KR102165643B1 - Concentrated liquid of glycosylated Lentinus edodes, Concentrated powder of glycosylated Lentinus edodes and Manufactured method of the same - Google Patents

Abstract

The present invention relates to a processed shiitake product and a method of manufacturing the same, and more specifically, to a shiitake mushroom saccharification concentrate containing a high content of amino acids, a shiitake mushroom saccharification concentrate powder, and a method of preparing them.

Description

Concentrated liquid of glycosylated Lentinus edodes, Concentrated powder of glycosylated Lentinus edodes and Manufactured method of the same}

The present invention relates to a shiitake mushroom saccharification concentrate rich in beneficial amino acids, a shiitake mushroom saccharification concentrate powder, and a method of preparing them.

Mushrooms have been widely used for food since ancient times because they contain nutrients such as sugars, lipids, proteins, minerals and vitamins evenly and have a unique taste and aroma, and in recent years, they have been in the spotlight as a pollution-free natural food.

In addition, basidiomycetes contain various medicinal ingredients and have been used as a folk remedy since ancient times. Therefore, in recent years, interest in the ingredients of mushrooms, the higher basidiomycete, has increased, and research has been actively conducted in terms of food and pharmacology, and nutritional and medicinal components of various mushrooms have been revealed. Among these ingredients, proteins and amino acids are nutrients that have a great influence on biological function and metabolism, and are ingredients that have a close relationship with flavor. Therefore, these ingredients are attracting attention from many scholars.

Mushrooms are higher fungi that form fruiting bodies among basidiomycete belonging to the fungus, and about 15,000 species are known worldwide. Of these, about 970 species are recorded in Korea, and about 20 species are cultivated. Mushrooms have recently been recognized for their pharmacological effects and are attracting a lot of attention as a functional food along with the pharmaceutical field. An important factor that attracts attention as a medicine and a new food ingredient is that mushrooms rarely have side effects even when taken for long periods of time for food or medical use. Mushrooms contain various nutrients that are important to the human body, such as vegetable protein, amino acids, enzymes, fat, iron, fiber, vitamins, and minerals. Because of their unique aroma and taste, people have enjoyed eating them since ancient times. It is known as a low-calorie food rich in protein. In addition, as a food material containing a lot of various health functional substances, it has physiologically active effects on the prevention of adult diseases such as antioxidant activity for aging prevention, anti-cancer, anti-diabetes, weight loss, immunity enhancement, and prevention of high blood pressure. It is widely used as a raw material for pharmaceuticals, and products for improving liver function and immunity using not only fruiting bodies but also mycelium are on the market. Recently, as mushrooms have been sufficiently recognized by consumers as a health food, they have reached the stage of securing a fixed consumer base. So far, most of the processes using basidiomyces as food and pharmaceuticals have been mainly made up of simple products using fruiting bodies or liquid culture of these myceliums. Mushrooms are one of the top 10 health foods in the world, and consumption is increasing in proportion to the degree of economic development. Developed countries eat more than 5kg per person per year. It is estimated that Korea consumes about 3kg, and consumption is expected to increase continuously as it is recognized as a functional food with low calorie suitable for the era of well-being.

Shiitake mushrooms, which are often found on our table because they are easy to obtain and inexpensive, are rich in nutrients as they have long been known as an excellent medicine for longevity. In'Donguibogam' and'Bonchogangmok', it is recorded that'it strengthens the energy and prevents hunger, thereby correcting the wind and helping blood circulation'. It has the effect of clearing the blood and boosting the appetite, and it is better absorbed when added together when cooking pork. The unique flavor of shiitake mushrooms is because guanylic acid is higher than that of other mushrooms, and guanylic acid has the property of lowering cholesterol, which is good for patients with high blood pressure and heart disease. In addition, lentinan in shiitake mushrooms is a powerful anticancer substance that activates the immune system. Therefore, it is effective not only for cancer, but also for viral diseases such as colds, high blood pressure, and diabetes. In addition, shiitake mushrooms are rich in various minerals and vitamins, and fiber helps digestion of the stomach and small intestine, which is good for obesity, diabetes, heart disease, and liver disease. In addition, it is rich in protein, calcium, phosphorus, iron, vitamin D, which strengthens bones, vitamin B, which is essential for hematopoiesis, and elitatenin, which helps blood metabolism, so it is good for growing children. Sun-dried shiitake mushrooms are twice as nutritious as fresh shiitake mushrooms. Especially, it has a lot of vitamin D, which helps absorb calcium, so it is known to strengthen it and prevent osteoporosis.

In Korea, wild and artificially cultivated mushrooms are widely edible, but systematic studies on the components of edible mushrooms are insufficient in terms of food. A study on the lipids of Kwon's mushrooms and shiitake mushrooms and 11 kinds of amino acid automatic analyzers There are some reports such as quantification of 17 kinds of free and total amino acids contained in edible mushrooms, TLC of amino acids by gourd and quantification by automatic amino acid analyzer, and the aroma components of edible mushrooms, composition of organic acids and fatty acids by the authors. There is a situation in which there is a lack of new application products using mushrooms.

