Part 80

=393. Diversity of Character.=—The proteids which occur in vegetable products are found in all parts of the tissues of the plants, but in cereals especially in the seeds. In grass crops and in some of the legumes, such as clover, the nitrogenous matters are chiefly found in the straw and leaves. The general classification of these bodies has already been given, but each kind of plant presents marked variations, not only in the relative proportions of the different classes, but also in variations in the nature of each class. For this reason the study of vegetable proteids is, in some respects, a new research for each kind of plant examined. There are, however, some general principles which the analyst must follow in his work, and an attempt will be made here to establish these and to construct thereon a rational method of conducting the investigation. In the separation and estimation of complex bodies so nearly related to each other, it is difficult not only to secure satisfactory results, but also to prevent the transformation of some forms of proteid matter into others nearly related thereto by the action of the solvents used for separation and precipitation.

=394. Separation of Gluten from Wheat Flour.=—The most important proteid in wheat is the body known as gluten, a commercial name given to the nitrogenous matters insoluble in cold water. The gluten thus obtained does not represent a single chemical compound, but is a complex consisting of at least two proteid bodies, which together form an elastic, pasty mass, insoluble in cold water containing a trace of mineral salts. This mass has the property of holding mechanically entangled among its particles bubbles of gas, which, expanding under the action of heat during cooking, give to bread made of glutenous flours its porous property.

In respect of proteids, the American wheats, as a rule, are quite equal to those of foreign origin. This is an important characteristic when it is remembered that both the milling and food values of a wheat depend largely on the nitrogenous matter which is present. It must not be forgotten, however, that merely a high percentage of proteids is not always a sure indication of the milling value of a wheat. The percentage of gluten to the other proteid constituents of a wheat is not always constant, and it is the gluten content of a flour on which its bread making qualities chiefly depend. The percentage of moist gluten gives, in a rough way, the property of the glutenous matter of absorbing and holding water under conditions as nearly constant as can be obtained. In general, it may be said that the ratio between the moist gluten and the dry gluten in a given sample is an index for comparison with other substances in the same sample. Upon the whole, however, the percentage of dry gluten must be regarded as the safer index of quality. In respect of the content of glutenous matter, our domestic wheats are distinctly superior to those of foreign origin. They are even better than the Canadian wheats in this respect. It may be fairly inferred, therefore, that while our domestic wheats give a flour slightly inferior in nutritive properties to that derived from foreign samples, it is nevertheless better adapted for baking purposes, and this quality more than compensates for its slight deficiency in respect of nutrition, a deficiency, which, however, is so minute as to be hardly worth considering.

The gluten is separated in this laboratory from the other constituents of a flour by the following process:

Ten grams of the fine-ground flour are placed in a porcelain dish, well wet with nearly an equal weight of water at a temperature of not to exceed 15°, and the mass worked into a ball with a spatula, taking care that none of it adheres to the walls of the dish. The ball of dough is allowed to stand for an hour, at the end of which time it is held in the hand and kneaded in a stream of cold water until the starch and soluble matter are removed. The ball of gluten thus obtained is placed in cold water and allowed to remain for an hour when it is removed, pressed as dry as possible between the hands, rolled into a ball, placed in a flat bottom dish and weighed. The weight obtained is entered as moist gluten. The dish containing the ball of gluten is dried for twenty hours in a steam-bath, again weighed, and the weight of material obtained entered as dry gluten. The determination of dry and moist gluten cannot in any sense be regarded as an isolation and estimation of a definite chemical compound. For millers’ and bakers’ purposes, however, the numbers thus obtained have a high practical value. A typical wheat grown in this country will contain about 26.50 per cent of moist and 10.25 per cent of dry gluten.

The gluten of wheat is composed of two proteid bodies, gliadin and glutenin. Gliadin contains 17.66 per cent, and glutenin 17.49 per cent of nitrogen. Gliadin forms a sticky mass when mixed with water and is prevented from passing into solution by the small content of mineral salts present in the flour. It serves to bind together the other ingredients of the flour, thus rendering the dough tough and coherent. Glutenin serves to fix the gliadin and thus to make it firm and solid. Glutenin alone cannot yield gluten in the absence of gliadin, nor gliadin without the help of glutenin. Soluble metallic salts are also necessary to the formation of gluten, and act as suggested above, by preventing the solution of the gliadin in water, during the process of washing out the starch. No fermentation takes place in the formation of gluten from the ingredients named.

The gluten, which is obtained in an impure state by the process above described, is, therefore, not to be regarded as existing as such in the wheat kernel or flour made therefrom, but to arise by a union of its elements by the action of water.

