JPS59175874A - Medium for yeast-lysing microorganism - Google Patents

Abstract

PURPOSE:To prepare a medium for the culture of yeast-lysing microorganisms, capable of actively proliferating the microorganisms and suppressing the proliferation of other general microorganisms, by adding a medium composition containing a phosphoric acid salt and a magnesium salt to yeast cells. CONSTITUTION:The objective medium can be prepared by adding a powdery or liquid composition containing a phosphoric acid salt such as dipotassium phosphate and one or more compounds selected from magnesium salts, ammonia, ammonium salts, amines, amine salts, peptides and proteins, to yeast cells. A sulfur-free amino acid, a nucleic acid-constituting base, etc. may be used as the amine as well as the conventional amine such as methylamine, dimethylamine, etc.

Description

[Detailed description of the invention] The present invention relates to a medium for yeast-dissolving microorganisms.

In recent years, searches for yeast lytic microorganisms have been conducted for the purpose of obtaining yeast cell wall lytic enzymes, preparing yeast protoplasts, and preparing yeast extracts, but the culture medium used for these searches does not contain yeast cells. When separating yeast-dissolving microorganisms from soil, sewage, activated sludge, etc., the sample When measuring the concentration of yeast-lytic microorganisms in yeast or when enriching yeast-lytic microorganisms, general microorganisms that are not yeast-soluble and have a fast growth rate will grow first, and the growth of yeast-lytic microorganisms will be suppressed and the purpose will be achieved. often cannot be reached. Furthermore, when attempting to pure isolate or pure culture yeast-dissolving microorganisms, yeast-dissolving microorganisms may not be obtained due to contamination with common microorganisms.

Therefore, in consideration of the above-mentioned situation, we used a medium for yeast lytic microorganisms mainly consisting of activated sludge extract and live yeast cells to suppress the growth of microorganisms that do not have yeast lytic activity, and at the same time to suppress the growth of yeast lytic microorganisms. Author 1-<Finding a way to promote 1
~Ta. (1939 patent application/9!; No. 37.2) 1 ~ ~, since activated sludge extract still contains impurities unnecessary for the growth of yeast-dissolving microorganisms, activated sludge extraction By preparing a medium in which only the substances necessary for the growth of yeast lytic microorganisms are extracted from the food, it is expected that the risk of contamination by other microorganisms during the cultivation of yeast lytic microorganisms will be reduced. When using yeast-dissolved microorganisms in activated sludge extract, it is better to have a clear culture medium composition from the viewpoint of food hygiene, and also to simplify culture medium preparation. A series of research efforts are underway to uncover growth-promoting factors. In other words, yeast lytic microorganism YLM-/(Feikoken Bibiri No. 67g/,
Hereinafter, as a result of searching for the yeast-dissolved microorganism growth promoting factor in the activated sludge extract using 0 named YLM-7-J, we found that magnesium salts, ammonium salts, amines, amino acids, peptides, It was revealed that proteins, bases, etc. are growth-promoting factors.In a medium composed of these factors, yeast-dissolving microorganisms, which are widely present in soil, various wastewaters, sewage, activated sludge, etc., proliferate well. On the other hand, it was discovered that the growth of other common microorganisms was also suppressed using activated sludge extract, and the present invention was completed.

A detailed description of the present invention follows below.

The medium for yeast lytic microorganisms in the present invention is prepared by adding the minimum necessary medium composition for the growth of yeast lytic microorganisms to yeast cells. The microorganism is allowed to grow aerobically at a temperature of about 30°C, preferably about 3θ°C, and at the same time the yeast cells are lysed.

