KR101935277B1 - A novel isolated microorganism having high-saccharifying and use thereof - Google Patents

Abstract

The present invention provides a new microorganism having high starcholytic activity and resistance to alcohol at a high concentration, which is isolated from a conventional yeast.

Description

A novel isolated microorganism having high saccharifying ability and high-saccharifying and use thereof,

The present invention relates to a novel microorganism and a use thereof, and more particularly to a novel microorganism having excellent starcholytic activity and glycosylation activity which are isolated from a conventional yeast, and a use thereof.

Starch saccharification is a process of decomposing starch stored in grains and converting it into a form that can be used by organisms. Starch saccharification using yeast in the traditional fermentation process is one of the most important factors determining the quality of traditional liquor. In general, the traditional nuruk is made by crushing cereals such as barley and wheat, molding it hard by adding water, and then exposing it to the natural environment to make various fermenting microorganisms grow. Therefore, the variety and microbial content of the koji in the koji , The temperature and the composition of the feedstock. The complexity of these yeast microorganisms has the advantage of diversifying traditional regions and making it possible to localize them, but it also makes the continuous production of products with uniform flavor and quality difficult to cause the competitive power of the traditional wine industry to deteriorate. The type and ability of the mold responsible for the glycation function in the yeast are particularly important. When the yeast lacking the glycation ability is used, the saccharification of the starch is slowed down and the whole fermentation period is prolonged, . Therefore, the use of yeast having excellent glycation ability is an essential process in the production of traditional sake, but there is a fatal disadvantage that it is difficult to maintain only a certain amount of fungi having excellent glycation ability in the conventional process of producing natural sake. In domestic breweries, amylase enzyme is added separately to supplement the uncertain saccharifying ability of this yeast, or the entry used for manufacturing in Japan is used. Therefore, molds having excellent saccharifying ability among yeast-derived fungi are selected , It is necessary for the development of the traditional wine industry to improve the microorganism suitable for the traditional fermentation process. In this regard, Korean Patent Laid-Open Publication No. 10-2016-0111621 discloses a novel strain of Aspergillus oryzae MBF378 having high alpha-amylase activity and glucoamylase activity.

However, although the activity of alpha-amylase and glucoamylase is very high in the above-mentioned prior art, when the concentration of glucose in the medium is increased, the amylase biosynthesis is inhibited. Therefore, after a certain degree of progress of the starch glycosylation, the production of amylase is inhibited, Is still slow.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a strain having high starch-decomposing activity and high resistance to alcohol. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a novel Aspergillus oryzae M63 strain having enhanced glycosylation, deposited with Accession No. KCTC13153BP.

According to another aspect of the present invention, there is provided a composition for starch saccharification comprising the strain, a disrupted solution of the strain, a culture supernatant of the strain, or a mixture thereof.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; And a saccharification step of saccharifying the starch in the starch-containing water by inoculating the starch-containing water with the yeast produced in the step of producing the yeast, thereby saccharifying the starch.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A saccharification step of saccharifying the starch in the starch-containing material by inoculating the starch-containing material with the yeast produced in the yeast-producing step; And an alcohol fermentation step in which an alcohol fermenting microorganism is inoculated into the saccharide produced in the saccharification step to perform alcohol fermentation.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; And a simultaneous fermentation step of simultaneously performing saccharification and alcohol fermentation by inoculating yeast and alcohol fermenting bacteria produced in the production step into a starch-containing product.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A saccharification step of saccharifying the starch in the starch-containing material by inoculating the starch-containing material with the yeast produced in the production step; An alcohol fermentation step of performing alcohol fermentation by inoculating an alcohol fermenting microorganism into the saccharide produced in the saccharification step; And a step of extracting the alcohol from the alcohol fermentation product produced in the alcohol fermentation step.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A simultaneous fermentation step of simultaneously performing saccharification and alcohol fermentation by inoculating yeast and alcohol fermenting bacteria produced in the yeast-producing step with starch-containing water; And a step of extracting the alcohol from the alcohol fermentation product produced in the simultaneous fermentation step.

