Investigating germination and outgrowth of bacterial spores at several scales Clément Trunet, Frederic Carlin, Louis Coroller

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Clément Trunet, Frederic Carlin, Louis Coroller. Investigating germination and outgrowth of bacte- rial spores at several scales. Trends in Food Science and Technology, Elsevier, 2017, 64, pp.60-68. ￿10.1016/j.tifs.2017.03.008￿. ￿hal-01551069￿

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Guti ver e fi loc com/ ‘ omics’ 208 errez-  eld, 6 si 4, 4, . on of theof on ma & & p. 60- ate/ rl . a eln . apr,M,OBin . eKse,C,Oms . ta.(2011). al. et S., Oomes, C., Koster, de A., O'Brien, M., Caspers, J., Beilen, van (2012). S., al. Brul, et P., Jasti, P., McClure, S., Kathariou, P., Cook, J., Bassett, S., Brul, hsae,A,Vtu,D,Ya,G-. omn . i,J,&Lsc,R 21) Broadly (2010). R. Losick, & J., Liu, T., Norman, G.-C., Yuan, D., Vitkup, A., Chastanet, ala,J,Sam,A,Vreln . ytnal,M,Dvigee . & F., Devlieghere, M., Uyttendaele, A., Vermeulen, A., Sharma, J., Daelman, rk,A (1999). A. Driks, rn,J .(04.A ciieoag pr germination spore orange Acridine An (2014). G. J. Bruno, hn . un,S,&L,Y 20) eltm eeto fkntcgriainand germination kinetic of detection Real-time (2006). Y. Li, & S., Huang, D., Chen, byna,W,d oig . rl . eKse,C 21) pr rtois The proteomics: Spore (2014). C. Koster, de & S., Brul, L., Koning, de W., Abhyankar, rnn .P,&Wlisn .(2008). M. Wilkinson, & P., U. Cronin, e,R,Hfmn . lmk,I 21) emnto n ampli and Germination (2012). I. Glomski, & P., Hoffman, R., Dey, rn,J,Cos . ek .(05.Aetrsi the in Apertures (2015). M. Peck, & K., Cross, J., Brunt, old,J,Fern J., Collado, (2000). al. et M., Brown, D., Kilsby, J., P. McClure, K., Davies, J., P. Hill, germination G., of Ciarciaglini, Levels (2014). D. Popham, & S., Melville, R., Helm, W., Ray, Y., Chen, byna,W,TrBe,A,Dke,H,Kr,R,Bu,S,&d otr .(01.Gel- (2011). C. Koster, de & S., Brul, R., Kort, H., Dekker, A., Beek, Ter W., Abhyankar, byna,W,Kmhrt . wre . a en . a e e,N,Bu,S., Brul, N., Wel, der van H., Veen, van B., Swarge, K., Kamphorst, W., Abhyankar, alad . Legu S., Gaillard, rnn . ikno,M 20) h s of use The (2007). M. Wilkinson, & U., Cronin, as,D,Cla . azl,S,Pgii . eldne . ocnel,P (2016). P. Cocconcelli, & M., Delledonne, E., Puglisi, S., Gazzola, technique F., SEM Colla, a D., of Bassi, combination A (2009). S. P. Cocconcelli, & F., Cappa, D., Bassi, Legu O., Couvert, L., Coroller, E., Baril, byna,W,Sedr . eKnn,L,d otr . rl .(2017). S. Brul, & C., Koster, de L., Koning, de S., Stelder, W., Abhyankar, aca . a e or,M,&Ae,T 21) oprtv nlssof analysis Comparative (2010). T. Abee, & M., Voort, der van D., Garcia, edr .D,Shlea,P,Lu,K,Hydik,M,Vneael,P,Mes . & L., Moens, P., Vandenabeele, M., Heyndrickx, K., Leus, P., Scheldeman, D., J. Gelder, e etn . rid . aal,H,Meear . weeig . be T. Abee, & M., Zwietering, R., Moezelaar, H., Gaballo, A., Arvind, H., Besten, den odce . hn,B,Glet .J,Tmis .M,MGvr,A,Aseg . tal. et B., Alsberg, A., McGovern, M., E. Timmins, J., R. Gilbert, B., Shann, R., Goodacre, lie,P .