Chapter 10

These constitutional problems are followed up in (3) by the indirect method of differentiating the relationships of these furfuroids to yeast fermentation, from those of the pentoses. Straw and esparto celluloses are subjected to the processes of acid hydrolysis, and the neutralised extracts fermented. With high furfural numbers indicating that the furfuroids are the chief constituents of the extract, there is an active fermentation with production of alcohol. The cupric reduction falls in greater ratio to the original (unfermented) than the furfural. Observations on the pure pentoses--xylose and arabinose added to dextrose solutions, and then exposed to yeast action--show that in a vigorous fermentation not unduly prolonged the pentoses are unaffected, but that they do come within the influence of the yeast-cell when the latter is in a less vigorous condition, and when the hexoses are not present in relatively large proportion.

(4) The observations on the growing plant were resumed with the view of artificially increasing the differentiation of the two main groups of carbohydrates. From a portion of a barley crop the inflorescence was removed as soon as it appeared. The crop was allowed to mature, and a full comparison instituted between the products of normal and abnormal growth. With a considerable difference in 'permanent tissue' (13 p.ct. less) and a still greater defect in cellulose (24 p.ct.), the constants for the furfuroids in relation to total carbohydrates were unaffected by the arrested development. This was also true of the behaviour of the hydrolysed extracts (acid processes) to yeast fermentation.

(5) The extract obtained from the brewers' grains by the process described in (2) was investigated in relation to animal digestion. It has been now generally established that the furfuroids as constituents of fodder plants are digested and assimilated in large proportion in passing through animal digestive tracts, and in this respect behave differently from the pentoses. The furfuroids being obtained, as described, in a fully hydrolysed condition (monoses) the digestion problem presented itself in a new aspect, and was therefore attacked.

The result of the comparative feeding experiments upon rabbits was to show that in this previously hydrolysed form the furfuroids are almost entirely digested and assimilated, no pentoses, moreover, appearing in the urine.

Generally we may sum up the present solution of the problem of the relationship of the furfuroids to plant assimilation and growth as follows:--The pentoses are not produced as such in the process of assimilation; but furfural-yielding carbohydrates are produced directly and in approximately constant ratio to the total carbohydrates; they are mainly located in the permanent tissue; in the secondary changes of dehydration, &c., accompanying maturation they undergo such differentiation that they become readily separable by processes of acid hydrolysis from the more resistant normal celluloses; but in relation to alkaline treatments they maintain their intimate union with the latter. They are finally converted into pentoses by artificial treatments, and into pentosanes in the plant, with loss of 1 C atom in an oxidised form. The mechanism of this transformation of hexoses into pentoses is not cleared up. It is independent of external conditions, e.g. fertilisation and atmospheric oxidations, and is probably therefore a process of internal rearrangement of the character of an oxidation.

ZUR KENNTNISS DER IN DEN MEMBRANEN DER PILZE ENTHALTENEN BESTANDTHEILE.

E. WINTERSTEIN (Ztschr. Physiol. Chem., 1894, 521; 1895, 134).

~ON THE CONSTITUENTS OF THE TISSUE OF FUNGI.~

(p. 87) These two communications are a contribution of fundamental importance, and may be regarded as placing the question of the composition of the celluloses of these lowest types on a basis of well-defined fact. In the first place the author gives an exhaustive bibliography, beginning with the researches of Braconnot (1811), who regarded the cellular tissue of these organisms as a specialised substance, which he termed 'fungin.' Payen rejects this view, and regards the tissue, fully purified by the action of solvents, as a cellulose (C_{6}H_{10}O_{5}). This view is successively supported by Fromberg [Mulder, Allg. Phys. Chem., Braunschweig, 1851], Schlossberger and Doepping [Annalen, 52, 106], and Kaiser. De Bary, on a review of the evidence, adopts this view, but, as the purified substance fails to give the characteristic colour-reactions with iodine, he uses the qualifying term 'pilzcellulose' [Morph. u. Biol. d. Pilze u. Flechten, Leipzig, 1884].

C. Richter, on the other hand, shows that these reactions are merely a question of methods of purification or preparation [Sitzungsber. Acad. Wien, 82, 1, 494], and considers that the tissue-substance is an ordinary cellulose, with the ordinary reactions masked by the presence of impurities. In regard to the lower types of fungoid growth, such as yeast, the results of investigators are more at variance. The researches of Salkowski (p. 113) leave little doubt, however, that the cell-membrane is of the cellulosic type.

