JPH05292984A - Production of microbial cellulose - Google Patents

Abstract

PURPOSE:To obtain the subject cellulose in high efficiency and improved yield by inoculating a microbial strain capable of producing microbial cellulose on a specific medium incorporated with n-paraffin and optionally a surfactant, culturing the strain under aeration and agitation and collecting the produced substance. CONSTITUTION:A microbial strain capable of producing microbial cellulose (e.g. Acetobacter pasteurianus ATCC 10245) is inoculated on a microbial cellulose production medium containing n-paraffin or n-paraffin and a surfactant and cultured at 30 deg.C for 5 days under aeration and agitation to effect the production and accumulation of microbial cellulose in the medium. The microbial cellulose accumulated in the medium is separated by filtration, immersed in 1% NaOH aqueous solution at room temperature for 24 hr to remove proteins and neutralized with 1% acetic acid solution to obtain a large amount of microbial cellulose at a low cost.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently producing microbial cellulose with a microorganism capable of producing microbial cellulose, and more specifically, culturing a microorganism capable of producing microbial cellulose by aeration stirring. The present invention relates to a method for producing microbial cellulose characterized by efficiently producing microbial cellulose.

[0002]

BACKGROUND OF THE INVENTION It has been known that microorganisms belonging to the genus Acetobacter have a high ability to produce microbial cellulose, and are used for industrial production of the substance. ing.

Usually, when producing the substance, a medium developed by Hestrin and Schlum (Bioche) is used.
m. J. , 58, 345 (1954)) was the most productive, and many researchers have used this medium. The yield of is usually 5 to 1
It was about 0%.

[0004] In the past, attempts have been made to improve the sugar yield by improving the composition of the medium. For example, the addition of inositol, phytic acid or pyrroloquinoline quinone has been shown to improve the productivity. (Japanese Patent Laid-Open No. 61-212295).

There is also an example in which the yield is increased by adding a small amount of a cellulase preparation while retaining the cellulase activity (JP-A-63-74490).

[0006] Further, there is a method in which the inactivated cellulase preparation is contained in the production medium (JP-A-2-23888).
8).

However, none of the methods has improved the yield so much, or even if the yield is high, the additives are very expensive, so that it is difficult to use them practically.

As a culture method, static culture is mainly adopted.

Microorganisms belonging to the genus Acetobacter are aerobic bacteria, and the better the oxygen supply, the better the growth.
Since the growth is several times faster than static culture, aeration and agitation culture is generally performed in acetic acid fermentation and the like, but static culture is more efficient when producing the substance. Can be produced in a regular manner.

[0010] This is because when microbial cellulose is produced using a microorganism belonging to the genus Acetobacter, the microbial cellulose is formed into flocs when aerated and agitated and cultivated. This is because as the oxygen supply rate to the center of the flocs becomes rate-determining as it becomes larger, the yield will eventually decrease.

The same applies to other microorganisms having the ability to produce microbial cellulose.

[0012] Therefore, since microorganisms belonging to the genus Acetobacter, which have the ability to produce microbial cellulose, grow faster when aerated and agitated culture, if aerated and agitated culture can be applied when producing microbial cellulose, It is expected that the substance can be efficiently produced.

It is an object of the present invention to provide a production method capable of efficiently producing the substance by aeration and agitation culture when the microbial cellulose is produced using a microorganism having an ability to produce the microbial cellulose.

[0014]

[Means for Solving the Problems] The present inventors diligently studied the conditions for producing microbial cellulose under aeration and stirring conditions,
When n-paraffin is added to the medium, since n-paraffin is a hydrophobic substance, it exists in the form of droplets in the medium, and microbial cellulose is produced with these oil droplets as nuclei, but n-paraffin is high. Since it has oxygen-retaining ability, when flocs composed of microbial cellulose are produced with these oil droplets as the nucleus, unlike ordinary aeration-agitation culture,
Since oxygen is also supplied from n-paraffin in the center of flocs, it was found that microbial cellulose can be produced more efficiently than simply performing aeration and stirring culture.

