Animal Protein Factor S ment Produced acteria
le-
J
PRODUCTION AND ,EVALUATION H. M. HODGE, C. T. HANSON, AND R. J. ALLGEHER U . S. Industrial Chemicals Co., Dioision of National Distillers Products Corp., Baltimore, M d . Some protein sources, such as dried skim milk, zein and alpha protein isolated from soybeans (Glidden & eo. product), were found to be unsatisfactory for this process. When a sterile high protein-low carbohydrate medium is seeded with an enriched culture of the previously described organisms, and the pH is held a t the neutral point or slightly on the alkaline side, a proteolytic fermentation occurs with the formation of vitamin Biz. Production of vitamin Blz under these conditions is rapid in a 3% soybean oil meal mash as shown in Table I.
ICROBIAL production of vitamin B I has ~ been reported by a number of workers using aerobic cultures and highly aerobic conditions. As an example of an aerobic BIZproduction process, the concurrent production of antibiotics and vitamin Bls using streptomyces strains is probably the most familiar. 8tokstad and coworkers (6) have reported BU production by a flavobacterium. Lewis (6) has developed a fermentation using Bacillus megatherium, and Hall and coworkers (5) have employed Flavobacterium devorans for Bu synthesis. Hendlin and Ruger (d), in studying the effect of cobalt on microbial synthesis of Lactobacillus lactis Darner (LLD) active substances, used shake flask technique with ten undesignated cultures. Halbrook et al. ( a ) reported synthesis of relatively small quantities of vitamin BIZin a semianaerobic medium by a number of isolates from poultry house litter and droppings. Apparently all of the above mentioned organisms are strict aerobes, and require highly aerobic conditions for maximum cell development and BLz production in commercially significant amounts. However, the formation of this vitamin in ruminants, and in such materials as fermented chicken droppings, takes place under anaerobic conditions. It is evident, therefore, that vitamin Blz can be formed under anaerobic conditions and it is the purpose of this paper to describe such a fermentation, together with the evaluation of the end product.
TABLEI. PRODUCTION OF VITAMINBln B Y MIXED CULTURE Fermentation Time, Hours 48 56 64 72 96
Vitamin BIZ,y per MI. 0,004 0.162
0.416 0.375 0.330
In this fermentation there was little production of vitamin Bltin the first 48 hours, during which time the carbohydrates in the mash were removed by a saccharolytic fermentation with Aerobacter aerogenes. The saccharolytic fermentation may be brought about by a variety of organisms and, therefore, gives the process flexibility in incorporating growth factors other than vitamin Btr which are produced by bacteria or yeast. The production of growth factors in addition to vitamin Blz is demonstrated in Table VIII. At 48 hours the sugar-free medium was inoculated with the proteolytic mixture and maintained under anaerobic and slightly alkaline conditions. The formation of vitamin B1L was rapid, reaching a maximum a t 64 hours, or a t the sixteenth hour after the proteolytic inoculum was added. The maximum vitamin BIZ value was usually obtained between the sixteenth and twenty-fourth hour after proteolytic fermentation is initiated. Approximately half of the total solids in the mash is consumed during this period of active bacterial proteolysis. The vitamin BIZ produced in this fermentation, as in other bacterial fermentations, is present in or on the cells, since it can be removed from the beer by filtering or centrifuging the cells, and is released from the cells by autoclaving. With this preliminary laboratory information as a basis, the fermentation was extended and adapted t o a large scale plant process with success. The beer is acidified and concentrated to a solids content of approximately 15% and then spray-dried to produce a stable powder. The potency of this dry supplement has been tested and remains constant for a long period of time. Assays of the vitamin B12 produced have been made by the L. leichmannii procedure and verified by chick tests.
