KR20150108574A - Method for producing Cheongtaejeon using fermentative microorganism - Google Patents

Abstract

The present invention relates to a manufacturing method of green moss tea using a green moss tea fermentation strain, capable of standardizing a manufacturing method of green moss tea and evenly producing green moss tea with excellent taste and quality, by manufacturing mother tea by inoculating tea leaves steamed after culturing microorganisms related to a fermentation process of green moss tea and manufacturing green moss tea by using the mother tea.

Description

TECHNICAL FIELD The present invention relates to a fermentative microorganism,

More particularly, the present invention relates to a method for preparing a seed culture by cultivating a pre-fermentation strain and culturing the seed culture, Mixing the finely ground and finely ground tea leaves with the tea seeds, molding the tea leaves into a preform shape, drying and storing the preform, and pre-fabricating the preform by the method.

Camellia sinensis LO Kuntze) is a subspecies of Camellia and is distributed in a very wide area. The cultivars are Chinese larvae ( var. macrophylla ), which is called carrots and rabbits, and Indian larvae ( var . asssamica ) And Chinese small leaflet ( var. Sinensis ).

Tea is variously called according to tea variety, production environment, harvest time, jade method, product type and flavor. It is not easy to classify them correctly, but it is generally classified according to harvest time and degree of fermentation. According to the time of harvesting, it is classified into a first tea, a first tea, a tea or tea, a spring tea, a summer tea, a fall tea, and a completely non-fermented tea having a fermentation degree of 0% Tea is classified as a green tea or oolong tea, and more than 80% fermented tea is classified as black tea, and the post fermented tea is classified as yellow tea or black tea. When the characteristics of green tea jade are classified according to the method of green tea, green tea jade uses the method of inactivating the enzyme present in the tea leaves in the early stage of jade and stopping the fermentation. . The taste is excellent and the tea leaves are green. The green tea which is fermented about 20 ~ 40% of half - fermented tea and the oolong tea which is fermented about 40 ~ 60% have excellent aroma.

The quality of tea is greatly influenced by the environment of tea leaves, maturity, variety, soil, weather, fertilization, and degree of fermentation, and the chemical composition of tea is greatly changed by these conditions.

Cheongtaejeon is our traditional car, which has been preserved for many years in the history of Korean tea culture. It was first introduced in the Party, and it was in the south coast region of Gangjin and Haenam mainly in Jangheung from the Three Kingdoms period to the late 1940s. In Jangheung province, a prototype of Jedah and a traditional car before Cheongtae were found in Borimsa until the Japanese colonial period, but it has been hidden since June 25th. Historically, the area near Jungchung-myeon, Bucheon-myeon, Jangheung County, which is regarded as the main site of Jeong-tae, is a place for tea and a somewhat existed area. The records of the cars in Jangheung area include 'Seojokseok' and 'Imha' written by Jae Won, who specifically mentioned Jangheung Volimsa 's tanks (Cheongtaejeon).

Jedah before jingtae picked a clear day from the end of April to the beginning of May, picked out the tea leaves, put the chunks of the ground tea leaves into the mold until the tea leaves turned yellow, and dried in a sunny day And a hole was made in the center to hold it in a slender kitten.

Korean Patent No. 1037915 discloses a method of manufacturing a pre-fermented product using a fermenter, and Korean Patent Publication No. 2010-0059278 discloses a method of producing a fermented tea using a lactic acid bacteria strain. However, as described in the present invention, there is no disclosure of a method for manufacturing pre-fermentation using pre-fermentation fermentation strains.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method of isolating and isolating microorganisms involved in the fermentation process before quenching and preparing a starter using the microorganisms, The present inventors have completed the present invention by providing a standardized pre-fermentation preparation method and flavor standardized fermentation process.

In order to solve the above problems,

(a) culturing the fermentation broth before fermentation, centrifuging to obtain a strain, and diluting the obtained strain with physiological saline to prepare a starter;

(b) washing the collected tea leaves, heating them with steam, and then milling them;

(c) inoculating the seeds of step (a) in the leaves of step (b), mixing and culturing the seeds to prepare a mother tea;

(d) adding tea leaves prepared in the same manner as in step (b) to tea leaves of step (c), mixing the tea leaves, molding and drying the tea leaves; And

(e) punching a hole in the preform of step (d), drying and storing the preform.

In addition, the present invention provides a cryopreservation prepared by the above method.

According to the present invention, it is possible to standardize the production method before quenching by cultivating microorganisms involved in fermentation before fermentation and then preparing a hybrid tea by inoculating the tea leaves with the fermentation, Thereby, it is possible to uniformly produce cheonggyejeon with excellent taste and quality.

