JP2019037252A - Novel koji mold, koji seasoning/koji beverage using koji mold, and process for producing koji seasoning/koji beverage - Google Patents

Abstract

To provide a low cost and simple production process in which koji seasoning/koji beverage or the like with few fine foreign substances and smooth tongue-texture can be obtained in a short time by an inexpensive and simple grating process.SOLUTION: Provided is a process for producing koji seasoning/koji beverage using koji mold, the process for producing koji seasoning/koji beverage comprises a step of saccharification of grains contained in feed raw material including a step of increasing the extracellular concentration of serine protease, in which the step of increasing the extracellular concentration of serine protease comprises adding serine protease as an enzyme agent.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a koji seasoning and koji beverage, a method for producing koji seasoning and koji beverage, and a novel koji mold suitably used in the manufacturing method.

Rice bran secretes enzymes such as α-amylase, glucoamylase, transglucosidase, acid protease, and acid carboxypeptidase in the saccharification process of rice, and decomposes starch and proteins of rice to produce grapes, etc. It is known to accumulate various essential amino acids, and amazake using the action of such koji molds on rice is eaten and consumed regardless of the season as one of excellent nutritional foods.

Amazake is mainly made from rice bran and rice, or sake lees, and the production method using koji and the method using sake lees are known. The method of using rice bran is obtained by decomposing rice starch and protein with enzymes produced by rice bran by mixing and fermenting rice bran and rice as raw materials. In addition, as a method of using sake lees, a method in which sake lees are dissolved in hot water and heated to add sweetness such as sugar or a combination thereof is also employed.

Regardless of the method used to produce amazake, if you cannot drink amazake, the solid content of rice (or rice bran) or sake lees will be uncomfortable and often shunned. The amazake paste is used. However, the amazake paste conventionally used for canned sake and kartkan amazake has a common problem that water-insoluble foreign matter is included, and the foreign matter feels bad and rough when eating and drinking. Therefore, in order to suppress this roughness, it has been proposed that the rice bran and / or sake lees paste is once dissolved in water to form a slurry and then processed by a mechanical method such as a mass collider, a homomixer, or a French press. (For example, Patent Document 1, Patent Document 2, etc.). However, this method is expensive and requires special equipment, and it takes much time to grind.

JP 2006-014701 A JP 2007-31648 A JP 2006-211966 A JP 2010-4760 JP 2007-82505 A JP 2009-095279 A

  An object of the present invention is to provide a low-cost and simple production method in which, in view of the above-mentioned present situation, a strawberry seasoning and a strawberry beverage with a small amount of fine foreign matter and a smooth touch can be obtained in a short time by an inexpensive and simple threading process. For the purpose.

  The inventors of the present invention have intensively studied to find that the cause of the rough texture is a foreign substance having a particle size of 0.2 mm or more and having a main component as a protein. When the enzyme agent was examined, it was found that the roughening component was decomposed by the addition of serine protease.

  Particularly in rice bran, no koji mold producing high serine protease has been known so far, and most proteases produce only acidic protease. In support of this, Patent Document 3 discloses a novel gonococci that has a significantly increased saccharide-degrading enzyme activity and / or proteolytic enzyme activity than the parent strain, and is useful as a practical strain in liquor brewing. However, the “proteolytic enzyme” here is limited to one or two of the enzyme group consisting of acidic protease and acidic carboxypeptidase, and no mention is made of serine protease. Patent Document 4 proposes a method for producing an alkaline protease by culturing a koji mold transformed with a vector consisting essentially of nucleotides derived from koji mold in a potato dextrose medium and a liquid medium. The medium is not rice bran. Patent Document 5 is an example in which a protein hydrolase is expressed on a bran medium using a transformed koji mold, but the production of serine protease on rice bran has not been verified. Patent Document 6 cultivates koji molds by irradiating heavy ion beams to obtain koji molds that achieve both high protease production and high glutaminase production, but the protease production ability is an evaluation of total protease activity. It does not suggest the production of serine protease on rice bran.

  Accordingly, one aspect of the present invention made to achieve the above object is a method for producing a koji seasoning and koji beverage using koji mold, and in the saccharification process of cereals contained in raw materials, a serine protease It is the manufacturing method of the strawberry seasoning and the strawberry drink including the step which increases the extracellular concentration of. By this method, it is possible to easily obtain a seasoning seasoning / beverage beverage with a small amount of fine foreign matter and a smooth touch without using expensive and special equipment such as a mascolloider in a short time. it can. Further, even when using a mass colloider, the processing speed can be greatly shortened, so that the manufacturing cost can be reduced.

