JP2003339392A - Method for producing bacterial cellulose - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing bacterial cellulose, capable of efficiently producing the bacterial cellulose by using glucose of low cost as a source of carbon. <P>SOLUTION: This method for method the bacterial cellulose includes culturing acetobacter in the presence of the glucose, wherein a glucose dehydrogenase gene of the acetobacter is disrupted. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing bacterial cellulose represented by Nata de Coco.

[0002]

2. Description of the Related Art Cellulose fibers produced by bacteria (hereinafter referred to as "bacterial cellulose") are cellulose produced by some bacteria (bacteria such as Acetobacter, agrobacterium, rhizobium and the like), and D-glucose is β- It is a fibrous polymer having a linear chain structure with 1,4 bonds. Bacterial cellulose has a network structure of extremely fine fibers, which is 1/100 to 1/1000 that of plant cellulose, and therefore has high elasticity, high water retention and emulsion stabilizing effects, and is useful as a new industrial material.

For example, Nata de Coco is a typical product using bacterial cellulose. Nata de Coco is produced by crushing the flesh of coconut using acetic acid bacteria, filtering it to obtain coconut milk, and fermenting this with water, sugar, acetic acid, and the like.

Bacterial cellulose is used as a material for high-performance speakers in addition to Nata de Coco because of its high elasticity, high water retention and emulsion stabilizing action.

Conventionally, acetic acid bacteria have produced bacterial cellulose using fructose as a main carbon source. When glucose (glucose) is used as a carbon source, the amount of bacterial cellulose is about 1/4 of that of fructose. Not produced and production efficiency is very poor. It is considered that this is because the acetic acid bacterium converts glucose into an organic acid such as gluconic acid by the action of the extracellular enzyme and cannot take glucose into the body.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing bacterial cellulose using inexpensive glucose as a carbon source.

[0007]

As described above, when acetic acid bacteria are cultured with glucose as the main carbon source, most of glucose is oxidized to gluconic acid outside the cells by the action of glucose dehydrogenase, Only the remaining glucose is used for bacterial cellulose production.
The present inventors predicted that more glucose could be utilized as a substrate for bacterial cellulose by blocking the oxidation reaction of glucose to gluconic acid, and produced an acetic acid bacterium that disrupted the glucose dehydrogenase gene. . Then, they found that bacterial cellulose can be efficiently produced by culturing this glucose dehydrogenase gene-disrupted strain in the presence of glucose, and completed the present invention.

That is, according to the present invention, there is provided a method for producing bacterial cellulose, which comprises culturing an acetic acid bacterium in which the glucose dehydrogenase gene has been destroyed in the presence of glucose.

Preferably, the acetic acid bacterium is Gluconacetobacter
xylinus. Preferably, an acetic acid bacterium in which the glucose dehydrogenase gene has been disrupted by incorporating the chloramphenicol acetyltransferase gene in the glucose dehydrogenase gene sequence can be used. Preferably, the culture can be performed in the presence of glucose and ethanol. Preferably, ethanol can be added sequentially.

According to another aspect of the present invention, using the method for producing bacterial cellulose of the present invention as described above,
A method of making de coco is provided.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below. In the present invention, bacterial cellulose is produced by culturing an acetic acid bacterium in which the glucose dehydrogenase gene has been destroyed in the presence of glucose.

The type of acetic acid bacterium used in the present invention is not particularly limited as long as it can produce bacterial cellulose. For example, Acetobacter xylinum ( Acet
bacterium xylinum ), acetobacter pasteurianus ( A. pasteurianus ) and other acetic acid bacteria (genus Acetobacter)
Can be used. Preferably, Gluconacetobac
ter xylinus can be used. More specifically, Gluconacetobacter xylinus BPR2001 strain is JCM9730
Easily available as ^T.

In the acetic acid bacterium used in the present invention, the glucose dehydrogenase gene is destroyed. Destruction of the gene can be performed using a gene recombination technique known to those skilled in the art. For example, the glucose dehydrogenase gene can be disrupted by incorporating the chloramphenicol acetyltransferase gene within the sequence of the glucose dehydrogenase gene.

Destruction of the glucose dehydrogenase gene of acetic acid bacterium (hereinafter referred to as "gdh gene") can be carried out, for example, by deleting the gdh gene by homologous recombination of DNA. Thus, g of acetic acid bacteria
Examples of the method of incorporating another gene into the dh gene include a method of incorporating a gene encoding chloramphenicol acetyltransferase (hereinafter referred to as “cat gene”).

