KR102218129B1 - Enzymatic Lysate of Velvet Antler Extract and Fermentation Product thereof - Google Patents

Abstract

The present invention relates to an enzymatic lysate of a velvet antler extract and a fermented product thereof. Particularly, the present invention relates to a method for producing an enzymatic lysate of a velvet antler extract, an enzymatic lysate of a velvet antler extract produced thereby, a method for producing a fermented product of a velvet antler extract using the same, and a fermented product of a velvet antler extract produced thereby. The production method according to the present invention can significantly increase the content of sialic acid, uronic acid, etc., which are functional components of a velvet antler extract, and thus the enzymatic lysate and the fermented product produced by the method can be widely used as general food, health functional food, pharmaceutical composition or the like.

Description

Enzymatic Lysate of Velvet Antler Extract and Fermentation Product thereof {Enzymatic Lysate of Velvet Antler Extract and Fermentation Product thereof}

The present invention relates to an enzymatic digestion product of antler extract and a fermentation product thereof, specifically, a method for preparing an enzyme digestion product of an antler extract and an enzyme digestion product of an antler extract prepared therefrom, and a method for preparing a fermented product of antler extract using the same It relates to a fermented product of the antler extract.

Antlers (velvet antlers) are antlers in the growth stage of deer and reindeer (Cervidae ) animals, and are collected and dried before keratinization. Deer antler has been used for the purpose of rejuvenation and appetite enhancement since ancient times, and the benefits of antler known through Donguibogam include treatment of anemia, improvement of liver function, prevention of arteriosclerosis, inhibition of osteoporosis, recovery of chronic fatigue, and improvement of diabetes . Antlers are horns in which antlers are keratinized, and are often used as a substitute for antlers, but their efficacy is known to be lower than that of antlers. Deer antler contains various functional substances such as sialic acid, hexose, pentose, hexoamine, uronic acid, prostaglandin, and ganglioside. It is known.

Sialic acid is a common name for neuraminic acid derivatives, and most of them exist in a form bound to glycosides. Sialic acid is itself a functional substance and is known as an indicator of ganglioside.

Ganglioside is known as a functional substance that is effective in strengthening immunity, anti-cancer effect, blood circulation, memory improvement, and joint health. Ganglioside is a combination of sphingosine, fatty acids, and complex polysaccharides and contains at least one molecule of sialic acid. Therefore, the measurement of the sialic acid content can indirectly determine the ganglioside content. Gangliosides are classified into various types, such as GM1 (monosialotetrahexosylganglioside), GM2, GM3, GD1a, and GT1b, depending on their structure.

GAG (glycosaminoglycan) is a negatively charged polysaccharide with a structure in which disaccharide units are repeated, and is a component of connective tissues such as skin and cartilage. GAG is classified into heparin, heparan sulfate, keratin sulfate, hyaluronan, and dermatan sulfate. S-GAG (sulfated-glycosaminoglycan) is a major component of cartilage and a functional component used as a joint nutritional supplement. Chondroitin sulfate is the main component of GAG, and accounts for 88% of the total uronic acid (acid polysaccharide) of antlers. In addition, the content of acid mucopolysaccaride in the velvet layer of antlers is about 0.16% by dry weight, which is a relatively small amount, and the proportion of acid mucopolysaccharides in the mucoprotein of the velvet layer of antlers is about 1.8%. Is being reported. As such, uronic acid is known as a constituent of antler GAG, and the increase in uronic acid and GAG content by fermentation can be predicted to promote osteoblast growth, prevent osteoporosis, and improve arthritis treatment effects.

As described above, deer antlers contain various functional ingredients, but conventionally, the functional ingredients of antlers cannot be sufficiently extracted using only heat extraction, and absorption efficiency into the human body is low. In addition, there is a problem in that the functional ingredients of antlers are lost when immersed in alcohol and minced to use antlers as herbal medicine. It is known that enzymatic treatment and fermentation of antler extract can increase the content of functional ingredients. According to Korean Patent Registration No. 10-1679388, a fermented antler fermented product with increased ganglioside and sialic acid content is disclosed by adding pectinase and cellulase and fermenting it with a Lactobacillus strain. However, there is still room for improvement in terms of discovering enzymes and strains that can further increase the content of functional ingredients.

Republic of Korea Registration Gazette No. 10-1679388 (2016.11.24)

According to an embodiment of the present invention, an object of the present invention is to provide a method for preparing an enzymatic decomposition product of antler or antler extract and an enzymatic decomposition product of an antler or antler extract prepared therefrom.

