JP2009278964A - Chitosan powder for salt-pickled food, salt-pickled food using the same and kimchi produced using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chitosan powder for salt-pickled food, standardized by difference in deacetylation degree and viscosity, a salted food using the same and kimchi produced using the same. <P>SOLUTION: The chitosan powder for salt-pickled food, having excellent antimicrobial property, oxidation resistance, mutation resistance and anti-cancer activity is any one or a combination of two or more selected from the group consisting of a water-soluble chitosan having a deacetylation degree of 50-90% and a viscosity of 1-10 cP, an insoluble chitosan having a deacetylation degree of 60-100% and a viscosity of 8-80 cP and its sitologically acceptable salt. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a chitosan powder for salting, a salted product using the same, and a kimchi produced using the same.

  Chitosan is a substance obtained by deacetylating chitin contained in crab, crayfish, squid and shrimp shells, and is an animal dietary fiber having a (+) charge. The structure of chitosan consists of a copolymer of N-acetyl-glucosamine (a chitin molecule) and glucosamine (a chitosan molecule) depending on the degree of deacetylation. Chitosan is known to activate aging cells to suppress aging, strengthen immunity, prevent disease, activate the body's natural healing ability, and regulate biological rhythms. However, the mechanism has not been fully elucidated. The effects of chitosan include anticancer action, antibacterial action, immune enhancement action, liver function enhancement, cholesterol suppression, intestinal effective bacteria increase, harmful bacteria suppression, blood pressure regulation effect, heavy metal and harmful component adsorption excretion, blood sugar level suppression, cell Activation is known. However, even though chitosan has various physiological activities, its molecular weight is very large, and our body does not have an enzyme that decomposes it, so its application range is limited, and when it is taken as it is Has the problem that most of it is not absorbed by the body and discharged outside the body.

  In general, the crustacean snow crab / snow crab is a typical special product produced in the clean waters of Gyeongsangbuk-do and occupies 30% of Korea's national production. Chitin and chitosan are resources that reach a production base annual production of 100 billion tons, and are comparable to the cellulose produced by plants. Therefore, chitosan has been mainly used in the agricultural field as a flocculant for wastewater treatment or as a soil conditioner, but in recent years, it has been applied to physiological synthetic detergents, pharmaceuticals, cosmetics, foods, etc. The scope of application has been expanded. However, polymeric chitosan is insoluble in water at neutral pH conditions and soluble in low-concentration organic acid solutions, but it has a bitter taste and a bitter taste, so it is somewhat difficult to apply to foods. There was difficulty. In addition, imports of chitosan products are increasing by 10% every year, but domestic production has declined since 2003.

  On the other hand, kimchi is a traditional fermented food unique to the Korean people, a source of various minerals and vitamins, and is widely known as a health food that helps the growth of effective bacteria in the intestine after ingestion. Kimchi is a product made by salting radish, Chinese cabbage, and pepper, mixing with spices such as chili, daikon, green onion, ginger, and salted spices, ripening by the production of lactic acid, and fermenting at a low temperature. An indispensable side dish at the table. Pickling kimchi is a means for storing sugar beet for a long period of time. Conventionally, in order to store kimchi for a long period of time, kimchi has been buried in the soil to extend the preservation period of kimchi. However, due to changes in the living environment, kimchi can often not be buried in the soil, and even if the kimchi is stored in the soil, the scent of kimchi that has aged after a long time has passed. I will. Therefore, research for storing for a long time while maintaining the unique taste of kimchi has been actively conducted. As a part of such research, recently, a method of adding chitosan as a natural additive of kimchi is known. Chitosan is soluble in weak acids, has a (+) charge in the molecule, and is known to have particularly high antibacterial properties, so it is often used in general foods as a natural preservative.

  As prior arts for improving the storability of kimchi using chitosan, Patent Document 1 and Patent Document 2 “Method for producing kimchi delay-improving kimchi” and Patent Document 3 “Manufacturing kimchi with extended shelf life” Method ", Patent Document 4," Method for producing kimchi containing chitosan and mineral substance ", Patent Document 5," Method for producing kimchi using chitosan-containing loess aqueous extract ", and Patent Document 6," Fresh grass There is a manufacturing method of kimchi as a main material. Further, as techniques for applying chitosan to the kimchi flavor, there are “Natural savory kimchi composition of instant kimchi and method for producing the same” in Patent Document 7, “Chitin chitosan kimchi” in Patent Document 8, and the like. Examples of techniques to which chitosan is applied include “Kimchi ripening delay method” in Patent Document 9, “Acid-delayed kimchi production method” in Patent Document 10, and “Kimchi freshness maintenance method” in Patent Document 11.

  As described above, various researches have been developed to improve the storage stability of kimchi and the condiment of kimchi and the technology that applies chitosan to kimchi itself, but standardized chitosan in order to use optimal conditions. There is still no research and development on the technology that applied this to Kimchi.

Therefore, when standardized chitosan is used to produce tailored chitosan for a specific food, it can contribute to national health by improving food storage period, reducing food waste, preventing food poisoning, etc. It is considered that functional kimchi with chitosan added can be produced to ensure competitiveness in the huge kimchi market.
Korean Patent Application No. 1999-23848 Korean Patent Application No. 1999-23849 Korean Patent Application No. 1998-20451 Specification Korean Patent Application No. 1999-25417 Korean Patent Application No. 2003-26731 Korean Patent Application No. 2001-42866 Specification Korean Patent Application No. 1994-34463 Specification Korean Patent Application No. 1997-24877 Specification Korean Patent Application No. 1997-81996 Specification Korean Patent Application No. 1996-33362 Korean Patent Application No. 1997-66558

  The inventors of the present invention standardized chitosan having various physiological activities and applied it to kimchi while standardizing chitosan according to the difference in the degree of deacetylation and the viscosity and using the standardized chitosan. The present invention was completed by confirming that the kimchi produced in this way has antibacterial activity, antioxidant activity, antimutagenic effect and anticancer activity even better than chitosan itself, and excellent storage stability and texture. .

  Then, an object of this invention is to provide the chitosan powder for salting standardized by the difference in a deacetylation degree and a viscosity, the salted material using the same, and kimchi manufactured using the same.

  In order to achieve the above object, according to an aspect of the present invention, water-soluble chitosan having a degree of deacetylation of 50 to 90% and a viscosity of 1 to 10 cP, a degree of deacetylation of 60 to 100% and 8 to Antibacterial, antioxidant, antimutagenic and anticancer activities which are any one or a combination selected from the group consisting of insoluble chitosan having a viscosity of 80 cP and food-acceptable salts thereof To provide excellent salted chitosan powder.

  The water-soluble chitosan or the insoluble chitosan can be used in the form of a pharmaceutically acceptable salt, and the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. Is useful. As the free acid, an inorganic acid and an organic acid can be used. As the inorganic acid, hydrochloric acid is preferable, and as the organic acid, acetic acid, citric acid, lactic acid and the like are preferable.

  The salted chitosan powder has antibacterial activity against 12 water-soluble chitosans (S-1 to S-12) and 11 insoluble chitosans (NS-1 to NS-11) having different degrees of deacetylation and viscosity The water-soluble / insoluble chitosan having excellent antioxidant activity, antimutation effect and anticancer activity was selected and normalized by the degree of deacetylation and viscosity. Among the chitosans, five types of water-soluble chitosan (S-2, S-9, S-10, S-11, S-12) and six types of insoluble chitosan (NS-2, NS-5, NS-8) NS-9, NS-10, NS-11) show excellent antibacterial activity against the strains classified by food group, and S-7, S-10 and NS-8 have excellent antioxidant activity and antimutation S-10 and NS-8 exhibit excellent anticancer effects. Accordingly, the chitosan according to the present invention comprises a water-soluble chitosan having a degree of deacetylation of 50 to 90% and a viscosity of 1 to 10 cP, and an insoluble chitosan having a degree of deacetylation of 60 to 100% and a viscosity of 8 to 80 cP. Screen and standardize by degree of deacetylation and viscosity. Among these, water-soluble chitosan (S-10) having 90% deacetylation degree and 3 cP viscosity and insoluble chitosan (NS-8) having 95% deacetylation degree and 22 cP viscosity are the most excellent. Exhibit antibacterial activity, antioxidant activity, antimutagenic effect and anticancer activity.

