JPS59179093A - Saccharification of starch - Google Patents

Abstract

PURPOSE:Saccharification of starch is carried out by adding glycoamylase as well as acidic protease to increase the alcohol yield in the alcohol fermentation. CONSTITUTION:A starch resource such as wheat, barley, oats, rye, rice, corn, sweet potatoes, cassva or their processed products is combined with an appropriate amount water and steamed. Then, glucoamylase and acidic protease are added to the product to effect saccharification at 30-50 deg.C for 50-200hr. Both of glucoamylase and acidic protease originate from microorganisms such as rhizopus or aspergillus and the amount of the former is 20-200 units per 1g. of the starch, while that of the latter is 100-400 units. A yeast is inoculated, after the saccharification, to effect alcohol fermentation, resulting in increase of alcohol yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel starch saccharification method and a non-cooking alcoholic fermentation method using the saccharification method.

Conventionally, in the non-cooking alcoholic fermentation method.

Amylases such as α-amylase [Starch Science.

2H3), 210-221 (1974)),
Glucoamylase (JP-A-57-102189. JP-A No. 57-102189.
-186494) to saccharify starch and carry out alcohol fermentation.

As a result of repeated studies on efficient non-cooking alcoholic fermentation methods, the present inventors found that adding acidic protease to glucoamylase improves saccharification efficiency and significantly improves alcohol yield in alcoholic fermentation. This discovery led to the completion of the present invention.

The present invention will be explained in detail below.

The present invention provides a method for saccharifying starch by adding glucoamylase and acid protease to starchy raw materials.

Examples of starchy raw materials include grain starch such as wheat, barley, oats, and rye, forged corn flour, sesame starch, and forged mackerel flour. As starchy raw materials, it is also possible to use powders obtained by pulverizing these raw materials and passing them through a sieve, or starch extracted therefrom by a known alkali extraction method. For alkali treatment, add 0.2 to 3 to 7 times the amount of starch.
% NaOH was added, stirred, cooled, allowed to stand, and centrifuged.
Remove the supernatant and repeat the NaOH treatment again. This operation is repeated 3 to 7 times, the powder is washed with distilled water until it becomes neutral, and the resultant product is vacuum dried and used as alkali-treated vehicle n#* powder.

As the glucoamylase, glucoamylase produced by microorganisms such as Rhizopus and Aspergillus is preferably used. The amount of glucoamylase used varies depending on the type of starchy raw material, but generally it is 2 g per 1 g of starchy raw material.
Use 0 to 200 units.

Glucoamylase activity is 5. Using soluble starch as a substrate,
1 per ml of reaction solution for 10 minutes at pH 4, 5, 40°C.
The amount of enzyme that can produce glucose in (2) is defined as 1 glucoamylase unit (AU).

As the acidic protease, acidic proteases produced by microorganisms such as Rhizopus and Aspergillus are preferably used. The amount of acidic protease used varies depending on the starchy raw material, the amount of glucoamylase, etc., but generally it may be added at a rate of 100 to 400 units per gram of starchy raw material, and 1 to 20 units per 1 unit of glucoamylase.

Protease activity uses milk casein as a substrate and p
H3, 0, 1 ml per ml of reaction solution for 60 minutes at 40°C.
The amount of enzyme capable of producing 00r tyrosine is defined as 1 protease unit (PU).

The saccharification method involves dissolving the starch raw material in an acetate buffer.

Add glucoamylase and acid protease.

This is done by treating at 30-40'c for 50-200 hours.

The present invention also provides a non-cooking alcoholic fermentation method using the saccharide obtained in 1 above.・Alcoholic fermentation method is 1
It is carried out according to conventional batch or continuous fermentation methods.

For fermentation, commercially available compressed baker's yeast and alcoholic yeast (Sa
ccharomyces Cerell131ae)
Fermentation is carried out at a pH of 3 to 5 and a temperature of 25 to 35°C.The pH is adjusted using phosphoric acid, citric acid, hydrochloric acid, etc., but phosphoric acid is preferable.

The production of alcohol is 1. For example, Alcohol Handbook (edited by the Fermentation Industry Association, 5th edition, April 15, 1978) 12.
The analysis may be performed according to the method described on page 8, and the amount of alcohol produced can be estimated by the reduction in carbon dioxide gas.

Examples of the present invention are shown below.

Example 1 Saccharification of wheat 'fB flour by glucoamylase and acidic protease; Wheat starch (manufactured by Katayama Chemical Co., Ltd.) 100Kr, 0.1
M acetate buffer (pH 4,5 > 8 ml + tll
a Glucoamylase (manufactured by Seikagaku Corporation, derived from Rhizopus bacteria)■, 9AU (contains no protease activity)
Acid protease (Agr, Biol, Che
16.4 PU (which does not contain glucoamylase activity) and a small amount of thymol (as a preservative) were mixed.

The reaction was carried out at 38°C for 100 hours. DNS method (J.

The degree of saccharification was determined by quantifying reducing sugars using Biol, Chem, 47.5 (+921)). As a control, a system containing no acid protease or glucoamylase and a system using wheat starch treated with 0.2% NaOH were treated in the same manner and the degree of saccharification was examined. The results are shown in Figure 1.

In Figure 1, A uses a combination of glucoamylase and acid protease, and B uses only glucoamylase.

C uses only acidic protease, triangle mark indicates o, 2%
The experimental results using NaOH-treated wheat starch are shown, and the circles indicate the experimental results using NaOH-untreated wheat starch.

From FIG. 1, it can be seen that the addition of acidic protease significantly promotes the saccharification of @flour.

