JPS639827B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing alcohol, and more particularly to a method for producing alcohol from starchy raw materials using a new bacterial species, Aspergillus K27, or an amylolytic enzyme produced by the bacterium.

In recent years, interest in alternative energy has been increasing due to the rise in oil prices due to the decrease in oil supply. In particular, the production of alcohol from agricultural products such as sugarcane, corn, mackerel, and tapioca is attracting attention.

The present inventor took advantage of the fact that Aspergillus K27 or the amylolytic enzyme produced by the bacterium has an extremely high ability to saccharify various starchy raw materials,
We have discovered a method to efficiently produce alcohol without steaming by allowing K27 or an amylolytic enzyme produced by the bacteria and a microorganism capable of alcohol fermentation to act on various starchy raw materials, and have completed the present invention. Ivy.

That is, the gist of the present invention is to develop a new species that belongs to the genus Aspergillus and has different mycological properties from Aspergillus fumigatus in that (1) the color of the conidiophore is colorless, and (2) the growth temperature range is 10 to 55°C. The present invention resides in a method for producing alcohol, which is characterized in that the fungus species Aspergillus K27 or an amylolytic enzyme produced by the bacterium and a microorganism having alcohol fermentation ability are allowed to act on a starchy raw material.

Aspergillus K27 used in the present invention is a new strain belonging to the genus Aspergillus, and one of the strains, Aspergillus K27 AC-1,
It has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as No. 568 (Original Deposit No. 7158). Details of this strain are described in Japanese Patent Application No. 144544/1982, and its mycological properties are shown below.

Growth status on each medium (1) Malt extract agar medium Growth is good, with approximately 50% growth on the 3rd day at 37°C.
reaching a diameter of mm.

The basal hyphal layer is thin and flat. The colony surface is velvety to woolly. Colonies are initially white in color and turn green to dark green when a large number of conidia are formed. The underside of the colony is initially colorless and later becomes pale yellow.

(2) Tuapetsk agar medium Growth is good, about 45% on the 3rd day at 37°C.
reaching a diameter of mm.

The basal hyphal layer is relatively thin and flat. The colony surface is velvety to woolly. The color of the colony is white at first, and turns green when many conidia are formed.
It turns dark green. The underside of the colony is initially colorless and later becomes pale yellow.

Physiological properties (1) Growth range (using malt extract medium) PH: 3-8 Temperature: 10-55℃ (2) Optimal growth conditions (using malt extract medium) PH: 4-7 Temperature: 35-45℃ Morphology Scientific properties Conidial head: cylindrical, length 120-200μ. Diameter 30~
60μ, light green.

Conidiophore: length 150-300μ, diameter 2.5-8μ. Branches and stands up from basal hyphae or aerial hyphae. Smooth surface, colorless.

Apical capsule: 15-28μ in diameter, flask-shaped, pale green, forming a phialide from about the upper half.

Metre: Metre is not formed.

Phialide: 6.5-9.5 x 2-2.5μ, pale green.

Conidia: 2.5-3.0 μ in diameter, spherical to sub-spherical, rough surface,
The agglomerates are dark green.

In order to produce an amylolytic enzyme using the bacterial species of the present invention, a culture medium normally used for amylase production is used at 10 to 55°C, preferably 25 to 45°C, pH 3 to 8,
Preferably, solid culture or liquid culture may be performed for 4 to 7 days, and a significant amount of amylolytic enzyme is accumulated in 3 to 7 days.

Examples of carbon sources used in the liquid medium used to produce amylolytic enzymes include various starches, starch hydrolysates, corn meal, wheat flour, and blackstrap molasses. Examples of the nitrogen source used include peptone, cottonseed residue, meat extract, yeast extract, casein, cornstarch liquor, malt extract, soybean oil, skim milk powder, inorganic ammonium salts, and inorganic nitrates. Others: KH ₂ PO ₄ , FeSO ₄ ,
Inorganic salts such as MgSO ₄ , KCl, CaCl ₂ , CoCl ₂ , MnSO ₄ , and, if necessary, organic trace nutrients can be added to the medium. Moreover, ordinary wheat flour or the like is used as the solid medium.

The culture solution obtained in this way, the bacterial cells separated from the culture solution, the culture solution, the koji malt, and the extract of the koji malt can all be used as a crude enzyme.

The activity of the amylolytic enzyme obtained as described above is measured as follows: The enzyme solution is applied to a 1% soluble starch solution dissolved in a pH 4.5 buffer at 45°C, and after 15 minutes, the amylolytic enzyme produced is Somogyi Nelson (Somogyi Nelson)
Nelson) method. The titer that produces 1 μmole of glucose per minute under these conditions is defined as 1 unit.

Conditions for alcoholic fermentation in the present invention, such as temperature and pH, vary depending on the type of microorganism used for fermentation, but are usually 25 to 55°C and pH 3.5 to 8.0.

The microorganisms used for alcoholic fermentation may be any microorganisms that have the ability to perform alcoholic fermentation from glucose, such as Saccharomyces.
Examples include yeasts of the genus Zymomonas and bacteria of the genus Zymomonas.

