THE USE OF AMYLOLYTIC YEASTS FOR THE PRODUCTION OF A NUTRITIONAL

PRODUCT

DISSERTATION

Presented in Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State

University

By

CALVIN CHARLES KUEHNER, B. Sc. in Agr., M. S.

The Ohio State University

1953

Approved by: - 6 - per cent, depending upon species of animal, product produced, level of production, feeding practice, length of the feeding period of the animal considered, and so forth. The higher percentage applies only to the milk cow only during the relatively short period of maximum milk production and not during an entire life span or any substantial part of that span.

Some workers have compared the production of protein by animals with protein "biosynthesis" by microorganisms. Yin (1949) compares the most efficient protein producer among farm animals, the pig, with yeast and finds that the pig retains 20 per cent to 40 per cent of the calories taken in as feed in the increase in body weight at a rate of

O.O56 grams to 0.073 grams of solid per calorie. Only a part of this weight is edible. Yeast shows an efficiency of 50 to 60 per cent and yields 0.125 grams to 0 .2 5 grams of solid, all of which is edible, corresponding to 0.6 to 1.1 calorie per calorie of intake. The effi­ ciency' of B-vitamin production by yeast is 10 to 200 times that of any animal product per calorie of feed. Thaysen (1943) estimates one acre of carbohydrate crop yields 840 pounds of protein as food yeast, but only 70 pounds as meat or milk protein.

Research in many countries has established that yeast is a nutritious substance, high in protein content and may be produced from cheap raw materials. That it has not met with outstanding success is due principally to the fact that it has been badly presented and marketed. Not only must food yeast be efficiently produced, it must Table 28. Comparative vitamin values of yeast products from various substrates (micrograms per gram, dry material). (continued)

Substrates Niacin Pantothenic Riboflavin Biotin Pyridoxine Thiamine Choline acid

White potatoes, no slop 29.0 11.0 1.0 0.1 3.0 10.0 1132.6 Amount synthesized 24.0 -16.0 0.0 0.1 -14.0 5.0 832.0 White potatoes + slop 274.0 147.0 18.0 0.6 27.0 11.0 1068.0 Amount synthesized 269.0 120.0 17.0 0.5 10.0 6.0 454.0

Citrus peel juice * 348.0 110.7 48.2 6.4 37.8 Fir wood sugar * 450.0 134.4 80.8 2.3 38.3 — — Pear waste * 346.0 40,1 44.9 3.1 36.6 — - Jamaican molasses * 402.0 50.8 39.8 3.4 43.3 — - Wood sulfite liquor ':B*417.3 37.2 45.0 2.3 33.4 5.3 -

* Wiley, A. J., G. A. Dubey, B. F. Lueck, and L. P. Hughes (1950). Inskeep, G. C., A. J. Wiley, J. M. Holderby, and L. P. Hughes (l95l). Table 29. Comparative amino acid values of yeast products from various substrates, (micrograms per gram, dry material).

Phenyl­ Threo­ Iso- Methio­ Substrates Arginine Leucine Lysine alanine Valine Histidine nine Leucine nine

Wheat, no slop 26.3 17.4 17.1 11.7 29.4 71.4 37.1 24.0 1.5 Amount synthesized 15.3 8.8 10.5 6.1 15.4 67.1 7.1 6.8 0 Wheat + slop 27.4 23.7 22.9 16.3 39.7 9.9 77.1 39.4 2.9 Amount synthesized 16.4 15.1 16.3 10.7 25.7 5.6 47.1 22.2 1.4 Wheat + slop (dried) 22.8 17.8 21.3 11.9 32.2 6.1 44.0 25.6 2.4

Sweet potatoes, no slop 12.5 11.9 14.8 7.1 23.1 4.8 19.0 23.8 1.7 Amount synthesized 2.0 6.6 4.5 3.7 12.1 3.1 7.1 19.5 1.5 Sweet potatoes + slop 13.4 12.3 ■M7 n * nf 8.7 24.7 4.5 39.6 25.3 1.9 Amount synthesized 2.9 7.6 7.5 1 5.3 13.7 2.8 27.8 21.1 1.7

(continued) Table 29. Comparative amino acid values of yeast products from various substrates, (continued) (micrograms per gram, dry material).

Phenyl- Threo­ Iso- Methio­ Substrates Arginine Leucine Lysine alanine Valine Histidine nine Leucine nine

White potato, no slop 22.8 20.4 24.4 11.2 31.6 7.6 60.0 36.4 2.4 Amount synthesized 15.8 15.8 16.4 7.9 19.1 5.3 41.7 26.6 1.6 White potato + slop 13.5 13.1 23.1 9.1 28.9 5.4 45.7 31.4 1.7 Amount synthesized 6.5 8.5 15.1 5.8 16.4 3.1 27.4 21.6 0.8

Wood sulfite liquor* 36.1 35.7 41.4 24.1 29.8 13.1 25.8 37.5 8.4

* Inskeep, G. C., A. J. Wiley, J. M. Holderby, and L. P. Hughes (1951). - 98 -

DISCUSSION

This study is believed to be the first in which amylase

synthesizing yeasts have been intentionally used for the production

of a nutritional product. Sanchez-Marroquin and Solorozano (1947) have studied the use of Endomycoipsis fibuliger and Saccharomyces

carba.jali. but their work was apparently confined to the alcoholic

fermentation of starchy substrates. In the present study yeasts of

the species E. fibuliger (Lindner) Dekker or E. chodati (Nechitch)

Wickerham and Burton were utilized to produce yeast preparations

directly from amylaceous substances. The extracellular enzyme

system produced by these yeasts converts starch to fermentable

sugars; these sugars are then available for cell growth by that same

organism or by any other organism which is present in the medium.

In this study a non-amylase synthesizing yeast, such as Torulopsis utilis (Henneberg) Lodder was cultured in combination with o ne of

the starch hydrolyzing yeasts.

The propagation was conducted in the presence of a source of

assimilable nitrogen, s\ich as urea or ammonium sulfate, together

with potassium phosphate, magnesium sulfate, and calcium carbonate.

The propagations were not unduly sensitive to variations in quantity

of such added mineral salts when a wheat mash was used as the medium. - 99 -

The lack of significant differences when phosphate, magnesium,

and calcium salts were added to the mash in varying amounts is not

surprising considering that a whole wheat mash, made with tap water, was used as the propagation medium. The tap water used contained

approximately 300 to 400 mg. per liter of calcium and magnesium

salts, and could be expected to furnish sufficient amounts of these

minerals for the growth of the yeast especially since the pH was

acid in all cases. Swanson (1912) has shown that approximately

0 .4 8 2 per cent of the dry weight of whole wheat is due to water-

soluble phosphorous compounds. If these phosphates are in an

assimilable form it may well account for the lack of significant

results when phosphate salts were added.

The wide range of pH values at which the propagation was

operable is important from an industrial standpoint. That yields

were lowest at pH 4.0 seems reasonable if it is assumed that the

pH optima for E. fibuliger amylases are similar to those of fungal

amylases. Tsuchiya, Gorman, and Koepsell (1950) report considerable

inactivation of alpha-amylase at pH 4.2. Alpha-amylase was staole

over a pH range of 4.75 to 7.25, according to these workers. An

operating pH of 5.0 seems to be the most suitable for the propa­

gation as alpha-amylase is quite stable at this value. A pH of

6 .0 might give slightly better growth of T. utilis, but in a con­

tinuous operation,where aseptic conditions are not observed, use of - 100 - medium adjusted to such a high pH value would increase the danger of bacterial contamination. Harris, et al. (1948) conducted a four-month continuous propagation of Torulopsis utilis without using aseptic conditions. No contamination was observed as long as the pH was held at 5.0; however, if the pH rose to 6.0 bacterial con­ tamination became evident. This contamination disappeared as soon as the pH was readjusted to 5.0.

Although it is necessary to carry out the propagations under aerobic conditions, the rate of aeration is not as critical a factor as is the degree of agitation which has a marked effect upon the results obtained in this process. *or example, the net yield of synthesized protein appears to depend upon the degree of agitation.

Optimum results are obtained under vigorous agitation in which the whole volume of mash is maintained in a state of continuous movement•

Hixson and Gaden (1950) have presented data obtained in studies of oxygen transfer which indidate that successful fermen­ tations require not only sufficient oxygen for metabolic activity, but intimate contact between organism and substrate. Gaden (1952) discussed the effects of agitation and mass transfer in fermentations.

Agitation by aeration alone may be used, but this is usually accom­ panied by a foaming problem. With mycelial organisms, mechanical agitation provides a great number of growth centers by causing fragmentation and a relative motion between the microorganism and the medium . At low agitation rates microorganisms may clump so - 101 - that the surface exposed to the medium is very small. Autolysis may occur if the cells settle to the bottom of the propagator or

fermenter. It is believed that since one of the organisms used in

the propagation is mycelial these observations of Gaden apply to

this propagation.

The process -was modified by the use of amylase-containing

liquid (backslop or slop) obtained from a previous propagation.

This liquid was mixed with starch-containing mash to accomplish

partial hydrolysis. This treated material was then used for the

propagation of the amylolytic and the ancillary yeast. Backslopping

will reduce the initial viscosity of the mash making it easier to

handle in pumps and other equipment usually found in a yeast

factory. The backslopping procedure may be operated continuously

as follows: The starch-containing material such as macerated

potatoes, ground corn, wheat, or similar material is first mixed

with an initial amount of slop and the mixture heated to optimum

temperatures to gelatinize the starch and to reduce viscosity.

The amylase in this first mixture will be inactivated, but more

backslop is added and the temperature reduced by this addition to

55° C., whereupon the amylase in the backslop will convert much of

the starch to sugar. The converted mash is then fed continuously

to the yeast propagators. The propagators also receive a continuous

supply of mineral salts and simple nitrogenous materials. Aeration

and agitation are provided at a rate known to permit the best growth

of the yeast cells. Liquid containing the yeast, amylase, and minor - 102- amounts of residual nutrients is continuously removed from the propagator to a second propagator or storage tank and from there it is led to centrifugals which separate the solids from the major part of the liquid. The solids may be dried for storage and later used as food or feed. The liquid is returned to the initial stage of the process as backslop.

The type of operation described above is especially important in that there is little disposal of liquid wastes required. When the osmotic pressure of the medium becomes too high to support good yeast growth, the entire mass may be concentrated and dried. At this stage some waste disposal may be necessary. The selection of an ancillary yeast which is tolerant of high osmotic pressures, such as Hansenula subpelliculosa, might be advantageous in this type of operation.

The efficiency of this process is difficult to determine because more than one nutritionally important compound is being produced. The value of the product should be based on its bio­ logical value as a feeding product. This value must be obtained by feeding studies.

White (194S) and Agarwal and Peterson (1949) observed that in the production of food yeast from natural substrates the yield of yeast is often calculated on the basis of the sugar supplied or utilized. These bases of calculation are misleading since they do - 103 - not account for the utilization of other organic compounds present in natural materials. Sperber (1945) made a carbon balance and found that more carbon was recovered as yeast and carbon dioxide than was present in the sugar of molasses. He attributed this excess carbon to non-sugar compounds which were used by the yeast in the synthesis of cell material.

Since the calculation of efficiencies upon the basis of sugar utilized or supplied is so common it may be of interest to discuss this procedure and to relate it to the present process. The theoretical yield of yeast (total dry weight) that can be expected has been discussed by several writers. Brahmer (1947) calculated the highest possible yield for a yeast containing 6 per cent nitrogen to be 83 per cent, by weight, reckoned on glucose. Effront (1927) stated that 2 .0 grams of glucose are required to form 1 .0 grams of yeast protein. The work of Claasen (1935) upheld the figures of

Effront. Claasen obtained 1.0 grams of yeast protein from 2.14- grams of glucose. He found, however, that the maximum practical yield of yeast obtainable from 100 grams of hexose sugar was 47.5 grams. Assuming yeast to be 55 per cent protein would give an expected yield of approximately 2 6 .1 grams of protein from 100 grams of hexose sugar.

According to White (1948) the average carbon content of yeast dry matter is about 47 per cent, so that 48 parts of carbon from

180 parts of hexose sugar will form 1 0 2 .1 parts of yeast dry matter. - 104 -

The maximum yield obtainable from 100 parte of hexose sugar is 56.7 parts of yeast dry matter. This would give a theoretical yield of

39.08 pounds of yeast dry matter from 100 pounds of wheat which contained 68.4 pounds of starch (calculated as glucose). Assuming yeast to be 50 per cent protein, the theoretical yield of protein would be 1 9 .5 pounds from 100 pounds of wheat, or 6 8 .4 pounds of starch, calculated as glucose. These figures of Claasen and White correspond approximately to the accepted yield of T. utilis from molasses, i.e., 25 parts of yeast protein from 100 parts of sugar.

The efficiencies of protein production, based on the sugar supplied and the sugar utilized, are presented in Table 30.

Efficiencies are calculated on the assumption that 2 parts of hexose yields 0 .5 parts of yeast protein.

The large discrepancies in the two sets of potato experiments requires further study. In the view of preliminary observations made by Wickerham (1949) that yeast grew poorly on medium made from old, sprouting potatoes as compared with medium made from unsprouted potatoes, when the peels were included in the medium, it may be of interest to determine whether such a relation also holds true for stored and freshly harvested potatoes as this study seems to indicate. If this is true, it should be determined whether such lessening of growth is due to a. "positive" cause, such as the production of a toxic substance, or to a "negative" cause such as the loss of some necessary growth factor upon storing. - 7 - be made appealing to the consumer. If people do not accept the new

food the product may be modified until it is acceptable. Another

procedure open to the food scientist is to adopt every means of modern

advertising to persuade the people that this new product is not only

something they need, but something that would be a valuable supple­ ment in their daily diet. An example of the eventual acceptance by

the general public of a food substance which was rejected upon its

introduction may be found in the story of the potato.•

The eating habits of the entire Irish nation, as well as much of

Europe, were changed radically by the importation of the potato from

South America by Sir Walter Raleigh and the Spanish (Salaman, 19^9)f

however, three centuries were required for the new food to become popular with the people. New food sources such as yeast may thus

become established in the world food industries. Economic pressures

and a program of education may reduce the time required to bring about

this adjustment. Meanwhile, yeast may be fed to animals to produce the * tasty meat products to which people in countries such as the United

States, who have not yet felt the pinch of widespread hunger, are

endeared. In this way there will be an alleviation of the protein

shortage as more edible crop plants are released for human instead of

animal feeding. Yeast may be produced, as will be demonstrated, from

inedible materials.