  The step of increasing the extracellular concentration of the serine protease may include inoculating the new gonococcus with the grains contained in the raw material. As a result, the amount of the enzyme agent to be added separately during the saccharification step can be reduced, desirably no additional addition is required, and the manufacturing cost can be reduced.

  The step of increasing the extracellular concentration of the serine protease may include adding serine protease as an enzyme agent. Thereby, an enzyme reaction advances more rapidly and productivity can be improved.

  Another aspect of the present invention made to achieve the above object is a koji mold which belongs to Aspergillus oryzae and produces a serine protease in rice bran. This koji mold is a novel koji mold that produces a serine protease that is not produced at all by any known species in rice koji. For example, it can suppress the production of foreign substances during the production of amazake, and can perform special mechanical treatment such as mass colloid. It is extremely useful in that it is unnecessary.

  The above koji mold is fractionally quantified by enzyme inhibitors in protease activity measurement (30 ° C., pH 6.0) by the casein forin method of an aqueous extract of rice bran cultured at an ambient temperature of 30 ° C. and a humidity of 90% or more for 2 days. It is preferable that the relative activity of the serine protease with respect to the acidic protease measured by is from 20% to 200%.

  Another aspect of the present invention made to achieve the above object is Aspergillus oryzae BA-10230 (NITE P-01871) or Aspergillus oryzae BA-11400 (NITE P-01872).

  Another aspect of the present invention made to achieve the above object is a method for producing a koji seasoning and koji beverage, which includes an operation of increasing the concentration of serine protease in the process of processing sake lees contained in the raw materials. According to this production method, even when rice bran is not used as a raw material to be fed, it is possible to easily obtain a strawberry seasoning, a strawberry beverage, etc. with a small amount of fine foreign matter and a smooth touch in a short time.

  The other aspect of this invention made | formed in order to achieve the said objective is the koji or koji seasoning and koji drink obtained by the manufacturing method of koji seasoning and koji drink containing rice koji or sake koji as a raw material. With this configuration, it is possible to obtain a strawberry seasoning / beverage beverage with a smooth texture.

  Another aspect of the present invention is a strawberry seasoning / beverage beverage comprising a serine protease having an enzyme activity or inactivated, or a fragment of serine protease. If the serine protease contained in strawberry seasonings and strawberry beverages has an enzymatic activity, for example, for the purpose of immersing meat and fish meat, the meat quality will be significantly softened in a short time with the penetration of taste, flavor and umami Bring effect. On the other hand, if the serine protease contained in strawberry seasonings and strawberry beverages is inactivated or decomposed, the progress of the enzyme reaction in the final product will lead to deterioration of the product. it can.

  According to the present invention, in the conventional saccharification process of rice with rice bran, the production of fine foreign matter that has been generated in the saccharified product is suppressed, and a strawberry seasoning / beverage beverage with improved texture of the tongue can be obtained in a short period of time. Therefore, productivity can be remarkably improved.

In Example 4, it is a photograph which shows the result of having soaked ham in the salt candy produced with the novel mutant strain BA-11400. In Example 4, it is a photograph which shows the result of having soaked ham in the salt candy produced with the novel mutant BA-10230. In Example 4, it is a photograph which shows the result of pickling ham in the salt candy manufactured with the commercially available salt candy made from M company. In Example 4, it is a photograph which shows the result of having soaked ham in the commercially available salt jar made from H company.

  The novel koji mold of the present invention determined the base sequence of 18S rDNA according to a conventional method and compared it with a known base sequence. As a result, it showed 99% or more homology with Aspergillus oryzae, so that Aspergillus oryzae ( Aspergillus oryzae) is a serine protease high-producing strain in rice bran. Examples of the acquisition method include screening of commercially available strains and isolated strains, and the action of mutation creation means such as physical ultraviolet (UV) irradiation and use of chemical mutagens. Of these, from the viewpoint of simplicity, screening for commercially available strains and UV irradiation are preferred. For example, the Aspergillus oryzae spores are irradiated with UV, then cultured on a plate, and a mutant having improved serine protease production ability is selected by enzyme fractionation quantification. A mutant of Orise is obtained.

