Method for controlling neutral polysaccharide production

Abstract

To provide a control method of a neutral polysaccharide production amount in which a production amount of neutral polysaccharide being one of extracellular polysaccharides (EPS) of lactic acid bacterium can be increased.SOLUTION: A control method of a neutral polysaccharide production amount includes a process of culturing lactic acid bacteria in a composition for lactic acid fermentation containing vitamin B2.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for controlling the production amount of neutral polysaccharides, which are one of the exopolysaccharides (EPS) of lactic acid bacteria. [Background technology]

[0002] Lactic acid bacteria are used worldwide for food fermentation, providing added value to foods by extending their shelf life, changing their texture, improving their nutritional value, etc. It is gradually becoming clear that exopolysaccharides (EPS), which are produced outside the cells of lactic acid bacteria, also contribute greatly to these added values.

[0003] Regarding the role of EPS in food, for example, it is known that EPS increases the viscosity of yogurt to prevent separation, and contributes to the smooth texture characteristic of yogurt. In addition, it has been revealed that EPS plays a role in protecting the bacterial cells from environmental stress due to its water retention, osmotic pressure resistance, and antibiotic resistance in the genera Lactobacillus and Bifidobacterium. It is thought that EPS contributes to the survival of lactic acid bacteria in food and the arrival of useful lactic acid bacteria in the intestinal tract. In addition, some lactic acid bacteria strains are known to produce EPS as a physiologically active substance that contributes to the maintenance and improvement of human health.

[0004] Furthermore, the types of EPS produced by lactic acid bacteria are known to be acidic polysaccharides and neutral polysaccharides, and neutral polysaccharides in particular are known to have health benefits including immunomodulatory effects.

[0005] As for techniques related to EPS production by lactic acid bacteria, for example, the techniques disclosed in Patent Documents 1 to 3 have been proposed as techniques for increasing the amount of EPS produced. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2017-216926 A [Patent Document 2] Re-table 2014 / 084340 [Patent Document 3] JP 2014-027925 A Summary of the Invention [Problem to be solved by the invention]

[0007] However, the techniques of Patent Documents 1-3 aim to increase the amount of EPS produced (total amount), and do not control the amount of production according to the type of acidic polysaccharide and neutral polysaccharide. In particular, if the amount of neutral polysaccharide produced as EPS can be increased, it is considered to be effective in enhancing health benefits.

[0008] The present invention has been made in consideration of the above circumstances, and has an objective of providing a method for controlling the production amount of neutral polysaccharides, which is one of the exopolysaccharides (EPS) of lactic acid bacteria, and which can increase the production amount of neutral polysaccharides. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention provides a method for controlling the production amount of neutral polysaccharides, which are one of exopolysaccharides (EPS) of lactic acid bacteria, comprising the steps of: The method is characterized by including a step of culturing lactic acid bacteria in a composition for lactic acid fermentation containing vitamin B2. Effect of the Invention

[0010] According to the method of the present invention for controlling the amount of neutral polysaccharide produced, it is possible to increase the amount of neutral polysaccharide produced among the exopolysaccharides (EPS) of lactic acid bacteria. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a method for extracting a crude polysaccharide (EPS) fraction from a sample in Examples 1 and 5. [Diagram 2] FIG. 2 is a graph showing the amount of EPS produced in each culture medium in Example 1. [Diagram 3] This figure shows the amount of EPS produced when the bacterial cell concentration was increased to 2-fold and 5-fold, and the culture supernatant of each was added to MRS medium for culture. [Figure 4] FIG. 13 shows the amount of EPS produced in a medium supplemented with vitamin B2. [Diagram 5] FIG. 1 shows the results of fractionating the EPS confirmed in Example 1 into acidic polysaccharides and neutral polysaccharides and examining the effects of adding an inducer. [Figure 6] FIG. 1 shows the results of EPS extraction using MRS medium supplemented with vitamin B2. [Figure 7] FIG. 13 shows the effect of vitamin B2 addition on the EPS structure, in which fractionated EPS was acid-hydrolyzed and the sugar composition was examined using HPLC. [Figure 8] FIG. 1 shows the results of an investigation into the effect of vitamin B2 on the amount of EPS produced in fermented soymilk. [Figure 9] FIG. 1 shows the results of an investigation into the effect of vitamin B2 on the EPS type produced in fermented soymilk. [Figure 10] FIG. 1 shows the results for fermented soy milk medium supplemented with vitamin B2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present inventors have discovered a novel finding that the addition of vitamin B2 to the culture of Lactobacillus delbrueckii subsp. delbrueckii (hereinafter sometimes referred to as "L. delbrueckii") strain TUA4408L (Tokyo University of Agriculture Public Access No. NRIC0701 strain) increases the production of neutral polysaccharides, which are one of the exopolysaccharides (EPS) of lactic acid bacteria, and have thus completed the present invention.

