INDUSTRI4L I S D ESGINEEHING CHEMISTRY
1032
T 4BLE I.
ITA AN IN
PRODUCT
.\SI\I~LS
F r e s h , Howard Supreme strawberries F r e s h Klondike strawberries Frozenstraaberries n i t h sugar; 2 parts berries t o 1 of sugar Strawberry ice cream, containing 30% frozen-pack berrier a s above Frozen strawberries; no added sugar S e g a t i v e control
c L4ss.4Y O F
3 3 3 3 2 2 2 4 4 3 3 3 2
FRESH A S D
DAILY DOS~GE Grams 2
4 2 4 3 6 9 10 15 9
3 4 0
FROZES STRAWBERRIES ASD
AT. G.us I N T~EIGHT Grams 300 28i 337
299 345 269 - 1.’ 320 222 425 285 270 -109
original antiscorbutic potency. I n spite of the beating in of air and agitation by the dasher of the freezer, the low temperature must exert a marked protective action on the vitamin C. In this laboratory similar treatment a t higher temperatures n i t h tomatoes and cranberries has caused marked losses of vitamin C. Attention is called to the depressing effect on growth of excessively large doses of either strawberries or strawberry ice cream. This effect has been repeatedly noted in other vitamin C assays and may be due to the disturbing effect on the digestive and nervous systems and to the unbalancing of the diet. I n spite of poor or even negative weight gains, no scurvy was found on autopsy in any animal in this expeiiment .
STRAM BERRY
.iv. S ~ R T I T A L SCURVY PERIOD S C O R E Dnus 70 0 io 0 72 0 72 0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 90 0 33
13
1-01, 2 5 . No. 9
ICECRE.LX1
Berries available only 70 d a y s Berries available only i 2 days
.......... . . . One animal died a t end of 41 day:: no w u r v y One animal died a t end of 26 d a y s ; i i o scurvy One animal sick during last 2 s-eeks of period ; no scurvy Severe s c u r v
ACKNOWLEDGMENT The Klondike frozen strawberries were packed, frozeii, and stored by the Birdseye Laborat,ories, Gloucester, 1\Ia.:,s. IJITERATURE C I T E D
(11 Kohnian, E. F.,Eddy, W. H., and IIalliday, S . . ISD. ESG. HEM., 20, 202-4 (1928). ( 2 ) Scheunert, .i., ” D e r Titamingehalt der d e u t s c h e i i Xiahriings-
mittel. I. Obst und Gzmuse,” Springer, 19L’!). (3) Sherman, H. C.‘., La Mer, V. K., and Campbell. H . L., J . -1m. (‘hem. SOC., 44, 165-72 (1922). (4) Smith, C. -I., Rergheim, O., a n d Hawk, P. B.. 1’i.o~. .Sue. E r p t l . id. .\led., 19,228 (1921). RECEIVED F e b r u a r y 17, 1933. Presented before t h e Division of Agricultural and Food Chemistry a t t h e 85th Meeting of t h e American Chemical Society, K a s h i n g t o n , D. C . , March 26 t o 31, 1933.
Yeast-Growth Stimulants in White Sugars 13. 13. HALL,LAWREKCE €1. JAMES.AXD L. s. STUART Bureau of Chemistry and Soils, Washington, D. C.
T
HE presence of stimulating substances in sugar and
other materials is highly important in fundamental studies of yeast metabolism, in commercial fermentation processes, and in many other instances in which the growth of such microorganisms is a vital factor. I n the commercial manufacture of beverages difficulty has arisen owing to the development of yeast precipitates or clouding in bottled goods during storage. This type of spoilage has frequently been attributed to sugars, and a n attempt to determine the extent and probable importance of nonsugar growth-stimulating substances has been made.