  One strain of Neisseria gonorrhoeae newly isolated by the above method was found to belong to Aspergillus oryzae by the following genetic analysis. However, since the serine protease production ability is remarkably high, Identified, named and labeled Aspergillus oryzae BA-10230, named NITE P-01871 at the Patent Microorganism Depositary Center (NPMD), and another strain named and labeled Aspergillus oryzae BA-11400. ) Is deposited as NITE P-01872.

The method and results of gene analysis are shown below.
(1) Acquisition of chromosomal DNA 100 ml of Sabouraud medium (glucose 4.0%, polypeptone 1.0%, pH 5.6) is placed in a 500 ml Sakaguchi flask, autoclaved, and Aspergillus oryzae BA-10230 or Aspergillus oryzae BA- 11400 was inoculated. After 4 days of shaking culture at 25 ° C., no. The cells were collected by filtration with 2 filter paper. Freeze the cells with liquid nitrogen, pulverize them to a fine powder in a mortar, slowly add 15 ml of DNA extraction buffer (5.0% SDS, 0.1 M, NaCl, 50 mM Tris-HCl, pH 8.0). Dissolved by shaking. The supernatant was obtained by centrifugation (5,000 rpm, 6 min, rt.), Phenol / chloroform extraction was performed three times, ether extraction was performed twice, and the ether was evaporated. Then, 1 M of 3M sodium acetate and 25 ml of ethanol were added, It was left at -30 ° C for 30 minutes. Chromosomal DNA was recovered by centrifugation (12,000 rpm, 10 min, 4 ° C.), washed with 70% ethanol, and dissolved in 400 μl of TE. Next, 10 μl (0.132 U) of RNase was added to the obtained DNA solution, treated with RNase at 37 ° C. for 1 hour, then 5 μl (0.6 U) of proteinase K was added, and treated at 50 ° C. for 1 hour. After phenol / chloroform extraction (twice) and chloroform / isoamyl alcohol extraction (once), DNA was collected by ethanol precipitation. After washing with 70% ethanol solution, ethanol was removed and dissolved in 200 μl of TE.
(2) Amplification of 18S rDNA gene fragment P1 5′-ATCTGGTTGATCCTGCCAGT-3 ′ (SEQ ID NO: 1)
P2 5'-AATGATCCCTCCGCAGGTTC-3 '(SEQ ID NO: 2)
Using the chromosomal DNA obtained by the above-described method as a template DNA, 25 cycles of PCR reaction were performed using a combination of P1 primer and P2 primer. The nucleotide sequence “SEQ ID NO: 3” (SEQ ID NO: BA-10230 18S rDNA base sequence) and “SEQ ID NO: 4” (SEQ ID NO: BA-11400 18S rDNA base sequence) of the obtained DNA fragment were determined.
When the base sequences shown in “SEQ ID NO: 3” and “SEQ ID NO: 4” were compared with a known base sequence database, both showed 99% or more homology with the 18S rDNA base sequence of Aspergillus oryzae. This strain was identified as Aspergillus oryzae.

  The novel koji mold of the present invention is a protease activity measurement by a casein forin method (30 ° C., pH 6.0) of an aqueous extract of rice koji cultured for 2 days under conditions of an atmospheric temperature of 30 ° C. and a humidity of 90% or more. It is preferable that the relative activity of serine protease with respect to acidic protease measured by fractional quantification with an enzyme inhibitor is 20% or more and 200% or less. The more preferable lower limit of the activity is 35%, and the more preferable upper limit is 180% or less, 150% or less, or 120% or less. Here, the activity is a proteolytic enzyme activity, which is a numerical value calculated per 1 g of rice bran, with the amount of enzyme that releases a non-protein substance equivalent to 1 μg of tyrosine per minute at 30 ° C. as one unit.

  The koji mold of the present invention can be widely used for producing koji beverages and koji seasonings with or without alcoholic fermentation by yeasts such as sake, shochu, mirin, rice miso, and salt koji, in addition to amazake.

  Hereinafter, the method for producing the koji seasoning and koji beverage of the present invention when the raw material is rice will be described, but the same applies to other grains such as barley, wheat, red beans, oats, koji, koji, and corn. is there. In the present specification, the “charged raw material” is a concept that includes a raw material for straw and a hanging raw material.

