KR100901138B1 - Method of extracting porcine placenta and health food comprising the extract - Google Patents

Abstract

A method for preparing swine placenta extracts using lipase and protease and a health food comprising the same are provided to reduce contamination in a production process and to increase the content of a bioactive substance by adding lipase and protease to the swine placenta. A method for preparing swine placenta extracts comprises the following steps of: pulverizing frozen swine placenta 2-5mm in size; adding water and lipase to the pulverized swine placenta such that the swine placenta reacts with the lipase at 39-45°C for 5-10 hours; adding an emulsifier to the reaction liquid and agitating the liquid to collect a first supernate; adding the water and protease to the residue so that the residue reacts with the protease at 28-40°C for 12-20 hours; obtaining a second supernate from the residue; and mixing the first and second supernates.

Description

Preparation method of pig placenta extract and health food comprising pig placenta extract {Method of extracting porcine placenta and health food comprising the extract}

The present invention relates to a method for producing a pig placenta extract and a health food comprising a pig placenta extract.

The placenta is an organ that develops in the womb of a pregnant mammal and nourish and sustain life through the umbilical cord.

The placenta has the effect of inhibiting the activity of the enzyme tyrosinase, which catalyzes the production of melanin granules from tyrosine, thereby inhibiting the production of melanin pigment and at the same time promoting the discharge of cells containing melanin pigment, preventing and improving blemishes. It has been reported to be effective in preventing and, therefore, whitening as well as preventing pigmentation (Mallick S et al., Pigment Cell Res. Feb 18 (1), 25-33, 2005).

In addition, the placenta contains essential amino acids, melatonin, RNA, DNA, and other nucleic acid components, the antioxidant enzyme SOD (Super Oxide Dismutase), hyaluronic acid (Hyaluronic acid), antioxidants, immune cofactor (cytokine), placental peptide, insulin-like growth Recovery factors such as growth factors and cytokines such as insulin-like-growth factor, epidermal growth factors (EGF) and unique senescent cell activating factors (SCAF) It is known to be useful for enhancing immunity.

On the other hand, the pig placenta was found to be remarkably similar to the molecular structure of the placenta in the placenta of mammals. Don placenta is an important component of protein, various nutrients, DNA and RNA. It has been reported as a source of active cytokine.

As an effective method of extracting the placenta, a method of obtaining the product by hydrolyzing the placenta using strong acid or strong alkali has been used, but in this method, physiological activities such as various amino acids of placenta in the process of high temperature treatment for a long time There is a problem that the material is destroyed.

In addition, by performing a degreasing process to remove fat using acetone or the like in the extraction process, there is a problem that the lipid active ingredient is also removed.

In addition, the conventional placental extraction method using protease takes a long time to extract the active ingredient, there is a problem that is easily contaminated during the thawing process of the placenta, it is necessary to develop a method to improve this.

An object of the present invention is to provide a method for producing placenta extract, which can reduce the contamination in the manufacturing process and increase the content of various active ingredients.

In addition, an object of the present invention is to provide a health food comprising a placenta extract prepared by the above method.

In order to achieve the above object,

According to one aspect of the invention,

i) crushing the frozen pig placenta to 2-5 mm in size;

ii) adding water and lipase to the crushed pig placenta and reacting at 39 to 45 ° C. for 5 to 10 hours;

iii) recovering the supernatant by adding an emulsifier to the reaction solution of step ii) and then stirring;

iv) recovering the supernatant by adding protease and water to the residue of step iii) for 12 to 20 hours at 28 to 40 ° C; And

v) a method for preparing placenta extract, comprising the step of mixing the supernatant of steps iii) and iv).

In addition, according to another aspect of the present invention,

Health foods comprising placenta extract prepared by the above method can be presented.

Hereinafter, the present invention will be described in detail.

In the present invention, the placenta is not particularly limited as long as it is a placenta of mammals such as humans, sheep, cows, dogs, and pigs. Preferably, the placenta can be used. Porcine placenta not only has the advantages of easy recovery and large amounts, but also has been found to be surprisingly similar to the molecular structure of placenta among mammalian placenta. Porcine placenta has also been reported as a source of bio-active cytokine, which controls cell differentiation and fetal development as an important component of proteins, various nutrients, DNA and RNA.

