JP3307627B2 - A method for producing a group of cytochalasin-like substances from the yeast Zygosaccharomycesrouxii. - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing a group of cytochalasin- like substances extracted from osmotic pressure-resistant yeast (Z. rouxii) which exists and is used in nature.

[0002] Conventionally, the ex-post Saccharomyces Roma Isis Rokishii has been one of the food spoilage cause, usage of this yeast was not been invented yet Tei. On the other hand, the cytochalasin substance first discovered by a British scientist in the 1960's has reversibility, antibacterial properties, and an effect of suppressing the division of animal cells, and the like. However, all the cytochalasin substances discovered so far existed only in molds.

[0003]

[Problems to be Solved by the Invention] "Moulds" are easily denatured due to their properties, and it is difficult to breed and handle safely and stably, and the production equipment is expensive. However, "Yeasts" are hard to denature and are safe and stable. Reproduction is possible and production equipment is inexpensive, and above all, there is no need to worry about the presence or absence of "mold toxicity". Cytochalasin and cytochalasin-like substances have been discovered from cytochalasin A and B, which were discovered earlier, to cytochalasin W, and are used in various fields as substances with their own functions (hereinafter, these sites). Kalasin Ano
Solstice W is referred to as “cytochalasin substance”) . Discovered ever
A group of the above-mentioned cytochalasin substances has a high possibility of containing a new substance, and requires a method of producing using a microorganism which has not been used conventionally . By the way, in the field of antibiotics and antibacterial substances, fungi have been extracted from molds and strong disease-causing bacteria have emerged against drugs already on the market .
Therefore , development of new chemicals by new methods is required.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems. After culturing yeast digosaccharomysis roxii in a medium containing a sugar such as glucose, the yeast is then dissolved in an organic solvent such as ethyl acetate. wherein the extracted, a method of making a cytochalasin-like substance groups to the organic solvent solution from cytochalasin like metabolites group obtained by completely skipping and obtaining the cytochalasin-like substance groups And

[0005]

[Aspect of the implementation of the present invention is Jigosakkaromai sheet scan
Roxyi is cultured in a medium containing sugar. The culture medium is a culture medium for culturing yeast usually, and the sugar is glucose, sucrose,
Maltose and the like.

[0006] The sugar concentration of the medium is from 2% to 40%.

The pH of the medium is between 4 and 7.

[0008] The culture temperature is from 10 ° C to 35 ° C.

[0009] The culturing time is from 1 to 7 days.

[0010] Harvesting of the supernatant after culturing is by centrifugation.

The method of extracting metabolites utilizes an organic solvent solution.

The above-mentioned organic solvent liquid is ethyl acetate, chlorofoam or the like.

In the subsequent operation, the solvent is completely removed, and the obtained substance is dissolved in ethyl alcohol, chloroform, or the like, and the resulting substance is harvested as a metabolite produced from Digosaccharomysis roxii.

[0014]

According to the above method, Digosaccharomysis
The amount of unpurified metabolites that can be recovered by culturing Roxyi is 1.0 g to 1.6 g per liter of medium.

Table 1 is a table showing the results of an investigation of antibacterial activity against bacteria of eight genera using the Paper disc method.

[Table 1]

An experimental method was as follows. A normal agar medium was placed in a chalet, a soft agar (0.5% agar) medium containing bacteria was inoculated thereon, and Z. This is a method in which a substance of the present invention (hereinafter, referred to as a sample) extracted from R. rouxii and a paper disc to which a control is attached are placed and cultured overnight at 30 ° C., and a transparent diameter appearing around the disc is measured. The sample is already dissolved in alcohol,
Pure alcohol was used as a control. If it has antibacterial properties, it is the logic that bacteria do not propagate around the disc and become transparent. As a result, it was found that the control ethanol had no effect, whereas the effect of the sample became stronger as the amount of the sample was increased.

FIG. 1 is a diagram showing the antibacterial activity against Bacillus toyoi (Bacillus, a kind of bacillus). In the experimental method, a normal liquid medium was placed in a test tube, and added so that the sample concentration was 1.8 mg / ml and 3.5 mg / ml. No sample (concentration 0mg
/ Ml) was used as a control. These are growth curves obtained by ingesting the target bacteria and culturing them at 30 ° C. for 12 hours, and measuring the growth of the bacteria with an absorbance meter. The graph in (a) shows the pH of the medium as 5.
5, and the graph in (b) was obtained by adjusting the pH to 6.0. As a result, pH 6.0
It can be seen that the growth of the bacteria was clearly suppressed at 5.5. It can be seen that when the concentration of the sample reached 3.5 mg / ml at pH 5.5, the bacteria did not grow at all.

