Mar.,
1920
T H E J O U R N A L O F I N D U S T R I A L A N D E N G I N E E R I N G CH E M I S T R Y
from compressed yeast. Half-normal sugar solutions were inverted a t about 30’ C. with varying amounts of this solution. The results are given in Table VII. TABLE VII-INVERSION CONSTANTS BY INVERTASE METHODOF HUDSON TIMEOF INVERSION FACTORS FOR VOLUME OF INVERTASE SOLUTION USED Hrs. 21 45 70
5 cc. 140.46 141.54 141.70
7.5 cc. 141.54 141.34 141.72
10 cc. 141.68 141.72
Time permitted only comparative tests by t h e invertase, Herzfeld and monochloroacetic methods on two samples of Cuba seconds and one of refinery barrel sirup. I n the invertase tests the solutions were carefully “deleaded” according t o directions by Browne.1 I n the other tests the minimum amount of basic lead acetate t o clarify was used. Table VI11 shows the comparative results by the three methods, making i t clear t h a t the monochloroacetic acid method gives results much closer to those obtained by the standard invertase method than does the Herzfeld method. A-Comparison
SAMPLE METHOD
TABLE VI11 of Double Polarization Methods rnn- __
centraInvert tion Polarization Per Direct of ( A l l p e a d - Fac- cent Polar- Solu- ingsin.N/2 tor of Suization tion Solution) Used crose
Cuban Second -14.10 141.70 88.24 Sugar No. 1438 Invertase 88.01 N Monochloroacetic 44.44 N / 2 -13.51 141 .OO 88.45 -14.38 142.66 88.63 Herzfeld 89.01 N Cuban Second -11.18 141.70 84.08 Sugar No. 1323 Invertase 88.40 N Monochloroacetic 44.00 N/2 -11.26 141.00 84.36 N , 7 1 2 . 1 6 142.66 85.04 Herzfeld 88.23 Barrel SiruD from Am-. Sug. Rfy. Invertase 34.63 N -6.65 141.70 36.35 Monochloroacetic 17.39 N/2 -6.46 141.00 36.41 Herzfeld 35.02 N -6.93 142.66 36.85 B-Percentage Difference between Per cent of Sucrose b y Three Methods Monochloroacetic Herzfeld Herzfeld minus minus minus SAMPLE Invertase Invertase Monochloroacetic 0.18 0.2 1 0.39 Cuban Second h’o. 1438 0.28 0.96 0.68 Cuban Second hlo. 1323 0.44 Barrel Sirup 0.06 0.50
The effect of the monochloroacetic acid method on commercial glucose readings was briefly investigated. Many analysts do not realize t h a t commercial glucose is not a well-defined chemical compound but may vary considerably in composition and physical characteristics. The average glucose of to-day is considerably lower converted than t h a t of a few years ago so t h a t t h e Ventzke reading of I 7 5 for the (sucrose) normal weight under standard conditions of polarizing is too low. I n fact, the sample of glucose used in t h e present investigation showed a Ventzke reading of I 77.8 j. TABLE IX-EFFECT O F MONOCHLOROACETIC ACID O N COMMERCIAL GI,UCOSE READINGSIN DOUBLE POLARIZATION METHOD Readings TOTAL Readings with 3 g. DIFFERMonochlorowith TEST MADE no Acid acetic Acid DIFFERENCE ENCE (b) 88.10 (a b) = 0.60 1 .89 Before heating ( a ) 88.70 (c)86.81 (b-6) = 1.27 .... 88.09 After heating (b) 88.05 ( a - b) = 0 . 4 4 1.99 ( a ) 88.49 Before heating (c)86.85 (b-C) 1.50 .... After heating 88.50 (b) 87.98 ( a - b ) = 0.54 1.85 Before heating ( a ) 88.52 (c) 86.67 (b- 6 ) = 1.31 . .. After heating 88.53
-
5
.
AVERAGE1.88
Table I X shows the polarization effect of monochloroacetic acid, as used in t h e double polarization method described, on an approximately I O per cent solution of this glucose.2 “Handbook of Sugar Analysis,” p. 276. a Weber and McPherson’s investigations.
(lags), 312, 320.
