82
INDUSTRIAL A N D ENGINEERING CHEMISTRY CONCLUSIONS
1. The combination of sulfuric acid with cellulose during cellulose acetate manufacture has long been known but has now been found to be quantitative during the intermediate stages of acetylation. Toward the completion of acetylation and with extended time of reaction, the combined sulfur is gradually replaced by acetyl. 2. The loss in sulfur content during isolation of samples from acetylation dopes is avoided by maintaining anhydrous conditione during the dilution prior to precipitation. , 3. Sulfuric acid combines with cellulose during acetylation to form a ceIlulose acetate acid sulfate. This acid sulfate is remarkably resistant toward hydrolysis with 14% ammonium hydroxide. 4. Samples high in combined sulfur can be reprecipitated without intermittent drying with only slight loss of sulfur. 5 . Extreme variations in combined sulfur content may be found during the early stages of hydrolysis. Rapid addition of water and low temperature favor the retention of combined sulfur. 6. During hydrolysis the combined sulfur drops rapidly to a minimum and then increases slightly on prolonged hydrolysis. The same !eve1 of sulfur content was introduced into a sulfur-free cellulose acetate when hydrolyzed with acetic acid, water, and sulfuric acid in amounts equal to those resulting from a cellulose acetylation with sulfuric acid catalyst. 7. The amount of combined sulfur a t the completion of acetylation and during hydrolysis is roughly proportional over a wide range to the amount of sulfuric acid catalyst used. I n the
Vol. 38, No. 1
event of partial neutralization of catalyst a t the start of hydrolysis, the amount of combined sulfur in the product is determined by the amount of soluble sulfate present during hydrolysis. 8. The conversion of sulfuric to sulfoacetic acid during these cellulose acetylations was negligible. Sulfoacetic acid is a very weak acetylation catalyst which does not combine appreciably with cellulose during acetylation. LITERATURE CITED (1) Caille, A., Chimie & industrie, 12, 441-8 (1924). ( 2 ) Ibid., 13, 11-13 (1925). (3) Clement, L., and RiviBre, C., Bull. SOC. chirn., 4,889-80 (1937). (4) Cross, C. F., and Bevan, E. J., “Researches on Cellulose, 19001905”, pp. 83-93, London, Longmans, Green and Co., 1906. ( 5 ) Deripaako, A.,Cellulosechem., 12,254-63 (1931).
(6) Fabriek van chemische Producten, French Patent 858,324, (Jan. 25, 1929), reproduced in Faust’s “Celluloseverbindungen”, pp. 848-50, Berlin, Julius Springer, 1935. (7) Genung, L. B., and Mallatt, R. C., IND. ENG. CHEM.,ANAL. ED., 13, 369-74 (1941). (8) Malm, C. J., and Tanghe, L. J., Ibid., 14,940-2 (1942). (9) Marschall, A,, and Stauch, H., J . mabromol. Chem., 1, 56-73
(1943).
(10) Ost, H., 2. angew. Chem., 32,66-70.78-9,82-9 (1919). (11) Taniguchi, M., J . SOC.Chem. Ind. Japan, 44, Suppl. binding, 83-5 (1941). on the program of the Division of Cellulose Chemistry of the 1845 Meeting-in-Print, A M E R I C A N CHmxrrcar, SOCIETY.
PEEsENTan
CONTROLLING ORANGE DECAY Thiourea, Thioacetamide, 2-Aminothiazole, and Quinosol in Aqueous Solution J. F. L. CHILDS AND E. A. SIEGLER U . S . D e p a r t m e n t of Agriculture, Orlando, Fla.