Korean Patent Registration Publication No. 10-1479209 (announcement date 2015.01.09) Korean Patent Registration Publication No. 10-08437886 (announcement date 2008.07.03)

The present invention is a new product using shiitake mushrooms, and an object of the present invention is to provide a processed shiitake mushroom that can maximize the amino acid content beneficial to the human body contained in the shiitake mushrooms. That is, the present invention is to provide a method for preparing a saccharified concentrate of shiitake mushrooms, which is a processed product of shiitake mushrooms, and a saccharified concentrate of shiitake mushrooms and a saccharifying concentrate of shiitake mushrooms prepared by this method.

In order to solve the above-described problems, the method for preparing a saccharified concentrate of shiitake mushrooms of the present invention is to prepare a saccharified concentrate of shiitake mushrooms by saccharifying the concentrate obtained by concentrating the extract of dried shiitake mushrooms with saccharification enzyme, and then reconcentrating the saccharified product. It aims to provide a method.

In addition, another object of the present invention is to provide a method of manufacturing a shiitake mushroom saccharification concentrate powder further performing a process of lyophilizing and pulverizing the shiitake saccharification concentrate to powder.

In addition, an object of the present invention is to provide a shiitake mushroom saccharification concentrate prepared by the above shiitake mushroom saccharification concentrate manufacturing method.

In addition, an object of the present invention is to provide a shiitake mushroom saccharified concentrated powder prepared by the method for producing the shiitake mushroom saccharified concentrated powder.

In addition, an object of the present invention is to provide a health functional food comprising the shiitake mushroom saccharification concentrate and/or shiitake mushroom saccharification concentrate powder.

The shiitake mushroom saccharification concentrate and shiitake mushroom saccharification concentrate of the present invention have a very high amino acid content, particularly contain essential amino acids in a high concentration, and have excellent anti-inflammatory effects, and can be applied as various health functional foods.

1 is a schematic process diagram of a method for preparing a saccharified concentrate of shiitake mushrooms of the present invention.
Figure 2 is a schematic process diagram of a method of preparing a saccharification concentrate using the ethanol extract of shiitake mushrooms of the present invention.
3 is a measurement result of cytotoxicity for Raw 264.7 cells of a saccharification concentrate using the hot water extract of Example 1 and the ethanol extract of Example 2. FIG.
4 is an anti-inflammatory measurement result of a saccharification concentrate using the hot water extract of Example 1 and the ethanol extract of Example 2 against Raw 264.7 cells.
5 is a result of measuring cytotoxicity of Raw 264.7 cells for each of Examples 3 to 7 of the saccharification-enriched powder prepared by varying the amount of saccharification enzyme used (50 ~ 500g) when preparing the saccharification-enriched powder using ethanol extract.
6 is an anti-inflammatory measurement result of Raw 264.7 cells for each of Examples 3 to 7 of the saccharification-enriched powder prepared by varying the amount of saccharification enzyme used (50 ~ 500g) when preparing the saccharified enriched powder using ethanol extract.

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

The shiitake mushroom saccharification concentrate of the present invention can be prepared by saccharifying the concentrate obtained by concentrating the extract of dried shiitake mushrooms with saccharification enzyme as shown in the schematic process diagram in FIG. I can.

The extract may be a hot water extract or an ethanol extract.

First, a description will be given of a method of preparing a saccharification concentrate of shiitake mushrooms using hot water extract as follows.

The method for preparing a saccharified concentrate using hot water extract includes the first step of preparing dry shiitake powder; A second step of preparing a hot water extract by performing a hot water extraction process on the dried shiitake mushroom powder; A third step of preparing a first concentrate by concentrating the hot water extract under reduced pressure; 4 step of preparing a saccharified product by mixing the first concentrate and saccharification enzyme to saccharify; And a 5 step of preparing a second concentrate by concentrating the saccharified product under reduced pressure.

The dried shiitake powder is obtained by pulverizing the dried shiitake powder after drying the shiitake mushrooms, and for example, the dried shiitake mushroom powder may be filtered through 40 to 60 mesh.

The hot water extraction process is performed by mixing 1,500 to 2,500 parts by weight of water, preferably 1,800 to 2,200 parts by weight of water, and more preferably 1,900 to 2,100 parts by weight, based on 100 parts by weight of dry shiitake powder, and then at a temperature of 75 to 90°C. In, preferably, the hot water extraction process may be performed at a temperature of 75 to 85° C. for 10 to 16 hours, preferably for 10 to 14 hours, and more preferably for 11 to 13 hours.

At this time, if the amount of water used is less than 1,500 parts by weight, the shiitake powder absorbs moisture and filtration may be difficult. If the amount of water used exceeds 2,500 parts by weight, there is a problem of excessive time and cost in the concentration process due to excessive use. In addition, when the temperature during hot water extraction is less than 75°C, there may be a problem that the amino acid content in the prepared concentrate is low, and when the temperature exceeds 90°C during hot water extraction, there may be a problem that the unique flavor of shiitake mushrooms decreases. It is preferable to perform the hot water extraction process in the above temperature range.

The concentration under reduced pressure in the third step can be performed by a general method used in the art. The vacuum concentration is good to be carried out so that the concentration of the first concentrate is about 8 to 20 brix, preferably 8 to 15 brix, more preferably about 9 to 13 brix, at this time, the concentration of the first concentrate is less than 8 brix In this case, there may be a problem of deterioration in the storage process before concentration, and if it exceeds 20 brix, there may be a problem that it is difficult to control the temperature during the concentration process.

