermore, the AL3G1 strain showed assimilation of the oligosaccharides stachyose, raffinose, and melibiose, whereas the L. coryniformis reference strain lacked assimilation of any of these oligosaccharides. The characteristic of assimilating high-molecular-weight oligosaccharides rather than lower-molecular-weight sucrose is a unique ecological feature seen in Bifidobacterium and other bacteria. Such characteristics are far removed from the general ecological features of lactic acid bacteria, and therefore, the AL3G1 strain can be said to be a novel lactic acid bacterium.

[0042] [Table 1]

[0043] [Example 3] To evaluate the applicability of the AL3G1 strain to soybean fermentation processing, fermentation tests were conducted using soy milk or similar liquid processed products made primarily from soybeans. These processed products differed in their main ingredients, including domestically produced soybeans, imported soybeans, and organically grown soybeans, as well as in the presence or absence of adjustments to their components and storage temperatures. The bacteria were inoculated into these soybean processed products using the same method as in Example 1, and the pH decrease was measured up to 96 hours at 30°C to evaluate whether lactic acid fermentation was progressing.

[0044] Table 2 shows the fermentation results using 10 different products. The AL3G1 strain showed a good decrease in pH regardless of which soybean liquid processed product was used, and coagulation was observed between 20 and 30 hours of fermentation.

[0045] Therefore, the AL3G1 strain is applicable to the fermentation processing of different types of soy products and is considered suitable for fermenting legume raw materials containing oligosaccharides such as stachyose and raffinose.

[0046] [Table 2]

[0047] [Example 4] To evaluate the ease of use of the AL3G1 strain as a food fermentation starter, we conducted tests to determine if it could be continuously and stably subcultured using soy milk as an example. Unsweetened soy milk from Company A and unsweetened soy milk from Company B were used, and the bacteria were inoculated in the same manner as in Example 1. After fermentation at 30°C for 24 hours, the fermented product was subcultured by adding it to fresh soy milk at a volume of 1%, and fermentation was carried out at 30°C for 24 to 48 hours. This operation was repeated up to the 10th generation, and thereafter the subculture amount was further reduced to 0.1%, and subculture was repeated up to the 20th generation. The pH reached by each fermented product was measured to evaluate whether there was a decrease in fermentation power.

[0048] Table 3 shows the changes in the target pH during 20 subculturing cycles. The AL3G1 strain did not show significant changes in the target pH of the fermented product even after repeated subculturing in unadjusted soy milk, and was able to stably repeat fermentation. This indicates that the nutrients required for the growth of the AL3G1 strain are sufficiently contained in soy milk, and that the AL3G1 strain can be easily maintained simply by using a culture medium with soy milk as the sole raw material.

[0049] [Table 3]

[0050] [Example 5] Using soy milk fermentation as an example, the effect of different fermentation temperatures on the fermentation rate of the AL3G1 strain was evaluated. Unsweetened soy milk (manufactured by Company A) was inoculated with the bacteria in the same manner as in Example 1, and fermentation was carried out at different temperatures ranging from 45°C to 15°C.

[0051] The results are shown in Table 4. The AL3G1 strain showed good fermentation rates at 37°C to 30°C, delayed fermentation at 25°C and 20°C, and extreme fermentation delay at 45°C and 15°C. Therefore, like typical starter cultures for fermented milk, the AL3G1 strain was shown to have an optimal fermentation temperature in the mesothermal range.

[0052] [Table 4]

[0053] [Example 6] The viability of the AL3G1 strain was evaluated using soy milk fermentation as an example. First, subfermentation was carried out using unsweetened soy milk (manufactured by Company A) in the same manner as in Example 4. Next, the fermented product from the third subfermentation was mixed and divided into 1g portions, which were stored at different temperatures: room temperature, +4°C, and -80°C. The number of viable AL3G1 strain cells before and after storage was measured using MRS agar medium, and the viability at each temperature was evaluated.

[0054] Table 5 shows the survival results. Compared to the initial sample, no significant decrease in viable cell count was observed during the test period at -80°C, confirming that the sample could be stored stably for a long period. Furthermore, while the viable cell count decreased rapidly at room temperature, it was possible to maintain the initial viable cell count for one week at +4°C, and even after two weeks, it showed a survival rate of over 20%.

[0055] From the above, it was shown that, similar to how starter cultures for general fermented milk are stored in skim milk culture medium, the AL3G1 strain can also be used to stably store fermented soy milk products as a stock for a long period of time.

[0056] [Table 5] [Accession Number]

[0057] NITE BP-04155

[0058] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. The strain of *Leugolactobacillus coliniformis* AL3G1 is identified by accession number NITE BP-04155.

2. A lactic acid bacteria starter containing the Leugolactobacillus coliniformis AL3G1 strain, identified by accession number NITE BP-04155.

3. A fermented product obtained by adding a lactic acid bacteria starter to a fermentation raw material and fermenting it, wherein the lactic acid bacteria starter is the AL3G1 strain of Lougolactobacillus coliniformis, identified by accession number NITE BP-04155.

4. The fermented product according to claim 3, wherein the fermentation raw material is a leguminous plant or a processed product thereof.

5. The fermented product according to claim 4, wherein the leguminous plant is soybean.

6. The fermented product according to claim 4, wherein the processed product of a leguminous plant is soy milk.

7. A method for producing a fermented product, comprising the step of adding a lactic acid bacteria starter to a fermentation raw material and fermenting it, wherein the lactic acid bacteria starter is the AL3G1 strain of Lougolactobacillus coliniformis, identified by accession number NITE BP-04155.

8. The method according to claim 7, wherein the fermentation raw material is a leguminous plant or a processed product thereof.

9. The method according to claim 8, wherein the leguminous plant is soybean.

10. The method according to claim 8, wherein the processed product of a leguminous plant is soy milk.

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