FIG. 1 is a schematic representation of a jade method of preparing a starter by adding starter.
Fig. 2 is a photograph of Cheong-tae, which is a traditional fermented tea of the pre-war type.

In order to achieve the object of the present invention,

(a) culturing the fermentation broth before fermentation, centrifuging to obtain a strain, and diluting the obtained strain with physiological saline to prepare a starter;

(b) washing the collected tea leaves, heating them with steam, and then milling them;

(c) inoculating the seeds of step (a) in the leaves of step (b), mixing and culturing the seeds to prepare a mother tea;

(d) adding tea leaves prepared in the same manner as in step (b) to tea leaves of step (c), mixing the tea leaves, molding and drying the tea leaves; And

(e) punching a hole in the preform of step (d), drying and storing the preform.

Specifically, in step (a), the pre-fermentation fermentation strain is cultured at 23 to 33 ° C for 20 to 50 hours, followed by centrifugation to obtain a strain, One strain may be diluted to a physiological saline at an OD ₆₀₀ of 0.25 to 0.45 to produce a seed. Preferably, the pre-fermentation strain is cultured at 25 to 31 ° C for 24 to 48 hours, followed by centrifugation to obtain a strain , And the obtained strain may be diluted with physiological saline at a concentration of 0.3 to 0.4 at OD ₆₀₀ to produce a seed culture. More preferably, the fungi and yeast in the pre-fermentation strain are maintained at 25 ° C, For a period of time, centrifuging the strain to obtain a strain, and diluting the obtained strain to physiological saline at a concentration of 0.4 at an OD ₆₀₀ to prepare a seed culture. However, the present invention is not limited thereto.

Specifically, in step (b), the collected tea leaves are washed, and the washed tea leaves are steamed for 0.5 to 2 minutes and then finely ground And may be, but not limited to, washing the collected tea leaves, finishing the washed tea leaves with steam for one minute, and crushing them.

The tea leaves can be collected from the early April to the late May, preferably from the end of April to the beginning of May, but the collection time is not limited thereto.

In step (c), the seeds of step (a) are inoculated in an amount of 8 to 12% based on the weight of the tea leaves of step (b) And then incubating the suspension at 33 ° C for 20 to 50 hours to prepare a seedlings and then storing the seedlings at -3 to 4 ° C. Preferably, the seedlings of step (a) %, And incubating the mixture at 25 to 31 ° C for 24 to 48 hours to prepare a homogenate and then storing the homogenate at -1 to 1 ° C. More preferably, the step (a) ), 10% of the weight of tea leaves, and the mixture is inoculated. The fungus and yeast strain are cultured at 25 占 폚, the bacterium is cultured at 31 占 폚 for 24 to 48 hours, But is not limited thereto.

In the pre-fermentation method according to an embodiment of the present invention, the step (d) may be carried out in such a manner that the tea leaves newly prepared in the same manner as in the step (b) (B), 2 to 5% by weight based on the leaf weight of the mixture, mixing the mixture with the mixture, adding the mixture to the mixture in an amount of 2 to 5% Is added in an amount of 3% based on the weight of the freshly prepared tea leaves, and the mixture is molded into a pre-formed shape and dried naturally, but the present invention is not limited thereto.

The addition of the seedlings may preferably be 3% of the weight of the ground tea leaves after the heat treatment, but the addition rate is not limited if the seedlings are added at such a rate that proper fermentation can take place during the drying process. In addition, the natural drying may be a daylight drying or an indoor drying, preferably a drying method in which sunlight drying and indoor drying are simultaneously performed, but the present invention is not limited thereto.

 In step (e) of the method according to an embodiment of the present invention, the step (e) is a step of piercing a hole in the tea of step (d), drying naturally and storing at 18 to 26 ° C Preferably, the step (d) may be carried out by punching holes in the preform-shaped tea, then naturally drying and storing at 23 to 25 ° C, and more preferably, the step (d) Dried, naturally wrapped in a broad cloth bag, placed in a jar, and stored at a room temperature of 23 to 25 ° C. However, the present invention is not limited to such a condition as long as fermentation before fermentation and maturation are appropriately performed.

In a method according to an embodiment of the present invention, the pre-fermentation strain is selected from the group consisting of Aspergillus niger) JMICTJ1 (accession number: KACC93189P), Bacillus amyl Lori rake Pacifico Enschede (Bacillus amyloliquefaciens JMICTJ2 (Accession No .: KACC91913P), Bacillus subtilis subtilis JMICTJ3 (Accession No .: KACC91914P) or Rhizopus oryzae JMICTJ4 (Accession No .: KACC93190P), but it is not limited to fermentation strains suitable for fermentation before fermentation.

In addition, the present invention provides a cryopreservation prepared by the above method.

