JP2018170996A - Culture medium for yeast culture, and yeast culture method, and proliferation method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium that can promote proliferation of yeast and can suppress mixture and proliferation of saprophytes.SOLUTION: A culture medium for yeast culture contains at least one of an inorganic nitrogen source and an organic acid and a salt thereof and has a pH of 2.5 or more and less than 4.0.SELECTED DRAWING: None

Description

  The present invention relates to a yeast culture medium and a yeast culture method and growth method using the same.

  Yeast is used for producing fermented foods, alcohols, organic acids and the like. Furthermore, yeast having oil / fat decomposability is used for decomposing oil and fat in wastewater in grease traps and the like.

  Wastewater from kitchens and food factories contains a large amount of oil, and a grease trap is provided to separate and discard these oil components that have accumulated and floated on the upper layer. However, the oil accumulated in the grease trap solidifies and remains as scum (lumps of oil) on the water surface of the grease trap, or it accumulates and adheres to the inner wall surface of the grease trap or inside the piping to block the piping. There is. At this time, the accumulated oil may be oxidized and spoiled and cause odors and pests. Also, if the accumulated oil is left unattended, the oil trap's ability to remove oil will be reduced, causing oil to flow into sewage and rivers. For this reason, when oil is accumulated in the grease trap, it is necessary to request a specialized supplier to remove the oil by vacuum treatment or high-pressure washing treatment, which increases costs.

  Therefore, in order to efficiently reduce the oil content in the grease trap, a method using yeast having oil / fat resolving power has been studied. For example, Patent Document 1 discloses a Yarrowia lipolytica that is excellent in the effect of reducing oil content in a grease trap.

Japanese Patent Laying-Open No. 2015-192611

  In yeast culture, a nutrient rich medium such as YM medium or YPD medium is usually used. Here, when the yeast described in Patent Document 1 is cultured in a YM medium or a YPD medium, it takes a long time (for example, about 72 hours) to grow to the maximum viable cell count. Therefore, a technique for growing yeast in a shorter time has been desired.

  In addition, when yeast is used in a grease trap, it may be necessary to periodically add yeast. Therefore, it is required to maintain the number of viable yeast grown without decreasing. However, when cultured in a YM medium, a YPD medium, or the like, there is a problem that not only yeast but also various bacteria are mixed and proliferated over time.

  Therefore, this invention is made | formed in view of the said situation, and it aims at providing the culture medium which can accelerate | stimulate the proliferation of yeast and can suppress mixing and growth of miscellaneous bacteria.

  In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, it has been found that the above-mentioned problems can be solved by a yeast culture medium containing an inorganic nitrogen source and at least one of an organic acid and a salt thereof and having a pH of 2.5 or more and less than 4.0. It came to.

  ADVANTAGE OF THE INVENTION According to this invention, the culture medium which can accelerate | stimulate the proliferation of yeast and can suppress mixing and growth of various bacteria is provided.

In an Example and a comparative example, it is a graph which shows the result of having investigated the change of viable count. In an Example and a comparative example, it is a graph which shows the result of having investigated the change of the contamination rate of various bacteria.

  Embodiments of the present invention will be described below. In addition, this invention is not limited only to the following embodiment.

  In the present specification, “X to Y” indicating a range means “X or more and Y or less”, and “weight” and “mass”, “wt%” and “mass%”, “part by weight” and “ “Part by mass” is treated as a synonym. Unless otherwise specified, measurement of operation and physical properties is performed under conditions of room temperature (20 to 25 ° C.) / Relative humidity 40 to 50% RH.

<Yeast culture medium>
According to one aspect of the present invention, there is provided a yeast culture medium comprising an inorganic nitrogen source and at least one of an organic acid and a salt thereof and having a pH of 2.5 or more and less than 4.0. With this configuration, yeast growth can be promoted, and contamination and growth of various bacteria can be suppressed.

(Inorganic nitrogen source)
The yeast culture medium of the present invention contains an inorganic nitrogen source as an essential component.

