Action of Koji Extract Upon Gelatinized Starch

From the point of view of the sake brewer, the change which koji solution produces in the nature of starch is of the utmost importance, and it will on that account be needful to enter into somewhat minute details concerning its action under varying conditions of time and temperature. That a remarkable change does take place will be evident to any one who adds a few cubic centimeters of a filtered solution of koji to a quantity of thick starch paste, especially if the latter be at a temperature of about 45° to 50° C. Within a very short time, a few minutes at most, the paste or jelly which before would not have moved on inverting the vessel containing it, will become as liquid as water, and if the flocks of cellulose be allowed to settle, as transparent as water. This cannot be observed in the ordinary process of manufacture, but the change takes place, it is only disguised by the presence of a considerable quantity of insoluble matter. In order, therefore, to understand the chemical reactions involved in sake brewing, the first point is to ascertain the composition of the clear, transparent solution obtained as above described.

Using malt extract instead of koji extract a similar change would be observed, and the nature of the resulting solution has been very thoroughly examined by O'Sullivan*, and more recently by Brown and Heron^. The result of their investigations has been to prove that dextrin and maltose are the only products of the solution of starch by malt extract, and that the change may be represented by definite chemical equations, which are different according to the temperature at which the conversion takes place. Thus according to O'Sullivan when the malt solution is allowed to act upon gelatinized starch at the ordinary temperature of the air, or at any temperature whatever below 63°C, the reaction is presented by his equation A.

That is to say that the products of the reaction contain 67.8% of maltose and 32.2% of dextrin, and have a specific rotatory power of 170.6°.

Between the temperatures 64° and 66°C, the reaction is represented by the B' equation.

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*. Journ. Chem. Soc, 1876, vol. II., p. 125, also 1879. Trans. p.770. t. Ibid., 1879 Trans. p. 596.

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*. Journ. Chem. Soc, 1876, vol. II., p. 125, also 1879. Trans. p.770. t. Ibid., 1879 Trans. p. 596.

Between 67° and 70°C, equation B represents the reaction.

Soluble starch Maltose g-dextrin i

Between 70°C and the point at which the activity of the malt diastase is destroyed, the reaction is expressed by equation C.

Soluble starch Maltose ^-dextrin

Brown and Heron agree with 0'Sullivan in finding only maltose and dextrin as the products of the action of malt extract upon starch, but their experiments lead them to represent the proportions formed at different temperatures a little differently. There is also a difference in their theoretic views as to the weight of the molecule of soluble starch and the nature of the dextrins, but we may leave that aside. They imagine that the conversion of starch into maltose and dextrin is to be represented by nine difference equations in the following manner:

1 10 (C12H20O10) + H20 = C12H220jj + 9 C12H20O10 (Erythro-dextrin a)

2 10 (C12H20O10) + 2 H20 = 2 C12H22011 + 8 C12H20O10 (Erythro-dextrin B)

3 10 (C12H20O10) + 3 H20 = 3 C12H22011 + 7 C12H20O10 (Achroo-dextrin a)

4 10 (C12H20O10) + 4 H20 = 4 C12H22011 + 6 C12H20O10 (Achroo-dextrin B)

The series is continued through the intermediate equations.

8 10 (C12H20O10) + 8 H20 = 8 C12H22011 + 2 C12H20O10 (Achroo-dextrin Z)

9 10 (C12H20O10) + 9 H20 = 9 C12H22011 + C12H20O10 (Achroo-dextrin h)

Of these, the most stable is number 8 which represents the manner in which the reaction takes place at and below 63°C. They consider also that they have definite evidence of the existence of equations 4, 3, and 2, and indications of 5 and 6.

It will be seen that the weight of maltose formed at low temperatures is always greater than at high temperatures, a circumstance which has to be carefully attended to in the process of making beer, because it depends upon the proportion of sugar in the wort whether the brewed liquid will contain much or little alcohol.

The above mentioned observers have been able to obtain definite chemical equations because of the absence of any hydrating action of malt extract upon maltose. As, however, it has been shown in Chapter 4 that the solution of koji hydrates maltose, we cannot expect, even if that sugar is formed, to obtain results of the same sharpness as where the products first formed are unacted upon.

