JP5463559B2 - Physiological activity regulator of mold and method of using the same - Google Patents

Description

  The present invention relates to a fungal physiological activity regulator containing rare sugars, which inhibits mold sporulation and exhibits fungal colony-like growth-inducing action, and a method for using the same, and is useful in the microorganism-related technical field using fungi A biological activity regulator is provided. The present invention provides a method by which fungi can be separated into a single colony when a fungal mutant or a gene is introduced, and the target mutant or gene can be easily isolated. It is possible to prevent the occurrence of various harmful effects such as contamination of the working environment in the fermentation industry, contamination of fermentation bacteria, etc. It provides useful technologies that are expected to develop.

  Mold is a kind of microorganism called “fungus” and has been used for the production of sake, soy sauce, miso, cheese, etc. for a long time. Currently, in practical use, it is used as a fermentation industry in the food manufacturing industry such as brewed foods, antibiotics and seasonings, and in the manufacture of chemicals. “Moulds” include, for example, filamentous fungi (growing while branching in filamentous cells called mold mycelia, creating spores at the tip, and spreading and spreading spores), mushrooms (mycelium concentrates in a fungus shape) ), Yeast (single cell that grows mainly by budding and division, has a fermenting action that decomposes into sugars, carbon dioxide, and alcohol, and is indispensable for brewing sake and making bread. And so on.

  Molds are used in the production of various foods. For example, tame soy sauce is mainly made from defatted soybean and whole soy protein raw materials, and during this tame soy production process, filamentous fungal spores grow in the koji making process using a koji making device. . Sake making for soy sauce is a well-known technique and is described in detail in Non-Patent Document 1, for example. In the chrysanthemum manufacturing process, a large amount of spores may be scattered in the air as the straw drops and moves at the input and discharge sites of the belt conveyor opened to the atmosphere. Therefore, conventionally, as a method for removing scattered spores, an apparatus for removing spores together with air has been provided, such as exhausting to the outside by ventilation with a large ventilation fan or recovery using a dust collector. As a method to prevent spores from being scattered, when transporting the spore deposited from the chrysanthemum apparatus to the next process equipment, the transport device is a sealed screw conveyor, and liquid is placed in the casing of the sealed screw conveyor. There has been proposed a spore scattering prevention soot conveying device provided with nozzles for spraying (Patent Document 1).

  In addition, when a large amount of spores are scattered in the air, the working environment is deteriorated, and if the spores enter the body of the worker, it becomes a causative substance that causes allergies, which may be a serious problem. Therefore, it was impossible to completely remove the scattered spores, and extra costs were required for the installation and operation of the equipment. Spores are much easier to disperse than bacterial cells and can survive even under conditions where vegetative cells easily die, making them a source of microbial contamination in culture, purification processes, and products. Inhibits spore formation and spore germination by allowing foods contaminated with microorganisms that form heat-resistant spores to be sterilized and preserved by treatment at the degree of pasteurization, avoiding the usual high-temperature sterilization conditions that cause quality degradation As an agent, a sporulation and germination inhibitor of spore bacteria containing 1,5-anhydrofructose has been proposed (Patent Document 2).

  Molds are used in genetic engineering experiments and cell engineering operations, and it is experimentally important to form a colony state by suppressing hyphal elongation. In order to separate and acquire microorganisms, microorganisms that meet the purpose are isolated from the isolated microorganism colonies by a method of isolating colonies formed on the plate. For example, a coryneform bacterium capable of producing biotin from pimelic acid transformed with a plasmid into which a gene of a gene group encoding an enzyme involved in biotin biosynthesis has been introduced. A plate medium containing pimelic acid and not containing biotin Cultured above, and selected according to the size of colonies grown on the medium, to isolate coryneform bacteria having high biotin-producing ability (Patent Document 3). In screening microorganisms, colony formation is an indispensable element for screening microorganisms, as proposed by a screening apparatus (Patent Document 4) that automatically performs colony position detection and the like.

Regarding the induction of filamentous colony-like growth, the effect of L-sorbose has been reported (Non-patent Document 2). Examples have been disclosed that allow Neurospora to grow as a single colony by adding sorbose to the medium in various plating media used to select improved host strains (patents) Reference 5). As an example of a colony forming medium, a medium composed of 50 ml of 4 × Frie medium stock solution, 10 g of sorbose, 2 g of sucrose, 12 g of agar, and 750 g of water is known. This method of selecting microorganisms also makes it possible to screen a large number of clonal colonies after mutagenesis.

