KR100666055B1 - Producing process for water soluble beta-glucan - Google Patents

Abstract

The present invention is a method for producing a water-soluble beta glucan by inoculating the Aureobasidium flulan seed in the medium, Aureobasidium flulans strains 20 ~ 25 ~ 35 ℃ in grain byproduct liquid medium based on bran Incubate for 72 hours, inoculate the enriched Aureobasidium flulans strain into a solid medium based on bran, ferment the solid medium for 20 to 72 hours at 20 to 40 ° C, and soluble in grain by-product solid medium. It relates to a method for preparing beta glucan.

The method of the present invention has the effect of obtaining a high concentration of water-soluble beta glucan by the solid phase culture method using a low-cost medium.

Solid State Fermentation, Water Soluble Beta Glucan, Grain By-Product Medium, Solid Medium, Fermentation

Description

Producing process for water soluble beta-glucan

The present invention relates to a method for producing a high concentration of water-soluble beta glucan by culturing and fermenting Aureobasidium pullulans (ATCC No.42023) in a grain by-product medium based on bran. Specifically, the aureobasidium flulans strain is cultured in a liquid medium made of grain by-products such as bran, powder, rice bran, and the like to be enriched in large quantities. It relates to a method for producing a water-soluble beta glucan.

Many functional polysaccharides of nature are known to exist. Glucan, a type of polysaccharide present in nature, is present in yeast cell walls, mushrooms, and grains, and is divided into α- or β-type according to the molecular structure. Beta glucan is divided into beta (1-3) glucan, beta (1-4) glucan, beta (1-6) glucan and the like depending on the shape of the sugar. Among the beta glucans, beta (1-3) glucan has an effect of strengthening immunity, and among these, beta (1-3) main chain has a beta (1-6) defect in a double-chain structure that is water-soluble. . Water-soluble beta glucan is contained in agaricus, ganoderma lucidum, and fingering mushrooms and is known to have pharmacological action such as antitumor activity. It is also produced by mushroom mycelium or by microbial fermentation in the aurebacidium.
Beta-glucan is classified into water-soluble and water-insoluble according to the bonding structure. In case of beta (1-3) or beta (1-4) monostructure, insoluble or beta (1-3) is mixed with beta (1-6) bond, and is water-soluble. 121 ℃, 30 minutes Stable under heating conditions In addition, due to different absorption in vivo, immune-related effects are superior to insoluble beta glucan than insoluble beta glucan. Among them, the water-soluble beta glucan produced by mushrooms is known to have excellent antitumor activity and antibacterial activity (Seljelid, et al: Association of macrophage Activation with Anti-tumor Activity by Synthetic and Biologic Agents. Cancer Res; 1989) , 37: 3338-3343) It is known to have a variety of pharmacological effects, such as hypoglycemic action, hypotension action, attracting attention as medicines, cosmetics, dietary supplements and feed additives.
Conventionally, beta glucan is extracted from mushroom cells or yeast, or from grains such as oats, barley, rye, or grains thereof.
Extraction from mushrooms is a method of culturing mushroom spawn for 1-6 months to extract beta glucan and then producing beta glucan using yeast to isolate and remove beta glucan in yeast cell wall by culturing yeast in liquid state. The method of purifying, and the method of producing beta glucan by using the grain by-product is to classify the grain by-product to extract and purify the beta glucan. This technology produces beta glucan through solid phase fermentation using aureobasidium flulans as a medium composed of grain by-products.
The method of producing beta glucan from mushrooms has the advantage of high productivity (20-30%) and water solubility, so that the immune boosting effect is high, but the production cost is high because the culture period of spawn is 1 to 6 months, and the liquid culture method using yeast Silver has a short shelf life of 3 to 4 days, but has low usability, low water solubility of beta glucan, and beta glucan separated and purified from grain by-products.

Aureobasidium flulan strain is a strain that is used to produce pullulan, which is used as a food additive, and is approved by the US Food and Drug Administration (FDA) for food manufacturing (GRAS Standard Title 21, vol 3). .

