JPH08278A - Production of cellulosic substance - Google Patents

Abstract

PURPOSE:To produce a cellulosic substance in high efficiency by flow-down culture. CONSTITUTION:Cells produced by liquid culture are scattered on a sheet-formed culture member 4 made of e.g. polypropylene nonwoven fabric. The culture member 4 is suspended on a supporting member 3 in a culture vessel 1 and a culture liquid 2 is sprayed to the top of the culture member 4 with a culture liquid feeding pump 8 and allowed to flow down along the surface. The microorganisms are brought into contact with the culture liquid 2 to produce a cellulosic substance.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for efficiently producing a cellulosic substance by flow-down culture.

[0002]

2. Description of the Related Art Conventionally, as a method for producing a gelled cellulosic substance from microorganisms, "Nata de coco production method" (Japanese Patent Laid-Open No. 61-149055) and "Microbe production of cellulosic material" (special Kaihei 5-682
36) and the like. First, the former is inoculated with acetic acid bacteria in a mixture of coconut juice and sugar, and by removing the acid component from the jelly-like solid product obtained by stationary culture under acidic conditions, nata de coco with good taste, texture and texture. Is a method of manufacturing. On the other hand, the latter is a method of inoculating a cellulosic substance-producing microorganism into a liquid medium and performing static culture while continuously or intermittently adding a liquid medium or a mixture of the microorganism and the medium to the cellulosic substance. Is a method of manufacturing.

[0003]

However, all of the above-mentioned conventional examples are static culture methods and cannot be said to be efficient methods. That is, in the stationary culture method, the produced cellulose forms a layer having a substantially uniform thickness, and the upper surface thereof floats to the same surface as the liquid surface. Fresh cellulose is produced at the top of the cellulose layer, which is most advantageous for oxygen inoculation, and is pushed down into the old cellulose, the liquid with the bottom side down in sequence. The nutrients required for the production of cellulose pass from the culture solution through the entire cellulose layer and move to the uppermost surface of the cellulose layer. Therefore, the thicker the formed cellulose layer is, the more the resistance to the bacterial cells is increased and the growth of the cellulose is inhibited in terms of nutritional supplementation. Further, in the same culturing method, there is a defect that the cellulose layer is not formed when the culture solution is stirred to promote the culturing, and the culturing cannot be controlled.

Therefore, the inventor of the present invention can easily supply oxygen and nutrients by arranging the culture member vertically or inclined, inoculating the bacterial cells into the culture member, and flowing the culture solution along the surface of the culture member. , The amount of culture solution supplied, nutritional components,
Alternatively, they have found that the culture conditions can be controlled by appropriately changing the temperature and the inclination of the sheet member. The present invention has been completed based on this finding.

That is, according to the present invention, a culture member on which a microorganism belonging to the genus Acetobacter and having a cellulosic substance-producing ability is grown is arranged vertically or inclined and the culture solution is supplied to the top of the culture member. It is a method for producing a cellulosic substance, characterized by producing a cellulosic substance by allowing it to flow down along the surface and contacting the microorganism with a culture solution.

[0006]

First, the microorganism used in the present invention may be any microorganism as long as it belongs to Acetobacter and produces a cellulosic, gel-like substance, for example, Acetobacter Pasteurianu.
s, Acetobacter Hanseni, and Acetobacter xylinum.

The liquid nutrient medium includes glucose, sucrose, fructose, maltose, starch hydrolysates, carbon sources such as molasses, ammonium salts such as ammonium phosphate, ammonium sulfate and ammonium chloride,
Organic or inorganic nitrogen sources such as nitrates, urea, peptone, organic trace nutrients such as amino acids, vitamins, fatty acids, nucleic acids, substances containing these, and phosphates, magnesium salts, calcium salts, iron salts, Examples thereof include synthetic or semi-synthetic medium in which inorganic salts such as manganese salt are appropriately combined. In addition, a natural liquid nutrient medium may be prepared by using a fruit juice such as coconut milk, a plant extract, a sugar solution, etc. as a main raw material.
These media are adjusted to pH 3 with the addition of organic or inorganic acids.
It is preferable to adjust to ~ 6 before use.

