JPS61212295A - Production cellulosic substance by bacterium - Google Patents

Abstract

PURPOSE:To obtain a cellulosic substance stably, rapidly and efficiently, by cultivating a bacterium belonging to the genus Acetobacter, capable of producing the cellulosic substance, in a specific medium. CONSTITUTION:A bacterium [(Acetobacter aceti subsp. xylinum) ATCC 10821, etc.] belonging to the genus Acetobacter, capable of producing a cellulosic substance, is cultivated in a medium containing inositol or phytic acid. Cellulose, a substance containing a heteropolysaccharide having cellulose as a main chain and a substance containing glucan such as beta-1,3-glucan, beta-1,2 glucan, etc., may be cited as the cellulosis substance.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing cellulosic substances produced by microorganisms belonging to the genus Acetobacter and having the ability to produce cellulosic substances.

This cellulosic substance is edible and used in the food field, and has excellent aqueous dispersibility, so it can be used to maintain the viscosity of foods, cosmetics, or paints, strengthen food raw materials, retain moisture, improve food stability, etc. It has industrial utility as a low-calorie additive or emulsion stabilization aid.

Based on its structural and physical characteristics, it has various industrial uses as a reinforcing material for polymers, especially water-based polymers.

Since such disaggregated products exhibit a high tensile modulus, materials obtained by solidifying the cellulosic disaggregated products into paper or solid forms are expected to have excellent mechanical properties based on the structural characteristics of microfibrils, and can be used as various industrial materials. There are applications as

(Prior Art) A method of producing cellulose using a microorganism belonging to the genus Acetobacter and having the ability to produce cellulose is conventionally known.

In these methods, expensive yeast extract, begtone, etc. are added to the culture medium, so the cellulose produced is expensive and the production rate is slow, and the production amount, q, is low, but not satisfactory. It's not something.

Furthermore, it is not known that specific components contained in yeast extract etc. are involved in promoting cellulose production.

(Problems to be Solved by the Present Invention) To develop a method for stably, painlessly, efficiently and inexpensively producing cellulosic substances using microorganisms belonging to the genus Acetobacter that have the ability to produce cellulosic substances. There is a particular thing.

We conducted research and found that by culturing microorganisms that belong to the genus Acetobacter and have the ability to produce cellulosic substances in a medium supplemented with inositol or phytic acid, it was possible to produce significantly more cellulose than with conventional production methods. I learned that the productivity of sexual substances is improved. The present invention was completed based on this knowledge.

The microorganism used in the present invention belongs to the acetovector group and may be any microorganism that produces cellulosic substances.

- Examples include Acetobacter aa@t, Acetobacter spicis, and xylinum.
i5ubsp.

xyllnum) ATCC10821.

salts or derivatives of phytin. Here, the phytin derivative is a substance produced in the process of decomposing phytic acid into inositol, and is a substance in which 1 to 5 phosphate groups are bonded to inositol.

The inositol or inositol used in the present invention may contain these substances in addition to being chemically pure.

As the carbon source, sucrose, glucose, fructose, mannitol, tulpitol, galactose, maltose, erythritol, cadnitol, glycerol ethylene glycol, ethanol, acetic acid, etc. are used alone or in combination. Furthermore, starch thawed products containing these substances, citrus molasses, beet molasses, beet juice, sugarcane juice, fruit juices including citrus fruits, etc. can be used.

Nitrogen sources include ammonium sulfate, ammonium chloride,
Ammonium salts such as ammonium phosphate, nitrates, urea, organic or inorganic nitrogen sources such as vitamins, fatty acids, nucleic acids, etc. It is also effective to add 2.7.9-tricardexy IH pyrrolo(2,3-5]-quinoline-4,5-dione.

When using an auxotrophic mutant strain that requires amino acids and the like for growth, it is necessary to supplement the required nutrients. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals, etc. are used.

The - of culture is controlled to 3 to 7, preferably in the range of 3.5 to 5.

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

The oxygen concentration supplied to the culture tank may be 5 to 100%, preferably 20 to 30%.

By culturing for 1 to 30 days, a cellulosic substance is produced on the surface layer of the side where oxygen is supplied to the culture solution in normal static culture.

The cellulosic substance produced by the method of the present invention may be collected as is, or may be further treated to remove substances other than the cellulosic substance, including bacterial cells contained in the substance.