This paper, then, will be devoted chiefly to a consideration of

the production of a nutritional yeast product from substrates .. Table 30. Comparative efficiencies of protein synthesis on various substrates based on carbon supplied and utilized.*

Substrates Lbs. starch Per cent Lbs. protein Per cent Per cent supplied (as starch synthesized efficiency efficiency glucose) per 100 utilized per 100 lbs. on starch on starch lbs. raw material raw material supplied utilized

Purified wheat starch + i 105 yeast extract 111.0 29.6 8.9 32.0 108.2

Whole wheat mash 70.6 87.4 4.4 25.0 28.4 Whole wheat mash. backslopped 70.6 91.7 7.3 41.4 45.0

White potato 40.4 59.5- 10.8 1 0 6 .8- 180.0 68.3 26.6 48.0 White potato, backslopped 40.4 84.9 11.1 110.0 129.0

* Efficiencies are based on the assumption that 1 lb. of hexose sugar gives (continued) 0.25 lb. of yeast protein. ( \ Table 30. Comparative efficiencies of protein synthesis on various substrates based on carbon supplied and utilized* (continued)

Substrates Lbs. starch Per cent Lbs. protein Per cent Per cent supplied (as starch synthesized efficiency efficiency glucose) per 100 utilized per 100 lbs. on starch on starch lbs. raw material raw material supplied utilized

Sweet potato 49.4 58.6 - 13.5 109.2- 186.2- 45.5 33.4 61.8 Sweet potato, 106 backslopped 49.4 73.4 8.9 47.0 78.4

Rice 75.6 25.2 1.0 5.0 19.6 Rice, backslopped 75.6 64.8 5.7 25.2 46.6

Corn 82.9 22.3 1.4 6.8 30.6 Corn, backslopped 82.9 33.6 3.5 16. 8 49.8

* Efficiencies are based on the assumption that 1 lb. of hexose sugar gives 0,25 lb. of yeast protein. - 107 -

It is evident from the data that the efficiencies of protein production on the basis of starch supplied or starch utilized, were quite low for all substrates used. It was observed that fermentation took place during the propagations. Evidence for this was the strong odor of esters or alcohol. Since 87 per cent of the starch in a 5 per cent wheat mash was utilized, without the addition of backslop, and 92 per cent of the starch was utilized when backslop was added, and the respective efficiencies were 1 4 .2 per cent and

2 2 .5 per cent, it seems evident that some non-protein carbon compounds were being formed. Whether or not most of this carbon was yeast material is not known. The difficulty of separating the yeast from the residual solids of the mash added to the complexity of the analytical problem.

Further studies on the physiology of the starch-hydrolyzing yeasts seem to be in order, especially as related to the amylolytic process.

This further knowledge may make possible the devising of a more efficient process. Simplification of the culture media by the use of purified starch, plus other necessary nutrient materials may be necessary in order to escape the effects of the many complex organic compounds, and their degradation products, found in raw materials such as wheat, corn, and potatoes.

Industrial applications of the use of amylolytic yeasts for the production of nutritive materials from starch-containing substrates could assume two forms: (l) a process in which a portion of a starch- - 108 -

containing crop, such as potatoes, is used for the production of food yeast, and (2) a process in which a starch-containing waste is utilized. In the first instance a mash concentration as high as

could efficiently by handled would be desired in order to make the most efficient use of the propagators. In regard to the second case,

that of disposal of starchy wastes such as are obtained from starch

processing plants, the utilization of large amounts of dilute materials,

often containing as little, as 1 per cent, or less, of solids would be

required. In view of the results obtained it appears that yields

increase in mashes above 5 per cent solids concentration. Whether

it would be desirable to operate at these lower solids concentrations

would depend upon the costs of operation with such dilute materials,

as well as the demand for yeast, and the efficiency of the yeast

process in reducing the biological oxygen demand. If dilute materials

had to be concentrated before propagation the power costs could be

expected to be excessive. Schleef (1948), in a study of the economics

of fodder yeast produced from sulfite waste liquor, has pointed out

that yeast production would not be considered an absolute solution to

the pollution problem since the biological oxygen demand of industrial

wastes would be reduced only a little over one-half. Further experi­

ence is necessary in the actual fermentation of waste materials by

yeast before the value of the process can be fully established.

Amino acid and vitamin analyses indicate that the product

obtained in this study contains all of the essential amino acids,

with the possible exception of tryptophane which was not assayed, and - 109 - varying amounts of the B-vitamins, niacin, pantothenic acid, riboflavin, biotin, pyridoxine, thiamine, and choline. No con­ clusion can be reached, however, as to the suitability of this product for inclusion in the animal diet until feeding tests have been conducted. These tests must be made in order to determine availability of the vitamins and amino acids and to eliminate any possibility of harmful effects due to the feeding of this yeast product.

Dirr (1942) described the significant increase in uric acid in the body fluids of humans due to the ingestion of yeast. Braude

(194S) points out, however, that in cattle and other animals uric acid is converted to allantoin. There is no danger, therefore, of uric acid appearing in the blood of these animals as a result of ingestion of yeast. There have been various reports in the literature of the production of liver necrosis by the ingestion of certain types of yeast (Cty-orgy, et al. 1950). This problem must be thoroughly investigated before the use as a feed of yeasts, which have not previously been so used, is advocated. ■»

According to the United States Pharmocopeia (1950), dried yeast must contain not less than 40 per cent protein, and, in each gram of dried yeast, the equivalent of not less than 0.12 mg. of thiamine hydrochloride, 0.04 mg. of riboflavin, and 0.25 mg. of niacin. The yeast products obtained from the various starchy substrates used in this study are all low in these three vitamins. In general, the - 110 -

amounts of niacin obtained approach more closely the required amounts than do the amounts of riboflavin and thiamine (Table 31).

Peterson (1948), among others, has pointed out the variation in the content of B-vitamins depending upon the strain of yeast and, more

especially, upon the cultural conditions used. No single set of figures

can be used to express the values. Fink and Just (1942) found that the

thiamine content of yeast depends largely upon the thiamine content of

the substrates and of the inoculum. Comparative amounts of the yeast

products obtained from various starchy substrates required to furnish

the minimum daily requirements of riboflavin, thiamine, and niacin

are presented in Table 31. The minimum daily requirements are those

recommended for adults by the National Research Council (1943).

Further study of operating conditions may make possible a process

in which the yields of vitamins are increased.

In regard to the selection of a suitable combination of organisms

for the propagation, there are other factors to be considered than

yields of protein, solids, and vitamins, although these are of prime

importance. For example, the separation of the solids, or product,

from the liquid at the end of the propagation period i3 an important

industrial consideration. The mycelial-type growth of the Endomycopsis

seems to simplify this task presumably by acting as a type of filter-

aid. The smaller cells are caught in the abundant bunches of - m -

Table 31. Comparative amounts of yeast products from various substrates required to furnish the minimum daily requirements of adult humans for three B-vitamins.*

Lake Starchy substrates States Wheat Sweet Potato White notato Yeast slop no slop no slop no Vitamin slop slop slop

Thiamine 339.6 300.0 163.6 360.0 450.0 180.0 163.7

Riboflavin 60.0 1350.0 142.1 207.6 158.0 2700.0 150.0

Niacin 42.2 84.2 57.0 104.1 87.1 606.8 64.2

* Minimum requirements assumed to be as follows: Thiamine 1,8 g., riboflavin 2.7 g., niacin 17.6 g. mycelial growth and the whole mass settles out leaving a clear supernatant liquid more readily than if these organisms were not used.

Differences have been noted in the rapidity of sedimentation of the solids from the mash after the propagation of the yeast. In some cases, notably with E. fibuliger NRRL Y-25, the solids began to separate from the liquid about 15 to 30 minutes after cessation of agitation and aeration. In all cases after holding the mash for 12 hours at 2° C. the solids settled out giving a "yeast cream" of about

10 to 12 per cent solids concentration. In industrial practice, according to Wiley, et al. (1951) the medium containing yeast goes to primary separators where it is concentrated to 10 to 12 per cent solids.

The yeast cream is then washed in fresh water, and finally concentrated to 14 to 18 per cent solids when it is delivered to the driers.

Primary separation is usually done by centrifuges, but it is evident that sedimentation would result in lower operating costs. - 112 -

SUMMARY

A process has been developed in -which a mixed culture consisting

of an amylase producing yeast, either Endomycopsis fibuliger (Lindner)

Dekker or Endomycopsis chodati (Nechitch) Wickerham and Burton, and

a non-amylase producing yeast such as Torulopsis utilis (Henneberg)

Lodder is cultured on a cooked, but not converted, starch mash such

as wheat, corn, rice, or potatoes. The entire mass of centrifugable

solids, after propagation, was harvested as the product. This

product,containing both yeast and residual material from the substrate

used, also contains nutritionally valuable materials such as protein and

various vitamins.

In general, yields of approximately 60 pounds of solids were

obtained from 100 pounds of raw material. The protein in these

solids varied from about 20 to 30 per cent. An effort to determine

the optimum operating conditions for the process was made by the

changing of a single variable in any one experiment.

The optimum concentration of wheat mash was 5 to 10 per cent.

Urea, incorporated in the mash as a nitrogen source, appeared to

give slightly better yields than ammonium sulfate, and urea had the

further advantage of facilitating maintenance of a constant pH. - 113 -

The addition o±' magnesium sulfate and calcium carbonate to the medium vras unnecessary under the conditions used. The process operated over a broad range of pH values* the optimum being pH 5 to 6. The total yields of product were approximately equal to agitation rates of 500,

650, and 800 RPM at all aeration rates tested between 0.5 and 2.0 volumes of air per volume of medium per minute, except at 350 RPM when the yields were lower. The per cent protein of the product varied with the rate of agitation. At 800 RPM the yield of protein ranged from 30 to 32 per cent, at 650 RPM the range was 28 to 30 per cent, at 500 it was 24 to 27 per cent, and at 350 it was 22 to 25 per cent. The best agitation and aeration rates, considering practical limitations, would appear to be 650 RPM and 2 volumes of air per volume of mash per minute.

A study was made of the use in a propagation of the diastatically active supernatant liquid ("slop11 or "backslop") obtained from a previous propagation containing a starch-hydrolyzing yeast. Use of this liquid was effective in reducing the initial viscosity of a starch mash by partially saccharifying the starch.

Filamentous fungus culture filtrates (Aspergillus niger) and living amylolytic fungi plus T. utilis gave better yields of protein but poorer total yields of product than were given by the starch hydrolyzing yeasts. The net yields of protein were approximately equal whether the "molds" or E. fibuliger were used in the process. - 114 - Evidence was obtained that the use of yeasts, such as Hansenula subpelliculosa, which are more tolerant of higher osmotic pressures than is T. utilis may be of value in a continuously operated process. - 8 - containing starch. The use for this purpose of species of yeasts vihich produce an extracellular amylase system comprises the experimental part of this work.

By the use of amylase-synthesizing yeasts a process may be devised for the utilization of starch -without a preliminary conversion step.

The use of a mixed culture is visualized. This would consist of a

"starch hydrolyzer", or yeast which produces sufficient enzyme to

convert the starch to fermentable sugars, and a "protein synthesizer",

or a yeast which would utilize the excess of fermentable sugar

produced in protein synthesis. The organisms could possibly be care­

fully selected so that each yeast would produce some nutritionally valuable substance which was not produced by the other organism. Thus,

a more complete feeding product could be obtained. Indeed, it might

even be possible to produce a feeding product "tailored" to fit certain

specialized requirements in human or animal feeding.

Fulmer (1930) has summed up the problem of mixed fermentations

quite well when he said, "Evidently pure culture industrial fermenta­

tions are not always the most desirable, and it is likely that some

of the most striking advances in zymotechnology lie in the utilization

of synergistic phenomena". - 115 -

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« i t Dirr, K. 1942. Uber den Wert der Wuchshefen fur die menschliche

it Ernahrung. II. Uber die biologischen Brauchbarkeit der Bergin

Holzzuckertrockenhefe. Biochem. Zeitschr. 312: 233 - 251. - 1 1 8 -

Dunn, C. G. 1952. Food Yeast. Wallerstein Lab. Communic. 15:

61 -79.

n Fink, H. and F. Just. 1942. Uber den Vitamin B-gehalt verschiedener

Hefen und seine Beeinflussung. V. Die Vitamin B^-Verluste bei II der Zuchtung von Hefen im Luftungsverfahren. Biochem. Zeitschr.

311: 61-72.

Floro, W. R., V. Williams, W. A. Flook, and J. S. Collier. 1947.

Food Yeast: Some aspects of its development and production.

Proc. British West Indies Sugar Tech. 1947: 38 - 53.

Frey, C. N. 1930. History and development of the modern yeast

industry. Ind. Eng. Chem. 22: 1154 - 1162.

______, G. W. Kirby, and A. Schultz. 1936. Yeast: Physiology,

manufacture, and uses. Ind. Eng. Chem. 26: 879 - 884.

Fulmer, E. I. 1930. The chemical approach to problems of

fermentation. Ind. Eng. Chem. 22: 1148 - 1150.

Gaden, E. L. 1952. Agitation and mass transfer in fermentation.

The study of effects on individual cells. Abstracts of Papers,

121st Meeting, Amer. Chem. Soc. - 119 -

Gontard, A. V. 1948. Yeast: Dried yeasts and their derivatives.

I. The story of yeast. Anheuser Busch Inc., St. Louis.

Greening, E. W. and E. 0. Greening. 1893. An improved method of

utilizing and means for treating yeast. Eng. Pat, 20,060.

Gyorgy, P., C. S. Rose, R. M. Tomarelli, and H. Goldblatt. 1950.

Yeast in the production of dietary massive hepatic necrosis in

rats. Jour. Nutrition 41: 265 - 278.

Hesse, A. 1949. Industrial biosyntheses. I. Fats. Adv. in Enzymol.

9: 653 - 704.

Harris, E. E., J. F. Saeman, R. R. Marquardt, M. L. Hannan, and S. C.

Rogers. 1948. Fodder yeast from wood hydrolysates and still

residues. Ind. Eng. Chem. 40: 1220 -1223.

Hayduck, F. 1915. Die Herstellung und Verwendung von Hefe als

it Nahrungs- und Futtermittel unter besonderer Berucksichtigung

der gegenwartigen Verhaltnisse. Zeitschr. Angew. Chemie

Wirtschaftlicher Teil 28: 697 - 699.

Hixson, A. W., and E. L. Gaden Jr. 1950. Oxygen transfer in submerged

fermentation. Ind. Eng. Chem. 42: 1792 - 1801. - 1 2 0 -

Hodge, J. E. and H. A. Davis. 1952. Selected methods for determining

reducing sugars. U. S. Dept. Agr. Bull. AIC - 333.

Horn, M. J., D. B. Jones, A. E. Blum. 1950. Methods for micro­

biological and chemical determinations of essential amino acids

in proteins and foods. U. S. Dept. Agr. Misc. Publication No. 696.

Horowitz, N. H. and G. V/. Beadle. 1943* A microbiological method

for the determination of choline by the use of a mutant of

Neurospora. Jour. Biol. Chem. 150: 325 -333.

Husain, S. 1946. Yeast as a food supplement in Indian diet. J. E.

Seagram and Sons, Inc. Louisville.

Inskeep, G. C., A. J. Wiley, J. M. Holderby, and L. P. Hughes. 1951.

Food yeast from sulfite liquor. Ind. Eng. Chem. 43: 1702 - 1711.

Kressel, E. 1895. The manufacture of a new alimentary extract.

Eng. Pat. 15,885.

Lindner, P. 1907. Endomyces fibuliger n. sp. ein neuer Garungspilz

und Erzeuger der sog. Kreidekrankheit des Brotes. Wochenschr.

Brau. 24: 469 - 474. - 121 -

Locke, E. G., J. F. Saeman, G. K. Dickerman. 1945. The production

of wood sugar in Germany and its conversion to yeast and

alcohol. FIAT Final Report No. 499.

Mather, K. F. 1952. The problem of antiscientific trends today.

Science 115: 533 - 537.

Maynard, L. A. 1946. The role and efficiency of animals in

utilizing feed to produce human food. Jour. Nutrition 32:

345 - 361.

National Research Council (U.S.). 1943* Recommended dietary

allowances. Reprint and Circular Series No. 122.

