August 15, 1942
ANALYTICAL EDITION
taken before the buret is removed from the titrated solution. and when the titration is resumed the buret must be read again after rejection of the solution contained in the tip.
(3) Kirk, P. L., I b i d . , 14,1 (1933). (4) Linderstrdrn-Lang, K.,and Holter, H., Compt. rend. trav. lab. Carlsberg, 19,No. 14 (1933). ( 5 ) Llacer. A. J.. and Sozzi. J. A.. see A. A. Benedetti-Pichler. “Introduction t o the Microtechnique of Inorganic Analysis”; New York, John Wiley & Sons, 1942. (6) Schwarz, K., Mikrochemie, 13, 1 (1933). (7) Struszynski, M.,Przemysl Chem., 20,53 (1936). I
Literature Cited (1) Heathy, N. G.,Mikrochemie, 26, 147 (1939). (2) Hybbinette, A,-G., and Benedetti-Pichler, A. A,, I b i d . , Emich Memorial Issue (1941).
663
,
Nicotinic Acid Content of Cereals and Cereal
Products Microbiological Method of Assay JOHN S. ANDREWS, HAROLD M. BOYD, AND WILLIS A. GORTNER General Mills Research Laboratories, Minneapolis, Minn.
T
HE dietary position of cereals and cereal products in
relation to their pellegra-preventive attributes has recently been given considerable prominence by the inclusion of nicotinic acid or niacin in the list of required components of enriched flour (3). (The word “niacin” has recently been accepted by the Federal Security Agency as a nontechnical synonym for the term “nicotinic acid”.) This action has focused attention not only on the natural nicotinic acid content of flours designed for enrichment but also on the relative position occupied by the whole wheat from which these flours are derived, together with the other products obtained from milling. TABLE I. NICOTINICACID ASSAYSOF EXTRACTS OF WHOLE WHEATFLOUR Type of Extraction
Nicotinic Acid Irg./g.
37 75 37 72 70
Search for information on this subject has been handicapped by the uncertainty of the suitability of the analytical methods used for estimating nicotinic acid, particularly chemical procedures. Kodicek (4) has emphasized the discrepancies observed in the analysis of cereals and pointed out the adverse role played by “chromogenic” substances of unknown composition. Similar observations have also been reported by Waisman and Elvehjem (IO). Melnick, Oser, and Siegel (6) have applied an adaptation of the MelnickField chemical method (6) to the determination of flour and bread and reported fairly good agreement between the values thus obtained and those resulting from the application of the microbiological method of Snell and Wright (9). This observation lends support to the validity of the assay data and suggests a satisfactory specificity for the two types of procedures when the specified precautions are followed in the chemical method. This compatibility leaves the analyst with some latitude in the choice of methodology, with reasonable assurance that the selection will not introduce serious errors into the final assay results. I n the authors’ laboratory the chemical and microbiological methods have been investigated for the purpose of comparing their applicability to cereal analyses, for both controlling the
manufacture of enriched flours and evaluating other grain products. While in general fairly good agreement has been observed between the two types of procedures, values obtained chemically tend to be high. This is due to the difficulty of ensuring the complete removal of or compensation for “chromogenic” substances which are naturally present or are formed during extraction processes. For this reason microbiological assays have appeared to be more acceptable in instances where large discrepancies are observed. Recent studies of the microbiological procedure have given rise to speculation about the accuracy of the nicotinic acid values thereby obtained. Depending on the procedure employed for extracting the cereal sample, assays varying more than twofold in magnitude can be produced. Snell and Wright (9) in their original description of the micrpbiological method reported that alkaline extraction of cereals, in contrast to animal tissues, gives somewhat higher values than those obtained when water ia employed for the extracting medium. They apparently attributed this to differences in extraction efficiency, since they found that finely ground samples gave comparable values by both extraction procedures. Oser, Melnick, and Siegel ( 7 ) also reported that alkaline extraction gives higher values but attributed this to hydrolysis rather. than extraction since treatment of aqueous extracts with alkali raises the apparent nicotinic acid content to that found by direct extraction with sodium hydroxide. This explanation assumes that hydrolysis imparts greater availability t o the test organism of some watersoluble derivative or precursor of nicotinic acid. The nature of this substance is unknown, since nicotinamide, cozymase, and nicotinuric acid all possess equivalent nicotinic acid activity to Lactobacillus arabinosus 17-5 (9). The remaining derivative, trigonelline, is inactive and is not readily converted to an active form. The authors’ observations have shown even wider differences than those previously reported. These results have necessitated a careful inquiry into the relative behavior of various cereal products and a more extended evaluation of extraction procedures. The data presented in Table I demonstrate the influence of aqueous, acid, and alkaline solvents on the microbiological assay of a typical whole wheat flour.
