Preparation of Koji

Starch is a substance insoluble in water and incapable of undergoing fermentation directly, that is, of being converted into alcohol. In beer-making countries the conversion of the starch into a sugar from which alcohol can be produced is effected by the use of malt, a body formed by allowing the embryo of the barley grain to become partially developed, by which a change in the character of the grain occurs, as the result of which it becomes possessed of certain properties attributed to the existence of a hypothetical substance known as "diastase". The peculiarity of "diastase" is that it is a body containing nitrogen and having the power of rendering thick starch-paste liquid owing to the formation from it of the sugar maltose together with dextrin. Other kinds of diastase occur, as for example in the saliva, and in the pancreas, and these forms, although they resemble in some respects the diastase contained in malt differ from it in other particulars. Thus, the diastase of malt is not able to cause maltose to take up water and so be converted into dextrose, but both the diastase of the saliva and of the pancreas effect the hydration of maltose and change it into dextrose. It is evident, therefore, that different kinds of diastase exist, and that it is not one substance only which possesses these properties. As the material koji is employed in the manufacture of sake, and as it is used for the same purpose as malt in beer breweries it becomes necessary to examine it in some detail that we may ascertain how far it agrees with, and how far it differs from other similar bodies.

Koji is prepared both in breweries and in special works, as it is used for various purposes besides sake making. It will be most convenient for us to examine the mode of manufacture in the special koji works, as there will be found the conditions essential to its successful production more readily than in the sake breweries. I am especially indebted to Mr. Jihei Kamayama of Yushima, Tokyo, for much information as well as for permission to investigate at his works the whole process of manufacture.

The essential part of the process is carried out in long narrow passages cut in the solid clay about 15 or 20 feet below the surface of the ground. The object of this is to have a chamber which being once heated will not easily lose its heat either by radiation or by conduction. That this result is produced by cutting the chambers in the clay is shown by the constancy of temperature which they are found to possess even when considerable changes take place in the temperature of the outer air. Clay is a very bad conductor of heat, and it is practically impossible for heat to be communicated either to or from these passages through the clay. The passages are about 25 or 30 feet in length, and each set is reached through a very low and narrow one---made so for the purpose of preventing as much as possible an exchange between the outer and inner air. The opening passage is not more than between 3 and 4 feet high and about 4 feet wide, and is usually closed with mats. It is approached by descending a shaft from the ground above, and at the other end it opens into a passage of somewhat larger dimensions, from which two others branch off nearly at right angles. It is in these innermost parts that the highest temperature is maintained. In the sake-breweries the warm chambers are less carefully constructed, being built near the surface of the ground of wooden planks coated with mud and thickly covered over with straw mats. This is evidently a less perfect method of keeping in the heat than that adopted in the koji works proper. Having described the apparatus used we may now consider how the rice is treated. It is brought to the works husked but not cleaned, and the process of cleaning or whitening, is done by the manufacturers. This consists in removing that thin outer skin, the testa, which, as we have seen, contains a large proportion of cellulose and mineral matter. It is removed by the brewers, as they say, because it would render the liquid brewed very liable to putrefy. In removing the bran the rice suffers a considerable loss of weight, owing not only to the loss of the testa, but also to the fact that many of the grains become broken and are rejected on that account. In most places the cleaning is effected by human labor. The rice to be cleaned is placed in a wooden mortar sunk in the ground, and a heavy wooden hammer supported upon a fulcrum is so arranged that on pressing down the side of the lever away from the mortar and then removing the pressure, the heavy end of the lever falls by its own weight into the mortar. As it falls it causes the grains of rice to rub against one another and so the skin becomes scraped off. The loss of weight varies according to the degree to which the cleaning is carried; that which is used for the preparation of koji and of moto (called moto-mi) loses from 30 to 40 percent of its volume, whilst the kake-mi, used in the stages designated soye, naka, and shimai, is not so thoroughly cleaned and loses only about 25 percent of its volume. The numbers given are, of course, only approximate for, in every operation the percentage of loss must be different. The pounded mass is separated into three portions---the whole grains---the broken grains, and the bran. The whole grains are employed in the manufacture of koji and sake, the broken grains are sometimes made into an inferior kind of koji, but generally, like the bran, are sold to other persons. The amount of bran obtained is said to be about 3 kuwamme (25 pounds) for every koku (4.96 bushels) of rice cleaned.

