KR101316086B1 - Novel Microorganism Degrading agar and carrageenan - Google Patents

Abstract

The present invention relates to novel microorganisms having agar and carrageenan resolution, and more particularly, to Bacillus alcalophilus having the ability to degrade agar and carrageenan.
According to the present invention, it is possible to produce reducing sugars such as galactose and glucose from agar and carrageenan, which are difficult to process by-products generated in the red algae processing process or during the red algae extraction process, and thus have an environmental protection effect. The effect can be recycled into.

Description

 Novel Microorganism Degrading agar and carrageenan

The present invention relates to novel microorganisms having agar and carrageenan resolution, and more particularly, to Bacillus alcalophilus having the ability to degrade agar and carrageenan.

Currently, researches on new and renewable energy such as bio, hydrogen, and solar are being actively carried out to develop stable and environmentally friendly energy that can replace petroleum and coal by strengthening climate change agreement and environmental regulation around the world. As raw materials for bioenergy production, research and production are conducted in leading countries of renewable energy production such as the United States, Brazil, and Germany, mainly using grains such as sugar cane and corn, and wood-based resources as forestry and agricultural by-products. However, due to problems such as food shortage, limitation of dominant area and nutrient supply according to crop types, and economical efficiency by additional pretreatment processes such as hemicellulose and lignin other than cellulose, the limitation of biofuel production using grains and wood-based resources Is revealed.

In order to solve these problems, marine algae having a cell wall composed of many fibers and various polysaccharides have recently attracted attention as a new bioenergy source. The main producers of seaweed are Asian countries such as Korea, Japan, China, and Indonesia, and the sea area is larger than that of land. In addition, algae grow faster than wood and plant-based cellulose, and absorb carbon dioxide in the air through photosynthetic reactions, thereby reducing greenhouse gases, and simplifying the pretreatment process due to the low content of lignin in algae. Large seaweeds contain about 60% to 95% water, and more than 50% of the remaining components are made up of carbohydrates. On the other hand, seaweed (P orphyra yezoensis ) culture is actively performed among red algae in Korea and Japan, and red algae account for more than half of the algae native to Korea. In addition, it has a wider range of habitat than brown algae and green algae, and grows from shallow depths to deep water rays, so it has more species than other algae, so it is excellent in terms of supply of raw materials. It contains many sugars, which can be converted to ethanol, which has advantages in terms of energy conversion. Large algae have various types and binding forms of polysaccharides according to species. Among them, red algae are agar, carrageenan, and other xylans, viscous polysaccharides having cellulose, which are structural polysaccharides in the inner wall of cell walls, and sulfates between outer and cells. , Met, etc. In particular, agar and carrageenan are 28% and 24%, respectively, and cellulose is 3 ~ 16%.

Agar, a representative by-product of red algae processing and extraction, is not a monosaccharide polysaccharide but a mixture of neutral polysaccharide agarose (60-80%) and acid polysaccharide agalopectin (20-40%). Rhodes repeats α-1,3 bonds between agarobioses, a disaccharide in which D-galactose and 3,6-anhydro-L-galactose (AG) are β-1,4 bonds. Β-1,4 between the disaccharide and neoagarobiose, a disaccharide in which 3,6-anhydro-L-galactose (AG) binds to α-1,3. Agaropectin has the same basic structure as agarose, but contains an acidic group such as sulfuric acid group, and thus has low gelation power, unlike agarose. Carrageenan is an acidic polysaccharide in which a sulfuric acid group is partially bonded to a polymer polysaccharide combined with D-galactose and 3,6-anhydro-D-galactose (AG), but the constituent sugar is similar to agar, but different from agar. Agar is 3,6-anhydro-L-galactose (AG), whereas carrageenan is 3,6-anhydro-D-galactose (AG), and the content of sulfuric acid is 3-6%. In comparison, carrageenan is present in extremely large amounts of 20-25%. In addition, agar has a strong coagulation force, whereas carrageenan has a weak coagulation force and a strong viscosity. Therefore, using microorganisms that decompose pure agar and carrageenan isolated from soil, mud flats, intestines of seabed animals, as well as the production of galactose or glucose required for ethanol production, peat sugar, samtang sugar, Degradative metabolites such as oligosaccharides can be useful bioactive substances. To date, algae and carrageenan, which are algae polysaccharides, have been extracted from red algae through processing such as alkali, acid, or enzyme treatment, and are widely used industrially as food and cosmetic additives and health food resources. However, due to the complicated structure of red algae itself, it is difficult to use it as a substrate for biofuel production, and treatment of by-products and wastes generated in addition to the materials currently used remains a problem. As a member of the OECD, Korea is obliged to follow the 96 Protocol adopted by the London Convention on the Regulation of Waste Dumping, which cannot be ruled out as a result of the disposal of seaweeds discarded after 2012.