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However, the method of obtaining beta glucan by inoculating aureobasidium flulans strain in a liquid medium composed of grain by-products and fermenting the liquid medium shows that the content of water-soluble beta glucan is low because it does not produce more than 2% by weight of the medium composition. There are disadvantages.

Therefore, in this field, in order to industrially utilize beta glucan, it is necessary to develop a method for producing beta glucan at low cost and with high efficiency.

It is an object of the present invention to provide a method for preparing water-soluble beta glucan at low cost and high efficiency.
The inventors of the present invention using aureusiobacidium flulans strain (ATCC No.42023) having the characteristic of secreting beta-glucan out of the cell, and in a liquid medium composed of low-cost grain by-products such as bran, powder, rice bran, oxime By enriching the Aureciobacidis flulan strain and inoculating the culture obtained by inoculation into a solid medium to obtain a culture having a water content of at least 2% by weight of water-soluble beta glucan, it is dried to obtain a water-soluble beta glucan at low cost and high efficiency. The present invention was completed by confirming that it can be obtained.

The present invention is a solid medium composed of grain by-products based on wheat bran to grow Aureobasidium flurans cells in a liquid medium composed of grain dispersion based on bran. It relates to a method for producing a high concentration of water-soluble beta glucan by fermentation.
The present invention is characterized in that beta-glucan is produced by inoculating a solid medium with Aureobasidium flulans, a test strain that secretes water-soluble beta-glucan extracellularly.

The present invention is characterized by using a solid phase fermentation method consisting of a liquid culture and a solid culture to produce a water-soluble beta glucan using the Aureobasidium flulan strain.
The present inventors have confirmed that there is a limit in increasing the content of beta glucan as a liquid culture method, and considered a culture method in a solid medium. However, the solid phase medium composed of grain by-products are mixed with various materials such as spores and shells of grains, so that it is difficult to completely sterilize, and thus it is not easy to grow and propagate spawn. For this reason, the conventional method of culturing beta glucan in a solid medium has not been used.
The present inventors pay attention to the fact that in the liquid culture method using the conventional aureobasidium strain, beta glucan is also possible in parallel with the production of the aureobasidium strain in the present invention, using the liquid medium for strain growth in the present invention and the liquid medium Inoculation and fermentation of aureobasidium strains grown in a specific population above in a solid medium was confirmed that can produce a high concentration of water-soluble beta glucan.

In the present invention, the liquid culture step is to multiply the number of cells of aureobasidium flulans in a relatively inexpensive liquid medium, and the solid culture step is a nutrient suitable for producing the water-soluble beta glucan by the aureobasidium flulans. There is provided a solid medium composition which is not high in raw material cost, which can be added to food, cosmetics, pharmaceuticals, and feed.

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

The present invention uses the Aureobasidium flulans strain, which secretes water-soluble beta-glucan extracellularly, produces low-cost, high-concentration, and difficult to industrialize solid culture conditions for the establishment of an immune enhancing effect, antitumor activity, antibacterial. This is to use beta glucan products with various pharmacological effects such as activity, skin regeneration effect, hypoglycemic action, blood pressure lowering action as raw materials for medicine, cosmetics, dietary supplement and feed additives.

The present inventors have endeavored to create a culture environment that satisfies the above conditions. As a result, the cultivation step is divided into liquid culture, solid culture, and drying stages. By setting it to commercialization, it is possible to produce a water-soluble beta glucan of 2% or more by weight in a solid medium composed of grain by-products, thus completing the present invention.

The liquid culture medium of the present invention contains nutrients for the growth of Aureobasidium flulans, and the unit cost is not high, and the medium raw material which can be used directly in food, cosmetics, medicine, feed, bran, powder, rice bran, etc. It is selected as a grain by-product of the mixture and mixed with groundwater in a total amount of 5% to 30% of the liquid medium in a single or mixed state, and the mixed liquid medium is sterilized at 121 ° C. for 30 minutes to 60 minutes in a culture tank. Aseptic seedlings were inoculated into the nutrient medium and incubated at 25 ° C. to 35 ° C. for 20 to 72 hours to show the same amount of cell growth as the medium commercially available.

