JPH05292983A - Production of microbial cellulose - Google Patents

Abstract

PURPOSE:To obtain the subject cellulose in high efficiency and improved yield by inoculating a microbial strain belonging to the genus Acetobacter and capable of producing microbial cellulose on a specific medium, subjecting the strain to standing culture while keeping the carbon source to a low concentration and collecting the produced and accumulated substance. CONSTITUTION:A microbial strain belonging to the genus Acetobacter and capable of producing microbial cellulose (e.g. Acetobacter pasteurianus ATCC 10245) is inoculated on a medium for the production of microbial cellulose and cultured by standing culture at 30 deg.C for 192 hr while keeping the concentration of the carbon source (e.g. glucose) in the medium at a low level (i.e., <=2 g/L) during the culture period and continuously supplying a liquid having a carbon source concentration of 40% at a rate of 0.26 mL/h for replenishing the consumed carbon source. The microbial cellulose produced and accumulated on the surface of the culture liquid is separated by filtration, immersed in 1% NaOH aqueous solution at room temperature for 24 hr to remove the protein and neutralized to obtain the objective microbial cellulose.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing microbial cellulose.

More specifically, it belongs to the genus Acetobacter,
When producing microbial cellulose by a microorganism having the ability to produce microbial cellulose, efficient production of microbial cellulose characterized by culturing while maintaining the carbon source concentration of the medium in static culture below a certain concentration Concerning the law.

[0003]

2. Description of the Related Art It has been conventionally known that microorganisms belonging to the genus Acetobacter have a high ability to produce microbial cellulose, and they are used for industrial production of the substance. ing.

Usually, when producing various substances, the 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%.

Therefore, by improving the medium composition,
Attempts have been made to increase the sugar yield, and for example, the addition of inositol, phytic acid, or pyrroloquinoline quinone has been shown to improve productivity (JP-A-61-212295).

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

There is also a method of incorporating an inactivated cellulase preparation in a production medium (JP-A-2-23888).
8).

However, in any of the methods, the yield has not been improved so much, or even if the yield is high, the additives are very expensive, so that it is difficult to use them practically.

On the other hand, attempts have been made to increase the yield by devising a culture method.

For example, in static culture, it has been found that the production rate of microbial cellulose is improved by continuously or intermittently adding a medium during the culture (JP-A-62-265990).

However, even with this method, the yield with respect to sugar was about 25%, which was insufficient.

Further, a method has been proposed in which the growth step and the microbial cellulose accumulation step are separated and the carbon source concentration in each step is controlled in aeration and agitation culture (JP-A-63-202394). The maximum rate was about 20% in terms of weight.

The present invention is to provide a production method capable of efficiently and inexpensively producing a microbial cellulose when the microbial cellulose is produced using a microorganism having the ability to produce microbial cellulose belonging to the genus Acetobacter. ..

[0014]

[Means for Solving the Problems] Since the yield of saccharides in static culture is high, the present inventors examined the culture conditions in static culture, and determined the yield of microbial cellulose as the sugar concentration of the medium. The lower is the better, the lower the sugar concentration in the medium is, the higher the yield will be. However, in reality, the sugar concentration to be maintained and the pH change during the culturing, resulting in a higher sugar metabolism rate. It has been found that proper culturing is very difficult to perform due to variations.

[0015] In Japanese Patent Laid-Open No. 62-265990, it is proposed to add a medium continuously or intermittently during the culture period of static culture, but the yield to sugar is only about 25%. ..

The reason for this is that the addition condition of the medium is set to be less than the maximum rate of the microbial cellulose production rate, and the carbon source concentration of the culture solution during the culture period is not taken into consideration. Although the production efficiency was improved as compared with the case where the medium was not supplemented, the present situation is that only the same level of performance has been achieved in terms of sugar yield.

Based on these findings, the effect of the sugar concentration of the medium in the stationary culture was earnestly studied, and the carbon medium concentration of the starting medium was lowered in the stationary culture to appropriately add carbon sources such as sugar during the culture period. It was found that the production yield of microbial cellulose is remarkably improved by supplementing and culturing while controlling the carbon source concentration below a certain concentration, and the present invention has been completed.

That is, according to the present invention, a microorganism having the ability to produce microbial cellulose belonging to the genus Acetobacter is inoculated into a microbial cellulose production medium and statically cultured to produce and accumulate the substance during the medium period, In a method for collecting a substance, the present invention relates to a method for producing microbial cellulose, which comprises culturing while maintaining a low carbon source concentration in a medium, and preferably controlling the carbon source concentration to 2 g / L or less.

