THE B VITAMINS IN HONEY1 Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021

GEORGE KITZES, H. A. SCHUETTE, AND G. A. ELVEHJEM Departments of Chemistry ana Biochemistry, University of Wisconsin, Madison

(Eeceived for publication March 25, 1943)

Honey is a widely used food —undoubtedly due to its sweetness, pleasant taste and aroma. Since honey is a natural substance and is food for the honeybee, there has been much speculation as to its value in the nutrition of man. Honey is prepared from the nectar of flowers by worker bees and stored in the honeycomb. Although honey is derived primarily from nectaries of flowers, we cannot overlook the part the honeybee plays in storing the nectar in the hive and in processing it to its final form. Thus honey should be considered the product of the bee processed from a plant product. The analysis of honey has shown that it consists of approxi mately 75-80% sugar, mainly invert sugar, which is a conver sion product of sucrose. Schuette et al. ( '32, '37, '38, '39) have shown the presence of mineral elements in honey. Notable among these are iron, copper, sodium, potassium, manganese, calcium, magnesium, and phosphorus, all of which have been shown to be essential to good nutrition of animals. Since honey is basically derived from the flower and moreover is a food for the bee, much speculation has arisen as to its vitamin content, especially because great emphasis has been placed on the im portance of vitamins in human nutrition. It seems that this problem has interested workers for two decades, many of whom have reported the absence of vitamins A, B, C, D, and E 1Published with the approval of the Director of the Wisconsin Agricultural Ex periment Station. Supported in part by a grant from the American Honey Institute. The authors are indebted to Dr. Frank M. Strong for valuable suggestions regard ing the microbiological assays. 241 242 GEORGE KITZES AND OTHERS in honey. These results may be interpreted in the light of the methods employed, which have been much improved in recent years. Recently chemical determinations of vitamin C in honey

have been made by Griebel ('38). He reported 160-280 mg. Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 ascorbic acid per 100 gm. of mint honey and 7-22 mg. per 100 gm. of other honeys. Griebel and Hess ( '39) identified the strong reducing substance in thyme and mint honeys as as corbic acid. They reported 311.2mg. and 102.6mg. of this vitamin per 100gm. of the respective honeys. Buckwheat honeys contained from 7.36-18.6 mg. ascorbic acid per 100 gm. They (Griebel and Hess, '40) determined also the ascorbic acid content of the nectar of the above plants. It was shown that there is a considerable loss of vitamin C when nectar is converted into honey. Schuette and Kaye ( '42) investigated a series of honeys and found 0.55-20.9 mg. of vitamin C per 100 gm. The degree of destruction of vitamin C in honey was also studied. It was noted that there was a decrease in the total ascorbic acid content of honey after pure ascorbic acid was added. The loss was greatly increased in the honeys placed in diffused light. Hay dak and Palmer ( '42 a) reported the vitamin content of honeys of varied nectar source and origin. They reported the following values per 100 gm. of honey : thiamine, 2.1-9.1 Mg-; riboflavin, 35-145 Mg-; pyridoxine, 210-480 Mg.; pantothenic acid, 25-192 Mg.; nicotinic acid, 4-94 mg. ; ascorbic acid, 0.6-5.4 mg. The authors of this paper will attempt to give a clear pic ture of the vitamins in honey obtained by the improved methods of vitamin assay. These include the more recent michrochemical and microbiological methods, some of which have been modified in the University of Wisconsin. Prelim inary investigations did not reveal the presence of the fat- soluble vitamins ; therefore only the contents of water-soluble vitamins were determined. Together with numerous samples of honey we have analyzed pollen and royal jelly, two natural substances also serving as food for bees. THE B VITAMINS IN HONEY 243