According to another embodiment of the present invention, an object of the present invention is to provide a method for producing a fermented product of antler or antler extract and a fermented product of an antler or antler extract prepared therefrom.

One aspect of the present invention provides a method for preparing an enzyme digestion product of an antler extract comprising the step of contacting the antler extract with a pancreatic enzyme complex.

The term "deer antler" as used in the present invention refers to antlers of deer and animals that have not been boned within 2 months of age. Nokgak is a keratinized horn of deer and animals. The manufacturing method may use not only antler extract but also antler extract.

In the present invention, the antler extract can be extracted using a conventional solvent under conventional temperature and pressure conditions according to a conventional method known in the art.

According to one embodiment, the antler or antler extract may be extracted using water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent. For example, using water as a solvent, or one or two or more solvents selected from the group consisting of methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate, and acetone A mixed solvent can be used. In this case, the amount of the solvent used may be 1 to 60 times, 10 to 50 times, 20 to 40 times, or 30 times compared to antler or antler.

The antler extract may be extracted using various methods such as hot water extraction, hot water extraction, cold needle extraction, reflux extraction, and ultrasonic extraction. The extraction conditions may be adjusted according to the extraction method, and the temperature may be 60 to 120°C, 70 to 110°C, 80 to 100°C, or 90°C, and the time may be 1 to 5 hours, 2 to 4 hours, or 3 It may be time, but is not particularly limited. Thereafter, the extracted antler extract may perform one or more of filtration, concentration, and drying to remove the solvent contained in the extract, and filtration, concentration, and drying may all be performed as necessary. For example, filtration may be performed by filtration by filter paper or by reduced pressure filtration using a reduced pressure filter, concentration may be performed by a vacuum concentration method, and drying may be performed by a freeze drying method, but is not particularly limited.

In the present invention, the pancreatic enzyme complex is composed of various types of enzymes made in the pancreas of animals, and has digestive power for carbohydrates, proteins and fats.

According to one embodiment, the pancreatic enzyme complex may be pancreatin or pancreatic enzymes derived from porcine pancreas. For example, the pancreatin may be composed of protease, amylase, and lipase.

When the antler extract is brought into contact with the pancreatic enzyme complex, some sialic acid is separated from gangliosides containing two or more sialic acids in the antler extract, and the isolated sialic acid binds to gangliosides without other sialic acids, thereby increasing the content of gangliosides bound to sialic acid. I can. Sialic acid or s-GAG is itself a functional substance and is known as an indicator substance representing the functional substance content of antler extract. According to an embodiment, as a result of analyzing the increase in sialic acid content by hydrolysis using various types of enzymes, it was found that the effect of increasing the sialic acid content was the most excellent when the pancreatic enzyme complex was treated.

According to one embodiment, the step of contacting the pancreatic enzyme complex is 60 minutes to 240 minutes, 80 minutes to 220 minutes, 100 minutes to 200 minutes at a temperature of 30 to 60 ℃, 35 to 55 ℃, or 40 to 50 ℃ , 120 to 180 minutes, or may be a contact for 160 minutes.

According to one embodiment, the amount of the pancreatic enzyme complex used is 0.5 to 8.5% (w/v), 1.0 to 8.0% (w/v), 1.5 to 7.5% (w/v), based on the total volume of the antler extract. 2.0 to 7.0% (w/v), 2.5 to 6.5% (w/v), 3.0 to 6.0% (w/v), 3.5 to 5.5% (w/v), or 4.0 to 5.0% (w/v) Can be

In addition, another aspect of the present invention provides an enzymatic digestion product of an antler extract prepared by the method for preparing an enzyme digestion product of the antler extract.

The enzyme decomposition product of the antler extract may have an enhanced content of sialic acid, s-GAG, and uronic acid compared to the antler extract.

According to an embodiment, the rate of increase in the sialic acid content of the enzyme decomposition product may be 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the antler extract.

According to an embodiment, the sialic acid content of the enzyme digest is 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, or It may be 90 μg/mL or more.

According to an embodiment, the uronic acid content of the enzyme digest is 20 µg/mL, 30 µg/mL, 40 µg/mL, 50 µg/mL, 60 µg/mL, 70 µg/mL, 80 µg/mL, or It may be 90 μg/mL or more.

On the other hand, another aspect of the present invention a) preparing an enzymatic digestion product of the antler extract prepared by the method for preparing the enzyme digestion product of the antler extract; And b) fermenting the enzyme decomposition product into a strain.

The term "fermentation" as used in the present invention refers to a process of generating useful substances by decomposing organic substances with microorganisms and their enzymes, and a substance produced by the fermentation process is called a fermentation product.