On the other hand, low-molecular-weight organic substances containing monosaccharides such as rare sugars are considered not to have physiological activity, and monosaccharides are known to be an energy source or a source of sweetness, but they do more than that. However, it has been proposed to provide agrochemicals using the effect of inducing the systemic acquired resistance of plants, use as a growth inhibitor of harmful microorganisms as well as plant pathogens (Patent Document 6) ) Various applications have been developed.
For example, D-psicose promotes insulin secretion from rat pancreatic β-cells (cell lines that secrete insulin), indicating the potential for treatment of insulin-dependent diabetes with D-psicose, and In animal experiments using rats, D-psicose was found to have a hypoglycemic effect, indicating the physiological activity and effectiveness of rare sugars.
However, since the physiological activity of rare sugars on microorganisms is not yet known in detail, new development of rare sugars on microorganisms has been demanded. Although the expression of the physiological action of sorbose on mold as described above has been publicly known, no detailed report on the physiological activity of other rare sugars has been known so far.

JP 2006-304627 A JP 2004-113181 A Japanese Patent Application Laid-Open No. 07-87963 Japanese Patent Application Laid-Open No. 08-205852 JP 11-510065 JP JP 2006-8669 A Edited by Toshiro Tochikura "Science and Technology of Soy Sauce" Japan Brewing Association, P.400-402 (1994) Michael H. Gold and Therese M. Cheng, Induction of colonial growth and replica platingof the white rot basidiomycete Phanerochaete chrysosporium, Applied and Environmental Micribioligy, Vol. 35, No. 6, 1223-1225 (1978).

  Under such circumstances, in light of the above-described conventional techniques, the present inventors have made extensive efforts with the goal of developing new technologies that apply the biological activity of rare sugars to microorganisms to microbial application technologies. The present inventors have found that sugar is effective in inhibiting mold spore formation and inducing fungal colony-like growth, and by further research, the present invention has been completed.

  The present invention provides a fungal physiological activity regulator containing rare sugars, which inhibits mold sporulation and induces fungal colony-like growth, and a technique for using the same, and provides genetic engineering operations and cell engineering. It provides a new technique that is useful in an operational operation. For example, in the past, when a fungal mutant strain or a gene-transferred strain is taken out, a single colony is formed in the microorganism, and the target mutant strain or gene-transferred strain is separated and collected from the colony. However, according to the present invention, the target mold can be separated and collected very easily. In addition, the ability to achieve colony-like growth induction by a simple method of adding a small amount of rare sugar to the medium widens the application range of the above-described engineering technique using mold.

  In addition, in the fermentation industry using mold, mold spreads widely by spreading spores, and various adverse effects may spread. The present invention can solve the problems that the fermentation process cannot produce the target substance due to mold contamination, the environment is polluted, and the health damage of employees occurs. According to the present invention, it was newly recognized in psicose and allitol that it was possible to suppress the generation of mold spores, so that the production of foods by fermentation, for example, the process of producing miso, soy sauce, liquor and the pharmaceutical by fermentation For example, the use of rare sugars in the manufacturing process of antibiotics, etc., can be expected to lead to new developments in the future microbial utilization industry.

The gist of the present invention for solving the above problems is the following fungal physiological activity regulator.
(1) Physiological activity of mold exhibiting mold colony-like growth induction and inhibition of mold sporulation selected from Aspergillus niger and Aspergillus oryzae, using D-psicose as an active ingredient Regulator.
(2) The mold physiological activity regulator according to (1) above, wherein the concentration of D-psicose in the culture solution to which the mold physiological activity regulator is applied is 0.2 to 5.0% by weight.
( 3 ) The mold physiological activity regulator according to (1) or (2 ) above, which contains sugars other than the D-psicose and rare sugars.
( 4 ) The mold physiological activity regulator according to any one of (1 ) to ( 3) , wherein a mixture of D-psicose and D-fructose is used as D-psicose.
( 5 ) The mold physiological activity regulator according to any one of (1) to ( 4 ) above, which contains D-psicose and deoxycholate.

