KR101087160B1 - Composition for Lowering Cholesterol Using Hydrolysed Squid by Protease - Google Patents

Abstract

The present invention relates to a composition for lowering cholesterol by enzymatic degradation of cuttlefish meat. More specifically, by adding proteolytic enzyme (alcalase) to the pre-treated squid meat to hydrolyze the squid meat, and then separated into liquid, solid or powder form to measure the cholesterol lowering effect of the squid meat enzyme decomposition products through animal experiments It relates to a composition for lowering blood cholesterol composed of steps.

The cuttlefish enzymatic digestion product of the present invention is a pretreatment step (1) of washing and dehydrating the cuttlefish meat from which the guts of the cuttlefish are separated, a step of heat treatment to heat and gelatinize the cuttlefish meat (2), and a step of grinding the heat treated cuttlefish meat ( 3) A composition for lowering cholesterol using an enzymatic product of squid meat, comprising the step (4) of adding an enzyme to squid meat and hydrolyzing it, and the step (5) of separating the hydrolyzate.

The present invention relates to a new invention of a functional material and its manufacturing method (Patent Application No. 10-2009-0028165) by enzymatic decomposition of squid meat filed on April 1, 2009 to the Korean Patent Office.

The present invention improves the enzymatic hydrolysis process by adopting a heating and stabilizing process for squid meat, so that squid meat is well decomposed into peptides of low molecular weight by protease, thereby reducing blood cholesterol.

Cuttlefish meat, heating and dehydration, protease, enzyme hydrolyzate, cholesterol lowering

Description

Composition for Lowering Cholesterol Using Hydrolysed Squid by Protease

The present invention relates to a composition for lowering cholesterol by enzymatic degradation of cuttlefish meat. More specifically, after hydrolysis of squid meat by adding proteolytic enzyme (alcalase) to the pretreated squid meat, taking the supernatant using a centrifuge, using hot air, freezing or spray-drying to use squid meat enzyme digest It relates to a composition for lowering cholesterol.

The internal organs of the raw material itself are maintained by removing and cleaning the guts of the squid and simply dehydrating it from the shaft. In order to disable the enzyme inhibitor in squid meat and activate the enzymatic reaction, raw meat is heated by autoclave and gelatinized. After grinding the squid meat, the proteinase is added, hydrolyzed and centrifuged to obtain the supernatant, followed by hot air, freezing, or spray drying to obtain squid enzyme.

The present invention is an invention related to a new material of the functional material and its manufacturing method (Patent Application No. 10-2009-0028165) by the degradation of the squid meat filed on April 1, 2009 to the Korean Patent Office.

The present invention relates to a composition for lowering cholesterol using squid meat hydrolyzate by enzymatic degradation of squid meat caught in large quantities off the coast of Korea.

The squid has a body of three parts: the head, the body and the legs. The head is between the legs and the body and has big eyes on both sides. It has two cuffs and eight legs and a tapered end with a sucker with a short bag inside. Between the third and fourth legs, there is a cuff, longer than the other leg, slightly wider at the end, and there is a sucker. Usually it is in a pocket and stretches when catching food. Squids live in the sea and live from coast to deep sea. Squids are edible and cuttlefish, pattern squid, fire squid, cuttlefish, sago squid, squid, white squid are especially important in fisheries. It is eaten raw but can also be processed into dried squid. The smallest squid is a small squid, only 2.5cm long, and the largest squid is Architheutis harveyi , a type of giant squid, which lives in the Atlantic Ocean and reaches 15.2m including the tip. Squids are carnivorous and feed on small fish, shrimp, crabs, etc., while feeding large fish, sea turtles, and seawater. The reproductive season is mostly from April to June, and is a fish species caught in large quantities off the coast of Korea.

