KR20050096450A - Enzyme with decomposable ability of laminaria japonica ,undaria pinnatifida and its origin new microbacterium sp. a2203 - Google Patents

Abstract

The present invention is a novel Microbacterium sp. A2203 (Accession Number: KACC 91096) strains and their enzymes. Microbacterium sp. A2203 (Accession No .: KACC 91096) quickly decomposes the skin of algae and lowers the viscosity of the decomposed products, thereby making it possible to increase the diversity of algae processing aptitude by suppressing the effect of alginic acid on algae processing. Also, it can be used as health functional raw material and cosmetic raw material due to low molecular weight of seaweed.

Description

Kelp, Enzyme with decomposable ability of Laminaria japonica, undaria pinnatifida and its origin new microbacterium sp. A2203}

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. Although furnaces are very vulnerable and processing is essential, it is no exaggeration to say that algae, a large-scale resource, are left almost untouched due to the current level of processing or 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- 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. Inoculate the culture medium adjusted to the concentration of 10 ⁹ or more into each kelp and seaweed cut to 3cm × 4cm, and measure the change in kelp and seaweed after 72 hours. Seventeen strains showing significant constitutional changes were selected for the second time. Among them, carboxymethyl cellulose degrading ability and alginic acid-containing medium were tested for 11 strains with high growth after incubation at 30 ° C for 24hr in nutrient agar containing 0.5% sodium alginate. Six strains showing depression symptoms were selected.

<Example 2>

Degradation strain identification and identification

One strain having the highest resolution among each strain was isolated and referred to as A2203. This strain was isolated from sewage of Ocheon Industrial Complex in Ocheon-dong, Yeosu. Independent colonies were obtained from Nutrient agar, stored on sloped medium and lyophilized, and used for identification. The biochemical properties of the degraded strains are shown in Table 1.

A2203 strain microscopy and IMVIC test results 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 result of A2203 strain Strain name A2203 Carbon source capacity:               cellobiose +++               xylose ++               sucrose -               fructose +               galactose ++               glucose +++               lactose -               rhamnoe +               maltose ++

The microorganisms were identified by 16S rDNA Sequencing by KCCM. Specifically, DNA was extracted from the strain, and the base of 16s rDNA corresponding to 16srDNA was amplified by a conventional polymerase chain reaction method and determined by a sequencer. As a result, Microbacterium sp. It was identified as Microbacterium A2203.

Example 3

Enzyme Isolation and Purification

Seaweed and seaweed were powdered in Nutrient broth to prepare a derivatizing 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 10000rpm was recovered, and the primary cells were removed using a 0.45um microfilter (tff method), and then the secondary molecular weight cut-off was performed at MW 1000,000. A crude enzyme solution of MW 100,000 or more was obtained and buffer exchange was performed with biomax 10 (Phamacia co. Ltd) with pH 4.8 buffer. SDS-PHAGE electrophoresis confirmed that MW 240,000, MW 320,000 and MW 420,000 bands were separated and inoculated with 0.2% enzyme solution in CMC 2% nutrient medium and NA-alinate 2% medium. Found.

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.

As a result of the experiment, kelp, A2203 of Microbacterium sp. With excellent seaweed resolution was deposited on March 26, 2004 to the Korea Agricultural Microbiological Conservation Center (KACC) and was assigned accession number KACC 91096.

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

As described above, the microbacterium sp. A2203 has excellent resolution of kelp and seaweed polysaccharides and crudes, and the enzyme solution obtained therefrom is mainly composed of extracellular enzymes. In contrast, kelp and seaweed dry weight of 97% or more can be commercialized, which will increase productivity, and the decomposition of polysaccharides by enzymatic degradation, kelp, seaweed, etc. can be used as a functional material in food, cosmetics, and pharmaceutical fields.

1 is a novel microbaterium sp. It is a tangle change in political culture of A2203.

2 is a novel microbaterium sp. Gram-stained micrograph of A2203.

3 is a novel microbaterium sp. A2203 KCCM Identification Request Form (item A).

4 is a novel microbaterium sp. Identification and structure diagram of A2203.

5 is a microbaterium sp. Na-alginate decay of A2203-derived enzyme.

6 is a microbaterium sp. A2203 Degradation time by enzymes.

Claims (3)

Kelp, a new strain A2203 belonging to the genus microbacterium with seaweed resolution (Accession Number: KACC 91096) An enzyme isolated from the strain A2203 described in claim 1 A process of degrading kelp and seaweed using the enzyme of claim 2.