Further, the present invention is to summarized as a method of culturing following microorganisms.
( 6 ) A method for culturing a microorganism, comprising performing a microorganism culturing step in the presence of the fungal physiological activity regulator according to any one of (1) to ( 5 ) above.
( 7 ) A method for culturing microorganisms, comprising controlling fungal spore formation and colony-like growth induction using the fungal physiological activity regulator according to any one of (1) to ( 5 ) above.

The following effects are exhibited by the present invention.
(1) It is possible to provide a mold physiological activity regulator that contains a rare sugar and inhibits mold spore formation and exhibits mold colony-like growth-inducing action.
(2) A mold culturing method in which mold spore formation is inhibited by carrying out a microorganism culturing step in the presence of the mold physiological activity regulator of the present invention can be provided.
(3) By carrying out the microorganism culturing step in the presence of the mold physiological activity regulator of the present invention, a mold culturing method in which mold colony-like growth is induced is possible.
(4) By making koji in the presence of the physiological activity regulator of the present invention, the environment in food production using koji can be protected from contamination by mold spores.
(5) A mold spore formation regulating method in which mold spore formation is controlled using the mold physiological activity regulator of the present invention can be provided.
(6) It is possible to improve the culture technology of microorganisms in genetic engineering operations and cell engineering operations and to promote the development of microorganism-related technologies.

The present invention is likely to become a source of microbial contamination in the culturing and purification steps when using microorganisms having spore-forming ability, for example, when transporting sputum with spores grown from a koji making apparatus to equipment in the next process. Is applied as a rare spore-forming inhibitor of mold to prevent it from being scattered in the air, and in mold genetic engineering experiments, it suppresses hyphal elongation to form a colony state. Is an operation that is important in the experiment, and is characterized by the application of a rare sugar to such an operation.
Regarding the induction of filamentous colony-like growth, only one case of L-sorbose has been reported for Phanerochaete chrysosporium (basidiomycetes). L-sorbose belongs to monosaccharides but is not classified as a rare sugar. The present invention is an epoch-making thing that has demonstrated the physiological activity of a rare sugar, which has been regarded as having no physiological activity, to microorganisms. In particular, the colony-like inducing action of D-psicose was found to be superior to that of L-sorbose, and L-psicose and L-tagatose were not as good as D-psicose, but showed colony-like growth inducing action. It has been found.

  Regarding the formation of mold spores, there have been no reports on the inhibitory action of other sugars including rare sugars so far. The title and the present invention have been completed. In particular, it was found that D-psicose, L-psicose and allitol are excellent in spore formation inhibitory action of microorganisms.

The “rare sugar” in the present invention can be defined as a monosaccharide that exists only in a small amount in nature. Examples of monosaccharides present in large amounts in naturalization include, for example, seven types of D-glucose, D-fructose, D-galactose, D-mannose, D-ribose, D-xylose, and L-arabinose. Other monosaccharides can be classified as rare sugars because of their low abundance in nature.
In the present invention, the term “colony-like” means that the growth of mold on a solid medium restricts the growth of mycelia, so that the mycelium does not diffuse and forms dots (like colonies formed by bacteria or yeast). It is a term that means growing.
“Physiological activity” means a property acting on a specific physiological regulatory function against mold, and in the present invention, it includes inhibition of mold sporulation function and induction of mold colony-like growth function. It is a term.

Rare sugars are well known as monosaccharides and are relatively easily available sugars. An example of a method for producing a rare sugar will be described below using D-psicose as an example.
In the production of D-psicose, for example, a microorganism producing an enzyme (DTE • D-tagatose epimerase) is first cultured on an agar medium, and this is used as a seed in a large jar fermenter. Since the microorganisms obtained in the culture solution are very small, the cells in the culture solution are collected and collected by high-speed rotation centrifugation about 10,000 times or more per minute in a high-speed cooling continuous centrifuge. The collected microorganism is crushed with a high-pressure homogenizer, and the enzyme (DTE / D-tagatose epimerase) contained in the microbial cells is collected. The recovered enzyme is immobilized on a bioreactor carrier, and fructose and D-fructose, which are raw materials of D-psicose, are passed through a column having a stationary carrier in the bioreactor, thereby allowing fructose and D- Fructose is converted to D-psicose. Since the produced D-sicose contains impurities, it can be separated and purified by a pseudo moving chromatographic separation apparatus and then concentrated to obtain highly pure D-psicose. As another method for producing psicose, a method of preparing a large amount of psicose by roasting and extracting coffee beans husk and silver skin, which are discarded in the coffee production process, is proposed. (Refer to Unexamined-Japanese-Patent No. 2004-143062).
D-psicose is highly safe based on the results of experiments in which large doses of D-psicose produced in this way were administered to animals at once, and experiments in which relatively small amounts were repeatedly administered over several weeks. Has been confirmed.

