KR102072226B1 - Preparation Method of Chitinase and Chitin Oligosaccharides from Bacillus thuringiensis - Google Patents

Abstract

The present invention relates to a method for preparing chitinase and chitin oligosaccharides. More specifically, the present invention relates to a method for preparing chitinase and chitin oligosaccharides using a Bacillus thuringiensis strain.

Description

Preparation method of chitinase and chitin oligosaccharides using Bacillus thuringiensis strains Bacillus thuringiensis

The present invention relates to a method for preparing chitinase and chitin oligosaccharide, and more particularly, to a method for producing chitinase and chitin oligosaccharide using Bacillus thuringiensis strain.

Chitin is a linear polymer polysaccharide composed of polysaccharides of N- acetyl-glucosamine. Chitin is often found in the exoskeleton of crustaceans and insects. It is also found in the cell walls of fungi and yeast. Chitin and its degradation products are known to be available in a variety of fields, such as food, pharmaceuticals, but their use is limited due to the low solubility of chitin.

Chitinases are known as enzymes that break down insoluble chitin into monosaccharides, disaccharides, and oligosaccharides, and are produced by organisms such as bacteria, fungi, insects, plants, and animals.

Bacillus thuringiensis is a bacterium of about 1 ㅧ 3μm that produces insecticidal toxins, isolated from insect pests, and widely used to control pests that cause pests or plant diseases in agriculture. It is a bacterium. Bacillus thuringiensis is currently classified into 19 serotypes by flagellar antigens. Although bacteriologically very similar to Bacillus cereus, the bacterium produces crystalline toxins (δ-endotoxins) of about 0.6 ㅧ 2μm of bi-pyramidals (such as spindles, quadrilaterals, and indeterminates) in apoptosis It is called. Crystalline toxin has strong toxicity against insects (especially black and white), but it is harmless to humans and plants, so it is mass-produced and its preparation is practically used as a microbial insecticide.

Since Bacillus thuringiensis was first separated in the early twentieth century, it has been a very diverse species. In addition to δ-endotoxins, Bacillus thuringiensis is a heat-stable β-exotoxin (Bond et al., 1969) and a heat-stable α-exotoxin known to affect cell membrane phospholipids that degrade or kill cell membranes (Krieg, 1971). Create Endotoxin crystals, which account for 20-30% of the total weight of spore formation, are produced in the form of paraaposomes in stages 3-4 of the period of sporulation (Bulla et al., 1977) and reported to be insecticidal to insects. (Angus, 1971; Paras ad et al., 1974).

Endotoxins have the form of double pyramid, double trifunctional, oblong, cubic, conical, amorphous (Grigorova et al., 1966; Bucher, 1966; Oh et al., 1985). It has a molecular weight of about 130KD, and this protein has been reported to be decomposed into polypeptides of about 68,000KD by protease secreted in the middle intestine of the twins, and then activated to destroy the epithelial tissue of the middle intestine, killing the larvae. Cooksey, 1971; Kurstak, 1982; Bulla et al., 1979). This active polypeptide is produced by a single gene and has been reported to have a gene on the plasmid DNA in this bacterium (Martin and Dean, 1981; Stahly et al., 1978; Schneph and Whitely, 1981; Gonzalez et al. , 1981).

Patent Publication No. 10-2014-0015822

The present inventors have completed the present invention by finding that including a colloidal chitin in the culture substrate when Bacillus thuringiensis is cultured can effectively induce chitinase production.

Accordingly, an object of the present invention is to provide a colloidal chitin as a substrate for the culture of Bacillus thuringiensis strains to induce more chitinase production, and to break down chitin using Bacillus thuringiensis. It is to provide an enzyme production method.

Another object of the present invention is a chitin using Bacillus thuringiensis and brown edible, which can produce a greater amount of chitin oligosaccharides when the chitinase of Bacillus thuringiensis is treated with colloidal chitin made of brown edible ( Tenebrio molitor ). It is to provide a method for producing oligosaccharides.

Another object of the present invention is a feed supplement and / or livestock containing a crystalline toxin that is potent toxicity against insects (especially the species of birch and bicot) as well as chitin oligosaccharides effective for immune immune system enhancement, but harmless to humans and plants To provide a plant disease control agent and a method for producing the same.