We have, therefore, in the first place to ascertain whether maltose is one of the products of the action of koji extract upon starch paste, and that it is so, will be shown by the following experiments.

To prove this an indirect method is resorted to, on account of the difficulty of isolating small quantities of maltose in a pure state from solutions containing much dextrin. The method adopted consists in determining the reducing action of the solution upon oxide of copper, and from the amount of cuprous oxide precipitated by a given weight of the starch products in solution (calculated from the specific gravity of the liquid) to find the weights of maltose and dextrin, assuming these to be the products. If no other substance is formed, the specific rotatory power of the solution calculated from the percentages of maltose and dextrin present will agree with the specific rotatory power of the solution actually observed. If they do agree the solution must contain the bodies assumed to be present, because if others were there, the specific rotatory powers would differ from one another. A detailed description of one experiment will render this more intelligible.

Experiment 7: A koji solution was prepared by digesting for a short time 25 grams of freshly prepared sample of koji in about 100 cc of water. The liquid was then filtered, the residue digested with a fresh quantity of water, and the whole thrown upon the filter and washed until the filtrate amounted nearly to 500 cc. The solution was then diluted exactly to 500 cc at 15°C. The filtration occupied three or four hours even with the assistance of a filter pump on account of the slimy nature of the insoluble matter. The solution so made contained in 100 cc 1.46 gram of solid matter calculated from the specific gravity (using the divisor 3.86), 1.0125 gram glucose, and caused an optical rotation of 8 divisions in a 200 mm tube. This gives a specific rotatory power:

5 grams of starch, previously dried at 100°C, were gelatinized with about 75 cc of water, the paste allowed to cool to 40°C, then mixed with 25 cc of the koji solution, and left for 25 minutes till it was quite clear. It was then rapidly heated to boiling, cooled, and diluted to 250 cc. 100 cc of this solution, after filtration, contained 2.15 grams of solid matter, and 0.63 gram glucose, determined by weighing the reduced cuprous oxide after ignition. The optical rotation in a 200 mm tube was 32.4 divisions.

As the koji solution contained in 250 cc of the starch products was 25 cc (i.e., one tenth) we must deduct the weight of solid matter and glucose contained in 10 cc of the koji extract from the weights above found in 100 cc of the liquid. The optical rotation must also be diminished by one tenth the amount caused by the koji solution alone. We thus get:

Solids in 100 cc formed from starch 2.15 - 0.146 = 2.004 grams Sugar, calculated as glucose 0.63 - 0.101 = 0.529 gram

Optical rotation 32.4 - 0.8 = 31.6 divisions

The percentage of sugar calculated as glucose is 26.39, and the maltose corresponding is 26.39 / 0.61 = 43.28%, and the dextrin, therefore, 100 - 43.28 = 56.72. If we calculate the specific rotatory power which a mixture of maltose and dextrin in these proportions ought to have we find it to be: [a] calculated = 216 x 0.567 + 150 x 0.433 = 187.4°

There is a difference between the two results of 3.4°, which is not more than might be caused by errors of experiment. If we assumed the solution to contain dextrin and dextrose, the specific rotatory power would be only 174°, a difference of nearly 17°. This experiment, therefore, shows that maltose and not dextrose, is formed.

Experiment 8: 5 grams of starch were gelatinized and after cooling to 40°C mixed with 25 cc of the same koji extract and kept at that temperature for 3/4 hour. An additional 25 cc of koji was then added and the whole allowed to remain at 40°C for 15 minutes longer, then boiled and diluted to 250 cc. After filtration the solution contained, deduction having been made for the koji added:

Solid matter 2.035 grams in 100 cc

Sugar (calculated as dextrose) 0.888 grams in 100 cc

Optical rotation 28.5 divisions

Hence [a]j observed 169.5°

The composition of the solution, assuming the sugar to be maltose is:

Maltose 71.54 %

Dextrin 28.46 %

The specific rotatory power calculated for this mixture is 168.8°, which agrees very closely with the observed number.

