This research bulletin reports on Department of Horticulture research project 434, Thermal Activation of Bacterial spores in Processed Foods--P. [2].Digitized 2007 AES.Includes bibliographical references (page 10)

Fields, Marion L.

Jenne, Robert Charles

University of Missouri: MOspace

RESEARCH BUllETIN 806 NOVEMBER, 1962 UNIVERSITY OF MlSSOURI COLLEGE OF AGRICULTURE AGRICULTURAL EXPERIMENT STATION EL\.IER R . KIEHL, DirtmJr STUDIES ON THE HEAT RESPONSES OF BACTERIAL SPORES CAUSING FLAT SOUR SPOILAGE IN CANNED FOODS II . Effect of Oidium /act;s on Therm al Activation and Recovery of Bacillus Coagulans Spores M . L FIELDS ANO R. C. )HNN~ (PubliCltiOD 1UthOrilCd Augwt 13, 1962) COLUMBIA, MISSOURI CONTENTS Introduction Materials Vld Methods Organism.. .. . . .. . . .. . . . ... Cleaning procedure for spores ......... . .... . ...... . Prepantion of the tomuo juice serum Prcp=tion of Oidium tomat(l juice serum and mycelial mat . . . Effects of living Oidium JaetlS on spore recoveries of .... ... 3 ..... ... 3 . .... . .. 3 . ...... 4 . . .4 4 Bacillus coaguJans in tomato juice . ... .. .. .. . . .4 Heat treatments without a holding period . . . .. .. . . . .4 Holding periods with and without prior heat treatments .. .. ........... . 5 Enumention of spores ..... , Results.......... . ........ . ..... "'..... . . . . . .... . . . ............ , Heat treatments widwut a holding period ... .... . ....... , Holding periods with and without prior hear treatments. . .. .6 Effect of living Oidium Iacti! on spore recoveries in tomato juice ....... 9 Discussion .. .. .... . .......... . . · ...................... 9 Literature cited .. . 10 ACKNOWLEDGMEN T This !es«reh bulletin reportS on Dep:mmenr of Honiculrure reseueh project 4'4, Therm1.i Activ: .. ion of Bacterial Spores in Processed Foods. STUDIES ON THE HEAT RESPONSES OF BACTERIAL SPORES CAUSING FLAT SOUR SPOILAGE IN CANNED FOODS II. Effect of Oidium lactis on T hermal Activation and Recovery of Bacillus Coagulans Spores M. L FIELDS AND R. C. JENNE II h1$ been shown previously (hat Oidium la(/is can r:I;S(C the pH of tomato juice in thin films (0 1 pH r~ng(" oplimum for sporubrion of Bacililis coagll/am in 24 hours 11 2~ ·C ~nd in 48 hours at 3' ·C (Fields. 19(2) In these studies, sporubtion of Baril/liS coagll/allJ ace!.ln...! in tomato juice in which dther dead or living Oidhtl'l u.ais wu prcsc:nl. Since WiJdnun and Oark (1947) have shown Thai Oidiu/1I t.ulis prod\ICC:S a slime on machinery in wmuo canning factories wh~rc stri« sanirary mndidons are not maintained. this organism m~y have a more imponlm role in "fun sour" spoilage than previously though I. considering the previously mcndoned sporu-lation studies. In thee elnning pl~m Ihen: arc nuny places along a rrunu&cn.uing line suifed (or fhe sporui3fion u .... ell as for g<crminacion fO occur_ So-Jic fanics, choppen, juic( exrDnon and fillers have ~n listed 1$ possibl( sourc!s ofbae-retial spores to be produced (Williams, 1940; Wessel and Benjamin, 19-41;:md Bohrer and Reed. 1949). Since rhesc ;ncrC1S<:S in numbers of spores do occur in rhe pbm, one may nise fhe <jucstion as to what happens to spores produced ;n fhe plant .... hen Ihey find rheir ..... ~y to v:lrious pbnt env;ronmems. Will many of these spores germinate fO produce more vegualive cdls :lOd bring about in-Cfcased spote ConCCflltations on equipment' This TC$C1rch .... 