P
RELIMINARY reports have been made recently on the effectiveness of thiourea (2)and several other organic compounds (3) in controlling decays of Florida orange fruits. Tests with thiourea over two seasons and additional tests with thioacetamide, 2-aminot%iazole, and quinosol @-hydroxyquinoline sulfate) confirm the results previously reported. Although the investigations are still in progress, the problem of decay control in citrus fruits is so important economically that it is desirable t o report at this time on the status of the work. The first comprehensive publication on control of orange decay appeared in 1908 (17). Since then citrus fruits have taken first place in economic value, and improved methods of handling and increased use of refrigeration in transit have decreased the loss from decay on a per box basis; nevertheless the total loss has increased with the tremendous increase in production. Refrigeration in transit, however essential for the delivery of fruit in a fresh condition to the wholesale markets, merely delays the incidence of decay and transfers the main loss to the retailer, the consumer, and ultimately back to the producer. For this reason there have been many attempts to develop a treatment which would decrease the spoilage of fruit after its arrival at the wholesale market. I n this problem most of the critical etiological factors have been known for many years. The stem-end rots are caused by the fungi D i p b d i a natalensis .and Phomopsis citri; the blue and green molds, respectively, are caused by the fungi Penicillium itaEicum and P. digitatum. A11 of these organisms are dissemiinated by spores. The stem-end rot organisms infect the LLbutton” (receptacle, calyx, and stem parts) of the fruits some time before picking but remain inactive until after harvest; the two Penicil-
l i u m species, whose spores are ubiquitous, generally infect
abrasions on the fruit after harvest. During the past thirty years a number of control measures have been advocated. Some of these have been of practicral value; others have not justified the expense involved. Decay of Florida grapefruit caused by Diplodia and Phomopsis has been greatly reduced by the general adoption of Winston’s recommendation (22) that the fruit be harvested by pulling, which separates the buttons from the fruits. Orange buttons are not easily removed, but it has been demonstrated (14) that pulling oranges is feasible in Florida a t certain seasons when the fruit is “tree ripe”. However, experience has shown that, when fruit is pulled, there is often increased loss from green mold. Despite the degree of control secured by remedial measures applied in the grove, the need for a method which will practically ensure 100% control is apparent when the problem is viewed from the standpoint of the various groups that comprise the citrua industry. Each group recognizes that intangible liabilities are inherent in fruit which arrives at the consumer market with latent, invisible infection; but since the loss by each group is only a fraction of the total, there has been little appreciation of the enormity of the aggregate loss. I n experiments at this laboratory over two seasons i t was found that oranges stored three weeks at 70” F. with about 70 to 80% relative humidity showed 20 to 60% decay. The major portion of such losses is borne by the consumer. One of the earliest control measures used commercially was the borax dip, developed about 1923 (8) This has remained the standard for comparative tests with hundreds of other antiseptics and fungicides. T o be most effective under Florida conditions, i t is essential that borax be applied to the fruit shortly after harvest
INDUSTRIAL AND ENGINEERING CHEMISTRY
January, 1946
and before exposure to ethylene gas (1). Since that procedure is not always convenient, control suffered and the method feil into disuse. A proprietary compound known as Dowicide A (sodium o-phenyl phenate) is used in some packing houses. The same material is also used (16) in conjunction with formaldehyde (to decrease rind injury). I n 1935 Tompkins reported (21)that paper wraps impregnated with diphenyl were effective. The vapors from this material are fungistatic in that they arrest the growth of the organisms causing decay (18,21). . The effectiveness of thiourea in decay control was brought out during a search for a control method that could be applied in the grove. -4 material was sought which would prevent further growth of the latent Diplodia amd Phomopsis infections or would cause abscission of the buttons after the fruit was picked. To this latter objective, many synthetic plant hormones and other physiologically active compounds were tested. F. D. Jones, of American Chemical Paint Company, suggested that benzothiazylthiazoglycolic acid and thiourea were worth a trial. I n field tests thiourea sprayed on the trees failed to cause abscission of the buttons and resulted in foliage injury. I n laboratory tests good decay control was obtained by treating the fruit after harvest. Examination of the literature revealed that thiourea decreased damping-off of cuttings in several susceptible plants, when applied with root-initiating hormones ( 6 ) . It has been reported toxic in vitro to several species of the dermatophyte%, Trichophyton and Achorion (10),and moderately active in the chemotherapy of pathogenic and saprophytic mycoses of animals (13). I t s presence is reported in several species of fungi (12) and in one species of angiosperm (16). Preliminary results with thiourea led to a search for other compounds of equal control value; three other materials, quinosol, thioacetamide, and 2-aminothiazole have given encouraging re-' sults. Though seldom used in agriculture, the fungicidal properties of quinosol are well known (19); it has been reported to control carnation wilt (7), and has been used in the treatment of Dutch elm disease ($4). No report was found in Chemical Abstracts of the use of thioacetamide or 2-aminothiazole as fungicides or bactericides subsequent t o 1935. As with many new pesticides, an important question is the possibility of deleterious effects when treated fruit is consumed. In this lreport there is no intentional implication that any of the four compounds that have given experimental control will successfully meet the public health requirements or other conditions necessary before a new treatment can be put into commercial practice. It should be mentioned, however, that the remarkable results obtained with a t least two of these materials may fore-
83
Thiourea and thioacetamide in 5 % and quinosol in 8% aqueous solution decreased decay of Florida orange fruits, caused by stem-end rot and the b h e and green molds, from approximately 40 to 2% or less, when applied as a momentary dip. Control with 5 % solution of 2-aminothiazole was not consistent. Five per cent of thiourea or of thioacetamide was equally effective in commercial trials when incorporated in the water phase of wax emulsions used on fruit. Similarly 5 % solution of quinosol or of thiourea gave excellent control when applied to the fruit prior to waxing by the solvent-wax process. Penetration of the fruit tissues by these compounds is closely associated with their effective action, and the presence of thiourea in the tissues'of treated fruit wab shown. In the case of thiourea and thioacetamide, the presence of both sulfur and amino groups in the molecule appears essential to effective action when applied as a dip.