In some works (sake-works) steam power is employed to work the cleaners, and in other places water power is used.

The rice is next placed in a tank, covered with water, and from time to time trodden upon by the workmen, the water being frequently changed. The fine dust which was adherent to the grain is carried away by the water, but the amount of matter thus lost, although sufficient to make the water milky is not known. After this washing the grains is left in steep for one night by which it becomes quite soft and is ready for steaming. The object of the steeping is merely to render the grain soft so that the subsequent steaming may be as short as possible. It is therefore, not analogous to the steeping of the barley-grain in making malt, an operation which is required to promote the germination of the embryo. In the case under consideration, indeed, the embryo has been completely destroyed by the rough beating, and no subsequent germination is possible. It is important to remember this, so that it may be clearly understood in what respects the manufacture of koji differs from that of malt. But even were the embryo not removed by the process of cleaning, it would be completely killed by the next operation, that of steaming. The soaked rice is placed in a large tub which is provided with a false bottom covered with cloth; the tub is then fixed upon an iron boiler full of water. When the water boils the steam passes through an opening in the true bottom of the tub, and as it ascends through the rice which is placed upon the cloth covering the fast bottom, it heats the grain and causes the starch to become gelatinized. The grains of steamed-rice are flexible and of a horny appearance, and must be the same throughout. In this state the rice is called mi. It is now spread out upon mats to cool, and during this time the workmen prevent the grains cohering by rubbing them between their hands. When the temperature has fallen to about 29°C the foreman mixes with the rice a small quantity of tane, a yellowish powder consisting of the spores of a fungus described by the late Mr. Ahlburg under the name of Euro-tium oryzeae (Ahlb).* The quantity employed is not exactly the same in different works, but averages about 3cc to 4 to (72 liters) of rice.

The subsequent operations vary a little in different works but not in any essential particulars. I shall, therefore, only describe them as carried out in the koji works at Yushima, Tokyo.

The spores are in the first place thoroughly mixed with two or three handfuls of the rice, and this mixture is then scattered over the whole quantity of steamed rice; the corners

*. Mittheilungen der deutschen Gesselschaft fur Natur and Volkerkuneostasiens. 16tes. Heft. 1878.

of the mats are turned up so as to collect the whole into a heap in the middle which is afterwards again spread out, and these operations are repeated several times to ensure that the spores shall be uniformly distributed. The rice mixed with fungus spores is then carried below to the front part of the chambers where the temperature is not high, and is there allowed to remain one day covered with mats. On the second day the temperature of the mass is about 25 or 26°C so that it is rather lower than when the spores were mixed with it. About noon of the second day (calling that on which the admixture with spores took place the first day) the rice is put into baskets and carried above where it is sprinkled with water. In the evening of that day the mixture is spread out in thin layers upon wooden trays called koji-buta which are carried to the innermost part of the subterranean passage and placed upon the floor underneath the benches which bear the koji of the third day. The trays are allowed to remain in this position from about 5 p.m. on the second day until about 5 a.m. on the third day, by which time the previous batch of koji on the benches has been removed, and the new batch is then put in its place. The mixture of rice and spores which was previously spread out in a think layer over the tray is at this time (5 a.m. third day) collected into a heap on each tray and left until between 9 and 10 a.m. During this time the temperature rises considerably and, by the vegetation of the fungus, the grains are bound together. In order to prevent the temperature rising so high as to injure the vitality of the plant, the workmen cools the mass by spreading it out in a thin layer and leaving it for some time. After it has become somewhat cooler he again collects it into heaps and leaves it until about 1 p.m. at which time it has once more attained a temperature nearly as high as at 9 or 10 a.m. after which it is spread out and repeatedly worked with the hands during the rest of the day. Between 8 o'clock in the evening of the third day and 5 a.m. of the fourth day the fungus still continues to grow, sufficiently to bind the whole mass together and to the tray. At 5 a.m. it is removed from the chamber and preserved on the trays until required for use.