15 insl;luled to ans-.... er these queStions. MATERIALS AN D MET HODS Org,znism Sf Din 21 of &d1J1IS l7I4gll/4tu which had previously been used in sporula-tion srudin (Fields, 19(2) wu used in Ihis tC$Cllrch. Sprnv Pr-oduaion Duplicate suspensions were prepared as follnws: spore production W15 obtain-ed by transferring a small amount of the srock cU]fure 10 a rcsr tube containing 10 ml of rhermouidurans broth. After 24·hour incubation al ,2"C, these broth cul· lUres were tDl\Sfen-ed ascptiaJly to Raux Hasks containing thermoacidur:ans agar, pH 7.0, that wu fortified wilh 10 ppm manganese sulf:tte. Afrer Ihe funsEe! W1IS eomplere, rhe Roux Basks were b"enrly agitated to insure even distribution of lhe bacleria over the surface of rhe agar. The flas ks w(-re then incubated. If '2°C. Spore stains .... ere made of Ihe cul tures at v:lrious limes during incubuion. The malachire green spole sraining method as proposed by Barrholome"" and Met!-wer (19'0) ""as used. T he culrures l\"Cre found to contain betw~n 20 and 4Q 4 MISSOURI AGRICULTURAL EXPBR1ME:>'"T STATION percent spores af[cr 3 dap incubation. This amoune of sporulation wu con· sidcred quite adequare for uSr in this study. Cleaning proUduTt for SportS A lysozyme dtlning procedure which has been p~iously dtscribcd (Finley and Fields, 1962 and J enne 19(2) was used in this research. Suspensions were designated as suspension No.1 and :2 and Stored at 4¢C uncil used. Preparation of ~he tomato jui~e serum The tomatO juice serum was prepared from tomatoes which had been care-fully washed, (rimmed, put through a cyclone finisher, and then hated to 190°F. This juice was then cooled and stored 1! O'C unril used. The [OmalQ juice W1\ removed from storage as needed, rh''''cd as n.pidJ)' :.IS possible, filtered fim through cheese cloth, .nd then ccmrifuged for 12 min to remove any luge pat· tides. The tomato juice was adjusted to the desited pH, using approximatel)' , N NaOH with a Beckman uro·madc g11s.! electtode pH.meter, Nine-ml sam· p ies were pipetted into tCSt tubcs and sterilized at 2~O°F for I~ min. Subse-qu. endy, it was stored at room temperature until used. The fin~l pH of the non· aJjusred tomato juice was 4. 1. Pnparation of Oidium tomato juiu u rum and mycelial mat 'Oidium tomato juice (O.T.]. ) serom was prepared by placing 1'0 ml of tomato juke serum in Roux flas ks and inoculating Ihis juice with a culture of Oidium Lutis. After, days of incubation, the juice was gently decanted leaving an in~ct mycelial mat. The O.T). w:as heated at 212°F and stored in a gallon jug at 34° F. Two lots "'ere ptepared having final pH ,",-lues of 6.4~ and 6.2~. When required, the juice was removed frortl storage, pipetted in 9·ml or 2.ml quanti. ties into test tubcs, plugged and autodaved at 2~O°F for I~ minutes Efftcts of living Oidium lactis on SPOrt /"tcovrrin of BaciIIus coagulans in tomato j uice The mycelial mat formed in the production of O.T). previously de-scribed was us.ed in this study. Spores of suspension No. I were mixed witb tOmatO juke pH 4.2' tv y!e1d COUntS of 21,700/ml. in lOO-ml quantities. This spore suspension was poured gently down the side of the Roux flask in order to flOlt the mycelial mat. Duplicate flasks were prepared for each tempera-ture and incubated at 4", 2'", and 3~"C for 24 hours. At the end of the incuba· tion period, the juice "'"1.5 dccanted from bcnC2th the mycelial mar, mixed thor-oughly, hC2ted to 9O"C for 10 min and plated out. Hrat 'reatmmt, without a holding period Heat treatments were carried out at 80° and 9O"C since previous experiments had shown that hC2t activation occurred u these temperamres while 100·( ",".,IS lethal Uenne, 1962). These temperatures are also possible plant temperatures. The SO" and 90°C heat treatments were performed in a wata barh. The test tubcs containing 9 ml of O.T.). :lnd tomato juice adjusted 10 pH RESEARCH BULLETIN 806 , 4.5, 5.5, 6.