JP2018171003A - Culture medium for yeast culture and yeast culture method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique that can improve yeast culture efficiency to increase an amount of bacteria collection per unit volume of a culture medium.SOLUTION: A culture medium for yeast culture contains a phosphorus source, a magnesium source, and solvent. The carbon source contains more than 100 g/L of glycerol, and also contains trace elements consisting of iron, calcium, copper, boron, and manganese in a total content of 0.0008 g/L or more in terms of elements.SELECTED DRAWING: None

Description

  The present invention relates to a yeast culture medium and a yeast culture 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, according to the study by the present inventors, when the yeast described in Patent Document 1 is cultured using a YM medium or a YPD medium, there is a problem that the amount of collected bacteria per unit volume of the medium is small. It has been found. For example, when the yeast described in Patent Document 1 was cultured in a jar fermenter using a YM medium, it was found that the cell weight obtained even when cultured for 72 hours was less than 40 g / L. Thus, if the amount of collected bacteria per unit volume of the medium is small, a large amount of medium is required to obtain the target amount of bacteria, which is extremely inefficient. Therefore, it has been desired to develop a technique for further increasing the culture efficiency (that is, increasing the amount of collected bacteria per unit volume of the medium).

  Therefore, the present invention has been made in view of the above circumstances, and an object thereof is to provide a technique capable of improving the culture efficiency of yeast and increasing the amount of collected bacteria per unit volume of the medium. .

  In order to solve the above-mentioned problems, the present inventors have intensively studied. As a result, by using glycerol as a carbon source, the concentration is made higher than a certain value, and a trace element (consisting of iron, calcium, copper, boron, and manganese) in an amount greater than a certain value is contained. Has been found to be solved, and the present invention has been completed.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to improve the culture | cultivation efficiency of yeast and to raise the amount of collected bacteria per unit volume of a culture medium.

  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 an aspect of the present invention, there is provided a yeast culture medium containing a carbon source, a nitrogen source, a phosphorus source, a magnesium source, and a solvent, wherein the carbon source contains more than 100 g / L of glycerol. A yeast culture medium is provided which contains trace elements composed of iron, calcium, copper, boron, and manganese in a total amount of these elements of 0.0008 g / L or more. By culturing yeast using a culture medium having such a configuration, it is possible to improve the culture efficiency of the yeast and increase the amount of collected bacteria per unit volume of the culture medium. Hereinafter, the yeast culture medium according to the embodiment will be described in more detail.

  First, the yeast culture medium according to this embodiment contains a carbon source, a nitrogen source, a phosphorus source, a magnesium source, and a solvent as a basic configuration. Since it contains a solvent, the yeast culture medium according to this embodiment is a liquid medium.

(Carbon source)
The yeast culture medium according to this embodiment essentially contains glycerol as a carbon source. And the content of glycerol is characterized by being over 100 g / L. Glycerol (C ₃ H ₈ O ₃ ) is a substance that is chemically reduced compared to glucose (C ₆ H ₁₂ O ₆ ), and can provide an improved reducing power in the metabolic process of yeast. it can. Further, since reducing power is often required in the metabolic process of yeast, it is considered that the culturing efficiency is improved by containing glycerol in an amount exceeding 100 g / L. The glycerol content in the yeast culture medium according to this embodiment is preferably 110 g / L or more, more preferably 120 g / L or more, still more preferably 130 g / L or more, and particularly preferably 140 g. / L or more, and most preferably 150 g / L or more. On the other hand, there is no particular limitation on the upper limit value of the glycerol content, but usually it may be about 250 g / L or less.

  In addition, the yeast culture medium according to this embodiment may further contain a substance other than glycerol as a carbon source. Examples of carbon sources other than glycerol include glucose, fructose, cellobiose, raffinose, xylose, maltose, galactose, sorbose, glucosamine, ribose, arabinose, rhamnose, sucrose, trehalose, α-methyl-D-glucoside, salicin, melibiose, lactose, Merezitose, inulin, erythritol, glucitol, mannitol, galactitol, N-acetyl-D-glucosamine, starch, starch hydrolysate, sugars such as molasses and molasses, natural products such as wheat and rice, alcohols such as 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. One or more carbon sources other than glycerol can be selected and used.

