detoxification of gossypol by rummen microorganism

8/14/2019 Detoxification of Gossypol by Rummen Microorganism

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The M echanism of Gossypol Detoxification byR um inant A nim als1RAYM OND REISER AND H WEI C. FUD epartm ent of B iochem istry and N utrition,T ex as A gr ic ultu ra l E xpe rim en t S ta tio n,C oll eg e S ta tio n, T ex as

It has been known for many years thatcottonseed m eal containing free gossypolproduces toxic symptoms if fed to swine(W ithers and Carruth, '15, '18) or chicks(Couch et al., '55; R igdon et al., '58) atsignificant levels, and that gossypol hasbeen dem onstrated to be toxic to other anim als (W ithers and C arruth,'18; S ch wartze,'24; Eagle, '50). But it has also been demonstrated that relatively large am ounts ofgossypol c ontained in cottonseed m eal m aybe fed to ruminant animals with no signof toxicity (C ranford, '10; Jones et al., '41;R am sey and M iles, '53).The m echanism whereby gossypol is detoxified by the ruminant animal has rem ained unknown. Since m ost differencesin m etabolism between rum inant and non-rum inant animals may be traced to theactivity of rum en m icroorganism s, the possibility was considered that these organisms might also act upon gossypol to destroy it in som e m anner.Previous work in this laboratory hasshown that dietary polyunsaturated fattyacids may be hydrogenated by rumenliquor,2 or in the rum en in vivo (Reiser andReddy, '56). This suggested that gossypolm ight also be reduced in the highly reducing environm ent of the rum en.

EXPERIMENTALPreparation of soluble gossypol. About50 mg of crystalline gossypol3 were dissolved in 15 m l of alcohol. Twenty milli-liters of glycerol were added and thealcohol evaporated on a steam bath underreduced pressure. The product is a clearsol which may be mixed with water in anyp ro po rtio n w ith ou t p re cip ita tio n.Determination of free and bound gossypol. Gossypol assays, free and bound,

were determ ined by the method of Ponsand H offpauir ('57 ).Determination of free t-amino group.This assay was made by the method ofB aliga et al. ('59).Preparation of rumen liquor. Rumenliquor was obtained from freshly slaughtered beefs, then strained through cheesecloth and used as soon as possible.Aerobic and anaerobic incubation. Todeterm ine whether anaerobic or aerobicorganisms have a different influence ongossypol, 3.5 m l of the strained liquor w eremixed with 1.25 ml of gossypol sol. Inone set of experiments 0.5 ml of 4% glucose and in another set 0.5 ml of 95%alcohol were added as a source of energy.The results of the study are given intable 1. There was no loss of total gossypol, but the free gossypol was significantly reduced, indicating a binding effectbut no destruction. There was probablyno significant difference betw een the aerobic and anaerobic cultures, bringing intoquestion bacterial participation in the freegossypo l disa ppearance. N everthele ss glucose apparently stim ulated m ore activitythan alcohol. However, different rum enliquors were used and there was no control run w ithout glucose or alcohol.Effect of energy source. The influenceof the nature of the added energy sourcewas further studied by anaerobic incubation. The results (table 2) do not supportthe superior activity of glucose. This sug-Received for publication Septem ber 18, 1961.1 Supported, in part, by a grant from the OscarJohns ton Foundat ion.- R eiser, R . 1951 Hydrog nation of polyunsaturated fatty acids by the rum inant. Federation Proc.,1 0: 2 36 (a bs tr ac t).3 G ossypol acetate w as sent to us from the SouthernU tiliza tio n R esea rch a nd D ev elo pm en t D iv isio n, U SDA,New Orleans, Louisiana by Dr. T. H . Hopper whosek in dn ess w e g ra tefu lly a ck no wled ge.