As a prior art using squid guts or squid meat, Korean patent registration 10-0001786 (processing method of squid) is to immerse the peeled squid in warm saline and smoke it, and then spray the aqueous solution such as seasonings such as salt and glycerin and spices. To dry. Korea Patent Registration 10-0130178 (processing method of squid) is mixed with acid, alcohol, salt mixture of squid meat, then immersed, dried, baked and rolled. Korean Patent Publication No. 10-1997-0042984 (Method for Separation of Polyunsaturated Lipids from Squid Processing By-products) is performed by grinding squid intestine, adding isopropyl alcohol, extracting by high speed stirring, methyl esterification, urea precipitation or chromatography. do. Korea Patent Registration 10-805918 (Bimbijang for noodles containing squid-incorporated autolysate and its manufacturing method) adjusts the ground squid gut to pH 2-5 and autolyzes by protease at 20-60 ℃ for 10-30 hours After heating, the mixture is heated at 90-110 ° C. for 20-30 minutes to obtain squid intestine autolysate. Korean Patent Registration 10-0815118 (Noodle soup composition containing squid gut hydrolyzate) is for noodles containing squid gut hydrolyzate, kelp extract, soy sauce, shrimp extract, leek extract, radish extract, shiitake extract, mirin and water Jangguk composition. Korean Patent Laid-Open Publication No. 10-2008-37082 (Method for manufacturing soft products using squid and soft products using squid) is composed of a compaction process of squid meat, a crushing process, and an organic acid addition process. The prior art published so far has many research reports using squid meat, but a composition or product for lowering blood cholesterol using enzyme hydrolyzate by heat treatment of squid meat is a new technology that has not been reported until now.

The squid meat of the present invention is well decomposed into peptides of low molecular weight by protease (alcalase), and thus, antioxidative activity, tyrosine inhibitory activity, ACE inhibitory activity, cholesterol reduction, etc. are expected as functional materials.

The present invention is a pretreatment step (1) for washing and dehydrating the cuttlefish cut off the internal organs of the squid, heat treatment to heat and gelatinize the cuttlefish (2), grinding the heat treated cuttlefish (3), squid meat It consists of the process (4) of adding an enzyme to hydrolysis, and the separation process (5) of a hydrolyzate. The enzyme hydrolyzate of the cuttlefish meat of this invention is used as a composition for cholesterol reduction.

Conventional squid meat is immersed and smoked and then seasoned and dried, or mixed with additives to squid meat, dried and baked and rolled, or mostly squid products or squid or squid salted fish. In addition, squid by-products separated unsaturated lipids to produce unsaturated fatty acids (EPA, DHA, etc.), or to produce enteric squid containing squid-intestinal degradation.

The present invention is difficult to consume the remaining squid in the conventional consumption patterns, such as dried squid, soft products, squid fish, salted fish, because the squid is captured in large quantities not only on the east coast, but also on the south and west coasts due to the rise of the sea temperature. There is a need to pioneer new uses because of the inevitably falling prices that inevitably reduce the income of fishermen.

The present invention uses the enzyme hydrolyzate of squid meat as a composition for lowering cholesterol using an enzyme hydrolyzate obtained by hydrolysis of squid meat using protease.

Conventional hydrolysis conditions were enzyme concentration 2.42%, reaction time 5.9 hours (hrs), pH 6.96, hydrolysis degree (DH) was 60.8%, but the present invention is optimal for hydrolyzing squid meat with Alcalase 2.4L enzyme at 55 ℃. The conditions were: enzyme concentration 1.12%, reaction time 5 hours, pH 7.26, DH was 62.94%, and the hydrolysis process was improved by heating and gelatinization, reducing enzyme concentration 53.7%, reducing reaction time 15%, and increasing hydrolysis degree 3.5%. .

Table 1. Hydrolysis Effects

division Prior art Invention Improvement effect (%) Enzyme concentration (%) 2.42 1.12 53.7% savings Response time (hr) 5.90 5.00 15% shorter Degree of hydrolysis (%) 60.80 62.94 3.5% increase Reaction temperature (℃) 55 55 same PH 6.96 7.26 Near neutral

In addition, the present invention shows that the squid meat group decreases 3.9%, 18.8% and 21.4% of the squid enzyme hydrolyzate group compared to 7.3% of total cholesterol, 8.3% of LDL-cholesterol and 8.1% of neutral lipid. appear. In the case of HDL-cholesterol, the general squid meat powder group increased 0.90mg / dL, while the experimental group increased 5.41mg / dL, and the squid meat hydrolyzate group increased the HDL-cholesterol by about 20%.

