KR100607371B1 - Use of Raja kenojei extract - Google Patents

Abstract

The present invention is to investigate the changes of the components, pH and ammonium ions according to the fermentation temperature and process of the hot water extract of the edible portion, viscera, brain and cartilage of the skate, and evaluated their antibacterial, anticancer and antioxidant physiological functions The present invention relates to providing an extract, and has an excellent effect of providing a hot water extract for each part of a skate which is fermented at 10 ° C. for 10 days having antibacterial, anticancer and antioxidant activity.

Skates, guts, edibles, brain, cartilage, antibacterial, antioxidant, anticancer

Description

Use of Raja kenojei extract

1 is a graph showing the pH change of the skate effluent with the fermentation temperature.

2 is a graph showing the change of ammonium ions of the skate effluent with the fermentation temperature.

Figure 3 is a graph showing the antibacterial activity of the skates visceral hot water extracts of various concentrations (a: 0.5%; b: 1%; c: 2%).

Figure 4 is a graph showing the antibacterial activity of the hydrothermal extract of the skates brain (a) and cartilage (b) of various concentrations.

Figure 5 is a graph showing the antioxidant activity of the hydrothermal extract of the decorative portion (a), viscera (b) and cartilage (c) of the skate.

Figure 6 is a graph showing the anticancer activity of hydrothermal extracts of the decorative part (a), viscera (b), brain and cartilage (c) of skate.

The present invention relates to the use of the skate extract, and more particularly, the present invention investigates the changes of the components, pH and ammonium ions according to the fermentation temperature and process of the hot water extract of the edible portion, gut, brain and cartilage of the skate , To evaluate the antimicrobial, anticancer and antioxidant physiological function of the present invention to provide the use of the skate.

Skates ( Skate, Raja kenojei Muller et Henle) are a stiletto cartilage and benthic fish belonging to the family Stingray, which lives up to about 3,000 m in the depths of most of the world's waters. As a unique group, about 280 species have been reported around the world, accounting for about a quarter of the total number of cartilage fish (900 to 1100 species). It is widely distributed in the southwest sea of Korea, the central south sea area of Japan, and the East China Sea, and is particularly caught in Mokpo, Yeonggwang, and Busan. The stocks of skates are squids, squids, crabs, crayfish, and so on. In addition, fermented as a traditional food in Mokpo, etc., and enjoyed a favorite, but now the favorite population is gradually increasing with a unique aroma and taste. Skate's unique aroma is because it contains a lot of urea and urea precursors inside for deep osmotic pressure control. In addition, as the fermentation proceeds, a nose-smelling and tangy taste is formed, and the produced ammonia inhibits the growth of harmful bacteria and gives off a distinctive smell of skate. In skates and its fermented products, free amino acids such as anserine, taurine, alanine, and lysin have been detected. It is known to be involved in the transport of iron ions for the activation of ATPase (myrosine ATPase) and the activation of cytochrome oxidase. Taurine is also produced more in the skate, which is said to have several physiological functions in addition to preventing cholesterol accumulation. At present, however, fatty acid contents such as stingray, mackerel, herring, salmon, cod, and shark have been reported, and fatty acid contents have been reported for skates. Did. In addition, although the skate is known as a traditional fish fermented food, the seafood processing plant still ferment the skate in a classic way. However, the demand for fermented skates is increasing, reaching about 20 billion per month only in Jeonnam area, and it is very necessary to scientifically and uniformize the fermented foods at the time of spread nationwide.

Accordingly, an object of the present invention is to investigate the changes in the components, pH and ammonium ions according to the fermentation temperature and process of hot water extracts for each portion of the skate, gut, brain and cartilage of skates, their antimicrobial, anticancer and antioxidant physiology By evaluating the functionality it is intended to provide the use of the skates extract.

The object of the present invention is to ferment the skate for a variety of temperatures and time to determine the optimum fermentation temperature after investigating changes in ingredients, pH and ammonium ions and to obtain the hot water extract for each part of the skate and their antimicrobial effect, antioxidant and anticancer Achieved by evaluating sex.

