KR100415283B1 - Method of producing cla and mushroom mycelium culture containing same - Google Patents

Abstract

The present invention relates to a method for producing CLA by culturing mushrooms, and to mushroom mycelium and culture containing the CLA produced thereby, and a method of using the same, the method comprising liquid culturing mushroom strains in a medium containing linolenic acid According to the method comprising a can effectively produce CLA (conjugated linoleic acid) having the effect of strong anticancer, immune enhancement, body fat reduction, mushroom mycelium and culture containing the CLA produced in this way Since the function of CLA is added to the physiological activity of the mycelium and the culture medium to exert a synergistic effect, it can be added to foods and the like and used for the production of functional foods.

Description

CLA production method and mushroom mycelium and culture containing same {METHOD OF PRODUCING CLA AND MUSHROOM MYCELIUM CULTURE CONTAINING SAME}

The present invention relates to a method for producing CLA by culturing mushrooms, mushroom mycelium and culture containing the CLA produced thereby, and a method of using the same.

To date, CLA (conjugated linoleic acid) is linolenic acid (linoleic acid) or chemically synthesized by the alkaline isomerization method from fat, or non-containing them. Ruminant microorganisms such as B. fibrisolvens, or L. It is produced by microorganisms such as L. reuteri . L. L. reuteri is a microorganism that produces CLA as a useful intestinal bacterium and has been used to produce fermentation products such as yogurt.

CLA synthesized in a large amount by chemical methods is used for health supplements and the like. In addition, CLA synthesized by microorganisms derived from the rumen is transferred to meat or milk and contained in a small amount in these products. L. CLA synthesized by L. reuteri is used to produce products such as milk and yogurt.

On the other hand, mushroom mycelium contains a polysaccharide such as β-D-glucan or has a physiological activity such as anti-cancer, immuno-enhancing, etc. due to a special material possessed by the mushroom mycelium itself. The mushroom mycelium culture medium produces various substances depending on the type of mushroom bacteria or the composition of the culture medium.

An object of the present invention is to provide a method for producing CLA with high efficiency by a method of culturing mushroom mycelium which is a higher fungus which has not been known as a CLA production method up to now.

Another object of the present invention is to provide a mushroom mycelium and culture containing CLA, and to provide a method of using the same for functional foods.

1 is a GC analysis chart of fatty acids extracted from a culture solution obtained by the liquid culture of mushroom mycelium according to the present invention,

2 is an MS analysis chart of c9, t11 CLA contained in a culture obtained by liquid culture of mushroom mycelium according to the present invention;

3 is a liquid culture of mushroom mycelium according to the present invention . GC analysis chart showing the effect of L. reuteri on c9, t11 CLA production.

CLA production method of the present invention for achieving the above object, characterized in that it comprises the step of liquid culture of the mushroom strain in a medium containing linolenic acid.

Here, the culture medium containing linoleic acid is preferred, including soybean and soybean meal hydrolyzate or enzyme, vegetable oil hydrolyzate, and El. It is more preferable to add L. reuteri and incubate for 1-7 days at 25-35 ° C. under shaking or aeration.

In addition, the present invention provides a mushroom mycelium and culture containing the CLA prepared by the above method and a method for producing a functional food by adding it to food.

In the present invention, CLA was produced by culturing mushroom bacteria in a specially prepared culture solution in a liquid culture method, and mushroom mycelium and culture containing the CLA thus produced could be prepared. At this time, L. mushrooms . The addition of L. reuteri is preferred because it can increase the amount of natural CLA produced.

The CLA contained in the mushroom mycelium and the culture thus produced may have a synergistic effect on the functionality of the mushroom mycelium or the mushroom mycelium solution or impart various physiological activities of the CLA. In other words, the mushroom mycelium contains a polysaccharide such as β-D-glucan or has a physiological activity such as anticancer activity or immunity enhancement due to a special substance possessed by the mushroom mycelium itself. Depending on the various materials are produced. If the CLA produced by the culture is included here, the function of CLA is added to the physiological activity of the mushroom mycelium, thereby enabling a synergistic effect.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only examples of the present invention, and the present invention is not limited thereto. Each example was repeated to confirm the reproducibility of the results.