SALTSAS POSSIBLE STIMULANTS Most syithetic media selected for investigations of thib nature contain sucrose as a basic nutrient. The possibility of salts (carried in the sugars as contaminants) acting as growthpromoting substances was considered, and an attempt was made to detect their presence by using the sugars in a synthetic medium which had been deprived of one or another of its necessary ions. I n the early part of this investigation each sugar mas added to Fulmer, Selson, and Sherwood’s medium F ( 2 ) from which first one and then another of the recommended salts had been eliminated. This medium F contains ammonium chloride, calcium chloride, dipotassium phosphate, calcium carbonate (precipitated), dextrin, and sucrose. The first test was to determine whether the phosphorus was adequate. Medium F was prepared in 100-cc. quantities with and without added phos-
pliater, and the different sugars were added. (I’otaAuiii n-as provided in the form of potassium carbonate.) The media. were sterilized and inoculated with 0.1 cc. of a water suspension of yeast cells from a 24-hour wort agar slant. (The yeast used in this study mas isolated from a beverage in which a sufficient crop had developed to cause a visible sediment. This culture was well suited to this study because of its apparent demand for the particular growth-promoting substance under investigation. It has been identified as a variety of Saccharomyces cerecisiae.) From three to ten thousand yeast cells were inoculated into each flask. The cells had been centrifuged and washed repeatedly. After 48 hours of incubation, total numbers of viable organisms were determined in wort agar plates, The results are shown in Table I. TaRLE
I. I-EAST GROWTHS OBTAINEDIN WITH
S U G . ~ R WITH SAMPLE A D D E D POa 1 4,820,000 2 2,120,000 3 7,840,000
.om
WITHOUT
\$’ITHOUT ~ D D E DPOI
2,640,000 900,000 630,000
&IEDICJI
PHOSPHATES
sUG,iR
SAMPLE 4 5 6
Tt-ITH .\DDED Po1
i,OOO,OOO 476,000 544.000
F
!\-ITHOCT .\DDED POL
33,000 7,000 3,000
I t is evident that the addition of phosphates to the modified medium (i. e., inediurn F complete) increased the growth with every sugar tested. This indicated that, in general, t h e sugars were not supplying sufficient amounts of this salt, although the amount of growth in sample l indicated that that particular sugar furnished sufficient phosphates for marked yeast development.
September, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
With '10 added phosphates, some growth took place, varying with difierent sugars. While a t first this might indicate that the sugars were supplying definite quantities of phosphates to the medium, it must be borne in mind that in a medium in which no phosphates were present some yeast development might be expected on the first subculture from a medium as rich in nutrients as wort agar. In general, however, it can be concluded that these sugars did not carry sufficient phosphates to be significant. I n a similar manner the same sugars were tested for their ability to provide the nitrogen necessary for growth. These results are shown in Table 11. OBTAINED IN MEDIUM F TABLE11. YEASTGROWTHS WITH AND WITHOUT NITROGEN WITH SUQAR SAMPLE ADDEDN 1 7,840,000 2 4,820,000 3 2,120,000
WITHOUT
ADDEDN 4,300,000 2,460,000 540,000
SEQAR WITH SAMPLEADDEDN 4 476,000 5 544,000 6 7,000,000
WITHOUT ADDEDN 220,000 15,000 13,000
These results indicate that some of the sugars contained ample nitrogen to support abundant yeast growth and others did not, suggesting that the stimulants were nitrogen compounds. However, these sugars were also under study in the Carbohydrate Division of this bureau, where exhaustive chemical analyses were being made. By a comparison between the analytical data available there and the results of the fermentation tests, it appeared that there was no direct correlation between the amounts of nitrogen present and the stimulating abilities of the sugars. ABILITYO F SUGARS
TO
SUPPORT
YEaST
GROWTH
It was observed, however, that the yeast used in this study grew in sterilized, 10 per cent solutions of many of the sugars without the addition of nutrient salts. As this solution offered a ready means of studying the abilities of different sugars to support yeast growth, it was used in place of medium F in the remainder of this investigation. The solutions were made up in 100-cc. quantities and sterilized in 300-cc. Erlenmeyer flasks a t 15 pounds for 20 minutes. With this new medium, slight modifications in inoculations and cell counting were adopted: All flasks were seeded with an inoculum prepared by transferring a portion of a 24-hour wort agar growth to about 100 cc. of sterile
distilled water. After vigorous shaking the number of cells per cubic centimeter of the water sus ension was determined by direct microscopical counting, w i t l a double-ruled Neubauer hemacytometer (eight hundred squares were always counted). (The yeast used in the above experiments was particularly well suited t o counting by this method, since any clusters formed were readily broken up by shaking.) The strength of the inoculum usually was then adjusted so that each cubic centimeter contained between three and five million cells. One cubic centimeter of this inoculum was added to the 100 cc. of sugar solution in each flask, producing an inoculation which led to moderate growth if nutrients were present. Both the inoculum and subsequent crops were readily counted on the Neubauer slide without dilution. The inoculated solutions were incubated at 30' C. for 72 hours and then counted. The multiplication of yeast cells was used as an arbitrary measure of the quantity of nutrients present,. A series of sugars was tested in 10 per cent solutions by the technic described above. Some did not support yeast growth, while others produced comparatively large yeast crops. Results typical of this part of the investigation are given in Table 111, in which the sugars are arranged in the order of ascending yeast counts. For convenience the number of cells per cubic centimeter of the original sugar solution immediately after inoculation is recorded as one unit, and the total crop after growth as a multiple of this number.