Hereinafter, the specific structure of this invention is demonstrated.

The present invention is a fermentation step of the skate in various temperature and time conditions; Investigating changes in the component, pH and ammonium ions of the skate; This study consists of evaluation of antimicrobial effect, antioxidant activity and anticancer activity of hot water extract of each part of skate.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1: Preparation of the present invention Skate hot water extract

Skate ( Raja kenojei Muller et Henle) used in the present invention was transported to the laboratory in the frozen state of storage in Yeongsanpo, Naju-si, Jeollanam-do, completely thawed and then washed the surface twice with tap water and once with distilled water. The surface was dried with sterile gauze and then fermented for up to 16 days at 4, 10 and 20 ° C. with humidity set at 60-90%. Since the fermentation time is slower at lower temperatures, 4 ° C. is fermented for 16 days, 10 ° C. for 8 days, and 20 ° C. for 6 days, before fermentation. The hot water extract sample is divided into parts according to the fermentation period (eg, edible part, internal organ, brain) and ground (HMF-340, Han-Il, Korea), and 30 mL of distilled water is added per 1g of sample (Lee, YO 1996. Studies on the antioxidative characteristics and antioxidative substance of Kimchi.p.51.Doctor thesis of Busan National University.) Reattach the liquid to the flask three times for 30 minutes at 100 ℃ and filter it with filter paper (Whatman No. 2). The filtered solution was lyophilized again to form hot water extracts for each part of the skate, and cartilage was directly taken from Youngsanpo Food Co., Ltd.

Experimental Example 1 Analysis of General Components of Skate Hot Water Extract with Fermentation Temperature and Time

Skates were harvested at 4, 10 and 20 ° C for 60 to 90% of humidity, and fermented under each condition. General component analysis is based on the AOAC method (1995. Official Methods of Analysis.Chapter 35, pp. 382-383. eds, Assoc. Off. Anal. According to Chem., Washington, DC, crude fat was analyzed by Soxhlet method, crude protein by micro-Kjeldahl method, and ash by 600 ° C direct ashing method.

% Change in fermentation at 4 ℃ Fermentation time (days) Crude protein Crude fat Crude ash 0 1 2 4 6 8 10 12 14 16 88.15 ± 0.92 ^{* 1)} 88.09 ± 1.89 88.20 ± 2.49 88.12 ± 0.21 87.87 ± 1.19 87.41 ± 2.18 86.85 ± 0.36 87.07 ± 1.25 87.04 ± 0.25 87.04 ± 2.45 2.24 ± 0.47 2.82 ± 0.21 2.35 ± 0.23 2.08 ± 0.09 2.20 ± 1.02 2.13 ± 0.42 2.01 ± 0.18 2.01 ± 0.79 2.04 ± 0.17 2.02 ± 0.64 9.63 ± 2.05 9.89 ± 2.63 9.59 ± 1.04 9.75 ± 2.12 9.99 ± 0.02 10.35 ± 3.04 10.86 ± 2.81 10.97 ± 3.02 10.97 ± 1.02 10.97 ± 0.02

*: Each value is the mean ± standard deviation.

1): The experiment was repeated three times.

% Change in fermentation at 10 ℃ Fermentation time (days) Crude protein Crude fat Crude ash 0 1 2 4 6 8 87.71 ± 2.82 ^{* 1)} 85.49 ± 0.08 86.88 ± 3.25 86.43 ± 3.04 86.51 ± 2.02 84.97 ± 6.81 2.82 ± 0.01 2.82 ± 0.02 2.81 ± 0.09 2.53 ± 0.02 1.93 ± 0.05 1.08 ± 0.04 9.56 ± 0.08 10.51 ± 2.69 11.03 ± 4.25 11.03 ± 4.02 11.60 ± 2.03 14.04 ± 3.16

*: Each value is the mean ± standard deviation.

