INDUSTRIAL AND ENGINEERING CHEMISTRY
1118
LITERATURE CITED SOC.(London), 123d,276
Burch, Proc. R o y . (1929). Bur. Mines, Tech. P a p e r 525 (1932). Carothers. J . Am. Chem. Soc.. 52. 5279 (1930). Fischer, Brennstoff-Chem,,1, 35 (1920) ; Ges. Abhandl. Kenntnzs Kohle, 5, 55 (1920). Fischer, Peters, and Cremer, Brennstof-Chem., 13,364 (1932). Fischer and Schrader, Ibid., 1, 22 (1920). Gillet, Proc. 3rd Intern. Conf. B i t u m i n o u s Coal 2, 1 (1931). Juettner and Howard, Carnegie Inst. Tech., Coal Research Lab. Contribution 8 (1934). Parks and Huffman, "Free Energy of Organic Compounds," p. 105, Chemical Catalog Co., New York, 1932. Stadnikoff, Proc. 3rd Intern. Conf. B i t u m i n o u s Coal 1, 674 (1931).
Swelling in Canned Chopped Hams L. B. JENSEN, I. H. WOOD,AND C. E. JAXSEN Swift & C o m p a n y Research Laboratories, Chicago, Ill.
F
OR a number of years the authors have observed that
certain test lots of canned spiced hams and luncheon meats (mixed cure), when placed in the standard incubator (99" F. or 37.2" C.), have often shown swells within 48 hours. Meats of these lots showing quick swelling have been prepared so that bacterial counts of composite samples show less than one hundred bacteria per gram. On the other hand, cans from trimmings purposely seeded with 5 to 10 million bacteria (many species) and cured, canned, and cooked, have remained for 30 t o 90 days under 37.2" C. incubation without signs of gas production in the cans. Ordinary plant lots of nitrate-nitrite-sugar cured spiced hams of every brand and size in the industry show, under incubation, keeping times ranging from 24 hours to 90 days. None of the brands is uniform in keeping time from, for example, month to month. * The purpose of this report is to indicate the research that has been done in this laboratory on these phenomena and t o incite more cooperative work along these lines.
BACTERIOLOGY The writers have studied especially the fermentative characters of the many bacteria isolat'ed from cooked and raw spiced ham. Of the hundreds of strains isolated from chopped hams, none1 fermented any sugar or carbohydrate (standard sugar broth) with gas formation. Where, then, does the gas in swelled cans originate? Analysis of the gas usually shows a predominance of carbon dioxide :
coz
%
75 to 97 0.1 to 5.8 N2, methane, 0 2 , etc. 1.4 to 20.0° 5 Mean of one hundred analysea.
Hn
I n this laboratory quantitative bacteriological determinations of kinds of microbes in incubated spiced ham and juice always showed the majority of species to be gram-positive, aerobic sporing rods. However, in none of the twenty test 1
Escherichia. Alrobacter, and Proleus groupa are rarely present.
Vol. 26, No. 10
(11) Washburn, B u r . Standards J . Research,
2,476 (1929). (12) Wheeler and Burgess, J. Chem. Soc., 99, 649 (1911); 105, 181 (1914); Fuel, 5, 65 (1926); Jones and Wheeler, J . Chem. Soc., 105, 140, 2562 (1914); 107, 1318 (1916); Tideswell and Wheeler, Ibid., 115, 619 (1919); Holroyd and Wheeler, Ibid.. 1928, 2669, 3197; 1929, 633; Fuel, 9, 40, 76, 104 (1930); Pictet and Ramseyer, Ber., 44,2486 (1911); Pictet and Bouvier, Compt. rend., 157, 779, 1436 (1913): Ber., 46, 3342 (1913), 48, 926 (1915); Chem-Ztg., 38, 1025 (1914); Pictet, Kaiser, and LabouchPre, Compt. rend., 165, 113 (1917); Pictet, Ann. chim., [9] 10, 249 (1918); Taylor and Porter, Bur. Mines, Tech. P a p e r 140 (1916). RECEIVED July 2, 1934. Presented before the Division of Gaa and Fuel Chemistry a t the 88th Meeting of the American Chemical Society, Cleveland, Ohio, September 10 t o 14,1934.