  In general, examples of a medium used for yeast culture include a YM medium and a YPD medium. When yeast is cultured using these media, there is a problem that the time required for growth to the maximum viable cell count becomes long (for example, about 72 hours). The present inventors have found that the growth of yeast can be promoted by using an inorganic nitrogen source as the nitrogen source of the medium. It is thought that growth is promoted by preferentially taking in nitrates, ammonium salts, and the like that yeast can easily use as a nitrogen source.

  Examples of the inorganic nitrogen source include nitrates such as sodium nitrate and calcium nitrate; ammonium salts such as ammonium nitrate, ammonium sulfate and ammonium chloride; nitrites such as sodium nitrite; ammonia; urea and the like. As the inorganic nitrogen source, one or more kinds can be selected and used. Of these, the inorganic nitrogen source is preferably selected from the group consisting of sodium nitrate, calcium nitrate, ammonium nitrate, ammonium sulfate and ammonium chloride, more preferably sodium nitrate, calcium nitrate and It is selected from the group consisting of ammonium nitrate, more preferably sodium nitrate. By using sodium nitrate as the inorganic nitrogen source, the maximum viable cell count can be increased as compared with other inorganic nitrogen sources.

  The compounding amount of the inorganic nitrogen source is, for example, 0.01 to 1.0 w / v%, preferably 0.1 to 0.5 w / v%, based on the total amount of the yeast culture medium.

(Organic acids and their salts)
The yeast culture medium of the present invention contains at least one of an organic acid and a salt thereof as an essential component. By using at least one of the organic acid and its salt, contamination and growth of various bacteria can be suppressed.

  Examples of organic acids and salts thereof include fumaric acid, sodium hydrogen fumarate, acetic acid, lactic acid, succinic acid, gluconic acid, pyrpinic acid, citric acid and the like. As an organic acid and its salt, it can select and use 1 type (s) or 2 or more types. The organic acid and its salt are preferably selected from the group consisting of fumaric acid, sodium hydrogen fumarate, and acetic acid, more preferably selected from fumaric acid and sodium hydrogen fumarate, and more preferably, from the viewpoint of suppressing contamination with various bacteria. Is fumaric acid. By using fumaric acid as the organic acid, contamination and growth of various bacteria can be suppressed even when the blending amount is smaller than that of other organic acids.

  The blending amount of the organic acid and its salt is, for example, 0.01 to 2.0 w / v% with respect to the total amount of the yeast culture medium. When fumaric acid is used as the organic acid, the amount of fumaric acid is preferably 0.05 to 0.40 w / v%, more preferably more than 0.05 w / v%, based on the total amount of the yeast culture medium. It is 0.40 w / v% or less, More preferably, it is 0.08-0.20 w / v%.

(Other nutrients)
The yeast culture medium of the present invention can contain a carbon source, an organic nitrogen source, and a mineral as needed in addition to the inorganic nitrogen source and at least one of an organic acid and a salt thereof.

  Carbon sources 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, glucitol, mannitol, galactitol, N-acetyl-D-glucosamine, saccharides such as starch, starch hydrolysate, molasses, and molasses, natural products such as wheat and rice, alcohols such as glycerol, methanol, and ethanol And hydrocarbons such as hexadecane. These carbon sources are appropriately selected in consideration of the assimilation by the yeast to be cultured. Moreover, the said carbon source can be used 1 type or 2 types or more selected.

  Organic nitrogen sources include yeast extract, wheat extract, meat extract, fish extract, peptone, polypeptone, malt extract, soybean hydrolysate, soybean powder, casein, milk casein, casamino acid, glycine, glutamic acid, aspartic acid, etc. Examples include amino acids, corn steep liquor, other animals, plants, and microbial hydrolysates. These organic nitrogen sources are appropriately selected in consideration of the assimilation by the yeast to be cultured. Further, one or more of the above organic carbon sources can be selected and used.

  Examples of the mineral include phosphorus, sulfur, potassium, calcium, magnesium, iron, sodium, cobalt, copper, zinc, nickel and the like. These minerals are appropriately selected in consideration of the assimilation by the yeast to be cultured. In addition, one or more of the above minerals can be selected and used.