An example of the composition of the medium composition is ro. Phosphate of about M, preferably dipotassium phosphate, 0.0 as magnesium ion. Preferably, a medium composition in which a magnesium salt such as magnesium sulfate and an ammonium ion are mixed to be about 1 ppm and an ammonium salt such as phosphoric acid and ammonium ion to be 7 mM or more is distilled. It is dissolved in water and used at pHg~/θ, preferably adjusted to about pH 7-9. Regarding the medium composition, it is effective to add only magnesium salt to phosphate or add only ammonium salt to phosphate, but Preferably, both salts are added. Instead of ammonium salts, amines or salts thereof or mixtures thereof may be used. The amines mentioned here include general amines such as monomethylamine, dimethylamine, trimethylamine, and tetramethylamine, as well as non-sulfur amino acids, peptides,
Refers to bases that are constituents of proteins and nucleic acids. Specific examples of non-sulfur amino acids include glycine, L-alanine, and L-alanine.
-Leucine, L-Isoleucine, L-Haijin, L-
Neutral aliphatic amino acids such as serine and L-threonine; acidic aliphatic amino acids such as L-aspartic acid and L-glutamic acid; basic amino acids such as L-arginine, L-lysine, and L-ornithine. Aliphatic amino acids; Heterocyclic amino acids such as L-proline, L-histidine, etc.; L-phenylalanine, L-tyrosine,
Examples include aromatic amino acids such as L-trypto7f7 and the like. A specific example of the peptide is L-arginyl-L-glutamic acid.

Specific examples of proteins include casein and albumin. Specific examples of bases that are constituents of nucleic acids include adenine, guanine, and
Examples thereof include nucleosides such as adenosine and guanosine, and nucleotides thereof such as adenylic acid and guanylic acid.

The above medium composition may be prepared with an aqueous solution at the time of use, and then adjusted to a desired pH;
After dissolving in the required amount of each salt with low hygroscopicity, adjust to the desired pH.
Add a powdered product with sodium carbonate to it at step 1 and dissolve it in a specified amount of distilled water just before use.
It can be used as

The yeast to be added to the medium composition can be of any genus or species, but since its lysis spectrum differs depending on the type of yeast-dissolving microorganism, the yeast is selected depending on the lysis spectrum of the target yeast-dissolving microorganism. The yeast concentration may be higher than /θ/rnl, but if the concentration is high (j×/θ〆rnl), the concentration of the medium composition can be increased in proportion to the yeast concentration to efficiently lyse the bacteria.

The medium can also be used as a solid medium by adding agar (approximately 0.7%), but if live yeast cells are added to the medium composition to make a solid medium, a heated solution of the medium composition and agar may be used. Cool to about txs℃ and then add live yeast cells.
It is necessary to create a flat plate immediately. When measuring lytic spots caused by yeast lytic bacteria on a plate medium, the yeast concentration should be
/♂~5xiJ"ime, lytic spots can clearly appear.

The following examples further illustrate the invention, and it is to be understood that the invention is not limited to these examples.

Example / (C) Preparation of yeast suspension YM medium (glucose/%, yeast extract 0.3%, malt extract 03, peptone 05%)/θθml
Place it in a flask and kill Hansenula at /2θ℃/θ2θ.
a) Y-/(Feikoken Bacterial Serial No. 3.!; No. 9'1) was inoculated with a platinum loop L and cultured with shaking at 3-°C for 2 days. After the culture is completed, the culture solution is centrifuged to collect the bacterial cells, and the cells are collected at about 70θ.
Wash twice with 1ml of sterile water. Wash the bacterial cells by approximately 2×/θ
It was suspended in sterilized water for 1 to 1 hour and the yeast suspension for 1 to 1 hour so that it was 9//me.

(YLM-7 preculture'//3M phosphate buffer Sml as MLSS/θ
Extract with hot water from activated sludge with θθθppm I ~ Restore to raw waste 1
- Add 2 ml of activated sludge extract, adjust p) I to θ, add distilled water to make it 9ru/3, put it into an L-shaped tube, sterilize it for 10 minutes at 2θ”C, and then -(1)
Add the yeast suspension/me obtained in
A loopful of the culture was inoculated and cultured with shaking at 1-13°C for 3 days.