A4icroorganisms belonging to the genus Bacillus are responsible f o r the formation of gas in nitrate-nitrite-sugar cured chopped hams. The bacilli will nol form carbon dioxide in ordinary carbohydrate media. Unless nitrate, sugar, and cured meat are present together, the bacilli will not ferment sugar with gas production. A medium is described to estimate these bacteria quantitatively so that they m a y be removed from the processing environment. The presence of these bacilli in canned spiced ham and the speed with which they f o r m carbon dioxide when incubated should not be taken as a n index of the sanitary quality of the food. Incubation tests should be supplanted by direct microbiological examinations of aseptically opened cans of chopped ham. sugar broths, inoculated with several varieties of these bacteria belonging to the genus Bacillus, did gas formation occur. The substrate used for growing these bacilli was a spiced ham medium prepared as follows : Ordinary culture tubes are approximately half filled with finely chopped or ground spiced ham (taken from a sound can) and then wetted down with a solution made of 0.4 per cent sodium nitrate and 2 per cent cane sugar (sucrose). The finished tube after autoclaving under 1 kg. per sq. cm. (15 pounds per square inch) pressure for 15 minutes should appear as follows: (a) No trapped bubbles of air should appear in the medium. ( b ) A quarter of an inch (0.635 om.) of liquid should cover the top of the
ground meat. .4 layer of fat from the spiced ham should effect a seal t o prevent (c) evaporation on storage. ( d ) The medium including meat and liquid should not fill the tube over half full.
To inoculate the medium, Pasteur pipets or small 1-cc. pipets are used, introducing inocula from bottom of medium to surface. No bubbles of trapped air should remain in the tube after inoculating. The medium is incubated at 37.2" and 48.9" C. (99' and 120' F.), More rapid gas production sets in when these bacilli (responsible for swells) are incubated at 48.9" C. In practice the inoculated tubes are incubated at 37.2' and 48.9" C. for 48 hours.
Gas production is indicated by the number of trapped bubbles in the medium, and rotating the tube helps to bring out these bubbles. Chemical analysis of the gas shows about 95 per cent carbon dioxide, 1 per cent hydrogen, and the remainder oxygen, methane, etc. (mean of one hundred analyses), Fermentation of the sugar is responsible for the
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 ST R Y
October, 1934
formation of carbon dioxide. [The sugar (sucrose) disappears. The Sorensen formaldehyde titration indicates that the protein is not attacked during fermentation which leads to swells in sealed cans and gas bubbles in the medium.] If cultures are carried out on nutrient agar or brain veal agar, juices from fermented swollen cans, fermented spiced ham, or the liquid from gassy meat tubes just described, after 24-hour incubation a t 37.2’ C. practically pure cultures of luxuriantly growing members of the genus Bacillus are observed. Some colonies cover the surface of the agar completely; all are usually large and mucoid for the first 24 hours of growth, but not necessarily so. These bacteria alone are responsible for gas formation in cans of mixed-cure chopped hams incubated at 37.2” to 48.9” C. There is no question as to the classification of the bacilli with the genus Bacillus, the type species being the “hay bacillus.” (The hundred strains described in this report were avirulent and nontoxigenic when g r o w 72 hours in veal infusion broth and 1.0 cc. of whole broth culture injected intraperitoneally into white mice.) Table I shows the approximate position of these bacilli according to Bergey’s criteria (1).
1119
MEASUREMENTS OF ELECTRICAL CHARGESOK BACILLISTRAINS
THE
The cataphoretic velocity was determined by the method of Jensen and co-workers ( 2 ) . The surface electrical negative charges on one hundred broth cultures (washed three times in conductivity water) measured in conductivity water were as follows (potential difference, 120 volts) : No.
NO.O F
CULTURES
1 10 46
CHARQE
p/sec. 4 2 . 0 (circa)
37.0 31.0
OF
CULTURES
CHARGEI dsec.
39 2 2
26 0 22 0 16.0
These data indicate, as do the fermentation data, that the group of one hundred strains is not to be considered homogenous but to be thought of as having many characteristics in common.
