KR20130086865A - Rice-nuruk prepared by using rhizopus oryzae ccs01 strain and makgeolli prepared by using the rice-nuruk - Google Patents

Abstract

PURPOSE: Makkoli (Korean rice wine) using rice nuruk (fermentation starter) with an enhanced activity of alpha-amylase is provided to have low acidity and high alcohol content and to remarkably increase preference with a low degree of browning. CONSTITUTION: Rhizopus oryzae CCS01 (KACC93118P) is used as a spawn for rice nuruk with an enhanced activity of alpha-amylase. A method for producing makkoli comprises the step of using grains and the rice nuruk. The grains are selected among white rice, brown rice, germinated brown rice, green rice, barley, buckwheat, and wheat. The makkoli has low acidity, high alcohol content, and a low degree of browning. [Reference numerals] (AA) Nuruk(fermentation starter) on sale; (BB) Rice nuruk made of R. Oryzae CCS01; (CC) Degree of browning(OD 420mm); (DD) Non-glutinous rice; (EE) Glutinous rice; (FF) Brown rice; (GG) Germinated brown rice; (HH) Green rice; (II) Barley; (JJ) Buckwheat; (KK) Milled wheat; (LL) Grains

Description

Rice- Nuruk with enhanced alpha-amylase activity prepared with Ryzopus duck CSC01 strain

The present invention is Lysopus duck CCS01 ( Rhizopus oryzae CCS01) alpha production by strain - this is the amylase activity enhancer ssalnuruk and palatability of the pH and assist browning prepared using the ssalnuruk relates to a superior rice wine.

Recently, with the spread of well-being and LOHAS trends, interest in Korean traditional liquor has increased, and rice wine, a muddy liquor with a unique refreshing taste and low alcohol content, is entering a new turning point.

Makgeolli is a Korean traditional liquor made from glutinous rice, non-glutinous rice, barley rice, brown rice, corn, sweet potato, wheat, and other ingredients. It is a refreshing drink with 2-8% alcohol content. Unlike other alcoholic beverages, Makgeolli is rich in various nutrients. It contains vitamins B, which are involved in human metabolism, essential amino acids such as lysine, leucine and arginine, flavors such as ethyl acetate, amyl acetate, ethyl caroate , An organic acid to relieve thirst with a sour taste, and acetylcholine to help liver function. Makgeolli is not only high in nutritional and functional value, but also contains fresh yeast and has a unique taste that cannot be compared with other alcoholic beverages. The distribution of microorganisms differs depending on the type of yeast, and accordingly the enzyme activity, yeast content, and alcohol fermentation power by microorganisms are different, and it is reported that yeast has a great influence on the quality characteristics such as volatile flavor components, taste, and color of makgeolli. have.

Yeast has been made by breeding many kinds of microorganisms such as mold, yeast and bacteria in the natural state. When manufacturing makgeolli using such yeast, it is difficult to homogenize the product and there is concern about deterioration. It is manufactured using Koji, which was developed in Japan. Makgeolli is strongly pointed out that our traditional stock, Makgeolli, is not Korean at a time when not only domestic consumption but also exports to Japan are rapidly increasing.

Therefore, there is a demand for the development of excellent yeast that is excellent in enzyme activity and alcohol generating ability and enhances palatability of makgeolli.

As a result of intensive research to meet the demands of the prior art, the present inventors have found / identified the strain of Raizus duck material CCS01, which makes it possible to prepare rice malt with excellent characteristics, and the rice malt has excellent palatability. It was confirmed that the present invention can be produced and came to complete the present invention.

It is an object of the present invention to provide rice malt with excellent enzyme activity and alcohol production ability, such as alpha-amylase activity, prepared with the Lysopus duck material CCS01 strain.

Still another object of the present invention is to provide makgeolli, which has low acidity, high alcohol content, and good color, made from rice malt as described above.

In order to achieve the above object, the present invention provides a rice malt with excellent enzyme activity and alcohol production ability, such as alpha-amylase activity prepared with the Lysopus duck material CCS01 strain.

Raizopus  Duck CCS01 ( Rhizopus oryzae CCS01 Strain

The present inventors have isolated / identified strains of Lysopus duck from CCN01 from conventional Nuruk, confirming that this strain has excellent characteristics such as producing yeast with enhanced alpha-amylase activity and the National Institute of Agricultural Science. The strain was deposited with the accession number KACC93118P on April 11, 2011 (Example 1).