As for the glycosylation enzyme of the fourth step, at least one selected from malt, yellow soup (Aspergillus oryzae) and black soup (Aspergillus niger) may be used, and preferably malt may be used. And, the saccharification is prepared by using a saccharification enzyme 5 to 40 parts by weight, preferably a saccharification enzyme 10 to 30 parts by weight, more preferably 15 to 25 parts by weight based on 100 parts by weight of the first concentrate The content of essential amino acids in the saccharification concentrate can be maximized.

In addition, the saccharification is a mixture of the first concentrate and the saccharification enzyme for 20 to 30 hours at 55 to 65°C, preferably at 57 to 63°C for 20 to 30 hours, more preferably 58 to 62°C. It is recommended to ferment for 20 to 28 hours under the condition to obtain a saccharified product. In this case, if the saccharification temperature is less than 55°C or exceeds 65°C, there may be a problem that the essential amino acid content in the saccharified product is reduced.

And, by concentrating the saccharified product in step 4 under reduced pressure to obtain a second concentrate of 35 to 50 brix, preferably 35 to 45 brix, more preferably 37 to 43 brix, shiitake mushroom saccharification using hot water extract A concentrate can be obtained. At this time, the concentration under reduced pressure may be performed by a general method used in the art, and in a preferred embodiment, it may be performed by concentration under reduced pressure under a pressure of 900 hpa to 950 hpa.

Next, a method of preparing a saccharified concentrate of shiitake mushrooms using ethanol extract will be described.

Shiitake mushroom saccharification concentrate using ethanol extract is 1 step of preparing dry shiitake powder; After performing the ethanol extraction process on the dried shiitake mushroom powder, a second step of removing the solvent to prepare a first ethanol extract; A third step of preparing a first concentrate by concentrating the first ethanol extract under reduced pressure; 4 steps of drying the first concentrate; 5 step of preparing a saccharified product by mixing the dried first concentrate and saccharification enzyme to saccharify; And a sixth step of preparing a second concentrate by concentrating the saccharified product under reduced pressure.

The dried shiitake powder is obtained by pulverizing the dried shiitake powder after drying the shiitake mushrooms, and for example, the dried shiitake mushroom powder may be filtered through 40 to 60 mesh.

The ethanol extraction process is performed by mixing 1,200 to 2,200 parts by weight of an aqueous ethanol solution, preferably 1,400 to 2,200 parts by weight of an aqueous ethanol solution, more preferably 1,600 to 2,100 parts by weight, and reflux extraction based on 100 parts by weight of the dry shiitake powder. The ethanol extraction process can be performed.

At this time, when the amount of the aqueous ethanol solution is less than 1,200 parts by weight, there may be a problem of gelation, and when the amount of the aqueous ethanol solution exceeds 2,200 parts by weight, there is a problem of residual solvent due to an excessive amount. In addition, the aqueous ethanol solution is preferably an aqueous ethanol solution having a concentration of 70 to 95% by volume, and preferably an aqueous ethanol solution having a concentration of 75 to 95% by volume. At this time, if the concentration of the aqueous ethanol solution is less than 70% by volume, the total amino acid content in the saccharification concentrate of shiitake mushrooms may be low, and if the concentration exceeds 95% by volume, acetaldehyde may be generated.

Then, a first ethanol extract is prepared by removing an aqueous ethanol solution as a solvent from the ethanol extract obtained through the ethanol extraction process.

The concentration under reduced pressure in the third step can be carried out by a general method used in the art, and in the vacuum concentration, the concentration of the first concentrate is 8 to 20 brix, preferably 8 to 15 brix, more preferably 9 to It is good to perform so as to be about 13 brix. At this time, if the concentration of the first concentrate is less than 8 brix, there may be a problem of deterioration during storage, and if it exceeds 20 brix, there may be a problem that caramelization occurs. Then, the obtained first concentrate is dried.

The saccharification enzyme of the 5th step may be used at least one selected from malt, yellow soup, and black soup, preferably malt. And, the saccharification is prepared by using 5 to 40 parts by weight of saccharification enzyme, preferably 10 to 30 parts by weight of saccharification enzyme, and more preferably 15 to 25 parts by weight based on 100 parts by weight of the first concentrate It can maximize the content of essential amino acids in the saccharification concentrate of mushrooms.

And, the saccharification of step 5 is a mixture of the dried first concentrate and saccharification enzyme for 20 to 30 hours at 55 to 65°C, preferably at 57 to 63°C for 20 to 30 hours, more preferably It is good to ferment at 58 to 62° C. for 20 to 28 hours to obtain a saccharified product. At this time, if the saccharification temperature is less than 55° C. or exceeds 65° C., there may be a problem that the essential amino acid content in the saccharified product decreases.

And, step 6 is to obtain a second concentrate of 35 to 50 brix, preferably 35 to 45 brix, more preferably 37 to 43 brix concentration by concentrating the saccharified product in step 5 under reduced pressure again, using an ethanol extract A saccharification concentrate of shiitake mushrooms can be obtained. At this time, the concentration under reduced pressure may be performed by a general method used in the art, and in a preferred embodiment, it may be performed by concentration under reduced pressure under a pressure of 900 hpa to 950 hpa.