The author's researches extend over a typical range of products obtained from _Boletus edulis, Agaricus campestris, Cantharellus cibarius, Morchella esculenta, Polyporus officinalis, Penicillium glaucum_, and certain undetermined species. The method of purification consisted mainly in (a) exhaustive treatments with ether and boiling alcohol, (b) digestion with alkaline hydrate (1-2 p.ct. NaOH) in the cold, (c) acid hydrolysis (2-3 p.ct. H_{2}SO_{4}) at 95°-100°, followed by a chloroxidation treatment by the processes of Schulze or Hoffmeister, and final alkaline hydrolysis.

The products, i.e. residues, thus obtained were different in essential points from the celluloses isolated from the tissues of phanerogams similarly treated. Only in exceptional cases do they give blue reactions with iodine in presence of zinc chloride or sulphuric acid. The colourations are brown to red. They resist the action of cuprammonium solutions. They are for the most part soluble in alkaline hydrate solution (5-10 p.ct. NaOH) in the cold. They give small yields (1-2 p.ct.) of furfural on boiling with 10 p.ct. HCl.Aq.

Elementary analyses gave the following results, which are important in establishing the presence of a notable proportion of nitrogen, which has certainly been overlooked by the earlier observers:--

_________________________________________________________ | | | | | | 'Cellulose' or residue from | C | H | N | |______________________________________|______|_____|_____| | | | | | | Boletus edulis (Schulze process) | 42.4 | 6.5 | 3.9 | | Boletus edulis (Hoffmeister process) | 44.6 | 6.3 | 3.6 | | Polyporus off. | 43.7 | 6.5 | 0.7 | | Cantharellus cib. | 44.9 | 6.8 | 3.0 | | Agaricus campestris | 44.3 | 6.6 | 3.6 | | Botrytis | 42.1 | 6.3 | 3.9 | | Penicillium glaucum | | | 3.3 | | Morchella esculenta | | | 2.5 | |______________________________________|______|_____|_____|

It is next shown that this residual nitrogen is not in the form of residual proteids (1) by direct tests, all of which gave negative results, and (2) indirectly by the high degree of resistance to both alkaline and acid hydrolysis. The 'celluloses' are attacked by boiling dilute acids (1 p.ct. H_{2}SO_{4}), losing in weight from 10 to 23 p.ct., the dissolved products having a cupric reduction value about 50 p.ct. that of an equal weight of dextrose. As an extreme hydrolytic treatment the products were dissolved in 70 p.ct. H_{2}SO_{4}, allowed to stand 24 hours, then considerably diluted (to 3 p.ct. H_{2}SO_{4}) and boiled to complete the inversion. The yields of glucose, calculated from the cupric reduction, were as follows:--

Boletus edulis 65.2 p.ct. Polyporus off. 94.7 " Agaricus campestris 59.1 " Morchella esculenta 60.1 " Cantharellus cib. 64.9 " Botrytis 60.8 "

It will be noted that the exceptionally high yield from the Polyporus cellulose is correlated with its exceptionally low nitrogen. By actual isolation of a crystalline dextrorotary sugar, by preparations of osazone and conversion into saccharic acid, it was proved that dextrose was the main product of hydrolysis. The second main product was shown to be acetic acid, the yield of which amounted to 8 p.ct. in several cases.

Generally, therefore, it is proved that the more resistant tissue constituents of the fungi are not cellulose, but a complex of carbohydrates and nitrogenous groups in combination, the former being resolved into glucoses by acid hydrolysis, and the latter yielding acetic acid as a characteristic product of resolution together with the nitrogenous groups in the form of an uncrystallisable syrup.

In the further prosecution of these investigations (2) the author proceeded from the supposition of the identity of the nitrogenous complex of the original with chitin, and adopted the method of Ledderhose (Ztschr. Physiol. Chem. 2, 213) for the isolation of glucosamin hydrochloride, which he succeeded in obtaining in the crystalline form. In the meantime E. Gilson had shown that these tissue substances in 'fusion' with alkaline hydrates yield a residue of a nitrogenous product (C_{14}H_{28}N_{2}O_{10}), which is soluble in dilute acids [Recherches Chim. sur la Membrane Cellulaire des Champignons, La Cellule, v. II, pt. 1]. This residue, which was termed mycosin by Gilson, has been similarly isolated by the author. It is proved, therefore, that the tissues of the fungi do contain a product resembling chitin. [See also Gilson, Compt. Rend. 120, 1000.] This constituent is in intimate union with the carbohydrate complex, which is resolved similarly to the hemicelluloses. Various intermediate terms of the hydrolytic series have been isolated. But the only fully identified product of resolution is the dextrose which finally results.