(03.Teftr snw igecl eoiso atraadarchaea. and bacteria of genomics Single-cell now: is future The (2013). C. P. Blainey, 68, & trends- ehooy6 21)60 (2017) 64 Technology lg;mlclrdfeecsbtena xrm etrssatsoeforming 221 spore resistant heat subtilis of extreme Bacillus analysis an between biology differences systems molecular ology; in advances and Challenges 27 assessment. Technology, risk microbial quantitative in technologies eeoeeu ciaino h atrrgltrfrsouainin sporulation for regulator master the subtilis of activation heterogeneous cell. vegetative the of emergence facilitate 51 to Microbiology, align exosporium and coat eeu epnet ettetetadlwtmeauestorage. low-temperature Membr and treatment heat 235 25(2), to Microbiology, response geneous treated 231 prsb oldeln amoebas. soil-dwelling 78 by spores eiw,63 Reviews, eeoeet fsingle of heterogeneity 107 America, paradox. spectral versus nltclCeity 78 Chemistry, Analytical at rsn n h future. the and present past, emnto of germination eciainof deactivation inhibitory spores. bacterial the 66 on Microbiology, treatment determine Environmental preservation combination to a route of effect a bioluminescence, Germination-induced 9 One, PLOS in proteins oeigha eitneof iformis resistance heat Modeling fraction. repoemcidenti proteomic free o atra pr tessria n outgrowth. and models 240 survival predictive Microbiology, stress of development spore the bacterial for for Proteomics stability: food microbial of composition in The (2016). al. et 1,53 on 105(1), pH Microbiology, medium Food recovery of of Journal the national time and temperature lag the regrowth time, the heating the of effects inlJunlo odMcoilg,140(2 Microbiology, Food of Journal tional ugot navgtbebsdfo model. food vegetable-based a in outgrowth of analysis of Transcriptome process germination spore the studying tyrobutyricum tridium for microanalysis X-ray and 30 ology, 1636 o,P .(07.Rmnsetocpcsuyo atra endospores. 389 bacterial Chemistry, of study Bioanalytical spectroscopic and Raman (2007). D. P. Vos, weihenstephanensis o uniyn orltosbtenclua niaosadaatv behavior. adaptive 5 and One, indicators PLoS cellular framework between A correlations robustness: quantifying bacterial for for biomarkers long-term and Short- (2010). ui-on yoyi assetoer n ore rnfr infrared transform Fourier and in spectrometry biomarker mass acid dipicolinic pyrolysis the Curie-point of Detection (2000). FEMS DOI 2) 8075 (22), e e nu . 227 240 Microbiology . ,J-.(03.Dvlpeto iet-eetgot oe o heat- for model growth time-to-detect a of Development (2013). J.-M. e, sc  : : prsa ucino prlto eprtr n pH. and temperature sporulation of function as spores ailscereus Bacillus rceig fteNtoa cdm fSine fteUie ttsof States United the of Sciences of Academy National the of Proceedings ,29 . rtois 11 Proteomics, . in 10. ri 1,1 (1), (10) 4,e95781 (4), - ne,A,Rdio . aas . oe,A 20) ieisof Kinetics (2003). A. Lopez, & J., Camats, M., Rodrigo, A., andez,  ailssubtilis Bacillus pt publis 1) 8486 (18), e e food rnl . ay . aat .(05.Qatfigtecombined the Quantifying (2005). P. Mafart, & N., Savy, I., erinel,  1016/ 36 ailssubtilis Bacillus 1,12 (1), 8081 . ailscereus Bacillus ailscereus Bacillus ,45 odioaeadalbrtr strain. laboratory a and isolate food e ailssubtilis Bacillus . ,11 20 eiw,37 Reviews, . e - fl BB prsotie tdfeettemperatures. different at obtained spores KBAB4 e science e . ec fsouaincniin ntesoeca protein coat spore the on conditions sporulation of uence 50 fi j.tifs.2017.03 . 18 24 aino the of cation e spores. e 2) 4541 (23), . 5,322 160(5), Microbiology, in Research 1) 6936 (19), . hed e Bacillus 68 8491 . . ora fFluorescence of Journal 243 ailsthuringiensis Bacillus ailscereus Bacillus omn,spromn,adgriaigspores. germinating and superdormant, dormant, . . ESMcoilg etr,358 Letters, Microbiology FEMS in nentoa ora fFo irbooy 165(3), Microbiology, Food of Journal International ailsweihenstephanensis Bacillus pr coat. spore 3,407e (3), spores. endospores. 9,3735 (9), ,2143 - prsb ae weesRmnspectroscopy. Raman tweezers laser by spores : e spores. and rnl . otle,F,Buas . ta.(2012). al. et C., Boulais, F., Postollec, I., erinel,  e

