WO2017065374A1 - Method for producing solid starter for unrefined rice wine using aspergillus oryzae 75-2 and indigenous spawn having excellent saccharification capability - Google Patents
Method for producing solid starter for unrefined rice wine using aspergillus oryzae 75-2 and indigenous spawn having excellent saccharification capability Download PDFInfo
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- WO2017065374A1 WO2017065374A1 PCT/KR2016/005344 KR2016005344W WO2017065374A1 WO 2017065374 A1 WO2017065374 A1 WO 2017065374A1 KR 2016005344 W KR2016005344 W KR 2016005344W WO 2017065374 A1 WO2017065374 A1 WO 2017065374A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/20—Malt products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/69—Aspergillus oryzae
Definitions
- the present invention is an indigenous spawn with excellent saccharifying power, aspergillus duck material (spe / // / "s oryzae) 75-2 (KACC93236P) method for producing a solid seed for takju, and the solid seed for takju prepared by the method It is about.
- Takju is the oldest liquor among traditional liquors, and it is a milky white turbid liquor that is commonly referred to as a rice wine, commonly enjoyed by the common people in sub-Seoul districts.
- the alcohol content ranges from 5 to 16 degrees, and usually 6 to 7 degrees. It is also.
- the brewing method of Takju which is usually called Makgegi, varies depending on the province, but the most common brewing method is made by mixing yeast and water in gouda rice made of semi-seed or semi-rice and dipping, saving water, and increasing it. The alcohol is fermented, soaked in a sieve and separated by sieving.
- Takju is the most populist traditional pop wine indigenous to our nation, which has few headaches and has a rich flavor and rich taste after drinking, but has difficulty in commercial production of Jong-Joo technology for the manufacture of Takju. Not only is there insufficient diversity, but also there is a problem in that subtractive loss occurs due to abnormal fermentation.
- 10-1297707 discloses a method of producing yeast and fermented wine using the yeast by inoculating Aspergillus Omose as a strain on wheat
- Korea Patent Document 10—2015-0090335 discloses a method for preparing a makgeolli fermenter using the fermenter spawn Aspergillus aurise S_ 2 (ACC93172P) which is used for brewing makgeolli, but Aspergillus duck 75 There is no known method for producing a solid terminal of -2 (KACC93236P). Therefore .
- the inventors of the aspergillus duck material is an indigenous spawn with excellent glycosylation power
- An object of the present invention is an indigenous spawn with excellent glycosylation power, aspergillus duck material (pe / // / i / s oryzae) 75-2 (KACC93236P) method for producing a solid seed for the sake. And to provide a solid seed for Takju prepared by the above method.
- step 2) increasing the rice of step 1), and then cooling to prepare a gourd rice; 3) Aspergillus duckwood in the gourd rice of step 2) oryzae) 75-
- the present invention provides a solid terminal produced by the above production method. ⁇ Effects of the Invention ⁇
- the present invention for the efficient preparation of Takju solid seed of Aspergillus duck material 75-2 ACC93236P), an indigenous seed having excellent saccharification ability, in the production of solid seed, inoculated 5% of liquid seed, and 6 days at 35 ° C.
- the production method is the optimum condition for producing solid species of Aspergillus duck material 75-2 with high enzymatic activity, high spore count, and low bacterial count. It can be usefully used.
- 1 shows the Aspergillus duck 3 ⁇ 406 > / ⁇ 77 / "5 oryzae) 75- of the present invention.
- 2 (I (ACC93236P) is a diagram showing the steps of the solid final production method.
- Figure 2 is a view showing the actual solid state production process of the present invention in a photograph.
- 3 is a view showing the change of the empire according to the seed starter content (seed starter content).
- FIG. 4 is a diagram showing the enzyme activity according to the liquid seed inoculation amount.
- 5 is a diagram showing the enzyme activity according to the imperial temperature.
- FIG. 6 is a diagram illustrating a change of an empire with an empire time.
- FIG. 9 is a view showing the spores of the solid species of the present invention.
- 10 is a diagram showing the number of spores according to A. inoculation amount, B. imperial temperature, and C. fermentation period.
- 11 is a diagram showing the number of bacteria according to A. inoculation amount, B. imperial temperature, and C. fermentation period.
- FIG. 12 is a view showing a freeze drying apparatus and a hot air drying apparatus used in the present invention.
- Figure 13 is a view showing the properties of the solid terminal according to the drying method and the addition of excipients.
- 14 is a view showing the moisture content of the solid terminal according to the drying technique.
- Figure 15 is a diagram showing the number of spores and viable count of solid seed in accordance with the drying technology.
- 16 is a diagram showing the bacterial contamination of the solid state according to the dryer after adding trehalose as an excipient.
- step 2) increasing the rice of step 1), and then cooling to prepare a gourd rice; 3) Aspergillus duckwood pev ⁇ J / s oryzae) 75-
- the immersion of step 1) is preferably immersed in water for 4 to 12 hours. Saving water is preferably put in a colander or sieve to drain the water for 30 minutes to 120 minutes, it is more preferable to drain the water for 1 hour.
- two steps) of the increase is preferably by using a steaming brush steamed for 40 minutes to 120 minutes, and the steam is more preferred for an hour, and cooling is the preferable, and 40 ° C cooling to 40 to 50 o C Cooling as soon as possible is most desirable.
- the Aspergillus duck material 75-2 (KACC93236P) of step 3) is preferably pre-cultured and main culture for 4 to 6 days at 25 to 30 o C, respectively, to prepare a seed, and the strain platinum is It is more preferable to incubate at 28 ° C for 5 days, and incubated for 6 days by inoculating 2% in the main culture medium. At this time, the humidity is preferably all 70%. Also.
- the liquid spawn of step 3) is preferably inoculated in gourd rice at a concentration of 2 to 5%.
- the culture is preferably incubated for 5 to 7 days at 35 to 40 ° C, for high enzymatic activity of the Aspergillus duck material 75-2 (KACC93236P) of the present invention inoculated 5% of the liquid spawn at 40 o C It is more preferable to incubate for 6 days, and for high spore count, inoculate 10% of liquid spawn at 40 o C More preferably incubated for 6 days.
- the drying of step 4) is preferably dried at 40 to 50 ° C. for 20 to 30 hours, more preferably at 45 0 C for 24 hours.
- the drying is preferably a method of hot air drying or lyophilization, and it is more preferable to use a hot air drying method for high spore count and viable cell count and low bacterial contamination.
- the present invention provides a solid terminal produced by the above production method.
- the inoculation amount of the liquid spawn in order to determine the optimum conditions for the production of solid seed of Aspergillus duck material dpe // // s oryzae) 75-2 (KACC93236P) of the present invention, the inoculation amount of the liquid spawn, imperial temperature, And as a result of analyzing the enzyme activity according to the empire time, as shown in Figures 4, 5, and 7, 5% inoculation of the liquid spawn, and incubated for 6 days at 40 ° C, the enzyme activity of the strain was confirmed to be high (Figs.
- freeze drying showed twice as much water content as hot air drying when dried for the same time (see FIG. 14), and freeze drying and Hot air drying showed a greater number than before drying (see FIG. 15), and the bacterial contamination was 1.9 times lower than that of freeze drying when hot air drying.
- skim milk skim mi lk
- hot air drying is In the case of adding trehalose (t reha lose) as an excipient, it was confirmed that the freeze-drying method is suitable for freeze-drying rather than hot air drying because the degree of contamination is about twice as low (see FIG. 16). Therefore, the production method of the Aspergillus duck material 75-2 solid seed station of the present invention can be usefully used to prepare a solid seed station having a high enzymatic activity, a high number of spores, and a small number of bacteria.
- Example 1 Aspergillus duck material 75-2 was selected and used as a saponified bearer of high saccharification power in strain cultured raw rice starch and luxury medium.