To remove impurities, water washing, pressure dehydration, dilute acid washing,
Alkaline cleaning, treatment with polar organic solvents such as toluene and ethyl acetate, treatment with bleaching agents such as sodium hypochlorite and hydrogen peroxide, treatment with cell lytic enzymes such as lysozyme, and interfaces such as sodium lauryl sulfate and desoxycholic acid. Impurities can be removed from the cellulose-like substance by treatment with an activator, heating washing at a temperature ranging from room temperature to 200°C, etc., either alone or in combination.

The cellulosic substance obtained in this way as used in the present invention refers to the following.

The cellulosic substances of the present invention are those containing cellulose and heteropolysaccharides with cellulose as the main chain, and β-1,3,
It contains glucans such as β-1 and β-2. Components other than cellulose in the case of heteropolysaccharides include mannose, fructose, galactose, xylose, arabinose,
These include hexoses such as rhamnose and glucuronic acid, and pentose acids.

Note that these polysaccharides may be a single substance, or two or more types of polysaccharides may be mixed together due to hydrogen bonding or the like.

Example 1 Sucrose 5%, potassium I phosphate 0.3% magnesium sulfate (heptahydrate) 0.05%, cabmino acid (Dif
(manufactured by Co., Ltd.) 400 #l of medium with a composition of 0.8 cm and pH 5.
! The mixture was poured into a 500 m-capacity slope flask and sterilized at 120°C for 30 minutes.

3 on a slanted agar medium with 2 pieces of agar added to the medium with the above composition.
Acetobacter aceti subspice xylinum ATCC 10821 grown for 6 days at 0°C was inoculated into the above sterilized medium and shaken at 30°C for 2 days). manufactured by )0
．． 05%, phytin calcium salt (manufactured by Tokyo Kasei) 0.
02%, phytin, 1 calcium, 4 magnesium, 2
100 m of medium added with 0.02 t of ammonia salt (manufactured by Organo)! The mixture was poured into a 500ml wide-mouth reagent bottle with a cotton stopper and sterilized at 120°C for 10 minutes.

Each bottle was inoculated with 5 g of the above seed mother, and 3
The cells were statically cultured at 0°C for 2 weeks.

) The cellulose substance produced in each bottle was thoroughly washed by repeated pressure dehydration washing, dried at 105° C. until it reached a constant weight, and measured.

The results are shown in Table 1.

As can be seen from the results in Table 1, the production of cellulose-like substances is significantly improved by the addition of inositol, phytic acid, phytic calcium salt, and phytic/(1 calcium, 4 magnesium, 2 ammonia salt).

Example 2 Table 4 Basic medium Sucrose 51 Potassium I phosphate 0.3%.

Soybean decomposition liquid, soybean whey,
Ajiso, rice bran decomposition liquid, corn soaked extract, dried yeast at 0.05'% total nitrogen, and 0.02% cabumino acid and phytic acid were added, and the medium was adjusted to -(5). The mixture was poured into a 500° wide-mouth reagent bottle with a 200ml/l cotton stopper and sterilized at 120°C for 20 minutes.

Acetobacter aceti subspice xylinum ATCC10 grown for 6 days at 30°C on a test tube slanted agar medium containing 0.5% yeast extract and 2% agar to the basal medium.
821 was inoculated into the above sterilized medium and statically cultured at 30°C for 5 weeks.

1 bottle - The cellulose-like material produced in each bottle was washed thoroughly by repeating the pressurized dehydration washing machine, dried at 105°C until it reached a constant weight, and then measured.

The results are shown in Table 2.

As can be seen from the results in Table 2 (soybean decomposition liquid known to contain phytic acid, flavoring liquid, wheat bran decomposition liquid, soybean whey, rice bran decomposition liquid, malt extract, corn soaking liquid) It can be seen that addition of natural substances such as yeast extract, yeast, and soyton improves the production of cell mouth 1) Joichisu-like substances.

Example 3 Casamino acid (DlfcrJ!!!) was added to the basal medium shown in Table 2.
Adjust the - of the medium containing 0.8% phytic acid and 0.05% phytic acid to 3.4, 5, 6.7, respectively, and make the medium 100 Wtl.
The mixture was poured into a 500 rfLt wide-mouth reagent bottle capped with cotton and sterilized at 120°C for 20 minutes.