FIG. 2 shows Escherichia coli.
It is a figure which shows the antibacterial property with respect to (Escherichia coli). The experimental method is the same as in FIG. 1 and shows the results at pH 5.5. When the sample concentration is 2.0 mg / ml, the pH is 5.
It can be seen that there is an effect on both the sample adjusted to 5 and the sample not adjusted (pH 4.6).

FIG. 3 shows Pseudomonas aerug.
It is a figure which shows the antibacterial property with respect to inosa (Pseudomonas aeruginosa). The experimental method and results are the same as those for E. coli in FIG.

FIGS. 4A and 4B show Salmonell.
It is a figure which shows the antibacterial property with respect to a enteritidis (Salmonella enteritidis which causes food poisoning). The experimental method is the same as in FIG. 1, (a) is pH 5.5, (b) is pH 6.
It is a growth graph at 0. It can be seen that the growth and growth of the bacteria were both suppressed at pH 5.5 to pH 5.5.

FIG. 5 shows a Micrococcus lute.
It is a figure which shows the antibacterial property at pH5.5 with respect to us (a kind of cocci). The experimental method was the same as in FIG. 1, but the culture time was up to 24 hours. In the test tube containing the sample concentration of 2 mg / ml, 2 bacteria
It turns out that it did not breed even after 4 hours.

FIG. 6 shows Staphylococcus a.
It is a figure which shows the antibacterial property at pH 5.5 with respect to ureus (Staphylococcus). The experimental method is the same as in FIG. It can be seen that the bacteria are not propagated up to 24 hours and the sample is effective, even when the sample is added at 2 mg / ml and the pH is not adjusted to 4.6 or the pH is adjusted to 5.5.

Table 2 shows the minimum growth inhibitory concentration (MIC) of each of the substances of the present invention in each of the bacteria.

[Table 2]

This table summarizes the results of the antibacterial experiments shown in FIGS. Bacteria are roughly classified into Gram-positive and Gram-negative. Unadjusted minimum growth inhibitory concentration (MIC) for each bacteria, pH, pH5.
5, expressed as pH 6.0. If the pH is not adjusted, the antimicrobial properties of the sample can be seen in a small amount, and it can be said that the effect of this sample appears as the pH is lowered. Unadjusted pH
The minimum growth inhibitory concentration (MIC) for Salmonella was 0.1%.
7 mg / ml, 1-2 mg / ml for staphylococci.
2.0 mg / ml for E. coli.

FIG. 7 shows a Micrococcus lute.
FIG. 4 shows that the antibacterial property of a sample in a us (a type of cocci) is reversible. The experimental method is the same as in FIG. Sample concentration of 0.5mg / ml and 1mg / ml
I made it. As a control, ethanol was added to a concentration of 20 μl / ml. In addition, the normal medium without addition is also available from Contr.
olI and ControlII were used. The bacteria grew as usual in the test tube to which ethanol was added, and reached a growth peak after culturing for 48 hours. 0.5 mg / ml sample
Although the growth of the bacteria was suppressed in the added test tube for up to 24 hours, it grew weakly after 24 hours. On the other hand, the test tube to which the sample was added at 1.0 mg / ml did not grow even after passage of the bacteria for 168 hours. ControlI
The growth of the bacteria was normal in the test tubes of Control II and Control II. After culturing them for 12 hours to increase the number of bacteria, samples were added at 0.7 mg / ml and 1.5 mg / ml, respectively. 0.7mg / ml of sample in medium with increasing number of bacteria (Control I)
Growth has been suppressed for a few hours and then increased to normal. On the other hand, when the sample is filled up to 1.5 mg / ml (Contro
1II) The growth of bacteria with an increased number was also suppressed for 168 hours or more. The Control II test tube and the test tube to which the sample was added at 1.0 mg / ml from the beginning were cultured for 168 hours.
The culture medium was centrifuged to remove the bacterial cells, washed twice with sterile water, returned to a normal medium, and the culture was continued. As a result, it was found that both bacteria grew normally. The conclusion is as follows. (1) The optimal use amount of the substance group of the present invention is suitable for the number of bacteria, and can be increased by increasing the concentration of the sample. (2) The substance group of the present invention can suppress the generation of bacteria for a long time depending on the amount of use. (3) The effect of the substance group of the present invention is reversible, and when the bacteria are returned to the original medium, they grow normally.

FIG. 8 shows a Micrococcus lute.
4 is a heterostratigraphy micrograph showing the effect of a sample on the growth of a us (a type of cocci). The photo of (a) is Micrococcus luteus
The photograph was taken after culturing the bacteria in a normal medium for 12 hours.
It can be seen that the fungus grows normally and cell division occurs normally. The photograph in (b) was taken after the culture was performed for 12 hours with the medium containing 2.5 mg / ml of the sample. It can be seen that the cells of the bacterium are expanding, and the cells are not completely divided, and the two cells are stuck together.