FERMENTATION AND ISOLATION OF VITAMIN Bn-PRODUCING ORGANISMS
While proceeding with a series of enrichments for the isolation
of facultative or anaerobic vitamin B12-producing organisms from soil, i t was noted that a mixed culture from one soil, in a sugarfree, high protein medium, produced the vitamin in small amounts -0.02 to 0.03 y per ml. Upon further subculture and enrichment of the mixture, the production of BIZas measured by Lactobanllus leichmannii assay increased to a level of 0.2 t o 0.5 y per ml. The organisms isolated from this mixture, by selective methods for each type, are listed according to their numerical predominance as follows: Pseudomonas sp.; Streptococcus bovis; Proteus vulgaris; and Clostridium putrijicum. A study of these isolated bacteria showed that in pure culture in an anaerobic neutral soybean oil meal or cooked meat medium, none of these types would produce vitamin B12 in appreciable quantity, but when grown as a mixture the Pseudomonas and Proteus strains formed Bl2 in substantial amounts. The production in y BIZper milliliter was Proteus sp., 0.13; Pseudomonas s p . , 0.03; and a mixed culture of !he two organisms, 0.32. A variety of proteins of vegetable or microbial origin are favorable substrates for Biz production using the above described organisms under anaerobic conditions. Plant proteins in cereal grains, such as cottonseed, soybean oil meal, copra meal, and proteins present in yeast and bacteria, are suitable substrates.
EVALUATION
Before marketing a new feed ingredient it is, of course, essentiat to test extensively for both toxicity and performance. A few
132
INDUSTRIAL A N D ENGINEERING CHEMISTRY
January 1952
of these tests conducted in this laboratory and independent laboratories are presented.
TOXICITY.Althou h it would not be ex ected that the t y es of organisms used in %is fermentation woufd be pathogenic, &is point was checked by feeding the unheated beer containing viable organisms to chicks. The unheated beer was mixed directly with dry vitamin B d e f i c i e n t &rowingmash to give a concentrac tion of 20 y of vitamin B12 per lulo of feed. In a &week chick experiment, growth was normal and there was no mortality. The dried animal protein factor supplement obtained from the above described bacterial fermentation has been fed to chicks and rats a t levels up to 10% with no evidence of toxicity and only minor variations from normal growth. Feeding a 10% level is approximately 50 times the recommended level for this material. Figure 1shows the results of a subacute oral toxicity test of three commercial, competitive animal protein factor products, on young rats as the test animal. The basal ration used was Purina Laboratory Chow Meal. At this abnormally high level, none of the supplements showed any marked depressing effect. However, one animal protein factor supplement (Brand B) showed a stunting of growth because of the fact that the feed containing the supplement was not readily consumed by the rats a t first, although they gradually became more adapted to it. There waa no mortality in any of the groups. The activity of the fermentation product waa PERFORMANCEI. determined with chicks. Practical tests have shown natural animal proteins to be quite variable in quality and vitamin Bl, potency. This, coupled with uncertainty in the supply of animal and marine protein, long used in manufactured feeds, and the rise in prices of these materials owing to shortages, stimulated the development of various types of animal protein factor supplements. Such supplements permit economies in formulations and also enable good feeds to be produced when fish meal and meat scrap are not available. The following tables show the results of a number of comparative tests of various animal protein factor supplements made in the majority of cases by independent laboratories. The composition of a ration specifically formulated by the Wisconsin Alumni Research Foundation to test for animal protein activity was: Mg
Lb. per
100'