The starchy raw materials used in the present invention include not only relatively pure starch (e.g., cornstarch, potato starch), but also grains, tubers, other seeds, etc. (e.g.,
It includes all kinds of starchy substances such as crushed, ground, and crushed products (corn, rice, wheat, potatoes, sweet potatoes, tapioca, white mackerel, ripe green bananas, and acorns).

The appropriate amount of amylolytic enzyme is 1 to 200 units per gram of starch.

Examples and Reference Examples are shown below, and isolation of the microorganism of the present invention from soil, production of an amylolytic enzyme, and production of alcohol will be specifically explained.

Reference Example 1 Soil collected at Gunmoto 1-chome, Kagoshima City was diluted 1,000 times with sterile physiological saline, 1 ml of which was mixed with 9 ml of the following isolation agar medium (), placed in a sterile shear dish, and incubated at 45℃ for 2 hours. Cultured for 1 day.

Isolation agar medium () % NH ₄ NO ₃ 0.1 MgSO ₄・7H ₂ O 0.02 KH ₂ PO ₄ 0.14 Yeast extract 0.01 α-RS ⁽¹⁾ 0.5 Agar 1.5 (PH6.1-6.3) Note (1) Wheat starch The insoluble starch obtained when the starch is liquefied is collected and freeze-dried.

Colonies generated by the above culture were transferred using a platinum loop to a slanted agar medium () having the following composition, and cultured at 45°C for 2 days.

Slanted agar medium () % Peptone 0.5 Yeast extract 0.3 Malt extract 0.3 Glucose 0.2 Agar 1.5 (PH7.0) Dilute one platinum loop of the bacteria grown on the medium by the above culture to 10,000 times with physiological saline, and add 1 ml of it. was mixed with 9 ml of the above isolation agar medium (2) and cultured at 45° C. for 2 days in a sterile petri dish, and it was confirmed macroscopically and microscopically that the multiple colonies that emerged were not different from each other.

Ten of the above colonies were each inoculated onto agar slants (2009) and cultured at 45°C for 2 days, and it was confirmed macroscopically and microscopically that the bacteria on the 10 agar slants were the same. Furthermore, it was confirmed that the properties and physiological properties on each medium of these 10 cultured bacteria were the same and as described above.

From this result, it can be seen that all 10 cultured bacteria are single bacteria that were purely isolated from nature.

Next, as described above, a protective agent (skim milk) was added to the pure cultured malt extract slanted agar medium.
10% and a 1% aqueous solution of sodium glutamate) to prepare a spore suspension. This spore suspension was dispensed into ampoules in 0.2 ml portions and freeze-dried.

The drying method is to slowly freeze to -30 to -40℃, then
Dry at room temperature at 0.03 torr for 18-20 hours. Then, it was vacuum sealed with a gas burner and stored at 4°C.

The freeze-dried bacteria thus obtained were reconstituted after 3 months. At this time, use sterile physiological saline as condensate.
A malt extract medium was used as the medium. The properties and physiological properties of the reconstituted bacteria in each medium were the same as before freezing.

Reference Example 2 Aspergillus K27 AC-1 was cultured on a slanted agar medium () at 45℃ for 2 days, and a loopful of the bacterial cells grown on the slant was taken and inoculated into 100ml of a medium with the following composition in a 500ml flask. did. 45℃
After culturing with shaking for 5 days, the culture solution was filtered and the amylolytic activity of the resulting solution was measured.
It was 12 units/ml.

Medium % Wheat starch 2.0 NH ₄ NO ₃ 0.1 Yeast extract 0.01 Corn staple liquor 0.08 KH ₂ PO ₄ 0.14 FeSO ₄・7H ₂ O 0.001 MgSO ₄・7H ₂ O 0.05 KCl 0.05 (PH6.1-6.3) Example 1 Raw 20 g of corn starch, 400 units of amylolytic enzyme obtained according to Reference Example 2, and 0.5 g of baker's yeast.
Pour into a 500ml fermenter and make up to 80ml with tap water.
The pH was adjusted to 4.6-6.0 and fermentation was carried out at 30°C.

The following shows changes over time in the fermentation ratio (produced alcohol amount/theoretical alcohol amount x 100).

Elapsed time (hr) 24 48 72 Fermentation ratio (%) 48.7 80.1 88.3 Example 2 33 g of ground corn, 1000 units of amylolytic enzyme obtained according to Reference Example 2, and 1.0 g of baker's yeast
was put into a 500ml fermenter, and the volume was made up to 100ml with tap water. The pH was adjusted to 4.6-6.0 and fermentation was carried out at 30°C.

The change in fermentation rate over time is shown below.

Elapsed time (hr) 24 48 72 Fermentation ratio (%) 56.2 80.7 86.5

Claims (1)

[Scope of Claims] 1 Belonging to the genus Aspergillus, Aspergillus
Fumigatos is a new species of Aspergillus K27 that has different mycological properties in that (1) the color of the conidiophores is colorless, and (2) the growth temperature range is 10 to 55°C, or the amylolytic enzyme produced by this fungus. A method for producing alcohol characterized by allowing microorganisms capable of alcohol fermentation to act on starchy raw materials.