~ and 7.0 were inoculated with 1 ml of the properly diluted spore suspension (~OO to 1000 spores/mL). The menstrua were then thoroughly mixed to guarantee proper disuibution of the spor~ throughout the liquid. Duplicate [ul>(s were heated at each pH unit and two tubes were used as non· heated controls. A ~·min come·up time was rC<juired to reach the tempe .... ture of heat shock at bath tempe .... tures. The timing was begun as soon as the desired temper~rure was reached. Tcst rubes con~ining the spore suspensions were removed at appropriate imeN:lls and immersed in a cold·water bath. The suspensions were cooled to room tempe .... · ture in about 2 min, depending on the rempe .... ture of heat shock. Holding periods with and without prior htar treatmmf$ O.T.)., pH 6.2~, and tomato juices wilh pH of 4.1 and 4.2~ were used in these studics. Suspension No. 1 was used to inoculate teSI rubes con· .... ining 9 ml of tomato juice, pH 4.1. Hi lf of these tul>(s were heated to SOo for 10 min prior to incubating ar 2~0 , 3~ 0 and ~2 ° C. The other half were in· cubated at the same temperature without any pr~hcating. Tubes were removed at 0, 1, 2, 6, 12, and 24 hours ro detnmine the number of viable or recove .... ble spores. Prior to pl~ting, the tubes were hcoted al 90°C for 10 min to kill any vegetative cdls. Two spore levds (21 x 10' and 11.6 x 10') were used ro determine the ef· feet of holding unheated sporcs in O.T.)., pH 6.2~, and (Omato juice, pH 4.2~, for 24 hours at 2~0 and 35°C. In these tests, the juices were prepared as 9-ml blanks prior to inoculation with spores. At the end of the holding period, the rubes were heated to 9O°C for 10 min. cooled rapidly and plated OUt immediately. Suspension No.2 W::IS used in a tCSt in which 1 ml of suspension was added to 2 ml of juices to give suspension of 7.3 x 10' spores/ml. These tubes w~re incubated at 25° illd 35·C for 24 and 48 hours. At the end of the5(' rimes, ruMS were heated at 9O°C for 10 min to kill \"e~e!'oltive cells. Spore st~ ins were made of each rube to determine if germination had occurred. Enum, ration of sportS Viable plate countS were made using triplicate plates for each tube Dextrose rryprone ag:u: W":I.S used as the plaling medium. All plales were incuinted u ~2·C for 48 hours. RESULTS Htat trtatmmts without a 1M/ding p , riod Four sepa .... t~ runs are summarized and shown in Table 1; there W":I.S no sig. nificant difference between Ihe spores heated in O.T). and tomato juice at the same pH's at both rempentures nor was there a signifiC2nt difference among pH 's at the 80°C hear shock At the 90· C heat shock, however. the percent germina tion at pH 6.~ W":I.S significantly higher Ihill at pH 4.~ for . bom O.T). and tomato juice. The percent germination at pH 7.0 was also sig· 6 MI.SSOURI AGRICULTURAL ExP£RtMENT STATION nificantly lower than I'<'rcent germination at pH 6,~ for O.T J. The significance of these data is [hat these spores were hear activated at both SO· and 90°C but a higher ~rcent aCtivation was attained al 90°C. TABLE i_ E FFECT OF "H ON SPORE GERMINATION AFTER A HEAT TREATMENT OF SO AND SOOc FOR is MllfUTES ill TOMATO J UICE ANDO. T.J. Percent" Per<:enta Germination after Germination after 8ooS; heat§hock 9QoC heat ~hook " OTJb T,o OTt' n' ••• m ,,. 118. ,~ ••• m '" 122.8 '" ••• m '" 146. () ". , .. m '" 101 ,3 132 a.n.e percent germination was based On non-heated samples whlch ~re ooD8ldered to be lGO%. To be slgnlll~ant by the Q test, dlfferencu between % germination must be grea.ter than 1M lollowlnjr. b , 37. 2; c , 35. 7; d , 25. 6 and e, 33. 2. Holding periodJ with a"d without p rior heat treatlllmts Da~~ in Table 2 show the inhibitory etfen of pH on spore recoveries. T~lI! were greater decrC:l.ses in counts at higher incubation !emper~tures in rorn.tO juice. pH 4.1. Horing rhe spores:l.