To date, microorganisms known to degrade agar or carrageenan by enzymatic methods include agar-decomposing microorganisms, Agarivorans albus (Agarivorans sp.) Having heat-resistant beta agarase for producing agar oligosaccharides in a high temperature solvo state. JA-1, KCCM10645P) (Korean Patent Registration No. 736,889), Thalassomonas agarivorans ( Thalassomonas sp. SL-5, KCCM 10790P) (Korean Patent Registration No. 794,593), galactose having beta agarase for producing agar oligosaccharides Saccharophagus degradans 2-40 ( Saccharophagus sp. AG21, KFCC 11416P) having both alpha agarase and beta agarase for the production of (Korean Patent Application No. 2008-0038244), and excellent agar resolution even above 40 ℃ It is possible to maintain a variety of agar oligosaccharides, which are high value-added products, and to add value industrially. Having the beta Niagara claim that can be produced at a high olrigodangreul neo agar Glaciecola mesophila AJ548479 have been reported, such as (Glaciecola sp. SL-12, KCCM 10945P) ( Korea Patent Registration No. No. 1,072,503).

As a microorganism having carrageenan resolution, Pseudomonas sp. (MTCC 5261) for the development of new media to maximize carrageenase production (Korean Patent Application No. 2008-7026331) and reports related to carrageenase production and carrageenan degradation (JP1006656, JP 2000116376) Although reported, microorganisms that decompose carrageenan have yet to be developed.

In addition, agarase- producing enzymes and agarase- producing microorganisms include Acinetobacter sp., Agarivorans sp., Alteromonas sp., Bacillus sp., Cytophaga sp., Flavobacterium flevense, Microbulbifer sp., Microbulbifer thermotolerans, Microsilla, Pseudoalteromonas sp ., Pseudoalteromonas antarctica, Pseudoalteromonas atlantica, Pseudomonas sp., Streptomyces coelicolor, Vibrio sp., Vibrio harveyi, Zobellia galactanivorans, etc., and enzymes that break the carrageenan, the microorganism producing the color group or claim Alatospora acuminata, Alatospora flagellata, Alteromonas, Anguillospora crassa, Anguillospora longissima, Articulospora tetracladia, Calcalispora hiemalis, Clavariopsis aquatica, Culicidospora gravida, Dactylella aquatica, Dendrospora erecta, Diadema setosum, Flagellospora penicillioides, Gyoerffyella gemellia thorella stella lla Lemonniera centrosphaera, Lemonnier a cornuta, Lemonniera terrestris, Margaritispora aquatica , Microbulbifer elongatus, Nectria lugdunensis, pleuropedium tricladioides, Pseudoalteromonas carrageenovora, Pseudomonas carrageenovora, Pyramidospora densa, Sigmoidea prolifera, Taeniospora gracilis, Tetrachaetum elegans, Tetracladium marchalianum, Tetracladium maxilliforme, Tetracladium setigerum, Tricellula aquatica, Tricladium splendens , Varicosporium elodeae, Vibrio sp., Volucrispora graminea, etc. (http://www.brenda-enzymes.info).

However, no microorganisms have been developed that directly decompose red algae containing agar and carrageenan, and at the same time, microorganisms that decompose agar and carrageenan together have not been reported.

