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INDUSTRIAL AND ENGINEERING CHEMISTRY
source of nicotinic acid activity. In patent flour the relation of thiamin and nicotinic acid is similar, since both contain approximately 10 per cent of the whole wheat total. However, because thiamin is more concentrated in the tissues represented by the second clear flour, red dog, and shorts, longer extraction flours will not show the large increases in nicotinic acid activity that they show in thiamin.
Summary The nicotinic acid assay of cereals and cereal products by the microbiological method is influenced by the type of extraction employed. Water and dilute acids yield lower values than stronger acids and alkali. This discrepancy can be attributed either to the formation of growth-stimulating substances by the latter solvents or to the liberation of active nicotinic acid compounds by hydrolysis of a less active or inactive precursor. The evidence presented, while not conclusive, suggests that the second explanation is more likely to be correzt. If this is true, the choice of extraction procedure required for evaluating cereals will depend on the nutritional availability of the activity liberated by hydrolysis. Wheat germ, unlike bran and endosperm, does not exhibit
Vol. 14, No. 8
enhanced activity on alkaline hydrolysis. In this respect it resembles yeast and animal tissues. The distribution of nicotinic activity in the various fractions obtained by commercial milling differs from that of thiamin and riboflzvin. Concentration is greatest in the bran.
Literature Cited (1) Andrews, J. S., Boyd, H. M.,and Terry, D. E., Cereal Chem., 19, 55 (1942). (2) Andrews, J. S.,Boyd, H. SI.,and Terry, D. E., IND.ENG. CHEM., ABAL.ED.,14, 271 (1942). (3) Federal Register, 6, No. 103, 2580 (May 27, 1941). (4) Kodicek, E., Biochem. J . , 34, 712, 722 (1940). (5) Melnick, D., and Field, H., Jr., J . Biol. Chem., 134, 1 (1940). (6) Sfelnick, D., Oser, B. L., and Siegel, L., IND.EKG. CHEM., ASAL. ED., 13, 879 (1941). (7) Oser, B. L., Melnick, D., and Siegel, L., paper presented before Division of Biological Chemistry at 102nd Meeting of AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J. (1941). ( 8 ) Sherwood, R. C., Nordgren, R., and Andrews, J. S.,Cereal Chem., 18, 811 (1941). (9) Snell, E. E., and Wright, L. D., J . Biol. Chem., 139, 675 (1941). (10) Kaisman, H. A . , and Elvehjem, C. A., IND.ENG.CHEM., ANAL. ED.,13, 221 (1941). PAPER 39, Journal Series, General Mills, Inc., Research Laboratories.
Growth Stimulants In the Microbiological Assay for Riboflavin and Pantothenic Acid J. C. BAUERNFEIND, A. L. SOTIER,
I
AND
C. S. BORUFF, Hiram Walker & Sons, Inc., Peoria, Ill.
T HAS been the observation of workers in this laboratory that a discrepancy in results exists when aqueous extracts
of certain foodstuffs are assayed microbiologically for their riboflavin and pantothenic acid content with and without the inclusion of the extracted residues. In the original microbiological riboflavin assay method, Snell and Strong (6) extracted the riboflavin by autoclaving the natural foodstuff in a large volume of water, or by boiling with dilute acid. The extracted residues were usually removed by centrifugation, but in some cases suspensions were assayed directly. Pennington, Snell, and Williams (6) in their microbiological pantothenic acid assay method make no definite suggestions for the removal of extracted residues from the autoclaved aqueous suspensions, but state that if necessary, fuller’s earth or kieselguhr may be used to clarify the extract. On the other hand, Strong, Feeney, and Earle ( 7 ) state that autoclaved aqueous suspensions of finely ground samples may be used without the removal of the extracted residues, in their recent microbiological assay for the determination of pantothenic acid.