Example 1 Composition and Culture of Basic Mushroom Mycelia Culture Solution

The basal medium and culture conditions used to test the production of CLA by liquid culture of mushroom mycelium are as follows.

① Mushroom Strains: Pleurotus ostreatus (PO) and 8 other species

② Medium composition: As the linolenic acid source, soybean and soybean meal containing a large amount of linolenic acid were soaked in water and sufficiently ground by a mixer. Enzymes, such as protease, cellulase, and lipase, were processed here (60 degreeC, 200 rpm, 1-5 hours). In the case of adding red seed oil or the like as a linolenic acid source, an edible oil hydrolyzate obtained by hydrolyzing the cooking oil with alkali was used. To this was added sulfur white sugar, MgSO ₄ , KH ₂ PO ₄ and adjusted to 1 L with distilled water. This was sterilized and used as basal medium.

③ Cultivation conditions: incubation for 1-7 days by shaking or aeration at 25 ~ 35 ℃.

Example 2 Identification and Quantification of CLA Produced by Mushroom Mycelia Culture

In order to investigate the presence of CLA in the cultured mycelium and the culture medium in Example 1, the mushroom mycelium was first recovered, followed by trituration with chloroform: methanol (2: 1, v / v) to extract fatty acids, methylation, and CLA with GC. Was analyzed.

1 is a GC analysis chart of fatty acids extracted from a culture solution obtained by the liquid culture of mushroom mycelium according to the present invention.

The peak was identified by MS on the GC chart and found to be CLA.

Figure 2 can be confirmed that the CLA in the MS analysis chart of the fatty acid extracted from the culture solution obtained by the liquid culture of the mushroom mycelium according to the present invention.

[Example 3] CLA production amount change according to the concentration of soybean meal

The concentration of soybean meal in the basal medium of Example 1 was cultured (25 ° C., 7 days, aeration), and the amount of CLA was measured by the method of Example 2.

Table 1 shows the effect of soybean meal content on the production of CLA, the unit is mg CLA / g fat. In the control group, no CLA was detected, and the concentration of CLA increased with the concentration of soybean meal. If the concentration of soybean meal was higher than 6%, no increase in CLA concentration was observed.