[0013] Hereinafter, one embodiment of the method for controlling neutral polysaccharide production amount of the present invention will be described.

[0014] The method of the present invention for controlling the amount of neutral polysaccharide produced is a method for promoting the amount of neutral polysaccharide produced, which is one of the exopolysaccharides (EPS) of lactic acid bacteria.

[0015] Specifically, the method of the present invention for controlling neutral polysaccharide production comprises the step of culturing lactic acid bacteria in a composition for lactic acid fermentation containing vitamin B2.

[0016] The lactic acid bacteria is preferably Lactobacillus delbrueckii subsp. delbrueckii (hereinafter, sometimes referred to as "L. delbrueckii") TUA4408L strain (Tokyo University of Agriculture Public Publication No.: NRIC0701 strain). The present applicant has fermented soy milk using this L. delbrueckii TUA4408L strain and has commercialized soy milk that is not flavored, that is, plain type lactic acid fermented soy milk (Marusanai Co., Ltd. Product name: Soy Milk Yogurt, Product name: Soy Milk Yogurt using soy milk from domestic soybeans) and lactic acid fermented beverage (Marusanai Co., Ltd. Product name: Soy Milk Drink Orange Yogurt Flavor, Soy Milk + Calcium 350), etc.

[0017] Other examples of lactic acid bacteria include Lactobacillus delbrueckii subsp. delbrueckii NRIC 0700 strain (accession number: NITE P-726), NRIC 0705 strain (accession number: NITE P-727), NRIC 0755 strain (accession number: NITE P-728), and NRIC 0756 strain (accession number: NITE P-729).

[0018] These lactic acid bacteria can be used in liquid, frozen or powder form, but it is more preferable to use powder form in order to adjust the number of bacteria and vitamin B2 concentration. The number of lactic acid bacteria (starter) to be added to the preparation should be 1 x 10 per 1 g. 5 ~1×10 8 This range is preferred.

[0019] The composition for lactic acid fermentation may contain various kinds of dairy ingredients, but when L. delbrueckii is used as the lactic acid bacteria, it is preferable to contain soy milk (e.g., soybean solid content 7.0% to 9.5%). Soy milk obtained by a conventional method from soybeans or dehulled soybeans can be used.

[0020] The content of vitamin B2 in the composition for lactic acid fermentation can be appropriately adjusted depending on the number of lactic acid bacteria, the amount of the composition for lactic acid fermentation, etc., but is preferably in the range of 0.33 to 0.85 μg / ml. Within this range, the production amount of neutral polysaccharides can be increased depending on the purpose.

[0021] Furthermore, to the composition for lactic acid fermentation, sugar, saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, sweeteners, organic acids (malic acid, citric acid, lactic acid, tartaric acid, etc.), flavorings, water, gelling agents (gelatin, agar, pectin, carboxymethylcellulose (CMC), etc.), thickeners, stabilizers, etc. can be added as necessary.

[0022] Regarding the method for controlling the production amount of neutral polysaccharides of the present invention, specific production and control steps are shown, for example, as follows, taking a lactic acid-fermented soy milk product as an example.

[0023] First, soy milk and a composition for lactic acid fermentation with an adjusted amount of vitamin B2 are sterilized at 90-95°C for 15-20 minutes, cooled to about 42-44°C, and a starter (a predetermined amount of lactic acid bacteria) is added. Approximately 400g of this composition for lactic acid fermentation containing lactic acid bacteria is filled per cup, and fermented for about 7 to 10 hours at a temperature suitable for the lactic acid bacteria. After fermentation is completed, the mixture is cooled in a refrigerator to obtain a lactic acid fermented soy milk product. The inclusion of vitamin B2 in the composition for lactic acid fermentation promotes the production of neutral polysaccharides by the lactic acid bacteria, and a lactic acid fermented soy milk product containing a large amount of neutral polysaccharides can be obtained.

