KR101710989B1

KR101710989B1 - A new strain having acid tolerances and a method fermenting dendropanax morbifera by using same

Info

Publication number: KR101710989B1
Application number: KR1020150077309A
Authority: KR
Inventors: 지연태
Original assignee: 전남대학교산학협력단
Priority date: 2015-06-01
Filing date: 2015-06-01
Publication date: 2017-02-28
Also published as: KR20160141518A

Abstract

The present invention relates to a new strain of galactomyces candidum derived from P. japonicus having excellent acid tolerance and laccase activity, a method of fermenting unripe fermented Hwangchulchuk extract using the same, The present invention relates to a fermentation broth of an extract of U. chrysanthemum which is obtained by removing uriciol, which is an allergen, and antioxidant function, which is obtained by the method according to the present invention. The Galactomyces candidum C27 (Den-1) strain of the present invention is excellent in acid tolerance, , It is possible to remove uriciol, which is an allergen causing substance, because of its excellent activity as a lacquer. Therefore, it is possible to ferment the extract of Ganoderma lucidum L. of strong acid using Galactomyces candidum C27 (Den-1) strain of the present invention and to maintain the inherent antioxidative effect while eliminating allergen induction. In addition, the extract can be fermented without further sugar addition by using the above-mentioned strains, so that various adult diseases can be prevented. Accordingly, it is expected that the fermented liquid of the extract of Hwangchujang fermented by using the above strain can be usefully used for various foods such as yogurt, beverage and the like without any burden on allergy or adult diseases.

Description

[0001] The present invention relates to a novel strain having an acid resistance and a method for fermenting it by fermenting dendropanax morbifera using same

The present invention relates to a novel strain having acid tolerance and a method for fermenting it by using the same. More specifically, the present invention relates to a novel strain having a high acid tolerance and having laccase activity, The present invention relates to a method for fermenting unfermented sugarcane extracts of the genus Galactomyces candidum and a fermentation broth of an extract of Wuchulcia bark extract having the antioxidant function of the allergy- .

Dendropanax morbifera LEV. Is an evergreen broad-leaved forest belonging to Araliaceae. It is known that the name Yellowlich is originated from a yellowish liquid when the skin is scratched, and the yellowish liquid has been used for coloring or surface processing when making woodwork.

This woody plant is a subtropical plant that grows and grows exclusively in the islands of Jeju, Jeolla, South and West coast of Korea, Wando, Bogil, Eocheido, Jindo, Hongdo, Geomundo, It reaches 15 m, young branches are green and have no hairs. Leaves are alternate phyllotaxis, ovate or ovate, and the flowers bloom in June with light yellowish green color, and they are bloomed and hang on a mountain type inflorescence. The fruit of the perennial tree is elliptical and opens in black in October. The juice or resin used for the traditional crafts of Huangchu is collected from August to September. The above-mentioned woody resin has a golden color, and is resistant to heat, durability and water resistance, and has good adhesion and gloss, and has been used as a natural paints and civilian medicine materials in traditional handicrafts since ancient times.

In addition, it has been focused on the development of medicines or foods using Hwangchujang extract because of its antioxidant activity, blood circulation effect, protection of liver function, hypertension improving effect and nervous stability efficacy. However, it is known that Uchilacchae contains urshiol, which is a cause of skin allergy. It causes allergies when it is consumed or applied to the skin. The extract of Huchilchia japonica which is currently marketed is obtained by hot water extraction, Such as dermatitis.

Therefore, there has been attempted to use a fermentation broth obtained by fermenting Hwangchu tree extract in order to remove the urushiol ingredient, which is an allergen, but since Hwangchu has a strong sterilization and antimicrobial activity and does not grow microorganisms for fermentation, There was difficulty. Therefore, there are no reports on the microorganisms or fermentation methods used for the fermentation of Hwangchu-gil. However, traditionally, in the private household, there is a so-called "enzyme fermentation", in which the leaves, fruits, and stems of Huangchuang are put into a container at a ratio of 1: 1 with sugar and fermented for 6 months to 1 year. It is too long and practical for practical use is difficult. This enzyme fermentation is not a true fermentation because it is not an environmental condition in which microorganisms can grow because the concentration of sugar in the medium is so high that the water activity is low, I can do it. In addition, in order to ferment Hwangchu-myeon extract, sugar such as glucose is added as a carbon source separately in the medium. These sugars remain after fermentation and cause various adult diseases.

