Lactic acid bacteria, fermentation liquid using the same, and method for producing the same

Abstract

To provide lactic acid bacteria with excellent antifungal properties, a fermentation liquid using the same, and a method for producing the same. [Solution] A fermentation liquid having antifungal properties containing a lactic acid bacterium, Lactobacillus paracasei strain RJ-84 (NITE P-03558), a culture medium for culturing the lactic acid bacterium, and a method for producing a fermentation liquid having antifungal properties, which includes culturing the lactic acid bacterium.

Description

Technical Field

[0001] The present invention relates to a lactic acid bacterium, a fermentation broth containing a medium obtained by culturing said lactic acid bacterium, and a method for producing a fermentation broth comprising culturing said lactic acid bacterium.

Background Art

[0002] Mold acts as a spoilage organism for most foods and is a major hazard to foods, therefore it is required to suppress its growth as much as possible in the food field, and the mold-proof performance of products is an important issue related to quality abnormalities. In addition, in recent years, there has been a strong demand for extending the expiration date of foods such as bread, and from this point of view, suppressing the growth of mold is also extremely important.

[0003] Conventionally, for example, it is known that for bread, mold-proofing properties are imparted by increasing the organic acid concentration in bread using brewed vinegar, propionic acid, fermentation broth, and the like. However, although brewed vinegar can impart mold-proof properties at low cost, it has sour taste and acid odor, so there is a problem that ensuring sufficient mold-proof properties strongly affects the taste of the product; while propionic acid is a synthetic additive, so there is a problem that its use is avoided.

[0004] Heretofore, there has been provided a mold-proof fermentation broth containing a medium obtained by culturing a lactic acid bacterium that releases a mold-proof substance into the medium, wherein the lactic acid bacterium is Lactobacillus reuteri, and the mold-proof substance is not reuterin or a peptide (see, for example, Patent Document 1), water, grain flour, 10 per 1 g of the mixture 5 ~10 7 pentose-assimilating facultative heterofermentative lactic acid bacteria, 10 per 1 g of the mixture 7 ~10 8Methods for producing a starter include a step of fermenting a mixture containing individual baker's yeast and 5 to 20 mg of pentose per gram of the mixture to obtain a starter (see, for example, Patent Document 2), and an antifungal composition containing Lactobacillus paracasei CHCC14676 strain deposited in the German Microbial Cell Culture Collection with acceptance number DSM25612 (see, for example, Patent Document 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2019-162065 [Patent Document 2] Japanese Patent Publication No. 2018-153150 [Patent Document 3] International Publication No. 2013 / 153070 [Overview of the project] [Problems that the invention aims to solve]

[0006] While the technologies described in Patent Documents 1 to 3 above offer a certain degree of mold prevention, further improvements are needed to meet the recent demand for extended shelf life.

[0007] The present invention aims to meet these demands, break the status quo, solve the aforementioned problems in the past, and achieve the following objectives. Specifically, the present invention aims to provide lactic acid bacteria with excellent antifungal properties, a fermentation liquid using the same, and a method for producing the same. [Means for solving the problem]

[0008] As a result of diligent research to achieve the above objective, the inventors discovered Lactobacillus paracasei strain RJ-84 (NITE P-03558), and found that a fermentation liquid using this strain can impart antifungal properties to food, thus completing the present invention.

[0009] The present invention is based on the inventors' aforementioned findings, and the means for solving the aforementioned problems are as follows: <1> This lactic acid bacterium is characterized by being Lactobacillus paracasei strain RJ-84 (NITE P-03558). <2> The aforementioned <1> This is a fungicidal fermentation liquid characterized by containing a culture medium in which the lactic acid bacteria described above are cultured. <3> The aforementioned <1> This is a method for producing a fermented liquid with antifungal properties, characterized by including the cultivation of the lactic acid bacteria described in [reference]. [Effects of the Invention]

[0010] According to the present invention, the aforementioned problems of the conventional method can be solved, and a lactic acid bacterium with excellent antifungal properties, a fermentation liquid using the same, and a method for producing the same can be provided. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows an example of the state of the bread after storage in Test Example 2. [Modes for carrying out the invention]

[0012] (lactic acid bacteria) The lactic acid bacterium of the present invention is Lactobacillus paracasei strain RJ-84. The RJ-84 strain was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) and was accepted as NITE P-03558 on November 15, 2021.

[0013] The method for producing said lactic acid bacteria is not particularly limited and may be appropriately selected. For example, as shown in the test examples described later, methods for production include selecting lactic acid bacteria that delay mold growth in the culture supernatant of lactic acid bacteria by using mold growth as an indicator, adding the fermentation broth of said lactic acid bacteria to a food and selecting lactic acid bacteria that delay mold growth in said food, and combining these methods.

[0014] Since said lactic acid bacteria have excellent antifungal properties, they can be suitably used in the antifungal fermentation broth and the method for producing the same described below.