As a result of analyzing the sequencing of 26S rDNA into the strain of Lycopus duck CCS01, it was determined to be 699 bp (FIG. 1). This sequence was analyzed by genotype 26S rDNA gene sequence homology with the strains of the genus Lyzopus on the NCBI database and the results are shown in Table 1.

Strain ^{2 )} 26S rDNA  Gene sequence Similarity (%) ^{One )} One 2 3 4 5 6 7 8 9 10 CCS01 100 2.AY213625 100 100 3.HQ435039   99.7   99.7 100 4.AB250191   93.7   93.7   93.4 100 5.AB250195   82.1   82.1   81.9   80.5 100 6.AB250198   93.4   93.4   93.4   97.2   80.0 100 7.AY213627   90.8   90.8   90.8   93.9   79.6   93.2 100 8.DQ466593   87.8   87.8   87.8   86.6   80.0   86.4   85.2 100 9.DQ466608   82.1   82.1   81.9   80.5   80.0   79.6   80.0   80.3 100 10.FN182234   93.2   93.2   93.2   97.7   80.0   93.6   86.7   99.0   80.0 100 ¹⁾ Calculated from the CLUSTAL W and PAM250 residue weight tables.
²⁾ R. oryzae CCS01 (CCS01), R. oryzae UWFP 846 (AY213625), R. delemar ATCC 34612 (HQ435039), R. casespitosus CBS 427.87 (AB250191), R. stolonifer CBS 319.35 (AB250195), R. homothallicus CBS 336.62 (AB250198), R. sexualis BCRC 32007 (AY213627), R. azygosporus BCRC 31158 (DQ466593), R. circinans BCRC 33727 (DQ466608), R. microsporus CBS 261.28 (FN182234).

Raizopus  Duck CCS01  Yeast production with strain

Rizopus duck material CCS01 strain is placed in sterile distilled water to suspend the spores to use as a spawn.

In the present invention, it is preferable to use rice which is not cooked as leaven.

The manufacturing process of the yeast can be performed according to a conventional method. Specifically, the rice (white rice or brown rice) is washed with water and then poured sufficient water, soaked for 4 to 5 hours at room temperature, and then drained by sieving through a burlap sieve. To the dry rice, add 1 ~ 10% by weight of Ryzopus duck material CCS01 spawn and add sterile distilled water and mix (dough). Fill the dough into the yeast mold, chop it, put the molded yeast into the tray, cover it with burlap, and mature for 4 to 10 days at 20 to 25 ℃ until the mycelia grow completely. The aged yeast is dried and dried at room temperature to remove mycelium and then powdered.

The yeast prepared according to the invention has excellent alpha-amylase activity (Example 3).

In addition, makgeolli with low acidity, high alcohol content, good color, and improved palatability manufactured by Nuruk are provided.

Raizopus  Duck CCS01  Rice wine production using yeast of strain

Makgeolli is prepared using the yeast and grain raw materials prepared according to the present invention.

As raw materials of grains, non-glutinous rice, glutinous rice, brown rice, germinated brown rice, green rice, barley, buckwheat or fine grains may be used alone or in combination of two or more. After washing grain raw material with water, pour water and immerse it sufficiently at room temperature, remove water, and use it after cooling by increasing the steam at 95 ~ 100 ℃ for 1 ~ 2 hours.

Makgeolli production is by a conventional method. For example, it can manufacture by the two stage forging method as follows. Nuruk and water are mixed at a weight ratio of 1: 1.5, and fermented at 25 ° C. for 20 to 28 hours to prepare a base liquor (single immersion). In one step soaking, alcoholic fermentation yeast may be further added as necessary to prepare the base liquor. Yeast that can be used is Saccharomyces cereviase ( Saccharomyces cereviase ), the addition amount is preferably 2 to 5% (v / v).

18 to 20% by weight of the prepared base material, 25 to 27% by weight of grain raw materials, 2 to 3% by weight of yeast and 52 to 53% by weight of purified water are mixed and fermented at 25 ° C. for 5 to 10 days (two-stage immersion).

