KR20120056138A - Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver - Google Patents

Abstract

PURPOSE: A composition containing fermented silkworm powder is provided to prevent and treat various adult diseases such as hyperlipidemia and fatty liver. CONSTITUTION: A method for preparing fermented silkworm powder comprises: a step adding sterilized water to silkworm powder and sterilizing; and a step of adding Bacillus subtilis or an Aspergillus kawachii culture liquid; and a step of fermenting at 37°C for 3-12 days and drying. A pharmaceutical composition for preventing or treating hyperlipidemia and fatty liver contains the fermented silkworm powder as an active ingredient. A pharmaceutical composition for antioxidation and thrombolysis effects contains the fermented silkworm powder as an active ingredient.

Description

Method for preparing fermented silkworm powder and composition comprising fermented silkworm powder for the preventing or treating of hyperlipidemia and fatty liver}

The present invention Bacillus in silkworm powder A method for producing fermented silkworm powder fermented with the addition of subtilis (Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus kawachi ) strain and a composition for preventing or treating various adult diseases such as hyperlipidemia and fatty liver including fermented silkworm powder It is about.

Recently, the number of patients with hyperlipidemia and fatty liver has been reported to increase rapidly due to the change of diet and excessive drinking due to the improvement of living standard. In particular, excessive intake of fat increases blood LDL cholesterol and causes the accumulation of fat in the body. In the end, it causes cardiovascular diseases such as atherosclerosis. Thus, there have been attempts to develop new materials to prevent and treat hyperlipidemia and fatty liver.

Insect-related industries are attracting attention as new biomass materials, and interest in silkworms and related by-products is increasing as a new natural material for developing new biomaterials.

The study showed that feeding rats with hyperlipidemia induced unsaturated fatty acid extracted from silkworm pupa resulted in a 30% reduction in atherosclerosis index. As a result, it has been reported that the skin moisturization is improved by more than 50% compared to the non-corrected skin. And it has been experimentally proved that male silkworm chrysalis extracts and isolates a protein that promotes the synthesis of cGMP, which is known as one of the male's erectile promoting ingredients, to rescue its structure and enhance its erectile promoting activity.

On the other hand, the main components of the mulberry leaf feeding silkworms are moisture, protein, carbohydrates, fats, inorganic salts, vitamins, etc., and the physiological activity of the mulberry leaves lowers the hypoglycemic effect and lowers the amount of neutral lipids that cause obesity. It has been reported to reduce the amount of, and inhibits the generation of active oxygen caused by aging about 20% and at the same time increases the activity of free radical scavenging enzyme by about 14% has been reported to be effective in inhibiting aging. In addition, silkworms contain abundant essential amino acids, and according to a report from the Korea Food Research Institute, it is reported that alanine in essential amino acids promotes alcohol metabolism to help hangover and improve liver function.

Republic of Korea Patent No. 10-0396986 relates to a method for producing a blood glucose lowering substance excellent in inhibiting the post-prandial blood sugar rise from silkworm powder, Republic of Korea Patent No. 10-0361085 is based on the ethanol extract of silkworm powder , Composition of the health supplements having anti-diabetic effect, containing the dry leaf extract extract, silk gourd dry extract extract, silk furotene, beta-carotene, ginkgo leaf extract extract, vitamin B1, B6, C and E and evening seed oil as supplementary ingredients It relates to a method, the Republic of Korea Patent No. 10-0721644 discloses a hangover rehabilitation health functional food comprising a silkworm pupa hot water extract as an active ingredient, but mainly relates to anti-diabetic effect or hangover efficacy, More research is needed to determine the efficacy of preventing and treating fatty liver and hyperlipidemia.

The present invention has been made in view of the above, fermented silkworm powder production method and fermented silkworm fermented by adding Bacillus subtilis ( Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus Kawachi) strain to silkworm powder An object of the present invention is to provide a composition for preventing or treating various adult diseases such as hyperlipidemia and fatty liver containing powder.

The object of the present invention is Bacillus subtilis ( Bacillus subtilis ) or Aspergillus in silkworm powder Fermented silkworm powder was prepared by adding kawachii (Aspergillus Kawachi) strain, followed by fermentation to identify its constituent proteins and amino acids, and for the components of liver, blood, lipids, alcohols, aldehydes, etc. It was achieved by confirming the effect of the fermented silkworm powder.

The present invention has an excellent effect showing improved hyperlipidemia and fatty liver prevention and treatment effect compared to the existing non-fermented silkworm powder by fermenting the silkworm powder using the strain.

1 is Bacillus subtilis and Aspergillus Native-PAGE electrophoresis of silkworm powder fermented by kawachii is shown.
2 is Bacillus subtilis and Aspergillus SDS-PAGE electrophoresis of silkworm powder fermented by kawachii is shown.
3 is Bacillus subtilis and Aspergillus It shows the free amino acid composition of silkworm powder fermented by kawachii .
4 is Bacillus subtilis and Aspergillus It shows the thrombolytic activity of silkworm powder fermented by kawachii .
5 is Bacillus subtilis and Aspergillus It shows DPPH free radical scavenging ability of silkworm powder fermented by kawachii . BHT stands for Butylated hydroxyl toluene and the other abbreviations are the same as in FIG. Data is shown as mean ± SD (n = 3).
6 is Bacillus subtilis and Aspergillus It shows the reducing power of silkworm powder fermented by kawachii . BHT stands for Butylated hydroxyl toluene and AA stands for Ascorbic acid. Other abbreviations are the same as in FIG. 4. Data is shown as mean ± SD (n = 3).
Figure 7 shows the effect of SP, BFSP and AFSP on the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) in the liver of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
Figure 8 shows the effect of SP, BFSP and AFSP by ADH and ALDH Western blot analysis in liver of alcohol administered rats.
9 shows the effects of SP, BFSP and AFSP on Zn and Fe concentrations in the liver of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
10 shows the effect of SP, BFSP and AFSP on TBARS in liver fraction and serum of alcohol-administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
FIG. 11 shows the effects of SP, BFSP and AFSP on glutathione concentrations in liver and serum of alcohol administered rats. Figures in other letters are statistically significant at p <0.05. (Mean ± SE, n = 6).
Figure 12 shows the effect of SP, BFSP and AFSP on the histopathologic changes of livers of alcohol-administered rats (magnification x 200). Hepatocyte staining was performed by H & E (hematoxylin and eosin) staining method.
Figure 13 shows the effect of SP, BFSP and AFSP on the histopathologic changes of liver of alcohol-administered rats (magnification x 200). Hepatocyte staining was performed by H & E staining method.

The present invention Bacillus in silkworm powder The present invention relates to a method for producing fermented silkworm powder fermented by adding subtilis (Bacillus subtilis ) or Aspergillus kawachii ( Aspergillus kawachi ) strains, and a composition comprising fermented silkworm powder.

The composition comprising the fermented silkworm powder of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the composition comprising the fermented silkworm powder of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium, phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used.

The composition comprising the fermented silkworm powder according to the present invention may be prepared by oral preparations such as powders, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to a conventional method; External preparations; Suppositories; And in the form of sterile injectables. The amount of fermented silkworm powder may vary depending on the age, sex, and weight of the patient, but may be administered once to three times daily in an amount of 10 to 35 mg / kg body weight, preferably 15 to 25 mg / kg body weight. . The dosage of the extracts and fractions can be increased or decreased depending on the type of disease administration route, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

The results obtained from the experiments were expressed as mean and standard error (mean ± SE) by one-way ANOVA test, and the significance test between each experimental group was Duncan's multiple range test.

Example  1. Preparation of Fermented Silkworm Powder

Silkworm powder, an experimental material, was hot-air dried and purchased from Suncheon Farming Corporation (Suncheon, Jeonnam). Bacillus for Time-Based Fermentation of Hot Air Dried Silkworm Powder subtilis KACC 91157 and Aspergillus kawachii KCCM 32819 strain was used from the Korea Microorganism Conservation Center. 50 g of each hot-air dried silkworm powder was put in a 500 ml beaker, and 1.5 times distilled water was added to the powder to absorb all the powder, followed by sterilization at 121 ° C. for 15 minutes. Bacillus precultured at room temperature The subtilis culture was well mixed with silkworm powder at 1% (v / w) level and then fermented at 37 ° C. for up to 12 days. Also precultured Aspergillus The kawachii mycelium was also mixed with silkworm powder at 5% (v / w) level and then fermented at 37 ° C for up to 12 days. At this time, in order to prevent drying during the fermentation process, the sterilized distilled water was sprinkled with a predetermined amount to proceed with the fermentation. The samples were taken one by one every three days while fermenting up to 12 days with two strains, and then completely dried and used for analysis.

Example  2. Over time fermentation of silkworm pH  And measurement of protein concentration

The fermented silkworms over time were added to distilled water at a concentration of 1% (w / v) and stirred, and the pH was measured directly with a pH meter (Methrohm 691, Swiss). The protein concentration of fermented silkworms over time was low at 540 nm by Lowry method (Lowry, OH, NJ Rosebrogh, AL Farr and RJ Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol . Chem . 193, 265-271) . Absorbance was measured and measured using bovine serum albumin as a standard.

Silk fibroin, a natural protein derived from gajam, and sericin protein from silkworm silk, have been reported to have high alcoholic liver toxicity and antioxidant effects. Bacillus and Aspergillus, which has a high protein breakdown, has been used as a high protein food material in previous experiments. In fermented silkworms obtained by fermentation of the genus microorganisms, physiological activities such as increased protein content, antioxidant activity, thrombolytic action, and tyrosinase activity inhibition tended to be higher than those before fermentation [Rahama, EH and MS Narasinga Rao. 1983. Effect of limitted proteolysis on the functional properties of cottonseed flour. J. Agric . Food Chem . 31, 356-361; Sekul, AA, CH Vinnett and RL Ory. 2000. Some functional properties of peanut protein partially hydrolyzed with papain. J. Agric . Food Chem . 26, 855-859. In addition, in the analysis of proteolytic pattern on SDS-PAGE, the protein of about 97-66 kDa was decomposed and showed the difference in band, which showed the difference in physiological activity by affecting amino acid and peptide composition by the action of proteolytic enzymes. It has been suggested [Rahama et al., 1983; Sekul et al., 2000].

