while particular examples have been selected for the purposes of illustration , one of ordinary skill in the art will recognize from the information set forth herein that the materials and methods of the present invention are applicable to a wide variety of treatment circumstances . accordingly , one of ordinary skill in the art will recognize that various modifications may be made to the specific materials and concentrations described hereinafter without departing from the scope of the present invention , which is defined more particularly in the appended claims . in accordance with the preferred embodiments of the present invention , sulfur dioxide , nitrogen , and ammonia gas , when applied are used in the following amounts , as measured at standard temperature and pressure : in testing the materials and methods of the present invention , various experimental techniques were employed to determine the effectiveness of control and test compositions on samples of moist wheat . unless otherwise specified , all wheat samples tested were adjusted to a hydration of about 40 % moisture . further all controls were surface sterilized with 5 % naocl , which is a normal procedure for sprouting studies to prevent microbial interference due to mold growth . such surface sterilization was not necessary to test samples due to the antimicrobial effects of the test treatments . the following tests on wheat correspond to similar results obtained in prior experimentation with corn . initial testing was carried out with hard wheat which was soaked in water to facilitate sprouting . the soaked wheat was then subjected to gas treatments and placed over moist filter papers in petri dishes . these petri dishes were incubated at 37 ° c . for two days , at the end of which the number of sprouted kernels were counted and percentage sprouting calculated . the results are set forth in table i . table i______________________________________example treatments per 100 g wheat % sprouting______________________________________1 control 702 100 ml so . sub . 2 03 50 ml so . sub . 2 04 50 ml nh . sub . 3 455 50 ml so . sub . 2 + 50 ml nh . sub . 3 1 . 36 50 ml nh . sub . 3 + 50 ml so . sub . 2 257 60 ml so . sub . 2 08 60 ml so . sub . 2 + 60 ml nh . sub . 3 09 70 ml so . sub . 2 010 70 ml so . sub . 2 + 70 ml nh . sub . 3 0______________________________________ as seen from example 1 ( control ), about 70 % sprouting will occur in the absence of an effective treatment . treatments with 50 ml of ammonia gas per 100 g of wheat reduced sprouting to 45 %. applications of sulfur dioxide gas or sulfur dioxide gas followed by equivalent volumes of ammonia gas , proved to be quite effective ( see examples 2 , 3 , 5 , 7 , 8 , 9 , and 10 ). the importance in the order of application of such gases is demonstrated by example 6 , wherein comparatively reduced effectiveness was demonstrated when an application of ammonia gas was immediately followed by sulfur dioxide . a similar experiment on wheat was conducted where wheat was allowed to sprout first , and then was subjected to the above treatments . the results obtained indicated that each of the above treatments were correspondingly effective in arresting further sprouting . to investigate the effect of the above - mentioned treatments on dry grain , wheat containing less than about 15 % moisture was treated with 50 ml of sulfur dioxide followed by 50 ml of ammonia gas per 100 g . the wheat was then soaked and permitted to incubate , whereupon 100 % sprouting was observed , as compared with 1 . 3 % for the pre - soaked wheat . accordingly , it was determined that higher amounts of gases may be needed to prevent 100 % sprouting when treating dry wheat . in order to further investigate the effects of applications of individual gases and gas combinations , soaked wheat was subjected to the following treatments and treatment combinations , as indicated in table ii . table ii______________________________________example treatments / 100 g wheat % sprouting______________________________________effects of single gases11 control 8412 co . sub . 2 ( 100 ml ) 7813 n . sub . 2 ( 100 ml ) 9014 nh . sub . 3 ( 100 ml ) 8015 so . sub . 2 ( 100 ml ) 016 so . sub . 2 ( 80 ml ) 017 so . sub . 2 ( 60 ml ) 018 so . sub . 2 ( 50 ml ) 019 so . sub . 2 ( 40 ml ) 020 so . sub . 2 ( 30 ml ) 321 so . sub . 