(3) YLM-/ in medium supplemented with magnesium salts
growth//magnesium sulfate in 3 rnl of 15M phosphate buffer, θ, 0. ! ,2,!
Add θ, /θθ, 2θ0.20θ0 and /θθθ0μg and adjust the pH to θ. Add distilled water to make 9ml of the culture medium composition in an L-shaped tube and sterilize at 726°C for 79 minutes.
- After Example/ - Yeast suspension obtained in (1)/ml
3, and further Y obtained in Example/-(,,l!l
LM-/ pre-culture solution/θθμl was inoculated from 1 to 1, cultured with shaking in Toyo Kagaku Sangyo Co., Ltd. Biophoto Recorder TN-//, 2D at 3 to 0°C, and absorbance was continuously measured. As a result, as shown in Table 1, the time required to reach the minimum absorbance indicating yeast lysis was the shortest in the medium containing magnesium sulfate and ro-:zoθμ. It was also confirmed by microscopic observation that the change in absorbance, yeast lysis, and YLM-/ growth occurred in parallel 1-, so from now on, we will use the time to reach the minimum absorbance to calculate the yeast lysis time and the growth of YLM-7. We decided to use time as an indicator.

Table 1 Examples! Magnesium sulfate is used as magnesium in phosphate buffer jme with low concentration! Add θμ2 and further add ammonium phosphate to θ, /1.2, '%, g, /co and 2θ, respectively.
After adding μmole and adjusting the concentration to θ, add distilled water to make θml, put the medium composition into an L-shaped tube /2θ℃/θ
After sterilization for 1 minute, culturing was carried out under the same conditions as in Example 1-(3). As a result, as shown in Table 2, the time to reach the minimum absorbance was determined by the amount of ammonium phosphate added.
In the case of 2θμmole, the time required to reach the minimum absorbance of the medium to which 2rnl of the activated sludge extract obtained in Example/-(, 2+) was added was almost comparable.

Table 2 Example 3 Culture was carried out under the same conditions as in Example 3, except that instead of ammonium phosphate, the 9 elemental compounds shown in Table 3 were added to the medium at a concentration of jμmole//θml medium. .

As a result, as shown in Table 3, jetanolamine, monomethylamine, dimethylamine, trimethylamine, tetramethylammonium chloride, β-phenethylamine,
Methionine, amino acids other than cysteine, L-arginyl-L-glutamic acid, casein, albumin, adenine, guanine, atenosine, guanosine, adenylic acid,
Addition of guanylic acid and cytosine reduced the time to reach minimum absorbance.

From this, not only ammonium ions but also a wide range of substances belonging to amines can be YLM-
It was found to have a proliferation promoting activity of 7.

Example medium composition number ■ Phosphate disu) IJum/dihydrate 7/jg, phosphate-potassium/, g f? , magnesium sulfate heptahydrate 20j ■ and monoammonium phosphate //3 mg medium composition number ■ dipotassium phosphate tag 7, magnesium sulfate heptahydrate 20.3 ■ and ammonium phosphate i/smg medium Composition number ■ Medium composition number ■ Each compound of medium composition plus 22 ml of sodium carbonate Medium composition number ■ Dipotassium phosphate /, / 3
S', magnesium sulfate heptahydrate;; o, s my and ammonium phosphate//
Jmq Medium composition number■ Medium composition number■ 10j of sodium carbonate is added to each compound of medium composition number■ Medium composition number■ Dipotassium phosphate 7g, magnesium sulfate heptahydrate, 20. j yy and glycine 7j7q Medium composition number ■ Sodium carbonate 6 for each compound of medium composition number ■
3 mti added Medium composition number ■ Dipotassium phosphate /, / 3!
, Magnesium Sulfate Heptahydrate 20, S yng and Glyci 77! ;'mg Medium Composition No. ■ 26 kg of sodium carbonate was added to each of the compounds in Medium Composition No. - ■ The above seven types of powdered medium compositions were thoroughly ground in a mortar and mixed 1-. As a result, the sample with medium composition number (■) had moisture absorption L and was no longer a dry powder. Medium composition number ■～■
Since it was a dry powder, I left it in a tessicator for 2 days, then poured it with distilled water. It was dissolved in The pT of these solutions was measured, and each rue was put into an L-shaped tube and sterilized at 7.2θ°C for 70 minutes.
-Culture was performed under the same conditions as C31. As shown in Table 9, the media using medium composition numbers ■, ■, ■, and ■ were the activated sludge extract obtained in Example/-(J,
The time to reach the minimum absorbance was approximately equal to that of the medium with m6 added. That is, it is possible to store the powders of medium composition numbers ■, ■, ■, and ■ in preparation 1-, dissolve them in water, and use them whenever necessary.This method reduces the labor involved in preparing the medium. The author can be omitted.