The method for incorporating the chloramphenicol acetyltransferase gene into the gdh gene is as follows:
It can be performed by a gene recombination technique known to those skilled in the art. For example, after cleaving the DNA of acetic acid bacterium with a restriction enzyme, a partial region containing the gdh gene (8
72 bp) is isolated and the DNA fragment is PCR amplified and cloned.

Then, ca is added to the DNA fragment obtained above.
Incorporates the t gene. The method for introducing another gene into a DNA fragment is also known to those skilled in the art. For example, the inside of the gene sequence in the DNA fragment is cleaved with a restriction enzyme, and another gene (for example, the cat gene) to be introduced is ligated to the cleaved site using a ligase to obtain a DNA.
Another gene can be incorporated into the fragment. The DNA fragment in which the cat gene is incorporated into the gdh gene by the above method is incorporated into a plasmid vector. The obtained recombinant plasmid vector is introduced into cells of acetic acid bacterium by a known method such as electroporation. As a result, homologous recombination occurs and c
An improved acetic acid bacterium having an inactivated gdh gene in which the at gene is integrated can be produced.

As described above, the acetic acid bacterium in which the glucose dehydrogenase gene has been disrupted can be grown only in the acetic acid bacterium having the DNA into which the cat gene has been introduced by culturing in a medium containing chloramphenicol. Thus, only acetic acid bacteria in which the glucose dehydrogenase gene has been destroyed can be selectively collected.

In the method of the present invention, the above-prepared glucose dehydrogenase gene-disrupted acetic acid bacterium is cultured in the presence of glucose, and bacterial cellulose accumulated in the medium is recovered. The culture of the acetic acid bacterium itself can be performed by a method known to those skilled in the art, as described below.

In the present invention, glucose is used as a carbon source for producing bacterial cellulose. However, this carbon source does not have to consist of glucose alone,
It may be a mixture with the following other sugars, for example, fructose, sugar, mannose and the like. It is also possible to add starch hydrolyzate, citrus molasses, beet molasses, beet juice, sugar cane juice, fruit juice, etc. containing the above-mentioned carbon source to glucose for use.

In culturing, glucose is dispersed in warm water. The ratio of glucose to water is 1 g / L to 2
A range of 00 g / L, particularly 10 to 80 g / L is preferable.

In addition to the above carbon sources in the present invention, those added to the medium required include pH adjusting agents, antifoaming agents, corn stipe liquor, various nitrogen sources such as amino acids and Na,
Metal ion sources such as K, Ca, Fe, Zn, Mn, Cu and Mg, or various vitamins can be added to the extent that they are usually added to the microorganism production medium.

Specifically, the compounds containing metal ions include FeSO ₄ , CaCl ₂ , Na ₂ MoO ₄ and ZnS.
Hydrated salts of O ₄ , MnSO ₄ , CuSO ₄ , MgSO _{4 and the} like can be mentioned.

As vitamins, inositol, nicotinic acid, pyridoxy hydrochloride, thiamine hydrochloride, calcium pantothenate, riboflavin, p-aminobenzoic acid,
Examples thereof include folic acid and biotin.

In the method of producing bacterial cellulose using the improved acetic acid bacterium of the present invention, it is preferable to add ethanol at the time of culturing because the production amount of bacterial cellulose is improved. It is preferable to add the ethanol sequentially, rather than all at once. The amount of ethanol added is preferably in the range of 1 g / L to 3 g / L.

The pH of the culture broth is not particularly limited as long as acetic acid bacteria can grow and bacterial cellulose can be produced, but it can generally be cultivated within a pH range of 3 to 7.
The pH is preferably 4 to 6, for example, a pH around 5
Can be set to. The pH can be adjusted using any alkali and acid, for example, an alkali such as sodium hydroxide, potassium hydroxide or sodium carbonate,
Also, acids such as acetic acid, citric acid, hydrochloric acid, sulfuric acid or nitric acid can be used.

The culture temperature for producing bacterial cellulose using the improved acetic acid bacterium of the present invention is usually in the range of 10 to 50 ° C, preferably 10 to 40 ° C, more preferably 20 to 40 ° C, and further preferably Is 25 to 35 ° C.

As a culture method, any microorganism culture method can be used, and static culture, shaking culture, aeration stirring culture, or the like can be used. As a culture operation method, a batch fermentation method, a fed-batch fermentation method, a repeated batch fermentation method, a continuous fermentation method, or the like can be used. As the stirring means, for example, an impeller (stirring blade), an air lift fermenter, a pump-driven circulation of a fermentation broth, or the like can be used alone or in combination.