In order to prepare the placenta extract of the present invention, the placenta must first be collected, and in particular, the normal placenta of swine, which has been verified by a veterinarian, is collected in a freezer (-20 ° C. or lower) and collected. The frozen placenta is crushed into sawdust size (2 to 5 mm), put in a washing machine, washed with brine and dehydrated. It is not preferable to use frozen placenta as it is, and to use it after thawing and then use it in a subsequent process to cause contamination problems in subsequent processes.

Then, 1 to 5% by weight of lipase and the remaining amount of purified water are added to 20 to 30% by weight of the dehydrated pig placenta, followed by lipolysis reaction at 39 to 45 ° C. for 5 to 10 hours, preferably 7 to 8 hours.

The range of placental content and lipase content was chosen as the preferred range in terms of lipolysis efficiency. The reaction time and temperature were selected in the preferred range in consideration of lipolysis efficiency and unit cost.

After the lipolysis reaction, 1 to 5% by weight, preferably 2% by weight of vegetable emulsifier is added and stirred for 1.5 to 3 hours, and then the supernatant (hereinafter referred to as 'supernatant 1') and the placental residue are separated.

Subsequently, 0.5 to 5% by weight of protease and the remaining amount of water are added to 30 to 40% by weight of the placental residue and reacted at 28 to 40 ° C for 12 to 20 hours, preferably 15 to 17 hours. The protease is derived from a microorganism, a plant or an animal, and is not particularly limited, but papain is preferred.

The range of placental residue content and protease content was selected as a preferred range in consideration of hydrolysis efficiency. The hydrolysis reaction is carried out for 12 to 20 hours, preferably 15 to 17 hours. When the reaction is carried out for less than 12 hours, the hydrolysis reaction does not occur sufficiently. When the reaction is carried out for more than 20 hours, it is more than the time required for the desired hydrolysis efficiency, resulting in high production cost, which is uneconomical.

28-40 degreeC is preferable and, as for the said reaction temperature, 30-35 degreeC is more preferable. In the case of reacting below 28 ° C, the hydrolysis reaction may not occur properly, and in excess of 40 ° C, the enzyme may be inactivated.

The hydrolysis reaction product is centrifuged to recover the supernatant (hereinafter referred to as 'supernatant 2'), and the supernatant 1 and the supernatant 2 are mixed to prepare the pig placenta extract of the present invention.

The placental extract of the present invention may further include a process of terminating the hydrolysis reaction by heating to 85 to 100 ° C. after mixing the supernatants 1 and 2. This process can be carried out, for example, at 95 ° C. for 30 minutes.

In addition, the odor may be removed by adding and stirring activated carbon, may be subjected to a filtration process using a filter, and the reaction product may be stabilized by adding trehalose, an edible stabilizer. These processes can be performed by selecting or combining.

In the filtration process, it is preferable to perform filtration one or more times using a 0.1 to 10 μm filter. If the pore size of the filter is less than 0.1 μm, the filter is very precise and extremely expensive (ultrafiltration), and thus, the economy is inferior. If the pore size is more than 10 μm, there is a problem in that a float is formed in the final product and the product quality is significantly decreased.

Placenta extract of the present invention obtained through the filtration was carried out for amino acid analysis by weighing the total amount of nitrogen. Table 1 shows the results.

To produce the finished product, the container is sterilized at 200 ° C. for 2 hours to fill the extract of the present invention in a bottle, and the sterilized container is filled with the extract of the present invention, followed by high-pressure steam sterilization at 121 ° C. for 15 minutes. Can be.