Mushroom Soybean meal concentration (%, w / v) 0.1 0.7 1.5 3.0 6.0 10.0 c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount Leaves mycelium 4.3 0.7 5 5.7 1.4 7.1 6.0 1.7 7.7 6.7 2.1 8.8 6.9 2.0 8.9 6.3 2.4 8.7 Culture 57.4 49.7 107.1 68.3 56.3 124.6 70.6 58.8 129.4 72.1 60.9 133 71.9 61.0 132.9 70.7 61.5 132.2 Cordyceps sinensis mycelium 6.7 1.5 8.2 10.9 3.0 13.9 11.7 3.5 15.2 13.0 4.2 17.2 12.8 3.9 16.7 12.1 3.5 15.6 Culture 14.1 8.2 22.3 19.4 11.9 31.3 20.4 12.7 33.1 23.0 13.5 36.5 24.0 12.7 36.7 23.5 11.5 35 spring and autumn mycelium 1.9 0.9 2.8 3.8 1.5 5.3 4.1 1.8 5.9 5.2 1.9 7.1 5.5 2.0 7.5 5.4 1.9 7.3 Culture 7.0 6.3 13.3 10.5 9.6 20.1 11.4 10.5 21.9 13.4 10.8 24.2 13.8 11.1 24.9 12.6 10.1 22.7 Sazooka mycelium 3.0 2.7 5.7 5.7 5.1 10.8 6.1 5.5 11.6 7.4 7.0 14.4 7.3 6.8 14.1 7.5 6.7 14.2 Culture 36.2 27.9 64.1 37.6 36.1 73.7 45.7 40.5 86.2 65.7 50.3 116 60.0 51.5 111.5 55.8 56.1 111.9 circle mycelium 0.7 0.1 0.8 1.4 0.1 1.5 1.7 0.1 1.8 2.1 0.1 2.2 2.0 0.2 2.2 2.1 0.1 2.2 Culture 3.0 1.0 4 5.2 2.8 8 5.8 3.1 8.9 7.3 3.8 11.1 7.2 3.7 10.9 7.8 2.8 10.6 Spirit mycelium 2.9 2.1 5 4.9 3.0 7.9 5.8 3.2 9 6.1 3.1 9.2 6.2 3.8 10 6.3 2.9 9.2 Culture 7.8 7.2 15 42.0 24.1 66.1 55.1 37.9 93 59.3 53.8 113.1 52.3 64.7 117 60.4 63.3 123.7 Elevation mycelium 2.3 1.1 3.4 4.4 2.1 6.5 5.5 2.9 8.4 6.4 4.5 10.9 7.2 3.6 10.8 8.3 3.5 11.8 Culture 21.0 20.8 41.8 50.2 45.7 95.9 67.8 65.4 133.2 78.9 65.4 144.3 70.1 67.2 137.3 80.5 78.6 159.1 situation mycelium 1.7 0.0 1.7 2.5 0.0 2.5 3.1 0.0 3.1 3.6 0.5 4.1 3.6 0.0 3.6 3.4 0.0 3.4 Culture 23.1 22.8 45.9 25.5 24.4 49.9 33.8 36.3 70.1 45.0 40.4 85.4 45.5 44.1 89.6 46.2 45.3 91.5 wisdom mycelium 11.8 18.7 30.5 15.0 24.1 39.1 16.8 25.2 42 18.0 26.9 44.9 18.1 27.1 45.2 18.9 26.0 44.9 Culture 10.9 7.1 18 15.1 10.9 26 16.0 11.8 27.8 16.7 12.4 29.1 16.6 12.3 28.9 16.3 12.1 28.4

[Example 4] CLA production amount according to the culture period and air inflow method

The concentration of soybean meal in the basal medium of Example 1 was fixed at 6.0%, and the content of CLA produced by varying the incubation period and aeration method at 25 ° C was investigated.

Table 2 shows the effect of the incubation period on the production of CLA, a culture in aeration, Table 3 shows the effect of the aeration method on the production of CLA, the result of incubation for 7 days. The unit is both mg CLA / g fat. In the conditions of the CLA production is the highest in 7 days of culture, and the aeration method in the aeration method, it can be seen that the amount of CLA production compared to the shaking culture.

Mushroom Incubation period (days) 0 3 7 10 c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount Leaves mycelium 0 0 0 3.2 0.9 4.1 6.3 1.9 8.2 7.7 2.1 9.8 Culture 0 0 0 36.7 29.7 66.4 70.6 59.4 130 82.3 61.9 144.2 Cordyceps sinensis mycelium 0 0 0 6.3 1.8 8.1 11.7 3.9 15.6 13.0 4.2 17.2 Culture 0 0 0 11.5 5.6 17.1 21.7 11.7 33.4 23.0 13.5 36.5 spring and autumn mycelium 0 0 0 2.9 0.5 3.4 4.8 1.0 5.8 5.2 1.9 7.1 Culture 0 0 0 7.2 4.7 11.9 12.2 9.2 21.4 53.4 8.8 62.2 Sazooka mycelium 0 0 0 4.7 2.0 6.7 9.5 4.7 14.2 10.4 5.0 15.4 Culture 0 0 0 20.4 15.9 36.3 67.3 38.7 106 61.5 52.0 113.5 circle mycelium 0 0 0 1.0 0.1 1.1 2.0 0.1 2.1 3.1 0.1 2.1 Culture 0 0 0 3.2 1.6 4.8 6.9 3.2 10.1 7.3 3.8 11.1 Spirit mycelium 0 0 0 3.6 1.3 4.9 6.2 2.7 8.9 6.1 4.3 10.4 Culture 0 0 0 34.5 23.2 57.7 56.9 53.2 110.1 49.1 57.3 106.4 Elevation mycelium 0 0 0 3.9 2.2 6.1 5.9 4.1 10 6.4 1.5 7.9 Culture 0 0 0 44.7 33.6 78.3 70.7 73.4 144.1 75.2 81.9 157.1 situation mycelium 0 0 0 1.7 1.8 3.5 3.0 1.1 4.1 3.2 3.5 6.7 Culture 0 0 0 14.3 14.2 28.5 46.1 40.4 86.5 49.8 37.6 87.4 wisdom mycelium 0 0 0 8.8 8.1 16.9 24.5 17.6 42.1 39.0 6.9 45.9 Culture 0 0 0 6.8 4.7 11.5 15.6 11.5 27.1 16.7 16.4 33.1