Washington, D. C.

Nechitch, A. 1904. Sur les ferments de deux levains de l'Inde, le

Mucor praini et le Dematium chodati. Action des sels sur la

fermentation alcoolique. Inst, botan. Univ. Geneve 5: 1-36.

Pasteur, L. 1876. Etudes sur la Biere. Masson, Paris. 1928.

Peterson, W. H. 194S. Vitamins and minerals of yeast. In ,rYeasts

in feeding: A symposium11. Brewers1 Yeast Council. - 122 - Roine, P. 1946. On the synthesis of nitrogenous compounds by yeast.

Suomen. Kemist. 19 B: 40.

Salaman, R. N. 1949. The history and social significance of the

potato. MacMillan Co. Toronto.

Sanchez-Marroquin, A. and M. Solorozano. 1947. Estudios sobre la

amilasa de Endomycopsis fibuliger. II. Poder amilolitico y

fermentativo en cultivos mixtos. Anales escuela nac'l cienc.

biol. (Mex.) 4: 311 - 324.

Schleef, M. L. 1948. Economics of fodder yeast from sulfite waste

liquor. “Washington State College Bur. Econ. Bus. Res. Bull. No.

7: 1 - 43.

Skeggs, H. R. and Li D. Wright. 1944. The use of Lactobacillus

arabinosus in the microbiological determination of pantothenic

acid. Jour. Biol. Chem. 156: 21 - 26.

Skoog, F. 1945. Food yeast production and utilization inGermany.

British Intelligence Objectives Sub-committee Report No. 21

(PB Report 2041).

Snell, E. E. and F. N. Strong. 1939. 4 microbiological assay for

riboflavin. Ind. Eng. Chem. (Anal. Ed.) 11: 346 - 350. - 9 -

REVIEW OF LITERATURE

The nutritional value of yeast in the diets of human beings and other animals has been the subject of much research in recent years. Excellent general reviews covering the nutritional studies of yeast, as well as methods of production have been presented by Braude (19^2), Dunn (1952), Hesse (19^9), Bunker (19^8), Colonial

Food Yeast Limited (19^), and the proceedings of the symposium "Yeasts in Feeding" (19^8). Skoog (19*4-5) has reported the importance and the development of the nutritional yeast industry in Germany, and Locke,

Saeman, and Dickerman (19*4-5) have discussed the production of feeding yeast from wood sugars.

To appreciate fully the development of the food yeast industry

it will be necessary to go back to the beginning of the yeast indus­ try in general. In reviews of the history of yeast production, Frey

(1930)j and Gontard (19*4-8), among others, have pointed out the various periods of development of the yeast industry. That the ancient peoples understood quite well the use of yeast in fermentations has been quite well established. The beginning of the scientific period as far as yeast is concerned may be said to be the recognition by Van Helmont

(1577 - l6Mi-) that fermentation was caused by a specific agent.

Following this time many workers applied themselves to the problem

and the modern theory of fermentation slowly developed. White (19*4-8),

describing the compressed yeast industry, stated that it is only - 123 -

Somogyi, M. 1945* A new reagent for the determination of sugars.

Jour. Biol. Chem. 160: 6l - 68.

Sperber, E. 1945. Studies in the metabolism of growing Torulopsis

utilis under aerobic conditions. Ark. Kemi. Min. Geol. 21 A:

1 - 136.

Stelling-Dekker, N. M. 1931. Die Hefesammlung des Centralbureau

voor Schimmelcultures. I. Teil. Die sporogenen Hefen.

Koninklijke Akademie van Wetenschapper, Amsterdam.

Stokes, J. L., M. Gunness, I. M. Dwyer, and M. C. Caswell. 1945.

Microbiological methods for the determination of amino acids.

II. A uniform assay for the ten essential amino acids.

Jour. Biol. Chem. 160: 35 - 49.

Stone, M. 1942. And the greatest of these is — yeast. Amer.

Brewer 75 (2): 3& - 39.

Swanson, C. 0. 1912. Acidity in wheat flour: its relation to

phosphorous and to other constituents. Ind. Eng. Chem. 4:

274 - 278. - 124 -

Thaysen, A. C. 1943* Value of microorganisms in nutrition (Food

Yeast). Nature 151: 406 - 408.

. and M. B. Morris. 1943* Preparation of a giant strain

of Torulopsis utilis. Nature 152: 526 - 528.

______. 1944. Production of strains of

yeast suitable for food purposes. British Patent 560,800.

Tsuchiya, H. M., J. Corman, and H. J. Koepsell. 1950. Production of

mold amylases in submerged culture. II. Factors affecting the

production of alpha-amylase and maltase by certain Aspergilli.

Cereal Chem. 27: 322 - 330.

United States Department of Agriculture. 1952. Crop Production:

Annual Summary. Crop Reporting Board, Washington, D. C.

United States Pharmocopeia. 14th Edition. 1950. 670,

it Voltz, W. and A. Baudrexel. 1911. Die Verwertung der Hefe im

menschlichen Organismus. Biochem. Zeitschr. 30: 457 - 473.

Von Loesecke, H. W. 1946. Controversial aspects: Yeast in human

nutrition. Jour. Amer. Dietitic Assoc. 22: 485 - 493. Wahl, R. and Henius, M. 1895. Process of making yeast food.

U. S. Patent 540,471.

White, J. 1948. The principle and practice of yeast production. I.

Historical. Yeast physiology and nutrition. Amer. Brewer 81:

21 - 23, 48, 52.

Wickerham, L. J. 1949. Unpublished data.

______, and K. A. Burton. 1952. Occurrence of yeast

mating types in nature. -Jour. Bact. 63: 449 - 451.

______, L. G„ Lockwood, 0. G. Pettijohn, and G. E. Ward.

1944. Starch hydrolysis and fermentation by the yeast

Endomycopsis fibuliger. Jour. Bact. 48 : 413 - 427.

Wiley, A. J. 1952. Personal communication.

______, G. A. Dubey, B. F. Lueck, and L. P. Hughes. 1950.

Torula yeast grown on spent sulfite liquor. Ind. Eng. Chem. 42

1830 - 1833.

______, J. M. Holderby, and L. P. Hughes. 1951. Food yeast

from sulfite liquor* Ind. Eng. Chem. 43• 1702 - 1711. - 126 - it Wochenschrift fur Brauerei. 1899. Ueberaichtliche zusaramenstellung

der Verfahren und Vorschlage zur Verv/erthung der Hefe, 1ns-

besondere zur Verarbeitung derselben zu Nahrungs- und Genuss-

bezw. zu Futtermitteln. 16 : 307 - 310.

Wright, L. D. and H. R. Skeggs. 1944. Determination of biotin -with

Lactobacillus arabinosus. Proc. Soc. Expt. Biol. Med. 56:

95 - 98.

Yin, H. C. 1949. Microbial farming. Econ. Bot. 3' 18J+ - 192. - 127 - GLOSSARY

Backslopping — The practice of adding slop obtained from one

experiment to the mash in a new experiment.

Mash — A mixture of grain, or tubers, and liquid consisting of water

or slop, or both.

Premalting — The technique of adding a small amount of amylase-

containing material to ground grain before cooking. Although

the amylase is inactivated when the cooking temperature is

reached, a certain amount of starch is saccharified before this

point, thereby reducing the viscosity of the mash.

Propagation — An aerobic process in which the emphasis is placed

upon obtaining optimal yeast growth and multiplication. This

term is used to differentiate the process from "fermentation" which

is usually considered to mean an anaerobic process for the production

of alcohol or other organic compounds.

Propagator — Any container in which yeast is propagated.

Slop — The supernatant liquid obtained by centrifugation of the

mash at the completion of the incubation period (propagation

period) of the yeast. - 1 2 8 -

Autobiography

X, Calvin Charles Kuehner, was born in Put-In-Bay, Ohio,

December 12, 1922. 1 received my secondary school education in the public schools of the city of Columbus, Ohio. My undergraduate training was obtained at The Ohio State University, from which I received the degree Bachelor of Science in Agriculture in 1949 after completing undergraduate requirements of the Plant Science curriculum.

After further study in the Botany Department, specializing in Mycology,

I was awarded the degree Master of Science from The Ohio State

University in 1950. While in residence at The Ohio State University

I assisted Dr. William D. Gray, Department of Botany and Plant

Pathology, during 1949 - 1950. I received a Reasearch Assistantship from the Ohio Agricultural Experiment Station during 1950 and 1951 which enabled me to study the physiological effects of radioactive isotopes upon fungi. My work was carried on in the Department of

Botany and Plant Pathology, The Ohio State University. In the autumn of 1951 I assumed the position of Zymologist at the Northern

Regional Research Laboratory, United States Department of Agriculture,

Peoria, Illinois, where I conducted off-campus research to complete my requirements for the degree Doctor of Philosophy. - 10 - . recently that the production of yeast in more or less pure culture has been effectively used for both baking and brewing. During the

nineteenth century yeast produced as a by-product of the brewing and distilling industries was used for bread making. Beginning about i860, more and more attention was paid to the possibility of improving yeast yields from brewing processes. The "Vienna" process, which was begun about that time, led to much larger yields of yeast than had been previously obtained. This process involved the gentle aeration of a malted grain infusion, a departure from the prevailing technique of using unaerated brews.

The studies of Pasteur (1876) laid the foundation for the study of the physiology of yeasts. Frey (195°) stated that Hansen of

Copenhagen, apparently stimulated by the researches of Pasteur, began his work on the culture of yeasts. He successfully applied the technique of obtaining pure cultures from single cells which had been developed by Brefeld in 1870 for use with molds. Frey has also emphasized the fact that the pure culture techniques of Hansen did not gain immediate acceptance, msorne situations it still has not been accepted but, as

White (19^8) pointed out, it is generally considered to be an important factor in the successful production of good quality compressed yeast.

Greening and Greening (1893) were among the early workers in the feed yeast industry, jn their patent they describe a method for passing waste brewers' yeast through steam heated rollers until the - 11 - moisture is reduced to less than 10 per cent. The flakes were then scraped from the rollers hy knives and ground into a meal or powder.

They stated, "Fresh yeast meal, being rich in albuminous matters, and the tonic principle of hops, is serviceable for admixture with other substances to increase their feeding value and flavour when used as feeding meals, or cakes, for cattle". They further proposed that dirjby _ II or stale yeast be used as fertiliser. In 1899 the Wochenschrift fur

Brauerei reviewed the literature on yeast as a foodstuff. This is believed to be the first review article on the nutritional yeast industry. In this article the processes for the utilization of yeast as a food are classified as follows:

1. Processes for the extraction of soluble constituents

from the yeast.

2. Processes which utilize the whole of the yeast

substance as a food.

3. The incorporation of yeast with fodder materials for

cattle.

Under the first category it seems that the first published process was an Austrian patent taken out by Bauer, Kanis, and Jahn in 1888. In this patent the substitution of a concentrated yeast decoction for meat extract was described. The first working process was an American patent taken out by Wahl and Henius (1895) in Chicago. In their patent the decoction was evaporated in vacuo to a syrup, or made into biscuits.

The next advance was by Kressel (1895) who proposed that the yeast be extracted at a temperature not exceeding 58° C. to obtain the coagu- lable protein. Although these, as well as many other workers, suggested - 12 — the use of yeast as a foodstuff, Voltz and Baudrexel (1911) were the first to actually conduct feeding experiments to determine .the food value of the yeast. They reported that human feeding experiments showed k6 per cent digestible protein in a yeast treated with sodium carbonate and water to remove the bitter taste of the hops. Eighty- six per cent of the protein was assimilated with 7^*8 per cent of the available energy in the protein being utilized. These results were obtained in nitrogen equilibrium experiments. These workers also reported the feeding to humans of 100 grams of ..yeast in a period of one to two hours without any apparent ill effects.

Czadek (1911), in Austria, reported dried brewers' yeast to be a stable feed containing crude protein which was 9 0 .8 per cent digest­ ible. He recommended the use of yeast in feeding because of the protein content and especially because of the high content of phosphoric acid.

In 1915 Crowther reported the first experiments carried out in

Great Britain. Brewers' yeast was fed to cows and pigs with varying degrees of success. Crowther further reported that he experienced difficulty in making the cows eat the yeast because of its bitter taste. He stated, "There is no reason to believe that dried yeast possesses special virtues which any other highly digestible food rich in albuminoids might not be expected to possess".

From these initial papers in the use of yeast in feeding, it is evident that the nutritional yeast industry of today has its roots in - 13 -

the brewing industry. These early writers have clearly indicated

that an attempt was being made to utilize surplus brewers1 yeast.

Protein-poor Germany with a widespread brewing industry could naturally be expected to be a pioneer in this field. The main

initial interest in developing a yeast feeding program could be

expected to come from the European countries which needed to explore

every possibility for increasing the food supply. In countries such as the United States where food was abundant and the pressure of

increasing populations and decreasing resources had not been felt to any appreciable extent, this question was not of vital interest.

The greatest advances in the yeast industry were made during the period of the first World War, when scarcity and high cost of cereal grains, together with the blockade of the coast of Europe, brought about extensive research by the Central Powers, on the production of yeast from substitute materials.

, ii n Hayduck (1915)# at the Institut fur Garungsgewerbe in Berlin, developed the details of processes for thed preparation of yeast using molasses as a raw material, with the addition of ammonia and ammonium salts to the fermenter or propagator as sources of nitrogen for the

synthesis of yeast protein. He used Torulopsis utilis, which was named the "mineral yeast" because it could be cultured on a sugar medium containing mineral salts. Hayduck introduced the idea of the

"zulauf" or differential feeding into practice. This was the technique of feeding wort and mineral salts into the fermenter at an increasing - Ik -

rate in order to keep pace with the yeast growth.

According to Skoog (19^5); large scale manufacture of yeast was planned in Germany using molasses as the raw material. Shortages of

sugar prevented these plans from being carried out on a significant-

scale, and the end of the war brought a cessation of interest in the

practical aspects of the problem. Research, however, continued in

this field. Von Loesecke (19^6) remarks that part of the failure of

the project was due to the overfeeding of yeast to humans.

The use of yeast as a food was reintroduced in Germany on a large

scale in 1936 as a part of the "Four Year Plan". It was considered

desirable to cover a large part of the lack in high quality protein

required for self-sufficiency by producing food yeast industrially

from waste products from the wood pulp, paper, and staple fiber indus­

tries as well as from other carbohydrate wastes. The yeast was to be

used by the army in the form of extracts to enrich low protein foods,

and in the form of "feed briquettes™, or feed concentrates, for horses.

The former usage was so successful that the import and use of meat

extracts was gradually eliminated bringing about a considerable

saving in meat and transportation facilities.

The extreme importance occupied by yeast in the food supply of

Germany is indicated by the fact that a plant expansion program was

especially approved by Hitler in 19^3> and an order by Roichsmars eftall - 15 - Goering in 1 9 ^ gave the yeast program the same priorities as the mineral oil program. In spite of the demand, and elaborate construc­

tion planning for a yearly production of approximately 130,000 tons,

the actual production of dried yeast reached an estimated maximum rate

of only 15,000 tons.

