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Whey Utilization. IV. Availability of Whey Nitrogen for the Growth of Saccharomyces Fragilis
W H E Y UTILIZATION. IV. AVAILABILITY OF W H E Y NITROGEN FOR THE GROWTH OF S A C C H A R O M Y C E S F R A G I L I S AARON E. WASSERMAN Eastern Regional Research Laboratory, USDA, Philadelphia, Pennsylvania
SUMMARY Saccharomyces fragilis utilized approximately 50% of the ammonia nitrogen, 25% of the organic nitrogen, and 25% of the total nitrogen in whey, and all of the added ammonia nitrogen. The available nitrogen was found to be in the organic fraction soluble after heat- and acid-precipitation of the whey proteins.
Whey contains approximately 0.14% nitrogen. The principal nitrogenous components are the heat- and acid-precipitable lactalbumin and lactoglobulin, whereas the nonprotein nitrogen fraction consists of proteose-peptones, amino acids, and other substances. Orla-Jenson et al. (4) reported that the noneoagulable N in heat- and acid-treated whey was not utilized for growth by various strains of lactic acid bacteria, and whey so treated was used as a base for the addition of nitrogen sources in a study of the metabolism of these organisms. These authors also showed that although the bacteria used casein as a source of N for growth, they were unable to use lactalbumin N. Leviton and Whittier (2), on the other hand, reported that the nonheat-eoagu]able N fraction of whey was utilized for growth and riboflavin production by Ashbyii gossypii. A study of the growth of Saccharomyces fragilis in whey showed that approximately 25% of the total whey nitrogen was used by the yeast (6). A further study, reported here, shows that the assimilable nitrogen was available only from the noneoagulable nitrogen fraction, and that the heat- and acid-precipitable proteins were not broken down by the actively growing yeast. 1VIATERIALS AND METI-IODS
S. fragilis, NRRL Yl109, was grown in a medium consisting of Cottage Cheese whey, 0.5% (NH4)eSO4, 0.5% K.~HP04, and 0.1% yeast extract. The whey pH was about 4.6, but following the addition of the salts was approximately 5.6, which was in the range for optimal yeast growth. The temperature was maintained at 32 ___ 1 °, and the 500-m]. quantities of media in the propagators were aerated at 2 liters/minute. Aliquots of the growing yeast suspensions were removed for the determination of the nitrogen fractions. The yeast was sedimented immediately by centrifugation and supernatants treated as described in the test. Total nitrogen (TN) was determined by micro-Kjeldahl distillation following digestion by the Miller and Miller modification (3) of the KochMcMeekin method (1). Ammonia nitrogen (AN) 1 was determined by microKjeldahl distillation, and the organic nitrogen (ON) fraction was calculated as the difference between TN and AN. Received for publication April 30, 1960. 1Ammonia N was determined by the addition of alkali to undigested samples. Traces of volatile protein degradation products also may be distilled over in this determination. 1231
12:32
AARON E. WASSERMAN
Deproteinated whey was prepared from whey clarified by passage through a medium sintered glass filter. The pH was adjusted to 6.0 with a few drops of concentrated NaOH, and the whey heated in a boiling water bath for 10 min. (500- and 1,000-ml. quantities of whey were heated in the autoclave at 121 0 for 10 min.). After cooling, the pH of the whey was adjusted to 4.5, and the precipitated protein removed by centrifugation.
Yeasts were grown in whey media in the presence or absence of (NH 4 hS04 as a source of inorganic nitrogen. The nitrogen composition of the media was determined after 4 hr. of growth. Results of the analyses are shown in Table 1. TABLE 1 Course of nitrogen utilization during growth of Saccharomyces [ragiUs in whey medium, with and without added ammonium sulfate Whey medium Minus (NH.).SO. Growth time
TotalN
(hr.)
o 1 2
3 4 Yeast yield
129 97 96 95 95
14 6 6 7 7 14.7 g. dry wtjliter
Organic N
TotalN
(mg. N /100 115 91 90 88 88
mZ.) 252 194 116 99 99
Plus (NH.)2S0. Organic NH.+N N 128 124 87 107 21 95 8 91 8 91 25.9 g. dry wtjliter
The total N of the medium without added (NH 4hS04 decreased to approximately 75% of its initial value within an hour after yeast growth began. Continued growth for several hours did not reduce further the nonutilizable N of the medium. When the yeasts were grown in the fully supplemented medium, the same pattern of N utilization was observed. The total N decreased to the same level as in the (NH4hS04-free medium, and was not consumed further in spite of continued yeast growth. These results had been observed previously (6). The total N was separated into the ammonia N and organic N components. The whey medium, in the absence of (NH 4)2S04' contains little AN, and the yeast used most of this nitrogen and about 25% of the ON fraction. The remainder of the ON was the nonutilizable residue. In the presence of added (NH 4 hS04, the yeast readily used the added AN, although some ON disappeared simultaneously. However, when the ammonia nitrogen had been consumed, the nonutilizable ON residue remained. The quantity of N in the whey available to the yeast was insufficient to support a good growth of yeast. In the presence of added nitrogen, the yeast yield obtained in 4 hr. of growth was almost twice as great as the yield in the absence of (NH 4hS04. Whey organic nitrogen is composed of two fractions: (1) heat- and acidprecipitable N, and (2) noncoagulable, or soluble, N. When the whey was
WHEY UTILIZATION.