The chelating agent may be in the form as shown in Fig. 2, and is preferably fermented by Aspergillus niger JMICTJ1, Bacillus amyloliquefaciens JMICTJ2, Bacillus subtilis JMICTJ3 or Raiofus oryzae JMICTJ4 It can be a car.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One. Cheongtaejeon  material

Cheongtaejeon, which was used in this experiment, was purchased from Jangheung special products store and used as a sample during the 2010 ~ 2012 year. The leaves of green tea were harvested in Jangheung - dowon in October, 2010. Frozen green tea leaves were collected from Jangheung Daewon and used as samples.

Example  2. Strain Isolation and Identification

One) Cheongtaejeon  Preparation of culture medium for strain isolation

Table 1 and Table 2 show the composition of the plate medium for separation of superior culture broth before quenching. The PDA (Potato Dextrose Agar) medium for yeast and fungi isolation and the NA (Nutrient Agar) medium for bacterial isolation were each sterilized at 121 ° C for 15 minutes, cooled to 45 ° C, shaken well and dispensed into a petri dish A plate culture medium was prepared and used.

Composition of PDA plate media division content (%) Potato starch 0.4 Dextrose 2.0 Agar 1.5

Composition of NA plate medium for the isolation of pre-ciliated bacteria division content (%) Beef extract 0.3 Peptone 0.5 Agar 2.0

2) Cheongtaejeon  Strain isolation

The prepared culture medium was preincubated with the crushed chrysanthemum powder, and the PDA medium and the NA medium were cultured at 25 ° C and 31 ° C for 24 to 48 hours, respectively. After confirming all the different types of colonies seen on the cultured plates, flame sterilized platelike colonies were collected on a new plate and plated. Thereafter, the PDA medium was cultured at 25 ° C and the NA medium was cultured at 31 ° C for 24 to 48 hours to isolate the strain pure.

3) 16S and 18S for isolating isolates rRNA  Analysis method

A) Preparation of template DNA

0.5 mL of sterile saline solution was placed in a 1.5 mL centrifuge tube and colonies were collected with a sterilized toothpick and dissolved in the solution. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was removed and the remaining pellet was dissolved in 0.5 mL of InstaGene Matrix (Bio-Rad, USA). After treatment at 56 ° C for 30 minutes, heat treatment at 100 ° C for 10 minutes was performed, and PCR was performed using the supernatant.

B) PCR

1 μl of the template DNA was added to 30 μl of the PCR reaction solution, and PCR was carried out using primers for prokaryotic 27F / 1492R and eukaryotic ITS1 / ITS4 primers at 94 ° C. for 45 seconds, 55 ° C. for 60 seconds and 72 ° C. for 60 seconds PCR amplification was carried out under the conditions that the amplification was performed once. The DNA fragment was amplified by about 1,400 bp.

C) Purification of PCR products

Residual primers or dNTPs not synthesized were removed from the PCR product using the Montage PCR Clean up kit (Millipore).

D) Sequence analysis

The purified PCR products of about 1,400 bp were subjected to sequencing using the primers shown in Table 3. Sequencing analysis was performed on Macrogen using Big Dye terminator cycle sequencing kit v.3.1 (Applied BioSystems, USA) and Applied Biosystems model 3730X automated DNA sequencing system (Applied Bio Systems, USA).

The primers used for amplification and base sequence Strain primer The base sequence (5 '- > 3') SEQ ID NO: Amplification Sequencing Prokaryotic 27F AGT GTT TGA TCM TGG CTC AG One ● 1492R TAC GGY TAC CTT GTT ACG ACT T 2 ● 518F CCA GCA GCC GCG GTA ATA CG 3 ● 800R TAC CAG GGT ATC TAA TCC 4 ● Eukaryotic ITS 1 TCC GTA GGT GAA CCT GCG G 5 ● ● ITS 4 TCC TCC GCT TAT TGA TAT GC 6 ● ●

4) Cheongtaejeon  Seeds for manufacturing ( Starter ) Selection of strain

The food microorganisms involved in the fermentation having the characteristics as shown in Table 4 were selected as pre-fermentation strain.

List of fermented seeds before fermentation Strain name Code number Characteristics Aspergillus or Lee
( Aspergillus niger ) JMICTJ1 Black germ, amylase enzyme production,
Mainly used in aerodrome when manufacturing alcohol Bacillus amyloliquefaciens
( Bacillus amyloliquefaciens ) JMICTJ2 Bacillus subtilis, excellent protease activity Bacillus Subtilis
( Bacillus subtilis ) JMICTJ3 Production of enzymes such as amylase and protease Rijo pus duck
( Rhizopus oryzae ) JMICTJ4 Production of enzymes such as amylase, excellent glycation ability

Example  3. Using selected strains Mocha  Manufacturing method

1) Culturing conditions for strain

Table 5 and Table 6 show the medium for the production of the seeds to be used for pre-fermentation using the selected strains. PDB (Potato Dextrose Broth) medium for yeast and fungal culture and NB (Nutrient Broth) medium for bacterial culture were sterilized at 121 ° C for 15 minutes. The fungi and yeasts such as Aspergillus were cultured at 25 ° C using PDB medium and the bacterium such as Bacillus were incubated at 31 ° C for 24-48 hours using NB medium.

