KR20200027482A - Novel chitinase-producing Salinivibrio sp. Kostichola BAO1801 strain and use thereof - Google Patents

Abstract

The present invention relates to a strain that produces a chitinase. More particularly, the present invention relates to a novel Salinivibrio sp. strain isolated from salted shrimp, a chitinase having heat stability and salt tolerance obtained from the strain, and a use thereof.

Description

S. coliola BAO1801 strain and use thereof {Novel chitinase-producing Salinivibrio sp. Kostichola BAO1801 strain and use thereof}

The present invention relates to a strain that produces a kinase, more specifically, a new strain of the genus Salinibibri isolated from salt shrimp, a kinase having heat stability and salt resistance obtained from the strain, and various uses thereof will be.

Salted fish is a traditional Korean salted fermented food used as a food additive and food. It is mixed with 10-30% (w / w) salt and stored at 15-25 ℃ for several months. Various fish, roe, shellfish and fishery , Oyster and shrimp (Guan et al. 2011). Of the various varieties, salted anchovies and small shrimps contain the highest levels of valuable oligosaccharides due to digestion of the scale and shell by chitinase produced by bacteria during fermentation (Adrangi and Faramarzi 2013; Chandrasekaran 2015). . Salted and fermented shrimp are rich in peptides, free amino acids, glucose and vitamins to protect digestive activity and antioxidant and angiotensin converting enzyme (ACE) inhibitors in the liver. These increased nutrients are due to the hydrolysis of complex compounds of tissue to oligomers or monomers by microorganisms during fermentation (Goo and Park 2014).

Chitinase (chitinase) is an enzyme involved in the decomposition and development of the insect exoskeleton, and is particularly essential for the rear generation of insects that cause molting and metamorphosis, and is also involved in the proliferation of various bacteria and fungi. The chitin component constituting the living body is a monosaccharide unit, glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), which form a glycosidic bond (glycosidic bond), and since individual chains are connected by hydrogen bonds, their physical strength is very high. It is a substance that is difficult to decompose because of its high level. Due to these physicochemical properties, chitin is present in the exoskeleton of arthropods (extracellular parasites, insects, crustaceans, etc.) and cell walls such as algae, fungi, yeast, etc. do. Kitinase is a major enzyme that primarily decomposes chitin, and converts chitin polymer into chitin-oligosaccharide, which enables the decomposition of chitin into GlcN and GlcNAc.

Recently, chitinase has attracted attention for its various functions and applications in various fields (Patel and Goyal 2017). Chitinase has been used to produce bioactive chitooligosaccharides with enormous drug potential. Some bioactive chitooligo saccharides have anti-inflammatory, antihypertensive, anti-tumor, antibacterial and antioxidant effects (Aloise et al., 1996; Park et al., 2000; Wang et al., 2006; Wang et al. Functional properties of chitooligosaccharides are strongly related to their molecular weights (Goo and Park 2014).

In order to obtain a chitin-degrading derivative, chitinase having good chitin-decomposing activity in various microorganisms and having substrate specificity is being developed. Most bacterial chitinase were produced in Serratia, Chromobacterium, Klebsiella, Bacillus and Streptomyces gena (Adrangi and Faramarzi 2013). Screening bacteria with high chitinase activity that converts chitin to COS has great potential to enhance kinase for food industry and medical applications (Shehata et al., 2018).

Korean Registered Patent No. 10-1658761

The present inventors have completed the present invention by isolating a new strain of Salinivibrio sp. That produces chitinase having excellent thermal stability.

Accordingly, it is an object of the present invention to provide a new strain of Salinivibrio sp. That produces a kinase having properties that are advantageous for the food industry and medical applications.

Another object of the present invention is produced by the Salinivibrio sp. Salinivibrio sp. Decomposes chitin at the temperature, pH and salinity of an area outside the active range, as well as the temperature, pH and salinity at which a typical chitinase has activity. It is to provide a kinase having a characteristic that can be done and a method for manufacturing the same.

Another object of the present invention is to provide a fermented food having an increased chito-oligosaccharide content and excellent antioxidant activity by including a food additive and its additives, including Salinivibrio sp.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention described above, the present invention provides a Salinivibrio costicola BAO1801 strain (Accession No: KACC 81071 BP) that produces a kinase.

In a preferred embodiment, the strain is cultured in NaCl 10-30% (w / v) medium.

In a preferred embodiment, the kinase has activity in a temperature range of 30 ° C to 80 ° C.

In a preferred embodiment, the kinase has activity at pH 2-9.

The present invention is also produced from the Salinivibrio costicola BAO1801 strain (accession number: KACC 81071 BP) to provide a kitase that has heat stability and flame resistance.

In a preferred embodiment, the chitinase is active at 10-30% (w / v) NaCl.

In a preferred embodiment, the kinase has activity in a temperature range of 30 ° C to 80 ° C.