Therefore, the present inventors have continued research to develop a novel strain capable of eliminating the uriciol component, which is an allergen causing substance, because it has excellent acid tolerance and is able to grow even in strong acid, but has excellent lactase activity. The strain Galactomyces candidum C27 (Den-1) was found to be resistant to allergenic substances such as uriciol, because it has excellent resistance to organic acids and can grow not only in strong acid but also in lacase activity. Thereby completing the present invention.

Therefore, a technical problem to be solved by the present invention is to provide a novel strain having an acid-resistant activity and excellent in lacase activity.

In addition, another object of the present invention is to provide a method for fermenting Hwangcholgak extract using the strain.

Another object of the present invention is to provide a fermentation broth of an extract of Fusarium oxyspermum fermented by using the above method.

In order to solve the above-mentioned technical problem, the present invention provides a method for producing a galactomycosis Candidomu ( Galactomyces) comprising a nucleotide sequence of SEQ ID NO: 1 having acid resistance and lacquer activity candidum ).

In order to solve the above-mentioned technical problems, the present invention provides a method for fermenting Hwangryeok tree using the strain.

Preferably, the present invention provides a method for fermenting Hwangchujang by sugar-free fermentation using the strain.

In addition, the present invention provides a fermentation broth of Hwangcholguk extract fermented according to the above method.

The Galactomyces candidum strain having acid tolerance and lacase activity of the present invention can be derived from D. fumigatus. Since the known strains of Bacillus sp. And Lactic acid bacteria were not grown in the extract of Hwangchujang, the leaves, stems or roots of the stained spots of the Hwangchunjung trunks were cleanly washed with sterilized water Next, a colony grown in a yellow agar medium was firstly selected in a colony grown on a LB agar medium, and then cultured on a minimal medium of Huangchil extract to isolate a strongly grown strain. Can be separated.

According to the present invention, all three strains (C2, C8, C27) isolated from H. grubu were Galactomyces candidum and Bacillus sp. And each showed 99% or more homology. Among the strains identified as Galactomyces sp., C27 (Den-1) strain showed the highest acid tolerance and lactase activity and showed the highest efficacy in the fermentation.

Galactomyces candidum is a strain approved by the US Food and Drug Administration (FDA) for Generally Recognized As Safe (GRAS). The European Food Safety Agency (EPSA) of Safety (QPS), which is a microbiological unit that can be added to food. Galactomyces candidum strains are used in the fermentation stage of cheese fermentation in France and elsewhere in Europe to promote aroma.

The 18s rRNA nucleotide sequence of the isolate having acid resistance and lacquer activity derived from Huaculgus chinensis isolated in the present invention was registered in GenBank of NCBI, USA (Accession Number KJ579946, SEQ ID NO: 1) Doom ( Galactomyces candidum ) C27 (Den-1), and deposited on March 24, 2015 as the deposit number KCCM11677P at the Korean Microorganism Conservation Center.

The Hwigaechuki extract may be a Hwangchu woody solvent extract obtained by extracting Huangchu tree with at least one extraction solvent selected from the group consisting of water, C1 to C4 linear or branched alcohols, and the like. The extraction solvent may be used in an amount of 10 to 30 parts by weight based on 1 part by weight of U. pergolas.

The extraction method for obtaining the extract of the present invention can be produced according to a known extraction method commonly used in the technical field of the present invention. Specifically, commonly used extraction methods such as cold precipitation method, heat extraction method, ultrasonic extraction method, filtration method, pressure extraction method, reflux extraction method, supercritical extraction method and electric extraction method can be used.

