JPS6018393B2 - Method for recovering β-amylase from wheat starch production waste liquid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for recovering or further purifying 3-amylase contained in wheat starch production waste liquid using simple equipment and a simple method, that is, simply and at low cost.

Currently, wheat starch production waste liquid must be purified by the coagulation sedimentation method or activated sludge method before being discharged.
Therefore, the processing requires a huge amount of cost.

The present inventors have made efforts to develop a method for recovering 8-amylase contained in 4. barley starch production waste liquid, and as a result, they have been able to efficiently collect 8-amylase using simple equipment and a simple method, and have been able to collect 8-amylase from wheat, which was an unused resource. This has led to the establishment of a method that effectively utilizes starch production wastewater and significantly reduces wastewater treatment costs. As patents related to this technical field, there are already Patent Publications No. 57-52836 and No. 5 Patent Publication No. 52836.
There are 7-489 mice.

However, Japanese Patent Publication No. 57-52836 is characterized in that 8-amylase is adsorbed onto synthetic aluminum silicate and then eluted with a phosphate buffer;
No. 54 relates to a method of utilizing 4. barley starch production waste liquid as an amylase source, and both are completely different in technical configuration from the present invention. The present inventors discovered the following fact and arrived at the present invention.

That is, ■ 4. Use the waste liquid (usually pH 5.5 to 7.5) produced as a by-product during wheat starch production as it is, or if the pH is lower than that, adjust the pH to approximately the above range, and then add about 1% to it. Add about 1 to 12%, preferably 3 to 8%, of a diluted sodium alginate solution to the waste liquid, and adjust the pH to 3.7.
If the base degree is adjusted to ~4.9, preferably around 4.0, the liquid will coagulate.

■ Most of the 8-amylase is transferred to this aggregate.

(2) After the aggregate is dehydrated by far-D separation or pressure flotation, the pH should preferably be about 5 to 7.5, and can be solubilized by returning it to around 6 to 7. (2) When a calcium compound such as calcium chloride or an aluminum compound such as alum is added to this solubilized liquid to neutralize the mixture, aggregates are formed, but most of the 8 amylase activity is transferred to the liquid part.

Next, we will discuss the wheat starch production method that is currently commonly used.

The commonly used method is similar to the martin method, and more specifically, for example, a dough is made by adding about the same amount of water to wheat flour, and then about 1" volume of flour is added in a washing machine. Wash with water and separate into starch milk and raw gluten.
The starch milk obtained is separated, the bran fraction is removed, and a nozzle separator and a screw are used. -When centrifuged in a decanter, it is separated into starch milk and waste liquid. The amylase according to the present invention is transferred to and contained in this waste liquid section when wheat starch is produced according to the above method using the unfloured flour separated from near the aleurone layer of wheat seed grains as a raw material. , its potency is about 16-2 mountain units, and its solids content is about 0.8-1.0%. The above manufacturing waste liquid contains 0 to 0.5% insoluble matter due to the manufacturing process, but it can be further removed using a centrifuge with high separation efficiency (Sharpless centrifuge or Q Laval centrifuge, etc.) or furnace filtration. By clarification, the amount of sodium alginate added can be reduced.

Sodium alginate is the best flocculant used in the process of flocculating waste liquid (flocculation process), followed by carrageenin, and CMC is also effective, but as shown in Reference Example 1, sodium alginate is the best flocculant. inferior in comparison. Furthermore, this effect is not observed in tamarind seeds and guar gum. Reference example 1 (Method) Wheat starch manufacturing waste liquid (solid content 0.9%, 20u/skin [
) 1% sodium alginate solution, 1% carrageenin solution, and 1% CMC solution were added to 20% to 80% of each solution (2 to 8%).
%), the pH was adjusted to 4.0 with 1M diluted hydrochloric acid to form flocs, and the flocs were centrifuged (40%).
0 pm, 5 minutes) and collected as a cake, and this cake was neutralized and dissolved (pH 6.5) with dibasic sodium phosphate solution to bring the total volume to 100.

Table 1 below shows the amylase activity of the centrifuged and neutralized lysates. Table 1/1.Wheat starch production waste liquid [6] Reference example 2 shows a mutual comparison between the wheat starch production waste liquid and the isolated soybean protein production waste liquid, which is also known as a source of 8 amylase.

Reference Example 2 (Method) Wheat starch manufacturing waste liquid (pH 5.8, 2 mounds/secretion, solid content 0.9%) or isolated soybean protein manufacturing waste liquid (pH 5.8,
After adding 20 to 80 g of 1% sodium alginate solution (2 to 8%) in the evening, adjust the pH to 4.0 with diluted hydrochloric acid and coagulate. , centrifugation (4
00 pm for 5 minutes), the floc was collected as a cake, and this cake was neutralized and dissolved with dibasic sodium phosphate solution to bring the total volume to 100 pm.