Accordingly, the present inventors have made efforts to develop a method for recycling agar and carrageenan, which are by-products generated in the red algae processing process or during the red algae extraction process, and as a result, the new microorganism Bacillus alkalophilus has excellent ability to simultaneously decompose agar and carrageenan. It was confirmed that the present invention was completed.

An object of the present invention is to provide a novel microorganism having agar and carrageenan degradability and a method for decomposing red algae or hancheong and carrageenan using the new microbe.

In order to achieve the above object, the present invention provides Bacillus alcalophilus KACC91705P having agar and carrageenan resolution.

The present invention also provides a method of degrading red algae using Bacillus alcalophilus KACC91705P.

The present invention also provides a method for decomposing agar and carrageenan using Bacillus alcalophilus KACC91705P.

According to the present invention, it is possible to produce reducing sugars such as galactose and glucose from agar and carrageenan, which are difficult to process by-products generated in the red algae processing process or during the red algae extraction process, and thus have an environmental protection effect, and produce bioactive substances and bioethanol. The effect can be recycled into.

1 shows six pure strains (1) SY1, (2) SY2, (3) SY3, (4) SYR1, (5) SYR2 and (6) SYY1 in a Kim powder solid flat medium at 37 ° C. for 4 days. After incubation, the formed colony (A) and Lugol's solution (Lugol's solution) was administered to compare the appearance of forming a clear zone (B) by the decomposition of gold.
2 shows six pure strains (1) SY1, (2) SY2, (3) SY3, (4) SYR1, (5) SYR2, and (6) SYY1 in agar solid plate medium, carrageenan solid plate medium. After culturing at 37 ℃ for one day, the resulting colony (A) and Lugol's solution (Lugol's solution) administered by comparing the appearance of forming a clear zone (clear zone) by agar decomposition, carrageenan decomposition.
FIG. 3 shows the results of laver decomposition of (a) 10 g · l ⁻¹ and (b) 5 g · l ⁻¹ isolates at different concentrations of laver powder when the isolate was cultured in a 250-ml flask for 8 days. It is a graph shown [pH (▲); Cell density (●); Total reducing sugars (·)].
4 shows the results of agar and carrageenan decomposition reactions of (a) 3 g · l ⁻¹ , and (b) 1 g · l ⁻¹ isolated strains according to concentrations of agar and carrageenan when the isolate was cultured in a 250 ml flask for 8 days. Graph shown [pH (▲); Cell density (●); Total reducing sugars (·)].
FIG. 5 shows the formation of a clear zone by agar decomposition when the isolated strains were cultured at 37 ° C. for 6 days in agar solid plate medium (A) and the isolated strains for 6 days in the carrageenan solid plate medium. It is the result which shows the state (B) which forms the clear zone by carrageenan decomposition when it incubated at ° C.

The present invention relates to Bacillus alcalophilus KACC91705P having agar and carrageenan resolution in one aspect.

To date, no microorganisms that directly decompose red algae containing agar and carrageenan have been disclosed. At the same time, no microorganisms that decompose agar and carrageenan together have been reported.

Bacillus alcalophilus KACC91705P strain of the present invention is a microorganism that directly decomposes agar and carrageenan-containing red algae, and also produces agarase and carrageenase, a novel strain that decomposes not only agar as well as carrageenan, It can deal with the problems of by-products or wastes from red algae processing and extraction processes and produce biofuels and bioactive substances.

In the present invention, microorganisms capable of efficiently decomposing agar and carrageenan, which are waste algae polysaccharides generated in red algae processing and extraction processes, are separated from samples collected in various environments such as soil, seawater, and tidal flats. It was confirmed that Calophilus KACC91705P degrades agar and carrageenan. In addition, when the agar and carrageenan are decomposed by the microorganisms, it was confirmed that reducing sugars of galactose, glucose and oligosaccharides are produced as degradation products.

In another aspect, the present invention relates to a method for decomposing red algae using Bacillus alcalophilus KACC91705P.