Mushroom Aeration Method Aeration concussion c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount Leaves mycelium 6.9 2.0 8.9 5.1 0.9 6 Culture 71.9 61.0 132.9 41.1 37.1 78.2 Cordyceps sinensis mycelium 12.8 3.9 16.7 9.2 2.3 11.5 Culture 24.0 12.7 36.7 17.3 10.2 27.5 spring and autumn mycelium 5.5 2.0 7.5 3.6 1.3 4.9 Culture 13.8 11.1 24.9 7.5 6.1 13.6 Sazooka mycelium 7.3 6.8 14.1 7.3 4.1 11.4 Culture 60.0 51.5 111.5 44.3 43.1 87.4 circle mycelium 2.0 0.2 2.2 1.6 0.1 1.7 Culture 7.2 3.7 10.9 5.2 1.7 6.9 Spirit mycelium 6.2 3.8 10 7.1 1.6 8.7 Culture 52.3 64.7 117 34.6 33.7 68.3 Elevation mycelium 7.2 3.6 10.8 7.4 2.4 9.8 Culture 70.1 67.2 137.3 55.7 53.8 109.5 situation mycelium 3.6 0.5 4.1 2.7 0.1 2.8 Culture 45.5 44.1 89.6 24.9 33.8 58.7 wisdom mycelium 18.1 27.1 45.2 12.4 18.5 30.9 Culture 16.6 12.3 28.9 13.5 10.0 23.5

Example 5 L. L. reuteri Changes in CLA Production with Addition

Based on the results obtained in Example 4, namely the result of incubation for 7 days while aeration in a basal medium in which the concentration of soybean meal was adjusted to 6.0% (1) basal + L. L. reuteri and (2) Basic + L. After culturing for 2 days at 37 ℃ with L. reuteri + MRS medium (0.1%), the amount of CLA production was investigated.

Table 4 shows L. The effect of L. reuteri on CLA production in mushroom mycelium was expressed in mg CLA / g fat. Here you see El. Terry flow was when the handle (L. reuteri) is CLA production increase than the basic group, in El. In the case of L. reuteri culture, the amount of CLA was higher when MRS was added.

3 is a liquid culture of mushroom mycelium according to the present invention . It shows the effect of L. reuteri on CLA production.

Mushroom Treatment Details basic Basic + L. reuteri Basic + L. reuteri + MRS Badge c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount c9, t11 t10, c12 Total amount Leaves mycelium 6.9 2.0 8.9 9.4 2.7 12.1 12.4 3.1 15.5 Culture 71.9 61.0 132.9 97.2 61.2 158.4 117.2 68.3 185.5 Cordyceps sinensis mycelium 12.8 3.9 16.7 17.6 6.7 24.3 19.3 7.4 26.7 Culture 24.0 12.7 36.7 28.2 15.4 43.6 29.1 16.2 45.3 spring and autumn mycelium 5.5 2.0 7.5 15.1 3.7 18.8 19.2 5.2 24.4 Culture 13.8 11.1 24.9 21.8 12.7 34.5 26.3 14.9 41.2 Sazooka mycelium 7.3 6.8 14.1 11.6 8.2 19.8 19.6 10.1 29.7 Culture 60.0 51.5 111.5 89.2 61.4 150.6 101.4 62.3 163.7 circle mycelium 2.0 0.2 2.2 4.2 0.3 4.5 9.9 0.6 10.5 Culture 7.2 3.7 10.9 13.4 3.7 17.1 19.4 4.1 23.5 Spirit mycelium 6.2 3.8 10 12.2 4.3 16.5 20.3 5.2 25.5 Culture 52.3 64.7 117 83.5 74.7 158.2 102.8 79.3 182.1 Elevation mycelium 7.2 3.6 10.8 14.7 5.3 20 21.9 6.4 28.3 Culture 70.1 67.2 137.3 93.9 69.7 163.6 112.1 76.8 188.9 situation mycelium 3.6 0.5 4.1 9.7 1.6 11.3 14.8 3.1 17.9 Culture 45.5 44.1 89.6 72.7 45.9 118.6 102.4 36.5 138.9 wisdom mycelium 18.1 27.1 45.2 26.5 25.9 52.4 35.6 27.1 62.7 Culture 16.6 12.3 28.9 28.3 12.6 40.9 35.1 14.8 49.9