J. A m . Chem. SOC.,11
253
SUMMARY
The following double polarization method is suggested as a result of these investigations. Dissolve the normal weight of sample in a IOO cc. flask, clarify with an appropriate amount of lead acetate, make up t o volume and filter (the usual procedure for commercial polarizations). Transfer 5 0 cc. of filtrate to a I O O cc. flask, add ~j cc. of a 20 per cent solution of monochloroacetic acid, make up t o volume with water, and polarize within I j min. after adding the acid. To invert, transfer about j o cc. of the solution t o a 5 0 cc. flask, stopper tightly by tying down the cork, and immerse flask in boiling water, maintaining active ebullition for 30 min., or for 6 0 min. for lowgrade products clarified -with a large amount of lead acetate. Remove flask, and cool quickly to room temperature. Allow to stand a t least 2 hrs. and polarize in a zoo mm. tube with thermometer.
s = -2 ( a - b ) t x 141 - 2 S = Per cent Sucrose a = Direct reading
IO0
b = Invert reading = Temperature
t
All solutions should be made and polarized as nearly as possible at 20’. The advantages of this method are: I-Direct and invert readings are made on a solution of unchanged acidity and sugar concentration. 11-Excess of basic lead acetate, equivalent t o one cc. in a half (sugar) normal solution, does not affect the inversion or produce troublesome precipitates. 111-It gives more accurate results than the Herzfeld method. IV-Inverted solutions of low-grade products are lighter in color than those inverted by t h e Herzfeld method, and therefore easier t o polarize. V-No error is introduced by making up t o volume after inversion. The chief disadvantage seems t o be the time required, but this is less than t h a t required by the invertase or the more rational modifications of the Clerget and Herzfeld methods in which t h e inversion is carried out a t room temperature, which requires a t least 2 2 hrs. The actual time required in manipulation is little, if any, more than t h a t taken by the usual methods.
T H E QUANTITIES OF PRESERVATIVES NECESSARY TO INHIBIT AND PREVENT ALCOHOLIC FERMENTATION AND THE GROWTH OF MOLDS’j2 By Margaret C. Perry and George D. Beal LABORATORY O F ORGANIC ANALYSIS,UNIVERSITY OB ILLINOIS, URBANA,
ILLINOIS
The practice of preserving perishable foodstuffs for longer or shorter periods is not new. The application of heat and cold are old family methods which 1 Abstracted from thesis submitted in partial fulfillment of the requirements for the degree of Master of Arts in Chemistry in the Graduate School of the University of Illinois. 3 Read a t the 58th Meeting of the American Chemical Society, Philadelphia, Pa., September 2 t o 6, 1919
2 54
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y 1
1
1
PLATE 1 YEAST Amount presermtiva required fwr cem@lete stcrilizof,bn Amounf prererrohvc requlrcd fomhibit okhotli- fermmfetion
Vol.
12,
No. 3
For carefully controlled experiments nothing should be left t o chance; in this series of determinations we have used positive inoculation of sterile media with a pure culture of the specific organism. Hence, the results give a definite measure of the amount of the preservative necessary for absolute preservation and also for the inhibition of gas formation and the visible growth of mold. EXPERIMENTAL
have been elaborated upon and improved in the more modern methods of canning and cold storage. The drying of fish, fruits, and meats, either in the sun or by artificial heat, has been known and utilized for centuries; probably the use of salt is nearly, if not quite, as old. The use of sugar either with or without the addition of vinegar and various spices is but another old family contrivance. The application of creosote from the smoke of incompletely burned wood is thought t o have been discovered accidentally in an attempt t o use artificial heat in drying. All of these methods, or a t least variations of the same principles, are in use today, and in addition we now use certain manufactured chemicals in the preservation of our perishable food materials. The object of this study was t o determine the amount of various food preservatives required t o check alcoholic fermentation, t o inhibit the decomposition of foodstuffs by molds, and t o destroy all living organisms in standard one per cent dextrose broth. Conditions under which food is kept in the home permit of chance inoculation. Some foods, especially those which are used as condiments, offer a greater opportunity than others for chance inoculation. The work of Bachmann' and, t o a greater extent, t h a t of Hoffmann and Evans2 depends partly upon chance inoculation. This affords a probability of a mixed culture, also the possibility of losing some of the preservative by evaporation, especially in the case of volatile preservatives, as will be explained later. 1
1
THISJOWRNAL, 8 (1916), 620. I b i d . , 3 (1911), 835.
