CA2059798A1

CA2059798A1 - Time temperature indicator with distinct end point

Info

Publication number: CA2059798A1
Application number: CA002059798A
Authority: CA
Inventors: Thomas J. Jalinski
Original assignee: Individual
Current assignee: Oscar Mayer Foods Corp
Priority date: 1991-01-31
Filing date: 1992-01-22
Publication date: 1992-08-01
Also published as: EP0497459A1; CZ25892A3; JPH0599754A; AU1055392A; AU644935B2; US5085802A; CN1068423A; HUT62412A; PL293329A1; NZ241438A; TW201817B; HU9200273D0; BR9200449A; KR920014433A

Abstract

ABSTRACT OF THE DISCLOSURE
A new and improved integrating indicator system operable to signal the attainment of one or more preselected time-temperature integrals which monitor the temperature and time history of a product utilizes a dual system of specific reaction pairs which simultaneously generate acid and alkali from two neutral substrates. One of the substrates is present in excess of the other. The preferred dynamic indicator system generates a constant pH
buffer in the alkali range that is maintained until one of the substrates is depleted. At that time, a rapid pH
change in the indicator solution to the acid range occurs, resulting in a very sharp visual color change in a pH-sensitive dye. In preferred embodiments, the specific reaction pairs are enzyme/substrate pairs, preferably urease/urea and yeast/triacetin. A preferred combination pH-sensitive dye package includes m-nitrophenol, p-nitrophenol and litmus to provide an indicator which changes from green to reddish pink upon the expiration of a given amount of time at constant temperature, or in a shorter period of time, upon exposure to elevated abuse temperatures. In especially preferred embodiments, one of the enzyme substrate pairs includes an enzyme component provided by a microorganism which has been shock treated prior to incorporation in the indicator to improve temperature sensitivity and provide extended half life.
The new and improved integrating indicators are adapted for use with packaged foodstuffs intended for refrigerated and room temperature handling and storage at temperatures between about 20°F to about 120°F.

Description

2~ 7~
Case 19323 TI~ T~P2RAT~R~ INDTCATOR ~IT~
- DI~TINCT ~ND POI~T
BACXGROUND OF THE INVENTION
Tho prosent invention relatQs to indieator systems for indicating time-temperature histories of a produet More particularly, it relates to a new and improved time-temperature integratlng indicator for monitoring the safe limits of refrigerated storage for a food product or other material The de~irability-o~ detecting whether or not a food produet has been subjected to adverse temperature conditions has been'recognized and num~rous indieator deviees of this kin~ are described in the patent literature one-éspeeially prevalent class of these indicators rdatesrto indicators for deteeting whether or not a frozen foo~ pr~duct has been allowed to thaw Typieally, the~~fFeeze indicators ineludo a frozen material whieh molts at some presel~etod tomperature so as to irreversibly aetivate an indicator, either chemically or physically Typieal of these deviees are those d-scribed ln US``3,43~,010 and elsewhere Most Or the ' frozen fo'od indieator'devices merely signal'a thaw, without maklng any'att' mpt to measure the period d~ring which th--produet $- thawed or th- tomp ratur- whieh the produet attai~ hll-~it is or wa~ thaw~d Anoth-r e~as~Tof ~nown indieators for ~ood prod~cts utiliz-- 'dl'f~u~ion~or capillary aetion with a wick or similar p-~eable ~e~bor to provido som- degree of gradation sueh a~ those shown in US 3,414,415 and US

3,479,877 to name but a few It'i'~ n'o~ r~eognlz-d that various natural and synthetie mat-rial~ d-teriorate with tho pa~sage of time, even wh-n taking sueh precautions as storing under refrigeration, packaging in an inert atmosphere, sterilization and adding spoilage retardants For examplo, foods, film, pharmaceuticals, biological ~ ~' .. : ~ ~:

~y F~ 9~ X~Ç

2 ~3 ?9 ~ '9 8 preparations and tho like may each d~mon~trate decompo~ition with th- passage of tim-, even when sterilizQd or maintained at sufficiently low tQmperature to preclude microbiological degradation D-eo~position may occur for various reason~ including strietly chemical r-actions sueh as oxidation and nzy~atie proe-sse~
Consequently, for eaeh typ~ of mat-rial th-re often exists a limit to ~ts permissibl- storag- lif~, after which time a dise-rnible ehanga in son- prop~rty oeeur~ A ~ystem whieh indicat-~ whon thi~ tim- linit ha- b ~n ~xeeeded is d~sired in the food paekaging industry The deterioration kinetic~ involv-d ~n tho~-process~s m~y b xe-~dingly eo~plicat~d Gonoral~y, dot~riorati-on i~ a funetion of t-~p-r~tur-, how-v-r, the rat- of det-rioration of-a produet ~ay vary with the t-~p-ratur-, so-that-on- rat- of d-t-rioration i~ ob~erved at a first t~p~ratur- and a diff-r-nt-r~t- of -~dQt-rioration i- ob~rv d at a ~-eond t-op r~turo The ov-rall or total a~nunt Or d-t-rioration will d-p-nd upon th- tix- ~t whieh th- produet is h-ld at aeh t-mp~rature, i e , an int-gral of t~n and t-~p raturo Th- praetical off-ets of thi~ aan b~----n, for -xu~pl-,--fro~ two identieal ~aopl-~ of food paekag d ~i~ult~n-ou~ly For both of th--- th-r- ay b a flnit- ti~- t-~p-rature int-gral unti} a~di-e-rnibl- ehang- in food quality oeeur- If-on- p~ek~g i- allow d to ri-- in t-up~rature by 10 or~20- or uor~ in th- eour~- of lt- di-tribution or storag , it-~quarity ~h lf lifa will b- r~due-d a-eo~par d with th- oth-r paekag- whieh i~ naintain d at an appropriat- low r r-frig-rat-d t-uperatur- for it~ ntire storag- lif- Con~u~ r-, about to pureha-- th--o paekag-s,~ both of w~ieh ar- now tor-d at a nor~
r~frig-ration display e~-- pr---ntly hav no way of ase-rtaining thi~ diff-r-ne- in th- t-~p ratur- hi~tori~s -~
of th- produets th-y aro buying Sy~to~s hav- b--n suggest~d for monitoring th-t~p~ratur~ hi~tory of a product US Pat-nt 2,C71,028 ~
~: -2 ~ rlJ ~ ~