=395. Extraction with Water.=—It is quite impossible to get an extract from fine-ground vegetable matter in pure water because the soluble salts of the sample pass at once into solution and then a pure water solvent becomes an extremely dilute saline solution. The aqueous extract may, however, be subjected to dialysis, whereby the saline matter is removed and the proteid matter, not precipitated during the dialytic process, may be regarded as that part of it in the original sample soluble in pure water. Nevertheless, in many instances, it is important to obtain an extract with cold water. In oatmeal the aqueous extract is obtained by Osborne as follows: Five pounds of fine-ground meal are shaken occasionally with six liters of cold water for twenty-four hours, the liquid removed by filtration and pressure and the extraction continued with another equal portion of water in the manner noted. The two liquid extracts are united and saturated with commercial ammonium sulfate which precipitates all the dissolved proteid matter. The filtrate obtained is collected on a filter, washed with a saturated solution of ammonium sulfate and removed as completely as possible from the filter paper by means of a spatula. Any residual precipitate remaining on the paper is washed into the vessel containing the removed precipitate and the undissolved precipitate well beaten up in the liquid, which is placed in a dialyzer with a little thymol, to prevent fermentation, and subjected to dialysis for about two weeks. At the end of that time, the contents of the dialyzer are practically free of sulfates. The contents of the dialyzers are then thrown on a filter and in the filtrate are found those proteids first extracted with water, precipitated with ammonium sulfate and redissolved from this precipitated state by pure water. In the case of oatmeal, this proteid matter is not coagulated by heat, and may be obtained in the dry state by the evaporation of the filtrate last mentioned at a low temperature in vacuo. It is evident that the character of the proteid matter thus obtained will vary with the nature of the substance examined. In the case of oats, it appears to be a proteose and not an albumin.

=396. Action of Water on Composition of Proteids.=—When a body, such as oatmeal, containing many proteids of nearly related character, is exposed to the action of a large excess of water, the proteid bodies may undergo important changes whereby their relations to solvents are changed. After oatmeal has been extracted with water, as described above, the proteid matter originally soluble in dilute alcohol undergoes an alteration and assumes different properties. The same remark is applicable to the proteid body soluble in dilute potash. Nearly all the proteid matter of oatmeal is soluble in dilute potash, if this solvent be applied directly, but if the sample be previously treated with water or a ten per cent salt solution the subsequent proportion of proteid matter soluble in dilute potash is greatly diminished. Water applied directly to the oatmeal apparently dissolves an acid albumin, a globulin or globulins, and a proteose. The bodies, however, soluble in water, exist only in small quantities in oatmeal. Experience has shown that in most instances, it is safer to begin the extraction of a cereal for proteid matter with a dilute salt solution rather than with water, and to determine the matters soluble in water alone by subsequent dialysis.

=397. Extraction with Dilute Salt Solution.=—In general, it is advisable to begin the work of separating vegetable proteids by extracting the sample with a dilute brine usually of ten per cent strength. As conducted by Osborne and Voorhees, on wheat flour, the manipulation is carried on as follows:

The fine-ground whole wheat flour, about four kilograms, is shaken with twice that weight of a ten per cent sodium chlorid solution, strained through a sieve, to break up lumps, and allowed to settle for sixteen hours. At the end of this time, about half of the supernatant liquid is removed by a siphon or by decantation and filtered. Two liters more of the salt solution are added, the mixture well stirred and the whole brought onto the filter used above. The filtrate is collected in successive convenient portions and each portion, as soon as it is obtained, is saturated with ammonium sulfate. All the proteid matter is precipitated by this reagent. The precipitate is collected on a filter, redissolved in a convenient quantity of the salt solution and dialyzed for fourteen days or until all sulfates and chlorids are removed. The proteid matter, which is separated on dialysis, in this instance, is a globulin.

The proteid matter not precipitated on dialysis is assumed to be that part of the original substance soluble in water.

A part of the water soluble proteid matter obtained as above is coagulated by heat at from 50° to 80°. The part not separated by heat gives a precipitate on saturation with sodium chlorid.

In wheat there are found soluble in water two albumins and a proteose.

In separating the albumin coagulating at a low boiling point from the dialyzed solution mentioned above, it is heated to 60° for an hour, the precipitate collected on a gooch, washed with hot water (60°), and then successively with ninety-five per cent alcohol, water-free alcohol and ether. On drying the residual voluminous matter on the filter over sulfuric acid, it becomes dense and horny, having in an ash free state, according to Osborne, the following composition:

Per cent. Carbon 53.06 Hydrogen 6.82 Nitrogen 17.01 Sulfur 1.30 Oxygen 21.81

=398. Treatment without Precipitation with Ammonium Sulfate.=—Where abundant means are at hand for dialyzing large volumes of solution, the preliminary treatment of the solution made with ten per cent sodium chlorid with ammonium sulfate may be omitted.

When the precipitated proteids are to be used for the estimation of the nitrogen therein contained, it has been proposed to substitute the corresponding zinc salt for the ammonium sulfate. This reagent has given satisfactory results in this laboratory and while a larger experience is desirable before commending it as an acceptable substitute in all cases, yet its obvious advantage, in being free of nitrogen for the use mentioned, entitles it to careful consideration.