As the device for culturing, various devices used for producing foods using ordinary microorganisms can be used. For example, a jar fermenter with an agitator that can keep the inside of the tank at a constant temperature can be used. The culture period is usually 1 to 6 days, preferably 2 to 4 days. By such culture, bacterial cellulose is produced at about 1 g / L to 10 g / L.

The bacterial cellulose produced by the method of the present invention may be recovered as it is, or may be treated to remove impurities other than the cellulosic material contained in the recovered product. To remove impurities,
Washing with water, pressure dehydration, dilute acid washing, alkaline washing, treatment with bleaching agents such as sodium hypochlorite and hydrogen peroxide, treatment with microbial enzyme such as lysozyme, surface activity such as sodium lauryl sulfate and deoxycholic acid Impurities can be removed from the cellulosic material by treatment with a chemical agent or washing with heat alone or in combination.

The bacterial cellulose thus obtained includes cellulose, a heteropolysaccharide having cellulose as a main chain, and a glucan such as β-1,3, β-1,2. In the case of the heteropolysaccharide, the constituent components other than cellulose are hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose and glucuronic acid, pentose sugars and organic acids.

Nata de coco can be produced using the bacterial cellulose produced by the method of the present invention.
Methods of making nata de coco using the method of the invention are also within the scope of the invention.

[0032]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 <Acquisition of improved strain> Gluconacetobacter xylinus BPR2001
The DNA of the strain (JCM9730 ^T ) is transferred to the Qiagen Genomic-tip System
(Manufactured by Qiagen), and the gdh gene is replaced with the enzyme Ampl
It was amplified by the PCR method using iTaq Gold (manufactured by Applied Biosystems).

Next, the PCR-amplified gdh gene was inserted into the plasmid vector pT7Blue (Takara Shuzo). Insertion reaction is L
igation Kit (Takara Shuzo) was used. The resulting plasmid was digested with the restriction enzyme BspT104I (Takara Shuzo)
The ends were blunted using an blunting kit (Takara Shuzo).

On the other hand, the cat gene is a plasmid vector pHS.
The cat gene on G398 (Takara Shuzo) was amplified by PCR. Next, the ends were blunted and inserted into the cleaved plasmid obtained as a result of the above steps using a Ligation kit (Takara Shuzo). With the above operation, the cat is
Plasmid pT7Blue-PGDHC containing DNA with inserted gene
Manufactured 1.

The plasmid pT7Blue-PGDH obtained by the above method
C1 was electroporated (equipment: Biorad Gen
It was introduced into acetic acid bacteria by e pulser II). In particular,
It was used to cause homologous recombination of the gdh gene at a certain ratio between the chromosomal DNA of the acetic acid bacterium and the plasmid.

The above-mentioned acetic acid bacterium in which the chloramphenicol resistance gene was incorporated was chloramphenicol at 250 μg /
1 and fructose are incubated in a lactate-ethanol medium containing 40 g / l. The acetic acid strain grown in this medium was designated as improved strain GD-1. This GD-1 was used in the subsequent bacterial cellulose production test. However,
The composition of the lactic acid-ethanol medium is as follows: Yeas
t Extract, 2.4g / l; KH ₂ PO ₄ 1g / l; MgSO ₄ / 7H ₂ O, 0.25g /
l; (NH ₄ ) ₂ SO ₄ , 3.3g / l; Salt Mixture Solution (FeSO ₄
/ 7H ₂ O, 360mg / l; CaCl ₂ / 2H ₂ O, 1470mg / l; Na ₂ MoO ₄ / 2H
₂ O, 242 mg / l; ZnSO ₄ / 7H ₂ O, 173 mg / l; MnSO ₄ / 5H ₂ O, 139 m
g / l; CuSO ₄ / 5H ₂ O, 5mg / l), 10ml / l; Vitamin Mixture So
lution (inositol, 200mg / l; nicotinic acid, 40mg / l;
Pyridoxine hydrochloride, 40 mg / l; Thiamine hydrochloride, 40 mg / l
l; calcium pantothenate, 20 mg / l; riboflavin, 2
0 mg / l; p-aminobenzoic acid, 20 mg / l; 10 mg / l folic acid aqueous solution, 20 mg / l; 10 mg / l biotin aqueous solution, 20 mg / l), 10 ml /
l; lactic acid, 0.3 ml / l; ethanol, 20 g / l.