  Moreover, this invention provides the salting liquid containing the chitosan powder for salting normalized by the difference in a deacetylation degree and a viscosity.

  The salted solution preferably contains chitosan powder in an amount of 0.2 to 1.0% by weight based on the total weight of the salted solution. If the chitosan powder is less than 0.2% by weight, the chitosan powder content is too low to improve the storage and texture of the salted product, and if the chitosan powder exceeds 1.0% by weight In addition, the storage properties and texture of the salted product are not improved further.

  In addition, the present invention provides a salted product produced by immersing sugar beet in the salted solution. The salted product is a generic term for substances produced by immersing not only vegetables, but also fruits, meats and seafood in salted solution, and examples of vegetables are Chinese cabbage, young radish, radish, pepper, onion, potato, chili. Etc. The salted product is preferably impregnated with 0.05 to 0.25% by weight of chitosan based on the total weight of the salted product.

  Moreover, this invention provides the kimchi manufactured using the said salted product.

  The present invention also provides a kimchi comprising 0.01 to 1.5% by weight of the salted chitosan powder based on the total weight of the kimchi.

  Kimchi containing chitosan powder according to the present invention can be manufactured using a standardized kimchi recipe.

  Chitosan-containing kimchi produced by a standardized method exhibits better antibacterial, antioxidant, antimutagenic and anticancer activities than chitosan itself, so that chitosan is well-ripened so that kimchi fermentation is slowed down. It shows excellent storage safety by extending the period to reach the season. In addition, chitosan-containing kimchi suppresses the growth rate of Leuconostoc sp. And Lactobacillus sp. Lactic acid bacteria and exhibits excellent storage and storage. In addition, vegetable cellulose is a negative (−)-charged dietary fiber, but chitosan is a positive (+)-charged dietary fiber. Therefore, when chitosan is impregnated in a suitable amount in a salted product, it becomes a positive dietary fiber. Since the structure of kimchi plant cellulose becomes dense, it improves the texture of kimchi and complements the functionality of the dietary fiber itself.

  In the salted chitosan powder according to the present invention, water-soluble / insoluble chitosan excellent in antibacterial activity, antioxidant activity, antimutagenic effect and anticancer activity was selected and normalized by the degree of deacetylation and viscosity. Thereby, the chitosan containing kimchi manufactured using this shows the antibacterial activity, the antioxidant activity, the antimutagenic effect, and the anticancer activity which were further superior to chitosan itself. In addition, the chitosan-containing kimchi of the present invention exhibits excellent storage stability by increasing the time to reach the appropriate maturity period so that the fermentation of kimchi is slow, and Leuconostoc (Leuconostoc sp.) and Lactobacillus sp. (Lactobacillus sp.), which suppresses the growth rate of lactic acid bacteria and exhibits excellent storability and preservability. Since the plant cellulose has a dense structure, it has the effect of improving the texture of kimchi and complementing the functionality of the dietary fiber itself.

  Hereinafter, preferred examples will be presented to help understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and do not limit the contents of the present invention.

Example 1: Production of chitosan 1-1. Manufacture of chitosan After drying the rice crabs rice husk obtained in Tokai, Korea, it was put into 5% NaOH aqueous solution and extracted at 100 ° C. for 5 hours to obtain a deproteinized rice husk. The deproteinized rice husk was placed in a 5% HCl aqueous solution, extracted at room temperature for 5 hours, and decalcified to obtain chitin. Thereafter, the reaction vessel was filled with a 45% NaOH aqueous solution, and the chitin obtained above was immersed therein, followed by temperature (80 ° C., 90 ° C., 100 ° C., 110 ° C.) and time (5, 10, 15, (20, 25, 30, 35, 40, 50 hours), chitosan was produced by deacetylation reaction. The degree of deacetylation and viscosity of the produced chitosan were measured and shown in Table 1. Deacetylation degree was measured using a colloid titration method (pvsk titration method). Viscosity was obtained by dissolving chitosan in acetic acid to make a 0.5% chitosan-containing acetic acid solution and storing it in a constant temperature bath at 20 ° C. for 2 hours. Measured under constant temperature.

  As shown in Table 1, the degree of deacetylation and the viscosity of chitosan differ depending on the temperature and time of the 45% NaOH aqueous solution. When the reaction is carried out at a high temperature for a long time, the degree of deacetylation increases, but the viscosity decreases, and when the reaction time is shortened, the viscosity increases, but the degree of deacetylation decreases. Moreover, when it was made to react at low temperature, although the time of deacetylation took long, it turned out that a viscosity falls slowly. Therefore, chitosan specifications are expressed in terms of deacetylation degree and viscosity.

1-2. Production of standardized chitosan In order to standardize the chitosan produced in 1-1, 12 types of water-soluble chitosan (S-1 to S-12) having different degrees of deacetylation and viscosity and 11 types of insoluble were obtained. Chitosan (NS-1 to NS-11) was prepared, and these are shown in Table 2 below. All chitosans are used while stored at room temperature in a plastic container, and the antibacterial activity, antioxidant activity, antimutagenic effect and anticancer activity of water-soluble / insoluble chitosan are measured by the methods of Experimental Examples 1 to 4 below. did. Of the 23 water-soluble / insoluble chitosans, chitosan having excellent antibacterial activity, antioxidant activity, antimutation effect and anticancer activity was selected and standardized.

  As a result of measuring the antibacterial activity, antioxidant activity, antimutagenic effect and anticancer activity of water-soluble / insoluble chitosan, five water-soluble chitosans (S-2, S-9, S-10, S-11, S) -12) and six insoluble chitosans (NS-2, NS-5, NS-8, NS-9, NS-10, NS-11) exhibit excellent antibacterial activity against the publicly reported strains of food groups, It was found that S-7, S-10 and NS-8 showed excellent antioxidant activity and antimutation effect, and S-10 and NS-8 showed excellent anticancer effect. Accordingly, the chitosan according to the present invention comprises a water-soluble chitosan having a degree of deacetylation of 50 to 90% and a viscosity of 1 to 10 cP, and an insoluble chitosan having a degree of deacetylation of 60 to 100% and a viscosity of 8 to 80 cP. Were selected and normalized by the degree of deacetylation and viscosity.

Example 2: Production of chitosan-containing kimchi 2-1. Chitosan material Among water-soluble / insoluble chitosan, water-soluble chitosan S-10 (deacetylation degree 90%, viscosity 3 cP) and insoluble chitosan NS, which are most excellent in antibacterial activity, antioxidant activity, antimutation effect and anticancer activity -8 (degree of deacetylation 95%, viscosity 22 cP) was used while being stored at 4 ° C.

2-2. Kimchi Ingredients Chinese cabbage was purchased from the Busan Fujeon market in Korea, salted spicy shikoshi sushi (Daisho Co., Ltd.), and red chili powder from Yongyang Agricultural Clean Red Chili Powder Processing Factory. Daikon, green onions, daikon and ginger were purchased from the Busan Fujeong market. The sugar used was white sugar, and the salt was sun salt (Gosen Co., Ltd.).

2-3. Manufacture of Kimchi Kimchi is 3.5 parts by weight of red pepper powder, 1.4 parts by weight of bonito, 0.6 parts by weight of ginger, 2.2 parts by weight of salty spice, 2.0 parts by weight of onion A standardized kimchi recipe was prepared by mixing at a ratio of 1 part by weight, 13.0 parts by weight of radish and 1.0 part by weight of sugar.