According to one embodiment of the present invention, in the present invention, an amount of 10 to 30 parts by weight (w / w) of purified water is added to 1 part by weight of a leaf, stem or root, and the mixture is heated at 90 to 130 캜 for 5 to 5 hours And then extracting it with heat to obtain an extract of Phellodendron chinensis.

The new strain of Galactomyces candidum C27 (Den-1), which has acid resistance and lacase activity, has excellent acid tolerance and is capable of growing in strong acid. As a result of measurement of laccase activity, bromophenol blue Bromophenol Blue, BPB).

Generally, the extract of Hwangchil-myeon is a strong acid with a pH of 4.0 or less, but the Galactomyces candidum C27 (Den-1) strain has excellent acid tolerance and can grow in strong acid. It can be fermented.

Accordingly, in one embodiment of the present invention, there is provided a method for fermenting a Hwangcholgak extract using the above-mentioned new Galactomyces candidum C27 (Den-1) strain.

Preferably, in the present invention, the strain is used to ferment without sugar additionally an additional saccharide.

According to the present invention, fermentation of Hwangchil-myeon extract has an effect of producing organic acids such as acetic acid, citric acid, lactic acid, succinic acid and the like, and the fermentation broth of Huangchil extract can maintain its antioxidant effect.

According to the above-mentioned method, the fermented broth of Fermented Houttuynia oryzae L. fermented with Galactomyces candidum C27 (Den-1) strain had excellent racaease activity and the uriciol, which is an allergen causing substance, was removed. Therefore, It can be processed into various foods such as beverages.

As described above, the Galactomyces candidum C27 (Den-1) strain of the present invention is excellent in acid tolerance and can grow in strong acid, and is excellent in lacase activity, so that uriciol, which is an allergen, can be removed. Therefore, it is possible to ferment the extract of Ganoderma lucidum L. of strong acid using Galactomyces candidum C27 (Den-1) strain of the present invention and to maintain the inherent antioxidative effect while eliminating allergen induction. In addition, the extract can be fermented without further sugar addition by using the above-mentioned strains, so that various adult diseases can be prevented. Accordingly, it is expected that the fermented liquid of the extract of Hwangchujang fermented by using the above strain can be usefully used for various foods such as yogurt, beverage and the like without any burden on allergy or adult diseases.

Fig. 1 is a photograph showing a strain grown by culturing in a medium of Huangchil extract.
Fig. 2 is a photograph showing the results of measurement of the activity of laccase in a strain derived from Japanese whiting tree.
FIG. 3 is a photograph of a fermentation broth of an extract of Hwangchulchuk extract obtained from a Hwangchulki-derived strain.
FIG. 4 and FIG. 5 are graphs showing the content of organic acid produced after fermentation of Hwanyeongseong extract.
6 is a graph showing a gallic acid standard curve.
FIG. 7 is a graph showing the total polyphenol content of the fermentation broth of Wortmannia sp.
8 is a graph showing a rutin standard curve.
FIG. 9 is a graph showing the total flavonoid content in the fermented broccoli extract.
FIG. 10 is a graph showing DPPH radical scavenging activity of the fermentation broth of P. forestry extract. FIG.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

<Example 1> Preparation of hot water extract

30 parts by weight of purified water was added to 1 part by weight of Huache trunk and extracted at 11 캜 for 6 hours. The hydrothermal extract of the obtained Hwanyeongsan stem showed a strong acidity at a pH of 3.8.

<Example 2> Isolation of Huangchil extract-producing strain

(Step 1) Selection of a strain

In order to isolate strains capable of fermenting Hwangchu tree extract, leaves and stem samples from injured parts of Hwangchung stem were cleaned with sterilized water, diluted with physiological saline, and colony grown on LB agar medium Colony grown on a yellow agar agar medium was firstly selected. Then, the strain was cultivated in a minimal medium of Huangchil extract, and the strains grown vigorously were secondly selected.