100 Lb. Wheat bran Wheat middlings Dehydrated alfalfa leaf meal Soybean oil meal Corn gluten meal Steamed bone meal Ground vellow corn Granite grits Limestone grits Iodized salt Fish oil Manganese sulfate Iodinated casein (protamone)
5 5
5 50 lo2 18 2 2
0.5 0.2 0.025 0.05
Thiamine Riboflavin Niacin Calcium pantothenate Pyridoxine Inositol Choline Aminobenzoic acid fZoBin Folic aeid Menadione Alpha tocopherol
4:
Ration 0.3 0.6
5.0
'
2.0 0.4
100 150 10 0.02 0.05
0.05
0.3
This ration is complete in all respects except for the animal protein factor. The demand for this factor is increasing by using a high protein diet and by the addition of Protamone (iodinated casein). Table I1 indicates that the bacterial-derived animal protein factor completely replaced fish solubles and compared favorably with an animal protein factor of antibiotic origin. Table I11 shows the results of a similar test using the same ration. Under the conditions of this experiment the bacterial animal protein factor supplement completely replaced 3% fish solubles and gave a better response than vitamin Bl2 alone. An all-vegetable protein ration, the Minnesota (E5)high energy-low cost starting ration ( I ) , was also employed as a basal ration to study the chick growth response of a number of animal protein factor supplements. Table IV shows the chick growth response of a number of animal protein factor supplements when they are added to the basal ration mentioned above. Undepleted Leghorns from a
133
commercial hatchery were used in this experiment a t the University of Minnesota. There is little difference in the growth response from the animal piotein factor supplements, regardless of source. 2%
201
I-
I
'3 W
3
isa
0
0
m
In I
5e
IOC
5 4
W
W
::
5c
w K
3 0
I
2
3
4 5 WEEKS
6
7
8
9
Figure 1. Subacute Oral Toxicity Test of Three Commercial Animal Protein Factor Products plemented basal ration (ooptrols) :3. Uneu IO%S rand A animal protein factor in basal ration 10% Brand B animal protein factor in basal ration 4.
10% bacterial animal protein factor in basal ration
Since this study with the Minnesota ration was of short durat i o n - 6 weeks-a further test was made with the same ration in the laboratory of U. S. Industrial Chemicals Co., employing undepteted New Hampshire chicks from a commercial hatchery (25 birds per group) for a 10-week period. These results are given in Table V.
ANIMAL PROTEIN FACTOR ACTIVITIES TABLE 11. COMPARATIVE OF FISHSOLUBLES AND FERMENTATION PRODUCTS Growth Response of ChicksD ResDonse, Using . Weight Positive Gain a t 4 Control Weeks, as loo%, Supplement Grams % Basal (negative control) 148 55 Positive control 3% fish solubles 270 100 Basal and 0.26% animal protein factor bacterial fermentation 277 105 Basalsnd 0.26% animal protein factor antibiotic ongin 247 81
Feed Consumed
B%i,
Poundo
1.25 1.07 1.15
1.33
a Test erformed by Wisconsin Alumni Research Foundation. shire chict's per group.
20 Leg-
FACTOR ACTIVITY TABLE111. ANIMALPROTEIN Growth Response of Chickso Response.
Supplement Neeative control Posltive control. 3% fish solubles 30 y Cr stalline Blr per kg. 0.26% acterial animal protein factor
5
Weight Gain a t 8 Weeks, Grams
568 716 703 747
Using
Posltlve Control as loo%,
%
0 100.0 91.2 121.0
Feed Consumed &?I Pounhe 5.82 5.85
7.25 6.77
0 Test performed by Wisconsin Alumni Research Foundation. 20 Legshire chicks per group.
INDUSTRIAL AND ENGINEERING CHEMISTRY
134
Vol. 44, No. 1
~~
TABLE IV.
C O M P A R A ~PERFORMANCE" VE OF ANIMAL PROTEIN TABLEVI. FACTQR SUPPLEMENTS
COMPARATJVE PERFORMANCE OF ANIMAL FACTOR SUPPLEMENTS~
6-Week Weight. G r a m Male Femele Basal ration (negative control), Basal and 16.5 Y Biz per kg. diet Basal and 0.06% animal protein factor, Brand C (antibiotic origin) Basal andO.15'7 animal rotein factor, Brand C Basal and 0.25%. animay protein factor, Brand B (antibiotic orion) Basal and 0.5% animal protein factor, Brand A (antibiotic ori 'n) Basal and 0.22#animal protein factor, direct bacterial fermentation Basal and 0.0625% animal protein factor. Brand D Test performed by University of Minnesota. ehicks per group. Q
TABLEV.