The protein concentrations of nonfermented silkworms were 48.6 ± 0.28% and 47.0 ± 0.08%, respectively (Table 1). Bacillus Protein concentration of fermented silkworms by strains showed a tendency to increase continuously with the time of fermentation, with a maximum content of 55.6% at 12 days of fermentation. Bacillus sp. Experimental results have also been reported to increase the protease enzyme activity in Cheonggukjang using b01 strain until 9 days of fermentation (In, JP, SK Lee, BK Ahn, IM Chung and CH Jang. 2002. Flavor improvement of cheonggkukjang by addition of Yucca (Yucca shidigera ) extract. Korean J. Food Sci Technol . 34, 57-64). Aspergillus The protein concentration of the fermented silkworm by the strain showed no significant change until the 9th day of fermentation, but the maximum content was 52.3% on the 12th day, but was lower than the protein concentration of the fermented silkworm by the Bacillus strain.

Bacillus sp. In the experiment for extracting insoluble protein contained in silkworm pupa with soluble protein using JH-209 strain, it showed the lowest value at pH 5.0 and showed a tendency to increase the amount of protein extraction as the pH increased. kwon, HJ, KH Lee, JH Kim, SS Chun, YJ Cho and WS Cha. 2006. Effect of protease on the extraction and properties of the protein from silkworm pupa. J. Korean Soc . Appl . Biol . Chem . 49, 304-308). In this experiment, the protein content increased over time as the pH value gradually increased during fermentation by Bacillus strains, and the activity of proteolytic enzymes became active (Table 1). In the fermentation with Aspergillus strain, there was no significant change in pH value until day 9, but increased protein concentration at day 12, which was similar to silkworm pupa protein concentration.

PH and Protein Concentration of Silkworms Fermented by Bacillus Subtilis and Aspergillus Kawachi with Different Fermentation Times Sample Time pH Protein content (%) Silkworm powder (SP) 0 day 7.11 48.60 ± 0.28 Bacillus subtilis 3 day 7.27 51.40 ± 0.38 6 day 7.43 52.51 ± 0.12 9 day 7.53 53.53 ± 0.18 12 day 7.97 55.60 ± 0.08 Aspergillus kawachii 3 day 6.55 49.60 ± 0.26 6 day 6.56 49.60 ± 0.28 9 day 6.74 50.10 ± 0.36 12 day 7.26 52.32 ± 0.38

Example  3. Over time Fermentation Silkworm Native - PAGE Protein pattern comparison

As the protein pattern of fermented silkworms over time, the gel concentration of slab-type native-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was 7.5% according to Davis method [7]. In other words, the separation gel was mixed with 30% acrylamide, 0.8% bis-acrylamide, 1.5 M Tris-HCl buffer (pH 8.8), 0.05% TEMED and 10% ammonium peroxide to make 1 mm thick. On a separation gel was prepared a 5% concentrated gel [30% acrylamide, 0.8% bis-acrylamide, 0.5 M Tris-HCl buffer (pH 6.8) to give 5 × sample buffer (312.5 mM Tris-HCl, pH 6.8), 50 % Glycerol, 0.05% bromophenol blue] solution was injected in a predetermined amount into the wall formed in the concentrated gel. Electrophoresis was performed through a constant current of 1.5 mA / gel (cm) under Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.8) and energized for 2 hours at 4 ° C. low temperature. After the electrophoresis was completed, the gel was stained with 1% Coomassie brilliant blue R-250 dissolved in 7% acetic acid, and then decolorized with 7% acetic acid, and dried and photographed.

As a result of comparative analysis by electrophoresis of silkworm protein over time with native-PAGE, Bacillus subtilis fermented silkworms were mostly decomposed and low-molecularized from day 3 of fermentation silkworm compared to the high concentration of 97-66 kDa band at the top of non-fermented silkworm protein. It appears that the outline is hard to see (FIG. 1). But Aspergillus Fermented silkworm protein bands by kawachii strains showed a tendency to gradually soften as the fermentation time elapsed, resulting in a significant difference between the two strains. In Kang et al., Native-PAGE comparisons of silkworm scenes and major hemolymph proteins of short-lived varieties showed a large number of bands at the beginning of 5 years of age. However, the overall increase in protein content is apparent (Kang, PD, HJ Yoon, KS Ryu, BH Sohn and HD Sohn. 1999. Electrophoretic patterns of hemolymph proteins of varieties with long and short life span in the silkworm Bombyx mori L. Korean J. Seric . Sci . 41, 1-8).

Example  4. Over time fermented silkworm SDS - PAGE Protein pattern comparison by

Over time fermented silkworm Protein pattern analysis was performed according to Weber and K. Osbom method (Weber, K. and M. Osborn. 1969. The reliability of molecular weight determination by sodium dodesyl sulphate-polyacrylamide gel electrophoresis. J. Biol . Chem . 244, 4406-4412) . The composition of SDS-PAGE was made of 10% acrylamide, 1.5 M Tris-HCl buffer (pH 8.8), 0.4% Sodium dodecyl sulfate (SDS), 10% ammonium peroxide and 0.05% TEMED. The composition of the concentrated gel was 5% acrylamide, 0.5M Tris-HCl buffer (pH 6.8), 0.4% SDS, 0.05% TEMED. SDS treatment of the sample protein was heat-treated at 100 ° C. for 5 minutes in a final concentration of 2% SDS, 14.4 mM β-mercaptoethanol, 60 mM Tris-HCl buffer (pH 6.8), 25% glycerol and 0.1% BPB. Electrophoresis was performed for 1 hour and 30 minutes through a constant current of 130-150 mA / gel (cm) under Tris-glycine buffer (25 mM Tris, 192 mM glycine, pH 8.3). After electrophoresis, the gel was added with 1% Coomassie Brilliant Blue R-250 to a fixed solution (methanol: acetic acid: water = 10: 10: 80) and stained for 10 hours, followed by a decolorizing solution (methanol: acetic acid: water = 10: 10: 80) was sufficiently decolored and dried and photographed.

As a result of comparing the electrophoresis pattern of fermented silkworm protein over time on SDS-PAGE (Fig. 2), Bacillus compared to the clear silkworm protein band of 97-66 kDa size before fermentation started. In the protein of fermented silkworm by the subtilis strain, since it was absent from the 3rd day of fermentation, it was mostly degraded by proteolytic enzyme. But Aspergillus Protein bands of 97-66 kDa size in fermented silkworms by kawachii strains gradually disappeared as fermentation time passed, and most of them appeared to be degraded up to 12 days. Therefore, in order to obtain bioactive substances such as amino acids or peptides from silkworm protein in industry, Aspergillus Bacillus rather than fermentation by kawachii strain Fermentation by subtilis strains may be more economical.

Example  5. Composition and Free Amino Acid Measurement of Fermented Silkworms

Constitutive amino acid analysis was performed by adding 15 ml of formic acid peroxide and 15 ml of 6N HCl to 0.2 g of the fermented silkworm sample on day 12, and acid hydrolyzed for more than 24 hours in a 110 ℃ dry oven. The decomposed sample was concentrated under reduced pressure in a 55 ° C water bath, and then a predetermined amount of the sample was filtered through a 0.2 μm membrane filter by applying a volumetric flask to 25 ml with a pH 2.20 citric acid dilution buffer (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK). Free amino acid analysis was carried out using fermented silkworm samples on day 12 with an amino acid autoanalyzer (Biochrom 30, Amersham Biosciences Ltd., Cambridge, UK).

The total content of constituent amino acids was 661,692 ppm and Bacillus of Aspergillus kawachii strain fermented silkworm compared to 354,610 ppm of unfermented silkworm Subtilis strain fermented silkworms were detected in significant amounts of 679,708 ppm (Table 2). Constituent amino acid concentrations of unfermented silkworms were analyzed in order of glycine (48,519 ppm), alanine (46,349 ppm), glutamic acid (42,402 ppm), aspartic acid (32,798 ppm), and serine (29,882 ppm) [3]. However, in this experiment, the most amino acid among the constituent amino acids of fermented silkworm by these two strains accounted for 63.25% -63.51% with glutamic acid, which was different from before fermentation. The constituent amino acid composition of Aspergillus kawachii fermented silkworms was as follows: glutamic acid 418,536, aspartic acid 32,335, serine 30,190, tyrosine 21,848, and leucine 21,145 ppm. Constituent amino acid composition of Bacillus subtilis fermented silkworm was found in the order of glutamic acid 431,660, aspartic acid 32,135, serine 29,195, lysine 24,270, histidine 23,857 ppm, and it was observed that there was a difference in the constituent amino acid composition of fermented silkworm by the two strains. In particular, glutamic acid, proline and histidine of non-fermented silkworms were significantly increased by fermentation of two strains, while glycine, alanine and valine were significantly decreased. Up to now, studies on alcoholic liver toxicity have been reported by single amino acid treatment such as glycine, glutamic acid, arginine, serine, aspartic acid (Lee, JH, NK Kim, DY Lee and CH Lee. 1999. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J. Food Sci . Technol . 31, 802-808; Yin, M., K. Ikejima, GE Arteel, V. Seabra, BU Bradford, H. Kono and I. Rusyn and RG Thurman. Glycine accelerates recovery from alcohol-induced liver injury. J. Pharacol . Exp . Ther. 286, 1014-1019). On the other hand, the amino acid composition of soybean sprouts, which are widely used to relieve hangovers, contained aspartic acid, glutamic acid, and arginine, which also showed high liver protection effects (Lee et al., 1999). Meanwhile, serine 31%, aspartic acid 17.8% and glycine accounted for 19.1% in the constituent amino acid composition of sericin protein, and alcoholic liver toxicity has been reported (Kato, N., S. Sato, A. Yamanaka). , H. Yamada, N. Fuwa and M. Nomura. 1998. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci . Biotechnol . Biochem . 62, 145-147). Therefore, since it contains many amino acids such as glutamic acid, serine and aspartic acid, which are effective for protecting the liver, it is expected to be useful as a health supplement material for improving liver function.

