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BULLETIN OF THE COLLEGE OF AGRICULTURE, TOKYO IMPERIAL UNIVERSITY, JAPAN
Vol. 2, No. 2, 1894-1897, P.68-72.

by K. Yabe

Since remote times these has been prepared in Japan from soya beans, a sort of vegetable cheese called natto.
The beans are fast boiled in water for five hours to render them exceedingly soft.
The still hot mass is in small portions wrapped in straw and the bundles thus formed, well tied at both ends are then placed in a cellar, in the middle of which a fire is kindled, whereupon the cellar is well closed.
The heat is left to act for twenty-four hours, after which the product is ready for consumption.
Although the moderate heat of the cellar acts only for twenty-four hours, there is still a considerable bacterial change going on.
The microbes may be derived either from the air or from the straw.
Of course it can nato be expected that bacteria on the surface of the soya beans would still very active.
They are probably killed by the five hour's boiling.
This product has a peculiar but not putrid smell.
The sort mase of beans is kept togather by a very thick viscid substance.
In this substance I have found four kinds of microbes present, and the chemical decomposition of proteids must be due to one or more of these microbes.

A trace of viscid liquid of the cheese was inoculated in a gelatine solution and a plate culture was prepared.
After a few days numerous colonies(1260) appeared, which 4 different kinds could be obserbed, and of these were prepeared pure cultures.
Three of these different cultures were formed by micrococci, and one by a small, not motile, bacillus liquifying gelatine and producing a greenish fluorescence.
It forms white flocculent masses on potatoes. and on soya beans it forms white colonies.
With regard to the micrococci the colonies of the three kinds can be distinguished by their colors: yellow, orange yellow, and white.

The yellow micrococcus belongs to the larger kinds.
In gelatine it forms white colonies along the canal.
At the end of canal concavity is formed.
The gelatine is in a small degree liquefied.
On agar, potatoes, and soya beans, it first forms white colonies that graduality turn yellowish.
On soya bensit develops the characteristic smell which is observed with natto itiself.
In 2% peptone solution it forms a white deposit.

The orange yellow micrococcus forms round colonies on geratine.
It has a general resemblance to the former but iti does not liquefy gelatine at all, and produces on soya beans a disagreeable smell,
While the former does not spread considerably on potatoes, this one does so forming a slimy cover.

The white micrococcus has a general resemblance, in regard to growth and development of its colonies, to the last named.
With convinced me that the above mentioned yellow micrococcus is the chief cause, while the regard the slimly substance which shows an enormous degree of viscidity farther experiments have to carried out; because the yellow micrococcus is not the cause of this viscidity.

As the soya bean is very rich in protein, the various decomposition products of proteids might be expected to be present in this cheese to a certain extent.
6.8 kilos. of the raw cheese were extracted with boiling water, and the aqueous solution precipitated with basic acetate of lead.
The filtrate from this precipitate was mixed withmercuric nitrate with the gradual additin of small quantities of soda as long as a precipitate was formed.
The precipitate, after being washed well on a filter, was decomposed by sulphuretted hydrogen, and the filtrate evaporated on the water bath, ammonia being, from time to time, added to keep the solution neutral.
In the concentrated liquid were formed after some time white crystalline masses composed of radiating needles of the forms characteristic of tyrosin.
They were easily soluble in dilute ammonia and in hydrochloric acid, slightly soluble in cold, and easily in hot, water.
they were purified by repeated recrystalizationm and then yielded the reaction of DIRIA WURSTER and HOFMANN for tyrosin.
The determination of nitrogen by KJELDAHL's method yielded 7.08%, while the theory require 7.75%.
Aldo the cooper compound was obtained by boiling solution with cooper hydrate and filtering while hot.
The total puanity obtained amounted to 3,212 grm.
The mother liquor from which the tyrosin was separated was further concentrated and divided two parts (a) and (b).
The Part (a) was precipitated with phosphotungstic acid after the addition of a little sulphuric ascid (c) and the filtrate mixed with caustic baryta to separate the sulphuric acid and phosphotungstic acid.
After the removal of the excess of baryta by a current of carbon dioxide, the filtrate was evaporated.
Numerous spherocrystals were obtained with the behavior of leucin mixed with the crystals of ammonium nitrate.
To remove the latter, a little baryta was added and by evaporations the ammonia was expelled.
When the residue was treated with alcohol, barium nitrate remained behind while the alcoholic solution on evaporation yielded crystals of leucin which were converted into the characteristic copper compound which yielded on analysis 19.76% copper, while the formula (C6H12NO2)2Cu requires 19.5%.