It is known that the extraction procedure varies in different laboratories employing these microbiological techniques, and while the present authors believe that little or no differences result from these varied extraction procedures in the case of many foodstuffs, they are convinced differences do occur in certain ones which are reported in this investigation. Procedure I n the microbiological riboflavin assays reported herein the procedure of Snell and Strong (6) was carried out, while the pantothenic acid assays were conducted according to the microbiological procedure described by Pennington, Snell, and Williams (5), with modifications which included the shaking of all assay tubes after 16 to 20 hours of incubation and the use of a stab culture medium which more closely resembles the assay media:
Stab Cu:ture Medium Glucose, grams Peptone solution, ml. Cystine solution, ml. Salt solution A, ml. Salt solution B, ml. Yeast extract (Difco), gram Agar grams A d j d t t o pH 6.7, dilute t o 200 ml., add agar, melt, and sterilize in tubes
2 20 20 1 1 1 3.5
The preparation of the above ingredients has been described by Snell and Strong (6). The test organism used in these assays was Lactobacillus casei. The growth response was determined by the titer of the lactic acid produced after a 72-hour incubation period, except where an occasional turbidimetric evaluation was made, using the Coleman Universal spectrophotometer at a wave length of 650 mp. Merck’s synthetic riboflavin and synthetic dextrorotatory calcium pantothenate served as the assay standards. RIBOFLAVIN ASSAYOF EXTRACTS WITH AND WITHOUT RESIDUES. Samples of dried skim milk, distillers’ dried solubles, meat scraps, fish meal, alfalfa leaf meal, and commercial liver meal in amounts of 0.5 to 1.0 gram were autoclaved with 25 to 35 ml. of distilled water for 15 minutes at 15 pounds’ pressure in conical 50-ml. centrifuge tubes. After cooling, the tubes were centrifuged and the supernatant liquid was decanted. Another 25- to 35-m1. portion of water nas added to the residue and the entire procedure was repeated. A third volume of water was then added t o the residue and the tubes were recentrifuged. The clear centrifuged wash water was combined with the supernatant liquid from the two extractions and made to volume. Sometimes a filtration was necessary. The final extracted residues were resuspended in distilled water and made to volume. Equal parts of the clarified extract and the suspended residues were combined and designated as the u ater extract with residual solids suspended. The results of the riboflavin assay of the clarified water extract, and the water extract with solids suspended, are presented in Table I.
A N A L Y T I C A L E D I T I 01N
August 15, 1942
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667 tion. Portions of the aqueous extracted residues were extracted with acetone and methanol, respectively, for 12 hours and portions of the acid residues were extracted with acetone and ethyl ether, respectively, for 16 hours, the resulting extracts and residues of which, likewise, were assayed for their growth-stimulating effect.
TABLE I. RIBOFLAVIN VALEESFOR FOODSTUFFS
Foodstuff Dried skim milk A Dried skim milk B Commercial liver meal A Commercial liver meal B Alfalfa leaf meal Meat scraps Fish meal Distillers’ dried solubles
-
Xcrobiological Assay
Clarified Water water extract extract with plus 40 t o 60 Clarified solids micrograms of water susmethyl palmiextract pended t a t e a per tube .Vicrograms o/ riboflavin p e r g r a m 19.7 19.8 28.9 .. 16.1 16.9 57.4 56.8 55.3 55.7 83.5 84.0 14.1 14.4 19.2 14.7 16.0 18.i 6.2 8.7 10.6 4.6 7.0 9.7 l5,9 22.0 23.8
m-ater extract of ether-extracted foodstuff with solids suspended
.... 16,4
5.4
16.9
0 . 9 5 % ethyl alcohol solution. b Carbon tetrachloride extracted.