cast the discovery of others that will meet all requirements, if one of these does not. Thus the fact that thiourea gave excellent coatrol was the opening wedge which led to tests with the other compounds. Of equal importance is the experimental evidence which offers a logical basis for the fungicidal activity of three of these compounds (3) and should serve as a helpful guide for future selections. The tests have been sufficiently comprehensive to include the salient points considered pertinefit to packing house operations in the event that any of these materials can meet legal requirements and be justified on the basis of cost. METHODS
Oranges used in this work were usually clipped directly from the trees, but several commercially picked lots were obtained from packing houses. The varieties were those grown commercially in Florida such as Parson Brown, Pineapple, Seedling (sweet orange), and Valencia. I n most cases the fruit was exposed t o ethylene vapors for approximately 40 hours before treatment, primarily to predispose it to Diplodia stem-end decay and also because this ('degreening" process is widely used in packing houses a t certain seasons of the year. After treatment the fruit was stored a t 70" F. for 3 weeks, and a t the end of that time the total number .of decayed fruit was determined. The temperature of 70" F. approximates that a t which the fruit would be held in the home and also is favorable for growth of all the decay organisms involved. No attempt was made to control humidity, which normally averaged about 70 to 80%. TABLE I. EFFECTOF CONCENTRATIONS OF COMPOUND ON CONI n the course of ,this work seventy-one compounds in the TROL OF STEM-END DECAYAND PENICILLIUM ROTOF ORANGES following groups were tested: eight inorganic sulfur compounds, -Number of Fruitnine organic acids, six iodine compounds (organic and inorganic), Organic Concn., Stem- PeniTotal Compound % Sound end oilhum Total Rot, % fourteen plant-hormonelike compounds, fifteen organic sulfur compounds, fifteen proprietary organic fungicides or synthetics, and four unclassified organic compounds. Unless otherwise specified, the immersion treatment in all experiments was a momentary dip of 5 seconds or less in water solutions of the several compounds. Vatsol OT, 0.05%, was used as wetting agent, except with quinosol with which it is incompatible. Tests showed that the addition of a wetting agent did not imThio10 87 0 0 87 0.0 acetamide Check 56 14 12 82 31.7 prove decay control, but its use was continued for the sake of 7 85 0 1 86 1.2 5 183 0 2 186 1.7 more uniform coverage and drain-off. Cheok 112 61 15 178 42.6 3
Quinosol
1 Check 8
4 2 1
Check
.
172 88 56 146 142 133 113 90
8 8 37 1 6 16 37 67
3 1 8 2 1 2 1 3
183
97 96 149 149
161
161 150
6.0 9.3 41.7 2.0 4.7 11.9 35.1 40 0
RESULTS
The concentrations of thiourea which control stem-end rots and blue and green mold decays were investigated in a series of tests in which solutions of 0.1 to 10.0% were used. The data in Table I are the aggregate of four to eight replicatidns a t each con-
INDUSTRIAL AND ENGINEERING CHEMISTRY
,84
Vol. 38, No. 1
THIOUREA ON RIND AND IN FRUIT
TABLE 11. Temp.,
F.
130 106 Check
,
5% 2-AMIXOTHIAZOLE ON CONTROI. STEM-END DECAY AND PENICILLIUM ROT E F F E C T OF
------Number Sound 285 269 222
Stemend 5 9
67
of Fruit--Penicillium 9 17 8
Total 299 297
OF
Total Rot, % 4.7 8.8 25 2
centration on several varieties of oranges, and show that with concentrations of 4Yo and above there was no material difference in effectiveness. A concentration of 1.07, shows roughly 50Yo control. Similar experiments mere made t,o determine the effective concentrations of thioacetamide and quinosol. The results with thioacetamide (Table I) indicate that i t closely parallels thiourea in effectiveness. Quinosol is slightly less effective in the control of both types of rots (Table I). Inconsistent results characterized the tests with 2-aminothiazole in that decay control fluctuated from poor to excellent. This compound is relatively insoluble (1.0% a t 130" F. is turbid) but a series of test's were made with 5.0% concentrations a t temperatures of from 82' to 130" F. Above 130" rind injury is encountered. There was no material increase in control from 82" to 106' F., but at 130" increased control was noted, as Table I1 shows. IN VITRO TESTS
No attempt is made to list all the compounds tested in vitro against the four fungi. It is sufficient to say that some showed high t,oxicity to several organisms and some showed toxicity t,o all four; but with the exception of the four compounds discussed in this paper, none showed any control of decay in dipping tests with orange fruits, I n this connection it should be mentioned that urea and thioacetic acid, closely related to thiourea and to thioacetamide, respectively, showed no measurable decay control in several tests. Urea was less toxic than thiourea to all four organisms in vitro; thioacetic was much more toxic than thioacetamide. This emphasizes the fact that the criterion of t,oxicity in vitro, so frequently used in the large-scale testing of fungicides, may have little relat,ion to effect,iveness in controlling decay and other diseases. EFFECT OF TIME OF IMMERSIOIV
The relation of the length of immersion to decay control was investigated by dipping lots of fifty oranges in a 1.0% solution of thiourea for (a) 5 seconds, ( b ) 1 hour, and (c) 4 hours. The experiment was repeated twice. The fruits wcre washed with water after dipping to remove any thiourea that had not penetrated. Total decay a t 3 weeks was ( a ) 31.4, ( b ) 3.9, ( c ) 0.7, and 31.9% in the check lots (Table 111). Under similar conditions of treatment with subsequent washing, 2% thiourea solution was effective with 30-minute immersion (4.6% decay), and 4yo solution Fas effective with 15-minute immersion (2.4Yo decay), whereas the controls averaged 31.6% decay.