UEBER DIE KOHLENHYDRATE D. HEFE.

E. SALKOWSKI (Berl. Ber., 27, 3325).

~ON THE CARBOHYDRATES OF YEAST.~

The author has isolated the more resistant constituents of the cell-membrane by boiling with dilute alkalis, and exhaustively purifying with alcohol and ether.

The residue was only a small percentage (3-4 p.ct) of the original, and retained only 0.45 p.ct. N.

It was heated in a digester with water at 2-3 atm. steam-pressure, and thus resolved into approximately equal portions of soluble cellulose (a) and insoluble (b). The latter, giving no colour-reaction with iodine, is termed achroocellulose; the former reacts, and is therefore termed erythrocellulose. The former is easily separated from its opalescent solution. It has the empirical composition of cellulose. In the soluble form it resembles glycogen. The achroocellulose is isolated in the form of horny or agglomerated masses. It appears to be resolved by ultimate hydrolysis into dextrose and mannose.

SECTION V. FURFUROIDS, i.e. PENTOSANES AND FURFURAL-YIELDING CONSTITUENTS GENERALLY

(1) ~Reactions of the Carbohydrates with Hydrogen Peroxide.~

C. F. CROSS, E. J. BEVAN, and CLAUD SMITH (J. Chem. Soc., 1898, 463).

(2) ~Action of Hydrogen Peroxide on Carbohydrates in the Presence of Ferrous Salts.~

R. S. MORRELL and J. M. CROFTS (J. Chem. Soc., 1899, 786).

(3) ~Oxidation of Furfuraldehyde by Hydrogen Peroxide.~

C. F. CROSS, E. J. BEVAN, and T. HEIBERG (J. Ch. Soc., 1899, 747).

(4) EINWIRKUNG VON WASSERSTOFFHYPEROXID AUF UNGESÄTTIGTE KOHLENWASSERSTOFFE.

C. F. CROSS, E. J. BEVAN, and T. HEIBERG (Berl. Ber., 1900, 2015).

~ACTION OF HYDROGEN PEROXIDE ON UNSATURATED HYDROCARBONS.~

The above series of researches grew out of the observations incidental to the use of the peroxide on an oxidising agent in investigating the hydrolysed furfuroids (102). Certain remarkable observations had previously been made by H. J. H. Fenton (Ch. Soc. J., 1894, 899; 1895, 774; 1896, 546) on the oxidation of tartaric acid by the peroxide, acting in presence of ferrous salts, the --CHOH--CHOH-- residue losing H_{2} with production of the unsaturated group, --OH.C=C.OH--. These investigations have subsequently been considerably developed and generalised by Fenton, but as the results have no immediate bearing on our main subject we must refer readers to the J. Chem. Soc., 1896-1900.

From the mode of action diagnosed by Fenton it was to be expected that the CHOH groups of the carbohydrates would be oxidised to CO groups, and it has been established by the above investigations (1) and (2) that the particular group to be so affected in the hexoses is that contiguous to the typical

| --CO

group. There results, therefore, a dicarbonyl derivative ('osone'), which reacts directly with 2 mol. phenyl hydrazine in the cold to form an osazone. This was directly established for glucose, lævulose, galactose, and arabinose (2). While this is the main result, the general study of the product shows that the oxidation is not simple nor in direct quantitative relationship to the H_{2}O_{2} employed. The molecular proportion of the aldoses affected appears to be in considerable excess, and the reaction is probably complicated by interior rearrangement.

In the main, the original aldehydic group resists the oxidation. But a certain proportion of acid products are formed, probably tartronic acid. On distillation with condensing acids a large proportion of volatile monobasic acids (chiefly formic) are obtained. The proportion of furfural obtained amounts to 3-4 per cent. of the weight of the original carbohydrate.

Since the general result of these oxidations is the substitution of an OH group for an H atom, it was of interest to determine the behaviour of furfural with the peroxide. The oxidation was carried out in dilute aqueous solution of the aldehyde at 20°-40°, using 2-3 mols. H_{2}O_{2} per 1 mol. C_{5}H_{4}O_{2}. The main product is a hydroxyfurfural, which was separated as a hydrazone. A small quantity of a monobasic acid was formed, which was identified as a hydroxypyromucic acid. Both aldehyde and acid appear to be the alpha beta derivatives. The aldehyde gives very characteristic colour reactions with phloroglucinol and resorcinol in presence of hydrochloric acid, which so closely resemble those of the lignocelluloses that there is little doubt that these particular reactions must be referred to the presence of the hydroxyfurfural as a normal constituent.