Table 2 below shows the 8 amylase activity of the supernatant after centrifugation and the neutralized lysate. 2. /・Misaki starch anti-I”
'' and mutual comparison of separated soybean protein production waste liquids As shown in Reference Example 2, the wheat starch production waste liquid was better than the separated soy protein production waste liquid.

This seems to be because the waste liquid from isolated soy protein production contains considerably more water-soluble impurities. In the case of isolated soybean protein production waste liquid, increasing the amount of sodium alginate added can improve the flocculating effect, but the disadvantage is that the viscosity of the solution increases, making subsequent separation operations difficult. The content of the present invention is as follows when considering the behavior of amylase 8.

Wheat starch production waste liquid usually has a pH of 5 to 7, and 3-amylase in this is mixed with sodium alginate and fed with 4-amylase. By keeping the reamylase close to , it aggregates in a form bound to alginic acid, and by recovering the aggregates by means such as centrifugation or pressure flotation, it becomes possible to easily recover the rearamylase from the waste liquid. This is the first invention. Of course, coagulation will not occur even if the pH is adjusted to 4.0 without adding sodium alginate to the waste liquid. In the second invention, amylase is solubilized while containing sodium alginate by adjusting the pH of the aggregate containing 3-amylase recovered as an insoluble matter to around neutrality. By drying and powdering this solubilized product as it is using a fly mist dryer, a coarse
Amylase can be produced. However, although it has been solubilized, the viscosity of the solution is high in this state, and the pH is 4.
If it is less than 5, it will become insolubilized again and it will not be able to be passed through a sterile oven. However, by adding a calcium compound such as calcium chloride and/or an aluminum compound such as alum to the above-mentioned solubilized product and bringing the pH to around neutrality, 8-amylase is liberated again, and sodium alginate is insolubilized as calcium alginate and/or aluminum alginate ( gelation). This is the third invention, and in this case, there is almost no a-amylase activity in the infusible calcium alginate and/or aluminum alginate, and by separating and removing this insolubilized material by means such as a pressing furnace, calcium alginate and/or alginate The 3-amylase moves into solution in a state free of aluminum. This liquid can be filtered through a sterile oven, or it can be further purified by ultraconcentration without becoming obsolete at pH 4.5 or lower. The calcium compound and/or aluminum compound used when releasing 8-amylase from the above-mentioned solubilized material is in the stable pH range of 8-amylase (pH 3.8 to 7.8).
) Any material may be used as long as it is used within the range of ), such as calcium chloride, emulsified calcium, calcium phosphate, potassium alum, ammonium alum, aluminum chloride, etc., but from the viewpoint of solubility, calcium chloride and/or ammonium alum is preferable. It is like that. In addition, the cake without P-amylase activity, that is, calcium alginate and/or aluminum alginate obtained by the pressing furnace, is subjected to acid treatment, water washing, alkali treatment, etc. to remove calcium and/or aluminum, and is recovered as sodium alginate and used again. I can do it. The amount of the calcium compound and aluminum compound added is required to convert the entire amount of sodium alginate into calcium alginate and/or aluminum alginate.

In other words, if the amount of calcium compound and/or aluminum compound added is small, the resulting gel becomes viscous, making it difficult to use in the pressing furnace. This presence will hinder the subsequent purification process. However, since the presence of a certain amount of calcium actually helps stabilize 8-amylase, in the case of a calcium compound, it is best to add the entire amount of sodium alginate in excess of the amount to make calcium alginate. The enzyme solution obtained in this way can be processed using the usual concentration method.
For example, concentration may be carried out by ultraconcentration, vacuum concentration, salting out, precipitation with an organic solvent, or other means.

The content of the present invention will be described in detail below with reference to Examples.