The present invention may further perform a process of lyophilizing and pulverizing the above-prepared shiitake saccharification concentrate to powder it to prepare shiitake mushroom saccharification concentrate powder. Explaining the method of preparing the saccharified powder of shiitake mushrooms using the ethanol extract, as shown in the schematic process diagram in Figure 2, the second concentrate obtained in step 6 is subjected to an ethanol extraction process to obtain an ethanol extract again. 7 steps to do; And an 8 step of lyophilizing and pulverizing the reacquired ethanol extract to be powdered. It can be further performed.

At this time, the ethanol extract of step 7 is mixed with an aqueous ethanol solution having a concentration of 70 to 95% by volume of the second concentrate, and preferably an aqueous ethanol solution having a concentration of 75 to 95% by volume, followed by reflux extraction to obtain the ethanol extract again. can do.

The shiitake mushroom saccharification concentrate and/or shiitake mushroom saccharification concentrate of the present invention thus prepared contains 15,500 to 20,000 mg% of total amino acids, more preferably 16,500 to 20,000 mg%, and more preferably 17,000 to 20,000 mg%. can do.

In addition, in the shiitake mushroom saccharification concentrate and/or the shiitake mushroom saccharification concentrate of the present invention, the essential constituent amino acid content relative to the total constituent amino acid content may satisfy Equation 1 below.

[Equation 1]

40% ≤ (essential amino acid content/total component amino acid content)×100% ≤ 47%, preferably 41.0% ≤ (essential component amino acid content/total component amino acid content) x 100% ≤ 44.5%, more preferably 42.0 % ≤ (essential constituent amino acid content/total constituent amino acid content)×100% ≤ 44.5%

In addition, the shiitake mushroom saccharification concentrate and/or shiitake mushroom saccharification concentrate powder of the present invention contains 1,600 to 3,000 mg% of total free amino acids, more preferably 1,800 to 2,800 mg%, and more preferably 2,000 to 2,750 mg%. I can.

In addition, the essential free amino acid content relative to the total free amino acid content of the shiitake mushroom saccharification concentrate and/or the shiitake mushroom saccharification concentrate powder of the present invention may satisfy Equation 2 below.

[Equation 2]

40% ≤ (essential free amino acid content/total free amino acid content) x 100% ≤ 62%, preferably 45.0% ≤ (essential free amino acid content/total free amino acid content) x 100% ≤ 60.0%, more preferably 50.0 % ≤ (essential free amino acid content/total free amino acid content)×100% ≤ 58.0%

The total constituent amino acids and total free amino acids are aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, and alanine. ), proline, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine.

The essential constituent amino acids and essential free amino acids may include threonine, valine, methionine, isoleucine, leucine, histidine and lysine.

As described above, the shiitake mushroom saccharification concentrate and/or the shiitake saccharification concentrate powder of the present invention containing a high content of various amino acids have excellent anti-inflammatory effects, and can be applied as ingredients such as health functional foods to produce various coagulating products. May be.

Hereinafter, the present invention will be described in more detail through examples, but the following examples do not limit the scope of the present invention, which should be interpreted to aid understanding of the present invention.

[ Example ]

Example One : Hydrothermal Shiitake mushroom through extract Saccharified Produce

1 kg of dried shiitake mushrooms were pulverized and powder passed through 50 mesh was prepared as a sample.

After mixing 1 kg of the shiitake powder and 20 kg of water, extraction was refluxed at 80° C. for 12 hours to perform hot water extraction to obtain a hot water extract.

Next, the hot water extract was concentrated under reduced pressure from 950 hpa to 900 hpa to obtain a first concentrate having a concentration of 10 brix.

Next, a mixture of the first concentrate and 200 g of malt, which is a saccharification enzyme, was fermented at 60° C. for 24 hours to prepare a saccharified product.

Next, the saccharified product was concentrated under reduced pressure from 950 hpa to 900 hpa to prepare a shiitake mushroom saccharification concentrate as a second concentrate having a concentration of 40 brix.

Example 2: Shiitake mushroom through ethanol extract Saccharified Produce

1 kg of dried shiitake mushrooms were pulverized and powder passed through 50 mesh was prepared as a sample.

After mixing 1 kg of the shiitake powder and 20 kg of an aqueous ethanol solution having a concentration of 85% by volume, extraction was refluxed at 30° C. for 12 hours to perform ethanol extraction to obtain an ethanol extract.

Next, an ethanol extract was prepared by removing an aqueous ethanol solution as a solvent from the ethanol extract.

Next, the ethanol extract was concentrated under reduced pressure at 900 hpa to obtain a first concentrate having a concentration of 10 brix.

Next, the first concentrate was freeze-dried to obtain a dried first concentrate.

Next, with respect to 100 parts by weight of the dried first concentrate, a mixture of 20 parts by weight of malt, which is a saccharifying enzyme, was fermented at 60° C. for 24 hours to prepare a saccharified product.

Next, the saccharified product was concentrated under reduced pressure at 900 hpa to prepare a shiitake mushroom saccharification concentrate as a second concentrate having a concentration of 40 brix.

Experimental example 1: Shiitake mushroom Saccharified Measurement of cytotoxic and anti-inflammatory effects

Cytotoxicity confirmation test and anti-inflammatory effect test in Raw 264.7 cells of the shiitake mushroom saccharified solution prepared in Examples 1 and 2 were performed.