TABLE 111. EFFECTO F IhlMERSION P E R I O D I N 1% THIOUREA ON CONTROLOF STEWENDDECAYAND PENICILLIUM ROT Immersion Time 5 8ec. 1 hr.
4 hr.
--
Sound 28 35 35 51
52 43 54
51
Check
44 27 35 38
Number of FruitStemPeniend cillium 22 0 16 0 5 2 3 1 0 0 0 2 0 0 0
0
0 24 16 6
1 0 0
1
Total 50 51 42 55 52
45
54 51
Total Rot, o/c 31.4 3.9
45 51
0.7
45
31.9
51
In, connection with the question of the deleterious,nature of thiourea-treated fruit for human consumption, it was desired to determine what quantity, if any, penetrates into the juice, what quantity penetrates into the rind, and how much remains on the outside. It was found that fifty dry, clean oranges (26.3 pounds), dipped in a 0.05% solut,ion of Vatsol OT and drained, had removed 33 rnl. of solution. Fifty clean, wet oranges (26.0 pounds) removed only 14 ml. Hence when dry fruit is dipped in a 5Yo solution plus 0.05% Vatsol OT, the thiourea is carried away from the dipping tank a t the rate of approximately .125 parts per million of fruit weight, and roughly half that if the fruit was wet prior t o dipping. Thiourea on the rind was determined colorimetrically 21 days after immersion in 5% thiourea plus 0.05% Vatsol OT, by washing off the deposit and analyzing by the Grote method (9). The deposit ranged from 32 to 46 p.p.m. of fruit weight on medium-size fruit (183 to 217 grams). The turbidity and color of orange juice rendered colorimetric determinations difficult; but by making a serics of thiourea dilutions in the juice of untreated fruits from t'he same lot, and matching the nearest concentrations in a Duboscq type colorimeter with a type G filter, indicative results were obtained. It was found that thiourea in concentrations of 9.7 to 12.4 p.p.m. was prcsent in the juice of similarly treated oranges 19 days after immersion. Only one series of orange rind determinations was completed, and the results indicated that thiourea was present.at 16.6 to 20.1 p.p.m. of rind weight. When the time elapsed and the handling involved in making decay counts are considered, the discrepancy between the total thiourea found by a.nalysis and that calculated on the basis of solution removed from the dipping tank is not considered serious. ADAPTATION TO PRACTICE
I n attempting to adapt thiourea and other compounds to commercial practice a number of questions arose, such as choice of concentrations, the possibility of using the compounds in wax emulsions, the effect on control of brushing or polishing before the freshly dipped fruit became dry, and the effect of rinsing off the deposit before waxing. Since a 5% solution of thiourea falls well within the range of effective concentrations, it was used almost entirely in packing house tests. The same is true of thioacetamide and, to a lesser degree, with the other two compounds. However, much of the early work and conscquent elucidation of practical principles was done wit,h thiourea. The natural waxy covering of citrus fruits is largely removed in washing and scrubbing; hence it is customary to wax and polish by one of several methods. A common method consists in dipping the fruit, in proprietary wax emulsions, known as waterwaxes. Thiourea, thioacetamide, and 2-aminothiazole are compatible wit,h several such waxes but quinosol is not. A series of tests were made with thiourea a t 5y0 of the water phase of a wax emulsion containing approximately 5% solids. Thc results in Table IV show that the total number of fruit rotted at the end of 3 weeks was 1.3Yo in the thiourea plus wat,er-wax treatments, and 30.9% in the checks; comparable experiment,s wcre pcrformed with thioacetamide and with 2-aminothiazole. Waterwaxes without thiourea or other decay inhibitors showed no cont,rol of decay. Quinosol, which is not compatible with the water-waxes tested, was used in conjunction with the solvent-waxing method in which the wax is dissolved in a volatile solvent such as bcnzene and applied as a mist-type spray. After momentary immersion in a 57, solut,ion of quinosol, the fruit was allow-ed to dry before exposure to the solvent-wax spray. Total decay in the treated lots was 6.270, and 34.593 in the checks a t the end of 3 weeks (Table V). Quinosol solutions do not wet the rind surface readily. Of the six emulsifiers and wett,ing agents tested, only Tween 80 was compatible with quinosol and was used in these tests a t 0.75