In the manufacture of koji for sake-making the sprinkling with water on the second day is omitted, and the project is then called ki-koji (raw koji).

The formation of koji is an illustration of the growth of the mycelium of a fungus which uses the starch of the rice grain as food. In plants which possess chlorophyll and develop in sunlight two processes go on, assimilation and respiration. The former is accompanied by a fixation of carbon contained in carbonic acid under the influence of the sun's rays, and by the simultaneous liberation of oxygen. In this way the majority of plants add to their substance. At the same time the second process, respiration, goes on, but to a smaller extent than the former: it consists of an oxidation of the tissues of the plant, carbonic acid being liberated. This is the only process which goes on in plants destitute of chlorophyll, the green coloring matter of plants, and it can be well observed to take place in the growth of the koji fungus. This process of respiration, or oxidation, as a chemist might call it, is accompanied by a remarkable development of heat sufficient to keep the temperature of the koji and of the chamber very high. The following temperature observations will show this---the first series was made in spring when the amount of koji being made was very small, and the outside temperature not very much below that of the chamber. In the second series of observations, made in December, the differences are much greater, the temperature of the outside air being very low, and that of the koji much higher. During the month in which these observations were made the amount of material produced is very large, and the chambers are kept fully worked: it is owing to this circumstance that the differences of temperature between the koji and that of the chamber are so much more marked than in May.

Table 5: Temperatures of Koji and Chambers in May. Koji of the Third Day Only

Date

Hour

Temperature

Temperature of the koji chamber

Temperature of the koji (3rd day)

of the outer air

Minimum

Maximum

May 18 th

8 a.m.

55.3° F.

72° F.

76° F.

No koji

6 p.m.

61.8

72

74

May 19 th

7 a.m.

59.0

72

77

89.6° F.

8 p.m.

64.0

74

76

May 20th

8 a.m.

57.7

76

77

84.2

9 p.m.

64.6

75

77

May 21st

7 a.m.

60.5

75

76

9 p.m.

65.0

74

76

86

May 22nd

9 a.m.

68.6

75

77

86

9 p.m.

60.0

76

79

89.8

May 23rd

7 a.m.

65.5

77

88

8 p.m.

65.0

79

82

95

May 24th

7 a.m.

64.0

80

81

102

8 p.m.

66.5

78

80

86

A careful examination of the second series of temperature observations will enable us to trace the growth of the fungus very clearly. The temperatures of the koji at various times in the day have been arranged and are given in the table below.

Until 1 p.m. in every case the temperature of the koji is above 100° F., and after 1 p.m. in every case it falls below that point. The period of most active growth is, therefore, in the morning, and corresponds with the time during which the material is heaped up in masses. The effect of opening out the masses of koji will be best seen in the temperatures taken on Dec. 6th. At 8 a.m. the temperature was 106.6° F. and it continued to rise a little until between 9 a.m. and 10 a.m. when the workman broke open the heaps and spread them out. The temperature taken at 10 a.m. shows that the mass had cooled down 5.6° F. After this

Table 6: Temperatures of Koji and Chambers in December. Koji of the Third Day Only

Date

Hour

Temperature of the outer air

Temperature of the koji chamber

Temperature of the koji (3rd day)

Minimum

Maximum

Observed

December 5th

8 a.m.

40.7° F.

-

-

82° F.

104.8° F.

2 p.m.

49.5

82° F.

83° F.

82

91.9

8 p.m.

42.5

81

83

81

88.8

December 6th

8 a.m.

41.5

80

83

83

106.6

10 a.m.

44.7

81.6

82

81.6

101.0

1 p.m.

50.0

81

82.5

81.5

104

December 7th

9 a.m.

38.5

80

82.5

81.5

104.2

2 p.m.

51.0

80.5

82

81.5

93.6

December 8th

8 a.m.