The microorganism used in the present invention may be any strain as long as it is a microorganism belonging to the genus Acetobacter and capable of producing a microbial cellulosic material. For example, Acetobacter Bastorianus ATCC.
10245 and Acetobacter sp. DA stock (FERM
P-12924) and the like are used.

Acetobacter sp. DA is a bacterium isolated from vinegar moromi, and its bacteriological characteristics were examined according to a general method for classifying microorganisms (for example, "Classification and Identification of Microorganisms" (Takehisa Hasegawa, Academic Society Publishing Center)).
(1) Gram-negative aerobic bacteria, (2) Oxidizing ethanol to produce acetic acid, (3) Being able to grow in a pH 4.5 medium, (4) Capable of assimilating acetic acid and lactic acid From this, the Barjay's Manual of Systematic Bacteriology Vol. 1 pp. 267-2
74, 1984 ("Bergy's Manual"
of Systematic Bacteriolo
gy "vol.1, P.267-2741984), it was found to be a microorganism belonging to Acetobacter.

This strain was deposited at the Institute for Microbial Industry of the Agency of Industrial Science and Technology, and was designated Microorganism Research Institute No. 12924 (FERM P-129).
24).

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

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

The concentration of these carbon sources is preferably 2 g / L or less.

As the carbon source to be supplemented during the culturing period, the above-mentioned carbon sources can be used, but during the culturing, the pH of the medium is not changed because it is not affected by the oxidizing action of the microorganism, and the conversion to microbial cellulose is made. Gluconic acid and acetic acid are also preferably used because of their high efficiency.

The carbon source to be supplemented may be one kind or a mixture of two or more kinds.

These carbon source supplements are appropriately added so that the concentration of carbon source contained in the medium does not exceed 2 g / L.

Therefore, it is desirable to appropriately measure the concentrations of sugar, gluconic acid and acetic acid during the culture period.

When acetic acid is used as a carbon source, acetic acid has a growth-inhibiting effect on the microorganism, and therefore a strain having resistance to acetic acid, for example, Acetobacter sp. It is desirable to use DA strain or the like.

Acetobacter sp. The DA strain is a strain of the genus Acetobacter having a normal microbial cellulose-producing ability, which shows a vigorous ability to produce the substance even in the presence of acetic acid at a concentration of 10 g / L or more, which significantly reduces the productivity of the substance. It is particularly preferable to use it for carrying out the present invention.

The method of supplementing the carbon source may be continuous or intermittent.

The nitrogen source contained in the medium may be any one that can be utilized by the microorganism, such as ammonium sulfate,
Inorganic nitrogen sources such as ammonium phosphate and nitrate, and organic nitrogen sources such as yeast extract, polypeptone and corn steep liquor are used.

As the inorganic salt, a phosphate, a magnesium salt, an iron salt, a calcium salt or the like is 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 culture method is static culture, but since the added carbon source is uniformly diffused throughout the culture medium during the culture, intermittent stirring or the like is carried out under conditions that do not affect the production of microbial cellulose during supplementation. It is desirable to do.

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

Although the pH of the medium changes during culturing as the microorganism grows, it is desirable to use an alkali or an acid to maintain the pH range suitable for the production of microbial cellulose.

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

Impurities contained in the substance collected by the above-mentioned 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. Alternatively, it is removed in combination.

[0040]

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

[0041]

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) 100 m
l is dispensed into a 300 ml wide-mouth Erlenmeyer flask with a plastic cap, and Acetobacter Bastorianus AT
CC 10245 was inoculated and statically cultured at 30 ° C. for 192 hours.

After completion of the culture, the microbial cellulose produced on the surface of the culture solution was taken out by filtration, immersed in a 1% NaOH aqueous solution for deproteinization treatment for 24 hours at room temperature, and then immersed in a 1% acetic acid solution for neutralization treatment. Was done.

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.

On the other hand, 50 ml of a medium which does not contain glucose at all and has a composition twice as high as that of HS medium except for glucose.
Dispense 300 ml into a wide-necked Erlenmeyer flask with a plastic cap, Acetobacter Bastorianus ATC
C10245 was inoculated and statically cultured at 30 ° C.

During the culture period, a liquid having a glucose concentration of 40% was continuously supplied at a rate of 0.26 ml / h, and the culture was completed 192 hours after the start of the culture.

When the glucose concentration during the culture period was measured by a conventional method, it was maintained at 1.5 g / L or less.

At the end of the culturing, the amount of the culture solution was 100 ml, and the added glucose amount was about 2 g when added up, which was the same as the case where the glucose was added from the start of the above culture.

After completion of the culture, the insoluble film containing microbial cellulose formed on the surface of the culture solution was subjected to deproteinization treatment in the same manner as above, dried and the amount of microbial cellulose produced was measured.

