Feed Yeast from Dairy By-Products1

F E E D Y E A ST FROM DAIRY BY-PRODUCTS 1 N A N D O R PORGES, J A N E T B. P E P I N S K Y

AND L E N O R E J A S E W I C Z

Eastern Regional Research Laboratory,2 Philadelphia 18, Pennsylvania

The possibility of producing feed yeast from dairy by-products and wastes was suggested from a previous study on the biochemical oxidation of dilute dairy waste. In that investigation Saccharomyces fragilis removed the lactose and casein, leaving in the effluent only a small amount of the original organic matter (10). As shown by Whittier (20) and Webb and Whittier (18), the use of whey in various fermentations is not novel, but its practicability depends upon economic considerations and the availability of suitable organisms. Various yeasts ferment lactose to alcohol (1, 11, 19), and details of such a process using Torula cremoris have been published (13). Good yields of whey yeast have been obtained with S. fragilis (2) ; in Germany, whey yeast plants operating in conjunction with dairies use Torula species (5). Yeasts, that grow on dairy products are higher in protein a n d amino nitrogen than other yeasts; they also are high in ascorbic acid, thiamin and riboflavin (14). Riboflavin apparently is synthesized by yeasts that utilize lactose (12). Thus, the propagation of yeasts upon whey, skimmilk and dairy wastes may offer a means of obtaining an enriched feed supplement from soluble dairy products and at the same time reduce the pollution load. PROCEDURES

Powdered skimmilk and powdered whey 5f the average composition shown in table I were dissolved in distilled water to the desired concentrations. All propagations were conducted at 30 ° C. Cultures used as inocula were cultivated for 65 hr. in Roux flasks containing 150 ml. of the indicated medium prior to use as seed. Filtered air was passed through the larger propagators at the rate of one volume of air per volume of fermenting solution per minute. Determinations were made for lactose (16), protein (7), chemical oxygen deman~l as a measure of organic matter (9), dried centrifuged solids and direct yeast count. Contamination was controlled by addition of 1 mh of concentrated nitric acid for every 2 1. of solution in the fermentor when contamination was noticed. Such treatment, which does not seem to interfere with the growth of yeasts, is used for controlling infection at the Wiirttemberg whey yeast plant (5). EXPERIlYIENTAL

Comparison of yeasts. A number of yeast cultures 3 grown on 0.1 per cent Received for publication Jan. 12, 1951. 1 Report of a study made under the Research and Marketing Act and presented a t the l l 8 t h Meeting, American Chemical Society, Chicago, Ill., Sept. 3-8, 1950. 2 One of the laboratories of the Bureau of Agri~fltural and Industrial Chemistry, Agricultural :Research Administration, U. S. D. A. s The yeasts were obtained through the courtesy of the Fermentations Division. N o r t h e r n l~egional Research Laboratory, Peoria, Ill. 615

616

NANDOR PORGES ET AL

s k i m m i l k solutions were used as inoeula. Twelve a n d a h a l f ml. of a c u l t u r e were used to seed 250 ml. sterile 0.1 p e r cent s k i m m i l k c o n t a i n e d in a 500-ml. gas washing bottle e q u i p p e d w i t h a f r i t t e 4 glass disc. A f t e r the solutions were a e r a t e d f o r 48 hr. at a r a t e of 125 ml. a i r p e r vessel p e r minute, d e t e r m i n a t i o n s were made to o b t a i n the d a t a p r e s e n t e d in table 2. These are the results o b t a i n e d with a selected few of the 20 yeasts examined. TABLE 1 Composition of powdered milk products used

Skimmilk

Whey

50.5 36.9 8.1 4.5 95.5 87.4

76.5 12.5 6.0 4.9 95.0 89.0

Lactose (%) Protein (%) Ash (%) Moisture (%) Total solids (%) Organic solids (%)