37.5

79

82.5

80

100.0

Table 7: Temperature of Koji on Third Day

Hour

Dec. 5

Dec. 6

Dec. 7

Dec. 8

8 a.m.

104.8° F.

106.6 ° F.

-

100.0 ° F.

9 a.m.

-

-

104.2° F,

-

10 a.m.

-

101.0

-

-

1 p.m.

-

104.0

-

-

2 p.m.

91.9

-

93.6

-

8 p.m.

88.8

-

-

-

the mixture was again made up into heaps and at 1 p.m. the temperature had again rise, though not quite so high as at 8 a.m. After the heaps have been broken down between 1 p.m. and 2 p.m. the rice continues to cool; on the 5th the temperature at 2 p.m. was 91.9° and at 8 p.m. had fallen to 88.8°F. The object, therefore, of the working of the mass is not so much to prevent the grains becoming too much matted together as to regulate the activity of the growth of the plant. If the grains were allowed to remain heaped up during the whole time, there would be a danger of the temperature rising to too high a point, and perhaps rendering the product useless, whilst if the grains were never collected into heaps, the temperature would not rise sufficiently high to allow the growth to go on vigorously.

The amount of heat generated during the growth of the fungus is remarkable, and will be best appreciated from the observations made in December. At that time the temperature of the open air in the shade varied between 38° and 51° F., whilst in the subterranean chamber the temperature of the air was very nearly constant and very much higher than that of the open air. The growing chamber is not artificially heated except at starting—that is, after having been disused for a considerable time. It is then heated by the introduction of barrels containing hot water, but after that, all the heat it receives is derived from the combustion of the rice, and the liberation of its carbon and hydrogen in the form of carbonic acid and water. That carbonic acid is formed in large quantity is shown by the rapid removal of the oxygen from a confined portion of air by the actively growing plant. A handful of the mixture on the trays was put into a bottle holding about 3 liters of air, and the bottle was then tightly closed with a cork through which tubes passed by means of which a sample of the air in the bottle could be forced out and collected for analysis. During the time the bottle remained in the chamber the ends of these tubes were closed with aoutchouc tubes and pinch-cocks. The bottle was allowed to remain at the temperature of the chamber for four hours, at the end of which time it was found that the whole of the oxygen in the three liters of air had been replaced by carbonic acid. The grains of rice in the bottle remained loose, whilst those on the trays exposed to the free air of the chamber were matted together. From this it may be inferred that the quantity of oxygen contained in the bottle was insufficient to generate the heat required by the fungus for growth, which, therefore, ceased as soon as all the oxygen was consumed.

The oxidation which goes on during the growth of the fungus, and by which the heat is generated, is effected mainly at the expense of the starch contained in the cells of the grain. Figure 2 represents a section of a grain of koji cut perpendicularly to the long axis, and shows that the cellular divisions at the circumference are almost lost, whilst in the center they are pretty distinct. Very few grains of starch, however, can be distinguished, only those which have resisted gelatinization during the operation of steaming: the starch is there, but cannot be distinguished, on account of its homogeneity. The following analysis of koji (A and B) will indicate its general composition, although as will be explained later on, the amount of the soluble matter varies under different treatment even with the same specimen a fact which accounts for the large percentage of starch in one specimen and the small amount in the other. The composition is given of the material after deducting the percentage of moisture lost by drying at 100°C.

Table 8: Composition of Koji Dried at 100° C.

Table 8: Composition of Koji Dried at 100° C.