Bacillus for 12 days of fermentation subtilis and Aspergillus Amino Acid Composition of Silkworms Fermented by kawachii (ppm) Amino acids Silkworm Bacillus subtilis Aspergillus kawachii L-Aspartic acid 32,498 32,335 32,135 L-Threonine 15,930 16,341 15,108 L-Serine 29,882 30,190 29,195 L-Glutamic acid 42,402 418,536 431,660 L-Proline 9,359 13,831 13,629 L-Glycine 48,519 8,270 9,256 L-Alanine 46,349 7,818 8,021 L-Valine 17,027 3,059 3,493 L-Isoleucine 13,103 14,256 13,822 L-Leucine 18,820 21,145 20,369 L-Tyrosine 20,114 21,848 23,557 L-Phenylalanine 13,769 14,926 15,349 L-Histidine 9,373 20,708 23,857 L-Lysine 19,613 20,526 24,270 L-Arginine 17,553 17,905 15,987 Total 354,610 661,692 679,708

Bacillus in this experiment subtilis and Aspergillus kawachii Among the free amino acids of fermented silkworms by the two strains, alanine contained 16.257% and 16.198%, respectively, followed by glutamic acid 12.540% and 12.678%, respectively (Table 3 and FIG. 3). Next, glycine, valine, leucine, and isoleucine were found in order. However, there was no significant difference in free amino acid composition by fermentation of the two strains.

Bacillus for 12 days of fermentation subtilis and Aspergillus Free amino acids of silkworms fermented by kawachii
Free amino acids Retention time Bacillus subtilis
(%) Aspergillus kawachii
(%) Phosphoserine 4.333 0.47 0.48 Taurine 6.933 0.36 0.75 Urea 10.566 0.052 0.052 Aspartic acid 20.799 4.028 3.580 Throneine 26.665 4.935 4.921 Serine 28.632 5.283 5.781 Glutamic acid 34.264 12.540 12.678 AAAA (α-Aminoadipic acid) 42.264 0.085 ND Proline 44.164 0.147 0.148 Glycine 45.964 8.048 8.533 Alanine 47.464 16.258 16.197 Citrulline 48.997 1.63 0.79 AABA (α-Aminobutyric acid) 50.830 0.164 0.063 Valine 53.697 6.470 6.611 Cystien 58.083 0.065 0.064 Methionine 60.563 0.624 0.776 Cystachionine 63.163 0.247 0.177 Isoleucine 65.296 4.137 4.096 Leucine 67.069 6.817 6.817 Tyrosine 70.196 2.793 2.750 β-Alanine 72.062 ND ND Phenylalanine 73.429 3.762 3.656 β-Aminoisobutylic acid 75.229 ND ND Homocysteine 77.395 ND ND Γ-Aminobutyric acid (GABA) 80.062 0.930 0.906 Ethanolamine 83.761 0.499 0.389 Ammonia 87.128 8.026 6.958 Ornithine 94.427 0.822 0.939 Lysine 97.360 4.398 4.510 3-Methyl-histidine 99.127 0.047 ND Histidine 100.560 2.338 2.460 Arginine 114.193 3.177 3.283

Example  6. Over time fermentation Measurement of thrombolytic enzyme activity of silkworm

Fibrinolytic activity over time of fermentation silkworm is fibrin plate method to transform the (Astrup, T. and S. Mullertz. 1991. The fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. Biophys 40, 346-351) lysed zone Measured by. Fibrin, fibrinogen, and thrombin used to measure thrombolytic activity are determined by Sigma Co. (St. Louis MO, USA) was purchased and used. Fibrin plates were dissolved in 0.06% fibrinogen in 0.2 M bromide buffer (pH 7.5), then dispensed in a 10 ml aliquot of Petri dishes and uniformly mixed with 40 units of thrombin (5,000 units). After forming (clot) and used for 30 minutes at room temperature. The sample was eluted with distilled water at a concentration of 1%, filtered (Whatman No. 2), and 50 μl was added dropwise onto the fibrin plate and reacted at 37 ° C. for 3 hours, and the diameter of the resulting transparent ring site was measured. The diameter was measured in two perpendicular to each other to obtain the area of the transparent zone and expressed in unit / ml.

When blood clots are made in blood vessels, it interferes with blood circulation, causing high blood pressure, arteriosclerosis, stroke, and angina pectoris. At this time, thrombolytic enzymes are known to be effective in preventing various circulatory diseases by inhibiting blood clot production. Bacillus in Nature subtilis , Fusarium pallidoroserum , Katsuwonus pelamis , Streptococcus It has been extensively researched and developed for the activity of thrombolytic enzymes derived from aureus microorganisms and traditional fermented foods such as cheonggukjang, soybean paste, anchovy salt, natto (nattokinase) and shiokara (katauwokinase). In addition, it has been reported that only a few insects are found in 76 species of Korean native insects, which have excellent antithrombotic activity in 10 kinds of extracts, including worms, dragonflies, and silkworms (Ryu, HY, JC Heo, JS). hwang, SW Kang, CY Yun, SH Lee and HY Sohn. 2008. Screening of thrombin inhibitor and its DPPH radical scavenging activity from wide insects. J. Life Sci . 18, 363-368). The 70% methanolic extract of lyophilized silkworm powder showed some thrombolytic activity, while the hot-air dried silkworm extract showed no activity. It was observed that it depends on the method of drying silkworm larvae. KE Eom, BK Park and YS Cho . 2009. Biological activity of fermented silkworm powder. J. Life Sci . 19, 1468-1477). In the previous experiment, the thrombolytic activity of fermented silkworms fermented with 1% inoculation of B. subtilis strains on hot air dried silkworms and fermented for 2 days was 2.2 unit, but it was 3.4 units for fermented silkworms fermented for 12 days with 5% inoculation of A. kawachii strain. some high fibrinolytic activity was observed (Cha, JY, Kim YS, PD Kang, HY Ahn, KE Eom and Cho YS. 2010. Biological activity and chemical characteritics of fermented silkworm powder by mold. J. Life Sci . 20, 237-244). In the present invention, hot air dried silkworms were inoculated with 1% of B. subtilis strains and examined for thrombolytic activity over time. The fermented silkworms fermented 12 days with 5% inoculation of A. kawachii strains were 5.83 units. Results similar to those of the fermented silkworms were observed (FIG. 4). However, the thrombolytic activity of fermented silkworms fermented with both strains showed a maximum activity at 6 days and then decreased. B. subtilis using Soybean grit The highest thrombolytic enzyme activity at 5 days in HA strain fermentation was reported to affect the enzyme activity according to fermentation period [Kim, JE and SB Lee. 2009.Production of bioactive components and anti-oxidative activity of soybean grit fermented with Bacillus subtilis HA according to fermentation time. Korean J. Food Sci . Technol. 41, 179-185]. Therefore, the antithrombotic activity of fermented silkworm fermented with A. kawachii strain was 15.02 uint on the 6th day of fermentation, and the fermentation of about 5-6 days was necessary for obtaining fermentation products with thrombolytic enzyme activity of fermented silkworm. It is feed.

Example  7. Over time fermentation of silkworms DPPH Determination of antioxidant activity by the method

Over time fermented silkworm Antioxidant activity was determined according to the method of Blois (Blois, MS 1958. Antioxidant determination by the use of a stable free radical. Nature 26, 1199-1204). That is, DPPH (α, α'-diphenyl-β-picrylhydrazyl) solution was prepared by dissolving DPPH 16 mg in 100 ml ethanol and then mixing 100 ml of distilled water and filtering with filter paper (Whatman filter paper NO. 2). 1 ml of a 0.1% (1 mg / ml) sample solution was mixed with 5 ml of DPPH solution for 30 minutes at room temperature, and then the absorbance was measured at 528 nm. As a control, commercial antioxidant BHT was added at 0.05%, and the absorbance decrease was measured in the same manner as above. DPPH free radical scavenging ability was expressed as a percentage (%) of the absorbance difference between the sample addition and no addition.

DPPH free radical scavenging activity (%) = 1-(Abs / Abc) × 100

Abc: Absorbance of control treatment at 528nm

Abs: Absorbance of sample treatment at 528nm

Polyphenol-based materials in foods are known as hydrogen donor antioxidants that have molecular structural characteristics including ring structures containing at least one hydroxy group and nonpolar groups such as methyl groups or nonpolar hydrocarbon chain structures. DPPH free radical scavenging antioxidant activity assay (Blois, 1958). DPPH is the most common antioxidant activity measurement based on the principle of measuring the difference in chromaticity when the specific color disappears due to non-radical formation when electrons are paired by electrons or hydrogen atoms. It is well known that the method is highly related to the antioxidant activity of natural products. Antioxidant activity was 42.12% in non-fermented silkworm water extract (1 mg / ml), and showed a high activity of 95.96% at the concentration of 1 mg / ml of the commercial antioxidant BHT used as a control (FIG. 5). Antioxidant activity of B. subtilis fermented silkworm water extract (1 mg / ml) was found to increase proportionally with longer fermentation period. It showed a tendency to increase slowly after 42.12% of fermentation showed more than 75.90% of activity on 9th day of fermentation. Antioxidant activity of A. kawachii fermented silkworm water extract showed no significant change until day 9 of fermentation, but showed a high activity of 73.99% at day 12 of fermentation (FIG. 5). Antioxidant activity has been reported to increase the antioxidant activity by compounds such as peptides and amino acids, which are metabolites produced during fermentation in the Korean traditional food, Doenjang, Okara koji and Indonesia thempeh (Matsuo, M). . 1997. In vitro antioxidant activity of Okara koli, a fermented Okara, by Aspergillus oryzae. Biothechol . Biochem. Bioch . 61, 1968-1972; Matsuo, MN Nakamura, Y. Shidoji and T. Osawa. 1997. Antioxidative mechanism and apoptosis induction by 3-hydroxy anthranilic acid, an antioxidant in Indonesian food, tempeh, in the hyamn hepatoma derived cell line, HuH-7. J. Nutr . Sci . Vitaminol . 43, 249-259; Shon, MY, J. Lee, JH Choi and SY Park. 2007. Antioxidant and free radical scavenging activity of methanol extract of chenoggkukjang . J. Food Compos . Anal. 20, 113-118). Marcuse's literature reported antioxidant activity in most amino acids except cystein (Marcuse, R. 1962. The effect of some amino acids on the oxidation of linoleic acid and its methyl ester. J. Am . Oil Chem . 39, 97-103). The increase of antioxidant activity in water extract of fermented silkworm compared to silkworm extract before fermentation is considered to be due to the metabolites produced during fermentation. Antioxidant activity was measured by DPPH scavenging activity among the 304 extracts of 76 Korean native insects, which were found to have antioxidant activity only in four species of worms, thorns, weeds, and leaves and pine larvae (Park, YJ, JC Heo, SM An, EY Yun, SM Han, JS Hwang, SW Kang, CY Yun and SH Lee. 2005. High throughput-compatible screening of antioxidative substances by insect extract library. Korean J. Food Preserv . 12, 482-488). As such, only a few insects have antioxidant activity, and the silkworm itself has an antioxidant activity and is more active by microbial fermentation.