(Nitrogen source)
It is preferable that the culture medium for yeast culture which concerns on this form contains an inorganic nitrogen source as a nitrogen source. In general, examples of a medium used for yeast culture include a YM medium and a YPD medium. When an inorganic nitrogen source is used as the nitrogen source of the culture medium for yeast, yeast growth can be promoted. This is considered to be promoted by preferentially taking in an inorganic nitrogen source that yeast can easily use as a nitrogen source.

  Examples of the inorganic nitrogen source include ammonium salts such as ammonium chloride, ammonium nitrate, and ammonium sulfate; nitrates such as sodium nitrate and calcium nitrate; 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 ammonium chloride, sodium nitrate, calcium nitrate, ammonium nitrate, and ammonium sulfate, more preferably ammonium chloride, sodium nitrate, from the viewpoint that yeast growth can be promoted. , Selected from the group consisting of calcium nitrate and ammonium nitrate, more preferably ammonium chloride or sodium nitrate, and particularly preferably ammonium chloride. By using ammonium chloride as the inorganic nitrogen source, the culture efficiency (the cell weight per unit volume of the medium) can be increased as compared with other inorganic nitrogen sources.

  Moreover, the culture medium for yeast culture which concerns on this form may further contain an organic nitrogen source as a nitrogen source. 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.

  The blending amount of the nitrogen source is, for example, 1.0 to 5.0 w / v%, preferably 2.0 to 4.0 w / v%, based on the total amount of the yeast culture medium. Among these, the compounding quantity of an inorganic nitrogen source is 0.5-3.0 w / v% with respect to the whole culture medium for yeast cultures, Preferably it is 1.0-2.0 w / v%.

(Phosphorus source)
As the phosphorus source, a phosphate can be suitably used. Examples of the phosphate as the phosphorus source include potassium monohydrogen phosphate, potassium dihydrogen phosphate, and ammonium phosphate. Among them, potassium dihydrogen phosphate can be preferably used.

  The amount of the phosphorus source is, for example, 0.1 to 0.5 w / v%, preferably 0.2 to 0.4 w / v% in terms of the weight of the phosphorus source compound relative to the total amount of the yeast culture medium.

(Magnesium source)
As the magnesium source, a magnesium salt can be suitably used. Examples of the magnesium salt as the magnesium source include nitrates and chlorides, and magnesium sulfate and hydrates thereof (for example, magnesium sulfate heptahydrate) can be preferably used.

  The compounding amount of the magnesium source is, for example, 0.05 to 0.3 w / v%, preferably 0.1 to 0.3 w / v% in terms of the weight of the magnesium source compound with respect to the total amount of the yeast culture medium.

(solvent)
As the solvent, water is usually used. Examples of water include pure water, distilled water, deionized water, and RO water, but are not particularly limited.

(Trace element)
Furthermore, the culture medium for yeast culture according to the present embodiment essentially contains five trace elements composed of iron, calcium, copper, boron, and manganese, and the total content in terms of these elements is 0. It also has a feature in that it is .0008 g / L or more. As described above, the glycerol content is set so as to exceed a predetermined value as described above while containing a predetermined amount of trace elements in a certain amount, thereby synergistically improving the culture efficiency of yeast. Thus, the weight of cells per unit volume of the medium can be dramatically increased. Although the mechanism by which such an effect is achieved is not completely clear, these predetermined trace elements act in a catalytic manner on enzymes that mediate the metabolic process of yeast. This is thought to be due to the fact that the metabolic processes of the cells are promoted in the direction of bacterial cell growth. In addition, the content of the predetermined (five types) trace elements (total content in terms of elements) is preferably 0.0009 g / L or more, more preferably 0.0010 g / L or more, and still more preferably. Is 0.0011 g / L or more. Moreover, the content of the predetermined (five kinds) trace elements (total content in terms of elements) is preferably 0.0008 to 0.0012 g / L. In addition, although there is no restriction | limiting in particular about the ratio (element conversion) of each content (element conversion) of these (5 types) trace elements, From a viewpoint of making the effect of this invention exhibit more, content of a boron atom is 1 mass. Parts, 5 to 9 parts by mass of iron atoms, 5 to 9 parts by mass of calcium atoms, 4 to 8 parts by mass of copper atoms, and 3 to 7 parts by mass of manganese atoms. More preferably, when the content of boron atoms is 1 part by mass, iron atoms are 6-8 parts by mass, calcium atoms are 6-8 parts by mass, copper atoms are 5-7 parts by mass, and manganese atoms are 4 to 6 parts by mass.