[0024] The method for controlling the production amount of neutral polysaccharides of the present invention is not limited to the above-mentioned embodiments. EXAMPLES

[0025] The method for controlling the amount of neutral polysaccharide produced of the present invention will be described below with reference to examples. However, the method for controlling the amount of neutral polysaccharide produced of the present invention is not limited to the following examples.

[0026] <Example 1> Culture supernatant addition test using the HSJ method The effect of adding culture supernatant on EPS production was examined using L. delbrueckii TUA4408L strain. The J culture supernatant prepared by the HSJ method (a three-stage culture method consisting of H culture, S culture, and J culture. In H culture, the cultured cells of H culture are collected by centrifugation, and the same amount of fresh liquid medium is added and cultured until stationary phase. In J culture, the cultured cells of H culture are collected by centrifugation, and the same amount of fresh liquid medium is added and cultured until stationary phase) was added to MRS medium at 1%, 3%, 5%, and 10%, and L. delbrueckii TUA4408L strain was cultured, and EPS was extracted as shown in Figure 1.

[0027] As a result, it was confirmed that the amount of EPS produced in all culture media tended to be higher than that in the control (no culture supernatant added) (Figure 2). Furthermore, with the exception of the 10% addition, the amount of EPS produced increased as the amount of culture supernatant added increased.

[0028] In addition, to investigate the effect of bacterial cell concentration during the culture supernatant addition test, the bacterial cell concentration was increased to 2-fold and 5-fold only during J culture using the HSJ method, and each culture supernatant was added to MRS medium and cultured.

[0029] As a result, it was confirmed that the sample to which culture supernatant with an increased bacterial cell concentration was added produced even higher EPS production (Figure 3).

[0030] Example 2: Search for inducers The MRS culture supernatant, which had a 5-fold increase in the bacterial cell concentration in Example 1, in which an increase in the amount of EPS produced was particularly observed, was subjected to LC-MS / MS to search for inducers.

[0031] As a result, it was revealed that the substance that had increased significantly compared to the MRS medium without the bacteria was riboflavin (hereafter referred to as vitamin B2).

[0032] We then used a modified Tamura-Kakuta synthetic medium to compare the growth and EPS production with and without the addition of vitamin B2 (V.B2). We found that while there was no significant change in growth in the medium containing five times the normal amount of vitamin B2, the amount of EPS produced was increased compared to the normal synthetic medium (Figure 4).

[0033] <Example 3> Effect of induction factor addition on EPS production It has been reported that polysaccharides produced by lactic acid bacteria are acidic and neutral polysaccharides. Therefore, the EPS confirmed in Example 1 was fractionated into acidic and neutral polysaccharides by anion exchange chromatography, and the effect of adding an inducer was examined.

[0034] As a result, it was confirmed that neutral polysaccharides increased with the addition of culture supernatant, suggesting that factors that induce EPS production affect neutral polysaccharide production (Figure 5). Furthermore, in samples in which EPS was extracted using MRS medium supplemented with vitamin B2, the amount of neutral polysaccharides produced increased depending on the amount of vitamin B2 added (Figure 6).

[0035] <Example 4> EPS composition analysis To examine the effect of vitamin B2 addition on the EPS structure, the fractionated EPS was acid hydrolyzed and the sugar composition was examined using HPLC.

[0036] As a result, the glucose content in the neutral polysaccharide fraction was increased by adding vitamin B2 compared to normal static culture. In the acidic polysaccharide fraction, although there was no change in the production amount under the same conditions, the fructose and mannose contents increased (Figure 7).

[0037] Example 5: Effect of vitamin B2 on the amount and type of EPS produced in fermented soy milk Since the presence of vitamin B2 was suggested as an inducer of EPS produced by lactic acid bacteria, we investigated whether the addition of vitamin B2 to soy milk is also involved in the production of EPS.

