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464
A comparison of Figure 3 with data from recent literature is presented in Table 111. I n addition, molecular weights were calculated from the following equation developed by Fenske, McCluer, and Cannon ( 3 ) : mol. wt. = 240
+
Saybolt viscosity at 100” E’. 28.0 305 - viscosity index
32,310 Iogio
(7.4)
for all samples shown in Tables I, 11,and I11 which fall within the recommended range for this equation (molecular weights of 300 to 425). Although it is not possible to calculate the respective probable accuracies of the two methods for predicting molecular weights, an inspection of the comparisons as presented indicates that Figure 3 covers a wider spread of both viscosities and viscosity slope factors with less probability for large errors. A very rough estimate of the probable error of values obtained from Figure 3 is about * 3 per cent. An attempt was made to correlate all of these molecular weight data with the viscosity a t one temperature and the specific gravity, substituting a viscosity-gravity function for the viscosity slope factor. Although a general trend could be shown, the viscosity-gravity function (as measured by Watson’s characterization factor, 11) was not sufficiently interchangeable with the viscosity slope factor to permit formulation of any definite relation. It was concluded that the slope of the viscosity-temperature curve was a more desirable variable for use in correlation of the molecular weights of petroleum fractions.
Conclusions 1. Molecular weights of petroleum fractions from two crude sources were related directly to the viscosities of the
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fractions a t 100’ F., a separate curve being obtained for each crude. 2. The differences shown by these two curves were used as a basis for estimation of the effect of the viscosity-temperature function on molecular weight. The final correlation was presented in a form to permit prediction of the molecular weight from viscosities a t 100’ and 210” F. 3. Molecular weight data from the literature were compared with this correlation, and it is concluded that the molecular weight of any petroleum fraction can be predicted with a probable error of *3 per cent. The actual error depends largely on the accuracy with which viscosities can be determined, but it appears that, if reasonable care is exercised, the predicted molecular weights will be well within the usual accuracy of engineering calculations.
Literature Cited (1) Bell, T. G., and Sharpe, L. H., Oil Gas J . , 32, No. 13,13 (1933). (2) Davis, G . H. B., and McAllister, E. N., IND.ENQ.C R ~ M 22, ., 1326 (1930). (3) Fenske, M. R., McCluer, W. B., and Cannon, M. R., Ibid., 26, 976 (1934). (4) FitzSimons and Thiele, Ibid., Anal. Ed., 7,11 (1935). ( 5 ) Gullick, N. J., J . Inst. Petroleum Tech., 17,541 (1931). (6) Huffman, H.M., Parks, G. S., and Daniels, A. C., J . Am. Chem. SOC.,52, 1547 (1930). (7) Huffman, H. M., Parks,G. S., andThomas, S. B., Ibid., 52, 1032 (1930). (8) Rotinjam, L., and Nagornow, N., 2.physik. Chem., A169, 2031 (1934). (9) Schottky, “Thermodynamik,” pp. 339-42 (1929). (10) Steed, A. H., J.Inst. Pekoleum Tech., 16,799 (1930).
(11) Watson, K.M., Nelson, E. F., and Murphy, G. B., IND.ENO. CHEM.,27, 1460 (1935).
RECEIVED October 8, 1936. Presented before the Division of PetroIeum Chemistry at the 92nd Meeting of the American Chemical Society, Pittaburgh, Pa., September 7 to 11, 1936.
CONTROL OF ROPE IN BREAD CHARLES HOFFMAN, T. ROBERT SCHWEITZER, AND GASTON DALBY Ward Baking Company, New York, N. Y.