  In the case of using solid rice bran, it includes a pretreatment step optionally applied to the raw materials, a steaming step for obtaining steamed rice, and a saccharification step.

  The pretreatment process includes a rice roasting process, a whitening process, a dipping process, and the like. The roasting step is optional, but may be performed on any grain raw material before or after whitening. In the milling process, the milling rate is optional, but is usually in the range of 40% to 90%.

  For the steaming process, either non-pressure steaming at 100 ° C. or lower and pressurized steaming at a temperature exceeding 100 ° C. can be employed.

  The saccharification process is a first stage in which rice bran obtained in the steaming process is inoculated with koji mold to obtain rice koji, and kake raw materials and / or salt and hot water, shochu or alcohol for brewing are added to the rice koji. And a second stage maintained at a predetermined temperature for a predetermined time. In the second stage, an amylase preparation may be added. In the present specification, the “saccharification step” means a series of steps for decomposing starch in cereal grains contained in raw materials to glucose. Therefore, the “saccharification process” is a concept including “liquefaction” used by those skilled in the art in the sense of degrading starch to a certain extent even if it does not reach glucose.

  The koji mold used in the first stage may be any of koji molds obtained by a conventionally known koji mold, the koji mold of the present invention, other mutations, screening, or genetic recombination techniques. Includes the step of increasing the secretion of serine protease and increasing the extracellular concentration of serine protease by inoculating the novel koji mold described above.

  As the rice used in the second stage, glutinous rice and low-glutelin rice can be used in addition to general brewed rice. Although the rice polishing rate is arbitrary, it is usually in the range of 40% to 90%. It is preferable to sterilize and gelatinize starch components in advance by a steaming method or roasting method. The predetermined temperature is usually 50 ° C. to 65 ° C., and the predetermined time is usually 5 hours or longer and is usually left overnight. However, depending on the product, it is aged for a long time (1 month to 1 year) at room temperature. . In the second stage, one or more amylases selected from the group consisting of α-amylase, glucoamylase, isoamylase, and pullulanase prepared separately may be supplementarily added.

  In the second stage, a commercially available serine protease prepared separately may be added to increase the extracellular concentration of serine protease. The serine protease used in the second step is not particularly limited. For example, subtilisin-like serine proteases such as subtilisin, thermitase, proteinase K, lantibiotic peptidase kexin; trypsin, chymotrypsin, thrombin, elastase And chymotrypsin-like serine proteases. These serine proteases may be derived from any bacteria.

  The step of increasing the extracellular concentration of serine protease in the first stage and the second stage is not an alternative and can be used in combination. Further, in the present specification, “increasing the extracellular concentration of serine protease” includes not only increasing the amount of serine protease from a state in which a predetermined amount of serine protease exists in advance outside the bacterial cell, but also serine protease. Increasing the serine protease from a zero concentration state in which the protease is substantially absent is also included.

  As another method for increasing the extracellular concentration of serine protease, in the first stage, the second stage, or as a subsequent process, serine protease accumulated in the bacterial body by crushing the bacterial body is used. It may include releasing to the outside.

  The enzyme contained in the product obtained by the saccharification step can be inactivated by appropriate heat treatment or addition of a deactivating substance. When serine protease is added, it is basically inactivated by heating, but it does not exclude maintaining the activity. The protease group containing serine protease produced by the novel koji mold has an option of maintaining activity and an option of deactivating depending on the product application. For example, when the product is amazake, it is preferably deactivated due to its characteristics. On the other hand, if the product is salted salmon or rice miso, it may be deactivated, and when meat, ham, fish meat, etc. are soaked, the enzyme will soften the meat quality along with the penetration of taste, flavor and umami The option of leaving the enzyme in the final product is also conceivable.

  In the above, the case where rice (rice bran) is used as the feed material has been described. However, when rice (rice bran) is not used as the feed material, for example, the concentration of serine protease is increased even when using sake lees. The same effect can be obtained by adjusting the optimum pH and temperature of activity. However, it is preferable to use a mechanical treatment such as a mass collider.

  The product form of the koji seasoning and koji drink obtained by the production method of the present invention is not particularly limited. For example, it can take various forms such as freeze-dried products, pastes, slurries, granules, powders, liquids, etc. In order to obtain a product form, a conventionally known processing method can be employed.