  In addition, there is no restriction | limiting in particular about the form in which the said predetermined | prescribed trace element is contained in a culture medium, As an iron source, divalent or trivalent iron chloride, sulfate, bromide, nitrate, etc. (for example, chloride chloride) And ferric sulfate, ferrous sulfate, and hydrates thereof. Examples of the calcium source include calcium chloride, carbonate, phosphate, sulfate, nitrate, and the like (for example, calcium chloride, calcium carbonate, calcium nitrate, and hydrates thereof). Examples of the copper source include copper sulfate, phosphate, acetate, bromide, and the like (for example, copper sulfate, copper phosphate, copper acetate, copper bromide, and hydrates thereof). Examples of the boron source include boric acid. Examples of the manganese source include manganese sulfates and nitrates (eg, manganese sulfate, manganese nitrate, and hydrates thereof).

  Moreover, the culture medium for yeast culture which concerns on this form may further contain trace elements other than the said predetermined (5 types) trace element. Examples of the trace element other than the predetermined trace element include, but are not limited to, sodium, cobalt, zinc, sulfur, nickel, and the like.

(PH)
Although there is no restriction | limiting in particular in the pH of the culture medium for yeast which concerns on this form, Since it is preferable to generally culture | cultivate yeast on weakly acidic conditions, Preferably it is 5.0-7.0, More preferably Is 5.5 to 6.5. Particularly, it is suitable for culturing Yarrowia genus such as Yarrowia lipolytica which is a yeast used in Examples described later. Here, the pH of the yeast culture medium can be measured using a pH meter. The pH of the medium should be adjusted by using an acid such as sulfuric acid, hydrochloric acid, nitric acid, acetic acid or citric acid; or a base such as sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate as a pH adjuster. Can do.

(yeast)
Yeast cultured in the yeast culture medium according to the present embodiment is not particularly limited, but includes Yarrowia, Candida, Pichia, Hansenura, and Saccharomyces. ), Kluyveromyces, Trichosporon, Lipomyces, Rhodotorula, Schizosaccharomyces n, Lactobacillus cc, Lactobacilli Yeast is preferably used.

  In the yeast culture medium of the present invention, among these yeasts, the culture efficiency of yeasts of the genus Yarrowia having a high oil content decomposition activity can be further improved. 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, etc. can be exemplified, but Yarrowia lipolytica is more preferable, and Yarrowia lipolytica LM02-011 (accession number NITE P-01813) is more preferable. . In addition, the above-mentioned yeast is available from culture collections, such as ATCC, NBRC, and DSMZ.

(Method for preparing yeast culture medium)
As described above, the yeast culture medium according to this embodiment is a liquid medium. The method for preparing the medium according to this embodiment is not particularly limited, and a conventionally known method can be used. For example, it can be prepared by dissolving a carbon source (including glycerol essential), a nitrogen source, a phosphorus source, a magnesium source, and the predetermined trace element in a solvent typified by water. When it is necessary to adjust the pH of the culture medium for yeast culture, the acid or alkali described above may be used as the pH adjuster. Moreover, you may add an antifoamer as needed.

<Culture method of yeast>
According to one aspect of the present invention, there is provided a yeast culturing method comprising culturing yeast in the 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.

  By using the yeast culture medium according to the present invention, the yeast culture efficiency can be increased, and the weight of the cells per unit volume of the medium can be increased. For example, when cultured by the method described in the examples described later, when Yarrowia lipolytica is cultured in a jar fermenter containing a conventionally used YM medium, the cells can be obtained even after 72 hours of culture. The weight is less than 40 g / L. In contrast, by using the yeast culture medium according to the present invention, the cell weight obtained under the same culture conditions can be increased by 7 times or more.

  As described above, the yeast culture medium according to the present invention can grow yeast with higher culture efficiency than conventional culture media.