(PH)
The yeast culture medium of the present invention has a pH of 2.5 or more and less than 4.0. If the pH is less than 2.5, the yeast cannot be efficiently propagated. A pH of 4.0 or higher is not preferable because there is a possibility that various bacteria may be mixed and proliferate. The pH is preferably 2.5 to 3.5, more preferably 2.5 to 3.0. The pH of the yeast culture medium can be measured using a pH meter.

  Since the culture medium for yeast culture of this invention contains at least one of an organic acid and its salt, the pH of a culture medium can be adjusted with the compounding quantity of an organic acid and its salt.

(yeast)
The yeast cultured in the yeast culture medium of the present invention is not particularly limited as long as it can grow in an environment having a pH of 2.5 or more and less than 4.0, but preferably Yarrowia, Candida, Pichia Genus Hansenula, Saccharomyces, Kluyveromyces, Trichosporon and the like.

  Specific examples of yeasts include Yarrowia lipolytica, Yarrowia sp., Candida famata, Candida bombicola, and Candida cerd. Candida rugosa, Candida utilis, Candida intermedia, Candida paralipolytica, Candida antarctica , Candida sp., Pichia farinosa, Pichia kluyveri, Pichia canadensis, Pichia promenta (Pichia promia) ), Pichia menbranaefaciens, Pichia rhodanensis, Hansenura anumala, Hansenura hansenuran Hansura sulata), Hansenula ciferrii (Hansenura ciferrii), Hansenula polymorpha (Hansenula polymorpha), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces Fell Mentha tee (Saccharomyces fermentati), Saccharomyces Norubenshisu (Saccharomyces norbensis), Saccharomyces Obiforumisu (Saccharomyces Oviformis), Kluyveromyces marxianus, Kluyveromyces lactis, Trichosporon brassicae (T ichosporon brassicae), Trichosporon capitatas, Trichosporon chuaneum, Trichosporon ferntan (Trichosporon fermentans), Trichosporon fermentans (Trichosporon fermentans) Species (Trichosporon sp. ) And the like.

  Among these yeasts, the yeast culture medium of the present invention can further promote the growth of Yarrowia yeasts. Yarrowia lipolytica ATCC 48436, Yarrowia lipolytica NBRC1548, Yarrowia lipolytica LM02-011 (Accession number NITE P-01813), Yarrowia lipolytica NBRC0746, Yarrowia lipolytica NBRC0746 ), Yarrowia sp. Such as Yarrowia YH-01 can be exemplified, Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-011 (accession number NITE P-01813) is more preferable. .

  The above yeast can be obtained from culture collections such as ATCC, NBRC, DSMZ and the like.

(Method for preparing yeast culture medium)
The yeast culture medium of the present invention can be used as a liquid medium or a solid medium. From the viewpoint of growing yeast, the yeast culture medium of the present invention is preferably a liquid medium.

  The method for preparing the medium is not particularly limited, and a conventionally known method can be used. For example, it can be prepared by dissolving an inorganic nitrogen source, an organic acid and a salt thereof, and if necessary, an organic nitrogen source, a carbon source and a mineral in water. When it is necessary to adjust the pH of the culture medium for yeast culture, it is preferable to use the organic acid and its salt as a pH adjuster. Moreover, acids other than the said organic acid and alkalis can also be used. The acid or alkali is not particularly limited, but acids such as hydrochloric acid, nitric acid, sulfuric acid, and acetic acid, and alkalis such as sodium hydroxide and potassium hydroxide are used. Moreover, you may add an antifoamer as needed.

<Culture method and growth method of yeast>
In one embodiment of the present invention, there is provided a method for culturing yeast, comprising culturing yeast in the above yeast culture medium.

  The yeast according to the present invention can be cultured by a usual method. Depending on the type of yeast, the cells are cultured under aerobic conditions or anaerobic conditions. In the former case, the yeast is cultured by shaking or aeration stirring. Alternatively, the yeast may be cultured continuously or in batches. The culture conditions are appropriately selected and are not particularly limited as long as the yeast according to the present invention can grow, and may be appropriately selected according to the type of yeast to be cultured. Moreover, it does not restrict | limit especially about the amount of microbial cells added to a yeast culture medium, It can adjust suitably.

