KR20180082860A - Manufacturing method for Cricket(Gryllus bimaculatus) Chitosan and anti-fungi agent using it - Google Patents

Abstract

The present invention is to produce a cricket chitosan having excellent antibacterial properties from a dried cricket at a high yield, and to provide a natural antibacterial agent using the same. A method for producing a cricket chitosan of the present invention comprises: a step of generating cricket powder by pulverizing a dried cricket into fine powder, and washing and drying the same; a cricket protein removal step of removing cricket protein by mixing and stirring the dried cricket powder with sodium hydroxide; a color removal step of removing a color by mixing, stirring, and immersing the residue having the cricket protein removed therefrom in an APS solution, and washing the same; a desalination step of immersing the residue having the color removed therefrom while stirring the same, and then washing, drying, and desalting the same; and a step of mixing the desalted residue in a sodium hydroxide solution, and filtering, washing, and drying the same for deacetylation.

Description

{Manufacturing method for Cricket (Gryllus bimaculatus) Chitosan and anti-fungi agent using it}

The present invention provides chitosan having excellent antimicrobial activity from a dried cricket (Gryllus bimaculatus; hereinafter referred to as cricket) at a high yield and to provide a natural antimicrobial agent using the chitosan.

Chitosan is a linear polysaccharide obtained by partial deacetylation of chitin with D-glucosamine and N-acetyl-D-glucosamine bonded to? -1,4 linked.

Such chitosan is found in many exoskeleton cells of arthropods such as fungi and shells of shrimp or insects.

Chitosan is widely used in a variety of fields such as wastewater treatment, food and beverage, cosmetic, pesticide, cell culture, textile and medical devices due to the remarkable biological properties such as natural antioxidants, antibacterial agents, hypocholesterolemia and immunostimulation.

Recently, chitin present in some insects has been concentrated, and it has been proposed to use these organisms as new quantum chitin raw materials. Insect chitin and chitosan are generally very similar to shrimp chitosan. Chitin occurs in alpha form and is known to have similar physico-chemical properties. However, one difference appears to be the generally low molecular weight of insect chitosan compared to shrimp chitosan. Insect chitosan has a low molecular weight from 2.6 kDa for Colorado potato beetle to 501 kDa for sulfhydryl chitosan.

To increase the stability of bioactivity during processing, encapsulation systems are used to protect sensitive physiologically active compounds from environmental factors such as light, moisture, oxygen and heat. Encapsulation, one of these efforts, is well known for preserving bioavailability and release control and for blocking offensive odors and tastes. Encapsulation of compounds such as microcapsules, beads and microemulsions is commonly used in the food and pharmaceutical industries, and nanocapsulation systems are increasingly used as delivery vehicles in delivery systems.

A problem to be solved by the present invention is to provide a food preserving agent which produces chitosan having excellent antibacterial activity from dried crickets in high yield and increases the antimicrobial activity when food is added and a natural antimicrobial agent having antimicrobial activity in packaging food will be.

The method for producing cricket chitosan according to the present invention comprises crushing dried crickets (hereinafter referred to as cricket) with fine powder, washing and drying the resulting crickets to produce cricket powder; A cricket protein removal step of mixing the dried cricket powder with sodium hydroxide and stirring to remove the cricket protein; Removing the cricket protein-free residue from the APS solution by mixing and stirring the mixture; A desalting step of immersing the color-removed residue in water while agitating, washing, and then desiccating to desalinate; Mixing the desalted residue with a sodium hydroxide solution, filtering and washing the solution, and drying and deacetifying the desalted residue.