[0038] (method) Lactobacillus delbrueckii NRIC 0701 (TUA4408L) was used as the test strain, and cultured in MRS medium and soy milk medium using the HSJ method, which is suitable for polysaccharide production. After washing the stationary phase cells, the whole cell mass was inoculated into an equal amount of new MRS medium and cultured at 37°C for 24 hours twice, and then inoculated into soy milk medium (Everyday Delicious Unadjusted Soy Milk) in the same way and cultured at 40°C for 8 hours to prepare the sample for polysaccharide analysis.

[0039] In addition, soy milk medium was supplemented with three levels of vitamin B2 (0.01μg / mL, 0.03μg / mL, 0.05μg / mL) to prepare test medium. The vitamin B2 content in soy milk is 0.03mg / 100g (=0.3ng / mg) (Japan Food Research Laboratories).

[0040] The crude polysaccharide fraction from the sample was collected by the method shown in Figure 1. The crude polysaccharide fraction was further fractionated into neutral and acidic polysaccharide fractions by anion exchange chromatography using DEAE TOYOPEAL 650M (φ2.4 × 25 cm). The amount of polysaccharide in each fraction was determined by the phenol-sulfuric acid method using freeze-dried samples, and the amount of reducing sugar was taken as the amount of polysaccharide.

[0041] (result) The results of EPS production are shown in FIG.

[0042] Although an increase in polysaccharides was observed after fermentation, no effect of vitamin B2 addition on EPS production was observed.

[0043] The results of investigating the type of polysaccharides produced with or without the addition of vitamin B2 to the fermented soy milk medium are shown in Figures 9 and 10.

[0044] As shown in FIG. 9, almost no polysaccharides were detected in the uninoculated soy milk medium, whereas acidic polysaccharides were the major polysaccharides in the fermented soy milk without added vitamin B2.

[0045] On the other hand, as shown in Figure 10, the amount of neutral polysaccharides increased and the amount of acidic polysaccharides decreased as the amount of vitamin B2 added increased. This was the same tendency as the results for the MRS medium in Example 3. However, the decrease in the amount of acidic polysaccharides with an increase in the amount of vitamin B2 added tended to be greater than that with the MRS medium. The reason why the total amount of sugars did not change much with vitamin B2 is presumably because there was no significant change in the total amount of neutral polysaccharides and acidic polysaccharides.

[0046] In addition, the total absorbance value of each fraction in Figure 10 was calculated. When the concentration of vitamin B2 added to the soy milk medium was 0.01 μg / mL, the amount of neutral polysaccharide produced was 0.9182, when the concentration of vitamin B2 added to the soy milk medium was 0.03 μg / mL, the amount of neutral polysaccharide produced was 5.9669, and when the concentration of vitamin B2 added to the soy milk medium was 0.05 μg / mL, the amount of neutral polysaccharide produced was 9.4173.

[0047] It was confirmed that when the vitamin B2 concentration was tripled (0.03 μg / mL), the amount of neutral polysaccharides produced increased 6.5-fold, and when the vitamin B2 concentration was increased five-fold (0.05 μg / mL), the amount of neutral polysaccharides produced increased 10.3-fold.

Claims

1. A method for controlling neutral polysaccharide production, which increases the production of neutral polysaccharide, which is one of exopolysaccharides (EPS) of lactic acid bacteria, comprising: The method includes a step of culturing lactic acid bacteria in a composition for lactic acid fermentation containing vitamin B2, The content of vitamin B2 is 0.03 to 0.85 μg / ml, A method for controlling neutral polysaccharide production, characterized in that the lactic acid bacterium is Lactobacillus delbrueckii subsp. delbrueckii.

2. The method for controlling neutral polysaccharide production according to claim 1, wherein the composition for lactic acid fermentation contains soy milk.

3. The number of lactic acid bacteria when added to the composition for lactic acid fermentation is 1×10 per 1 g of the composition for lactic acid fermentation. 5 pieces ~ 1×10 8 The method for controlling the production amount of neutral polysaccharides according to claim 1 or 2, characterized in that the neutral polysaccharide production amount is within the range of 100 μg / ml.

4. The method for controlling neutral polysaccharide production amount according to any one of claims 1 to 3, characterized in that the Lactobacillus delbrueckii subsp. delbrueckii is Lactobacillus delbrueckii subsp. Delbrueckii TUA4408L strain (NRIC0701 strain).

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