S. f r a g i l i s , N R R L 1109, gave the highest y i e l d of solids. I t removed large a m o u n t s of lactose a n d p r o t e i n f r o m the solution. A f t e r the solution was centrifuged, a s u p e r n a t a n t solution low in organic m a t t e r , as m e a s u r e d in terms of chemical oxygen d e m a n d (C.O.D.), remained. C a n d i d a l y p o l y t i c a a n d T. u t i l i s u t i l i z e d only small a m o u n t s of these solubles a n d the solids y i e l d s were low. The other organisms m a y find a p p l i c a t i o n , b u t S. f r a g i l i s was selected f o r f u r t h e r s t u d y a n d T. c r e m o r i s was used l a t e r f o r some c o m p a r a t i v e tests. The percentage of the o r i g i n a l C.O.D. f o u n d in the solids (table 2) a p p r o x i m a t e d the percentage of t o t a l solids recovered as c e n t r i f u g e d solids. I t is i n t e r e s t i n g to note t h a t S.

TABLE 2 Avtlon o/ yeasts on 0.1% skimm41k solution after gg-hr, aerationa

Organism

S. Z. Z. T. C. T.

fragilis ................................. laetis ...................................... casei ....................................... cremoris .............................. lypolytica ........................... utilis .......................................

NRRL strain

1109 1114 1564 1131 1094 900

Lactose used

Total nitrogen removed

C.O.D. supernarant

(%) 79 86 85 59 7

(%) 78 75 71 57 22

(%b) 27 25 26 34 82

(%b) 50 46 34 46 6

(%¢) 48 45 39 43 15

9

95

5

5

0

C . O . D . Solids solids recovered

a Solution had 547 ppm. lactose, 370 ppm. protein and 1,117 ppm. C.O.D. b Based on the original chemical oxygen demand of the skimmilk. e Based on total solids originally in the skimmilk solution. f r a g i l i s d i s s i p a t e d only a b o u t 23 p e r cent of the o r i g i n a l C.O.D., possibly as C02. Higher milk concentrations and ammonium sulfate additions. A d d i t i o n of

11.25 p p m . of n i t r o g e n to a 0.1 p e r cent skimmilk solution i n o c u l a t e d with S. I n c r e a s i n g the milk solids as shown in table 3 r e q u i r e d a d d i t i o n a l amounts of (NH4)2S04 for' more complete u t i l i z a t i o n of the lactose a n d f o r m a t i o n of removable solids. I n the

f r a g i l i s r e s u l t e d in good removal of organic m a t t e r (10).

617

FEED YEAST

24-hr. period, the number of yeast cells increased from 2.9, 7.3 and 14.5 million for the respective milk concentrations to the numbers given in the table. Although skimmilk contains nitrogen, it is a p p a r e n t that a more readily available source is required for increased activity. Batch production. Using the f e r m e n t o r described by Humfield (4), 12-1. fermentations were made on a 2.5 per cent skimmilk solution and on a 2.5 per cent whey solution. (NH4)2S04 equal to 281 ppm. nitrogen was added. One 1. of culture grown on the skimmilk solution was used for the former, and the same volume of culture grown on the whey solution was used for the latter. The yeast counts in the f e r m e n t o r at the start were 9.7 million per milliliter for the milk and 3.9 million for the whey, reaching 525 million and 460 million at the end of the fermentations 13 and 16 hr. later, respectively. These values are comparable to those obtained with S. cerevisiae on grain wort (15). Sugar was utilized as rapidly as it was by T. utilis on peanut protein waste water, when 700 ppm. TABLE 3

Action of S. fragilis grown for 24 hr. on s~immil~ of different concentrations w~th added nitrogen (The % values are based on the amount of that constituent present at the start) Skimmilk solids

I~itrogen added as (NH4) 2SO,

Lactose used

Total nitrogen removed

Solids recovered

C.O.D. supernatant

Cells/

(g./1.)

(ppm.)

(%)

(%)

(%)

(% )

(millions)

(fold)

10 10 10

0. 11.3 113.

30 49 98

26 37 88

19 24 50

50 26 11

49 62 123

17 21 42

25 25 25

0. 11.3 282.

39 46 98

49 60 88

13 33 42

49 32 13

100 122 169

13 16 22

50 50 50

0. 11.3 563.