In the conventional proteolytic reaction using strong acid or strong alkali, physiological activities such as various amino acids, growth promoting factors, cytokines, hyaluronic acid, FGF (fibroblasts), epithelial cell promoting factor (EGF), etc. Since there is a problem that the substance is inactivated or disappeared, the extraction method using the lipase and protease of the present invention has an advantage that can greatly solve this problem. In addition, the conventional placental extraction method using the protease takes a long time to extract the active ingredient, there is a problem that the lipid active ingredient is removed by performing protein hydrolysis after the degreasing process, the present invention is to solve such problems I can solve it.

In addition, according to another aspect of the present invention, a pig placenta extract prepared by the above-described manufacturing method and a health food containing the same can be presented.

The placenta extract by the preparation method of the present invention was found to have a significantly increased amino acid content than the placenta extract according to the conventional protease method. In addition, since the content of physiologically active substances such as hyaluronic acid, glycosaminoglycans is high, the composition of the present invention can be usefully used as a functional food raw material for the purpose of improving health.

The placental extract of the present invention may be appropriately determined in content depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the manufacture of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight, based on the total raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages, vitamin complexes, and the like, and includes all of the functional foods in a conventional sense.

The health functional food of the present invention may contain various flavors or natural carbohydrates and the like as additional ingredients, as in general beverages.

As described above, the production method of the present invention can be usefully used in the manufacture of health functional foods, etc., since the placenta is hydrolyzed using lipase and protease to increase the content of the active ingredient.

Hereinafter, the configuration and operation of the present invention through the preferred embodiment of the present invention will be described in more detail. However, this is presented as a preferred example of the present invention and in no sense can be construed as limiting the present invention. Details that are not described herein will be omitted since those skilled in the art can sufficiently infer technically.

Example  One:

Frozen pig placenta was crushed into sawdust size (2-5 mm) with a shredder (Ilsung biaxial shredder ISM-410, Ilsung Cycling Co., Ltd.) and washed with brine. 2 wt% of lipase (Lot # 18525, 2,000 U / mg, Sigma-Aldrich) and 73 wt% of water were added to 25 wt% of the dehydrated placenta and reacted at 43 ° C. for 8 hours.

Subsequently, 2% by weight of a vegetable emulsifier (green tea concentrate, an eco-friendly green tea emulsifier, and a halo-green farming method) was added, followed by stirring at 65 ° C. for 2 hours. After centrifugation, the supernatant 1 and the placental residue were separated, and to 35% by weight of the placenta residue, 3% by weight of papain (Lot # 1095981, 30 U / mg, Sigma-Aldich) and 62% by weight of water were added. After reacting at 35 ° C. for 16 hours, the supernatant 2 was separated by centrifugation. The supernatant 1 and the supernatant 2 were mixed and heated at 95 ° C. for 30 minutes, and 0.4% activated carbon was added at 65 ° C. to remove the odor while stirring, followed by filtration using a 1 μm filter and a 0.45 μm filter.

The composition from which the solid material was removed through the filtration was weighed in total nitrogen (final nitrogen was 2.06 mg / ml), and an amino acid analysis was performed (see Table 1). The total amount of nitrogen refers to the input of placenta prescribed by Korea Food and Drug Administration (KFDA). If this value is the same, the input of the first placenta can be regarded as the same.

Comparative Example 1 :

Frozen pig placenta was crushed to a sawdust size (2 to 5 mm) with a shredder (Ilsung biaxial shredder ISM-410, Ilsung Cycling Co., Ltd.) and washed with brine. 480 g of acetone and 79.52 kg of purified water were added to 20 kg of the washed and dehydrated placenta and reacted at 40 ° C. for 3 to 5 hours. This was dried for 8 hours at 80 ° C. to obtain 500 g of degreasing powder. 43 g of papain (Lot # 1095981, 30 U / mg, Sigma-Aldich) and 885 g of water were added to 500 g of the placenta powder to make the weight ratio 35: 3: 62 as in Example 1, and then reacted at 55 ° C. for 15 hours. Then, it heated at 95 degreeC for 30 minutes.

The complete protein hydrolysis was confirmed by the burette reaction and trichloroacetic acid reaction. It was concentrated under reduced pressure until it gelled at 65 ° C. and filtered sequentially using a 1 μm filter and a 0.45 μm filter. After measuring the total amount of nitrogen (the total amount of nitrogen was 2.24 mg / ml), amino acid analysis was performed.