Preferably, the cricket protein removal step is performed by adding dry cricket powder produced using dried crickets to sodium hydroxide (NaOH), removing proteins contained in the cricket, filtering the reaction mixture, and washing the residue with a pH of 5.5 The residue after removing the cricket protein was added to the APS solution and stirred. After the filtration, the filtrate was washed with distilled water , And the desalting step is carried out by immersing the colorless residue in the oxalic acid solution at room temperature while stirring the residue, washing the residue after filtration with distilled water to a pH of 5.5, drying it, Is prepared by immersing the demineralized residue in a sodium hydroxide solution having a concentration of 50-67% by weight and chitin-deacetylating with stirring, and stirring the mixture with distilled water Followed by pretreating and drying.

The present invention has the advantage of producing cricket chitosan having high antimicrobial activity from dry crickets superior to chitosan extracted from excellent shrimp with high yield.

In addition, the cricket chitosan of the present invention can be used as a food additive which can increase the preservation period of food by minimizing the occurrence of microorganisms as compared with chitosan extracted from shrimp by adding food.

In addition, the cricket chitosan of the present invention has an advantage that it can be used as a natural antimicrobial agent which can increase the freshness of instant food by suppressing microbial generation of food than the packaging material coated with ordinary chitosan when it is applied to the packaging material of instant food.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a process diagram of a method for producing cricket chitosan according to the present invention
2 is a FT-IR graph of cricket chitosan and commercial chitosan according to the present invention.
3 is an XRD pattern graph of cricket chitosan and commercial chitosan according to the present invention.
4 is an electron micrograph of cricket chitosan and commercial chitosan according to the present invention.
Figure 5 is a molecular weight graph of cricket chitosan according to the present invention.
Fig. 6 is a photograph of fresh shrimp packed with cricket-chitosan and commercial chitosan-impregnated packaging material according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.

1, the cricket chitosan manufacturing method according to the present invention includes a cricket powder producing step S01, a cricket protein removing step S02, a color removing step S03, a desalting step S04, (S05).

In the cricket powder producing step S01, crushed dried crickets are pulverized into fine powder in a pulverizer, and the pulverized pulverized powder is washed with distilled water to remove impurities. The pulverized powder is heated to about 60 ° C and dried to produce cricket powder .

In the cricket protein removal step (SO2), the dried cricket powder produced using the dried crickets is added to sodium hydroxide (NaOH), and then the cricket protein is removed by stirring at about 95 DEG C and about 130 rpm for about 6 hours . The reaction mixture is then filtered through a 100-mesh sieve and the residue is washed with distilled water to a pH of 5.5 to produce a protein-free residue.

The color removal step (SO3) is performed by adding the residue from which the cricket protein has been removed to the APS solution, stirring in a shaking water bath at about 50 DEG C and about 130 rpm for about 6 hours, filtering through a 100 mesh sieve, Wash with distilled water until it is 5.5 to remove color from the protein-free residue. At this time, the APS solution is preferably an APS solution (50% (w / v)) produced by mixing distilled water and APS at a weight ratio of 1: 1 and dissolving at about 50 ° C for 30 minutes.

The desalted step (SO4) was carried out by immersing the colorless residue in a solution of oxalic acid at room temperature with stirring for about 3 hours, filtering through a 100-mesh sieve, washing the residue with distilled water to a pH of 5.5, Deg.] C and desalted.

The deacetylation step (SO5) was carried out by immersing the desalted residue at 95 DEG C in a high concentration of sodium hydroxide solution (50% -67%), chitin deacetylating by stirring at 130 rpm, washing with distilled water And then dried at about 60 ° C.

In addition, the cricket chitosan produced by the method of the present invention has high antimicrobial activity and can be used as a natural antimicrobial agent applied to the packaging material of instant food, thereby extending the shelf life of the food.

Hereinafter, embodiments of the present invention will be described in detail.

<Extraction of cricket chitosan>

Gryllus bimaculatus was pulverized according to the above-mentioned cricket chitosan extraction method, and 40 g of cricket powder was carried in 400 mL of 1M NaOH. After stirring for 6 hours at 95 rpm at 130 rpm, the reaction mixture was filtered with a 100-mesh sieve, The cricket protein was removed by washing with distilled water until the pH reached 5.5.