The data are presented in Table 111. It will be observed that the aqueous
22.6 17.2 31.6 33.8 15.4
and acid-extracted residues exerted no growth-stimulating effect in the absence of riboflavin in the assay medium, but were found to have a 6.0 considerable stimulatory action, being greater for the acid-extracted residues, 5.5 in the presence of 0.10 microgram of 13.6 riboflavin per tube. This stimulatory action was not due to the mere presence of sumended solids. for the stimulatory agent could be extracted from the aqueous and acid residues by organic solvents. Additional experimentation on the saponification of a n ether extract of distillers’ dried solubles has revealed the stimulatory activity to be present in both the unsaponifiable and saponifiable fractions, the latter fraction, however, having the major part of the activity. STIMULATORY ACTIVITY O F PHOTOLYZED EXTRACTS. The stimulatory phenomenon was also studied in another fashion. It has been known for some time that riboflavin in alkaline solution is destroyed by irradiation. Aqueous suspensions of four foodstds mentioned above were autoclaved for 15 minutes, cooled, and adjusted to pH 8.0 to 9.0. The suspensions were then introduced into 1-liter Erlenmeyer flasks, plugged with cotton, and autoclaved for 5 more minutes. After ,cooling, these flasks were suspended over 100 to 200-watt electric light bulbs and irradiated through the bottom of the flasks for 24 to 30 hours. Following the sterile irradiation period, the contents of the flasks were adjusted to pH 6.6 to 6.8, and made to volume, and aliquots were assayed microbiologically. The results are shown in Table 11. Little or no growth response was obtained by incorporating the photolyzed
34.5,33.86 42.2 13.6
5.2
Fluorometric Deterniination (31
Good agreement mas obtained between the riboflavin values for dried skim milk, alfalfa leaf meal, and one sample of liver meal by the two methods of extract preparation. Higher riboflavin values were obtained on meat scraps, fish meal, distillers’ dried solubles, and a second sample of liver meal by assay of the extracts with solids suspended as compared t o the values on the clarified extracts. STIMULATORY ACTIVITY OF EXTRACTED RESIDUES.I n order to ascertain whether or not all the riboflavin was extracted from the last-mentioned foodstuffs, the waterextracted and mashed residues were assayed for their riboflavin content in the presence and absence of added synthetic riboflavin. The results of this experiment are shown in Table 11. The addition of the extracted residues to the basal medium produced no significant increase in the growth of L. casei over that of the basal medium, as shown by titration values, except in the case of meat scraps where apparently all of the riboflavin was not removed. I n the presence of 0.10 microgram of riboflavin per tube the extracted residues of the liver meal and meat scraps produced a n appreciable growth-promoting effect. It is therefore observed that these extracted residues, which appeared to contain no riboflavin, exerted a growthTABLE11. STIWJLATORY ACTIONOF AQUEOVSEXTRACTED RESIDUESAND PHOTOstimulating action on the microLYZED .4QUEOrS SOSPENSIOXS O F FOODSTUFFS organism when incorporated in the ( I n t h e presence of suboptimum amounts of riboflavin) medium with a suboptimum amount Titration Values a s M I . of 0.1 .V Alkali Micrograms of of synthetic riboflavin. The exRiboflavin Xfg. Basal tracted residue of dried skim milk per Tube medium RePer of plus Added covered Cent was microbiologically inert, while Original Basal preparaper per StirnulaMaterial Foodstuff medium tion tube tube tion the residue of alfalfa leaf meal exerted a little stimulatory action. Aqueous Extracted Residues The extracted residue of distillers’ Dried skim milk 4.0 0.75 0.85 0.10 0.102 2 6.0 0.75 0.90 0.10 0.103 3 dried solubles will also produce the Commercial liver meal B 1 . 0 0 . 8 0 0 . 8 0 0.10 0.159 59 stimulatory phenomenon shown 2.0 0.80 0.80 0.10 0.172 72 above. Alfalfa leaf meal 6.0 0.75 0.70 0.10 0.111 11 Two samples of 4 grams each were extracted with a total of 800 ml. of water and a total of 660 ml. of 1.0 -V hydrochloric acid, respectively, in four separate autoclaving extraction periods of 10 minutes each. After each autoclaving period, the suspensions were centrifuged, the supernatant liquid was decanted, and the residues were reextracted with another portion of solvent. After the final extraction, the residues were dried and tested for their riboflavin content and stimulatory ac-