The present invention is also a Salinivibrio genus Costiola ( Salinivibrio costicola ) BAO1801 strain (accession number: KACC 81071 BP) culturing in a medium containing chitin; a method for producing a kinase comprising the kinase; Provides a method for producing a kinase, characterized in that it has flame resistance and heat stability.

In addition, the present invention provides a food additive comprising any one of the aforementioned Salinivibrio costicola BAO1801 strains (Accession No: KACC 81071 BP) or cultures thereof.

In addition, the present invention provides a fermented food product comprising the food additive described above.

In a preferred embodiment, the fermented food is fermented seafood containing chitin, and the seafood is one or more selected from the group consisting of shrimp, crab, and crayfish.

In addition, the present invention is any one of the above-mentioned Salinivibrio costcoola ( Salinivibrio costicola ) BAO1801 strain (accession number: KACC 81071 BP), the culture of the strain, any one of the above described kinase and the method described above It provides an eco-friendly microbial agent containing at least one of the obtained kinase.

In a preferred embodiment, the eco-friendly microbial agent inhibits the mycelial growth of fungal plant pathogens by decomposing the mycelium of plant pathogens that make up chitin as a cell wall.

In a preferred embodiment, the fungal phytopathogen is one or more of Rhizoctonia solani , Aspergillus niger , Fusarium oxysporum .

In addition, the present invention provides a plant disease control method comprising the step of treating the above-mentioned environmentally friendly microbial agent to the soil, plants or seeds of plants.

In a preferred embodiment, the plant disease is caused by fungal plant pathogens that make up chitin as a cell wall.

In addition, the present invention provides a food preservative comprising at least one of the aforementioned kinase and the kinase obtained by the method described above.

First, according to the present invention, it is possible to provide a new strain of Salinivibrio sp. That produces a kinase having properties that are advantageous for the food industry and medical applications.

In addition, the kitinase of the present invention is produced by a new strain of Salinivibrio sp., And not only at the temperature, pH and salinity at which the general chitinase has activity, but also at the temperature, pH and salinity of the region outside the active range. Since it exhibits excellent properties capable of decomposing chitin, it can be widely applied to the food industry and the medical industry.

In addition, according to the present invention, as well as to provide a food additive containing Salinivibrio sp. ( Salinivibrio sp.) New strains or cultures thereof, by providing the additive, the content of chito-oligosaccharides is increased and a fermented food with excellent antioxidant activity is provided As it can, it has the potential as a starter culture in the food industry, especially in fermenting seafood containing chitin.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a phylogenetic diagram of related species by 16S rRNA gene sequence analyzed in a strain of Salinivibrio costicola BAO1801 , a new strain according to the present invention.
Figure 2 is a graph showing whether or not the production of kinase according to the culture medium of the Salinivibrio cost of Cola ( Salinivibrio costicola ) BAO1801 strain according to the present invention.
Figure 3 is a photograph of the results of SDS-PAGE analysis of the purified kinase from the Salinivibrio costicola BAO1801 strain according to the present invention. Here, Lanes M is a labeled protein, lane 1 is a crude extract, and lane 2 is a purified kinase after DEAE chromatography.
Figure 4 is a graph showing the effect of temperature and pH on the activity and stability of the kinase produced in the strain Salinivibrio costicola BAO1801 according to the present invention.
Figure 5a and 5b is a result of analyzing the substrate decomposition pattern of the kinase produced in the strain Salinivibrio costicola BAO1801 according to the present invention by TLC TLC.
6 is a graph showing the results of HPLC analysis showing the concentration of hydrolysis products when the substrate of the kinase produced in the strain Salinivibrio costicola BAO1801 according to the present invention is 2% colloidal chitin.
7 is a graph showing the effect of inhibiting spore germination of fungal plant pathogens of chitinase produced in the Salinivibrio costicola BAO1801 strain according to the present invention.
8 is a graph showing the antioxidant activity test results of 80% ethanol extract of salt and fermented shrimp treated with the Salinivibrio costicola BAO1801 strain according to the present invention.

The terminology used in the present invention is selected from the general terms that are currently widely used, but in certain cases, there are also terms that are arbitrarily selected by the applicant. In this case, the meanings described or used in the detailed description of the invention are not considered as simple term names. Therefore, the meaning should be grasped.

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

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numerals used to describe the present invention throughout the specification indicate the same components.

The technical features of the present invention are advantageous in the food industry and the medical industry, etc., because the general chitinase has activity capable of decomposing chitin at the temperature, pH and salinity of an area outside the active range, as well as temperature, pH and salinity of the activity. Salinivibrio sp., Which produces a kinase having properties, is to develop a new strain and various uses thereof.