2 ( 20 ml ) 2022 so . sub . 2 ( 8 ml ) 3823 so . sub . 2 ( 4 ml ) 72effects of gas combinations24 co . sub . 2 ( 50 ml ) + so . sub . 2 ( 20 ml ) 2025 co . sub . 2 ( 50 ml ) + so . sub . 2 ( 30 ml ) 626 co . sub . 2 ( 50 ml ) + so . sub . 2 ( 50 ml ) 027 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 20 ml ) 628 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 30 ml ) 029 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 50 ml ) 030 nh . sub . 3 ( 50 ml ) + co . sub . 2 ( 50 ml ) 2531 nh . sub . 3 ( 80 ml ) + so . sub . 2 ( 40 ml ) 25 + co . sub . 2 ( 40 ml ) 32 so . sub . 2 ( 30 ml ) + n . sub . 2 ( 50 ml ) 833 so . sub . 2 ( 10 ml ) + n . sub . 2 ( 50 ml ) 3534 so . sub . 2 ( 20 ml ) + n . sub . 2 ( 40 ml ) 1235 so . sub . 2 ( 20 ml ) + n . sub . 2 ( 20 ml ) 436 so . sub . 2 ( 20 ml ) + n . sub . 2 ( 10 ml ) 937 so . sub . 2 ( 10 ml ) + n . sub . 2 ( 50 ml ) 7338 so . sub . 2 ( 10 ml ) + n . sub . 2 ( 40 ml ) 7539 so . sub . 2 ( 10 ml ) + n . sub . 2 ( 100 ml ) 7540 so . sub . 2 ( 50 ml ) + nh . sub . 3 ( 50 ml ) 041 so . sub . 2 ( 40 ml ) + nh . sub . 3 ( 40 ml ) 142 so . sub . 2 ( 30 ml ) + nh . sub . 3 ( 30 ml ) 743 so . sub . 2 ( 20 ml ) + nh . sub . 3 ( 20 ml ) 8044 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 40 ml ) 1 + nh . sub . 3 ( 40 ml ) 45 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 30 ml ) 6 + nh . sub . 3 ( 30 ml ) 46 so . sub . 2 ( 20 ml ) + n . sub . 2 ( 50 ml ) + 1 nh . sub . 3 ( 20 ml ) 47 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 20 ml ) 13 + nh . sub . 3 ( 20 ml ) 48 so . sub . 2 ( 40 ml ) + n . sub . 2 ( 40 ml ) + 4 nh . sub . 3 ( 40 ml ) 49 so . sub . 2 ( 30 ml ) + n . sub . 2 ( 30 ml ) + 36 nh . sub . 3 ( 30 ml ) 50 so . sub . 2 ( 20 ml ) + n . sub . 2 ( 20 ml ) + 82 nh . sub . 3 ( 20 ml ) ______________________________________ ( 1 ) applications of air , carbon dioxide , nitrogen and ammonia gases ( examples 11 - 14 ) are not particularly effective to prevent sprouting of moist wheat ; ( 2 ) applications of sulfur dioxide in amounts above 30 ml per 100 g are effective to prevent wheat sprouting ( see examples 15 - 23 ); ( 3 ) carbon dioxide does not effect sprouting inhibition caused by sulfur dioxide ( examples 24 - 26 ); ( 4 ) prior applications of nitrogen gas were shown to enhance the sensitivity of wheat to sprouting inhibition caused by sulfur dioxide ( compare examples 27 - 29 to examples 21 , 20 and 18 ); ( 5 ) applications of ammonia gas and carbon dioxide are more active in combination than either of those gases alone ( compare example 30 to examples 12 and 14 ); ( 6 ) when sulfur dioxide gas follows the application of ammonia and preceeds the application of carbon dioxide , the combination is not as effective in preventing sprouting as a sole application of the corresponding volume of sulfur dioxide gas ( compare example 31 to example 19 ); ( 7 ) when nitrogen applications follow applications of sulfur dioxide , some improvement in wheat sprouting inhibition percentages may be obtained , particularly when corresponding volumes of sulfur dioxide and nitrogen gas are utilized ( compare examples 34 , 35 and 36 to example 21 ). such improvements are dependent upon concentration ( amount ) and are particularly exhibited with applications of 20 ml of sulfur dioxide . at 10 ml of sulfur dioxide subsequent applications of nitrogen gas produced inferior results ( compare examples 33 and 37 - 39 to example 22 ). therefore , when only nitrogen gas and sulfur dioxide are utilized to treat the subject grain , sulfur dioxide applications preferably should be in the amounts of about 15 - 35 ml per 100 g of grain , and more preferably 20 - 30 ml per 100 g of grain . in these instances , 10 - 40 ml of nitrogen gas , preferably 20 - 30 ml per 100 g of grain should be utilized in these treatments ; ( 8 ) the effects of ammonia gas treatment when preceeded by sulfur dioxide and nitrogen gas applications are demonstrated in examples 44 - 50 . when sulfur dioxide gas in amounts of 20 - 30 ml ( examples 46 , 49 and 50 ) preceeded the application of 50 ml of nitrogen gas which in turn preceeded the application of 20 - 30 ml ammonia gas , the best results were obtained ( example 46 ). this may be explained by the fact that the nitrogen gas , in addition to sensitizing the wheat to sprouting inhibition , also aids in the distribution of sulfur dioxide within the grain mass . when nitrogen gas was applied prior to so 2 better results were obtained when ammonia gas was not utilized as part of the treatment . compare examples 45 and 47 to examples 27 and 28 . however , in view of the lowered corrosiveness of the treatments , and color , flavor , and taste advantages gained thereby , tests utilizing ammonia gas compare favorably to tests of corresponding amounts of just sulfur dioxide ( compare examples 44 , 45 , and 47 to examples 19 , 20 , and 21 , respectively ). ( 9 ) except at low amounts ( i . e . 