  That is, after preparing a 10% salted solution with sun-dried salt, Chinese cabbage was soaked for 10 hours. The salted Chinese cabbage was washed 3 times with tap water and drained for 3 hours. Use water-soluble chitosan (S-10) powder and insoluble chitosan (NS-8) powder as sub-materials in an amount of 1 part by weight and 0.5 parts by weight for 100 parts by weight of salted Chinese cabbage. Combined. Cut the radish and green onion, add red chili powder dissolved in water to the shredded radish, mix well, and then mix the salted pepper, daikon and ginger well, and adjust the saltiness with each salt. Kimchi was manufactured.

Example 3 Production of Kimchi Using Chinese cabbage Soaked in Chitosan-Containing Salting Solution Insoluble chitosan NS-8 was dissolved in acetic acid to produce a 2% chitosan solution. 10% salted solution by adding sun salt to water added using the chitosan solution so that the chitosan content is 0.05, 0.15, 0.3, 0.5% with respect to the total weight of the salted solution. Manufactured. Chinese cabbage was soaked in the chitosan-containing salted solution for 10 hours. The salted Chinese cabbage was washed 3 times with tap water and drained for 3 hours. Then, chop the radish and green onion, add red chili powder dissolved in water to the shredded radish, mix well, and then mix the salted pepper, daikon and ginger well. Adjusted to produce kimchi.

Example 4: Preparation of Kimchi used after rinsing salted Chinese cabbage with a rinsing solution containing chitosan Insoluble chitosan NS-8 was dissolved in acetic acid to prepare a 2% chitosan solution. Using the chitosan solution, water was added so that the chitosan content was 0.1, 0.2, 0.3, and 0.5% with respect to the total weight of the rinsing solution to prepare a chitosan-containing rinsing solution. After preparing a 10% salted solution with sun-dried salt, Chinese cabbage was soaked in this salted solution for 10 hours. The salted Chinese cabbage was washed twice with tap water, rinsed with the chitosan-containing rinse for 3 minutes in the final rinsing process, and then drained for 3 hours. Then, chop the radish and green onion, add red chili powder dissolved in water to the chopped radish, mix, and then mix the salted pepper, daikon and ginger, and adjust the saltiness with each salt. Kimchi was manufactured.

Example 5: Production of Kimchi Using Chitosan-Containing Seasoning After producing a 10% salted solution with sun salt, Chinese cabbage was soaked in this salted solution for 10 hours. The salted Chinese cabbage was washed 3 times with tap water and drained for 3 hours. The water-soluble chitosan (S-10) powder and the insoluble chitosan (NS-8) powder were mixed at a weight ratio of 1: 1, and then 0.1.0.25, 0.00 with respect to 100 parts by weight of the salted Chinese cabbage. 5, 1.5 parts by weight of chitosan was used as an auxiliary material and mixed with the seasoning. Cut the radish and green onion, add red chili powder dissolved in water to the shredded radish, mix well, and then mix the salted pepper, daikon and ginger well, and adjust the saltiness with each salt. Kimchi was manufactured.

Experimental Example 1: Measurement of antibacterial activity To confirm the antibacterial activity of water-soluble / insoluble chitosan, the following experiment was conducted.

1-1. Strain and culture medium As the pathogenic microorganisms and spoilage microorganisms for searching antibacterial activity, 9 kinds of gram-negative bacteria and 6 kinds of gram-positive bacteria were used and are shown in Table 3 below.

  Listeria monocytogenes ATCC 19115, Staphylococcus aureus ATCC 29737, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 21366, Aeromonas hydrophila subsp. Lactobacillus plantrarum ATCC 8014, Serratia marcescens ATCC 990, Escherichia coli ATCC 8739, Vibrio parahaemolyticus ATCC 17802, and Vibrio vulnificus ATCC 27562 were used after being sold by the Korean Microbiology Conservation Center and Salmonella typhimurium ATCC 13311.

  As the culture medium, tryptic soy broth (TSB, Difco, USA), Brain Heart Infusion (BHI, Difco, USA), MRS culture solution (Difco, USA) is used, and chitosan is used. Muller Hinton broth (MHB, Merck, Germany) was used as a medium for searching for antibacterial activity.

1-2. Antibacterial activity depending on the type of chitosan For use in this experiment, water-soluble chitosan is completely dissolved in distilled water and insoluble chitosan is completely dissolved in 1% (v / v) acetic acid to give a 1% (w / v) concentration of chitosan stock solution. Manufactured.

  The antibacterial activity of chitosan was determined by examining the presence or absence of the formation of a growth-inhibiting ring (clear zone) for the published strain by the paper disc method, and the final concentration of the medium used was 0.1% in MHA. The chitosan stock solution was added as described above. At this time, NS-1, NS-3, and NS-4 chitosan among the insoluble chitosans did not dissolve, so they were not used in this experiment. The pH of the chitosan-added medium used in this experiment was corrected to pH 5.9 using 1N HCl and 1N NaOH. The antibacterial activity of 0.1% water-soluble chitosan against the official strains is shown in Table 4, and the antibacterial activity of 0.1% insoluble chitosan against the official strains is shown in Table 5.

  As shown in Table 4, the antibacterial activity of 0.1% water-soluble chitosan varied depending on the strain, but S-12 exhibited antibacterial activity against 13 strains out of 15 publicly known strains. Among the 15 chitosans, the antibacterial spectrum was the widest, and S-10 and S-11 also showed antibacterial activity against 9 strains. S-2 and S-9 showed antibacterial activity against 8 strains, S-3 and S-4 against 7 strains, S-1 and S-7 against 6 strains, S- 8 showed antibacterial activity against 5 strains, and S-5 and S-6 showed antibacterial activity against 4 strains, respectively. That is, the degree of antibacterial activity varied depending on the type of chitosan. Proteus vulgaris was inhibited by all water-soluble chitosan, and Aeromonas hydrophila, Salmonella typhimurium and Erwinia carotovora were also inhibited by most water-soluble chitosan. Bacillus cereus was also inhibited by all chitosans except S-8.

  Moreover, as shown in Table 5, 0.1% insoluble chitosan showed antibacterial activity against all the publicly known strains. At the same concentration, the antibacterial activity of chitosan was significantly higher for insoluble chitosan than for water-soluble chitosan.

1-3. Growth inhibitory activity of chitosan against pathogenic microorganisms and spoilage microorganisms Water-soluble chitosan S-2, S-9, S-10, S-11, S-12 showing high antibacterial activity and insoluble chitosan NS- showing high antibacterial activity 2, NS-5, NS-8, NS-9, NS-10, NS-11 were selected and examined for growth inhibitory activity against 15 publicly-known strains.

  In order to examine the antibacterial activity of the selected chitosan, a 1% (w / v) chitosan stock solution was added to MHB, and the final concentrations were 0.1% for water-soluble chitosan and 0.1% for insoluble chitosan. It was used after adjusting to 05%. The pathogenic microorganism is TSB, and the lactic acid bacteria are subcultured twice in MRS culture solution twice, respectively. After inoculating 0.05 mL of the chitosan-added medium into the medium supplemented with chitosan, the culture is shaken at 37 ° C. for 24 hours, and then peptone water is used. The number of viable bacteria (log No. CFU / mL) was measured by appropriately diluting with the use of a bacterium. Lactic acid bacteria were measured by virtue of the pour plate method using MRS agar, Erwinia carotovora subsp. Carotovora using BHI agar, and other spoilage and pathogenic microorganisms using TSA. The growth inhibitory activity of 0.1% water-soluble chitosan against the official strain is shown in Table 6, the growth inhibitory activity of 0.1% insoluble chitosan against the official strain is shown in Table 7, and the growth of 0.05% insoluble chitosan against the official strain is shown. The inhibitory activity is shown in Table 8.

