KR101435580B1 - Method for Producing Bacterial Cellulose Using Gluconacetobacter sp. KCG326 Strain Using Optimal Carbon Source and Nitrogen Source - Google Patents

Abstract

The present invention discloses a method for producing bacterial cellulose using a strain of KCG326 in the presence of a gluconacetobacter. Specifically, the present invention discloses a method for producing bacterial cellulose which can increase bacterial cellulose production yield by using a sucrose as a carbon source and a yeast extract or a soybean as a nitrogen source by using a strain of KCG326 in a glucone acetobacterium.

Description

TECHNICAL FIELD The present invention relates to a method for producing bacterial cellulose by using KCG326 strain in a glucone acetobacterium using an optimal carbon source and nitrogen source. KCG326 Strain Using Optimal Carbon Source and Nitrogen Source}

The present invention relates to a method for producing bacterial cellulose by using Gluconacetobacter sp. KCG326 strain (KACC 91555P) using an optimal carbon source and nitrogen source.

Recently, a lot of attention has been focused on the production of natural polysaccharides, and these polysaccharides are called biopolymers or biodegradable polymers. Among these polymers, bacterial cellulose derived from a microorganism has been found to be bound to β-D-glucopyranose by β-1,4-glucoside (β-1,4-glucoside) The long chains are in the form of ribbine-like bundles, which are held in hydrogen bonds, and show structural differences from plant-derived cellulose, which is in the form of bundles of microfibrils.

Unlike plant-derived cellulose, bacterial cellulose does not contain other ingredients besides cellulose, so it is easy to separate and purify. In other words, the plant has a crystalline structure including not only cellulose but also hemicellulose or lignin. In the case of bacterial cellulase, however, since there are no other impurities besides the cellulose produced by the bacteria, And purification is easy. Bacterial-derived cellulose is the only natural cellulose that can be obtained on a film, and its properties have attracted attention, and various studies have been conducted as useful materials.

At present, strains producing celluloses include Acetobacter sp . , Acetic acid bacteria), Agrobacterium sp . ), Rhizobium sp . ), Genus Pseudomonas sp . ), Sarcina ( Sarcina sp . ), And in particular, Acetobacter sp . ), ≪ / RTI > Acetobacter xylium , A. pasteurinanus , and Acetobacter hansenii are well known. Recently, 16S rRNA base sequence has been used to classify the cell systematically. Cell strains that produce high purity cellulose are morphologically characterized as intestinal bacterium and strong acid-resistant bacteria, which secrete cellulose from the cells .

Since acetic acid bacteria produced bacterial cellulose by Brown in 1886, acetic acid bacteria has been the subject of major research because of its excellent ability to produce cellulose. Bacterial cellulose has a higher degree of crystallization than cellulose produced by general plants, and has excellent physical properties such as mechanical strength, adsorption, water retention, suspension stability, and binding property, and practical application of materials in various fields such as food additives, industrial materials and medical materials It is progressing. Therefore, studies are underway to increase productivity by developing strains with high cellulose yield in political culture, developing strains suitable for aeration culture, and establishing effective culture conditions.

Strains producing bacterial celluloses usually use glucose, sucrose, molasses, fructose, mannitol, glycerol or the like as a carbon source during growth, and bacterium and persimmon 75, 1993, 18-22), casein, peptone, peptone, and the like as a supplementary carbon source (S. Masaoka et al., J. Ferment Bioeng. Yeast extract is used as a nitrogen source (KV Ramana et al., World J. Microbial Biotechnol., 16, 2000, 245-248; YK Yang et al., J. Ferment. 312-317). The optimum carbon source, auxiliary carbon source and nitrogen source are different depending on the strain.

It is not only costly to establish optimal culturing conditions by choosing the optimal one among various kinds of carbon source, supplementary carbon source and nitrogen source. It is also important to control the yield lowering due to lack of nutrients, byproducts It is important because it can improve the yield through the inhibition of accumulation. Korean Patent Laid-Open Publication No. 2003-0088596 discloses a condition in which the yield is improved by using 0.5% glucose and 1.5% fructose as a carbon source at the time of culturing the strain KJ of Acetobacter jirium, Korean Patent Publication No. 1997-0001555 Discloses a culture method of producing cellulose by using fruit juice containing acetic acid or 20% glucose in Acetobacter jirium ATCC 53582 strain. European Patent No. 279506 also discloses a method of increasing the yield by limiting the concentration of carbon sources according to the growth stage.

