Yeast medium

Abstract

To provide a medium that can quickly culture Asterotremella humicola 2-141-1 strain and can maintain the increased cell density.SOLUTION: A yeast medium contains carbon source, nitrogen source, fumaric acid, and ascorbic acid and is used for the yeast 2-141-1 strain deposited as the accession number NITE BP-02641.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a medium for Asterotremella humicola 2-141-1 strain, which is a type of yeast. [Background technology]

[0002] Wastewater from food factories and other facilities may contain large amounts of oil and fat. In Japan, the Sewerage Act and the Water Pollution Control Act set standards for the amount of oil and fat contained in wastewater from business establishments, and treatment facilities equipped with oil separators, flow adjustment tanks, and other treatment tanks are provided. To maintain the removal performance of the treatment facilities, the accumulated grease must be periodically removed, which is both time-consuming and costly. Therefore, in order to reduce the amount of grease accumulated in the treatment tank and the frequency of maintenance, methods of biodegrading the accumulated grease with microorganisms are being investigated.

[0003] The pH of wastewater may change significantly depending on the food residues discharged. Therefore, microorganisms for biodegrading oils and fats are required to be able to decompose oils and fats in water environments with a wide range of pH (e.g., pH 2.0 or higher and lower than 11.0). The applicant has reported Asterotremella humicola 2-141-1 strain (accession number NITE BP-02641, hereinafter also referred to as 2-141-1 strain), which is a yeast that can decompose oils and fats under a wide range of conditions, from pH 2 to lower than 11 (Patent Document 1).

[0004] Here, when the 2-141-1 strain in the treatment tank is added, the 2-141-1 strain will flow out little by little with the wastewater, so it is necessary to periodically add a certain amount of the 2-141-1 strain to the treatment tank. Therefore, it is necessary to culture the strain in order to ensure the amount of bacteria to be added. In order to reduce the frequency of culture, it is necessary to multiply the bacterial cells in a short period of time and to maintain the concentration of the cultured bacterial cells for about a week. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 220831 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a medium in which Asterotremella humicola 2-141-1 strain can be rapidly cultured and the increased cell concentration can be maintained. [Means for solving the problem]

[0007] 1. Contains a carbon source, a nitrogen source, fumaric acid, and ascorbic acid. A yeast medium, which is for the yeast strain 2-141-1 deposited under accession number NITE BP-02641. 2. The yeast medium according to 1, further comprising a milt-derived protein as an antibacterial agent. 3. The yeast medium according to 1 or 2, characterized in that the fumaric acid concentration is 0.03% by weight or more and 0.6% by weight or less. 4. The yeast medium according to any one of 1. to 3., wherein the ascorbic acid concentration is 0.03% by weight or more and 1.0% by weight or less. 5. It is in powder form and contains a carbon source, a nitrogen source, fumaric acid, ascorbic acid, citric acid, and citrate. A powdered medium for the yeast strain 2-141-1 deposited under accession number NITE BP-02641. Effect of the Invention

[0008] The yeast medium of the present invention allows the Asterotremella humicola 2-141-1 strain to grow rapidly and maintain the increased cell concentration for about one week. The yeast medium of the present invention is less susceptible to contamination (bacterial proliferation) and is suitable for culturing the 2-141-1 strain. The yeast medium of the present invention allows the Asterotremella humicola 2-141-1 strain to grow rapidly and maintain a high cell concentration for about one week. For example, the culture is prepared once a week and a certain amount of the culture is added to a treatment tank every day, thereby maintaining a high biodegradation treatment capacity. Therefore, the frequency of preparing the culture can be reduced. The powdered medium of the present invention can be stored in a dry state for a long period of time, and can be used by dissolving it in water when necessary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to a medium for Asterotremella humicola 2-141-1 strain, which is a yeast strain deposited under the accession number NITE BP-02641. In this specification, the expression "A to B" (A and B are numerical values) means A or more and B or less.

[0010] (2-141-1 stock) The Asterotremella humicola 2-141-1 strain was deposited internationally as NITE BP-02641 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD), 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan (postal code 292-0818) on February 21, 2018.