utilizes an onzym- such as p2psin in indica~or sy~toms US Pat-nt 3,751,382 disclose~ an enzymatic indicator in which ureas~ d-composes ur-a with the reaction products causing a chang- in the pH of th- syst-m The activity of the enzyme and thus th- rata of dQcomposition is dependent on t~mp~rature, 80 that the chang- in pH resulting from the activity of the enzym- can be monitored by conventional acid-bas- indicators Another system is d-scrib-d' in US 3,~68,976, wh-r-in ti~- teop-rature integraticn i8 achiev~d by monitoring oxygcn perm-ation through a film through th- U8- of a r-dox dy-F$nally in US 3,9~2,467, a ti~ -tomperatur-integrating indleator is d-s~rib d inelud$ng an organic acid g-n-rating compon-nt wh~ch i8 ~uk~et to solvoly~is at a flr t r~act-ion-ra~ to g-nerat- known a~ounts of acid Th~ solv-nt or solvolysis m-diu~ is provided with known guantit$-s of alkalin- nat-rial~ ~urflci-nt to neutralizo acid g~norat-d by solvoly~is for a giv n time p-riod at a giv-n roaetion t-op-ratur- Th- solvolytic r-action rat- $ner-aa-- w$th an incr-as- in t-operature A pH s-nsit$v dy- $s prov$dad to indieat- wh~n a suffic$-nt amount of aeld has b--n g-n-rat-d to n-utralize all of th- al~al$n- ~at-rial In a pr-f-rr d abodiment, two s-parat~ al~a~ln- mat $als Or d$rr-r-nt ba-icities ar- mploy-d in ~ lnation with mor- than on- pH
~ n~itiv dy~ rn aeeord~ne- with th$~ pr-r-rr-d bod$~ nt,--th- lndleating ~olution indleat-~ a f$rst eolor unt$1 th~ f$r~t alkalin- p-ei-s ha~ b -n n-utrallz d to an xt-nt ~uffici-nt to d-er-a~- th- pH of th~ indleator to a r$r~t int-rm d$ato rang-, th-r~by cau~ing a eolor~ehang- to a s-eond $nt-ro d$at- eolor Th- s-eond eolor r- a$na unt$1 th- s-eond alkalin mat-rial i~ n-utraIiz-d, ~hifting th- indieator pH into th- acid rang-, wh-r-upon the ov~rall indieator c~anges to ~' a third eolor In accordanco with thi~ prior art indicator, gradual change~ in color ar- observed and tim- d-lay- in ~59'~8 gen-rating a discernible color change may tak- longer than is de~irable or reguired for certain indicator applieations ~oreover, as is evident from th- patent, the solvolysis of the acid pr~cursor materials r-quires the USQ of special solvent combinations to increase the half lif~ of the solvolytic reaction- This i~ ;
undesirable in today's context, wh-r-in th- co-solvents Quggested in th- patont include, for exa~plQ, dioxane These co-solv-nts are generally to b- u--d ~paringly A basic problem in d-veloping satisfaetory time-tcmperatur- int-grating indicators is th- fact that the second derivative of time-temperature deeo~position (the change in rate per unit of temperatur- change), differs for different produet~ Thus, th- chang- in th- rate of deterioration p-r unit of temperatur- change for certain fruits and berri-s i9 vastly differ-nt fro~ th- change in rat- for l~an m-ats Th- valu-s for dairy produets are diff-rent fro~ both b rrie~ and m-ats Con~-gu-ntly, a system whieh is d-p nd-nt on a ingl- nzy atie r-aetion or th- permeability of a given film would b suitable as an indicator only for thos- mat-rials having a similar slop- for the r-lation-hip of chang- of rat- of d-eompo-ition of a eertain Xind with resp et to t~op-ratur-Tim t-op ratur- integrating indieator sy~t-c- are -not li~it-d in applieation to monitoring long torag-p r$od- at b low room temperatur- Tb- am-eon~id rat~on- apply to ~horter tim- p riod~ and to high t-np-ratur~s Tb- ystems can b us-d to ensur-, for ~ ~
exampl-, that produets have b--n ad quat-ly h-at ~ ;
sterilized Th- indieator i~ thu~ ad~irably suited to n~ur- that cann-d good- which ar- autoelav-d hav- b-en -subj-et~d to th- appropriat- tim- and t-~p-ratur- integral r-quir-d to obtain a n-ee~sary d gr-- of ~ieroorganism kill In this ca--, th- firing of th- indieator is used ~ ~ -as the signal that the necessary sterilizing parameters hav be-n reached or exc-eded Similarly, indicators of ~ ----~ 2~^~t~7~8 thi~ type can be used to ensure that surgical in~tru~ents have be-n subj-cted to appropriate sterilization conditions or that pharmaceuticals hav- not b-en stored for p-riod~ in OXC-S8 of that which is p~r~issible Similarly indicators of this type are useful in the dairy industry to indicate that dairy produets have been properly Pasteuriz-d and th- lik-A major probl-m with prior art ti~o tQoperatur-int~grating indicators i~ that th-ir ti~ r--pon~ is g-n~rally unacc-ptabl- FrequQntly th- d-v-lop~ent of - sufficient color to b- diff-r~ntiabl- by th- viewer may take as long as ~0 to 100~ of th- lif- of th- indicator At pres-nt, with th- advent-of ~odifi-d atmosphere paekag s and n-w-r af-r pr-s-rvativ- -p ei-s for ext-nding th- roou te~p-rature ~h-lf life of eooked fresh foods the consu~ing publie ha- co~- to njoy a nunber of high quality fr-sh tasting food produet~ which heretofore w-r- unavailabl- Inst-ad of providing th- food in frozen form or in a can eook d fr-ah food~ ~ay b- tr-at-d and torod in ~odifi-d at~o-ph-r- paekages and stor-d for sufficiently long p-riod~ und-r r-~rig-rated eonditions to now p~r~it th-~- fr-ah-r foods to b- availabl- to the consuuer Luneh-on traya ineluding a vari-ty of lunc~eon m ats sueh as turk-y and han and eh--ses ar- an illustrativ ~a~pl- Th- i~prov ~ nt- ~aking th~
produet- poa-ibl- hav now xt-nd-d th- roo~ te~p~rature ~torag- atability of th- produet~ in th--- packag-- fro~ a ~att-r o~ hour~ to a day, to -v ral daya to a w -k This p-r~it~ th- diatribution chain rOr the food produet- to maXe th- food produet--available to th- eonau~ r for imo-diatQ eon-u~ptlon vithin th-ir improv-d and ~xt~nded sh-lf lif-In theory, th- ratio of th- rato of ebang- at one temperature of a prop rty of a tored artiel- who~-detorioration i~ b ing monitored to th- rat- of chang- at a low-r temp-ratur- differ~ for different ~atQrials This valu~ i~ often expre~-d for 10 increment- and i~