9.0
Meat scraps
Dried skim milk Commercial li\.er meal B Alfalfa leaf meal Meat scraps
10.0 15.0
0.75 0.90 0.90
Photolyzed Aqueous 4.0 0.90 6.0 0.90 1.0 0.90 2.0 0.90 6.0 0.90 9.0 0.90 10.0 0.90 15.0 0.90
0.70 1.40 1.40
0.10 0.10 0.10
0.114 0.158
0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
0.101 0.104 0.146 0.176 0.109 0,122
0.15s
14
58 58
Suspensions 0.90 0.90 1.15 1.35 1.00 0.90 1.00 1.00
0.142 0,156
1 4
46 76 9 22 42 56
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 8
The addition of 20 micrograms or more per 10 ml. of medium of calcium stearate, palmitic acid, linoleic (In the presence of suboptimum amounts of riboflayin) acid, or diglycol laurate in the presMg. Titration Values a s ence of suboptimum amounts of riboper Tube MI. of 0 . 1 S Alkali of Basal flavin produced marked stimulation, Original midiulll Micrograms of plus Riboflavin Per while in the absence of riboflavin no Foodhdded Recovered Cent extracstuff growth response occurred over that Stimulation ExBasal per per tube tube residue 1-0, Extracted Residue tracted medium tion obtained on the basal medium alone. 4.0 0.80 0.10 0.128 1 Distillers' dried soiubies 1.0 28 Calcium oxide, n-caproic acid, or (extracted 4 times with water) 6.0 0.80 0.10 0.134 1.0 34 ethyl laurate did not produce sig0 80 0.10 0.148 2 -4cetone extract of 1 4.0 1.0 48 nificant growth-stimulatory response 62 6.0 1.o 0 90 0.10 0.162 either in the absence or presence of 1.0 1 00 0.10 0.108 8 3 dcetone residue from 2 4.0 added synthetic riboflavin. The high... .. ... 6.0 1.0 34 est level of linoleic acid markedly 0 io 0.10 0.134 4.0 4 Methanol extract of 1 1.0 0.60 0.10 0.134 6.0 1.0 34 inhibited growth. This marked in0.10 0.108 4.0 1.o 0.80 5 Methanol residue from hibition has been observed in only 6 8 6.0 1.0 0.90 0.10 0.106 4 one other instance when 160 micro0.80 0.10 0.137 37 0.95 6 Distillers' dried solubles 4.0 grams per 10 ml. of medium of (extracted- 4 times 63 0.80 0.10 0.163 6.0 0.95 with 1.0 h HCI) duodecyl alcohol were added in the 0.10 0.166 66 4.0 0.95 0 80 7 Acetone extract of 6 presence of 0.10 microgram of ribo0.95 80 0.10 0.180 6.0 0.60 12 flavin per tube. 0.10 0.112 0.95 1.00 8 .4cetone residue from 7 4.0 0.10 0.109 0.95 6.0 1 .oo 9 Up to this point the effect of a 0.95 0.90 4.0 9 E t h y l ether extract of 6 0.10 0.10 0.202 0.177 102 77 stimulatory compound has been shown 0.95 1.00 6.0 only in the absence of riboflavin and 0.95 0.70 4.0 0.10 0 106 6 10 E t h y l ether residue 6.0 0.95 0.70 from 9 0.10 0.101 1 the presence of 0.10 microgram of riboflavin per 10 ml. of medium. Figure 1 demonstrates the typical reaction on the addition of graded amounts aqueous suspensions of the foodstuffs in the medium, thus of a stimulatory compound (in this case Merck's 90 per indicating the absence of riboflavin. However, in the prescent lecithin was used) to the medium with increasing levels ence of the suboptimum amount of 0.10 microgram of riboof riboflavin until the optimum riboflavin requirement for the flavin per tube, photolyzed aqueous suspensions of liver meal, growth of the organism was reached. and meat scraps, produced considerable stimulatory action, The figure shows that lecithin is not a vitamin or growth dried skim milk no stimulation, and alfalfa leaf meal a small factor for Lactobacillus casei, for its addition to the medium amount. These results are in general agreement with those in the absence of riboflavin or in the optimum amount of obtained on the aqueous extracted residues also reported in riboflavin resulted in no additional growth response. It Table 11. does, however, appear to assist the microorganism to make STIMULATORY ACTIVITYOF KNOWNCOMPOUXDS. Based more efficient use of suboptimum amounts of riboflavin and therefore may be said to have riboflavin-sparing action. on the knowledge obtained in previous work a number of known compounds J+ere tested for their stimulatory power. The space required for the manifestations of the effect of Water-insoluble salts, glass, etc., were finely ground before all compounds tried is not warranted here. However, all compounds used (Table V) have been divided into two they ryere added to the medium. The water-soluble compounds \yere dissolved and made to volume. Water-insolugroups: (1) those displaying appreciable