1233
IV.
submitted to the heat and acid treatment described in Methods, 52% of the ON was noncoagulable (Table 2). T h e presence of the medium supplements during the heat and acid treatment did not significantly affect the q u a n t i t y of ON precipitated. However, it was noted that after the heat treatment, the p H of the whey alone remained at the initial value of 6.0, while the p i t of the whey medium changed to 5.3. TABLE 2 Effect of heat- and acid-deprotelnatlon on the nitrogenous components of whey alone and whey plus the medium salts Whey Treatment
Total N
NH, + N
)~'one Heat and acid
128 74
25 20
Whey medium Organic 29
Total N
NH,+ N
Organic N
I41 139
104 59
(rag. N/IO0 ,m~.) 103 54
245 198
Since the ON component of whey is composed of almost equal quantities of the noncoagulable and the heat- and acid-precipitable N fractions, it was of interest to determine which fraction was used b y the yeast. Therefore, the growth of yeast in deproteinated whey medium was compared with growth in the regular whey medium. To 500-ml. quantities of deproteinated whey and untreated whey were added the salts and yeast extract necessary to formulate the media. A f t e r inoculation with S. fragilis, an aliquot was removed from each propagator, clarified by centrifugation, and the supernatants divided into two portions. One portion, labeled Pre-treatment, was refrigerated. The second portion was adjusted to p H 6.0, submerged in a boiling water bath for 10 rain., and cooled. A f t e r the p H was adjusted to 4.5, any precipitate formed was removed by centrifugation. The supernatant solutlon, labeled Post-treatment, was refrigerated. The yeasts were grown in the two media for 3 hr., at which time all the lactose had disappeared and the maximum yield of yeast had been reached. Samples were again taken from the two propagators, and treated as described above. The analysis of the N components of the various samples is shown in Table 3. The initial heat- and acid-treatment of the whey removed approximately 53% of the whey organic nitrogen. D u r i n g the 3-hr. growth period all of the ammonia N added to both the deproteinated and regular whey was TABLE 3 Nitrogen disappearance in regular whey and heat- and acid-treate(l (deproteinated) whey media during growth of Saeeharoqnyees fragilis Deproteinated whey medium
Whey medium Growth time
Sample
Total N
NHJ N
247 178 104 38
149 128 9 4
(hr.) 0 3
Organic ~
Total N
NH~+ Organic N N
(rag. N/IO0 ~L) Pre-treatment :Post-treatment Pre-treatment Post-treatment
] 08 50 95 34:
180 176 37 36
130 131 4 3
50 45 33 33
1234
AARON
E. WASSERM:AN
assimilated by the yeast, and in both media approximately the same quantity of nitrogen (15 rag/100 ml.) disappeared from the ON. Since, in the deproteinated whey medium the precipitable N had already been removed, the nitrogen utilized by the yeast originated in the remaining noncoagulable portion. In the regular whey nledium, the ON utilized could have come from either the precipitable protein or the noneoagulable N component. The heat- and acidtreatment of the exhausted medium after 3 hr. of yeast growth revealed that the nitrogen used by the yeast in the whey medium also originated in the noucoagulabte organic nitrogen fraction. REFERENCES (1) K0c~, F. C., A~D MCMEEKI~, T. L. A New Direct Nesslerization Micro-Kjeldahl Method and a Modification of the Nessler-Folin Reagent for Ammonia. g. Am. Chem. Soc., 46: 2066. 1924. (2) LEVlTO~, A., AND WI~ITTIER, E. O. The Utilization of Whey in the Microbiological Synthesis of Riboflavin. J. Dairy Sci., 33:402. 1950. (3) MILLER, G. L., A~D MILLS,R, E. E. Determination of Nitrogen in Biological Materials. Anal. Chem., 20: 481. 1948. (4) O~J~A-JE~SE~, S., O T ~ , N. C., AN]) S~[oG-KJAnR, A. Die Stickstoffnahrung der Milchsi~urebakterien. Zentr. Bakteriol. II, 94:460. 1936. (5) WASSZRMAN, A. E., I-IOPKINS, W. J., ANI) PORC,ES, ~N. Whey Utilization: Growth Conditions for Saccharomyces fragilis. Sewage and Industrial Wastes, 30: 913. 1958. (6) WASSERMAN, A. E., HOPKINS, W. J., AND P0tCGES, N. Rapid Conversion of Whey to Yeast. Proc. X V t h Intern. Dairy Congr., 2:1241. 1959.