  The culture temperature is usually 15 to 50 ° C, preferably 25 to 40 ° C.

  The culture time is not particularly limited, and varies depending on the culture conditions, the usage form of the yeast after the culture, and the like.

  When cultivating yeast in the yeast culture medium of the present invention, various forms of yeast can be used. For example, yeast can be suspended in a culture solution, recovered as a solid content from the culture solution, dried, or immobilized on a carrier.

  When using yeast recovered as a solid content from the culture solution, any means known to those skilled in the art can be employed as the recovery method. For example, the yeast culture liquid cultured by the above-mentioned method can be obtained by solid-liquid separation by centrifugation, filtration, etc., and collecting the solid content. If this solid content is dried (for example, freeze-dried), dried yeast can be obtained.

  When using yeast in a state of being immobilized on a carrier, the carrier for immobilizing the yeast is not particularly limited as long as it can immobilize the yeast, and is generally used for immobilizing microorganisms. The carrier to be used is used in the same manner or appropriately modified. For example, a method of including and fixing on a gel material such as alginic acid, polyvinyl alcohol, gellan gum, agarose, cellulose, dextran, and a method of adsorbing and fixing to a surface of glass, activated carbon, polystyrene, polyethylene, polypropylene, wood, silica gel, etc. Can be used.

  Further, the method for immobilizing yeast on a carrier is not particularly limited, and a general method for immobilizing microorganisms is used in the same manner or appropriately modified. For example, an immobilization method by pouring a yeast culture solution into a carrier, an immobilization method by pouring the yeast culture solution into a carrier using an aspirator, and a medium in which the yeast culture solution is sterilized For example, a method of pouring into a mixture of a carrier and a carrier, culturing with shaking, and naturally drying the carrier taken out of the mixture.

  In a preferred form of the present invention, the yeast is used in the form of a live bacterial preparation from the viewpoint of the viability of the yeast during storage. The yeast can be used as the yeast. When the yeast is used, the viable preparation includes a yeast-containing layer formed on a porous carrier, and the yeast-containing layer is 0.8 parts by weight with respect to 1 part by weight of the yeast as dry cells. More than 1.5 parts by weight of trehalose, 0.01 to 5 parts by weight of milk protein, 0.03 to 10 parts by weight of amino acid, and 0.02 to 10 parts by weight of metal salt are contained. When trehalose is contained in the yeast-containing layer in an amount of 0.8 parts by weight or more with respect to 1 part by weight of the yeast as dry cells, the survival rate of the yeast during formulation can be improved. Moreover, the fall of the survival rate of the yeast during the preservation | save period of a living microbe formulation can be prevented by containing less than 1.5 weight part. From the viewpoint of a further suitable balance between the survival rate during the formulation of yeast and the survival rate during the storage period, the trehalose content of the yeast-containing layer is preferably 1 part by weight of yeast in terms of dry cells. Is more than 0.9 parts by weight and less than 1.5 parts by weight, more preferably 1 part by weight or more and 1.4 parts by weight or less.

  In addition, disaccharides other than trehalose, for example, disaccharides other than trehalose such as sucrose, lactose and maltose may be used alone or in combination of two or more and used together with trehalose. In this case, the content of disaccharides other than trehalose is, for example, 0.01 parts by mass or more and less than 0.8 parts by mass with respect to 1 part by weight of non-spore bacteria as dry cells, preferably 0.1 parts by mass. Part to 0.5 parts by weight.

  It does not restrict | limit especially as a milk protein in a yeast content layer, The material containing various conventionally well-known milk proteins can be used. Although raw materials with a high milk fat content such as whole milk powder can be used, when producing a live bacterial preparation for fat and oil decomposition such as that used in a grease trap, it is preferable that the oil content in the preparation is low, for example Preferred examples include casein, sodium caseinate, whey, concentrated whey, whey protein concentrate (WPC), whey protein isolate (WPI), whey powder, skim milk, milk protein concentrate (MPC), nonfat dry milk, α -Lactalbumin, (beta) -lactoglobulin etc. can be illustrated, These can be used individually by 1 type or in mixture of 2 or more types. Milk protein is 0.01-5 weight part with respect to 1 weight part of said yeast as a dry microbial cell in a yeast content layer, Preferably it is 0.02-1 weight part, More preferably, it is 0.05-0. By including 5 parts by weight, the survival rate of yeast during formulation can be improved.