Dextrose

25.02 %

58.10 %

Soluble in water

Dextrin

3.88

(B) 69.45%

Soluble ash

0.52

0.54

Soluble Albumenoids

8.34

6.40

1.83

Insoluble ash

0.09

0.04

Insoluble in water

Starch

56.00

26.2

Cellulose

4.20

1.94

Fat

0.43

0.50

99.98

99.96

Water in original koji 25.82 28.10

Water in original koji 25.82 28.10

Figure 2: Section of the koji grain perpendicular to the long axis. x 364

Figure 2: Section of the koji grain perpendicular to the long axis. x 364

Comparing these with the analyses of whitened rice given on page 10 it will be observed that the amount of starch present is much reduced. This is due to its conversion into dextrose and dextrin, which has been mainly effected during the solution in water, owing to an active agent contained in the koji of which more will be said hereafter. The percentage of starch which would correspond to the dextrose, dextrin. and starch given in the first analysis is 82.4%, a number very closely agreeing with that which the rice dried at 100° C actually contained. The actual loss of material during the growth of the fungus cannot be determined, therefore, by an analysis of the koji, although the increase in the total amount of albumenoids indicates that there has been a loss of some of the other constituents of the grain. The large proportion of soluble albumenoids will strike every one, but as this is connected with the existence of a kind of "diastase" contained in the koji, it will be referred to in connection with the properties of that body.

The loss of material caused by the growth of the fungus is evident when we consider the weight of koji formed from a given weight of rice. Mr. Jihei Kamayama was kind enough to make careful weighings of the rice used and of the resulting koji. The result obtained was that 3 to of whitened rice which weighed 11.43 kuwamme yielded 12.38 kuwamme of koji, or 100 parts by weight of the rice gave 108.3 parts by weight of koji. The rice contained 14.2% of water, and the koji contained 29.5%, therefore, deducting the water from each, we find that 85.8 parts of dry rice gave 76.4 parts of dry koji, equal to 89%, or in other words, 11% of material was lost by the dry rice. This loss is probably nearly all starch, and if so, every 100 parts of rice converted into koji would evolve nearly 18 parts of carbonic acid. Now 107 pounds of dry rice are converted into koji every day in each chamber, and thus evolve 19.2 pounds of carbonic acid measuring 2240 liters. The total capacity of each chamber cannot be more than 20,000 liters, and therefore in order to remove the carbonic acid formed a constant circulation of air is necessary. If this were not provided for the air would not only become irrespirable by the workmen, but would also become unfit for the growth of the plant which requires a supply of oxygen. At the same time care has to be taken that the current of fresh air is not sufficiently rapid to lower the temperature of the air within the chamber. The mode of ventilation depends upon the difference in temperature between the inner and the outer air, the inner air being warmer rises up a square shaft at the front end of the series of passages, whilst the cold air bringing fresh oxygen enters and flows along the floors of the chambers, until in its turn it is warmed and rises through the shaft to the air above. This method is amply sufficient during winter when the difference of temperature between the air outside and inside is about 40°F, but when, as in the spring and early summer the difference becomes less than 10°F, frequent stoppages occur. This, perhaps, might be remedied by burning a small fire at the foot of the shaft, and thus artificially causing a draft, but as a smaller quantity of koji is required in summer, it is not of so much importance.

In the germination of barley Day* has shown that an amount of oxygen is absorbed by the grain greater than is required to produce the carbonic acid liberated and he concludes that this increased absorption of oxygen is not connected with the liberation of carbonic acid. Whether a similar absorption occurs in the present case is not known, but if, as is not improbably, it does occur, the amount of starchy material lost by the rice during the conversion into koji will be even greater than that given above. The amount of carbon oxidized during the germination of the barley grain is said by Day to be about 2.5 percent., and he finds that there is a pretty constant relation between the carbon oxidized and the water formed, which averages 12 carbon to 18.28 water. Thus for every atom of carbon oxidized one molecule of water is liberated, a ratio which would agree with the formula for dextrose C6H12O6 or in its simplest form CH2O. Possibly a similar relation may be observed in the case of koji; that a large liberation of water does occur is evidenced by the increased percentage contained by the koji compared with that in the rice, and also by the moisture of the atmosphere in the chamber. If however, a fixed relation were to exist it would be hidden owing to the moistening of the rice which takes place on the second day; in the instance just discussed the ratio between the weight of carbon burnt and water contained by the koji in excess of that contained in the rice at starting is very nearly 12:24 or 3 atoms of carbon to 4 molecules of water; an amount of water greater than corresponds to the formula for dextrose.

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