HE development of rope in bread has been for many years one of the most troublesome problems of the baker. The disease develops in various types of homemade as well as commercial bread. Lloyd and McCrea (la), Cohn e t al. (5), and Bunzell and Forbes (3), among others, studied rope in the h i s h e d loaf. The work of Laurent (IO), Vogel ( I S ) , Fuhrmann (S), and Lloyd et al. (11) showed that several strains of spore-forming bacilli were responsible for the disease. These organisms have come to be regarded generally as members of the B. mesentericus group. The spores of these bacilli are very resistant to heat and thus are able to survive baking temperatures and subsequently grow in the bread during hot and humid weather. Although rope has been definitely proved to be the result of the growth of bacterial spores, the sources of infection have never been clearly shown. Watkins (20) contributed a method of inoculating sterile bread slabs with a boiled flour mixture containing the suspected rope organisms. Lloyd e t al. ( 1 1 ) and Brahm (8) developed plating methods which
T
Other ingredients than flour, particularly yeast, malt, and malted products are the chief sources of rope in this country. Rope can be avoided by the selection of ingredients through careful laboratory control. Ingredients of unknown origin and characteristics should be subjected to careful laboratory examination before being used in bread making. were not suitable, however, for materials containing solid particles. Voitkevich (19)developed a method using loaves of bread for the test media. Little was accomplished through use of these methods, and no one has demonstrated scientifically all of the important sources of the rope organism. Russell (16) in 1898 suspected yeast but had no evidence showing the degree of infection of the yeast or other ingredients. Russell’s important observations appear to have been ignored by early investigators. Flour was blamed, and the literature freely referred to this ingredient as the source of the disease. European investigators were in agreement with this viewpoint. Watkins (20) regarded flour as the only material responsible for the appearance of rope in bread. So
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strong was this belief that the baking industry appears to have taken for granted that rope was largely due to infected flour. A change of flour did not always appear t o eliminate the trouble, and some investigators therefore felt that unsanitary bakeshop conditions were contributing and aggravating causes of rope infection. Amos and Kent-Jones (1) studied the rope spore content of flour, using as a basis for their technic the unpublished method of the present authors for the quantitative estimation of rope spores in flour. This method had been privately communicated to Kent-Jones in 1928. The general acceptance of the viewpoint that rope infections were to a large extent unavoidable and that rope organisms were normally present in flour led to extensive studies of the most effcient methods of delaying or stopping the growth of the bacilli in bread. Neumann and Knischewsky (i4), Neumann et al. (i5), Zeckendorf (22), Henderson (Q), Williams (M), Cohn and Henderson (4),Morison and Collatz (IS),and Fisher and Halton (7) showed that a definite relation existed between the acidity of the bread and the growth of the rope bacteria. Watkins (20) studied the use of lactic acid as a preventative of the disease. In a more recent paper Fisher (6)discussed the use of vinegar and acid phosphate as remedies for rope in bread. Stuchlik (i7)found that rope bacilli will not grow in a medium having a greater acidity than a p H of 4.6. The present tendency of bakers to increase the richness of their bread by the use of larger quantities of milk solids is favorable to the growth of the rope organism. Other factors remaining the same, the pH of the bread becomes higher as the quantity of milk solids is increased: % ' Skimmed Milk Solids (Based on Flour)
pH of Bread
3.0 6.0 9.0 12.0
5.37 5.58 5.68 5.70
Milk acts as a buffer in the dough, resists development of acidity, and provides greater fermentation tolerance. The effectiveness of vinegar and other acid materials is decreased as the percentage of milk solids in bread is increased. Experience in this laboratory with various procedures indicated that the estimation of the bacterial spores by a dilution method offered the most promising approach. The rope bacilli formed a pellicle or surface growth on standard bouillon, a cultural characteristic which served to differentiate between rope organisms and most of the other types of spore-forming bacteria. The enumeration by this procedure will include not only the various strains of rope spores but also the spores of such bacteria as have the ability to produce a pellicle or surface growth in nutrient brdth. I n the early stages of this work each bouillon tube that developed a pellicle was examined microscopically, and sterile pieces of moist bread in test tubes were inoculated with the sporeforming culture, incubated, and observed. Since the pellicleforming spores in only rare instances proved to be other than rope spores, the completed test with sterile bread was omitted. This simplified the procedure and also saved time which is of importance in control work. I n its simplified form the method here described is actually a presumptive quantitative test for rope spores, and the results obtained are designaked as the "rope spore counts."
Dilution and Inoculation Method Weigh two grams of a representative sample of the material to be examined under as nearly aseptic conditions as possible, and transfer to a 250-ml. glass-stoppered bottle. Add approximately 10 grams of sea sand. Measure 96 ml. of distilled water in a 100-cc. graduate and pour into the dilution
465
bottle. This provides a dilution of 1 to 48 and is used to determine spores up to two hundred per gram. Shake the 1-48 dilution vigorously B t y times; each shake should be an up-and-down excursion of about one foot. Immediately after the sand has settled, pipet 4.8 ml. of the 1-48 dilution into another 250-ml. glass-stoppered bottle containing 95.2 ml. of distilled water. This provides a dilution of 1 to 1000 and is used to determine the presence of spores in numbers between 250 and 20,000 per gram. Pipet the following amounts of the 1-48 dilution into test tubes containing sterile broth: M1.
Dilution
M1.
Dilution
MI.