stimulation when ble, alcohol-soluble organics were first dissolved in ethyl added t o the medium in the presence of 0.10 microgram of alcohol and water was added until the solution was 0.95 per cent ethyl alcohol. Usually a colloidal solution resulted. The amount of alcohol introduced in this manner before sterilization of the assay tubes was tried repeatedly without the observation of significant stimulation or inhibition. T h e compounds were added in the absence of r i b o f l a y i n a n d in t h e presence of 0.10 niicrogram of riboflavin per tube. Sufficient data are presented in Table I T on a few of the compounds 0 I 1 I to show the results of 0 " " 0 ' 5 " ' ' IO I " "21 their addition under the MICROGRAMS RIBOFLAVIN PER IO ML MEDIUM conditions of the experiOF L. casei TO LECITHIX IR THE PRESEXCE OF RIBOFLAVIN ment . FIGURE 1. RESPOYSE TABLE 111. STIJICLATORY ACTION OF EXTRACTED RESIDCESOF DISTILLERS' DRIED SOLUBLES
' '
August 15, 1942
ANALYTICAL EDITION
669
aqueous liver meal extract. Failures of stimulation of the clarified extracts of this product occurred in five other trials using other known stimulants. It is suggested that already the value obtained on the clarified extract is a stimulated value (not a true riboflavin value) and that the addition of more stimulating agent is noneffective. It has been demonstrated many times that the per cent stimulation per unit of compound decreases sharply to the point where additional units are without effect. The suggestion mentioned above also involves the water-solubility of these stimulating agents. Approximately 1 per cent of lecithin mas mixed with samples of dried skim milk, meat scraps, and fish meal. These samples and controls without lecithin a 0.95% ethyl alcohol solution. were given the usual autoclaving b E t h y l alcohol added in same fashion as in a. aqueous extraction, and after cooling, the solids were removed by aravitv filtration through filter DaDer. TABLE V. COMPOUNDS EXHIBITING APPRECIABLESTIMULATION ON THE GROWTH OF L. casei IN THE PRESENCE OF SUBOPTIMUM The filtrates of all samples were assayed -for ribdflavin AMOUNTSOF RIBOFLAVIN microbiologically. The samples containing lecithin assayed (Assayed in amount of 20 t o 160 micrograms per 10 ml. of medium) 13 to 52 per cent higher in apparent riboflavin content than Duodecyl alcohol5 Palmitic acid the non-lecithin-containing samples, thus demonstrating the S-octadecyl alcohol Stearic acid passage of the stimulant through the filter as a true or a Diglycol laurate Undecylenic acid Methyl palmitate Oleic acid finely colloidal solution. Another experiment with methyl Eleostearic acid Methyl stearate Linoleic acid Calcium stearate palmitate showed no appreciable stimulation in any of the Ricinoleic acid E t h y l oleate filtrates. F a t t y acid mixtureb Methyl oleate Lecithin (90%) Duodecyl aldehydea Since it has been demonstrated that the stimulatory agent 7-Undecalactonec of the acid residue of distillers’ dried solubles could be exS o n s t a n d a r d chemicals. tracted by ethyl ether, the six original dried foodstuffs u-ere ether-extracted for 6 to 8 hours and dried, and the autoclaved Florasynth Laboratories, New York, S . Y. b Neo fat No. 23, Armour & Co., Chicago, Ill. (15 minutes a t 15 pounds’ pressure) aqueous extracts n ith Felton Chemical Co., Inc., New York. N. Y. residual solids suspended were microbiologically assayed for riboflavin content. The data of this study are presented in Table I as well as riboflavin determinations on the original riboflavin per tube and (2) those showing little or no stimufoodstuffs by the fluorometric method of Hodson and Sorris lation. (S), using the Coleman Universal spectrophotometer with The following materials exhibited little or no stimulation; the mercury vapor lamp attachment. ground glass, glass beads, asbestos, sodium chloride, calcium The results show that the stimulatory agents are apparently chloride, calcium oxide, calcium carbonate, calcium lactate, extracted from the original dried foodstuffs with ethyl ether calcium phytate, calcium gluconate, butyric acid, valeric and that autoclaved aqueous suspension of ether-extracted acid, n-caproic acid, malic acid, citric acid, pimelic acid, material may be used in the bacterial assay procedure. sebacic acid, salicyclic acid, cinnamic acid, furoic acid, furylThe microbiological assay results of ether-extracted liver acrylic