Extraction Procedures The aqueous extracts were prepared by autoclaving 1-gram samples with 90 ml. of water for 15 minutes at 6.8-kg. (15-pound) pressure. After cooling to about 50” C., 1 ml. of 6 per cent takadiastase solution was added, and the mixture was allowed to cool at room temperature (30 to 40 minutes), made up to 100 ml., and filtered.
664
INDUSTRIAL AND ENGINEERING CHEMISTRY
Alkaline extraction employed 50 ml. of 1.5 per cent sodium hydroxide. After autoclaving, the suspensions were neutralized to pH 6.5 with hydrochloric acid, made up t o 100 ml., and filtered. Digestion with takadiastase after neutralization facilitates subsequent filtration and yields the same results. Where alkaline hydrolysis was carried out, 50 ml. of the filtered aqueous extracts were treated with 2.5 ml. of 30 per cent sodium hydroxide and autoclaved for 15 minutes at 6.8-kg. (15-pound)pressure. After neutralizing they were made up to 100 ml. Acid extracts were prepared by autoclaving 1-gram samples with 50 ml. of 0.1 N sulfuric acid or 25 ml. of 3 N hydrochloric acid. The resulting suspensions were neutralized t o pH 6.5 with sodium hydroxide, made up to 100 ml., and filtered. No digestion with takadiastase is required t o facilitate filtration or improve extraction. The assay values given in Table I show that water and dilute acid are comparable extraction media. Alkali and stronger acids are also comparable but yield twofold higher results. In confirmation of the observation reported by Oser, Melnick, and Siege1 (7), alkaline hydrolysis of the clear aqueous extracts raises the nicotinic acid assays to essentially those obtained by direct extraction with alkali. Thus the factor responsible for the increase in apparent nicotinic acid is water-soluble but requires the action of alkali to exert its growth-promoting effect on the test organism. The fact that 3 N acid also yields high values demonstrates that the unknown factor is also susceptible to acid hydrolysis. This observation is of interest since it suggests that trigonelline is not the substance responsible for the increased values. In order to confirm this suggestion trigonelline was subjected to the aqueous, acid, and alkaline extractions described above. I n no instance was any nicotinic acid activity observed. Since these studies indicated that fairly strong acid was required to produce the “hydrolytic effect”, the action of alkali was examined. Autoclaved aqueous suspensions of a whole wheat flour were treated for various periods of time with different concentrations of sodium hydroxide a t room temperature and on the steam bath. The results are shown in Table 11.
Vol. 14, No. 8
grade flours, wheat germ, starch, and gluten. For this reason it is doubtful if the effect of alkali noted above can be attributed to nonnicotinic acid growth-stimulating substances. In view of the possibility that stimulators might exert a variable influence on the test organism over a prolonged growth period, a comparison of the assay values of aqueous and alkaline extracts of whole wheat flour was made a t intervals during 78 hours’ incubation a t 37” C. Titration of acidity was carried out after 23, 30, 47, 54, 71, and 78 hours. The average for the aqueous extract was 31.5 micrograms per gram, and for the alkaline extract, 72 micrograms per gram. Since individual values did not show variations greater than * 5 per cent it is obvious that growth rates in the extracts are very similar to those in the nicotinic acid standards. If any stimulator is present in the extracts it is not revealed by this type of test. TABLE111. DIALYSIS OF AQ~JEOUS EXTRACTS OF WHOLE WHEAT FLOUR Nicotinic Acid in Dialyzate Ba Ratio B/A
Dialysis Time of
Aa
0 1 2 3 4 24
6.‘1 4.7 3.4 2.8
a
..
9.4 6.1 4.6 3.8
..
1:54 1.30 1.35 1.36
..
Nicotinic Acid in Undiffused Extract A B Ratio B/.4
26.7 20.0 16.5 12.6 10.4 5.0
61.7 47.3 41.5 36.7 30.5 17.2
2.31 2.36 2.52 2.92 2.93 3.45
A Total B
26.7 26.1 27.3 26.8 27.4
..