In Great Britain before and in the early days of World War II,

some attention was given to the possibility of using dried yeast as

a food for livestock. Thought was later given to the use of yeast as

a food especially to supplement the diet of certain people in the

Colonial possessions. Thaysen and Morris (19^3; 19^*0 developed a

rapidly growing strain of Torulopsis utilis (Torulopsis utilis var.

thermophila) which can be propagated in tropical countries. Torulopsis

utilis var. thermophila has an optimum growth temperature of k0° C. as

compared with temperatures of 25 ° to 30°C^'commonly used in yeast

production.

In Britain, according to Stone (19^2), surplus brewers1 yeast was put to many uses. It went to make "meat extracts", packet gravies,

and soups. The demand for "marmite" and similar yeast products

exceeded the supply. Yeast was also incorporated in poultry feed and

has been used to supplement the oat ration of horses if added to the

extent of one-eighth of the oat ration, with a small addition of salt.

Floro, Williams, Flook, and Collier (19^7) give a description of

the production of Torulopsis utilis, for nutritional purposes, which Acknowledgement s

I wish to express my gratitude first of all to Dr. R. W.

Jackson, Head, Fermentation Division, to Dr. Kenneth B. Raper, formerly In Charge, Culture Collection Section, and to Dr. L. J.

Wickerham, ZymologLst, Culture Collection Section, Northern

Regional Research Laboratory, Peoria, Illinois, for permission to use laboratory facilities for the conduct of this research. I am especially indebted to Dr. Wickerham, who formulated the problem, for his advice and helpful criticism during the course of the research. I wish to thank Dr. Harlow H. Hall, In Charge, Feeds and

Vitamins Section, for permission to use the facilities of his section for the analytical work, to Miss Carolyn E. Smith, Mr. John 0. Pavich

Miss Margaret Shekleton, and Mr. Kermit A. Burton for their assistance in the conduct of much of the routine work, analytical and otherwise, to Mr. Jesse R. Dilley for making the drawings, and to Mr. Roland W.

Haines for the photographs.

I wish also to thank Dr. William D. Gray, my advisor, for all the assistance and advice he has given me which, directly or indirectly, has enabled me to complete this work. Finally, I would like to express ay thanks to Bonnie M. Kuehner, ay wife, for her assistance in typing and reading the manuscript, and for her patience during the course of this work.

a 00122 - 1 6 - was "begun at the Frome factory of the British West Indies Sugar

Company Limited, in Jamaica. The work done by Thaysen and co-workers at Teddington, England was applied commercially at this place. A complete discussion of the work done on a laboratory and pilot plant scale at Teddington may be found in a publication by the Colonial

Food Yeast Limited (1944).

In the United States methods were developed for the production of food yeast from citrus waste, fruit juices, vegetable wastes, whey, wood sugars, and wood sulfite liquor. The work on the utilization of wood wastes is especially noteworthy as it has resulted in a profita­ ble industry. The Lake States Yeast Corporation at Rhinelander,

Wisconsin is presently producing Torulopsis utilis for feeding purposes

from sulfite waste liquor, a by-product of the paper industry. The present capacity of this plant is approximately 14,000 pounds of yeast per day (Wiley, 1952). - 17 -

MATERIALS AND METHODS

Microorganisms:— All of the microorganisms used in this study

were obtained from the stock culture collection of the Northern

Regional Research Laboratory, Peoria, Illinois. Some of the yeasts

were isolated and identified by Dr. L. J. Wickerham of this laboratory, while others were originally obtained from other workers.

Wickerham, Lockwood, Pettijohn, and Ward (1944) reported

diastatic activity by strains of the yeast species Endomycopsis

fibuliger (Lindner) Dekker. Lindner (1907) originally isolated,

from spoiled bread, a filamentous yeast which he named Endomyces

fibuliger. This yeast was later classified by Stelling-Dekker (1931)

as Endomycopsis fibuliger. Wickerham, et al (1944) obtained four­

teen strains of this species from Dr. Norman C. Laffer, who had

isolated them from macaroni and from flour used in the preparation

of macaroni. The strain of E. fibuliger used most often throughout

this work, NRRL Y-1062, was one of Laffer's strains. Other strains

of E. fibuliger obtained from the stock culture collection for use

in this work were NRRL Y-3, NRRL Y-25, and NRRL Y-77.

A second species of Endomycopsis was discovered and classified

as E. chodati (Nechitch) Wickerham and Burton. This organism was

first isolated from fermenting rice by Nechitch (1904) who named it

Dematium chodati. Berkhout (1923) recognized it as a yeast and gave - 1 8 - it the name Candida chodati. Wickerham and Burton (1952) observed the sexual stage of this organism and placed it in the genus

Endomycopsis. Although this organism is closely related to

Endomycopsis fibuliger it differs from it in several ways. One important difference is that E. chodati synthesizes methionine whereas E. fibuliger is deficient for methionine. This capacity could be the determining factor in the selection of E. chodati for use on substrates deficient in vitamins or amino acids. Strains of

E. chodati used in this study were obtained as follows:

NRRL Y—193S Received from Dr. Robert W. Stone, Department of

Bacteriology, Pennsylvania State College, and was

isolated from silage.

NRRL Y-2051 Isolated from spots on the surface of the bark of

a fig tree in New Orleans, Louisiana by Miss

Dorothea Teunisson. The surface of the spots was

quite hard for a depth of approximately l/l6 inch.

Under this hard surface there was about l/B inch

of soft, moist, rotted material from which the

yeast was isolated.

NRRL Y-2052 Isolated from a mass of roach remains, roach eggs,

and cobwebs obtained from wounds in a Royal Palm

tree in New Orleans, Louisiana by Miss Teunisson.

Two strains of Aspergillus niger van Tieghem, NRRL M-330 and

NRRL M-337, were obtained from the culture collection of the

Northern Regional Laboratory for comparison with the amylase-producing - 1 9 - yeasts.

The following yeasts were obtained from the Northern Regional

Laboratory culture collection and were used as ancillary yeasts in the food yeast propagation.

NRRL Y-900 Torulopsis utilis (Henneberg) Lodder.

NRRL Y-1084 Torulopsis utilis (Henneberg) Lodder, var. major

Thaysen et Morris.

NRRL Y-1094 Candida lipolvtica (Harrison) Diddens et Lodder. ii NRRL Y-1109 Saccharomvces fragilis Jorgensen.

NRRL Y-1591 Rhodotorula mucilaginosa var. plicata Lodder.

NRRL Y-1683 Hansenula subpelliculosa Bedford.

NRRL Y-2020 Candida arborea nom. nud.

NRRL Y -2053 and NRRL Y-2054 Ballistospore-producing pink

yeasts giving a good mirror image.

During the course of this work the non-amylase-synthesizing

organisms were kept on malt extract— yeast extract (MY) agar. This

medium contained the following constituents in grams per liter of

distilled water: 3 grams malt extract, 3 grams Difco yeast extract,

5 grams peptone, 10 grams glucose, 20 grams agar. The pH, which was

not adjusted, varied between 5 and 6 depending upon the particular

batch of ingredients. - 20 -

The amylase-synthesizing organisms were maintained on slants of a soluble starch— malt extract— yeast extract (SSMY) agar. The composition of this medium was follows: 3 grams yeast extract,

3 grams malt extract, 5 grams peptone, 20 grams agar, 10 grams soluble starch, and distilled water to make 1000 ml. All of the ingredients except the soluble starch were mixed together and heated for 10 minutes, or until the agar was completely melted. The soluble starch, suspended in a little cold water, was then added to the melted agar. The resulting mixture was stirred until the starch was thoroughly dissolved in the agar. The resulting medium was dispensed into culture tubes which were plugged with cotton and sterilized.

Sterilization was effected by autoclaving for 20 minutes at 15 pounds pressure.

Plates were prepared by aseptically pouring sufficient sterile, melted agar, prepared as described above, to evenly cover the bottom of sterile petri plates to a depth of approximately 3 mm.

Preparation of inoculum:— A 24-hour-old culture was used as the primary inoculum in all experiments. If there had been a period in which daily transfers of the organisms under investigation had not been made, the cultures were not used until at least three transfers

24 hours apart had been made.

Inoculum of the starch-hydrolyzing organisms was prepared by inoculating four 80 ml. portions of sterile wheat mash with 1 ml. of - 21 - a 70 per cent light-transmission suspension of the organism. The yeast suspension was prepared by aseptically pipetting 2 ml. of

sterile distilled water onto the 24-hour-old culture of the appropriate organism which was growing on a SSMY agar slant. One ml. of this suspension was placed in 9 ml. of sterile distilled water and the percentage light transmission measured on a Lumetron photoelectric colorimeter using the blue filter. The suspension was adjusted, if necessary, with sterile distilled water to 70 per cent light transmission. Suspensions were always more dense than

70 per cent light transmission, never requiring the addition of more cells.

Flasks so inoculated were placed on a Gump rotary shaker operating at 200 RPM and describing a 2.25 inch arc. These cultures were incubated on the shaker for two days at 28° C. The entire contents of each flask were then aseptically transferred to a 3 liter

Fernbach flask containing 800 ml. of sterile mash of the same com­ position as that in the small flask. These cultures were d.so placed on the Gump shaker at 28° C. for 24 hours. At the end of this period these cultures were used to inoculate the propagators.

They contained approximately 2.2 x 10^ cells per ml. The culture was added to the propagators in the amount of 40 per cent of the total mash volume, or 8.8 x 10^ cells per ml. of mash. This comparatively high concentration of inoculum was used to provide a high initial population in the propagators. Approximately 50 per

cent of the total inoculum added consisted of E. fibuliger, the - 22 - remaining 50 per cent consisted of T. utilis or other non-amylolytic yeasts. Since aseptic techniques were not observed during the propagation period, and since a comparatively short ( 8 hour) propa­ gation period was used, this high initial seeding was used to pre­ vent contamination as well as to ensure rapid substrate utilization.

A continuous or semi-continuous propagation in which the starch- containing material and nutrients are introduced into a high initial yeast population is visualized for this process.

Non-amylase-synthesizing organisms for inoculum were cultured on the following medium:

C e r e l o s e ...... 3.0 per cent

KH PO...... 0.15 " " 2 4 MgSO, • 7H 0 ...... 0.05 !l » 4 2 Urea ...... 0.20 " "

Difco yeast extract...... 0.25 " "

Distilled water — to make to desired volume.

The pH of this medium was measured, but not adjusted. It varied between 5.5 and 6.0 after autoclaving, depending upon the batch of yeast extract used. Eight hundred ml. of this medium was placed in each of two three liter Fernbach flasks. The flasks were covered with cotton and cheesecloth caps tied in place. Sterilization was accomplished by autoclaving at 15 pounds gauge pressure for 20 minutes.

After sterilization and cooling, each flask of medium was - 23 - inoculated with 0.2 ml. of a 26 per cent light transmission suspension of the appropriate organism prepared in the same way that has been described for the starch hydrolyzing yeasts. Cultures were incubated for 40 hours on the Gump shaker at 28° C. The cells centrifuged from 200 ml. of this culture were used to inoculate each propagator. "When different organisms were compared in a single experiment, the various inocula were standardized so that equivalent amounts of each organism would be used. Ten ml. samples of the cultures to be used for inoculum were centrifuged in graduated centrifuge tubes and the volumes of wet cells were read directly.

The volume of each culture required to give equivalent volumes of wet cells was calculated and the appropriate amounts were centri­ fuged and added to the propagators.

In the latter part of this study a mixed culture inoculum was prepared by inoculating 80 ml. portions of a sterile wheat mash in

300 ml. Erlenmeyer flasks with a 70 per cent light transmission suspension of the amylase-synthesizing organism as above described.

After 1.5 days of incubation on the Gump shaker at 28° C. this culture was aseptically transferred to an 800 ml. portion of sterile medium in a Fernbach flask. At the same time 1.0 ml. of a 24-hour- old culture of Torulopsis utilis var. major, Y-1084, or other non­ amylase-synthesizing organism, was added to the flask. .This mixed culture was incubated on the Gump shaker at 28° C, for 24 hours.

At the end of this time 820 ml. of this culture was used to inocu­ late each propagator containing 1230 ml. of mash. This procedure - 24 - was initiated in order to simplify the process by eliminating the preparation of two kinds of medium. It was also considered to be desirable to culture T. utilis for inoculum in the type of medium in which it was to be propagated.

Preparation of mash and inoculum for the comparison of

Aspergillus niger with Endomycopsis fibuliger;— Since strains of

Aspergillus niger which synthesize extracellular amylase habe been used for the conversion of starchy materials to sugar, it was considered desirable to compare the yields of yeast that could be obtained when strains of this organism were used as the saccharifying agents instead of E. fibuliger or E. chodati. The saccharifying agents which were compared were two strains of Aspergillus niger. NRRL M-330 and NRRL M-337, Endomycopsis fibuliger NRRL Y-1062, and a filtrate of known amylase activity of A . niger M-337.

Two series of propagations were made. In the first propagation the filtrate, mold, and yeast cultures were compared by using them to convert a wheat mash under conditions which were optimum for the conversion by A. niger cultures or filtrates. In this experiment a

20 per cent mash was prepared by autoclaving a wheat and tap water slurry for 30 minutes at 25 pounds gauge pressure. After this time the mash was cooled until the temperature reached 77° C. when the converting agents were added at the rate of 20 ml. of mold or yeast culture, or filtrate, per 50 grams of wheat on a dry weight basis.

The mash was held at a temperature of 58° C. for 30 minutes to assure - 25 - as complete a conversion as possible. During the conversion period the mash was stirred continuously with a Lightnin Mixer, Model L, to remove all lumps and to distribute the converting agent evenly throughout the mash. After the conversion period, during which the living organisms were killed by the 58° C. temperature, the mash was cooled, diluted to a 5 per cent concentration with cold tap water, and inoculated with double the standard volume of inoculum of T. utilis var. major.

Tsuchiya, Corman, and Koepsell (1950) have described the production of fungal amylase preparations by the use of corn and distillers’ thin stillage obtained from the alcoholic fermentation of a corn mash. Essentially the same technique was used in this

experiment except that wheat and dried distillers’ solubles obtained

from an alcoholic fermentation of wheat were used in place of corn.

A medium consisting of 5 per cent finely ground wheat and 5 per cent dried distillers' solubles suspended in tap water was used. The

grain and solubles were mixed with the water until the slurry was as homogenous as possible. The mash was sterilized by autoclaving at

15 pounds pressure for 20 minutes. Two 300 ml. Erlenmeyer flasks

containing 80 ml. portions of sterile mash were inoculated with a

small portion of A. niger mycelium taken with a flamed loop needle

from a 24-hour-old culture growing on a SSMY agar slant. A third

flask of sterile mash was inoculated with a small amount of E.

fibuliger, NRRL Y-25, taken from a 24-hour-old culture, also growing

on a SSMY agar slant. These flasks were placed on the Gump shaker TABLE OF CONTENTS

Page

Introduction ...... 1

Review of Literature...... 9

Materials and Methods ...... 17

Microorganisms used ...... 17

Preparation of inoculum...... 20

Preparation of mash and inoculum for the comparison

of Aspergillus nigerwith Endomycousis fibuliger 24

Laboratory propagators...... 26

Ha'shing of g r a i n s ...... 31

Mashing of tubers...... 34

Analytical procedure ...... 35

Calculation of yields and efficiencies ...... 38

Results...... 40

Survey of c u l t u r e s ...... 40

Comparison of E. fibuliger and A. niger as

saccharifying agents...... 47

Comparison of single and mixed propagations .... 49

Effect of the variation of propagation conditions . 54

P h o s p h a t e ...... 54

Magnesium s u l f a t e ...... 55

Calcium carbonate ...... 55

Comparison of urea and ammonium sulfate as

nitrogen sources...... 59

- ii - - 26 - and incubated for 24 hours at 28° C. At the end of this time 10 mX.« portions of each culture were transferred to 1 liter Erlenmeyer flasks containing 200 ml. of the same sterile medium. These flasks were incubated on the Gump shaker for three days at 28° C. At the end of this period they were added to the mash in the conversion step previously described.