acid, glutamic acid hydrochloride, glycerol, cinnamic meal and the fluorometric results on the original liver meal alcohol, furfuryl alcohol, 2-alpha-furfuryl ethanol, tetrasupport the contention that stimulatory compounds may hydrofurfuryl alcohol, geraniol, inositol, cholesterol, acrolein, occur not only in the extracted residues of foodstuffs but in furfural, ethyl-n-caprylate, ethyl-n-caproate, isoamyl-n-capthe extracts. roate, ethyl laurate, ethyl sebacate, geranyl valerate, methyl Several of the compounds which were shown to have stimucinnamate, enanthyl cinnamate, butyrolactone, alpha-angellatory action in the microbiological riboflavin assay were also ica lactone, choline chloride, biotin concentrate, p-aminotested in the microbiological pantothenic acid assay, in the benzoic acid, thiamin hydrochloride, calcium pantothenate, absence of pantothenic acid, and in the presence of the subnicotinic acid, pyridoxine, maleic anhydride, tetrahydrooptimum amount of 0.08 microgram of pantothenic acid per furfurylbenzyl ether, and asparagine monohydrate. 10 ml. of medium. Table T’I contains the results of this The stimulatory effects of known compounds have all been experiment. shown in the presence of synthetic riboflavin. I n Table I The titration values obtained when fusel oil residue, methyl the data demonstrate that the assay value of a clarified palmitate, oleic acid, and lecithin were introduced in the aqueous extract of a foodstuff can be enhanced by the addibasal medium are more irregular than were obtained in the tion of methyl palmitate to the assay tube. The results microbiological riboflavin assays heretofore presented. Khen obtained thus simulate the effect of the water-extracted solids these compounds n-ere assayed with the suboptimum amount of those foodstuffs containing stimulants. One exception to of pantothenic acid, their stimulatory action was significant, this statement is the case of commercial liver meal A, where thus indicating that the same type of stimulatory compounds neither the extracted liver meal solids nor methyl palmitate which interfere in the microbiological riboflavin assay a1.o had any stimulatory action on the assay results of the clarified interfere in the microbiological pantothenic acid assay. The
T A B L E Iv. STIhlUL.4TORY ACTIOXO F K S o W N CO&IPOUNDs ( I n the presence of suboptimum amounts of ribo5avin) Titration Values a s MI. Per Cent Stimulation in Presence of of 0.1 .V Alkali Basal medium of 0.10 Microgram Mirrograms Ba$al plus of Riboflavin per Compound per Tube medium compound Tube 20 1.0 0.90 22 Calcium Etearate 160 1.0 49 0.70 20 1.0 1.00 2 Calcium oxide 160 1.0 0.95 -8 20 0.8 0.75 37 Palmitic acid0 160 0.8 0.70 80 30 0.9 0.95 0 n-Caproic acida 160 0.9 0.85 1 5 0.8 0.70 8 Linoleic acid* 10 0.8 0.65 13 20 0.8 0.60 28 160 0.8 0.50 - 33 E t h y l IaurateQ 20 0.8 0.75 2 160 0.8 0.76 4 0 . 8 0.65 16 Diglycol laurateo 20 160 0.8 0.60 108 O.i 0.70 5 E t h y l alcoholb ... (0.95:;) ... 0.7 0.70 3
0 C
670
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 8
It has been the authors' experience that agreement of result's a t (In t h e presence of suboptimum amounts of pantothenic acid) different levels of assay is not alTitration Values as NI. Per Cent Stiniulaways a good index of the presence of 0.1 A' Alkali tion in Presence of Basal medium 0 . 0 8 llicrogram of of stimulatory action. Apparently, Micrograms Basal plus Pantothenic Acid the amount of stimulatory agent Compound per Tube medium compound per Tube Fusel oil residuea 20 1.1 2.4 29 present in the foodstuff is a decid120 1.1 2.4 131 ing factor. If similar stimulation Methyl palmitates 40 2.0 2.1 15 occurs at different levels of assay 120 2.0 3.2 25 along the linear portion of the Oleic acid" 20 1.1 1 .i 12 curve, agreement of results be120 1.1 1.6 66 34 tween levels of assay may occur. If Lecithin0 40 2.0 1.6 120 2.0 1.4 76 these conditions are not met disagreement is evident. 0 0 . 