INDUSTRIAL AND ENGINEERING CHEMISTRY
January, 1946
TABLE IV. EFFECTOF 5% ORGANICCOMPOUNDS IN WATERWAX EMULSION ON CONTROL OF STEM-END DECAY AND PENICILLIUM
-~ Treatment Thiourea
Sound
0 0 1 0 0
162 39 44 44 239
__
--
Total Check
Total Thioacetamide
Check
Total 2-Aminothiazole
Total Check
165 40 45 44 241
6
535
125 33 29 31 150
--
26 7 13 12 61
__
14 0 2 1 28
165 40 44 44 239
368
119
45
532
3
44 48 45 43 49 240
1
2 0 0 0
0 4 0
0 0 1
-
-
5
480
31 36 30 32 29 155
11 3 8 3 11 50
5 12 5 8 9 28
47 51 43 43 49 233
-
-
-
-
313
86
67
466
41 3s 36 213
5 11 8 9
2 8 3 16
48 52 47 238
__
-
-
323
33
29
385
31 36 30 153
11 3 8 '64
5 12 5 22
47 51 43 239
250
-_
86
Checks
Thiourea, air-dried solvent-Ax
Total Checks
32 9
-
-_
44
380
Thiourea, brushdried, solventwax 16 1
-
4
23
435
39 36 34 32 135
7 3 9 3 77
2 12 1 8 23
48 51 44 43 235
-
-
_-
99
46
421
59 97 44 41 40 43 38
0 2
0
59 99 45 42 41 43 39
Total
to 1% concentration. The other three compounds can also be adapted to the solvent-wax method, as indicated by the results with thiourea (Table V); the treated lots show 1.6% decay and the checks, 35.4%. Fruit treated with solvent-wax alone has shown no evidence of decay control in comparison with untreated fruit. I n commercial practice, however, fruit to be treated by the solvent-wax must first be dried and polished. This is customarily accomplished by passing the wet fruits over a long series of roller brushes while exposed to currents of warm air. To determine whether such a drying procedure would decrease control of decay, a series of experiments was made with fruit which was brush-dried after treatment and then sprayed with solvent-wad. The results (Table V) show 6.5% decay in the treated fruits and 32.7% in the checks. When the fruits were rinsed before brush drying, there was a further decrease in control with 19.6% decay in the treated fruits and 26.1% in the check lots (Table V). As will be discussed later, a slight modification of the treatmen$ in the solvent-wax method should result in better control. An important question is the length of the period of protection afforded by these chemical treatments. Although most of the experiments were concluded at the end of 3 weeks, occasional lots of thiourea-treated fruit were stored at 38" and 50" F. for 2 weeks, then removed to 70" F. storage, and held for 3 additional weeks for decay development-5 weeks in all. There was no increase in decay over that found in the usual holding period of 3 weeks at 70" F. I n one of the experiments with thiourea the check lot showed 37.2% rot at the end of 3 weeks and was discarded; the thiourea-treated lot, which showed 0.8% decay in this same period, was held an additional 3 weeks. At that time two more fruits had rotted (with green mold), an increase of only
Checks
,
0 1 1 0 1
-_
0 1 0 0 0 0
'
-
-
362
5
1
368
27 76 35 22 21 30 22
28 24 7 18 17 9 16
1 0
56 100 44 41 39 40 39
2 2 1 1
-
-
-1
__
233
119
8
359
1 1 4 0 2 2 3 3
1 1 5 0 3 1 3 0
36 38 30 59 49 53 . 8i 109
-_
-_
432
16
14
462
22 18 32 27 37 42 45 78
15 21 11 28 15 7 16 19
2 1 0
39 40 43 56 56 54 62 97
1 0
'
-
-_
-
--
Total
301
132
14
447
Thiohrea, rinsed, brushed, solvent-wax
52 37 36
14 12 4
2 1 0
68 50 40
-_
-_
-
-
Total
125
30
3
158
Checks
45 37 34
16 15 5
1 4 0
62 56 39
-Total
116
__ 36
_
5
6.2
34.5
1.6
.
35.
64 112
-
1 4 5
Total Rot, %
-
276
34 7
Total 48 55 45 44 243
__
_-
2 3
Number of Fruit-StemPeniend cillium 0 1 2 6 1 0 9 0 2 6
408
-_ Total
30 9
Total
_-
Sound 47 47 44 35 235
__
Total
1 3
47 53 47 43 49 241
6
'
Treatment Quinosol, solventwax
-
469
-
Total Rot, %
-_
1
Total
Total
3 1 0 0 2
-
TABLEV. EFFECTOF 5% CONCENTRATION OF COMPOUND, FOLLOWED BY VARIOUS TREATMENTS, ON CONTROL OF STEM-END AND PENICILLIUM ROTS ,-.-
528
Total
ROT
Number of Fruit-StemPenicillium end
85
6.5
32.