- the Gomwang was separated from the traditional fermented food Nuruk and secured in Rural Chong fermented food and fermentation resource laboratory _ medium bran medium (bran bran (7.5%) prepared in the composition of Table 1 in 100 mL of distilled water and ultra-high pressure sterilizer (121 Sterilization with 0 C, 15 min) or after passage using PDA medium. Used while storing at 4 ° C.
- Solid seed was prepared by the process shown in Figure 1 using a small imperial machine (Mini-15, Yaekagi, Japan) to prevent contamination of various bacteria, the specific method is as follows and shown in Figure 2:
- Takju solid seed soup is prepared in powder and preparation form (FIG. 2).
- the humidity of the small imperial machine of ⁇ Example 2> was kept constant at 70%, the temperature conditions of step 4) were changed, and according to the same manufacturing method, Enzyme activity was analyzed.
- Aspergillus duck material 75-2 ACC93236P showed the highest acidic protein enzymatic activity at 40 o C, and it was found that 35 to 20% of mycelia and spores could grow better than low temperature (20 to 30 o C). By adjusting the optimum temperature to 40 ° C it was confirmed that the enzyme activity is more than two times higher (Figure 5). ⁇ 1-3> Analysis of enzyme activity according to empire time
- Aspergillus duck material 75-2 showed a tendency to increase the enzyme activity with the passage of the fermentation time, in particular, was confirmed to increase rapidly at 96 hours, in the case of ghicoamyl ase activity, even after 96 hours It was confirmed that the increase showed the highest activity at 96 144 hours and thereafter tended to decrease (FIG. 7).
- the switch Imperial fermented material duck depart at 4 days (96 hours), but the difference in the "inoculum size and the amount of seasoning Imperial conditions of the strain isolated from Korean traditional Phitsanulok based on.
- the enzyme activity is the most Optimal conditions were sought on the sixth day of good empire.
- Liquid seed inoculation amount, empire temperature, and the number of bacteria according to empire time were measured by the following method.
- the spawn company in Japan releases products on the average, ignoring the contamination of about 10 2 bacteria, but the experimental example of the present invention results in a slightly higher number of bacteria than in Japan, but establishes a mass production system for the commercialization of spawn for Takju.
- pollution can be reduced like a spawn company in Japan.
- the pore of the present invention finally provides a method of drying for industrial mass production in the future. In order to establish the optimum conditions, the experiment was conducted.
- the freeze drying apparatus is Freezing dryer MCID8518 (Ilshin bio base Co., Ltd. South Korea) and the hot air drying apparatus is Heated-air dryer DS-80-3 (Dasol scientific Co., Ltd. South Korea) was used.
- Solid freeze of the present invention the rapid freeze at -80 ° C and then lyophilized for 24 hours using a freeze-drying device, or hot air drying for 24 hours at 45 ° C using a hot air drying device, Moisture content, viable cell count, and bacterial contamination were analyzed.
- skim milk Skim milk
- the degree of contamination of bacteria before drying was 2.0 ⁇ 10 2 ⁇ 0.6, 5.0 ⁇ 10 2 ⁇ 2.5 during lyophilization, and hot air drying was 1.0 ⁇ 10 2 ⁇ 0.5, and when trehalose was added as an excipient, Before, 3.0 ⁇ 10 2 ⁇ 0.7. Lyophilized 5.0 x 10 2 ⁇ 1.0. It is judged that freeze drying is more suitable than hot air drying because the method of freeze drying to 8.0 x 10 2 ⁇ 1.5 at the time of hot air drying has twice the pollution degree (FIG. 16).
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Abstract
The present invention relates to a method for producing a solid starter for unrefined rice wine using Aspergillus oryzae 75-2 (KACC93236P) and indigenous spawn having an excellent saccharification capability, and to a solid starter for unrefined rice wine produced thereby. Particularly, in producing the solid starter of the strain, the following findings were confirmed: enzyme activity of the strain was high when 5% of a liquid starter was inoculated and cultivated for six days at 35°C; the number of spores of the solid starter was high when 10% of the liquid starter was inoculated and cultivated for six days at 40°C; and the number of bacteria in the solid starter was lower, the lower the amount of liquid strain inoculation and the longer the fermentation period. Therefore, the production method can be useful as the optimal condition for producing the solid starter of Aspergillus oryzae 75-2 having low bacteria count.
Description
【명세서】 【Specification】
【발명의 명칭】 [Name of invention]
당화력이 우수한 토착 종균, 아스퍼질러스 오리재 75-2를 이용한 탁주용 고체종국 제조방법 Indigenous spawn with excellent saccharifying power, Takju solid seed production method using Aspergillus duck material 75-2
【기술분야】 Technical Field
본 발명은 당화력이 우수한 토착 종균, 아스퍼질러스 오리재 ( spe/ // /"s oryzae) 75-2(KACC93236P)를 이용한 탁주용 고체종국의 제조방법, 및 상기 방법으로 제조된 탁주용 고체종국에 관한 것이다. The present invention is an indigenous spawn with excellent saccharifying power, aspergillus duck material (spe / // / "s oryzae) 75-2 (KACC93236P) method for producing a solid seed for takju, and the solid seed for takju prepared by the method It is about.
【배경기술】 Background Art
탁주는 전통주 가운데서 가장 역사가 오래된 술이며, 주로 서울이남 지역에서 서민층이 주로 즐겨 마셨던 술로 농주 (農酒)라고도 불리워지는 유백색의 탁한 액으로서 , 알코올 도수는 약 5~ 16도의 범위이며 보통은 6~7도이다. 통상 막걸리라 불려지는 탁주의 양조법은 지방에 따라 다르기는 하나 , 가장 일반적인 양조법은 참쌀 또는 템쌀을 세미하고 침지, 절수 및 증자를 하여 만든 고두밥에 누룩과 물을 섞어 만든 것으로 7~ 10일 정도 경과하면 알코올 발효가 되어 술덧을 떠서 체로 걸러 탁주로 분리하게 된다. 탁주는 음주 후, 두통이 거의 없고 그옥한 풍미와 감칠맛이 일품인 우리 민족 고유의 가장 서민적인 전통 대중주이나, 탁주 제조를 위한 종국제조 기술의 상업적 생산에 어려움이 있고, 제품의 불균일성 및 맛의 다양성이 미흡할 뿐만 아니라 또한 이상 발효로 인한 감 산패가 발생한다는 문제점이 있다. Takju is the oldest liquor among traditional liquors, and it is a milky white turbid liquor that is commonly referred to as a rice wine, commonly enjoyed by the common people in sub-Seoul districts. The alcohol content ranges from 5 to 16 degrees, and usually 6 to 7 degrees. It is also. The brewing method of Takju, which is usually called Makgegi, varies depending on the province, but the most common brewing method is made by mixing yeast and water in gouda rice made of semi-seed or semi-rice and dipping, saving water, and increasing it. The alcohol is fermented, soaked in a sieve and separated by sieving. Takju is the most populist traditional pop wine indigenous to our nation, which has few headaches and has a rich flavor and rich taste after drinking, but has difficulty in commercial production of Jong-Joo technology for the manufacture of Takju. Not only is there insufficient diversity, but also there is a problem in that subtractive loss occurs due to abnormal fermentation.
따라서 탁주의 품질 고급화 및 표준화를 위한 종균제조 기술은 국가주도의 운영이 필요하며 , 이는 우수한 특성을 가진 종균을 보다 안정적으로 유지 및 공급하는 기술이 필요하다.