The present invention also relates to cultivation of optimal carbon source, nitrogen source and the like which can increase production yield of bacterial cellulose when producing bacterial cellulose by using KCG326 (accession number: KACC 91555P) in the genus of gluconacetobacter, which is confirmed to have the ability to produce bacterial cellulose Condition.

It is an object of the present invention to provide a method for producing bacterial cellulose by glucone acetobacterium KCG326.

Other and further objects of the present invention will be described below.

The inventors of the present invention have found that, in the production of bacterial cellulase using the strain KCG326 in the genus Acacobacterium, sucrose, glucose, lactose, fructose, maltose and mannitol as a carbon source, The yield of bacterial cellulose was examined at a concentration of 10% (w / w) in a medium consisting of yeast extract 1% (w / w) and purified water. The yield of bacterial cellulose in sucrose was 27.2 g / m ² The yield was high. These experimental results are different from those in which a common carbon source in bacterial cellulosic production is known as glucose (Masaoka S, et al., J. Fermen. Bioeng., 75, 18-22, 1993) It can be said that the most suitable carbon source when producing bacterial cellulose using KCG326 strain is sucrose.

The KCG326 strain in Gluconacetobacter is a strain isolated from the fermented product of black tea mushroom by the RDA and deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, under accession number KACC 91555P.

Further, when the production yield of the bacterial cellulose was confirmed by varying the concentration of the medium containing sucrose having the highest production yield of the bacterial cellulose on the basis of the above-mentioned experimental results, the inventors found that the bacterial cellulose production amount at the addition concentration of 9% (w / 33.0 g / m < ² >

Furthermore, the inventors of the present invention have found that a yeast extract, polypeptone, tryptone, malt extract, corn streep liquor and soytone can be used to identify the most suitable nitrogen source for the production of bacterial cellulase by the strain KCG326 The yield of bacterial cellulose was 40.0 g / m ² in yeast extract treatment and 33.8 g / m ² in soytone treatment. The yield of bacterial cellulose was 0.1% (w / w) in the medium consisting of sucrose 9% (w / when g / m ² was the number of check indicate, the process to the optimum of 0.05% to determine the concentration (w / w) to 0.45% (w / w) range of these nitrogen sources, in the case of yeast extract, 0.1% ( in the process the concentration of w / w) and in the case of soy tone was confirmed respectively show the production yield of 40.0g / m ^2, and 43.2g / m ² at a concentration of 0.20.

The present invention provides a method for producing bacterial cellulose using the strain KCG326 of the present invention comprising the steps of: (a) preparing a culture medium for bacterial cellulose containing a carbon source and a nitrogen source; b) inoculating the culture medium with a strain of KCG326 in the presence of gluconacetobacter, (c) culturing under aerobic conditions after inoculation, and (d) obtaining bacterial cellulose from the culture medium, wherein the bacterial cellulose The preparation medium is characterized in that it contains sucrose as a carbon source and at least one of yeast extract and soybean as a nitrogen source.

As used herein, "yeast extract" refers to the genus Saccharomyces spp . ) Means a degradation product obtained by chemical decomposition after mechanical or chemical destruction of yeast cells. Specifically, it refers to a hydrolyzate of acid hydrolyzate of yeast cells or a hydrolyzate obtained by autolysis of endogenous enzymes of yeast after mechanical or chemical destruction of yeast cells. To increase the endogenous enzyme activity, an exogenous enzyme such as protease, peptidase or nuclease is added The yeast extract is meant to include a yeast degradation product obtained by adding such an exogenous enzyme. The degradation products obtained by autolysis are usually pasteurized and concentrated after cell debris is removed. The yeast extract generally contains salts such as amino acids, proteins, peptides, vitamins, carbohydrates, nucleotides, phosphoric acid and the like. Specific details concerning the production of yeast extracts are disclosed in Kelly, M. (1982) Yeast Extract Industrial Enzymology, Godfrey, T. ed.), Chae, HJ et al. (2001), Bioresource Technology 76: 253-258). Preferably refers to a powdery decomposition product obtained by autolysis of endogenous enzyme of yeast after the destruction of yeast cells used in the following examples.