In the present invention, the step of preparing the enzymatic digest of step a) may be performed by the above-described method for preparing the enzymatic digest of an antler extract.

In the present invention, the step of fermenting the enzyme decomposition product of step b) may be performed by inoculating a strain or adding and culturing a strain culture solution.

According to one embodiment, the strain may be one or more selected from the group consisting of Lactobacillus curvatus and Enterococcus faecium. Preferably, it may be Lactobacillus curbatus HY7602 or Enterococcus facium HY5004. The Lactobacillus curbatus HY7602 (accession number: KCTC 14154BP) and Enterococcus facium HY5004 (accession number: KCTC 14153BP) were selected as strains having an excellent effect of increasing sialic acid content by the present inventors, and the Korea Research Institute of Bioscience and Biotechnology 2020 It was deposited as of March 11, 2010.

According to one embodiment, when using the two strains, the mixing ratio of the strains is Lactobacillus curbatus HY7602: Enterococcus facium HY5004 = 1: 0.1 to 2 weight ratio, 1: 0.5 to 1.5 weight ratio, or 1 : It may be a weight ratio of 1.

According to one embodiment, the amount of the strain may be 10 ⁶ to 10 ¹² , 10 ⁷ to 10 ¹¹ , 10 ⁸ to 10 ¹⁰ , or 10 ⁹ , and the unit may be CFU/mL or CFU/g.

According to one embodiment, the amount of the strain is 0.01 to 10% (v/v), 0.05 to 5% (v/v), 0.1 to 1% (v/v), or 0.5 based on the total volume of the antler extract. May be %(v/v).

According to one embodiment, in the fermentation conditions, the temperature may be 30 to 60°C, 35 to 55°C, or 37°C. The time may be 1 to 72 hours, 2 to 60 hours, 4 to 48 hours, or 6 to 36 hours of incubation.

In addition, another aspect of the present invention provides a fermented product of an antler extract prepared by the method for producing an enzyme fermented product of the antler extract.

The enzyme fermented product of the antler extract may have an enhanced content of sialic acid, s-GAG, and uronic acid compared to the antler extract or the enzyme decomposition product of the antler extract.

According to an embodiment, the increase rate of the sialic acid content of the fermented product may be 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the enzyme decomposition product of the antler extract.

According to an embodiment, the sialic acid content of the fermentation product is 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, or 90 It may be greater than or equal to μg/mL.

According to an embodiment, the increase rate of the uronic acid content of the fermented product may be 50%, 60%, 70%, 80%, 90%, or 100% or more compared to the enzyme decomposition product of the antler extract.

According to an embodiment, the uronic acid content of the fermentation product is 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, or 90 It may be greater than or equal to μg/mL.

The method for preparing an enzyme decomposition product of antler antler extract according to an embodiment of the present invention and a method for producing a fermented product can significantly increase the content of sialic acid and uronic acid, which are functional components of antler extract, and Fermented products can be widely used as general foods, health functional foods, or pharmaceutical compositions.

1 shows the change of sialic acid content by Pancrypsin concentration/time for an antler extract according to an embodiment of the present invention.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Confirmation of increase in sialic acid content by enzyme for antler extract

Antler extract was prepared by hot-water extraction with 30 times the amount of water at 90° C. for 3 hours. The prepared antler extract was treated with various enzymes at 40-50° C. for each hour as shown in Table 1 below to check the content of sialic acid. Each enzyme treatment amount was 2.5% (w/v). The enzymes used are all edible enzymes, and were purchased and used from Vision Biocam. As a control, an antler extract without enzyme treatment was used.

Sialic acid content analysis was performed using a Sialic acid fluorescence labeling kit (Takara, Cat. #4400), which is mainly used in the food industry. The test method followed the basic procedure enclosed in the kit. Specifically, 100 µl of antler extract sample and 100 µl of 25 mM HCl were added to the EP tube, and heat-blocked at 80 to 100°C. The sample was cooled, 50 μl of the sample cooled in the tube and 200 μl of the mixed solution (DMB solution:coupling solution:distilled water = 1:5:4) were added and reacted at 50° C. for 2.5 hours. The reaction was terminated by cooling for 5 minutes, and the sialic acid content was measured by HPLC. The results are shown in Table 1.