The object of the present invention is not limited to the above-mentioned object, and even if not explicitly mentioned, the object of the invention that can be recognized by those skilled in the art from the description of the detailed description of the invention will be naturally included. .

In order to achieve the above object of the present invention, the present invention comprises the steps of preparing a culture medium by adding colloidal chitin; Inoculating the culture with Bacillus thuringiensis strain ( Bacillus thuringiensis ); And providing a chitinase by purifying the culture solution.

In a preferred embodiment, the colloidal chitin is a brown edible ( Tenebrio molitor ) colloidal chitin or crab shell colloidal chitin.

In a preferred embodiment, the brown meal colloidal chitin is obtained by treating brown mealworm larvae.

In a preferred embodiment, the chitinase is a recombinant protein obtained by expressing the chitinase gene of the Bacillus thuringiensis strain.

In addition, the present invention comprises a chitin oligosaccharide comprising at least one of sugar, disaccharide, trisaccharide, and six sugars by treating the chitinase prepared by any one of the above-described method to colloidal chitin. It provides a method for producing chitin oligosaccharides, which is obtained.

In addition, the present invention comprises the steps of preparing a culture solution by adding colloidal chitin; It provides a method for producing livestock feed comprising a; and inoculating the culture solution Bacillus thuringiensis strain ( Bacillus thuringiensis ).

In a preferred embodiment, the colloidal chitin is brown meal colloidal chitin or crab shell colloidal chitin.

In addition, the present invention is prepared by the above-described manufacturing method provides a feed for livestock containing chitin oligosaccharides.

In addition, the present invention comprises the steps of preparing a culture solution by adding colloidal chitin; And inoculating Bacillus thuringiensis strain ( Bacillus thuringiensis ) in the culture solution and culturing.

In a preferred embodiment, the colloidal chitin is brown meal colloidal chitin or crab shell colloidal chitin.

The present invention also provides a plant bottle control agent prepared by the above-described manufacturing method comprising chitin oligosaccharides.

According to the method for producing chitinase of the present invention described above, colloidal chitin may be provided as a culture substrate in the culture of Bacillus thuringiensis strain to induce more chitinase production.

In addition, according to the method for preparing chitin oligosaccharide of the present invention, chitin oligosaccharides can be produced when the chitinase of Bacillus thuringiensis is treated with colloidal chitin made of brown rice wine ( Tenebrio molitor ).

In addition, the animal feed and / or plant disease control agent of the present invention is obtained by culturing Bacillus thuringiensis in a culture solution containing colloidal chitin, so that chitin oligosaccharides are effective for enhancing immune domains, as well as insects (especially saponaceae Contains toxic toxins, but harmless to toxins and plants.

These technical effects of the present invention are not limited to the above-mentioned range, and even if not explicitly stated, the effects of the invention that can be recognized by those skilled in the art from the description of the specific contents for the implementation of the following invention are also mentioned. Of course included.

Figure 1 is a manufacturing process showing the process of producing a brown meal colloidal chitin in the present invention.
Figure 2 is chitin lyase ( Bt chitinase and r Bt chitinase) of the Bacillus thuringiensis strain according to the present invention by using the crab shell colloidal chitin and brown meal colloidal chitin respectively as a substrate (1, 2 and 3 days) TLC analysis of the chitin oligosaccharides produced by the photograph.
Figures 3a to 3d is the chitinase ( Bt chitinase and r Bt chitinase) strains of Bacillus thuringiensis strains according to the present invention by using the crab shell colloidal chitin and brown-faced colloidal chitin respectively as a substrate (1, 2) , Day 3) is a graph showing the HPLC analysis of the chitin oligosaccharides produced.
Figure 4 is a chitinase of the Bacillus thuringiensis strains according to the present invention ( Bt chitinase and r Bt chitinase) of the shell of the chitin oligosaccharides produced by using the colloidal chitin and the brown-collar colloidal chitin as a substrate, respectively, HPLC The result is analyzed.
5 is chitinase ( Bt chitinase and r Bt chitinase) of the Bacillus thuringiensis strains according to the present invention by using the bark of the colloidal chitin and brown rice fed colloidal chitin as substrates (0.05, 0.1, 0.2, respectively) The chitin oligosaccharides produced by the unit) were analyzed by TLC.
6a to 6d are chitinases ( Bt chitinase and r Bt chitinase) of Bacillus thuringiensis strains according to the present invention, respectively. It is a graph showing the result of HPLC analysis of chitin oligosaccharides produced by stars (0.05, 0.1, 0.2 unit).