[0011] The 2-141-1 strain showed a 1.5 × 10 6At an inoculation amount of CFU / mL, it is possible to decompose 50% by weight or more of fats and oils in 24 hours at a pH of 2 or more but less than 11 and in the range of 15 to 35°C, and it is possible to decompose 90% by weight or more of fats and oils in 24 hours at a pH of 2.5 or more but less than 9.0 and inclusive at 30°C. In the treatment tank, the bacteria gradually flow out along with the wastewater and decrease in number, so the bacteria must be replenished periodically. However, the 2-141-1 strain is extremely practical, as it can demonstrate a high rate of reduction in oil and fats just 24 hours after replenishment.

[0012] (Culture medium) The yeast medium of the present invention is for the 2-141-1 strain and contains a carbon source, a nitrogen source, fumaric acid, and ascorbic acid. Carbon source There are no particular limitations on the carbon source that can be used as long as it can be assimilated by the 2-141-1 strain. Specific examples of such sugars include glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin, melibiose, lactose, melezitose, inulin, erythritol, ribitol, xylitol, glucitol, mannitol, galactitol, inositol, N-acetyl-D-glucosamine, starch, starch hydrolysates, molasses, and blackstrap molasses; natural products such as wheat and rice; alcohols such as glycerol, methanol, and ethanol; organic acids such as acetic acid, lactic acid, succinic acid, gluconic acid, glucuronic acid, pyruvic acid, and citric acid; and hydrocarbons such as hexadecane. These sugars may be used alone or in combination of two or more. Among these, glucose, galactose, sorbose, glucosamine, arabinose, rhamnose, sucrose, maltose, trehalose, α-methyl-D-glucoside, cellobiose, salicin, melibiose, lactose, melezitose, starch hydrolysates, glycerol, erythritol, ribitol, xylitol, glucitol, mannitol, galactitol, inositol, gluconic acid, glucuronic acid, lactic acid, succinic acid, citric acid, gluconic acid, and ethanol are preferred. The amount of carbon source (the total amount when two or more types are used) is, for example, 0.01 to 20.0 w / v%, preferably 0.1 to 10.0 w / v%, and more preferably 0.5 to 3.0 w / v%, based on the total amount of the yeast culture medium.

[0013] Nitrogen source Examples of nitrogen sources include organic nitrogen sources such as meat extract, fish meat extract, peptone, polypeptone, tryptone, yeast extract, malt extract, wheat extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acid, various amino acids such as glycine, glutamic acid, aspartic acid, corn steep liquor, other animal, plant, and microbial hydrolysates, and inorganic nitrogen sources such as ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, and ammonium chloride, nitrates such as sodium nitrate and calcium nitrate, nitrites such as sodium nitrite, and urea, and one or more of these may be used in combination. Among these, fish meat extract, tryptone, yeast extract, malt extract, and ammonium chloride are preferred. The amount of the nitrogen source to be added is preferably 0.01 to 5.0 parts by mass, and more preferably 0.1 to 2.5 parts by mass, per 1 part by mass of the carbon source.

[0014] Fumaric acid, ascorbic acid The medium of the present invention contains both fumaric acid and ascorbic acid, which suppresses the proliferation of various bacteria (contamination) and enables the 2-141-1 strain to be cultured efficiently. The fumaric acid concentration is preferably 0.03% by weight or more and 0.6% by weight or less, more preferably 0.55% by weight or less, and even more preferably 0.5% by weight or less. The ascorbic acid concentration is preferably 0.03% by weight or more and 1.0% by weight or less, more preferably 0.8% by weight or less, and even more preferably 0.6% by weight or less.

[0015] Milt-derived protein The medium of the present invention preferably contains milt-derived protein as an antibacterial agent. Milt-derived protein is a protein obtained from the milt of fish, particularly herring, salmon, etc., and is known to exhibit a wide range of antibacterial properties against fungi including yeast and bacteria. Although the 2-141-1 strain is a yeast, it is hardly affected by the milt-derived protein. Therefore, by using a medium containing the milt-derived protein as an antibacterial agent, the 2-141-1 strain can be efficiently cultured while suppressing the proliferation (contamination) of various bacteria. The concentration of the milt-derived protein is not particularly limited as long as it is within a range that does not inhibit the growth of the 2-141-1 strain and suppresses the proliferation of miscellaneous bacteria, but is, for example, from 0.001% by weight to 0.05% by weight, preferably from 0.002% by weight to 0.03% by weight.

