KR20230104528A - Method for preparing fresh antler velvet fermentation freeze-dried antler with enhanced antler functional indicator component and use of product obtained therefrom - Google Patents

Abstract

The present invention relates to a method for preparing fermented freeze-dried deer antler of fresh deer antler enriched with functional indicator components of deer antler and to fermented freeze-dried deer antlers prepared by the method.
The fermentation freeze-dried deer antler manufacturing method of the present invention has higher useful components than the conventional fermented deer antler or its extract, so that functional fermented freeze-dried deer antlers can be effectively prepared.

Description

Method for preparing fresh antler velvet fermentation freeze-dried antler with enhanced antler functional indicator component and use of product obtained therefrom}

The present invention relates to a method for fermenting and fermenting freeze-dried deer antlers of fresh deer antler enriched with functional indicators of deer antler, and to fermented freeze-dried deer antlers prepared by the method.

As of 2019, the domestic raw material consumption market for deer antlers is about 63 billion won, or 374.7 tons. Among the 20 or so countries that consume deer antlers, Korea is the second largest consumer of deer antlers in the world after China. Imported deer antlers distributed in Korea as herbal medicines are mainly from New Zealand (Red Deer) and Russia (Elk). In 2019, the amount of imported antlers in Korea was 306 tons (52.3 billion won), and about 68.6 tons of domestically produced deer antlers were produced. It is becoming. 47.0 tons of elk (large-sized) and 18.5 tons of plum blossom (small-sized) are produced, and these deer antlers are mainly used as health foods using antler extract, and are used as pouches, sticks, powder, and liquid powder products. there is.

Deer antler contains various physiologically active ingredients, and has traditionally been known as a supplement for strengthening and improving weakness. Deer antlers help with anemia, rheumatism, stroke, spasm healing and anti-inflammation, antioxidant, anti-cancer, anti-virus, anti-aging, growth improvement, muscle strength improvement, skin regeneration, memory enhancement, sexual function recovery, fatigue improvement, post-illness recovery, wound healing, etc. has been known to For this reason, antler is the most widely used animal herbal medicine in Korea. However, due to physicochemical thermal denaturation during drying, the insolubility of antler-specific physiologically active components is greatly increased.

Recently, the demand for various fermented deer antler products is increasing for health, but special attention is required because useful components and efficacy differ significantly depending on the quality (grade, cut, part) of deer antler. The effect of fermented deer antler cannot overcome the difference in useful components unique to antler by quality (grade, part, aging) of input antler material. In other words, fermented antler using only aged antler (lower antler or antler, old antler) has a lot less useful components than fermented antler made from whole antler (whole antler) of normal excellent antler (grades 1 to 3). In addition, fermentation of dried deer antler is fermented with dry raw materials that have already undergone a lot of insolubility of the unique physiologically active ingredients of deer antler due to physicochemical thermal denaturation, so the fermentation effect is inevitably insufficient compared to fermentation in the fresh raw material state. there is.

Therefore, in order to minimize the insolubilization of physiologically active substances generated during the processing of dry antler velvet (DA), the present researchers performed fermentation in the raw antler state, and then developed a technology to produce fermented freeze-dried antler and from this The present invention was completed by optimizing the functional components of the obtained fermented freeze-dried antler and developing conditions and methods suitable for product use.

One aspect is 1) fermenting by inoculating a strain into an antler raw material; and 2) freeze-drying the fermented antler.

Another aspect is to provide a fermented freeze-dried antler prepared by the above method.

Another aspect is to provide a functional indicator material and/or antioxidant composition comprising the fermented freeze-dried deer antler, an extract thereof, or a fraction thereof.

Another aspect is one or more strains selected from the group consisting of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae; culture of the strain; Concentrates of the above strains or cultures; And to provide a composition for fermentation of antler, comprising at least one selected from the group consisting of dried products thereof.

One aspect is 1) fermenting by inoculating a strain into an antler raw material; and 2) freeze-drying the fermented antler.

The term "velvet antler" in this specification refers to a medicinal material obtained by collecting and processing young horns of a deer that have not hardened. Young horns that have not yet been ossified or partially ossified are cut and dried, called deer antlers, and horns that have become keratinized in autumn are called antlers.

Deer antlers of medium and large species have seasonal breeding characteristics. Deer antlers grow externally even after the summer solstice (longest amount of sunlight), but due to rapid ossification, the aging state in which useful functional substances decrease occurs, and the quality deteriorates. In Korea, normal products are classified into grades 1 to 5, and abnormal products are classified into non-standard grades according to the cutting time and aging state of deer antler to classify such quality. In addition, the parts of the antler are classified into tip, bone, upper, middle, and lower parts based on the point of maximum growth, and a significant decrease in useful functional substances occurs as it progresses from the tip to the lower part. Other antlers do not fall into the category of antlers as the entire antler has undergone keratinization.

Deer antler contains various active ingredients, and in particular, has been known as a representative herbal medicine for treating various weaknesses because of its tonic effect. Deer antler is used to treat and prevent diseases such as epilepsy, ulcer, anemia, rheumatism, and stroke, improve symptoms such as insomnia, fatigue, headache, and convulsions, promote gastrointestinal movement, increase appetite, treat and improve nervous breakdown, recover after illness, prevent aging, It is known to improve memory, promote sexual function recovery, and promote wound healing. For this reason, antler is the most consumed animal herbal medicine in Korea.

The raw material for antler may be raw antler, aged antler, dried antler or fermented antler. The fresh velvet antler refers to horns in which the antler has not yet undergone a drying process, and is stored in a frozen state after removing hair or washing three or more times in hot water at 90° C. or higher.

In one embodiment, the raw material for the deer antler may be a raw deer antler or an aged deer antler obtained by aging the fresh deer antler.

In the above method, step 1) may include: A) aging the raw material for deer antler; and B) inoculating the strain into the aged antler and fermenting it.

The step of aging in step A) may be a step of aging at low temperature for a long time.

The aging of step A) may be performed at 0 ° C to 15 ° C, specifically 0 to 15 ° C, 0 to 13 ° C, 0 to 11 ° C, 0 to 9 ° C, 0 to 8 ° C, 0 to 7 ° C. °C, 0 to 6 °C, 0 to 5 °C, 2 to 15 °C, 2 to 13 °C, 2 to 11 °C, 2 to 9 °C, 2 to 8 °C, 2 to 7 °C, 2 to 6 °C, 2 to 5 °C ℃ 4 to 15 ℃, 4 to 13 ℃, 4 to 11 ℃, 4 to 9 ℃, 4 to 8 ℃, 4 to 7 ℃, 4 to 6 ℃, 4 to 5 ℃, 6 to 15 ℃, 6 to 13 ℃ , 6 to 11 ° C, 6 to 9 ° C, 6 to 8 ° C, 6 to 7 ° C, 7 to 15 ° C, 7 to 13 ° C, 7 to 11 ° C, 7 to 9 ° C or 7 to 8 ° C. it may be

In addition, the aging of step A) may be performed for 12 to 72 hours, specifically 12 to 72 hours, 12 to 66 hours, 12 to 60 hours, 12 to 54 hours, 12 to 48 hours, 18 to 72 hours hour, 18 to 66 hours, 18 to 60 hours, 18 to 54 hours, 18 to 48 hours, 24 to 72 hours, 24 to 66 hours, 24 to 60 hours, 24 to 54 hours, 24 to 48 hours, 30 to 72 hours hour, 30 to 66 hours, 30 to 60 hours, 30 to 54 hours, 30 to 48 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 36 to 54 hours, 36 to 48 hours, 42 to 72 hours time, 42 to 66 hours, 42 to 60 hours, 42 to 54 hours, 42 to 48 hours, 48 to 72 hours, 48 to 66 hours, 48 to 60 hours, or 48 to 54 hours.

The aging may be sterilization and/or aging using alcohol.

The step of sterilizing and/or aging the raw antler may be using 50 to 95% alcohol, specifically 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 60 to 95%, 60% alcohol. to 90%, 60 to 85%, 60 to 80%, 70 to 95%, 70 to 90%, 70 to 85%, or 70 to 80% of alcohol may be used.

The step of sterilizing and/or maturing the raw antler may be to treat the raw antler with alcohol in an amount of 1 to 5% based on the weight of the fresh antler. 3.5% dose, 1 to 3% dose, 1 to 2.5% dose, 2 to 5% dose, 2 to 4.5% dose, 2 to 4% dose, 2 to 3.5% dose, 2 to 3% dose, 2 to 2.5% It may be treated with a dose, 2.5 to 5% dose, 2.5 to 4.5% dose, 2.5 to 4% dose, 2.5 to 3.5% dose, or 2.5 to 3% dose.

The term "fermentation" in the present specification means that microorganisms do not decompose organic substances using their own enzymes. Fermentation 　reaction and decomposition reaction proceed by similar processes, but when a useful substance is produced as a result of decomposition, it is called fermentation, and when a bad smell or harmful substance is produced, it is called decomposition. "Fermented product" means any material produced through the above fermentation. The fermented product may include a fermentation broth, a diluted or concentrated solution of the fermentation broth, a dried product obtained by drying the fermentation broth, or a crude or purified product thereof, or a fraction obtained by fractionating the same.

In the method, the strains inoculated to ferment the antler raw material are Lactobacillus, Bacillus, Saccharomyces, Bifidobacterium, Streptococcus, Leucono It may contain one or more selected from the group consisting of stock (Leuconostoc), Pediococcus (Pediococcus) and Lactococcus (Lactococcus) genera.

The strain is Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus rhamnosus ( L. rhamnosus ), Lactobacillus acidophilus ( L. acidophilus ), Lactobacillus casei ( L. casei ), Lactobacillus fermentum ( L. Fermentum ), Lactobacillus bulgaricus ( L. bulgaricus ), Lactobacillus delbruekhi subspecies lactis ( L. delbruekii subsp. lactis ), Lactobacillus gasseri ( L. gasseri ), Bifidobacterium breve ( Bifidobacterium breve ), Lactobacillus helveticus ( L. helveticus ), Lactobacillus salivarius ( L. salivarius ), Lactobacillus reuteri ( L. reuteri ), Lactobacillus crispatus ( L. crispatus ), Lactobacillus johnsonii ( L. strains of the genus Lactobacillus, such as johnsonii ), strains of the genus Bacillus, such as Bacillus subtilis , or strains of the genus Bacillus, such as Bifidobacterium breve ( Bifidobacteriumbreve ), Bifidobacterium longum ( B. longum ), Bifidobacterium infant This ( B. infantis ), Bifidobacterium adolescentis ( B. adolescentis ), Bifidobacterium bifidum ( B. bifidum ), Bifidobacterium catenulatum ( B. catenulatum ), Bifidobacterium pseudo Catenulatum ( B. pseudocatenulatum ), Bifidobacterium angulatum ( B. angulatum ), Bifidobacterium gallicum ( B. gallicum ), Bifidobacterium lactis ( B. lactis ), Bifidobacterium no Strains of the genus Bifidobacterium such as B. animalis , Saccharomyces cerevisiae, Saccharomyces sake (Sacchoromyses sake), Saccharomyces ellipsoidus, Saccharomyces Strains of the genus Saccharomyces such as Saccharomyces formosensis, Saccharomyces coreanus and Saccharomyces boulardii may be used.