한편. 본 발명의 아스퍼질러스 오리재의 고체종국과 관련한 선행기술로서, 대한민국 등록특허 10-1297707는 밀에 균주로서 아스퍼질러스 오뫼제를 접종하여 누룩 및 상기 누룩을 이용한 발효주의 제조방법을 개시하였고, 대한민국 공개특허 10— 2015-0090335는 막걸리 양조에 이용되는 발효제 종균 아스퍼질러스 오리제 S_ 2( Aspergi l l us oryzae S-2 ; ACC93172P) 이를 이용한 막걸리 발효제 제조방법에 대하여 개시하였으나, 아스퍼질러스 오리재 75-2(KACC93236P)의 고체종국 제조방법에 대해서는 현재까지 알려진 바가 없다. 이에 . 본 발명자들은 당화력이 우수한 토착 종균인 아스퍼질러스 오리재Therefore, the spawn production technology for quality improvement and standardization of Takju needs to be led by the state, which requires more stable maintenance and supply of spawn with excellent characteristics. Meanwhile. As a prior art related to the solid state of the Aspergillus duck material of the present invention, Korean Patent No. 10-1297707 discloses a method of producing yeast and fermented wine using the yeast by inoculating Aspergillus Omose as a strain on wheat, Korea Patent Document 10—2015-0090335 discloses a method for preparing a makgeolli fermenter using the fermenter spawn Aspergillus aurise S_ 2 (ACC93172P) which is used for brewing makgeolli, but Aspergillus duck 75 There is no known method for producing a solid terminal of -2 (KACC93236P). Therefore . The inventors of the aspergillus duck material is an indigenous spawn with excellent glycosylation power
75-2( ACC93236P)의 탁주용 고체종국의 효율적인 제조를 위해 연구하던 중, 고체종국 제조에 있어, 액체종균을 5% 접종하고, 350C에서 6일 동안 배양하였을 때, 상기 균주의 효소활성이 높음을 확인하였고, 액체종균을 10% 접종하고. 40oC에서 6일 동안 배양하였을 때. 상기 고체종국의 포자수가 높음을 확인하였으며, 액체종균의 접종량이 적을수록, 발효기간이 길어질수록 고체종국의 세균수가 감소하는 것을 확인함으로써. 상기 제조방법은 효소활성이 높고, 포자수가 많으며, 세균수가 적은 형태의 아스퍼질러스 오리재 75-2의 고체종국을 제조할 수 있음을 확인함으로써, 본 발명을 완성하였다. 【발명의 상세한 설명】 During the study for the efficient preparation of 75-2 (ACC93236P) Takju solid seed, in the production of solid seed, the enzyme activity of the strain when inoculated 5% of the liquid seed and incubated for 6 days at 35 0 C Confirmed high, and inoculated with 10% of the liquid spawn. Incubated at 40 o C for 6 days. By confirming that the number of spores of the solid seed is high, the less the inoculation amount of the liquid seed, the longer the fermentation period by confirming that the bacterial count of the solid seed decreases. The production method completed the present invention by confirming that the solid final station of Aspergillus duck material 75-2 in the form of high enzymatic activity, high spores, and low bacterial counts could be prepared. [Detailed Description of the Invention]
【기술적 과제】 [Technical problem]
본 발명의 목적은 당화력이 우수한 토착 종균, 아스퍼질러스 오리재 ( pe/ ///i/s oryzae) 75-2(KACC93236P)를 이용한 탁주용 고체종국의 제조방법. 및 상기 방법으로 제조된 탁주용 고체종국을 제공하기 위한 것이다.
【기술적 해결방법】 An object of the present invention is an indigenous spawn with excellent glycosylation power, aspergillus duck material (pe / // / i / s oryzae) 75-2 (KACC93236P) method for producing a solid seed for the sake. And to provide a solid seed for Takju prepared by the above method. Technical Solution
상기 목적을 달성하기 위하여 , 본 발명은 In order to achieve the above object, the present invention
1 ) 도정된 쌀을 세척 후, 침지시킨 다음 절수하는 단계; 1) washing the milled rice, dipping and then saving water;
2) 상기 단계 1 )의 쌀을 증자시킨 후, 방냉시켜 고두밥을 제조하는 단계; 3) 상기 단계 2)의 고두밥에 아스퍼질러스 오리재
oryzae) 75-2) increasing the rice of step 1), and then cooling to prepare a gourd rice; 3) Aspergillus duckwood in the gourd rice of step 2) oryzae) 75-
2(KACC93236P)의 액체종균을 흔합한 후, 뒤집기와 뒤섞기를 하면서 배양시키는 단계; 및 Mixing 2 (KACC93236P) liquid spawn and incubating with inverting and mixing; And
4) 상기 단계 3)의 배양된 고두밥을 건조시킨 후 분상 또는 조제 형태로 제조하는 단계를 포함하는 아스퍼질러스 오리재 75-2(KACC93236P)를 이용한 고체종국의 제조방법을 제공한다. 4) It provides a method for producing a solid seed using Aspergillus duck material 75-2 (KACC93236P) comprising the step of drying the cultured gourd rice of step 3) in the form of powder or preparation.
또한, 본 발명은 상기 제조방법으로 제조된 고체종국을 제공한다. 【발명의 효과】 In addition, the present invention provides a solid terminal produced by the above production method. 【Effects of the Invention】
본 발명은 당화력이 우수한 토착 종균인 아스퍼질러스 오리재 75- 2 ACC93236P)의 탁주용 고체종국의 효율적인 제조를 위하여, 고체종국 제조에 있어, 액체종균을 5% 접종하고, 35°C에서 6일 동안 배양하였을 때. 상기 균주의 효소활성이 높음을 확인하였고, 액체종균을 10% 접종하고, 40oC에서 6일 동안 배양하였을 때, 상기 고체종국의 포자수가 높음을 확인하였으며, 액체종균의 접종량이 적을수록, 발효기간이 길어질수록 고체종국의 세균수가 감소하는 것을 확인함으로써, 상기 제조방법은 효소활성이 높고, 포자수가 많으며, 세균수가 적은 형태의 아스퍼질러스 오리재 75— 2의 고체종국을 제조하기 위한 최적조건으로 유용하게 사용될 수 있다. 【도면의 간단한 설명】 The present invention for the efficient preparation of Takju solid seed of Aspergillus duck material 75-2 ACC93236P), an indigenous seed having excellent saccharification ability, in the production of solid seed, inoculated 5% of liquid seed, and 6 days at 35 ° C. When incubated for It was confirmed that the enzyme activity of the strain was high, 10% inoculation of the liquid spawn, and when incubated at 40 o C for 6 days, it was confirmed that the spore count of the solid seed is high, the less inoculation of the liquid spawn, the fermentation By confirming that the longer the period, the fewer the bacterial counts of the solid species, the production method is the optimum condition for producing solid species of Aspergillus duck material 75-2 with high enzymatic activity, high spore count, and low bacterial count. It can be usefully used. [Brief Description of Drawings]
도 1은 본 발명의 아스퍼질러스 오리재 ¾06>/ §77 /"5 oryzae) 75-
2(I(ACC93236P) 고체종국 제조방법의 단계를 나타낸 도이다. 1 shows the Aspergillus duck ¾06 > / §77 / "5 oryzae) 75- of the present invention. 2 (I (ACC93236P) is a diagram showing the steps of the solid final production method.
도 2는 본 발명의 고체종국 제조 공정을 실제 사진으로 나타낸 도이다. 도 3은 액체종균 접종량 (seed starter content)에 따른 제국의 변화를 나타낸 도이다. Figure 2 is a view showing the actual solid state production process of the present invention in a photograph. 3 is a view showing the change of the empire according to the seed starter content (seed starter content).
도 4는 액체종균 접종량에 따른 효소활성을 나타낸 도이다. 4 is a diagram showing the enzyme activity according to the liquid seed inoculation amount.
도 5는 제국 온도에 따른 효소활성을 나타낸 도이다. 5 is a diagram showing the enzyme activity according to the imperial temperature.
도 6은 제국 시간에 따른 제국의 변화를 나타낸 도이다. 6 is a diagram illustrating a change of an empire with an empire time.
도 7은 제국 시간에 따른 효소활성을 나타낸 도이다. 7 is a diagram showing the enzyme activity according to the empire time.
