KR100625299B1 - Microorganism for decomposing Laminaria japonica and Undaria pinnatifida and a method for decomposing Laminaria japonica and Undaria pinnatifida using the same - Google Patents

Abstract

The present invention relates to a novel Microbacterium sp. A2203 (Accession No .: KACC 91096) strain and its enzyme having seaweed resolution such as seaweed and seaweed. Microbacterium sp. A2203 (Accession No .: KACC 91096) rapidly decomposes the skin of algae and lowers the viscosity of the degradant, thus reducing the alginic acid temperature during seaweed processing, thereby improving the diversity of seaweed processing suitability. We can plan. Also, it can be used as health functional raw material and cosmetic raw material due to low molecular weight of seaweed.

Kelp, Seaweed, Microbacterium, Seaweed, Seaweed Functional, Seaweed Degradation

Description

Microorganisms for Degrading Seaweed and Seaweed and Method for Degrading Seaweed and Seaweed Using the Same {Microorganism for decomposing Laminaria japonica and Undaria pinnatifida and a method for decomposing Laminaria japonica and Undaria pinnatifida using the same}

Figure 1 is a micro tumefaciens in (Microbacterium sp.) The crude seaweed change in static culture of A2203.

 2 is a Gram-stained micrograph of the genus A2203 of Microbacterium.

 3 is a KCCM Identification Request Form (Item A) of A2203 genus Microbacterium.

 4 is a structural diagram showing the identification of A2203 of the genus Microbacterium.

 Figure 5 is a Na-alginate decay experiment results of the enzyme A2203 derived from the microbacterium genus.

 Figure 6 is a picture of the decomposition time by the enzyme derived from A2203 genus microbacterium.

Algae production in Korea is a large-scale fishery resource, accounting for about 25% of total aquatic production, but the processing method mainly consists of simple processing such as small items, dried products, salted products, and seasoned roasted products. It is not an exaggeration to say that although algae distribution is so weak that processing is essential, the algae, a large-scale resource, is left almost untouched due to the current level of processing or the development of processing technology.

Most carbohydrates in algae are non-digestible polysaccharides that are not hydrolyzed by human digestive enzymes. They are relatively stable to acids and alkalis and are difficult to degrade without special bacterial enzymes. Digestive absorption rate in the human body is poor. Particularly, the structural polysaccharides, which are the strongest part of brown algae represented by kelp and seaweed, are the main problem in processing, mainly composed of cellulose, and the content is about 5.71 ～ 14.3%.

The cells of kelp and seaweed are composed of viscous polysaccharides such as alginic acid, fucoidan and laminaran. Alginic acid contains 10-30% D- D-mannuronic acid and L-guluronic acid are β-1,4-linked acidic polysaccharides with molecular weights ranging from 32,000 to 200,000. Fucoidan is synthesized in the intracellular Golgi apparatus and is present as intercellular tissues of all brown algae, and it is assumed that the brown algae prevents drying when exposed to the low tide.

 Until now, as a processing method to efficiently use seaweed, most of the methods of extracting and processing seaweed from useful components by hot water extraction, alkali, acid or enzyme treatment, etc., but various bioactive substances included in seaweed during this extraction process. , And deterioration and destruction simultaneously, and it is a softening method to increase the processability of kelp.It shows excellent softening effect by treating at 0.3% concentration of acetic acid for more than 1 hour and phosphate at 0.2% for 30 minutes. However, prolonged cooking process caused the loss of minerals contained in kelp seaweeds.Thus, seaweed and seaweed are excellent health food resources, but the most serious problem in processing with seaweed is the intercellular filling of cell walls due to the strength of the seaweed. Difficult to extract useful components of polysaccharides, The Department should also requires a lot of money as well as have not been properly utilized in degradation of palatability.

Extraction and decomposition techniques of useful seaweeds have been proposed at home and abroad, such as thermochemical processes such as pyrolysis, gasification, and liquefaction, and microbial processes using enzymes produced from microorganisms such as hydrolysis and fermentation. However, it has not been put to practical use because it has not achieved great effectiveness.One of the biggest reasons for this failure is that most studies on the extraction and hydrolysis of seaweed useful components have been conducted. This is because only specific components are targeted.

Therefore, in the present invention, instead of degrading the specific components as described above, the method of using microorganisms and their enzymes to decompose the whole seaweed and seaweed components and studied the method, and as a result isolate and identify strains with high degradability, obtain enzymes and decompose Through the production of a high yield of low viscosity decomposition solution through the present invention.

Seaweed, seaweed resolution was selected from the samples collected from about 200 natural places, and microorganisms with excellent activity were selected and isolated. Among them, a large amount of extracellular protein secretion was selected to separate and purify the enzymes, and then subjected to digestion experiments on wet-crushed substrates of kelp and seaweed to determine the yield and residue content to explore the commercial potential of the new strain derived from mycobacteria. Was intended.

Example 1

Sampling and Microbial Separation

In April 2003, 10 g of each sample was collected from 200 natural places such as soil, sewage, seawater, fresh water, sewage and rice straw in Yeosu, Jeollanam-do, inoculated in 90ml of sterile peptone water. After incubating the culture medium adjusted to the concentration of 10 ⁹ or more into each kelp and seaweed cut to 3cm × 4cm, and measuring the change of kelp and seaweed body after 72 hours, 33 samples were first screened and the above procedure was repeated twice. Seventeen strains showing significant constitutional changes were selected for the second time, and eleven strains with high growth after 30 hr 24hr culture in nutrient agar containing 0.5% sodium alginate were selected. Six strains with carboxymethyl cellulose (CMC) resolution and decay in alginic acid-containing media were selected.