The study of these oxidations was then extended to typical unsaturated hydrocarbons--viz. acetylene and benzene. (4) From the former the main product was acetic acid, but the attendant formation of traces of ethyl alcohol indicates that the hydrogen of the peroxide may take a direct part in this and other reactions. This view receives some support from the fact that the interaction of the H_{2}O_{2} with permanganates has now been established to be an oxidation of the H_{2} of the peroxide by the permanganate oxidation, with liberation, therefore, of the O_{2} of the peroxide as an unresolved molecule [Baeyer].

Benzene itself is also powerfully attacked by the peroxide when shaken with a dilute solution in presence of iron salts. The products are phenol and pyrocatechol, with some quantity of an amorphous product probably formed by condensation of a quinone with the phenolic products of reaction.

* * * * *

These types of oxidation effects now established give a definite significance to the physiological functions of the peroxide, which is a form of 'active oxygen' of extremely wide distribution. It would have been difficult _a priori_ to devise an oxidant without sensible action on aldehydic groups, yet delivering a powerful attack on hydrocarbon rings; or to have suggested a synthesis of the sugars from tartaric acid with a powerful oxidising treatment as the first and essential stage in the transformation.

Our present knowledge of such actions and effects suggests a number of new clues to genetic relationships of carbon compounds within the plant. The conclusion is certainly justified that the origin of the pentoses is referable to oxidations of the hexoses, in which this form of 'active oxygen' plays an important part.

We must note here the researches of O. Ruff, who has applied these oxidations with important results in the systematic investigation of the carbohydrates.

UEBER DIE VERWANDLUNG DER _D_-GLUCONSÄURE IN _D_-ARABINOSE (Berl. Ber., 1898, 1573).

~CONVERSION OF _D_-GLUCONIC ACID INTO _D_-ARABINOSE.~

* * * * *

_D_ UND _L_ ARABINOSE (_Ibid._ 1899, 550).

* * * * *

ZUR KENNTNISS DER OXYGLUCONSÄURE (_Ibid._ 1899, 2269).

~ON OXYGLUCONIC ACID.~

Ruff in these researches has realised a simple and direct transition from the hexoses to the pentoses. By oxidising gluconic acid with the peroxide the beta --CHOH-- group is converted into carbonyl at the same time that the terminal COOH [alpha] is oxidised to CO_{2}. The yields of the resulting pentose are large. Simultaneously there is formed an oxygluconic acid, which appears to be a ketonic acid of formula --CH_{2}OH.CO.(CHOH)_{3}.COOH--.

From these results we see a further range of physiological probabilities; and with the concurrent actions of oxygen in the forms of or related to hydrogen peroxide on the one side, and ozone on the other, we are able to account in a simple way for the relationships of the 'furfuroid' group, which may include a number of intermediate terms in the hexose-pentose series.

Following in this direction of development of the subject is a study of the action of persulphuric acid upon furfural.

EINWIRKUNG DES CARO'SCHEN REAGENS AUF FURFURAL.

C. F. CROSS, E. J. BEVAN, and J. F. BRIGGS (Berl. Ber., 1900, 3132).

Regarding this reagent as another form of 'active oxygen,' it is important to contrast its actions with those of the hydrogen peroxide. Instead of the beta-hydroxyfurfural (_ante_, 115) we obtain the delta-aldehyde as the first product. The aldehydic group is then oxidised, and as a result of attendant hydrolysis the ring is broken down and succinic acid is formed, the original aldehydic group of the furfural being split off in the form of formic acid. The reactions take place at the ordinary temperature and with the dilute form of the reagent described by Baeyer and Villiger (Ber. 32, 3625). These results have some special features of interest. The alpha delta-hydroxyfurfural has similar colour reactions to those of the alpha beta-derivative, and may also therefore be present as a constituent of the lignocelluloses. The tendency to attack in the 1·4 position in relation to an aldehydic group further widens the capabilities of 'active oxygen' in the plant cell. Lastly, this is the simplest transition yet disclosed from the succinyl to furfural grouping, being effected by a regulated proportion of oxygen, and under conditions of reaction which may be described as of the mildest. In regard to the wide-reaching functions of asparagin in plant life, we have a new suggestion of genetic connections with the furfuroids.

