Researches on Cellulose Part 13

BARLEY CROP, WOBURN, 1894.

________________________________________________________________________ [G] _____________ [A] [B] [C] [D] [E] [F] [H] [I] [J] _________ __________ _____ ______ ______ ______ ______ ______ __________ May 7 6 weeks 1 19.4 7.0 53.4 12.7 6.8 1.03 : 1 6 14.7 7.0 55.9 10.3 5.7 1.23 : 1 June 4 10 weeks 1 17.6 7.7 52.9 11.6 6.1 1.26 : 1 6 13.5 8.1 58.5 13.4 7.8 1.04 : 1 July 10 15 weeks 1 42.0 9.0 65.7 9.8 6.4 1.40 : 1 6 32.9 10.6 65.7 12.5 8.2 1.30 : 1 Cut 21 weeks 1 64.0 11.9 70.0 14.5 10.1 1.18 : 1 Aug. 21 6 64.6 13.4 70.5 15.0 10.6 1.26 : 1 Carried 22 weeks 1 84.0 12.7 75.0 16.5 12.4 1.02 : 1 Aug. 31 6 86.4 12.4 78.4 15.1 11.8 1.05 : 1 BARLEY CROP, WOBURN, 1895. May 15 7 weeks 1 20.6 6.6 53.9 10.2 5.5 1.20 : 1 6 17.8 5.8 56.7 9.6 5.4 1.07 : 1 June 18 12 weeks 1 34.6 8.0 38.2 14.7 5.6 1.42 : 1 6 33.4 7.6 44.5 15.0 6.7 1.14 : 1 July 16 16 weeks 1 52.8 12.1 55.6 16.3 9.1 1.33 : 1 6 54.4 10.6 46.2 19.1 8.8 1.20 : 1 Aug. 16 20 weeks 1 66.8 9.2 49.1 17.0 8.3 1.10 : 1 6 65.0 9.8 49.8 19.1 9.4 1.04 : 1 Sept. 3 22 weeks 1 84.3 10.4 45.7 17.6 8.0 1.31 : 1 6 86.3 10.2 45.3 17.3 7.8 1.30 : 1 _________ __________ _____ ______ ______ ______ ______ ______ __________

The variations exhibited by these numbers are significant. It is clear, on the other hand, that the a.s.similation of the furfuroids does not vary in any important way with variations in conditions of atmosphere and soil nutrition. They are essentially _tissue_-const.i.tuents, and only at the flowering period is there any acc.u.mulation of these compounds in the alkali-soluble form. It has been previously shown (ibid. 27, 1061) that the proportion of furfuroids in the straw-celluloses of the paper-maker differs but little from that of the original straws. For the isolation of the celluloses the straws are treated by a severe process of alkaline hydrolysis, to which, therefore, the furfuroid groups offer equal resistance with the normal hexose groups with which they are a.s.sociated in the complex.

The furfuroids of the cereal straws are therefore not pentosanes. They are original products of a.s.similation, and not subject to secondary changes after elaboration such as to alter either their const.i.tution or their relationship to the normal hexose groups of the tissue-complex.

(1) CONSt.i.tUTION OF THE CEREAL CELLULOSES

(Chem. Soc. J. 1896, 804).

(2) THE CARBOHYDRATES OF BARLEY STRAW

(Chem. Soc. J. 1896, 1604).

(3) THE CARBOHYDRATES OF THE CEREAL

STRAWS (Chem. Soc. J. 1897, 1001).

(4) THE CARBOHYDRATES OF BARLEY STRAW

(Chem. Soc. J. 1898, 459).

C. F. CROSS, E. J. BEVAN, and CLAUD SMITH.

These are a series of investigations mainly devoted to establishing the ident.i.ty of the furfural-yielding group which is a characteristic const.i.tuent.

