347 



bacteria. So it is inevitable that the tormation of indigo-reil is very f^reat in the case 

 vi the bacterial fermentation of the indican, while it is possible to rcduce its aniount 

 practicall)- to zero in the case of chemical deconiposition. As it is besides hardly 

 possible to esparate the indigo-blue from the substance of the bacteria, only an inipure 

 indigo can be obtained by nieans of their action. 



In consequence of the growth of Acrobactcr the reaction of plant extractions, 

 particularly of the indigo-plants, first becomes feebly acid, later feebly alkaline by 

 the formation of free alkali. This is also prejudicial to the prodiiction of indigo, as 

 in acid solutions the indoxyl oxidises very slowly, by which again much indigo-red is 

 fornied, vvhile at the same time part of the indoxyl gets lost in another way. 



Worthy of note is the influence of various sugars on the indican deconiposition 

 by Acrobacter. Mr. van Hasselt found that already.34 pCt. glucose, as well in liquid 

 cultures as in gelatine experiments, prevens deconiposition, while niuch larger quan- 

 tities, even to lOpCt. of cane-sugar, maltose and lactose have no effect at all and 

 levulose but very little. Evidently the very sugar produced by the splitting counter- 

 acts this splitting, while other sugars have not this efïect, or in less degree. To this 

 rule mannose makes an exception, as indican deconiposition is in the sanie way 

 counteracted by it as by glucose. This opposing influence gives consequently only 

 partly and not completely the answer to the question after the nature of the sugar 

 separated out of the glucoside by bacteria ')■ * 



There are however fornis of Acrobactcr which, in ferment-experinients, producc 

 unequal quantities of carbonic acid und hydrogen from glucose and mannose, and hy 

 their help it is proved that the sugar fornied from indican can only be glucose. 



Nitrates, also, have a remarkably opposing influence on the production of indigo 

 by Aërobacter. Common saltpetre is active already at '/ao pCt., which is in perfect 

 accordance with the antifermenting action of this salt in general, on which reposes 

 its use in the dairy industry, to prevent one the most important defects of cheese, in 

 Holland called »rijzers«. 



6. Indicaii-decomposition by the Indigo-ensymes. 



The indigo-enzvmes prepared from Iiidigofcra leptaslacliya, Polygoniim tiiic- 

 torium, Pliajus grandiffonis, aethyl-acetate-yeast (Saccharomyces sphaericus) and 

 emulsine of sweet aimonds, have been compared as to their intensity of action on 

 indican at difïerent temperatures, for which notable differences have been found. No 

 other group of enzynies is known to lead with equal ease and certainty to the deter- 

 mination of these relations as this group of the indigo-enzvmes. 



The experiments wereconducted as f ollows. Of solutions of abont 0.5 pCt. indican") 

 10 cc. were passed into equal test-tubes selected for the purpose. After heating them 

 te the required teniperature in a large beakerglass, arranged as waterbath, with 



') Mr. van Hasselt prepared the osazmi from the indican-sugar and found. after 

 recrystallisation from alcohol, the mclting point to be at io5° to 100° C, that is nearly 

 the same as that of glucosazf>n, which is 204°to205»C. But the melting point of 

 mannosazon is about as high. 



"■) Stronger solutions give no more exact results. 