Example 6 Preparation of CLA-containing culture extract (CLA-culture extract) and CLA-containing culture extract extract (CLA-extract)

The whole liquid culture of Examples 3 and 5 was sterilized and extracted at high temperature and high pressure to obtain a CLA-culture extract. This extract was concentrated under high pressure vacuum to give a concentrated CLA-extract.

Example 7 Preparation of CLA-containing Mycelium Concentrate (CLA-mycelium Concentrate) and CLA-containing Mycelium Powder (CLA-Mycelium Powder)

CLA-mushroom mycelium can be obtained by centrifugation or filtration in Examples 3 or 5 or as a residue remaining after high pressure extraction in Example 6.

The mycelium was powdered as it was, or the mycelium was treated with Novozyme 234 (manufactured by Novo Corporation) (48 hours at 60 ° C., 200 rpm shaking) to be centrifuged or filtered to obtain a CLA-mycelium filtrate and a residue. The filtrate was concentrated in vacuo to give a CLA-mycelia concentrate. In addition, the concentrated solution was vacuum lyophilized or hot air dried to obtain CLA-mycelium powder.

Example 8 Use of CLA-Culture Extract, CLA-Extract, CLA-Mycelium Concentrate and CLA-Mycelium Powder as Food Ingredients

Extracts, extracts, concentrates and powders obtained in Examples 6 and 7 were added at concentrations of 0.1 to 5% by weight, respectively, to prepare products such as functional biscuits, cakes, gums, candy, cookies, etc. containing CLA-mushroom mycelium or concentrate. .

In addition, extracts, extracts, concentrates and powders were each added at a concentration of 0.05 to 100% by weight to prepare functional drinks containing CLA-mushroom mycelium or concentrate according to a conventional beverage preparation method.

Example 9 Preparation of Capsules, Granules, and Tablets Using CLA-Culture Extract, CLA-Extract, CLA-Mycelium Concentrate and CLA-Powder

Extracts, extracts, concentrates and powders obtained in Examples 6 and 7 were added at a concentration of 0.1 to 100% by weight to prepare functional capsules, granules, tablets and the like containing CLA-mushroom mycelium or concentrate according to a conventional production method.

As described above, according to the present invention, CLA (conjugated linoleic acid) having effects such as strong anticancer, immune enhancement, and body fat reduction can be efficiently produced by a mushroom culture method.

Mushroom mycelium and culture containing the CLA produced as described above can be used in the production of functional food by adding to food and the like, because the CLA function is added to the physiological activity of the mushroom mycelium can exhibit a synergistic effect.

Claims (6)

CLA production method comprising the step of liquid culture of the mushroom strain in a medium containing linolenic acid. The method of claim 1 wherein the medium comprising linolenic acid comprises an enzymatic digest of soybean and soybean meal or an edible oil hydrolyzate. The method of claim 1 or 2, wherein L. Culture method by adding L. reuteri . The method according to claim 1 or 2, wherein the method is incubated for 1-7 days at 25-35 ° C. under shaking or aeration. delete delete