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprise" or "have" are intended to indicate that there are features, numbers, steps, actions, components, parts, or combinations thereof described in the description of the invention, one or more other It should be understood that it does not exclude in advance the possibility of the presence or addition of features or numbers, steps, actions, components, parts or combinations thereof.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and are not construed in ideal or excessively formal meanings unless expressly defined herein. Do not.

In interpreting a component, it is interpreted to include an error range even if there is no separate description. In particular, where the terms "about", "substantially" and the like are used, they may be interpreted as being used at or near that numerical value when a manufacturing and material tolerance inherent in the stated meaning is given. .

In the case of a description of a temporal relationship, for example, if the temporal after-term relationship is described as 'after', 'following', 'after', 'before', etc. This includes non-consecutive cases unless' is used.

Hereinafter, with reference to the accompanying drawings and preferred embodiments will be described in detail the technical configuration of the present invention.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

Technical features of the present invention is made in chitin-decomposing enzyme with the Bacillus tube Lindsay N-Sys strain Based that there Bacillus tube Lindsay N-Sys (Bacillus thuringiensis), incorporation of strain the colloidal chitin during culture to a culture substrate can lead to chitin-decomposing enzyme produced effectively Method and a chitin oligosaccharide manufacturing method that can produce more chitin oligosaccharides by treating the chitinase of Bacillus thuringiensis prepared by the above method to colloidal chitin made of brown rice wine ( Tenebrio molitor ).

Therefore, the method for preparing chitinase of the present invention comprises the steps of preparing a culture solution by adding colloidal chitin; Inoculating the culture with Bacillus thuringiensis strain ( Bacillus thuringiensis ); And purifying the culture solution to obtain chitinase.

Here, the colloidal chitin may be all known colloidal chitin, and in one embodiment, may be a brown edible ( Tenebrio molitor ) colloidal chitin or crab shell colloidal chitin. In particular, brown mealy colloidal chitin is obtained by treating brown mealworm larvae, which in one embodiment can be obtained by treatment as shown in FIG. 1. In some cases, the chitinase may be a recombinant protein obtained by expressing the chitinase gene of the Bacillus thuringiensis strain.

In addition, the chitin oligosaccharide manufacturing method of the present invention comprises the step of treating the chitinase prepared by any one of the above-described method to colloidal chitin, at least one of one sugar, two sugars, three sugars, six sugars A chitin oligosaccharide comprising is obtained.

In addition, the method for preparing livestock feed and plant disease control method of the present invention comprises the steps of preparing a culture solution by the addition of colloidal chitin; And inoculating Bacillus thuringiensis strain ( Bacillus thuringiensis ) in the culture solution. Here, the colloidal chitin may be all known colloidal chitin, and in one embodiment, may be brown meal colloidal chitin or crab shell colloidal chitin.

Example 1

1. Preparation of Colloidal Chitin-containing Cultures

① Crab shell colloidal chitin preparation

Crab shells are subjected to acid treatment (5% hydrochloric acid, 1 to 5 hours, room temperature) → alkali treatment (2 to 5% caustic soda, 1 to 5 hours, 90 degrees to 100 degrees) to obtain chitin derived from crab shells. At room temperature, chopped crab shell-derived chitin is added to the mortar and then a small amount of acetone is added to make it like porridge. Afterwards, slowly add 5-10 volumes of chilled concentrated hydrochloric acid to the mortar. It was poured well with a 50% ethanol solution (with a magnetic stirrer) and dispersed well. It was precipitated and again dispersed in water several times to remove acid and alcohol. Through this, crab shell colloidal chitin (CC) was prepared.