Fig. 1 is a photograph showing a strain grown by culturing in a medium of Huangchil extract. As shown here, Galactomyces candidum C18 and C27 (Den-1) strains were actively propagated.

(Step 2) Measurement of Laccase Enzyme Activity of Strain

The activity of laccase was determined by the resolution of Bromophenol Blue (BPB). That is, 1% of BPB was added to YM (Yeast-Malt) medium and cultured at 30 ° C for 48 hours.

The results are shown in Fig. Fig. 2 is a photograph showing the results of measurement of the activity of laccase in a strain derived from Japanese whiting tree. Herein, the first tube is Galactomyces candidum C27 (Den-1) strain, the second tube is Galactomyces candidum C18, the third tube is Jan-1, the fourth tube is A81, the fifth tube is C89, Tube # 6 is Bacillus sp. A1, and tube 7 is the control group. As a result, it was found that the tubes 1 to 4 had an excellent BPB resolution of 99% or more.

(Step 3) Identification of the strain

The isolates were purified using DNA isolation and purification kit, and the 18s rRNA gene sequence of yeast was analyzed, the 16s RNA gene sequence of bacteria was analyzed, and the homology of DNA sequence in NCBI BLAST was analyzed and compared .

As a result, Galactomyces candidum in all three yeast strains (C2, C8, C27) and Bacillus sp. And each showed 99% or more homology. The most efficacious strain of Galactomyces candidum The 18s rRNA sequence of the C27 (Den-1) strain was registered in the GenBank of NCBI, USA (Accession Number KJ579946). On March 24, 2015, it was deposited with the Korean Microorganism Preservation Center as accession number KCCM11677P.

(Step 4) Observation of acid tolerance of Galactomyces candidum C27 (Den-1) strain

Citric acid (YEC medium) or malic acid (YEM medium) as a carbon source was added to 0.02% of yeast extract to a pH of 2.8 or less to prepare Galactomyces candidum C27 (Den-1) %, And cultured at 30 ° C for 2-4 days. The results are shown in Table 1 below.

As shown in Table 1, it can be seen that the growth of Galactomyces candidum C27 (Den-1) strain is very good.

<Example 3> Fermentation of Fusarium oxysporum extract

After adding 0-30% of glucose to 1L of the Huangchil extract obtained in Example 1, 1-20% of seeds were added and cultured at 30 ° C for 2-7 days.

The results are shown in Fig. FIG. 3 is a photograph of a fermentation broth of an extract of Hwangchulchuk extract obtained from a Hwangchulki-derived strain. Here, the first tube is Galactomyces candidum C18, the second tube is Jan-1, the third tube is Galactomyces candidum C27 (Den-1) strain, the fourth tube is A81, and the fifth tube is C89.

As shown in FIG. 3, the color of the fermentation broth varied from reddish brown to golden depending on the type of microorganism. In particular, fermentation was successfully performed in tube No. 3 in which Galactomyces candidum C27 (Den-1) I could.

<Example 4> Measurement of organic acid content of fermented broccoli extract

The content of organic acids in the fermentation broth was determined by GC / MS. Solid-phase-micro extraction method was used for organic acid extraction and collection. The GC / MSD device used HP6890 / HP5973. Sample 1 was a fermentation broth of Hulchicorn extract using a strain of Galactomyces candidum C18. Sample 2 was a fermentation broth of Hulchillus japonica extract using Jan-1 strain. Sample 3 was a fermentation broth of Haulchill tree extract using Galactomyces candidum C27 (Den-1) And Sample 4 is a fermentation broth of Hwangchujang Extract using strain A81. Sample 5 is a fermentation broth of Hwachulchuk extract using C89 strain.

The results are shown in Figs. 4 and 5, and Table 2. FIG. 4 and FIG. 5 are graphs showing the content of organic acid produced after fermentation of Hwanyeongseong extract.