Example 2゜Alcoholic fermentation of barley powder by adding glucoamylase and acidic protease: Barley powder (Amagi 2-row Saga variety)
30 g of powder (pulverized using a sieve of 20 ml), glucoamylase agent (“GR-2” manufactured by Amano Pharmaceutical Co., Ltd., derived from Rhizopus spp.) 1
B or 36AU, acidic protease agent (Amano Pharmaceutical Co., Ltd. “Neurase G” 5 derived from Rhizopus spp.) O or 3
8PU, 3g of pressed yeast (manufactured by Oriental Yeast Industry Co., Ltd.),
Mix 0.05 g of potassium metabisulfite (as a preservative) and 80 ml of tap water, adjust the pH to 4.5 with 10% phosphoric acid, and shake at 38°C for 5 days (reciprocating shaking 100-11
0 times/min), and the decrease in weight due to the generation of CO2 was determined by weighing every 24 hours, and was converted to the amount of ethanol.

The results are shown in Figure 2. In the figure, D stands for glucoamylase*
18ΔU, mu 36AIJ), indicating that no acidic protea base agent was added.

From FIG. 2, it can be seen that the addition of acidic protease has a significant effect on alcoholic fermentation.

Example 3 Alcoholic fermentation of wheat starch; Wheat starch (treated once with 0.2% NaOH) 10
g, enzyme agent, tap water 50ml. 2g of pressed yeast.

Mix 0.025 g of potassium metabisulfite, 10%
Adjust the pH to 4.5 with phosphoric acid and shake at 38°C for 6 days (
(Reciprocating shaking 100-110 times/min) Lower fermentation.

The amount of ethanol was determined in the same manner as in Example 2.

The enzyme used has the following composition.

Table 1 Activity GHI Gluco7 Miller f (AU) 41.9 51.0 45
．． 6 acidic protease (PI) 72.8 16
．． 9 -α-amylase (LAu') 1425 1
411-* α-amylase activity was determined by reaction using potato starch as a substrate at pH 4, 5, and 40°C for 10 minutes.

Blue Value Method (J, Biochem, 4
1.583 (1954). 1α-amylase unit (LAU).

1% starch paste liquid 10i1 Blue Value 40
The amount of enzyme was determined to be reduced by 1% per minute at ℃.

The results are shown in Figure 3. In the figure, G, H, and I indicate tests using the enzyme compositions shown in Table 1 above.

The results show that with purified glucoamylase alone (1), almost no increase in alcoholic fermentation is observed, and a comparison of G and H reveals that acidic protease contributes to alcoholic fermentation.

Corn starch (manufactured by Katayama Chemical Co., Ltd.) instead of wheat starch
25mg, cassava starch 50■, sweet potato starch (manufactured by Katayama Chemical Co., Ltd.) 50■, rice starch [prepared according to the method of Starch Science, 20.99 (1973)] 25■ were used, and as the acid protease, Neurase G was used as Agr. , Biol, Chem, 31.7
]0 (1967) (1■
The degree of saccharification of these starches was examined in the same manner as in Example 1, except that 0.08 units of glucoamylase was used. The results are shown in Figures 4-6.

Figure 4 shows an example of the use of corn starch.
, 1 unit) is used together with glucoamylase (0.5 units), and L is the case where only acidic protease (4.1 units) is used.

Figure 5 shows an example of the use of rice starch. In Figure 1, M indicates glucoamylase (0.5 units) and acid protease (4.1 units).
N indicates the use of only glucoamylase (0.5 units), and 0 indicates the use of only acid protease (4.1 units).

Figure 6 shows an example of the use of cassava starch and sweet potato starch. In Figure 1, the circle mark is charas mackerel starch, the triangle mark is sweet potato starch, and P is glucoamylase (3,0 units) and acidic protease (
Q uses only glucoamylase (2,4 units), R uses acidic protease (4,9 units)
The case where only is used is shown.

[Brief explanation of drawings]

FIG. 1 shows the time course of wheat starch saccharification in Example 1. In the figure, A is a combination of glucoamylase and acidic protease, and B is a combination of glucoamylase and acid protease. C indicates the use of acidic protease only, and the triangle mark indicates 0.
2% NaOH-treated starch is used, and the circle indicates an example where NaOH-untreated starch is used. FIG. 2 shows the time course of alcohol fermentation using barley powder in Example 2. In the figure, glucoamylase (
・18AU, mu36AU) and acidic protease (38P
U) Combination 5 E is glucoamylase (018AU, △3
6AU) is used. F indicates that no enzyme agent was added. FIG. 3 shows the time course of alcohol fermentation using wheat flour powder in Example 3. G and H in the figure. (2) shows an example of the enzyme composition shown in Example 3. FIG. 4 shows the change in saccharification over time when corn starch is used. FIG. 5 shows the time course of saccharification when using rice starch. FIG. 6 shows changes over time in the saccharification of cassava starch and sweet potato starch. Patent Applicant (102) Kyowa Hakko Kogyo Co., Ltd. No. 1 (hours) No. 2 (hours) No. 81 (days) No. 3 (days) No. 001 20 40 GO8o 100
11 minutes (poetry) 5th ward I j island day 1 hour C hours 61 audience oo 1-20 Q 60 90
10° warped opening (time) 457-

Claims (4)

[Claims] (1) A method for saccharifying starch, which comprises adding glucoamylase and acidic protease to a starchy raw material to saccharify starch. (2) Starchy raw materials are wheat, barley, oats, and rye. 2. The method according to claim 1, wherein the method is selected from rice, corn, sweet potato and cassosapa, or processed products thereof. (3) A non-cooking alcohol fermentation method characterized by saccharifying starch by allowing glucoamylase and acidic protease to act on a starchy raw material, and performing alcohol fermentation using this as a raw material. (4) Starchy raw materials are wheat, barley, oats, and rye. 4. The method according to claim 3, wherein the material is selected from rice, corn, sweet potato, canola mackerel, or processed products thereof.