The “mold” used in the present invention is a kind of microorganism and called “fungus”, and has been used for the production of sake, soy sauce, miso, cheese, etc. for a long time. Currently, in practical use, it is used as a fermentation industry for the production of brewed foods, antibiotics, seasonings and the like. “Moulds” include, for example, filamentous fungi (growing while branching in filamentous cells called mold mycelia, creating spores at the tip, and spreading and spreading spores), mushrooms (mycelium concentrates in a fungus shape) ), Yeast (single cell that grows mainly by budding and division, has a fermenting action that decomposes into sugars, carbon dioxide, and alcohol, and is indispensable for brewing sake and making bread. And so on.
Among them, Aspergillus, also called Aspergillus, is one of the most common molds found in nature. Neisseria gonorrhoeae is a general term for useful molds isolated from cocoons. The most commonly used koji mold in Japanese koji is a mold called Aspergillus oryzae , and all koji molds used in sake and miso are classified as Aspergillus oryzae. Furthermore, more than 90% of Aspergillus oryzae used for soy sauce production are Aspergillus oryzae. [ Aspergillus niger ] is excellent in the ability to produce citric acid from glucose, and is therefore used in the production of citric acid using molasses as a raw material. Pectin-degrading enzyme (pectinase) obtained from Aspergillus niger is used for clarification of fruit juice and fruit species. In addition, Shiitake fungi ( Lentinula edodes ), Enokitake mushrooms ( Flammmulina velutipes ), Hiratake fungi ( Pleurotus ostreatusu ) and the like are adjusted for physiological activity by rare sugars.

As a result of experiments using the following 8 types of ketohexose (6 types are rare sugars), it was found that the rare sugars induce colony-like growth and suppression of sporulation of molds ( Aspergillus niger and oryzae ). An example of the experimental conditions performed is shown below.
Monosaccharides used: D-psicose, L-psicose, D-fructose, L-fructose, D-tagatose, L-tagatose, D-sorbose, L-sorbose Test strains: Aspergillus niger NBRC 6341, Aspergillus oryzae NBRC 4134
Medium used: 1% monosaccharide, 0.1% yeast extract, 2% agar
Culturing method: Spore suspensions of each bacterium were prepared, and these were inoculated on an agar plate medium having the above composition, followed by culturing at 28 ° C.

Rare sugars exhibited a physiological activity-regulating action, but the action of D-psicose, L-psicose, L-tagatose, allitol and saccharides combining them was particularly remarkable.
Regarding the induction of colony-like growth, D-psicose induced colony-like growth in Aspergillus niger (FIG. 1). At present, only one case of L-sorbose action on Phanerochaete chrysosporium (basidiomycetes) has been reported for the induction of filamentous colony-like growth, but the effect of D-psicose exceeds that of L-sorbose. Was revealed by this experiment. Although L-psicose and L-tagatose were not as much as D-psicose, they showed an effect of inducing colony-like growth.
Regarding Aspergillus oryzae , it was revealed that L-psicose and D-psicose induce colony-like growth although the effect of L-sorbose is somewhat inferior (see FIG. 3). In addition, D-psicose and L-psicose showed a spore formation inhibitory effect.

Regarding the inhibition of sporulation, in Aspergillus oryzae , among rare sugars, it was observed that D-psicose significantly inhibited sporulation, especially in the presence of 3% D-psicose. It was. Moreover, allitol significantly inhibited sporulation, and no sporulation was observed in the presence of 1% allitol.
In addition, inhibition of sporulation by rare sugars was also confirmed in Aspergillus niger . In addition, it has been clarified that allitol significantly inhibits sporulation in the presence of sodium deoxycholate.