20 ml ), applications of so 2 gas followed by ammonia gas produced sprouting percentages comparable to those of just sulfur dioxide gas ( compare examples 40 - 42 to examples 18 - 21 ). thus , table ii clearly indicates that nitrogen gas , sulfur dioxide gas and ammonia gas , the orders of introduction of these gases , and the relative amounts of these gases introduced into the grain mass to be treated , all bear upon the effectiveness of such treatments in preventing and arresting sprouting . from table ii it may also be noted that a substantially concurrent application of ammonia gas with carbon dioxide results in a substantial reduction in sprouting percentages . in order to investigate the effect of the timing of applications of certain of the above - mentioned gases on sprouting , tests were conducted with controlled time intervals between applications of the below - indicated gases . the results of these tests are set forth in table iii . table iii______________________________________time interval for reversibility of sprouting by nh . sub . 3example treatments / 100 g wheat % sprouting______________________________________51 control 8052 so . sub . 2 ( 100 ml ) 053 nh . sub . 3 ( 100 ml ) 8054 so . sub . 2 ( 40 ml ) 055 so . sub . 2 ( 40 ml ) + nh . sub . 3 ( 40 ml ) 80 immediate56 so . sub . 2 ( 60 ml ) + nh . sub . 3 ( 60 ml ) 60 immediate57 so . sub . 2 ( 100 ml ) + nh . sub . 3 ( 100 ml ) 0 immediate58 so . sub . 2 ( 40 ml ) + 10 min . + 10 nh . sub . 3 ( 40 ml ) 59 so . sub . 2 ( 60 ml ) + 10 min . + 0 nh . sub . 3 ( 60 ml ) 60 so . sub . 2 ( 40 ml ) + 30 min . + 0 nh . sub . 3 ( 40 ml ) 61 so . sub . 2 ( 60 ml ) + 30 min . + 0 nh . sub . 3 ( 60 ml ) ______________________________________ examples 51 - 54 in table iii are provided as controls . as seen in examples 55 and 56 , simultaneous applications of sulfur dioxide gas and corresponding amounts of ammonia gas cause inhibition of the otherwise expected effect of that concentration of sulfur dioxide gas . as the amount of applied so 2 increases , simultaneous introduction of a corresponding amount of ammonia gas is less effective to inhibit the action of so 2 . when ammonia gas application follows the application of sulfur dioxide gas by 10 to 30 minutes the &# 34 ; interference &# 34 ; between so 2 and ammonia gas is not observed ( compare examples 55 and 56 to examples 58 - 61 ). thus , it may be concluded that delays of at least 10 minutes following the application of sulfur dioxide gas are preferred prior to the introduction of ammonia gas into the grain mass to be treated . in order to investigate the mechanism by which nitrogen gas application sensitizes grain to the action of sulfur dioxide , wheat ( after naocl surface sterilization ) was transferred to a round bottom flask and enough sterile water was added to rehydrate the wheat to 40 % moisture . the air from the round bottom flask was removed by means of a syringe and nitrogen was injected into the round bottom flask , after which the flask incubated over night . the next morning , wheat was removed from the round bottom flask and was subsequently subjected to the following treatments , and plated on petri dishes to sprout . the results are set forth in table iv : table iv______________________________________effects of soaking the wheat in nitrogenatmosphere vs air atmosphere % sproutingexample treatments / 100 g wheat nitrogen air______________________________________62 control 96 8263 so . sub . 2 ( 20 ml ) 28 1964 so . sub . 2 ( 30 ml ) 0 065 so . sub . 2 ( 40 ml ) 0 066 so . sub . 2 ( 20 ml ) + nh . sub . 3 ( 20 ml ) 69 7067 so . sub . 2 ( 30 ml ) + nh . sub . 3 ( 30 ml ) 15 1068 so . sub . 2 ( 40 ml ) + nh . sub . 3 ( 40 ml ) 8 369 n . sub . 2 ( 50 ml ) + so . sub . 