Loss of cell constituents on reconstitution of active dry yeast

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

63, 131-143 (1956)

Loss of Cell Constituents on Reconstitution of Active Dry Yeast T. Herrera and W. H. Peterson From the Department

of Biochemistry, University Madison, Wisconsin

of Wisconsin,

and E. J. Cooper and H. J. Peppler From the Research Laboratories, Red Star Yeast and Products Milwaukee, Wisconsin

Company,

Received January 19, 1956

INTRODUCTION Active dry yeast (ADY) is the product obtained when the growth of Saccharomyces cerevisiae is washed and pressed to a solids content of 30-32 %, extruded, and dried under controlled conditions of temperature and humidity to 92% solids. Representative samples of high quality ADY contain about 18-20 billion live cells/g. When rehydrated under suitable conditions, ADY is employed in the same manner as compressed yeast. Since the end of World War II, when no ADY was employed in commercial bakeries in this country, more than 500 bakeries, including some of the largest establishments in the United States, have adopted active dry yeast exclusively. The baking strength of ADY can be influenced markedly by the temperature of rehydration. A recent report (1) demonstrated a progressively lower baking strength of active dry yeast when subjected to increasingly lower temperatures of rehydration. Water suspensions of ADY held at 4-15°C. for short periods exhibited decreased viable cell counts and poor performance in standard straight dough fermentations. In preliminary experiments initiated to explain the effect of low-temperature rehydration, a chance observation revealed that approximately 75 % of the oxidized diphosphopyridine nucleotide (DPN) was extracted at 4.5”C. as compared to 15 % at 43°C. Subsequent quantitative studies 131

132

HERRERA, PETERSON, COOPER AND PEPPLER

showed that DPN represented only a minor portion of the solids extracted and that ADY rehydrated at low temperatures lost as much as 25 % of its cell contents. In the present study the analyses of the extractable contents of normal ADY have been extended to provide evidence regarding the nature of the major cell components removed at rehydration temperatures which are the extremes for poor and optimum bakeshop performance. EXPERIMENTAL

Preparation of Yeast Extract-s Commercial samples of active dry yeast made by three different manufacturers were chosen for this work. The yeasts were reconstituted by suspending 10 g. ADY in 100 ml. of distilled water at either 4.5” (40°F.) or 43°C. (110°F.). These temperatures were selected to avoid freezing and heat inactivation in the extraction. In addition, 43°C. has been widely accepted as a near-optimal temperature for ADY rehydration. Since the particles of ADY were more resistant to suspension at 4.5” than at 43”, frequent hand stirring or continuous mild mechanical agitation for 15 min. at the lower temperature was employed to obtain effective dispersion. Vigorous mechanical agitation was undesirable because of its temperature effect and cell-fracture possibilities. Samples prepared at 43°C. were stirred continuously for 2-3 min. and then cooled in an ice-water bath to minimize autofermentation and to facilitate sedimentation of the cells during centrifugation. Cooling the extracts after the initial rehydration of 43” did not affect the quantity of solids extracted. Suspensions thus prepared were centrifuged at 4.5”C. and 1000 X g and aliquots of the supernatants were analyzed. Tests with 0.1 g.phosphate buffer at pH 7.0 or with 0.85% NaCl showed that the same quantity of nitrogen was extracted with these solutions as with distilled water. Hence the loss of cell constituents was not a pH or salt effect. All of the work was done on portions of the same lots of yeast. Many rehydration experiments were made and small variations in the composition of the extracts were observed. In a given series of analyses these were all done on the same extract, for example, Tables III, IV, and V.