As shown in Table 6, growth of A. hydrophila was not observed in the S-11 and S-12 chitosan addition groups, and the other three types of chitosan grew about 4-5 log cycles compared to the control group. Was suppressed. The selected chitosan showed strong antibacterial activity against E. carotovora, but no growth was observed especially in S-2, S-10 and S-11, and about 10 ^{1 in} S-9 and S-12. CFU / mL was shown and growth was suppressed by about 7 log cycles compared to the control group. P. fluorecens, V. parahaemolyticus and V. vulnificus showed 10 ⁸ CFU / mL strain in the control group, and the selected chitosan showed 10 ⁷ CFU / mL strain, which was about 1 log cycle compared to the control group. The strain was low in degree, and the growth inhibitory effect by chitosan was very low. B. subtilis showed no bacterial growth in the remaining chitosan other than S-9, and S-9 showed a bacterial count of 10 ¹ CFU / mL or less. B. cereus shows strains of 10 ¹ CFU / mL or less in S-9, S-10, and S-12, and no bacterial growth was observed in S-2 and S-11. L. curvatus, which is a lactic acid strain, has not been observed to grow in all of the selected chitosans, and L. plantarum has not been observed to grow in any chitosan other than S-11. It showed strong growth inhibitory activity against the strain. In the case of L. monocytogenes and S. aureus, no fungus growth was observed in S-12, and the remaining chitosan showed 10 ⁷ to 10 ⁸ CFU / mL, indicating fungal growth similar to the control group, and 6 species Among the gram-positive bacteria, the growth inhibition rate was the lowest for chitosan. As described above, the antibacterial activity of water-soluble chitosan was higher than that of Gram-negative bacteria against Gram-positive bacteria, and among the water-soluble chitosans, S-12 chitosan showed a broad antibacterial activity against the publicly known strains.

As shown in Table 7, in the case of A.hydrophila, the control group showed 10 ⁸ CFU / mL, NS-11 showed 10 ⁵ CFU / mL, and NS-2 showed 10 ⁴ CFU / mL. The growth of about 4 to 5 log cycles was suppressed, and the remaining chitosan also showed the number of bacteria of about 10 ² to 10 ³ CFU / mL, and the growth of about 5 to 6 log cycles was suppressed. P. vulgaris and E. carotovora showed 10 ² CFU / mL fungal growth in NS-10 after 24 hours of culture, and no fungal growth was observed in the remaining chitosans other than NS-10 chitosan. V. vulificus showed a growth of 10 ² CFU / mL with NS-5, and no growth was observed with other chitosans. L. monocytogenes, S. aureus, P. fluorecens, S. marcescens, E. coli, V. parahaemolyticus, B. subtilis, B. cereus, L. curvatus and L. plantarum are all in chitosan at 24 hours in culture. No fungal growth has been observed. As described above, insoluble chitosan showed strong antibacterial activity in all of Gram positive bacteria and Gram negative bacteria, and showed higher antibacterial activity against Gram positive bacteria.

  In the case of A. hydrophila, S-11 and S-12, which are water-soluble chitosans, showed no antimicrobial growth and superior antibacterial activity than insoluble chitosan. Insoluble chitosan showed a broad antibacterial spectrum compared to water-soluble chitosan, and also showed high bacterial growth inhibition.

  Insoluble chitosan showed high antibacterial activity at a concentration of 0.1%, but no difference in antibacterial activity depending on the type of chitosan was observed. No difference in the selective antibacterial activity of microorganisms was observed.

Moreover, as shown in Table 8, in the case of S. typhymurium, NS-2, NS-5, NS-8 showed 10 ⁸ CFU / mL at 24 hours of culture, indicating a similar strain to the control group, NS -11 showed 10 ⁵ CFU / mL growth and the remaining chitosan did not show growth. S. marcescens showed a bacterial count of 10 ¹ to 10 ² CFU / mL in all chitosans at 24 hours of culture and its growth was suppressed by about 6 to 7 log cycles compared to the control group. The difference in the degree of growth inhibition due to is not observed. Similar to S. marcescens, A. hydrophila has no difference in the degree of growth inhibition depending on the type of chitosan, but the number of bacteria is reduced by about 4 log cycles compared to the control group. Regardless of the high growth suppression degree. Among Gram-positive bacteria, L. monocytogenes and S. aureus were N-2, B. cereus was only N-9 and N-10 chitosan and showed less than 10 ¹ CFU / mL growth. No fungal growth was observed in chitosan. L.curvatus is ¹⁰ 1 CFU / mL showed bacterial growth on the order of less than or ¹⁰ 1 CFU / mL, ^L.plantarum show no bacteria growth ¹⁰ 1 ^~10 3 CFU / mL in the remaining chitosan except NS-11 The bacterial growth was remarkably suppressed as compared with the control group, but in the case of the lactic acid strain, the growth inhibitory effect of chitosan tended to be somewhat lower than that of other Gram-positive bacteria. NS-11 showed the highest growth inhibitory effect of 0.5% insoluble chitosan against the publicly reported strain, and NS-9 showed a slightly lower effect. The growth inhibitory effect of insoluble chitosan on the publicly reported strain was higher in Gram-positive bacteria than in Gram-negative bacteria, and in Gram-positive bacteria there was no difference in growth inhibitory effect depending on the type of chitosan, but in the case of Gram-negative bacteria, chitosan deacetylation The higher the degree, the higher the growth suppression effect.

1-4. Minimum inhibitor concentration (MIC) against pathogenic and spoilage microorganisms
Water-soluble chitosan S-2, S-9, S-10, S-11, S-12 showing high antibacterial activity, and insoluble chitosan NS-2, NS-5, NS-8, NS- showing high antibacterial activity 9, NS-10 and NS-11 were selected and the minimum growth inhibitory concentrations for these 15 publicly known strains were measured.

  In order to determine the minimum growth inhibitory concentrations of water-soluble chitosan and insoluble chitosan for the publicly known strains, the agar diffusion method was used, and after culturing at the appropriate growth temperature for each of the publicly known strains for 72 hours, the minimum growth inhibitory concentrations were measured. did.

  The MIC measurement results for water-soluble chitosan are shown in Table 9, and the MIC measurement results for insoluble chitosan are shown in Table 10. Moreover, the high antibacterial activity chitosan standard group according to food groups is shown in Table 11.

  As shown in Table 9, the MIC range of water-soluble chitosan for the official strains was 0.05-0.8%,> 0.8%, but in the case of lactic acid strains, the MIC was lower than other test strains showed that. In the case of L. curvatus, S-2 and S-11 showed 0.05% MIC, and the remaining chitosan showed 0.08% MIC. L. plantarum showed the highest MIC of 0.1% for S-10, 0.0-12% for S-12, 0.05% for S-2, S-9 and S-11. The MICs of B. cereus and B. subtilis were 0.1-0.3%, depending on the type of chitosan, but the MICs of B. subtilis were slightly lower than those of B. cereus. In the case of E. carotovora, the MIC of S-11 and S-12 is 0.08%, indicating a very low MIC compared to other chitosans. E. carotovora is the lowest MIC among gram-negative bacteria showed that.

  Further, as shown in Table 10, the MIC range of insoluble chitosan for the publicly reported strains is NS-2, NS-5 for A.hydrophila, NS-2 for P.vulgaris, NS-5 for E.coli, Sal.typhimurium. NS-2, NS-5, NS-9, NS-11 each show> 0.1% MIC, and the remaining MIC range of chitosan is 0.03-0.1%, water soluble It showed a very low MIC value compared to chitosan. The MIC range of Gram-positive bacteria was 0.03 to 0.08%, and the MIC of insoluble chitosan against the published strain was mostly 0.05%. In contrast, the MIC range for Gram-negative bacteria is 0.05-1% or> 1%, indicating a broad MIC value compared to the Gram-positive group, and the MIC of insoluble chitosan for Gram-negative bacteria is mostly 0. The MIC of insoluble chitosan was also lower for Gram-positive bacteria than Gram-negative bacteria, indicating 08%.