  Amino acids in the yeast-containing layer are, for example, glycine, alanine, valine, leucine, isoleucine, phenylalanine, glutamine, glutamic acid, asparagine, aspartic acid, cysteine, lysine, serine, threonine, methionine, tryptophan, histidine, algin, proline, tyrosine, And derivatives thereof. Examples of amino acid derivatives include amino acid salts (for example, potassium salts and sodium salts), ester forms, hydrates, solvates and the like. The above amino acids can be used alone or in combination of two or more. An amino acid is 0.03-10 weight part with respect to 1 weight part of said yeast as a dry microbial cell in a yeast content layer, Preferably it is 0.1-5 weight part, More preferably, it is 0.1-3 weight By including a part, the survival rate of the yeast at the time of formulating can be improved. In addition, when 2 or more types of amino acids are contained in an oil decomposition layer, the said numerical value shows a total amount of 2 or more types.

  Examples of the metal salt in the yeast-containing layer include sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), nickel (Ni), zinc (Zn), Sulfate, sulfite, hyposulfite, persulfate, thiosulfate, carbonate, phosphate, pyrophosphate, hydrochloride, nitrate, nitrite, acetate, propion of metal elements such as copper (Cu) Examples thereof include, but are not limited to, acid salts, butyrate salts, citrate salts, oxalate salts, halides (eg, fluorides, chlorides, bromides, iodides). More specifically, the metal salt is, for example, sodium sulfate, sodium sulfite, sodium hyposulfite, sodium thiosulfate, sodium carbonate, sodium persulfate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium acetate. , Sodium nitrate, sodium nitrite, sodium acetate, sodium citrate, sodium oxalate, sodium chloride, potassium sulfate, potassium sulfite, potassium hyposulfite, potassium thiosulfate, potassium carbonate, potassium persulfate, monopotassium phosphate, phosphoric acid Dipotassium, tripotassium phosphate, potassium acetate, potassium nitrate, potassium nitrite, potassium acetate, potassium citrate, potassium oxalate, potassium chloride, magnesium sulfate, magnesium sulfite, magnesium thiosulfate, magnesium carbonate, primary li Magnesium phosphate, dibasic magnesium phosphate, tribasic magnesium phosphate, magnesium pyrophosphate, magnesium nitrate, magnesium nitrite, magnesium acetate, magnesium citrate, magnesium oxalate, magnesium chloride, calcium sulfate, calcium sulfite, calcium thiosulfate, Examples include calcium carbonate, calcium nitrate, calcium nitrite, calcium acetate, calcium citrate, calcium oxalate, and calcium chloride. Said metal salt can be used individually by 1 type or in mixture of 2 or more types. The metal salt in the yeast-containing layer is 0.02 to 10 parts by weight, preferably 0.05 to 1.5 parts by weight, and more preferably 0.1 parts by weight with respect to 1 part by weight of the yeast as dry cells. By including ˜1 part by weight, it is possible to prevent a decrease in the survival rate of the yeast during the storage period of the viable preparation. In addition, when a yeast content layer contains 2 or more types of metal salts, the said numerical value shows 2 or more types of total amount.

  From the viewpoint that the survival rate of yeast during the storage period is particularly excellent, sulfate, carbonate, phosphate, or a combination thereof, wherein the metal salt is an element selected from the group consisting of Na, K, Mg and Ca It is preferable that it is Mg and / or Ca sulfate. The “metal salt” includes hydrates and solvates of metal salts.

  In addition to the above components, the yeast-containing layer optionally includes, for example, monosaccharides such as glucose and fructose; disaccharides other than trehalose such as sucrose, lactose and maltose; cyclodextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxy Polysaccharides such as methylcellulose, crystalline cellulose and corn starch; proteins such as soy protein; protein hydrolysates and peptides such as soy peptide, gelatin, peptone, and tryptone; fats and oils such as soybean oil, rapeseed oil, palm oil, sesame oil, olive oil; ascorbine Acids, salts thereof, vitamins such as tocopherols; surfactants such as polyglycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters; may include polyethylene glycol, glycerin, etc. .