1): The experiment was repeated three times.

% Change in fermentation at 20 ℃ Fermentation time (hr) Crude protein Crude fat Crude ash 0 1 2 4 6 88.09 ± 2.68 ^{* 1)} 87.27 ± 4.79 86.89 ± 0.58 83.32 ± 3.51 86.04 ± 6.11 2.29 ± 0.03 2.65 ± 0.28 2.28 ± 0.06 2.57 ± 0.08 2.72 ± 1.06 9.55 ± 0.24 9.97 ± 0.02 10.87 ± 0.08 14.24 ± 0.90 11.31 ± 0.80

*: Each value is the mean ± standard deviation.

1): The experiment was repeated three times.

As shown in Tables 1, 2 and 3, at 4 ° C. (Table 1), there was little change in composition, which is thought to be due to fermentation at low temperature. 10 ℃ fermented skates showed a slight increase in ash content of 9.59% -14.04% during fermentation, based on 100 g of dry sample. This resulted in the decrease of the protein and fat content and the increase in the ash content of the main solids due to the outflow of the leachate. (Lee, KA 1999. Extractive nitrogenous constituents of fermented commercial skate, Raja kenojei . P.9.Master thesis of Yosu National University.).

In addition, the fat content tended to decrease little by little as the fermentation progressed. The protein content was 87.71-84.97%, but there was no significant change, but it decreased slightly. The protein content of 20 ℃ fermented skates was relatively similar to the average protein content of 88.09-86.04% and 2.29-2.72% fat, based on 100g of dry sample, and the ash content was 9.55-11.31%, similar to 10 ℃ fermented skates. It was. Therefore, there was no significant difference in the change of the general components according to the fermentation temperature and duration.

Experimental Example 2: Measurement of pH and Ammonium Concentration

Measurement of pH and ammonia concentration according to the temperature of the effluent collected while fermenting skates at 4, 10, and 20 ° C was performed by taking 20 mL of the leaching liquid while fermenting skates, and diluting it with 180 mL of distilled water. The pH of the sample solution was measured with a pH meter (Model 735P, Korea Istech Incorporated), and the concentration of ammonium was measured by the Ammonium Electrode (Model 735P, Korea Istech Incorporated) and the Phenali method (Arnold EG, R. Chairman, T). Rhodes and SC Lenore. 1985. Standard methods for the examination of water and wastewater, pp. 382-383. 16th eds, APHA, Washington.

As shown in Figure 1, in the case of 4 ℃ seemed to increase slightly up to 4 days of fermentation, but the rise of the pH was very low compared to 10, 20 ℃, which is a low fermentation at a low temperature, so the production of ammonium also increases the pH This is believed to be low. In the case of 10 ° C, the pH rapidly increased from 6.3 to 7.65 in one day, and then remained constant until 5 days of fermentation, but then increased secondly to pH 9.05 at 8 days of fermentation. In the case of 20 ° C., the pH increased sharply from the beginning of fermentation, and then reached 8.93 on the fourth day of fermentation. In addition, it was found that the fermentation progressed at a higher temperature because the fermentation rate is faster than the 10 ° C 20 ℃. In general foods, as fermentation progressed, the pH increased in skates, as opposed to the decrease in pH due to the action of microorganisms and rot bacteria. This is because the skates contained urea and urea precursors in the body for the purpose of controlling osmotic pressure.