J . N UTRIT ION, 7 6: " 62 215


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216 RAYMOND REISER AND HWEI C. FUTABLE 1

Effect of aerobic and anaerobic incubation of rum en fluid and gossypol on the disappearanceof total and free gossypolD isappearance ofossypolIncubationconditions^Anaerobic

AerobicTestFree% 62 .9s752.1J.3Total%00TestFree% 29 2. 6364.123,4Total%001 Incubation at 37C for 48 hours.2 Test 1. Rumen liquor, 3.5 ml; 4% glucose, 0.5 ml; and gossypol solution, 1.25 ml.3 Each value is the result of 4 replicate tests.4 Test 2. Rumen liquor, 3.5 ml; 95% alcohol, 0.5 ml; and gossypol solution, 1.25 ml.ta nd ar d d ev ia ti on .

TAB LE 2Effect of incubating gossypol and rumen liquor anaerobically with glucose or alcohol

Mediaino.i23Disappearance ofossypolTest1Free%

859.0* 70 1.88 14.6Total%

000Test

22Free%

80 .233.45410Total% 000

1 Incubation at 37C for 48 hours.2 Each value is the result of 4 replicate tests.3 Medium no. 1. Rumen liquor, 5 ml; gossypol solution, 0.2 ml.4 S ta nd ar d d ev ia tio n.5 Medium no. 2. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 2% glucose, 2 ml.6 Medium no. 3. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 95% alcohol, 1 ml.TABLE 3

Effect of centrifugation of rumen fluid at various speeds on the formationof the bound gossypol

Centrifugationspeed(20min.)None5001,0002,0002,5004,5005,0006,0006,50010,00010,500Disappearanceo f f re eossypolTest

I%49485855Test30033

2.5482.5305455Test

3362.2292.4398.3313.1Test

4*679.1673696640Tests590590590

1 Test 1. Rumen liquor, 10 ml; gossypol solution, 0.2 ml; incubated aerobically at 37C for 24hours; one tube at each speed.2 Test 2. Rumen liquor, 5 ml; gossypol solution, 0.2 ml; and 1% glucose, 2 ml; incubated anaerobically at 37" C for 48 hours, in duplicate.3 Test 3. Rumen liquor, 5 ml and gossypol solution, 0.2 ml; incubated anaerobically at 37C for4 8 h ou rs , in trip lic ate .*Test 4. Rumen liquor, 5 ml and gossypol solution, 0.4 ml; heated for 20 minutes at 70C ina water bath, in duplicate.5 Same as test 4, except that a different sample of rumen fluid was used, in duplicate.6 S ta nd ar d d ev ia ti on .


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G O SSY PO L D ET O XI FI CA TI ON 21 7gests that the reaction is probably not dueto a reduction of the gossy pol to a colorlessderiv ativ e by bacteria and again suggeststhat the disappearance of f ree gossy pol isnot due to m icrobial action.Activity of centrifugea rumen liquor.To determ ine w hether the gossypol isbound by bacteria or by soluble protein,sam ples of rum en liquor w ere centrif ugedat various speeds up to 10,500 rpm andthen incubated w ith gossypol sols for 48hours at 37 C.T he results (table 3) showclearly that centrifugation had no inf luence on the percentage of gossypol thatw as bound. A lthough bacteria are dif f icult to rem ove com pletely f rom rum enliquor by centrif ugation, there are relativ ely few rem aining in the supernatantaf ter 20 m inutes at 10,500 rpm , and oneshould expect a graded decrease in numbers from uncentrifuged liquor. If thegossypol had been bound by the bacteriaone should ex pect a decrease in the degreeof binding in the centrifuged specim ens.B ecause no decrease occurred, the tentativ e conclusion w as m ade that the gossy polw as bound by soluble protein.Nonbacterial nature of the reaction. Asecond test to determ ine w hether the gossypol is bound by rum en bacteria or soluble protein w as m ade by adding gossypolsol to the liquor and im m ediately im m ersing in a hot w ater bath at 70 Cfor 10, 20,30, or 60 m inutes.