Further, when not only n-paraffin but also a surfactant is added at the same time, the added oil droplets of n-paraffin are atomized and well dispersed in the medium.
The inventors have found that microbial cellulose can be produced more efficiently, and have completed the present invention.

That is, the present invention is a method of inoculating a microbial cellulose-producing medium with a microorganism capable of producing microbial cellulose, culturing with aeration and stirring to produce and accumulate the substance in the medium, and collecting the substance in the medium. The present invention relates to a method for producing microbial cellulose, which comprises culturing by adding either n-paraffin or n-paraffin and a surfactant.

The microorganisms used in the present invention are not particularly limited as long as they are microorganisms capable of producing microbial cellulose, and Acetobacter, Gluconobacter, Sarsina, Agrobacterium, Pseudomonas, Rhizobium. Examples include microorganisms belonging to the genus.

In particular, microorganisms belonging to the genus Acetobacter have high production capacity of microbial cellulose and are preferably used. For example, Acetobacter pastorianus ATCC 102
45 or the like is used.

The carbon source is not particularly limited as long as it can be assimilated by the microorganism, and is usually sucrose, glucose,
Fructose and the like are used alone or in combination.

Further, a starch hydrolyzate or a cellulose hydrolyzate containing these carbon sources can also be used.

Any nitrogen source can be used as long as it can be utilized by the microorganism, and inorganic nitrogen sources such as ammonium sulfate, ammonium phosphate and nitrate, and organic nitrogen sources such as yeast extract, polypeptone and corn steep liquor are used.

As the inorganic salt, phosphate, magnesium salt, iron salt, calcium salt and the like are used appropriately.

When the microorganism requires vitamins, nucleic acids, amino acids, etc. as micronutrient sources, it is necessary to supplement the required nutrient sources.

The n-paraffin added to the medium should be of high purity unless it contains a component that affects the growth of microorganisms capable of producing microbial cellulose and the ability to produce microbial cellulose. There is no.

The amount added to the medium is 0.10 to 0.50, preferably 0.15 to 0.4 by volume ratio with respect to the amount of the medium.
It is 0.

The method of addition to the medium is not particularly limited, and it may be added to the medium in advance before the start of the culture, or may be added to the medium after the start of the culture in a total amount or in divided portions.

The yield is further improved by adding a surfactant together with the n-paraffin.

The type of surfactant to be added is not particularly limited as long as it does not affect the growth of microorganisms capable of producing microbial cellulose or the ability to produce microbial cellulose. For example, Tween 80 (manufactured by Atlas Powder Co., Ltd.) is used. ), Span 80 (manufactured by Kao Corporation) and the like are preferably used.

The amount added to the medium is 0.01 to 10.0, preferably 0.03 to 4.5 by volume ratio with respect to the amount of the medium.
The concentration of n-paraffin coexisting at this time is 0.10 to 0.50 in volume ratio with respect to the amount of medium, preferably 0.1.
It is 5 to 0.40.

The method of addition to the medium is not particularly limited, and n
-It may be added to the medium in advance with the paraffin before the culture is started, or after the culture is started, a mixed solution with n-paraffin may be prepared, and the mixed solution may be added to the medium in the whole amount or in divided portions.

The initial pH of the medium is preferably 3 to 7, preferably 5 to 6.

The culture temperature is 10 to 40 ° C., preferably 25 to 35 ° C.

As a culturing method, it is necessary to appropriately supply oxygen to the growth of the microorganism, and it is usually produced in the culture medium by aeration stirring culture. The state of supply of oxygen to the microorganism is aeration stirring. It can also be produced in the culture medium by shaking culture, which is almost the same as the culture.

Furthermore, impurities contained in the substance collected by the above operation are washed with water, washed with an alkali, treated with an organic solvent such as toluene, treated with sodium hypochlorite, treated with a surfactant such as sodium lauryl sulfate. Are removed by themselves or in combination.

[0035]

EXAMPLES The present invention will be described in detail below with reference to examples.

