Mold Agents in Conversion of Starch

H. M. Tsuchiya, R. H. Blom.

The bases for industrial processes of saccharifying starch that is, converting it to sugar were laid early in the nineteenth century.

The first announcement that it was possible to saccharify, or hydrolyze, starch came in 1811, when G. S. C. Kirchoff, a Russian chemist, reported that starch would yield sugarlike substances on treatment with acid. Five years later, M. Kirchhoff stated that starch could also be degraded by a treatment with diastase, an enzyme from plant sources.

Both procedures have been adapted to industrial operations. In the method that involves acid hydrolysis, a starch slurry is treated, at high temperatures, with hydrochloric acid or nitric acid as a catalyst. The conditions of saccharification, the neutralization of acid, and the removal of residual solids by filtration vary according to the product desired.

In the enzymatic hydrolysis, water is added to starch or starch-containing material, the slurry is heated to gelatinize the starch, and an enzyme (an organic catalyst) is added. Depending on the product desired, the conversion conditions in the enzymatic saccharification of starch may be varied.

Diastases, or amylases, are the enzymes that hydrolyze starch to dextrins and two sugars, maltose and dextrose.

These amylolytic enzymes are of two types: The dextrinogenic enzymes, which primarily convert starch to dextrins ( carbohydrates intermediate in molecular size between starch and sugar) ; and the saccharogenic enzymes, which saccharify the higher polymers of dextrose to sugars.

The dextrinogenic enzyme can hydrolyze the two types of molecules in starch amylose (a straight-chain or linear molecule) and amylopectin (a branched-chain or ramified molecule) . The saccharogenic enzyme can split off sugars from the terminals of amylose and amylopectin molecules. Neither enzyme can hydrolyze the chemical linkages at the branch points in amylopectin. The dextrinogenic enzyme, however, can cut the molecular chain of glucose units in amylopectin at other points. The products formed, like those resulting from the activity of the dextrinogenic enzyme on amylose, are dextrins. The saccharogenic enzymes in malt then attack the free terminal ends of dextrin molecules and split off more sugar molecules.

AMYLASES are found in the seeds of such plants as barley, wheat, and soybeans and also in animal glands, such as the pancreas, and in body fluids, such as saliva, blood, and urine. Diastases occur in micro-organisms, such as bacteria and fungi.

Of the higher plant sources, barley seed is used most commonly on an industrial scale to saccharify starch. Because activity is enhanced by malting the grain, the seed is steeped in water, allowed to sprout, and then dried. When the grain germinates, the dextrinogenic activity of the material is increased substantially. Apparently the dextrinogenic, or a-amylase, enzyme is synthesized during germination. Moreover, the saccharogenic enzyme, or B-amylase, is released from its bound. or inactive, form. The germinated and dried barley seed is known as malt. In the fermentation of grain to ethyl alcohol, the conversion of starch to maltose has depended almost entirely on the use of barley malt, at least in this country. Other amylolytic materials have been considered from time to time, however.

Both a- and B-amylases are found in wheat and rye malts in amounts and proportions approximately comparable to those in barley malt. They are also present in ungerminated cereals like barley, wheat, and rye.

In an attempt to exploit the amylases in ungerminated cereals, the Balls-Tucker process was developed to extract and utilize the enzymes in wheat. The process involves the extraction of ground wheat with a weak solution of sodium sulfite, and the addition of the sulfite-diastase mixture thus obtained to cooked grain to saccharify the starch. The amylolytic activity in the soybean and sweetpotato is of the B-amylase type.

Pancreatic diastase is the only amylolytic enzyme of animal origin now used to any extent in industry. This enzyme displays a-amylase activity but not the saccharogenic characteristics of B-amylase.

CERTAIN MICRO-ORGANISMS produce enough starch-hydrolyzing enzymes under favorable conditions to merit industrial consideration. Some of the bacteria, especially certain members of the genus Bacillus (gram-positive, aerobic, sporeforming rods), elaborate -amylase when they are propagated in either submerged or surface cultures. The dextrinogenic enzymes of bacteria display extraordinary stability in heat; some can withstand boiling temperature for short periods and still rapidly liquefy starch.

Some mold species also produce amylolytic enzyme systems that degrade starch to dextrins and fermentable sugars. The organisms in the crude amylase preparations used in the Orient are primarily of the Aspergillus flavus-oryzae group. The mold-bran process, a refinement of the Oriental practice of propagating molds on moistened rice and other grains, has been developed in the United States. L. A. Underkofler and his associates at Iowa State College have tested the organisms used in the process for their ability to convert starch to fermentable sugars, as measured by the production of alcohol from grain mashes saccharified with mold bran.

The 27 mold cultures tested included strains from 4 genera Aspergillus, Mucor, Penicillium, and Rhizopus. All strains of Aspergillus oryzae were effective in the conversion of starch. Although Mucor rouxii and M. circinelloides were as effective as the Aspergilli, the strains of M. javanicus and of Penicillium chrysogenum and P. purpurogenum were inferior. Of the 14 strains of Rhizopus tested, all but 1 produced active mold-bran preparations.

In the amylo process for the production of alcohol, Rhizopus japonicas, Mucor rouxianus, and other related molds have been used to convert starch to sugar. When investigations were originally undertaken at the Northern Regional Research Laboratory to develop a submerged-culture process for the production of fungal amylase, more than 350 strains of 5 genera Aspergillus, Rhizopus, Mucor, Penicillium, and Monilia were tested for their ability to elaborate a-amylase.

Of 278 Aspergillus strains, representing 41 species, only 34 produced dextrinogenic amylase under mold-culture conditions. As was to be expected, the enzyme production varied among the strains within a species. Certain strains of A. niger, A. oryzae, and A. zventii were particularly active. Of 80 Penicillum cultures, 8 displayed dextrinogenic activity of low order. Of eight cultures of Rhizopus, Mucor, and Monilia, none demonstrated any marked u--amylase activity, but one strain of Rhizopus was capable of saccharifing grain mashes.

Other strains of Rhizopus and Mucor behaved the same way, suggesting that those organisms possess amylolytic systems other than the dextrinogenic or liquefying enzyme. Some of the properties of the diastatic systems elaborated by molds in submerged culture weir, then studied. The properties were compared to those of other starch-hydrolyzing enzymes, particularly barley malt.

Early in the investigation on fungal amylase, chemists observed that the yield of alcohol from grain mashes saccharified with mold-enzyme preparations did not correlate with the a-amylase activity of the preparations. Although a low, but definite, concentration of the dextrinogenic amylase was required and., indeed, essential for satisfactory alcohol production, the alcohol yield depended also on the presence of other amylolytic enzymes. B-Amylase was absent in fungal-amylase preparations, but at least two other enzymes, which displayed maltase activity, were present. Evidence has accumulated that "maltase" can hydrolyze the higher polymers of glucose directly to the simple sugar. As it hydrolyzes maltose as well as the higher carbohydrates, this enzyme is most conveniently measured by its action on the disaccharide. The importance of maltase lies in the fact that alcohol yields from grain mashes saccharified with fungal amylase correlate well with the maltase activity of mold preparations, provided a low but significant activity of a-amylase is also present.

There are certain fungal-amylase preparations of high maltase activity that can hydrolyze isomaltose, a disaccharide that differs from maltose in that the chemical linkage between the glucose residues is of the a-1,6 type in isomaltose, whereas it is of the -1,4 type in maltose.