[0015] (Fermentation Broth and Method for Producing the Same) The antifungal fermentation broth of the present invention (hereinafter sometimes referred to as "fermentation broth") comprises at least a medium obtained by culturing the lactic acid bacteria of the present invention, and further comprises other components as necessary. Said fermentation broth can be suitably produced by the method for producing a fermentation broth of the present invention. Hereinafter, the fermentation broth of the present invention will be described together with the description of the method for producing the fermentation broth of the present invention.

[0016] <Method for Producing Fermentation Broth> The method for producing a fermentation broth of the present invention comprises at least a culturing step of culturing the lactic acid bacteria of the present invention, and further comprises other steps as necessary.

[0017] <Culturing Step> Said culturing step is not particularly limited as long as the lactic acid bacteria of the present invention are cultured, and a known culturing method may be appropriately selected. In said culturing step, if necessary, the lactic acid bacteria of the present invention may be cultured together with lactic acid bacteria other than the lactic acid bacteria of the present invention.

[0018] The medium used for said culture is not particularly limited, and can be appropriately selected from among media generally used for culturing lactic acid bacteria. There are also no particular limitations on the components added to the medium, and components that can generally be added to a medium during fermentation of lactic acid bacteria can be appropriately selected, and examples thereof include amino acid sources, carbon sources, various inorganic salts, vitamins, and cereal flours. These may be used alone, or two or more of them may be used in combination.

[0019] The temperature for said culture is not particularly limited, and a temperature generally used for culturing lactic acid bacteria can be appropriately selected; for example, the temperature may be 20 to 40°C. The time for said culture is not particularly limited, and a time generally used for culturing lactic acid bacteria can be appropriately selected; for example, the time may be 12 to 48 hours. Other culture conditions such as the culture mode and the inoculation amount of lactic acid bacteria are also not particularly limited, and modes and conditions generally used for culturing lactic acid bacteria can be appropriately selected.

[0020] <Other Steps> Said other steps are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected; examples thereof include a removal step of removing cell residues and the like by centrifugation or the like, and a heat sterilization step of heat-sterilizing the obtained fermentation broth.

[0021] According to the method for producing a fermentation broth of the present invention, a fermentation broth having excellent antifungal properties can be produced simply and efficiently.

[0022] The fermentation broth of the present invention is not particularly limited as long as it comprises a medium obtained by culturing the lactic acid bacteria described above, and may contain other components as necessary.

[0023] The use of the fermentation broth of the present invention is not particularly limited and can be appropriately selected; for example, it can be suitably used for food and the like. The type of the food is not particularly limited as long as it is a food for which imparting antifungal properties is useful, and breads can be exemplified as a suitable example.

[0024] The fermented liquid may be used alone or in combination with other ingredients, such as shelf-life improving agents.

[0025] There are no particular restrictions on how the fermentation liquid is used, and it can be appropriately selected depending on the type of food. For example, in the case of bread, it can be added to the ingredients of the dough. There are also no particular restrictions on the amount of the fermentation liquid used, and it can be appropriately selected depending on the type of food. For example, in the case of bread, it can be used in an amount of, for example, 0.5 to 20% by mass relative to the flours used.

[0026] The fermentation liquid of the present invention is excellent because it can impart antifungal properties to foods such as bread, and does not have the adverse effects on food such as sourness or sour odor that were problems when conventional acetic acid was used. Therefore, the present invention also relates to a method for imparting antifungal properties to food using the fermentation liquid of the present invention. [Examples]

[0027] The present invention will be explained below with reference to test examples, but the present invention is not limited in any way to these test examples.

[0028] (Example Test 1: Selection of Lactic Acid Bacteria) Using the applicant's library of lactic acid bacteria, a screening for fungicidal lactic acid bacteria was performed as follows.

[0029] Three types of culture media with the following compositions were prepared. (1) 125 mM phosphate buffer MRS agar MRS medium 5.5g Agar-agar ··· 2g 125 mM phosphate buffer (pH 6.25) ... 100 mL (2) 200 mM phosphate buffer MRS agar MRS medium 5.5g Agar-agar ··· 2g 125 mM phosphate buffer (pH 6.5) ... 100 mL (3) MRS agar medium supplemented with 0.5% calcium carbonate MRS medium 5.5g Agar-agar ··· 2g Calcium carbonate ··· 0.5g 125 mM phosphate buffer (pH 6.25) ... 100 mL

[0030] The test strains were inoculated from glycerol stocks in loops of one platinum loop onto MRS liquid medium and incubated statically at the optimal growth temperature for 48 hours. 5 μL of the culture solution was spotted onto each of the above-mentioned MRS agar plates in n=2 portions, dried thoroughly, and then incubated statically at the optimal temperature for 48 hours.

[0031] Black mold (IAM-2020, Aspergillus niger) spore stock solution (8.5 × 10 8 (cfu / mL) with a final concentration of 8.5 × 10 4 The solution was mixed onto PDA agar (prepared with 200 mM phosphate buffer) to a concentration of cfu / mL, and 10 mL was layered onto each plate in which lactic acid bacteria were grown. When adding chloramphenicol to the PDA agar medium, a 10,000 ppm chloramphenicol solution was added to achieve a final concentration of 100 ppm.