0h 3h 6h Control 6.75 6.75 6.75 Prozyme Exp 5000 5.86 6.38 7.25 Prozyme PR 8.44 7.12 7.41 Plantase PT 7.55 8.41 6.82 Pancrypsin 10.99 20.64 17.21 Pluszyme 6.78 6.81 6.56

The unit of the numbers shown in Table 1 is µg/mL. Prozyme EXP5000 is a protease derived from the fungus Aspergillus oryzae (exo peptidase activity), Prozyme PR is a bacterial-derived protease complex (endo protease activity), Plantase PT is a cellulase complex (non-starch polysaccharide degradation activity), and Pluszyme is β derived from Aspergillus niger. -glucosidase (non-starch polysaccharide decomposition activity), and Pancrypsin is a pancreatic enzyme (exo peptidase activity) derived from Hog pancreas glands.

As a result of the experiment, it was confirmed that the effect of increasing the sialic acid content in the case of Pancrypsin treatment was superior to that of the treatment with protease and cellulose derived from mold and bacteria. In particular, the content of sialic acid increased about 2 times compared to 0 hours after 3 hours of pancrypsin treatment, and slightly decreased after 6 hours compared to 3 hours. As a result, it was confirmed that Pancrypsin is an effective enzyme for increasing the sialic acid content of antler extract.

Example 2: Confirmation of increase in sialic acid content by Pancrypsin concentration/hour for antler extract

In order to confirm the optimal conditions for each concentration and time of the enzyme, Pancrypsin was treated with each concentration/time in the antler extract, and the change in sialic acid content was compared. The concentration of the enzyme was 0.5%, 1%, 2.5%, 5%, 7.5% (w / v), except that the reaction for 0, 1, 2, 3, 4, 5 hours, the same as in Example 1 It was done by the method.

Sialic acid content analysis was measured in the same manner as in Example 1, and the results are shown in FIG. 1.

As a result of the experiment, it was confirmed that the effect of increasing the sialic acid content was the most excellent when Pancrypsin was treated with 5% (w/v). In particular, the sialic acid content was highest when 4 hours elapsed after pancrypsin treatment, and the sialic acid content increased about 3 times compared to 0 hours.

Example 3: Confirmation of increase in uronic acid content by enzyme for antler extract

In the same manner as in Example 1, the antler extract and the enzyme were treated by time to confirm the uronic acid content. As a control, an antler extract without enzyme treatment was used.

The uronic acid content analysis was performed in the following manner. _{First, 1.2 mL of H 2} SO ₄ /sodium tetrabrorate solution was added to an EP tube, and 200 µl of a sample was dispensed. After lightly vortexing and placing on ice, it was treated with a bath in water at 100°C for 5 minutes. Placed on ice again, cooled for 10 minutes, and 20 µl of m-hydroxydiphenyl (dyeing reagent) was added. 200 µl was dispensed into a microplate and absorbance was measured at 520 nm. The results are shown in Table 2 below.

0h 3h 6h Control 10.6 10.6 10.6 Prozyme Exp 5000 65.4 120.8 98.6 Prozyme PR 112.9 113.9 112.6 Plantase PT 33.3 19.7 68.5 Pancrypsin 19.7 68.5 55.2 Pluszyme 30.2 28.9 21.5

The unit of the numbers shown in Table 2 is µg/mL.

As a result of the experiment, the uronic acid content of Prozyme PR did not change according to the treatment time, and the content of Pluszyme was rather decreased. The uronic acid content of Prozyme EXP 5000, Plantase PT, and Pancrypsin increased with treatment time. In particular, when 3 hours elapsed after treatment with Pancrypsin, the uronic acid content increased by about 3.5 times, and the effect of increasing the uronic acid content was found to be the most excellent.

Example 4: Fermentation strain search of antler extract

4-1. Fermentation strain search

Antler solid medium was prepared by adding 1.5% agar to the antler extract, and 12 kinds of bacteria having smooth colonies were selected by streaking the candidate microorganism group on the antler solid medium. Candidate microorganisms are listed in Table 3 below.

number Strain name One Lactobacillus fermentum 2 Lactobacillus brevis 3 Lactobacillus delbrueckii subsp. bulgaricus 4 Lactobacillus curvatus 5 Lactobacillus curvatus 6 Enterococcus faecium 7 Enterococcus faecium 8 Enterococcus faecium 9 Enterococcus faecium 10 Enterococcus faecium 11 Enterococcus faecium 12 Enterococcus faecium 13 Enterococcus faecium

4-2. Confirmation of sialic acid content change by strain

The antler extract was treated with Pancrypsin 2.5% (w/v) at 40 to 50° C. for 4 to 5 hours, and the obtained enzyme digestion product was reacted at 95 to 100° C. for 5 minutes to inactivate the enzyme. Thereafter, the 12 strains of Table 3 were inoculated into 40 mL of the enzyme digestion product at 10 ¹⁰ CFU/mL, respectively, and cultured at 37° C. for 36 hours, and the sialic acid content of the fermentation was analyzed. As a control, an enzyme digestion product not treated with the strain was used.