  In order for the rare sugar to exhibit colony-like growth induction and sporulation inhibition in mold culture, the rare sugar concentration is preferably in the range of 0.2 to 5.0 wt%, and more preferably in the range of 0.5 to 4 wt%. It is more preferable that If it is below the lower limit of this range, the degree of expression of the action effect due to the rare sugar is slightly impractical, and if it exceeds this range, it is not practical due to economic reasons, and harmful effects due to a large amount of rare sugar occur. Sometimes.

By adding 0.001 to 0.10 wt% of deoxycholate, for example, sodium salt to the medium for culturing mold, the physiological activity regulating action by rare sugar can be amplified. FIG. 1 shows the test results of culturing Aspergillus niger using a medium containing no sodium deoxycholate, and FIG. 2 showing a medium containing 0.01 wt% sodium deoxycholate. It can be seen that the induction of colony-like growth is further promoted. It is clear by comparing FIG. 3 and FIG. 4 that similar results were obtained with Aspergillus oryzae .

  Among the rare sugars, allitol has a remarkable inhibitory effect on mold spore formation, and when 0.2 to 3 wt% is added to the medium, spore formation is inhibited, but in particular, the concentration is 0.8 wt% or more. Is added to completely prevent spore formation. D-psicose and L-psicose show similar inhibitory effects, although slightly inferior to allitol. For example, a medium supplemented with 3 wt% D-psicose can completely prevent mold sporulation.

The action of controlling the physiological activity of fungi of rare sugars as described above is useful in genetic engineering operations and cell engineering operations using fungi, for example, brewing industry using fungi, and pharmaceutical production by fermentation. . In the sense of protecting mold spores from being scattered, it is possible to prevent the fermentation process from being contaminated by spore dispersion in the microbial industry such as the brewing industry, greatly improving the fermentation process, and reducing environmental contamination and fermentation in the fermentation process. The effect of improving the health management of engaged people is great, and it is applied to new aspects of the fermentation industry, including the production of food and medicine.
Next, although the detail of this invention is demonstrated by an Example, this invention is not limited at all by these Examples.

In this example, the effect of rare sugars on the growth of Aspergillus niger , which is a representative mold, was examined, particularly the effect on the induction of colony-like growth.
The following eight types of ketohexose were selected from the monosaccharides, and a medium containing 1 wt% of each saccharide was prepared.
D-glucose, D-fructose, L-sorbose, D-sorbose, L-psicose, D-psicose, L-tagatose, and D-tagatose each contain 1% monosaccharide, yeast extract 0.1%, agar 2% Eight culture media were prepared. A spore suspension of Aspergillus niger IFO 6341 was prepared as a test strain, which was inoculated on each agar plate medium, and then observed for a colony-like induction state after culturing at 28 ° C. for 6 days. The test results are shown in FIG.
From the photograph in FIG. 1, it was demonstrated that D-psicose, L-psicose, and L-tagatose have an inducing effect on colony-like growth of Aspergillus niger IFO 6341. In contrast to the L-sorbose test results shown in FIG. 1, the action of inducing colony-like growth of these three rare sugars showed not only inferior results to L-sorbose, but also D-psicose. Then, it became clear that the effect which exceeds the well-known L-sorbose was shown.

Aspergillus niger IFO 6341 was cultured for 6 days in the same manner as in Example 1 except that 0.01 wt% of sodium deoxycholate was further added to the medium, and the results are shown in the photograph of FIG. As a result, it was clarified that D-psicose, L-psicose, and L-tagatose have an effect of inducing colony-like growth of Aspergillus niger . It was found that by adding ± sodium deoxycholate, the colony-like diameter was small and the surrounding area became clearer and the effect of inducing colony-like growth was enhanced.

In the same manner as in Example 1, Aspergillus oryzae IFO 4134 was cultured, and the effect of inducing the colony-like growth of rare sugars was tested. The result is shown in FIG. Although it was somewhat inferior to the action effect of L-sorbose, it became clear that L-psicose, D-psicose, L-tagatose and D-tagatose have an action of inducing colony-like growth.

  Aspergillus oryzae IFO 4134 was cultured in the same manner as in Example 3 except that 0.01 wt% of ± sodium deoxycholate was further added to the medium, and the effect of inducing colony-like growth of rare sugars was tested. The result is shown in FIG. It was found that the effect of inducing colony growth was enhanced by adding ± sodium deoxycholate.