19.6
__ 157
26.1
0.8%. The fruit was in reasonably good condition both in appearance and for consumption. DISCUSSION AND CONCLUSIONS
Some of the factors that affect decay control should be reviewed to provide a perspective for evaluating the results. These factors are best considered in relation to their effect on the type and amount of decay encountered. Although all four types of fungi are more abundant in the older groves, there is considerable fluctuation in the prevalence of individual fungus species. The interrelation of grove site, season of harvest, maturity of picked fruit, and effect of gassing with ethylene is shown by the various types and amounfs of decay in different samples of fruit. I n view of this wide variability, the uniformly high degree of control in the experiments reported here is surprising. There was little tendency in these tests for the total decay of the treated lots to fluctuate as it did in the untreated lots. Fortunately the total percentage of decay was remarkably uni-
86
INDUSTRIAL AND ENGINEERING CHEMISTRY
form in randomized samples of fruit from a single picking in a given grove. Occasional fruit rot (less than 1% of the total) caused by various other organisms was classified as side rots, but data are omitted from the tables for the sake of brevity since their control parallels that of the other rots. The experiments on the relative effectiveness of thiourea, thiacetamide, and quinosol a t different concentrations (Table I) provided the basis for the selection of concentrations for use in small-scale packing house tests. It is seen that 5% is well within the range of effective concentrations. At this concentration a slight dilution in the course of an experiment, occasioned by dipping fruit still wet from a previous rinsing, should affect the results very little and a t the same time permits a short immersion period. It may prove feasible under commercial conditions, however, to decrease concentrations somewhat without detriment except for an occasional lot of heavily infected fruit. With lower concentrations i t should be possible to regulate penetration and consequent amount of decay by varying length of immersion period as indicated in Table 111. If tolerance limits for the chemical are established, this method might serve a useful purpose. Penetration of fruit tissues is definitely associated with the highly effective action of these compounds in controlling citrus decay. It was noted earlier that infection by the stem-end rot fungi takes place some time prior to picking, and the fungi are beyond the reach of strictly protective fungicides a t that t i e , as indicated by lack of control by dipping in solutions of limesulfur, HE-175, Puratized LN, or 604. Borax may penetrate citrus fruit tissues to a moderate degree, judging by the results obtained commercially. I n the borax treatment ( 1 ) satisfactory control i s obtained only when the fruit is dipped soon after picking. If treatment is delayed several days by necessary packing house handling, the degree of control drops to a point of questionable benefit. The colorimetric analyses, in which approximately 12 p.p.m. of thiourea was found in the juice and 20 p.p.m. io the rind of treated oranges, constitute direct evidence that thiourea penetrates into orange tissues. Supporting evidence is the fact that prolonged immersion (I hour) in 1.0% solution gave control whereas momentary dipping a t this concentration did not (Table 111). The striking results obtained with thiourea and‘ with thioacetamide incorporated in water waxes (1.3 and 2.3% decay, respectively, in treated fruit and 30.9 and 32.9%, respectively, in the checks, Table IV) are believed due in part to the degree of penetration which this method of waxing allows. Here the treated fruit is dried slowly by currents of warm air as it is conveyed to the polishing brushes. By the time it reaches the brushes, sufficient penetration has taken place so that removal of the excess thiourea or thioacetamide in polishing does not impair control. I n thc solvent-wax method the immiscibility of thiourea solution with the solvent requires that it be applied as a separate treatment. The fruit was dipped momentarily, drained, then rapidly brushed dry, and polished before exposure to the wax ’spray. Hence much of the thiourea was removed before i t could penetrate sufficiently and decay control was impaired (6.5% decay in treated lots and 32.7% in checks, as is shown in Table 11). On the basis of limited tests (two), if the treated fruit is air-dried before brushing and polishing, excellent control can be obtained with the solvent-wax method (1,3y0 decay in treated and 34.0% in check, for a total of 314 oranges). From the results obtained with these materials in conjunction with the water-wax and the solvent-wax processes, there is reason to believe that one or more of the compounds can be adapted for use with other fruit waxing and polishing procedures. I n order to remove all excess thiourea from the rind surface, in view of possible future tolerance limits, oranges were rinsed immediately after dipping in 5 % solution, then dried, polished, and waxed by the solvent-wax method. Decay control was greatly decreased (Table V), and again the limiting factor appeared to be