The manipulation, with the exception of the precipitation with ammonium sulfate, is the same as that described in the preceding paragraph. The globulins are completely precipitated when the dialysis is complete and may be separated from the soluble albumins and proteoses by filtration.

=399. Separation of the Bodies Soluble in Water.=—Albumins.—By the methods of treatment just described, the proteid matters soluble in ten per cent sodium chlorid solution are separated into two classes, viz., globulins insoluble in pure water and albumins and proteoses soluble in pure water. The aqueous solution will also contain any amids or nitrogenous bases soluble in the dilute saline solution and in water. Osborne and Voorhees have found that the best way of separating the albumins in the pure aqueous solution is by the application of heat. By means of a fractional coagulation the albumins are divided into classes, viz., those separating at from 60° to 65° and those remaining in solution at that temperature but separating up to 85°. The respective quantities of these albumins are determined by collecting them in a filter and estimating the nitrogen therein by moist combustion in the usual way. Even a larger number of albumins may be secured, as in the maize kernel, by such a fractional precipitation by means of heat. Chittenden and Osborne find in this instance that the precipitation begins at about 40°.

Proteose.—After the separation of the albumins by heat the filtrate may still contain proteid matter. This matter belongs to the proteose class. It may be partially secured by concentrating the filtrate, after the removal of the albumins, to a small bulk when a part of the proteose body will separate. It may be thrown out entirely by treating the filtrate above mentioned with fine-ground salt until it is saturated or by adding salt until the solution contains about twenty per cent thereof and precipitating the proteose by acetic acid.

=400. Separation of the Globulins.=—The globulins which are extracted with ten per cent solution of sodium chlorid and precipitated on dialysis may be separated by fractional solution into several bodies of nearly related properties. This solution is conveniently accomplished by saline solvents of increasing strength. In the case of the maize globulins, Chittenden and Osborne employ dilute solutions of common salt for effecting the separation, beginning with a quarter of a per cent and ending with a two per cent mixture.

=401. Proteids Soluble in Dilute Alcohol.=—Some of the proteid bodies which are soluble in dilute salt solution and in water are also soluble in alcohol. Since these bodies are more easily identified by the processes already described, attention will be given in this paragraph solely to those proteid bodies which are insoluble in water or dilute salt solution and are soluble in dilute alcohol.

For the extraction of these bodies, the residue, left after extraction with a ten per cent solution of sodium chlorid or with water, is mixed with enough strong alcohol to secure by the admixture with the water present in the sample an alcohol of about seventy-five per cent strength. The mixture is well shaken and digested for some time, at a temperature of about 46°, and thrown on a filter which is kept at about the same temperature. The residue is again mixed with alcohol of the same strength (seventy-five per cent) using about four liters for two and a half kilos of the original material. During the second digestion the temperature is kept at about 60°. The latter operation is repeated three times and in each case the filtrate obtained is evaporated separately. This process is especially applicable to the meal from maize kernels, which contains a high relative percentage of an alcohol soluble proteid, zein.

The chief part of the zein is found in the first two extracts, obtained as described above. On evaporation, the zein separates as a tough, leathery, yellow-colored mass on the walls of the containing vessel. It is cut into small pieces and digested for several days in cold, pure alcohol. This is followed by digestion with a mixture of ether and pure alcohol, and finally with pure ether. By this treatment a part of the zein becomes insoluble in seventy-five per cent alcohol. The part soluble in dilute alcohol is precipitated by pouring it into water.

Another method of preparing zein is to extract the meal with seventy-five per cent alcohol after it has been treated with a ten per cent salt solution.

In this case the extraction is continued with seventy-five per cent alcohol in successive portions until no more proteid matter passes into solution. The several extracts are united and the alcohol removed by distillation, by which process the zein is separated. It is washed with distilled water, until the sodium chlorid is removed, dissolved in warm alcohol of about eighty per cent strength and any insoluble matter removed by filtration. On evaporating the filtrate nearly to dryness, the zein is separated and pressed as free of water as possible, yielding a yellow, elastic substance resembling molasses candy. This preparation is purified by digestion with pure alcohol and ether in the manner described. The two zeins which are secured by the treatment, one soluble and the other insoluble in alcohol, are practically identical in composition.

Zein freshly precipitated by pouring its alcoholic solution in water is wholly insoluble in water, and, on boiling therewith, is changed into the variety insoluble in dilute alcohol. Boiled with dilute sulfuric acid, six in 300 cubic centimeters of water, it melts, forming a gummy mass, which is very slowly attacked by the acid yielding proteoses and peptones. Heated with stronger sulfuric acid it undergoes decomposition, yielding leucin, tyrosin, and glutamic acid.