The solid culture of the present invention is 50% to 70% of acid (HCl, 10ppm) of a single or a mixture of bran, powder, rice bran, octane with nutrient-containing, low-cost, edible conditions of the strains grown in the liquid culture The water containing the inoculated 30% to 60% inoculated medium at 1 to 2 hours at 100 ℃ to 110 ℃ to increase the incubation at 20 ℃ to 40 ℃, 20 hours to 72 hours. Subsequent incubation periods maintain sufficient conditions for Aureobasidium flulans to produce water-soluble beta glucan.

The drying process of the present invention is dried 12 hours to 20 hours by hot air of 60 ℃ to 80 ℃ through a 30-mesh crushing process to facilitate drying the solid medium containing aureobasidium flulans after the culture is completed It is a process to complete.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following examples and experimental examples.

Experimental Example 1
<Enrichment test of Aureobasidium flulan strain>
In cultivation of microorganisms, generally, sterilized liquid medium is mainly used.
The present inventors carried out this enrichment test to determine whether the aureobasidium strain can be grown in a solid medium medium to a probable concentration. The population of aureobasidium strains that can stick on solid media should be at least 10 ⁸ per ml.
As the experimental medium, those that are traded under the trade names YM broth, NB broth, Czapeck broth, and potato broth are mainly used.
Y-Mbros is composed of 0.3wt% yeast extract per 10 liters of water, malt extract 0.3wt%, peptone 0.5wt%, textose 1wt%, envibros is 0.3wt% beef extract, peptone 0.5wt% per liter of water, Zapekkbros is composed of 2.5wt% NaNO ₃ , 0.1wt% K ₂ HPO ₄ , 0.05wt% KCl, 0.05wt% MgSO ₄ , 0.0001wt% FeSO ₄ , 0.5wt% peptone, 3wt per liter of water. It is composed of%, potato broth is composed of 0.4wt% of starch per 1 liter of water, 2wt% of dextrose, it is sterilized for 15 minutes at 121 ℃ each used as a liquid medium.
To compare the culture performance of aureobasidium flulans in liquid byproducts of grain by-products, 900 g of water was mixed with 100 g of a mixture of 50% by weight of bran, 25% by weight of powder, and 25% by weight of rice bran, The liquid medium was prepared and their culture performance was compared with those of YEMBROS, ENVIBROS, ZAPECBROS, and potato broths, and the results are shown in the following [Table 1].
In Table 1, Y-Mbros is a yeast extract 0.3wt%, malt extract 0.3wt%, peptone 0.5wt%, dextrose 1wt% and the rest is a liquid medium composed of water, Envibros is 0.3kg% peptone 0.5 wt% and the rest is a liquid medium composed of water, Rapekkbros is 2.5wt% NaNO ₃ , 0.1wt% K ₂ HPO ₄ , KCl 0.65wt%, MgSO ₄ 0.05wt%, FeSO ₄ 0.001wt%, Peptone 0.5wt %, Glucose 3.0wt% and the remainder is a liquid medium composed of water, PDIDOS is 0.4wt% starch, dextrose 2wt% and the remainder is a liquid medium composed of water.
In case of artificial medium such as YEMBRO, sterilization of grain by-product medium for 15 minutes at 121 ℃ and 30 minutes at 121 ℃, inoculated with Aureobasidium flulans spawn and incubated for 48 hours, followed by Aureobasidium flulans spawn The population of was investigated and shown in the following [Table 1].
As shown in Table 1, the largest number of Aureobasidium flulan strains was found in the grain by-product medium.
It was found that efficient culture of aureobasidium flulans was possible even in a low-cost grain by-product medium without using a commercially available medium.