Remove the guts of squid and wash it to dehydrate it to keep freshness of raw squid meat. After squid meat or heat-protected squid meat is added and reacted with the addition of protease, the supernatant is taken by using a centrifugal separator, followed by hot air, freezing, or spray drying to obtain squid meat enzyme decomposed product. The squid meat enzymatically decomposed product is mixed with a food, health functional food or pharmacologically acceptable excipient to provide a composition for lowering blood cholesterol.

In the present invention, the squid meat powder enzymatically decomposed compared to the general squid meat powder was significantly reduced in blood total cholesterol, LDL-cholesterol and triglyceride, and the HDL-cholesterol level was significantly increased. The total cholesterol and triglyceride levels in liver were also significantly decreased. Also, fecals were collected to confirm the concentrations of cholesterol and bile acids. It is believed that cholesterol and bile acids in the liver are synthesized and excreted into the stool, thereby lowering the cholesterol level in the body.

The composition for lowering cholesterol by enzymatically degrading squid meat of the present invention is prepared by the following method. A pretreatment step (1) for washing, dehydrating and cutting the cuttlefish cut off the internal organs of the cuttlefish (1), the step of heat-treating the cuttlefish meat (2), the step of grinding the heat-treated cuttlefish meat (3), and the cuttlefish meat It consists of the process (4) which adds an enzyme and hydrolyzes, and the separation process (5) of a hydrolyzate.

In the above pretreatment, the cuttlefish is cut into 2-4 × 2-4 cm and dehydrated for 10 to 30 minutes at room temperature over the shell, and the heat treatment heats the fresh cuttlefish meat at 120 to 125 ° C. for 13 to 18 minutes in an autoclave or a hot air balloon. After sterilization and gelatinization, the heat-treated squid meat is ground and then enzymatically decomposed. Enzymatic digestion is 1.5 to 2.5 times purified water per weight of squid meat, and 1.1 to 1.5% of Alcalase is added as enzyme to react with a pH of 7.0 to 7.5 at 55 ° C. for 4.5 to 5.5 hours to obtain squid hydrolyzate.

The present invention represents a method of using the squid meat hydrolyzate prepared by the above-mentioned method as a health functional food.

The present invention shows a method of using a squid meat hydrolyzate prepared by the above-mentioned method with an excipient acceptable in the health functional food method and using it as a health functional food.

The present invention shows a method of using squid meat hydrolyzate prepared by the above-mentioned method as a food.

The present invention refers to a method of mixing a squid meat hydrolyzate prepared by the above-mentioned method with an excipient acceptable in the food sanitation method and using it as a food.

The present invention shows a method of using a squid meat hydrolyzate prepared by the above-mentioned method as a medicine.

The present invention refers to a method of mixing squid meat hydrolyzate prepared by the above-mentioned method with excipients that are acceptable under the Pharmaceutical Law.

The present invention is a pretreatment step (1) for washing and dehydrating the cuttlefish cut off the internal organs of the squid, heat treatment to heat and gelatinize the cuttlefish (2), grinding the heat treated cuttlefish (3), squid meat An enzymatic product of squid meat, which is prepared by adding an enzyme to the hydrolyzate (4) and a hydrolyzate separation step (5), is prepared.

Enzymatic hydrolyzate of the present invention has been demonstrated the effect of lowering cholesterol can be used as food, health functional food materials and pharmaceutical materials.

Hereinafter, the present invention will be described in more detail by each process.

Heat Treatment Process of Squid Meat

The squid cut into 2 to 4 cm in size by washing with water, and sterilized by heating and sterilizing for 13 to 18 minutes at 120 to 125 ℃ in an autoclave. If the temperature is low, it takes a long time. If the temperature is high, the protein may be denatured. Therefore, the heat treatment is preferably performed at 121 ° C. for 15 minutes.

<Manufacturing process of hydrolyzate by enzymatic decomposition>

The ground squid meat was tested for optimal hydrolysis conditions by Response Surface Methodology using 2.4L of commercial protease Alcalase. The reaction temperature of the enzyme was fixed at 55 ℃, which is the optimum temperature for enzyme activity, and the degree of hydrolysis increased with increasing reaction time, pH, and enzyme concentration.