  In addition, as shown in Table 11, 5 types of water-soluble chitosan (S-2, S-9, S-10, S-11, S-12) and 6 types of insoluble chitosan (NS-2, NS-5) NS-8, NS-9, NS-10, NS-11) show excellent antibacterial activity against the public strains classified by food group, so that it can improve the shelf life of foods, such as seafood, meat, and vegetables. It is effective in preventing caused corruption and food poisoning.

  According to the above results, it is related to the antibacterial activity of chitosan against the publicly known strain, and in the case of water-soluble chitosan, the degree of deacetylation is 60% or more, and in the case of insoluble chitosan, the degree of deacetylation is 89% or more, It showed very good antibacterial activity. Moreover, the growth inhibitory activity and the minimum growth inhibitory concentration of the selected chitosan against the publicly known strain were higher in the insoluble chitosan than in the water-soluble chitosan, and more effective in the gram positive group than in the gram negative group.

Experimental Example 2: Measurement of antioxidant activity In order to confirm the antioxidant activity of water-soluble / insoluble chitosan, the following experiment was conducted.

2-1. DPPH (1,1-diphenyl-2-picrylhydrazyl) elimination effect The electron donating ability of water-soluble / insoluble chitosan to DPPH was measured by the method of Blois et al. 10 μL of water-soluble / insoluble chitosan diluted with methanol (1 mg / mL, 0.5 mg / mL) was added to a 96-well plate, 100 μL of 60 μm DPPH reagent solution was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then absorbance at 540 nm. Was measured.

  The antioxidant effect of water-soluble chitosan against DPPH free radicals is shown in FIG. 1, and the antioxidant effect of insoluble chitosan against DPPH free radicals is shown in FIG.

  As shown in FIGS. 1 and 2, water-soluble chitosan S-9 and S-10 showed 79% and 74% radical scavenging ability at a concentration of 1 mg / mL, respectively, and even at a low concentration of 0.5 mg / mL. High antioxidant activity of 77% and 72%, respectively. It was found that the antioxidant activity between these two samples did not vary greatly with concentration. However, the S-4 sample showed 83% activity at a concentration of 1 mg / mL, but showed 48% activity at a concentration of 0.5 mg / mL. From these results, it can be seen that the change in antioxidant activity of water-soluble chitosan is greatly influenced by the concentration. On the other hand, insoluble chitosan showed an antioxidant activity of 40 to 50% without significant change in the antioxidant activity.

2-2. Antioxidation experiment in cellular system 1) Cell types and reagents LLC-PK ₁ (porcine renal epithelial cell) is ATCC (Solon, Ohio, USA), DMEM (Dulbecco's modified Eagal medium) and FBS (fetal bovine) for culture. serum) is Invitrogen CO. Used from (Grand Island, NY). The SNP used to induce oxidative stress is a product of Wako (Tokyo, Japan), SIN-1 (3-morpholinosydnonimine), AAPH [2,2'-Azobis (2-aminopropane) dihydrochloride], pyrogallol. (pyrogallol), MTT [3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide] is available from Sigma Chemical Co. (USA) product was used.

2) Cell Culture LLC-PK ₁ cells were cultured in a 37 ° C., 5% CO ₂ incubator using DMEM containing 100 units / mL phenicillin-streptomycin and 5% FBS. The cultured cells were re-supplied 2-3 times a week, and after primary washing with phosphate buffered saline (PBS) around 6-7 days in culture, 0.05% trypsin-0.02% After detaching the adherent cells with EDTA and centrifuging, the accumulated cells were put into a medium, mixed well with a pipette so that the cells were uniformly dispersed, and used for experiments while being subcultured every 6 to 7 days. At the time of subculture, the number of passages was recorded, and when the number of passages was 10 times or more, new cells were cultured for experiments.

3) Cell survival When cells are in a confluence state, peroxyl radicals are used to induce oxidative stress after planting in 96-well plates at 1 × 10 ⁴ cells / mL per well and culturing for 2 hours. (LOO ⁻ ), ONOO ⁻ , NO, O ₂ ⁻ donors AAPH (10 mM), SIN-1 (1 mM), SNP (1.2 mM) and pyrogallol (1.2 mM) are added and cultured for 24 hours. did. After inducing oxidative stress, S-10 and NS-8 were treated with different concentrations (50, 100, 500, 1000 μg / mL) and cultured for 24 hours, and then 1 mg / mL MTT solution was injected into each well. After re-culturing at 37 ° C. for 4 hours, the produced formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at 540 nm (Mosmann, 1983).

Table 12 shows the results of the antioxidant activity of water-soluble chitosan (S-10) and insoluble chitosan (NS-8) on LLC-PK ₁ cells treated with AAPH according to the concentration, and SIN-1 was treated. Table 13 shows the results of the antioxidant activity of water-soluble chitosan (S-10) and insoluble chitosan (NS-8) against LLC-PK ₁ cells according to concentration.

As shown in Table 12, water-soluble chitosan (S-10) dissolved in PBS and insoluble chitosan (NS-) dissolved in 1% acetic acid were selected through a screening process for LLC-PK ₁ cells treated with AAPH. As a result of considering the antioxidant activity according to 8) by concentration, the cell viability of the control group treated with only AAPH was 23.6%, but the cells after treatment with S-10 and NS-8 by concentration were each The survival rate increased in a concentration-dependent manner. In the case of water-soluble chitosan, the cell survival rate was 67.1% at 1000 μg / mL, and increased compared to the control group. Therefore, water-soluble / insoluble chitosan showed an excellent improvement effect on oxidative stress of LLC-PK ₁ cells by AAPH, and particularly water-soluble chitosan showed further superior antioxidant activity than insoluble chitosan.

Moreover, as shown in Table 13, the control group treated with SIN-1 alone decreased the cell viability to 20.4% due to cell damage due to oxidative stress, whereas S-10 and NS-8 The cell viability after treating chitosan according to concentration increased in a concentration-dependent manner, and was 72.9% and 64.2%, respectively, when treated with 1000 μg / mL. This seems to have shown an ameliorating effect on oxidative stress through direct erasing ability against ONOO ⁻ .

Experimental Example 3: Measurement of antimutagenic effect The following experiment was conducted to confirm the antimutagenic effect of water-soluble / insoluble chitosan and chitosan-containing kimchi.

1-1. Reagents and strains D-biotin, L-histidine.HCl (monohydrate), D-glucose-6-phosphate (monosodium salt), and NADP (sodium salt) are available from Sigma Chemical Co. (USA) and Bact nutrient cultures (dehydrated) and Bitek agar were purchased from Difco Laboratories (USA). Further, as a mutagenesis source, MNNG (4-methyl-N′-nitro-N-nitrosoguanidine), which is a direct mutagenesis, is obtained from Aldrich Chemical Co. (USA), dissolved in sterilized distilled water and used for experiments. AFB ₁ (aflatoxin B ₁ ), an indirect mutagen, was obtained from Sigma Chemical Co. (St.Louse, MO, USA) was used by dissolving in DMSO.

  The strain used in the experiment was Salmonella typhimurium TA100, which is Ames, B., University of California, USA. N. Used as provided by Dr. These strains were used after confirming genetic traits such as histidine requirement, deep rough (rfa) mutation, uvrB mutation, and R-factor immediately before the experiment.