[0036]

Example 1 Hestrin-Schlum medium (HS medium)
(2.0 g of D-glucose, 0.5 g of bactopeptone (manufactured by Difco), 0.5 yeast extract (manufactured by Difco)
g, citric acid 0.115 g, disodium hydrogen phosphate 0.27 g, distilled water 100 ml, pH 6.0).
5 ml was dispensed into a test tube (length 140 mm, inner diameter 16 mm) and inoculated with a culture solution of Acetobacter pastorianus ATCC 10245, which had been shake-cultured in HS medium for 5 days in advance, amplitude 20 mm, shaking speed 280 spm.
Culturing was carried out at 30 ° C. for 5 days under the above conditions.

At the same time, while keeping the total liquid volume at 11.5 ml,
Acetobacter pastorianus ATCC 10245 was inoculated into a medium prepared by mixing the concentrated liquid of the above HS medium and the liquid of n-paraffin at the ratio shown in Table 1, and cultured under the same conditions.

In this case, the concentration of each medium component in the concentrated liquid of the HS medium is set to 11.5 ml so that the medium component becomes the same as that in the case where n-paraffin is not added, and the concentration ratio of n-paraffin is adjusted. What was suitably prepared according to it was used.

After completion of the culture, the microbial cellulose produced in the culture solution was taken out by filtration, immersed in a 1% NaOH aqueous solution and subjected to deproteinization treatment at room temperature for 24 hours, and then 1
% Of acetic acid solution and neutralized.

After the neutralization treatment, the product was thoroughly washed with water and dried, and the dried product was weighed to obtain the amount of microbial cellulose produced.

The amount of microbial cellulose produced was 6 mg in the HS medium, whereas when n-paraffin was added, it was as shown in Table 1 and when the ratio of n-paraffin was 0.26, it was 14 mg. Was about twice that of the case without addition.

[0042]

[Table 1]

[0043]

[Example 2] Hestrin-Schlum medium (HS medium)
(D-glucose 2.0 g, bactopeptone (manufactured by Difco) 0.5 g, yeast extract (manufactured by Difco) 0.5)
g, citric acid 0.115 g, disodium hydrogen phosphate 0.27 g, distilled water 100 ml, pH 6.0).
5 ml was dispensed into a test tube (length 140 mm, inner diameter 16 mm) and inoculated with a culture solution of Acetobacter pastorianus ATCC 10245 which had been shake-cultured in HS medium for 5 days in advance, and the actual volume was 1 L and the aeration rate was 0.1 vvm. Culturing was carried out at 30 ° C. for 5 days under the condition of stirring at 250 rpm.

At the same time, with the total liquid volume kept at 1 L, the above H
Tween 80 (manufactured by Atlas Powder) or n-paraffin and Span was added to a medium in which the concentrated liquid of S medium and n-paraffin were mixed in the ratio of Table 2 in the same manner as in Example 1.
80 (manufactured by Kao Corporation) was added, and Acetobacter pastorianus ATCC 10245 was inoculated into this medium and cultured under the same conditions.

After completion of the culture, the microbial cellulose produced in the culture solution was taken out by filtration and immersed in a 1% NaOH aqueous solution for 24 hours for deproteinization treatment, and then 1
% Of acetic acid solution and neutralized.

After the neutralization treatment, the product was thoroughly washed with water and dried, and the dried product was weighed to obtain the amount of microbial cellulose produced.

The amount of microbial cellulose produced was as shown in Table 2 and was higher than that of n-paraffin alone by Tween 8
In the case of adding 0 or Span 80, the production amount was improved under the condition that the ratio of n-paraffin was low, or the microbial cellulose production amount which could not be achieved by n-paraffin alone was obtained.

[0048]

EFFECTS OF THE INVENTION According to the present invention, microbial cellulose can be efficiently produced by aeration and agitation culture, which has been difficult in the past, and microbial cellulose can be supplied inexpensively and in a large amount.

[Table 2]

[Table 3]

Claims (1)

[Claims] 1. A method of inoculating a microbial cellulose-producing medium with a microorganism capable of producing microbial cellulose, culturing with aeration and stirring to produce and accumulate the substance in the medium, and collecting the substance, wherein n-paraffin is added to the medium. Alternatively, a method for producing microbial cellulose, which comprises culturing by adding either n-paraffin or a surfactant.