As shown in Figure 2, the fermentation temperature at 4 ℃ did not show a significant difference in the ammonium ion concentration content over time, which is a result of fermentation at a relatively low temperature compared to 10, 20 ℃ time to reach the maturity more than 15 days It appeared to take. Optimal maturation of skate fermentation has not been scientifically identified, but the sensory evaluation results of researchers in the laboratory of the present inventors (Yeosu University, Department of Biotechnology) (not shown) showed very good results at around 10.0 mg / mL of ammonium. I made it a standard. When fermented at 10 ℃ began to increase sharply from day 5 to 14.2 mg / mL on the 8th and then slightly increased. Therefore, 5 days is the early stage of fermentation, 6 days is the early stage of maturation and 9 days are the late stages of fermentation at 10 ℃. At 20 ° C, the ammonium ion concentration reached 10.3 mg / mL at 4.5 days, and the time to reach the beginning of fermentation was 12 hours faster than 10 ° C. However, fermentation characteristics according to the skate fermentation temperature are considered to take too long fermentation time and low economic feasibility at 4 ℃. In the case of 20 ℃, fermentation proceeds in a short time, but the tissue becomes soft and fermented at high temperature. It was found to fall compared to 10 ° C. Therefore, it was shown that the skate fermentation is best fermented at 10 ° C.

From the above results, the degree of fermentation progress could be estimated by measuring the pH and ammonium ion concentration of the skate effluent, and it is thought that the degree of fermentation can be controlled by using the same.

Example 2: Identification of Physiological Functionality

At the fermentation temperature of 10 ℃ obtained in Example 1 to investigate the antimicrobial effect, antioxidant and anticancer activity of the hot water extract of each part of the skate according to the fermentation period to determine the physiological function.

Experimental Example 1 Antibacterial Effect Measurement

The final concentration of the hot water extract for each part of the skate for the antibacterial activity was 0.5%, 1%, and 2%, respectively, and sterilized after adding to NB (Nutrient Broth, USA Diffco Lab) medium. ( E. coli (KCTC No. 1039)) was inoculated at a final concentration of 3.6 × 10 ⁵ CFU / mL and cultured in a shaking water bath at 37 ° C. and 120 rpm for 22 hours to monitor the growth of microorganisms. Absorbance (660 nm) was measured at 2 hour intervals using a spectrophotometer (UVS-30NP, Korea Sunil Optron).

The antibacterial effect of the hot water extract of skates was found to be little (not shown). It is thought that the antimicrobial effect is very low due to the degeneration of proteins and medium-chain fatty acids having 12 to 18 carbon atoms in small amounts in animal and plant tissues.

As shown in Figure 3, the antimicrobial activity of the skates visceral hot water extract according to ripening was added 0.5% (Fig. 3 (a)), the fermentation 0 days (control group) was 36.6% in 10 hours of culture and then cultured 18 By time, the antimicrobial effect gradually decreased. On the fourth day of fermentation, the bacterial growth was 11.9% at 8 hours, after which the growth inhibition effect was gradually decreased. In addition, 8 days of fermentation showed an antimicrobial effect of about 15.5% from 8 hours to 18 hours of cultivation, and 10 days of fermentation showed the highest antimicrobial effect of 40.5% at 8 hours of cultivation. At the addition of 1% concentration (Fig. 3 (b)) 0. fermentation, 29.2% after 18 hours incubation, 32.0% on the 4th day of fermentation, 38.0% on the 8th day of fermentation, 35.0% on the 10th day of fermentation . In the group added with 2% sample concentration (Fig. 3 (c)), the 0 day fermentation sample showed a maximum antimicrobial effect of up to 35.5%, and a low antibacterial effect of up to 23.4% for 4 days of fermentation. Up to 43.3% in 8-day samples and 38.3% in 10-day samples. Therefore, the antimicrobial effect of the skates gut was higher, and the higher the concentration of the sample, the higher the fermentation of the skates gut.

In the group added 0.5% of the samples according to the concentration of the skate hot water extract (Fig. 4 (a)) in the group was added 23.0% at 4 hours of bacterial culture, the antimicrobial effect increased until 12 hours, but slightly decreased. In the group with 1% sample concentration, the antimicrobial effect was up to 36.1% at 4 hours of cultivation, and up to 41.0% in the group with 2% sample concentration. It was reduced to about 30%. Therefore, the antimicrobial effect of the skate was higher as the sample concentration, like the skate intestine, was better, and the growth inhibition effect was higher at the early stage of microbial culture.