In the present specification, "soiton" refers to a decomposition product obtained after decomposition of defatted soybean meal, which is a by-product of soybean oil, remaining as pancreatic enzymes or pancreatic juice, Refers to a decomposition product in powder form obtained by concentrating the degradation product used in the following examples. Pancreatic enzymes include lipase such as lipase, protease such as trypsin, and sugar-enzyme such as? -Amylase, and pancreatic juice is mostly composed of pancreatic enzyme.

In this specification, "% (w / w)" means a concentration (w / w) expressed in terms of weight percents, Weight solute. For example, 10% (w / w) means that 10 g of the solute is dissolved in 100 g of the solution. Therefore, in the method of the present invention, when sucrose is added to the medium in an amount of 9% (w / w), 91 wt% (w / w) of other medium components such as purified water and 9 wt% Can be understood as meaning.

In the method of the present invention, it is preferable that the culture medium for producing bacterial cellulose in step (a) is prepared by containing sucrose in an amount of 9% (w / w) for the reasons described above.

In the method of the present invention, when the culture medium for producing bacterial cellulose in step (a) contains only yeast extract as a nitrogen source, it is preferably contained in an amount of 0.1% (w / w) .

In the method of the present invention, it is preferable that the culture medium for the production of bacterial cellulose in step (a) contains 0.20% (w / w) of soybean as a nitrogen source.

In the method of the present invention, the culture medium for bacterial cellulase production in step (a) is preferably a medium having a pH in the range of 3 to 7, particularly 5 to 6, in view of the fact that the microorganism to be inoculated is a strain of KCG326 in the glucone acetobacterium .

In addition, in the method of the present invention, the culture medium for producing bacterial cellulose in step (a) may further comprise an auxiliary carbon source. The auxiliary carbon source is not essential for the growth of bacterial cellulosic producing microorganism KCG326 or the production of bacterial cellulose by KCG326 strain of glucone acetobacterium. However, the auxiliary carbon source is not essential for bacterial cellulosic producing microorganism KCG326 of Gluconacetobacter Citric acid, acetic acid, ethanol, formic acid, malic acid, skin acid, and the like may be used as a component added to promote the production of bacterial cellulose by growth and / or KCG326 strain in the glucone acetobacterium, Further, it has been found that a juice which originally contains at least one of these components or at least one of these components is artificially added (Kim SY, et al., Appl Biochem Biotechnol., 129-132, 705-15, 2006), persimmon SY, et al., Appl Biochem Biotechnol., 129-132, 705-15, 2006), apple juice (Lee OS, et al., J. Kor. Soc. 12 (5), 722-728, 2002), beer wastewater (CJ, et al., J. Appl. Microbiol. Biotechnol., 12 (5), 722-728, (Jonas R, et al., Polym Degrad Stab., 59, 101-106, 1998), and coconut by-products (Park JK, et al., Kor Chem Eng Res., 44 (1), 52-57, 2006) Pineapple juice (see Japanese Patent Application No. 1999-299034), and the like.

In the method of the present invention, the culture medium for bacterial cellulose production in step (a) may further comprise inorganic salts and trace elements. Examples of the inorganic salts that can be used include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybdate salt and chelate metal salt. Examples of trace salts include amino acids, vitamins, fatty acids, Sulfite pulp waste liquid, and the like. These inorganic salts and trace elements may be used in admixture of one or more species and may be included in the medium in the range of 0.001% (w / w) to 3.0% (w / w).

In the method of the present invention, after the culture medium for producing bacterial cellulose is prepared in the step (a), a sterilization step may be further included. This sterilization step is intended to prevent contamination of other microorganisms, and as a result, to increase bacterial cellulose production yield. As the sterilization method, any method known in the art can be used such as a high-temperature and high-pressure sterilization method, ultraviolet irradiation, and the like.

In the method of the present invention, it is preferable that the strain KCG326 in the step (b) is inoculated after pre-culture for activation thereof. The following examples can be referred to for the preferable medium components in the pre-culture, the composition of the components, the pre-culture conditions, the pre-culture period, the inoculum amount of the pre-culture solution and the like.

In the method of the present invention, the culturing temperature in step (c) is preferably in the range of 10 to 40 ° C, more preferably 25 to 35 ° C, and the culturing period is preferably 5 to 20 days.