[0032] After solidifying the PDA agar medium, it was incubated at 30°C for 24 hours. The growth of fungal hyphae was visually observed, and the antifungal activity of each lactic acid bacterium was determined based on the presence or absence of inhibition zones.

[0033] As a result, the following four strains of lactic acid bacteria were selected as having antifungal properties. • Lactobacillus pentosus strain RJ-61 (hereinafter sometimes referred to as "RJ-61") • Lactobacillus paracasei strain RJ-84 (hereinafter sometimes referred to as "RJ-84") • Lactobacillus pentosus strain RJ-91 (hereinafter sometimes referred to as "RJ-91") • Lactobacillus pentosus strain RJ-99 (hereinafter sometimes referred to as "RJ-99")

[0034] (Test Example 2: Evaluation of mold prevention properties through the production of fermentation liquid and bread-making tests) Fermentation solutions were prepared using four lactic acid bacteria strains selected in Test Example 1 and Lactobacillus reuteri L-015 strain (hereinafter sometimes referred to as "L. reuteri") described in Japanese Patent Publication No. 2019-162065, and their antifungal properties were evaluated through bread-making tests.

[0035] <Manufacturing of fermented liquid> Lactic acid bacteria were pre-cultured by inoculating one loopful (1 platinum loop) of glycerol stock onto MRS medium and allowing it to stand at 30°C for 24 hours. In this culture, a fermentation medium that had been heat-sterilized at 70°C for 10 minutes was used. 25 mL of the fermentation medium was dispensed into a 50 mL Falcon tube, and the lactic acid bacteria pre-culture solution described above was added to a total of 1% by mass. The culture was then incubated at 30°C for 48 hours with shaking at 150 rpm to produce the fermentation liquid. The composition of the fermentation medium and the lactic acid bacteria pre-culture solution (hereinafter sometimes referred to as "raw materials for the fermentation liquid") was as follows. -Composition of the raw materials for the fermentation liquid- · Flour ··· 5.0% by mass • Skim milk powder • 5.0% by mass Glycerol ··· 2.0% by mass • Glucose ··· 3.0% by mass • Yeast extract ··· 1.0% by mass · Lactic acid bacteria preculture solution ··· 1.0% by mass · Water ··· 83.0% by mass

[0036] Table 1 shows the results of the analysis of the pH, lactic acid content, and acetic acid content of the obtained fermentation liquid.

[0037] [Table 1]

[0038] <Bread Making Exam> Using the fermentation liquid prepared as described above, white bread was produced using the sponge and dough method. The ingredients and process are as follows. A white bread prepared in the same manner except for the absence of the fermentation liquid was used as a control (hereinafter sometimes referred to as "additive-free"). [Composition] Chutane book edition · Wheat flour 70 parts by mass 30 parts by mass • Bread quality improver 0.1 parts by mass - • Yeast 2.2 parts by mass - · Sugar - 5 parts by mass • Skim milk powder - 2 parts by mass • Salt - 2 parts by mass • Fermentation liquid - 1.5 parts by mass · Fats and oils - 5 parts by mass · Water 40 parts by mass 30 parts by mass [Process] Chutane book edition Mixing: L2 min M2 min L2 min M4 min ↓ M2 min H1 min · Kneading temperature 24℃ 27℃ Fermentation (floor) time: 4 hours 16 minutes · Split weight - 220g x 6 • Bench time - 17 minutes • Proofing conditions - 38°C, relative humidity 85% 2.5cm below the mold • Baking conditions - 210°C, 35 minutes • Cooling - Room temperature, 90 minutes In the above process, L represents low speed, M represents medium speed, H represents high speed, and ↓ represents the addition of oil or fat.

[0039] -evaluation- Two black mold cells (IAM-2020, Aspergillus niger) were inoculated per spot into the crumb portion of the obtained loaves of bread, and the bread was stored at 28°C for 96 hours to observe mold growth. The degree of mold growth was evaluated by the number of "+" signs (more "+" signs indicate greater mold growth). The results of the mold growth evaluation, along with the analysis of the pH, lactic acid content, and acetic acid content of the obtained loaves of bread, are shown in Table 2. An example of the condition of the bread after storage is shown in Figure 1.

[0040] [Table 2]

[0041] The results of Test Example 2 confirmed that among the lactic acid bacteria selected in Test Example 1, Lactobacillus paracasei strain RJ-84 had the strongest antifungal effect.

[0042] The aforementioned Lactobacillus paracasei RJ-84 strain was deposited with the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) and was accepted as NITE P-03558 on November 15, 2021. [Accession Number]

[0043] NITE P-03558

Claims

1. A lactic acid bacterium characterized by being Lactobacillus paracasei strain RJ-84 (NITE P-03558).

2. A fermentation liquid having antifungal properties, characterized by containing a culture medium for culturing the lactic acid bacteria described in claim 1.

3. A method for producing a fungicidal fermentation liquid, characterized by comprising culturing the lactic acid bacteria described in claim 1.

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