Composition of PDB medium for cultivation of fermented seed culture division content (%) Potato starch 0.4 Dextrose 2.0

Composition of NB medium for culture of fermented seed culture division content (%) Beef extract 0.3 peptone 0.5

2) Production method of seed culture

Strain cultivation is completed, it was prepared by adjusting the then using a centrifuge to remove cells and cell culture medium in a concentration of 0.4 at OD ₆₀₀ in 0.85% saline.

3) Method

The tea leaves collected in the morning were washed and then steamed for 1 minute and milled with mortar. The bacterium such as bacillus was incubated at 31 ° C for 24 to 48 hours to stabilize and strengthen the forces of the bacterium. After the seedlings were inoculated at 10% 1 < [deg.] ≫ C.

Example  4. Inoculation of seed Mocha  Used Cheongtaejeon  Manufacturing standardization

The method for standardization of pretreatment manufacturing using seedling seeding inoculum is shown in Fig. In the pre-treatment of the present invention, Jade standardization was performed by a method of inoculating a seed with reference to a conventional Jeda method before the present invention.

That is, tea leaves were collected in the morning, washed, steamed for 1 minute, and finely crushed using a mortar. Then, the inoculated seeds prepared in Example 3 were heated and finely ground, and 3% of the prepared tea leaves were inoculated. Coarse drying was performed by natural drying, which combined with daylight drying and indoor drying. After drilling a hole in the center of the dried pre-shaped tea lump, it was finally dried, wrapped in a broad cloth bag and stored in a jar at 23 ~ 25 ℃.

Agricultural Biotechnology Research Institute KACC93189P 20131203 Agricultural Biotechnology Research Institute KACC91913P 20131203 Agricultural Biotechnology Research Institute KACC91914P 20131204 Agricultural Biotechnology Research Institute KACC93190P 20131203

<110> JANGHEONGGUN <120> Method for producing Cheongtaejeon using fermentative          마이크로organism <130> PN12378 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 ccagcagccg cggtaatacg 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 taccagggta tctaatcc 18 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 tccgtaggtg aacctgcgg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 tcctccgctt attgatatgc 20

Claims (4)

(a) culturing the fermentation broth before fermentation, centrifuging to obtain a strain, and diluting the obtained strain with physiological saline to prepare a starter;
(b) washing the collected tea leaves, heating them with steam, and then milling them;
(c) inoculating the seeds of step (a) in the leaves of step (b), mixing and culturing the seeds to prepare a mother tea;
(d) washing freshly collected tea leaves, adding steam to the finely ground tea leaves after adding steam to the tea leaves, mixing the tea leaves with the seeds of step (c), shaping the leaves and drying them; And
(e) drilling a hole in the preform of step (d), and drying and storing the preform. The method according to claim 1,
(a) culturing the pre-fermentation fermentation broth at 23 to 33 ° C for 20 to 50 hours, centrifuging the strain to obtain a strain, and diluting the obtained strain to physiological saline at an OD of ₆₀₀ to 0.25 to 0.45 to prepare a seed step;
(b) washing the collected tea leaves, heating the leaves for 0.5 to 2 minutes with steam and finely grinding;
(c) The seeds of step (a) are inoculated by 8 to 12% of the weight of the tea leaves of step (b), mixed and cultured at 23 to 33 ° C for 20 to 50 hours, &Lt; / RTI &gt;
(d) The freshly collected tea leaves are washed and heated with steam. The leaves of step (c) are added to the finely ground tea leaves in an amount of 2 to 5% based on the weight of the finely ground tea leaves. Natural drying step; And
(e) punching holes in the tea of step (d), drying naturally and storing at 18 to 26 ° C. The method according to claim 2, wherein the pre-fermentation strain is selected from the group consisting of Aspergillus niger JMICTJ1 (Accession No .: KACC93189P), Bacillus amyloliquefaciens JMICTJ2 (Accession No .: KACC91913P), Bacillus subtilis Bacillus subtilis JMICTJ3 (Accession No .: KACC91914P) or Rhizopus oryzae JMICTJ4 (Accession No .: KACC93190P). 4. A method according to any one of claims 1 to 3,

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