 In the culture of step (c) of the present invention, it is preferable that the oxygen concentration is in the range of 1 to 100% (w / w), particularly 20 to 80% (w / w) for making an aerobic condition.

In the culturing in the step (c) of the present invention, the culture method may be either a static culture method or a continuous culture method, and preferably a static culture method may be used.

The present invention relates to a bacterial cellulose obtained by the above-mentioned process for producing a bacterial cellulase. The bacterial cellulose of the present invention is produced by a strain of KCG326 in the genus Aconitobacterium. As in the case of general bio-cellulose, the bacterial cellulose of the present invention can be used as a drug for drug delivery such as burn treatment, wound treatment, ulcer treatment, inflammation treatment, antibiotics and anesthetic, A cosmetic product, a foodstuff for enhancing a texture, and a paper for a filler application.

As described above, according to the present invention, it is possible to provide a method for producing bacterial cellulose using a strain of KCG326 in the genus Gluconacetobacter. According to the present invention, sucrose as a carbon source and yeast extract or a soybean as a nitrogen source have an effect of increasing bacterial cellulose production yield by a strain of KCG326 in a glucone acetobacterium.

FIG. 1 is a graph showing the yield according to the concentration of sucrose added as a carbon source in the production of bacterial cellulose by the strain KCG326 in the presence of glucone acetobacterium.

Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these embodiments.

< Example  1> Carbonic  Depending on the type Gluconacetobacter sp . Production yield of bacterial cellulose of KCG326

The type of carbon source is Gluconacetobacter sp. The effect of KCG326 on the yield of bacterial cellulose was examined.

The basal medium for the preservation and preincubation of the strains was the 272 medium (10% (w / w) glucose, 1% (w / w) yeast extract, 2% w / w CaCO ₃ ), 1.5% (w / w) of agar, and purified water, pH 6.8).

For pre-culture, 200 ml of 272-medium containing SPL Incu Tissue for plant tissue culture was inoculated with a platinum strain preserved in a plate agar medium and incubated at 27 ° C for 48 hours.

The culture medium for the bacterial cellulase production was prepared from 10% (w / w) of various carbon sources, 1% (w / w) of yeast extract and purified water of the remaining amount as shown in Table 1 below. In this culture, 1% (w / w) of the preculture was inoculated on a tray containing 1,300 ml of the present culture, and the mixture was incubated at 27 ° C for 14 days.

The amount of bacterial cellulose produced was examined as follows. The resulting bacterial cellulose was recovered and sufficiently washed with water, then immersed in 0.5 N NaOH solution and treated at 90 ° C for 2 hours to dissolve the cells. These were washed with distilled water until neutral, dried at 105 ° C until the weight became constant, and the dry weight was measured. Absorbance was measured at 660 nm using a UV-visible spectrophotometer (Ultrospec 4000, Pharmacia Biotech, England). The yield of bacterial cellulose is expressed in g / m &lt; ^{2 &} gt ;. The experiment was repeated twice and the result was the average value.

The production yield of bacterial cellulose according to various carbon sources is shown in Table 1 below.

Production yield of bacterial cellulose according to the type of carbon source Carbon source Production of bacterial cellulose (g / m ² ) Sucrose 27.2 Glucose 23.0 Lactose 3.0 Fructose 22.1 Maltose 2.6 Mannitol 2.0

Referring to the results of Table 1 above, it can be seen that the amount of bacterial cellulose produced in the medium supplemented with sucrose is the highest at 27.2 g / m ² , which indicates that the common carbon source for bacterial cellulose production is glucose (Masaoka S, et al., J. Fermen. Bioeng., 75, 18-22, 1993).

< Example  2> Sucroose  Depending on treatment concentration Gluconacetobacter sp . Production yield of bacterial cellulose of KCG326

In Example 1, sucrose was added to Gluconacetobacter sp. As it was confirmed that KCG326 is the most suitable carbon source for the production of bacterial cellulose, the treatment concentration of sucrose in the medium is lower than that of Gluconacetobacter sp. The effect of KCG326 on bacterial cellulosic yield was investigated.