The strain may include one or more selected from the group consisting of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, specifically Lactobacillus plantarum; a combination of Lactobacillus plantarum and Bacillus subtilis; or a combination of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae. In addition, the Lactobacillus plantarum may be Lactobacillus plantarum LP20B49 (accession number: KACC92397P).

The strains are Lactobacillus rhamnosus JLFR LR04 (Accession Number: KACC92399P), Lactobacillus sakei JLFR LS07 (Accession Number: KACC92398P) and Lactobacillus plantarum LP20B49 (Accession Number) : KACC92397P), Bacillus subtilis, and Saccharomyces cerevisiae.

In the above method, the step of fermenting the antler may be performed at a temperature condition of 5 to 45 ° C, specifically 5 to 45 ° C, 5 to 42 ° C, 5 to 40 ° C, 5 to 38 ° C, 5 to 45 ° C 37 ℃, 15 to 45 ℃, 15 to 42 ℃, 15 to 40 ℃, 15 to 38 ℃, 15 to 37 ℃, 25 to 45 ℃, 25 to 42 ℃, 25 to 40 ℃, 25 to 38 ℃, 25 to 37 °C, 35 to 45 °C, 35 to 42 °C, 35 to 40 °C, 35 to 38 °C or 35 to 37 °C.

In the above method, the step of fermenting the antler may be performed for 24 to 144 hours, specifically 24 to 144 hours, 24 to 132 hours, 24 to 120 hours, 24 to 108 hours, 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 36 to 144 hours, 36 to 132 hours, 36 to 120 hours, 36 to 108 hours, 36 to 96 hours, 36 to 84 hours, 36 to 72 hours, 48 to 144 hours, 48 to 132 hours, 48 to 120 hours, 48 to 108 hours, 48 to 96 hours, 48 to 84 hours, 48 to 72 hours, 60 to 144 hours, 60 to 132 hours, 60 to 120 hours, 60 to 108 hours, 60 to 96 hours, 60 to 84 hours, 60 to 72 hours, 72 to 144 hours, 72 to 132 hours, 72 to 120 hours, 72 to 108 hours, 72 to 96 hours, or 72 to 84 hours. .

The method includes: a) a step of testing deer-derived diseases and harmful pathogens on raw materials for deer antler; b) a process of thawing raw materials for deer antlers at a low temperature for a long time; c) a process of washing raw materials for deer antler; and d) a process of sterilizing raw materials for antler antler.

In one embodiment, the method comprises: ① performing an examination for deer-derived diseases and harmful pathogens to ensure food safety of the raw material for antler antler; ② Thawing the antler raw material at low temperature for a long time; ③ Washing the thawed antler in hot water; ④ sterilizing the washed antler; ⑤ aging the sterilized antler; ⑥ Fermenting the aged antler; and ⑦ freeze-drying the fermented antler.

The step of performing the deer-derived disease and harmful pathogen test may be to examine the presence or absence of one or more selected from the group consisting of Mycobacterium tuberculosis , Brucella species and deer chronic wasting disease (CWD). there is.

Examining whether or not Mycobacterium tuberculosis or tuberculosis disease has occurred may be confirming whether or not 16S rRNA of Mycobacterium tuberculosis is detected/expressed.

Examining the presence or absence of Brucella or Brucella disease may be to determine whether the BCSP-31 gene of Brucella or the protein expressed therefrom is detected/expressed.

Examining whether or not the deer chronic wasting disease has occurred may be to check whether the PRNP gene or the prion protein expressed therefrom is detected/formed.

In one embodiment, the test for confirming the presence or absence of tuberculosis disease or Mycobacterium tuberculosis may be performed through a PCR test, and specifically, a PCR test is performed using the sequences of SEQ ID NOs: 1 and 2 as forward and reverse primers. can

In one embodiment, the test for confirming the presence or absence of Brucellosis or Brucellosis may be performed through a PCR test, and specifically, the PCR test is performed using the sequences of SEQ ID NOs: 3 and 4 as forward and reverse primers. it may be

In one embodiment, the test for confirming the presence or absence of the deer chronic wasting disease may be performed through a PCR test, specifically performing a PCR test using the sequences of SEQ ID NOs: 5 and 6 as forward and reverse primers it could be

The step of thawing the antler raw material at low temperature for a long time is a process performed when the antler raw material is in a frozen state, and may be performed for 1 to 6 days under a condition of about 0 ° C to 15 ° C.

The thawing may be performed at 0 ° C to 15 ° C, specifically 0 to 15 ° C, 0 to 13 ° C, 0 to 11 ° C, 0 to 9 ° C, 0 to 8 ° C, 0 to 7 ° C, 0 to 6 ℃, 0 to 5 ℃, 2 to 15 ℃, 2 to 13 ℃, 2 to 11 ℃, 2 to 9 ℃, 2 to 8 ℃, 2 to 7 ℃, 2 to 6 ℃, 2 to 5 ℃, 3 to 15 °C, 3 to 13 °C, 3 to 11 °C, 3 to 9 °C, 3 to 8 °C, 3 to 7 °C, 3 to 6 °C or 3 to 5 °C may be performed under temperature conditions.

The thawing may be performed for 1 day to 6 days, specifically 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 2 days to 6 days, 2 days It may be carried out for 5 days, 2 days to 4 days, 2 days to 3 days, 3 days to 6 days, or 3 to 5 days, 3 days to 4 days.

The thawing step is to thaw the cryopreserved raw antler to a thawed state to the extent that the external rust blood does not flow or to a slightly frozen state, and to thaw gently to minimize physical damage to the ice crystal tissue of the ice crystal thawing zone of the antler. it could be

The step of washing the thawed antler in hot water may be washing 1 to 5 times using hot water of 90 ° C or higher.

The washed raw antler may further include slicing to a thickness of 0.5 to 5 mm, specifically 0.5 to 5 mm, 0.5 to 4.5 mm, 0.5 to 4 mm, 0.5 to 3.5 mm, 0.5 to 3 mm, 1 to 5 mm, 1 to 4.5 mm, 1 to 4 mm, 1 to 3.5 mm, or 1 to 3 mm thickness.

The step of sterilizing the washed raw antler may be using 50 to 95% alcohol, specifically 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 60 to 95%, 60 to 90% alcohol. %, 60 to 85%, 60 to 80%, 70 to 95%, 70 to 90%, 70 to 85% or 70 to 80% alcohol.

The step of sterilizing the raw antler may be to treat the alcohol in an amount of 1 to 5% based on the weight of the fresh antler, specifically, 1 to 5%, 1 to 4.5%, 1 to 4%, 1 to 3.5%, to 3% dose, 1 to 2.5% dose, 2 to 5% dose, 2 to 4.5% dose, 2 to 4% dose, 2 to 3.5% dose, 2 to 3% dose, 2 to 2.5% dose, 2.5 to 5 % dose, 2.5 to 4.5% dose, 2.5 to 4% dose, 2.5 to 3.5% dose, or 2.5 to 3% dose.

Freeze-drying the fermented deer antler may include rapid freezing at a temperature of -20 ° C or lower for 6 to 24 hours, followed by drying at a core temperature of -30 to -70 ° C for 6 to 48 hours. .

The quick freezing may be performed at a temperature of -20 ° C or less, specifically -20 ° C or less, -30 ° C or less, -40 ° C or less, -45 ° C or less, -20 to -70 ° C, -20 to - 60℃, -20 to -50℃, -20 to -45℃, -30 to -70℃, -30 to -60℃, -30 to -50℃, -30 to -45℃, -40 to -70 ℃, -40 to -60 ℃, -40 to -50 ℃, -40 to -45 ℃, -45 to -70 ℃, -45 to -60 ℃ or -45 to -50 ℃ temperature conditions may be carried out there is.

The quick freezing may be performed for 6 to 24 hours, specifically 6 to 24 hours, 6 to 21 hours, 6 to 18 hours, 6 to 15 hours, 6 to 12 hours, 9 to 24 hours, 9 to 21 hours time, 9 to 18 hours, 9 to 15 hours, 9 to 12 hours, 12 to 24 hours, 12 to 21 hours, 12 to 18 hours, or 12 to 15 hours.

The drying may be performed at a raw material center temperature of -30 to -70 ° C, specifically -30 to -70 ° C, -30 to -65 ° C, -30 to -60 ° C, -30 to -55 ° C, - 40 to -70 °C, -40 to -65 °C, -40 to -60 °C, -40 to -55 °C, -45 to -70 °C, -45 to -65 °C, -45 to -60 °C or -45 It may be carried out under -55 ° C conditions.

The drying may be performed for 6 to 48 hours, specifically 6 to 48 hours, 6 to 36 hours, 6 to 24 hours, 6 to 21 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours , 12 to 21 hours, 18 to 48 hours, 18 to 36 hours, 18 to 24 hours, 18 to 21 hours, 21 to 48 hours, 21 to 36 hours, or 21 to 24 hours.

It may further include a step of sterilizing after the step of freeze-drying, wherein the step of sterilizing is performed by fermenting the freeze-dried antler at 50 to 70 ° C, 50 to 65 ° C, 55 to 70 ° C, or 55 to 65 ° C. to 5 hours, 1 to 4 hours, 2 to 5 hours, or 2 to 4 hours.

The method includes sialic acid (Glycosaminoglycan, GAG), uronic acid, gamma-aminobutyric acid (γ-aminobutyric acid. GABA), amino acids, essential amino acids and aromas in antler. It may be to enhance the content of at least one selected from the group consisting of compounds, and specifically, the fermented freeze-dried antler prepared by the above method is a general dried antler, a freeze-dried antler, a freeze-dried antler after aging only without fermentation, and/or the above It may be that the content of the above ingredients is increased compared to the fermented freeze-dried antler that is not prepared by the method.

The amino acids are selected from the group consisting of glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, asparagine, aspartic acid, lysine, glutamine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, tryptophan and cysteine. It may contain at least one selected from the group consisting of isoleucine (Ile), lysine (Lys), and histidine (His). In addition, the amino acids may include essential amino acids.

The aroma compound includes at least one selected from the group consisting of 2-butanone, acetic acid butyl ester, 2-furanmethanol, and benzaldehyde. can

In one embodiment, a method comprising fermentation using Lactobacillus plantarum followed by freeze-drying or the fermentation freeze-dried antler prepared by the method is glycosaminoglycan, gamma-aminobutyric acid, 2-butane at least one content selected from the group consisting of one, acetic acid butyl ester, benzaldehyde, alanine, serine, proline, valine, threonine, leucine, isoleucine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, tryptophan, and essential amino acids This may be enhanced, and specifically, the content of the above ingredients may be increased compared to normal dried antler, freeze-dried antler, and/or antler lyophilized after maturation without fermentation.