In the present invention, the enzyme concentration was 2.44% when the squid meat was hydrolyzed at 55 ° C. with enzyme 2.4L enzyme, but the enzyme concentration was 1.44%, and the use of enzyme was reduced by half, and the reaction time was 5.9 hours. It was shortened to 5 hours, but the pH was 6.96 but was 7.26. In addition, the degree of hydrolysis (DH) was 60.8, but increased by 2.14% to 62.94%. In the heating and steaming process of the present invention, the hydrolysis process is significantly improved more efficiently than the conventional raw squid meat enzyme treatment process, such as an enzyme concentration of 54%, a reaction time of 15%, and a degree of hydrolysis of 2.14%.

<Separation process of enzyme hydrolyzate>

After squid is hydrolyzed with enzyme, the supernatant is taken by centrifugation at 8,000-12,000 × g for 15-30 minutes using a centrifuge. The centrifuge is powdered by removing hot water by hot air, freezing, or spray drying.

Measurement of the solubility of hydrolyzate

As a result of measuring the solubility with various pH and time of the hydrolyzate is shown in FIG. Solubility was found to be higher than 75% over a wide pH range. Solubility decreased above pH 3 and above pH 10 in the alkali range. This is thought to have caused a decrease in solubility because of the high content of the peptide near pH 3, the isoelectric point of the peptide. As a result, high solubility in a wide range (pH 3-10) is thought to be widely used not only in the food and dietary supplement industry but also in the pharmaceutical industry.

The amino acid content of squid hydrolyzate hydrolyzed using 2.4L Alcalase was aspartic acid (12.3%), Glutamic acid (11.5%), Leucine (10.1%), Lysine (10.0%), Arginine ( 8.0%), and their amino acid composition accounted for about 50% of the total amino acid content. In addition, the relationship between taste and amino acid composition shows that the content of sweet Gly, Pro, Ala and Ser is about 24%, and the content of Glu and Asp, which is the rich and sour amino acid, is about 24% and 48% of the total amino acid content. Was good in terms of taste, and the content of hydrophobic amino acids such as Val, Met, Leu, Ile, Phe and Arg related to bitterness was 29%. Free amino acids are rich in sweet Proline and Glycine amino acids, which makes them excellent raw materials for seasoning.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

EXAMPLES Enzyme Degradation of Squid Meat

In the pretreatment process of squid meat, 5kg of frozen squid is put in cold water below 10 ℃ and thawed while removing the intestines and washing several times. The washed squids were cut into sizes suitable for heat treatment. It is preferably cut into 2-4 × 2-4 cm and naturally dehydrated at room temperature over a sieve for 10-30 minutes to maintain the freshness of the raw material itself and the proper moisture content.

In the heat treatment process of squid meat, the cut squid meat was heat-treated at 121 ° C. for 15 minutes in an autoclave. Preferably, it is heat sterilized and gelatinized at 119-123 degreeC for 13 to 18 minutes.

In the process of grinding the heat-treated squid meat, the squid meat cooled to room temperature was put in a grinding blender and ground at a maximum strength (2: Hi) for 2 minutes. Preferably, it grinds for 1-3 minutes.

When squid meat was hydrolyzed with Alcalase 2.4L enzyme at 55 ° C, the enzyme concentration was 1.12%, the reaction time was 5 hours, and the pH was 7.26. The degree of hydrolysis (DH) was 62.94%.

After squid is hydrolyzed with enzyme, the supernatant is taken by centrifugation at 8,000-12,000 × g for 15-30 minutes using a centrifuge. Preferably, the supernatant is taken by centrifugation at 10000 × g for 20 minutes using a centrifuge. The centrifuge is liquid, or hot, frozen or spray dried to form a solid or powder.

<Experimental Example>; Cholesterol lowering experiment

The cholesterol lowering experiment of the present invention was performed by the following materials and methods.