BACILLIAS SOLE CAUSEOF SWELLS Examination of one thousand cans of spiced ham and luncheon meat (of all manufacturers’ brands) coming out of incubation as swells have shown these bacilli to be present. Inoculation of pooled broth cultures of these bacilli into TABLE I. BASICCULTURAL CHARACTER? twenty-five cans standing up under 90-day incubation has (All are aerobic and motile; no indole is formed; all liquefy ge!atin. all digest resulted in sm-elling these cans in 24 hours. Twenty-five casein, no H?S is formed, all are usually gram positive and sporing rods) incubated cans so treated with mixed cultures of micrococci NITRATES THIMP. COLOR did not swell. A test lot of trimmings from hams handled RE- DEXMILE FOR ON TCBES DCCED TROSE STARCH in aseptic manner, cured, and sugared in dust-proof jars, GROWTH AGAR c. and stuffed directly into 3- and 6-pound cans without adding Coagulation E . tumescens 37 White spices or wrapping in paper, processed according to the peptonization Acid slims Acid schedule of this company, showed no gas-forming bacilli ( 3 0 Yellow- Peptonization B . cohaerens 1 3 5 1 ish present. These twenty-four cans did not swell after 90-day ( 40 slimy Peptonization .4cid incubation. Ten cans of this batch of practically sterile B . megatherium ( 3 5 Br%?”ham, spiced and wrapped in paper, were processed and then slimy Acid coamlaAcid incubated a t 37.2’ C. All ten cans swelled in 4 days. tion peptoB . ruminatus The spices added to chopped hams are usually coriander nization Acid coagula+ Acid i= 100 strains of spiced 30 White and white pepper. Examination of the spice mixture showed tion peptoham organism slimy five million spores of the gas formers per gram. Every nization batch of spice tested here was badly contaminated with These strains from the chopped hams may be distinguished spores of carbon-dioxide-forming bacilli. Examinations of from some of the other species by their ability to reduce the paper used by all manufacturers to wrap hams before canning showed the presence of spores of these bacilli in nitrates. To show the biochemical characters of the gas formers, every square inch of paper. Thus, if the meat was sterile, the action of one hundred bacillary strains was studied on the spices and paper would make for poor “keep-tests,”-i. e., dextrose, levulose, maltose, galactose, salicin, arabinose, incubation tests. The problem of eliminating these bacteria rhamnose, xylose, inositol, raffinose, lactose, mannitol, sor- from paper and spices lies in the province of the manubitol, dulcitol, sucrose, mannose, trehalose, dextrin, and facturers concerned. About one hundred cans of bacillus-free spiced ham have glycerol. Of these nineteen test substances, dulcitol, glycerol, and inositol were not attacked by any of the one hundred been inoculated with various races of yeast, torulae, microstrains. Fourteen fermentation groups were observed. KO cocci, gram-negative lactose fermenters, Clostridia, and gas was produced in any sugar broth or sugar agar medium. Pseudomonas isolated from swelled cans of spiced ham. I n While this classification is not of great practical importance, no instance has a snrell at 37.2” C. incubation for 90 days in a mixed-cure product with these inocula been obtained. the data are listed here as a matter of record (Table 11). When the mixed-cure meat is free from members of the genus Bacillus, cans of spiced ham or luncheon meat will not TABLE 11. FERMENTATION FORMULA swell under 37.2” or 48.9’ C. incubation. (All fermented dextrose, levulose, salicin, mannitol, mannose, trehalose, dextrin; = acid production)
+
No. OF
CWL- MAL-GALACARAUI-R a . 4 ~ -X u - RAFFI. LAC- SORBI- Sn-
GROUPT C R E S TOSE TOSE I 2 0 + + I1 I + + 111 l o + + IV
V
VI VI1 VI11 IX X XI XI1 XI11
XIV
5 4 4
1 3 1 1
1
I
+
++
13 2
+ + + +
+ + +
. 6
5 + +
+
-
+
+ + +
-
+
NOSE
+
-
+
+
+ + + +
-
NOSE
+
+ + -
+
+ + -
+
+
+
-
-
-
-
-
+
LOSE
+ + + -
+
-
-
-
+
NO0E
-
-
+ + + +
-
+ -
-
-
TOSE
--
TOL
++-- +- + ++- -++ +- -- -- --
CROSE
+++ + +++ + +++ ++
MECHANISM OF CARBON DIOXIDEFORMATION Nitrate, sugar, and cured meat must be present together before the bacilli will ferment m-ith gas production. If the set-up is made with c. P. nitrite without nitrate, no carbon dioxide gas forms in this system. If the sugar is omitted, no carbon dioxide gas, or very little, is formed. If cured meat is omitted and nitrate, sugar, and any soluble proteinpeptone materials are present, no carbon dioxide gas forms. Some preliminary experiments, using in the test tube a medium of sugar, nitrate, and extracted nitrosohemochromogen or nitrosohemoglobin (the cured “cooked” and “raw”
I N D U S T R I A L A ND E N G I N E E R I N G C H E M I S T R Y
1120
pigments, respectively), indicate that carbon dioxide gas may be formed by the bacilli under these circumstances. It is common knowledge that omission of nitrate in curing chopped hams will result in finished canned products which do not show the usual fermentative carbon dioxide swells. Upon incubation a t 37.2' C. the reduction potential of such a product meets the requirements for optimum conditions for growth of Clostridia and other anaerobic forms. This type of spoilage, not often rewlting in swells, impresses everyone with the need for sufficient nitrate in the cure. Kitrate, according to a new concept of potentials, is an oxidizing agent ( 3 ) . It is an easily proved fact that nitratenitrite-salt-sugar cure favors most races oi aerobic bacteria, whereas nitrite-salt-sugar cure always inhibits fermentation and aids in putrefaction. CRITERIA
SAXITART QU.4LITY OF CANXED SPICEDHAMS As shown in this work, incubation of cans a t 37.2" and 48.9" C. affords no certain data for judging the sanitary qualities of this food. Cans of meat prepared from practically sterile trimmings but spiced with contaminated spice or wrapped with contaminated paper will swell when incubated a t 37.2" and 48.9' C. Trimmings showing five to ten per gram of the bacilli OF
Vol. 26, No. 10
described and a total count (of all viable microorganisms) of one hundred per gram will give a canned product that swells after several days of incubation, whereas trimmings showing no ('carbon dioxide gas bacilli," but having an initial count of ten million bacteria per gram may give a product that will not swell in 90 days a t 37.2" C. Hence incubation tests of these products should be supplanted by direct bacteriological examinations of the meat from aseptically opened cans. Since we have a medium to detect the bacilli responsible for gas production, obviously we can find them and eradicate them from the environment of processing. With trimmings free from these bacteria, sterilized spices, and microbially clean wrapping paper, a spiced ham may be produced with nitrate-nitrite-sugar cure that will not swell at 37.2' and 48.9" c. LITERATURE CITED (1) Bergey. Manual of Determinative Bacteriology, 4th ed., Williams 8;: Wilkins Co , Baltimore, 1934. ' 2 ) Jensen,L. B . , e t a]., J. Bact., 15, 367-450 (1928); Collected Papers Mayo Chnzc, 1930. 898-900; .J. Inf~ctiousDiseases, 52, 167-
85 (1933). 13) Stephenson, Marjory, "Bacterial Metabolism," p. 74, Longmans, Green &- Co., New York, 1930.