Methods of Analysis Moisture and Solids. Samples were dried at 100°C. for 16 hrs. in a forced draft oven. Ash. Samples were heated at 500°C. for 16 hr. Phosphorus was determined by the method of Bolin and Stamberg (2). Nitrogen Compounds. Total nitrogen was obtained by the macro-Kjeldahl method and amino nitrogen by the Van Slyke procedure. Amino acids were determined before and after hydrolysis according to the microbiological method of Barton-wright (3). Hydrolysis of the extracts was accomplished by adding HCl to

ACTIVE DRY YEAST

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make a 2.5 N solution and autoclaving at 15 lb. pressure for 10 hr. HCl was removed by evaporating the hydrolyzate to dryness at a low temperature. The dry solids were resuspended in a suitable volume of water, adjusted to a pH of 6.8-7.0, and made to volume, and aliquots were taken for assay. Chromatographic separation of amino acids in unhydrolyzed extracts was carried out with the procedure of Clayton and Strong (4). Nucleic acid was determined on the cells before and after reconstitution by the method of DiCarlo and Schultz (5). Fat. A weight of 1.5 g. ADY was hydrolyzed in 75 ml. of 1:l HCl in a steam bath for 3 hr.; the suspension was evaporated to dryness on a steam bath and further dried at 100°C. for 1 hr. The cake was transferred to an extraction thimble and refluxed in a Soxhlet extractor with ether for 16 hr.; the ether was evaporated and the dried solids were weighed. Carbohydrate. Total carbohydrate was determined with the anthrone reagent according to the procedure of Trevelyan and Harrison (6). All values are expressed in terms of glucose. Trehalose was separated on the s-collidine paper chromatogram of Partridge (7), and the paper was stained wit,h the silver nitrate reagent of Trevelyan et al. (8). Vitamins. Extracts prepared at 43°C. were concent.rated fourfold by evaporation under vacuum at 60°C. before analysis. The extracts prepared at 4.5%. were used without further concentration. Niacin was determined by the method of Snell and Wright (9) ; thiamine, by the procedure of Hennessy and Cerecedo (10); riboflavin, by the method of Snell and Strong (11). CO* Production. Duplicate samples of 4.5 g. ADY were reconstituted in 20 ml. of water at 4.5” and another pair at 43°C. One sample of each pair was subsequently diluted to a final volume of 100 ml. The other sample was centrifuged, the supernatant was decanted, and the cells were washed with 50 ml. of 4.5”C. water and centrifuged again. The washed cells were resuspended to a final volume of 100 ml. with 4.5”C. water. A lo-ml. aliquot of each sample was used for measuring anaerobic CO, production in a complete fermentation medium (12). RESULTS

Composition of Yeasts The summary of analytical data on the three ADY samples (Table I) reveals a similarity in gross composition. Sample Z was somewhat lower in ash, fat, and niacin than the other two yeasts. The totals for the analyses exceed 100 % because of the summations of the individual items and partly because of the method of calculation. All of the nitrogen is expressed as crude protein, and the carbohydrate as glucose. Most of the latter is trehalose, and hence the figures are about 5 % too high. The data in Table I are used in calculating the losses incurred on reconstitution of the yeasts.

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TABLE I Composition of Active Dry Yeasts (All values are expressed on dry solids basis) Analyses

Sample x

Sample Y

Sample z

Moisture, To Ash, % Fat, % Crude protein, To Carbohydrate as glucose, ye

8.4 5.3 5.6 45.6 39.3

8.5 4.6 6.5 43.0 41.8

8.4 4.4 4.6 43.4 41.7

104.2 0.88 48 37 469

104.4 0.77 21 44 422

102.5 0.86 22 38 342

Total, % Phosphorus, y. Thiamine, Pg./g. Riboflavin, pg./g. Niacin, pg./g.

TABLE II Composition of Extracts (All values are expressed on dry solids basis) Analyses

T

Sample x

Ecgaction

temperature,

4.5

43

= I _-

Sample Y 4.5

43

-Ash, mg./g. Crude protein (N X 6.25)) mg./g. Carbohydrate, as glucose, mg./g. Total, mg./g. Solids extracted, w.lg. Phosphorus, mg./g. Niacin, pg./g.