The nuruk according to the present invention is excellent in alpha-amylase activity and alcohol producing ability, so that makgeolli having excellent palatability is produced when it is used for making makgeolli. In addition, the nuruk according to the present invention can produce makgeolli having excellent palatability without limitation to the grain raw materials used.

Makgeolli prepared according to the present invention has a low acidity, a high alcohol content, and especially a low browning degree, which significantly enhances palatability.

Figure 1 is a 26S rDNA nucleotide sequence of Lysopus duck duck CCS01 strain.
Figure 2 shows the alpha-amylase activity of yeast and conventional yeast according to the present invention.
Figure 3 is a manufacturing process of the yeast using the Lysopus duck material CCS01 strain.
Figure 4 shows the pH of the yeast grain and rice grains prepared by using the conventional yeast in accordance with the present invention.
Figure 5 shows the acidity of the yeast grain and the eight grain rice wine prepared using the conventional yeast in accordance with the present invention.
Figure 6 shows the brix sugar content of the eight grains rice wine produced by using the yeast and conventional yeast according to the present invention.
Figure 7 shows the alcohol content of the yeast and eight grain rice wine prepared using the conventional yeast according to the present invention.
Figure 8 shows the browning degree of the 8 kinds of grain rice wine made by using the yeast and conventional yeast according to the present invention.

The present invention will be explained in more detail by the following examples. These examples are for illustrating the present invention, and the scope of the present invention should not be limited by them.

Example

Example  One. Raizopus  Duck CCS01 Separation and identification

10 g of Korean Nuruk (Jeju Nuruk, Pearl Grain Co., Ltd.) was added to 90 mL of sterile distilled water, shaken vigorously, and left to stand for 1 hour. The supernatant was dissolved in potato dextrose agar (PDA, Difico, USA) (1.5 mg / mL). After plating for one week and incubating for one week, only spores of the form of lycopus with typical black spores were selected and spores were centrally placed in a medium containing fresh potato dextrose agar chloramphenicol (1.5 mg / mL). Inoculated and incubated at 25 ° C. for 5 days. This process was repeated 2-3 times to isolate pure Lazopus strain.

Spores from pure isolates were suspended in sterile distilled water, spores were taken in a 1.5 mL centrifuge tube, about 5 mL, centrifuged, spores collected, and genomic DNA was isolated using DNAzol kit (Invtrogen, USA). .

Using the isolated genomic DNA as a template, a denatured 0.6 kb fragment was amplified by performing 30 cycles of denaturation at 94 DEG C for 1 minute, 52 DEG C for 30 seconds, and 72 DEG C for 30 seconds. The amplified 26S rDNA PCR product was electrophoresed in 1% agarose, recovered and purified 0.6 kb fragment, cloned using pGEM-T Easy (Promega, Madison, USA) and Escherichia coli After transforming to DH5a, transformants were randomly selected for pure separation, inoculated in pure LB liquid medium containing 50 ㎍ of ampicillin, and cultured for 16 hours at 37 ° C. Plasmids were isolated / purified as described in Purification Kit (Intron, Suwon, Korea).

The isolated plasmid was used as sequencing template strand. Nucleic acid sequences were analyzed using dideoxy chain termination using PRISM Ready Reaction Dye terminator / primer cycle sequencing kit. The 26S rDNA sequence was finally identified in alignment with those of the BLAST network service and the US National Bioinformatics Center (NCBI) species (Table 1). The identified strains were named as lycopus duck CCS01, and deposited on April 11, 2011 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and received accession number KACC93118P.

Example  2. Raizopus  Duck CCS01 Strains  Nuruk Manufacturing

Lysopus duck material CCS01 strain plate according to Example 1 was put into 100 ml of sterile distilled water to suspend the spores were used as spawn.

2 kg each of white and brown rice was washed with water, poured with sufficient water and soaked for 4 hours at room temperature. To each of the white rice and brown rice, which had been drained, 1 to 10 wt% (w / w) of Ryzopus duck material CCS01 was added, and 200 mL of sterile distilled water was added and kneaded. After filling the dough in the yeast mold and chopped, the molded yeast was put in a tray and covered with burlap, and aged at 20 to 25 ° C. until the mycelium was completely grown. The aged yeast was naturally dried at room temperature and then mycelia were removed and powdered, and stored in a 4 ° C. refrigerator.