TABLE111.
SAMPLE
a
1053
CELLS SOLUTIOXS
?*lULTIPLICATIOS O F PE.4ST
PER CENT
SUGhR
MULTIPLEO F COUXT hFTER INOCUL.4TlON
SAMPLE
IN
10
MULTIPLE OF COUNTAFTER
INOCELATION
1 indicates no increase in numbers.
One hundred seventeen samples of white sugars have been studied in this way, the samples being collected during a period of three years. Marked differences in ability to support yeast growth were evident, both between sugars collected in the same year and between those collected in different years. Larger yeast crops than those recorded in Table I11 have been noted, especially during the earlier part of this investigation. The lesser stimulation in the more recent samples may be due to improvement in quality, or to changes in the growth and metabolism of the yeasts after they have been maintained on laboratory media for some time.
STUDY OF STIMULANTS Since it was found that some sugars did not support growth, while others produced excessive crops, attention was directed to a study of the stimulants. Several sugars, some of which promoted yeast growth and some not, were extracted with alcohol in a manner similar to that described by Devereux and Tanner ( I ) , which was designed for the removal of yeast growth-promoting substances from plant materials. The sugars were dissolved in hot distilled water until saturated solutions resulted. After the solutions had cooled, the saturated sirups were removed, and to them sufficient 95 per cent ethyl alcohol was added to result in an alcoholic concentration of 80 per cent. The sugar crystallized out from these solutions. The crystallization was usually complete within 96 hours. The alcoholic liquor was then removed from the crystallized sugar by decantation and reserved for future studies. The sugar was dried, powdered, and bottled as alcohol-purified sucrose. The growth-promoting abilities of the alcohol-purified sugars were then compared with those of the unpurified sugars in 10 per cent solutions. The same method of testing was used as previously outlined. The results given in Table IV show the influence of alcohol purification in reducing the total yeast crops. TABLEIV. YEASTCROPSOBTAINED WITH SUGARS USEDBEFORE AND AFTER PURIFICATION MULTIPLEOF YEAST IXOCULUM~
Unpurified SAMPLE suear
1 2 3 4 5 6
1 1 3 11.3 22.6 24.0
a Inocula strength,
Purified suear
1 1
1 6.6 3.0 9.6
MULTIPLE OF YEAST IXOCULUM~
Unpurified SAMPLE sugar I
7 8 9 10 11 12
27.0 31.0 34.5 44 59 87.8
Purified
suear 9.5 10.0 1.3 3.0 12.0 1.0
1.
The reductions in yeast crops obtained by alcohol purification of the sugars are similar to those obtained by Funk and Freedman (3) and suggest that the growth-promoting factors may be related to the more commonly known growth stimulants from plant materials. However, since minute quantities of certain salts may show stimulative effects upon cell growth, it appeared advisable to test their influence on yeast development. The ash from a sugar which showed marked growth stimulation was added to 10 per cent solutions of a nonstimulating sugar. Counts on the yeast crops obtained showed that the salts in the ash had exerted no stimulative influence. This, with the information gained previously, led to the conclusion that the
10.5.2.