도 8은 제국 시간에 따른 품온 변화를 나타낸 도이다. 8 is a view showing a change in product temperature with the empire time.
도 9는 본 발명의 고체종국의 포자를 나타낸 도이다. 9 is a view showing the spores of the solid species of the present invention.
도 10은 A.접종량, B.제국 온도, 및 C.발효기간에 따른 포자수를 나타낸 도이다. 10 is a diagram showing the number of spores according to A. inoculation amount, B. imperial temperature, and C. fermentation period.
도 11은 A.접종량, B.제국 온도, 및 C.발효기간에 따른 세균수를 나타낸 도이다. 11 is a diagram showing the number of bacteria according to A. inoculation amount, B. imperial temperature, and C. fermentation period.
도 12는 본 발명에서 사용한 동결건조 장치 및 열풍건조 장치를 나타낸 도이다. 12 is a view showing a freeze drying apparatus and a hot air drying apparatus used in the present invention.
도 13은 건조방법과 부형제 첨가에 따른 고체종국의 성상을 나타낸 도이다. 도 14는 건조기술에 따른 고체종국의 수분함량을 나타낸 도이다. Figure 13 is a view showing the properties of the solid terminal according to the drying method and the addition of excipients. 14 is a view showing the moisture content of the solid terminal according to the drying technique.
도 15는 건조기술에 따른 고체종국의 포자수 및 생균수를 나타낸 도이다. 도 16은 부형제로서 트레할로스 (trehalose)를 첨가한 후, 건조기 에 따른 고체종국의 세균 오염도를 나타낸 도이다. Figure 15 is a diagram showing the number of spores and viable count of solid seed in accordance with the drying technology. 16 is a diagram showing the bacterial contamination of the solid state according to the dryer after adding trehalose as an excipient.
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
이하, 본 발명을 상세히 설명한다.
보 반명으 Hereinafter, the present invention will be described in detail. Bo half name
1 ) 도정된 쌀을 세척 후, 침지시킨 다음 절수하는 단계; 1) washing the milled rice, dipping and then saving water;
2) 상기 단계 1 )의 쌀을 증자시킨 후, 방냉시켜 고두밥을 제조하는 단계; 3) 상기 단계 2 )의 고두밥에 아스퍼질러스 오리재 pev^J/ s oryzae) 75- 2) increasing the rice of step 1), and then cooling to prepare a gourd rice; 3) Aspergillus duckwood pev ^ J / s oryzae) 75-
2( KACC93236P)의 액체종균을 흔합한 후, 뒤집기와 뒤섞기를 하면서 배양시키는 단계; 및 Mixing 2 (KACC93236P) liquid spawn and incubating with inverting and mixing; And
4) 상기 단계 3)의 배양된 고두밥을 건조시킨 후, 분상 또는 조제 형태로 제조하는 단계를 포함하는 아스퍼질러스 오리재 75-2( KACC93236P)를 이용한 고체종국의 제조방법을 제공한다. 4) It provides a method for producing a solid seed using Aspergillus duck material 75-2 (KACC93236P) comprising the step of drying the cultured gourd rice of step 3), and then prepared in powder or preparation form.
상기 방법에 있어서, 단계 1 )의 침지는 물에 4 내지 12시간 동안 담그는 것이 바람직하고. 절수는 소쿠리 또는 체에 넣고 30분 내지 120분 동안 물을 빼주는 것이 바람직하며, 1시간 동안 물을 빼주는 것이 더욱 바람직하다. 또한, 단계 2)의 증자는 찜 솔을 이용하여 40분 내지 120분 동안 찌는 것이 바람직하고, 1시간 동안 찌는 것이 더욱 바람직하며, 방냉은 40 내지 50oC가 되도록 식히는 것이 바람직하고 40°C가 되도록 빨리 식히는 것이 가장 바람직하다. In the method, the immersion of step 1) is preferably immersed in water for 4 to 12 hours. Saving water is preferably put in a colander or sieve to drain the water for 30 minutes to 120 minutes, it is more preferable to drain the water for 1 hour. In addition, two steps) of the increase is preferably by using a steaming brush steamed for 40 minutes to 120 minutes, and the steam is more preferred for an hour, and cooling is the preferable, and 40 ° C cooling to 40 to 50 o C Cooling as soon as possible is most desirable.
또한, 단계 3 )의 아스퍼질러스 오리재 75-2(KACC93236P)는 25 내지 30oC에서 4 내지 6일 동안 각각 전배양 및 본배양을 시켜 종균을 제조하는 것이 바람직하고, 상기 균주 1백금이를 28°C에서 5일 동안 전배양 하고, 이를 본배양 배지에 2% 접종하여 6일 동안 배양시키는 것이 더욱 바람직하다. 이때, 습도는 모두 70%인 것이 바람직하다. 또한. 단계 3)의 액체종균은 2 내지 5%의 농도로 고두밥에 접종시키는 것이 바람직하고. 배양은 35 내지 40°C에서 5 내지 7일 동안 배양하는 것이 바람직하며, 본 발명의 아스퍼질러스 오리재 75-2(KACC93236P)의 높은 효소활성을 위해서는 액체종균을 5% 접종하고 40oC에서 6일 동안 배양하는 것이 더욱 바람직하고 , 높은 포자수를 위해서는 액체종균을 10% 접종하고 40oC에서
6일 동안 배양하는 것이 더욱 바람직하다. In addition, the Aspergillus duck material 75-2 (KACC93236P) of step 3) is preferably pre-cultured and main culture for 4 to 6 days at 25 to 30 o C, respectively, to prepare a seed, and the strain platinum is It is more preferable to incubate at 28 ° C for 5 days, and incubated for 6 days by inoculating 2% in the main culture medium. At this time, the humidity is preferably all 70%. Also. The liquid spawn of step 3) is preferably inoculated in gourd rice at a concentration of 2 to 5%. The culture is preferably incubated for 5 to 7 days at 35 to 40 ° C, for high enzymatic activity of the Aspergillus duck material 75-2 (KACC93236P) of the present invention inoculated 5% of the liquid spawn at 40 o C It is more preferable to incubate for 6 days, and for high spore count, inoculate 10% of liquid spawn at 40 o C More preferably incubated for 6 days.
또한, 단계 4)의 건조는 40 내지 50oC에서 20 내지 30시간 동안 건조시키는 것이 바람직하며 , 450C에서 24시간 동안 건조시키는 것이 더욱 바람직하다. 상기 건조는 열풍건조 또는 동결건조의 방법인 것이 바람직하며, 높은 포자수 및 생균수 및 낮은 세균 오염도를 위해서는 열풍건조의 방법을 사용하는 것이 더욱 바람직하다 . In addition, the drying of step 4) is preferably dried at 40 to 50 ° C. for 20 to 30 hours, more preferably at 45 0 C for 24 hours. The drying is preferably a method of hot air drying or lyophilization, and it is more preferable to use a hot air drying method for high spore count and viable cell count and low bacterial contamination.