The components of the liquid medium were YM broth (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, dextrose 1%), enbi broth (0.3% beep extract, peptone 0.5%), japek broth (NaNO ₃ 2.5%, K ₂ HPO ₄ 0.1%, KCl 0.05%, MgSO ₄ 0.05%, FeSO ₄ 0.0001%, peptone 0.5%, Glucose 3%), potato broth (potato starch 0.4%, dextrose 2%) , By varying the composition of grain by-product medium mixed with rice bran, 121 ℃, 15 minutes for artificial medium in a 5L fermenter, high temperature sterilization of 121 ℃, 30 minutes to 60 minutes in a by-product medium, Aureobasidium influenza Lances were inoculated and liquid cultured for 12 to 72 hours.
<Proliferation strain identification test>
YM agar medium (YM agar, yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, dextrose 1%, agar 2%, YM broth with agar added), en-agar medium (beef extract 0.3% , peptone 0.5%, agar 2%), zapek-agar medium (NaNO ₃ 2.5%, K ₂ HPO ₄ 0.1%, KCl 0.05%, MgSO ₄ 0.05%, FeSO ₄ 0.0001%, peptone 0.5%, Glucose 3%, agar 2%), potato-agar medium (potato starch 0.4%, dextrose 2%, agar 2%), grain by-products (grain by-product with 50% bran, agar 2%) obtained in the above experiment After plating the culture solution 0.1ml, and confirmed the population growth of the seed after 48 hours, the grain by-product medium mixed with bran, powder, and rice bran was almost the same or higher than that of the artificial medium Aureobasidium flulans.

Badge composition YM NB Czapeck Potato Bran, powder, rice bran Population of Aureobasidium flulans {A. pullulans cfu / ml} 2.1 × 10 ⁸ 1.8 × 10 ⁸ 2.4 × 10 ⁸ 2.0 × 10 ⁸ 2.5 × 10 ⁸
As a result of the experiment, it was confirmed that the aurevacidial flulan strain can be cultured in a solid medium in a grain by-product liquid medium consisting of bran, powder, and rice bran.

Example
<Production of Liquid Medium>

Cereal by-products (grain by-products with wheat bran at 50%) agar %) After 48 hours, the population growth of the seed was confirmed, and the maximum cell mass was shown from 20 hours to 72 hours.
Incubation time (hours) 12 20 36 48 72 86 A. pullulans cfu / ml 1.3 × 10 ⁸ 2.0 × 10 ⁸ 1.9 × 10 ⁸ 2.0 × 10 ⁸ 2.1 × 10 ⁸ 1.8 × 10 ⁸

Incubation time (hours) 12 24 36 48 72 86 A. pullulans cfu / ml 1.3 × 108 2.0 × 108 1.9 × 108 2.0 × 108 2.1 × 108 1.8 × 108

<Liquid culture temperature>

The culture temperature of the Aureobasidium flulans was changed to 20 ° C. to 40 ° C. in a grain by-product liquid medium subjected to high temperature and high pressure sterilization at 121 ° C. for 30 to 60 minutes, followed by incubation for 20 hours to 72 hours. 50% of the grain by-product, agar 2%) was provoked after 48 hours to confirm the population growth of the seed showed a maximum cell mass at a temperature from 25 ℃ to 35 ℃.
Culture temperature (℃) 20 25 30 35 40 A. pullulans cfu / ml 1.3 × 10 ⁸ 2.2 × 10 ⁸ 2.1 × 10 ⁸ 2.1 × 10 ⁸ 1.1 × 10 ⁸

Culture temperature (℃) 20 25 30 35 40 A. pullulans cfu / ml 1.3 × 108 2.2 × 108 2.1 × 108 2.1 × 108 1.1 × 108