RECEIVBDJune 8 , 1934
Lubricating Oils from Ethylene R. G. ATKINSON AND H. H. STORCH, U. S.Bureau of Mines Experiment Station, Pittsburgh, Pa.
T
KE a m o u n t of olefins analysis in an Orsat apparatus The experiments described below indicate p r e s e n t i n commercial (in volume per cent): that it should be possible to produce a good gases m a y exceed two Oxygen 0.39 "light" lubricant f r o m the lower members of Nitrogen 1.60 billion cubic meters per year. Saturatea 1.00 the olefin series by a two-stage process, the first Ethylene 97.11 In addition, an enormous supply slep being a thermal polymerization to a liquid of olefins is obtainable by dehyFigure 1 is a flow sheet of d r o g e n a t i n g paraffinic gases boiling in the gasoline range. The material t h e a p p a r a t u s in which the (6,7). I n a review of the literathermal polymerization was acproduced in this manner can then he polyture on olefin polymerization, complished. merized by aluminum chloride to a viscous liquid Egloff, S c h a a d , a n d L o w r y It consists of a cylindrical suitable f o r a lubricant. (3) have estimated the potenalloy-steel bomb 1.35 liters in While there are not enough data available tial production of olefins from volume, copper- pl a t ed inside. natural gas, cracking still gas, Three tubes enter through conto enable one to predict anything about the nections on the head of the bomb. waste gas from natural gasoline commercial applicability of the process, it is One serves as a thermocouple plants, and other refinery gases well, one t o conduct the incoming thought that further investigations should prove to amount to f o u r t e e n billion gas to the b o t t o m of the bomb, of value. cubic meters in one year. Findand t h e t h i r d t o remove t h e polymerized gas and vapors from ing a means of more profitable udization of olefinic gases in large quantities is of much the top of the bomb. The outlet tube leads t o a coil cooled with iced water, immediately under which is a trap to catch the concern to the industrial chemist. condensed liquid. The pressure is reduced at the outlet of this Lubricating oils obtained by polymerizing ethylene with trap. The wet gas from the trap is led through another trap aluminum chloride have been investigated by Nash, Stanley, chilled with carbon dioxide snow and then through a column and Bowen (4), and also by Sullivan, Voorhees, Neeley, and packed with activated charcoal. The autoclave was heated to 371" C. and maintained at this ShankIand (9), who reported these Iubricants to have a very temperature throughout the experiment. Ethylene was let inferior viscosity-temperature coefficient. The latter authors into the system rapidly until the pressure built up t o 70.6 kg. have shown that the viscosity index (1) of an oil produced by per sq. cm.; then the valve controlling the outlet was cracked, polymerization increases proportionally to the length of the and a constant rate of flow through the gas meter was maintained. After 60 minutes the flow of gas was stopped, and all straight carbon-atom chain in the starting material. Hence liquid products obtained were collected. it was thought advisable to investigate the possibility of obThe data for seven experiments are listed in Table I. taining a good lubricant by first polymerizing ethylene thermally to liquid of low molecular weight and then treating Column 7 gives the volume per cent of the residual gas this material with aluminum chloride to yield a viscous liquid. absorbed by 1.6-gravity sulfuric acid. The absorbable material was thought to be largely propene. PRODUCTION OF LIQUIDBY THERMAL POLYMERIZATION It was observed that, when the reaction temperature was is, Commercial-grade ethylene was used throughout the in- 410" C. or higher, the ethylene usually "flashed"-that vestigation. A 587-cc. sample of the gas gave the following reacted very rapidly as evidenced by a sudden rise in pressure