In one embodiment, a method comprising fermentation using Lactobacillus plantarum followed by freeze-drying or the fermentation freeze-dried antler prepared by the method is glycosaminoglycan, gamma-aminobutyric acid, 2-butane One or more contents selected from the group consisting of one, 2-furanmethanol, serine, threonine, glutamic acid, arginine, tyrosine, and tryptophan may be enhanced. content may be enhanced.

In one embodiment, a method comprising fermenting a combination of Lactobacillus plantarum and Bacillus subtilis followed by freeze-drying, or the fermented freeze-dried deer antler prepared by the method contains sialic acid, Cannes, gamma-aminobutyric acid, 2-butanone, acetic acid butyl ester, benzaldehyde, glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, The content of one or more selected from the group consisting of tryptophan and the total amount of essential amino acids may be increased, and specifically, the content of the above ingredients is increased compared to normal dried antler, freeze-dried antler, and/or freeze-dried antler after aging only without fermenting. it could be

In one embodiment, a method comprising fermentation using a combination of Lactobacillus plantarum and Bacillus subtilis followed by freeze-drying, or the fermented freeze-dried deer antler prepared by the method is sialic acid, acetic acid butyl ester, The content of one or more selected from the group consisting of proline, leucine, methionine, and phenylalanine may be increased, and specifically, the content of the above components may be increased compared to antler lyophilized after fermenting using another strain.

In one embodiment, a method comprising fermenting using a combination of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae and then freeze-drying or fermentation freeze-drying prepared by the method Deer antler contains sialic acid, glycosaminoglycan, gamma-aminobutyric acid, 2-butanone, acetic acid butyl ester, benzaldehyde, glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, asparagine, lysine, glutamic acid , methionine, histidine, phenylalanine, arginine, tyrosine, tryptophan, and at least one content selected from the group consisting of the total amount of essential amino acids may be increased. The content of the above components may be increased compared to lyophilized antler.

In one embodiment, a method comprising fermenting using a combination of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae and then freeze-drying or fermentation freeze-drying prepared by the method The deer antler has an increased content of at least one selected from the group consisting of uronic acid, 2-butanone, benzaldehyde, glycine, alanine, proline, valine, isoleucine, asparagine, aspartic acid, lysine, histidine, phenylalanine, and the total amount of essential amino acids. Specifically, the content of the above components may be increased compared to freeze-dried deer antler after fermenting using another strain.

The method may be to enhance the antioxidant activity of the antler, and specifically, the fermented freeze-dried antler prepared by the method or the method is a general dried antler, a freeze-dried antler, a freeze-dried antler after only maturing without fermentation, and/or Antioxidant activity may be enhanced compared to fermented freeze-dried antler not prepared by the above method.

The method may be to increase the lactic acid bacteria content of the antler, and specifically, the fermented freeze-dried antler produced by the method or the method is a general dried antler, a freeze-dried antler, a freeze-dried antler after aging without fermentation, and/or It may be that the content of lactic acid bacteria is increased compared to the fermented freeze-dried antler not prepared by the above method.

The method may further include extracting, concentrating, or pulverizing the fermented freeze-dried antler.

The method may improve extraction efficiency and/or concentration efficiency of antler, and specifically, may improve production efficiency compared to general dried antler or freeze-dried antler. Therefore, when an extract or concentrate is prepared from the fermented freeze-dried antler prepared by the above method, remarkably excellent production efficiency can be exhibited.

In one embodiment, when extracts and concentrates were prepared from the above method or the fermented freeze-dried antler prepared by the above method, as a result of concentration based on the same solid content concentration, it was found that production efficiency was superior to that of normal dried antler and freeze-dried antler. Confirmed.

Another aspect is to provide a fermented freeze-dried antler prepared by the above method. The same parts as those described above also apply to the above.

The fermented freeze-dried deer antler contains sialic acid (Glycosaminoglycan, GAG), uronic acid, gamma-aminobutyric acid (GABA), amino acids and aromas in the deer antler. It may be to enhance the content of at least one selected from the group consisting of compounds, and specifically, the fermented freeze-dried antler prepared by the above method is a general dried antler, a freeze-dried antler, a freeze-dried antler after aging only without fermentation, and/or the above It may be that the content of the above ingredients is increased compared to the fermented freeze-dried antler that is not prepared by the method.

The fermented freeze-dried deer antler may have enhanced antioxidant activity, and specifically, the fermented freeze-dried deer antler prepared by the above method is a general dried deer antler, a freeze-dried deer antler, a freeze-dried deer antler after only maturing without fermentation, and/or the method described above. It may be that the antioxidant activity is enhanced than that of fermented freeze-dried antler that is not prepared with

The fermented freeze-dried deer antler may have an increased lactic acid bacteria content. Specifically, the fermented freeze-dried deer antler prepared by the above method is a general dried deer antler, a freeze-dried deer antler, a freeze-dried deer antler after aging only without fermentation, and/or the method described above. It may be that the content of lactic acid bacteria is increased than that of fermented freeze-dried deer antler that is not manufactured.

The fermented freeze-dried deer antler may have improved extraction and/or concentration efficiency. Specifically, the fermented freeze-dried deer antler prepared by the above method is a general dried deer antler, a freeze-dried deer antler, a freeze-dried deer antler after aging only without fermentation, and / Or it may be one with improved extraction and / or concentration efficiency than fermented freeze-dried antler not prepared by the above method.

Another aspect is to provide a method for producing a fermented freeze-dried deer antler extract comprising obtaining an extract from the fermented freeze-dried deer antler prepared by the above method. The same parts as described above are also applied to the method.

As used herein, the term "extract" refers to a product such as a liquid component obtained by immersing a target material in various solvents and then extracting at room temperature or a warm state for a certain period of time, and a solid component obtained by removing the solvent from the liquid component. can mean In addition, in addition to the above results, it is intended to include the extracts of all formulations that can be formed using the extracts themselves and extracts such as dilutions of the results, concentrates thereof, crude products thereof, purified products, dried products, powders, or mixtures thereof. can be interpreted comprehensively.

The extraction solvent of the extract may be a protic polar solvent or an aprotic polar and non-polar solvent. The protic polar solvent may be water, methanol, ethanol, propanol, isopropanol, or butanol. The aprotic polar solvent may be dichloromethane, tetrahydrofuran, ethyl acetate, acetonitrile, dimethylformamide, dimethylsulfoxide, acetone, 2-butanone, or hexamethylphosphoramide. The non-polar solvent may be pentane, hexane, chloroform, or diethyl ether. The non-polar solvent excludes benzene. The solvent is a C1-C6 alcohol, a C3-C10 ester, such as a C3-C10 acetate, a C3-C10 ketone, a C1-C6 unsubstituted or halogenated hydrocarbon, a C2-C10 ring ether, a mixture thereof or a mixture of one or more of the above solvents and water. The solvent may be ethanol, propanol, acetonitrile, ethyl acetate, acetone, 2-butanone, chloroform, dichloromethane, hexane, a mixture thereof, or a mixture of one or more of the above solvents and water. The hydrocarbon may be an alkane, alkene, or alkyne.

The extract is water; C1 to C4 lower alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as glycerine, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane. It may be extracted with one or more solvents selected from the group.

The extract may be extracted by one or more methods selected from the group consisting of reduced pressure and high temperature extraction, hot water extraction, reflux extraction, hot water extraction, cold needle extraction, room temperature extraction, ultrasonic extraction, steam extraction and fractional extraction.

The extraction may be carried out at 60 °C to 100 °C, preferably at 70 °C to 100 °C, and more preferably at 85 °C to 100 °C, but is not limited thereto. Extraction may be carried out for 4 hours to 72 hours, preferably for 4 hours to 48 hours, more preferably for 4 hours to 24 hours, but is not limited thereto.

The term "fraction" in this specification refers to a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

A fractionation method for obtaining the fraction is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity or a chromatographic fractionation method that performs separation by affinity), a combination thereof, and the like.

The type of fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. The fractionation solvent is water; C1 to C4 lower alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as glycerine, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane. It may contain one or more solvents selected from the group.

Another aspect is to provide a fermented freeze-dried antler extract or a fraction thereof prepared by the above method. The same parts as those described above also apply to the above.

Another aspect is to provide an antioxidant composition comprising the fermented freeze-dried antler, an extract thereof, or a fraction thereof. The same parts as described above also apply to the composition.

As used herein, the term "antioxidation" means an action of inhibiting oxidation. Oxygen that enters the body through respiration has a beneficial effect on the body, but in this process, active oxygen is produced, and biolipids are oxidized by active oxygen. It is being studied to induce diseases such as circulatory system disease or cancer, and aging. Therefore, proper maintenance of active oxygen can be treated as a major principle to prevent cell oxidation and cell aging.

The composition may be a pharmaceutical composition.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms. Here, the meaning of "pharmaceutically acceptable" means that the application (prescription) does not have toxicity more than is adaptable without inhibiting the activity of the active ingredient. Any type of carrier that can be used in the pharmaceutical composition may be used as long as it is commonly used in the art and is pharmaceutically acceptable. Non-limiting examples of the carrier include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or Mineral oil etc. are mentioned. These may be used alone or in combination of two or more. The pharmaceutical composition may be prepared as an oral formulation or parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods.

The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. When formulating the pharmaceutical composition, it may be prepared by adding diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants.

When the pharmaceutical composition is prepared as a dosage form for oral use, formulations such as powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer, etc. can be manufactured with Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, Celluloses such as sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil etc. are mentioned. In the case of formulation, if necessary, diluents and/or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included.

When the pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. In the case of formulation as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine or sterilization for injection water, isotonic solutions such as 5% dextrose, and the like can be used. When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated into suppositories, the base is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like may be used.

The pharmaceutical composition may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit/risk ratio applicable to medical treatment or prevention. , the effective dose level depends on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the present used It may be determined according to factors including drugs used in combination or simultaneous use with the composition of the invention and other factors well known in the medical field. The pharmaceutical composition may be administered alone or in combination with ingredients known to exhibit known therapeutic effects. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

The dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present invention is particularly Although not limited, it may be administered once a day or administered several times by dividing the dose. The dosage or frequency of administration is not intended to limit the scope of the present application in any way.

The pharmaceutical composition may be administered singly or in multiple doses in a pharmaceutically effective amount. At this time, the composition may be formulated and administered in the form of a liquid, powder, aerosol, injection, infusion (intravenous gel), capsule, pill, tablet, suppository or patch. The administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.