SUMMARY Saccharomyces fragilis utilized approximately 50% of the ammonia nitrogen, 25% of the organic nitrogen, and 25% of the total nitrogen in whey, and all of the added ammonia nitrogen. The available nitrogen was found to be in the organic fraction soluble after heat- and acid-precipitation of the whey proteins.
Whey contains approximately 0.14% nitrogen. The principal nitrogenous components are the heat- and acid-precipitable lactalbumin and lactoglobulin, whereas the nonprotein nitrogen fraction consists of proteose-peptones, amino acids, and other substances. Orla-Jenson et al. (4) reported that the noneoagulable N in heat- and acid-treated whey was not utilized for growth by various strains of lactic acid bacteria, and whey so treated was used as a base for the addition of nitrogen sources in a study of the metabolism of these organisms. These authors also showed that although the bacteria used casein as a source of N for growth, they were unable to use lactalbumin N. Leviton and Whittier (2), on the other hand, reported that the nonheat-eoagu]able N fraction of whey was utilized for growth and riboflavin production by Ashbyii gossypii. A study of the growth of Saccharomyces fragilis in whey showed that approximately 25% of the total whey nitrogen was used by the yeast (6). A further study, reported here, shows that the assimilable nitrogen was available only from the noneoagulable nitrogen fraction, and that the heat- and acid-precipitable proteins were not broken down by the actively growing yeast. 1VIATERIALS AND METI-IODS
S. fragilis, NRRL Yl109, was grown in a medium consisting of Cottage Cheese whey, 0.5% (NH4)eSO4, 0.5% K.~HP04, and 0.1% yeast extract. The whey pH was about 4.6, but following the addition of the salts was approximately 5.6, which was in the range for optimal yeast growth. The temperature was maintained at 32 ___ 1 °, and the 500-m]. quantities of media in the propagators were aerated at 2 liters/minute. Aliquots of the growing yeast suspensions were removed for the determination of the nitrogen fractions. The yeast was sedimented immediately by centrifugation and supernatants treated as described in the test. Total nitrogen (TN) was determined by micro-Kjeldahl distillation following digestion by the Miller and Miller modification (3) of the KochMcMeekin method (1). Ammonia nitrogen (AN) 1 was determined by microKjeldahl distillation, and the organic nitrogen (ON) fraction was calculated as the difference between TN and AN. Received for publication April 30, 1960. 1Ammonia N was determined by the addition of alkali to undigested samples. Traces of volatile protein degradation products also may be distilled over in this determination. 1231
12:32
AARON E. WASSERMAN
Deproteinated whey was prepared from whey clarified by passage through a medium sintered glass filter. The pH was adjusted to 6.0 with a few drops of concentrated NaOH, and the whey heated in a boiling water bath for 10 min. (500- and 1,000-ml. quantities of whey were heated in the autoclave at 121 0 for 10 min.). After cooling, the pH of the whey was adjusted to 4.5, and the precipitated protein removed by centrifugation.
Yeasts were grown in whey media in the presence or absence of (NH 4 hS04 as a source of inorganic nitrogen. The nitrogen composition of the media was determined after 4 hr. of growth. Results of the analyses are shown in Table 1. TABLE 1 Course of nitrogen utilization during growth of Saccharomyces [ragiUs in whey medium, with and without added ammonium sulfate Whey medium Minus (NH.).SO. Growth time
TotalN
(hr.)
o 1 2
3 4 Yeast yield
129 97 96 95 95
14 6 6 7 7 14.7 g. dry wtjliter
Organic N
TotalN
(mg. N /100 115 91 90 88 88
mZ.) 252 194 116 99 99
Plus (NH.)2S0. Organic NH.+N N 128 124 87 107 21 95 8 91 8 91 25.9 g. dry wtjliter
The total N of the medium without added (NH 4hS04 decreased to approximately 75% of its initial value within an hour after yeast growth began. Continued growth for several hours did not reduce further the nonutilizable N of the medium. When the yeasts were grown in the fully supplemented medium, the same pattern of N utilization was observed. The total N decreased to the same level as in the (NH4hS04-free medium, and was not consumed further in spite of continued yeast growth. These results had been observed previously (6). The total N was separated into the ammonia N and organic N components. The whey medium, in the absence of (NH 4)2S04' contains little AN, and the yeast used most of this nitrogen and about 25% of the ON fraction. The remainder of the ON was the nonutilizable residue. In the presence of added (NH 4 hS04, the yeast readily used the added AN, although some ON disappeared simultaneously. However, when the ammonia nitrogen had been consumed, the nonutilizable ON residue remained. The quantity of N in the whey available to the yeast was insufficient to support a good growth of yeast. In the presence of added nitrogen, the yeast yield obtained in 4 hr. of growth was almost twice as great as the yield in the absence of (NH 4hS04. Whey organic nitrogen is composed of two fractions: (1) heat- and acidprecipitable N, and (2) noncoagulable, or soluble, N. When the whey was
WHEY UTILIZATION.