Dilution
4.8 2.4 1.6
1-10 1-20 1-30
1.2 0.8 0.6
1-40 1-60 1-80
0.48 0.32 0.24
1-100 1-150 1-200
Shake the 1-1000 dilution twenty-five times as previously described. Prepare a third dilution of 1 to 100,000 by pipetting 1 ml. of the 1-1000 dilution into 99 ml. of water. Use this dilution for determining spores when present in numbers between 25,000 and 100,000. Pipet the following amounts of the 1-1000 dilution into test tubes containing nutrient broth: M1.
Dilution
M1.
Dilution
4.0 2.0 1.0 0.4
1-250 1-500 1-1,000 1-2,500
0.2 0 1 0.05
1-5,000 1-10 000 1-2o:ooo
Shake the 1-100,000 dilution twenty-five times. Pipet the following amounts of this dilution into test tubes containing nutrient broth. M1.
Dilution
4.0 2.0 1.0
1-25.000 1-50.000 1-100,000
Heat the tubes of nutrient broth in the Arnold sterilizer for 30 minutes a t 100" C. The time of heating is taken from the time the temperature of the sterilizer reaches 100" C. Incubate the tubes of nutrient broth for 48 hours a t 37.5' C. Carefully examine the tubes a t the end of 48 hours for the presence of a surface growth. The presence of a pellicle or any surface growth constitutes a presumptive test for rope spores. Record results as plus or minus for each dilution, depending on whether a surface growth is present or absent. The number of rope spores per gram of material tested is taken as the reciprocal of the highest dilution giving a positive result. An exception is made in the case where a negative result is obtained in a lower dilution than the highest dilution giving a positive result. Then the result should be recorded as indicating a number of rope spores equal to the reciprocal of the dilution next lower than the highest one giving a positive result. The quantitative estimation is not complete unless the two highest dilution tubes show negative results. It will not be necessary in all cases to make long series of dilutions. The work may be shortened considerably for routine examinations and for the sorting out of suspicious samples. The 1 4 8 dilution is adequate for materials which consistently show negative or low rope counts. Strict bacteriological technic must be observed throughout the test. All glassware and media must be sterile and the work must be carried on as far removed from dust and drafts as possible. The preparation of the 1 4 8 dilution will vary slightly, depending on the nature of the material to be tested. I n the case of liquid materials such as water and milk, 2 ml. of the material to be examined are diluted with 94 ml. distilled water. I n the case of solid materials such as flour, yeast, sugar, etc., 2 grams of the sample are diluted with 96 ml. water.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Coarse materials such as grains should be ground to pass a t least a 20-mesh sieve before using in dilutions. It is permissible to use larger quantities of sand than are specified, provided the material is of such a nature that ordinary shaking with 10 grams of sand fails to break up the materials properly. It is essential that the time of heating of the bouillon tubes in the Arnold sterilizer be adequate to destroy all vegetative bacteria. If larger quantities of media than are here described are used to make the tests, the period of heating must be increased. This would apply in the case where special tests are applied to flour and water. When flour shows less than ten rope spores per gram, a special examination may be conducted as follows: Weigh 1-, 2-, 5-, and 10-gram portions of flour under as aseptic conditions as possible and deposit them in Erlenmeyer flasks containing 100 ml. of previously sterilized standard bouillon. Mix the flour and bouillon with a whirling motion, being careful not to wet the cotton plug. Heat the flasks in the Arnold sterilizer at least one hour after the sterilizer has reached a temperature of 100" C. Incubate at 37.5" C. for 48 hours. After the incubation period each flask is used to inoculate a test tube of sterile nutrient broth, several platinum wire loops of material being used for each inoculation. Heat the inoculated tubes for 30 minutes a t 100" C. in the Arnold sterilizer, and incubate for 48 hours. Absence of a pellicle or surface growth in the test tubes is recorded as zero spores per 10 grams of flour. The development of a pellicle in any of the test tubes is recorded as indicating one rope spore per quantity of flour used in the Erlenmeyer flask from which the inoculation had been made. An exception is made in the case where a negative result is obtained in a larger quantity of flour than the next smaller quantity showing a positive result. Then the result should be recorded as indicating one rope spore per quantity of flour used in the next larger quantity than the smaller amount showing a positive result.