  The material of the porous carrier that can be used in the viable bacterial preparation is not particularly limited. For example, diatomaceous earth, perlite, vermiculite, talc, clay, zeolite, bentonite, kaolin, calcium carbonate, activated clay, titanium dioxide Silica sand, pumice, activated carbon, carbon black, graphite, polylactic acid, polystyrene, polyvinyl chloride and the like can be exemplified, but are not limited thereto. The material of the porous carrier is preferably diatomaceous earth from the viewpoint of the survival rate of the yeast during formulation.

  In addition, “porous” in the porous carrier that can be used for the above-mentioned viable microbe preparation refers to a state having a large number of small cellular voids on the surface of the carrier. Preferably, the porous carrier has a bulk density (tapping bulk density) of 0.01 to 2 g / ml. By using a porous carrier having a bulk density as described above, the survival rate of the yeast after the formulation step can be further improved. The bulk density of the porous carrier is more preferably 0.05 to 1.5 g / ml, still more preferably 0.2 to 1 g / ml. In addition, said bulk density is the value measured by the tapping bulk density measuring method.

  From the viewpoint of the survival rate of yeast during formulation, the carrier is preferably in the form of particles having an average particle diameter (diameter, volume basis) of 1 to 300 μm, and the average particle diameter (diameter, volume basis) is 10. More preferably, it is in the form of particles of ˜200 μm, and more preferably 30 to 150 μm. The particle size of the carrier is a value measured by a laser diffraction method.

  The viable bacterial preparation can be formulated by spraying a spray liquid onto a porous carrier that can be used in the live bacterial preparation. The spray solution can be prepared by adding the above-described yeast and trehalose, milk protein, amino acid, metal salt and other components as necessary to water and stirring as necessary. As the water used for the preparation of the spray liquid, any of tap water, distilled water, ion exchange water, pure water, ultrapure water, etc. may be used, but distilled water, ion exchange water, pure water with less impurities. It is preferable to use ultrapure water. A mixed solution in which a lower alcohol such as methanol, ethanol or propanol or a polar solvent such as acetone is appropriately added to water may be used.

  Spraying of the spray liquid onto the porous carrier that can be used in the above-mentioned viable cell preparation can be performed using, for example, a fluidized bed granulator. By formulating with a fluidized bed granulation method, the yeast-containing layer can be formed on the porous carrier at a relatively low temperature. For this reason, it is advantageous from a viewpoint of the improvement of the survival rate at the time of formulating. Further, it is advantageous in that the above-mentioned yeast-containing layer can be uniformly formed on the porous carrier by formulating with a fluidized bed granulation method.

  The spray amount of the spray liquid with respect to the porous carrier can be arbitrarily set, and is not particularly limited. However, for example, 0.1 to 10 weights as the solid content of the spray liquid with respect to 1 part by weight of the porous carrier. The ratio is preferably 0.5 to 2 parts by weight.

  The spray liquid is usually sprayed onto the porous carrier at a rate of about 50 to 300 g / min.

  In the case of formulation by a fluidized bed granulation method, the temperature of the hot air may be appropriately set in consideration of the heat resistance of the yeast, etc., for example, the inlet temperature is 30-60 ° C., preferably 35-50 ° C. It is. The temperature of the fluidized bed may be appropriately set in consideration of the heat resistance of yeast and the like, and is, for example, a temperature of 30 to 60 ° C, preferably 35 to 50 ° C. After spraying, the porous carrier sprayed with the spray liquid may be dried as it is in a fluidized bed granulator, or may be dried separately in a dryer.

  The powdered viable bacterial preparation may be partially or entirely coated with a known coating agent depending on the use. In addition, powders such as lubricants (for example, talc, mica, silica, magnesium stearate), desiccants (calcium oxide, silica gel), fillers, etc. may be mixed with the viable cell preparation at an arbitrary ratio to form a mixed preparation. . Further, the preparation may be pulverized or classified so as to have a desired particle size.