1) Experimental Animal and Diet

The experimental animals purchased 30 Sprague-Dawley male rats weighing 175 ± 10g at 5 weeks of age from Central Experimental Animals, and were supplied with general solid feed (purina, Korea) for a week, followed by a one-week adaptation period. Ten animals were divided into three groups and individually bred in a stainless steel wire cage. Breeding environment was maintained at a constant temperature of 22 ± 3 ℃, 50 ± 10% of humidity, the contrast was repeated every 12 hours (light = 03:00 ~ 15:00), water was supplied without restriction. All groups were fed a high-cholesterol diet (AIN-93G Purified Diet with 1% cholesterol & 0.5% cholic acid), and 5% of general squid meat lyophilized powder was added to the diet of the general squid meat group. To the diet of the meat enzyme hydrolyzate group, 5% of squid meat enzyme hydrolyzate powder was added and fed.

2) weight gain and dietary efficiency

Body weight was measured by taking blood every week from the first week of the experiment, and daily weight gain was divided by the period of weight gain minus the first week of the experiment. The food efficiency ratio (FER) was calculated by dividing the total weight gain during the experiment by the total dietary intake.

The effects of high cholesterol diet, squid meat lyophilized powder, and squid enzyme hydrolyzate powder on the weight change of rats fed different diets during the 10-week period are shown in Table 3 below. Body weight gain was not significantly different between the control group and the group fed normal squid meat, but the group fed the squid enzyme hydrolyzate showed a significant difference. 102.74 ± 10.57g was the highest in the group without squid meat, and 100.47 ± 4.28g was the highest in the group ingesting normal squid meat powder, but 93.81 ± 5.04 in the group ingesting squid enzyme hydrolysis powder. g was significantly lower (p <0.05). As a result, the actual dietary efficiency (FER) was also the highest in the control group and the lowest in the squid enzyme hydrolyzate powder group (p <0.05). This suggests that the squid enzyme hydrolyzate prevented the absorption of lipids in the body, resulting in low weight gain.

Table 2. Weight gain in rats

division Initial weight (g) Final weight (g) Total weight gain (g) Daily gain (g / d) Control group 223.82
± 12.81 ^a3) 326.56
± 17.43 ^a 102.74
± 10.57 1.83
± 0.19 ^a Common Squid 222.94
± 9.62 ^a 323.41
± 9.00 ^a 100.47
± 4.28 ^a 1.79
± 0.08 ^a Enzyme digestion 225.61
± 9.34 ^a 319.42
± 12.66 ^a 93.81
± 5.04 ^b 1.68
± 0.09 ^b

¹⁾ Values are mean ± SD of 10 rats per group.

²⁾ *: cholesterol diet + no supplemented squid

**: cholesterol diet + 5% normal squid

***: cholesterol diet + 5% hydrolysates of squid

³⁾ Values within the same column with different superscripts are significantly different at p <0.05 by Duncan's multiple-test.

3) Determination of blood cholesterol content

Serum total-cholesterol, triglyceride and HDL-cholesterol concentrations were measured by the kit of Asan Pharmaceutical. Blood was fasted 12 hours before collection, anesthetized with zoletil 50 (Virbac, Korea) and collected via eyeball. Blood was collected in an anticoagulant-treated heparin tube (FUZIFILM, Japan), which was centrifuged at 3,000 rpm for 10 minutes to collect serum from the supernatant. Collected serum was collected in a test tube and stored frozen at -20 ℃ until analysis. The quantification of serum total cholesterol is formed by reacting Δ ⁴ -cholesterol, which is produced by making ester-type cholesterol in serum into free cholesterol by cholesterol esterase, and then reacting cholesterol oxidase with peroxidase and phenol + 4-amino-antipyrine. The quinone type red pigment was measured at the absorbance at 500 nm using a spectrophotometer to measure the colorimetric. The concentration of triglyceride was 550 nm using a spectrophotometer on a quinone pigment obtained by reacting free glycerol with triglyceride and lipoprotein lipase with glycerol kinase and L-γ-glycerophosphoic acid, L-γ-glycerophosphooxidase and amino antipyrine. Absorbance was measured at and colorimetric quantification. HDL-cholesterol precipitates the low-density lipoprotein (LDL) and the very low density lipoprotein (VLDL) containing apo-lipoprotein B among lipoproteins by the action of phosphotungstic acid and magnesium cations in the separation solution. Cholesterol in high-density lipoproteins was measured at 500 nm using a spectrophotometer in the same way as serum cholesterol. LDL-cholesterol was calculated by Friedewald equation ¹⁾ , and the formula is as follows.