27.3 62.4

Sample Z

-

8.1 19.1

23.9 47.4

-43

4.5

-

-11.6 31.8

27.5 53.1

10.0 28.7

52.7

170.0

52.3

35.0

164

253.7 227

62.2 53

235.3 224

96.1 100

250.6 245

91.0 93

1.8 238

0.54 53

1.6 209

0.94 107

1.9 201

0.80 79

164

--

-

.-

--

-

Composition of Extracts Data on the composition of the water extracts (Table II) show that nearly 25% of the solids of the yeast cell was removed at 4.5”C. The loss was made up largely of ash, crude protein, and carbohydrate. No figures are given for fat, because negligible quantities of ether-soluble material were found in the solids of the extracts. The totals for ash, crude protein, and carbohydrate in most cases exceed the direct deter-

ACTIVE

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mination of solids for the same reasons as were given for the totals of Table I. Based on the totals in the yeast (Table I), the loss was greatest for ash, 52-62 % ; intermediate for carbohydrate, 40-42 % ; and least for crude protein, ll-14%. At the higher temperature of reconstitution the losses from the cells were conspicuously lower t,han at 4.5”C. In most cases they were less than one-half and in some cases less than one-fourth of those incurred at 4.5”C. Calculation of the percentage composition of the extracted solids shows that in t’he extract obtained at 4.5”C. protein (N X 6.25) makes up from 21-28 % of the total, ash about 11 %, and carbohydrate 62-66 %. At 43°C. a larger proportion of solids consists of protein (around 33 %), Dhe ash is about the same, and a considerable fluct,uation occurs in the proportion of carbohydrat’e (51-66 %). Phosphorus losses follow in general the same pattern as those of the major constituents: 2-4 times as much at 4.5”C. as at 43°C. The percentage of the total that is extracted is only about half as much for phosphorus as for ash, e.g., 20 vs. 52%, 6 vs. 15%, etc. The percentage figures, however, follow those for total solids rather closely. The proportions of the total niacin lost in the extracts are among the highest in the table. They are of the same magnitude as those for ash. Some preliminary data on the loss of thiamine and riboflavin showed much smaller losses for these two vitamins t’han for niacin. Preliminary analyses, including one-dimensional paper chromatograms, show that trehalose accounted for approximately 95 % of the carbohydrates extracted at either temperature. Forms of Nitrogen in Extracts The amount and composition of t’he nitrogen obtained in typical extra&s prepared at the two temperatures are compared in Table III. From 11.1 to 13.7% of the total nitrogen in the yeasts was extracted at 4.5”C. and from 4.2 to 7.4% at 43°C. In two other experiments the figures mere somewhat higher at 4.5”C. (12.3-15.6%) and lower at 43°C. (3.7-5.7 %). The loss at the lower temperature appears to represent most of the mobile nitrogen of the cell. The soluble nonprotein nitrogen, for example, obtained from cells disintegrated by freezing, grinding, and extracting with water was only about 15% of the t’otal nitrogen. The figures are also in the same order of magnitude as those reported by ot#her investigators for soluble nonprotein forms of nitrogen. From frozen baker’s

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-_-

AND

PEPPLER

TABLE III

Forms of Nitrogen in Extracts

Series 1

Analyses Extraction

temperature, ‘C.

Nitrogen in yeast, mg./g. Nitrogen extracted, mg./g. of yeast Per cent of total in yeast NH2-N, y. of N extracted Before hydrolysis After hydrolysis NHI-N, y. of N extracted Before hydrolysis After hydrolysis