Example  8. Over time fermentation of silkworm Fe  Reducing power measurement

Fe-reduction of fermented silkworms over time was measured according to the method of Zhu et al. [31]. Take 0.75 ml of 0.1% water extract from fermented silkworm, mix 1.25 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 1.25 ml of 1% (w / v) potassium ferricyanide [K ₃ Fe (CN) ₆ ] at 50 ° C. The reaction was shaken for 20 minutes. The reaction solution was acidified by adding 1.25 ml of 10% trichloroacetic acid (w / v), and centrifuged at 3,000 rpm for 20 minutes. 2.5 ml of the supernatant was taken, and 2.5 ml of distilled water and 0.5 ml of 0.5% ferric chloride (FeCl ₃ ) were mixed, and reacted at room temperature for 10 minutes to measure absorbance at 700 nm of a Spectrophotometer (HITACHI U-2900). At this time, ascorbic acid and BHT, which are widely used as commercial antioxidants, were compared with the sample to measure reducing power, and the absorbance was measured by the same method. Reducing power shows strong reducing power as the absorbance is increased in the sample reaction solution.

The degree of Fe reducing power activity is the absorbance value indicating the reduction of metal ions by the chain reaction of free radicals providing hydrogen atoms. The effect of reducing power is that the higher the absorbance value of the reactant, the higher the antioxidant activity. To indicate. Fe-reducing power showed absorbances of 1.29 and 1.21 at concentrations of 0.1% of BHT and ascorbic acid, which are commercial antioxidants used as controls (FIG. 6). The reducing power of the non-fermented silkworm water extract (1 mg / ml) was 0.41, which was three times lower than the absorbance of commercial antioxidant BHT or ascorbic acid used as a control (FIG. 6). However the B. subtilis fermentation silkworm water extract (1 mg / ml) showed a tendency to decrease gradually while increasing the fermentation day 6 0.56, A. kawachii fermentation silkworm water extract (1 mg / ml) in a large change in fermentation to 9 days After 12 days of fermentation, the highest activity was 0.47. The absorbance measured by reducing power in hot water extract (1 mg / ml) was 0.34, whereas the activity was increased by 0.65 in fermentation, which is consistent with the results of the present invention (Park, SJ). , SW Song, DH Seong, DS Park, SS Kim, J. Gou, JH Ahn, WB Yoon and HY Lee.2009.Biological activities in the extract if fermented Codonopsis lanceolata.J . Korean Soc.Food Sci.Nutr. 38, 983-988).

In the above experiments, the reducing power and thrombolytic activity of B. subtilis fermented silkworms were highest at 6 days of fermentation and the highest at 12 days of fermentation, and the electrophoretic analysis on SDS-PAGE showed 96-66 kDa size at 3 days of fermentation. Most of the protein had been degraded. The reducing power, antioxidant activity and protein concentration of A. kawachii fermented silkworms were highest at day 12 of fermentation.In the electrophoresis analysis of SDS-PAGE, 96-66 kDa-sized proteins began to degrade gradually from day 3 of fermentation. Mostly done. Therefore, it was suggested that the fermentation time should be changed according to the species used in order to expect a specific physiological activity in the fermented silkworm using microorganisms.

Example  9. Animal Experiment

Experimental material

Precultured Bacillus subtilis The strain was inoculated into sterilized hot air dried silkworm powder, fermented at 37 ° C. for 48 hours, and then hot air dried for 6 hours to be used in experimental animals. Aspergillus kawachi strains were also inoculated in sterilized hot-air dried silkworm powder for 72 hours at 150 rpm and 30 ℃ in PD liquid medium, fermented at 30 ℃ for 12 days, and then fermented silkworm powder sample obtained by hot-air drying for 6 hours. Used for animal experiments.

Diet  And Experimental group

Dietary composition is shown in Table 4, normal group (N), alcohol administration control group (C), alcohol + hot air drying silkworm powder administration group (SP), alcohol + Bacillus subtilis Fermented silkworm powder group (BFSP), alcohol + Aspergillus kawachi The experiment was divided into fermented silkworm powder administration group (AFSP). Determination of the amount of fermented silkworm powder in the diet is described by Yoon et al. In Yoon JW, Rhee SK, Lee KB. Effects of silkworm extract powder on plasma lipids and glucose in rats. Korean J Food Nutr 18: 140-145 (2005)], wherein the fermented silkworm powder added in the diet was replaced with casein and sugar as a protein source.

Experimental dietary composition (%) Ingredients Normal Alcohol Control SP ^{1 )} BFSP ^{2 )} AFSP ^{3 )} Casein 20 20 17 17 17 Corn starch 15 15 15 15 15 Sucrose 55 55 53 53 53 Cellulose 5 5 5 5 5 Corn oil 10 10 10 10 10 Mineral mixture ^{4 )} 3.5 3.5 3.5 3.5 3.5 Vitamin mixture ^{5 )} One One One One One Choline bitartrate 0.2 0.2 0.2 0.2 0.2 DL-Methionine 0.3 0.3 0.3 0.3 0.3 SP 0 0 5 0 0 BFSP 0 0 0 5 0 AFSP 0 0 0 0 5

¹⁾ SP: Silkworm powder.

²⁾ BFSP: Bacillus subtillis fermented silkworm powder.

³⁾ AFSP: Aspergillus kawachii fermented silkworm powder.

⁴⁾ AIN 93 M-MX mineral mix, MP Biomedicals, Illkirch, France.

⁵⁾ AIN 93 VX vitamin mix, MP Biomedicals, Illkirch, France.

Experimental Animals and Breeding Conditions

The experimental animals were purchased for 6 weeks old Sprague-Dawley male rats. In this experiment, body weight was equally classified into randomized complete block design, and the room temperature (22 ± 2 ℃), humidity (50 ± 5%) and contrast cycle (light cycle: 07: 00 ~ 19: 00) It was raised in a controlled animal breeding room. Dietary intake was measured every day during the breeding period, and body weight was measured at a fixed time once a week. At this time, the alcohol administration was administered together with the sample at the beginning of the experiment, starting with the first 10% alcohol concentration step by step to a final 30% concentration to prevent acute toxicity by alcohol in the experimental animals.

Animal Testing, Sampling and Analytical Sample Preparation

Animal experiments were reared for four weeks with feeding for each group, followed by fasting for more than 12 hours on the last day of the experiment, and dissected by light anesthesia with ether. Blood was collected from the abdominal aorta after laparotomy, blood was collected, and the serum obtained by centrifugation at 3,000 rpm for 20 minutes after being left at room temperature for about 30 minutes was used for serum biochemical analysis. After blood collection, each tissue was extracted, washed with cold 0.9% saline solution, dried with filter paper, weighed, and provided as an analytical sample.

Example  10. Evaluation of animal test results

Changes in weight, diet and drink intake

It was shown that the weight gain did not increase much by the alcohol intake for 4 weeks compared with the normal group, and that the growth was influenced by the alcohol, and the dietary intake was also decreased at the same time (Table 5). On the other hand, alcohol decreases the absorption of nutrients or dietary components in the small intestine, which is a digestive organ, resulting in an effect imbalance, leading to a decrease in weight gain. However, dietary supplementation of SP, BFSP, and AFSP in alcohol-fed rats showed an increase in body weight due to increased dietary intake (Table 5).

Effects of SP, BFSP and AFSP on Body Weight, Diet and Drink Intake, and Relative Tissue Weight in Alcoholic Rats Group Body weight gain
(g) Food intake
(g / day) Water consumption
(mL / day) Normal 212.19 ± 7.55 ^a 19.83 ± 0.17 ^a 35.50 ± 1.28 ^a Control 99.04 ± 17.17 ^b 13.00 ± 0.63 ^b 14.50 ± 0.67 ^b SP 119.92 ± 18.90 ^b 12.83 ± 0.40 ^b 15.67 ± 0.92 ^b BFSP 116.34 ± 13.13 ^b 12.83 ± 0.54 ^b 17.67 ± 0.92 ^b AFSP 115.63 ± 13.46 ^b 12.50 ± 0.43 ^b 16.83 ± 1.47 ^b

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p <0.05. (mean ± S.E., n = 6).

NS: Not significant different.

Absolute and relative weight of each organ

The absolute increase and relative weight of each organ with respect to body weight are shown in Table 6 and Table 7, respectively. Absolute weight showed a significant difference in all experiments + group compared to normal group (Table 6).