아을러, 본 발명은 상기 제조방법으로 제조된 고체종국을 제공한다. 본 발명의 구체적인 실시예에서, 본 발명의 아스퍼질러스 오리재 dpe/ /// s oryzae) 75-2(KACC93236P)의 고체종국 제조의 최적화 조건을 구명하기 위하여, 액체종균의 접종량, 제국 온도, 및 제국 시간에 따른 효소활성을 분석한 결과, 도 4, 도 5, 및 도 7에 나타난 바와 같이, 액체종균을 5% 접종하고, 40oC에서 6일 동안 배양하였을 때, 상기 균주의 효소활성이 높음을 확인하였고 (도 4, 도 5, 및 도 7), 포자수의 경우, 표 2 내지 표 4, 및 도 10에 나타난 바와 같이 액체종균을 10% 접종하고, 40oC에서 6일 동안 배양하였을 때, 상기 고체종국의 포자수가 높음을 확인하였다 (표 2 내지 표 4. 및 도 10). 또한, 표 5 내지 표 7, 및 도 11에 나타난 바와 같이, 액체종균의 접종량이 적을수록, 발효기간이 길어질수록 고체종국의 세균수가 감소하는 것을 확인하였다 (표 5 내지 표 7, 및 도 11). In addition, the present invention provides a solid terminal produced by the above production method. In a specific embodiment of the present invention, in order to determine the optimum conditions for the production of solid seed of Aspergillus duck material dpe // // s oryzae) 75-2 (KACC93236P) of the present invention, the inoculation amount of the liquid spawn, imperial temperature, And as a result of analyzing the enzyme activity according to the empire time, as shown in Figures 4, 5, and 7, 5% inoculation of the liquid spawn, and incubated for 6 days at 40 ° C, the enzyme activity of the strain Was confirmed to be high (Figs. 4, 5, and 7), and in the case of spores, 10% inoculation of the liquid spawn as shown in Tables 2 to 4, and 10, and incubated at 40 ° C. for 6 days. When confirmed, it was confirmed that the number of spores of the solid seed state (Table 2 to Table 4 and Figure 10). In addition, as shown in Table 5 to Table 7, and Figure 11, the smaller the inoculum of the liquid spawn, the longer the fermentation period was confirmed that the number of bacteria in the solid final station reduced (Table 5 to Table 7, and Figure 11) .
또한. 동결 및 열풍건조에 따른 고체종국의 건조방식을 비교 검토한 결과, 동일한 시간 동안 건조하였을 때, 동결건조가 열풍건조보다 2배 높은 수분함량을 나타내었고 (도 14 참조), 포자수의 경우 동결건조 및 열풍건조는 모두 건조 전보다 많은 수를 나타내었으며 (도 15 참조), 세균 오염도는 동결건조보다 열풍건조 시 1.9배 정도 오염도가 낮음을 확인하였다. 또한, 상기 고체종국의 보호를 위해 탈지유 (skim mi lk)를 부형제로서 첨가하고 건조하였을 때 , 열풍건조가
적합하였고, 트레할로스 ( t reha lose)를 부형제로서 첨가한 경우에는 동결건조하는 방법이 2배 정도의 오염도가 낮아 열풍건조보다는 동결건조가 적합함을 확인하였다 (도 16참조) . 따라서 , 본 발명의 아스퍼질러스 오리재 75-2 고체종국의 제조방법은 효소활성이 높고, 포자수가 많으며, 세균수가 적은 형태의 고체종국을 제조하는데 유용하게 사용될 수 있다. Also. As a result of comparing and reviewing the drying method of solid state according to freeze and hot air drying, freeze drying showed twice as much water content as hot air drying when dried for the same time (see FIG. 14), and freeze drying and Hot air drying showed a greater number than before drying (see FIG. 15), and the bacterial contamination was 1.9 times lower than that of freeze drying when hot air drying. In addition, when the skim milk (skim mi lk) is added as an excipient and dried for the protection of the solid final state, hot air drying is In the case of adding trehalose (t reha lose) as an excipient, it was confirmed that the freeze-drying method is suitable for freeze-drying rather than hot air drying because the degree of contamination is about twice as low (see FIG. 16). Therefore, the production method of the Aspergillus duck material 75-2 solid seed station of the present invention can be usefully used to prepare a solid seed station having a high enzymatic activity, a high number of spores, and a small number of bacteria.
이하, 본 발명을 실시예 및 실험예에 의해서 상세히 설명한다. Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해서 한정되는 것은 아니다. However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
<실시예 1>균주 배양 생쌀 전분 및 호화배지에서 당화력이 높은 탁주용 곰광이로 아스퍼질러스 오리재 75-2(KACC93236P)를 선정하여 사용하였다. 상기 곰광이는 전통 발효식품인 누룩에서 분리하여 농촌진홍청 발효식품과 발효자원연구실에서 확보하여 하기 _표 1의 조성으로 제조한 밀기울 배지 (밀기울 (7.5%)을 증류수 100 mL에 넣고 초고압멸균기 ( 1210C , 15분)로 살균) 또는 PDA 배지를 이용하여 계대배양한 후. 4°c에 보관하면서 사용하였다. Example 1 Aspergillus duck material 75-2 (KACC93236P) was selected and used as a saponified bearer of high saccharification power in strain cultured raw rice starch and luxury medium. The Gomwang was separated from the traditional fermented food Nuruk and secured in Rural Chong fermented food and fermentation resource laboratory _ medium bran medium (bran bran (7.5%) prepared in the composition of Table 1 in 100 mL of distilled water and ultra-high pressure sterilizer (121 Sterilization with 0 C, 15 min) or after passage using PDA medium. Used while storing at 4 ° C.
【표 11 배지 조성 (%) Table 11 Medium Composition (%)
PDA 배지 MEA 배지 맥아 추출물 (ma l t PDA medium MEA medium malt extract (ma l t
감자 전분 (potato starch) 0.4 3 Potato starch 0.4 3
ext ract ) 덱스트로스 (dextrose) 2 펩톤 (peptone) 0.5 한천 (agar ) 1.5 한천 ( agar ) 1.5
<실시예 2>고체종국 제조 ext ract) dextrose 2 peptone 0.5 agar 1.5 agar 1.5 Example 2 Production of solid seed station
고체종국은 잡균의 오염 등을 방지하기 위해서 소형 제국기 (Mini -15 , Yaekagi , 일본)를 이용하여 도 1에 나타난 과정으로 제조하였으며 , 구체적인 방법은 하기와 같으며 도 2에 나타내었다: Solid seed was prepared by the process shown in Figure 1 using a small imperial machine (Mini-15, Yaekagi, Japan) to prevent contamination of various bacteria, the specific method is as follows and shown in Figure 2:
1 ) 도정된 쌀을 깨끗이 씻고 물에 4~12시간 담근다; 1) Wash the milled rice and soak in water for 4-12 hours;
2) 물이 축적된 쌀을 소쿠리에 1시간 정도 넣어 두어 물을 뺀다; 2) the rice with accumulated water is put in a colander for about 1 hour to drain the water;
3) 쌀을 찜 솔에 넣고 1시간 정도 찐 뒤. 40oC가 되도록 빨리 식힌다; 3) Put rice in a steaming brush and steam for 1 hour. Cool to 40 o C as quickly as possible;
4) ME 배지 (맥아 추출물 3% 및 펩톤 0.5%)에 아스퍼질러스 오리재 75-2를 1 백금이 접종하여 28°C에서 5일 동안 전배양 한 후, 본배양 배지에 2%를 접종하여 4) Inoculated with Aspergillus duck 75-2 1 ME in ME medium (3% malt extract and 0.5% peptone), pre-incubated at 28 ° C for 5 days, and then inoculated 2% in the main culture medium
6일 동안 더 배양한 액체종균을 고두밥에 5% 뿌려 섞은 후, 종균을 배양시킨다; Sprinkle 5% of the liquid spawn incubated for 6 more days in the soybean rice, and then culture the spawn;
(배양조건 : 36°C , 수분 50%(5시간) 배양 → 38°C , 수분 70%( 16시간) 배양) ; (Culturing conditions: 36 ° C., 50% water (5 hours) culture → 38 ° C., 70% water (16 hours) culture);
5) 배양하면서, 상기 고두밥을 2~3일 간격으로 뒤집기와 뒤섞기를 수행한다;5) incubation, flipping and shuffling the gourd rice at 2-3 days intervals;
6) 4일째 제국기에서 꺼내어 , 450C에서 24시간 동안 건조시켜 사용한다: 및 7) 탁주용 고체종국을 분상 및 조제형태로 제조한다 (도 2) . 6) Take out from the Imperial Day on Day 4, dry at 45 0 C for 24 hours, and use: and 7) Takju solid seed soup is prepared in powder and preparation form (FIG. 2).