Acetobacter aceti subspice xylinum ATC grown for 6 hours at 30°C on a slanted agar medium prepared by adding 2 g of agar to the medium with the above composition and adjusting the pH to 5.
C10821 was inoculated into the above sterilized medium and incubated at 30°C for 2 hours.
Cultured for a week.

Example 1 Cellulose-like material produced in each bottle
It was measured using the method.

The results are shown in Table 3.

As can be seen from Table 3, the initial stage of the culture medium is 4 to 5. It can be seen that the production amount of lurose material is high.

Example 4 5 pieces of issue clause, phosphoric acid! Add 50 Qm of medium containing 0.05 potassium to a small jar fermenter (total volume: 100 Qm).
0 inl) and sterilized at 120°C for 30 minutes.

Acetobacter aceti subspice was precultured for 3 days in a Sakaro flask using the above medium.

Xylinum ATCC 10821 was inoculated into the above-mentioned small jar fermenter, and the temperature was maintained at 30°C with low oxygen supply (2
Culture was carried out for 120 hours under conditions of 0 Orpm, 1/20 v, v, m ) and high oxygen supply (1200 rpm, 1/2 v, v, m ).

The cellulose substance produced in each jar of the I storage was measured by the method of Example 1.

The results are shown in Table 4.

Table 4 1. .

As can be seen from Table 4, hepta-.--sulfate substances are produced under both low-oxygen supply conditions and high-oxygen supply conditions, but the productivity of cellulose-like substances is higher under high-acid supply conditions.

Example 5 Saccharose 5T, phytic acid 0.10%, Cabumino acid (manufactured by Dife'>) 1.0%, 1 potassium phosphate 0.1
04.0.0 of magnesium sulfate heptahydrate to the basic medium of
Add each to make 3ch, 045%, 2ch, 4% p
70 m of each culture medium adjusted to H4 and H5 was placed in a glass petri dish with a diameter of 15 squares and sterilized at 120°C for 10 minutes.

Acetobacter aceti-y′buspicis xylinum ATCC precultured in a Sakalo flask using basal medium
10821 was inoculated at 10% volume into each of the above-mentioned Petri dishes and statically cultured at a culture temperature of 30° C. for 7 days.

) The cellulose-like material produced in each petri dish was thoroughly dialyzed in running water to remove anti-acid, and then dried at 105°C until it reached a constant weight, and then measured.

The results are shown in Table 5.

Table 5 As can be seen from the results in Table 5, magnesium sulfate (heptahydrate)
The production of cellulose-like substances increased with the addition of .

Example 6 Various sugar-containing natural squeezed juices in terms of sugar: 5 g, 1 potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05 g, general amino acids (taste chordates) 0.1%, phytic acid 0.11p)
Is medium 20011Lt was placed in a 5ooMl wide-mouth reagent bottle and 120tl was sterilized for 10 minutes.

The cells were inoculated according to Example 5 and statically cultured for 3 weeks at a culture temperature of 28°C.

) The main cellulose-like material produced was determined according to Example IK.

The results are shown in Table 6.

As can be seen from the results in Table 6, various natural juices It can be seen that it is used in the production of loin-like substances.

Example 7 Acetobacter xylinum IFo 3288 Acetopacter acetii IFo 3284 Acetobacter aceti subspicis aceti ATCC23747 Acetopacter aceti subspicis xylinum ATCC2
3769 Acetobacter acetisubspice xylinum ATCC23770 was inoculated by the method of Example 1.
The cells were statically cultured at ℃ for 4 weeks.

◇The production amounts of cellulose-based substances and insoluble polysaccharides were measured by the method of Example 1.

The results are shown in Table 7.

As can be seen from the results in Table 7, Acetobacter a productivity is improved.

Example 8 Yeast extract 0.3%, phytic acid 0.02%, monopotassium phosphate 0.3%, magnesium sulfate heptahydrate 0
．． Medium 2001, which was prepared by adding various saccharides and their mixing ratios to a basic medium adjusted to -5 with a composition of 0.3%, was placed in a 500-gold reagent bottle with a cotton stopper and sterilized at 120°C for 10 minutes.

Acetobacter aceti subspice xylinum AT precultured in a Sakaro flask using the basic medium of Example
Inoculate CC10821 into each wide-mouth reagent bottle and incubate at temperature 3.
The cells were statically cultured at 0°C for one month.