Sample x

4.5

43

72.9

72.9

10.0

3.1

13.7

= I

T

Sample Y

-

4.5

Sample 2

-

43

4.5

68.8 7.6

68.8 5.1

69.5

69.5

8.5

4.6

4.2

11.1

7.4

12.2

6.6

65.7 71.3

71.2 72.0

67.8 70.6

66.5 70.2

65.9 71.8

64.3 74.0

0.0 9.4

0.0 10.5

0.0 8.5

0.0 10.7

0.0 9.8

0.0 10.5

3-

-I

_-

-

.-

_-

-

43

yeast, Roine (13) obtained 15.5% of the total nitrogen by extraction with water and 12.7 % with cold 8% trichloroacetic acid. Lindan and Work (14) reported that 13.5% of the total nitrogen in dried baker’s yeast and 17.7 % of that in dried brewer’s yeast, were extracted by 70 % cold ethyl alcohol. Considerably lower figures (7-9 %) w&e reported by Miettinen (15) for ethanol extraction of a torula yeast. The nature of the nitrogen extracted is indicated by the high amino nitrogen content of the extracts. This accounted for 64-71% of the total nitrogen before hydrolysis and only 70-74 % after hydrolysis. Therefore, there was very little peptide nitrogen, and hence free amino acids should make up most of the nitrogen extracted. More data in support of this interpretation will be presented later. No ammonia was found in the extracts, but, a considerable amount was produced by hydrolysis. Some of this may have come from amide nitrogen, e.g., glutamine, but it probably resulted from the decomposition of amino acids during hydrolysis. With large amounts of carbohydrate in the extract, the decomposition of amino acids is increased. Occurrence of Free Amino Acids in Extracts The presence of free amino acids percentage of free amino nitrogen mensional paper chromatography corresponding to the control strips

in the extracts suggested by the large was verified qualitatively by one-diof the extracts. Spots on the paper (run with known amino acids) indi-

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YE.4ST

cated the presence of the following acids: tryptophan, phenylalanine, isoleucine and/or leucine, valine, proline, alanine, glutamic acid, glycine, and lysine; two poorly defined spots were also observed. Quantitative data on five of these amino acids were obtained by microbiological assay of the extracts from one of t’he yeasts. The data are given in Table IV. There is considerable variation in the ratio of the free to the combined form of the amino acids. The ratio varies from 25 to 75 for glycine in the 4.5%. extract to 82 to 18 for glutamic acid in the 43°C. extract. (The ratios given are the percentage distribution of t’he two forms of amino acid calculated from the data in Table IV. For example, the glycine calculations are: 86 X 100/348 = 25, free amino acid; 100 - 25 = 75 combined amino acid.) The totals for the five amino acids show that the ratio of free to combined amino acid was 67 lo 33 in the 4.5%. extract and 75 to 25 in the 43°C. sample. These figures are in t,he same range as the figures for cz-XHZ-K (Table III). They are based of course on the assumption that the assay microorganisms responded only to the free amino acids, which is not necessarily correct. However, taken together wit’h t*he high percentage of a-amino nitrogen in the extracts, and t’he evidence from the paper chromatograms, it seems cert,ain that a very large proportion of the amino acids was present in the free state. TABLE Occurrence

of Free and Combined

IV Amino

Acids in Yeast Extracts

Series 2 Amino acid0

Alanine, Alanine,

before hydrolysis after hydrolysis

Glutamic Glutamic Glycine, Glycine,

acid, before hydrolysis acid, after hydrolysis before hydrolysis after hydrolysis

In extract per loo g. of dry yeast At 4.YC., mg. At 43°C.. mg.

478 661

127 197

2649 3689

725 883

86 348

33 80

Lysine, Lysine,

before hydrolysis after hydrolysis

78 141

26 47

Valine, Valine,

before hydrolysis after hydrolysis

184

318

75 100

3475 5157

986 1307

Total, Total,

before hydrolysis aft,er hydrolysis

a The values as representing

and those after before hydrolysis are taken as “free” both free and combined forms of the amino acid.

hydrolysis

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Amino Acid Composition of the Hydrolyzed Extracts