Next, the present invention will be specifically described with reference to the accompanying drawings. In FIG. 1, 1 is a box-shaped, hermetically-formed culture tank in which a culture solution 2 serving as a nutrient source for bacterial cells is stored at the bottom. In the upper part of the inside of the culture tank 1, support members 3 each having a round bar shape in the front-rear direction in FIG. 1 are installed at equal intervals in parallel to the ceiling of the culture tank 1, and the support members 3 are inoculated with cells. The culture member 4 is suspended. In the present embodiment, the culture member 4 shown in the form of a sheet as the culture member 4 is formed of, for example, a non-woven fabric, a cloth material, a filter cloth material, a canvas, or the like, and has a roughness so that the bacterial cells can grow. The material does not matter as long as it has Alternatively, a member having rigidity such as metal or plastic may be attached to the surface of the member, or the surface of metal or plastic may be roughened.

Reference numeral 5 denotes a distribution pipe for the culture solution 2, which is used as the support member 3
2, a spray nozzle 6 is provided on the surface facing the support member 3 as shown in FIG. 2, and the culture solution 2 is supplied to the top of the culture member 4 suspended by the support member 3. Is configured. The distribution pipes 5 are connected to each other by a connecting pipe 7. Reference numeral 8 denotes a culture solution supply pump for supplying the culture solution 2 to the culture member 4, and the suction port 9 is connected to the bottom of the culture tank 1 so as to be able to suck the culture solution 2.

On the other hand, the discharge port 10 of the culture solution supply pump 8
Is connected to the distribution pipe 5 via a filter 11 and a heat exchanger 12. The filter 11 is installed to remove contaminants from the culture medium 2, and the heat exchanger 12 is installed to control the temperature of the culture medium 2.
An air supply port 13 is opened on one side of the lower part of the culture tank 1, and an air discharge port 14 is opened on the other side of the upper part of the culture tank 1. The air supply port 13 has a sterile filter 15 on its suction side. And blower 17 equipped with heat exchanger 16
Is provided and is configured to supply temperature-controlled oxygen to the bacterial cells.

Thus, the cellulosic substance is produced by the microorganisms using the apparatus shown in FIGS.
The culture member 4 is inoculated with the bacterial cells. At this time, it is preferable that the cellulosic substance is evenly generated on the entire surface of the culture member 4. From this point, it is possible to spray the liquid-cultured bacterial cells on the culture member 4 or soak the member 4 in a liquid medium. Etc. In addition, when the bacterial cells are cultivated in agar medium or the like, there is a method of applying them to the culture member 4.

[0012] The microbial cells are added to the culture member 4 in the range of 1 × 10 ² to 1 × 1
It is sufficient to inoculate about 0 ⁶ cells / cm ² . The concentration of the culture solution 2 is 1 to 15% (V / V) as the sugar concentration, and the temperature is 20 to 3
Hold at 5 ° C. The ratio of the culture solution 2 supplied to the culture member 4 is 0.1 per unit area of the culture member 4.
-10 ml / hr · cm ² is suitable.

Next, FIG. 3 shows an example in which the culture member 4 is installed while being inclined. In the case of this embodiment, an upper support member 18 installed in the same manner as in FIG. 1 and a lower support member 19 installed right below the upper support member 18 in FIG. It is installed in a sliding table. Then, the culture member 4 is adhered to the upper surface of the support plate 20, the cells are inoculated into the culture member 4, and the culture solution is supplied from the top of the culture member 4.
Thereafter, the cells are cultured in the same manner as in the example of FIG. 1 to produce a cellulosic substance. In the present embodiment, the culture surface of the culture member 4 is reduced, but there is an advantage that the flow rate of the culture solution 2 can be controlled in a wider range.

Next, FIG. 4 shows an example in which the culture member 4 is formed in a cylindrical shape. In the figure, reference numeral 25 is a column formed of a plastic or iron material in a cylindrical shape, the upper part of which is opened, the lower part of which is provided with a passage port 26 for the culture solution 2 and which is erected at the bottom of the culture tank 1. ing. The culture members 4 are installed on the outer circumference and the inner circumference of the column 25, and from the top thereof, the spray nozzles 6 are provided corresponding to the respective culture members 4 as in FIG. The culture solution 2 supplied to the inside 27 of the culture member 4.
Are configured to flow down along the culture member 4 and circulate through the liquid passage port 26.