The APS solution (50% (w / v)) was newly prepared by dissolving 400 g of APS in 400 g of distilled water at 50 캜 for 30 minutes. Subsequently, the residue in which the cricket protein was removed was immersed in a shaking water bath, After stirring at 130 rpm for 6 hours, the mixture was filtered through a 100-mesh sieve and washed with distilled water until the pH reached 5.5 to remove the color.

Further, the decolorized residue was immersed in a solution of oxalic acid, stirred at room temperature for 3 hours, filtered with a 100-mesh sieve, washed with distilled water to pH 5.5, and desiccated by drying overnight at 60 ° C in an air oven.

Finally, chitin deacetylation was carried out at 95 ° C with a high concentration of sodium hydroxide solution (concentration of 50-67%), and the reaction was shaken at 130 rpm while shaking the water over time. The cricket chitosan was filtered, washed with distilled water to pH 5.5, and the air was oven-dried overnight at 60 ° C to produce chitosan.

As shown in Table 1, about 4.01 g of cricket chitin produced from the cricket powder of 40 g was extracted to account for about 10.11% of the dry weight, and about 2.20 g of chitosan was extracted, Was 55.0%. The yield of chitosan was found to be significantly higher than that of 15% in cicada and 20% in silkworm pupa.

&Lt; FT-IR analysis >

FT-IR is a widely used analytical method for identifying specific functional groups in a compound. Using 0.01 g each of the prepared cricket (Gryllus bimaculatus) chitosan and commercial chitosan (shrimp extracted chitosan), the Nicolet iS50 FT-IR and Nicolet iS50 ATR spectrometer (Thermo Fisher Scientific Inc., USA) was used to confirm the FT-IR and is shown in the graph of FIG.

As can be seen from the graph of FIG. 2, the FT-IR spectra of cricket chitosan and commercial chitosan are very similar in wavelength and transmittance. Also, cricket chitosan showed a strong band of 3355cm ^-1 and 3292cm ^-1 corresponding to the stretching vibrations of the budding of the OH and NH, commercial chitosan showed a strong band at 3354cm ^-1 and 3290cm ^-1, Cricket chitosan and FT-IR spectrum of a commercial chitosan showed a band of 2872cm ^-1 and 2871cm ^-1 corresponding to the (CH ₂₎ vibration of the CH ₂ OH group. In addition, a clear attenuation of the band at 1652 cm ^-1 (C = O) of the NHCOCH ₃ group (amide I band) was observed, indicating successful deacetylation. In addition, characteristic NH bending vibrations of the NHCOCH ₃ group (amide II band) were detected at 1591 cm ^-1 .

Thus, it was confirmed that the FT-IR spectra of cricket chitosan and commercial chitosan have considerable similarities.

<Confirmation of deacetylation>

Table 2 shows the DA of chitosan as a function of reaction time and NaOH concentration (%) to confirm deacetylation of cricket chitosan.

As shown in Table 2, when the extracted cricket chitosan reacted in 50% of the NaOH solution for 9 hours, it was deacetylated to 66.54%. However, as a result of treating 55% NaOH solution, the DA increased sharply to 75.18% and 77.63% after 9 hours and 12 hours, respectively. Also, treatment with 67% NaOH for 9 hours showed deacetylation of cricket chitosan up to 84.98%.

&Lt; X-ray diffraction analysis (XRD) >

Crystallinity of cricket chitosan is determined by XRD. The XRD pattern of cricket chitosan was compared with commercial chitosan. The XRD patterns were collected at scan angles between 5 and 45 using D8 ADVANCE equipped with DAVINCI system and are shown in the graph of FIG.

As shown in FIG. 3, the XRD peaks of the cricket chitosan were observed at 10.50 ° and 20.07 °, and the XRD peaks of the commercial chitosan were observed at 10.33 ° and 19.90 °, respectively.