  The kimchi sample was prepared by squeezing the initial kimchi of the chitosan-containing kimchi produced in Example 2 and the ripening kimchi (pH 4.3) with a juicer (NUC, Korea) and two kinds Of methanol extract was used. The sap sample was collected in the form of a sap, and then centrifuged at 4 ° C. and 9000 rpm for 15 minutes, and the supernatant was collected by filtering through a Millipore filter (0.20 μm) to be sterilized. used. The methanol extract was prepared by grinding each chitosan kimchi fermented to the initial stage and the optimum ripening period of pH 4.3, lyophilized, and then grinding the sample to prepare a powder. / V) methanol was added, and the mixture was stirred twice for 12 hours, filtered, and then concentrated by a rotary vacuum concentrator. The extract was diluted with DMSO and used for experiments.

1-2. Antimutagenic effect on direct mutagen (MNNG) 0.1 mL of a strain (1-2 × 10 ⁹ cells / mL) cultured overnight in 0.5 mL phosphate buffer (direct mutagen), diluted sample 50 μL And 50 μL of the mutagen was added to the cap tube in an ice bath and vortexed lightly and pre-incubated at 37 ° C. for 30 minutes. Here, 2 μL of 45 ° C. top agar (histidine / biotin solution) was poured into each pre-cultured tube, vortexed for 3 seconds, smeared on a minimal glucose agar plate, and cultured at 37 ° C. for 48 hours. The number of revertants was counted. The inhibition rate was calculated according to Equation 1. The antimutagenic effects of water-soluble and insoluble chitosan against mutations induced directly in Salmonella typhimurium TA100 by mutagen MNNG (0.4 μg / plate) are shown in Table 14 and Table 15, respectively, and methanol extraction of chitosan-containing kimchi The antimutagenic effect of the product is shown in Table 16 and FIG.

Inhibition rate (%) = [(ab) / (ac)] × 10 (Expression 1)
a: the number of back mutations induced by the mutation source,
b: number of back mutations when the sample was processed,
c: Number of spontaneous reversion mutations in the absence of mutation source and sample.

  As shown in Tables 14 and 15, water-soluble chitosan had a higher antimutagenic effect at a higher concentration of 2.5 mg / plate than at a lower concentration of 1.25 mg / plate. When water-soluble chitosan was treated at a concentration of 2.5 mg / plate, S-7 showed an antimutagenic effect of 55%, whereas S-10 had a higher anti-mutation effect depending on the concentration added. Mutation effect was shown. Among the water-soluble chitosans, the antimutagenic effect of S-7 and S-10 was as high as 55%, and among the insoluble chitosans, NS-8 had a suppression rate of 70% and the antimutagenic effect was the highest. . Most water soluble / insoluble chitosans also significantly increased the antimutagenic effect with increasing concentration. In addition, the water-soluble / insoluble chitosan increased the antimutagenic effect as the degree of deacetylation increased and the viscosity decreased. This experiment provides an important basis for screening 23 chitosans.

  Further, as shown in Table 16 and FIG. 3, when Kimchi supplemented with 1% insoluble chitosan NS-8 was treated at a concentration of 1.25 mg / plate, an antimutagenic effect of 64% was exhibited. This was higher than the antimutagenic effect of standardized kimchi (41%). In addition, when chitosan-containing kimchi was treated at a concentration of 2.5 mg / plate, 1% of water-soluble chitosan S-10 and 1% of insoluble chitosan NS-8 were 77%. Mutation effect was shown. This was also higher than the antimutagenic effect of standardized kimchi (58%). Thus, it was found that water-soluble / insoluble chitosan-added kimchi exhibits a higher antimutagenic effect than standardized kimchi. In addition, kimchi with insoluble chitosan NS-8 shows higher antimutagenic effect than kimchi with water-soluble chitosan S-10, and 1% chitosan is added to kimchi with 0.5% chitosan. Kimchi showed high antimutagenic effect.

1-3. Antimutagenic effect on indirect mutagen (MNNG) To activate the indirect mutagen, an S9 mixture, a microsomal enzyme mixture of liver, was prepared by the method of Maron and Ames. For induction of liver enzymes in approximately 200 g of male SD (Sprague-Dawley) rats, Arochlor 1254, a polychlorinated biphenyl (PCB) mixture, was diluted once at a concentration of 200 mg per mL of corn oil and injected once (500 mg / kg). ) After 5 days, the liver was removed. The liver removed aseptically at 4 ° C. was washed several times with 0.15 M KCl, a 0.15 M KCl solution corresponding to 3 times the weight of the liver was added, and the mixture was homogenized (Potter-Elvehjem apparatus, USA). Homogenized. This was centrifuged at 9000 × g for 10 minutes to obtain an S9 fraction as a supernatant. Thereafter, 1 to 2 mL was dispensed into a cryo tube and rapidly frozen with dry ice, and then stored in a liquid nitrogen tank at −180 ° C. and used for experiments. The S9 fraction (10%) was mixed with MgCl—KCl salt (2%), 1M glucose-6-phosphate (0.5%), 1M NADP (4%), 0.2M phosphate buffer (pH 7.4). ) And sterilization number to prepare S9 mixture.

The experiment was carried out in the same manner as in the above method 1-2 except that the S9 mixture (indirect mutagenesis source) was used instead of the phosphate buffer (direct mutagenesis source). The antimutagenic effect of chitosan-containing kimchi methanol extract against mutations induced by the indirect mutagen AFB ₁ (0.5 μg / plate) in Salmonella typhimurium TA100 is shown in Table 17 and FIG.

As shown in Table 17 and FIG. 4, kimchi to which 0.5% of insoluble chitosan NS-8 was added at a low concentration of 1.25 mg / plate showed a suppression rate of 67% and had the highest antimutagenic effect. Kimchi to which 1% of water-soluble chitosan S-10 was added showed an inhibition rate of 63%. This was higher than the antimutagenic effect of standardized kimchi (53%). When chitosan-added kimchi was treated at a concentration of 2.5 mg / plate, kimchi to which 0.5% of water-soluble chitosan S-10 was added showed a suppression rate of 92%, and the antimutagenic effect was very large. Kimchi to which 1% of water-soluble chitosan S-10 was added showed an inhibition rate of 87%. This is higher than the antimutagenic effect of standardized kimchi (77%). Therefore, kimchi to which water-soluble / insoluble chitosan was added showed a tendency similar to the antimutagenic effect in MNNG in the antimutagenic effect on AFB ₁ which is an indirect mutation source.

Experimental Example 4: Measurement of antibody activity The following experiment was performed to confirm the anticancer activity of water-soluble / insoluble chitosan and chitosan-containing kimchi.

4-1. Cell culture RPMI1640, FBS, 0.05% trypsin-0.02% EDTA and 100 units / mL phenicillin-streptomycin were purchased from GIBCO (USA) and used for cell culture. A 5% CO ₂ incubator (Forma, model MC096, Japan) was used for cell culture. AGS human gastric cancer cells (AGS human gastric adenocarcinoma cells) and HT-29 human colon adenocarcinoma cells (HT-29 human colon adenocarcinoma cells) were used in the experiments after being sold by the South Korea Cell Line Bank (Seoul Medical University).

AGS human gastric cancer cells and HT-29 human colon cancer cells were cultured in RPMI 1640 containing 100 units / mL phenicillin-streptomycin and 10% FBS in a 5% CO ₂ incubator. Each cultured cancer cell was re-fed 2-3 times a week and washed with PBS on days 6-7, and then the adherent cells were detached with 0.05% trypsin-0.02% EDTA and centrifuged. After separation, put the accumulated cancer cells in a medium and mix well with a pipette so that the cancer cells are uniformly dispersed, and inject a fixed number of 10 mL into a 75 mL cell culture flask every 6-7 days Were used for experiments while being subcultured. The number of passages was recorded at the time of subculture, and when the number of passages was 10 times or more, new cancer cells were taken out of the liquid nitrogen tank and cultured again for experiments.