LDL-cholesterol = Total cholesterol- (HDL-cholesterol + TG / 5)

Table 3. Composition of AIN-93G Rat Diet Controlled by Cholesterol and Choline Acid

Ingredients g / kg Casein (high nitrogen) 200 L-Cystine 3 Sucrose 85 Corn starch 397 Dyetrose 132 Soybean oil 70 t-Butylhydroquinone 0.014 Cellulose 50 Mineral Mix ¹⁾ 35 Vitamin Mix ²⁾ 10 Choline bitartrate 2.5 Cholesterol 10 Cholic acid 5

¹⁾ AIN-93G Mineral mix (g / kg): Ca Carbonate 357, Potassium Phosphate 196, C ₆ H ₅ K ₃ O ₇ xH ₂ O 70, NaCl 74, K ₂ SO ₄ 46, MgO 24, Ferric Citrate 6, ZnCO ₃ 1.7, Manganous Carbonate 0.63, Cu Carbonate 0.3, Potassium Iodate 0.01, Na Selenate 0.01, Ammonium Paramolybdate 4H2O 0.008, Na Metasilicate 9H2O 1.45, Chromium Potassium Sulfate 12H ₂ O 0.28, LiCl 0.02, Boric Acid 0.08, Sodium Flouride 0.06, Nickel Carbonate 0.03, Ammonium Vanadate 0.007, Sucrose finely powdered 221.

²⁾ AIN-93G Vitamin mix (g / kg): Niacin 3, Ca Pantothenate 1.6, Pyridoxine HCl 0.7, Thiamine HCl 0.6, Riboflavin 0.6, Folic Acid 0.2, Biotin 0.02, Vitamin E Acetate (500IU / g) 15, Vitamin B12 (0.1%) 2.5, Vitamin A Palmitate (500,000 IU / g) 0.8, Vitamin D3 (400,000 IU / g) 0.25, Vitamin K1 / Dextrose Mix (10 mg / g) 7.5, Sucrose 967.

4) changes in blood cholesterol levels

Serum total cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceride levels are shown in Table 4. Compared with the control group, total cholesterol, LDL-cholesterol and triglyceride levels decreased by 13.60, 12.60 and 5.58 mg / dL, respectively, compared to the control group, and 25.83, 28.29 and 14.79, respectively. mg / dL decreased. This resulted in a 13.9%, 18.8% and 21.4% reduction in the squid enzyme hydrolyzate group compared with the general squid group, which decreased 7.3% of total cholesterol, 8.3% of LDL-cholesterol and 8.1% of neutral lipids. In the case of HDL-cholesterol, the general squid meat powder group increased 0.90mg / dL, while the experimental group increased 5.41mg / dL, and the squid meat hydrolyzate group increased the HDL-cholesterol by about 20%. In conclusion, it was observed that hydrolysis of general squid meat is effective in lowering blood cholesterol.

Table 4. Changes in Cholesterol in Squid Meat Hydrolyzate

Group ²⁾ Total cholesterol (mg / dl) HDL cholesterol (mg / ㎗) LDL cholesterol (mg / dl) Triglyceride (mg / ㎗) Control ^* 185.75
± 21.21 ^a3) 21.42
± 4.92 ^b 150.51
± 20.67 ^a 69.15
± 19.79 ^a Normal squid ** 172.15
± 8.77 ^ab 22.32
± 3.76 ^ab 137.91
± 8.62 ^b 59.57
± 10.83 ^ab Hydrolysates of squid *** 159.92
± 15.51 ^b 26.83
± 6.09 ^a 122.22
± 18.10 ^c 54.36
± 10.94 ^b

¹⁾ Values are mean ± SD of 10 rats per group.