<실험예 1>탁주용 곰팡이를 이용한 고체종국 최적화 조건 구명 <Experimental Example 1> Optimization of solid final conditions using Takju mold
<1-1> 액체종균 접종량에 따른 품질 분석 <1-1> Quality analysis according to liquid seed inoculation amount
탁주 제조용 곰광이 고체종국 최적화 조건을 구명하기 위하여 액체종균의 접종량에 따른 효소활성을 분석하였다 . The purpose of this study was to analyze the enzyme activity according to the inoculation amount of liquid spawn to investigate the optimum condition of solid Gokwang.
구체적으로, ME 배지에 아스퍼질러스 오리재 75-2(KACC93236P)를 각각 1 백금이 접종하고, 28°C에서 5일 동안 전배양 한 후, 본배양 배지에 2%를 접종하여 6일 동안 더 배양한 액체종균을 고두밥에 각각 2 , 5 , 및 10%를 살포하여 섞은 후, 상기 <실시예 2>의 소형 제국기를 이용하여 습도를 70%로 유지하며 종국을 배양시켰다.
*그 결과, 도 3에 나타난 바와 같이, 제조된 고체종국의 효소활성은 액체종균의 접종량이 5%일 때 효소활성이 가장 높았고, 2% , 10% 순서로 낮아짐을 확인하였다 (도 3) . Specifically, 1 platinum each of Aspergillus duck 75-2 (KACC93236P) inoculated in ME medium, pre-incubated for 5 days at 28 ° C, then inoculated 2% in the main culture medium for 6 more days The cultured liquid spawn was mixed by sprinkling 2, 5, and 10% of the soybean rice, respectively, and the final culture was carried out while maintaining the humidity at 70% using the small empire of <Example 2>. As a result, as shown in Figure 3, the enzyme activity of the prepared solid seed was the highest enzyme activity when the inoculation amount of the liquid spawn 5%, it was confirmed that the 2%, 10% lower in the order (Fig. 3).
<1-2>제국온도에 따른품질 분석 <1-2> Quality Analysis by Imperial Temperature
제국 온도에 따른 품질을 분석하기 위하여, 상기 <실시예 2>의 소형 제국기의 습도를 70%로 일정하게 유지하고, 단계 4)의 온도 조건을 변화하며 이하 동일한 제조방법으로 제국 온도의 설정에 따른 효소활성을 분석하였다. In order to analyze the quality according to the imperial temperature, the humidity of the small imperial machine of <Example 2> was kept constant at 70%, the temperature conditions of step 4) were changed, and according to the same manufacturing method, Enzyme activity was analyzed.
그 결과, 아스퍼질러스 오리재 75-2 ACC93236P)는 40oC에서 산성 단백질 효소활성이 가장 높게 나타나, 낮은 온도 (20~30oC ) 보다는 곰광이 균사체와 포자가 잘 생육할 수 있는 35~40oC로 최적온도를 조정함으로써 효소활성이 2배 이상 높은 것을 확인하였다 (도 5) . <1-3>제국시간에 따른 효소활성 분석 Aspergillus duck material 75-2 ACC93236P) showed the highest acidic protein enzymatic activity at 40 o C, and it was found that 35 to 20% of mycelia and spores could grow better than low temperature (20 to 30 o C). By adjusting the optimum temperature to 40 ° C it was confirmed that the enzyme activity is more than two times higher (Figure 5). <1-3> Analysis of enzyme activity according to empire time
상기 균주를 이용한 고체종국의 최적 발효시간을 구명하기 위하여, 상기 In order to determine the optimal fermentation time of the solid final state using the strain,
<실시예 2>의 제조방법으로, 도 6에 나타난 바와 같이 7일간 배양하면서 효소활성을 분석하였다. As a preparation method of <Example 2>, the enzyme activity was analyzed while culturing for 7 days as shown in FIG.
그 결과. 도 7에 나타난 바와 같이 , 아스퍼질러스 오리재 75-2는 발효시간이 경과할수록 효소활성이 증가하는 경향을 보였고, 특히 96시간에 급격히 증가함을 확인하였으며, ghicoamyl ase 활성의 경우, 96시간 이후에도 증가하여 96 144시간에 가장 높은 활성을 나타내고 이후에는 감소하는 경향을 나타냄을 확인하였다 (도 7) . 종래에 일본에서 도입된 국법의 경우, 아스퍼질러스 오리재의 제국 발효는 4일 (96시간)째에 출국을 하지만, 우리나라 전통누록에서 분리한 '상기 균주의 접종량과 양조미 제국조건에 따른 차이를 바탕으로. 효소활성이 가장
우수한 제국 6일째로 최적 조건을 구명하였다. As a result. As shown in Figure 7, Aspergillus duck material 75-2 showed a tendency to increase the enzyme activity with the passage of the fermentation time, in particular, was confirmed to increase rapidly at 96 hours, in the case of ghicoamyl ase activity, even after 96 hours It was confirmed that the increase showed the highest activity at 96 144 hours and thereafter tended to decrease (FIG. 7). For the national laws introduced into a conventional in Japan, the Aspergillus, the switch Imperial fermented material duck depart at 4 days (96 hours), but the difference in the "inoculum size and the amount of seasoning Imperial conditions of the strain isolated from Korean traditional Phitsanulok based on. The enzyme activity is the most Optimal conditions were sought on the sixth day of good empire.
<실험예 2> 고체종국의 포자수 확인 액체종균 접종량, 제국 온도. 및 제국 시간에 따른 고체종국의 포자수 및 세균수를 하기의 방법으로 측정하였다. 구체적으로, 시험재료 1~2 g을 정확히 준비하여 5% 트원 (Tween) 용액 10 mL에 \ 메틸렌 블루 (Methylene blue) 2-3 방울을 가하고 묽게 희석한 후, 혈구 계산판 (Heniocytonieter)에 떨어뜨리고, 현미경을 이용하여 검정하였다 (도 5). 현미경으로 반복하여 측정한 수를 a라 하고 , 포자수는 하기의 식으로 구하였다. 포자수 = (측정한 수 (a) X 묽게 한 배수) /시험 재료량 (g) 그 결과, 표 2 내지 표 4, 및 도 10에 나타난 바와 같이, 액체종균을 10% 접종하였을 때, 제국 온도 40oC, 및 제국 6일째에 가장 많은 포자를 생성함을 확인하였다 (표 2 내지 표 4. 및 도 10). 【표 2】 액체종균 접종량에 따른 고체종국의 포자수 <Experimental example 2> Spore count confirmation of solid seed | species liquid spawn inoculation amount, empire temperature. And the number of spores and the number of bacteria in the solid species according to the empire time was measured by the following method. Specifically, 1 to 2 g of the test material is accurately prepared, add 2-3 drops of \ methylene blue to 10 mL of a 5% Tween solution, dilute thinly, and drop it into a hemocytonieter. Assay using a microscope (Fig. 5). The number measured repeatedly by the microscope was called a, and the number of spores was calculated | required by the following formula. Pojasu = (number of measuring (a) X dilute multiple) / test amount of material (g) The results, Tables 2 to 4, and as shown in Figure 10, when inoculated with the liquid spawn 10%, Imperial temperature 40 o It was confirmed that the most spores were produced on C, and the empire day 6 (Tables 2 to 4. and FIG. 10). [Table 2] Spore Count of Solid Seeds according to Liquid Seed Inoculation Volume
【표 3】 제국 온도에 따른 고체종국의 포자수 종균 제국 온도 (°C) [Table 3] Spores of different species according to imperial temperature Seed spawn imperial temperature ( ° C)
20 30 35 40 20 30 35 40
A oryzae A oryzae
0.4±7.9xl06 6.8土8.9><106 11.1±2.4X106 12.5±6.7X106 0.4 ± 7.9xl0 6 6.8土8.9> <10 6 11.1 ± 2.4X10 6 12. 5 ± 6.7X10 6
75-2
【표 4】 75-2 Table 4
제국 시간에 따른 고체종국의 포자수 Spore counts of solid species over empire time
<실험예 3>고체종국의 세균수 확인 Experimental Example 3 Confirmation of Number of Bacteria in Solid State Countries
액체종균 접종량 , 제국 온도 , 및 제국 시간에 따른 세균수를 하기의 방법으로 측정하였다. Liquid seed inoculation amount, empire temperature, and the number of bacteria according to empire time were measured by the following method.