The results are shown in Table 4 below, and based on this, strains 1, 3, 4 and 13 having excellent effects of increasing sialic acid content were selected.

number Strain name Concentration (㎍/mL) Compared to the control group
Increase/decrease rate (%) 0 Control 20.04 - One Lactobacillus fermentum 24.72 23.35 2 Lactobacillus brevis 22.99 14.72 3 Lactobacillus delbrueckii subsp. bulgaricus 24.69 23.20 4 Lactobacillus curvatus 24.01 19.81 5 Lactobacillus curvatus 21.05 5.04 6 Enterococcus faecium 24.44 21.96 7 Enterococcus faecium 23.53 17.42 8 Enterococcus faecium 20.08 0.20 9 Enterococcus faecium 19.58 -2.30 10 Enterococcus faecium 19.61 -2.15 11 Enterococcus faecium 23.24 15.97 12 Enterococcus faecium 25.91 29.29 13 Enterococcus faecium 26.08 30.14

4-3. Comparison of sialic acid content change during fermentation of 4 strains

Fermentation was performed again in the same manner as in 4-2 using the selected four strains to measure the sialic acid content of the fermented product. The sample at 0 hours of each strain was used as a control enzyme digestion product not inoculated with the strain. The results are shown in Table 5 below.

Sample name Content (ug/mL) Increase or decrease rate compared to the control group (%) no. 1
L. fermentum 0h 6.68 - 36h 7.84 17.37 number 3
L. bulgaricus 0h 8.32 - 36h 9.65 15.99 Number 4
L. curvatus 0h 7.69 - 36h 15.31 99.09 Number 13
E. faecium 0h 7.76 - 36h 17.84 129.90

As a result of the experiment, strains 1 and 3 are The change in sialic acid content was relatively insignificant. On the other hand, strains 4 and 13 had a large amount of sialic acid generated, and the sialic acid content increased by about two times, respectively. Therefore, strain 4 L. curvatus HY7602 (accession number KCTC 14154BP) and 13 E. faecium HY5004 (accession number KCTC 14153BP) were selected as final candidates and deposited with the Korea Research Institute of Bioscience and Biotechnology on March 11, 2020.

4-4. Confirmation of change in uronic acid content by strain

The uronic acid content of the fermented fermented product for 36 hours was measured for two types of lactic acid bacteria ( L. curvatus HY7602, E. faecium HY5004) in which an increase in sialic acid content was confirmed. As a control, an enzyme digestion product not inoculated with the strain was used. The results are shown in Table 6 below.

Strain name density Increase or decrease rate compared to the control group (%) Control 16.00 - L. curvatus HY7602 27.77 73.56 E. faecium HY5004 27.01 68.81

As a result of the experiment, it was confirmed that both L. curvatus HY7602 (KCTC 14154BP) and E. faecium HY5004 (KCTC 14153BP) increased uronic acid content, and the uronic acid content increased to 73.56% and 68.81%, respectively, compared to the control group.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC14153BP 20200311 Korea Research Institute of Bioscience and Biotechnology KCTC14154BP 20200311

Claims (11)

a) preparing an enzyme digestion product of the antler extract by contacting the antler extract with the pancreatic enzyme complex; And
b) fermenting the enzyme digestion product into a strain,
The pancreatic enzyme complex of step a) is pancreatin derived from porcine pancreas,
The strain of step b) is one or more selected from the group consisting of Lactobacillus curvatus HY7602 (accession number: KCTC 14154BP) and Enterococcus faecium HY5004 (accession number: KCTC 14153BP). Enzymatic fermentation production method of antler extract.
The method according to claim 1,
The antler extract is extracted using water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent.
delete The method according to claim 1,
In the step a), the antler extract and the pancreatic enzyme complex are contacted at 30 to 60° C. for 60 to 240 minutes.
The method according to claim 1,
The amount of the pancreatic enzyme complex to be used is 0.5 to 8.5% (w/v) based on the total volume of the antler extract.
delete delete delete The method according to claim 1,
The amount of the strain is 10 ⁶ to 10 ¹² CFU / mL.
The method according to claim 1,
In the step b), the enzyme digestion product and the strain are cultured at 30 to 60° C. for 1 to 48 hours.
Fermented product of antler extract prepared by the method of claim 1.

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