Laboratory propagators:— Four stainless steel propagators were designed for the laboratory propagation of yeast. A photograph of one of these propagators is presented in Figure 1, while detailed drawings of the propagators are presented in Figure 2. Figure 3 shows the propagators in place and in operation. These propagators consist of stainless steel tubes 22 1/8 inches in height with an outside diameter of 4 inches, and a 0.065 inch thick wall. For aeration, each propagator was fitted with a ring sparger, 1 6/8 inches in diameter, constructed of stainless steel tubing 3/8 inches in diameter. Eleven pairs of holes l/64 inch in diameter were spaced

0.2 inchesapart completely around the ring. The propagators were fitted with propellors for agitation of the medium. The propellors

consisted of four stainless steel blades with dimensions of 39 x

15 mm. fitted to a stainless steel shaft so that the blades were inclined to give an upward lift to the medium, rotating it upward, then outward and downward, in the hope that the entrained air would remain longer in contact with the medium. Baffles were fitted to the

sides of the propagators to prevent a vortex from forming when the mash was agitated. - 27 -

Figure 1. Laboratory-scale yeast propagator (approximately l/5

actual size). Heading from left to right the parts are

as follows: (l) baffle plates, (2) propagator, (3)

agitator, and (4) ring sparger. I!-", i] 1

rr? - r roj^.. t.. ; .t ; r-'

>

<— r C . v & Figure 3. Yeast propagators in operation - 30 -

The agitators were driven by a 0.25 horsepower variable-speed motor which was fitted with a tachometer. The tachometer was adjusted to read directly in revolutions of the propellors per minute.

Airflow was measured with a Fischer and Porter Flowrator.

Air was introduced to the flowrators, and then to the propagators,

after first passing through a humidifier and a cotton filter. The

humidifier was necessary to prevent undue evaporation from the

medium in the propagators due to high degrees of aeration. The

cotton filter was used to keep oil and grease from the air lines

from entering the flowrators, as well as to prevent dirt from

entering the medium. The usual rate of aeration was one volume of

air per volume of medium per minute. Higher rates of aeration made

foam control extremely difficult.

A constant temperature was maintained by placing the propa­

gators in a water bath fitted with a thermostatically controlled

heating unit. The heat exchange was sufficiently good through the

stainless steel walls of the propagators to prevent any undue rise

in temperature of the medium due to heat evolved during the propa­

gation. Hydrogen-ion concentration was measured with a Beckmann

battery operated glass electrode. The pH of the medium was adjusted

with 10 N sulfuric acid or 40 per cent potassium hydroxide.

All cell counts were made using a Spencer bright-line

haemocytometer. - 31 -

Mashing of Grains:— Mash percentages were based on the dry weight of grain in the final volume of mash, that is, a 5 per cent mash was composed of 5 grams of finely ground grain (dry basis) in

100 ml. of mash. Moisture determinations were made on all starch-

containing raw materials and the weight to be used to obtain the

desired mash concentration was calculated therefrom.

Coarse grinding of the wheat facilitates feed recovery, and

is usually used in fermentation processes where the recovery of

the grains is a problem. Since in this process the entire solids

content after propagation, both the wheat and the yeast, comprised

the product, a fine grind was used to increase the availability of

the starch to the organism. The grain was ground on a 12 inch

Raymond Mill using a l/32 inch screen.

Methods of mash preparation have varied slightly throughout

the experiments. In the beginning the desired amount of finely

ground grain was weighed and placed in enough cold tap water to

make a d_urry of approximately 30 per cent concentration. The

grain was mixed into the'water with a Lightnin Mixer, Model L,

until the lumps were broken up and the slurry was homogenous. Hot o (60 C.) tap water was added in a quantity sufficient to bring the

solids concentration of the mash to approximately 10 per cent.

The temperature of the mixture was then raised to 80° C. by heating

the mash in a water bath. During the heating period the mash was

continuously stirred to prevent the formation ofHumps as the starch - 3 2 - gelatinized. At the end of the 15 minute cooking period enough cold tap water was added to make a mash of approximately 5 per cent

concentration. The temperature of the mash dropped to about 55° C., the optimum temperature for amylase activity according to Corman and Langlykke (1948)• Mechanical stirring was continued at this temperature for 15 minutes. This latter step was followed because in the backslopping experiments, amylase-containing agents were added to partially convert the gelatinized starch. The 15 minute mixing period was observed even when no converting agents were added in

order to make the mashing conditions comparable in all of the

experiments. At the end of' this: second 15 minute period, the mash was cooled to approximately 30° C. in a water bath. The pH was then measured and adjusted to 5.0 by the addition of 10 N sulfuric acid.

The original pH of cooked wheat mash was between 7.0 and 7.5. After

the pH was adjusted the mash was measured, made to the desired volume

by the addition of cold tap water, and placed in suitable containers.

The mash was usually dispensed into flasks as follows:

800 ml. of mash was jLaced in each of four 3-liber Fernbach flasks,

80 ml. of mash into each of four 300 ml. Erlenmeyer flasks, and the re­

mainder (usually 6000 ml.) in a 10 liter Pyrex carboy. The con­

tainers were pLugged with cotton and sterilized by autoclaving at 15

pounds gauge pressure for one hour. The autoclave was equipped with

a compressed air line which permitted the maintenance of the pressure

at 15 pounds per square inch during the cooling of the mash. This

procedure prevented the boiling over of the medium which usually - 33 - occurred if grain mashes were sterilized in the usual manner.

Wheat mashes so prepared proved unsatisfactory due to the tendency of the mash to form a hard cake and become viscous during autoclaving and subsequent cooling. Handling of this type of mash was difficult since accurate volume measurements could not be made

due to the large amount of air entrapped in the mash. Since

retrogradation takes place during the aging of cooked starch mashes, it was considered desirable to use the mashes as soon as they were

prepared.

After the first one-third of the work was completed, mashes

were prepared as previously described, except for autoclaving, and

they were used as soon as they were prepared. Mashes were cooked

only to 80° C. for 15 minutes in order to gelatinize the starch,

making it susceptible to enzyme action. After the pH was adjusted,

and the mash was made to volume, nutrients were added, the mash

was inoculated and placed immediately in the propagators. Mash

for use as inoculum-substrate was placed in flasks and sterilized

as previously described. For convenience these flasks stood at

least 24 hours after sterilization before inoculation.

Mashing procedure for other grains was the same as that used

for wheat. Appropriate amounts of ground corn or rice were used

to give a mash containing 5 per cent solids. The wheat used was

a soft red winter variety containing 67.& per cent starch, 11.6 - 3 4 - per cent protein, and 11.3 per cent moisture. A yellow dent variety of corn was used which contained approximately 77.6 per

cent starch, 10.2 per cent protein, and 9.2 per cent moisture.

A non-waxy variety of rice containing 68.5 per cent starch, 6.0 per cent protein and 10.8 per cent moisture was used.

Mashing of tubers:— The potatoes used were (l) Puerto Rico,

a variety of sweet potato containing 45 per cent starch, 7 per cent

protein, and 78.7 per cent water, and (2) White Cobbler, a white

potato containing 26 per cent starch, 7 per cent protein, and 70.0

per cent water. In the preparation of mash using sweet or white

potatoes, unpeeled tubers were washed in cold tap water and ground

through a ’’Universal" home food grinder using the fine adjustment,

the mash was brought to approximately a 20 per cent solids concen­

tration with cold tap water. The potato and water mixture was

autoclaved for 15 minutes at 15 pounds pressure to gelatinize the

starch and cook the potato to a soft mass. After autoclaving, the

solid portion of the mash readily settled out leaving a fairly clear

supernatant liquid which was poured off and reserved. The remaining

pulp was homogenized in a Waring Blender for 2.5 minutes. The homo­

genized pulp was then recombined with the supernatant liquid. The pH

was adjusted to 5.0 with 10 N sulfuric acid and the mash concentration

was brought to approximately 5 per cent by the addition of cold tap

water. The potato mashes were prepared the day before they were used

in an experiment and sterilized by autoclaving for 20 minutes at 15

pounds gauge pressure. No difficulty was experienced in handling these Page

Selection of optimum amounts of u r e a ...... 61

Effect of variation of pH ...... 64

Effect of temperature v a r i a t i o n ...... 64

VJheat mash concentration...... 66

Aeration and agitation rates ...... 66

Ratio of T. utilis to E. fibuliger inoculum .... 74

Length of propagation t i m e ...... 76

Backslopping ...... 76

Effect of backslopping on yeast y i e l d s...... 82

Recycling of i n o c u l u m ...... 85

Incremental and continuous addition of mash .... 87

Comparison of various starchy substrates ...... 89

Vitamin and amino acid analysis of the yeast product . . 93

Discussion...... 98

S u m m a r y ...... 112

Literature cited ...... 115

Glossary...... 127

Autobiography ...... * ...... 128

- iii - mashes since extremely high viscosities did not occur as they did in wheat mashes.

Mineral salts were added and the mash was inoculated immedi­

ately before the mash was added to the propagators. Inoculum was

prepared as described for the wheat runs; however, potato mash was used for the inoculum instead of wheat mash.

Analytical procedures:— Routine analyses were made on the uninoculated mash, the inoculated mash immediately after the

addition of nutrients and inoculum, and on the mash after the

completion of the propagation period. The types of analyses

carried out may be summarized as follows:

Whole mash (uninoculated, inoculated, and after propagation)

1. Total solids.

2. Total nitrogen in the mash and in the supernatant

liquid.

3. Free reducing sugars in the mash and the supernatant

liquid.

4. Total reducing sugars, after acid hydrolysis, in the

mash and the supernatant liquid.

Centrifugable solids after propagation. (Product)

1. Dry weight,

2, Total nitrogen. - 36 -

3. Total carbohydrate, after acid hydrolysis. This

included any residual starch or acid-hydrolyzable

polysaccharide component of the wheat as well as the

yeast cell material.

Supernatant liquid from mash after propagation.

1. Total nitrogen.

2. Free reducing sugars.

3. Total sugars, after acid hydrolysis.

Amounts of reducing sugars were measured by the method of

Somogyi (1945) as described by Hodge and Davis (1952) for use in this laboratory.

Total Kjeldahl nitrogens were determined as described in the official methods of the Association of Official Agricultural

Chemists (1940). 'When the size of the sample was insufficient to permit macro-Kjeldahl determinations, the micro-Kjeldahl method described by E. P. Clark (1941) was used.

The amount of urea remaining in the supernatant liquid or the centrifugable solids were measured by the method described by the

Association of Cereal Chemists (1947)• - 37 -

All assays for B-vitamins were microbiological with the exception of the thiamine assay which was fluorometric. The thiamine assay was conducted as described by the American Associ- tion of Cereal Chemists (1947). A Coleman Electronic Photo-

fluorometer, Model 12, using filters and PC-1, was used in the

assay.

Riboflavin was measured by the method of Snell and Strong

(1939) using Lactobacillus casei, NRRL B-442, as the test organism.

The biotin assay used was described by Wright and Skeggs (1944).

It employed Lactobacillus arabinosus, NRRL B-531* as the test

organism. This organism was also used for the niacin assay as

described by Snell and Wright (1941).

Pyridoxine was measured by the use of Saccharornyces

carlsbergensis as described by Atkins, Schultz, Williams, and

Frey (1943).

The amount of pantothenic acid was ascertained by the use of

Lactobacillus arabinosus« NRRL B-531* as described by Skeggs and

Wright (1944).

Choline was measured by the method of Horowitz and Beadle

(1943). The test organism was Neurospora crassa, 34486, ATCC No.

9277. - 38 -

All of the amino acid assays were conducted according to the procedure outlined by Horn, Jones, and Blum (1950), the only exception being in the hydrolysis of the samples. Yeast samples were hydrolyzed by autoclaving them with hydrochloric acid in sealed ampules for 15 hours according to the method of Stokes, et al

(1945). Leuconostoc mesenteroides NRRL B-1150 was used for the assay of threonine, histidine, lysine, leucine, and phenylalanine.

Lactobacillus arabinosus NRRL B-531 was employed for the assay of arginine, methionine, valine, and isoleucine.

Calculation of yields and efficiencies:— The amount of crude

protein in the uninoculated wheat mash was calculated by multiplying the total nitrogen value by 5.7, the "protein conversion factor11 for

wheat. Where other starchy materials such as potatoes were used

the conversion factor was the usual 6.25. This factor of 6.25 was

also used in the calculation of protein in the mash after propagation.

It was assumed that most of the protein present after propagation was

yeast protein.

Amounts of starch were calculated by subtracting the amount of

free sugar, as glucose, from the amount of total sugar, as glucose,

and multiplying the remainder by 0.9, the "starch conversion factor."

All yields were calculated on the basis of centrifugable

solids (product) obtained from 100 pounds of raw material (dry - 39 - weight basis)

The efficiency of the process was determined in two ways: (l) the efficiency based on the yield of solids (dry weight) from 100 pounds of raw material (dry weight), and (2) the per cent increase in protein due to processing, calculated by dividing the pounds of protein synthesized from 100 pounds of raw material by the pounds of protein originally in 100 pounds of raw material. - 40 -

RESULTS

Survey of cultures:— Before any attempt was made to study the

effect of variables on the propagation, a survey of organisms was made in order to select the most suitable yeasts for the production

of nutritional yeast.

The survey was divided into two sections, the selection of an

amylase-synthesizing strain of yeast, and the selection of an ancillary

yeast for protein and vitamin synthesis.

Selection of an amylase-synthesizing yeast consisted of a starch

plate survey in which yeasts producing extracellular amylase in

appreciable quantities were selected. These species were subjected

to further testing in the yeast propagators using wheat mash.

In the starch plate survey, soluble starch— malt extract agar

plates were prepared as previously described. Enough plates were

prepared so that there would be two sets of duplicate plates for

each organism tested. Organisms in which the production of extra­

cellular amylase was suspected were spotted in the center of these

plates using a flamed loop needle. After 4 days of incubation at

28° C. one set of the plates was flooded with 6 ml. of iodine

solution prepared by diluting 5 nil. of iodine stock solution with

300 ml. of distilled water. The width of the clear zones around

the colonies were immediately measured. All measurements were - 41 - made from the margins of the colonies. The second set of plates was tested after five days of incubation.

Four strains of Endomycopsis fibuliger and 24 strains of

Endomycopsis chodati were tested. Of the strains of E, fibuliger all four strains formed zones ranging between 12 and 16 mm. in width at the end of five days. Strains of E. chodati formed zones ranging from 2 to 11 mm. in width at the end of five days. Five of the most promising strains of E. chodati were used in further studies.

They were NRRL Y-1933, NRRL Y-1938, NRRL Y-1988, NRRL 1-2051 and

NRRL Y-2052.

A preliminary selection of non-amylase-synthesizing yeasts was made from among organisms which had been surveyed at this laboratory and had been found to produce good yields of vitamins and protein.