The food material used for this work was a 2 per cent dextrose br0th.l A t first we added t o I O cc. of this broth I O cc. of distilled water, minus the volume of the preservative, autoclaving a t I O lbs. for I O min. t o insure complete sterilization, and then adding t h e required amount of preservative. Later we found t h a t better results were obtained by sterilizing a known volume of this double concentration broth and adding sterile water and preservative t o double the original volume, thus giving a one per cent dextrose broth as a culture media. After the addition of the preservative, the tubes were allowed t o stand for a short time before inoculating with a pure culture of Saccharomyces cerevisiae,2 or the green mold, Penicillium glaucum.a The cultures were used from 3 t o j days after transplanting on dextrose agar slants. After inoculation the tubes were allowed t o stand a t room temperature for a period varying from 4 t o 8 days, according t o the time required t o produce visible growth in the check tubes, Determinations were made in duplicate and always accompanied b y duplicate checks, using I O cc. of the double concentration broth, adding I O cc. of sterile distilled water, and inoculating with the specific organism. The tubes showing positive gas formation were considered t o have undergone alcoholic fermentation, and in case of no gas formation, agar plates were made t o determine, if possible, the point a t which the organisms were killed. I n the same way plates were made from the tubes showing no visible growth of mold, These plates were allowed t o stand a t room temperature until the plates made from the check tubes showed a positive growth of the organism with which it had been inoculated. ABSOLUTE A L c 0 H o L - h the first set of determinations absolute alcohol was used as the preservative. When the tubes were covered with rubber caps, t o eliminate evaporation, the tubes inoculated with Sacc. cercvisiae showed gas formation up t o a concentration of 11 per cent alcoh01;~no living organisms were found a t 1 5 per cent. The tubes inoculated with P. g l a u c u m showed a visible growth up t o 8 per cent and no living organisms were found above 14 per cent. Where no attempt was made t o prevent evaporation, i. e., 1. 20 g. of dextrose, 6 g . of meat extract, and 1000 cc. of distilled water were heated on a water bath for 20 min., 10 g. of peptone were then added, the broth filtered, tubed, and autoclaved a t 10 lbs. for 10 min. 2 The culture of Sacc. cereuisiae was obtained by plating out from Fleischmann's yeast and isolating a pure culture on a dextrose agar slant. 8 The culture of P . glaucum was obtained by evaporating a known volume of a certain proprietary remedy to a thick sirup, making up to the original volume with distilled water, and allowing this t o stand until a growth of mold was obtained. The mold was then plated out and a pure culture isolated on a dextrose agar slant. 6 Per cent by volume.
Mar.,
1920
R-
m[]
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
tubes not capped, the results were not uniform and a higher concentration of alcohol was required: 16 per cent t o prevent gas formation and 2 0 per cent t o kill Sacc. cerevisiae; 8 per cent t o prevent visible growth of mold, and I 7 per cent t o completely destroy P . g l a u c u m . SODIUM SALICYLATE-Yeast tubes, t o which sodium salicylate‘ had been added, showed gas in one of the one per cent2 tubes, and a concentration of 9 per cent was necessary t o kill the organisms. Sodium salicylate seemed much more efficient as a preservative against molds, as a visible growth of mold was obtained up t o a concentration of 3 per cent, and no living organisms werefound above j per cent concentrations of the salt. S O D I U M BENZOATE-Dextrose broth preserved with sodium benzoate and inoculated with Sacc. cerevisiae showed no gas formation beyond 0.5 per cent, and no living organisms were found above 3 per cent. A j per cent solution of sodium benzoate was sufficient t o kill the P . g l a u c u m , and visible growth was absent in 0 . 