Aspergillus niger IFO 6341 and Aspergillus oryzae IFO 4134 were cultured in a medium containing D-glucose, D-fructose, L-sorbose, L-psicose, D-psicose and allitol, respectively, in the same manner as in Example 1 to form spores. The state of inhibition was tested. As a result, the following situation was found.
In Aspergillus oryzae IFO 4134, D-psicose was found to significantly inhibit the spore formation of the bacterium, and in particular, no spore was formed in the presence of 3% D-psicose. Furthermore, allitol significantly inhibited the spore formation of this bacterium, and no sporulation was observed even in the presence of 1% allitol.
Further, in Aspergillus niger IFO 6341, D-psicose inhibited sporulation of the bacterium, but sporulation was further significantly inhibited in the presence of 1% D-psicose + sodium deoxycholate. It has been clarified that allitol inhibits the spore formation of the bacterium, but significantly inhibits the spore formation of the bacterium in the presence of allitol and sodium deoxycholate.
Thus, it was revealed that D-psicose and L-psicose have an action of inhibiting the formation of spores against the above mold. This result is expected to be applied to new aspects in the microbial industry, such as the brewing industry, in the sense that it protects against mold spores.

The present invention relates to a fungal physiological activity regulator characterized by containing a rare sugar, inhibiting fungal sporulation and inducing fungal colony-like growth, and fermentation using the fungal physiological activity regulator The present invention relates to a method, a method for protecting an environment in food production using cocoons from contamination by mold spores, and a method for controlling mold spore formation.
The test results of the present invention suggest that, in particular, D-psicose has a great influence on the cell differentiation and maturation mechanism of C. cinereus. It is expected to lead to new aspects of research and its applications.

In addition, as a result of examining the effects of rare sugars on the growth of typical molds, Aspergillus niger and Aspergillus oryzae , in particular, the induction of colony-like growth and the effect on sporulation, D-psicose was added to the above mold. In contrast, it was found to induce colony-like growth. In the process of genetic engineering operation and cell engineering operation for fungi, it is important to grow fungi in a colony-like manner, and currently L-sorbose is used for that purpose. Showed that rare sugars such as D-psicose were useful as an alternative to L-sorbose, and it was possible to make new developments in genetic engineering and cell engineering operations.
Furthermore, among the rare sugars, it was revealed that D-psicose and allitol have spore formation inhibitory activity against the above-mentioned mold. This result is expected to be applied to new aspects in the microbial industry, such as the brewing industry, in the sense that it protects against mold spores. Thus, the present invention provides a technique useful for new development of rare sugars and development of new techniques.

The photograph which shows the colony-like growth condition at the time of culture | cultivating Aspergillus niger with the culture medium containing a rare sugar. The photograph which shows the colony-like growth condition when Aspergillus niger was cultured with the culture medium containing a rare sugar and sodium deoxycholate. The photograph which shows the colony-like growth condition at the time of culture | cultivating Aspergillus oryzae with the culture medium containing a rare sugar. The photograph which shows the colony-like growth condition at the time of culture | cultivating Aspergillus oryzae in the culture medium containing a rare sugar and sodium deoxycholate.

Claims (7)

An agent for controlling the physiological activity of fungi, which comprises D-psicose as an active ingredient and exhibits fungal colony-like growth induction and inhibition of fungal sporulation selected from Aspergillus niger and Aspergillus oryzae . The mold physiological activity regulator according to claim 1, wherein the concentration of D-psicose in the culture medium to which the mold physiological activity regulator is applied is 0.2 to 5.0% by weight. The fungal physiological activity regulator according to claim 1 or 2 , comprising a sugar other than the D-psicose and the rare sugar. The fungal physiological activity regulator according to any one of claims 1 to 3 , wherein a mixture of D-psicose and D-fructose is used as D-psicose. The mold physiological activity regulator according to any one of claims 1 to 4 , comprising D-psicose and deoxycholate. A microorganism culturing method, wherein the microorganism culturing step is carried out in the presence of the fungus physiological activity regulator according to any one of claims 1 to 5 . A method for culturing a microorganism, comprising controlling fungal spore formation and colony-like growth induction using the fungal physiological activity regulator according to any one of claims 1 to 5 .