4550 ailscereus Bacillus 6941 ailssubtilis Bacillus ple n niomna Microbiology, Environmental and Applied e odMcoilg,20 Microbiology, Food -technology . 2151 e 427 .0 . e irba hsooyadMetabolism, and Physiology Microbial 3742 prsatraha treatment. heat a after spores ) 146 3), 08 irbooyadMlclrBiology Molecular and Microbiology yoer ,71A A, Cytometry . . odMcoilg,55,73 Microbiology, Food

. ,1 pr ie emnto n cell and germination life, spore e fl e 58 wctmtyt td the study to cytometry ow e noprsehbtahetero- a exhibit endospores lsrdu sporogenes Clostridium 153 nentoa ora fFood of Journal International noul pr otprotein coat spore insoluble 6 . . odMcoilg,28(2), Microbiology, Food . fl rnsi odScience Food in Trends oecnemicroscopy uorescence Bacillus Bacillus and 5,545 (5), e 2,137 (2), (3), fi 329 aino anthrax of cation ailslichen- Bacillus 1 . odMicrobi- Food 43 prsusing spores pr physi- spore e e “Omics ple and Applied e 144 548 153 Analytical e “Omics Interna- Bacillus Bacillus 85 . . . spore Inter- Clos- Food Food ” . for & ” Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript germination andoutgrowth ofbacterial sporesatseveral scales.Trends inFood Science andTechnology, Journal homepage rends inTrends Food Science Technology& Ve ot . 're,A,vnSokm . oe,S,Cilad . elnwr,K,e al. et K., Hellingwerf, W., Crielaard, S., Oomes, I., Stokkum, van A., O'Brien, R., Kort, Membr eje,B,TrBe,A,Rued,H,Shrn . otj,R,vndrSe,H,e al. et H., Spek, der van R., Montijn, F., Schuren, H., Rauwerda, A., Beek, Ter B., Keijser, (2006). al. et R., J. Strahler, N., Patel, K., A. Walker, R., Zielke, G., Michailidis, P., Jagtap, of origin the On (2006). (2009). S. T. Brul, & Abee, W., Kallemeijn, & J., W., Smelt, A., Vos, Beek, Ter de L., Hornstra, J., H. M. Wells-Bennik, Y., Vries, Biolu- de (1994). L., al. Hornstra, et W., Waites, P., Setlow, C., Soper, D., Vinicombe, L., Hall, P., Hill, alr . rwzk . eJn,A,Mdl,K,Hfmn,T,Lu,M,e l (2016). al. et M., Laue, T., Hoffmann, K., Madela, A., Jong, De A., Krawczyk, K., Nagler, Legu L., Venaille, A.-G., Mathot, C., Trunet, N., Mtimet, (2011). T. Abee, & R., Moezelaar, M., Tempelaars, M., Groot, Nierop C., Melis, van tee,E,Hn,J,Qae . edetr .(06.Micro (2006). J. Leadbetter, & S., Quake, J., Hong, E., Ottesen, & E., Stackebrandt, H.-J., Mollenkopf, P., Rettberg, G., Horneck, R., Moeller, og . on,C,Sto,P,&L,Y 21) oioigrtsadhtrgniyof heterogeneity and rates Monitoring (2014). Y. Li, & P., Setlow, C., Doona, L., Kong, spore the of function and assembly, Structure, (2007). C. Moran, & A., Henriques, Guti eJn,A,vndrMue,S,Kies .P,&Kk .(05.TRx Transcriptome T-REx: (2015). J. Kok, & P., O. Kuipers, S., Meulen, der van A., Jong, de v Legu apparent the between Relationship (2000). P. Mafart, & O., Couvert, I., Leguerinel, in Changes (2017). P. Setlow, & Q., Li, L., Rao, B., Setlow, G., Korza, adugt . elw . ail,W,Hoe,D,Ppfaku . elw P. Setlow, & E., Papafragkou, D., Hoover, W., Daniels, B., Setlow, M., Paidhungat, a ei,C,Amia . ot . ro,M,&Ae,T 21) emnto inhi- Germination (2012). T. Abee, & M., Groot, R., Kort, C., Almeida, C., Melis, van aat . Legu & P., Mafart, Whit V., Baranov, O., Ornatsky, D., M. Leipold, cekn . il . anr . al . os .