The pharmaceutical composition is not particularly limited thereto, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. It can be administered by route. However, oral administration can be administered in an unformulated form, and since the active ingredients of the pharmaceutical composition can be denatured or decomposed by gastric acid, the oral composition is formulated to coat the active agent or protect it from degradation in the stomach. It can also be administered orally in the form of a localized form or an oral patch. In addition, the composition may be administered by any device capable of transporting active substances to target cells.

The composition may be a food composition.

The term "food" in this specification refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcoholic beverages. , vitamin complexes, health functional foods and health foods, etc., and include all foods in the conventional sense.

The food can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the manufacture. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The food composition can be prepared in various types of dosage forms, and, unlike general drugs, has the advantage of using food as a raw material and has no side effects that may occur when taking drugs for a long time and has excellent portability, so the food composition of the present invention It can be taken as an adjuvant to enhance the effect of improving bone-related diseases.

The food composition may be a health functional food composition.

In this specification, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act No. 6727, and 'functional' refers to the human body. It means to obtain useful effects for health purposes such as regulating nutrients for the structure and function of food or physiological functions.

The health food (health food) means food that has an active health maintenance or promotion effect compared to general food, and health supplement food (health supplement food) means food for the purpose of health supplement. In some cases, the terms health functional food, health food, and dietary supplement may be used interchangeably. Specifically, the health functional food is a food prepared by adding the composition to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, suspended, etc., and when ingested, it has a specific effect on health It means coming, but unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material.

Since the food composition can be consumed on a daily basis, a high effect on improving efficacy can be expected, so it can be used very usefully.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used. Specifically, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agent (tar colorant, etc.), coloring agent (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen tartrate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

The food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, based on the food or beverage when preparing food or beverage. However, when ingested for a long period of time for health and hygiene purposes, the amount below the above range may be included, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

An example of the food composition may be used as a health beverage composition, and in this case, as in conventional beverages, various flavoring agents or natural carbohydrates may be included as additional components. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health drink composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The food composition may contain the extract of the present invention in various weight% if it can exhibit an anti-aging improvement or preventive effect, and specifically, the extract is 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition. %, but is not limited thereto.

The composition may be a feed composition.

The term "feed" in this specification may mean any natural or artificial diet, meal, etc., or a component of said meal, for or suitable for eating, ingesting, and digesting by an animal.

The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feeds such as proteins, inorganic materials, oils and fats, mineral oils, oils and fats, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

The feed composition may further include known additives that can be added to improve or prevent obesity and lipid-related metabolic diseases depending on the formulation. The feed composition may be in the form of a highly concentrated solution, powder or granules. The feed composition may further include any protein-containing organic flour commonly used to meet the dietary needs of animals. The feed composition may be added to animal feed by dipping, spraying, or mixing.

The feed composition may further include substances exhibiting various effects such as supplementation of nutrients and prevention of weight loss, enhancement of digestibility of fiber in feed, improvement of oil quality, prevention of reproductive disorders and improvement of conception rate, and prevention of high temperature stress in summer. For example, mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, trace mineral preparations such as zinc, copper, cobalt, and selenium, kerotene, vitamin E, vitamins A, D, E, and nicotinic acid. , Vitamins such as vitamin B complex, protected amino acids such as methionine and lysic acid, protected fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast cultures, live bacteria such as mold fermented products, yeasts, etc. can be included

The feed composition can be applied to a number of animal diets including mammals and poultry, that is, feed and drinking water.

The feed composition may include all substances added to feed (ie, feed additives), feed raw materials, or feed itself fed to the individual.

The composition may be a cosmetic composition.

The formulation of the cosmetic composition is not limited as long as it is a conventional cosmetic formulation, but for example, skin softening lotion, astringent lotion or nutrient lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing It may be water, pack, powder, body lotion, body cream, body oil or body essence.

The cosmetic composition may add materials selected arbitrarily according to the cosmetic formulation or purpose of use, for example, preservatives, stabilizers, surfactants, solubilizers, humectants, emollients, ultraviolet absorbers, preservatives, bactericides, antioxidants, pH Adjusting agents, organic and inorganic pigments, fragrances, cooling agents or antiperspirants, and the like may be further included. The blending amount of the additional components can be easily selected by those skilled in the art within a range that does not impair the objects and effects of the present invention.

When the formulation of the cosmetic composition is a paste, cream or gel, as a carrier component, for example, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide etc. may be included.

When the formulation of the cosmetic composition is a powder or spray, it may include, for example, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component, and in particular, in the case of a spray, additionally chlorofluoro propellants such as rhohydrocarbons, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition is a solution or emulsion, as a carrier component, for example, a solvent, a solubilizing agent, or an emulsifying agent may be included, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol , benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the cosmetic composition is a suspension, as a carrier component, for example, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspension such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester agent, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like.

When the formulation of the cosmetic composition is surfactant-containing cleansing, as a carrier component, for example, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosyl nates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters, and the like.

Another aspect is one or more strains selected from the group consisting of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae; culture of the strain; Concentrates of the above strains or cultures; And to provide a composition for fermentation of antler, comprising at least one selected from the group consisting of dried products thereof. The same parts as described above also apply to the composition.

The antler may be fresh antler, aged antler, dried antler or fermented antler.

The composition may be a composition for fermenting antler to prepare a fermented freeze-dried antler.

The fermentation freeze-dried deer antler manufacturing method of the present invention contains higher useful components than conventional dried deer antlers or extracts thereof, so that functional fermented freeze-dried deer antlers can be effectively prepared.

1 shows a process chart of the fermentation freeze-dried antler manufacturing method of the present invention.
Figure 2 shows a control group and various fermented freeze-dried antler experimental groups.
3 shows an example of the PMCA method.
4 shows the analysis results of the sialic acid content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
5 shows the results of analyzing the GAG content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
6 shows the analysis results of the uronic acid content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
7 shows the results of analyzing the GABA content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
8 shows the analysis results of the general strain and lactic acid bacteria content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
9 shows the results of analyzing the antioxidant activity of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
10 shows the analysis results of the aroma compound content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
11 shows the results of analyzing the low molecular weight compound content of antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
12 shows the analysis results of the lysine content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
13 shows the analysis results of isoleucine and histidine contents of antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
14 shows the analysis results of the total amino acid content of the antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.
15 shows the results of analyzing the yield of antler prepared by the fermentation freeze-dried antler manufacturing method of the present invention.

It will be described in more detail through examples and experimental examples below. However, these examples and experimental examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Method for manufacturing fermented freeze-dried antler of the present invention

In the present invention, in order to minimize the insolubility of physiologically active substances and organic substances that occur after drying the antler, aging and fermentation were performed in the raw antler state. It is a manufacturing technology for growth.

The technical point of the present invention is to minimize rapid physicochemical changes, minimizing rapid temperature changes in each step of thawing, aging, fermentation, and freeze-drying, while taking a long time within the food safety tolerance range. LTLT technology (Low Temperature Long Time) technology and Hurdle Technology & Food Engineering, which apply intensive management technology in each process step, are applied.

There are 7 hurdle steps in each process, as follows:

1) Pre-inspection process for food safety of raw antler raw materials to ensure food safety - After sampling, livestock diseases (tuberculosis, CWD, Brucella, etc.) are tested, and PMCA techniques using immunostaining and Western blotting are applied as analysis methods ( Step 1).

2) Low-temperature long-term thawing process - Depending on the cryopreservation state of raw materials, it is carried out at 3-7℃ for 2 to 5 days. It is thawed to a state where external rust blood does not flow or to a state where it is slightly frozen, and gradual thawing is performed to minimize physical damage to the ice crystal tissue of the ice crystal thawing zone of the antler (second step).

3) Washing and slicing process - Wash 3 times in hot water over 90 ℃, slice 1~3mm, and maintain the temperature of the workshop at 10~15℃ (3rd step)

4) Sterilization process of raw materials - Spread raw antler slices evenly on a sieve and treat them with 75% alcohol and 2.5% volume. (Step 4)

5) Low-temperature long-term (LTLT) maturation process - Aging in a dark room at 7℃ for 24 h to 48 h depending on the raw material. (Step 5)

6) Optimum fermentation process - Depending on the state of the raw material, the strain is inoculated and then fermented for 2 to 5 days. It may be necessary to alternate temperature between 5 and 37 ° C to prevent blood from flowing (Step 6)

7) Freeze-drying process; After quick freezing at -45 ℃ or less for 12 hours, dry at -45 ~ -55 ℃ for 21 hours, and sterilize at 60 ℃ for 3 hours. (Step 7)

Detailed descriptions of each step refer to FIG. 1 .

The ripening fermentation freeze-drying technology of fresh antler of the present invention minimizes the insolubility of useful components unique to fresh antler through the livestock disease prevention technology, probiotics technology, and hurdle technology technology, and enhances acquired fermentation functionality and improves food safety.

Example 2: Deer Antler Material

As for the antler material used in the experimental examples and examples of the present invention, 30 kg (n = 30) of live antler (FA) produced in 2020 ( Cervus canadensis, Elk ) were collected from the Korea Soil Yangrok Agricultural Cooperative Federation. Grade 2 cuts (within 7cm in depth of each tube branch) were used at 75 to 80 days of age, when the physiological activity content is at its peak, and tip bone and partner were used for fresh antler velvet (FV) parts.

First, the raw material for raw antler was tested for disease to ensure food safety (step 1). If raw antler was frozen, it was thawed at 7 ° C for 2 days in a slightly frozen state to the extent that blood did not flow (step 2), washed three times with hot water at 90 ° C or higher, and then sliced to a thickness of 1-3 mm, The raw materials were evenly mixed and used without any bias, and the temperature in the workplace was maintained below 15 °C (step 3). The raw material was evenly spread on a tray, sterilized by spraying 2.5% of 70% alcohol (step 4), and aged for 48 hours in a dark room at 5 ° C to 7 ° C (step 5). Lactobacillus plantarum LP20B49 (accession number: KACC 92397P, date of deposit: November 22, 2021), Saccharomyces cerevisiae , and Bacillus subtilis strains selected from Seoul National University and Jeonju University were inoculated differently for each treatment group, and at 72 Time The raw antler was fermented in sequence (step 6), and the fermented fresh antler was quickly frozen at -40℃ or less for 12 hours, and then used in a freeze dryer (KK-300, Beomil Co., Ltd.) at a core temperature of -45 After drying at -55 ° C for 21 hours, sterilization was performed at 60 ° C for 3 hours (step 7). Dry antler velvet (DA) in the control group applied the conventional method.

In order to confirm the functional indicator substance of the fermented freeze-dry Antler velvet extract (FFAE) that has undergone the above step-by-step process, dry Antler velvet extract (DAE) prepared in a traditional way was used as a control and compared. In addition, using TOWER KS-220 (Kyungseo E&P Co., Ltd., Incheon), clean water based on twice the raw material for each test section was added, and hot water extraction was performed at 85 ~ 100 ° C. for 20 hours.