1233
IV.
submitted to the heat and acid treatment described in Methods, 52% of the ON was noncoagulable (Table 2). T h e presence of the medium supplements during the heat and acid treatment did not significantly affect the q u a n t i t y of ON precipitated. However, it was noted that after the heat treatment, the p H of the whey alone remained at the initial value of 6.0, while the p i t of the whey medium changed to 5.3. TABLE 2 Effect of heat- and acid-deprotelnatlon on the nitrogenous components of whey alone and whey plus the medium salts Whey Treatment
Total N
NH, + N
)~'one Heat and acid
128 74
25 20
Whey medium Organic 29
Total N
NH,+ N
Organic N
I41 139
104 59
(rag. N/IO0 ,m~.) 103 54
245 198
Since the ON component of whey is composed of almost equal quantities of the noncoagulable and the heat- and acid-precipitable N fractions, it was of interest to determine which fraction was used b y the yeast. Therefore, the growth of yeast in deproteinated whey medium was compared with growth in the regular whey medium. To 500-ml. quantities of deproteinated whey and untreated whey were added the salts and yeast extract necessary to formulate the media. A f t e r inoculation with S. fragilis, an aliquot was removed from each propagator, clarified by centrifugation, and the supernatants divided into two portions. One portion, labeled Pre-treatment, was refrigerated. The second portion was adjusted to p H 6.0, submerged in a boiling water bath for 10 rain., and cooled. A f t e r the p H was adjusted to 4.5, any precipitate formed was removed by centrifugation. The supernatant solutlon, labeled Post-treatment, was refrigerated. The yeasts were grown in the two media for 3 hr., at which time all the lactose had disappeared and the maximum yield of yeast had been reached. Samples were again taken from the two propagators, and treated as described above. The analysis of the N components of the various samples is shown in Table 3. The initial heat- and acid-treatment of the whey removed approximately 53% of the whey organic nitrogen. D u r i n g the 3-hr. growth period all of the ammonia N added to both the deproteinated and regular whey was TABLE 3 Nitrogen disappearance in regular whey and heat- and acid-treate(l (deproteinated) whey media during growth of Saeeharoqnyees fragilis Deproteinated whey medium
Whey medium Growth time
Sample
Total N
NHJ N
247 178 104 38
149 128 9 4
(hr.) 0 3
Organic ~
Total N
NH~+ Organic N N
(rag. N/IO0 ~L) Pre-treatment :Post-treatment Pre-treatment Post-treatment
] 08 50 95 34:
180 176 37 36
130 131 4 3
50 45 33 33
1234
AARON
E. WASSERM:AN
assimilated by the yeast, and in both media approximately the same quantity of nitrogen (15 rag/100 ml.) disappeared from the ON. Since, in the deproteinated whey medium the precipitable N had already been removed, the nitrogen utilized by the yeast originated in the remaining noncoagulable portion. In the regular whey nledium, the ON utilized could have come from either the precipitable protein or the noneoagulable N component. The heat- and acidtreatment of the exhausted medium after 3 hr. of yeast growth revealed that the nitrogen used by the yeast in the whey medium also originated in the noucoagulabte organic nitrogen fraction. REFERENCES (1) K0c~, F. C., A~D MCMEEKI~, T. L. A New Direct Nesslerization Micro-Kjeldahl Method and a Modification of the Nessler-Folin Reagent for Ammonia. g. Am. Chem. Soc., 46: 2066. 1924. (2) LEVlTO~, A., AND WI~ITTIER, E. O. The Utilization of Whey in the Microbiological Synthesis of Riboflavin. J. Dairy Sci., 33:402. 1950. (3) MILLER, G. L., A~D MILLS,R, E. E. Determination of Nitrogen in Biological Materials. Anal. Chem., 20: 481. 1948. (4) O~J~A-JE~SE~, S., O T ~ , N. C., AN]) S~[oG-KJAnR, A. Die Stickstoffnahrung der Milchsi~urebakterien. Zentr. Bakteriol. II, 94:460. 1936. (5) WASSZRMAN, A. E., I-IOPKINS, W. J., ANI) PORC,ES, ~N. Whey Utilization: Growth Conditions for Saccharomyces fragilis. Sewage and Industrial Wastes, 30: 913. 1958. (6) WASSERMAN, A. E., HOPKINS, W. J., AND P0tCGES, N. Rapid Conversion of Whey to Yeast. Proc. X V t h Intern. Dairy Congr., 2:1241. 1959.