② Preparation of Crab Shell Colloidal Chitin-containing Culture Solution

0.5% of crab shell colloidal chitin prepared in culture for Bacillus thuringiensis strains (K₂HPO, (0.1g), MgSO₄7H₂O (0.05g), Yeast extract (0.05g), trace elements (0.1mL)) Crab shell colloidal chitin-containing cultures were prepared by addition to concentration.

2. Inoculation and Cultivation of Bacillus thuringiensis strains

1,000 μl of the prepared 0.5% crab shell colloidal chitin-containing culture was inoculated with 100 μL of Bacillus thuringiensis strain (KACC 10168) and incubated at 30 ° C. for 6 days.

3. Purification of Chitinase

Bt chitinase was purified through a chitin affinity column from the supernatant obtained by centrifugation of the Bacillus thuringiensis strain.

Example 2

Except for using the crab shell colloidal chitin but not the brown goose ( Tenebrio molitor ) colloidal chitin prepared in the same manner as in Example 1 to obtain a chitinase ( Bt chitinase).

Mealworm (Tenebrio molitor) colloidal chitin (Tenebrio molitor colloidal chitin: TC) is 95 degrees for 3 hours the reaction after 3 hours at 2N hydrochloric pulverized mealworm larvae, as illustrated in Figure 1 in a 5% sodium hydroxide solution The reaction obtained by reacting for a while was dried. The dried product was reacted in 3% hydrogen peroxide solution for 1 hour at room temperature to dry the reaction. The dried product was reacted with a strong hydrochloric acid solution at room temperature for 1 hour, and then 70% ethanol was added to adjust the pH to 6-7.

Example 3

To obtain a recombinant protein of chitinase of Bacillus thuringiensis strain (KACC 10168) was carried out as follows using a molecular cloning technique. The primary PCR for cloning used forward primer (5'-GGGCCATGGCTATGAGGTCTCAAAAATTC-3 ') and reverse primer (5'-GGGCTCGAGCTAGTTTTCGCTAATGACGGC-3'), and the DNA obtained from Bacillus thuringiensis strain was used. ). The enzyme used in the PCR process was used pfu polymerase (Bioneer). To clone into Escherichia coli expression vector pET28a (+) (Novagen), double digestion of PCR product and expression vector pET28a (+) was performed using two restriction enzymes, N co I and X ho I. Ligation was performed using Ligation Mix (Takara). The ligated vector was transformed into DH5α competent cells, and sequencing was performed by plasmid DNA extraction after mass culture of positive cells. After verifying that there was no error in the nucleotide sequence, the extracted plasmid DNA was transformed again into BL21 competent cells. The positive cells were cultured in large quantities until OD600 became 1, and then treated with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 hours to produce chitinase recombinant protein (r Bt chitinase).

Experimental Example 1

The activities of chitinases ( Bt chitinase and r Bt chitinase) of Bacillus thuringiensis strains (KACC 10168) obtained in Examples 1 to 3 were measured as follows.

500 μL of 0.5% crab shell colloidal chitin, 400 μL of 50 mM sodium acetate buffer (pH5), and 100 μL of chitinase were reacted at 37 ° C. at 50 rpm for 1 hour. After 1 hour, the reaction was terminated by mixing 200 μL of 1N sodium hydroxide, and centrifuged at 10,000 rpm for 5 minutes, 500 μL of the supernatant was mixed, mixed with 1 mL of Schales' reagent, and heated in boiling water for 15 minutes. After 15 minutes, the absorbance was measured at 420 nm to calculate the activity of chitinase. The unit is used as an active unit of chitinase, and means an amount of an enzyme that produces 1 μmol of GlcNAc per hour.

Experimental Example 2

As the substrates of chitinases ( Bt chitinase and r Bt chitinase) of Bacillus thuringiensis strains (KACC 10168) obtained in Examples 1 to 3, crab shell colloidal chitin and brown scale colloidal chitin were used for each day (1, 2, 3 days) The production of chitin oligosaccharides was tested as follows.

0.05 unit chitinase ( Bt chitinase and r Bt chitinase) was mixed on the colloidal chitin substrate and reacted at 37 ° C. at 50 rpm for 1, 2 or 3 days. As for the colloidal chitin substrate, the crab shell colloidal chitin and the brown meal colloidal chitin were used. The reaction solution was centrifuged at 10,000 rpm for 5 minutes, and the separated supernatant was used for TLC analysis and HPLC analysis.