<< Production of Bacterial Cellulose >> GD-1
Culture of the strain was performed as follows. The medium is ethanol (1
A corn steep liquor (CSL) -containing medium (pH 5.0) containing g / L) was used. Corn steep liquor (CSL) -containing medium (pH 5.
Composition of 0) glucose 40 g / L KH ₂ PO ₄ 1 g / L MgSO ₄ .7H ₂ O 0.25 g / L (NH ₄ ) ₂ SO ₄ 3.3 g / L salt mixed solution 10 ml / L vitamin Mixed solution 10 ml / L Corn steep liquor (CSL) 40 ml / L 10% antifoam aqueous solution 10 ml / L

Salt mixed solution FeSO ₄ .7H ₂ 0 360 mg / L CaCl ₂ .2H ₂ 0 1470 mg / L Na ₂ MoO ₄ .2H ₂ 0 242 mg / L ZnSO ₄ .7H ₂ 0 173 mg / L MnSO ₄ .5H ₂ 0 _{139mg / L CuSO 4 · 5H 2} 0 5mg / L

Vitamin mixed solution Inositol 200mg / L Nicotinic acid 40mg / L Pyridoxine hydrochloride 40 mg / L Thiamine hydrochloride 40mg / L Calcium pantothenate 20mg / L Riboflavin 20 mg / L p-aminobenzoic acid 20 mg / L 10 mg / L folic acid aqueous solution 20 mg / L 10 mg / L biotin aqueous solution 20 mg / L

A strain (1 ml of glycerol stock) was inoculated into a flask with a vent cap containing 100 ml of the medium, and statically cultured at 30 ° C. for 72 hours. Then, 12.5 ml of this culture solution was added to medium 1 in an Erlenmeyer flask with a baffle.
13 ml was inoculated and shake-cultured at 30 ° C. for 96 hours.

<Method for measuring bacterial cellulose production amount> The bacterial cellulose production amount was determined by filtering the culture solution in a bag made of a polypropylene mesh sheet, washing the solid content with distilled water and 0.2N NaOH aqueous solution with heating, It was dried and weighed.

<< Culturing Results >> Table 1 shows the results of the above culturing.

Example 2 The procedure of Example 1 was repeated, except that ethanol was not added to the medium. The amount of bacterial cellulose produced was measured as in Example 1. The results are shown in Table 1.

Comparative Example 1 Culture was carried out in the same manner as in Example 1 except that the wild strain (BPR2001) was used as the acetic acid bacterium. The amount of bacterial cellulose produced was measured as in Example 1. The results are shown in Table 1.

Reference Example 1 Culture was carried out in the same manner as in Comparative Example 1 except that fructose was used as the carbon source. The amount of bacterial cellulose produced was measured as in Example 1. The results are shown in Table 1.

[0046]

[Table 1]

[0047]

Industrial Applicability According to the method of the present invention, bacterial cellulose can be efficiently produced in a large amount using inexpensive glucose as a carbon source, which is advantageous in industrial production.
Bacterial cellulose produced by the method of the present invention is cut into a desired shape, and is mixed in various foods and drinks such as confectionery, frozen desserts, yogurt, almond tofu, honey beans, seasonings, dairy drinks and fruit drinks. .

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA05 BA77 CA06 DA05 EA04                       FA01 FA10 GA14 GA19                 4B041 LC10 LD01 LE10 LH16 LK10                       LP15                 4B064 AF11 CA02 CA19 CB30 CC03                       CC24 CD06 CD09 DA10

Claims (6)

[Claims] 1. A method for producing bacterial cellulose, which comprises culturing an acetic acid bacterium in which a glucose dehydrogenase gene is disrupted in the presence of glucose. 2. The method for producing bacterial cellulose according to claim 1, wherein the acetic acid bacterium is Gluconacetobacter xylinus. 3. An acetic acid bacterium in which the glucose dehydrogenase gene has been disrupted by incorporating the chloramphenicol acetyltransferase gene into the glucose dehydrogenase gene sequence is used.
The method for producing bacterial cellulose according to 1. 4. The method for producing bacterial cellulose according to claim 1, wherein the culture is performed in the presence of glucose and ethanol. 5. The method for producing bacterial cellulose according to claim 4, wherein ethanol is sequentially added. 6. A method for producing Nata de coco using the method for producing bacterial cellulose according to claim 1.