According to one embodiment of the present invention as described above, it is possible to achieve a strain producing effect that exhibits high starch decomposition activity under high glucose and starch conditions. Of course, the scope of the present invention is not limited by these effects.

1 is a photograph showing an Aspergillus oryzae M63 strain exhibiting a high starch decomposing activity by treating a mother strain with a low concentration of a mutagenic agent according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing a process for selecting a hyperglycosylated A. oryzae strain based on amylase activity using a laboratory evolution technique according to an embodiment of the present invention.
FIG. 3 is a graph showing the amylase activity of a hyperglycemia-producing strain according to an embodiment of the present invention.
FIG. 4 is a graph illustrating the amylase production stability of the M63 strain according to an embodiment of the present invention. FIG.
FIG. 5 is a graph comparing the temperature and pH characteristics of the strain M63 with that of the parent strain (MS2-12) and the control strain (Rib40) according to an embodiment of the present invention.
FIG. 6 is a graph comparing the saccharifying activity of the M63 strain with the parent strain (MS2-12) and the control strain (Rib40) according to an embodiment of the present invention.
FIG. 7 is a graph showing the amylase activity (1), glucose (2) and final alcohol concentration (3) in alcohol fermentation using M63 strain, parent strain (MS2-12) and control strain (Rib40) according to an embodiment of the present invention ), Which is a comparative analysis result.

Definition of Terms:

As used herein, the term "nuruk" is an essential fermentation agent for makgeolli, which is a typical traditional Korean wine, and is responsible for the starch saccharification and alcohol fermentation in the grain, which is a raw material of traditional rice wine. Therefore, it is one of the most important factors determining the quality of traditional wine in the traditional fermentation process. A mold containing the yeast may have a high ability Aspergillus spp., Such as glycosylation Rhizopus oryzae, Absidia corymbifera (Lichtheimia corymbifera ), Lichtheimia ramosa, Mucor indicus , etc. zygomycota (Zygomycota) and Aspergillus oryzae, A. niger, A. luchuensis . Among them, A. oryzae is the most isolated fungus in yeast. It secretes various amylase enzymes such as alpha-amylase and glucoamylase and plays an important role in starch glycation function of yeast.

As used herein, the term " starch containing substance " is intended to mean a starch-containing substance that is a saccharide, such as rice, wheat, barley, oats, buckwheat, sorghum, corn, rye, It also includes sweet potatoes, potatoes, cassava, arrowroot, accidental palms, taro, acorns, chestnuts, mung bean, buckwheat, banana and yam, all of which contain starch.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a novel Aspergillus oryzae M63 strain having enhanced glycosylation, deposited with Accession No. KCTC13153BP.

According to another aspect of the present invention, there is provided a composition for starch saccharification comprising the strain, a disrupted solution of the strain, a culture supernatant of the strain, or a mixture thereof.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; And a saccharification step of saccharifying the grain, grain powder or starch by inoculating the starch-containing material with the yeast produced in the step of producing the yeast, thereby saccharifying the starch.

In the saccharification method of the starch, the starch-containing material may be a grain, a grain powder, or a non-grain crop including starch, and the grain may be rice, wheat, barley, oats, buckwheat, sorghum, corn, rye And the starch may be a sweet potato, a potato, a cassava, an arrowroot, an accidental palm, a taro, an acorn, a chestnut, a mung bean, Or yam.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A saccharification step of saccharifying the starch in the starch-containing material by inoculating the starch-containing material with the yeast produced in the yeast-producing step; And an alcohol fermentation step in which an alcohol fermenting microorganism is inoculated into the saccharide produced in the saccharification step to perform alcohol fermentation.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; And a simultaneous fermentation step of simultaneously performing saccharification and alcohol fermentation by inoculating yeast and alcohol fermenting bacteria produced in the production step into a starch-containing product.

In the method for producing the fermented wine, the starch-containing material may be a cereal grain, a grain powder, or a non-grain crop including starch, and the grain may be rice, wheat, barley, oats, buckwheat, sorghum, corn, rye And the starch may be a sweet potato, a potato, a cassava, an arrowroot, an accidental palm, a taro, an acorn, a chestnut, a mung bean, Or yam.