Vol. 38, No. 1
insufficient time for penetration. There is evidence, however, that with longer immersion to permit adequate penetration, rinsing would not materially lessen control. Tests with thioacetamide, 2-aminothiazole, and quinosol were not so extensive as those with thiourea. In so far as the conditions of the tests with thioacetamide and with quinosol were similar to those with thiourea, the results were also similar. The importance of the relation between chemical structure and fungicidal activity warrants discussion. It was noted previously (2, 3) that thiourea, thioacetamide, and 2-aminothiazole are characterized by the presence of divalent sulfur and one or more amino groups in the molecule, and that in the case of thiourea and thioacetamide, a t least, the presence of both is necessary for decay control. Whether fungicidal activity is dependent on ( a ) the tautomerization of these compounds to the thiol form, ( b ) the formation of a ring structure, such as HN=C-XH, for
\/
S thiourea, as hypothesized by Cristol (4, or ( c ) the fact that there is a constant oscillation between two possible structures, is uncertain. It is of interest that the three most effective materials are highly water soluble, whereas many fungicidal compounds which are relatively insoluble in water have given little or no control of orange decays; in this connection Rivier’s observation (20) that thiourea tautomerizes to the thiol form only in water solution is noted. The fact that the sulfur in 2-aminothiazole is tied up in a rather stable ring structure may account in part for the mediocre control obtained with it. It was noted that the very volatile thioacetic acid is highly toxic to all four fungi when in contact with them in vitro. When fruit was dipped in water solutions (1.25, 2.5, 5%) of thioacetic acid, however, no control was obtained. It may be that t h e fungicidal activity of thiourea, for example, is due simply to t h e presence of the sulfur but that the amino group irj essential for penetration. Du Buy (6) found that “mixtures of hormones with substances which improve penetration (urea, thiourea, etc.) increase the uptake and translocation as measured on oat coleoptiles with the marker method.” The same happens when t h e NHI group is incorporated in the hormone (6). Zentmeyer advanced the theory ( W ) ,and has given evidence to support it, that the fungistatic action of quinosol is due to i t s quality of forming insoluble salts with the trace elements essential for the enzymatic action of the fungus. Horsfall and Zentmeyer found that many of the reagents, including thiourea, which are used in the determination of essential metals, are. fungistatic (11). Although the data and conclusions have been discussed at length, one of the main purposes of this paper is to point out that three materials are now known to give excellent control of orange rots on clipped fruit. When fruit is pulled, Penicillium rots are generally increased, as noted above, due t o frequent tearing of the rind and consequent exposure of freshly wounded surfaces. Control treatments on such fruit would not necessarily be 80 effective as those reported here on clipped fruit. At present the commercial use of any of these compounds is ill-advised since there is some question as to their complete harmlessness. Steps are being taken, however, to investigate the toxicological properties of the amounts of thiourea and thioacetamide found in treated citrus fruits.
LITERXTURE
CITED
(1) Brooks,Chas., J. Agr. Research, 68, 363 (1944). (2) Childs, J. F. L., and Siegler, E. A,, Phytopathology, 34, 98% (1944). (3) Childs, J. F. L., and Siegler, E. A., Science, 102, 68 (1945). (4) Cristol, P., Signeurin, R., and Fourcade, J., Compt. rend., 200, 2223 (1935). ( 5 ) Du Buy, H. G . , Am. J. Botany, 25, 11s (1938). (6) Du Buy, H. G., Chronica Bctan., 6 , 80 (1940). (7) Fron, G., Rev. hort. (Paris), 109, 647 (1937).
(8) Fulton, H. R., and Bowman, J. J., J. Agr. Reasarch, 28, 96L (1924).
January, 1946
INDUSTRIAL AND ENGINEERING CHEMISTRY
(9) Grote, I. W., J . Biol. Chem., 93, 25 (1931). (10) Hachiya, T., and Nishimura, J., J . Pharm. SOC.J a p a n , 52, 756 (1932) (Eng. trans., pp. 89-91). (11) Horsfall, J. G., and Zentmeyer, G. A,, Phytopathology, 34, 1004 (1944). (12) Klein, G., and Farkass, E.,-Oesterr. Botan. Gaz., 79 (2), 107 (1930). (13) Mayer, R. L., Rev. mddicale, franc., 1941, 3-19. (14) Meckstroth, G. R., Citrus Znd., 25, 9 (1944). (15) Miller, E. V., Proc. Florida State Hort. Soc., 57, 144 (1944). (16) Ovcharov, K. E., Compt. rend. m a d . sci. U.R.S.S., 16, 461 (1937).
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(17) Powell, G. H., et al., U. S. Dept. Agr., Bur. Plant Ind., Bull. 123 (1908). (18) Ramsey, G. B., Smith, M. A., and Heiberg, B. C., Botan. Gaz., 106 ( l ) , 74 (1944). (19) Rigler, N. E., and Greathouse, G. A., IND. ENQ.CHEM.,33, 693 (1941). (20) Ilivier, H., and Borel, James, Helv. Chim. Acta, 11, 1219 (1928). (21) Tompkins, R. G., Rep. Food Inv. Bd. 1935, Dept. Sci. Indus, Res. London, p. 129 (1935). (22) Winston, J. R., U. S. Dept. Agr., Circ. 396 (1936). (23) Zentmeyer, G. A.,'Science, 100 (2596), 294 (1944). (24) Zentmeyer, G. A., and Horsfall, J . G., Phgtopathology, 33, 16 (1943).