<Scanning Electron Microscope Analysis>

Surface morphology of cricket chitin and chitosan was observed by SEM and compared with commercial chitosan. Prior to microscopic examination, cricket chitin and chitosan were thoroughly ground and covered with gold under vacuum using an ion sputter (S-2380, HITACHI, Japan). SEM was performed using Bio-SEM (E-1010, HITACHI, Japan). The sample was photographed at a magnification of 1000 times and 10000 times and is shown in Fig.

As can be seen from the photograph of FIG. 4, the surface of cricket chitosan and commercial chitosan are very similar.

<Mass analysis of chitosan>

The molecular weight of cricket chitosan was measured using an AXIMA mass spectrometer (SHIMADZU-Biotech Co., Tokyo, Japan) and is shown in Fig.

As shown in FIG. 5, the Mw of the cricket chitosan of the present invention was found to be 8,302 Da or less. Strong signals were observed in the range of 550-1500 and weak signals were detected up to 8302. The strong peak indicated that the product was composed of chitooligosaccharides with a degree of polymerization of 2.8. The weak peak indicates the molecular weight of the chitosan polymer. The Mw of cricket chitosan was lower than that of commercial chitosan of 50-190 kDa. It has been studied that low molecular weight (LMw) chitosan (average molecular weight in the range of 5000-10000 Da) has strong sterilization and excellent biological activity when compared to high molecular weight (HMw) chitosan. The inventive croquet chitosan also has excellent sterilization and biological activity . &Lt; / RTI &gt;

<Color measurement, zeta potential and pH value analysis>

For color measurement, 0.5 g of cricket chitosan and commercial chitosan were placed in a transparent Petri dish. The color of the sample was measured using a colorimeter (Minolta CR-400) standardized as an orthodontic white plate (L ^* = 96.04, a ^* = 0.20, b ^* = 2.01). Each sample was measured three times individually and the parameters were recorded as L ^* , a ^* and b ^* and shown in Table 3. In addition, to measure the pH value and zeta potential, commercial and cricket (Gryllus bimaculatus) chitosan 10 mL was dissolved in a 0.2% acetic acid aqueous solution at a concentration of 2.0 mg / mL. Zeta potentials were measured using a Zetasizer Nano ZS operating at 25 ± 1 ° C in multiple narrow modes and are shown in Table 3.

As shown in Table 3, the L ^* , a ^* and b ^* values of cricket chitosan and commercial chitosan were 72.16 ± 0.12, 1.96 ± 0.06, 11.83 ± 0.04 and 77.92 ± 0.51, 0.51 ± 0.06 and 13.06 ± 0.05, respectively. The two chitosan samples showed different results. L ^* and b ^* values of cricket chitosan was significantly lower than the L ^* and b ^* values of commercial chitosan. In contrast, the a ^* value of cricket chitosan was higher than that of commercial chitosan. At this time, the L ^* value (darkness at brightness), the a ^* value (green to red), and the b ^* value (blue to yellow).

The pH value of cricket chitosan was about 3.84, which was similar to the pH value of commercial chitosan of about 3.88.

The zeta potential of cricket chitosan and commercial chitosan also showed strong cationic charge and the zeta potential was very similar.

<Nano encapsulation of cricket chitosan>

 Cricket Chitosan NP is made from ionic gelation technology using cricket chitosan and TPP (tripolyphosphate). Cricket chitosan and commercial chitosan were dissolved in a 0.2% acetic acid aqueous solution at a concentration of 1.44 mg / mL. Centrifugation (5,000 rpm, 4 ° C, 15 min) was used to remove residues of insoluble particles. 2, 4 and 6 mL of aqueous TPP solution were added to 10 mL of chitosan solution at a flow rate of 1 mL / min using a Pasteur pipette under magnetic stirring (800 rpm) at room temperature. The reaction was carried out for 10 minutes. The particle size, zeta potential and PDI of cricket chitosan and commercial chitosan were measured using dynamic light scattering (DLS) analysis and are shown in Table 4.