49 63 99

24 48 83

11 15 46

56 51 9

194 250 290

12 16 20

ml.

Increase in yeast

sugar were consumed in 5 hr. (6). The solids, recovered with a Sharples centrifuge, showed a yield of 46 per cent for skimmilk and 20 per cent for whey. Apparently, some of the protein in the skimmilk was precipitated at the acidity of the fermentation liquor (about p H 4). Continuous propagation on skimmilk and whey. A 2-1. f e r m e n t o r (3) t h a t had been employed successfully in yeast production studies on f r u i t juices (17) was used in the following series of experiments. The starter was p r e p a r e d in the fermentor, by aerating gently for 40 hr. the culture contents of one Roux flask and the indicated quantities of media. The feed to the fermentor was added from a calibrated funnel at such a rate as to keep the sugar in the fermenting solution below 10 rag. per milliliter. Antifoam (corn oil and lard) was added when necessary. The p H was maintained between 4 and 5 by the addition of Ntt4OH. F i g u r e 1 shows the course of a fermentation in a solution containing 50 g. of skimmilk and 2.65 g. of (NH~)2S04 per liter (530 ppm. nitrogen). The starting volume was 450 ml. containing 130 million yeast cells per milliliter, or a total

618

NANDOR PORGES ET AL

of 58 × 109. A t the end of the fermentation, 16 hr. later, the direct cell count was 514 million per milliliter, which for the 2-1. volume was a total of 1,028 × 109, a 17-fold increase. The p H gradually dropped to 4.2 at the completion of the run. The yield of dried recovered solids, when corrected for absorbed antifoam, was 45 per cent, based on the total milk solids. A Kjeldahl determination showed a protein content of 67 per dent, indicating a portion probably was precipitated casein. W h e n the centrifuged solids were treated with N a O H to phenolphthalein pink, about one-half went into solution, indicating precipitated protein. 2.0

I

~-/ ~x

I

I

0')

- 1.5 . "

/

I 0

I

-75

I

4 @ 12 PROPAGATION. HOU RS Continuous p r o p a g a t i o n of S. fragilis on skimmilk.

FIo. 1. plus 2.65 g. (NH4)2SO 4 per liter.

I00

,

o 16 Feed contained 50 g. solids

A r u n made on a solution containing 50 g. of whey and 5.3 g. of (NH4)2S04 per liter (1,060 ppm. nitrogen) proceeded readily. I n this case, the starter had a volume of 750 ml. and a yeast count of 500 million per ml. I n 13 hr. when the r u n was completed, the yeast count was 633 million per ml. The total number of yeast cells had increased to 1,265 × 109 from the initial 381 × 109 cells. The dried solids showed a corrected recovery of 24 per cent, calculated to the original solids present. The protein content was 57 per cent, and treatment with N a O H showed the absence of soluble protein. Foaming. Foaming was a major difficulty and necessitated constant surveillance during a propagation. Reference has been made to this same difficulty in the German whey plants (5). The low yields obtained in the present trials may have been caused by the means taken to control foaming. At times, the rates of air flow and agitation were markedly reduced, and the odor of alcohol was dis-

FEED

619

YEAST

tinct. The a n t i f o a m agents used were not satisfactory. The silicones worked well with a 0.1 per cent skimmilk solution but not in higher solids concentration. Combinations of silicone and p e t r o l a t u m p r o v e d ineffective when used in small amounts in the batch fermentations. Mixtures of corn oil a n d l a r d were not successful in the continuous fermentations because of the large amounts required. The removal of the foaming p r o p e r t i e s b y coagulation was unsuccessful in the the following experiments. Continuous propagation on clarified whey. W h e y was clarified b y r u n n i n g steam into a 20-1. bottle charged with 1,000 g. of whey a n d 10 1. of water. Steaming was continued for 20 min. a f t e r the t e m p e r a t u r e reached 99 to 100 ° C. A f t e r cooling and a d j u s t i n g the whey solution to the 20-1. volume, the p r e c i p i t a t e d proteins were removed by filtering t h r o u g h p a p e r pulp. F o r each 40 g. of lactose TABLE 4

PropagationofS. fragilisonc~rifiedwhey Hour

Yeast count × lO~/ml.