( 80°C for 10 min >1Ccder:ned this decr= in COunts. Further studies using higher spore loads are shown in T.ble }_ There "'-:1.5 not any significant difference between the control CountS (21 X 10' Or 11.6 x 10") and the COUntS ar 2~o and 3~oC in O .T). It would 2ppcar !h2! the spores were dormant during this 24·hour inCUNtion period since there was no d= indicating germin3tion. However, when these spores were placed in tomato juice, the COUntS "''<:f<: signifio.ndy lowered, illus!r:oting the inhibitory role of pH. In order ro csr:oblish whether true germination .nd/or inhibition was occur· ring. spores were used in an increased concenrrHion 50 that smears could be m.de .nd visu31 observuion could be performed. "s shown in Table 3. the spore COunt decr<:ased from 7.3 x 10' ro 3.8 x 10' within 24 hours ar 2'·C .nd to 1.2 x 10" in 48 hours in O_T). at n·c '"~ ~~oC the spore count of '.2 x 10" was obtained in O .T). in 24 hours while there was an increase to 6.3 x 10" within 48 hours in O .T.). Numerous vegetative cells were observed in the smears of the O.T). This increase in CountS is in agreemenr with previous work (f ields, 1962) that sporulation can occur in O.T.). at 3'°C. A spore count of 2.8 x 10" was obtained when the spores were held for 24 hours u 2' ·C in tomatO juice while coums of 1.6 x 10' md 1.3 x 10" were determined in 24 and 48 hours re-spectively in tomato juice. Acrual spor<: countS of smors ""ere m.de of the rubes of tomato juice held at 24 and 48 hours at 2~oC and 24 .nd 48 hours at n°C. Both the 24 and 48 hour tubes held ar 2~·C had 100 percent spores, indicating mat the decline in spore counts was the result or the inhibitory role of pH which is in agreement with Pederson and Becker (1949) and Knock tI at (19'9). Tubes ." !~ g""'''''' --• . " 'i. ............. d · . . . . . ...... _00 0 • " 0 • ~ " ., 0 . , ~ i'~ 8:i ....... oO • ...... "" 0000 " o • • · . . . . . x~ :8::::°000 , -H 8 :i = ~ .,," • ." i. .......... .,. ~~ · . . . . 0 .............. 0 • ~ 1~~ 8 ... ~.., .. -........... • ,,~ i. ..... on .. '" :8',.;.,;..; .... ...... -i! g:S:;:S;l(l<7J -. 08 • " 0 " ~ ~ i "_0 00. • 0": .. ":0.., " .. ............... -~ • g i! • '~ g:8~:8:;O'I; " • • " •• • • ~ • i...,ONOr> "" • q · . . . . ,il ~ :g-g-"'" ........... ~'O • · , " '.1 ......... <D ...... JJ "---TABU S-GElU4INATION, INHIBITION, AND APPARENT DORMANCY OF SPORES OF B. OOAGULAN8 Control lncubo.Uon ,,,.,, n _ 2· T •J • O.T. J . lu1ceb , Zl .: 103 24 1Iou ... 17. 7.:103 23. 1 x 103 6.5xl03 , 11.6.:103 Z4 hou .. 13. 6.: 103 14.8x l 03 7.8 x 103 6. 5 x103 • 1. 3 .:108 .. .... ~ 3. 6.:108 5.2xl0' 2.1 x 10$ 1.6x106 • 7. 3.: 108 Ubou ... 1. 2xl08 6.3.: 106 I,G ':106 1. 3.:106 ~II 6. Z5 bpll 4 . 25 RESEARCH B ULlETIN 806 9 held at 3'~C for 2~ and ~8 hours had spore (oum! of 97.9 and 91 percell1 re-speCtively. The slight amoum of germination here was probably due to incl'C:lsN tempc:12tute but the major ef!"«t was the inhibitory tOle of pH. Eff«1 of living Oidium la(fiJ on spore recoveries in tomato juiu. The effect of living Oidium l4criJ on spore recoveries of &uitJltJ Mtgllla/IJ in tOmatO juice is shown in Table ~. Since the pH of the tomato wu rai~d to ~ . 2' at 2'·C and since there "'-ere only 1,000 speas dif!"= between recoveries TABLE 4-THE SPORE RECOVERltS "", ... TemE!n.tuH No Stor~ , Control CO\IDt "",0 .' ", , .. 'or l,ginal pH 4.25 bValUU ,. 102 SOOA C~. ",» ~ >0' '" "-.-.;ent pH after lneub&l lon ReeoVfl!I I1!r lode '" .. 4. 36 .. 5.U " 4.98 ~t n"C and "·C with a pH of " .