The antler concentrate was filtered through 100 mesh, and then concentrated to the same concentration of 18% solid content (D/M 18%) in a solid content measuring device (ATAGO PAL-2), and used as an analysis sample. In addition, fermented freeze-dry Antler velvet powder (FFAP) was prepared to measure the viable and total bacterial counts, and powders that passed 100 mesh in each test section were obtained.

Example 3: Food safety analysis of raw materials for raw antler

In order to ensure food safety for fresh antler (FA) raw materials before fermentation, deer-derived diseases and harmful pathogens were tested. Specifically, each (n = 80) was sliced at a thickness of 1 to 3 mm from the bottom of the battery and analyzed. There are no standardized tests for tuberculosis bacillus and brucellosis on deer antler samples and, specifically, deer Chronic Wasting Disease (CWD) test methods. In the present invention, PMCA (Protein misfolding cyclic amplification) technique, which is a highly sensitive assay for PrP ^c , was applied to the causative protein for detection using immunostaining and Western blotting (FIG. 3).

Example 3-1: DNA extraction

DNA extraction for genetic testing was performed using the Genomic DNA Extraction kit (Bioneer, Korea), and the method recommended by the manufacturer was applied. A brief description of the extraction process is as follows.

First, about 30 mg of an antler sample was collected, put into a 1.5 ml tube, and 200 μl of a tissue lysate was added. 20 μl of Proteinase K and 10 μl of RNase A were immediately added and mixed well. The antler tissue was completely dissolved while sensitizing in a constant temperature water bath at 60 °C for 1 hour. After adding 200 μl of Binding buffer and mixing well, 400 μl of ethanol was added, mixed well, and then placed in a Binding column tube and centrifuged at 8,000 rpm for 1 minute. 500 μl of washing solution was added, centrifuged at 8,000 rpm for 1 minute, and washed twice. After drying at room temperature for 5 minutes, 200 μl of the extract was added and centrifuged at 13,000 rpm for 1 minute to recover DNA, which was then stored at -20 ° C or less and used for testing.

Example 3-2: Preparation of primers and PCR conditions for detecting pathogens

Base sequences of primers for detecting target genes for each disease are listed in Table 1 below. Specifically, tuberculosis disease detects 16S Rrna of H37Rv strain, a standard strain of Mycobacterium tuberculosis complex, and brucellosis detects BCSP-31 of Brucella species and deer chronic wasting disease (CWD) tried to detect PRNP (prion protein gene).

disease name purpose
gene Product size (bp) Gen Bank Primer sequences sequence number tuberculosis disease 16S rRNA 543 bp NR102810 F ACG GTG GGT ACT AGG TGT GGG TTT C One R TCT GCG ATT ACT AGC GAC TCC GAC TTC A 2 brucellosis BCSP-31 440 bp MG457036 F GGG CAA GGT GGA AGA TTT 3 R CGG CAA GGG TCG GTG TTT 4 deer chronic
wasting disease Elk PRNP 184 bp EU082291 F GGA ACA AGC CCA GTA AAC CA 5 R TGG TTG GGG TAA CGG TAC AT 6

The PCR reaction conditions of the primers were performed under the conditions described in the following tables, respectively (Table 2: Tuberculosis disease, Table 3: Brucella disease, Table 4: Deer chronic wasting disease).

condition Temperature (℃) hour cycle Initial Denaturation 94 5 minutes 1 cycle Denaturation 94 1 min 25 cycles Annealing 60 1 min Extension 72 1 min final extension
(final extension) 72 10 minutes 1 cycle

condition Temperature (℃) hour cycle Initial Denaturation 95 1 min 1 cycle Denaturation 95 30 seconds 35 cycles Annealing 58 30 seconds Extension 72 1 min final extension
(final extension) 72 10 minutes 1 cycle

condition Temperature (℃) hour cycle Initial Denaturation 95 1 min 1 cycle Denaturation 95 30 seconds 35 cycles Annealing 58 30 seconds Extension 72 1 min final extension
(final extension) 72 10 minutes 1 cycle

Example 3-3: Decalcification, Immunohistochemical stain

In order to perform an immunochemical experiment using antler, it is necessary to demineralize the antler fixed in 10% neutral formalin solution. As the decalcification solution for removing calcium, Gooding and Stewart's decalcification solution was used, and the components are shown in Table 5. In this experiment, 5% formic acid was used.

Calcium was removed by treating the deer antler tissue with demineralization solution for 3 days at room temperature. Before entering the dehydration process, it was soaked in 5% sodium sulfate solution for 12 hours to improve stainability, and after that, it was neutralized by soaking in 70% ethanol for 12 hours, and then went through a general tissue specimen production process. A Vector ABC kit (Vector Laboratories, Peterborough, UK) was used and the experimental procedure was performed according to the manufacturer's instructions.

ingredient Formic acid Formalin D.W. Volume (%, ml) 5% - 25%
(5 ml - 25 ml) 5%
(5ml) 70% - 90%
(70 ml - 90 ml)

Example 3-4: CWD test method for antler samples (immunoblotting, Western blotting)

(1) Sample pretreatment

① Weigh about 200 μl of the pulverized sample and add it to the sample tube.

② Mount the sample tube on the ribolyser and homogenize slowly.

③ Take 250 μl from the homogenized sample, taking care not to contain solids, and transfer to an Eppendorf tube.

④ Add 250 μl of Proteinase K solution and mix well (close the tube cap and shake up and down about 10 times),

⑤ After mixing well, react quickly at 37℃ for 10 minutes in a constant temperature water bath.

⑥ After completion of the reaction, add 250 μl of Reagent B and mix well until the entire solution turns blue.

⑦ After mixing well, centrifugation was performed at 15,000 × g at 20 ° C for 7 minutes.

⑧ After centrifugation, discard the supernatant, turn the tube upside down and place it on paper for 5 minutes to dry to remove additional supernatant.

⑨ After drying, 25 μl of Reagent C was added.

⑩ Quickly react at 100℃ for 5 minutes in Heat Block.

⑪ Take out the micro tube from the heat block and mix well by vortex.

(2) Western blotting PrP extraction reagent:

① TN buffer: 100mM NaCl, 50mM Tris-HCl (pH7.5),

② Detergent buffer: 4% Zwittergen 3-14, 4% Sarkosyl 100mM NaCl, 50mM Tris-HCl (pH7.5) (※ per 1㎖ of detergent buffer: 10× TN buffer 100μl, 30% zwittergen 133.5 μl, 25% sarkosyl 160.0 μl),

③ Butanol-Methanol solution: 2-Butanol : Methanol = 5 : 1,

④ Proteinase K: 1mg/mL in 50mM Tris-HCl (pH8.0), 1mM CaCl2,

⑤ Pefablock : 0.1M in DDW,

⑥ Collagenase 20mg/㎖ in DDW,

⑦ DNase I: 10mg/ml in 50% glycerol, 10mM Tris-HCl (pH7.5), 10mM MgCl2,

⑧ 2×Sample buffer (up to 20㎖): 1M Tris-HCl (pH6.8) 1.25㎖ Glycerol 1㎖, 0.5M EDTA (pH8.0) 120㎖, β-Mercaptoethanol 800㎖, 1% bromophenol blue 800㎖ SDS 1g, Urea 4.8g (Small amount can be stored at room temperature during use, long-term storage at 4℃)

(3) Immunoblotting (Western blotting) extraction:

① Mix 250 μl of detergent buffer with 250 μl of tissue emulsion.

② Add 25 μl of 2-Buthanol, mix, and then carry out sonication.

③ Add 12.5 μl collagenase (20 mg/ml) and mix.

④ React at 37℃ for 30 minutes.

⑤ Add 10 μl 0.1M pefablock and mix.

⑥ Add 2 μl DNase I (10 mg/ml) and react at room temperature for 5 minutes.

⑦ Add 250 μl of Butanol-methanol solution and mix.

⑧ Centrifugation at 20,000g at 20℃ for 10 minutes.

⑨ Remove the supernatant and dry the precipitate.

⑩ Add 100 μl of sample buffer and boil at 100℃ for 5 minutes.

(4) Immunoblotting (Western blotting) electrophoresis:

① Prepare an electrophoresis tank, remove the white tape at the bottom of the gel, and attach the gel to the tank.

② After removing the plastic combs corresponding to the gel, fill each well with running buffer using a 1 ml pipette.

③ 10 μl of sample and 5 μl of marker were loaded on the first gel, and the samples were diluted step by step along with the marker on the second gel and loaded into each well.

④ Fill the upper buffer chamber (the space between gel and gel) with running buffer, and pour running buffer so that the lower buffer chamber is filled 3 cm from the bottom of the gel.

⑤ After adding 500 μl of antioxidant to the upper buffer chamber, cover the tank, connect the socket of the lid to the power supply, and turn it down at 150V for about 50 minutes.

⑥ While sample is unloaded, fill the transfer tank with transfer buffer for transfer and cool the transfer buffer by connecting the cooling device. In addition, cut the PVDF membrane and filter paper according to the gel, soak the membrane in methanol for 15 seconds to hydrate, discard the methanol, and soak in transfer buffer.

⑦ After the electrophoresis is finished, take out the gel from the electrophoresis tank, then take the gel out of the plastic frame of the precast gel and soak it in the transfer buffer.

⑧ Open the transfer cassette, place it on an appropriate tray, pour transfer buffer, place a sponge on one side of the cassette, place filter paper on it, and place the pre-hydrated membrane on it again.

⑨ Place the gel on the Membrane, remove air bubbles, place the filter paper and sponge in order, close the cassette, and attach it to the transfer tank.

⑩ Close the lid of the transfer tank, connect the socket to the power supply so that the membrane comes to the positive charge side, and transfer at 400mA for 1 hour and 30 minutes.

(5) Immunodetection through Western blotting:

① Blocking: After putting the blocking buffer in the membrane so that it is submerged, react while shaking at room temperature for 30 minutes.

② 1st antibody: Dilute 6H4, 8E74 or S1 antibody 3,000-fold in TBST, add the membrane so that it is submerged, and react at room temperature for 1 hour while shaking or overnight at 4℃.

③ Washing: Washing with TBST 3 times for 5 minutes.

④ 2nd antibody: After diluting the secondary antibody 3,000 times and putting it on the membrane, react at room temperature for 30 minutes while shaking.

⑤ Washing: Washing with TBST three times for 5 minutes.

⑥ After adding Luminiscence buffer, react for 5 minutes.

⑦ Dilute CDP-star (12.5mM) 50 times with Luminiscence buffer (CDP-star final concentration: 0.25mM).

⑧ Place the membrane on the OHP film, pour diluted CDP-star, and react at room temperature for 5 minutes.

⑨ Remove excess liquid from the membrane and cover the membrane with OHP film.

⑩ After attaching X-ray film to the membrane for 5 to 10 minutes in a dark room, immerse it in the developer and fixer in order to check the signal.