Bacillus alkalophilus KACC91705P of the present invention can decompose red algae containing agar and carrageenan, in one embodiment, as a result of culturing Bacillus alkalophilus KACC91705P in a solid medium containing seaweed powder, As a result of confirmation, it was confirmed that seaweed forms a transparent ring.

In another aspect, the present invention also relates to a method for decomposing agar and carrageenan using Bacillus alcalophilus KACC91705P.

Bacillus alkalophilus KACC91705P of the present invention can decompose agar and carrageenan, in one embodiment, the result of culturing Bacillus alkalophilus KACC91705P in each solid medium containing agar and carrageenan, respectively, through the Lugol solution As a result, it was confirmed that agar and carrageenan are decomposed to form a transparent ring.

Bacillus alkalophilus KACC91705P of the present invention decomposes agar and carrageenan contained in red algae, and as a degradation product, reducing sugars of galactose, glucose and oligosaccharides are produced. Such reducing sugars can be used for bioethanol production, and as a result, the Bacillus alkalophilus strain of the present invention can be very useful for producing bioethanol using red algae.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Pure Separation of Agar and Carrageenan Degrading Microorganisms

Bacteria that effectively degrade agar and carrageenan were isolated from samples taken from the soil, tidal flats, and septic tanks of marine animals. ·

Samples were collected and inoculated into a 100 ml flask of lavering medium (1.5 g of laver itself, Sea water, pH 7.42) and shaken at 21 ° C., 100 rpm for 21 days, or a laver powder medium of 250-ml flask (10 g of laver powder). L ^-1 , NH ₄ Cl 1 g.l ^-1 , Tap water, pH 6.8) and incubated at 37 ° C., 180 rpm for 7 days, 1% seaweed powder medium (NH ₄ Cl 1 g, respectively) ^1-1 , Tap water, pH 6.8) was added to a solid medium to which 1.5% agar was added. Each of the cultured microorganisms was continuously plated on a new solid medium until pure colonies were separated, and each strain was finally purified. Six strains forming pure colonies were identified, and the isolated six strains were named SY1, SY2, SY3, SYR1, SYR2 and SYY1, respectively. The isolated pure strains were subcultured every 2 weeks with storage at 4 ℃.

In order to screen useful microorganisms in the isolated pure strains, the indicator Bromocresol Purple (0.5 g ·) in a laver powder medium (steam powder 10 g · l ⁻¹ , NH ₄ Cl 1 g · l ⁻¹ , Tap water, pH 6.8) ^1-1 ) and the pure strain was plated in a solid medium to which 1.5% agar was added.

As a result, among the six strains, strain SYR1 showing a change in medium color according to pH change was confirmed in the laver powder agar medium to which the indicator was added.

Example 2: Screening of Agar and Carrageenan Degrading Microorganisms

In order to confirm the decomposition of seaweed out of the six pure strains isolated in Example 1 was plated in a solid powder medium laver and incubated for 1 to 2 days at 37 ℃, each strain 5 ml seaweed powder liquid medium in a 10 ml tube It was inoculated in the shaking culture for 1 to 2 days at 37 ℃, 180 rpm, 10 μl of each strain culture solution was dropped into a laver powder solid plate medium and dried, and then cultured at 37 ℃ for 4 days. After confirming colonies on the medium formed by each strain, 10 ml of Lugol's solution (Lugol's solution) (KI 10 g · l ^-1 , I 5 g · l ^-1 ) was applied to the laver powder plate medium for 5 minutes. Then, the solution was discarded, and then the resolution of the seaweed was examined by checking the clear zone by the seaweed decomposition.

As a result, it was confirmed that, among the six strains isolated, the strain showing the highest resolution in the laver powder solid plate medium was SYR1 (FIG. 1).