  A strawberry seasoning and strawberry beverage having serine protease activity is also one aspect of the present invention. The serine protease is not necessarily the same as the serine protease added in the above-described production method or the serine protease derived from the screening strain or the mutant strain. For example, trypsin, chymotrypsin, thrombin, plasmin, Streptomyces genus alkaline protease, Aspergillus Any of alkaline genus derived from the genus, alkaline protease derived from the genus Bacillus subtilis may be used.

  The serine protease or serine protease fragment having the enzyme activity is preferably contained in a significant amount at least so that it can be determined that it has been added from the outside or produced by the strain, and the atmospheric temperature is 30 ° C. and the humidity is 90% or more. 1 U / g of serine protease activity measured by fractional quantification using an enzyme inhibitor in the protease activity measurement (30 ° C., pH 6.0) of the aqueous extract of rice bran cultured under the above conditions for 2 days by the casein forin method It is preferable that it is contained above. A method for separating and detecting serine protease remaining in the product or a fragment thereof is not particularly limited, and for example, an enzyme activity or a Western blotting method can be employed.

  The koji seasoning and koji beverage of the present invention includes rice-derived koji seasoning and koji beverages such as sake, salt koji, mirin, mirin-like seasoning, rice miso, etc. in addition to amazake.

(Selection by screening of high serine protease producing strain)
About 200 strains of commercially available seed koji and Ichibiki isolates (isolated from nature such as soil and rice) were smeared on a PDA plate medium, and the grown koji molds were solid-cultured using 90% polished rice (2 at 30 to 35 ° C). Day). 10 g of the resulting rice bran was extracted and filtered with 50 ml of water at room temperature for 3 hours, and then 0.4 ml of the supernatant was subjected to a fractional quantification method using an enzyme inhibitor to select a serine protease high-producing strain.

(Creation of a serine protease high-producing mutant)
A spore suspension of the serine protease high-producing strain obtained by the above screening was prepared, and UV irradiation (15 W germicidal lamp, 7 minutes, lethality 95%) was performed according to a conventional method to induce mutation. The UV-treated spore suspension was smeared on a PDA plate medium, and the grown candidate strains were fished on a PDA slant medium and cultured at 30 ° C. for 3 days. The obtained mutant strain was subjected to solid culture (2 days at 30 to 35 ° C.) using 90% polished rice. 10 g of the resulting rice bran was extracted and filtered with 50 ml of water at room temperature for 3 hours, and then 0.4 ml of the supernatant was subjected to a fractional quantification method using an enzyme inhibitor to select a serine protease high-producing mutant.

(Measurement of enzyme activity and calculation of relative activity by enzyme fractionation method)
Evaluation of the enzyme activity in rice bran of the serine protease high-producing strain obtained by the above screening or mutation method was performed according to the following procedure.
To 0.4 ml of the crude enzyme solution obtained above, 0.1 ml of various inhibitor solutions were added to a final concentration of 0.1 mM and left at room temperature for 10 minutes. Ten minutes later, 1.0 ml of a 2% milk casein solution adjusted to pH 6.0, which had been kept at 30 ° C., was added and reacted at 30 ° C. for 20 minutes. The reaction was stopped by adding 2.5 ml of 0.4 M trichloroacetic acid solution and left at room temperature for 10 minutes. After 10 minutes, no. Filter through 6 filter paper to obtain a filtrate. Add 0.5 ml of 0.4M sodium carbonate solution and 0.5 ml of Folin reagent (1N) to 0.5 ml of the filtrate, mix well, and let stand at room temperature for 10 minutes. The absorbance at 660 nm was measured using a microplate reader (Sunrise Rainbow manufactured by Tecan).

Various protease activities under these conditions were defined as follows.
(1) Total protease activity: The above procedure was performed using water as an inhibitor solution, and the amount of enzyme that liberates non-proteinaceous substances corresponding to 1 μg of tyrosine per minute was evaluated as 1 unit.
(2) Acid protease activity: Pepstatin A is used as an inhibitor solution, and the above procedure is performed. Compared with the total protease activity, the proteolytic activity inhibited by pepstatin A is non-protein equivalent to 1 μg of tyrosine per minute. The amount of enzyme liberating the substance was evaluated as 1 unit.
(3) Serine protease activity: Using phenylmethylsulfonyl fluoride (PMSF) as an inhibitor solution, the above operation is performed, and the proteolytic activity inhibited by PMSF is reduced to 1 μg of tyrosine per minute compared to the total protease activity. The amount of enzyme that liberates the corresponding non-proteinaceous substance was evaluated as 1 unit.
(4) The relative activity of serine protease activity to acidic protease activity was calculated by the above serine protease activity / acidic protease activity × 100 (%).