  From the viewpoint of the survival rate of yeast during the storage period, the water content of the viable bacterial preparation after formulation is preferably 3 to 8% by weight, and more preferably 4.5 to 7.5% by weight. .

  By using the yeast culture medium of the present invention, the time for the yeast to grow to the maximum viable cell count can be shortened. For example, in the case of culturing by the method described in Examples described later, it takes about 72 hours for the YM medium conventionally used to grow Yarrowia lipolytica to the maximum viable count. On the other hand, by using the yeast culture medium of the present invention, if cultured for about 24 to 32 hours, Yarrowia lipolytica can be grown to the maximum viable cell count.

  Further, in the yeast culture medium of the present invention, it is not necessary to perform strict aseptic operation because contamination and growth of various bacteria can be suppressed. For example, after the yeast culture medium is prepared, the yeast culture medium may be cultured without sterilizing the yeast culture medium using an autoclave or the like. Moreover, it is not necessary to use an apparatus for performing aseptic operation such as a clean bench. Furthermore, since contamination and growth of miscellaneous bacteria can be suppressed, the maximum viable cell count can be reduced for a long time (for example, 12 to 72 hours or more) even if the culture is continued after the yeast has reached the maximum viable cell count. Almost can be maintained.

  As described above, the yeast culture medium of the present invention can grow yeast in a shorter time than conventional culture media. Therefore, in one embodiment of the present invention, there is provided a method for promoting the growth of yeast, comprising culturing yeast in the yeast culture medium.

  The effects of the present invention will be described using the following examples and comparative examples. However, the technical scope of the present invention is not limited only to the following examples.

(Preparation of culture medium for yeast culture)
Each component was melt | dissolved in distilled water so that it might become a composition of following Table 1, and the culture medium for Yeast culture | cultivation of Examples 1-5 and YM culture medium (comparative example) were prepared. The pH of the YM medium was adjusted using hydrochloric acid. In addition, the pH of the culture medium for yeast of Examples 1-5 is not adjusted. Moreover, "-" in Table 1 shows that it has not mix | blended.

  In Table 1 below, B-317F (manufactured by ADEKA Corporation) was used as the antifoaming agent.

(Preparation of viable bacterial preparation)
Yarrowia lipolytica LM02-011 strain (As of March 6, 2014, deposited with the National Institute of Technology and Evaluation Technology Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture) The deposit number is NITE P-01813.) Was inoculated into the following liquid medium and cultured at 30 ° C. for 48 hours (stirring speed: 300 rpm) with a jar fermenter to obtain a culture solution. .

Preparation method of liquid medium: final concentration is 0.18% (w / v) polypeptone, 0.12% (w / v) meat extract, 0.07% (w / v) NaHCO ₃ , 0.005% (w / V) NaCl, 0.002% (w / v) KCl, 0.002% (w / v) CaCl ₂ .2H ₂ O, 0.003% (w / v)) MgSO ₄ .7H ₂ O So that it was dissolved in pure water. The liquid medium was adjusted to pH 5 with hydrochloric acid and then autoclaved.

The obtained culture broth was centrifuged (× 10,000 g) for 10 minutes, and the cells were collected. 11% by weight of yeast, 14% by weight of trehalose, 3% by weight of skim milk (1% by weight as milk protein), 3% by weight of glycine and 1% by weight of MgSO ₄ .7H ₂ O %, CaSO ₄ .2H ₂ O was added to distilled water so as to be 1% by mass, and mixed to prepare a spray solution. As milk protein, skim milk containing 35% by weight of protein and 52% by weight of lactose in the solid content was used.

  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.) in a fluidized bed granulator (FA-LAB-1, Paulek Inc.) Set. 1077 g of the spray solution was sprayed on diatomaceous earth at a rate of 143 g / min, and the contents were dried by blowing hot air at 40 ° C. After spraying, hot air at 40 ° C. was then blown for 40 minutes in the fluidized bed granulator to dry the granulated product. As a result, a viable cell preparation was obtained. The water content of the viable preparation was 5.5% by weight.