Accordingly, the present invention provides a Salinivibrio costicola BAO1801 strain (accession number: KACC 81071 BP) that produces chitinase.

et al., 2017)에서 분리된다. 본 발명의 살리니비브리오 속 코스티콜라(Salinivibrio costicola) BAO1801 균주는 염장 및 발효된 새우에서 추출한 것이다.Salrini Vibrio genus of bacteria (family Vibrionaceae, class Gammaproteobacteria) are currently three kinds of species as thermophilic bacteria (Lopez-Hermoso et al., 2017) has been Salinivibrio paper in June, including S. costicola with. These bacteria are commonly salted foods and heavy environments (L

et al., 2017). The Salinivibrio costicola BAO1801 strain of the present invention is extracted from salted and fermented shrimp.

In general, the kinase is Bacillus sp. That grows well in an environment of 37 ℃ ~ 40 ℃. And reported in Serratia marcescens, etc., chitinase activity is mainly composed of chitinase that activity is strongly exhibited at 37 ℃ ~ 40 ℃, Salinivirbrio sp. Of the present invention ( Salinivirbrio sp.) By the coscola BAO1801 strain The produced kinase has excellent properties in which activity is strongly exhibited even in a temperature range of 30 ° C to 80 ° C, a pH range of pH2-9, and a salinity of 10 to 30% NaCl.

In addition, the method of producing the kinase of the present invention includes a culturing step of culturing the Salinivibrio costicola BAO1801 strain (accession number: KACC 81071 BP) in a medium; the medium used in the culturing step is Chitin may be contained, but may be contained. If necessary, the culture step may be performed in a medium having a salinity of 10 to 30% (w / v) NaCl. To obtain only the kinase, the step of purifying the kinase from the culture obtained in the culture step may be further included. That is, the culture obtained in the culturing step includes Salinivibrio costicola BAO1801 strain (Accession No: KACC 81071 BP), the kinase produced by the strain, and the chitin decomposition product decomposed by the kinase. Because it is included.

As described above, the chitinase produced by the Salinivibrio costicola BAO1801 strain (Accession No .: KACC 81071 BP) of the present invention has a temperature range of 30 ° C to 80 ° C, a pH range of 2-9, and Since chitin decomposes even at a salinity of 10 to 30% of NaCl to produce a chitin decomposition useful product such as chitin oligosaccharide, it is possible to decompose chitin of a polymer to a low molecule under a fairly wide range of temperature and pH conditions. That is, since the chitin derived from mushroom cell wall, chitin derived from yeast cell wall, chitin derived from fungal cell wall, chitin derived from insect exoskeleton, and chitin derived from crustacean exoskeleton, the kinase of the present invention includes the fields of application of commonly reported chitin oligosaccharides. It can be used in various fields such as food, medical, and eco-friendly microbial products.

Particularly, when the food additive containing Salinivirbrio sp. Of the present invention ( Salinivirbrio sp.), Or B.C. coli BAO1801 strain (accession number: KACC 81071 BP) or a culture thereof is utilized, the content of chito-oligosaccharides is increased and the antioxidant activity is excellent. Since it can provide fermented food, it has the potential as a starter culture in the food industry, especially in fermenting seafood containing chitin. Here, the seafood is not limited as long as the body tissue contains chitin, but may be one or more selected from the group consisting of shrimp, crab, and crayfish.

In addition, the Salinivirbrio sp.Coscola BAO1801 strain (accession number: KACC 81071 BP), an eco-friendly material that inhibits the growth of fungal plant pathogens when one or more of its culture and chitinase is irrigated to the soil As a microbial agent, excellent efficacy can be expected.

Therefore, the eco-friendly microbial agent of the present invention is a plant pathogen that comprises chitin as a cell wall, for example, Rhizoctonia solani , Aspergillus niger , Fusarium oxysporum , and bo Since the mycelial growth of fungal plant pathogens including Botrytis cinerea , Alternaria brassicicola , etc. can be suppressed, the eco-friendly microbial agent of the present invention can be used as a seed for soil, plants or plant seeds. Plant disease caused by the fungal phytopathogens constituting chitin as a cell wall can be effectively controlled, including the step of treating the cells.

Example 1. Isolation and identification of the new Salinivibrio genus Costiola BAO1801 strain

The Salinivibrio genus Costicoola BAO1801 strain of the present invention was isolated through the following process.

1.Material

Salted and fermented shrimp were obtained from Greenmin Food co located in Yeosu, Korea. Colloidal chitin was prepared from shrimp shell chitin (Sigma, USA). Briefly, 10 g of chitin powder was added to 100 ml of 10 N HCl to mix and incubated overnight at 4 ° C. After incubation, the mixture was added to 50% ethanol to precipitate colloidal chitin. The mixture was then centrifuged at 8000 x g for 15 min at 4 ° C. The colloidal chitin precipitate was then neutralized with distilled water to pH 7.0. It was then freeze dried to make a powder and stored at 4 ° C. Marine media was purchased from Difco (Detroit, MI, USA). N-acetyl glucosamine and N-acetyl chito oligosaccharides were purchased from Cayman (Ann Arbor, MI, USA). The PageRuler prestained protein ladder was purchased from Thermo Fisher Scientific (Schwerte, Germany). Other chemicals used in this study were purchased from Sigma (St. Louis, MO, USA).