279 392
309 390
383 428
374 408
400
388
41 1
392
423
...
374
10 White Leghorn male
COMPARATIVE PERFORMANCE^ OF ANIMALPROTEIN FACTOR SUPPLEMENTS Feed per. Weight in Grams 6 Weeks 8 Weeks 10 Weeks
Basal (negative control) 4% !ish rne.a! and 4% meat scrap (positive control) Basal and 0.175% animal PTOtein factor, direct bactenal fermentation Basal and 0.175% animal protein factor, Brand B (antibiotic origin)
Lb'aOft 10 Weeks. Lb.
634
795
1309
3 61
690
1074
1456
3.27
717
1118
1502
2.94
714
1115
1478
3.20
Test performed b y U. S. Industrial Chemicals, Inc. shire c h c k s per group. a
25 New Hamp-
The addition of both animal protein f x t o r supplements t o the Minnesota ration resulted in slightly improved growth and better feed e5ciency as compared t o animal protein supplements. The supplement of bacterial origin gave a response slightly greater than that of an antibiotic derived supplement. As additional evidence for the value of animal protein factor supplements in an all-vegetable protein ration containing alfalfa meal, fermentation solubles, and choline, the following ration was used i n a test a t the University of Arkansas: B.ass1 Rstion, Conventional Broiler Ingredients Ground yellow corn Groiind oata - - _- - - -.. Soybean meal (solvent proc:ess) Alfalfa meal (20% rotein) Fish meal (Menha& Mqat scrap (50% protein) Dried fermentation solubles (BY-500) Dicalcium phosphate Calcium carbonate Salt Choline chloride 25% dry mix) Vitamjn A, 6000 per gram V i t a m n D, 2000 U per gram Manganese sulfate
-
b
Pounds 57.35 36 2
1 1.6 1.3 0.5 0.1 0.1 0.05 0.016 100.016
Ration, Pounds Bo 3 23 3 4 4 1.5 0.6
0 -.. -5
0.1 0.1 0.05 0.016 __ 99.866
Normal stock White Wyandotte broiler chicks were used in this experiment. The test period was 10 weeks and 126chicks were in each group. The purpose of this experiment was to evaluate various commercial animal protein factor supplements on a vegetable protein ration as compared with a conventional broiler mash and to determine whether litter will supply sufficient animal protein factor for normal growth. Since no previous work has been reported on the effect of feed supplements on dressing grades, this factor was also investigated in the experiment. The results of this study are given in Tables VI and VII. The animal protein factor supplements were added on an equivalent cost basis. The addition of 2y0 dried whey demonstrated a slight additive effect over and above animal protein factor supplementation. Feeding animal protein factor supplements t o chicks on litter was effective in reducing mortality and up-grading the dressed poultry. In this experiment an animal protein factor
Feed per Lb. Weight, Pounds of Gain, Males Females Pounds Basal and 0.5% animal protein factor, Brand A (antibiotic origin) Basal and 0.1% animal protein factor. Brand C (antibiotic
PROTEIN
Mortality,
%
3.06
2.62
3.15
4.24
3.08
2.53
3.19
3.38
3.05
2.47
3.18
4.16
2.81 origin) 2.38 3.24 4.03 Basal and 0.1% animal protein factor, Brand C, and 2% dried 3.20 3.02 1.63 2.69 whey Basal and 0.1% animal protein factor. Brand C. chicks on old 3.09 2.61 litter 3.07 3.22 Conventional broiler ration 2.63 2.27 3.44 3.25 Basal chicks on old litter 2.20 1.76 3.77 19.51 Basal: chicks on new litter 2.36 1.78 3.83 27.35 a All chicks (126 per group) were on litter in open pens. Unless otherwise stated new litter was used. Test performed by University of Arkansas.