²⁾ *: cholesterol diet + no supplemented squid

**: cholesterol diet + 5% normal squid

***: cholesterol diet + 5% hydrolysates of squid

³⁾ Values within the same column with different superscripts are significantly different at p <0.05 by Duncan's multiple-test.

5) Determination of cholesterol content in liver tissue

After collecting blood, rats sacrificed under ether were dissected to remove liver, washed with 0.9% NaCl, dried with filter paper, and stored in a freezer at −70 ° C. until analysis. The extraction of cholesterol and triglycerides from liver tissue was applied to Folch method ²⁾ . That is, 3 g of liver sample was taken, homogenized by adding 30 ml of chloroform: methanol (2: 1, v / v) mixed solution, sonicated for 1 hour, filtered through Whatman No. 1 filter paper, and the filtrate was dried under reduced pressure. After adding 5 ml of ethanol to dissolve lipids, colorimetric quantification was performed according to the enzyme method in the same manner as in the analysis of serum cholesterol and triglyceride.

Cholesterol and triglyceride contents of liver were increased by cholesterol diet in liver tissue. Total cholesterol content of normal squid meat powder group was decreased 1.01mg / g, compared to control group. Silver significantly decreased 4.89 mg / g. In the experimental group of squid enzyme hydrolyzate powder, the total cholesterol level was reduced by 9.18mg / g and decreased by 39.2% compared to the control group, and the neutral lipid was reduced by 13.08mg / g and decreased by 35.1% (Table 6). ). As a result of this experiment, squid meat is decomposed into di-peptide, tri-peptide and oligo-peptide by hydrolase and digested and absorbed. Therefore, it is thought that it is effective in lowering total cholesterol and triglyceride.

Table 5. Changes of total-cholesterol and triglyceride during rat diet of cuttlefish enzyme

Group ²⁾ Total cholesterol
(mg / g) Triglyceride
(mg / g) Control ^* 23.39
± 12.15 ^a3) 37.25
± 13.57 ^a Normal squid ** 22.38
± 12.00 ^ab 32.36
± 14.60 ^ab Hydrolysates of squid *** 14.21
± 1.32 ^b 24.17
± 2.65 ^b

¹⁾ Values are mean ± SD of 10 rats per group.

²⁾ *: cholesterol diet + no supplemented squid

**: cholesterol diet + 5% normal squid

***: cholesterol diet + 5% hydrolysates of squid

³⁾ Values within the same column with different superscripts are significantly different at p <0.05 by Duncan's multiple-test.

6) Immediately excrete cholesterol and bile acid excretion

Feces were collected three days before the end of the experiment to separate cholesterol according to Czubayko et al. ³⁾ , and then quantitatively analyzed cholesterol using GC. In other words, freeze-dried stools were finely ground in a mortar and then 1g of 5α-cholestane and 500μl of cyclohexane were added, 1N NaOH 10mL was added and mixed well for 1 hour in a waterbath and 5mL of distilled water and 7mL of cyclohexane were added at 3,120 Cholesterol was isolated by centrifugation at rpm for 5 minutes. The separated upper layer was dried under reduced pressure, dissolved in 600 µl of cyclohexane, and quantified by GC. The content of Bile acid was colorimetrically measured using a Runpia bile acid kit (Tokyo, Japan) to the enzymatic method according to the method ^4), such as by taking the lower layer the aqueous layer separated from the cholesterol extraction process according to method ^3), such as Czubayko Crowell . In other words, 2 mL of 10N NaOH was added to the lower aqueous phase obtained by centrifugation, and saponification was carried out at 15 psi in an autoclave for 3 hours, followed by acidification to pH 2 by adding 0.2 mL of conc-HCl. Then, 40 mL of CHCl ₃ : MeOH (2: 1) mixture was added thereto, mixed well, and the supernatant was separated by centrifugation at 3,120 rpm for 5 minutes, the aqueous layer was extracted and reextracted with 20 mL of chloroform, and dried under reduced pressure. 25 mL of CHCl ₃ : MeOH (2: 1) mixture was added to dissolve the solution, and centrifuged for 5 minutes at 3,120 rpm, 5 ml of the mixture was dried under reduced pressure, dissolved in 1 mL of MeOH: H ₂ O (5: 1) mixture, and stored. . In the analysis, samples were diluted 10-fold and colorimetrically determined using Runpia bile acid kit (Tokyo, Japan). The measurement principle is that the bile acid, 3α-hydroxysteroid, becomes 3α-ketosteroid by the action of 3α-hydroxysteroid dehydrogenase, where NAD + becomes NADH as a hydrogen receptor. This NADH, by the action of diaphorase, donates hydrogen to nitrotetrazolium blue chloride to produce a blue-purple pigment diformazan. This was measured by colorimetric measurement at a wavelength of 540 nm to obtain a bile acid value.