In order to confirm the degradation of agar and carrageenan of the SYR1 strain among the six pure strains isolated based on the above results, plated in a laver powder medium and incubated for 1 to 2 days at 37 ℃, each strain was a 10 ml tube After inoculation into 5 ml laver powder liquid medium in shaking culture for 1 to 2 days at 37 ℃, 180 rpm, 10 μl of each strain culture solution was dropped into agar solid plate medium and carrageenan solid plate medium and dried. , And incubated at 37 ℃ for 4 days. After confirming colonies on the medium formed by each strain, 10 ml of Lugol solution was administered to agar and carrageenan plate medium and stained for 5 minutes, then discarded. Then, the clear zone was confirmed by agar and carrageenan decomposition. The resolution of agar and carrageenan was tested.

As a result, it was confirmed that the strain showing the highest resolution in the agar and carrageenan solid flat medium among the six strains isolated (S2).

Example 3 Identification of Agar and Carrageenan Degrading Microorganisms

SYR1 strains showing the highest agar and carrageenan resolution in Example 2 were identified primarily through Gram staining, catalase test, colony morphology and microscopic examination.

The SYR1 strain has various characteristics depending on the type and condition of the medium. It is a gram-positive bacillus with active motility in nutrient solid media, 0.5 to 1.2 μm wide and 2 to 4 μm long, with optimal degradation capacity between pH 6.8 and pH 8. Orange colonies, pink colonies and white colonies were formed when cultured in 0.8% nutrient solid medium, 1% laver powder solid medium, 1% agar solid medium, and 1% carrageenan solid medium at 37 ° C. for 2 days, and catalase test. The results indicated that it was positive.

badge Kim Nutrition medium
(Nutrient broth) Agar Carrageenan Badge status solid Liquid solid solid Liquid solid Liquid Area (㎛) 0.5-1.2 0.5-1.2 1-1.2 0.8-1 0.8-1 0.8-1 0.8-1 Length (㎛) 3-4 2-4 2-4 2 2-3 2-3 2-3 Spore form Oval Oval Oval Oval Oval Oval Oval Spore location end end end Close to the center
or
end Close to the center
or
end Close to center Close to the center
or
end motility + + + - + - + Colony color Pale pink - Pale orange White - White - Gram stain + Catalase +

Secondary identification was performed by analyzing 16S-rRNA sequences. The chromosomal DNA of each strain was isolated using AccuPrep chromosome DNA extraction kit (Bionia, Korea), and PCR was performed using the following primers with the isolated DNA as a template. The 16SrRNA gene of 1500 bp fragment was amplified.

518F primer: 5'-CCAGCAGCCGCGGTAATACG-3 '(SEQ ID NO: 1)

800R primer: 5'-TACCAGGGTATCTAATCC-3 '(SEQ ID NO: 2)

After requesting 16SrRNA sequencing analysis (Macrogen, Korea) for PCR product of DNA, the result of 16SrRNA partial sequence determined by macrogen analysis was shown in SEQ ID NO: 3 of the 16S-rRNA sequence of the SYR1 strain.

The results were analyzed using GenBank's Advanced BLAST (http://www.ncbi.nlm.nihm.nih.gov) using the BioEdit Sequence Alignment Editor (ver. 5.0.9, Hall, T., Nucleic Acids Symposium Series , 41: 95, gov) (Altschul et al ., Nucleic Acids Res. , 25: 3389, 1997). As a result, the SYR1 strain was identified as Bacillus alcalophilus species.

microbe 16S-rDNA
size GenBank
Accession No. Strain name Homology SYR1 1560 bp EU231621.1 Bcillus alcalophilus 97%

The SYR1 strain was deposited with the accession number KACC 91705P at the National Institute of Agricultural Science, January 20, 2012.

Example 4 Decomposition of SYR1 by Concentration of Seaweed Powder

In this example, the SYR1 strain decomposed laver at different laver powder concentrations of 5 and 10 g · l ⁻¹ to confirm the possibility of producing useful substances.

SYR1 strain to be used in the Example was inoculated in 1% laver powder liquid medium and cultured for 1 to 2 days at 37 ℃, 180 rpm, was used when the logarithmic growth phase is over.