TABLE VII. EFFECT OF ANIMAL PROTEIN FACTOR SUPPLEMENTS ON DRESSINQ GRADESBY U.S.D.A. STANDARDS SHOWINQ PERCENTAGE IN EACHGRADE" B
A Basal ?nd 0.5%. animal protein factor, Brand A (antibiotic origin) Basal and 0 . 1 % animal protein factor, Brand C (antibiotic origin) Basal and 0.4% animal protein factor (bacterial origin) Basal and 0.4T0 animal protein factor, Brand B (antibiotic origin) Basal and 0.1% animal protein faoto'r, Brand C, and 2 7 dried whey Basal a n 2 0.1% animal protein factor, Brand C, chicks on old litter Conventional broller ration Basal, chicks on old litter Basal, chicks on new litter Test performed by University of Arkansas.
97
3
97
3
C
Reject
100 97
3
90
10
97 73 7 43
3 24 43 21
43 29
3 7
7
supplement from direct bacterial fermentation gave the best grade of dressed broilers of all the rations studied. Although vitamin B,z is the most important growth factor in commercial animal protein factor supplements, current interest is mainly in factors other than vitamin BJ2. For example, it has been demonstrated recently that certain antibiotics and phenylarsonic acid have a stimulating effect on chick growth. There is some confusion in this field and performance varies considerably with the quality of the components of the ration and with the animals tested. To obtain information on factors other than vitamin Bi2, the practical chick starter ration shown below was employed a t Wisconsin Alumni Research Foundation: Pounds Wheat bran Wheat middlings Dehydrated alfalfa leaf mea Soybean oil meal Steamed bone meal Ground yellow corn Granite grits Limestone grits Iodized salt Fish oil Manganese sulfate
Riboflavin Choline
5 5 5 32 2 46.3 2 2 0.5 0.2 0.025 100.025
Mg. per 100-Gram Ration 0.15 100
Normal Legshire chicks were used in this experiment and the test wm continued for 8 weeks. The results are given in Table VIII. The data indicate growth stimulation above that obtained with fish solubles and vitamin Bl2 alone. It may be concluded from these results that unidentified growth factors, other than antibiotics, are produced by bacterial fermentation.
January 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
135
of Minnesota, and Carl Krieger of the Wisconsin Alumni Re-
TABLEVIII.
RESPONSETO
GROWTHFACTORSOTHERTHAN search Foundation for permission to u8e unpublished data origi-
VITAMINBI24
Supplement
VitaminBI8 Weight at 8 y/Kg. die{ Weeks, Grama Trace 818 15-20 890 ao 9ao Trace 890
ao arsenic acid Basal and 0.45% aqimal protein factor, Brand A (antibiotic origin)b Basal and 1% qnjmsl protein factor, Brand A (antibiotic or1 in) b Basal and 0 , 2 6 6 animal protein factor (bac20 terial origin) Test erformed by Wisconsin Alumni Research Foundation. shire chi& per group. b Vitamin Bit content was not determined.
963
910 990
980 20 Leg-
ACKNOWLEDGMENT
The authors wish to acknowledge and thank Edward Stephenson of the University of Arkansas, George Briggs of the University
nating in their laboratories. LITERATURE CITED
(1) Briggs, G. M., Hill, E. G., and Giles, M. J., Poultry Sci., 29, No. 5. 723 - - (1950). (2) Halbrook, E. R., Cords, Fay, Winter, A. R., and Sutton, T. El., J . Nutritwn, 41,565 (1950). (3) Hall, H. H., Benjamin, J. C., Bricker, H. M., Gill, R. J., Hayne8, W. C.. and Tsuchiua. H . M.. Bact. Proc.. Abstracts 21 (1950). paper presented a t t h e 60th Meeting of the Society of American Bacteriologists. (4) Hendlin, D., and Ruger, M. L., Science, 111,542 (1950). (5) Lewis, J. C., Ijicki, K., Snell, N. S., and Garibaldi, J. A., U. S. Bur. Agr. Ind. Chem., A I C 254 (October 1949). (6) Stokstad, E. L. R., Page, A. C., Pierce, J., Franklin, A. L., Jukes, T. H.. Heinie. R. W.. Epstein. M.. and Welch, A. D., J . Lab. Clin. Med., 33,860 (1948).