The contents of mutant cholesterol and bile acid showed a significant difference (Table 7). In comparison with the control group, the content of cholesterol and bile acid increased by 21.89 mg / day and 5.37 μmol / day, respectively, by 14.2%. And 6.1% increase, and the squid enzyme hydrolyzate group of the experimental group increased 28.86 mg / day and 15.10 μmol / day, showing an increase of 18.0% and 15.6%, respectively. This showed that the squid enzyme hydrolyzate group of the experimental group increased the content of stool cholesterol 3.8% more and the bile acid content 9.5% more than the general squid meat powder group. This is believed to lower cholesterol in the body by synthesizing bile acids and cholesterol in the liver and excreting it into feces.

Table 6. Water Seafood Squid hexavalent dietary cholesterol and bile acid change in the salary of ^one side of the ^rat)

Group ²⁾ Total cholesterol
(mg / day) Bile acid
(μmol / day) Control ^* 131.88
± 19.76 ^b3) 81.56
± 5.59 ^b Normal squid ** 153.77
± 26.76 ^ab 86.93
± 26.76 ^b Hydrolysates of squid *** 160.74
± 26.44 ^a 96.66
± 10.92 ^a

¹⁾ Values are mean ± SD of 10 rats per group.

²⁾ *: cholesterol diet + no supplemented squid

**: cholesterol diet + 5% normal squid

***: cholesterol diet + 5% hydrolysates of squid

³⁾ Values within the same column with different superscripts are significantly different at p <0.05 by Duncan's multiple-test.

7) Statistical Processing

The mean and standard deviation were calculated for each experimental group, and the significance between the experimental groups was statistically analyzed by ANOVA and minimum significance test using SASprogram. The significance level is (P <0.05).

As described above, the present invention has been described with reference to the preferred embodiments and experimental examples, but those skilled in the art may vary the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. It will be understood that modifications and changes can be made.

In the present invention, by introducing a heating and enzymatic process in the enzymatic hydrolysis of squid, the hydrolysis process such as reduction of enzyme concentration, reaction time and increase of hydrolysis degree is significantly improved, compared to conventional raw squid enzyme treatment process. This is big. In particular, the squid enzyme hydrolyzate powder group reduced the total cholesterol level in liver tissue by 39.2% compared to the control group, and neutral lipid decreased by 35.1%, which is effective in removing blood cholesterol. There is an advantage to widen.

1 is a manufacturing process diagram showing an example of the manufacturing process of the present invention.

Figure 2 shows the dietary efficiency after 10 weeks of dietary feeding to rats.

3 is a blood cholesterol lowered picture.

Claims (5)

delete delete A drug for lowering cholesterol comprising a composition for lowering cholesterol containing a squid enzyme degradable product which is obtained by gelatinizing squid meat and hydrolyzing with an enzyme of Alcalase 2.4L (Alcalase 2.4L) and a pharmaceutically acceptable excipient. Composition. Health for lowering cholesterol, including cholesterol-lowering composition and food-acceptable excipients, consisting mainly of squid degrading enzymes, which have been isolated by hydrolyzing with squid meat and hydrolyzing with Alcalase 2.4L enzyme. Nutraceutical composition. Food for cholesterol lowering, comprising a composition for lowering cholesterol comprising the squid enzyme enzymatic product, which has been isolated by hydrolysis with alkalase 2.4L enzyme after gelatinizing squid meat, and excipients acceptable under the Food Sanitation Act Composition.

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