(00.Eohsooyo food- of Ecophysiology (2010). T. Ross, & A., cytometric Dahl, Flow M., Wagner, (2007). C., Hill, D. T., McMeekin, Knorr, & V., Heinz, M., Bull, B., Chapman, A., Mathys, alzi . iwnta,V,&Vdna,G 21) Spore-forming (2010). G. Vedantam, & V., Viswanathan, M., Mallozzi, nMls .C . e etn .M . irpGot .N,&Ae,T (2014). T. Abee, & N., M. Groot, Nierop W., M. H. Besten, den J., C. C. Melis, an r si 20) seseto etrssac fbceilsoe rmfo product food from spores bacterial of by resistance heat isolates of Assessment (2005). tems hogotislf yl svgttv el rsoe sn rwhboundaries. growth using spores or 48,153 cells Microbiology, vegetative Food as cycle life of its throughout behavior the Modeling (2015). assessment. niiulspores. individual 20) nlsso eprlgn xrsinduring expression gene outgrowth. temporal and germination of Analysis (2007). proteomics. titative of events Early 134(1 Microbiology, Food of resistance (preservation) in heterogeneity 72 Microbiology, Environmental and of receptors germination of Characterization processes. inimical 129S 76, of Bacteriology, indicators Applied biological as spores and minescence Quanti Identi 227 28 biology, acid-stressed sorbic of outgrowth cereus and germination of Characterization 177 ology, of heterogeneity ugot nhg-aiiyevrnet sn N sequencing. RNA using 7 environments Microbiology, high-salinity in outgrowth and dormant from RNA extracting for method A (2006). germinating L. W. Nicholson, ihpesr emnto of germination high-pressure layers. surface 331 (pp. quanti absolute for spectroscopy. nlsswbevrfrRAsqepeso data. expression RNA-seq for webserver analysis uaintmeaueuo h siae etrssac fheated of resistance heat estimated the spores. upon temperature cubation 223 medium. recovery not the is of resistance synthesis heat protein that Evidence germination. spore temperatures: sub-lethal for extended essential various after outgrowth to and exposure germination rRNA, molecules, small 3556 71(7), Microbiology, Environmental nbe utgn nlsso niiulevrnetlbacteria. environmental individual of analysis 314 multigene enables iinof bition compounds. equation. linear-Bigelow a by 63 spores ence, of discrimination. resistance heat bacterial on for methods cytometry 419 mass Biochemistry, of ment on ahgn:Esnilkoldet mrv odsafety. food 139 improve ,S64 Microbiology, to Food of knowledge Journal Essential pathogens: borne of assessment Clostridia Science re-gir,I,Ra I., errez-Aguirre,  rnl . ovr,O,&Mfr,P 20) oeln h in the Modelling (2006). P. Mafart, & O., Couvert, I., erinel,  on définitive du du m on définitive ,J-. ulo,S 21) aetdvlpet nfobreptoe risk pathogen foodborne in developments Latest (2016). S. Guillou, & J.-M., e, 50) 1464 (5804),  e e ” 231 227 fi nlssapoc fbceilsoeoutgrowth. spore bacterial of approach analysis fi TC159soe:Peoyeadtasrpoeanalysis. transcriptome and Phenotype spores: 14579 ATCC aino ifrnilyepesdgnsduring genes expressed differentially of cation 1,17 e 43(1), Microbiology, Applied in Letters & 1,6 (1), aino h mato igeadmlil idsrse noutgrowth on stresses mild multiple and single of impact the of cation Trunet, C., Carlin,F.,Coroller, L.(Auteur decorrespondance) (2017). Investigating e . . ailscereus Bacillus ,57 4) e ok Springer York: New 347). nhmndisease. human in mrigTcnlge,8 Technologies, Emerging 2,275 (2), fl urn pno nFo cec,8 Science, Food in Opinion Current ailssubtilis Bacillus nentoa ora fFo irbooy 160 Microbiology, Food of Journal International rnl .