-.
.----,
RECEIVED February 3, 1951. Presented before the Division of Agricultural CHPIMICAL and Food Chemistry at the 118th Meeting of the AMERICAN SOCIETY, Chicago, IU.
Stabilization of Dielectrics Operating under Direct Current Potential H. A. SAUER, D. A. MCLEAN, AND L. EGERTON Bell Telephone Laboratories, Inc., Murray Hill, N. J.
T
HE effectiveness of stabilizers in electrical capacitors impregnated with chlorinated aromatic compounds has been fully established. Of the stabilizers discovered, anthraquinone (9, fO) has been most widely used commercially, although its chloro derivatives and azoxybenzene have also been employed. Anthraquinone possesses the desirable characteristics of low volatility, low toxicity, high stabilizing effectiveness, and commercial availability in pure form at low cost. All quinones tested to date have been found to have stabilizing action. Other compounds found to be effective are the nitroaromatics (6),maleic anhydride (6),sulfur (4,aromatic acid anhydrides, b e n d , and aromatic azo and azoxy compounds (I). In addition, it is claimed by Church and Garton (3)and by Church (2) that unsaturated aliphatic hydrocarbons which are readily reduced are stabilizers, octadecene in particular being mentioned. They indicate that the life of Chlorinated diphenyl capacitors has been extended more than ten times by the addilion of azobenzene or octadecene but under the conditions of the present work no stabilizing effect of octadecene could be noted, Much of the previous work has been rather exploratory in nature and in particular where test capacitors have been subjected t o accelerated aging tests, the number of samples has usually been small. This approach had no serious disadvantages in the early stages of the investigation since the effects observed were large and the question of precision therefore relatively unimportant. For example, ratios of the Iife of stabilized to unstabilized capacitors ranging from Bfold to about 1WfoId have been reported. This exploratary approach pennitted covering 8 wide range of compounds and selection of very effective stabilizers for commercid we at an early stage of the work. However, more thorough and precise experiments are necessary in order that both the fullest and most, effective use be ma&eof
stabilizers and that completely reliable data for testing the various theories of stabilization be obtained. The work reported here, while still limited by the inherent wide dispersion of life test results, is an attempt to determine certain relationships with more accuracy than most previous work. It can be divided into three phases: a study of the effect of concentration of anthraquinone on accelerated life test performance of chlorinated naphthalene-impregnated paper capacitor units; a comparison of the effectiveness of several stabilizers at 1% concentration; and investigation of the effect of stabilizers on the direct current life test performance of mineral oil-impregnated capacitor units. EXPERIMENTAL METHOD
The present work was facilitated by recent developments in testing technique which involve testing at higher temperature with a consequent higher factor of acceleration. The temperature of test is 130° C., this high temperature being justified because the deterioration process under direct current potential hss been shown to be predominantly chemical and electrochemical; 1 therefore, the life is an exponential fanction of p (I, IO). If the exponent in the function is the same for various groups of samples representing different conditions of impregnant, stabilizer, and paper, then their relative performance should be the same a t high as at low temperature. That this is approximately true is indicated by recent experimental results (fS). Like results of m y highly accelerated test, these should be checked under conditione more nearly approximating operating conditions before designs are established. In this connection, caution should be exercised in extrapolating to conditions where the state of the dielectric changes, such as from the tiquid to the solid state, or in changing