VERGLEICH DER PENTOSEN-BESTIMMUNGSMETHODEN VERMITTELST PHENYLHYDRAZIN UND PHLOROGLUCIN.

M. KRÜGER (Inaug.-Diss., Göttingen, 1895).

~COMPARISON OF METHODS OF ESTIMATING FURFURAL AS HYDRAZONE AND PHLOROGLUCIDE.~

The author traces the development of processes of estimating furfural (1) by precipitation with ammonia (furfuramide), (2) by volumetric estimation with standardised phenylhydrazine, (3) by weighing the hydrazone.

In 1893 (Chem. Ztg. 17, 1745) Hotter described a method of quantitative condensation with pyrogallol requiring a temperature of 100°-110° for two hours. The insoluble product collected, washed, dried at 103°, and weighed, gives a weight of 1.974 grm. per 1 grm. furfural.

Councler substitutes phloroglucinol for pyrogallol, with the advantage of doing away with the digestion at high temperature. (_Ibid._ 18, 966.) This process, requiring the presence of strong HCl, has the advantage of being applied directly to the acid distillate, in which form furfural is obtained as a product of condensation of pentoses, &c. A comparative investigation was made, precipitating furfural (a) as hydrazone in presence of acetic acid, and (b) as phloroglucide in presence of HCl (12 p.ct). In (a) by varying the weights of known quantities of furfural, and using the factor, hydrazone × 0.516 [+ 0.0104] in calculating from the weights of precipitates obtained, the maximum variations from the theoretical number were +1.71 and -1.74. In (b) it was found necessary to vary the factor from 0.52 to 0.55 in calculating from phloroglucide to furfural. The greatest _total_ range of variation was found to be 2.5 p.ct. The phenol process is therefore equally accurate, has the advantages above noted, and, in addition, is less liable to error from the pressure in the distillates obtained from vegetable substances of volatile products, e.g. ketonic compounds, accompanying the furfural.

This method has been criticised by Helbel and Zeisel [Sitz.-ber, Wiener Akad. 1895, 104, ii. p. 335] on two grounds of error, viz. (1) the presence of diresorcinol in all ordinary preparations of phloroglucinol, and (2) changes in weight of the precipitate of phloroglucide on drying. The process was carried out comparatively with ordinary preparations, and with specially pure preparations of the phenol. The quantitative results were identical. The criticisms in question are therefore dismissed. Although the process is to be recommended for its simplicity and the satisfactory concordance of results it is to be noted that it rests upon an empirical basis, since the phloroglucide is not formed by the simple reaction 2 [C_{5}H_{4}O_{2} + C_{6}H_{6}O_{3}] - H_{2}O = C_{22}H_{18}O_{9}, but appears to have the composition C_{16}H_{12}O_{6}.

In part ii. of this paper the author discusses the question of the probable extent in the sense of diversity of constitution of furfural-yielding constituents of plant-tissues. Glucoson was isolated from glucosazon, and found to yield 2.9-3.6 p.ct. furfural. Gluconic acid distilled with hydrochloric acid gave traces of furfural; so also with sulphuric acid and manganic oxide.

Starch was oxidised with permanganate, and a mixture of products obtained of which one gave a characteristic violet colouration with phloroglucol, with an absorption-band at the D line. On distilling with HCl furfural was obtained in some quantity. The product in question was found to be very sensitive to the action of bases, and was destroyed by the incidental operation of neutralising the mixture of oxidised products with calcium carbonate. It was found impossible to isolate the compound.

UNTERSUCHUNGEN UEBER DIE PENTOSANBESTIMMUNG MITTELST DER SALZSÄURE-PHLORO-GLUCIN-METHODE.[8]

E. KRÖBER (Journ. f. Landwirthschaft, 1901, 357).

~INVESTIGATION OF THE HYDROCHLORIC ACID-PHLOROGLUCINOL METHOD OF DETERMINING PENTOSANES.~

This paper is the most complete investigation yet published of the now well-known method of precipitating and estimating furfural in acid solution by means of the trihydric phenol. In the last section of the paper is contained the most important result, the proof that the insoluble phloroglucide is formed according to the reaction

C_{5}H_{4}O_{2} + C_{6}H_{6}O_{3} - 2H_{2}O = C_{11}H_{6}O_{3},

also, by varying the proportions of the pure reagents interacting, that the condensation takes place invariably according to this equation.