4-2. MTT assay The cultured cancer cells were dispensed into a 96-well plate at 180 μL at a concentration of 1 × 10 ⁴ cells / mL per well, and 20 μL of each sample was added according to the concentration, followed by incubation at 37 ° C. in a 5% incubator. Cultured for 72 hours. To this, 20 μL of MTT (Sigma, USA) solution produced at a concentration of 5 mg / mL was added to phosphate physiological saline, and further cultured for 4 hours under the same culture conditions. After centrifugation at 2000 rpm for 10 minutes, the supernatant was discarded, 150 μL of DMSO was added, and the plate was shaken for 30 minutes. At this time, the produced formazan crystals were dissolved in DMSO, and the absorbance was measured at 540 nm using an ELISA reader. Cytotoxicity (%) was calculated using Equation 2.

  Cytotoxicity rate (%) = [(absorbance of control group−absorbance of sample treatment group) / absorbance of control group] × 100 (Equation 2)

  The growth inhibitory effect of water-soluble chitosan on AGS human gastric cancer cells is shown in Table 18, the growth inhibitory effect of water-soluble chitosan on HT-29 human colon cancer cells is shown in Table 19, and insoluble chitosan on HT-29 human colon cancer cells. The growth inhibition effect is shown in Table 20. The growth inhibitory effect of chitosan-containing kimchi methanol extract on AGS human gastric cancer cells is shown in Table 21, and the growth inhibitory effect of chitosan-containing kimchi methanol extract on HT-29 human colon cancer cells is shown in Table 22.

  As shown in Table 18, the anticancer effect of water-soluble / insoluble chitosan on AGS human gastric cancer cells showed that S-10 showed the highest anticancer effect at a high concentration of 5 mg / mL, and as the concentration increased, the anticancer effect was increased. The effect also increased rapidly from 8 to 88%.

  Further, as shown in Table 19 and Table 20, HT-29 human colon cancer cells showed the same tendency as AGS human gastric cancer cells, with a high concentration of chitosan of 5 mg / mL compared to a low concentration of 1 mg / mL of chitosan. It showed a high anticancer effect. Water-soluble chitosan S-7 and S-8 showed the highest anticancer effect of 43 to 44%, and insoluble chitosan NS-8 and NS-9 showed very high anticancer activity of 83%.

  Further, as shown in Table 21, the effect of suppressing the growth of cancer cells on AGS human gastric cancer cells by the methanol extract of chitosan-containing kimchi was 1% S-10-containing kimchi and 1% NS-8 at a high concentration (200 μg / mL). In the case of containing kimchi, it was 66% and 89%, respectively, and showed higher cancer cell growth inhibitory effect than standardized kimchi (38%). Further, as shown in Table 22, the effect of suppressing the growth of cancer cells on HT-29 human colon cancer cells by the methanol extract of chitosan-containing kimchi was 1% S-10-containing kimchi and 1% at a high concentration (200 μg / mL). In the case of NS-8 containing kimchi, 47% and 63%, respectively, showed a higher cancer cell growth inhibitory effect than standardized kimchi (32%). As described above, it can be seen that kimchi to which insoluble chitosan is added exhibits a superior anticancer effect than kimchi to which water-soluble chitosan is added.

Experimental Example 5: Storage stability verification of chitosan-containing kimchi In order to confirm the storage stability of chitosan-containing kimchi according to the present invention, fermentation characteristics were observed every 5 days while kimchi was fermented at 4 to 15 ° C.

5-1. Measurement of changes in pH and acidity during kimchi fermentation As the sample, the chitosan-containing kimchi produced in Examples 2 to 5 was squeezed with a squeezer (NUC, Korea), and the juice was used. The pH was measured at room temperature with a pH meter (Corning 220, USA). The acidity was measured by AOAC method after taking 20 mL of a sample 20 times with distilled water and taking 10 mL from it. At this time, 1 mL of 0.1% phenolphthalein was added as an indicator, 0.1N NaOH was titrated, and the point of pink color was defined as the end point. The titration value was converted to lactic acid using Equation 3 and expressed as% content.

  Lactic acid (%) = {(mL of 0.1N NaOH × normal concentration of NaOH) / weight of sample (g)} × 9 (Equation 3)

  Changes in pH and acidity of water-soluble / insoluble chitosan-containing kimchi fermented at 15 ° C. (Example 2) are shown in Table 23, and kimchi produced using Chinese cabbage soaked in a salted solution containing chitosan fermented at 4 ° C. The changes in pH and acidity of (Example 3) are shown in Table 24. The pH and acidity of Kimchi (Example 4) produced after rinsing Chinese cabbage fermented with salt fermented at 4 ° C. with a rinsing solution containing chitosan. Changes are shown in Table 25, and changes in pH and acidity of Kimchi (Example 5) produced using chitosan-containing seasonings fermented at 4 ° C. are shown in Table 26.

  As shown in Table 23, considering the change in pH immediately after making kimchi, kimchi to which insoluble chitosan was added maintained a remarkably high pH. As the fermentation progresses, the fermentation rate of kimchi with chitosan added is slower than the fermentation rate of standardized kimchi, and the pH of kimchi with 1% S-10 added is 3.91 when the pH on the seventh day is compared. The pH of standardized kimchi was as low as 3.71. Further, the lower the concentration, the lower the pH, and the pH of kimchi with 0.5% NS-8 added was 3.73, which was almost similar to standardized kimchi. The kimchi to which chitosan was added passed through a suitable maturity period on the fourth day, and the fermentation proceeded more rapidly. On the seventh day of fermentation, the pH showed a similar value. Also, in terms of changes in acidity, the acidity of insoluble chitosan-containing kimchi was initially significantly lower than that of water-soluble chitosan-containing kimchi, but the acidity change was similar as fermentation progressed. As described above, the addition of chitosan in the early stage of fermentation slows down the rate of kimchi fermentation, which seems to be due to the effect of chitosan suppressing the initial fermentation.

  Further, as shown in Table 24, as the chitosan concentration in the salt water in which Chinese cabbage was soaked was increased to 0.05%, 0.15%, 0.3%, and 0.5%, the pH was slowly decreased. In the case of kimchi produced using Chinese cabbage pickled with 0.3% chitosan-containing salted solution, pH 4.2-4.4 and acidity 0.6-0.7% are maintained until 15-25 days of fermentation. In the case of kimchi made with Chinese cabbage soaked in 0.5% chitosan-containing salted solution, the optimal maturity period is reached only on the 35th day, and the pH remains below 4.0 even on the 35th day. It did not decrease. This shows that when compared with the fermentation aspect of standardized kimchi, the storability increases and the edible period becomes longer. In contrast, in the case of kimchi produced using Chinese cabbage soaked with 0.05% and 0.15% chitosan-containing salted solution, the optimal maturity period was reached at a time similar to standardized kimchi.

  Also, as shown in Table 25, kimchi produced after rinsing Chinese cabbage soaked in salt with a rinsing solution containing chitosan also has a greater storage than standardized kimchi, as it reached the proper maturity on the 15th. I understand. However, there is no difference between the chitosan addition concentrations.

  Also, as shown in Table 26, kimchi produced using a water-soluble / insoluble chitosan-containing (0.1, 0.25, 1.5%) seasoning reached the appropriate maturity on the 15th, It was confirmed that the kimchi produced using the seasoning containing 5% water-soluble / insoluble chitosan reached the suitable maturity period (pH 4.34) on the 10th day like the standardized kimchi. The acidity also shows a tendency similar to pH, and it can be seen that kimchi produced using a water-soluble / insoluble chitosan-containing spice has an increased storability over standardized kimchi. However, there is no difference between the chitosan addition concentrations.