Cultivation was performed using a 250 ml flask, inoculated with 2% of the SYR1 strain in 50 ml liquid medium containing seaweed powder at different concentrations, and performing a decomposition reaction for 8 days at 30 ° C. and 150 rpm.

During the biodegradation reaction as described above, samples in the flask were taken at two-day intervals to analyze the properties of the degradation reaction. The change in pH in the culture medium during the degradation reaction was measured by using a pH meter (ISTECH, Korea) and adjusted to pH 7 and pH 8 using sterilized NaOH and HCl at 3 day intervals. The concentration of the microorganism was measured by dividing the sample diluted three times in a cuvette, and then using a spectrophotometer (Hanbit Nano Biotech, Korea) at a specific wavelength (400 nm). The total reducing sugar concentration produced in the decomposition reaction was uniformly mixed with 100 μl of the supernatant from which the culture was centrifuged (7000 rpm, 10 minutes) and 1 ml DNS (3.5-dinitrosalicylic acid) solution using vortex. After reacting for 10 minutes in boiling water, and then cooled in cold water for 5 minutes, the value was measured at 570 nm using a spectrophotometer.

In the results of the decomposition test for each seaweed powder concentration, pH values of 7.8 at the initial culture at 5 g · l ⁻¹ and 10 g · l ⁻¹ seaweed powder concentrations were 8.26 and 8.19 after 8 days of final culture, respectively, and the density of microorganisms Was the highest when the culture was 6, and the total reducing sugars produced were 0.76 g · l ⁻¹ and 1.8 g · l ⁻¹ , respectively (FIG. 3).

The results of decomposition of seaweed powder showed that better decomposition reaction occurred at a seaweed powder concentration of 10 g · l ^-1 , which was higher than the low concentration.

Example 5 Decomposition of SYR1 with Agar and Carrageenan Concentrations

In this example, the SYR1 strain decomposes agar and carrageenan at different agar and carrageenan concentrations of 1 and 3 gl ⁻¹ to confirm the possibility of producing useful substances.

SYR1 strain to be used in the experiment was incubated for 1 to 2 days at 37 ℃ in 1% seaweed powder solid medium, was used when the logarithmic growth phase is over.

50 ml agar liquid medium at different concentrations (Agar, NaNO ₃ 2 g · l ⁻¹ , K ₂ HPO ₄ 0.5 g · l ⁻¹ , MgSO ₄ 7H ₂ O 0.5 g · l ⁻¹ , KCl 0.5 g · l ⁻¹ , CaCl ₂ 2H ₂ O 0.1 g · l ^-1 , FeSO ₄ 7H ₂ O 0.02 g · l ^-1 , Yeast extract 0.5 g · l ^-1 , Tap water, pH 7.8) and 50 ml Carrageenan liquid medium (Carrageenan, NH ₄ Cl 1 g · l ⁻¹ , K ₂ HPO ₄ 0.3 g · l ⁻¹ , KH ₂ PO ₄ 0.1 g · l ⁻¹ , Yeast extract 0.4 g · l ⁻¹ , Tap water, Using a 250 ml flask containing pH 7.8), the SYR1 strain was inoculated and incubated at 30 ° C. and 150 rpm for 8 days, and the experiment was performed using the same analysis method as in Example 4.

As a result of the decomposition test for each agar concentration, pH values of 7.8 at the initial stage of culture at 1 g · l ⁻¹ and 3 g · l ⁻¹ agar concentrations were 7.93 and 7.77, respectively, after 8 days of final culture, and the density of microorganisms was cultured. After 2 days, almost constant concentration was maintained and the highest value was obtained at 8 and 4 days of culture, respectively. The total reducing sugars produced were 0.24 g · l ⁻¹ and 0.41 g · l ⁻¹ , respectively (FIG. 4).

Decomposition results of each carrageenan concentration showed that the pH value of 7.8 at the initial stage of culture at 1 g · l ⁻¹ and 3 g · l ⁻¹ carrageenan concentrations was 7.72 after 8 days of final culture, and the density of microorganisms after 2 days of culture. Almost constant concentration was maintained and the highest value was observed at 8 days and 2 days of culture, respectively. The total reducing sugars produced were 0.21 g · l ⁻¹ and 0.38 g · l ⁻¹ , respectively (FIG. 4).