A second series of extracts was made, and after hydrolysis the extracts were assayed for 18 amino acids by microbiological methods. The weight bf amino acid found and the per cent of the total nitrogen (Kjeldahl) accounted for by each amino acid and the total nitrogen recovered are given in Table V. Glutamic acid accounts for about 30 % of the nitrogen in the extracts, alanine furnishes lo-12 %, and arginine 6-8 %. Glycine ranks fourth and valine fifth as major constituents of the extracted nitrogen. The amount of nitrogen supplied by the other 13 amino acids ranges from 0.1 to 0.2 % for methionine to around 3-4 % for lysine. As compared to the data of Lindan and Work (14) for ethanol extracts of dried baker’s and brewer’s yeasts and Miettinen’s (15) figures for the ethanol extracts of typical fresh Torula utilis, our values are higher for -glutamic acid, glycine, and valine, about the same for alanine, and lower for arginine and lysine. Although there are differences in detail, the general picture is about the same. In all cases, the six amino acids named contain the bulk of the nitrogen extracted. The total for all the amino acids is around 75% of the nitrogen extracted. With the ammonia formed during hydrolysis, 85% of the total nitrogen is accounted for. These figures are in the same range as given by Miettinen but somewhat higher than those reported by Lindan and Work. The unaccounted-for nitrogen (15 %) is probably contained in humin nitrogen, undetermined amino acids, perhaps purines and pyrimidines, and probably unknown compounds. Lindan and Work found 5 % of the nitrogen present as ornithine in the extract of brewer’s yeast and 0.3-l % as a- and r-aminobutyric acids and /3-alanine in the extract of baker’s yeast. Miettinen identified ethanolamine and citrulline in his extracts by means of paper chromatography. Both he and Lindan and Work have noted several unknown ninhydrin-positive compounds on their papergrams. No evidence was found in the reconstituted yeasts of a loss of nucleic acids as measured by the method of DiCarlo and Schultz (5). E$ect of Leaching on Activity

The loss of such a large proportion of the soluble cell constituents as occurs at 4.5”C. should be reflected in the metabolic activity of the cells. Carbon dioxide production was taken as a measure of such activity. The data are given in Table VI. Cells reconstituted at 4.5”C. produced only

ACTIVE

DRY

TABLE

V

Amino Acid Comvosition of Hudroluzed

Amino acid

Alanine

Arginine

acid

Cystine

Glutamic

43T. extract

~ X

Glycine

Histidine

-

12.6 12.0 13.0

-

240 159 195

7.0 5.7 6.7

45.9 77.7 48.6

5.7 8.4 6.1

244 113 144

2.3 1.3 1.6

93.8 88.8 82.6

3.7 3.1 3.4

0.11 0.23 0.18

17.3 19.2 19.3

0.75 0.73 0.85

3856 3014 3012

32.4 32.2 31.3

794.0 866.0 738.0

28.6 27.4 27.4

358 336 365

5.8 6.9 7.1

64.5 68.5 76.9

4.6 4.2 5.6

1.0 0.6 0.7

10.5 10.9 12.9

1.1 1.0 1.3

1.4 1.3 1.7

40.8 58.2 45.7

1.6 2.1 1.8

0.84 1.0 0.65

29.2 43.7 32.2

1.2 1.5 1.3

2.9 3.4 3.5

51.7 66.5 55.0

3.7 4.2 4.0

90.2 82.5 58.7

Lysine

9.7 11.0

Per cent of leached N”

10.5

152 113 153

Leucine

mg./lOO g.

212.0 230.0 221.0

41.9 21.6 26.1

Isoleucine

Per cent of leached N’=

704 640 636

14.3 17.7 14.8 acid

Extracts

-

ADY

mg./lOO g.

Aspartic

139

YEAST

174 160 176

TABLE

V-Continued

4.YC. extract Amino acid

43’C. extract

ADY mg./lOO g.

mg./lOO g.