As shown in Table 4, the physical properties of nanoparticles (NP) of cricketed chitosan (A) and commercial chitosan (B) NP were shown. Compared with commercial chitosan, the particle size of cricket chitosan was smaller than that of commercial chitosan. In addition, as the TPP volume increased, the particle size of cricket chitosan and commercial chitosan NP increased. The sizes of the two chitosan NPs ranged from 174 to 245 nm and 196 to 331 nm, respectively. The zeta potentials of the two chitosans exhibit strong cationic charge, ranging from 30.02 to 36.12 mV and 33.46 to 38.14 mV, respectively. The PDI values ranged from 0 to 1 and from 0 to 0.3, indicating a uniform nanosuspension. At this time, a higher value of PDI is associated with less uniform particle size distribution. As a result, it was confirmed that the NP of cricket chitosan had a more stable particle size distribution and stable NP than the commercial chitosan NP.

As described above, the molecular weight of cricket chitosan of the present invention was lower than that of commercially available chitosan, and showed that the molecular mass was less than 8,032 Da and mainly distributed in the range of 550-1,500 Da. The degree of deacetylation, FT-IR spectrum, XRD pattern and SEM image were very similar to that of commercial chitosan. Cricket Chitosan has a smaller particle size and PDI value and more homogeneous particle size distribution compared to commercial chitosan. Thus, cricket chitosan can be used as a chitosan source for the food, cosmetic and pharmaceutical industries.

<Antibacterial activity of cricket chitosan>

<Sample Preparation>

After weighing the rice, weigh the weights to be used, add chitosan, and mix (chitosan is evenly poured on the rice to make it look like antimicrobial rice). Water is added, the beads are packed, cooked and cooked with rice bran .

Also, after removing the blood of the pork ribs, marinate in the spices to be used, weigh only the chili and then weigh the chitosan. (If you add chitosan to the spice itself, you can not know exactly how much it is.) After the fortune is seasoned, only the amount of pork is calculated and chitosan is added.

Chitosan was prepared by weighing 30 g of sample and 1.5 g of chitosan content (w / v) of 1%.

<Experimental Results>

Cooked rice cooked with cricket chitosan and steamed pork were cooked at a cooking temperature of 85 ° C for 1 hour and 30 minutes, and bacterial counts and pH were measured for each period. Commercial chitosan was added as a control or cooked in the same manner without any addition. The bacterial count was measured in a constant environment using commercial 3M medium of common bacteria, Bacillus cereus, Staphylococcus aureus . The change in pH by period was measured and shown in Table 5.

<Antimicrobial Results>

As a result of the measurement, the antimicrobial activity of cricket chitosan was apparent in the untreated control group, the commercial chitosan added group, and the cricket chitosan added group. (2.69 ± 0.01 log CFU / g) and steamed pork (4.08 ± 0.08 log CFU / g, respectively) before cooking (p <0.05). The number of bacteria was not measured until 30 days after cooking by high temperature and heating time, and the number of bacteria was measured from the 35th day.

The average bacterial counts were 6.20 ± 0.00 log CFU / g, 6.28 ± 0.01 log CFU / g, 6.42 ± 0.10 log CFU / g, and 6.40 ± 0.10 log CFU / g, respectively, on the 35th day of the storage period. (Infinity). The number of bacteria was not measured in the chitosan-free group but the number of effective bacteria in the commercial chitosan group was less than 30, while the number of effective bacteria in the commercial chitosan group was not measured by the standard plate method according to the food circulation .

Bacillus cereus (Bacillus cereus) was not measured as an effective value on days 35 and 40, and was measured to be 10-15 or less in rice and pork steamed in the control and commercial chitosan groups.