In this experiment, the medium for the present culture was prepared from sucrose 1% (w / w) to 10% (w / w), yeast extract 1% (w / w) and remaining amount of purified water. The remaining pre-culture conditions, pre-culture period, amount of inoculation of the pre-culture medium into the present culture medium, the present culture conditions, the period of the present culture, and the method for measuring the amount of bacterial cellulose produced are the same as those in Example 1 above.

The results are shown in Fig. Referring to FIG. 1, when sucrose was added at 9% (w / w), the yield of the bacterial cellulose was the highest, and the yield was 33.0 g / m ² .

< Example  3> Depending on the type of nitrogen source Gluconacetobacter sp . Production yield of bacterial cellulose of KCG326

Depending on the type of nitrogen source, Gluconacetobacter sp. It was tested whether the production yield of bacterial cellulose of KCG326 was changed.

In this experiment, the medium used for the present cultivation was sucrose 9% (w / w), the various nitrogen sources 0.1% (w / w) shown in Table 2 below, and the remaining amount of purified water. The remaining pre-culture conditions, pre-culture period, amount of inoculation of the pre-culture medium into the present culture medium, the present culture conditions, the period of the present culture, and the method for measuring the amount of bacterial cellulose produced are the same as those in Example 1 above.

The results are shown in Table 2 below.

Production yield of bacterial cellulose by type of nitrogen source Nitrogen source Production of bacterial cellulose (g / m ² ) Yeast extract 40.0 Polypeptone 12.0 Tryptone 17.6 Malt extract 7.6 Corn streep liquor 22.0 Soytone 33.8 None 1.5

Table 2: references in look, yeast extract in the highest, and then the soy tone (soytone) is 33.8g / m ² to yield when a 40.0g / m ² is used as a nitrogen source High.

< Example  4> Yeast extract or Soiton  Depending on treatment concentration Gluconacetobacter  sp. Production yield of bacterial cellulose of KCG326

In Example 3, yeast extract and soytone were mixed with Gluconacetobacter sp. It was confirmed that KCG326 was the most suitable nitrogen source for the production of bacterial cellulose, so that the treatment concentration of yeast extract or soytone in the medium of Gluconacetobacter sp. The effect of KCG326 on bacterial cellulosic yield was investigated.

In this experiment, the medium used for the present culture was a medium prepared from sucrose 9% (w / w), yeast extract or soy protein 0.05% (w / w) to 0.45% (w / . The remaining pre-culture conditions, pre-culture period, amount of inoculation of the pre-culture medium into the present culture medium, the present culture conditions, the period of the present culture, and the method for measuring the amount of bacterial cellulose produced are the same as those in Example 1 above.

The results are shown in [Table 3] and [Table 4].

Production yield of bacterial cellulose according to treatment concentration of yeast extract Treatment concentration of yeast extract Production of bacterial cellulose (g / m ² ) 0.05 35.8 0.10 40.0 0.15 38.4 0.20 37.2 0.25 37.0 0.30 36.6 0.35 36.2 0.40 35.6 0.45 35.0

Production yield of bacterial cellulose according to treatment concentration of soytone Soitone treatment concentration Production of bacterial cellulose (g / m ² ) 0.05 24.5 0.10 33.8 0.15 38.6 0.20 43.2 0.25 37.4 0.30 34.3 0.35 31.4 0.40 27.6 0.45 23.5

As shown in Table 3 and Table 4, when the yeast extract was added at 0.1% (w / w), the yield was the highest at 40.0 g / m ^{2. In} the case of soytone, the yield was 0.20% (w / w) was the highest at 43.2 g / m ² .

Claims (7)

(a) preparing a medium for bacterial cellulose production containing sucrose 8% (w / w) to 10% (w / w) as a carbon source and 0.10% (w / w) to 0.30% ,
(b) inoculating this medium with a strain of KCG326 in the presence of a gluconacetobacter,
(c) culturing in an aerobic condition after inoculation, and
(d) obtaining bacterial cellulose from the culture. The method for producing bacterial cellulose using the strain KCG326 in the presence of glucone acetobacterium.
delete delete delete The method according to claim 1,
Wherein the culture medium for the production of bacterial cellulose in step (a) further comprises at least one of an auxiliary carbon source, inorganic salts and trace elements.
The method according to claim 1,
Wherein the culturing step of step (c) is performed by culturing for 5 to 20 days at a culturing temperature in the range of 10 to 40 占 폚.
delete

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