  As a result of observing the change in pH and acidity, kimchi produced using Chinese cabbage pickled with a chitosan-containing salted solution contains kimchi produced after rinsing salted Chinese cabbage with a rinsing solution containing chitosan and water-soluble / insoluble chitosan-containing It showed the best storage and storage compared to kimchi produced with spices.

  As described above, chitosan-containing kimchi has a longer time to reach a suitable maturation period than fermented kimchi because chitosan slowly ferments kimchi and increases its storage and storage. It can be seen that the storage stability is excellent.

5-2. Measurement of the number of lactic acid bacteria during kimchi fermentation The effect of chitosan-containing kimchi produced in Examples 2 to 5 on the growth of kimchi lactic acid bacteria, namely Leuconostoc sp. And Lactobacillus sp. .

  Kimchi improves the taste and flavor of kimchi while Leuconostoc sp. Lactic acid bacteria are produced in the early stages of fermentation, and suppresses the growth of aerobic bacteria, which are miscellaneous bacteria, giving a refreshing taste. As it progresses, Lactobacillus sp. Lactic acid bacteria that give a sour taste increase, pH decreases, and acidity increases.

The number of lactic acid bacteria during kimchi fermentation was measured using a plate count technique. Changes in the number of microbial bacteria during kimchi fermentation and aging were performed by diluting ¹ mL of the mixed solution in a stepwise manner to 10 ¹ to 10 ⁷ with sterilized distilled water, and heating and thawing 0.1 mL of each diluted solution in advance. After charging and mixing in 10 mL of MRS medium cooled to 45 ° C., a plate was made on a Petri dish and cultured for 48 hours in a 37 ° C. incubator, and the number of colonies that appeared was counted as the number of lactic acid bacteria. The medium used was an MRS agar medium mainly used for isolation of lactic acid bacteria. The composition of the MRS agar medium is shown in Table 27. Lactobacillus medium is a modified LBS agar medium (m-LBS medium, Table 28) in which acetic acid and sodium acetate are added to Lactobacillus selective medium (LBS medium) to inhibit the growth of Pediococcus. ) For 3 days at 30 ° C. As a Leuconostoc selective medium, a phenylethyl alcohol sucrose agar medium (PES medium, Table 29) supplemented with phenylethyl alcohol and sucrose was plated at 20 ° C. for 3 days.

  Changes in the numbers of Leuconostoc sp. And Lactobacillus sp. During fermentation of chitosan-containing kimchi (Example 2) at 15 ° C. are shown in FIG. 5, and Chinese cabbage pickled in a salted solution containing chitosan The changes in the number of Leuconostoc sp. And Lactobacillus sp. During fermentation at 4 ° C. of kimchi produced using sucrose (Example 3) are shown in FIG. Changes in the numbers of Leuconostoc sp. And Lactobacillus sp. During the fermentation at 4 ° C. of kimchi produced after rinsing with a chitosan-containing rinsing solution are shown in FIG. Changes in the numbers of Leuconostoc sp. And Lactobacillus sp. During fermentation at 4 ° C. for kimchi produced using a chitosan-containing spice (Example 5) are shown in FIG.

  As shown in FIG. 5 to FIG. 8, the chitosan-containing kimchi according to the present invention suppresses the growth rate of Leuconostoc sp. And Lactobacillus sp. It turns out that it shows preservability.

Experimental Example 6: Measurement of texture of chitosan-containing kimchi The following experiment was performed to confirm the texture of the chitosan-containing kimchi according to the present invention during the storage period.

The kimchi produced in Example 3 fermented for 3 weeks and 4 weeks (using salted solution containing 0.3% and 0.5% chitosan) was cut into 10 cm ² pieces to prepare specimens, and the cutting strength of the specimens Was measured to judge the sense of organization. The results are shown in Table 30.

  As shown in Table 30, it can be seen that the chitosan-containing kimchi according to the present invention has a much better texture than the standardized kimchi.

Experimental Example 7: Measurement of chitosan content of salted Chinese cabbage soaked in chitosan-containing salted solution and kimchi produced using the salted Chinese cabbage soaked in chitosan-containing salted solution according to the present invention, and kimchi produced using the same In order to confirm the chitosan content, the following experiment was conducted.

  The chitosan content of the salted Chinese cabbage soaked in the salted solution containing 0.3% and 0.5% chitosan in Example 3 and the kimchi produced using the salted Chinese cabbage was measured. The results are shown in Table 31.

  As shown in Table 31, the salted Chinese cabbage soaked in the salted solution containing chitosan and the kimchi produced using the same are impregnated into the salted Chinese cabbage by the salting concentration, thereby improving the texture of the kimchi and the dietary fiber itself. Complement the functionality of. That is, vegetable cellulose is a negative (−)-charged dietary fiber, but chitosan is a positive (+)-charged dietary fiber. Therefore, when chitosan is impregnated in a proper amount in a salted product, it becomes a positive dietary fiber. Since the structure of kimchi's vegetable cellulose becomes dense, it is considered that the texture of kimchi is improved and the functionality of the dietary fiber itself is complemented.

It is a figure which shows the antioxidant effect of water-soluble chitosan with respect to a DPPH free radical. It is a figure which shows the antioxidant effect of insoluble chitosan with respect to DPPH free radical. FIG. 6 shows the antimutagenic effect of chitosan-containing kimchi methanol extract against mutations induced by MNNG (0.4 μg / plate) as a direct mutagen in Salmonella typhimurium TA100. FIG. 2 shows the antimutagenic effect of chitosan-containing kimchi methanol extract against mutations induced by AFB ₁ (0.5 μg / plate) as an indirect mutation source in Salmonella typhimurium TA100. It is a figure which shows the change of the number of the genus Leuconostoc (Leuconostoc sp.) And the genus Lactobacillus (Lactobacillus sp.) During fermentation at 15 degreeC of chitosan containing Kimchi (Example 2). The figure which shows the number change of Leuconostoc sp (Leuconostoc sp.) And Lactobacillus sp (Lactobacillus sp.) During the fermentation at 4 degreeC of kimchi (Example 3) manufactured using the Chinese cabbage pickled with the salt solution containing chitosan. It is. Changes in the numbers of Leuconostoc sp. And Lactobacillus sp. During the fermentation at 4 ° C. of kimchi produced after rinsing salted kimchi with a rinsing solution containing chitosan FIG. It is a figure which shows the number change of Leuconostoc sp. (Leuconostoc sp.) And Lactobacillus sp. (Lactobacillus sp.) During fermentation at 4 degreeC of kimchi (Example 5) manufactured using chitosan containing seasoning.

Claims (8)

  Water-soluble chitosan having a degree of deacetylation of 50-90% and a viscosity of 1-10 cP, insoluble chitosan having a degree of deacetylation of 60-100% and a viscosity of 8-80 cP, and its food acceptable A salted chitosan powder excellent in antibacterial, antioxidant, antimutation and anticancer activity, which is one or a combination of one or more selected from the group consisting of salts.   2. The salted chitosan according to claim 1, wherein the foodically acceptable salt is any one selected from the group consisting of hydrochloride, acetate, citrate and lactate. Powder.   A salting solution containing 0.2 to 1.0% by weight of chitosan powder for salting according to claim 1 or 2 based on the total weight of the salting solution.   A salted product produced by immersing a vegetable in the salted solution according to claim 3.   The salted product according to claim 4, wherein the sugar beet is at least one selected from the group consisting of Chinese cabbage, young radish, radish, pepper, onion, daikon and chili.   The salted product according to claim 4, wherein the salted product is impregnated with 0.05 to 0.25% by weight of chitosan based on the total weight of the salted product.   Kimchi manufactured using the salted thing of any one of Claims 4-6.   Kimchi containing 0.01 to 1.5% by weight of the salted chitosan powder according to claim 1 or 2 based on the total weight of the kimchi.

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