2. Isolation of strains

Microorganisms showing a clearing zone were selected from marine medium supplied with 0.5% colloidal chitin to salted and fermented shrimp, and the chitin decomposition activity was measured. The strain showing the highest kinase activity was isolated and designated BAO-1801. Strain BAO-1801 is a gram-positive, rod-shaped and spore-free bacterium.

3. Identification of strains through 16s-rRNA sequencing

Strain BAO-1801 was identified as Salinivibrio sp. Based on 16S rRNA sequence analysis as shown in FIG. 1. Figure 1 is a phylogenetic tree (A phylogenetic tree) built based on 16S rRNA sequence with related species using neighbor-joining, strain BAO-1801 is Salinivibrio sp strain YM-12 (KX099322) as shown in Figure 1 And highest sequence similarity. The 16S rRNA sequence of strain BAO-1801 was deposited with GenBank under accession number KX990293, and the Salinivibrio costicola BAO1801 strain was sent to the National Academy of Agricultural Science Microbial Bank (Agricultural Biotechnology Research Institute, KACC) in May 2018. It was deposited on the 4th and was given the accession number of KACC 81071 BP.

Example 2. Kitinase preparation

1. Kitinase production

For the production of kinase, Salinivibrio sp. BAO-1801 was grown in marine medium with 0.5% colloidal chitin in a 37 ° C. shake incubator for 24 hours until OD600 reached 0.5. Then incubated overnight (1%) in 100 mL of the same medium and grown at 37 ° C. for 3 days at 200 rpm in an orbital shaker / incubator. Subsequently, the culture medium was centrifuged at 12,000 g for 20 minutes, and then the kinase was collected.

2.Kitinase purification

The cell-free culture was precipitated with saturated (80%) ammonium sulfate. The precipitate was collected after centrifugation at 6198 xg for 30 minutes. This was dissolved in 50 mM sodium phosphate buffer (pH 7), and dialyzed against the same buffer with continuous stirring at 4 ° C. for 24 hours. The dialysate was then loaded onto a DEAE-Sepharose CL-6B anion chromatography column (5 cm x 30 cm) equilibrated with 50 mM sodium phosphate buffer (pH 7), washed with the buffer, and 0-1 M NaCl Elution was with the containing buffer. Kitinase was then eluted in 4 ml fractions. Protein content was determined by Bradford assay (Thermo Fisher Scientific, Rockford, IL, USA).

Example 3. Kitinase preparation

The kinase was prepared by performing the same method as in Example 2, except that the marine medium without 0.5% colloidal chitin was used.

Experimental Example 1.

As in Example 2 and Example 3, kinase production was measured using a strain BAO-1801 grown in a medium with or without colloidal chitin, and the results are shown in FIG. 2. As shown in Figure 2, the growth of strain BAO-1801 was the same in both media (with or without colloidal chitin). Even in the absence of colloidal chitin, chitinase activity was detected in the medium. However, the addition of 0.5% colloidal chitin significantly increased the production of chitin degrading enzyme at 12 hours, which was maintained at 16 U / ml during the fixed growth phase.

Experimental Example 2

The purification results of the purified kinase in Examples 2 and 3 are shown in Table 1 below.

Purification step Total protein (mg) ^b Total activity (U) ^c Specific activity (U / mg) Purification (fold) Recovery (%) Culture
supernation 77.0 84.7 1.1 One 100 (NH ₄ ) ₂ SO ₄
precipitation 15.5 82.3 5.3 4.8 97.2 DEAE-Sapharose 0.8 29.9 39.7 36.2 35.3

Total protein (mg) ^b was measured by Bradford assay (Thermo Fisher Scientific, Rockford, IL, USA).

Total activity (U) ^c was measured in 50 mM sodium phosphate buffer (pH 7) at 40 ° C using colloidal chitin as substrate.

From Table 1, it can be seen that the crude extract precipitated with ammonium sulfate increased the non-specific activity of chitinase by 4.8 times, and after DEAE-Sepharose chromatography, the eluate increased by about 36.2 times with a yield of 35.3%.

Experimental Example 3

The purified enzyme was analyzed by SDS-PAGE using 4% polyacrylamide stacking gel and 12% degrading gel. Protein bands were visualized using Coomassie Brilliant Blue R-250 staining. The molecular weight of the purified chitinase was determined using standard proteins. The N-terminal amino sequence of the purified chitinase was analyzed by Edman digestion using a Procise Model 492 protein sequencer (Applied Biosystems, CA, USA) after electroblotting the protein onto a polyvinylidene difluoride membrane. .

As shown in FIG. 3, after SDS-PAGE, after staining the gel, a single band appeared with a molecular weight of about 94.2 kDa. This indicates that the kinase is a monomeric protein.