2 ( 30 ml ) 3 570 so . sub . 2 ( 30 ml ) + n . sub . 2 ( 50 ml ) 1 2______________________________________ when compared to controls which were subjected to similar treatments in air , it will be seen that prior soaking of the grain in nitrogen may slightly stimulate sprouting ( see example 62 ), but otherwise does not exert any substantial effect on any of the indicated treatments . thus , it has been concluded that nitrogen gas should be applied to the grain mass substantially concurrently with the application of sulfur dioxide . preferably , nitrogen gas should be applied to the treated grain mass no earlier than 1 hour before or later than 1 hour after the application of sulfur dioxide , and preferably no earlier than 30 minutes before to 15 minutes after the application of sulfur dioxide . other treatments have also been investigated to determine whether sprouting of wheat can be inhibited through the application of other compounds . in table v , treatments with ammonium bisulfate - acidified ammonium bisulfite and sodium bisulfate - acidified sodium bisulfite were found to be effective except when immediately followed by applications of ammonia gas . table v______________________________________example treatments / 100 g wheat % sprouting______________________________________effects of nahso . sub . 3 ( 10 % solution - 8 % hso . sub . 3 . sup .- ion ) 71 control 8072 nahso . sub . 3 ( 2 . 8 ml ) ph 4 . 0 673 nahso . sub . 3 ( 2 . 8 ml ) ph 3 . 0 674 nahso . sub . 3 ( 2 . 8 ml ) ph 4 . 0 + 77 nh . sub . 3 ( 60 ml ) 75 nahso . sub . 3 ( 2 . 8 ml ) ph 3 . 0 + 72 nh . sub . 3 ( 60 ml ) 76 nahso . sub . 3 ( 5 . 6 ml ) ph 4 . 0 077 nahso . sub . 3 ( 5 . 6 ml ) ph 3 . 0 078 nahso . sub . 3 ( 5 . 6 ml ) ph 4 . 0 + 37 nh . sub . 3 ( 120 ml ) 79 nahso . sub . 3 ( 5 . 6 ml ) ph 3 . 0 + 39 nh . sub . 3 ( 120 ml ) effects of ammonium bisulfite ( 50 % solution ) -(≈ 41 % hso . sub . 3 . sup .- ion ) 80 nh . sub . 4 hso . sub . 3 ( 0 . 4 ml ) ph 4 . 0 181 nh . sub . 4 hso . sub . 3 ( 0 . 4 ml ) + nh . sub . 3 ( 60 ml ) 44______________________________________ by way of comparison , the effects of other salts on inhibition of sprouting is set forth in table vi . table vi______________________________________effects of other salts on inhibition of sproutingexample treatments / 100 g wheat % sprouting______________________________________82 control 8583 10 % na . sub . 2 so . sub . 3 ( 1 . 7 ml ) ph 9 . 6 8384 10 % nahso . sub . 4 ( 1 . 85 ml ) ph 1 . 0 8485 10 % k . sub . 2 s . sub . 2 o . sub . 5 ( 1 . 5 ml ) ph 3 . 9 4086 20 % na . sub . 2 so . sub . 4 ( 2 . 2 ml ) ph 8 . 5 8587 10 % na . sub . 2 so . sub . 3 ( 3 . 4 ml ) + 9 nh . sub . 3 ( 60 ml ) 88 10 % nahso . sub . 4 ( 3 . 7 ml ) + 84 nh . sub . 3 ( 60 ml ) 89 10 % k . sub . 2 s . sub . 2 o . sub . 5 ( 3 . 0 ml ) + 43 nh . sub . 3 ( 60 ml ) 90 20 % na . sub . 2 so . sub . 4 ( 4 . 4 ml ) + 24 nh . sub . 3 ( 60 ml ) ______________________________________ as seen from table vi , only examples 87 and 90 show substantial improvements in percent sprouting . as seen from the above , numerous simple , inexpensive methods of treating grain , particularly wheat , are disclosed which are effective in preventing and arresting sprouting . when nitrogen gas is utilized in combination with sulfur dioxide gas , surprisingly low volumes of sulfur dioxide gas are effective in preventing sprouting . when ammonia gas is utilized following an application of either sulfur dioxide gas or sulfur dioxide gas and nitrogen gas , a non - corrosive , sprouting - inhibited product fit for human consumption results . applications of ammonium or sodium bisulfite solutions similarly have been found to prevent sprouting .
novel methods for treating grain , particularly wheat , to prevent sprouting are disclosed . these methods include applications of sulfur dioxide gas to grain in amounts which are effective to prevent sprouting . in an alternate method , nitrogen gas is applied to grain to enhance the anti - sprouting activity of sulfur dioxide . ammonia gas may also be subseqently perfused into the treated grain mass in amounts which are effective to preserve the color , odor and flavor of the grain , and to minimize the corrosiveness of the aforementioned sulfur dioxide treatment . concurrent application of carbon dioxide gas and ammonia gas is also disclosed as reducing sprouting .