EXPERIMENTAL The honeys under investigation were obtained from the col lection in the Laboratory of Foods and Sanitation of the Chemistry Department of the University of Wisconsin, from local grocery stores, and from beekeepers in various localities Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 of the United States. Many of these honeys were received in the comb and extracted in the laboratory by straining through layers of cheesecloth. The wax was washed with distilled water and dried for further analysis. The nicotinic acid assays were made by the microbiological method of Snell and "Wright ('41). Samples were prepared as follows : 25 gm. of honey were diluted to 75 ml. with distilled water, autoclaved for 15 minutes at 15 pounds pressure, and then diluted to the necessary volume. It was found that aque ous extraction gave the same results as acid or alkali. The latter tended to produce excessive caramelization of the sugars in the honey. The effect of the large percentage of sugar on the assay method and also the effect of caramelization were investigated. The sugar that was being introduced by the sample of honey had no apparent effect on the assay, but the caramelization which resulted from acid or alkali treatment interfered very much with the determination of the end-point in the titration of the lactic acid produced. Recovery of added nicotinic acid ranged from 90% to 110%. Pantothenic acid was determined by the microbiological method of Strong, Feeney and Earle ('41). Preparation of the sample was carried out as follows : 25 gm. of honey were diluted to 75 ml. and neutralized to pH 7.0 with NaOH. It was autoclaved for 15 minutes at 15 pounds pressure and diluted to the necessary volume. Enzyme digestion of the honey with clarase did not produce a significant change in the results. Ether extraction of the diluted honey did not remove any stimulatory or inhibitory substances. Recovery of added pantothenic acid ranged from 90% to 110%. Values are ex pressed in micrograms of calcium pantothenate per 100 gm. 244 GEORGE KITZES AND OTHEIÃŽS

Riboflavin was determined by the microbiological method of Snell and Strong ( '39). Preparation of the sample was as follows : 25 gm. of honey were diluted to 75 ml. with distilled water and autoclaved for 15 minutes at 15 pounds pressure.

The use of acid was avoided for the reasons explained for the Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 nicotinic assay. Enzyme digestion with clarase did not alter the value significantly. It should be mentioned that the natural pH range of honey is 4-5 ; therefore the honey dilution was acid during the autoclaving, thereby reducing any possible destruction of the riboflavin. Ether extraction of the honey dilution did not remove any stimulatory or inhibitory substances. Thiamine was determined by the Hennessy-Cerecedo thio- chrome method as modified by the Research Corporation Com mittee on the Thiochrome Method (Hennessy, '42). A Coleman photofluorometer was used to measure the fluorescence of the thiochrome produced by the oxidation of the thiamine. This instrument can accurately determine 0.02 pg- of thiamine. Recovery of added thiamine ranged from 85% to 105%. Values are expressed in micrograms of thiamine-hydrochloride. Pyridoxine was determined by the microbiological method of Atkin, Schultz, Williams, and Frey ('43). In this method the response of a strain of yeast to pyridoxine is measured by turbidity readings. The range of sensitivity of the assay is 5 to 40 mpg. Enzyme digestion was used to release any bound pyridoxine. Recovery of added pyridoxine ranged from 95% to 105%. Values are expressed in micrograms of pyridoxine- hydrochloride per 100gm. Biotin was determined by the microbiological method of Snell, Eakin, and Williams ('40), using yeast and measuring the growth of the organism by turbidity readings. The assays were conducted by Miss Josephine Gardner of the Biochemis try Department of the University of Wisconsin. Folie acid was determined by the method of Mitchell and Snell ('41), using the streptococcus lactis organism. The as says were conducted by Mr. T. D. Luckey of the Biochemistry Department of the University of \\7isconsin. The folie acid THE B VITAMINS IN HONEY 245 values are expressed in this paper as micrograms of folie acid based on a solubilized liver standard with a potency of 40,000. The samples of pollen which were analyzed were obtained from Professor Farrar of the Department of Economic En tomology of the University of Wisconsin. One sample of pollen was a mixture from an area around Madison, Wiscon Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 sin. The other sample was a mixture of pollen from Baton Rouge, Louisiana. The sample of royal jelly was obtained in a dry form from Dr. H. B. Parks of the Texas Agricultural Experiment Station.