61.7 56.7 57.0 56.8 54.4
..
A, before hydrolysis: B, after hydrolysis with alkali.
Several attempts have been made to separate the naturally active nicotinic acid compounds from the factor which is activated by hydrolysis. Extraction of whole wheat flour with alcohol has been tried by a variety of methods: Soxhlet extraction, direct heating with solvent, and addition of large volumes of alcohol to the sample after gelatinization in water and digestion with takadiastase. In all instances a separation into alcohol-soluble and alcohol-insoluble fractions was obtained but both exhibited nicotinic acid activity which was OF TIME,TEMPERATURE, AND CONCENTRATABLE11. EFFECT markedly enhanced by treatment with alkali. TION OF ALKALIox THE NICOTINIC ACIDASSAY In another experiment acidified aqueous and alkaline exNicotinic Acid tracts were shaken with Lloyd’s reagent. The adsorbates 30 minutes on 5 minutes a t room Concentration of NaOH temperature steam bath were washed thoroughly and assayed directly by suspending % Microgram8 per Oram in the culture medium. That obtained from the aqueous .. None 28 extract gave an assay value of 38 micrograms per gram (based 67 66 0.25 67 on the whole wheat flour) while that from the alkaline extract 66 0.50 72 66 1.00 gave a value of 73 micrograms per gram. The solutions 70 70 1.50 remaining after adsorption showed no nicotinic acid activity. Even after alkaline hydrolysis no growth was observed with the aqueous extract which had been treated with the Lloyd’s It is apparent that the effect of alkali is very pronounced, reagent. This demonstrates that adsorption of the naturally since the lowest concentration exerts in 5 minutes the same active nicotinic acid compounds and the unknown factor, both effect as six times this concentration for much longer periods before and after hydrolysis, occurs simultaneously and coma t higher temperatures. Thus the factor produced by hypletely under the experimental conditions employed. For drolysis is formed easily and completely by alkali in contrast this reason separation of these constituents cannot be accomto the slower production by acid. plished by such treatment. In order to gain some insight into the probable nature of Dialysis experiments have given the most promising rethis factor, recovery experiments were carried out using each sults. Aqueous extracts of whole wheat flour were dialyzed of the several extraction procedures. While such experiagainst water by means of cellophane membranes. The data ments are of limited value for critically evaluating methodobtained from a typical experiment are shown in Table 111. ology, it was expected that variable recoveries would result if It is evident that both nicotinic acid factors diffuse through the factor was a growth-promoter unrelated to nicotinic acid. the cellophane (No. 450), but a t different rates. The natuSimilar studies demonstrated the presence of nonflavin rdly active components dialyze more rapidly, resulting in an growth factors in the microbiological assay of riboflavin in appreciable increase in the ratio of the hydrolyzable factor to cereals ( 2 ) . the “free” nicotinic acid compounds in the undiffused fraction. Regardless of the type of extraction used, recoveries of The four dialyzates represent the amounts diffused during added nicotinic acid were quantitative within the limits of experimental error. This finding mas observed on a variety the first, second, third, and fourth hours, respectively. The slower diffusion rate of the hydrolyzable factor is reflected in of cereal products, including whole wheat, patent and low-
August 15, 1942
ANALYTICAL EDITION
the lower ratio between this and the naturallyvactive portion. Dialysis in these preliminary tests has thus reduced the concentration of hydrolyzable factor in the dialyzate to 40 per cent of the free nicotinic acid, and has concentrated it in the original extract to nearly 200 per cent. Studies are now under way to improve this separation further, so that each factor can individually be studied physiologically. While most of the authors’ studies have been confined to whole wheat flour, a few assays have been carried out on other cereal products, including a complete set of the fractions separated in the commercial milling of wheat (Table IV). The values found by the two extraction procedures are of interest since they indicate the ratio of natural nicotinic acid activity to the total activity after hydrolysis. While the limit of experimental error urges caution in interpreting a significant differentiation between the three flours and the three feeds, the position of germ is definitely outstanding. The response shown by treatment with alkali is much below that observed for the other wheat fractions. Since the germ sample contained appreciable quantities of other portions of wheat, which should contribute to the effect produced by alkaline extraction, it seemed probable that a more highly purified preparation would give the same assay value regardless of the extraction procedure. Such actually proved to be the case. A sample of germ of 85 to 90 per cent purity gave by aqueous extraction a value of 42.4 micrograms per gram; b y alkaline extraction, 43.8 micrograms per gram. It seems rather significant that one part of the wheat berry should differ so markedly from all the rest, but no explanation can be offered in the light of our present knowledge. This behavior of wheat germ is analogous to that exhibited by animal tissues, since Snell and Wright (9) have shown that alkali does not increase the assay values of a variety of animal organs. Nor is yeast influenced by the extraction procedure since practically identical values of 215 and 204 micrograms per gram were obtained by aqueous and alkaline extraction, respectively.