(Example 1: Production of amazake)
The polished rice obtained by milling at 90% of the polished rice ratio was washed according to a conventional method, immersed in water for 20 hours, drained for 1 hour, and then steamed by pressureless steaming for 30 minutes. 200 g of the obtained steamed rice is inoculated with a commercially available seed meal prepared in advance, and cultured for 2 days under conditions of an atmospheric temperature of 30 ° C. and a humidity of 90% or more. 410 g of cooked rice, 320 g of hot water, 0.5 g of amylase preparation, and 0.4 g of commercially available serine protease (trade name: Orientase 22BF, manufactured by HBI) were added and saccharified overnight at 50-60 ° C. The obtained saccharified product was subjected to a miso-sleeving machine (screw extrusion type, through-hole diameter 0.4 mm), and then heat-treated at 85 ° C. to obtain a sweet sake paste. When the resulting amazake paste was dissolved in water and eaten, there was no rough texture.

(Example 2: Production of amazake)
Amazake paste was obtained in the same manner as in Example 1 except that seed pods containing Aspergillus oryzae mutant BA-10230 (NITE P-01871) prepared in advance were inoculated. The relative activity of serine protease with respect to the acidic protease of the obtained rice bran was 55.1%, and when the obtained amazake paste was dissolved in water and eaten, there was no rough texture. Similarly, amazake paste was obtained in the same procedure as in Example 1 except that seed pods containing Aspergillus oryzae mutant BA-11400 (NITE P-01872) were inoculated. The relative activity of serine protease with respect to the acidic protease of the obtained rice bran was 54.8%. When the obtained amazake paste was dissolved in water and eaten, there was no roughness of the touch.

(Reference example: Amazake production)
The rice bran obtained in Example 1 was further cultured and cultured for 4 days. As a result, the rice bran obtained had green spores and its commercial value as amazake was lost. However, the relative activity of acid protease and serine protease was 195% for BA-10230 and 186 for BA-11400. % And the relative activity of serine proteases was greatly increased. When the amazake paste prepared using the obtained rice bran was dissolved in water and eaten, there was no rough texture. By further introducing a mutation into the gonococcal mutant strain to make it difficult for spore to grow, whitening the color of the spore, or increasing the growth rate, a mutant strain having a relative activity of nearly 200% can be put to practical use. It has been suggested.

(Comparative Example 1: Production of amazake)
Amazake paste was obtained in the same manner as in Example 1 except that acidic protease (trade name: Orientase AY, manufactured by HBI) was added instead of the commercially available serine protease. When the resulting amazake paste was dissolved in water and eaten, a rough texture was still felt and the presence of foreign matter was confirmed.

(Comparative Example 2: Production of amazake)
An amazake paste was obtained in the same manner as in Example 1 except that metal protease (trade name: Nuclidein, manufactured by HBI) was added instead of the commercially available serine protease. When the resulting amazake paste was dissolved in water and eaten, a rough texture was still felt and the presence of foreign matter was confirmed.

(Comparative Example 3: Production of amazake)
Amazake was obtained in the same manner as in Example 1 except that no commercially available serine protease was added. The obtained amazake was crushed with a mass colloid (model: super mass colloid MKCA6-2Jα, grinding wheel: MKE) (clearance: -100 μm from light contact, rotational speed of rotating grindstone: 1,800 rpm). By this method, the crushing processing speed for obtaining amazake paste that does not remain on the mesh even at 70 mesh is 118 kg / h, and the processing speed for producing the same level by the method of Example 1 is 296 kg / h. Yes, by using the present invention, the milling speed was improved approximately 3 times.