As shown in Figure 4 (b), the antibacterial effect of the skate cartilage showed 13.3% antimicrobial effect at 8 hours of incubation in the group added 0.5% sample, and then gradually decreased. In the group to which 1% of the sample was added, the antimicrobial effect of 19.4% was shown at 8 hours of incubation, and as in 0.5%, the antimicrobial effect gradually decreased. In the group added 2% of the sample, 26.5% showed antimicrobial effect at 8 hours of incubation. Unlike the group added with 0.5% and 1% of sample, the antimicrobial effect increased as the culture time elapsed. It was constant after increasing to%. Therefore, the antimicrobial effect of skate cartilage also showed higher antimicrobial effect, and the antimicrobial effect was sustained as sample concentration was higher along with skates gut and hot water extract of brain.

Experimental Example 2: Antioxidant (hydrogen donating ability) measurement

Dissolve 16 mg of α, α'-diphenyl-β-picrylhydrazyl (DPPH) in 100 mL ethanol and mix 100 mL distilled water to Whatman filter paper (Whatman filtered with filter paper No. 2). After mixing 1 mL of the sample prepared in 5 mL of this filtrate, the decrease in absorbance was measured at 528 nm. As a control group, BHA (t-buthyl hydroxy anisole, US Sigma Chemical Corporation), which is known as an antioxidant, was prepared at a concentration of 0.02%, and the blank was tertiary distilled water.

As shown in FIG. 5 (a), the antioxidant activity of the skated hot water extract of the skate was highest at 33.6% on the 0th day of fermentation in the group added with 1% of the sample, on the 4th, 8th and 10th days. 16.8%, 13.8%, and 14.3%, respectively, were the highest at 61.2% on day 0 of fermentation, followed by 21.3% on day 10, 14.8% on day 8, and 12.0% on day 4. Hence, the antioxidant activity of skated hot water extract was highest at day 0 of fermentation, but decreased rapidly from day 4 of fermentation and showed low antioxidant activity until day 10. Urea (urea) is a substance that has been recognized for its antioxidant protection. In particular, it is known that the content of uric acid is high in the platelets of marine cartilage, such as skate, because the excretion of marine cartilage is uric acid and TMAO (trimethylamine oxide) and is used as an osmotic regulator.

Therefore, on the 0th day of fermentation, it shows strong antioxidant power, and when used as a natural additive and food preservative or functional food material for cooking and processing of food, its effect is expected to be high. As shown in FIG. 5 (b), the antioxidant activity of the skated viscera hot water extract was the highest at 48.8% on the 8th day of fermentation when 1% of the sample was added, and 35.3% and 34.7% on the 0th, 4th and 10th day of fermentation, respectively. , Showed an antioxidant effect of 14.5%. When 2% of the sample was added, the highest value was 54.4% at day 0 of fermentation, followed by 54.2% at day 8 of fermentation, 45.5% at day 4 of fermentation, and 41.7% at day 10 of fermentation. In addition, the antioxidant activity of the H. gut hot water extract was high at both fermentation day 0 and fermentation day 8. The antioxidant effect of the skate water extract was found to be ineffective. It is thought that there is no antioxidant effect because many lipid components and proteins of brain are easily denatured and oxidized by heating during hot water extraction. In addition, it is believed that some of these components may be acting to promote rancidity because they do not have antioxidant power or have anti-oxidative effects, but the anti-oxidative effect of other coexisting substances is stronger. Therefore, it is considered that bioactivity should be examined from low molecular weight peptides obtained by enzymatic degradation of animal proteins. The antioxidant properties of the skate cartilage hydrothermal extract shown in Figure 4 (c) was relatively low as 14.4% when 0.5% of the sample was added. The highest was 16.7% when 1% of sample was added and 25.0% when 2% of sample was added.