구체적으로, 시료 10 g을 취하여 멸균 백에 넣고, 90 niL의 멸균 생리식염수를 사하여 균질기로 용해하고 멸균 생리식염수로 단계별로 희석한 후. PCA 배지에 도말하고. 나타난 세균수를 CFU/g으로 표기하였다. Specifically, take 10 g of the sample, put it in a sterile bag, dissolve in a homogenizer using 90 niL of sterile saline, and dilute stepwise with sterile saline. Smear on PCA medium. The number of bacteria shown is indicated as CFU / g.
그 결과. 표 5 내지 표 7 , 및 도 11에 나타난 바와 같이, 세균의 오염 수치는 102~103이었고, 액체종균의 접종량이 증가할수록, 세균수는 적게 나타났고, 제국온도가 증가함에 따라 증가하는 경향을 나타냈으며, 제국 기간이 길어지면서 세균의 수가 감소하는 것을 확인하였다 (표 5 내지 표 그 및 도 11 ) . As a result. As shown in Tables 5 to 7, and FIG. 11, the bacterial contamination levels were 10 2 to 10 3, and as the inoculation amount of the liquid spawn increased, the number of bacteria appeared less and tended to increase as the empire temperature increased. It was confirmed that the number of bacteria decreased as the empire period became longer (Tables 5 to 11 and FIG. 11).
【표 5】 Table 5
【표 6】 Table 6
제국 은도에 따른 고체종국의 세균수
제국 은도 (°C) Bacterial Counts of Solid Species According to Imperial Silver Road Imperial Silver Road ( ° C)
-οΊΣ 20 30 35 40 -οΊΣ 20 30 35 40
A. oryzae A. oryzae
1.43土 4.4X102 1.75±14.6X102 4·57±6.1Χ103 1.27±1.2X103 1.43 土 4.4X10 2 1.75 ± 14.6X10 2 4 · 57 ± 6.1Χ10 3 1.27 ± 1.2X10 3
75-2 75-2
【표 7] 제국 시간에 따른 고체종국의 세균수 [Table 7] Number of Bacteria in Solid Species According to Empire Time
특히, 일본의 종균 회사의 경우, 평균 102개 정도의 세균의 오염은 무시하고 제품을 출시하는데, 본 발명의 실험예의 결과는 일본 보다 세균수가 조금 높지만, 탁주용 종균 산업화를 위한 대량생산 시스템 구축 (Clean room) 된 종균회사에서 생산을 하면 일본의 종국회사처럼 오염도를 낮출 수 있다. In particular, the spawn company in Japan releases products on the average, ignoring the contamination of about 10 2 bacteria, but the experimental example of the present invention results in a slightly higher number of bacteria than in Japan, but establishes a mass production system for the commercialization of spawn for Takju. When produced by a clean room spawn company, pollution can be reduced like a spawn company in Japan.
<실험예 4> 제국 시간에 따른 품은 변화 <Experiment 4> Change of Bearishness According to Empire Time
상기 균주를 이용한 고체종국을 상기 <실시예 2>의 방법으로 7일간 제국하면서, 이의 품온 변화를 조사하였는데, 제국기의 온도를 일정하게 유지함으로써, 상기 균주가 생육하면서 내는 품온의 변화도 일정하게 유지하였고. 그 결과는 도 8에 나타내었다. <실험예 5> 탁주 제조용 곰팡이 종균의 건조 방법 최적조건의 확립 During the seven days of the solid seed station using the strain was empire by the method of <Example 2>, the change in the temperature of the product was investigated, by maintaining the temperature of the imperial period, the change in the product temperature during the growth of the strain is also kept constant And The results are shown in FIG. Experimental Example 5 Establishment of Optimum Conditions for Drying Molds of Takju
본 발명의 공광이 종국에 대하여 동결 및 열풍건조에 따른 고체종국의 건조방식을 비교 검토함으로써, 향후 산업용 대량생산을 위한 건조 방법의
최적조건을 확립하고자 하기 '의 실험을 수행하였다. By comparing and examining the drying method of the solid state according to freeze and hot air drying, the pore of the present invention finally provides a method of drying for industrial mass production in the future. In order to establish the optimum conditions, the experiment was conducted.
구체적으로, 도 12에 나타난 건조 방법 장치를 이용하여, 동결건조 장치는 Freezing dryer MCID8518( I lshin bio base Co. , Ltd. 대한민국)이고 열풍건조 장치는 Heated-air dryer DS-80-3(Dasol scientific Co. , Ltd. 대한민국) 제품을 사용하였다. 본 발명의 고체종국을 -80°C에서 급속 동결한 다음 동결건조 장치를 사용하여 24시간 동안 동결건조 하거나, 열풍건조 장치를 사용하여 45°C에서 24시간 동안 열풍건조 하여, 제조된 고체종국의 수분함량, 생균수, 및 세균 오염도를 분석하였다. Specifically, using the drying method apparatus shown in Figure 12, the freeze drying apparatus is Freezing dryer MCID8518 (Ilshin bio base Co., Ltd. South Korea) and the hot air drying apparatus is Heated-air dryer DS-80-3 (Dasol scientific Co., Ltd. South Korea) was used. Solid freeze of the present invention the rapid freeze at -80 ° C and then lyophilized for 24 hours using a freeze-drying device, or hot air drying for 24 hours at 45 ° C using a hot air drying device, Moisture content, viable cell count, and bacterial contamination were analyzed.
그 결과, 도 13 내지 도 16에 나타난 바와 같이, 동일한 시간 동안 건조하였을 때, 동결건조는 5.6%, 열풍건조는 2.8%의 수분함량을 나타내었고 (도 14), 포자수의 경우, 동결건조는 16.5χ106±1.5개, 열풍건조는 22.0χ 106±8.1개로 건조 전 13.8χ106±6.1개보다 많은 수를 나타내었으며 (도 15), 세균 오염도는 동결건조 4.8χ102±4.2개보다 열풍건조 시 2.5χ102±3.5개로 1.9배 정도 오염도가 낮음을 확인하였다. As a result, as shown in Figure 13 to 16, when dried for the same time, lyophilization was 5.6%, hot air drying showed a water content of 2.8% (Fig. 14), in the case of spores, lyophilization is 16.5 χ 10 6 ± 1.5 and hot air drying was 22.0 χ 10 6 ± 8.1, which was more than 13.8 χ 10 6 ± 6.1 before drying (Fig. 15), and bacterial contamination was higher than 4.8 χ 10 2 ± 4.2 of freeze drying. It was confirmed that the pollution degree was 1.9 times lower with 2.5 χ 10 2 ± 3.5 during hot air drying.
또한, 상기 고체종국의 보호를 위해 탈지유 (Skim milk)를 부형제로서 첨가한 후. 건조 전 세균의 오염 정도는 2.0χ102±0.6개, 동결건조 시 5.0χ102±2.5개고, 열풍건조는 1.0χ102±0.5개였으며, 트레할로스 (trehalose)를 부형제로서 첨가한 경우에는 건조 전에 3.0χ102±0.7개였고. 동결건조 시 5.0χ102±1.0개. 열풍건조 시 8.0χ102±1.5개로 동결건조하는 방법이 2배 정도의 오염도가 낮아 열풍건조보다는 동결건조가 적합하다고 판단되어 진다 (도 16).