Example  3. Raizopus  Duck CCS01  Alpha yeast strain and conventional yeast Amyla Aze activity

Alpha-amylase activity was measured by measuring the amount of soluble starch remaining through the iodine-starch reaction, and one unit of amylase was defined as the amount of enzyme that degrades 1 μg of soluble starch in one minute.

5g each of powder of white rice and brown rice yeast according to Example 2 and acid yeast produced by Sango Gok Co., Ltd. which is currently commercially available yeast, Songhak Nuruk produced by Song Hak Gok Co., Ltd. and Jinju Gok Co., Ltd. 5 g of each pearled malt was added to a 250 ml flask, and 100 ml of 0.5% saline was added thereto, followed by leaching at 20 ° C. for 20 hours at 20 ° C., followed by centrifugation to use the supernatant as an experimental enzyme solution (5% enzyme solution). .

Soluble starch was dissolved in boiling water to make a 2% soluble starch solution, and then an equal amount of 0.1 N acetic acid buffer solution (pH 5.0) was added to make a 1% starch solution at pH 5.0 to use as a substrate.

0.5 mL of 1% starch solution and 0.5 mL of 40 mM phosphate buffer (pH 6.0) were added to a test tube and preheated in a 37 ° C. constant temperature water bath. 50 μl (25 μl) of the enzyme solution was added to the test tube and allowed to react for 5 minutes, and then 1 mL of 0.1 N HCl was added to stop the reaction. 0.5 mL of the reaction was added to a 5 mL iodine solution (0.5 g of I _{2 and} 0.5 g of KI in 100 mL of distilled water) and the absorbance was measured at 700 nm. The results are shown in FIG. 2.

As can be seen in Figure 2, white rice or brown rice yeast prepared with the strain of Ryzopus duck material CCS01 exhibited significantly higher alpha-amylase activity compared to conventional yeast (acidic, pine, pearl). In particular, white yeast was the highest with 498.2 units.

Example  4. Raizopus  Duck CCS01  Preparation and Characteristics of Eight Grain Makgeolli Made with Strain Nuruk and Conventional Nuruk

The eight cereals used were white rice (non-glutinous and glutinous rice), brown rice, germinated brown rice, green rice, barley, buckwheat, and wheat.

White rice and brown rice were purchased from Hamyang Agricultural Cooperative Rice Processing Plant, germinated brown rice, green rice, and barley were purchased from oil price cooperative (Geochang), and buckwheat was purchased from Bongpyeong Young Agricultural Cooperative (Bongpyeong). Wheat was used by cutting only the bark of Uri wheat grown in Banseong-myeon, Jinju.

200 g each of white and brown rice, germinated brown rice, and green rice were washed with water, and after pouring enough water, they were soaked for 12 hours at room temperature, and 200 g of barley, buckwheat, and fine grains (7 min.) After pouring, it was immersed at room temperature for 4 hours, dried for 30 minutes, and each grain raw material was cooked at 100 ° C. for 1 hour and then cooled to use as a grain material.

The lycopus duck CCS01 strain white rice yeast prepared in Example 2 and conventional commercial yeast (acidic yeast, control) were added to purified water and 1.2 liter glass fermenter as shown in the following Table 2, respectively, and the yeast S. cereviase KCCM 12684 was 5.0% (v / v) inoculated and fermented at 25 ° C. for 1 day to prepare the base liquor (single immersion), and then add cooked grain raw materials, yeast and purified water as shown in Table 2, and then fermented at 25 ° C. for 7 days (stage 2). Immersion was completed.

1 soaking  leaven
 yeast
Purified water       4 mL
     56 g
     80 mL       0.52%
      7.27%
     10.39% 2 soaking Cooked cereals
yeast
Purified water 200 g
20 g
410 mL 25.97%
2.60%
53.25% Sum     770 mL (g)     100.00%

For the makgeolli, pH, acidity, brix sugar, alcohol content, and browning were analyzed. The fermented makgeolli sample was filtered through four gauze and used for analysis.

The pH is shown in FIG. 4 by measuring 50 mL of the filtered sample using a pH meter (model 3510, Jenway, UK), and the acidity was neutralized to pH 8.3 ± 0.1 with 10 mL of 0.1N-NaOH solution. After calculating the number of mL of 0.1N-NaOH consumed, it was shown in FIG. 5 in terms of acetic acid.