Example 4: Preparation of fermented freeze-dried antler

Fermented Freeze-dry Antler velvet (FFA) proceeds in the order of slicing, sterilization, aging/fermentation, and freeze-drying of selected raw antler velvet. Specifically, as described in Example 2 above, ① fresh antler without livestock disease was washed three times in hot water at 90 ° C, thawed, and then sliced into 1-3 mm thick slices, ② raw antler slices were spread so as not to overlap, and then 2.5% of the raw material was added. The corresponding 75% alcohol was sufficiently sprayed to sterilize, and ③ aged for 48 hours in a dark room at 5 ° C to 7 ° C. ④ Prebiotic minimum media as a fermentation medium for fresh deer antler (based on 1L: peptone 5g, sodium acetate 2.5g, 1M magnesium sulfate 7H ₂ O 0.5mL, 1M manganese sulfate 4H ₂ O 0.5mL, Tween 80 5mL, diammonium citrate 1g, dipotassium phosphate 1g, Glucose 20g, L-glutamic acid 1g) was prepared and sterilized under high pressure at 121 °C for 15 minutes, and strains finally selected from Seoul National University and Jeonju University were used for fermentation. ⑤ After proliferating the strains to be used for each test section, the MRS Broth was centrifuged, the supernatant was removed, and a prebiotic minimal medium was added to the remaining pellet. ⑥ Treatment was performed so that the sum of single or complex strains reached 7.5%, and the experimental group was untreated (T1), aged (T2), and after aging, Lactobacillus plantarum LP20B49 alone (T3), and after aging Lactobacillus plantarum LP20B49 and 2 types of Bacillus subtilis treated (T4), after aging Lactobacillus plantarum LP20B49, 3 types of Bacillus subtilis and Saccharomyces cerevisiae After treatment (T5), lyophilization was performed, and the test group was compared with a normal dried antler (control group, CON). ⑦ In the above T3 to T5, fermentation in each test zone was carried out at 5 ~ 37 ℃ for 72 hours. ⑧ Freeze-drying was carried out in a KGT-1200 (Hwajin Biotech Co., Ltd., Yeong-Gwang) at -45 ~ -55 ° C for 21 hours and sterilization at 60 ° C for 3 hours. was obtained (Fig. 2).

Example 5: Sialic acid measurement (Sialic aicd assay)

In order to measure the sialic acid content of each experimental group, the following experiment was performed.

First, in order to extract sialic acid in the sample, 0.1 mL of a 0.2NH ₂ SO ₄ solution was added to 0.1 mL of a fermented freeze-dried antler extract (FFAE) sample, hydrolyzed at 85 °C for 1 hour, and then 20 μL periodate ) solution (prepared from 0.25M NaIO ₄ in 9M H ₃ PO ₄ ) was added and reacted at room temperature for 20 minutes. Next, a 0.1mL m-arsenite preparation (10% NaAsO ₂ in 0.1NH ₂ SO ₄ with 0.5M Na ₂ SO ₄ ) solution was added to reduce excess periodate, followed by 0.25mL TBA solution ( 0.6% TBA in 0.5M Na ₂ SO ₄ ) was added, the color was developed at 85° C. for 15 minutes, cooled at room temperature, cyclohexanone 1mL was added, and centrifugation (900g, 7min) was performed. The supernatant was transferred to a 96-well plate and absorbance was measured at 549 nm.

Example 6: Measurement of glycosaminoglycan (GAG assay)

Glycosaminoglycan was measured by dimethylmethylene blue assay.

Specifically, based on DMMB solution 1L, DMMB 16mg, glycine 3.04g, NaCl 1.6g, 0.1M acetic acid 95mL was added, pH was adjusted to 3, and after filtering with a 0.45μm syringe filter, filtered with a whatman filter immediately before use. used after. After adding 200 μL of DMMB reagent to 20 μL of sample in a 96-well plate, shaking for 5 seconds, the absorbance was measured at 525 nm.

Example 7: Uronic acid assay

Dilute fermented freeze-dried deer antler extract (FFAE) sample 100 times, put 100 μL each in an EP tube, add sodium tetraborate at a concentration of 25 mM, add 400 μL of a solution dissolved in sulfuric acid, heat at 100 ° C for 10 minutes, and incubate at room temperature radiated from When the sample reached room temperature, 100 μL of a solution in which 0.125% carbazole was dissolved in absolute ethanol was added and reacted by heating at 100 ° C for 10 minutes. After cooling sufficiently to room temperature, 100 μL at a time was transferred to a 96-well plate and absorbance was measured at 550 nm. The content of uronic acid was calculated from a standard curve prepared with D-glucuronic acid.

Example 8: GABA (γ-aminobutyric acid; gamma-aminobutyric acid) measurement

After mixing 3 mL of fermented freeze-dried antler extract sample for each test section, 1.5 mL of 0.5M sodium bicarbonate, and 0.5 mL of 0.715 mg/ml 1-fluoro-2,4-dinitrobenzene, react at 60 ° C for 1 hour, then at room temperature. cooled in After filtering using a 0.22 μm syringe filter (PTFE, Advantec, Dublin, CA, USA), GABA in the obtained extract was analyzed by Shimazhu 20A series HPLC. For the column, a Symmetry C18 column (5μm, 3.9 x 150 mm, Waters, Milford, MA, USA) was used, and two mobile phases, ammonium acetate aqueous solution and acetonitrile were used in an oven at 30 ° C in a ratio of 85:15 (v / v ), elution was carried out for 20 minutes under isocratic conditions. The injection volume was 10 μL, and the wavelength was analyzed using 360 nm.

Example 9: Electrophoresis (Tris-glycine SDS-PAGE) measurement

In order to identify low-molecular-weight proteins and peptides according to the treatment conditions of each test section, fermented freeze-dried antler extract samples were mixed with 2X Tricine sample buffer at a ratio of 1:1, heated at 85 ° C for 2 minutes to denature proteins, and each sample After loading 50 ug each in wells of a 16.5% polyacrylamide gel, Tricine SDS PAGE buffer was added and electrophoresis was performed at 80V. The gel was separated from the glass plate, stained with Coomassie blue for 1 hour, and then bleached for 24 hours using a solution containing methanol:acetic acid:water (1:1:8), and then the band was confirmed.

Example 10: Aerobic Count (AC) Analysis

10 g of fermented freeze-dry Antler velvet powder (FFAP) was taken from each test plot and added together with 90 mL of saline solution in a sterile plastic pouch. After homogenization for 1 minute, the homogenized samples were serially diluted with saline to determine the total number of aerobic plates. For counting, 1 mL of the diluted sample was spread on APC Petrifilm and Mold and Yeast Petrifilm (3M Healthcare., Paul, MN, USA) and incubated at 37°C for 48 hours. Colonies on the plate were counted and calculated as log10 colony forming units per sample (CFU/g). Each sample was measured in triplicate.

Example 11: Lactic acid bacteria (LAB) measurement

In order to investigate the growth of lactic acid bacteria in the fermented freeze-dried antler powder in each test group, the following experiment was performed.

First, after serial dilution of 100 μL of the culture medium of the experimental bacteria grown in MRS broth with 0.1% peptone water, 10 μL of 10 μL was dropped 3 times on an MRS agar plate, followed by incubation at 37 ° C for 48 hours. The number of colonies formed was counted to determine the initial number of viable cells.

Next, 1 g of fermented freeze-dried deer antler inoculated with 2.5% of the test bacteria and fermented for 5 days (2 days of maturation, 5 days of fermentation) was serially diluted with 0.1% peptone water, and then dispensed by 100 μL on MRS agar plates and smeared at 37 ° C. After culturing for 48 hours, the number of viable bacteria in freeze-dried antler fermented on the 5th day was measured.

Next, after serially diluting 100mL of the fermented product of the experimental bacteria and deer antler with 0.1% peptone water, 10mL of 10mL each was dropped 3 times on the MRS agar plate, incubated at 37 ° C for 48 hours, and then fermented on the 2nd and 5th days respectively. The viable cell count was measured. Compared to the initial viable cell count, the daily increase and decrease in the viable cell count were investigated.

Example 12: Evaluation of antioxidant ability using DPPH radical scavenging activity

DPPH assay was performed to determine the antioxidant activity of fermented freeze-dried antler extract (FFAE).

* 100 로 계산하였다.First, FFAE was diluted with distilled water to four concentrations before use. 1.9 mL of 95% ethanol and 0.1 mL of 0.5 mM DPPH were mixed and mixed with 0.1 ml of FFAE samples prepared for each of the four concentrations, reacted at room temperature for 30 minutes, and then transferred to a 96-well plate by 100 mL and the absorbance was measured at 517 nm. . Standard curve equations and positive controls were prepared using diluted Trolox at various concentrations. The DPPH radical scavenging activity of FFAE was calculated and expressed as μmol Trolox equivalent (TE) per gram of FFAE (μmol TE / g FFAE). A standard curve was prepared using L-ascorbic acid, and the DPPH radical scavenging activity (%) was

* Calculated as 100.

Example 13: Antioxidant ability evaluation using ABTS radical scavenging activity

ABTS assay was performed to determine the antioxidant activity of fermented freeze-dried antler extract (FFAE).

Specifically, ABTS + cation radicals were generated by the reaction between a 2 mM ABTS solution and 2.45 mM potassium persulfate (1:1 ratio) in water. The reaction mixture was stored in the dark at room temperature for 12-16 hours before use. The ABTS/radical solution was diluted with 95% ethanol to obtain an absorbance of 0.73 units at 734 nm using a spectrophotometer. 0.1 mL of FFAE at different concentrations was mixed with 1.9 mL of ABTS radical solution, incubated in a dark room at room temperature for 10 minutes, and then absorbance was measured at a wavelength of 734 nm using a spectrophotometer. Trolox prepared in various concentrations was used as a positive control. The ABTS free radical scavenging activity of the extract was calculated and expressed as μmol Trolox equivalent (TE) per gram of FFAE (μmol TE/g FFAE).

Example 14: Amino acid determination

In order to analyze the content of amino acids or essential amino acids present in fermented freeze-dried deer antler extract (FFAE), the following experiment was performed.

Specifically, each sample (2.5 g in powder form) was homogenized with 5 mL distilled water. After filtration through a 0.45 μm filter membrane (Merk Millipore Ltd., Carrigtwohill, Cork, Ireland), the filtrate was used for amino acid analysis. Amino acids were analyzed using ultra-high performance liquid chromatography (UPLC, Waters, Milford, USA) coupled to an Intrada amino acid column: 2 × 50 mm, 3 μm (Imtaka, Uphur St, Suite A, Portland). Clematographic separation was performed using solvent A (ACN: 100 mM ammonium formate = 20:80 v/v) and solvent B (ACN: tetrahydrofuran: ammonium formate: formic acid = 9:75:16:0.3 v/v). . Separation condition settings were 100% B for 3 min, 83% B for 3.5 min, 100% A for 3.5 min, 100% B for 7 min, and equilibrated again before the next sample injection. Amino acids were analyzed based on retention times of standard amino acid mixtures, and free amino acid values were expressed in μmol/g.