9 5 % ethyl alcohol solution. Asparagine produced no stimulation even when included a t a concentration of 5 mg. of the monohydrate per 10 ml. of medium and therefore the stimulation latter part of this statement is also supported by other assays herein described differs from that reported by Feeney and not reported here, in which the aqueous extracted residues of Strong (6). liver meal, fish meal, distillers' dried solubles, and meat scraps The presence of a factor in blood which enhances bacterial have been demonstrated to possess stimulatory activity in growth activity of riboflavin has been reported by Eckhardt, suboptimum amounts of pantothenic acid. Gyorgy, and Johnson ( 1 ) . These workers have irradiated To demonstrate the effect of adding graded amounts of a diluted alkaline blood to destroy its riboflavin and then atstimulatory compound (lecithin) to the assay medium in the tempted to recover added synthetic flavin by the Snell and presence of graded levels of pantothenic acid, Figure 2 is given. Strong microbiological assay method. They found that the The greatest effect of the stimulant occurs in the presence of grou-th of the microorganism was stimulated in the presence suboptimum amounts of the vitamin, as was also shown in of suboptimum amounts of riboflavin. The authors are able Figure 1. to offer confirmatory evidence of this stimulatory phenomenon. At the time of this investigation they had in the laboraDiscussion tory a sample of dried blood meal which was extracted with water, and the extracted residue incorporated into the meCertain foodstuffs contain compounds which exert a stimudium equivalent to 5 to 20 mg. of the original meal along with lating effect on the growth of L. casei in suboptimum concensuboptimum amounts of riboflavin. Stimulation was obtrations of riboflavin and pantothenic acid. Usually these served to the extent of 34 to 46 per cent. compounds are not extracted in the aqueous autoclaving exEckhardt, Gyorgy, and Johnson ( 1 ) suggested that subtraction procedure and therefore can exert their maximum stances to be assayed for riboflavin should be tested for this effects only when aqueous extracts with residual solids susstimulatory action through irradiation or fuller's earth treatpended are assayed. ment of extracts. The authors wish to repeat their advice This stimulatory action on the growth of the microorganism for the microbiological procedures and state that after a mais evidenced by either criteria of growth response, lactic acid terial is known to contain growth stimulants, it may be titration, or turbidity determination (cell production). avoided through one or more of the following treatments, deSnell and Strong (6) state that when recoveries of pure ribopending on the nature of the foodstuff: flavin were attempted in the presence of the residue from a hvdrochloric acid extraction of liver, the results were high and variable, although the residue showed a riboflavin content of zero. I n view of the authors' results, IO it is believed that their observation can be explained as the action of these growth stimulants. The authors' research confirms the observations of Snell and Strong (6) that the incorporation of inert solid material or milk solids in the assay medium did not affect the recovery of added riboflavin. Pennington, Snell, and Williams ( 5 ) Amumt Of Lecithin Per 10M l Medium reported that urine, oysters, and mush160 Micrograms rooms offered difficulty in that the pantothenic acid assay figures did not agree 4 0 Micrograms satisfactorily when calculated from different levels of assay, but that this diffi2 0 Micrograms culty could be overcome by the incorporation of a n acid-treated supplement of None the material to be assayed in the basal I medium. Research has been reported by Isbell, Wooley, and Fraser (4) demonstrating the inhibiting effect of urea on the growth of L. casei in the presence of OF L. casei TO LECITHIN IN THE PRESENCE OF PANTOFIGURE 2. RESPONSE THENIC ACID riboflavin. TABLEVI. STIMULATORY ACTION OF CERTAIS COMPOTXDS
- --------
1
I-
ANALYTICAL EDITION
August 15, 1942
The use of clarified aqueous extracts. The preliminary extraction of the dried sample with lipoid solvent, after which an autoclaved aqueous suspension of the product may be used for assay. The inclusion of photolyzed extracts of the product to be assayed in the riboflavin assay medium, or the inclusion of an alkaline or acid-treated extract of the product to be assayed in the pantothenic acid assay medium, as has been previously suggested by other workers.
Summarv Growth stimulants have been shown to exist in certain foodstuffs for the growth of L. casei in the microbiological assavs for riboflavin and Dantothenic acid when suboptimum amounts Of the are present in the assay medium* and +‘ttention i’ directed to the presence Of the treatments are discussed to avoid them.