T he results (table 4) show clearly thatthe binding ef fect cannot be due to the m etabolic action of bacteria because the reaction w as com pleted at 70 C in 10 m inutesor less. T he reaction is thus chem ical.TABL E 4Effe ct o f hea ting g os sypo l-rumen fluid so lutionsfor varying periods of time at 70C onthe disappearance of total andfre e gos sypol

Heatingperiodminutes10203060Disappearanceofossypol'-2Free811.7379

2.5804.4827.4Total0000

Comparis on o f ba cte ria l inc uba ti on andhot water temperatures. To obtain somecom parison betw een the hot w ater bathand incubation reactions, 10-m l sam plesof rum en liquor w ere m ix ed w ith 4 m l ofgossypol sol and either held at 70 Cfor20 m inutes, aerobically , or incubated at37 C for 48 hours anaerobically . Duplicate tests gav e identical results, the 70 Creaction binding som ew hat m ore gossy polthan the 37 C incubation reactions(table 5).Relation between binding of free gossypo l a nd disa ppea ra nce o f lysine -aminogroups. A final test of whether the gossypol is bound by rum en liquor protein isthe sim ultaneous disappearance of f reegossy pol and ly sine e-am ino groups. S amples of rum en liquor w ere incubated at37 Cf or 48 hours w ith the ratios of rum ento gossypol sol of 5 to 0.4 and 10 to 0.4.One sam ple w as heated at 70 C for 20m inutes w ith the ratio of rum en to gossypol sol of 5 to 0.4. T he m illigram s off ree gossy pol and ly sine c-am ino groups,th at d isap pe ared w e re d eterm in ed (tab le 6 ).T he m ole ratio of bound gossy pol to boundly sine w as 1:2 in three of the 4 tests.Influence o f pro teo lytic enzymes o n thesta bility o f rumen liquo r bound g ossypo l.To test the ef fect of digestion upon thestability of the ly sine-gossy pol com plex ,5 m l of rum en liquor w ere incubated for24 hours at 37 Cw ith 0.2 m l of gossy polsol. One m illigram of the enzym es w asadded and incubation continued for anadditional 24 hours. T he percentage off ree gossypol that disappeared w as thendeterm ined. T he addition of enz ym es hadno inf luence on the gossypol binding activ ity of the liquor (table 7).

TABL E 5Comparison of bound gossypol formation underdi ffe re nt type s o f inc uba tio n

D is ap p ea r a n ce o f g os sy p ol '- 2T yp es ofincuba t ion F r ee T ota l

A e robic w ater bath at7 0 Cor 2 0 m i nu te sA n aero bic at 3 7 Cf or 4 8 h ou rs 7503

640001 D et er m in ed in t rip lica te.2 Th e test solu t ion s con ta in ed 0.4 m l of gossypolsolu t ion and 5 m l of r um en flu id .3 S ta n da r d d ev ia tio n.

1 I n d u p li ca t e.2 Th e test sam ples con ta in ed 10 m l of r um en liq u oran d 4 m l of gossypol solu t ion .3 S ta n da r d d ev ia tio n.


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21 8 RAYMOND REISER AND HWEI C. FUTAB LE 6The simultaneous disappearance of the free c-amino group of lysine and of free gossypolduring incubation of rum en fluid-gossypol solution

Test'no.Disappearance o f f reelysine c-amino groupDisappearance o f f ree gossypo lMoleratioo f b oundgossypolboundlysine1234mg0.53