[0016] The medium of the present invention may further contain trace elements such as magnesium, manganese, calcium, sodium, potassium, phosphorus, boron, sulfur, copper, iron, and zinc, as well as inorganic salts such as phosphates, hydrochlorides, sulfates, acetates, carbonates, magnesium salts, calcium salts, chlorides, and halides thereof.

[0017] The medium of the present invention may be in any form, such as liquid, solid, or solid, but is preferably in liquid form because it is easy to culture in large quantities. The medium of the present invention preferably has a pH in the range of 2.5 to 4.2. By keeping the pH in this range, the proliferation (contamination) of various bacteria can be suppressed for a longer period of time, and the 2-141-1 strain can be efficiently cultured. The pH of the medium can be adjusted by any acid or salt thereof, such as inorganic acids, such as hydrochloric acid, nitric acid, carbonic acid, and sulfuric acid, and organic acids, such as citric acid and lactic acid. Among these, citric acid and citrate (sodium salt) are preferably used because they can be used as a powdered medium. Powdered medium can be stored in a dry state for a long period of time, and requires little space for storage. When used, it is preferable to dissolve it in water and use it as a liquid medium.

[0018] ·Culture method The 2-141-1 strain can be cultured by a conventional method, and either a liquid medium or a solid medium can be used, but it is preferable to culture in a liquid medium suitable for large-scale culture. There are no particular limitations on the water used to prepare the liquid medium, and pure water, distilled water, deionized water, etc. can be used. Furthermore, since the use of the medium of the present invention can suppress the growth of various bacteria, tap water can also be used as is. The culture conditions are not particularly limited as long as the 2-141-1 strain can grow, but for example, the culture temperature is usually 15 to 35° C., and preferably 20 to 30° C. The pH at the start of the culture is preferably 2.5 to 4.2. The pH changes during the culture, but the pH may or may not be maintained within this range during the culture. EXAMPLES

[0019] (Preparation of live bacterial preparations) The liquid medium was prepared by dissolving in pure water to a final concentration of 0.18% (w / v) polypeptone, 0.12% (w / v) meat extract, 0.07% (w / v) NaHCO3, 0.005% (w / v) NaCl, 0.002% (w / v) KCl, 0.002% (w / v) CaCl2·2H2O, and 0.003% (w / v) MgSO4·7H2O. The pH was adjusted to 5 with hydrochloric acid, and the liquid medium was sterilized by autoclaving. The 2-141-1 strain was inoculated into a liquid medium and cultured in a jar fermenter at 30° C. for 48 hours (agitation speed: 300 rpm) to obtain a culture solution.

[0020] The culture solution was centrifuged (×10,000 g) for 10 minutes to collect the bacterial cells (solid content 30% by weight). The obtained bacterial cells (solid content 30% by weight) were added to distilled water so as to have a concentration of 37% by weight, trehalose at 15% by weight, and skim milk at 1% by weight, and mixed to prepare a spray liquid.

[0021] 300 g of diatomaceous earth (average particle size 75 μm, bulk density 0.32 g / ml, Radiolite (registered trademark) #3000, Showa Chemical Industry Co., Ltd.) was set in a fluidized bed granulator (FA-LAB-1, Powrex Corporation). 1077 g of the above spray liquid was sprayed onto the diatomaceous earth at a rate of 143 g / min, and hot air at 40°C was blown to fluidize the contents and dry them. After spraying, hot air at 40°C was blown for 40 minutes in the fluidized bed granulator to dry the granulated material. This resulted in a live bacterial preparation. The moisture content of the live bacterial preparation was 5.5% by weight.

[0022] (Yeast culture 1) The yeast was cultured by the following method. (1) Each component was dissolved in distilled water to obtain the composition shown in Table 1, to prepare a yeast medium. (2) 200 mL of each yeast medium was dispensed into a 500 mL tall beaker. (3) Add 6 mg of the live bacterial preparation (2-141-1 strain: 8.0 × 10 4 CFU / mL) was added. (4) The culture was incubated aerobically at 30°C for 168 hours. [Table 1]

[0023] (Calculation of viable bacteria count) The culture medium was sampled every 24 hours from the start of culture, serially diluted with 5% Tween 80 solution, and applied to the surface of NB medium solidified with agar. After application, the medium was cultured at 30°C for 48 hours, and the number of colonies of 2-141-1 strain and other bacteria (miscellaneous bacteria) formed on the medium was counted, and the contamination rate (miscellaneous bacteria colony count / total colony count x 100) was calculated from the ratio of miscellaneous bacteria to the total amount of bacteria. In addition, the pH of the culture medium was measured every 24 hours from the start of culture. The results are shown in Table 2.