Example 15: Analysis of volatile aroma compounds (GC/MS) and sensory evaluation

The extraction of aroma compounds from the fermented freeze-dried antler extract (FFAE) was performed using SPME (Solid Phase Micro-Extraction) technology, and the extracted compounds were analyzed using Gas Chromatography/Mass Spectrometry (GC/MS).

Specifically, 1.0 ml of FFAE was placed in a 20 ml head space vial and 1.0 μl internal standard (2-methyl-3-heptanone, 816 mg/mL in methanol) was added. After that, PTFE-Silicone Lid was used for vial extraction. Extraction, absorption and desorption of aroma compounds were analyzed using a SPME sample preparer equipped with Carboxene-Polydimethylsiloxane (75μm) fiber (Supelco, USA) connected to Gas Chromatography (7890B GC) using mass spectrophotometry (5977B MSD, Agilent Technologies, Santa Clara, CA, USA). The extraction was adsorbed at 60 °C for 60 min and desorbed at 250 °C in the inlet port for 5 min with a split flow of 10 mL/min of volatile-containing fiber. The separation of volatiles was carried out in a capillary column (30 m x 0.25 mm i.d. x 0.25 μm film thickness) at a constant flow rate of 1 mL/min. The oven temperature was held at 40°C for 5 minutes and then increased to 250°C at a rate of 8°C/min and held for 5 minutes. Volatile aroma compounds were identified by Mass Spectrum Library (Agilent Technologies) comparison and retention times compared to external standards. Identified compounds were quantified by comparing the peak area of the internal standard to the sample peak area. For sensory evaluation of aroma compounds, food-related workers and college students (n=30) were selected and palatability was measured by a 9-point scale method after sensory evaluation training.

Example 16: Analysis of bioactive compounds (UPLC-Q-TOF-MS/MS)

For the analysis of bioactive substances, the following experiments were performed. Specifically, 1 g of each fermented freeze-dried antler extract (FFAE) was dissolved in distilled water and then filtered through a 0.45 μm filter membrane (Merck Millipore). Bioactive compounds were separated and detected in FFAE using Ultra high Performance Liquid Chromatography Tandem Mass (UPLC-Q-TOF-MS/MS, Xevo TQ-5, Waters, Milford, USA). Chromatographic separation was analyzed by injecting each FFAE in an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) at 40 °C at a flow rate of 0.5 ml/min. The mobile phase consisted of solvent A (distilled water + 0.1% formic acid) and solvent B (ACN + 0.1% formic acid). The elution component was set to 97% phase A for 0-5 minutes. 3-100% Liner Gradation Step B for 5-16 minutes; 100% phase B for 16-17 minutes; 100-3% step B for 17-19 minutes; 97% A phase for 19-25 minutes. Compounds eluted from the column were detected with a high-resolution tandem mass spectrometer SYNAPT G2 Si HDMS QTOF (Waters) in positive (+) and negative (-) mode. The capillary voltage and cone voltage of positive ion mode were set to 2 kV and 40 V, respectively. Negative ion models were set at 1 kV and 40 V, respectively. Mass spectrometry data were collected using the Centroid MS mode. The primary scan range was 50 to 1200 Da and the scan time was 0.2 sec. All ions were fragmented using 20 to 40 eV. The information acquisition (scan) time of all pieces is 0.2 seconds. During the data acquisition process, LE signals were acquired every 3 seconds for real-time quality correction. Monitoring positive ([M+H]+= 556.2771) and negative ([M-H]-= 554.2615) using leucine encephalitis as a locking mass by a locking spray interface at a flow rate of 10 μL/min for accurate mass acquisition did. Ion mode data collection and analysis were controlled and statistically analyzed by UNIFI V1.71 software (Waters), and individual peaks were identified by screening against appropriate scientific libraries in UNIFI V1.71 (Waters). The reliability of the indicator substances of fermented freeze-dried antler extract (FFAE) and dried antler extract (DAE) was confirmed through PCA analysis.

Example 17: Production Yield Analysis

In order to analyze the production yield of fermented freeze-dried antler extract (FFAE), the following experiment was performed.

Specifically, the analysis was performed by comparing Control (general dry antler, Dry Nokyong) and T1 (freeze-dry Nokyong) with fermented freeze-dried antler (T35, T45, T55). Each test section was measured 3 times, and the raw material based on 2,250g of fresh antler was added to each test section, and the input amount of dry antler with 9% moisture was 818g. 12,000 ml of purified water was added to each test section, hot water extraction was performed by heating at a temperature of 90-100 ° C. for 20 hours, and each extract was equally concentrated to a solid content of 18%, and the yield was compared and analyzed based on the weight of the extract.

Example 18: Statistical Analysis

Data were analyzed using the one-way ANOVA procedure from the Statistical Analysis System (SAS Institute, Cary, NC, USA, 2007) and means and standard errors were calculated for the variables. Origin of VA was considered as the main effect in the model and means were compared using Duncan's multiple range test. Significance was defined at p < 0.05.

Experimental Example 1: Food safety analysis of raw antler

In order to secure the food safety of fresh antler (FA) raw materials (n = 80) with an antler history number, harmful pathogens were tested for deer-derived tuberculosis, brucellosis, and deer chronic wasting disease (CWD), and were not detected in all samples.

sample
number velvet
history number Tuberculosis
PCR result Brucella
PCR result CWD
result sample
number Deer Antler History Number Tuberculosis
PCR result Brucella
PCR result CWD
result One 19-3454 not detected not detected not detected 41 19-2037 not detected not detected not detected 2 19-3009 not detected not detected not detected 42 19-2178 not detected not detected not detected 3 19-2011 not detected not detected not detected 43 19-2075 not detected not detected not detected 4 19-2082 not detected not detected not detected 44 19-1077 not detected not detected not detected 5 19-1002 not detected not detected not detected 45 19-1005 not detected not detected not detected 6 19-1010 not detected not detected not detected 46 19-3238 not detected not detected not detected 7 19-2065 not detected not detected not detected 47 19-1078 not detected not detected not detected 8 19-2112 not detected not detected not detected 48 19-1031 not detected not detected not detected 9 19-2061 not detected not detected not detected 49 19-2108 not detected not detected not detected 10 19-3375 not detected not detected not detected 50 19-3397 not detected not detected not detected 11 19-2159 not detected not detected not detected 51 19-1037 not detected not detected not detected 12 19-1075 not detected not detected not detected 52 19-1043 not detected not detected not detected 13 19-3383 not detected not detected not detected 53 19-1013 not detected not detected not detected 14 19-1023 not detected not detected not detected 54 19-1012 not detected not detected not detected 15 19-2039 not detected not detected not detected 55 19-1061 not detected not detected not detected 16 19-2006 not detected not detected not detected 56 19-2196 not detected not detected not detected 17 19-1006 not detected not detected not detected 57 19-3441 not detected not detected not detected 18 19-1029 not detected not detected not detected 58 19-2063 not detected not detected not detected 19 19-2085 not detected not detected not detected 59 19-2064 not detected not detected not detected 20 19-2095 not detected not detected not detected 60 19-2012 not detected not detected not detected 21 19-1036 not detected not detected not detected 61 19-2051 not detected not detected not detected 22 19-2073 not detected not detected not detected 62 19-3394 not detected not detected not detected 23 19-1047 not detected not detected not detected 63 19-1003 not detected not detected not detected 24 19-3225 not detected not detected not detected 64 19-2035 not detected not detected not detected 25 19-3352 not detected not detected not detected 65 19-3009 not detected not detected not detected 26 19-1054 not detected not detected not detected 66 19-3298 not detected not detected not detected 27 19-2004 not detected not detected not detected 67 19-3070 not detected not detected not detected 28 19-2049 not detected not detected not detected 68 19-3022 not detected not detected not detected 29 19-2059 not detected not detected not detected 69 19-1083 not detected not detected not detected 30 19-1008 not detected not detected not detected 70 19-3285 not detected not detected not detected 31 19-2089 not detected not detected not detected 71 19-1018 not detected not detected not detected 32 19-2080 not detected not detected not detected 72 19-1004 not detected not detected not detected 33 19-2026 not detected not detected not detected 73 19-3119 not detected not detected not detected 34 19-2169 not detected not detected not detected 74 19-2038 not detected not detected not detected 35 19-2168 not detected not detected not detected 75 19-3170 not detected not detected not detected 36 19-2180 not detected not detected not detected 76 19-3042 not detected not detected not detected 37 19-2109 not detected not detected not detected 77 19-3249 not detected not detected not detected 38 19-2096 not detected not detected not detected 78 19-3413 not detected not detected not detected 39 19-3104 not detected not detected not detected 79 19-2194 not detected not detected not detected 40 19-2195 not detected not detected not detected 80 19-2070 not detected not detected not detected

Experimental Example 2: Analysis of sialic acid (mg/mL) content

Based on the method described in Example 5, the results of analyzing the sialic acid content of fermented freeze-dried deer antler are shown in Table 7 and FIG. 4.

As a result, it was confirmed that the sialic acid content increased in all samples analyzed when compared to the control group, and in particular, it was confirmed that T22 and T52 increased by 333% and 353%, respectively, compared to the control group. In addition, it was confirmed that T45 was specifically increased over time compared to T42.

mg/mL stdev Increase rate compared to control group control group 0.043 0.008 100% T10 0.047 0.004 109% T22 0.143 0.014 333% T25 0.062 0.002 144% T32 0.062 0.004 144% T35 0.051 0.006 119% T42 0.109 0.004 253% T45 0.121 0.006 281% T52 0.152 0.001 353% T55 0.078 0.002 181%

Experimental Example 3: Analysis of glycosaminoglycan (mg / mL) content

Based on the method described in Example 6, the results of analyzing the glycosaminoglycan (GAG) content of the fermented freeze-dried deer antler are shown in Table 8 and FIG. 5.

As a result, it was confirmed that the GAG content increased by 351%, 333%, and 281% in T52, T22, and T45, respectively, compared to the control group. Specifically, T45 was continuously increased over time compared to T42.

mg/mL stdev increase rate Control 6.360 0.144 100% T15 5.277 0.668 109% T22 5.881 0.063 333% T25 1.465 0.036 144% T32 3.798 0.361 144% T35 6.777 0.308 119% T42 4.881 0.063 253% T45 4.777 0.072 281% T52 10.679 1.449 353% T55 3.735 0.072 181%

Experimental Example 4: Analysis of uronic acid (mg / mL) content

Based on the method described in Example 7, the results of analyzing the uronic acid content of the fermented freeze-dried antler are shown in Table 9 and FIG. 6.