Effects of SP, BFSP and AFSP on Tissue Weight in Alcohol-administered Rats Group Liver Kidney Spleen Heart Testis Epididymal
WAT Perirenal
WAT Normal 11.48
± 0.22 ^a 3.31
± 0.10 ^a 0.79
± 0.05 ^a 1.57
± 0.04 ^a 3.32
± 0.13 ^a 8.12
± 0.39 ^a 8.54
± 0.40 ^a Control 8.89
± 0.65 ^b 2.84
± 0.09 ^b 0.59
± 0.04 ^b 1.31
± 0.05 ^b 2.96
± 0.09 ^b 4.57
± 0.66 ^b 3.96
± 0.89 ^b SP 9.04
± 0.45 ^b 2.63
± 0.05 ^b 0.51
± 0.04 ^b 1.23
± 0.06 ^b 3.10
± 0.05 ^ab 4.90
± 0.57 ^b 4.10
± 0.75 ^b BFSP 9.71
± 0.60 ^b 2.86
± 0.20 ^b 0.56
± 0.03 ^b 1.17
± 0.06 ^b 3.10
± 0.06 ^b 4.94
± 0.42 ^b 3.97
± 0.53 ^b AFSP 9.23
± 0.37 ^b 2.67
± 0.07 ^b 0.54
± 0.03 ^b 1.23
± 0.04 ^b 3.01
± 0.10 ^ab 4.64
± 0.27 ^b 3.82
± 0.55 ^b

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p <0.05. (mean ± SE, n = 6).

In addition, the relative weight of each organ to body weight was not significantly different among the experimental groups in the liver, kidney, heart, and testicles, but there were slight differences among the groups in the spleen, the surrounding adipose tissue and the surrounding testicular adipose tissue. 7).

Effect of SP, BFSP and AFSP on Relative Tissue Weight of Alcohol-administered Rats Group Liver Kidney Spleen Heart Testis Epididymal
WAT Perirenal
WAT Normal 2.65
± 0.04 ^NS 0.76
± 0.03 ^NS 0.18
± 0.01 ^a 0.75
± 0.40 ^NS 0.78
± 003 ^NS 1.87
± 0.08 ^a 1.97
± 0.09 ^a Control 2.62
± 0.07 0.85
± 0.03 0.18
± 0.01 ^a 0.39
± 0.01 0.88
± 0.07 1.33
± 0.14 ^b 1.13
± 0.21 ^b SP 2.57
± 0.09 0.76
± 0.05 0.14
± 0.01 ^b 0.35
± 0.03 0.92
± 0.06 1.37
± 0.11 ^b 1.13
± 0.17 ^b BFSP 2.76
± 0.07 0.82
± 0.04 0.16
± 0.01 ^ab 0.33
± 0.02 0.91
± 0.02 1.40
± 0.07 ^b 1.12
± 0.13 ^b AFSP 2.66
± 0.05 0.77
± 0.03 0.16
± 0.01 ^{a b} 0.36
± 0.01 0.85
± 0.06 1.34
± 0.08 ^b 1.08
± 0.13 ^b

Relative tissue weight means the percent of the liver weight in the body weight.

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are significantly different at p <0.05. (mean ± S.E., n = 6).

NS: Not significant different.

Example  11. Liver tissue and serum lipid concentration and biochemical indicator analysis

The homogenate fraction preparation from the liver tissue was prepared by taking an amount of liver and adding ice-cold potassium phosphate buffer solution (0.1 mol / L potassium phosphate, pH 7.4 containing 1 mmol / L sodium EDTA and 1 mmol / L dithiothreitol). A homogenate was prepared by adding 9 times the amount using an IKA-ULTRA-TURRAX T25 basic homogenizer (IKA-WERKE GMBH & CO., KG, Staufen, Germany). The homogenization solution was first centrifuged at 800 xg for 10 minutes, and the supernatant was centrifuged at 10,000 x g for 20 minutes in a high-speed centrifuge (VS-24SMTi, Vision Scientific Co., Ltd.) to precipitate the pellet as a mitochondrial fraction. . At this time, the supernatant containing microsomes was ultracentrifuged at 105,000 xg for 60 minutes to obtain the cytosol fraction and precipitated microsomal fraction of the upper layer. Homogenizer, Mitochondria And Protein concentration of the microsomal fraction was measured using bovine serum albumin as a standard according to the Lowry method (Lowry OH, Rosebrough NJ, Farr AL, Randall RS. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 ( 1951)).

Serum total lipid, triglyceride, total cholesterol, HDL-cholesterol, free fatty acid, total protein, albumin concentration, and ALT, AST, γ-GTP, LDH activity were investigated at Neodin Medical Research Institute (Seoul, Korea). Requested analysis.

Neutral lipid concentration in liver tissue and blood

The liver is known to be an important tissue involved in many lipids and lipid metabolism, and blood triglyceride levels are closely related to hepatic triglyceride metabolism. In particular, triglyceride accumulation among liver lipids is well known as a causative agent for non-alcoholic or alcoholic fatty liver. In the present invention, the alcohol triglyceride concentration was significantly increased and the characteristics of alcoholic fatty liver were well represented (Table 8), which is consistent with the previous experimental results (Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet). JC.Usodeoxycholate alleviates alcoholic fatty liver damage in rats.Alcohol Clin Exp Res 22: 1538-1543 (1998)). Accumulation of hepatic lipids by hepatotoxicity in rats is known to be somewhat improved by amino acids with antioxidant and hepatotoxicity-improving effects such as glycine, arginine, taurine, cysteine and alanine. BFSP administration, which has high antioxidant activity, has been shown to significantly reduce the accumulation of hepatic triglycerides by alcohol (Table 8). In particular, hepatic triglyceride levels in the BFSP-administered group were reduced to normal levels. . Alcohol consumption has been reported to increase blood triglyceride levels (Oliva L, Beauge F, Choquart D, Montet AM, Guitaoui M, Montet JC. Ursodeoxycholate alleviates alcoholic fatty liver damage in rats.Alcohol Clin Exp Res 22: 1538 -1543 (1998)), the results of the present invention were inconsistent with these results (Table 8).

There was no significant difference in serum triglyceride levels between the normal group and the alcohol control group. Serum triglyceride concentrations tended to decrease in the AFSP-treated group and markedly decreased in the BFSP-administered group, showing differences among the strains used during fermentation. The decrease of triglyceride in blood by the administration of BFSP is thought to be due to the reduction of free fatty acid.

Effects of SP, BFSP, and AFSP on Neutral Lipid Concentrations in Liver and Serum of Alcoholic Rats Serum Triglyceride
(mg / 100 mL) Liver triglyceride
(mg / g) 46.67 ± 3.38 ^a 19.71 ± 1.38 ^a 51.33 ± 9.74 ^a 53.13 ± 3.54 ^b 52.80 ± 13.14 ^a 31.35 ± 3.14 ^ab 35.50 ± 8.01 ^ab 33.50 ± 8.01 ^ab 19.00 ± 2.22 ^b 21.10 ± 1.22 ^b

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Changes in blood lipid concentration

Blood lipid levels are used as diagnostic indicators of cardiovascular diseases such as atherosclerosis, hypertension, heart disease, and hyperlipidemia. And new clinical guidelines have been established in the US and elsewhere. Recently, attempts have been actively made to reduce blood cholesterol levels as well as neutral lipid concentrations and increase HDL-cholesterol levels in natural plant resources. Serum HDL-cholesterol concentration is an indicator of anti-arteriosclerosis. Unlike other lipoproteins, serum HDL-cholesterol concentration separates and transports LDL-cholesterol deposited on the walls of blood vessels to the liver for energy use or to promote excretion in vitro. It is widely known as a beneficial cholesterol that can reduce the risk of causing cardiovascular disease. Increasing total cholesterol levels in blood is one of the common phenomena seen in alcoholic animals. In this experiment, the total cholesterol and free fatty acid concentrations were significantly increased in the alcohol-administered group compared to the normal group. However, the total lipid concentration in blood was significantly decreased in silkworm powder and fermented silkworm powder administration group compared to alcohol control. Blood free fatty acid concentrations were only reduced by BFSP administration. Therefore, fermented silkworm powder was observed to have a strong effect of reducing the total cholesterol concentration by alcohol.

Effects of SP, BFSP and AFSP on Serum Lipids in Alcohol-administered Rats Group Total lipid
(mg / 100 mL) Free fatty acid
(nmol / L) Cholesterol
(mg / 100 mL) HDL
-cholsterol
(mg / 100 mL) Normal 216.8 ± 7.9 ^NS 0.82 ± 0.09 ^a 52.00 ± 1.93 ^a 21.00 ± 0.89 ^NS Control 249.0 ± 21.7 1.12 ± 0.12 ^b 64.67 ± 1.38 ^b 21.33 ± 0.76 SP 234.0 ± 27.0 1.30 ± 0.05 ^b 51.00 ± 4.85 ^a 21.60 ± 1.76 BFSP 211.0 ± 24.4 0.87 ± 0.06 ^a 50.33 ± 4.59 ^a 21.00 ± 1.39 AFSP 222.5 ± 19.3 1.15 ± 0.08 ^b 48.83 ± 1.78 ^a 21.50 ± 0.72

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

NS: Not significant different.

Blood ALT , AST , γ- GTP  And LDH  Active change

Table 10 shows the results of verifying liver function efficacy by measuring ALT, AST, γ-GTP, and LDH activity, which are used as clinical indicators of liver damage. Serum ALT and AST activity is an important clue of liver damage indices because transaminase is released into the blood as hepatic necrosis and hepatic tissue destruction progress due to liver injury.

Previous experiments in this experiment [Cha JY, Heo JS, Cho YS. Effect of zinc-enriched yeast FF-10 strain on the alcoholic hepatotoxicity in alcohol feeding rats. Food Sci Biotechnol 17: 1207-1213 (2008); Cha JY, Kim HS, Kang SC, Cho YS. Alcoholic hepatotoxicity suppression in alcohol fed rats by glutathione-enriched yeast FF-8 strain. Food Sci Biotechnol 18: 1411-1416 (2009)], AST, γ-GTP activity was significantly increased in the alcohol-administered group, but ALT activity tended to increase without statistically significant difference (Table 10). The increase in AST and γ-GTP activity in the alcohol-fed control group was found to be markedly decreased by BFSP and AFSP administration (Table 10). This effect suggests that liver protection effect is better by BFSP administration than AFSP.