  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 yeast culture medium≫
Example 1
Each component was dissolved in distilled water so as to have the composition shown in Table 1 below, and the yeast culture medium of Example 1 was prepared. For trace elements (Fe, Ca, Cu, B, Mn), Fe 0.03 w / v%, Ca 0.02 w / v%, Cu 0.02 w / v%, B0.005 w / v% with respect to 100 mL of water. By preparing a concentrated solution in which the components described in Table 1 below are dissolved in advance so that the concentration becomes Mn 0.02 w / v%, and adding this so as to achieve the concentration described in Table 1 below, A medium was prepared.

(Comparative Example 1)
Each component was dissolved in distilled water so as to have the composition shown in Table 2 below, and a yeast culture medium (YM medium) of Comparative Example 1 was prepared.

(Comparative Example 2)
A yeast culture medium of Comparative Example 2 was prepared in the same manner as in Example 1 described above, except that no trace element was added.

(Comparative Example 3)
A yeast culture medium of Comparative Example 3 was prepared in the same manner as in Example 1 described above except that the amount of the trace element was reduced to half.

(Comparative Example 4)
A yeast culture medium of Comparative Example 4 was prepared in the same manner as in Example 1 except that the concentration of glycerol was 1/3 (50 g / L (= 5 w / v%)).

(Comparative Example 5)
A yeast culture medium of Comparative Example 5 was prepared in the same manner as in Comparative Example 2 described above except that the concentration of glycerol was 1/3 (50 g / L (= 5 w / v%)).

(Comparative Example 6)
A yeast culture medium of Comparative Example 6 was prepared in the same manner as in Comparative Example 3 described above except that the concentration of glycerol was 1/3 (50 g / L (= 5 w / v%)).

≪Seed culture≫
100 mL of each of the media prepared in Examples and Comparative Examples prepared above was placed in four Sakaguchi flasks and sterilized.

  Next, yeast (Yarrowia lipolytica) was inoculated into each flask one platinum ear at a time, and cultured with shaking at 30 ° C. for 72 hours.

≪Culture in jar fermenter≫
About each of the culture medium of the Example adjusted above and each comparative example, culture | cultivation using a jar fermenter was performed with the following method.

  First, 5 L of medium was placed in a jar fermenter and sterilized. Next, 400 mL of the culture solution subjected to seed culture as described above was added and cultured at 30 ° C. for 72 hours. At this time, the pH was adjusted using sodium hydroxide and sulfuric acid so that the pH was 6 ± 0.1.

≪Bacteria collection≫
About the culture | cultivation by a jar fermenter, after precipitating a microbe using a centrifuge, the supernatant was thrown away and the cell weight was measured. The results are shown in Table 3 below.

  As can be seen from the results shown in Table 3, the culture efficiency (bacterial cell weight) was significantly increased in Example 1 as compared with each of Comparative Examples 1 to 6. In particular, it can be seen that the culture efficiency (bacterial cell weight) has been dramatically increased by 7 times or more compared to the case where the YM medium that has been conventionally used as a yeast culture medium is used.

Claims (9)

A yeast culture medium containing a carbon source, a nitrogen source, a phosphorus source, a magnesium source, and a solvent,
The carbon source comprises more than 100 g / L glycerol, and
A yeast culture medium comprising a trace element composed of iron, calcium, copper, boron, and manganese in a total amount in terms of these elements of 0.0008 g / L or more.   The culture medium for yeast culture | cultivation of Claim 1 whose content of the said glycerol is 150-250 g / L.   The culture medium for yeast culture of Claim 1 or 2 whose content of the said trace element is 0.0008-0.0012g / L.   When the content of the trace element (in terms of element) is 1 part by mass of the boron atom, the iron atom is 6 to 8 parts by mass, the calcium atom is 6 to 8 parts by mass, and the copper atom is 5 to 7 parts. The culture medium for yeast culture of any one of Claims 1-3 whose mass part and manganese atom are 4-6 mass parts.   The yeast culture medium according to any one of claims 1 to 4, wherein the nitrogen source is an inorganic nitrogen source.   The culture medium for yeast culture according to claim 5, wherein the inorganic nitrogen source is selected from the group consisting of ammonium salt, nitrate, nitrite, ammonia and urea.   The yeast is one 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 7 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 8.