0 1 2 4 5 6 7 9 10 11 12 13 14

42 47 44 45 56 47 48 44 48 49 52 48 54

Lactose

Propagation volume

(g./loo ml.)

(mz.)

3.63 1.99 2.60 2.16 1.56 1.40 1.22 .96 .68 .92 .79 .92 .44

200 240 330 380 490 580 645 730 810 1045 1195 1305 1505

in this solution, 1.2 g. ( N H 4 ) 2 H P 0 4 , 3.2 g. (NH4)2S04, a n d 1.55 ml. 28 p e r cent ~NHa (2) were-added. The NH3 was a d d e d a f t e r the solution was Sterilized in liter flasks p r i o r to use. The cultured contents of three R o u x flasks were aerated overnight. The yeasts were collected aseptically in centrifuge bottles, and the s u p e r n a t a n t was replaced with 200 ml. of fresh medium. This served as the s t a r t i n g volume, which was increased b y additions to a final volume of 1,500 ml. Tables 4 a n d 5 show data obtained on r u n s in which S. fragilis 1109 and T. cremoris 1131 were used. S. fragilis increased about 10-fold a n d T. cremoris about 17-fold; however, t h e latter f o r m e d m u c h smaller cells. Again, as shown earlier, S. fragilis utilized more sugar. Clarification of the whey did not seem to decrease its f o a m i n g properties. The r a p i d C.O.D. test showed t h a t the yield calculated on the s u g a r utilized was 40 p e r cent, but based on the s u g a r available the yields were 35 p e r cent a n d 29 per cent f o r S. fragilis a n d T. cremoris, respectively.

620

NANDOR BORGES ET AL

DISCUSSION The use of yeast for the disposal of dilute dairy waste by fermentation does not seem feasible. The considerable amount of oxygen-demanding substances that remains after the separation of solids still has a high percentage of the original pollution load. Further, fermentations of this type would require pure culture techniques with the necessity of pretreating the dilute wastes. On the other hand, the propagation of desirable yeasts on skimmilk and whey offers a means of converting the soluble lactose and proteins to insoluble proteinaceous material. The water-soluble vitamins found in the by-products are not lost but are recovered in the enriched final product (8). Vigorous aeration poses a problem when milk products with their high foam-forming ability are used as nutrients for the yeast. Improved antifoam substances or fermentation e q u i p m e n t of the W a l d h o f t y p e (5) will be r e q u i r e d f o r efficient y e a s t product i o n . F o a m i n g is r e d u c e d o n l y s l i g h t l y b y b o i l i n g w h e y a n d r e m o v i n g the eoagulum. TABLE 5 Propagation of T. cremoris on clarified whey Hour 0 2 3 5 6 8 9 10 11 12 13 14 15