(Example 3: Production procedure of salted salmon)
The polished rice obtained by milling at 90% of the polished rice ratio was washed according to a conventional method, immersed in water for 20 hours, drained for 1 hour, and then steamed by pressureless steaming for 30 minutes. 4 kg of the obtained steamed rice was inoculated with a seed meal containing Aspergillus oryzae mutant BA-10230 (NITE P-01871) or Aspergillus oryzae mutant BA-11400 (NITE P-01872) prepared in advance, Culturing was performed for 2 days under the conditions of an atmospheric temperature of 30 ° C. and a humidity of 90% or more, and 550 g of hot water and 111 g of sodium chloride were added to 450 g of the obtained rice bran and aged at 30 ° C. for 1 week. The obtained salted salmon was not inferior to ordinary salted salmon in color, taste and aroma. When the protease activity in the salted salmon obtained was separately quantified, the relative activity of serine protease with respect to acidic protease was 54.1% and 50.2%, respectively.
As a comparison, commercially available salt koji (manufactured by H company, manufactured by M company) was measured in the same manner, but serine protease activity was not observed (relative activity of less than 10%).

(Example 4: Ham experimental procedure)
Cut 4 slices of ham (made by Itoham Co.) without using starch as a binder, add 5 g of salt candy from Example 3 to this, and immerse it at 5 ° C for 2 days. It is. As a comparison, sliced ham was soaked in the same manner using commercially available salted salmon (manufactured by Company H, Company M).
As a result, as shown in the photograph, the sliced ham soaked in the salt koji containing the serine protease obtained in Example 3 was decomposed until it became tattered, but the acidic protease activity was the same level. In addition, none of the salt candy produced by Company M, which had about half the amount of acidic protease, caused significant degradation of sliced ham.

(Example 5: Experiment of immersing pork loin)
Put pork loin (about 100 g) cut to a thickness of 1 cm into the bag with 20 g of salted salmon obtained in Example 3 or commercially available salted salmon and mix well with the meat. Dipping was carried out at 3 ° C. for 3 hours. After 3 hours, the pork was lightly wiped with a cloth to wipe off moisture and salt. Thereafter, the meat was replaced with a bag, and after sealing, it was heated in a 75 ° C. water bath for 30 minutes. The heated loin meat was cut into a width of 1.5 cm, and the softness was compared. As a result, as described below, the pork loin soaked in salted salmon obtained in Example 3 was clearly evaluated to be softer than that soaked in commercially available salted salmon. The softness was evaluated by blinding all samples and then by sensory evaluation by 10 panelists. The softness criteria were evaluated in 7 stages, and the average score of each panel was compared. Note that the closer the score is to 7, the softer the score.

  As mentioned above, although the Example of this invention was described, this invention is not restricted to these Examples, In the range which does not deviate from the summary, it can implement with a various form further.

  INDUSTRIAL APPLICABILITY The present invention can be particularly suitably used for the production of koji seasonings and koji beverages and the production of pickled products such as meat, fish meat and vegetables using these.

Claims (5)

A method for producing a koji seasoning and koji beverage using koji molds,
In the saccharification process of the grains contained in the raw materials to be fed, including the step of increasing the extracellular concentration of serine protease,
The step of increasing the extracellular concentration of serine protease includes adding serine protease as an enzyme agent, and a method for producing a koji seasoning and koji beverage.   A method for producing a koji seasoning and koji beverage, which includes an operation of increasing the concentration of serine protease in a process for processing sake lees contained in raw materials. Furthermore, the crushing process speed | rate for obtaining the amazake paste which does not remain on a mesh even if 70 meshes is included including crushing processing by a mascoloider, The seasoning seasoning of Claim 1 or Claim 2 exceeding 118 kg / h -A method for producing a coffee beverage.   In the protease activity measurement of the aqueous extract by the casein forin method (30 ° C., pH 6.0), the relative activity of serine protease with respect to acidic protease measured by fractional quantification with an enzyme inhibitor is 20% or more and 200% or less.糀 Condiments and beverages.   A seasoning / beverage beverage containing a serine protease having an enzyme activity or inactivated or containing a serine protease fragment, the amount of foreign matter having a water-insoluble protein having a particle size of 0.2 mm or more as a main component is commercially available at the time of filing The rice bran was cultivated for 2 days on steamed rice with a brewing rate of 90% under the conditions of an ambient temperature of 30 ° C. and a humidity of 90% or more using the seed bran prepared, and the amount of rice bran relative to the total amount A rice cake or rice bran seasoning that is less than the amount of foreign matter contained in the product obtained by adding saccharified overnight at 50-60 ° C. by adding steamed kake rice and hot water in an amount of 17%. Food and beverages.