Decomposition experiments for each concentration of agar and carrageenan showed better degradation at 3 g · l ^-1 agar and carrageenan concentrations than at lower concentrations.

Example 6 Determination of Agar and Carrageenan Degradability

In Example 2, it was confirmed that the isolated strain SYR1 has the ability to degrade carrageenan, which is a representative polysaccharide of red algae, in addition to agar.

In this example, it was confirmed that the isolated SYR1 strain has the ability to decompose agar and carrageenan and observed over time.

Agar medium (Agar 10 g · l ⁻¹ , NaNO ₃ 2 g · l ⁻¹ , K ₂ HPO ₄ 0.5 g · l ⁻¹ , MgSO ₄ 7H ₂ O 0.5 g · l ⁻¹ , KCl 0.5 g · l ⁻¹ , CaCl ₂ 2H ₂ O 0.1 g · l ^-1 , FeSO ₄ 7H ₂ O 0.02 g · l ^-1 , Tap water) and carrageenan medium (Carrageenan 10 g · l ^-1 , NH ₄ Cl 20 g of SYR1 strain culture medium cultured in a laver powder liquid medium (1%) in the center of 1 g · l ⁻¹ , Tap water) was dropped and dried, and then cultured in a 37 ° C. incubator. After the SYR1 strain forms colonies on the medium, 10 ml of Lugol's solution (KI 10 gl ^-1 , I 5 gl ^-1 ) was stained for 5 minutes after agar plate agar, and then discarded. The formation of orange rings around colonies on agar plate media stained with and the change of the ring size over time were observed. Carrageenan solid flat media consisting of agar-like components were also stained with 10 ml of Lugol solution (KI 10 g · l ^-1 , I 5 g · l ^-1 ) for 5 minutes, then discarded. Due to the presence of yellow rings around the colonies on the carrageenan plate medium stained with brown color and the change of the ring size with time (Saraswathi, S. et al., African Journal of Microbiology Research . 5: 2960, 2011).

As a result of the experiment, the SYR1 strain was formed over 2 days, 4 days, and 6 days after the colonies cultured in agar plate medium with orange rings of 0.8 cm, 1.15 cm, and 1.6 cm in radius. It was confirmed that the size of the ring gradually increased by the decomposition of agar, and the radius of the ring was 1 cm due to the decomposition of the carrageenan of the SYR1 strain as the yellow ring around the colonies passed over 2 days, 4 days, and 6 days in the carrageenan plate medium. , 1.45 cm, 1.9 cm was found to gradually increase (Fig. 5).

It was confirmed that the purely isolated SYR1 strain has the ability to degrade carrageenan other than agar among polysaccharides contained in red algae.

Example 7 Determination of Agar and Carrageenan Degradation Capacity of Supernatant of Seaweed Culture

In this example, the supernatant of the isolated SYR1 strain culture solution was confirmed whether the ability to decompose agar and carrageenan.

After centrifugation (7000 rpm, 10 minutes) of 20 ml of the supernatant of the SYR1 strain cultured in a laver powder liquid medium (1%) in the center of agar medium and carrageenan medium for 2 days, drop and dry 3 Placed in a 37 ° C. incubator for one day. After 3 days, the experiment was carried out using the same degradation capacity measurement method as in Example 6.

As a result of the experiment, the clear zone was not formed by decomposition of the agar and carrageenan of the supernatant.

As a result, it was confirmed that the purely isolated SYR1 strain had intracellular agar and carrageenan degrading enzyme.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Agricultural Biotechnology Research Institute KACC91705P 20120207

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Claims (3)

Bacillus alkalophilus with agar and carrageenan resolution KACC91705P.
Method for digesting red algae using Bacillus alcalophilus KACC91705P.
Decomposition of agar and carrageenan using Bacillus alcalophilus KACC91705P.

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