665
applied to the evaluation of cereals and cereal products. The resistance of the hydrolyzable factor to dilute acids suggests that gastric digestion would fail to liberate activity while the sensitivity to alkali would operate favorably in subsequent digestive processes. This assumes, of course, that the unknown factor exerts activity comparable to that displayed toward the Lactobacillus arabinosus.
Go
80 ’O
D€AC€NT
90
I J
EXTQACTfON
OF VITAMINS IN PRODCCT~ OF FIGURE 1. DISTRIBUTION WHEATMILLING
In the event that only the naturally active nicotinic acid and nicotinic acid derivatives are of nutritional value, careful consideration must be given to the condition employed for extraction. While the authors’ data are not yet complete, it is apparent that the less active form is hydrolyzable even by water. In one experiment 15 minutes’ autoclaving gave a TABLE IV. DISTRIBUTION OF SICOTINIC ACIDIN THE PRODUCTS value of 33 micrograms per gram; extending the autoclaving OF WHEATMILLIXG time to one hour increased the value to 45 micrograms per (As determined b y microbiological assay of aqueous and alkaline extracts) gram. For this reason more drastic extraction conditions Ratio of % of Total may be expected to yield high results, intermediate between Nicotinic Acid Alkaline t o N. A. in Mill Mill Fraction Aqueous Alkaline Aqueous Fraction those representing natural activity and total activity after PQ.10. PLl./L7. complete hydrolysis. If, on the other hand, the total activity 1.83 10.2 Patent flour 12 6.6 is nutritionally available, extraction should be performed 1.94 3.2 13.4 26 First clear flour 1 .so 4.7 46 Second clear flour 83 either with alkali or fairly strong acid in order to ensure com74 120 1.62 7.6 Red dog plete utilization by the test organism. 1.66 17.8 96 159 Shorts 1.68 56.3 197 Bran 330 Returning to the assays of the mill fractions, the values 1.21 0.2 56 Germ 68 1.82 100.0 Wheat 38 70 expressing per cent of total nicotinic acid are averages of those calculated from the assays of each type of extract. This While in such materials as yeast, germ, and animal organs seemed entirely justified, since the two sets were very similar. both vegetable and animal sources are represented and The greatest variation was observed in the case of patent accordingly cannot be differentiated on such a basis, it is flour, where the aqueous assay values showed it to contain 9.8 rather striking that in all cases we are dealing with “living” per cent of the total nicotinic acid; alkaline assay values or potentially living tissues. On the other hand, such prodwere 10.6 per cent. This agreement between the two exucts as bran and flour entirely lack this attribute and are traction procedures is further indication that a homogeneous present in wheat primarily to protect the germ and supply it relation exists between the two forms of nicotinic acid activwith food during the early stages of germination. This ity, since it is difficult to conceive of a condition where nonsuggests a possible differentiation of two forms of nicotinic nicotinic acid factors would so closely parallel the nonuniform acid activity based on the cellular aggregates with which they distribution of nicotinic acid activity in the various fracare associated, biologically active tissues possessing a more tions. Once again, however, the evidence requires more direct readily available form than “storage” tissues where hydrolysis substantiation. is required to liberate complete activity. The distribution of nicotinic acid (Figure 1) in the products For the purpose of estimating the importance of cereals as of milling differs from that of either thiamin or riboflavin dietary sources of nicotinic acid, it is of vital importance to ( I , 8). The germ is relatively low in nicotinic acid content, decide the nutritional roles played by each of these types of since thiamin and riboflavin are present in six- to eight-fold activity. -4re both fully utilized by animal organisms, or is concentrations of those existing in whole wheat. This is only the naturally active form available? This question compensated for by the relatively higher concentration in the must be answered before present analytical methods can be bran. which of all the commercial mill fractions is the richest
666
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.