TLC analysis of the reaction product was carried out on silica gel plates by n-propanol: tertiary distilled water: ammonia water (70: 30: 1, v / v) according to the method of Huang et al. (1996). It was detected by reacting with the color reagent according to the method and the results are shown in FIG. 2.

Further, chitin oligosaccharides were detected by HPLC using 70% acetonitrile (0.5 mL / min) and NH 2 P-50 column from 10 μL of the reaction product, and the results are shown in FIGS. 3A to 3D, FIG. 4, Table 1, and Table. 2 is shown.

Reaction
time (day) Substrate N -acetyl-chitooligosaccharides (ppm) GlcNAc (GlcNAc) ₂ (GlcNAc) ₃ Total 0 CC 0.0 0.0 0.0 0.0 TC 0.0 0.0 0.0 0.0 One CC 25.9 39.2 3.3 68.4 TC 35.4 88.1 0.0 123.5 2 CC 28.2 56.4 3.6 88.3 TC 42.8 101.1 0.0 143.9 3 CC 27.9 59.2 4.3 91.3 TC 43.8 112.0 0.0 155.7

Reaction
time (day) Substrate N -acetyl-chitooligosaccharides (ppm) GlcNAc (GlcNAc) ₂ (GlcNAc) ₃ Total 0 CC 0.0 0.0 0.0 0.0 TC 0.0 0.0 0.0 0.0 One CC 27.0 72.6 6.8 106.4 TC 33.1 75.0 7.6 115.7 2 CC 28.2 89.1 8.2 125.6 TC 39.6 103.0 10.2 152.8 3 CC 27.4 90.0 8.6 126.0 TC 45.5 104.7 10.5 160.7

From Figure 2, it can be seen that the main sugar produced by the chitinase produced by the Bacillus thuringiensis strain is a disaccharide [(GlcNAc) ₂ ]. In addition, the production of sugars was also confirmed when the brown-collar colloidal chitin was used as a substrate, and the amount of sugar produced was greater when the brown-collar colloidal chitin was used than when the crab shell colloidal chitin was used.

As can be seen from FIGS. 3a to 3d, the main sugar produced by the chitinase produced by the Bacillus thuringiensis strain was 2-saccharides, and when the brown-ringed colloidal chitin was used as a substrate, 112.2 ppm and 104.7 ppm, respectively, were used. Was created. When the crab shell colloidal chitin was used as a substrate, 59.2 ppm and 90.0 ppm were produced for each enzyme. In the case of using Bt chitinase, trisaccharide [(GlcNAc) ₃ ] was produced using the crab shell colloidal chitin as a substrate, and in the case of r Bt chitinase, both substrates produced trisaccharide. .

Figure 4 shows the results of day 3 of the reaction of Figs. 2 and 3a to 3d, when comparing the detection time of chitin oligosaccharide standard, GlcNAc and disaccharide were detected in all results, Bt chitinase Trisaccharides were also detected in all results except when using brown-colloid colloidal chitin. Table 1 shows the same results as FIGS. 3A and 3B and Table 2 shows the same results with FIGS. 3C and 3D.

Experimental Example 3

As the substrate of the chitinases ( Bt chitinase and r Bt chitinase) of the Bacillus thuringiensis strains obtained in Examples 1 to 3, crab shell colloidal chitin and brown-faced colloidal chitin were used, respectively (0.05, 0.1, 0.2 unit) The production of chitin oligosaccharides was tested as follows. 0.05, 0.1, 0.2 unit chitinases ( Bt chitinase and r Bt chitinase) were mixed on a colloidal chitin substrate and reacted at 37 ° C. at 50 rpm for 3 days. TLC analysis and HPLC analysis of the reaction product were carried out in the same manner as in Experimental Example 1. The TLC analysis of the reaction product is shown in FIG. 5. HPLC results of the reaction products are shown in FIGS. 6A to 6D, Table 3 and Table 4.