These organisms were tested for their suitability in the production

of nutritional yeast from wheat by culturing them in combination with E. fibuliger or E. chodati. The stainless steel propagators were used in this survey.

Conditions for the propagator survey were more or less

arbitrarily chosen. A 5 per cent wheat mash was prepared as

described previously. Mineral salts were added as follows on the

bapis of the final volume of inoculated mash: - 42 -

KHoP0 0.05 per cent

MgSO^ • 7 H20 0.05 11

Urea . 0.25 11

0.25 11

CaCO 0.001 »

The pH of the medium was maintained at 5.0 throughout the propagation. Temperature was held at 33° C. Air was supplied at the rate of one volume of air per volume of mash per minute.

Agitation was 650 RPM. The results of the survey are presented in Table 1.

"When various combinations of non-amylase synthesizing organisms and E. fibuliger NRRL Y-1062 or E. chodati NRRL Y-2051 were compared, the best yields of solids and per cent increase in protein due to processing were always obtained with T. utilis var. ma.jor NRRL

Y-1084. Repeated experiments indicated, however, that experimental variation of the yield of solids was of the order of 6 per cent.

The variation that could be obtained in the per cent protein in the solids was as high as 2 per cent. Considering these experimental errors, it would appear that T. utilis var. ma.jor and H. sub- nenieiil osa NRRL Y-1683 gave similar yields when cultured with

E. fibuliger Y-1062. Table 1. Survey of cultures

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-1062 E. fibulieer + Y-1084 T. utilis var. ma.ior 77.0 8.5 27.1 68.7

Y-1062 E. fibulieer + Y-1683 H. suboelliculosa 73.7 8.2 27.8 66.2

Y-1062 E. fibulieer + Y-1591 R. mucilaeinosa var. plicata 62.5 6.1 26.1 59.6 Y-1062 E. fibulieer + Y-1094 C. lipolytica 63.3 5.9 25.4 57.8

Y-1062 E. fibulieer + Y— 900 T. utilis 59.6 5.1 ■ 26.5 47.6

(Continued) Table 1. Survey of cultures, (continued)

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-1062 E. fibulieer + Y-1109 S. fragilis 53.3. 3.1 24.5 31.0

Y-25 E. fibulieer + Y-1084 T. utilis var. ma.ior 64.0 9.6 32.3 87.5

Y-25 E. fibulieer + Y-2020 C. arborea 45.3 9.1 44.4 82.6

Y-77 E. fibulieer + Y-1084 T. utilis var. ma.ior 62.7 7.1 . 29.4 62.2

(continued) THE USE OF AMYLOLYTIC YEASTS FOR THE PRODUCTION OF A NUTRITIONAL

PRODUCT

INTRODUCTION

Famine has been evident in the whole history of man. Because of these set-backs, man has developed food plant cultivation and animal husbandry as the most practical means of obtaining food supplies. As the population of the world increases the supply of food produced by these methods does not seem to be increasing at a sufficiently rapid rate to offset widespread hunger.

Obviously, the only solution to the underproduction of food must be either a relative, increase in productive capacity, or a relative decrease in the population. The latter solution suggests the justifi­ cation of war or other methods of controlling or decreasing the population. One of the most logical approaches seems to be the ex­ pansion of the potential food supply.

Mather (1952) has stated that at the present time kO per cent of the earth's inhabitants appear to be living under conditions to which the Malthusian principle of external restraints does not apply.

In the case of the other 60 per cent the population is, however, under Malthusian restrictions. These latter people are concentrated chiefly in Asia, Africa, Europe, and a few localities in the Western

- 1 - Table 1. Survey of cultures, (continued)

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-2051 E. chodati + Y-1084 T. utilis var. ma.ior 65.5 10.0 30.6 86.2

Y-2051 E. chodati + Y-2020 C. arborea 63.8 5.9 28.6 48.0

Y-2051 E. chodati + Y— 900 T. utilis 63.4 7.3 30.9 58.9

Y-2052 E. chodati + Y-1084 T. utilis var. ma.ior 61.4 10.3 30.5 88.7

Y-1938 E. chodati + Y-1084 T. utilis var. ma.ior 67.8 11.7 29.8 100.9 - 46 -

After several propagations the use of E. chodati Y-2051 and

Y-2052 -was discontinued in the survey work because of the tendency of these organisms to cause excessive frothing during the propagation.

This frothing could not be controlled by the use of any of the anti­ foam agents which were tried. Results obtained with this species, however, indicated that the yields were equal or superior to yields obtained with E. fibuliger Y-1062. The per cent increase in protein was close to 100 per cent whenever these organisms were used. The yields of solids were somewhat lower than yields of solids obtained by use of JE. fibuliger. This fact suggests an inverse relation between the per cent protein and the total yield of solids.

Although E. fibuliger Y-25 and Y-77, plus T. utilis var. ma.ior gave a greater per cent of protein in the solids than did E. fibuliger

Y-1062 and T. utilis var. ma.ior. the total yield of solids was much higher in the latter case. There was no significant difference in yields among the three strains of E. chodati: Y-2051, Y-2052, and

Y-1938.

E. fibuliger Y-1062 and T. utilis var. major Y-1084 were

Chosen as the test organisms to be used in the remainder of this study. - 47 -

Comparison of Endomycopsis fibuliger and Aspergillus niger as saccharifying agents.: — Two sets of experiments were conducted. In the first experiment a separate conversion step using cultures of

A. niger M-330 and M-337 and E. fibuliger Y-25, and a filtrate of

A. niger M-337 was employed for the saccharification of the starch.

During the conversion process the organisms were killed. In the second experiment living cultures of the two organisms were used.

Comparisons were made of the yields of solids and of protein obtained using T. utilis var. ma.ior in both cases.

In the first propagation, there was no significant difference in the yields of solids obtained (Table 2). The per cent of protein in the solids obtained by use of 3,molds" or ''mold11 filtrates was nearly twice as great as that obtained by the use of E. fibuliger.

The propagation period was 8 hours at 33° C. An aeration

rate of one volume of air per volume of mash per minute was used.

The agitation rate was 650 RPM, and the pH was held at 5.0 by the

periodic addition of 10 N sulfuric acid or approximately 40 per cent

potassium hydroxide. In this first experiment the inoculum was

cultured in a manner known to be optimum for amylase production by

Aspergillus niger, and which was described in the previous section. Table 2. Comparison of E. fibuliger with A. niger as saccharifying agents.

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Filtrate A. niger + Y-1084 T. utilis var. ma.ior 50.6 10.3 45.7 80.5

Y-25 E. fibuliger + Y-1084 T. utilis var. ma.ior 52.0 0.8 26.2 6.3

M-330 A. niger + Y-1084 T. utilis var. ma.ior 55.7 9.9 40.7 77.1

M-337 A. niger + Y-1084 T. utilis var. ma.ior 53.6 11.2 45.0 87.5 - 49 -

The second propagation in which molds and yeasts were compared

consisted of a mixed propagation of the starch-saccharifying organisms with T. utilis var. ma.ior. In this propagation a 40 per cent inoculum

of each of the starch saccharifying organisms was used, together with a standard T. utilis inoculum. Mash and inoculum were prepared as

described previously as the standard procedure used in experiments

with yeasts alene.

Results of the second propagation, which are presented in Table

3, indicate that a mixed propagation markedly increases the per cent

of.protein in the solids and the amount of protein synthesized when

E. fibuliger is used, but does not improve the amount of protein

synthesized when A. niger is used.

Comparison of single and mixed propagations:— Yields obtained

with E. chodati, NRRL Y-2051 and E. fibuliger Y-1062 were compared

when each organism was cultured alone and when each was cultured with

T. utilis var. ma.ior NRRL Y-1084. Table 4 presents the results

obtained in this experiment. Both organisms showed a higher yield

of protein and of solids when cultured in a mixed propagation than

when they were cultured alone. E. fibuliger gave a higher per cent

increase in protein when it was cultured alone than did E. chodati. Table 3. Comparison of E. fibuliger and A. niger in a mixed propagation.

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-25 E. fibuliger + Y-1084 T. utilis var. major 64.0 9.6 32.3 87.5

Y-25 E. fibuliger + Y-2020 C. arborea 45.3 9.1 44.4 82.6

M-330 A. niger + Y-1084 T. utilis var. ma.ior 46.9 9.6 44.0 87.2

M-337 A. niger + Y-1084 T. utilis var. ma.ior 47.7 9.6 43.3 87.3 Table 4. Comparison of E, fibuliger and E. chodati in single and mixed propagations.

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-2051 E, chodati alone 58.9 5.2 28.6 54.1

1-2051 E. chodati + Y-1084 T. utilis var. ma.ior 61.6 8.9 30.1 92.5

Y-1062 E. fibuliger - alone 64.0 6.1 24.6 63.8

Y-1062 E. fibuliger + Y-1084 T. utilis var. ma.ior 72.1 11.3 29.0 117.2 - 52 -

Yields were good enough in all cases not to warrant the exclusion of the possibility of a single propagation using E. fibuliger or E. chodati♦ The next step in the comparison of a single with mixed cultures was the comparison of the growth of T. utilis in a mash

converted by the use of amylase-containing slop from a previous

experiment in which E. fibuliger was used.

In order to further compare the effectiveness of mixed propagations, a mash containing 60 per cent slop was prepared. One portion of the mash was inoculated with T. utilis var. ma.ior. the other portion was inoculated with a mixed culture of T. utilis var. ma.ior and E. fibuliger Y-1062. Results are presented in Table 5.

The per cent protein in the solids is considerable higher

when T. utilis and E. fibuliger are cultured in combination than

when T. utilis is cultured alone on slop-converted mash. A inverse

relation between the yield of solids and the per cent protein is

again evident. Table 5. Comparison of T. utilis var. ma.ior in single and mixed propagations on backslopped mash.

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Y-1084 T. utilis var. ma.ior 56.0 1.3 23.1 11.1 alone

Y-1062 E. fibulieer + Y-1084 T. utilis var. major 51.0 4.2 31.1 36.2 - 54 -

Effect of the variation of propagation conditions:— At the completion of the survey of cultures it was necessary to establish the optimum conditions for the operation of the processing

Endomycopsis fibuliger NRRL Y-1062 in a mixed culture with

Torulopsis utilis var. ma.ior NRRL Y-1084. Where propagator space permitted, chiefly in the temperature experiments, Endomycopsis chodati NRRL Y-2052 was also compared in order to obtain as much data as possible on the suitability of this organism for use in the process.

Attention was first turned to the mineral salts which it was necessary to add to the medium in order to obtain optimal growth.

Later, conditions such as the concentration of the mash, pH, temperature, aeration and agitation, ratio of inoculum, and the propagation time were studied. Finally the use of "backslop", or the diastatically active supernatant liquid from a previous propagation of E. fibuliger or E. chodati, in the medium and the use of starch substrates other than wheat were considered. Vitamin and amino acid analyses were made on the solids obtained from various experiments in order to gain some information as to the food value of the product.

Phosphate:— Potassium di-hydrogen phosphate was supplied to the propagation at levels of 0.00, 0.05, 0.10, and 0.15 per cent of the 2000 ml. of a 5 per cent wheat mash which was added to each propagator. - 2 -

Hemisphere. They are not, according to Mather, taking advantage of the opportunities offered to them by modem, science and technology for improving their means of subsistence.

The fact remains that whatever means are taken to increase the production of food crops, the amount of tillable land is finite.

Although the assumption that the world's population will continue to increase indefinitely unless limited by the food supply is highly debatable, it must be granted that it is a possibility. In this case can anything be done to substantially increase the total food supply?

It is true that the productivity of many agricultural lands is de­ creasing chiefly because of mismanagement. Whether this productivity decrease will continue despite the possibilities of increases through more careful soil handling, and the use of hybrid crops, is a question that cannot be definitely answered at this time.

In the present system of food production thed necessary food materials — carbohydrates, fats, proteins, and vitamins — are obtained either from green plants, or by the feeding of green plants to animals, which, in turn, are consumed by humans. A very small portion of the total food supply is derived from the non-green plants.

Even these plants must, with few exceptions, depend upon the green plants for carbon. How then can larger food supplies be created if a situation is postulated in which ali of the arable land is being utilized to its fullest capacity? Yin (19^9) has pointed out that

an alga, Chlorella pyrenoidenosa, shows characfberistics which - 55 -

The per cent of protein, and the total protein synthesized, decreased as the level of phosphate increased (Table 6). There was no significant difference in the yields of solids at any of the levels of potassium phosphate used. On the basis of these experi­ ments there was no apparent benefit derived from the addition of phosphate.

Magnesium sulfate:— Magnesium sulfate (MgSO^ • 7H20) was added at the rate of 0.00, 0.05, 0.5, and 1.0 per cent of the volume of inoculated 5 per cent wheat mash. Results of this experiment are presented in Table 7.

There was no significant difference in the per cent protein in the solids at any level of magnesium sulfate employed. The highest per cent of protein was obtained where no magnesium sulfate was added. The yield of solids, and the total protein synthesized, decreased with an increasing amount of magnesium sulfate in the medium. The highest yield of solids, as well as of protein, was obtained where magnesium sulfate was excluded from the medium.

Calcium carbonate:— Additions of calcium carbonate at the levels of 0.00, 0.001, 0.01, and 0.10 per cent of the volume of inoculated mash gave no significant differences at any level of calcium carbonate supplied (Table 8). Table 6. Effect of variation in amount of phosphate supplied on yields of yeast solids and protein,

Per cent Lbs. solids Lbs, protein % protein % increase k h 2p o . from 100 lbs. synthesized in solids in protein added raw material from 100 lbs. due to raw material processing

None 65.4 3.7 23.5 31.5

.05 67.1 4.0 23.4 34.1

.10 64.5 2.5 22.0 21.3

.15 66.1 1.8 20.5 15.6 Table 7. Effect of the addition of magnesium sulfate on yields of yeast solids and protein

Per cent Lbs. solids Lbs. protein % protein % increase MgSCh *7H20 from 100 lbs. synthesized in solids in protein added raw material from 100 lbs. due to raw material processing

None 63.0 8.2 30.2 75.4

0.05 62.6 7.2 28.9 66.7

0.5 61.5 6.3 27.9 58.3

1.0 5 8.4 5.9 28.7 54.4 Table 8. Effect of the addition of calcium carbonate on the yields of yeast solids or protein.

Per cent Lbs. solids Lbs. protein % protein % increase CaCOo from 100 lbs. synthesized in solids in protein added raw material from 100 lbs. due to raw material processing

None 59.9 7.0 28.3 69.8

.001 59.9 7.6 29.4 76.6

.01 57.8 6.8 29.1 68.4

.10 58.4 7.8 30.3 77.7 - 59 -

Comparison of urea and ammonium sulfate as nitrogen sources:—

A combination of equal parts, by weight, of ammonium sulfate and urea had been used in the survey as a source of nitrogen for the yeast.

Nitrogen sources were supplied so as to give a nitrogen concentration of 0.14 grams per 100 ml. This corresponds to 2.4 per cent nitrogen if calculated on the basis of the weight of wheat in a 5 per cent mash. White (1948) states that industrially a nitrogen concentration of 1.8 per cent based on molasses weight is used. Since wheat contains amino acids and other assimilable nitrogen compounds this amount of nitrogen was considered to be more than sufficient.

An experiment was set up in which the ratio of urea and ammonium sulfate was varied as follows: urea alone, two parts of urea to one of ammonium sulfate, one part of urea to two parts of ammonium sulfate, and ammonium sulfate alone. In all cases the nitrogen concentration was 0.14 grams per 100 ml. of medium.