Experiment 9: With a solution prepared from different koji, using 50 cc of the koji solution containing 1.206 gram of solid matter per 100 cc, the following results were obtained from 5 grams of gelatinized starch kept for 2 hours at 10-15°C.

Maltose 70.0 %

Dextrin 30.0 %

Specific rotatory power observed is 174.0°.

Specific rotatory power calculated is 169.8°.

The two last experiments give results which correspond nearly with Brown and Heron's equation number 7:

10 (C12H20O10) + 7 H2O = 7 C12H22O„ + 3 C^H^O^ which requires 70.9 % of maltose and [ajj calculated = 169.2°.

Solutions in which maltose can be detected can only be obtained by making use of dilute solutions of koji and in comparatively small quantity. In by far the greater number of experiments the maltose which is at first formed is hydrated to dextrose by the excess of diastase present in the koji solution and as in the brewing operations a very large excess of koji is used, the brewer of sake has practically nothing to do with maltose, but only with dextrose. In this respect the brewing process in Japan differs from beer brewing in Europe and America, where the alcohol is fermented for the most part from maltose.

The following experiments will serve to illustrate the production of dextrose and dextrin only. The mode of recognizing the nature of the products is the same in principle as that used to identify maltose vis a comparison of the observed specific rotatory power with the number calculated from the percentages of sugar and dextrin, assuming in this case the sugar to be dextrose with a specific rotatory power [ajj = 59°.

Experiment 10: 20 grams of dry starch gelatinized and 200 cc of a solution of koji (prepared from 50 grams in 500 cc of water) diluted to one liter were heated to 30°C for 6 hours, then allowed to stand for 2 hours at 15°C. The solution contained in 100 cc (deduction having been made for the koji extract) 1.96 gram of solid matter and 1.68 gram glucose, with a rotation of 12.8 divisions. This gives:

Dextrose 85.7 %

Dextrin 14.3 %

Specific rotatory power observed is 79.0°. Specific rotatory power calculated is 81.4°

Experiment 11: 4 grams of gelatinized starch and 96 cc of koji solution (20 grams of koji in 500 cc of water) were heated at 45°C for 3-1/2 hours, then evaporated to about 200 cc and diluted to 250 cc. The composition of the solid matter in solution, after deducting the koji extract, was:

Dextrose 86.0 %

Dextrin 14.0 %

Specific rotatory power observed is 85.7°.

Specific rotatory power calculated is 81°.

In both experiments, therefore, dextrin and dextrose are the only products.

Experiments were next directed towards ascertaining the degree of rapidity with which the conversion of starch into dextrose took place at different temperatures. For this purpose it was necessary to allow the mixture of starch and koji solution to react for some time, and to ascertain the composition of the solution, or its specific rotatory power, at different stages. Setting out in one direction the duration of the digestion, and in a direction at right angles to this the specific rotatory power of the solution at stated intervals, the progress of the action can be represented by a curved line, using the specific rotatory power as a measure of the change which has occurred in a given time.

The first series of experiments was carried out at the temperature of the air, which at that time varied between 4° and 10°C.

Experiment 12: 450 cc of starch paste containing 11.43 grams of dry starch were mixed with 50 cc of koji extract. The whole was allowed to stand at this temperature with an occasional shaking and samples were withdrawn after 48, 120, 192, and 240 hours, respectively. After making a deduction for the 50 cc of koji solution used, the amount of solids in 500 cc and the optical rotation were found to be as follow:

Table 15: Action of Koji on Starch at 4-10°C

Time

Total starch in solution (koji deducted)

Specific rotatory power of starch

48 hours

9.714 grams

109.6°

120 hours

9.904 grams

100.2°

192 hours

10.369 grams

90.4°

240 hours

10.450 grams

80.4°

The curve illustrating this series of experiments is seen in Figure 4. The action upon the starch, as indicated by the fall in the specific rotatory power, is more rapid at the begin ning of the experiment, but afterwards proceeds in a regular and continuous manner during the remainder of the experiment.

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