Comparative example  2:

The placenta extract was prepared in the same manner as in Example 1 except that the frozen placenta was thawed as a raw material of pig placenta, and then cut into pieces of 3-5 cm.

The extract of Comparative Example 2 caused a problem of contamination in the manufacturing process (which is judged to be contamination during thawing) and caused an error in the component analysis test, and thus the following figures were not shown.

Test Example  1: Component Analysis and Quantitative Test

1-1: Amino Acid Analysis

Amino acid quantification of the compositions of Example 1 and Comparative Example 1 of the present invention was carried out using the AccQ-Tag Chemistry Package of Waters. This method is a precolumn derivatization method to analyze peptides and proteolytic amino acids.

The results are shown in Table 1, whereby, the placenta extract by the method of the present invention can be seen that the content of several amino acids significantly increased than the placenta extract obtained by the conventional protein hydrolysis method.

TABLE 1

amino acid Protease Extraction (Comparative Example 1) Lipase + Protease Extraction (Example 1) L-Aspartic acid 1.03 2.16 L-Serine 0.46 0.84 L-Glutamic acid 1.36 2.54 Aminoacetic acid 2.53 3.86 L-Arginine 0.80 1.54 L-Threonine 0.40 0.78 L-Alanine 1.06 2.04 L-Proline 1.33 2.51 L-Valine 0.53 1.03 L-Lysine 0.63 0.94 L-Leucine 0.70 1.21

Hereinafter, the samples of 1-2 to 1-5 were dissolved in 30g of distilled water 5g of the powder of Example 1 and Comparative Example 1 lyophilized powder, and then ultrasonically extracted for 30 minutes, and the supernatant was lyophilized by filtering the 0.22 ㎛ filter.

1-2: Uronic acid  Quantitative Carbazole assay )

Uronic acid is a sugar derivative having an aldehyde or carboxyl group of sugars and plays an important role in detoxification of the human body, and is also important as a component of viscous polysaccharides such as hyaluronic acid and cord leucine sulfate.

Absorbance was measured using UV spectrophotometer at 530 nm by carbazole reaction using D-glucuronic acid lactone as standard. The sample was dissolved in distilled water, and the absorbance was measured using a UV spectrophotometer at 530 nm by Cabazole reaction, and the content was determined by calculating the content of D-glucuronic acid lactone.

1-3: Glycosaminoglycans  Quantitative Carbazole assay )

GAG is a component of protein sugar that accounts for most of the complex protein in connective tissue, and is currently increasing interest as an indicator of arthritis therapeutics.

Absorbance was measured by UV spectrophotometer at 530 nm by carbazole reaction using NZP chondroitin sulfate as standard. The sample was dissolved in distilled water and the absorbance was measured by UV spectrophotometer at 530 nm by carbazole reaction, and the content was determined by calculating the content of NZP chondroitin sulfate.

1-4: Sialic Acid Determination ( Warren assay )

Sialic acid is an amino sugar that is well known as a glycoprotein or glycolipid constituent sugar, and is particularly contained in mucin of the submandibular gland, and has an important physiological significance as an infection defense factor that helps immune function by inhibiting hemagglutination by influenza.

Using N-acetylneuraminic acid as a standard, the absorbance was measured by using a UV spectrophotometer at 549 nm by the Warren method. After the sample was hydrolyzed with 0.1 N sulfuric acid at 80 ° C. for 1 hour, the absorbance was measured using a UV spectrophotometer at 549 nm by the Warren method, and the content was determined by calculating the content of N-acetylneuraminic acid.

1-5: by enzyme Chondroitin  Sulfuric acid ( Chondroitin sulfate ) And hyaluronic acid ( Hyaluronic  acid)

Chondroitin sulfate is a sulfated mucopolysaccharide that makes up connective tissues such as cartilage, blood vessel walls, and tendons. In vivo, it binds to proteins and together with collagen forms a major component of intercellular matter. Hyaluronic acid is a basic substance that makes up the living body and is the central substance for making skin. In particular, it acts to weave the skin cells and keeps the moisture of the skin as it attracts water molecules that are 214 times larger than its own.