Example 1 Flour Blue Ginkgo 500 manufactured by Nisshin Seifun Co., Ltd.
Add 400ml of water to 9 and knead with a kneader to make a dough 3
After being left for 0 minutes, it was transferred to a washing machine and washed with 4,000 sleeves of water to obtain 259 kg of raw gluten and approximately 3,700 sleeves of crude starch milk with a starch concentration of approximately 6.8%. This coarse starch powder is made from centrifugal milk (120
The red lees fraction was removed using a mesh), and then about 1,700 liters of starch milk with a starch concentration of about 14% and about 2,000 liters of waste liquid were obtained using a nozzle separator. This waste liquid has an amylase titer of 2
The solid content per mountain is 0.9%. This waste liquid (pH
5.7) Add 1% sodium alginate solution, which was previously dissolved in a separate pot, and adjust the concentration to 4.0 by thoroughly crossing the ocean with hydrochloric acid previously diluted, and then centrifuge this solution through a non-porous wall. A dehydrated cake 19k9 with a solid content of 25% was obtained through a machine, and dried in a vacuum dryer to obtain 4.7k9 (45%) of bran 8 amylase powder.
0 skin/obtained. Example 2 500 k9 of wheat flour made by Nisshin Seifun Co., Ltd. and 40 k9 of water
Add 0 soybeans and knead with a kneader to make a dough. Leave it for 30 minutes, then transfer to a washing machine and wash with 4000 ml of water.
Crude starch milk with 9k9 and starch concentration of about 6.8% approx. 3700
I got that.

The red lees was removed from this coarse starch milk using a centrifuge (120 mesh), and then about 1,700 ml of starch milk with a starch concentration of about 14% and about 2,000 ml of waste liquid were obtained using a nozzle separator. This waste liquid has an amylase titer of 20 units and a field type content of 0.9%. To this waste liquid (pH 5.7), add 1% sodium alginate solution 72Z, which was previously dissolved in a separate pot, and add the bait 4. while stirring thoroughly with diluted hydrochloric acid.
After adjusting to 0, the aggregates were recovered by a normal pressure flotation process to obtain 80 g of a paste-like liquid with a solid content of 6%. Add 400 ml of dibasic sodium phosphate to this and dissolve (axis 6.8, solid content 6.
6%, viscosity 80 specific p activity, 300u/night) dried in a nozzle dryer to obtain crude amylase powder 5.2k9 (41
Obtained 00u/. Example 3 400 g of dibasic sodium phosphate was added to 80Z of a paste-like liquid obtained by the same treatment as in Example 2 and dissolved (volume 6.8, solid content 66%, viscosity 80 specific p activity, 300 u / evening), then add 33ml of 20% calcium chloride solution to adjust the pH to 7.
After adjusting to 0, 85 ml of imari clarified liquid was obtained using a compressor filter.

Next, 35 liters of water was added to the residual liquid and squeezed again using the compressor filter to obtain 35 liters of clear liquid. Combine the clarified liquid (120 mm) using an ultraconcentrator (Kanebuchi Chemical Co., Ltd. PS-15 polysulfon membrane,
The standard molecular weight cutoff was 15,000) to obtain a concentrated solution, which was passed through a sterile oven and then dried in a spray dryer (inlet temperature: 15yo,
Purified 8 amylase powder 31k dried at outlet temperature 70℃)
g (6320 u/night) was obtained. Example 4 Add 400 g of dibasic sodium phosphate to 80 g of the paste-like liquid obtained by processing in the same manner as in Example 2 and dissolve it (volume 6.8, solid content 6.6%, viscosity 80 ratio p, After adjusting the pH of this liquid to 68 and gradually adding 33 g of 8% ammonium alum solution, it was filtered through a press furnace to obtain 85 g of clear liquid.

Next, 35 g of water was added to the residual liquid, and the mixture was filtered through a press furnace again to obtain 35 g of clarified liquid. Combine the clarified liquid (120) using an ultraconcentrator (Asahi Kasei Corporation SIP-3013 molecular weight cutoff 60)
00) to obtain a concentrated solution, which was dried in a sterile oven and a mist dryer (inlet temperature 155°C, outlet temperature 7°C) to obtain purified 3-amylase powder 2.0k9 (9310u).
'Evening) was obtained. (Note) Amylase titer 2% soluble starch 20 secretion Quantitated as glucose by the Fehringlehmann-Shaul method Reference: 1 unit indicates the titer that produces one glucose per gram of enzyme per minute.

Claims (1)

[Claims] 1. Adding sodium alginate to wheat starch production waste liquid,
A method for recovering β-amylase from wheat starch production waste liquid, which comprises adjusting the pH of the liquid to around 3.7 to 4.5 to aggregate β-amylase. 2 Add sodium alginate to the wheat starch production waste liquid,
The pH of the solution is adjusted to around 3.7 to 4.5, β-amylase is recovered as an aggregate, and the pH of this product is returned to around neutrality to solubilize it. How to recover amylase. 3 Add sodium alginate to the wheat starch production waste liquid,
The pH of the solution is adjusted to around 3.7 to 4.5, β-amylase is collected as an aggregate, the pH of this is returned to around neutrality and solubilized, and the pH is maintained at 5 to 7.5. A method for recovering β-amylase from wheat starch production waste liquid, which comprises adding a calcium compound and an aluminum compound to the waste liquid and purifying it to remove insoluble matter.