(1) Cytotoxicity confirmation experiment was used in the experiment by culturing Raw 264.7 cells in a 96 well plate at a concentration of 1 × 10 ⁵ cells/mL. Then, the saccharification solutions of Examples 1 and 2, which are samples, were treated in Raw 264.7 cell culture medium at concentrations of 10, 50, 100, 500 µg/mL, and after 24 hours, the medium was removed and a new medium After 10 uL MTS was added to the mixture and reacted for 4 hours, absorbance was measured at 540 nm. The cell viability of each sample was calculated relative to the sample untreated group as 100%, and the results are shown in FIG. 3.

(2) In the experiment to confirm the anti-inflammatory effect, Raw 264.7 cells were dispensed in a 96 well plate at a concentration of 1 Х10 ⁵ cells/mL and cultured for 24 hours. Next, it was treated with the saccharification solution of Examples 1 and 2 and Lipopolysaccharide (LPS). At this time, LPS was treated at a concentration of 1 μg/mL, respectively, and each of the saccharified solutions of Examples 1 and 2, which are samples, was diluted to a concentration of 10, 50, 100, and 500 μg/mL and added to the cells.

After 24 hours, the cell culture solution was recovered and the NO production inhibitory ability was measured using a NO assay using the Griess reagent system, and the results are shown in FIG. 4.

Referring to Figure 3, when applied at a concentration of 500 μg / mL showed a cytotoxic result. In contrast, cytotoxicity was not observed at 50 to 100 μg/mL.

Referring to FIG. 4, as the saccharification solution concentration increased, the NO production inhibitory ability was significantly increased. In addition, the saccharification solution of the ethanol extract of Example 2 showed superior NO production inhibitory ability than the saccharification solution of the hot water extract of Example 1.

Through the above experiment, the saccharification solution using the hot water extract or ethanol extract of the present invention is less than 500 μg/mL concentration, preferably 50 to 400 μg/mL concentration, and has excellent NO production inhibitory ability without cytotoxicity. I could confirm.

Example 3-1: Shiitake mushroom through ethanol extract Of saccharified powder Produce

1 kg of dried shiitake mushrooms were pulverized and powder passed through 50 mesh was prepared as a sample.

After mixing 1 kg of the shiitake powder and 20 kg of an aqueous ethanol solution having a concentration of 85% by volume, extraction was refluxed at 30° C. for 12 hours to perform ethanol extraction to obtain an ethanol extract.

Next, a first ethanol extract was prepared by removing an aqueous ethanol solution as a solvent from the ethanol extract.

Next, the first ethanol extract was concentrated under reduced pressure at 950 hpa to obtain a first concentrate having a concentration of 10 brix.

Next, the first concentrate was freeze-dried to obtain a dried first concentrate.

Next, with respect to 100 parts by weight of the dried first concentrate, a mixture of 20 parts by weight of malt, which is a saccharifying enzyme, was fermented at 60° C. for 24 hours to prepare a saccharified product.

Next, the saccharified product was concentrated under reduced pressure at 950 hpa to prepare a second concentrate having a concentration of 40 brix.

Next, the second concentrate was mixed with an aqueous ethanol solution having a concentration of 90% by volume, followed by reflux extraction to prepare a second ethanol extract.

Next, the second ethanol extract was lyophilized at -40°C and then pulverized to prepare a saccharified concentrated powder of shiitake mushrooms.

Example 3-2 ~ Example 3-5

In the same manner as in Example 3-1, shiitake mushroom saccharification concentrated powder was prepared, but the amount of saccharification enzyme malt used during saccharification was varied as shown in Table 1 below to prepare shiitake mushroom saccharification concentrated powder, respectively, in Examples 3-2 to 3 Each of -5 was implemented.

division 1st concentrate (parts by weight) Glycated enzyme
(Malt, parts by weight) Example 3-1 (A) 100 5 Example 3-2 (B) 100 10 Example 3-3 (C) 100 20 Example 3-4 (D) 100 30 Example 3-5 (E) 100 50

Experimental example 2: Shiitake mushroom Of saccharified powder Measurement of cytotoxic and anti-inflammatory effects

The cytotoxicity test and anti-inflammatory effect test in Raw 264.7 cells of the shiitake mushroom saccharified powder prepared in Examples 3-1 to 3-5 were performed.

In the cytotoxicity test, Raw 264.7 cells were cultured in a 96 well plate at a concentration of 1×10 ⁵ cells/mL and used in the experiment. And, each of the saccharification-enriched powders of Examples 3-1 to 3-5, which are samples, were treated in Raw 264.7 cell culture medium at concentrations of 10, 50, 100, and 500 µg/mL, and after 24 hours, the medium was removed. Then, 10 uL MTS was added to the new medium, followed by reacting for 4 hours, and absorbance was measured at 540 nm. The cell viability of each sample was calculated relative to the sample untreated group as 100%, and the results are shown in FIG. 5 and Table 2 below.

Cell viability
(%) Addition amount of saccharified concentrated powder 10
μg/mL 50
μg/mL 100
μg/mL 500
μg/mL Example 3-1 (A) 98.4% 96.4 94.9 92.8 Example 3-2 (B) 93.5 90.2 89.1 82.4 Example 3-3 (C) 98.5 95.2 93.3 90.9 Example 3-4 (D) 95.8 91.0 88.6 86.3 Example 3-5 (E) 88.3 83.2 75.3 66.3

In the case of Example 3-5, the cell viability was 75.3% at a concentration of 100 µg/mL and 66.3% at a concentration of 500 µg/mL, showing toxicity. Unlike Examples 3-1 to 3-4, overall low Cell viability was shown. On the other hand, in the case of Examples 3-1 to 3-4, a cell viability of 86% or more, preferably 90% or more was shown, and in particular, Example 3-1 and Example 3-3 added range of the saccharified concentrated powder All of them showed a high cell viability of more than 90%.