Per cent of leached N’=

Methionine

X Y Z

20.5 9.5 10.0

0.17 0.10 0.10

5.6 7.3 5.1

0.20 0.23 0.19

Phenylalanine

X Y Z

51.2 46.4 48.5

0.37 0.43 0.43

18.5 28.3 24.4

0.60 0.80 0.77

Proline

X Y Z

278 110 108

3.0 1.5 1.4

82.7 47.1 25.6

3.8 1.9 1.2

Serine

X Y Z

115 93 95

1.3 1.4 1.3

24.0 23.0 21.0

1.1 1.0 1.0

Threonine

X Y Z

106 63 69

1.1 0.8 0.85

32.0 36.0 42.0

1.4 1.4 1.9

Tryptophan

X Y Z

14.7 11.7 12.8

0.17 0.17 0.17

4.9 5.2 4.8

0.23 0.23 0.23

Tyrosine

X Y Z

45.3 33.3 40.6

0.30 0.31 0.32

9.7 10.4 8.8

0.28 0.27 0.30

Valine

X Y Z

326 316 323

3.4 4.1 4.1

96.7 99.0 96.0

4.3 4.0 4.5

X Y Z

-

9.4 8.5 9.8

-

10.5 9.7 10.5

X Y Z

-

82.7 81.0 82.4

-

86.1 84.2 85.3

Ammonia

N

nitrogen Total recovered

~1The percentages given are based on the total nitrogen (Kjeldahl) of this series of extracts, mg. N/100 g. dry yeast: at 4.5”C., X = 1139; Y = 903; Z = 952. At 43”C., X = 265; Y = 300; Z = 259. 140

ACTIVE

TABLE E$ect of Reconstitution

VI

Temperature

on CO2 Production

Series 4

..___ Milliliters Extraction

141

DRY YEAST

-___---

CO* evolved by 0.45 g. yeast in 2.5 hr. -__-___

___--

._~~~~~~-.~

43°C.

4S”C.

temperature

_.--.-~ Fraction -____

Yeast Yeast Yeast

X Y Z

Cells

Cells and extract

Cells

Cells and extract

70

52

315

350

from 11 to 22% as much CO2 in a given t,ime as cells reconstituted at 43°C. Large variations were obtained in replicate runs with cells rehydrated at 4.5%. Therefore, the data should be considered as indicative rather than as an absolute measure of the performance of such cells. It was t’hought) that the erratic behavior of the 4.5”C. cells might be corrected if the cells and extracts were used together. Somewhat surprisingly, the addit’ion of the ext’racts depressed CO2 production in two of the test)s. Presumably the compounds lost in reconst’itution are unavailable t’o the cells because repeated runs, although not always resulting in decreased CO2 production, failed to demonstrate stimulation of gas formation. Ext’racts obtained at 43°C. had no significant effect’ on CO2 production. DISCUSSION

The reconstitution of the yeast apparently involves two events: the rehydration of the cell wall and the uptake of water by the cell constituents. The cell wall of active dry yeast must be badly disorganized and may present a discontinuous surface. It must be rehydrated to give a uniform semipermeable membrane that retains the constituents of the cell but’ allows nutrients to enter. The formation of such a membrane at 4.5”C. is apparently slow and allows the soluble constituents to escape. Conversely, at 43°C. the formation of the hydrated membrane should be more rapid and t.hus provide quickly a barrier that stops the further loss of valuable constituents. Simultaneously, or perhaps somewhat later than t,he reorganization of the cell wall, the proteins within the cell must be rest,ored to the physical condition in which they normally exist. While this restoration is taking place, the soluble compounds of small molecular weight are

142

HERRERA, PETERSON, COOPER AND PEPPLER

probably free to diffuse outward through the “porous” cell wall. Current views on the interrelation of protein and amino acids suggest that their interaction within the cell accounts for the higher concentration of amino acids inside than outside the cell. Gale’s review (16) on the uptake and retention of glutamic acid by bacteria suggests this relationship. Accumulation of glutamic acid in bacterial cells does not occur without the metabolism of glucose or some other source of energy. In the dried yeast the combination between protein and amino acids may have been broken and must be reformed before the latter can be retained within the cell. To set up and maintain this combination may require metabolic activity, and this occurs more rapidly at 43’ than at 4.5”C. In restoring both the cell wall and the cell constituents to their normal physical and biological state, water is as important as the solids since it is an integral part of the living cell. This view has been admirably set forth by Gortner and Gortner (17). A question that inevitably arises is: Do the leachings come from living cells as well as from dead cells? The answer to this question is that both types of cells must contribute to the leachings. For example, the soluble nonprotein nitrogen obtained from disintegrated cells is only about 15 % of the total nitrogen in the cells. This is not much more than that extracted at 4.5”C. Since most of the cells are capable of growth if rehydrated under suitable conditions, the living cells must provide some of the nitrogen in the water extract. However, only about 66% as many cells are alive when rehydrated at 4.5”C. as are alive when rehydrated at 43°C. (1). There does not seem to be any easy way of determining how much of the diffusible constituents can be lost and still retain viability because the loss comes from both living and dead cells. It is possible that the nonviable cells are not really dead. If the leachings could re-enter the cells, perhaps they could multiply again. The enzymes of the cell appear to be retained but the substances on which they operate are lost. ACKNOWLEDGMENTS The authors wish to thank Joseph Amsz, Jr., for assisting with the earlier analytical procedures and Dr. C. H. Keipper and staff for the chemical and vitamin analysis. Part of this work was done under a Point IV Grant to T. Herrera by the United States Department of Agriculture, Foreign Agricultural Service. SUMMARY