Next, comparative examples will be shown to numerically show the superiority of the present invention. A. Method of the present invention 1. Device: The device shown in FIGS. 2. Culture material: Rayon non-woven fabric Area: 80 cm ² (one side) Number of installations: 1 unit (one culture surface) Installation form: vertical 3. Cells: Acetobacter xylinum ATCC 10821 5 × 10
⁴ cells / cm ² inoculation 4. Culture medium: Coconut milk filtrate, sucrose 10%, ammonium phosphate 0.5%, acetic acid 1.0
%, Adjusted to pH 4.0. 5. Culture conditions Culture temperature: 30 ° C. Medium supply: 0.5 ml / hr · cm ² (per unit area of culture member) Oxygen concentration: Normal concentration in air Culture period: 14 days

B. Conventional method (stationary culture method) 1100 ml of the same culture solution as the method of the present invention was used to prepare a solution of 23.
Put in a 3 cm Petri dish (surface area 426 cm ² , depth 2.6 cm) and incubate with slant for 3 days, then pH
Acetobacter xylinu shaken in the same culture solution of 5.0 for 1 day
m ATCC 10821 was inoculated at 8.7 × 10 ⁴ cells / ml. The unit surface area is 2.3 × 10 ⁵ pieces / cm ² . Three
After static culture at 0 ° C. for 14 days, the culture solution has a thickness of 1.8 c on the surface.
m of gel-like cellulose was produced. This was collected and washed with running water for 3 days to remove impurities such as medium and cells. The wet weight was weighed, further dried at 105 ° C. until a constant weight was obtained, and the dried amount was weighed.

[0017] As shown in Table 1, the present invention produces a higher yield in the wet form and a higher ratio in the dry form than the conventional method.

Next, the present invention will be described more specifically with reference to examples. Example 1 First, Acetobacter xylinum liquid-cultured on a culture member
Spray 2 × 10 ⁵ pieces of ATCC 10821 / cm ² . And
In the culture tank shown in FIG. 1, 1600 cm ² (culture area 1
Ten sheet-shaped culture members made of polypropylene non-woven fabric are suspended at 500 cm ² ) on a support member. Next, 7.5 l / hr of the culture solution containing coconut milk as a main component is sprayed onto the culture member from the top of the culture member by the culture solution supply pump. At this time, the culture solution is kept at 28 ° C. by a heat exchanger. On the other hand, air controlled at 28 ° C. is supplied by a blower. After culturing for 16 days in the above state, a cellulosic material of 25 mm on average was produced on the surface of the culture member.

Example 2 First, Acetobacter xylinum liquid-cultured on a culture member
Spray 7 × 10 ⁴ pieces / cm ^{2 of} ATCC 10821. And
In the culture tank shown in FIG. 3, 5 culture members formed of nylon filter cloth at 1600 cm ² are attached to the surfaces of the support plates supported by the upper and lower support members. At this time, the culture member was erected at 45 degrees from the horizontal plane. Next, 3.8 l / hr of the culture solution containing coconut milk as a main component was sprayed onto the culture member from the top of the culture member by the culture solution supply pump. At this time, the culture solution is kept at 30 ° C. by a heat exchanger. on the other hand,
The air controlled at 30 ° C. is supplied by a blower. After culturing for 17 days in the above state, a cellulosic material having an average thickness of 19 mm was produced on the surface of the culture member.

[0019]

EFFECTS OF THE INVENTION The invention of the present application is constructed as described above, and it is possible to control the supply amount, concentration, temperature, etc. of the culture broth, and it is possible to culture the bacterium under optimum conditions. The yield is high, and the gelled cellulosic material produced is rich in cellulose content.

[Brief description of drawings]

FIG. 1 is a front sectional view of an apparatus according to the present invention.

FIG. 2 is a 2-2 view of FIG.

FIG. 3 is a front cross-sectional view of another embodiment.

FIG. 4 is a diagram of another embodiment of the culture member.

[Explanation of symbols]

 1 Culture Tank 2 Culture Liquid 4 Culture Material 6 Spray Nozzle 8 Culture Liquid Supply Pump 11 Filter 12 Heat Exchanger 15 Aseptic Filter

Claims (3)

[Claims] 1. A culture member on which a microorganism belonging to the genus Acetobacter and capable of producing a cellulosic substance is grown is arranged vertically or slanted, and a culture solution is supplied to the top of the culture member along the surface thereof. A method for producing a cellulosic substance, which comprises allowing the microorganism to flow down and bringing the culture solution into contact with the culture medium to produce a cellulosic substance. 2. The method for producing a cellulosic material according to claim 1, wherein the culture member has a sheet shape. 3. The method for producing a cellulosic material according to claim 1, wherein the culture member has a tubular shape.