<The composition of the solid medium>

The composition of the solid medium is 50% by weight of bran, and the amount of water is 10% to 80% by varying the composition of powder, rice bran, and oxime, and the medium is increased at 100 ° C to 110 ° C for 1 hour to 2 hours. Inoculated with spawn by 10% to 70% cultured at 25 ℃ to 35 ℃, 20 hours to 72 hours in a grain by-product liquid medium subjected to high temperature and high pressure sterilization for minutes to 60 minutes fermented at 15 ℃ to 50 ℃, 12 hours to 84 hours Results The highest yield of water-soluble beta glucan was found in medium cultured with bran, powder, rice bran, and oxime.
Badge composition One 2 3 4 5 6 7 Beta Glucan Content (%) 1.8 1.8 1.7 2.0 1.9 2.0 2.2
※ Medium composition 1: 100wt% bran
Medium composition 2: bran 50wt%, powder 50wt%
Medium composition 3: bran 50wt%, rice bran 50wt%
Medium composition 4: Bran 50wt%, Oxy 50wt%
Medium composition 5: bran 50wt%, powder 25wt%, rice bran 25wt%
Medium composition 6: bran 50wt%, powder 25wt%, octave 25wt%
Medium composition 7: Bran 50wt%, powder 16.7wt%, rice bran 16.7wt%, octave 16.6wt%

ingredient bran Bran powder Bran Rice Bran Bran Octopus Bran Powder Rice Bran Bran Powder Octopus Bran Powder Rice Bran Octopus Beta Glucan Content (%) 1.8 1.8 1.7 2.0 1.9 2.0 2.2

<Water content of solid medium>

25 ℃ to 35 ℃ in the grain by-product liquid medium subjected to high-temperature and high-pressure sterilization for 30 minutes to 60 minutes in a medium which was increased from 1% to 2 hours at 100 ° C to 110 ° C by varying the water content of the solid medium from 10% to 80%. 10% to 70% seedlings incubated for 20 hours to 72 hours were fermented at 15 ° C to 50 ° C for 12 hours to 84 hours. As a result, the production capacity of water-soluble beta glucan in solid medium containing 50% to 70% of water was increased. High.

Moisture content (%) 10 20 30 40 50 60 70 80 Beta Glucan Content (%) 0.6 2.1 2.0 2.0 2.5 2.5 2.3 1.7

<Fermentation Temperature of Solid Medium>

Grain by-products subjected to high-temperature and high-pressure sterilization for 30 minutes to 60 minutes in a medium in which the fermentation temperature of the solid medium was increased from 15 ° C. to 55 ° C. and the water content was 50 to 70% for 1 hour to 2 hours at 100 ° C. to 110 ° C. As a result of incubating 12% to 84 hours by inoculating 10% to 70% of the spawn grown in a liquid medium at 25 ° C. to 35 ° C. for 20 to 72 hours, beta glucan production capacity was increased at 20 ° C. to 40 ° C. The highest was confirmed.
Culture temperature (℃) 15 20 35 40 55 Beta Glucan Content (%) 0.5 2.4 2.1 1.2 0.8

Culture temperature (℃) 15 25 35 45 55 Beta Glucan Content (%) 0.5 2.4 2.1 1.2 0.8

<Starting vaccination concentration in solid medium>

High-pressure sterilization was carried out for 30 minutes to 60 minutes in medium in which the amount of water was inoculated in the solid medium to 10% to 70%, and the water content was increased from 50 to 70% and 100 to 110 ° C for 1 to 2 hours. Inoculated with 10% to 70% of the seed culture incubated at 25 ℃ to 35 ℃, 20 hours to 72 hours in the grain by-product liquid medium, incubated for 20 hours to 40 ℃, 12 hours to 84 hours to confirm that the water-soluble beta glucan The highest production capacity was between 30% and 60% spawn inoculation.

Liquid medium supply (%) 10 20 30 40 50 60 70 Beta Glucan Content (%) 2.0 2.0 2.6 2.5 2.4 2.2 2.1

<Fermentation of Solid Medium>

In the grain by-product liquid medium subjected to high-temperature and high-pressure sterilization for 30 minutes to 60 minutes in a medium in which the amount of water was increased from 50 to 70% and from 1 to 2 hours at 100 to 110 ° C by varying the solid fermentation time from 12 to 84 hours. Inoculated with 10% to 70% spawn incubated at 25 ℃ to 35 ℃, 20 hours to 72 hours and incubated at 20 ℃ to 40 ℃ as a result of high beta glucan production capacity in the fermentation for 20 hours to 72 hours Confirmed.