Chitinase activity (unit) Substrate N -acetyl-chitooligosaccharides (ppm) GlcNAc (GlcNAc) ₂ (GlcNAc) ₃ (GlcNAc) ₄ (GlcNAc) ₅ (GlcNAc) ₆ Total 0.05 CC 25.9 39.2 3.3 0.0 0.0 0.0 68.4 TC 35.4 88.1 0.0 0.0 0.0 0.0 123.5 0.1 CC 25.3 34.6 2.9 0.0 0.0 0.0 62.8 TC 39.3 126.4 0.0 0.0 0.0 0.0 165.7

Chitinase activity (unit) Substrate N -acetyl-chitooligosaccharides (ppm) GlcNAc (GlcNAc) ₂ (GlcNAc) ₃ (GlcNAc) ₄ (GlcNAc) ₅ (GlcNAc) ₆ Total 0.05 CC 27.0 72.6 6.8 0.0 0.0 0.0 106.4 TC 33.1 75.0 7.6 0.0 0.0 0.0 115.7 0.1 CC 28.3 87.4 6.2 0.0 0.0 0.0 121.9 TC 40.7 132.8 10.0 0.0 0.0 20.8 204.3 0.2 CC 27.1 62.9 0.0 0.0 0.0 0.0 90.0 TC 45.9 171.2 7.7 0.0 0.0 0.0 224.8

As can be seen from Figure 5, the main production sugar produced by the chitinase produced by Bacillus thuringiensis strains was [(GlcNAc) ₂ ] per disaccharide, the amount of disaccharides produced when using the brown mealy colloidal chitin More crab shell colloidal chitin was used. However, there was no difference according to the amount of chitinase.

As can be seen from Figure 6a to 6d, 1, 2, 3 sugars were detected when the crab shell colloidal chitin was digested using Bt chitinase. When r Bt chitinase was used, 1, 2 and 3 sugars were detected except that 0.2 units of r Bt chitinase were used. When Bt chitinase was used to break down brown colloidal chitin, sugar and disaccharide were detected. r When using Bt chitinase 1, 2, 3 have been detected per, specific enough [(GlcNAc) _6] were detected per 20.8 ppm 6 when using the r Bt chitinase of 0.1 units. The amount of chitin oligosaccharides produced was more frequently used as the substrate of the brown meal colloidal chitin. The contents of Table 3 are the same as those of FIGS. 6A and 6B, and the contents of Table 4 are the same as those of FIGS. 6C and 6D.

According to the experimental results described above, colloidal chitin is added as in the present invention in the culture of Bacillus thuringiensis strains which show strong toxicity against insects (especially, biennial and biennial) but which are harmless to humans and plants. It can be seen that the Bacillus thuringiensis strain can induce and promote chitinase production. Therefore, the culture solution obtained by culturing Bacillus thuringiensis strains in a culture solution containing colloidal chitin according to the present invention, including chitin oligosaccharides effective for enhancing the immune region, as well as crystalline toxins, boosters such as animal feed, plant disease control agents, etc. It is expected to be widely used in food and agriculture.

Although the present invention has been shown and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

Claims (11)

Preparing a culture solution by adding colloidal chitin;
Inoculating the culture with Bacillus thuringiensis strain ( Bacillus thuringiensis );
Purifying the culture solution to obtain chitinase; And
And treating the purified chitinase to colloidal chitin.
The colloidal chitin is a brown goose ( Tenebrio molitor ) colloidal chitin, chitin oligosaccharide obtained is a chitin oligosaccharide manufacturing method characterized in that it comprises a sugar and a disaccharide.
delete The method of claim 1,
The method for producing chitin oligosaccharides, characterized in that the brown goose colloidal chitin is obtained by treating the brown goose larvae.
The method of claim 1,
The chitinase is a chitinase production method characterized in that the recombinant protein obtained by expressing the chitinase gene of the Bacillus thuringiensis strain.
Producing chitinase, a recombinant protein, using the chitinase gene of the Bacillus thuringiensis strain as a template; And
And treating said chitinase to colloidal chitin.
The colloidal chitin is a brown goose ( Tenebrio molitor ) colloidal chitin, chitin oligosaccharide obtained is a chitin oligosaccharide manufacturing method, characterized in that it comprises a sugar, disaccharides, trisaccharides, and 6 sugars. delete delete delete delete delete delete

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