67 1
Literature Cited (1) Eckhardt, R. E., Gyorgy, P., and Johnson, L. V., Proc. SOC. Ezptl. Bid. Med., 46, 405 (1941). (2) Feeney, R. E., and Strong, F. M . , J . B i d . Chem., Proc. xxxviii (1941). (3) Hodson, A. Z., and Norris, L. C., J . Bid. Chem., 131,621 (1939). (4) Ishell, H., Wooley, J. G., and Fraser, H. F., U. S. Pub. Health Service, Pub. Health Rept., 56, 282 (1941). (5) Pennington, D., Snell, E. E., and Williams, R. J., J . BbZ. Chem., 135.213 (1940). (6) Snel1,’E.E., and Strong, F. M . , IND. & ENG.CHEM.,ASAL. ED., 11, 346 (1939). (7) Strong, F. M., Feeney, R. E., and Earle, Ann, Ibid., 13, 566 (1941). PRESENTED before t h e Divisions of Agricultural and Food Chemistry, Biological Chemistry, and Medicinal chemistry, program on Vitamins, a t t h e 102nd Meeting of t h e AMERICANCHEMICAL SOCIETY, Atlantic City, h-.J .
Extraction and Assay of Nicotinic Acid J
from Animal and Plant Tissues Comparison of Methods VERNON H. CHELDELINI
AND
ROBERT R. WILLIAMS, 297 Summit .4ve., Summit, N. J.
I S C E the discovery of the vitamin properties of nicotinic acid in 1937 (6) considerable interest has been focused upon the estimation of this substance. Its widespread importance to clinicians and students of nutrition has made obvious the desirability of having standardized procedures for its quantitative determination wherever it may occur. The practical use of such methods for the control and the manufacture of enriched bread and enriched flour is especially timely a t present. At the present time some 25 methods or modifications of methods for the determination of nicotinic acid have appeared in the literature] and several points of controversy exist as t o what experimental steps are t o be preferred. The present paper is a report of progress with various aspects of this problem as applied to a number of natural substances.
After digestion the samples were steamed 30 minutes t o inactivate the enzymes and remove the benzene. Each sample was filtered through a very thin layer of kieselguhr on a Hirsch filter and the residue was washed with a volume of water equal to about twice that of the filtrate. The combined washings and filtrates were diluted, usually to a concentration of 1 ml. per 25 mg. of fresh tissue. The extracts were placed in tubes or flasks stoppered with cotton plugs, steamed 5 to 10 minutes, and stored in the refrigerator until used. Materials which were received in a homogeneous state, such as milk powder, flour, and other cereals, were not ground or mixed thoroughly prior to the preparation of extracts. The samples of whole wheat flour and bread, white flour, enriched white bread, and dry yeast were obtained as “collaborative samples” from the Research Corporation Committee on Assay Methods. The other materials were obtained in nearby stores and markets.
Enzyme Digestion
Table I shows that in general maximum amounts of nicotinic acid are extracted from tissues by digesting with takadiastase and papain, either separately or in combination. (Since this combination has been found effective in releasing other vitamins from tissues, 2, the authors have employed it routinely in the preparation of extracts for nicotinic acid assays.) Other means, such as autolysis or brief hot water extraction] are somewhat less effective. Extraction with hot water has been deemed insufficient for chemical analysis, because of incomplete hydrolysis of nicotinamide or nicotinuric acid (7, 12, 14). This objection does not hold for the microbiological assays by the Snell and Wright method, since both of the above compounds as well as cozymase are as effective as nicotinic acid in promoting bacterial growth ( I Q ) , but it would seem, then, that nicotinic acid may exist in non-growth-promoting combinations from which it cannot be freed completely by hot water. The utility of nicotinuric acid as a pellagra preventive is at present uncertain, owing to the contradictory findings of Woolley and eo-workers (22) and of Dann and Handler (5) regarding the antiblacktongue potency of this compound. Since its presence in measurable quantities seems to be con-
S
For the liberation of nicotinic acid from possible complexes, autolysis or digestion with added enzymes seemed an attractive procedure. Accordingly, a number of materials were treated with enzymes and the extracts were assayed according to the Snell and Wright microbiological method (19). Seven enzymes were employed: takadiastase, malt diastase, pancreatic amylase, papain, pepsin, trypsin, and pancreatin.
Procedure Samples of fresh tissue were ground several times in a ment chopper, mixed thoroughly, and weighed into sterile tubes or flasks. Each portion was then suspended in ten times its weight of 0.1 per cent buffer solution, the pH of which was chosen to coincide xyith the supposed “optimal pH” for activity of the enzyme. A weighed amount of enzyme equal to 2 per cent, of the wei ht of the tissue sample was added to each flask, a few drops of benzene were added, and the samples were allowed to digest under specified conditions (see Table I). 1 Present address, Austin, Texas.
Department of
Chemistry, University of
Texas,
Results of Enzyme Digestion
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