(2) In the experiment to confirm the anti-inflammatory effect, Raw 264.7 cells were dispensed in a 96 well plate at a concentration of 1 Х10 ⁵ cells/mL and cultured for 24 hours. Next, it was treated with the concentrated saccharified powder of Examples 3-1 to 3-5 and Lipopolysaccharide (LPS). At this time, LPS was each treated at a concentration of 1 μg/mL, and each sample, each of the glycosylated concentrated powders of Examples 3-1 to 3-52, was diluted to a concentration of 10, 50, 100, and 500 μg/mL to Added.

After 24 hours, the cell culture medium was recovered and the NO production inhibitory ability was measured using a NO assay using the Griess reagent system, and the results are shown in FIG. 6.

Referring to FIG. 6, when Example 3-3(C) was treated at a concentration of 500 μg/mL, the NO production rate was 76.9%, and in the case of Example 3-5(E), 71.4 at a concentration of 500 μg/mL % NO production rate was shown, but it is considered to be caused by a decrease in cell viability due to toxicity. When Example 3-1 (A), Example 3-2 (B), and Example 3-4 (D) were treated at a concentration of 500 μg/mL, respectively, NO production rates of 94.1%, 83.1%, and 80.2% Indicated.

Experimental example 3: Shiitake mushroom Of saccharified concentrated powder Composition and free amino acid determination

The constituent amino acids and free amino acids of each of the saccharified concentrated shiitake powders of Examples 3-1 to 3-5 were measured by the following method, and the results are shown in Tables 4 and 5, respectively.

(1) Constituent amino acids

For amino acid analysis, put 0.5 g of the sample pulverized sample into a test tube, add 10 mL of 6 N-HCl, call the tip of the test tube, melt it into an ampoule, seal it, and hydrolyze it at 110°C for 24 hours in an autoclave, break the ampoule, and use a filter paper. While filtrating, dilute with 50 mL of methanol, concentrate under reduced pressure, and dilute with 5 mL of 20 mM HCl. Next, a fixed amount of the filtrate obtained by filtration through a 0.45 μm membrane filter was taken, derivatized using AccQ-Tag reagent, and analyzed by HPLC. Analysis conditions are shown in Table 3.

(2) free amino acids

Free amino acid analysis was carried out by adding 25 mg of sulfosalicylic acid to 10 mL of the filtrate obtained by the same method as for free sugar quantification, allowing it to stand at 4°C for 4 hours, and then centrifuging (50000 rpm, 30 minutes) to remove proteins, etc. , The filtrate obtained by filtering the supernatant through a 0.45 μm membrane filter was taken, derivatized using AccQ-Tag reagent, and analyzed by HPLC. Analysis conditions are shown in Table 3.

Item Analysis condition device Agilent Technologies 1200 Series Detector Agilent Technologies 1200 Series FLD column AccQ-Tag?? (Waters Co., 150 mm L×3.9 mm I.D.) Column temperature 37℃ Buffer solution
(Buffer solution)

A: AccQ-Tag Eluent A (acetate-phosphate buffer)
B: AccQ-Tag Eluent B (100% acetonitrile)
C: DW (Distilled water)

flux 1.0 mL/min Injection volume 5 μL

Configuration
amino acid
(mg%) Example
3-1 Example
3-2 Example
3-3 Example
3-4 Example
3-5 Aspartic acid 580.51±90.29 ⁴⁾ 670.70±98.64 892.32±148.92 520.07±13.62 340.45±27.42 Serine 1,129.91±171.34 1,056.33±54.64 1,248.67±98.96 1,063.44±13.67 882.62±44.40 Glutamic acid 2,494.07±385.59 2,536.40±252.17 2,819.69±344.82 2,222.43±7.12 1,479.30±80.82 Glycine 808.77±117.99 792.74±53.02 990.09±97.98 770.22±8.04 586.75±37.67 Histidine 873.64±128.45 960.89±58.55 990.02±79.49 850.22±29.26 855.83±49.90 Arginine 3,719.45±553.89 3,778.64±166.94 4,122.51±293.38 3,617.84±91.31 3,288.80±108.35 Threonine 1,101.95±168.61 1,014.08±72.09 1,298.33±122.92 1,012.61±17.23 715.53±64.71 Alanine 166.81±9.47 143.00±7.09 169.93±10.08 156.32±0.91 124.04±3.55 Proline 575.87±80.45 536.50±22.78 642.84±45.89 577.77±5.14 448.82±31.85 Tyrosine 408.41±54.14 484.48±33.10 504.48±40.50 389.57±19.04 344.39±2.08 Valine 1,352.82±203.42 1,081.68±16.25 1,211.05±74.61 1,213.95±8.07 1,173.39±40.77 Methionine 232.30±33.64 251.79±20.81 277.92±26.82 222.34±18.75 212.72±14.05 Lysine 686.51±114.36 681.97±103.77 844.24±132.74 522.47±57.86 272.57±39.42 Isoleucine 999.83±149.61 896.51±21.33 1,089.08±56.25 951.10±17.20 726.71±55.69 Leucine 1,049.79±158.54 1,069.11±96.19 1,349.35±145.10 998.12±15.60 785.04±53.51 Phenylalanine 1,050.53±143.42 1,197.54±70.60 1,248.51±103.10 1,057.02±67.88 1,131.03±20.56 TAA ^{1 )} 17,231.14±563.22 17,152.38±573.99 19,699.01±821.54 16,145.49±120.30 13,367.98±674.75 EAA ^{2 )} 7,347.35±502.05 7,153.57±459.60 8,308.48±541.01 6,827.83±31.13 5,872.80±338.60 EAA/TAA(%) ^{3 )} 42.64±0.26 41.77±0.12 42.29±0.15 42.29±0.12 43.90±0.32 ¹⁾ TAA: total amino acid
²⁾ EAA: Essential amino acid = Tyrosine + Valine + Methionine + Isoleucine + Leucine + Histidine + Lysine
³⁾ EAA/TAA (%) is measured based on Equation 1 below
[Equation 1]
(Essential amino acid content/total amino acid content)×100%
⁴⁾ Average value (±SD)