Proteus Chondroitinase ABC (C2905, Sigma) derived from vulgaris was dissolved in 50 mM Tris-HCl 60 mM Sodium acetate buffer (pH 8.0) to 1U / ml. Using NZP CS as a standard, the standard and sample were dissolved at 10 mg / ml, mixed with buffer, 30 mU of enzyme, and reacted at 37 ° C. for 12 hours to analyze the content of chondroitin sulfate.

Strepromyces hyalurolyticus the Hyaluronidase (H1136, Sigma) Hyaluronidase 62 U Dissolve the standard and sample by using Hyaluronic acid as a standard, using a 62 U to 10 mg / ml to be 50 mM Sodium phosphate buffer (pH 7.1 ) containing 0.15 M NaCl derived from The reaction was carried out at 60 ℃ for 12 hours to analyze the content of hyaluronic acid.

The reaction was detected at UV 232 nm while maintaining a flow rate of 1 ml / min by connecting the SAX-HPLC column to AKTA Purifier (Amersham Phaimacia) with gradient NaCl 0M ~ 2M (pH 3.5).

Table 2 Unit% (w / w)

  sample Sialic acid GAG Uronic acid Chondroitin sulfate Hyaluronic acid Example 1 0.001 ± 0.000 2.109 ± 0.896 0.053 ± 0.04 1.16 2.82 Comparative Example 1 0.0001 ± 0.0000 0.108 ± 0.096 0.003 ± 0.01 0.14 0.83

As shown in Table 2, the pig placenta extract prepared by the lipase and proteolytic enzyme treatment of the present invention compared to the pig placenta extract prepared through the conventional acetone degreasing process includes sialic acid, glycosaminoglycan, right It can be seen that the contents of various bioactive substances useful in the human body, such as lonic acid, chondroitin sulfate or hyaluronic acid, have been greatly increased.

Test Example  3: On the rat  Acute toxicity test for oral administration

In order to determine whether the extract of the present invention has acute toxicity, the following experiment was performed.

Acute toxicity test was performed using 6 week-old SPF rats. 1 g / kg / 10 of the composition of Example 1 of the present invention (5 g of the lyophilized powder of Example 1, dissolved in 30 ml of distilled water, sonicated for 30 minutes, and supernatant was lyophilized through a 0.22 μm filter) in 8 animals. Single oral administration at a dose of ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed. As a result, all animals treated with test substance showed no clinical symptoms and no dead animals, and no toxic changes were observed in weight change, blood test, blood biochemistry, and side findings. As a result, the composition of the present invention did not show a change in toxicity in rats up to 1 g / kg, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of at least 1 g / kg or more.

While the embodiments of the present invention have been described with reference to the accompanying tables, the present invention is not limited to the above embodiments, but may be embodied in various forms, and a person of ordinary skill in the art to which the present invention pertains. It will be appreciated that the present invention may be embodied in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (5)

i) crushing the frozen pig placenta to a size of 2 to 5 mm; ii) adding water and lipase to the crushed pig placenta and reacting at 39 to 45 ° C. for 5 to 10 hours; iii) recovering the supernatant by adding an emulsifier to the reaction solution of step ii) and then stirring; iv) recovering the supernatant by adding protease and water to the residue of step iii) for 12 to 20 hours at 28 to 40 ° C; And v) mixing the supernatant of steps iii) and iv); Placenta production method comprising a. The method of claim 1, wherein the reactant of step ii) comprises 20 to 30% by weight of pig placenta, 1 to 5% by weight of lipase, and a balance of purified water. The method of claim 1, wherein the reactant of step iv) comprises 30 to 40 wt% of placental residue, 0.5 to 5 wt% of protease and residual amount of purified water. Placenta extract prepared by the method of claim 1. Health functional food comprising the placenta extract of claim 5.