FIG. 9A is a photograph of Escherichi.
a coli (E. coli) and (b) are Salmon
11 is an electron micrograph showing the effect of a sample on the growth of each of S. enteritidis (Salmonella enteritidis). 2.5 mg / ml for both samples
Photographs taken after culturing the bacteria in the added medium for 12 hours show that the cell walls of the bacteria expand and partially protrude in both cases.

The specificity of the application is described below. In the food sector, the use of “antibacterial agents” in livestock and marine products is
It is a factor that promotes the transformation of bacteria and Salmonella into harmful bacteria. By adding the "inventive substance" to the feed, "propagation of harmful bacteria and variation of harmful bacteria" can be prevented. Pesticide use can be reduced. At present, about 130 kinds of food additives are approved and used in the food field, and the "antibacterial property" as the main ingredient can be made safe by using the "inventive agent". In preserving the foodstuffs of the primary product, the main ingredient "antibacterial growth inhibitor" is used, but the "inventive agent" enables safe rot prevention. In the field of cosmetics, "fungicides" in the toiletry field include those that are not necessarily harmless to the human body. The quality-maintaining ingredients of various cosmetics that adhere to the skin are not necessarily safe and non-irritating. This problem can be solved by "adding an inventive agent". In the field of pharmaceuticals, the substances already discovered and invented from A to W are all made from molds and are very expensive. The production of safe and inexpensive new antibiotics can be made by using yeast. The function of inhibiting cell division (proliferation) is
Fields that have the potential to inhibit the division and proliferation of AIDS virus, brain cells, etc. It is clear from the data and photographs that the present invention inhibits the propagation of disease-causing bacteria, and is a field where natural healing therapy by "autoimmune function" is expected.

The cytochalasin-like substance group (exactly the same substance group or a new substance group with similar properties is not known) was all extracted from molds. The medical field already has various possibilities. However, the present invention is characterized by its ability to be applied to a wide range of foods, demonstrating its antibacterial properties, cell division inhibitory function and its reversibility as the first substance of "yeast origin" and its application to cosmetics and pharmaceuticals. It can be used for high value-added fields.

[Brief description of the drawings]

FIG. 1 is a diagram showing antibacterial activity against Bacillus toyoi (Bacillus, a kind of bacillus).

FIG. 2 Escherichia coli (E. coli)
It is a figure which shows the antibacterial property with respect to.

FIG. 3. Pseudomonas aeruginosa
It is a figure which shows the antibacterial property with respect to (Pseudomonas aeruginosa).

FIG. 4 (a) and (b) are Salmonella en
teritidis (Salmonella enteritidis causing food poisoning)
It is a figure which shows the antibacterial property with respect to.

FIG. 5 is a diagram showing the antibacterial activity of Micrococcus luteus (a type of cocci) at pH 5.5.

FIG. 6: Staphylococcus aureus
It is a figure which shows the antibacterial property at pH 5.5 with respect to (Staphylococcus).

FIG. 7 is a diagram showing that the antibacterial property of a sample in Micrococcus luteus (a kind of cocci) is reversible.

FIG. 8 shows a coccus (IFO 12708 <Mi) of an inventive substance (extracted substance from yeast Digosaccharomysis roxii).
crococcus luteus>), in which (a) was taken after culturing for 12 hours in a normal medium, and (b) for 12 hours after culturing in a medium containing 2.5 mg / ml of the inventive substance. It is a thing.

FIG. 9 (a) shows Escherichia coli (IFO 33) photographed after culturing for 12 hours in a medium containing 2.5 mg / ml of an inventive substance (extract from yeast Digosaccharomysis roxii).
01 <Escherichia coli>, and (b) shows Salmonella enteritidis (IFO) photographed after culturing for 12 hours in a medium containing 2.5 mg / ml of an inventive substance (extract from yeast Digosaccharomysis roxii). 3313 <Salmonella ente
litidis>).

Continuation of the front page (72) Inventor Fumio Hashinaga 1-21-24 Gunori, Kagoshima City, Kagoshima Prefecture Kagoshima University Faculty of Agriculture (56) References JP-A-54-86696 (JP, A) Japan Society for Food Science and Technology 46th Meeting Abstracts (1999) p. 50, 2Ea5 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 17/18 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A method for producing a group of cytochalasin- like substances, comprising culturing yeast Digosaccharomysis roxii in a medium containing a sugar such as glucose, and then extracting an organic solvent such as ethyl acetate from a culture solution separated from the medium. Extracting a crude cytochalasin-like metabolite group by extracting it with a liquid and completely removing the organic solvent liquid from the yeast. Digosaccharomysis roxii .
How to make a group .