RESULTS AND DISCUSSION Table 1 gives the results of analyses of forty samples of honeys from different regions of the United States. Although the source of the honey is also listed, it is understood that honey is usually a mixture of nectars. The floral source listed, however, is the predominant one. An examination of the val ues obtained for the five members of the B-complex showed great variation among the samples. The greatest variation was in the nicotinic acid content. In one instance, a sample of honey contained nine times as much nicotinic acid as an other. The least variation occurred in -the thiamine and pyri- doxine values, but even here the variation was as much as fivefold. An attempt was made to correlate the vitamin con tent with floral source, origin, and color-grade. This proved fruitless for we did not have a sufficiently large number of samples of honey of one locality to enable us to say that one set of data is typical of that source. The wide variation of the vitamin contents precluded any attempt to correlate the values with the color-grade of the honeys. Table 2 presents a summary of the vitamin content of the honeys examined. It also shows the differences in the vitamin content of the old honeys as compared with the new samples. In the course of the investigation for pantothenic acid, it be came apparent that the pantothenic acid values for a large number of honeys were lower than those of the rest. It was found that the group of honeys that had been obtained from Vï * •* ^ •CO N CO 01 *CC -OWOOiOiOO^IMW t>. _ GO CO . tO OìCO O Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021

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246 THE B VITAMINS IN HONEY 247 the Chemistry Department contained 50% less pantothenic acid than those received from various beekeepers. The former were of the crops of 1935-1939 and had been stored in a dark closet at room temperature. The honeys obtained this year were of the crops of 1940-1942. Many of these samples were received in the comb; others had been extracted before ship Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 ment. The difference in the pantothenic acid content of these two groups may be explained by the fact that this vitamin is unstable in an acid solution. Honey is naturally acid, having a pH range of 4-5. This acidity is not very great, but any

TABLE 2 Comparison of aged and new honey«. All values are in micrograms per 100 gm.

BIBO- nvr NICOTINIO TH1A- PYRI- FbAVIN ACID MINE DOXINK

Honeys of year» 1935-1939 Mean 21.8 ±3.0 20.4 ±2.4 124.4 ±11.6 3.5 ±.25 7.6 ±.6fi Range 9-64 9 —60 63 —600 1.4 —6.2 4 —14

Honeys of years 1940-1942 Mean 26.3 ±2.1 54.4 ±3.6 108.5 ±9.1 4.4 ±.50 10.0 ±1.1 Ranfre 7 —60 20 _ 360 72 —590 2.2 —12 4 —27 pantothenic acid that might be in the honey could be destroyed slowly over a period of years. This is of little significance to the beekeeper or the housewife who do not keep honey for long periods of time. A few honey samples were analyzed for their biotin and folie acid content, two new members of the B-complex whose importance in nutrition is being investigated. Traces of biotin and folie acid wore found, with the average values of 0.066 Mg. and 3 Mg.per 100 gm., respectively. Since the amounts found were very small as compared to other foods, we did not examine many honeys. Table 3 presents a comparison of the vitamin content of the samples of honey of the years 1940-1942 with pollen and royal jelly. It may be noted that the royal jelly, a milky-white sub- 248 GEORGE KITZES AND OTHERS stance secreted by the worker bee is unusually high in panto- thenic acid and biotin. This may be related to the rapid meta bolism in the young bee larvae which depend on this substance for sustenance the first 3 days after emergence from the egg.

Comparison of the vitamin content of pollen with that of honey Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 revealed that in general pollen was a hundred times richer in the B vitamins than honey. This suggested that the vitamins in the honey may be due partially to the pollen present in each sample. All natural honeys contain a certain amount of pollen, which at times is very small in quantity. Haydak and Palmer ('42a) have found that a commercial process of clarifying

TABLE 3 Comparison of honey with pollen and royal jetty. All values are in mic'rograms per gram.

NO. PANTO- NICO- OF THENIC - TINIC DX« TD SAMPLES ACID '"VIN MINE DOXINE ACID

HonevPollenBoyal

jelly19210.5527.0320.00.2616.728.01.1100.0111.00.0446.018.00.109.010.20.000660.254.10.030.5 honey reduces the vitamin contents 33-50% of the original values. When we filtered diluted honey, through Whatman no. 40 filter paper subsequent to its preparation for assay, we found that the riboflavin content was reduced 40% and the nico- tinic acid values 25%. It is suggested that the large variation in the vitamin content of the honeys may be due in part to the variable amount of pollen present. The values reported in this publication compare favorably with those of other workers. Pearson ( '42) reports an aver age value of 30.3 \ig. of pantothenic acid per gram of pollen and 511 pg. per gram (dry weight) of royal jelly. Cheldelin and Williams ( '42) have analyzed royal jelly for the vitamins of the B complex. We find that our results agree very well with those of the Texas workers. Haydak and Palmer ('42b) report that chemical determinations did not show even traces of pyridoxine either in bee bread or royal jelly. The values THE B VITAMINS IN HONEY 249 they obtained for the amount of nicotinic acid and pyridoxine found in honey are many times higher than our results. The discrepancy is so great that it is believed to be due to the methods of analyses employed. It is significant to note that Haydak and Palmer used chemical methods for both the nico tinic acid and pyridoxine determinations. It is probable that Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 the treatment of the honey sample necessary to free the vita mins gave rise to interfering substances.