[0024] [Table 2]

[0025] When the medium of Comparative Example 1, which did not contain ascorbic acid, and the medium of Comparative Example 2, which did not contain fumaric acid, were used, contaminating bacteria began to grow from about 96 hours, and after 168 hours, approximately 30% of the total bacterial count was contaminating bacteria. In contrast, the media of Examples 1 to 8 of the present invention were able to preferentially culture the 2-141-1 strain, and were able to maintain a high bacterial cell concentration up to 168 hours of culture. In particular, no contaminating bacteria were observed in the media of Examples 1 to 6 and 8, which contained 0.32% by weight (=0.02×16%) or more of milt-derived protein. In Example 7, a small amount of contaminating bacteria grew, but the proportion of contaminating bacteria was about 3% even after 168 hours.

[0026] (Yeast culture 2) The 2-141-1 strain was cultured and the viable cell count was calculated in the same manner as in Example 1 above, except that a yeast medium in which the pH was adjusted by varying the amounts of citric acid and sodium citrate used was used as shown in Table 3 below. The results are shown in Table 3 together with those of Example 1. [Table 3]

[0027] [Table 4]

[0028] In Examples 1, 9, and 10, in which the pH at the start of the culture was 2.9 to 3.72, the proliferation of unwanted bacteria was suppressed even after 168 hours of culture, and the 2-141-1 strain was preferentially grown. In Example 11, in which the pH at the start of the culture was 4.38, the proportion of unwanted bacteria was about 11% after 168 hours of culture, but the proliferation of unwanted bacteria was almost completely suppressed up to 96 hours. In addition, in all of the Examples, the bacterial cell concentration of the 2-141-1 strain was able to be maintained at a high level up to 168 hours of culture.

[0029] (Yeast culture 3) S. cerevisiae strain BY4741 (ATCC 201388) P.pastoris CBS7435 strain (ATCC 76273) Yeast cultivation and viable cell count calculation were performed in the same manner as in Example 1 above, except that Y. lipolytica PO1f (ATCC MYA-2613) was used. The results are shown in Table 5 together with those of Example 1.

[0030] [Table 5]

[0031] When cultured in the medium for the 2-141-1 strain of the present invention, the 2-141-1 strain grew smoothly, and its numbers could be maintained for 168 hours (one week). In contrast, when cultured using the medium for the 2-141-1 strain of the present invention, the other yeasts were unable to increase their cell mass compared to the 2-141-1 strain, and the increase was approximately 6% for the CBS7435 strain in Comparative Example 4, and approximately 3% for the BY4741 and PO1f strains in Comparative Examples 3 and 5.

Claims

1. It contains carbon source, nitrogen source, fumaric acid, ascorbic acid, and milt-derived protein as an antibacterial agent. The fumaric acid concentration is 0.03% by weight or more and 0.6% by weight or less, The ascorbic acid concentration is 0.03% by weight or more and 1.0% by weight or less, The concentration of the protein derived from the milt is 0.001% by weight or more and 0.05% by weight or less, pH is 2.5 or more and 4.2 or less, A yeast medium, which is for the yeast strain 2-141-1 deposited under the accession number NITE BP-02641.

2. It is in powder form and contains a carbon source, a nitrogen source, fumaric acid, ascorbic acid, milt-derived protein as an antibacterial agent, citric acid, and citrate salts. The composition contains, relative to 1 part by weight of a carbon source, 0.05 parts by weight or more and 0.5 parts by weight or less of fumaric acid, 0.1 parts by weight or more and 0.5 parts by weight or less of ascorbic acid, and 0.0016 parts by weight or more and 0.0064 parts by weight or less of milt-derived protein, A powder medium for yeast strain 2-141-1 deposited under accession number NITE BP-02641.

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