Experimental Example 4

The result of N-terminal sequencing for purified chintinase showed the sequence of AAPSTPSLDWKPQNYSFVEVD. To confirm the internal peptide sequencing homology, homology with other chitinase deposited in the NCBI database was compared as shown in Table 2.

The results in Table 2 show that the sequence of AAPSTPSLDWKPQNYSFVEVD shares homology with other chitinase deposited in the NCBI database. In particular, the chitinase sequence produced by BAO-1801 shared high sequence identity with chitinase ChiC from Thalassomonas actiniarum (90%) and Pseudoalteromonas spp. (81%), Colwellia sp. 12G3 (76%). These results indicate that Salinivibrio sp. BAO-1801 shows that it is a kind of ChiC chitinase.

Enzyme Origin NH ₂ -terminalaminoacid Identity (%) ChiC (this work) Salinivibrio sp. AAPSTPSLDWKPQNYSFVEVD - ChiC (WP_044834007) Thalassomonas actiniarum AAPSTPSLDWQPQNYSFVEVV 90 ChiC (WP_010362144) Pseudoalteromonas citrea AAPSTPSITWKPQNYSFVDVN 81 ChiC (WP_077537999) Pseudoalteromonas aliena AAPSTPSINWEPQQYSFVEVD 81 ChiC (WP_101232706) Colwellia sp. 12G3 AAPSAPSIDWTPQSYSFVEVN 76 ChiA (ADM13645) Pseudomonas sp. TxG6-1 AAPGKPTIAWGNTKFAIVEVD 43 ChiA (ADR30609) Serratia marcescens AAPGKPTIAWGNTKFAIVEVD 43 ChiB (AAM94336) Vibrio harveyi AVDCTPLEAWDSSKVYNGGDQ 19

Experimental Example 5

The optimal temperature for chitinase activity was determined by incubating purified chitinase with substrate at different temperatures (30-90 ° C.) under standard analytical conditions. To study thermal stability, the purified enzyme was pre-incubated in 50 mM sodium phosphate buffer (pH 7) at a temperature of 30 to 90 ° C. for 1 hour. After the indicated incubation time, the residual activity of the enzyme was measured under standard conditions, and the results are shown in FIG. 4.

In addition, the optimal pH of the kinase activity is potassium chloride-hydrochloric acid (pH 2.0-3.5), phosphate-citrate (pH3.0-4.5), acetate (pH 4.5-6.5), 3- (N-morpholino) propanesulfonic acid (MOS) (pH 6.5-8.0), and N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) (pH 8.0-10.0) were determined using different buffers of 50 mM pH 2-10. To confirm the stability of the kinase at different pH, the purified enzyme was dialyzed against the buffer for 30 min at 40 ° C. After cooling on ice for 10 minutes, the residual activity of the kinase under standard conditions was measured and the results are shown in FIG. 4.

Here, the chitin degrading enzyme activity was determined by measuring GlcNAc generated from hydrolysis of the substrate according to a known method (Miller 1959) with slight modification. Briefly, the same amount of 1% (w / v) colloidal chitin and diluted enzyme were mixed and incubated at 40 ° C for 30 minutes. One unit of kinase activity was defined as the amount of releasing 1 mole of GlcNAc per minute under analytical conditions.

As can be seen in Figure 4A, the activity of the kinase of the present invention was gradually increased from 30 ℃ to 50 ℃. However, 85 to 90 ° C was deactivated. The optimum temperature for other bacterial chitinase is 30 ℃ ~ 50 ℃. FIG. 4B shows that the enzymes of the present invention maintained enzyme activity of 82.6% and 78.7% at 55 ° C and 60 ° C, respectively, suggesting that the kitinase of the present invention is very thermally stable.

FIG. 4C shows that the maximum activity of the kinase of the present invention is observed at pH 7. In addition, D of FIG. 4 shows that the kinase of the present invention was relatively stable at pH 4 to 8.5 while maintaining 80% or more of the initial activity. The kinase of the present invention was denatured at pH 9 or higher.

Therefore, the characteristic of having the activity in the wide temperature range of the chitinase (BAO-1801 chitinase) obtained from the B.C. 180 strain of the genus Saliniviribrio of the present invention (Salinivibrio costicola) is a food product in which high temperature is used to reduce the occurrence of microbial contamination. It is suitable for industry and also has the advantage of reducing the processing time.

Experimental Example 6

The substrate specificity of the BAO-1801 chitinase of the present invention was tested using various substrates including colloidal chitin, glycol chitin, soluble chitosan, glycol chitosan, chitosan oxide and shrimp powder, and the results are shown in Table 3. Enzyme activity was measured under standard conditions.