SUMMARY Microchemical and microbiological determinations showed the presence in honey of thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine, biotin, and folie acid. The varia tion among samples was very large, due perhaps to the source of the honey and the number of pollen grains present. Com parison of new and aged honeys revealed a decrease in the pantothenic acid content of the latter. Pollen and royal jelly have also been assayed for these vitamins and have been found to be good sources of the B-complex. Royal jelly is very rich in biotin and pantothenic acid, which may be significant in the metabolism of the young bee.

LITERATURE CITED ATKIN, L., A. S. SCHULTZ,W. L. WILLIAMSANDC. N. FREY 1943 Yeast micro biological method for the determination of pyridoxine. Ind. and Eng. Chem. (anal.), vol. 15, p. 141. CHELDELIN,V. H., ANDR. J. WILLIAMS 1942 The B vitamin content of foods. The University of Texas Publication No. 4237, p. 105. GRIEBELC. 1938 Vitamin C-enthaltende Honige. Ztschr. Untersuch. Lebensm., vol. 75, p. 417. GBIEBEL,C., ANDG. HESS 1939 Vitamin C-enthaltende Honige. Ztschr. Unter such. Lebensm., vol. 78, p. 308. 1940 Der C-Vitamingehalt des Blutennecktars bestimmter Labiaten. Ztschr. Untersuch. Lebensm., vol. 79, p. 168. HAYDAK,M. H., ANDL. S. PALMER 1942 a Vitamin content of honeys. 'J. Nutri tion, vol. 23, p. 581. — 1942 b Royal jelly and bee bread as sources of vitamins B„B„B„ C, nicotinic acid and pantothenic acid. J. Econ. Entom., vol. 35, p. 319. HENNESSY,D. J. 1942 The determination of thiamin in cereal products. "Cereal Chemists Bull. ' ', vol. 2. 250 GEORGE KITZES AND OTHERS

SCHUETTE,H. A., ANDD. J. HuENiNK 1937 Mineral constituents of honey. II. Phosphorus, calcium, magnesium. Food Research, vol. 2, p. 529. SCHUETTE,H. A., AND F. KATE 1942 Master's Thesis. Univ. Wisconsin. Un published. SCHUETTE,H. A., AND KATHOKAREMY 1932 Degree of pigmentation and its

probable relationship to the mineral constituents of honey. J. Am. Downloaded from https://academic.oup.com/jn/article/26/3/241/4726305 by guest on 27 September 2021 Chem. Soe., vol. 54, p. 2909. SCHUETTE,H. A., ANDR. E. TRILLER 1938 Mineral constituents of honey. III. Sulfur and chlorine. Food Research, vol. 3, p. 543. SCHUETTE,H. A., ANDW. W. WOESSNER 1939 Mineral constituents of honey. IV. Sodium and potassium. Food Research, vol. 4, p. 349. SNELL, E. E., R. E. EAKIN ANDR. J. WILLIAMS 1940 A microbiological method for the determination of biotin. J. Am. Chem. Soc., vol. 62, p. 175. SNELL,E. E., ANDF. M. STRONO 1939 A microbiological assay for riboflavin. J. Ind. Eng. Chem. (anal.), vol. 11, p. 346. SNELL, E. E., ANDL. D. WRIGHT 1941 A microbiological method for the deter mination of nicotinic acid. J. Biol. Chem., vol. 139, p. 625. STRONG,F. M., R. E. FEENET ANDA. EARLE 1941 Microbiological assay for pan- tothenic acid. J. Ind. Eng. Chem. (anal.), vol. 13, p. 566.