In experiments fed glutathione, S-adenosylmethionine, amino acids, and zinc-rich yeast, experiments showing strong hepatoprotective effects by reducing blood ALT, AST and γ-GTP activity were performed [Cha et al., 2008 ; Cha et al., 2009; Izu H, Shobayashi M, Manabe Y, Goto K, Iefuji H. S-adenosylmethionine (SAM) -accumulating sake yeast suppresses acute alcohol-induced liver injury in mice. Biosci Biotechnol Biochem 70: 2982-2989 (2006). In addition, carbon tetrachloride-induced hepatotoxicity model supplemented with glutathione-containing yeast S. cerevisiae has been reported to reduce ALT and AST activity in previous experiments [40]. Sugimura and Yamamoto, Sugiyama Y, Yamamoto K. The protective effect of glutathione-enriched yeast extract on acetaminophen-induced liver damage in rats. J. Jpn. Soc. Nutr. Food 51: 189-193 (1998)] showed that AST and ALT activities were reduced in a concentration-dependent manner by the administration of glutathione-containing yeast in acetaminophen-induced hepatotoxicity experiments. It was. This effect is shown to improve liver toxicity by increasing glutathione concentration in liver cells by administration of glutathione-containing yeast, and this study was also supported by the result that the intracellular glutathione concentration was increased by fermented silkworm powder administration. (FIG. 10).

It has been reported that blood LDH and ALP activity, which is used as a clinical functional indicator of liver, is increased by alcohol intake and has a close correlation with the induction of alcoholic liver toxicity [Cha et al., 2008; Sugiyama et al., 1998]. In this experiment, LDH activity was also increased in the alcohol intake group, but this increase by alcohol was significantly decreased by BFSP and AFSP administration and decreased by silkworm powder administration (Table Table 10). . Therefore, BFSP can be used as a dietary supplement for improving alcoholic liver disease by significantly reducing ALT, γ-GTP, and LDH activity.

Effect of SP, BFSP and AFSP on AST, ALT, γ-GTP and LDH Activity in Alcohol-administered Rats Group AST
(IU / L) ALT
(IU / L) γ-GTP
(IU / L) LDH
(IU / L) Normal 123.17 ± 5.80 ^a 36.50 ± 1.93 ^NS 0.28 ± 0.05 ^a 3191 ± 348 ^a Alcohol 149.00 ± 7.75 ^b 42.33 ± 3.66 0.60 ± 0.09 ^b 4243 ± 532 ^b SP 118.97 ± 6.16 ^a 38.00 ± 5.05 0.38 ± 0.07 ^ab 3096 ± 183 ^a BFSP 103.33 ± 9.58 ^a 34.00 ± 2.03 0.30 ± 0.05 ^a 1407 ± 461 ^c AFSP 111.67 ± 12.86 ^a 40.00 ± 3.30 0.65 ± 0.30 ^b 2100 ± 613 ^c

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

AST: aspartate aminotransferase, ALT: alanine aminotransferase, γ-GTP: γ-glutamyltranspeptidase, LDH: lactate dehydrogenase.

Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

NS: Not significant different.

Blood proteins, albumin and Globulin  Concentration change

Serum total protein concentration increased in chronic infections, chronic liver disorders, cirrhosis, etc., but significantly decreased in all experimental groups treated with alcohol compared to the normal group (Table 11). Albumin is a blood protein synthesized in the liver, which is important to account for about 60% of the total plasma protein, and is used as an important liver disease indicator that is reduced in severe liver disease. In this experiment, albumin concentrations were significantly decreased in all groups treated with alcohol compared to the normal group (Table 11). In addition, blood globulin concentration accounts for most of serum proteins except albumin in blood, and increases in infectious diseases, inflammatory diseases and malignancies. Significant decrease or decrease in alcohol control group and silkworm powder and fermented silkworm powder administration group compared to normal group Results were obtained (Table 11).

Effects of SP, BFSP, and AFSP on Concentrations of Total Protein, Albumin, and Globulin in Serum of Alcoholic Rats Group Protein
(g / 100 mL) Albumin
(g / 100 mL) Globulin
(g / 100 mL) Normal 6.02 ± 0.13 ^a 2.83 ± 0.09 ^a 3.18 ± 0.05 ^a Control 5.57 ± 0.12 ^b 2.57 ± 0.05 ^b 3.00 ± 0.08 ^ab SP 5.37 ± 0.07 ^b 2.48 ± 0.03 ^b 2.88 ± 0.04 ^b BFSP 5.57 ± 0.08 ^b 2.57 ± 0.03 ^b 3.00 ± 0.05 ^ab KWSP 5.30 ± 0.09 ^b 2.43 ± 0.03 ^b 2.87 ± 0.08 ^b

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

Example  12. Blood alcohol and Acetaldehyde  Concentration measurement

Alcohol and acetaldehyde concentrations in serum were measured using a commercial UV-Test kit (Darmstadt, Germany) of R-Biopharm. In other words, the blood alcohol concentration was measured by adding 0.1 mL of serum to a 3 mL mixture of NAD and phosphate buffer solution (pH 9.0), reacting for about 3 minutes, measuring absorbance at 340 nm, and adding 0.05 mL of ADH to the mixture for 10 minutes at 20 ° C. The change in absorbance according to the concentration of NADH produced at room temperature and absorbance at 340 nm was observed. The alcohol concentration in blood (%) was calculated using a standard solution.

Serum acetaldehyde concentration was measured by adding 0.2 mL of serum to a 3 mL mixture of NAD and phosphate buffer (pH 9.0), reacting for about 3 minutes, measuring absorbance at 340 nm, and adding 0.05 mL of ALDH to the mixture at After reacting for 5 minutes, the change in absorbance according to the concentration of NADH produced at absorbance 340 nm was observed. At this time, the acetaldehyde concentration in the blood was calculated using a standard solution.

Alcohol and its primary metabolite, acetaldehyde, are the main causative agents of experiencing hangover after drinking. They are potent toxins that act as a major mediator of liver damage through condensation reactions with active amines in vivo. It is metabolized by ALDH to form acetic acid and then hydrolyzed with carbon dioxide and water to undergo a complete decomposition process. Toxicity of acetaldehyde has been reported to impair cirrhosis and heart and brain function due to mitochondrial impairment. In general, alcohol and fruit juices, spices, and various herbal medicines appear to have different odors when drinking, which can be considered that alcohol metabolism was promoted by various substances in alcohol. Recently, it has been reported that red ginseng extract promotes alcohol metabolism in animal experiments with alcohol. Also, fermented red ginseng fermented red ginseng was confirmed to have a hangover effect.

Blood alcohol concentration was not detected in blood of the normal control group, but blood alcohol was detected in all experimental groups to which alcohol was administered (Table 12). The blood alcohol concentration was significantly increased in the alcohol control group compared to the normal group, and this increase was significantly decreased at the same concentration in the SP, BFSP, and AFSP groups. Plasma acetaldehyde concentration was significantly increased in the alcohol control group compared to the normal group, and the increase in the alcohol group tended to decrease in the silkworm powder and fermented silkworm powder administration groups without significant difference, respectively (Table 12). Fermented silkworm powder has been shown to have an effect of decreasing acetaldehyde concentration as well as blood alcohol concentration, which was shown to be due to the increase in the activity of alcohol-related enzymes ADH and ALDH in liver tissue. Previous studies have reported that soymilk supplemented with B. breve strains improves alcoholic hepatotoxicity due to a decrease in acetaldehyde concentration due to increased ALDH activity (Kano M, Ishikawa F, Matsubara S, Kikuchi-Hayakawa H, Shimakawa Y. Soymilk products affect ethanol absorption and metabolism in rats during acute and chronic ethanol intake.J Nutr 132: 238-244 (2002)). Silkworm powder or Aspergillus in this experiment Bacillus rather than fermented silkworm powder Fermented silkworm powder was administered to improve alcoholic hepatotoxicity, especially Bacillus The silkworm fermentation of the strain was shown to increase the efficacy of improving liver toxicity.

Effects of SP, BFSP, and AFSP on Serum Concentrations of Alcohol and Acetaldehyde in Alcohol Administration Rats Group Serum alcohol
(mg / 100 mL) Serum acetaldehyde
(mg / 100 mL) Normal 0.000 ± 0.00 ^a 2.63 ± 0.44 ^a Control 0.055 ± 0.01 ^b 5.68 ± 0.74 ^b SP 0.037 ± 0.00 ^c 3.03 ± 0.56 ^a BFSP 0.037 ± 0.00 ^c 2.76 ± 0.40 ^a AFSP 0.037 ± 0.00 ^c 2.84 ± 0.39 ^a

SP: Silkworm powder.

BFSP: Bacillus subtillis fermented silkworm powder.

AFSP: Aspergillus kawachii fermented silkworm powder.

Values with different letters are the significantly different statistically at p 〈0.05. (mean ± SE, n = 6).

Example  13. ADH  And ALDH  Enzyme Activity Measurement

Determination of ADH activity from liver tissue was performed by Bergmeyer's method (Bergmeyer HU.Methods of Enzymatic Analysis. Academic Press, New York, pp. 28, (1974)) to measure the rate of NADH production at absorbance 340 nm. In other words, the composition of the reaction solution was adjusted to 1.5 ml of distilled water, 0.75 ml of 1.0 M Tris-HCl buffer (pH 8.8), 20 mM NAD ⁺ 0.3 ml, 0.3 ml of ethanol, and 0.15 ml of enzyme source in a cuvette to a total of 3 ml. After 5 minutes of preincubation at 30 ° C., the change in absorbance at 340 nm was measured for 5 minutes.