As shown in Table 2, it was found that acetic acid, lactic acid, succinic acid, citric acid, and the like were produced in the fermentation broth of Hwangcholguk extract using the strains of Samples 1 to 5.

Example 5 Measurement of Antioxidative Activity of Fermented Extract of Green Leaves

(1) Total polyphenol content measurement

Total polyphenol content was measured by Folin-Denis method. To 100 μL of 10 mg / mL sample solution, 900 μL of distilled water was mixed and 100 μL of Folin-Ciocalteu's phenol reagent was added and reacted at room temperature for 5 minutes. 300 uL of 20% Na2CO3 was added thereto, and the mixture was adjusted to 2 mL with distilled water. This solution was reacted at room temperature for 2 hours and then dispensed into a 96-well plate and absorbance was measured at 760 nm using a microplate reader. Standard curves were prepared using gallic acid as the reference material (reliability r ² = 0.99). The results are shown in Figures 6 and 7. FIG. 6 is a graph showing a gallic acid standard curve, and is a graph showing the total polyphenol content of a fermentation broth of Horticultural extract. Here, A is a control group, B is a fermentation broth of Hwangchujang extract using Jan-1 strain, C is a fermentation broth of Ganoderma lucidum extract using Galactomyces candidum C27 (Den-1) strain, D is a Jan- E is a fermentation broth of Hulchicorn extract obtained from strain A81, F is a fermentation broth of Hulchillus japonica extract using C89 strain, and G is a fermentation broth of Hulchillus japonica extract using C100 strain.

As a result, the total polyphenol content was 1.5 times higher than the sample solutions after fermentation in the hot water extract before fermentation. In particular, the fermentation broth (C) of the extract of Ganoderma lucidum using Galactomyces candidum C27 (Den-1) showed the highest total polyphenol content in the sample solution after fermentation.

(2) Total flavonoid content

Flavonoids are yellow in the alkaline solution, hesperidin and naringin such as flavan, or water soluble flavonol glycosides. The absorbance at this time was measured and the total flavonoid content in the sample was calculated from the standard curve.

The sample solution was diluted with distilled water to a concentration of 10 mg / mL, and then 100 μL was taken. 1 mL of diethylene glycol and 100 μL of 1N-NaOH were mixed and reacted at 37 ° C. for 1 hour in a water bath. After the reaction, the plate was dispensed into a 96-well plate and absorbance was measured at 415 nm using a microplate reader. As a standard, a standard curve was created using rutin (Rutin hydrate sigma Lot # BCBJ4086V) (reliability r ² = 0.99).

The results are shown in Figures 8 and 9. FIG. 8 is a graph showing the rutin standard curve, and FIG. 9 is a graph showing the total flavonoid content of the fermentation broth. Here, A is a control group, B is a fermentation broth of Hwangchujang extract using Jan-1 strain, C is a fermentation broth of Ganoderma lucidum extract using Galactomyces candidum C27 (Den-1) strain, D is a Jan- E is a fermentation broth of Hulchicorn extract obtained from strain A81, F is a fermentation broth of Hulchillus japonica extract using C89 strain, and G is a fermentation broth of Hulchillus japonica extract using C100 strain.

As a result, the total flavonoid content was 291.4 ug / mL in the hot water extract before fermentation, which was twice as high as the sample solutions after fermentation. Especially in the sample solution after fermentation, 140.4 ug / mL was found to be the highest in the sample solution in the fermentation broth (C) of the extract of Ganoderma lucidum using Galactomyces candidum C27 (Den-1) strain. In addition, B showed 136.6 ug / mL and D was 128.4 ug / mL. Total flavonoid contents were 120.4 ug / mL in F, 121.8 ug / mL in F and 117.4 ug / mL in G.