In addition, after the addition of skim milk (Skim milk) as an excipient for the protection of the solid end. The degree of contamination of bacteria before drying was 2.0 χ 10 2 ± 0.6, 5.0 χ 10 2 ± 2.5 during lyophilization, and hot air drying was 1.0 χ 10 2 ± 0.5, and when trehalose was added as an excipient, Before, 3.0 χ 10 2 ± 0.7. Lyophilized 5.0 x 10 2 ± 1.0. It is judged that freeze drying is more suitable than hot air drying because the method of freeze drying to 8.0 x 10 2 ± 1.5 at the time of hot air drying has twice the pollution degree (FIG. 16).
Claims
【청구항 1】 [Claim 1]
1 ) 도정된 쌀을 세척 후, 침지시킨 다음 껄수하는 단계; 1) washing the milled rice, immersing and washing it;
2) 상기 단계 1 )의 쌀을 증자시킨 후, 방넁시켜 고두밥을 제조하는 단계; 2) increasing the rice of step 1), and then leaving it to stand to prepare gourd rice;
3 ) 상기 단계 2 )의 고두밥에 아스퍼질러스 오리재
oryzae) 75- 2(KACC93236P)의 액체 종균을 흔합한 후, 뒤집기와 뒤섞기를 하면서 배양시키는 단계; 및 3) Aspergillus duckwood in the gourd rice of step 2) oryzae) mixing 75-2 (KACC93236P) liquid spawn and incubating with inverting and mixing; And
4) 상기 단계 3)의 배양된 고두밥을 건조시킨 후, 분상 또는 조제 형태로 제조하는 단계를 포함하는 아스퍼질러스 오리재 75-2( ACC93236P)를 이용한 고체종국의 제조방법. 4) Method of producing a solid seed using Aspergillus duck material 75-2 (ACCC93236P) comprising the step of drying the cultured gourd rice of step 3) in the form of powder or preparation.
【청구항 2】 [Claim 2]
제 1항에 있어서, 상기 단계 1 )의 침지는 물에 4 내지 12시간 동안 담그는 것을 특징으로 하는 아스퍼질러스 오리재 75-2(KACC93236P)를 이용한 고체종국의 제조방법. The method according to claim 1, wherein the immersion of step 1) is immersed in water for 4 to 12 hours. Aspergillus duck material 75-2 (KACC93236P) manufacturing method of the solid terminal.
【청구항 3】 [Claim 3]
제 1항에 있어서 , 상기 단계 1 )의 절수는 소쿠리 또는 체에 넣고 30분 내지 120분 동안 물을 빼주는 것을 특징으로 하는 아스퍼질러스 오리재 75- 2 ACC93236P)를 이용한 고체종국의 제조방법 . The method of claim 1, wherein the water of step 1) is put into a colander or sieve and water is drained for 30 to 120 minutes. Aspergillus duck material 75-2 ACC93236P).
【청구항 4】 [Claim 4]
제 1항에 있어서, 상기 단계 2 )의 증자는 찜 솔을 이용하여 40분 내지 120분 동안 찌는 것을 특징으로 하는 아스퍼질러스 오리재 75-2(KACC93236P)를
이용한 고체종국의 제조방법 . The aspergillus duck material 75-2 (KACC93236P) is steamed for 40 minutes to 120 minutes using a steaming brush. Manufacturing method of solid final state using.
[청구항 5】 [Claim 5]
제 1항에 있어서. 상기 단계 2)의 방넁은 40 내지 50oC가 되도록 식히는 것을 특징으로 하는 아스퍼질러스 오리재 75-2(KACC93236P)를 이용한 고체종국의 제조방법. The method of claim 1. The method of step 2) is a method for producing a solid terminal using Aspergillus duck material 75-2 (KACC93236P), characterized in that the cooling to 40 to 50 ° C.
【청구항 6】 [Claim 6]
제 1항에 있어서, 상기 단계 3)의 아스퍼질러스 오리재 75-2( KACC93236P)는 25 내지 30oC에서 4 내지 6일 동안 각각 전배양 및 본배양을 시켜 종균을 제조하는 것을 특징으로 하는 아스퍼질러스 오리재 75-2(KACC93236P)를 이용한 고체종국의 제조방법. According to claim 1, Aspergillus duck 75-2 (KACC93236P) of the step 3) is pre-cultivation and main culture for 4 to 6 days at 25 to 30 o C, respectively, characterized in that to produce the seed A method for producing a solid final product using Aspergillus duck material 75-2 (KACC93236P).
【청구항 7】 [Claim 7]
제 1항에 있어서, 상기 단계 3)의 액체종균은 2 내지 5%의 농도로 접종시키는 것을 특징으로 하는 아스퍼질러스 오리재 75-2 ACC93236P)를 이용한 고체종국의 제조방법. The method according to claim 1, wherein the liquid spawn of step 3) is inoculated at a concentration of 2 to 5%. Aspergillus duck material 75-2 ACC93236P).
【청구항 8】 [Claim 8]
제 1항에 있어서, 상기 단계 3)의 배양은 35 내지 40oC에서 5 내지 7일 동안 배양하는 것을 특징으로 하는 아스퍼질러스 오리재 75-2 ACC93236P)를 이용한 고체종국의 제조방법. The method according to claim 1, wherein the culturing of step 3) is carried out at 35 to 40 o C for 5 to 7 days.
【청구항 9】 [Claim 9]
제 1항에 있어서, 상기 단계 4)의 건조는 40 내지 50oC에서 20 내지 30시간
동안 건조시키는 것을 특징으로 하는 아스퍼질러스 오리재 75-2(KACC93236P)를 이용한 고체종국의 제조방법. The method of claim 1, wherein the drying of step 4) is performed at 40 to 50 ° C. for 20 to 30 hours. Method of producing a solid terminal using Aspergillus duck material 75-2 (KACC93236P), characterized in that for drying.
【청구항 10] [Claim 10]
제 1항 내지 제 9항 중 어느 한 항의 제조방법으로 제조된 고체종국.
A solid seed produced by the method of any one of claims 1 to 9.
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KR20120071596A (en) * | 2010-12-23 | 2012-07-03 | 대한민국(농촌진흥청장) | A quick nuruk manufacturing method using aspergillus oryzae and a quick nuruk thereby |
KR101274638B1 (en) * | 2011-11-22 | 2013-06-13 | 주식회사농심 | Method or manufacturing improved nuruk for raw rice wine |
KR101297707B1 (en) * | 2011-10-19 | 2013-08-22 | 한국식품연구원 | Method of preparing nuruk and fermented alcohol using the same by inoculating aspergillus oryzae into wheat |
KR20150090335A (en) * | 2014-01-28 | 2015-08-06 | 사단법인 경기막걸리 세계화사업단 | Aspergillus oryzae s-2 kacc93172p of ferment spawn used in brewing rice-wine, and manufacturing method thereof |
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KR20030075730A (en) * | 2002-03-20 | 2003-09-26 | (주)진바이오텍 | Novel Aspergillus oryzae GB-106 mutant and method for producing soybean enzymatic food-stuff using thereof |
KR20120071596A (en) * | 2010-12-23 | 2012-07-03 | 대한민국(농촌진흥청장) | A quick nuruk manufacturing method using aspergillus oryzae and a quick nuruk thereby |
KR101297707B1 (en) * | 2011-10-19 | 2013-08-22 | 한국식품연구원 | Method of preparing nuruk and fermented alcohol using the same by inoculating aspergillus oryzae into wheat |
KR101274638B1 (en) * | 2011-11-22 | 2013-06-13 | 주식회사농심 | Method or manufacturing improved nuruk for raw rice wine |
KR20150090335A (en) * | 2014-01-28 | 2015-08-06 | 사단법인 경기막걸리 세계화사업단 | Aspergillus oryzae s-2 kacc93172p of ferment spawn used in brewing rice-wine, and manufacturing method thereof |
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