(98.Mdln obndefcso eprtr n pH and temperature of effects combined Modeling (1998). I. erinel,  e e uorescence 1,119 72(1), Chemistry, Analytical Bacillus 62 . nulRve fMcoilg,61 Microbiology, of Review Annual 8 ailsanthracis Bacillus e . 1,1 (1), . 1467 ailscereus Bacillus e 1,345 80(1), Microbiology, Environmental and Applied ailscereus Bacillus fi  283 k,N,De,T,&Rvia,M 21) rpe iia PCR Digital Droplet (2015). M. Ravnikar, & T., Dreo, N., cki, rtois 6 Proteomics, aino ahgn.I .Lcme(Ed.), Lacomme C. In pathogens. of cation e . pr epnet ihpesr n heat. and pressure high to response spore : 8 . prs mato h iohlccaatro inhibiting of character lipophilic the of Impact spores: e http://www. . nentoa ora fFo irbooy 55(1 Microbiology, Food of Journal International anu ) 9 2), monitoring e

162 tan18endospores. 168 strain uueMcoilg,5 Microbiology, Future e ora fBceilg,189 Bacteriology, of Journal e Bacillus 134S sc . 15 9.JB.00583 199(9). Bacteriology, of Journal 1) 5199 (19), spores. prsadtep n alcnetainof concentration NaCl and pH the and spores emnto identi germination 64, 60-68. DOI :10.1016/j.tifs.2017.03.008 r . 4,519 (4), . 1,44 (1), ebclu stearothermophilus Geobacillus it . p e fdpclncai release. acid dipicolinic of prsb hs-otatmcocp of microscopy phase-contrast by spores S78 ublié e nentoa ora fFo Microbi- Food of Journal International 3564 journals. e e fi . Comment citer cedocument: e 53 527 l,C,&Nt,M 21) Develop- (2011). M. Nitz, & C., eld, ailscereus Bacillus 5211 e . e e Bacillus ,120 127 . . .Tue ta./Ted nFo Science Food in Trends / al. et Trunet C. 1,555 (1), dan . 21 M eois 16 Genomics, BMC . 7,1109 (7),

e . 17 (20 urn irbooy 53(3), Microbiology, Current rnl . oolr . tal. et L., Coroller, I., erinel,  fi 126 s s / Fi db iecus quan- time-course by ed prs nu o a for Input spores: elsevier. 9,3624 (9), nentoa ora of Journal International . e ailssubtilis Bacillus ailssubtilis Bacillus 588 2,124 (2), TC14579. ATCC e ), Vol. Vol. ), ora fFo Sci- Food of Journal fl l nal 1123 fl ii iia PCR Digital uidic . ec ftein- the of uence

ln pathology Plant noaieFood Innovative e Bacillus . ver e 3634 International TC12980 ATCC loc com/ ple and Applied odMicro- Food ,663 rnir in Frontiers 13 Bacilli 6 Analytical e ora of Journal si 0 4, 4, 353 . . Science, Bacillus Bacillus Applied e on of theof on ma . spore spore spore 16 “ p. . e sys- and . 3), 60- ate/ 68, trends- elw .(02.Dnmc fteasml facmlxmcooeua structure macromolecular complex a of assembly the of Dynamics (2012). P. Setlow, elw .(03.Soegermination. Spore Molecular (2013). (2003). S. Ben-Yehuda, P. & L., Setlow, Sinai, G., Mamou, A., Rosenberg, E., Segev, high- vitro In (2007). A. Malkin, & H., Hill, K., Wheeler, T., Leighton, M., Plomp, elw .(03.Sme etn 2013 meeting Summer (2013). P. Setlow, al. et T., Das, H., Chandra, P., Gupta, G., Dubey, Prakash A., tempe Goyal, bean M., Soya (2010). Zaman, T. Saif Abee, & M., Nout, E., Dalmas, P., Hil, den Roubos-van enk,K,Mty,A,Hiz . nr,D 21) ehnsso endospore of Mechanisms nucleoid (2013). D. of Knorr, Analysis & V., (2000). Heinz, P. A., Mathys, Setlow, K., & Reineke, M., Ross, A., Cowan, K., Ragkousi, al. et Pinz E., Manders, R., Kemperman, J., Smelt, N., Vischer, A., Beek, Ter R., Pandey, kne,M,Uio,A,Sen . ugl,C,&Hle,I 20) Bos:Anext- A JBrowse: (2009). I. Holmes, & C., Mungall, L., Stein, A., Uzilov, M., Skinner, ml,J,Srne,S,&Bu,S 21) eaiu fidvda prso o pro- non of spores individual of Behaviour (2013). S. Brul, & S., Stringer, J., Smelt, of spores of Properties (2001). P. Setlow, & E., Melly, B., Setlow, of spores of Germination (2014). P. Setlow, tigr . eb . ere . i,C,&Pc,M 20) eeoeet ftimes of Heterogeneity (2005). M. Peck, & C., Pin, S., George, M., Webb, the S., Stringer, of assembly Non-uniform (2007). C. Turnbough, & J., Kearney, C., Steichen, ohv,E,L E., cryotomography.Tocheva, Electron (2010). G. Jensen, & Z., Li, E., Tocheva, (2006). P. Luskinovich, & A., Kretushev, V., Tichinsky, ml,J,Bs . ot . rl .