Vitamin Retention in Processed Meat i EFFECT
. .
OFSTORAGE
J. F. FEASTER AND J. M. JACKSON American Can C o m p a n y , Maywood, Ill.
D. A. GREENWOOD AND H. R. KRAYBILL American Meat I n s t i t u t e , Universiey of Chicago, Ill. Determination of the B vitamin contents of samples of processed pork luncheon meat during storage for a year at 45", 70°, and 98" F. indicated no significant losses of riboflavin, niacin, or pantothenic acid. Thiamine retention during the year was excellent at 45' F. (89 to 100~o), fair at 70" (59 to 7601,), and relatively poor at 98" (12 to 20y0). The percentage of thiamine retained during storage was found to be independent of the initial thiamine content and independent of the heat processing schedule employed for the preservation of the product.
T
HE annual production of approximately two billion pounds
of canned meats, packed under federal inspection in 1942, 1943, and 1944, is more than double the prewar figure. I n view of the increased consumption by the armed forces and civilian populations, information regarding the influence of storage times and temperaturea on the nutritive values of canned pork luncheon meat is of timely interest. A previous paper in this series (9) presented the effects of heat processing on the B vitamins of pork luncheon meat, according to various time and temperature schedules. Certain lots of canned meat from that study were stored under controlled conditions, and the changes found in vitamin values during storage are reported in this paper. Published data demonstrating the effects of storage on the vitamin contents of canned meats are meager. The changes in vitamin contents of canned pork, dehydrated pork, and dehydrated beef during storage a t temperatures ranging from -20" t o 145" F. (-29" to 63" C.) were studied by Rice and Robinson (8). At the end of 10-month storage a t 38", 80' and 98" F. (3", 27", and 37' C.) the canned product retained 100, 56, and 29% of the thiamine present in the product immediately after canning. At higher temperatures there was almost complete destruction of thiamine after 10-month storage. During storage the thiamine of dehydrated pork and beef was less stable than the thiamine in canned pork. I n coqtrast with thiamine, the niacin, riboflavin, and pantothenic acid contents of these meat products stored for 10 months a t temperatures up to 99' F. (37" C.) were found to be essentially 100% of that contained in the meat after processing. Rice and co-workers (6) found that dehydrated mixtures of 67% pork and 33% cereals (wheat, barley, or soybean flour) or dehydrated milk yielded higher retentions of thiamine than dehydrated pork alone. Products rich in carbohydrate materials
stabilized the thiamine in dehydrated pork. Furthermore, the retention of thiamine in dehydrated pork was correlated with the moisture content of the samples between 0 and 6% moisture levels. Low moisture content was correlated with high thiamine retention during storage [e.g., 0, 2, 4, 6, and 9% moisture i n dehydrated pork gave 91,60,23,9, and 11%retention of thiamine during 7-day storage a t 120" F. (49' C.)]. Recent reports suggest that the type of product and time and temperature under which canned foods are stored may be important factors influencing the degree t o which the B vitamins are retained- Data presented by Knott (4) BUggeSt that 50% of the thiamine in evaporated milk may be lost during storage for one year. High-temperature storage appears to be unfavorable to retention of the vitamins in tomato juice, lima beans, and whole-kernel corn (3). The degree to which thiamine was re-, tained varied from product to product, with 20 t o 35% remaining after one year a t 110' F. (43" C.). Riboflavin, niacin, and pantothenic acid were retained to the extent of 75% or more in these vegetables stored for one year a t temperatures up t o 110' F. A d a m and Smith (1) summariaed the United States AgriculturaI Experiment Station research on the vitamin content of preserved foods. The extent of vitamin loss during storage depends upon the storage conditions and the vitamin. Ascorbic acid and thiamine are the vitamins more likely to be affected by processing and storage a t higher temperatures. Information has not been previously reported regarding the effects of variations in heat treatments, concentration of the vitamin in the product, or size of the can in which pork luncheon meat is packed on retention of the B vitamins during storage. The experiments described in this paper were designed t o study the effect of time and temperature of storage on the B vitamins in pork luncheon meat which had been packed in various sized cans and heat-processed according t o different time and temperature schedules. MATERIALS AND ANALYTICAL METHODS
The samples of canned pork luncheon meat were prepared from one of the lots of b e a t described previously (8). The heat processing e uipment, methods, and analytical procedures employed were %e same as those used before. The heat processin schedules are listed in Table I. The samples coded K, L, and were procesded according to a time-temperature schedule calculated t o simulate the heat treatments received by meat in different positions in a 21/~-pound (404 X 510) can (8) processed
id!