The Phosphotungstic precipitate (c) was first washed with cold water containing some sulphuric acid and then decomposed in the usual way with caustic baryta and the filtrate after removal of the excess of baryta evaporated, whereby a syrupy liquid was obtained.
I searched here for lysin, lysatinin (both discovered by DRECHSEL) and arginine (discovered by SCHULZE), which are the decomposition products of the proteins of the germinating lupine, but all my effort were vain.
The syrup gave, however, all reactions of peptone and consisted for the most part of this substance.

The above mentioned part(b) was treated with ammoniacal solution of silver nitrate whereby a small amount of a white precipitate was obtained which was collected on a filter and washed with dilute ammoniacal solution of silver nitrate then dissolved in warm nitro acid of sp. gr. 1,1 with addition of little urea Upon cooling, microscopical needles were obtained, which proved to be a mixture of the silver compound of guanine and of hypoxanthine.
After the removal of the silver with sulphuretted hydrogen, filtering and evaporating with the addition of a little ammonia, a residue was obtained, soluble with great difficult in water and alcohol but easily soluble in mineral acids.

It was treated with ammonia whereby a part was dissolved and a part not.
The latter gave the sharpe reaction of GAPRANICA for guanine.
When dried with nitric acid in a platinum dish it gave a yellow residue, which turned red on the addition of soda.
The former, i.e., the soluble part was obtained by evaporating the annmoniacal liquor.
When evaporated in platinum dish with nitric acid, and the residue treated with caustic potash, no coloration tookolace.
The reaction of WEIDEL and of CAPRANICA did not leave any doubt that this substance was hypoxanthine, but there was contained also xanthine in the cheese.
This was obtained by adding ammonia to the filtrate separated from the first crystallization of the guanine and hypoxanthine silver compounds.
By adding ammonia, a yellowishflocculent precipitate was obtained from which the silver was removed by sulphuretted hydrogen.
The filtrate then evaporated to dryness left a faintly yellowish powder slightly soluble in water, insoluble in alcohol and ether, but easily soluble in alkalies and acids.
On treating it with nitric acid a yellow residue was obtained turning red upon the addition of soda and purple on heating.
The reaction HOPPE-SEYLER and WEIDEL left no doubt that this substances xanthin.
Whether these substance of the xanthine series were formed by the bacterial action during twenty-four hours in the warmed cellar is doubtful.
I think it is more probable that they were originally present in the soya bean.
But there van be no doubt that large portion of peptone, and also leucin and tyrosin were products of the bacterial action.
Considering the high temperature of the warmed cellar, the considerable extent of the bacterial decomposition is not very surprising.
There can hardly be any doubt that natto-preparation is more easily digestible than the original soya bean, as its ice very soft and contains more peptone.
We add finally the determination of the different forms of nitrogen in soya bean and in natto.

	Soya bean	Natto Prepared from the same soya bean.
Total Nitrogen	7,355%	7,542%

Surface culture on agar: white, without lustre.
Stab culture in agar: much development on the surface, but very little along the needle track; no gas bubbles visible.
On potate: light brown, with minute foldings. In bouillon: kahmhaut white and lusterless, breaking easily on shaking.

The second microbe forms small colonies on bouillon agar.
It reality liquefies gelatin producing thereby an acid reaction.
Stab culture in agar: developing along the track, with chalk-like white color and showing formations like the Bac. mycoides.
On potato: grayish, with irregular foldings.
In bouillon: developing on surface white and instreless.
Solution remains clear.
On shaking, the skin does nat break and it sinks.

1) The composition of Miso was studied by O.KELLNER and M.NAGAOKA. Bul. Col. of Agr. Tokyo, Vol.1., No.6; and that of Natto by K.YABE., Vol.2, No.2

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