1. The cellulose material produced was measured according to Example 1.

The results are shown in Table 8.

As can be seen from the results in Table 8, cellulose substances are produced by each saccharide alone and in combination.

Example 9 Saccharose 5%, phytin 0.02%, cademyl pentanoic acid (manufactured by Difc Juku) 0.5%, phosphate 1potassium 0.
1%, magnesium sulfate heptahydrate 0.5% in a basic medium with a composition of 2.7.9-)
3-5:)-quinoline-4,5-dione (hereinafter abbreviated as PQQ, collected from Methylomonas Methylopura AJ 11131) 0.001% and culture medium 100F! Ll was placed in a 500 d wide-mouth reagent bottle with a cotton stopper and sterilized at 120°C for 10 minutes.

Acetobacter aceti subspice xylinum ATCC10 precultured in a Sakalo flask using basal medium
821 was inoculated into a wide-mouth reagent bottle and statically cultured at a culture temperature of 30° C. for 14 days.

A medium without PQQ was cultured in the same manner as a medium without PQQ.

) The produced cellulose ridge material was thoroughly washed by dialysis in running water, dried at 105° C. until it reached a constant weight, and then measured.

The results are shown in Table 9.

As can be seen from the results in Table 9, it can be seen that the production of cellulose-rich substances increases with the addition of PQQ.

Example 10 Sucrose 51 yeast extract 0.5%, phosphate I
0.5 chips of potassium, 0.1 chips of magnesium sulfate heptahydrate
3000 Wtl of medium with H5 composition, area 42 x 37 tan,
The mixture was placed in a stainless steel container with a height of 20 crn, covered with eight layers of gauze, and sterilized at 120° C. for 20 minutes.

Acetobacter aceti sutusbisis xylinum ATC precultured in a Sakaro flask using the same medium as above
C10821 was inoculated and statically cultured for one month at a culture temperature of 30°C.

Cut the cellulose material (approximately 1.2 cm thick) produced in the I-group into 5 cm x 5 crs, and add a 25an2 test piece of cellulose material, which had been subjected to the following various settling treatments, and 400 ml of water at approximately ℃. The mixture was placed in a 500R 1-capacity beaker and kept at temperature for one day (abbreviated as Method A). The test piece was placed in 1-NaOH and treated at 100°C for 2 hours (B
Abbreviated as law. ). The test piece that had been dehydrated under pressure was immersed in a 1% ethyl acetate solution and kept at 40°C for one day.

(Abbreviated as C method.) The pressurized and dehydrated test piece was immersed in a 10x Kitchen Haiku (manufactured by Kao Soap) solution for one day. (Abbreviated as method D.) A test piece that had been dehydrated under pressure was immersed in a 4% lauryl sulfuric acid no-liquid solution and kept at 50°C for one day. (abbreviated as method E) A pressurized and dehydrated test piece was immersed in a solution containing 0.1% lysozyme, 0.001% lysonuclease (manufactured by Sigma), and 0.005% deoxyrinuclease (manufactured by S1gma) Q at 40°C. After that, pronase P (manufactured by Kaken Chemical Co., Ltd.) was added at a concentration of 0.1% and kept for 16 hours. (Abbreviated as method F) A pressurized and dehydrated test piece was immersed in a -4 solution containing 100 r of penicillin G and kept at 35°C for 1 day. Then, sodium dodecyl sulfate was added to the solution at 1 liter and kept for 1 day. (
It is abbreviated as G method. ) The pressure-dehydrated test piece was immersed in a 1% dichloroacetic acid solution, treated at 100°C for 30 minutes, then washed with pressure-dehydrated water, immersed in a 0.01% trypsin solution, and kept at 40°C for 1 day. (abbreviated as H method) The results are shown in Table 10.

The cleaning results were visually observed and expressed using a four-step method.

Display method -: No calming effect +: Slight K leg + 2 I leg + 1+: Extremely leg As can be seen from the results in Table 10, the cellulose of the present invention is small! It will be appreciated that the cleaning of food materials and the removal of impurities can be accomplished in a variety of ways.

Claims (1)

[Claims] Cellulose, which is characterized by culturing microorganisms belonging to the genus Acetobacter and having the ability to produce cellulose substances in a medium supplemented with inositol or phytic acid, producing and accumulating cellulose and cellulosic substances in the medium, and collecting the substances. manufacturing method