0.08"1.240.260.980.0450.990.0045moles0.00340.00840.00670.0068mg1.480.112.280.161.5801.750moles0.00310.00440.00310.00331111:1.2:1.9:2.1:2.01 All tes ts w ere ru n in d uplica te .2 T est 1 . F iv e m illiliters o f ru me n liq uo r inc ub ated a nae rob ica lly w ith 0 .4 m l o f g ossy po l-g ly ce ro ls olu tio n a t 3 7 Cor 4 8 h ou rs .3Standa rd deviat ion .4 T est 2 . T en m illiliters o f ru me n liq uo r in cu ba te d an ae rob ica lly w ith 0 .8 m l o f g oss yp ol s olu tio nat 37 Co r 48 h ours.5 T est 3. T en m illilite rs o f ru me n liq uo r inc ub ated an aero bic ally w ith 0 .4 m l o f eo ssy po l s olu tio nat 37 Co r 48 h ours.' T est 4. T en m illiliters of rum en liquor incubated aerobically w ith 0.4 m l of gossypol solutionat 70 Cn w ater bath for 20 m inutes.

TABLE 7E ffe ct o f p ro teo lytic en zy mes o n th e d is ap pe ar an ceof free gossypol bound in rumenf lu id -gossypol so lu t ion1

Enzymes addedTest'NoenzymePancreatinRhozyme-6Rhozyme-pfTest

2NoenzymePepsinTrypsin324.1338

2.5386.125079

1.6842.9774.91 The basic incubation medium in each tube was

5 m l of rum en fluid and 0.2 m l of gossypol solution.A fter incubation for 24 hours at 37 C ,one mi ll ig ramof the enzym e w as added to the m edium and incubatio n co ntin ued fo r an a dditio nal 2 4 h ou rs.2 In dupl ica te .' S ta nd ar d d ev ia ti on .4 I n t ri pl ic at e.S UMMARY AND CONCLUS IONThe indifference of the degree of gossypol binding of rumen liquor to aerobicor anaerobic incubation, high or low temperature, centrifugation or proteolytic enzymes, and the simultaneous disappearance of tw o m oles of lysine e-am ino groupsto each mole of gossypol, demonstratesconvincingly that the m echanism of rum inant detoxification of gossypol is by binding to soluble proteins, and that the bondis perm anent during protein digestion.

L IT ERATURE C IT EDBaliga, B. P ., M . E. Bayliss and C. M . Lyman1959 Determination of free lysine aminogroups in cottonseed meals and prelim inarystudies on relation to protein quality. Arch.Biochem. Biophys., 84: 1.Couch, J. R ., W . Y. Chang and C. M . Lyman1955 The effect of free gossypol on chickgrowth. Poultry Sci., 34: 178.Crawford, A . C. 1910 A poisonous principle incertain cottonseed m eals. J. Pharm acol. E xper.Therap., 1: 519.Eagle, E. 1950 Effect of repeated doses ofgossypol on the dog. A rch. Biochem., 26: 68.Jones, J. H ., J. M . Jones and J. J. Bayles 1941Utilization of home grown feeds in fatteningsteers in the trans-pecos region. Texas Agr.E xp. Sta. B ull. 604, C ollege Station, T exas.Pons, W . A., Jr., and C. L. Hoffpauir 1957Determination of free and total gossypol inmixed feeds containing cottonseed meals. J.A ssoc. O fficial A gr. Chem ists, 40: 1068.Ramsey, D. S., and J. T. M iles 1953 Cottonseed vs. cottonseed meal and corn as proteinsource in a concentrate m ixture for dairy cows.J. Dairy Sci., 36: 1308.Reiser, R ., and H. G. R. Reddy 1956 The hydrog nationof dietary unsaturated fatty acids. J.Amer. Oil Chemists' Soc., 33: 155.Rigdon, R. H., G . Cross, T. M . Feguson and J. R .Couch 1958 Effects of gossypol in youngchicks with the production of a ceroid likepigment. A . M . A. Arch. Pathol., 65: 228.Schwartze, E. W . 1924 Pharmacology of gossypol, J. Agr. Res., 28: 191.W ithers, W . A., and F. E. Carruth 1915 Gossypol, the toxic substance in cottonseed. J.

Agr. Res., 12: 83.1918 Gossypol, the toxic substance incottonseed. Ibid., 12: 83.

detoxification of gossypol by rummen microorganism

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