(08.Mdligteefc fsblta)heat sub(lethal) of effect the Modelling (2008). S. Brul, & R., Kort, A., Bos, J., Smelt, ai,F,&By,E 21) h fet fwtha ramn ntesrcua and structural the on treatment heat wet of effects The (2010). E. Buys, & F., in Tabit, water of state physical The (2009). B. Halle, & L., of Hederstedt, spores P., individual Setlow, in E., variability Sunde, time Lag (2011). M. Peck, & M., Webb, S., Stringer, and treatment heat of effects Contrasting (2009). M. Peck, & M., Webb, S., Stringer, e ek . onta .M,Pne,R,Kleej,W . ml,J,Mnes E., Manders, J., Smelt, W., W. Kallemeijn, R., Pandey, M., L. Hornstra, A., Beek, Ter ady . ipr . e ek . ice,N,Set . adr,E,e l (2015a). al. et E., Manders, J., Smelt, N., Vischer, A., Beek, Ter G., Pieper, R., Pandey, ady . e ek . ice,N,Set . rl . adr,E 21) iecell Live (2013). E. Manders, & S., Brul, J., Smelt, N., Vischer, A., Beek, Ter R., Pandey, & DOI ehooy6 21)60 (2017) 64 Technology h oto prso h bacterium the of 241 spores 83(2), Microbiology, of Molecular coat the - 550 spores. bacterial reviving of kinetics 307e 66(6), Technique, and Research spores. 9644 104(23), bacterial Sciences, of germinating Academy National single the of of ceedings dynamics structural resolution aino prsof spores of nation of analysis and Imaging (2005). 137 e 109(1), Microbiology, against Applied of activity Journal antibacterial show extracts 5,373 22(5), Recognition, Molecular of of killing and Journal germination of study microscopy force nciainudrhg pressure. high under inactivation of spores of outgrowth and germination during morphology specifi of 45 effect Microbiology, Food of the outgrowth of and germination analysis Quantitative (2015b). e58972 eeaingnm browser. genome generation germination. spore in stage 183 intermediate an at blocked 1251 teolytic 34 128(1), ology, cells. vegetative growing exponentially know. not eurdfrgriainadotrwhfo igesoe fnonproteolytic of 4998 spores botulinum single from outgrowth Clostridium and germination for required the 64 Microbiology, of assembly Non-uniform outgrowth: Bacillus 1) 5556 182(19), Bacteriology, of Journal a fFo irbooy 140 Microbiology, Food of nal esetvsi ilg,2 Biology, in Perspectives scale sub-nanometer Physics/0608093 at print displacements translational of Measurements effect odCnrl 29 Control, Food of treatment chemical 19334 spores. bacterial botulinum Clostridium 75 of Microbiology, spores individual of outgrowth nonproteolytic and germination on temperature incubation od:Tost td ehnsso aaeadrepair. and damage of mechanisms in study 28 spores microbial to stressed) Tools (thermally of behaviour foods: the of Models (2011). al. et uniyn h feto obcai,ha n obnto fbt ngermi- on both of combination and of heat outgrowth acid, and sorbic nation of effect the Quantifying of germination of pressure. high induction of Mechanisms (2002). mgn fgriainadotrwho individual of outgrowth and germination of imaging 52 Microbiology, nAag,P,Shl,G,Ngrjn . el,C,&Cmsn,T 20) Atomic (2009). 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