(3) DPPH free radical scavenging ability

The DPPH free radical scavenging activity was evaluated by diluting the prehydrolysis solution and the fermented sample solution of Hwangjak tree hydrothermal extract to the concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL and 10 mg / mL, respectively. 100 μL of the sample solution and 400 μL of 2,2-diphenyl-1-picrylhydrazyl solution were reacted in a 37 ° C. water bath for 30 minutes. After the reaction, the mixture was dispensed into a 96-well plate and reacted at 517 nm with a microplate reader. As a control, distilled water was used and the absorbance was expressed as a percentage based on the following equation (1): L-ascorbic acid 1 mg / mL was used as a control group.

In the above equation (1)

A _control is the absorbance of the reaction liquid which is reacted by adding purified water instead of the sample,

A _sample is the absorbance of the reaction solution reacted with the sample solution.

FIG. 10 is a graph showing the DPPH radical scavenging ability of the extract of P. japonicus extract. The DPPH radical scavenging ability was ascertained by concentration and the radical scavenging ability was increased in a concentration-dependent manner. D (91.3%) and F (91.6%) samples showed better antioxidant activity at 10 mg / mL compared to standard (L-ascorbic acid)

When DPPH radical scavenging activity was detected, the standard L-ascorbic acid activity was 95.7% at a concentration of 1 mg / mL. At the concentration of 10 mg / mL, the hot water extract showed 88.9% activity before fermentation and the sample solutions after fermentation had higher activity than the hot water extract before fermentation. Among them, D (91.3%) and F (91.6%) were the highest. Also, the concentration for showing the maximum radical scavenging ability was confirmed to be 5 mg / mL. Until the concentration of the sample solution was 5 mg / ml, the elimination efficiency of the sample solutions after fermentation was higher, but the decrease in the concentration of 1 mg / ml was smaller in the hydrothermal extract before fermentation. At the concentration of 0.1 mg / ml, C showed the highest elimination ability as 23%.

&Lt; Formulation Example 1 > Production of yogurt using a fermented liquid of extract

Yulchulchul fermented yogurt was fermented for 1-5 days by adding 0.1 ~ 30% of fermented fermented milk to milk.

Thus, the Galactomyces The candidateum C27 (Den-1) strain is excellent in acid tolerance and can grow in strong acid, and is excellent in lacase activity, so that uriciol, which is an allergen, can be removed. Therefore, it is possible to ferment the extract of Ganoderma lucidum L. of strong acid using Galactomyces candidum C27 (Den-1) strain of the present invention and to maintain the inherent antioxidative effect while eliminating allergen induction. In addition, the extract can be fermented without further sugar addition by using the above-mentioned strains, so that various adult diseases can be prevented. Accordingly, it is expected that the fermented liquid of the extract of Hwangchujang fermented by using the above strain can be usefully used for various foods such as yogurt, beverage and the like without any burden on allergy or adult diseases.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> A new strain having acid tolerances and a method fermenting dendropanax morbifera by using same <130> PA-15-0053 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 373 <212> DNA <213> Galactomyces candidum strain 18s rRNA <400> 1 tccgtaggtg aacctgcgga aggatcatta agaattgata atatttgtga aatttacaca 60 gcaaacaata attttataat caacacaaaa ataatcaaaa cttttaacaa tggatctctt 120 ggttctcgta tcgatgaaga acgcagcgaa acgcgatatt tcttgtgaat tgcagaagtg 180 aatcatcagt ttttgaacgc acattgcact ttggggtatc ccccaaagta tacttgtttg 240 agcgttgttt ctctcttgga attgcattgc ttttctaaaa tttcgaatca aattcgtttg 300 aaaaacaaca ctattcaacc tcagatcaag taggattacc cgctgaactt aagcatatca 360 ataagcggag gaa 373

Claims

Galactomyces candidum strain containing the nucleotide sequence of SEQ ID NO: 1 with acid tolerance and laccase activity and deposited with the deposit number KCCM 11677P.

delete

A method for fermenting a Hwangcholguli extract using the strain according to claim 1.

The method of claim 3,
&Lt; / RTI > without sugar addition.

A fermentation broth of an extract of Tochigi pallidum fermented according to the method according to claim 3 or 4.