This 'furfuroid' while equally resistant to alkalis as the normal cellulose group with which it is a.s.sociated, is selectively hydrolysed by acids. Thus straw cellulose dissolves in sulphuric acids of concentration H_{2}SO_{4}.2H_{2}O - H_{2}SO_{4}.3H_{2}O, and on diluting the normal cellulose is precipitated as a hydrate, and the furfuroid remains in solution. But this sharp separation is difficult to control in ma.s.s. By heating with a very dilute acid (1 p.ct. H_{2}SO_{4}) the conditions are more easily controlled, the most satisfactory results being obtained with 15 mins. heating at 3 atm. pressure.

(1) Operating in this way upon brewers' grains the furfuroid was obtainable as the chief const.i.tuent of a solution for which the following experimental numbers were determined:--Total dissolved solids, 28.0 p.ct. of original 'grains'; furfural, 39.5 p.ct. of total dissolved solids, as compared with 12.5 p.ct. of total original grains; cupric reduction (calc. to total solids), 110 (dextrose = 100) osazone; yield in 3 p.ct. solution, 35 p.ct. of weight of total solids.

Pentosazone a.n.a.lysis N 17.1 17.3 17.07 C 62.5 62.3 62.2 H 6.4 6.5 6.1 Melting-point 146-153

From these numbers it is seen that of the total furfuroids of the original 'grains' 84 p.ct. are thus obtained in solution in the fully hydrolysed form, which is that of a pentose or pentose derivative. It was, however, found impossible to obtain any crystallisation from the neutralised (BaCO_{3}) and concentrated solution, the syrup being kept for some weeks in a desiccator. It was noted at the same time that the colour reaction of the original solution with phloroglucol and hydrochloric acid was a deep violet, in contradistinction to the characteristic red of the pentoses. On oxidation with hydrogen peroxide, in the proportion of 1 mol. H_{2}O_{2} to 1 mol. of the carbohydrate in solution, carbonic anhydride was formed in quant.i.ty = 20.0 p.ct. of the latter.

Fermentation (yeast) experiments also showed a divergence from the resistant behaviour of the pentoses, a considerable proportion of the furfuroid disappearing in a normal fermentation.

(2) The quant.i.tative methods above described were employed in investigating the barley plant at different stages of its growth. The green plant was extracted with alcohol, the residue freed from alcohol and subjected to acid hydrolysis.

The hydrolysed extract was neutralised and fermented. In the early stages of growth the furfuroids were completely fermented, i.e.

disappeared in the fermentation. In the later stages this proportion fell to 50 p.ct. In the earlier stages, moreover, the normal hexose const.i.tuents of the permanent tissue were hydrolysed in large proportion by the acid, whereas in the matured straw the hydrolysis is chiefly confined to the furfuroids. In the early stages also the permanent tissue yields an extract with relatively low cupric reduction, showing that the carbohydrates are dissolved by the acid in a more complex molecular condition.

These observations confirm the view that the furfuroids take origin in a hexose-pentose series of transformations. The proportion of furfuroid groups to total carbohydrates varies but little, viz. from 1/3 in the early stages to a maximum of 1/4 at the flowering period. At this period the differentiation of the groups begins to be marked.

Taking all the facts of (1) and (2), they are not inconsistent with the hypothesis of an internal transformation of a hexose to a pentose-monoformal. Such a change of position and function of oxygen from OH to CO within the group --CH.OH-- is a species of internal oxidation which reverses the reduction of formaldehyde groups in synthesising to sugars, and appears therefore of probable occurrence.

These const.i.tutional 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 const.i.tuents 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 inst.i.tuted 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 const.i.tuents of fodder plants are digested and a.s.similated in large proportion in pa.s.sing 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 a.s.similated, 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 a.s.similation and growth as follows:--The pentoses are not produced as such in the process of a.s.similation; 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 CONSt.i.tUENTS 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, Morch.e.l.la esculenta, Polyporus officinalis, Penicillium glauc.u.m_, 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 a.n.a.lyses 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 glauc.u.m 3.3 Morch.e.l.la 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 "

Morch.e.l.la 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 const.i.tuents 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 ident.i.ty 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 const.i.tuent 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.