Determination of ALDH activity from liver tissue was performed using Koivula et al. (Koivula T, Koivusalo M, Lindros KO.Liver aldehyde and alcohol dehydrogenase activities in rat strains genetically selected for their ethanol preference.Biochem Pharmacol 24: 1807-1811 (1975)). A slight modification was used to generate NADH from NAD as an enzyme that produces acetate in acetaldehyde. That is, the composition of the reaction solution was distilled water 2.2 ml, 1.0 M Tris-HCl buffer (pH 8.0) 0.3 ml, 20 mM NAD ⁺ 0.1 ml, 0.1 M acetaldehyde 0.1 ml, 3.0 M KCl 0.1 mL, 0.33 M 2-mercapto 0.1 ml of ethanol and 0.1 ml of enzyme source were adjusted to a total of 3 ml, placed in a cuvette, preincubated at 30 ° C. for 5 minutes, and then the change in absorbance at 340 nm was measured for 5 minutes.

Western blot analysis was conducted to see the effect of fermented silkworm powder administration on the expression of ADH and ALDH enzyme proteins in alcoholic liver disease. Quantified ADH and ALDH enzyme proteins were electrophoresed 20 and 100 μg for each well of 10% SDS-polyacrylamide gel as well (Koivisto T, Eriksson CJ. Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol). -avoiding rat lines.Biochem Pharmacol 48: 1551-8155 (1994)). The isolated protein was transferred to nitrocellulose membrane for 16 hours at 4 ° C. under 300/240 mA / cm 2 condition. The membrane was blocked with non-specific binding protein for 1 hour at room temperature with TBST buffer solution (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 10% nonfat milk as a blocker. Wash for 5 minutes with buffer. The blocked membrane was reacted with primary and secondary antibodies for 1 hour at room temperature and then washed with TBST buffer. The washed membranes were overnight at 4 ° C. with rabbit ADH or ALDH1A1 conjugated HRP antibody (diluted at 1: 5,000, Abcam, England). Again washed with TBST buffer and incuvation with goat anti-mouse IgG-HRP (diluted 1: 2,000, DB USA). Proteins on the nitrocellulose membrane were detected by image analysis of SuperSignal West Pico Chemiluminescent Substrate. Relative densities of the reacted ADH or ALDH protein bands, respectively, were quantified by densitometer (Lumi-Imager F1, Roche, Switzerland).

Most of the alcohol absorbed into the body is absorbed by the stomach and small intestine, moved to the liver and then metabolized and broken down. Chronic alcohol intake can cause nutrient absorption and impair liver function. Alcohol metabolism in the liver is first converted to acetaldehyde by alcohol dehydrogenase (ADH) and cytochrom P-450, which increases acetaldehyde by MEOS (microsomal ethanol oxidizing system) during chronic alcohol intoxication. Done. Acetaldehyde, which accumulates in liver tissue mainly after chronic alcohol intake, is affected by ADH and ALDH activity. Since these ADH and ALDH enzyme activities play an essential role in the body's alcohol metabolism, the possibility of increasing the activity by natural bioactive components has been raised. Chronic alcohol administration in experimental animals increases ADH activity and decreases ALDH activity, suggesting the possibility of acetaldehyde accumulation in biological tissues by prolonged ingestion of alcohol. In the animal experiments, ADH activity in liver tissue showed a tendency to increase slightly in the alcohol control group compared to the normal group, but was significantly increased by the administration of SP, BFSP, AFSP (Fig. 7). ALDH activity was not different between the normal group and the alcohol control group, but the SP, BFSP, AFSP administration group was found to increase the activity (Fig. 7). These results show that fermented silkworm powder effectively decreases the blood alcohol and acetaldehyde concentrations in alcohol-administered rats because it promotes alcohol metabolizing enzymes ADH and ALDH activity in liver tissue. In particular, the ADH and ALDH activity in the BFSP-administered group was the highest, indicating that the alcohol and acetaldehyde concentration in the blood was reduced by using the alcohol in the body as a substrate.

ADH and ALDH protein expression levels are shown in FIG. 8. BFSP-administered group showed the highest ADH expression and AFSP-administered group showed relatively high ADH expression. These results show a high positive correlation in ADH enzyme activity and protein expression. However, the BFSP-administered group did not appear to have a significant effect on the ALDH expression level, which is another alcohol metabolism-related enzyme, suggesting that the BFSP did not necessarily match the enzyme activity and protein expression level. As in the previous results, it is reported that ADH enzyme activity and protein expression are greatly affected by alcohol and other dietary components. However, ALDH enzyme is slightly unaffected by alcohol and other dietary components. Reported to change gradually. Therefore, simultaneous administration of BFSP in alcohol-fed rats had more effect on ADH enzyme activity and protein expression related to alcohol metabolism than silkworm powder or AFSP, but not on ALDH protein expression.

Zinc is recognized as a dietary ingredient that plays an important physiological and biological role as an essential trace ingredient, and is known to have an effect on improvement of alcoholic liver toxicity. Zinc is a mineral component bound to many enzymes, and ADH enzymes are known to have a direct dependency on zinc. In a previous experiment, the administration of zinc-rich yeast as a catalytic element of ADH enzymes in alcohol-administered rats resulted in the promotion of ADH enzyme activity (Cha et al., 2008). For this reason, the concentration of zinc in the liver tissues was measured for this reason, but there was no statistically significant difference in the BFSP-administered group, indicating the highest zinc concentration, which was highly correlated with ADH enzyme activity (FIG. 9).

In addition to the mineral components, amino acid components such as asparagine, aspartic acid and arginine are also suggested as factors influencing ADH and ALDH activity related to alcohol metabolism. Addition of plum juice, a major component of hangover, and aspartic acid, its major constituent amino acids, increased ADH and ALDH activities (Hwang JY, Ham JW, Nam SH.Effect of Maesil ( Prunus mume) ) juice on the alcohol metabolizing enzyme avtivities.Korean J Food Sci Technol 36: 329-332 (2004)). In this experiment, plum juice promoted the decomposition of acetaldehyde produced by alcohol decomposition by increasing ADH activity and promoting alcohol degradation as well as ALDH activity. Aspart, the major constituent amino acid of plum juice Similar good effects were obtained with acid. In particular, aspartic acid and arginine among the amino acids promote the regeneration of NAD, which promotes alcohol degradation by promoting ADH activity, thereby lowering the toxicity of alcohol, and asparagine reacting with acetaldehyde to produce adducts while lowering the concentration of acetaldehyde, thereby reducing biological toxicity. It is suggested to weaken. In this experiment, the silkworm powder or fermented silkworm powder contains many of these amino acids, which may have the effect of improving alcoholic hepatotoxicity. This possibility appears to be higher because fermented silkworm powder contains taurine, cysteine and methionine which have inhibitory activity (Table 13).

Free Amino Acid Content (ppm) in SP, BFSP and AFSP Amino acids SP BSSP AKSP Phosphoserine 183 96 80 Taurine - 74 125 Phosphoethanolamine 579 - - Urea - 6 5 L-Aspartic acid 477 522 237 L-Threonine 1988 272 768 L-Serine 2003 970 496 Asparagine 1644 - - L-Glutamic acid 6294 2642 3056 L-α-Aminoadipic acid - 105 57 L-Proline 774 608 380 L-Glycine 1904 583 426 L-Alanine 6602 1823 1751 L-Citrulline - 334 132 L-α-Aminobutyric acid 42 - - L-Valine 2994 2174 1314 L-Cystine - 74 37 L-Methionine - 209 11 L-Isoleucine 2342 1549 1089 L-Leucine 3407 2368 1421 L-Tyrosine 2271 1477 925 L-Phenylalanine 2724 2163 1392 β-Alanine - - - DL-β-Aminoisobutyric acid - - - Γ-Aminobutyric acid (GABA) 40 110 84 L-Orinithine 218 670 2269 L-Lysine 1071 337 46 1-Methyl-L-Histidine - 1049 293 L-Histidine 1102 277 227 3-Methyl-L-Histidine - - - L-Carnosine - - - L-Arginine 3190 71 73 Total 41,809 20,447 16,606

-: Not detected.

Example  15. Changes in the concentration of lipid peroxides and antioxidants

Lipid peroxide measurement

Lipid peroxide content of the homogenized and microsomal biofilm fractions was quantified according to the Telegram method (Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem 95: 351-358 ( 1979)). In other words, 2 ml of TBA (thiobarbituric acid) reagent was added to 1 ml of a fraction solution containing 1 mg of protein, and the mixture was mixed well, and reacted in boiling water for 30 minutes, cooled at room temperature, and centrifuged at 3,000 rpm for 10 minutes. The supernatant was measured for absorbance at 535 nm. Lipid peroxide content was expressed in nmol / g of malondialdehyde.

Mineral content measurement

The mineral content of liver tissue was determined according to the AOAC analysis method (AOAC Official methods of analysis. 12th ed., Association of official analytical chemists. Washington, DC, USA (1975)). That is, 1 g of liver tissue was incubated for 3 hours in each 550 ° C. incineration furnace, dissolved in 6 N HCl, and completely acid-decomposed to completely remove the acid in a water bath, followed by addition of 3 N HCl to Whatman No. The filter was filtered through 4 filter papers and diluted at a constant ratio according to the type of element, and measured using an atomic absorption spectrophotometer (AAnalyst 300, Perkin Elmer, Norwalk CT, USA).

Glutathione  Content measurement

The glutathione content of liver tissue was mixed by adding 0.2 ml of tertiary distilled water and 0.5 ml of 0.4% sulfosalicylic acid to 0.2 ml of liver homogenate fraction, centrifuging, and then adding DTNB (5,5'-dithiobis (2-nitrobenzoic acid) Content was calculated by the standard calibration curve of glutathione, measured at 412 nm absorbance with the addition of a coloring reagent and expressed in mg per gram of liver tissue (Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione). J Lab Clin Med 61: 882-888 (1963).