Amino acids are important nutrients that make up a living body, and must be ingested from the outside to maintain them. Among a total of 16 amino acids, the main amino acids were arginine, glutamic acid, and serine, and the total amino acid content of Example 3-3 was the highest at 19,699.01 mg%, In Example 3-1, 17,231.14 mg%, in Example 3-2, 17,152.38 mg%, in the order of high, and Example 3-5 was the lowest at 13,367.98 mg%.

The main amino acids were arginine, a basic amino acid, and glutamic acid, a type of acidic amino acid. Arginine content was 4,122.51 mg% in Example 3-3, 3,778.64 mg% in Example 3-2, 3,719.45 mg% in Example 3-1, 3,617.84 mg% in Example 3-4, and Example 3- It was highest in the order of 5 to 3,288.80 mg%.

And, the content of glutamic acid was 2,819.69 mg% in Example 3-3, 2,536.40 mg% in Example 3-2, 2,494.09 mg% in Example 3-1, 2,222.43 mg% in Example 3-4, and Examples In Example 3-5, the order was 1,479.30 mg%.

Example 3-3 using 20 parts by weight of saccharification enzyme showed the highest amino acid content of constituent amino acids according to the amount of saccharification enzyme added, and Example 3-3 also showed the highest content of arginine, the main amino acid related to .

And, EAA/TAA (%) was 40% or more, preferably 41 to 44.5%, more preferably 42 to 44.5%, and the essential amino acid content ratio was very high.

Free amino acids
(mg%) Example
3-1 Example
3-2 Example
3-3 Example
3-4 Example
3-5 Aspartic acid 53.00±2.37 ⁴⁾ 34.98±4.00 35.03±2.80 47.55±5.30 41.26±2.36 Serine 64.60±0.44 125.99±5.52 127.43±3.82 75.72±4.24 17.39±1.26 Glutamic acid 526.96±21.47 352.25±43.81 505.56±35.10 389.53±58.58 149.36±10.02 Glycine 45.39±0.46 54.97±1.92 50.96±0.92 27.88±1.42 11.74±0.74 Histidine 452.01±10.08 679.99±33.79 931.79±34.13 723.51±40.10 19.05±1.17 Arginine 67.67±2.94 207.20±4.33 167.12±7.25 127.92±4.06 48.32±2.47 Threonine 64.69±0.31 121.25±5.42 113.44±3.94 71.56±3.25 69.47±4.21 Alanine 136.98±3.33 130.30±13.44 136.19±8.01 96.63±9.72 117.27±8.64 Proline 21.06±1.51 28.84±1.49 18.62±0.08 26.95±2.09 31.17±2.75 Tyrosine 25.98±0.82 42.52±8.70 51.57±0.89 40.56±0.35 8.41±0.64 Valine 41.38±1.60 84.06±2.52 68.14±3.96 44.52±2.08 43.27±1.48 Methionine 8.08±1.06 17.90±1.17 15.04±1.19 10.98±0.80 11.71±1.27 Lysine 61.85±2.29 125.43±11.66 130.61±11.33 88.46±9.78 48.07±3.21 Isoleucine 28.18±1.75 53.30±0.50 41.80±3.02 27.94±0.24 16.08±1.24 Leucine 33.98±2.92 83.39±1.02 68.49±5.20 41.56±0.46 36.27±3.27 Phenylalanine 41.00±5.30 100.80±4.05 79.87±5.30 54.00±3.80 21.64±2.16 TAA ^{1 )} 1,672.82±1.89 2,243.16±129.86 2,541.67±126.92 1,895.29±136.16 690.48±42.19 EAA ^{2 )} 731.18±24.69 1,266.12±46.65 1,449.19±68.05 1,062.54±50.37 265.56±19.64 EAA/TAA(%) ^{3 )} 43.71±1.53 56.48±1.19 57.02±0.17 56.11±1.37 39.08±0.11 ¹⁾ TAA: total amino acid
²⁾ EAA: Essential amino acid = Tyrosine + Valine + Methionine + Isoleucine + Leucine + Histidine + Lysine
³⁾ EAA/TAA (%) is measured based on Equation 2 below
[Equation 2]
(Essential free amino acid content/total free amino acid content) × 100%
⁴⁾ Average value (±SD)

Looking at Table 5, a total of 16 kinds of free amino acids were detected in all the shiitake mushroom saccharified concentrate powders of Examples 3-1 to 3-5, and the total free amino acids were 2,541.67 mg%, the highest in Example 3-3, and implemented. It was found to be 2,243.16 mg% in Example 3-2, 1,895.29 mg% in Example 3-4, and 1,672.82 mg% in Example 3-1 in that order. And, as major amino acids, histidine and glutamic acid were the highest.