Substrate Relative activity (%) Colloidal chitin 100 Glycol chitin 89.8 Colloidal chitin azure 42.3 Soluble chitosan 11.2 Glycol chitosan ND Oxidized chitosan ND (GlcNAc) 6 134.1 (GlcNAc) 5 112.6 (GlcNAc) 4 86.1 (GlcNAc) 3 11.5 (GlcNAc) 2 ND GlcNAc ND

From Table 3, it can be seen that the BAO-1801 chitinase of the present invention showed high activity on the colloidal chitin and glycol chitin (89.8%) substrates. However, the other polysaccharides tested could not be hydrolyzed. Moreover, this enzyme was very active against (GlcNAc) 6 and (GlcNAc) 5, but did not show activity against small dimer substrate (GlcNAc) 2. These results indicate that the specificity depends on the size and physical shape of the substrate, which can affect the rate of enzymatic hydrolysis. The BAO-1801 chitinase of the present invention may be useful for producing chitooligosaccharides in foods with high chitin content, such as fermented seafood.

Experimental Example 7

The degradation pattern of colloidal chitin and N-acetyl-chitooligosaccharides (GlcNAc) n (n = 2-5) substrates by BAO-1801 chitinase of the present invention was investigated using TLC analysis, and the results are shown in FIGS. 5A and 5B. Did.

As shown in Fig. 5a, the main products by degradation of colloidal chitin were GlcNAc2 and small amount of GlcNAc. A minor product, GlcNAc3, was also produced. Hydrolysis was performed with the main product (GlcNAc2) and GlcNAc as the incubation time was extended.

The BAO-1801 chitinase of the present invention hydrolyzed (GlcNAc) 5 and (GlcNAc) 2 and (GlcNAc) 3 at 1 hour. (GlcNAc) 3 was converted to (GlcNAc) 2 and GlcNAc after 8 hours (FIG. 5B). (GlcNAc) 4 degrades to GlcNAc and (GlcNAc) 2 during the hydrolysis period, while (GlcNAc) 4 is only converted to (GlcNAc) 2. However, the chitin degrading enzyme did not digest (GlcNAc) 2.

Experimental Example 8

The degradation pattern of colloidal chitin and N-acetyl-chito oligosaccharides (GlcNAc) n (n = 2-5) substrates by BAO-1801 chitinase of the present invention was investigated using HPLC analysis as follows, and the results are shown in FIG. 6. Shown.

To determine the hydrolysis properties of the enzyme, GlcNAc2, GlcNAc3 GlcNAc4 and GlcNAc6 were used as substrates. The reaction mixture consisted of equal volume of purified enzyme (0.2 U / ml) and 2% (w / v) substrate suspension. Then incubated at 40 ° C. for 8 hours. Aliquots were taken at different time intervals and centrifuged at 12000 xg for 10 minutes to remove undigested substrate. A 10 µl aliquot was loaded onto a silica gel 60 plate and developed in a solvent system containing 1-butanol-acetic acid-water (2: 1: 1, v / v). A mixture of 0.5% (w / v) GlcNAc and other (GlcNAc) n (n = 2-6) was used as standard. After development, the plate is sprayed with aniline-diphenyl amine reagent and heated at 100 ° C. for 5 minutes to visualize the hydrolysis product. The concentration of hydrolyzed N-acetyl-chito oligosaccharide is 40oC at a flow rate of 1 ml / min by HPLC analysis (Shimadzu LC-10) equipped with a Shodex NH2P-50 4E column using acetonitrile at 70 ° C as the mobile phase. Set and measured at 210 nm on a refractive index detector.

From Figure 6 showing the HPLC results confirming the TLC analysis results using colloidal chitin as a substrate, the degradation of (GlcNAc) 6 and (GlcNAc) 5 in contrast to the increased amounts of (GlcNAc) 2 (GlcNAc) 3-4 The pattern acted like an intermediate product. These were further decomposed into (GlcNAc) 1-2. The final yields of GlcNAc and (GlcNAc) 2 were 10.5% and 71.5%, respectively. These results may show that the BAO-1801 chitinase of the present invention shows a random catalytic mode on the chitin substrate having a high ability to form (GlcNAc) 2, and thus has the potential to improve food function.

Experimental Example 9

The antifungal activity of the BAO-1801 chitinase of the present invention was measured as follows using published methods (Kim et al., 2017). Pathogenic fungi including Aspergillus niger, Fusarium oxysporum, and Rhizoctonia solani were inoculated into potato dextrose agar (PDA) obtained from the Korean Agricultural Culture Center (KACC) and cultured at 25 ° C. to produce conidia. Spores were collected, filtered, and adjusted to 10 ⁴ spores / ml in potato dextrose broth. The reaction mixture consisted of 10 μl of a spore suspension and a dilution of BAO-1801 chitinase and incubated at 25 ° C. for 24 hours. The number of spore germination was measured under an inverted microscope (Olympus IX70, Melville, NY, USA), and the results are shown in FIG. 7.