Rope Spores i n Flour Using this simple method, hundreds of flour samples were examined during 1926, 1927, and 1928. These included patent, clear, rye, and whole wheat. The rope spore counts ran as high as one hundred fifty per gram of flour although many samples showed negative results. I n the summer of 1928 rope spore counts of all the flours gave negative results. I n the eight years since that time, no rope spores have been found in any flour sample examined. Even when tests were made with a maximum of 10 grams of flour the characteristic pellicle-forming organisms were absent. We are, however, constantly watching for flours containing rope spores because such flour would introduce considerably greater number of rope organisms in the doughs than any other ingredient with the same rope spore count. Improved methods of harvesting and milling of American grains appear to have had a beneficial effect on the flour as far as the presence of rope bacilli is concerned. No foreign flours were tested, and the rope problems of other countries are not known to the writers. If rope is present in such flours, it may be due to methods used in harvesting the wheat.
Rope Spores i n Other Ingredients It was obvious that the examination of the flour alone failed to show all the possible sources of rope contamination. I n the summer of 1928 rope developed in the bread even though rope spores were not present in the flour. I n order to explain this condition which was contrary to the generally accepted beliefs, counts were made on all ingredients used in the bread, The milk, sugar, salt, yeast, malt, shortening,
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and water were examined quantitatively for rope spores. Rope spores were found in yeast and malt in significant numbers but were absent in the other ingredients. Daily counts were made thereafter on yeast shipments. These counts showed a decided variation and ran as high as 20,000 per gram. Some typical counts on compressed yeast shipments of 1928 are cited. Although these counts represent one particular brand, similar variations also were observed in other brands of yeast: Date of ReRope ceipt of Spores Yeast in per Gram Aug., 1928 of Yeast 20 500 21 500 22 5,000 23 20.000
Date of Re- Rope oeipt of Spores Yeast in per Gram Aug., 1928 of Yeast 24 1,000 25 250 28 6,000
Date of ReRope ceipt of Spores Yeast in per Gram Aug., 1928 of Yeast 29 500 30 40 31 250
These counts helped to explain not only how rope development in bread could vary greatly in intensity from day to day but also why the usual expedients appeared to be more effective on some days than on others. The effect of the rope spore counts of yeast on rope development was observed in wrapped loaves of bread incubated a t 37.5" C. These experiments were conducted in conjunction with plant operations, and the observations were made on loaves taken from average runs of bread produced in the plant. A definite relation was found to exist between the rope spore count of the yeast and the development of rope in the incubated loaves. When the rope spore count of the yeast increased materially, the bread developed an objectionable ropy condition on incubation. These observations and tests with commercial bread were further confirmed by laboratory experiments. The pellicleforming cultures obtained from the yeast were introduced into laboratory doughs, and the resulting loaves were incubated and compared with bread made from the same dough without the culture. The control loaves were normal, whereas the inoculated loaves developed rope in 24 hours. Further confirmatory tests were made by comparing bread made with yeast having a low rope spore count with yeast having a high spore count. These results also confirmed the observations made on commercial runs of bread. When the manufacturers supplying the Ward Baking Company with compressed yeast were informed of these facts, they cooperated readily in efforts to reduce the rope spore content of their products. A large percentage of shipments during the past four years have shown ten or less rope spores per gram, and instances in which .the counts reach hhe one hundred mark have been rare. Malt extracts are also dangerous potential sources of rope infection. Samples examined showed considerable variation, some testing as high as 10,000 rope spores per gram. Manufacturers of malt have corrected this condition to the extent that this product can now be purchased relatively free from rope spores. As high as one hundred thousand rope spores per gram have been found in other materials subjected to the malting process. No evidence has been found of rope spores in milk products, in salt, or in water used in bread manufacture. Molasses and sugar a t times have shown small numbers of rope spores. Little difficulty, however, has been experienced with these products.
Interpretation of Rope Spore Counts Tentative standards have been set to meet the conditions under which the Ward Bakeries operate, and these standards have been in use for some years. Counts of twenty per 100 grams of flour, one hundred per gram of yeast or malt, and ten per gram of other ingredients are considered objectionable.
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Yeast and malt require special care on the part of the manufacturers in order to meet these standards. The additive effect of rope spores when present in a number of ingredients is a condition which must be given consideration. Buch a condition, although highly improbable, could easily cause development of rope in bread.
Literature Cited (1) Amos and Kent-Jones, Analyst,
55, 248-68 (1930); 56, 572-86 (1931). (2) Brahm, 2. ges. Getriedew., 13, 105-13 (1921). (3) Bunzell and Forbes, Cereal Chem., 7, 465-72 (1930); 9, 161-8 (1932). (4) Cohn and Henderson, Science, 48,501-5 (1918). (5) Cohn, Wolbach, Henderson, and Cathcart, S. Gen. Physiol., 1, 221-30 (1918). (6) Fisher, Northwestern Miller and Am. Baker, 11, 24-7 (1934).