Three different commercial samples of active dry yeast were rehydrated at two temperatures, 4.5”C. and 43”C., and the amounts of cell

rlCTIVE

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143

constituents passing into the water phase were determined. About 12 a/u of the protein, 20% of the phosphorus, 40% of the carbohydrate, and 50-60% of the ash were leached out at 4.5”C.; this is from two to four times that lost at 43°C. Little or no loss of riboflavin took place, but 50 % or more of the niacin was lost at the low temperature. Free amino acids accounted for most of the nitrogen lost, but small amounts of peptides also appeared in the leachings. The six most abundant amino acids in extract’s made at 4.5”C. and then hydrolyzed mew, in descending order: glutamic acid, alanine, glycine, valine, aspartic: acid, and arginine. The nitrogen in these amino acids accounted for about 60% of the total extracted. Smaller amounts of twelve other amino acids were also present. Approximately the same order of amino acids but in much smaller quantities was found in the 43°C. extracts. The loss of such large amounts of important cell constituents at 4.5”C. is reflected in the poor CO2 production (baking quality) of the reconstitut,ed yeasts. \‘arious factors that, may be responsible for the leaching phenomenon are discussecl. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

PEPPLER, H. J., AND RUDERT, F. J., Cereal Chem. 30, 146 (1953). BOLIN, D. W., AND STAMBERG, 0. E., Id. Eng. Chem., Anal. Ed. 16,345 (1944). BARTON-WRIGHT, E. C., Analyst 70, 283 (1945); 71, 267 (1946). CLAYTON, R. A., AND STRONG, F. M., Anal. Chem. 26, 1362 (1954). DICARLO, F. J., AND SCHULTZ, A. S., Arch. Biochem. 17, 293 (1948). TREVELYAN, R. S., AND HARRISON, J. S., Nature 170, 626 (1952). PARTRIDGE, S. M., Biochem. J. 42, 238 (1948). TREVELYAN, W. E., PROCTOR, D. P., AND HARRISON, J. S., Nafwe 166, 444 (1950). SNELL, E. E., AND WRIGHT, L. D., J. Biol. Chem. 139, 675 (1941). HENNESSY, D. J., AND CERECEDO, L. R., J. Am. Chem. Sot. 61, 179 (1939). SNELL, E. E., AND STRONG, F. M., Znd. Eng. Chem., ilnal. Ed. 11, 346 (1939). SCHULTZ, A. S., ATKIN, L., AND FREY, C. N., Ind. Eng. Chem., Anal. Ed. 14,35 (1942). ROINE, P., Ann. Acad. Sci. Fennicae, Ser. A, ZZ, No. 26 (1947). LINDAN, O., AND WORK, E., Biochem. J. 46, 337 (1951). MIETTINEN, J., Ann. Acad. Sci. Fennicue, Ser. A, II, Ko. 68 (1954). GALE, E. F., Advances in Protein Chem. 8, 287 (1953). GORTNER, R. A., JR., AND GORTNER, W. A., “Outlines of Biochemistry,” 3rd ed., pp. 243-56. John Wiley and Sons, Inc., New York, 1949.