Experimental Example 3: Anticancer Evaluation

MTT ((3- [4,5-dimethyl thiazol] -2,5-Diphenyltetrazolium bromode) assay was performed to evaluate the anticancer activity of hot water extracts for each area of skate. HepG2 (Hepatocellular carcinoma, human, ATCC No. HB-8065) The cells were seeded in 96-wells at 1 x 10 ⁴ cells per well and attached for 24 hours.Sample samples of hot water extracts for each site of skate were 125 mg / mL, 250 mg / mL, 500 mg / mL and 1,000 mg / mL were added to the medium and incubated for 72 hours, 20 mL of MTT solution was added thereto, and further incubated for 4 hours under the same culture condition.After 4 hours, 150 mL of isopropanol was added to insoluble formazan. Was dissolved in the microplate reader (Germany Benchmark Biorad Corporation), the absorbance was measured at 570 nm to show the death of cancer cells as follows.

Anticancer activity = (absorbance of control-absorbance of control / absorbance of control) x 100

As shown in FIG. 6 (a), the anticancer properties of the hot water extract of skated cabbage were 41.5%, 32.9%, 51.1%, and 49.6% at 0, 4, 8, and 10 days of fermentation, respectively, when the sample was 500 mg / mL. It showed a slight increase as the fermentation progressed, and the highest was 52.7% at 8 days of fermentation when the sample was 1,000 mg / mL.

The anticancer activity of the skated viscera hydrothermal extract shown in FIG. 6 (b) was 58.3% at 10 days of fermentation, 57.9% at 4 days, 56.8% at 8 days of fermentation, and 46.7% at day 0 of fermentation at 1,000 mg / mL of the sample. This was comparable to 60% anti-cancer activity against HepG2 at the same concentration of hot water extract. Therefore, the anticancer activity of the H. gut hot-water extract sample was high, but it showed a tendency to increase slightly as fermentation progressed.

As shown in FIG. 6 (c), the anticancer activity of the HSK brain water extract was 12.2% at 250 mg / mL of sample, 37.9% at 500 mg / mL, and 41.5% at 1,000 mg / mL of sample. appear. In addition, the anticancer properties of the skate cartilage hydrothermal extracts were 26.8%, 28.1%, and 33.6% in samples 125, 250, and 500 mg / mL, respectively, and were highest at 38.3% at 1,000 mg / mL. Chondroitin sulfate (chondroitin sulfate) is contained in the cartilage and skin of skates. Chondroitin sulfate is a viscous polysaccharide widely distributed in connective tissues in living organisms. In addition, chondroitin sodium is present in vertebrate cartilage, connective tissue, support tissues such as bone, aorta, heart valve, tendon, skin, cornea, etc., and has physiological activities such as anti-aging, bone formation, anti-tumor and anti-arteriosclerosis. It is excellent and especially effective in preventing and treating degenerative arthritis. It is used as a cosmetic, health supplement and medicine. In addition, six kinds of A, B, C, D, E, and K are reported depending on the position of the sulfate group, which is composed of D-glucuronic acid, N-acetylgalactosamine and a sulfate group. Studies on the antimutagenic and anticancer effects of glycoproteins and chondroitin sulfate in marine animals showed that the antimutagenic and anticancer effects of extracted chondroitin sulfate and glycoprotein showed strong antimutagenic and anticancer effects with increasing concentrations. However, in the present invention, the anti-cancer activity of viscera and edibles was superior to skate cartilage at 1000 mg / mL. Therefore, it is thought that functional protein components in not only skate cartilage but also edible part and visceral part of the hot water extract of each skate are considered, but more in-depth study should be conducted to identify anticancer substances.

As described through the above examples, the present invention relates to the use of the skate extract, the present invention is a hot water extract for each part of the skates fermented for 10 to 10 days at 10 ℃ having antimicrobial, anticancer and antioxidant activity There is an excellent effect to provide. Therefore, the present invention skate extract is a very useful invention in the functional food industry.

Claims (3)

delete delete A composition for anticancer drugs, comprising 250-1000 mg / mL of hot water extract of skated intestine fermented at a humidity of 60-90% and a temperature of 10 ° C. for 8-10 days.

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