The microbial cellulose produced was 0.20 g when glucose was added at the start of the culture, whereas it was supplemented with glucose during the culture period.
Although it was 0.24 g, the yield of microbial cellulose with respect to the amount of glucose consumed was 20% when glucose was added at the start of the culture, whereas it was 28% when glucose was supplemented during the culture. %Met.

[0050]

Example 2 100 ml of medium in which the cumulative glucose amount of the HS medium of Example 1 was changed to a concentration as shown in FIG. 1 within the range of 0.4 g / L to 80 g / L was placed in a 300 ml wide-mouth Erlenmeyer flask with a plastic cap. Dispense and inoculate Acetobacter Bastorianus ATCC 10245,
Static culture was performed at 30 ° C. for 192 hours.

On the other hand, as in Example 1, the medium prepared by adding medium components other than glucose in the HS medium at a high concentration according to the amount of glucose solution to be supplemented was subtracted from 100 ml of the amount of the glucose solution to be supplemented. The amount was dispensed into a wide-necked Erlenmeyer flask with a 300-ml plastic cap, Inoculated with Acetobacter Bastorianus ATCC 10245, and static culture was performed at 30 ° C.

During the culture period, glucose concentration 40% or 8
0% liquid is supplied, and the total amount of glucose in the glucose solution is low at an addition rate such that the medium amount for 192 hours becomes 100 ml and at the same time the integrated amount of supplemented glucose is in the range of 0.4 g / L to 80 g / L. In some cases, intermittently, or continuously when the integrated glucose level is high, 1
The culture was completed at 92 hours.

After completion of the culture, the insoluble membrane containing microbial cellulose formed on the surface of the culture solution was subjected to deproteinization treatment in the same manner as in Example 1, dried and the amount of microbial cellulose formed was measured.

FIG. 1 shows the relationship between the yield of microbial cellulose with respect to the amount of glucose produced and the amount of glucose consumed.
It was shown to.

At any glucose concentration, the yield of microbial cellulose produced and the yield of microbial cellulose relative to the amount of glucose consumed were higher in the culture supplemented with glucose than in the batch culture in which glucose was added at the start of the culture.

Particularly, in the supplemental culture, the culture in which the integrated glucose concentration was 10 g / L, which was maintained so that the glucose concentration was 2 g / L or less during the culture period, the yield of 25% or more was obtained. I got high grades that I couldn't achieve.

[0057]

Example 3 Similar to Example 1, glucose was not contained at all, and other medium components were placed in two 300 ml plastic wide-caps Erlenmeyer flasks with plastic caps, in which 50 ml of a medium having a composition twice that of HS medium was dispensed. Acetobacter
p. Inoculated with DA (FERM P-12924), 30
Static culture was performed at ℃.

After the start of culture, the concentration of acetic acid was adjusted to 4 in two flasks.
0% liquid was continuously added, but one was 0.26 ml /
At a rate of h, the other one was added at a rate of 0.39 ml / h. The culture was continued for 192 hours.

At the end of culturing, the amount of culture solution was 100 ml, and the cumulative amount of acetic acid was about 2 g.

When the acetic acid concentration during the culture period was measured by a conventional method, it was approximately 1.8 g at a rate of 0.26 ml / h.
/ L or less, but when acetic acid was supplied at a rate of 0.39 ml / h, 4.5 g at the end of the culture
Was / L.

After the culture was completed, the insoluble film containing microbial cellulose formed on the surface of the culture solution was subjected to deproteinization treatment in the same manner as in Example 1, dried and the amount of microbial cellulose formed was measured.

The microbial cellulose produced was 0.26 m.
It was 0.9 g at the addition rate of 1 / h, whereas it was 0.3 g at the supply rate of 0.39 ml / h.

[Brief description of drawings]

FIG. 1 shows that, in Example 2, the glucose concentration in the medium was changed and the batch culture and the glucose supplementation culture were performed.
It is the figure which measured the production amount of produced | generated microbial cellulose, and represented the production yield of microbial cellulose with respect to the amount of glucose consumed.

Claims (3)

[Claims] 1. A method for inoculating a microbial cellulose production medium with a microorganism capable of producing a microbial cellulose belonging to the genus Acetobacter, statically culturing to produce and accumulate the substance in the medium, and collecting the substance, A method for producing microbial cellulose, which comprises culturing while maintaining the carbon source concentration of the medium at a low concentration during the culturing period. 2. The method for producing microbial cellulose according to claim 1, wherein the carbon source concentration is 2 g / L or less. 3. The microorganism of claim 1 is a new strain Acetobacter sp. A method for producing microbial cellulose which is DA.