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(7) Fisher and Halton, Cereal Chem., 5, 192-208 (1928). (8) Fuhrmann, Zentr. Bakt. Parasitenk., 2 (14),385-99 (1905). (9) Henderson, Science, 48,247 (1918). (10) Laurent, Bull. acad. sci. helg., [3]10,765 (1885). (11) Lloyd, Clark, and McCrea, S. Hug., 19, 380 (1921). 48, (12) Lloyd and McCrea, Rept. to Food ( W a r ) Comm. Roy. SOC., (1918). (13) Morison and Collatz, Am. Inst. Baking, Bull. 5 (1921). (14) Neumann and Knischewsky, 2. ges. Getreiedew., 3,187-91 (1910). (15) Neumann, Mohs, and Knisohewsky, Ibid., 4, 127-32 (1912). (16) Russell, Wis. Agr. Expt. Sta., Ann. Rept., 15, 110 (1898). (17) Stuchlik, Chem. Obror, 10,4-6 (in English) (1935). (18) Vogel, 2. Hug., 26,398 (1897). (19) Voitkevich, Biol. Abstracts, 3, 1388 (1929). (20) Watkins, J. SOC.Chem. Ind., 25,350-7 (1906). (21) Williams, Biochem. Bull., 1, 529-34 (1912). (22) Zeckendorf, NatZ. Assoc. Master Bakers Proc., 16, 66-78 (1913). RECEIVEID November 7, 1936.
Rate of Linseed Oil Oxidation with Driers Measurements of the rate of absorption of oxygen a t 98.5” C. by linseed oil containing oleates and linoleates of cobalt, manganese, and lead are reported. The concentration of drier used is, in general, 0.1 per cent of metal based on total weight ofoil. The length of the induction period is used as a basis of comparison. The linoleates of cobalt and manganese have the same induction periods as the oleates of these metals. The linoleates of cobalt and manganese have shorter induction periods than the linoleate of lead. Lead salts of phenylpropiolic, salicylic, and acetic acids are, in contrast t o the lead
salts of linoleic and oleic acid, not appreciably soluble in linseed oil and have longer induction periods. Decrease of metal content in the cases of cobalt linoleate and lead acetate causes a n increase in the length of the induction period. Induction periods for linoleic and oleic acid a t a concentration of 1.0 per cent in linseed oil are practically the same as that for raw oil. In the case of linoleic acid alone, the induction period is very short with a high oxygen absorption: in the case of oleic acid there is no pronounced induction period and very little oxygen is absorbed.
F
A. J. CURRIER AND I. HARVEY KAGARISE OR many years it has been The present i n v e s t i g a t i o n deals with the rate of oxidation known that the presenceof The Pennsylvania State College, State College, Pa. of linseed oil with various driers certain substances in lina t the temperature of boiling seed oi1,accelerates the rate of water. This temperature was chosen because the rapid rate oxidation or “drying” of the oil. Such substances, commonly of oxidation at this point makes it possible fox a given exknown B.S“driers,” apparently act as catalysts in the oxidation periment to be completed within 1.5 or 2 hours. Rhodes and process. Many investigations have been made to determine Chen’s measurements (9) were made a t 30” C.; Long and the controlling factors and the effectiveness of driers. A survey of about twenty-five articles in the literature shows Chataway (5) worked a t 160” C. that three general lines of procedure have been followed : 1. Determination of the time required for a known weight of oil t o reach a definite temperature, when absorbed on cotton (Mackey oil tester)(6). 2. Determination of the amount of oxygen absorbed as a function of time : a. Volume or per cent of oxygen absorbed b y a film of oil of known weight on cloth or paper (3,9, 10). b. Volume or per cent of oxygen absorbed by a known weight of oil (shown b y change in iodine number) when “blown” in bulk b y a stream of conditioned air (6, 11). 3. Tests on increase in weight, plasticity, or hardness of a film of oil spread upon a surface in contact with air or oxygen (4, 12).
Apparatus and Materials
The type of apparatus used (Figure 1) was somewhat similar to that used by Rhodes and Chen (9): It consisted of two similar units in which two measurements of the rate of oxidation (absorption of oxygen) could be made simultaneously. Each unit was made up of a ~ ~ O - C C wide-mouth ., bottle containing a small frame of copper wire upon which a strip of cloth exactly 2 X 10 cm. was stretched. A known weight of linseed oil or oil containing the drier was absorbed upon this cloth. A known weight ( 5 grams) of soda lime was placed in the bottom of the bottle t o absorb carbon dioxide, water, and volatile f a t t y acids formed during the oxidation of the oil ($. The wide-