In the method for producing the fermented wine, the alcohol may be methanol or ethanol, but preferably it is ethanol. The alcohol fermenting bacteria may be fungi or bacteria, and the fungi may be yeast, especially Saccharomyces , Brettanamyces , Zygosaccharomyces or Schizosaccharomyces And the bacterium may be Zymomonas mobilis .

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A saccharification step of saccharifying the starch in the starch-containing material by inoculating the starch-containing material with the yeast produced in the production step; An alcohol fermentation step of performing alcohol fermentation by inoculating an alcohol fermenting microorganism into the saccharide produced in the saccharification step; And a step of extracting the alcohol from the alcohol fermentation product produced in the alcohol fermentation step.

According to another aspect of the present invention, there is provided a process for producing koji comprising: a koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 to a solid medium for koji; A simultaneous fermentation step of simultaneously performing saccharification and alcohol fermentation by inoculating yeast and alcohol fermenting bacteria produced in the yeast-producing step with starch-containing water; And a step of extracting the alcohol from the alcohol fermentation product produced in the simultaneous fermentation step.

The starch-containing material may be a cereal grain, a powder of the cereal grain, or a non-grain crop including starch, and the cereal grain may be rice, wheat, barley, oats, buckwheat, sorghum, maize, rye And the starch may be a sweet potato, a potato, a cassava, an arrowroot, an accidental palm, a taro, an acorn, a chestnut, a mung bean, Or yam.

In the method of producing the alcohol, the alcohol may be methanol or ethanol, but preferably it is ethanol, and the alcohol fermenting bacteria may be fungi or bacteria, and the fungi may be yeast, especially Saccharomyces , Brettanamyces , Zygosaccharomyces or Schizosaccharomyces And the bacterium may be Zymomonas mobilis .

The present inventors have found that the use of a yeast having an excellent saccharifying ability is an essential process in the production of traditional sake. However, there is a problem in that it is difficult to maintain only a certain amount of fungi having excellent saccharifying ability in the conventional process of producing natural sake yeast. Aspergillus oryzae strain, which is a fungal strain, has a disadvantage in that the amylase production is suppressed and the saccharification rate is slowed down after a certain degree of starch glycosylation is suppressed because the amylase biosynthesis is repressed when glucose concentration is increased in the medium. We attempted to construct A. oryzae using the laboratory evolution technique, which retains amylase biosynthesis ability even when present. The laboratory evolution technique is a method of improving single cell microorganism strains with high cell division rate such as recently used bacteria. However, it is difficult to apply the laboratory evolution technique applied to bacteria directly to the fungi improvement since the fungus fungus can not grow into single cells and the cell division rate is slow. Therefore, the inventors of the present invention have succeeded in improving the evolution rate of the laboratory by treating the low concentration mutagen inducing agent to the spore stage which is the only single cell type in the life history of the germplasmic fungi, and repeatedly cultivating the improved strain having excellent starch resolution in the starch medium + As a method for screening, a fungus was developed as a strategy for developing a hyperglycosylated Aspergillus oryzae (Fig. 1).

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Aspergillus oryzae  Laboratory Evolution of Strain

According to one embodiment of the present invention, strain MS2-12 isolated from the yeast was used as a parent strain and the strain was improved through a laboratory evolution technique.