<Example 2>

Degradation strain identification and identification

One strain having the highest resolution among each strain was isolated and named as Microbacterium sp. A2203. This strain was isolated from sewage of Ocheon Industrial Complex in Ocheon-dong, Yeosu. This was obtained from independent colonies (Nutrient agar) (Nutrient agar), stored on slope medium and lyophilized and used for identification. The biochemical properties of the degraded strains are shown in Table 1.

Microscopic observation and IMVIC test results of A2203 strain of Microbacterium spp. Strain name A2203 Colony mode yellow-short, rod Size (um) 1.53 motility + Gram staining + Oxidase test + Catalase test + MR test - VP test -

In addition, the hydrolysis and growth characteristics of the strain are shown in Table 2.

Hydrolysis and Growth Characteristics Strain name A2203 Hydrolysis Characteristics: Cellulose +                Starch -                Element -         toluene +                Casein - Alginate (Na) +        gelatin + Optimum growth temperature 25 ℃ Optimum growth pH 6.5-7 Optimal salt concentration 0.2%

Table 3 shows the carbon source capacity.

Carbon Source Utilization Results of A2203 Strains of Microbacterium Species Strain name A2203 Carbon source capacity:               cellobiose +++               xylose ++               sucrose -               fructose +               galactose ++               glucose +++               lactose -               rhamnose +               maltose ++

The microorganisms were identified by 16S rDNA sequencing by the Korea Microbiological Conservation Center (KCCM). Specifically, DNA was extracted from the strain, and the base of 16S rDNA corresponding to 16S rDNA was amplified by a conventional polymerase chain reaction method and determined by a sequencer. As a result, it was found to be Microbacterium sp., Which was named as Microbacterium sp.

Example 3

Enzyme Isolation and Purification

Seaweed and seaweed were powdered in nutrient broth (Nutrient broth) to prepare an inducer medium with a total content of 1%. After culturing in a fermenter at 25 ° C. and pH 7.0 for 30 hours, the supernatant centrifuged at 10000 rpm was recovered, and the primary cells were removed using a 0.45 um microfilter (tff method), and then the second molecular weight cut-off was performed at MW 100,000. off) and MW 100,000 or more enzyme solution was obtained and buffer exchange was performed with biomax 10 (biomax 10; Phamacia co. Ltd.) pH 4.8 buffer. SDS-PAGE electrophoresis confirmed the bands of MW 240,000, MW 320,000 and MW 420,000, which were separated and nutrient medium containing 2% carboxymethyl cellulose (CMC) and 2% containing alginate (Na-alinate). Inoculation of 0.2% of the enzyme solution into the medium revealed a significant depression.

Example 4

Kelp, the decomposition of seaweed

Wash the kelp and seaweed firstly to remove salts, soften the crude powder by wet grinding, and then add buffer solution adjusted to pH 4.8 to 28 times the weight of kelp and 30 times the weight of seaweed. After 7% of the dry weight of the substrate was added to the enzyme solution, the solution was digested at a temperature of 45 ° C. 20 hours at 100 rpm, sterilized at 100 ° C. for 20 minutes, and filtered through 100 mesh to obtain a residue and a decomposed product. The residue accounted for 5% of the weight of seaweed and 3% of seaweed on dry basis, and the degradation rate of 97% at 80mesh. The brix of cracked liquor was 6.2% and 4.7% for kelp and seaweed, respectively, which was significantly higher than the conventional manufacturing methods.

Table 4 shows the change in viscosity of kelp and seaweed decomposition process.

As the decomposition proceeds, a marked decrease in viscosity occurs, which indicates that there is an excellent effect on the decomposition of viscous polysaccharides such as alginic acid and fucoidan.

The results kelp, seaweed one resolution is excellent in micro tumefaciens (Microbacterium sp.) A2203 - March 26, 2004 was deposited with the Agricultural Korea Culture Center of Microorganisms (KACC) was given the accession number KACC 91096.

        Change in Degradation Viscosity of Seaweed and Seaweed Sample Viscosity (cp) Instrument: Brookfield LV, rpm: 30, temp: 30 ° C, period: 30second, spindle: 34 Decomposition 7529 Decomposition 10hr 2540 Decomposition 15hr 854 Decomposition 18hr 154 Decomposition 20hr 137 After sterilization 56 After filtration 24

The micro tumefaciens in selection as a result of the present invention as described above (Microbacterium sp.) A2203 is seaweed, coenzyme fraction of a resolution is excellent, and obtained therefrom to the seaweed in the viscous polysaccharides, and a crude product is because the bacteria in vitro enzyme achieving the main Enzyme production can be done inexpensively, and the decomposition process by enzymes is also possible compared to the existing production process, kelp, seaweed dry weight of 97% or more can be commercialized, and will increase the productivity, and by the decomposition of polysaccharides by enzymatic decomposition , Seaweed, etc. can be used as a functional material in food, cosmetics, pharmaceuticals.

Claims (4)

Microbacterium sp. Microorganisms (KACC 91096) that degrade kelp and seaweed. delete The method of claim 1, comprising the step of culturing the microorganism of the genus microbacterium mixed with kelp or seaweed. delete

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