I N D U S T 1l I A 1, A N D E N G I N I$ E I( 1 N G C 1%E M I S 'T H Y
stimulating substances contained nitrogen, were organic, and were soluble in 80 per cent alcoliol. Further studies on the relation of the growth-proniating factors to known growth stimulants of plant oridn, on the possible importance of carbons, fitter aids, etc., as~wellas the importance of such stimulants to bacteriological fermenbation tests, arc now under invcstigation. ACKNOWLEDGMENT
A11 of the sugar samples used in this investigation wcre furnislied by the Carbohydrate Division of this bureau. The
Voi. 25, No. 9
authors appreciate the advice a.nd asvistalice given by H. S. Paine, J. A. Ambler, aiid ot.her members of the Carbohydrate Division. I>lTEllATURE
Clren
(1) Ucuareux. E. I>,, and T1~imer.F.C., 1.Bacl., 14, 317-33 (1927). (2) Fulrnor. E. 1.. Nelson. V. 13,. arid Sherrrood, F. F..J . d m . Ckem. Soe.. 43,186--91 (1921). ( 3 ) Funk. C., and Freedmun. 1... 1.B i d . ('hem., 56, X51.-80(1923). ~ t r c r ~ February vs~ 1 1 . 1933
Beating Properties of Egg White W. C. HENRYAND A. D. BAHBOUR, Ontario Research Fouridat,ion, T ~ r o i i l .Ontario, ~, Canada
1
K TUE course of an irivestigation the use of beaten egg white in a commercial candy product, it \\-as necessary to deterlninet~le effect of various conditions on volume of the heat.cii white and tiicrelativestabilityof the foam. There is litt.le iiiforniation of a q u a n t i t a t i v e character to he found in the literature and no
Mdhods huce been devised for studying lhe iricrease in c&me of egg while on beating, and the stabilily of the beaten product. The efects of rate and time of beating, temperalure, hydrogen-ion concentration., and additions of water and oil have been determined, and also the ~mmparative resulls obtairiuble ruilli ,fresh and defrosled storage white.
foam for tile lrieasurelnell~ of St. Jolin F ] (6)~ used ~ amethocl ,.olumeor foam which consisted in beating the egg white with a hand beater llntilan apparentmaximum volume was obtained, drying tho foam in an oven at 60" C. to a solid condition, breaking it into sniall pieces, and measuring the volumc approximately in a graduated cylinder. They obtained better results with the thin than with the thick white, and better results with storage than
bfwnions AND d r r a i t ~ ~ u s It was felt that ll,ucll more couid be obtained with a m e c h a n i c a l beater with speed control, in place Of the hand beater by the preceding workers. The a p p a ' a t n s used i n a l l the folloving determinations was a Sunheani Mixmaster k i t c h e n beater, manufacturett by the Chicago Flexible Shaft Company. It, was provided with a rheost.at permitting its use at four different speeds. The increasc in volume on beating the egg white was determined as follows: smd1 glass cylinders, upm at both eads, of inside dimensions shout 17 X 50 mm., were used for the determinations. The volume of these cyiinders was carefully measured, and the weight of liquid egg white which they X-ould contain was caleulrtted, assuming t,he specificgravity of the white t,o be 1.035. To determine the volume increase oi the egg white after heating, one of these tubes was pushed into the heaten ~ R B S unt,il eomplet,ely 6llui, care bein taken to exclude large sir hubbles and to leave no part, of t i e tube unfilled. The excess w m removed from the ends of the tube by scraping off with a
FIGURE 1. APPARATUS fresh eggs. They also found that contamination of the wliitcs with a trace of yolk has a detrimental efect on the beating qualities, a fact which has long been known in corniriercial practice. Peter and Bell (J), using a penetrometer method, compared tlie stability of foanls made by beating solutions of dried whey protein and of dried egg albumin. In the case of tlie ~vheyprotein they obtained increased stability by small additions of calcium hydroxide and subsequent neutralization; by successivesmall additions of acid or alkali during whipping; and hy the addition of tannic acid, saponin, sodium sulfite, or sodium disulfite. KO attempt was made by these workers to measure the beaten volume. So far as can be learned from their published results, none uf the abovc investigators attempted to determine the most satisfactory time of beating, speed of agitation, or temperature during heating, and to standardize their procedure accordingly. d i i
s
600
$
8
TOO
zoo
% ffINUTE5
TOTAL
2
nfl€Of
4
6
BLr/lT/NG
Fx~une 2. COXPAHATWEVOLUME INCKEI~E or F i i ~ s eEGGWHITEA N D DEFROSTED %OHAGE EGG W H m :