Like ALT and AST activity, which are indicators of early acute hepatotoxicity, lipid peroxide concentrations in liver tissues are also used as indicators of hepatotoxicity. Lipid peroxide concentration, a measure of oxidative stress in liver tissue, is known to be closely associated with chronic alcohol intake. Chronic alcohol intake is known to result in excess production of lipid peroxide and free radicals in tissues and a decrease in the concentration of glutathione, an antioxidant in the body. This result was also confirmed in the experiment of the present invention in which the lipid peroxide concentration was increased in the homogenate, mitochondria and microsomal fractions of liver tissue after alcohol intake (FIG. 11).

Lipid peroxide levels were significantly decreased in liver tissue fractions by administration of BFSP in alcohol-ingested rats, and significantly decreased in mitochondrial and microsomal fractions by AFSP administration, but decreased in homogenate fractions. Means that fermented silkworm powder has antioxidant effect in liver tissue. Serum lipid peroxide concentration was also significantly increased by alcohol intake, significantly decreased by fermented silkworm powder administration (FIG. 11).

Iron, an essential trace element, is known as a substance that promotes fatty acid formation. It is a component of catalase that removes H ₂ O ₂ from the body and affects lipid peroxide reaction by the difference in the concentration of vitamin C in the body and the concentration of H ₂ O ₂ . In this study, the measurement of non-heme iron content is suggested as an important factor in the investigation of lipid peroxide reaction in vivo. The heme iron content in the liver tissue was significantly increased in the alcohol group compared to the normal group, but significantly decreased in the BFSP group (Fig. 9). Meanwhile, zinc concentrations in liver tissues (Goel A, Dani V, Dhawan DK.Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity.Chem Biol Interact 156: 131) -140 (2005)) was slightly higher in the BFSP-administered group, although there was no statistically significant difference between the experimental groups (FIG. 9).

The liver plays a central role in alcohol metabolism and causes tissue damage by the production of free radicals, and at the same time mitigates by acting as an intracellular antioxidant defense system. Glutathione in liver tissues is an antioxidant of nonprotein thiol tripeptide consisting of L-glutamate, L-cysteine and glycine and is known to play an important role in the antioxidant defense system through detoxification reactions in animal liver tissues in recent studies. Glutathione levels in tissues are closely associated with antioxidant systems that defend cells from the toxic effects of lipid peroxidation concentrations, active free radicals and other oxidative groups. Alcohol intake in rats lowers liver tissue and blood glutathione concentrations compared to normal group (FIG. 10). However, the decrease in liver tissue and blood glutathione concentration by alcohol intake was found to be restored to the normal group level by administration of BFSP (FIG. 10). Previous studies have reported that high levels of glutathione-containing yeast effectively prevent liver damage from carbon tetrachloride and alcohol administration [Cha et al., 2009; Shon MH, Cha JY, Lee CH, Park SH, Cho YS. Protective effect of administrated glutathione-enriched S accharomyces cerevisiae FF-8 against carbon tetrachloride (CCl ₄ ) -induced hepatotoxicity and oxidative stress in rats. Food Sci Biochenol 16: 967-974 (2007). Decreased lipid peroxide levels due to BFSP administration in alcohol-fed rats seemed to be closely associated with increased antioxidant activity. The hepatoprotective effect of unfermented silkworm powder is also believed to be due to its constituents such as amino acids. Methionine or cysteine as a precursor of glutathione metabolism in liver tissue has recently been reported to play an important role in endogenous glutathione synthesis [Peters TJ. Ethanol metabolism. Bri Med Bull 38: 17-20 (1982); Stipanuk MH, Coloso RM, Garcia RAG, Banks MF. Cysteine concentration regulates cysteine metabolism to glutathione, sulfate and taurine in rat hepatocytes. J Nutr 122: 420-427 (1992)], reported to increase glutathione concentrations in liver and kidney tissues by administration of cysteine and cysteine containing components [Hsu CC, Hiang CN, Hung YC, Yin MC. Five cysteine-containing compounds have antioxidative activity in Balb / cA mice. J Nutr 134: 149-152 (2004).

Recently, silk fibroin consisting of amino acids of 42% glycine and 32% alanine has been reported to improve alcoholic hepatotoxicity [Kang GD, Lee KH, Do SG, Kim CS, Suh JG, Oh YS, Nham JH. Effect of silk fibroin on the protection of alcoholic hepatotoxicity in the liver of alcohol preference mouse. Int. J Indust Entomol 2: 15-18 (2001). Amino acids such as serine, glycine, histidine and tyrosine are also known to improve galactosamine- and alcohol-induced hepatotoxicity [Lee JH, Kim NK, Lee DY, Lee CH. Protective effect of selected amino acids and food extracts on ethanol toxicity determent in rat liver. Korean J Food Sci Technol 31: 802-808 (1999); Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998). The free amino acid composition of Aspergillus- and Bacillus -fermented silkworm powders in this experiment were mainly glutamic acid (3056 ~ 2642 ppm), leucine (1421 ~ 2368 ppm), valine (1314 ~ 2174 ppm), alanine (1751-1823 ppm), And tyrosine (925-1477 ppm), respectively (Table 13), and fermented silkworm powder mainly composed of these amino acids is considered to have a high possibility of reducing alcohol-induced oxidative stress. Sericin proteins consisting of amino acids L-serine, L-glycine and L-alanine have also been reported to reduce hepatotoxicity induced by free radicals [Ming Y, Ikejima K, Arteel GE, Seabra V, Bradford BU, Kono H, Rusyn I, Thurman RG. Glycine accelerates recovery from alcohol-induced liver injury. J Pharmacol Exp Ther 286: 1014-1019 (1998); Inkeles S, Eisenberg D. Hyperlipidemia and coronary atherosclerosis. Medicine Baltimore 60: 110-123 (1981); Petushok NE, Miskevich D. The influence of the some amino acids on the antioxidant status in the liver of rats. Toxicology Letters 144: S116 (2003). Combination of ascorbic acid with arginine, histidine, taurine, and lysine amino acids resulted in a 70% increase in glutathione concentrations in liver tissue homogenization fractions, and a nearly double increase in glutathione system (glutathione reductase / peroxidase) activity in rat liver. It has been shown to act to significantly increase the antioxidant system of the tissue (Petushok et al., 2003).

In addition, hydroxy amino acids such as serine and threonine are known to exhibit antioxidant activity by forming chelating with trace elements such as copper and iron [Kato N, Sato S, Yamanaka A, Yamada H, Fuwa N, Nomura M. Silk protein , sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci Biotechnol Biochem 62: 145-147 (1998). In this experiment, the Bacillus -fermented silkworm powder contains more amino acids such as serine, glycine, alanine, tyrosine and histidine, rather than Aspergillus -fermented silkworm powder, which may not be related to having strong antioxidant activity (Table 2). Therefore, in this experiment, Bacillus -fermented silkworm powder contains more amino acids that are highly related to antioxidant activity, so that the protective effect against lipid peroxidation in liver tissue and blood is greater than silkworm powder or Aspergillus -fermented silkworm powder.

Example  16. Liver tissue  Histopathological observation

Immediately after animal dissection, the extracted liver was perfused with cold saline to remove blood, and a portion of the tissue was removed and fixed in a 10% neutral formalin solution, followed by paraffin embedding, which is a conventional tissue treatment. H & E (hematoxylin and eosin) staining was performed and observed with an optical microscope (Olympus BX41, Olympus Co., Tokyo, Japan) [Cha JY, Jun BS, Cho YS. Prevention of orotic acid-induced fatty liver in rats by capsaicin. Food Sci Biotechnol 13: 597-602 (2004)].

Fatty liver is the starting point of liver toxicity caused by excessive alcohol intake and is known to cause oxygen or nutritional imbalance in liver cells. Accumulation of Lipids in Liver Cells Fatty liver is the most common symptom of alcoholic liver toxicity early in life. The liver tissues were removed and histologic examination immediately after dissection of the animals. The control group maintained well the hepatic lobule structure centered on blood vessels. It was well maintained (FIG. 12). In the fermented silkworm powder group, the alcoholic liver toxicity was improved by decreasing blood ALT and AST activity and decreased lipid peroxide concentration in liver tissue.

Fatty liver induction due to the increase of fat globules in most liver cells by alcohol administration was also observed in this experiment. Alcohol is high in calories without nutrients like carbohydrates and proteins, which can eventually lead to fat accumulation in liver tissue. Meanwhile, in the fermented silkworm powder-administered groups, fat accumulation in hepatocytes by alcohol was improved, and liver histological observation showed a very similar appearance to the normal group, which is evidence of the hepatocellular protective effect of fermented silkworm powder (FIG. 13).

Claims (11)

Adding distilled water to the silkworm powder and sterilizing it; And
Bacillus subtilis culture or Aspergillus fermentation at 37 ° C. for 3 to 12 days after the addition of kawachii culture, followed by drying
Fermented silkworm powder manufacturing method prepared including. The method of claim 1, wherein 1% (v / w) Bacillus relative to silkworm powder subtilis Culture medium Fermented silkworm powder production method, characterized in that the addition. The method of claim 1, wherein 5% (v / w) of spergillus relative to silkworm powder kawachii Culture medium Fermented silkworm powder production method, characterized in that the addition. The method for producing fermented silkworm powder according to claim 1, wherein the silkworm is fermented for 6 days to 12 days. Fermented silkworm powder prepared by the method of claim 1. A pharmaceutical composition for preventing or treating hyperlipidemia and fatty liver, comprising the fermented silkworm powder of claim 5 as an active ingredient. A pharmaceutical composition having antioxidant efficacy, comprising the fermented silkworm powder of claim 5 as an active ingredient. A pharmaceutical composition having a thrombolytic effect comprising the fermented silkworm powder of claim 5 as an active ingredient. A dietary supplement for improving hyperlipidemia and fatty liver comprising the fermented silkworm powder of claim 5. A health supplement composition having antioxidant efficacy, comprising the fermented silkworm powder of claim 5. A dietary supplement having a thrombolytic effect comprising the fermented silkworm powder of claim 5.

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