Specifically, the spore was recovered by culturing the MS2-12 strain in a potato-dextrose agar (PDA) for 1 week. The recovered spores were suspended in potato-dextrose broth (PDB) at a concentration of 10 ⁷ per ml, and then swelled by incubation at 25 ° C for 5 hours. Then, 0.1% (v / v) of methylmethanesulfonate (MMS), a mutagenic agent, was treated at room temperature for 30 minutes and centrifuged and washed three times with sterile distilled water. MMS-treated fungal spore liquid was prepared by adding 10 or more spores per well to a 96-well plate with starch-glucose liquid medium (starch 10 g / L, glucose 10 g / L, beef extract 3 g / And the cells were cultured at 30 DEG C for 48 hours. After the incubation, the α-amylase activity was measured in each culture medium, and a single colony was isolated from the culture medium containing the fungus containing the highly active fungus on a PDA medium. Then, the amylase activity of each of the isolated fungi was measured to select the strains having excellent activity. The amylase activity was measured by incubating each of the above fungi strains in a 5 mL starch liquid medium containing starch and beef extract at 10 g / L and 3 g / L at 30 ° C for 48 hours Respectively. Then, the culture was centrifuged at 13,000 rpm for 5 minutes to allow the remaining starch and hyphae to settle, and the supernatant was taken. 180 mL of water was added to 20 mL of the supernatant and 200 mL of starch (1 g / L) dissolved in 30 mM PBS (pH = 6.0) was added and reacted at 30 ° C. for 20 minutes. After the reaction, the reduced reducing sugar was dissolved in dinitrosalicylic acid acid) method to measure amylase activity. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 micromole of glucose per minute, and the strain with excellent amylase activity selected was inoculated into the PDA medium for one week after the inoculation, and the spores were recovered. The spore was subjected to the mutation- 5 times, the strains showing the highest amylase activity were finally selected (Fig. 2).

As a result, an Aspergillus oryzae M63 strain having an α-amylase activity of 3,890 unit / L was finally selected (FIG. 3).

Example 2: Stability of the modified M63 strain

The safety of the improved strain M63 having excellent amylase activity selected in Example 1 was investigated according to an embodiment of the present invention.

Specifically, in order to investigate amylase activity after the continuous cultivation of the modified strain M63 on a solid medium, a minimum medium containing 1% starch (starch 10 g / L, K ₂ HPO ₄ 1.74 g / L, KH ₂ PO ₄ 1.36 g / L, NaCl 0.14 g / L, MgSO 4 o _{7H 2 o 0.492 g / L,} CaCl 2 0.077 g / L, FeSO 4 o _{7H 2 o 0.0025 g / L,} (NH 4) 2 SO 4 0.528 g / L , YNB 1 g / L, pH 6.5) and incubated at 30 ° C for 5 days. After the incubation, the agar block was collected from the edge of the solid medium and transferred to a new medium for 50 times. In order to test the stability of the strain, the fungus which occurred in each subculture was cultured for 2 days in a liquid minimum medium without agar, and then the amylase activity in the culture medium was measured.

As a result, it was confirmed that amylase production ability of the improved strain M63 was stably maintained (FIG. 4).

Example 3: Growth characteristics of the modified M63 strain according to the culture conditions

In order to investigate the growth characteristics of the modified M63 strain selected according to one embodiment of the present invention, an agar block having a predetermined size and containing a pre-cultured fungal strain was placed on a starch solid medium, , 30 ° C and 40 ° C for 2-5 days and compared with parental strain MS2-12 and control strain RiB40. The growth of the fungus was measured by the diameter of the agar mycelium, and the effect of pH on the starch agar medium adjusted to pH 4.5, 5.5, and 6.5 of the agar medium, agar block And cultured at 30 ° C for 2-5 days. The growth of the fungi was measured by measuring the diameter of hyphae on the agar medium.

As a result, as shown in FIG. 5A, the optimum temperature of the modified strain M63 was 30 ° C., and the optimum pH of the modified strain M63 was 5.5, as shown in FIG. 5B, This result is the same as that of the strain RiB40.

Example 4: Amylase production characteristics of the modified M63 strain

In order to investigate the amylase production characteristics of the modified M63 strain selected according to one embodiment of the present invention, the strain M63 was cultured in a starch liquid medium (starch 10 g / L, beef extract 3 g / L) at 30 ° C for 60 hours Were compared with the parent strains MS2-12 and the control strain RiB40. In addition, in order to observe the decrease of amylase activity after 24 hours of incubation, the pH in the medium was examined and the reduction of the pH in the medium was activated by the acidic protease produced by the fungus to decompose the protein present in the medium. The pH was adjusted to 6.5 by adding 50 mM of phosphate buffer to the liquid medium, and then cultured to investigate the amylase activity.