Table 9 Example 5 (1) Preparation of medium composition A Medium composition A Example/- Activated sludge extract obtained in (, 2) θθ
5M phosphate buffer (pH 7θ) per ml
After adjusting the pH to θ from 1 to 20 θ, the entire volume was adjusted to 20 θ and purified powdered agar (Bacto Agar manufactured by Difco) 77
After adding the agar, dissolve the agar by heating and put the resulting medium into a large test tube (/grnl! The medium composition A and 1~ were dispensed over a period of time and sterilized for /θ°C/θ minutes.

Media Composition B 46M phosphate buffer (pH 7θ) ml of magnesium sulfate heptahydrate, 2 o, s m! Add i+ and p
After adjusting the H concentration to θ, the total volume was made up to 2θθml, and purified powdered agar (Bacto Agar manufactured by Difco) was added.
Place the agar in a heating bath Ml- and the medium in a large test tube (/grrrm
Dispense /gm/: into /2θ℃/θ
The 2θ sterilized medium was mixed with medium composition B.

C. Medium composition C ///sM phosphate buffer (20.3 mg of magnesium sulfate heptahydrate and phosphoric acid-
Ammonium 37! ; After adding my and adjusting the pH to θ, the total volume was made up to 2θOml, and after adding 7 g of purified powder agar (Bacto Agar manufactured by Difco), the agar was heated to dissolve 1-
Transfer the culture medium to a large test tube (/g = ×2θOvan)/g
The medium composition C was prepared by dispensing ru/3 and sterilizing the medium for 10 minutes at 2θ''C.

(Separation of ,21YLM-/, measurement medium composition A,
B and C each/g ml! After heating and melting, heat at 5°C.
Add YLM-/bacterial solution to the sterilized physiological saline solution.
Appropriately diluted 1-diluent jθμ), and Example/-(1)
Quickly add 2 ml of the yeast suspension described in 1-1 and mix well.
After that, they were poured into sterilized Petri dishes and solidified into flat plates, forming flat plates A1B and C and 1-, respectively. As shown in Table 5, these plates were cultured at 3θ°C, and as shown in Table 5, lytic spots caused by lysis of viable yeast cells appeared on plate A in 3 to 3 days, and lysis of viable yeast cells appeared on plate Bdj. Lytic plaques caused by lysis of viable yeast cells appeared on day 1 to 3, and lytic spots caused by lysis of viable yeast cells appeared on plate C. The shredded, lytic science is a flat plate A.
1 The number of plates B and C was almost the same.

Table 9 Example 6 When the waste liquid from a sake factory was cultured on plates A and C in the same manner as in Example j instead of the YLM-/bacteria liquid, the 6th
As shown in the coating, lytic spots caused by lysis of viable yeast cells appeared on both plates A and C after 3j days, and the number of lytic bacteria was almost the same in both plates A and C. General microorganisms σ
Regarding the number of colonies, Plate C is 3 to 3 years old compared to Plate A. It decreased by 1-.

Claims (1)

[Scope of Claims] (1) Adding to yeast cells a medium composition containing seven or more types selected from phosphates, magnesium salts, ammonia, ammonium salts, amines, amine salts, peptides, and proteins. (2) The amine and amine salt are selected from primary amines, secondary amines, tertiary amines, and tertiary amines.
(3) The culture medium for yeast-dissolving microorganisms according to claim 1, characterized in that the amine and amine salt are present in the molecule. The medium for yeast-dissolving microorganisms according to claim 7, characterized in that it contains seven or more amino acids selected from non-sulfur amino acids that do not contain yellow, and amino acid salts. Claim 1, characterized in that the salt is seven or more types of bases, nucleosides, nucleotides, and salts thereof selected from purine bases having amine groups, pyrimidine bases, their nucleosides, and nucleotides. (5) The medium for yeast-lysing microorganisms according to claim 1, wherein the medium composition is in the form of a powder or liquid (B) The medium for yeast-lysing microorganisms is a liquid medium or The medium for yeast-dissolving microorganisms according to claim 1, which is a solid medium.