The sugar content was shown in FIG. 6 by centrifuging the filtered sample with a centrifuge (Hanil micro-12, Korea) and taking the supernatant to measure sugar content using a refractometer (N-1a, Atago Co., Tokyo, Japan). .

Alcohol content was measured by distillation after adding the same amount of distilled water to 100 mL of the filtered sample, and calibrated to 15 ℃ using a Gay Luccac Table and the results are shown in FIG.

The lactic acid bacteria count was properly diluted with sterile physiological water, smeared on an MRS plate medium containing 0.02% bromocresol purple (BCP), incubated at 30 ° C for 48 hours, and counted as yellow lactic acid bacteria. , The yeast water was measured in colonies generated after incubation for 48 hours at 30 ℃ after plating in a medium containing chloramphenicol (1.5 mg / mL) in PDA. Each experiment was repeated three times and expressed as an average value, expressed as colony forming units per mL of sample (CFU / mL) and the results are shown in Table 3.

Yeast / Grains Number of bacteria (log CFU / mL) ²⁾ Lactic acid bacteria leaven Conventional Yeast (Acid Yeast) Rice (NGUR) 7.32 6.59 Glutinous Rice (GUR) 7.20 6.86 Brown Rice (BR) 7.72 6.71 Germinated brown rice (GBR) 7.75 6.98 Green Rice (GRR) 7.61 6.91 Barley (BL) 7.34 6.93 Buckwheat (BW) 7.71 6.49 Precision (PW) 7.49 7.00 Rice malt made with lycopus duck CCS01 Rice (NGUR) 6.72 6.77 Glutinous Rice (GUR) 6.59 6.40 Brown Rice (BR) 7.04 6.57  Germinated brown rice (GBR) 7.28 6.32 Green Rice (GRR) 9 7.41 6.43 Barley (BL) 7.28 6.43 Buckwheat (BW) 7.54 6.59 Precision (PW) 7.08 6.63

Browning degree was measured at 420 nm using a spectrophotometer (Spectronic 2D, Thermo Electron Co., Califonia, USA) after centrifugation of the filtered sample with a centrifuge (Hanil micro-12, Korea) and taking the supernatant. Is shown in FIG. 8.

As can be seen in Figure 5, the makgeolli produced using the yeast according to the present invention had a low acidity compared to the makgeolli prepared using the conventional yeast, the taste was good. In particular, the Nuruk according to the present invention makes it possible to manufacture rice wine with almost constant acidity even if all grain raw materials are used in addition to white rice. In contrast, in the case of the conventional yeast, the acidity was very high when all grain raw materials except for white rice were used.

In addition, as shown in Figure 6 and 7, makgeolli prepared using the yeast produced by the strain of Ryuspus duck material CCS01 has a lower sugar content and higher alcohol content than the makgeolli prepared by the conventional yeast, Nuruk according to the present invention It can be seen that the alcohol production capacity is excellent.

In particular, as shown in Figure 8, the browning degree of the eight grains rice wine made with Nuruk according to the present invention was significantly lower than the eight grain rice wine made with conventional Nuruk. The lowest browning degree of makgeolli prepared by conventional yeast was 0.357 as non-glutinous rice (NGUR), and the highest was 0.568 as buckwheat (BW). The lowest browning degree of makgeolli was 0.094 for non-glutinous rice (NGUR) and 0.250 for buckwheat (BW). That is, the makgeolli made with the yeast according to the present invention showed a nearly white color to improve palatability.

National Institute of Agricultural Science, Genetic Resources Center KACC93118P 20110325

Claims (6)

Raizopus duck CCS01 ( Rhizopus oryzae CCS01) Rice malt with enhanced alpha-amylase activity, which is based on KACC93118P. The rice malt of claim 1, wherein the rice is white or brown rice. As a method of manufacturing makgeolli from raw grains,
A method using the rice malt according to claim 1. 4. The method of claim 3, wherein the cereal is at least one selected from the group consisting of white rice, brown rice, germinated brown rice, green rice, barley, buckwheat, and wheat. Makgeolli produced by the method according to claim 3, characterized by low acidity, high alcohol content, low browning degree. The rice wine according to claim 5, wherein the browning degree is 0.25 OD 420 nm or less.

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