Yeast count × 107/ml. 54

56

86 83 84 68 93 110 96 87 88 91 122

Lactose

Propagation volume

(g.liO0 ~nl.) 3.40 2.41 1.90 1.22 1.80 1.46 1.28 1.26 1.64 1.41 1.39 1.37 1.19

(~nl.) 200 385 410 410 620 800 800 875 1060 1270 1400 1500 1500

T h e selected yeast, S. fragilis, g r e w r e a d i l y o n u n c l a r i f i e d a n d clarified whey a n d skimmilk. T h e r e c o v e r e d m a t e r i a l was h i g h i n p r o t e i n . I t h a d o n l y negligible a m o u n t s of B12, 2-6~, p e r cent. ~ Y e a s t y i e l d s v a r y i n g f r o m 24 to 45 p e r c e n t were o b t a i n e d f r o m 5 p e r cent s o l u t i o n s of w h e y a n d s k i m m i l k , w i t h a b o u t 40 p e r cent c o n v e r s i o n of the u t i l i z e d s u g a r i n a clarified whey. T h e possibility is s u g g e s t e d of p r o d u c i n g a n e n r i c h e d p r o t e i n p r o d u c t b y such f e r m e n t a t i o n s . I~EFERENCES (1) BROW~E,It. H. Ethyl Alcohol from Fermentation of Lactose in Whey. Ind. Eng. Chem. News ed., 19: 1272, 1276. 1941. (2) EI~EBO,L.~ LUNDIN, I-I., AND :~¢~YRB.~CK,K. Yeast from Whey. Svenk Kemisk Tidskrift, 53: 96-108, 137-147. 1941. (3) FEUSTEL,I. C., AND HUMFELD, H. A New Laboratory ~ermentor for Yeast Production Investigations. J. Bact., 52: 229-235. 1946. 4 Vitamin B12 tests were made through the courtesy of J. C. Lewis, Biochemical Div. Western Regional l~esearch Laboratory, Albany, Cal.

FEED YEAST

621

(4) HUI~IFELD,H. An Improved Laboratory Scale Fermentor for Submerged Culture Investigations. J. Bact., 54: 689-696. 1947. (5) KIEFER, F. Some Details Concerning the Production and Utilization of Yeast in Germany for Nutritional and Chemical Purposes. FIAT, Final report no. 1274, (PB 97924). April 30, 1948. (6) KLATT, T. J., BARKER, E. D., POMES, A. F., AND PORGES, N. Food Yeast Growth on Peanut Protein Waste Liquors. Oil and Soap, 22: 319-321. 1945. (7) KOCH, F. C., AND MCMEEKIN, T . L . A New Direct Nesslerization Micro-Kjeldahl Method and a Modification of the Nessler-Folin Reagent for Ammonia. J. Am. Chem. Soc., 46: 2066-2069. 1924. (8) MYERS, R. P , AND WEISBERG, M. Treatment of Milk Products. U. S. Patent 2,128,845. Aug. 30, 1938. (9) POROES, N., PEPINSKY~ J. B., HENDLER~ N. C.~ AND HOOVER, S . R . Biochemical Oxidation of Dairy Waste. I. Methods of Study. Sewage and Ind. Wastes, 22: 318-325. 1950. (10) PORGES, N., PEPINSKY, ¢]-. B., HENDLER, N. C., AND HOOVER, S . R . Biochemical Oxidation of Dairy Wastes. I I . Comparative Study of Yeasts. Sewage and Ind. Wastes, 22: 888-892. 1950. (11) ROOOSA,M. Mechanism of the Fermentation of Lactose by Yeasts. J. Biol. Chem., 175: 413-423. 1948. (12) ROGOSA, M. Synthesis of Riboflavin by Lactose-fermenting Yeasts. J. Bact., 45: 459460. 1943. (13) ROGOSA,M., BROWNE, H. H., AND WHITTIER, E. 0. Ethyl Alcohol from Whey. J. Dairy Sei., 30: 263-269. 1947. (14) SPRIN~ER, 1~. Whey Yeast. Lebensm. Untersuch. u. Forch., 89: 353-363. 1949. (15) STARK, W. H., KOLACH0V, P. J., AND WILLKIE, H. F. Some Factors Affecting Yeast Propagation. Proc. Am. Soc. Brew. Chem., Fourth Ann. Meet., 49-56. 1941. (16) STILES, tI. R., PETERSON, W. H., AN]) FRED, E. B. A Rapid Method for the Determination of Sugar in Bacterial Cultures. J. Bact., 12: 427-439. 1926. (17) STUBRS, J. J., NOBT.E, W. M., AND LEWIS, J . C . F r u i t Juices Yield Food Yeast. Food Ind., 16: 694-696, 751. 1944. (18) WEEB, B. H., AND WHITTIEE, E . O . The Utilization of Whey. A Review. J. Dairy Sci., 31: 139-164. 1948. (19) WILHARI~, G., AND SACK, U. Ober die Leistunger eininger milchzuckerverg~irender Helen. Die Milchwissensehaft~ 2: 382-389. 1947. (20) WmTTIER, E. O. Lactose and its Utilization. A Review. J. Dairy Sci., 27: 505-537. 1944.