As shown in Figure 7, the purified chitinase of BAO-1801 of the present invention showed different degrees of inhibition against spore germination of three fungal phytopathogens. BAO-1801 chitinase inhibited spore germination from all test strains at high concentrations (> 200 μg / ml). However, F. oxysporum and R. solani were more sensitive to lower concentrations of BAO-1801 chitinase (> 50 μg / ml) than other strains. These results confirm that the antifungal activity of chitinase depends on the cell wall structure of spores. Although many other bacterial chitinase have been reported to inhibit spore germination, the antifungal activity of chitinase C-based enzymes produced by the strains of the genus Salinivibri like the present invention is not known so far.

Experimental Example 10

Antioxidant activity of the extract of the fermentation product obtained by treating the salted and fermented shrimp of Salinivibrio costicola BAO1801 strain of the present invention as follows and the results are shown in Tables 4 and 8 Did.

1. Fermentation and extraction of salted and fermented shrimp

Salinivibrio sp. BAO-1801 was incubated overnight at 37 ° C in 10 mL of MRS broth to reach 0.5 OD. The cultured suspension was added to the salted and fermented shrimp paste at 106 CFU / g. Fermentation was maintained at 20 ° C. for 0, 1, 2, 3, 4 months.

To prepare for extraction, 5 g of the fermented sample was suspended in 80% ethanol (1: 5 ratio w / v) and self-degraded by incubation at 30 ° C. for 1 hour with constant stirring. The suspension was centrifuged (10,000 x g, 30 min, 4 ° C) and the supernatant was collected as a crude extract.

2.Antioxidant of salt and fermented shrimp

DPPH scavenging capacity was slightly modified in Wang et al. (2010) method. Deionized water and DPPH solution were used as control samples. The test sample is for crude extracts only. After centrifugation at 4,000 × g (Hettich, EBA21, Germany) for 15 minutes, absorbance was measured 3 times using 517 nm. Activity was expressed in μmol trolox equivalent (TE) / g samples, and inhibitory activity was reported as a percentage of scavenging activity.

ABTS radical scavenging ability is described in Binsan et al. (2008). Activity was expressed in μmo trolox equivalent (TE) / g samples, and inhibitory activity was reported as a percentage of scavenging activity.

Ferric reduction / antioxidant power (FRAP) analysis was previously described in Binsan et al. (2008). Activity was expressed in μmol trolox equivalent (TE) / g samples.

3. Results

As shown in Table 4, shrimps fermented for 4 months showed the highest DPPH radical scavenging activity (4.30 μmol TE / g), and fresh shrimp (0 months) showed the lowest DPPH radical scavenging activity (1.47 μmol TE / g) (P <0.05). ABTS scavenging activity increased with increasing fermentation period.

Fermentation period (month) DPPH activity
(μTE / g sample) ABTS activity
(μTE / g sample) FRAP activity
(μTE / g sample) 0 1.47 ± 0.23 ^d 7.99 ± 0.34 ^d 16.03 ± 0.90 ^b One 1.67 ± 0.27 ^d 9.20 ± 0.10 ^c 16.77 ± 0.12 ^b 2 2.57 ± 0.40 ^c 10.60 ± 0.53 ^b 21.73 ± 0.84 ^a 3 3.40 ± 0.20 ^b 11.73 ± 0.32 ^a 22.03 ± 0.21 ^a 4 4.30 ± 0.22 ^a 12.33 ± 0.25 ^a 23.13 ± 0.31 ^a

It can be seen from FIG. 8A that the IC50 values of the shrimp ferment extract were 231.15 mg / mL, 193.35 mg / mL, 165.40 mg / mL, 137.86 mg / mL and 101.59 mg / mL for 0-4 months of fermentation.

As shown in B of FIG. 8, there was no difference in ABTS activity of the 3- and 4-month fermentations with IC50 values of 188.27 and 159.76 mg / mL, and the FRAP activity from C of FIG. 8 did not change after 2 months of fermentation and in the final experiment It can be seen that it showed the highest activity (23.13 μmol TE / g sample).

Experimental Example 11

The antifungal activity of the extract of the fermentation product obtained by treating the salt and fermented shrimp obtained in Experimental Example 10 with the Salinivibrio costicola BAO1801 strain of the present invention was measured as follows, and the results are shown in Table 5. .

이 한천에서 염장 및 발효된 새우의 항균 활성을 평가하기 위해 사용되었다. 즉 멸균 여과지를 각 조 추출물 50 mL로 함침시키고 미리 선택된 병원균을 접종한 한천 플레이트 표면에 놓았다. 한천 플레이트를 37 ℃에서 24 시간 동안 배양했다. 배양 후, 억제 영역의 디스크 직경을 측정 하였다(mm 단위). 각 측정은 두 번씩 수행되었다.Diffusion method is a known method using sterile filter paper (5mm diameter, Whatman No. 1)

It was used to evaluate the antibacterial activity of salted and fermented shrimp in this agar. That is, sterile filter paper was impregnated with 50 mL of each crude extract and placed on the surface of an agar plate inoculated with preselected pathogens. Agar plates were incubated at 37 ° C for 24 hours. After incubation, the disc diameter of the inhibition region was measured (in mm). Each measurement was performed twice.