2 j per cent. According t o Held,s only a small part of the benzoic acid is utilized as a preservative, as the greater part is bound by the protein of the media; if a stronger acid is used to satisfy this acid binding power of the protein, the effective disinfecting concentration of the benzoic acid is lessened. The disinfecting power seems t o be due wholly t o free benzoic acid, even when added in the form of salts, although the action is not due t o the mere acidity, i. e., the concentration of the hydrogen ion.4 S O D I T J N suLFITE-Yeast tubes containing 0.6 per cent sodium sulfite5showed no gas formation, and living organisms were found through 1 2 per cent; there was a visible growth of P . g l a u c u m through a concentration of 1 2 per cent; no higher concentrations were used. SODITJM ACID SULFITE-111 broth preserved with sodium acid sulfite no gas was formed and no visible growth was found even in a concentration of 0 . 2 5 per cent although living mold organisms disappeared only a t 6 per cent; and living organisms of Sacc. cerevisiae disappeared in tubes containing I O per cent sodium acid sulfite. FORMALDPHYDE-TUbeS to which formaldehyde6 had been added showed no gas formation and no living organisms a t concentrations as low as 0 . 2 5 per cent; no visible growth of P. g l a u c u m was found a t 0 . 2 5 per cent although living organisms were found t o persist up t o 0.4 per cent. CONCLUSIONS
Under temperature conditions a t which foodstuffs 1 Percentage purity of sodium salicylate and of sodium benzoate was determined by dissolving a known weight of the salt in distilled water, adding an excess of sulfuric acid, igniting at a low red heat in a platinum crucible, and weighing the sodium sulfate formed. * Per cent by weight. 3 Arch. H y g . , 84 (1915), 289; Chem. Abs., 10 (1916), 2358. 4 Marshall, “Microbiology,” 1912, 408. 5 Percentage purity of sodium sulffite and acid sulfite was determined according to the method outlined by Treadwell-Hall, “Analytical Chemistry,” 3rd Ed., 2, 692. 6 Concentration of formaldehyde determined by oxidation with iodine in alkaline solution, Romijn’s method, as outlined in Sherman’s “Organic Analysis,” 2nd Ed., 40.
PLATE 2 MOLD c1 hcomp/etc Amwnt arenrrative steriiiration required
2.55
Amount prrsor vative rtquired
--
t o rupprers v;slbCgrow+h
are kept the greater part of the time (room temperature) and under other conditions most favorable t o the growth of the specific organisms, i. e., the use of media best fitted t o the organism in question, sterilization of media, and inoculation with a pure culture, we obtained the following results which are expressed in both graphic (Plates I and 2 ) and tabular form: TABLE 1-CONCENTRATIONS NECESSARY TO INHIBITAND PREVENT GAS FORMATION AND THE GROWTH OF MOLDS Penicillium glaucum Saccharomyces cerevisiae N o LIVING GAS N o LIVING GAS FORMATION ORGANISMSFORMATIONORGANISMS Per cent Per cent PRESERVATIVE Per cent Per cent 8 17 20 Alcohol without cap 16 I 14 Alcohol: with cap 11 15 3 5 Sodium salicylate 1 9 None in 0 . 2 5 5 0.5 3 Sodium benzoate 12Y0 not 12% not 12% not Sodium sulfite 0.6 sufficient sufficient suacient 6 None in 0 . 2 5 Sodium acid sulfite None in-0.25 10 0.4 None in 0.25 None in 0 . 2 5 None in 0 . 2 5 Formaldehyde I
{
1
t
Formaldehyde and sodium acid sulfite exerted the same inhibitive effect toward Sacc. cerevisiae and were the most efficient; next in order were sodium benzoate, sodium sulfite, sodium salicylate, and the least efficient of all, alcohol. For absolute sterilization this order did not hold, the most efficient was formaldehyde, then sodium benzoate, sodium salicylate, sodium acid sulfite, alcohol, and sodium sulfite. There was no visible growth of P . g l a u c u m in concentrations as low as 0 . 2 5 per cent of sodium acid sulfite, formaldehyde, or sodium benzoate; 3 per cent of sodium salicylate or 8 per cent alcohol were required; 1 2 per cent of sodium sulfite were not sufficient t o inhibit growth. Practically the same order held for complete sterilization: formaldehyde, sodium benzoate, sodium salicylate, sodium acid sulfite, alcohol, and sodium sulfite.