(Calculation of the number of viable bacteria in the viable cell preparation)
The number of viable bacteria per 1 g of the viable cell preparation was calculated by the following method. In the examples and comparative examples, the viable cell counts calculated before the implementation of the examples and comparative examples (within 48 hours) were used. The storage of the viable cell preparation was carried out at 23 ° C. in a light-shielding glass vial.

  The viable bacterial preparation is serially diluted by stirring for 1 minute at 25 ° C. in 100 times amount of distilled water, and the resulting diluted solution is added to the agar medium (composition: the above liquid medium to agar concentration so that the final concentration is 20% by weight). Applied to the surface). After culturing the bacteria at 30 ° C. for 48 hours, the number of colonies formed on the agar medium was counted. From the number of colonies, the number of viable bacteria per 1 g of the preparation was calculated.

(Yeast culture)
Yeast was cultured by the following method.
(1) 500 mL of the above yeast culture medium or YM medium was dispensed into a 1 L tall beaker.
(2) 15 mg (yeast: 8.5 × 10 ⁷ CFU / L) of the viable preparation prepared above was added to the tall beaker of (1).
(3) After the viable cell preparation was added, the tall beaker of (2) was aerobically cultured at 30 ° C. for 96 hours to obtain a culture solution.

(Calculation of the number of viable yeast and calculation of contamination rate)
In Examples 1 to 5 and Comparative Example, the culture solution was sampled every 12 hours from the start of culture, diluted serially with distilled water, and applied to the surface of a broth medium solidified with agar. After application, the cells were cultured at 30 ° C. for 48 hours, and the number of LM02-011 strains and bacteria colonies formed on the medium were counted. The number of viable bacteria of LM02-011 strain was calculated from the number of colonies of LM02-011 strain. The results are shown in FIG. Moreover, the contamination rate of various bacteria was calculated using the following formula. The results are shown in FIG.

  In addition, the colony form of LM02-011 strain is a cream color and a circular dome shape, and can be clearly distinguished from colonies of various bacteria.

  As shown in FIGS. 1 and 2, in Examples 1 to 5, it can be seen that the growth rate of the LM02-011 strain can be improved by using nitrate as compared with the comparative example. Moreover, it turns out that mixing of miscellaneous bacteria can be suppressed for 72 hours or more by using fumaric acid.

  In Example 1, compared with Example 2 and 3, it turns out that the maximum viable cell count of yeast can be increased more by using sodium nitrate as an inorganic nitrogen source.

Claims (10)

  A culture medium for yeast culture comprising an inorganic nitrogen source and at least one of an organic acid and a salt thereof, and having a pH of 2.5 or more and less than 4.0.   The culture medium for yeast culture according to claim 1, wherein the organic acid and its salt are selected from the group consisting of fumaric acid, sodium hydrogen fumarate and acetic acid.   The yeast culture medium according to claim 2, wherein the organic acid is fumaric acid.   The culture medium for yeast culture according to any one of claims 1 to 3, wherein a blending amount of the organic acid and a salt thereof is 0.05 to 0.40 w / v% with respect to a total amount of the culture medium for yeast culture. .   The culture medium for yeast culture according to any one of claims 1 to 4, wherein the inorganic nitrogen source is selected from the group consisting of nitrate, ammonium salt, nitrite, ammonia and urea.   The culture medium for yeast culture according to claim 5, wherein the inorganic nitrogen source is selected from the group consisting of sodium nitrate, calcium nitrate, ammonium nitrate, ammonium sulfate and ammonium chloride.   The yeast culture medium according to any one of claims 1 to 6, wherein the yeast is a yeast that can grow in an environment having a pH of 2.5 or more and less than 4.0.   8. The yeast according to claim 1, wherein the yeast is selected from the group consisting of Yarrowia, Candida, Pichia, Hansenula, Saccharomyces, Kluyveromyces, and Trichosporon. A culture medium for yeast culture according to 1.   The culture medium for yeast culture | cultivation of Claim 8 whose said yeast is Yarrowia lipolytica (Yarrowia lipolytica).   A method for culturing yeast, comprising culturing yeast in the culture medium for yeast culture according to any one of claims 1 to 9.