As a result, it was confirmed that the uronic acid content increased by 192%, 177%, and 131% in T22, T42, and T55, respectively, compared to the control group, and specifically, compared to T52, T55 increased continuously over time. .

mg/mL Stdev increase rate Control 1.200 0.170 100% T10 1.740 0.396 145% T22 2.300 0.226 192% T25 1.980 0.085 165% T32 1.010 0.014 84% T35 0.530 0.014 44% T42 2.120 0.537 177% T45 1.000 0.085 83% T52 1.560 0.226 130% T55 1.570 0.354 131%

Experimental Example 5: Analysis of gamma-aminobutyric acid (mg/mL) content

Based on the method described in Example 8, the results of analyzing the content of γ-aminobutyric acid (GABA) in the fermented freeze-dried antler are shown in Table 10 and FIG. 7.

As a result, it was confirmed that the GABA content increased in all analyzed samples compared to the control group, and in particular, 460%, 346%, and 291% respectively in T35, T45, and T55 compared to the control group.

mg/mL stdev increase rate Control 0.096 0.011 100% T10 0.151 0.016 157% T22 0.129 0.027 134% T25 0.196 0.009 204% T32 0.243 0.027 253% T35 0.442 0.047 460% T42 0.365 0.016 380% T45 0.332 0.008 346% T52 0.213 0.004 222% T55 0.279 0.036 291%

Experimental Example 6: Measurement of the total number of lactic acid bacteria and general bacteria

Based on the method described in Examples 10 and 11, the results of measuring the total number of lactic acid bacteria (LAB) and the total number of general bacteria (AC) in fermented freeze-dried deer antler are shown in FIG. 8.

As a result, all samples except T52 were measured higher than the control group. In addition, the number of lactic acid bacteria and general bacteria was increased in T45 and T55 compared to T42 and T52, and it was confirmed that the number was the highest in T55 compared to other samples. Specifically, both LAB and AC were rather decreased at T35 than at T32.

Experimental Example 7: Evaluation of antioxidant activity (DPPH, ABTS)

Based on the method described in Examples 12 and 13, the antioxidant activity of fermented freeze-dried deer antler was measured based on DPPH radical scavenging activity and ABTS radical scavenging activity, and the results are shown in FIG. 9 .

As a result, the DPPH radical scavenging ability was significantly increased compared to the control group, and the ABTS radical scavenging ability was increased in all except for T22 and T55. Specifically, at T25, it was confirmed that both DPPH and ABTS increased by more than 200%.

Experimental Example 8: Aroma Compound Analysis (GC/MS)

Based on the method described in Example 15, the results of analyzing the aroma compounds of the fermented freeze-dried deer antler by GC/MS are shown in FIG. 10.

As a result, it was confirmed that the contents of aroma compounds such as 2-Butanone, Acetic acid butyl ester, 2-Furanmethanol, and Benzaldehyde all increased compared to the control group. In particular, 2-Furanmethanol, known as an aroma compound that affects the sweetness of brown sugar, increased by 540% and 318% at T25 and T35, respectively. 430% increase. In addition, 2-Butanone, known as freshly extracted Arabica coffee aroma, increased by 195% in T55, and acetic acid butyl ester, known as fermented apple juice and fermented ham aroma, increased by 279% in T52.

Experimental Example 9: Analysis of small molecule proteins and peptides

Based on the method described in Example 9 above, the results of measuring the levels of low-molecular-weight proteins and peptides performed by diluting the samples 200-fold are shown in FIG. 11.

As a result, it was confirmed that low-molecular-weight peptide conversion was progressing in T10, T32, T42, T52, and T55 compared to the control group.

Experimental Example 10: Amino Acid Analysis

Based on the method described in Example 14, an amino acid analysis experiment was performed to measure the amino acid content of fermented freeze-dried deer antler during fermentation, and the results are shown in Table 11 and FIGS. 12 to 14.

As a result, the total amount of total essential amino acids increased by about 20% or more at T3, T4 and T5 compared to the control group, and increased by 146%, 133% and 131% at T45, T55, T45 and T32, respectively. In addition, the contents of Ile, Lys, and His were increased by more than 200% in T55 and T45.

Control T10 T22 T25 T32 T35 T42 T45 T52 T55 Glycine (Gly) 97.5 111.3 80.8 93.4 111.6 89.3 101.7 101.3 108.8 113.4 Alanine (Ala) 101.9 101.5 87.3 96.5 122.8 125.7 113.7 125.3 121.0 139.0 Serine 50.5 59.3 38.1 46.8 79.4 61.1 71.3 58.0 68.4 60.0 Proline (Pro) 39.4 41.9 36.4 26.4 29.0 46.9 45.3 48.7 45.1 48.7 Valine (Val) 35.4 36.0 31.0 33.0 43.0 56.8 44.9 57.2 46.8 57.5 Threonine (Thr) 18.9 18.3 15.8 14.7 19.5 20.8 21.0 19.1 20.3 18.0 Leucine (Leu) 22.3 20.0 21.7 24.0 22.4 34.3 19.9 35.3 18.5 25.6 Isoleucine (Ile) 9.3 11.2 8.9 6.9 11.5 20.2 15.6 18.4 15.1 25.9 Asparagine (Asn) 5.0 5.6 3.3 6.7 10.3 6.0 7.3 6.0 5.8 8.1 Aspartic acid (Asp) 14.0 15.9 11.2 25.4 23.9 18.2 20.2 16.6 13.0 23.5 Lysine (Lys) 42.5 52.7 35.5 52.7 103.0 97.4 86.3 112.8 77.5 138.0 Glutamine (Gln) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 Glutamic acid (Glu) 57.8 60.6 57.4 31.4 54.4 56.8 62.9 51.0 64.7 54.0 Methionine (Met) 5.9 5.7 5.8 4.1 5.6 6.6 5.9 6.8 6.3 6.6 Histidine (His) 12.5 13.3 8.0 16.4 33.0 17.2 20.8 25.1 15.2 31.1 Phenylalanine (Phe) 12.6 12.6 12.0 11.8 14.0 14.9 13.8 15.1 13.7 15.1 Arginine (Arg) 27.2 25.5 18.6 30.8 43.2 44.9 34.0 39.7 34.2 43.0 Tyrosine (Tyr) 13.7 13.2 12.6 11.8 14.5 18.1 15.8 17.7 15.8 16.6 Tryptophan (TRP) 3.1 3.1 2.7 1.8 3.8 6.0 4.3 5.3 4.2 5.2 Cys*2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Total essential amino acids 569.6 607.8 487.2 534.7 745.0 741.3 704.8 759.5 694.6 829.4 Control Contrast Ratio (%) 100% 107% 86% 94% 131% 130% 124% 133% 122% 146%

Experimental Example 11: Deer Antler Concentrate Extraction Yield

Based on the method described in Example 17, in order to measure the production yield of fermented freeze-dried antler, the weight of the antler concentrate for each treatment method (three repetitions) was compared, and the results are shown in FIG. 15.

As a result, the yield of antler concentrate (D/M 18%) was extracted in the order of raw antler (1,783g), fermented freeze-dried antler (1,636g), freeze-dried antler (1,587g), and general dried antler (1,466g), respectively. As confirmed, it can be seen that the production yield of fermented freeze-dried antler is higher than that of freeze-dried antler and general dried antler.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

This patent was researched with the support of the high value-added food technology development project of the Agriculture, Forestry and Food Technology Planning and Evaluation Institute with funding from the Ministry of Agriculture, Food and Rural Affairs [Research Project No. 321033-3 (321033031SB010)].

In addition, this patent is the result of Assignment No. 321033-3, Professor Young-Hoon Kim of Seoul National University, Professor Sang-Nam Oh of Jeonju University, Professor Bo-Young Jeon of Yonsei University, Dr. Shin-Seok Kang, Researcher Dong-Hwi Yeom of Korea Deer Association, Professor Sang-Woo Kim of Chonbuk National University, Professor In-Ho Hwang of Nonghyup Feed, and Byeong-Min Min of Nonghyup Feed Division , Shinhan University Professor Lee Myeong-ho, Korea Soil Lok Nonghyup Dr. Kim Yong-an, and Dr. Kim Dong-hoon are the result of joint research.

Rural Development Administration National Academy of Agricultural Sciences Microorganism Bank (KACC) KACC92397P 20211122 Rural Development Administration National Academy of Agricultural Sciences Microorganism Bank (KACC) KACC92398P 20211122 Rural Development Administration National Academy of Agricultural Sciences Microorganism Bank (KACC) KACC92399P 20211122

Claims (15)

1) fermenting by inoculating a strain into an antler raw material; and
2) Freeze-drying the fermented antler
A fermentation freeze-dried antler manufacturing method comprising a. The method of claim 1,
Wherein the raw material for antler is raw antler, aged antler, dried antler or fermented antler. The method according to claim 1, wherein step 1)
A) aging an antler raw material; and
B) fermenting by inoculating the strain into the aged antler
That is, the manufacturing method comprising a. The method according to claim 3, wherein the aging of step A) is performed at 0 ° C to 15 ° C conditions. The method according to claim 1, wherein the strain is Lactobacillus, Bacillus, Saccharomyces, Bifidobacterium, Streptococcus, Leuconostoc, Pedio A manufacturing method comprising at least one selected from the group consisting of Pediococcus and Lactococcus. The method according to claim 1, wherein the strain comprises at least one selected from the group consisting of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae phosphorus, manufacturing method. The method of claim 1, wherein the method
A) A process of testing deer-derived diseases and harmful pathogens on raw materials for deer antler;
b) a process of thawing raw materials for deer antlers at a low temperature for a long time;
c) a process of washing raw materials for deer antler; and
D) Process of sterilizing raw materials for antler
To further comprise one or more steps selected from the group consisting of, the manufacturing method. The method according to claim 1, wherein the method is performed by adding sialic acid (Glycosaminoglycan, GAG), uronic acid, gamma-aminobutyric acid (γ-aminobutyric acid. GABA), amino acids in antler. And to enhance the content of one or more selected from the group consisting of aroma compounds, a manufacturing method. The method according to claim 8, wherein the aroma compound is one selected from the group consisting of 2-butanone, acetic acid butyl ester, 2-furanmethanol, and benzaldehyde The manufacturing method comprising the above. The method according to claim 1, wherein the method enhances the antioxidant activity of the antler. The method according to claim 1, wherein the method improves the extraction efficiency of the antler. The method according to claim 1, wherein the method is to increase the lactic acid bacteria content of the antler. Fermented freeze-dried antler prepared by the method of any one of claims 1 to 12. An antioxidant composition comprising the fermented freeze-dried deer antler of claim 13, an extract thereof, or a fraction thereof. At least one strain selected from the group consisting of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae; culture of the strain; Concentrates of the above strains or cultures; And a composition for fermentation of antler, comprising at least one selected from the group consisting of dried products thereof.

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