It may be inferred from the results of this experiment which are presented in Table 9, that there was no significant difference in the yield of solids obtained. There was an indication that with increasing amounts of ammonium sulfate, or decreasing amounts of urea, the per cent protein in the solids decreased. Since the differences showed that the per cent increase in protein due to processing was the highest when urea was used alone, and since pH control was made easier by the use of urea, it was decided that urea would be used by itself in future experiments. Table 9. Effect of using various ratios, of urea and ammonium sulfate in the propagation of Endomycopsis fibuliger. Y-1062 and Torulopsis utilis. Y-1084.

Amount of Lbs. solids Lbs. protein % protein % increase nitrogen- from 100 lbs. synthesized in solids in protein source added raw material from 100 lbs. due to raw material processing

Urea 60.7 9.2 34.1 79.3 alone

2 parts urea 1 part (NHj^SO^ 63.0 8.9 34.3 77.2

1 part urea 2 parts SO^ 60.3 8.2 32.7 70.7

(ffi4)2S0 alone 60.3 7.5 31.5 64.8 - 61 -

The use of urea, instead of ammonium sulfate, as the sole source of nitrogen made pH control easier since urea gave no re­ sidual acid as the nitrogen-containing portions of the molecule -were utilized. It should be emphasized, however, that either of the two salts may serve quite well as nitrogen sources. The selection of the optimum amount of urea to be furnished was the next consideration.

Selection of optimum amounts of urea;--Several experiments were conducted in which various amounts of urea were supplied to the propa­ gation. Typical results of these experiments are presented in Table

10. It is evident from the data obtained in these experiments that with an increase in the amount of nitrogen added, as urea, there was an apparent increase in the amount of crude protein in the solids.

It must be emphasized that these results were obtained with unwashed

centrifugable solids. When the solids were washed with distilled water made to pH 4.6 with hydrochloric acid, the results were markedly changed (Table ll). The per cent "protein” in the solids was lowered from 2 to 4 per cent simply by washing the cells with water which was near the isoelectric point of protein. Yields of

solids lowered from 3 to 6 per cent by water washing.

Urease was used to determine the amount of urea removed from

the solids by water washing. Use of this technique indicated that

of the water soluble nitrogen removed from the cells, 90 to 100 per

cent is due to urea. These experiments illustrated the necessity

for extreme caution when "crude protein” values for yeast products Table 10. Comparison of the effect upon yields of yeast product of the addition of various levels of urea.

Per cent Lbs. solids Lbs. protein % protein % increase urea from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

.2 61.2 3.$ . 24.7 29.8

.3 $6.6 2.5 24.9 21.4

•4 $4.2 2.4 2$.9 20.6

.5 $3.1 2.0 2$.6 16.9 Table U . Comparison of apparent yields of yeast product and protein obtained on unwashed and washed centrifugable solids.

Per cent Lbs. solids Lbs. protein % protein % increase urea from 100 lbs. synthesized in solids in protein raw materials from 100 lbs. due to raw material processing

Unwashed solids. .2 66.0 4.8 24.8 40.9 .3 59.8 4.6 27.2 39.7 .4 58.5 4.9 28.4 42.4 .5 59.1 6.0 29.9 51.7

Washed solids. .2 61.2 3.5 24.7 29.8 .3 56.6 2.5 24.9 21.4 .4 54.2 2.4 25.9 20.6 .5 53.1 2.0 25.6 16.9 — 6 4 — are obtained by the conversion of the total nitrogen values.

On the basis of duplicate experiments, using washed cells, optimum amounts of urea appear to be between 0.2 and 0.3 per cent of the total volume of inoculated mash. At this point the per cent protein in the solids, and the per cent increase in protein due to processing, seems to be the highest. The yields of solids dropped

sharply between 0.2 and 0.3 per cent urea levels. There was no significant difference in yields of solids among other levels used.

Effect of variation of pH:— Experiments were conducted in which an attempt was made to hold all of the conditions constant, with the exception of the pH during the course of the propagation.(Table 12).

The pH values were varied as follows: 4.0, 4.5, 5.0, 5.5* and 6.0.

The lowest per cent protein, 31.4, was obtained at pH 5-0. There

seemed to be no significant difference between the yield of solids for any of the pH levels used. A second experiment confirmed these findings.

Effect of temperature variation:— The temperature of the propagation was varied as follows: 25° C., 30° C., 33° C., and 37° C.

Endomycopsis fibuliger Y-1062 and E. chodati Y-2052 were compared in this experiment. Both were cultured with T. utilis var. ma.ior.

The results of this single series of experiments indicated

that the process is operable over a wide range of temperatures, i .ti., - 3 - indicate that it might be cultured in quantity, artifically, and used as a source of carbon. The raw materials would be carbon dioxide and water, the product would be sugar and other organic com­ pounds. Bush (1952) has described the production of algae for food purposes in which the organisms are cultured in plastic bags contain­ ing a suitable culture solution. Adams (1952) states that a yield of

15 dry tons per acre ha3 been obtained by culturing Chlorella in shallow pans. This may be compared with an average yield of one ton, dry weight, of corn per acre. This yield is based on the average yield of corn for the United States of ^0,6 bushels per acre as given by the

United States Department of Agriculture (1952). Moisture content of the corn is assumed to be 10 per cent and 56 pounds of corn is assumed per bushel.

Under the present conditions of production and distribution it is apparent that in certain areas of the world there is an abundance of carbohydrate material and a scarcity of proteins so vital to the human animal body. Data presented by the Food and Agriculture

Organization of1he United Nations presents a comprehensive review of the pre-World War II food situation. Booher (19^8) has compared these data with the estimated population of each country. The conclusion is clear that if the world’s entire food protein supplies were pooled and distributed among the world'3 population according to allowances recommended by the National Research Council (19^3), the protein supplies would in no way be adequate. Husain (19^6) has shown that more than 80 per cent of the total calories in the Indian diet is derived Table 12, Effect of the variation of pH, upon yields of yeast product and of protein.

pH. Lbs, solids Lbs. protein % protein % increase from 100 lbs, synthesized in solids in protein raw material from 100 lbs, due to raw material processing

4.0 69.5 6.7 25.6 59.7

4.5 66.0 7.2 27.7 64.2

5.0 62,6 8.5 31.4 76.5

5.5 67.1 6.7 26.1 60.6

6.0 65.8 8.0 26.8 79.0 - 66 -

25° C. to 37° C., although the highest yields of solids and the high­ est per cent of protein in the solids -were obtained at 25° C. (Table 13).

This compares quite well with the second experiment which was conducted at 25° C., the results of which are presented in Table 14.

Wheat mash concentration:— In attempts to select the optimum wheat mash concentration for the production of food yeast by E. fibuliger and T. utilis, the concentration of wheat was varied from

2 per cent to 10 per cent. Results are presented in Table 15.

Mineral salts were added on the basis of the wheat supplied. Amounts proportional to the weight of grain in a 5 per cent wheat mash were used.

The results of three experiments in which the mash concentration was varied indicated that no significant difference in the number of pounds of solids obtained at the different levels used. The amount of protein synthesized was highest at mash concentrations above 5 per cent. The high yields of solids and the corresponding low per cent of protein in the solids obtained in Experiment C, was another example of the inverse relation observed between the yield of solids and protein yield.

Aeration and agitation rates:— A comparison was made of yields obtained at agitation rates of 800, 650, 600, and 350 RPM and aeration rates of 0.5, 1.0, 1.5, and 2.0 volumes of air per volume of medium per minute. The results are presented in Table 16. Table 13. Effect of variation of temperature upon yields of yeast product and protein.

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs, due to raw material processing

2 f C. Y-2052 E. chodati + 1-1084 T. utilis var. ma.ior 60.7 10.7 36.6 92.63

Y-1062 E. fibuliger + Y-1084 T. utilis var. ma.ior 63.2 10.6 35.2 92.1

?0° C. Y-2052 E. chodati + Y-1084 T. utilis var. ma.ior 55.0 5.4 29.5 49.4

Y-1062 E. fibuliger + Y-1084 T. utilis var. ma.ior 61,8 6.1 27.4 55.8

(continued) Table 13. Effect of variation of temperature upon yields of yeast product and of protein (continued)

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

32° c. Y-2052 E. chodati + 60.2 5.2 32.0 57.5 Y-1084 T. utilis var. ma.ior

Y-1062 E. fibuliger + Y-1084 T. utilis var. Siajor 64.0 8.3 29.7 76.6

37° C. Y-2052 E. chodati + Y-1084 T. utilis var. ma.ior 58.0 5.5 27.9 51.3

Y-1062 E. fibuliger + Y-1084 T. utilis var. ma.ior 57.1 5.9 29.1 55.0 Table 14. Effects upon yield of yeast product and of protein by the propagation of various yeasts at low temperature (25° C.).

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw materials from 100 lbs. due to raw material processing

Y-2054 Unidentified pink yeast + Y-25 E. fibulieer 86.1 7.5 22.9 61.0

Y-2053 Unidentified pink yeast + Y-25 E. fibulieer 92.7 7.5 21.3 61.4

Y-1591 R. mucilaeinosa var. nlicata + Y-25 E. fibulieer 93.0 7.0 20.6 57.2

Y-1084 T. utilis var. ma.ior + Y-25 E. fibuliger 100.8 8.0 20.2 65.4 Table 15. Effect of the concentration of wheat mash upon yields of yeast solids and of protein.

Per cent Lbs. solids Lbs, protein % protein % increase wheat from 100 lbs, synthesized in solids in protein concentration raw material from 100 lbs. due to raw material processing

Exneriment A 9.5 79.8 10.5 28.5 86,2 7.2 81.6 10.7 27.7 89.5 5.2 70.2 8.1 26.8 75.7 3.1 50.7 5.5 31.3 52.4

Experiment B 7.6 66,0 5.6 24.9 52.3 6.0 63.7 5.0 24.8 46.0 4.1 64.2 4.8 23.9 31.4 2.2 63.5 1.6 21.8 12.7

(continued) Table 15. Effect of the concentration of wheat mash upon yields of yeast solids and of protein, (continued)

Per cent Lbs. solids Lbs. protein % protein % increase wheat from 100 lbs. synthesized in solids in protein concentration raw material from 100 lbs. due to raw material processing

Exoeriment C. 10.0 86.5 3.5 16.9 31.4 5.5 85.4 4.4 17.5 41.9 4.0 85.7 2.6 15.8 24.1 2.3 84.8 1.1 13.6 10.8 Table 16. The effect of different rates of aeration and agitation upon yields of yeast solids and protein.

Volume of air Lbs. solids Lbs. protein % protein % increase per volume of from 100 lbs. synthesized in solids in protein mash per minute raw material from 100 lbs. due to raw material processing

800 RPM 0.5 58.5 7.9 32.0 73.4 1.0 60.0 8.5 32.2 79.1 1.5 60.5 8.5 31.9 78.7 2.0 61.1 7.9 30.6 73.4

650 RPM 0.5 62,6 6.8 28.6 60.8 1.0 61.3 6.7 28.7 61.2 1.5 62.2 7.4 29.5 67.7 2.0 63.7 8.3 30.2 75.8

(continued) Table 16. The effect of different rates of aeration and agitation upon yields of yeast solids and protein (continued)

Volume of air Lbs. solids Lbs. protein % protein % increase per volume of from 100 lbs. synthesized in solids in protein mash per minute raw material from 100 lbs. due to raw material processing

500 RPM 0.5 58.9 4.0 24.9 37.7 1.0 61.0 4.8 25.3 44.7 1.5 61.5 5.0 25.5 47.0 2.0 60.4 5.5 26.8 51.7

350 RPM 0.5 52.0 2.1 25.1 19.1 1.0 57.2 2.2 23.1 20.5 1.5 59.5 2.5 22.6 22.8 2.0 62.7 3.7 23.4 33.8 - 74 -

An increase in the rate of agitation increased the amount of protein synthesized as vrell as the per cent of protein in the solids, whereas an increase in aeration rates appeared to have comparatively little influence. The one exception to this statement occurred when

2 volumes of air per volume of medium per minute was used with agitation rates of 800 and 650 RPM. There was no significant difference in yields of protein between the two agitation rates used at this degree of aeration. The yields of solids, in general, were not significantly different among the various agitation or aeration levels used.

"With the completion of these experiments on the composition of the medium, and other variables, it was concluded that a simple process for the production of yeast from wheat, using a mixed propa­

gation, was possible. It was necessary, however, to consider whether or not an increase in the propagation period or a variation

of the ratio of T. utilis to E. fibuliger inoculum would be of any value in the process.

Ratio of T. utilis to E. fibuliger inoculum:— In an effort to

ascertain whether or not the ratio of inoculum, i.e., the amounts of

E. fibuliger and T. utilis which were added to the medium had a major effect upon the yields the amount of E. fibuliger added was

kept constant, but 0.5, 1.0, 1.5, and 2.0 times the usual amount of

T. utilis inoculum was added. The results are presented in Table 17.

There was no significant difference in yields obtained among the

various ratios tested. _ 1* _ from rice. He points out that in India there are no available sub­ stitutes for cereals. The widespread culture of plants possessing a high protein content presents formidable difficulties in that country.

A similar situation undoubtedly exists in the other Oriental countries.

The Colonial Food Yeast Limited (19*4-9) has outlined the problem existing in the British possessions in the West Indies. Conditions of protein-scarcity existing in Germany during the two World Wars have been described by Skoog (19*4-5), and others, together with reports covering the measures taken to overcome this scarcity.

The increased production of protein is not the solution of the entire food problem since the production of carbohydrates, fats, and vitamins must also be increased. In view of the existing scarcities of protein and the relative abundance of carbohydrate materials, both edible and inedible, together with the existing methods of vitamin synthesis, it is profitable to consider the ways in wiiich the amount of protein in the world may be increased.

The primary source of proteins is the plant kingdom. Plants possess the ability to synthesize protein from simple components such as carbon dioxide, minerals, water, and simple nitrogen compounds.

The animal body cannot, as a rule, synthesize protein from any such simple materials, but is dependent upon plants for food protein supplies. Table 17. Effect of varying the ratio of T. utilis to E. fibuliger * upon yields of yeast solids and protein.

Amount of Lbs. solids Lbs. protein % protein % increase T. utilis from 100 lbs. synthesized in solids in protein inoculum per raw material from 100 lbs. due to 2 liters of mash raw material processing

100 ml. 62.4 7.5 29.0 70.0

200 ml. 62.1 7.1 28.5 66.3

250 ml. 60.1 7.8 30.8 73.4

400 ml. 61.2 8.2 30.8 77.1

* E. fibuliger inoculum constant. - 76 -

Length of propagation time:— In duplicate experiments the length of the propagation period was increased from 8 to 12 hours.

The results of these two experiments (Table 18) may be taken to indicate that the increase of the propagation time to 12 hours has no significant effect upon the yields of solids or of protein.

Backslonning:— In order to eliminate as much as possible the necessity for disposing of liquid waste materials from this process, all of the supernatant liquid, or slop, which had been separated from the solids, or product, by centrifugation of the mash after propagation of the yeast was added to the wheat as the mash for a new experiment was prepared. Since this slop contained some amylolytic enzymes it was hoped that a "premalting" effect would be obtained in which the viscosities of the wheat mash would be reduced. In plant practice high-viscosity mashes are difficult to pump.