Incubation time (hours) 12 24 36 48 60 72 84 Beta Glucan Content (%) 0.6 2.5 2.9 2.8 2.8 2.7 2.3

Experimental Example 1. Cell count analysis test of Aureobasidium flulans

In an embodiment of the present invention was tested as follows to determine the propagated population of the Aureobasidium flulans strain.

The liquid medium used was incubated for 25 minutes to 35 ℃ for 12 hours to 72 hours with a grain by-product medium mixed with YM broth, Envy broth, Zapek broth, potato broth and commercial bran and bran, powdered rice and rice bran Each culture solution is diluted eight times by collecting 1 ml of the solution aseptically and diluting it in 9 ml of distilled water sterilized at 121 ° C. for 15 minutes, taking 1 ml of the diluted solution and diluting it in 9 ml of sterile distilled water. This was again YM agar (with 2wt% agar added to YM broth), NB-agar, Zapek agar (with 2wt% agar added to Zapek broth), potato agar (0.1% of agar was added to the potato broth), 0.1 ml of the culture solution diluted to 10 ⁸ was added to the grain by-product (to which 2 wt% of the agar was added to the grain by-product liquid medium). Incubate for 48 hours at the temperature of ℃ to check the number of spawns, 0.1 ml of each culture solution diluted to 10 ⁸ in a grain by-product (grain by-product with a 50% bran, agar 2%) in a flat medium for comparison Smear and incubate for 48 hours to compare population numbers.

Experimental Example 2. Beta-glucan content test

In order to determine the water-soluble beta glucan content in the culture prepared in the embodiment of the present invention was tested as follows.

1 g of the culture of each example is suspended in phosphate buffer (potasium phosphate buffer, pH 6) and centrifuged at 10,000 rpm for 10 minutes. 1 ml of the supernatant was mixed with 1 ml of 0.2% pancreatin enzyme solution and reacted at 37 ° C. for 1 hour, and the reaction solution was mixed with 1.5 ml of ethanol and left at 8 ° C. for 12 hours at 4 ° C. Discard the supernatant by centrifuging again at 12,000 rpm for 10 minutes, and completely pellet the pellet in 0.05 M phosphate buffer, and then treat 1 ml 0.1% laminariase (an enzyme that breaks down beta-glucan into sugars) to 40 ° C. The substrate was allowed to react until the substrate was decomposed, and the reducing sugar was quantified by the finished sample.

Enzymes and substrates used in the experiment were purchased from Sigma, USA.

Experimental results showed that the solid phase culture had a water-soluble beta glucan content higher than the liquid culture, and the solid phase culture was higher than the liquid single culture.

The method of the present invention has the effect of obtaining a high concentration of water-soluble beta glucan using a low-cost medium.

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Claims (3)

Inoculating Aureobasidium flulan seed in a medium to produce a water-soluble beta glucan, incubating the aureobasidium flulan strain for 20 to 72 hours at 25-35 ° C. in a grain by-product liquid medium based on bran Inoculate the enriched Aureobasidium flulans strain into a solid medium based on bran, and ferment the solid medium at 20-40 ° C. for 20-72 hours to produce high concentration of water-soluble beta glucan in the grain by-product solid medium. How to manufacture. The method of claim 1, Grain by-product, liquid medium is made by mixing 80 to 90% by weight of water to 10-20% by weight of a mixture of 50% by weight of wheat bran and 50% by weight of a mixture of powder and rice bran. High concentration water-soluble betaglucan in grain by-product solid medium which is made by mixing 50-70% by weight of water to 30-70% by weight of 50% by weight of wheat bran, 50% by weight of powdered rice, rice bran and oxime uniformly. How to prepare. delete