In addition, in Example 3-3 to which 20 parts by weight of a saccharification enzyme (malt) was applied, the essential amino acid content was the highest.

In addition, EAA/TAA (%) was 40% or more, preferably 45 to 60%, more preferably 50 to 58%, and the essential free amino acid content ratio was very high.

Through the above Examples and Experimental Examples, it was confirmed that the saccharification concentrate of shiitake mushrooms and the saccharification concentrate of shiitake mushrooms prepared by the method of the present invention have excellent anti-inflammatory properties and have a very high content of essential amino acids.

Claims (14)

delete Prepared by saccharifying the concentrate obtained by concentrating the extract of the dried shiitake mushroom powder with saccharification enzyme, and then reconcentrating the saccharified product,
1 step of preparing dry shiitake powder;
After mixing 1,500 to 2,500 parts by weight of water with respect to 100 parts by weight of dry shiitake powder, a second step of preparing a hot water extract by performing a hot water extraction process at a temperature of 75 to 90°C for 10 to 16 hours;
A third step of preparing a first concentrate by concentrating the hot water extract under reduced pressure;
4 steps of preparing a saccharified product by mixing and saccharifying 5 to 40 parts by weight of a saccharification enzyme based on 100 parts by weight of the first concentrate; And
5 step of preparing a second concentrate by concentrating the saccharified product under reduced pressure; It is a hot water extract prepared by performing a process including,
The first concentrate has a concentration of 8 to 20 brix, the second concentrate has a concentration of 35 to 50 brix,
The saccharification of step 4 is performed by fermenting a mixture of the first concentrate and saccharification enzyme at 55 to 65° C. for 20 to 30 hours,
The prepared shiitake mushroom saccharification concentrate contains 1,600 to 3,000 mg% of total free amino acids,
The total free amino acids are aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, and proline. proline), tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine. Manufacturing method.
Prepared by saccharifying the concentrate obtained by concentrating the extract of the dried shiitake mushroom powder with saccharification enzyme, and then reconcentrating the saccharified product,
1 step of preparing dry shiitake powder;
After mixing 1,200 to 2,200 parts by weight of an aqueous ethanol solution with respect to 100 parts by weight of dry shiitake powder, after performing the ethanol extraction process at a temperature of 25 to 30°C for 8 to 12 hours, the solvent is removed to prepare an ethanol extract. step;
A third step of preparing a first concentrate by concentrating the ethanol extract under reduced pressure;
4 steps of drying the first concentrate;
5 steps of preparing a saccharified product by mixing and saccharifying 5 to 40 parts by weight of a saccharifying enzyme based on 100 parts by weight of the dried first concentrate; And
6 step of preparing a second concentrate by concentrating the saccharified product under reduced pressure; It is an ethanol extract prepared by performing a process including,
The first concentrate has a concentration of 8 to 20 brix, the second concentrate has a concentration of 35 to 50 brix,
The saccharification of step 4 is performed by fermenting a mixture of the first concentrate and saccharification enzyme at 55 to 65° C. for 20 to 30 hours,
The prepared shiitake mushroom saccharification concentrate contains 1,600 to 3,000 mg% of total free amino acids,
The total free amino acids are aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, and proline. proline), tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine. Way.
delete delete The method of claim 2 or 3, wherein the saccharification enzyme comprises at least one selected from malt, yellow soup (Aspergillus oryzae), and black soup (Aspergillus niger), and the saccharification is 20 to 30 at 55 to 65°C. Method for producing a saccharified concentrate of shiitake mushrooms, characterized in that carried out for a period of time.
delete delete A method for producing a saccharified concentrated shiitake powder, characterized in that the second concentrate of claim 2 or 3 is freeze-dried and pulverized to powder.
The second concentrate of claim 3 is subjected to an ethanol extraction process to obtain an ethanol extract again, and then freeze-dried and pulverized to form a powder.
Shiitake mushroom saccharification concentrate, characterized in that prepared by the method of claim 2 or 3.
Shiitake mushroom saccharified concentrated powder, characterized in that produced by the method of claim 9.
The method of claim 12, wherein the essential constituent amino acid content relative to the total constituent amino acid content satisfies Equation 1 below,
The essential constituent amino acids include threonine, valine, methionine, isoleucine, leucine, histidine and lysine,
The total amino acids are aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, and proline. proline), tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine;
[Equation 1]
40% ≤ (essential constituent amino acid content/total constituent amino acid content)×100% ≤47%
The method of claim 12, wherein the essential free amino acid content relative to the total free amino acid content satisfies Equation 2 below,
The essential free amino acids include threonine, valine, methionine, isoleucine, leucine, histidine and lysine,
The total free amino acids are aspartic acid, serine, glutamic acid, glycine, histidine, arginine, threonine, alanine, and proline. proline), tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine;
[Equation 2]
45% ≤ (essential free amino acid content/total free amino acid content) × 100% ≤ 62%

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