Viscosity measurements were conducted by using a 10 ml. tip-

delivery pipette which was filled and held in a vertical position.

The time required for the liquid to travel a distance of 10 cm. at

28° C. was measured with a stop-watch. Results are presented in

Table 19. Viscosities are reduced by approximately 50 per cent by

the use of slop. This is especially important in the 10 per cent

mashes since these are particularly difficult to handle and would

undoubtedly present problems in an industrial process. Table 18. Effect of the length of the propagation period on the yield of yeast solids and of protein.

Length of Lbs. solids Lbs. protein % protein % increase propagation from 100 lbs. synthesized' in solids in protein raw material from 100 lbs. due to raw material processing

First run 8 hours 62.1 7.6 31.3 62.2 12 hours . 59.4 7.4 32.0 63.4

Second run 8 hours 65.0 6.2 27.7 53.3 12 hours 61.0 5.1 27.4 40.4 Table 19. Effect of backslopping on the viscosity of wheat mashes of various concentrations.

Mash Non-propagated mash Propagated mash concentrations Non- Backslopped Non- Back- backslopped backslopped slopped

10 per cent 30 seconds * 15 seconds 2 seconds 2 seconds

6 » " 25 " 5 " 2 » 2 »

4 " " 3 " 2 " 2 " 2 "

2 " " 2 « 2 " 2 " 2 »

Distilled water 2 »

* Time required for mash to flow a distance of 10 cm. at 23° C - 79 -

Experiments were conducted on the slop to determine the optimum temperature and pH for the digestion of the starch by the enzymes in the slop. A 10 per cent mash was prepared in which 30 per cent slop was used. The starch was gelatinized by heating at

80° C. for 15 min. After this cooking period, enough cold slop was added to bring the mash concentration to 5 per cent; 100 ml. samples were then held at various temperatures in a water bath for a 15 min. conversion period. A whole mash sample was taken for a check immediately after the final addition of the slop. The optimum pH of digestion was determined by the use o.f a mash made in the same way as was described for the temperature experiments. Adjustments

of pH were made with 10 N sulfuric acid or 40 per cent potassium hydroxide. The same amount of liquid was added in all cases by making to volume with distilled water where necessary. Results of these experiments are presented in Tables 20 and 21.

Since only 60 per cent of the fermented mash could be separated

as slop, and since in certain experiments it was desired to add slop

at a level of 80 per cent of the total mash volume, a stock supply

of slop was prepared to supplement the 60 per cent slop obtained in

any one experiment. To give a large quantity of supernatant liquid

of the same composition, a run was made in the pilot plant using two

20 gallon propagators. Ten liters of mash were placed in each vat.

Due to mechanical limitations, air could be supplied at a rate of

0.5 volume per volume of mash per minute, and agitation, at a rate

of 350 RPM. The slop from this propagation was used to make up - 80 -

Table 20. Effect of temperature upon conversion of wheat starch by enzymes.*

Temperature Grams starch Per cent starch remaining converted after conversion

50° C. 2.6 44.6

55° C. 2.8 40.4

60° C. 2.8 40.4

65° C. 3.2 31.9

70° C. 3.3 29.7

75° C. 3.5 23.4

unconverted wheat mash 4.7

* pH 5.0, conversion time 15 minutes - 81 -

Table 21. Effect of pH upon the conversion of wheat starch by- enzymes in slop.*

pH Grains starch Per cent starch remaining converted after conversion

4*0 4.3 8.5

4.5 3.8 19.1

5.0 2.0 57.4

5.5 2.1 55.3

6.0 2.5 46.8

6.5 3.0 36.1 unconverted wheat mash 4.7 —

* Temperature 50° C., conversion time 15 minutes. - 82 - the mash for another propagation under the same conditions. At the end of this propagation the separated liquid was frozen in stainless steel containers at 0° F. until needed.

Effect of backslopping upon yeast yields:— A series of experiments was made in which all of the liquid separated from the processed mash from each run was used in the mash for the following run. (The amount of slop used amounted to 60 per cent of the final mash volume.) Results are presented in Table 22. This experiment duplicated the results obtained in another series of experiments of the same type.

With the increase of recycling times of the supernatant liquid, the yields of solids and the per cent protein in the solids increased until the fifth recycling when they began to decrease. In the seventh recycling a comparison was made between T. utilis var. ma.ior and an osmophilic yeast, Hansenula subpelliculosa NRRL Y-1683. It was believed that an organism more tolerant of higher osmotic pressures might give better yields in media containing much re­ cycled supernatant liquid. Results of this comparison are presented in Table 23.

There is an indication that H. subpelliculosa surpasses

T. utilis var. ma.ior in media containing slop. In media which had been recycled only once, H. subpelliculosa synthesized twice the amount of protein that T. utilis synthesized. On a medium containing Table 22. Effect of backslopping in six consecutive runs, using E. fibulieer. Y-1062 and T. utilis. Y-1084.

Backslopping Lbs, solids Lbs. protein % protein % increase cycle from 100 lbs. synthesized in solids ' in protein raw material from 100 lbs. due to raw material processing

0 51.9 1.9 21.1 22.6

1 68.5 7.3 27.6 62.6

2 62,1 7.8 31.3 62.2

3 65.0 6.2 27.7 53.3

4 56.7 6.9 32.7 59.4

5 59.8 4.8 27.5 41.1

6 52.5 1.5 25.0 12.9 Table 23. Comparison of T. utilis var. ma.ior. Y-1084 and Hansenula subpelliculosa. Y-1683, on backslopped and non-backslopped medium*

Culture No. Name of organism Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

No backslop Y-1683 H. subpelliculosa 73.7 8.2 27.6 66.2 Y-1084 T. utilis var. ma.ior 77.0 8.5 27.1 68.7

Backslop - once recycled Y-1683 H. subpelliculosa 56.9 9.8 32.4 112.7 Y-1084 T. utilis var. raa.ior 49.7 5.6 28.6 69.3

Backslop - six times recycled Y-1683 H. subpelliculosa 54.6 3.1 27.7 31.0 Y-1084 T. utilis var. ma.ior 50.0 1.3 26.0 11.5

* E. fibuliger. Y-1062, used in all cases - 5 -

Microorganisms are known to synthesize protein within the animal

"body. Black, -.Warner, and Wilson (1951) have shown that urea and other simple nitrogenous compounds, when fed to cattle, sheep, and other ruminants, are converted Into protein by the bacterial population of the digestive tracts of the animals. The ingested food undergoes extensive fermentation in the rumen, reticulum, and omasum. According to Booher (1948), the bacterial synthesis of proteins in the digestive tract is not limited to ruminants, but because of the extensive bacterial activity in the esophageal pouches of such animals, and the correspondingly greatzr opportunity for digestion and absorption, this process would seem to be of much greater importance in ruminants than in non-ruminants. Apparently the feeding of urea or ammonium salts has not been employed to any important degree, and the feeding of protein derived directly or indirectly from the higher plants still re­ mains the chief source of protein in animal nutrition.

Many writers have pointed out the inefficiency of using crop plants for the indirect production of protein by animal feeding. Christensen

(1948) states that livestock provides about one-third of all the food energy contained in the natural diet, but that these livestock consume three timps as much food energy as do all the people in the United

States. Only about one-ninth of the calories consumed by livestock are available for human consumption. Maynard (1946) points out that

80 per cent of all farm land used to produce the national food supply

is devoted to livestock production. Booher (1948) estimates that the efficiency of protein conversion by animals ranges between 8 and 45 - 85 - six-times recycled slop, the amount of protein obtained by the use of H. subpelliculosa -was three times the amount of protein obtained by the use of T. utilis. Comparison of the two organisms on media * containing no slop showed no significant difference in yields between the two yeasts.

Recycling of inoculum:— An effort was made to ascertain whether or not the yields will be affected by the inoculation of mash with part of the processed mash obtained from a previous experiment. The mash used for inoculum was kept under refrigeration at 2° C. for four days before use. Forty per cent of the final mash volume was supplied as this processed mash containing both E. fibuliger and T. utilis cells.

Yields of both solids and protein were among the highest obtained in any of the experiments in which slop was used. (Table 24).

The conclusion that processed mash may be used as inoculum for the initiation of new propagations seems to be justified. There was no appreciable contamination evident. Less than 1 per cent of the colonies appearing on streak plates after 4 days of incubation at

28° C. were mold colonies. No bacterial contaminants were found.

The second recycling of inoculum showed no decrease in per cent protein in the solids, or in total protein synthesized. A slight increase in the yield of solids was noted. Table 24. Effect of recycling inoculum upon yields of yeast solids and of protein.

No. of Lbs. solids Lbs. protein % protein % increase recycling from 100 lbs. synthesized in solids in protein times raw material from 100 lbs. due to raw material processing

1 61.3 8.1 32.1 69.4 a> 2 64.2 8.6 31.5 73.9 i - 87 -

Incremental and continuous addition of mash:— Since the presence of an excess of fermentable material leads to fermentation and wastage of carbohydrate in yeast propagations, the wheat mash containing mineral salts was added to the 40 per cent inoculum in approximately equal increments over the course of 7 hours of the

8 hour propagation period. Additions were made at the beginning of each hour. The aeration rate was adjusted hourly so it would be equivalent to 1 volume of air per volume of medium per minute at all times. Results of duplicate experiments are presented in Table 25.

Two experiments were run in which the mash was added continuously to the propagators at the rate of 3.0 ml. per minute for approximately

6.5 hours of an 8-hour propagation period. A simple feeding device consisting of a 2 liter side-arm suction flask was used. The mash was placed in this flask which was fitted with a one-hole rubber stopper and a piece of glass tubing. The glass tubing extended to the bottom of the flask. Air was introduced through the side-arm to build up pressure inside the flask and force the mash into the propagators through the glass and rubber tubing. Results of these experiments are also presented in Table 25.

There was no significant difference in yields obtained between incremental additions, continuous additions, or batch cultures. Table 25. Effect upon yields solids and protein of increment and continuous addition of mash*

Type of Lbs. solids Lbs. protein % protein % increase feeding from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Increment 70.5 8.1 28.0 69.7 Non-increment 68.5 7.1 27.6 62.6

Continuous 53.3 1.2 24.1 10.6 Non-continuous 52.5 1.5 25.0 12.9

Continuous 62.9 6.6 29.0 56.8 Non-continuous 59.7 5.1 28.0 43.9 - 89 -

Comparison of various starchy substrates:— A further attempt was made to assess the value of the process by comparing the growth of the yeasts on a medium consisting of purified wheat

starch, with the growth obtained on a whole wheat mash.

A 3 per cent wheat starch medium was prepared which contained

0.5 per cent Difco yeast extract (based on the weight of the starch

supplied). The yeast extract was added because neither E. fibuliger nor T. utilis will grow on purified wheat starch alone. A wheat mash containing 3 per cent starch was prepared and one portion was

supplemented with the same amount of yeast extract that was added

to the starch medium. Results are presented in Table 26.

Yields of solids obtained on a purified starch medium were equal

to those obtained using wheat mash with no added yeast extract. The

per cent protein in the solids was markedly lower than the per cent

protein obtained with wheat, but the amount of protein synthesized

was approximately equal to that obtained with wheat alone. There

was no significant difference in the per cent protein obtained with

wheat whether yeast extract was added or not added.

Comparisons of the yields obtained on potato, corn, and rice

media are presented in Table 26. Table 26, Comparison of yields obtained on purified wheat starch with those obtained on whole wheat mash.

Substrate Lbs, solids Lbs. protein % protein % increase from 100 lbs, synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Starch + Yeast extract 47.0 6.9 18.9

Wheat + Yeast extract 64.4 10.8 27.3 161.1

Wheat alone 47.5 7.5 29.8 ,110.7 - 91 -

The most striking differences were obtained in the experiments in which tubers were compared with the grains (Table 27). The quantity of protein synthesized from 100 pounds of raw material ranged between 10.8 to 13.5 pounds for white and sweet potatoes as compared with 0.9 to 5.6 pounds for corn and rice.

The efficiency based upon the yields of solids ranged between

5 1 .4 and 91.8 per cent for the tubers as compared with 37.2 to 59.7 per cent for corn and rice. There appeared to be a reduction in the yield of solids when backslopping was practiced for both the tubers and the grains.

The per cent protein in the solids showed the following increases upon backslopping: sweet potatoes, 11.1 per cent; white potatoes, 6.0 per cent; corn, 9.5 per cent; rice, 14.5 per cent.

An experiment using potatoes which had been stored for several months was conducted and the results compared with the results obtained using freshly dug potatoes. Reference to Table 27 will show that the yields of solids and of protein were considerably lower when stored potatoes were used. Table 27. Comparisons of yields of yeast solids and protein obtained by the use of various starch substrates.

Substrate Lbs. solids Lbs. protein % protein % increase from 100 lbs. synthesized in solids in protein raw material from 100 lbs. due to raw material processing

Sweet potatoes 91-8 - 35.2 * 13.5 - 3.4 * 22.4 - 21.5 * 192.1 - 79.2*

Sweet potatoes backslopped 47.8 - 38.5 * 8.9 - 6.0 * 33.5 - 27.2 * 126.7 -155.0*

White potatoes 60.7 - 31.5 * 10.8 - 3.4 * 30.5 - 41.8 * 141.5 - 34.7*

White potatoes backslopped 51.4 - 29.9 * 11.1 - 0. * 36.5 - 26.6 * 144.5 - 0. *

Corn 59.7 1.4 19.5 13.8

Corn backslopped 45.8 3.5 29.0 33.9

Rice 44.4 0.9 15.5 15.5

Rice backslopped 37.2 5.7 30.0 95.0

* The second figure presented for potatoes was obtained from a second experiment in which potatoes which had been stored for 4 months were used. Potatoes for the first experiment were freshly dug. - 93 -

Vitamin and amino acid content of the yeast product;—

Comparisons of the amounts of seven B-vitamins and nine amino acids obtained on various substrates are given in Tables 28 and 29. Yields obtained by other workers for various substrates are also presented*

In general, yields were increased by the use of backslopping.

The amounts of the vitamins and amino acids synthesized indicate that the propagation is of value in enhancing the nutritional value of the appropriate substrate as regards these materials. Where negative values are shown for the amount synthesized, there was a loss of the amount shown from the original substrate. These losses occur only in a propagation where backslopping was not practiced.

With the addition of slop, increases in these compounds were obtained instead of losses. Table 28. Comparative vitamin values of yeast products from various substrates (micrograms per gram, dry material).

Substrates Niacin Pantothenic Riboflavin Biotin Pyridoxine Thiamine Choline acid

Wheat, no slop 209.0 89.0 2.0 0i6 11.0 6.0 1310.0 -Amount synthesized 154.0 66.0 0.0 0.5 7.0 2.0 — Wheat + slop 331.0 133.0 19.0 1.0 9.0 11.0 2407.0 Amount synthesized 276.0 110.0 17.0 0.9 5.0 7.0 --- Wheat + slop (dried) 184.0 23.0 12.0 0.4 1.0 7.0 3.0

Sweet potatoes, no slop 169.0 57.0 13.0 0.4 23.0 5.0 — Amount synthesized 93.0 24.0 10.0 0.2 10.0 4.0 — Sweet potatoes + slop 202.0 113.0 17.0 0.7 18.0 4.0 7S3.0 Amount synthesized 146.0 80.0 14.0 0.5 5.0 3.0 —

(continued)