~ ', pH does not leem to be the only factor operating in these flxoveries. Thcsr data suggesl that an inhibitor to germiru-tion may be produced by Oir/hlm 1M/is and this inhibitor may be removed pat. tidly by inculntion at 35-C sinct the hight"St recovery of spores was obtained at this tempenrure bUI with a lower pH (4.95 ). If true germination were occur-ring, men recoveries at ,,· C would havc been lower Ihan al 25·C 10 be in agree-ment with Otba data in this study (Table 3). This thcory is mCIIglhened. by the f:!.ct that there was nor any differenct between O.T.). and 10mllO )l.Iicc when both were held in these media for a short time (Table I). In these tC$ts, the O.T.). had been autodaved, a process whiCh could have driven of!" a volatile inhibitor. DISCUSSION These srudies and the previous one (Fields, 1962) indicate that Oidium t.utis can play an important role in spore bl.lild-up in a processing plant. This oc-ganism can utilize the acid in the 61ms of tomato juices and nise the pH in short times (24-48 hours) which is of ptactical 'ignificance. Sporulation can oc-CUt either in the ptesence of dead or living Oidillm l/Utis (Fields, 1962). The data here suggest that, if spores find their WlIIy into juice films in which Oidium farris is not present, they may be removed from the potClltial spore build-up by being inhibited and dat the longer this a$$OCiarion exis{$ bcrwecn a low pH and sport: the les.s spores arc tc<:overN_ On the other hand, if sporcs arc in O.T.]. at 2'·C germinllion will occur but the tt:mpcnrurc is not high MISSOURI AGRICULTURAL E XPERIMENT STATION enougb to . lIow sporulation to occur in 48 boun (Tobie 3). If. bowever. tbe spores are in an environment of 3~·C, germi>l1.icn 1nd sporula tion on occur and inerease tbe spore build-up. There was some inhibition of spores in romaro juic~ wirh living Oidil(f/I u.air which was prob. bly due to pH and an inbibitory substance. Still, from a praeriol poim of view, most of tbe spores wben pl.ced in sucb an cnvironment would become residual spores wbicb could add to the gtowing "pool" in tbe spore build.up within a plant. UTERA T U RE CITED Bartholomew, J. W. and T. Mittwer. 19~O. Simplified BaCterial Spore Suin. Stain Techno!. 2J: 1~3-156. Becker. M. E. and C. S. Pederson. 19~O. The Physiological CbU1cten of &rillw CIhlg"f" nJ (BJcillus rbermoacidurans) J. Bacterio!. J9: 7l7·n~ . Bohrer, C. W. and J. M. R~d. 1949 Tomato Juice Spoilage Studies in 1948. Convemion i~ue information Lett~r No. 1219, N . ,'!. Canners' Assoc. Fields, M. L. 1962. T be Effect of Oidium f"ais on tbe Sporulation of Bttcill", (oag,,/ttm in Tomato Juice. App!. Microbiol. 10: 70.73. Finley, N. 1nd Fields, M. L. 1962. Hea t Actintion and Hea' Induced Formancy of Bacillus Stearorhermophilus Spores. AppJ. Mictobiol. IO:n1 ·236 Jenne, R. 1962. Tbe Effect of Environmenul Facton On Germinarion and Out-growth of &leillu, (o"g"I"" Spores. M. S. Thesis University of Missouri, Columbia, Missouri. Knock, G. G., M. L. Lambrechts, R. C. Humer, and F. R. Riley. 1 9~9 Souring of South Arrion Tomato Ju ice by Badffw coaguf"nJ. J. S<:i. Food Agrie. 10: 327·342. Pederson, C. S. and M. E. Becker. 1949. Flat Sour Spoilage of Tom.to Juices. N. Y. Sure Agr. Expt. St1. Tech. Bull. No. 287:1·22. Rice, A. and Pederson, C. S. 19~4. hetors InRuencing Growtb of &riff", trlttg'" 14m in Canne<l Tomato J uice. II . Acidic constituents of tomato juice 1nd specific orynic acids. Food Re5(arcb 19: 124·133. Wessel, D. J. and H. A. Benjamin. 1941. Process Control of Heat Resisr1Ilt Spoilage Organisms in Tomato J uice. Fruit Prods. J. 20: 178-180, 189. Wildm1Il, J. D. and P. B. Clark. 1947. Some Examples of tbe Occurrence of lI-b· cbinery Slime in Canning Faclories. J. Assoc. Offici . Agr. Cbemists. 30: ~82· ~8~. Williams, O. B. 1940. Sources of Contaminarion in the Manufacture of Tomato Juice. Frui t Prods. J. 19: 209, 2B. 

Effects of Odium lactis on thermal activation and recovery of Bacillus coagulans spores.

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