As a result, all the strains of the test group showed the highest amylase activity 24 hours after the start of culture, and gradually decreased with the progress of culture. The M63 strain showed about 3,899 units / L of amylase activity when cultured for 24 hours and the MS2-12 and RiB40 strains showed 2,770 and 1,868 units / L, respectively, lower activity than the modified strains, and the modified strains showed the highest (Fig. 6A). The pH of the culture medium decreased progressively as the culture proceeded, and the pH of the culture medium decreased to 3.1 after 36 hours (Fig. 6C). In addition, the pH of the medium was kept constant until 36 hours after the pH was adjusted to 6.5, and the amylase activity was maintained for up to 36 hours. The amylase activity was maintained for up to 36 hours after reaching the peak at 24 hours. Thereafter, as pH decreased, the pH began to decrease and at the same time the amylase activity decreased (FIGS. 6B and D). Based on the above results, it was estimated that the reduction of amylase activity in the starch medium was due to the activation of acidic protease with decreasing pH.

Example 5: Alcohol fermentation characteristics of the modified M63 strain

To investigate alcohol fermentation characteristics of the modified M63 strain selected according to an embodiment of the present invention, M63 and RiB40 strains were inoculated into 400 g of high-fat rice and fermented for 24 hours, followed by drying at 50 ° C for 24 hours, . 600 g of rice cake, 0.25 g of yeast, and 1.5 L of water were added to 400 g of each of the above-prepared entrants to conduct starch saccharification and alcohol fermentation experiments.

As a result, the amylase activity in the medium containing M63 strain reached its maximum value (Fig. 7A) and the concentration of glucose from the decomposition of starch also reached a maximum value of 100 g / L (Fig. 7B) . Thereafter, the amylase activity and glucose concentration plummeted, and at the same time, the alcohol concentration rapidly increased to reach an alcohol concentration of 18.5% after 5 days of fermentation (FIG. 7C). On the other hand, in the case of the control bacteria, the starch saccharification ability was remarkably decreased, and the maximum alcohol concentration was also 13.4%, confirming that the M63 strain selected according to the embodiment of the present invention has superior performance.

In conclusion, the M63 strain, which has been modified by the laboratory evolution technique of Aspergillus oryzae MS2-12 strain isolated from domestic brewer's yeast, has excellent ability to produce amylase, and thus has excellent glycation ability of starch, The starch can be saccharified without enzyme addition, and the excellent glycosylation ability of M63 strain can accumulate glucose at a high concentration in a short time compared to conventional processes, minimizing contamination of germ, which is a problem of the traditional liquor process, and accomplishing alcohol fermentation based on this Therefore, there is an advantage of minimizing the pollution of germ, which is a problem of the traditional liquor processing. In particular, since the saccharification of starch is an important process not only in the production of traditional liquor but also in the production of bioenergy, the M63 strain of the present invention is a strain highly industrially utilizable.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Korea Institute of Bioscience and Genetic Resources Center KCTC13153BP 20161115

Claims (5)

Deposited as accession number KCTC13153BP, the novel Aspergillus enhanced ability glycosylated's duck material (Aspergillus oryzae ) strain M63. A composition for starch saccharification comprising the strain of claim 1, a disruption of said strain, a culture supernatant of said strain or a mixture thereof. A koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 into a solid medium for koji; And
And saccharifying the starch in the starch-containing water by inoculating the starch-containing water with the yeast produced in the step of producing the yeast. A koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 into a solid medium for koji;
A saccharification step of saccharifying the starch in the starch-containing material by inoculating the starch-containing material with the yeast produced in the yeast-producing step; And
And an alcohol fermentation step of inoculating an alcohol fermenting microorganism to the saccharide produced in the saccharification step to perform alcohol fermentation. A koji-producing step of producing koji by inoculating a fresh Aspergillus oryzae M63 (KCTC13153BP) strain of claim 1 into a solid medium for koji; And
And a simultaneous fermentation step of simultaneously performing saccharification and alcohol fermentation by inoculating the starch-containing water with the yeast and alcohol fermentation bacteria produced in the production step.

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