Staphylococcus aureus was significantly increased at 3.04 ± 0.01 log CFU / g of rice in the control group on the 35th day of storage and 3.60 ± 0.35 log CFU / g in the control group. Staphylococcus aureus was also not effective, but bacteria were also found in rice and pork steamed in the control, commercial chitosan and cricket chitosan groups. However, the control group showed less than 20 rice and pork steamed rice and 10-15 rice and pork steamed commercial chitosan group, while the cricket chitosan group had less than 8 rice and 5 pork chopped rice. , The antimicrobial activity against Staphylococcus aureus was higher than that of other strains.

<pH result>

As shown in Table 5, the pH change started to appear from the 30th day in the rice control group, and the pH of the chitosan group was steadily decreased. There was no significant pH change in the steamed pork.

As a result, it was confirmed that the antimicrobial activity of cricket chitosan was higher than that of commercial chitosan in rice and pork steamed which could be applied as instant food.

<Confirmation of antimicrobial properties of packaging materials>

The dot packing materials dotted with cricket chitosan and commercial chitosan were modeled, and the packaging material was antimicrobially confirmed by ATP bioluminescence after packaging the raw shrimp as shown in FIG. 6, a and a 'are packaging materials filled with commercial chitosan, where a is the head of the raw shrimp to the left, and a' is the head to the right. In addition, 'b' and 'b' are packed with cricket chitosan, where a is the head of the raw shrimp to the left, and a 'is the head to the right.

As shown in Table 6, the commercial chitosan showed a RLU of 513,940 on the 5th day from 14.553 on the 1st day, and 236,838 RLU on the 5th day from 16,097 on the day of cricket chitosan.

Accordingly, there is a possibility that the application of cricket chitosan is antimicrobial to foods, and it can be applied to food industries such as food additives and packaging materials which reduce the occurrence of microorganisms added to foods.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that within the scope of the appended claims, various changes and modifications may be made.

Claims (6)

Crushing dried crickets (hereinafter referred to as cricket) with fine powder, washing and drying the resulting cricket powder;
A cricket protein removing step of mixing the dried cricket powder with sodium hydroxide and stirring to remove the cricket protein;
Removing the cricket protein-free residue by mixing and agitating the APS solution, washing and washing to remove color;
A desalting step of immersing the color-removed residue in water while agitating, washing, desiccating and desalting;
Comprising the steps of: mixing a desalted residue with a sodium hydroxide solution, filtering, washing and drying to deacetate. The method of claim 1, wherein the cricket protein removal step comprises: adding dry cricket powder produced using dried crickets to sodium hydroxide (NaOH), removing proteins contained in the cricket, filtering the reaction mixture, Lt; / RTI &gt; to 5.5, to yield a protein-free residue,
In the color removal step, the cricket protein-free residue is added to the APS solution and stirred, and the residue after filtration is washed with distilled water until the pH is 5.5 to remove color,
The desalting step is carried out by immersing the decolorized residue in a solution of oxalic acid in a solution of oxalic acid at room temperature, washing the residue after filtration with distilled water to a pH of 5.5,
The deacetylation step comprises immersing the demineralized residue in a sodium hydroxide solution having a concentration of 50-67% by weight, stirring, chitin deacetylating, washing with distilled water until a pH of 5.5 is reached, and drying Process for the production of cricket chitosan. The APS solution according to claim 1, wherein the APS solution in the color removal step is an APS solution (50% (w / v)) produced by mixing distilled water and APS at a weight ratio of 1: 1 and dissolving at about 50 ° C for 30 minutes Process for the production of cricket chitosan. A food additive comprising cricket chitosan prepared by the method of any one of claims 1 to 3 as an active ingredient. A natural antimicrobial agent comprising cricket chitosan prepared by the method of any one of claims 1 to 3 as an active ingredient. The natural antimicrobial agent according to claim 5, wherein the natural antimicrobial agent is applied to a packaging material of an instant food.

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