From Table 5 showing the results of paper diffusion analysis, the antimicrobial activity of salt and fermented shrimp extract treated with the BAO1801 strain of Salinivibrio costicola of the present invention was treated with 4 different food-borne pathogens among the two Gram bacteria. It was sensitive. Among them, the crude extract is Yersinia enterocolitica ssp. It showed excellent activity in enterocolitica KACC 15320. In addition, the longer the fermentation time after treatment with the Salinivibrio costicola BAO1801 strain of the present invention, the higher the antibacterial activity.

Pathogens Diameter (mm) of inhibition zone by crude extract from salted shrimp fermentation Control 1 month 2 months 3 months 4 months Gram-positive bacteria Staphylococcus aureus KCCM 11335 1 ± 0.4 1 ± 0.8 1 ± 0.6 1 ± 0.2 2 ± 0.2 * Listeria monocytogenes KACC 10764 - - - 1 ± 0.5 2 ± 1.4 * Bacillus cereus KACC1124 1 ± 0.2 1 ± 0.4 1 ± 0.4 1 ± 0.6 1 ± 0.2 Gram-negative bacteria Yersinia enterocolitica ssp. enterocolitica KACC 15320 1 ± 1.4 1 ± 0.4 1 ± 0.8 3 ± 0.5 * 4 ± 0.6 * Salmonella choleraesuis KCTC2932 1 ± 0.2 1 ± 0.2 1 ± 0.3 1 ± 0.4 1 ± 0.2 Escherichia coli K99 KCTC 2617 - - 1 ± 0.2 2 ± 0.4 * 3 ± 0.8 * * P <0.05

The present invention has been shown and described with reference to preferred embodiments as described above, but is not limited to the above-described embodiments and is within the scope of the present invention to those skilled in the art to which the present invention pertains. By this, various changes and modifications will be possible.

Agricultural Biotechnology Research Institute KACC81071 20180504

Claims (17)

Salinivibrio costicola BAO1801 strain producing kitinase (accession number: KACC 81071 BP).
The method of claim 1,
The strain is 10 to 30% NaCl (w / v) Salinivibrio genus Costiola ( Salinivibrio costicola ) BAO1801 strain, characterized in that cultured in a medium.
The method of claim 1,
The chitinase is a Salinivibrio costicola BAO1801 strain, characterized in that it has activity in a temperature range of 30 ° C to 80 ° C.
The method of claim 1,
The chitinase is characterized in that it has an activity at pH 2-9 , Salinivibrio costicola BAO1801 strain.
Kitiniase , produced from the Salinivibrio costicola BAO1801 strain (Accession No .: KACC 81071 BP), has thermal stability and flame resistance.
The method of claim 5,
The kinase is a kinase characterized in that it has activity at 10 to 30% (w / v) of NaCl.
The method of claim 5,
The kinase is a kinase characterized in that it has activity in the temperature range of 30 ℃ to 80 ℃.
Culture step of culturing Salinivibrio costicola BAO1801 strain (Accession No .: KACC 81071 BP) in a medium containing chitin; a method for producing a kinase comprising, wherein the kinase is resistant to salt and heat Method of producing a kinase, characterized in that it has stability.
A food additive comprising the Salinivibrio costicola BAO1801 strain (accession number: KACC 81071 BP) or a culture thereof according to any one of claims 1 to 4.
A fermented food product comprising the food additive of claim 9.
The method of claim 10,
The fermented food is fermented seafood containing chitin, wherein the seafood is one or more selected from the group consisting of shrimp, crab, and crayfish.
Salinivibrio costicola BAO1801 strain (accession number: KACC 81071 BP) of any one of claims 1 to 4, culture of the strain, kitty of any one of claims 5 to 7 An eco-friendly microbial agent comprising at least one of a kinase and a kinase obtained by the method of claim 8.
The method of claim 12,
The eco-friendly microbial agent is an eco-friendly microbial agent characterized by inhibiting the mycelial growth of fungal plant pathogens by decomposing the mycelium of plant pathogens comprising chitin as a cell wall.
The method of claim 13,
The fungal phytopathogen is an eco-friendly microbial agent characterized in that at least one of Rhizoctonia solani , Aspergillus niger , Fusarium oxysporum .
A method for controlling plant diseases comprising the step of treating the eco-friendly microbial agent of claim 12 on soil, plants or seeds of plants.
The method of claim 15
The plant disease is a plant disease control method characterized in that caused by fungal plant pathogens that constitute chitin as a cell wall. A food preservative comprising at least one of the kinase of any one of claims 5 to 7 and the kinase obtained by the method of claim 8.

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