2 ~ 3 ~

repr---nt-d by th- symbol Qlo for the CQ1~1U- ~cale and a10 for th- Fahr~nh-it scale For examplo, wlthin tho range of 0 to -20 C raw fatty meat and precook~d fatty mQat have r~ncidity Qlo values of about 3, wh-r-a~, raw lean ~Qat and precooked loan meat hav- rancidity Qlo values of betwe-n 5 and 6 V-g-tables g~n~rally havo a ~poiling Qlo of b-tweon 7 and 8, wh-r-a~ fruits and b rries have a spoiling Qlo Of approximately 13 Th- formulation of compon-nt~ of any indicator ~y~t-~ should b ~ cted so that th- chang- in th- rat- of d-v-lop~ nt of ~ignal per unit change in t-~p-ratur- should b- co~patiblo with the paramoter~ desired in th- article b~ing ~onitored The Q1o value for th- indicator g-n-rally should approximate the Qlo valuo of a proporty of a giv-n food cla~ boing mea~ur-d To maka this ~atch, th- prop-r ~ ction of the ~-~
indicator ingr~di-nts and appropriato manipulation of the r-lativ- conc~ntratlon of th-~- ingrodient~ n--d~ to be p rforo~d g-n-rally in accordanc- with the method~
d-~cribod in ~S 3,9~2,467, th- t-aching~ of which are specifically inco^rporat-d hor-in by r~f-ronc- -Accordinqly~ it i~ an ob~oct of th- pr-~ent inv~ntion to provid~ a n-w and i~prov d ti~ t-~p-ratur- int-grating indicator for u~- with food product~ adapt d for roo~
t-~p-ratur- or r-frig ratod ~torag- wh$ch provid-- an - , i~ diat- vi-uai lndlcation of prod,uct ~afoty and guality It i- anot~ r'~b~-ct of th pr-s-nt inv ntion to provid a ti~ 't-~p-ratur- int-grating indicator for u~o with food-tuff~ whlch'rapidly d-v-lop- a di~tinct ~nd point lgna~ a~'-oon a- ~afo ~toraq- li~it~ havo b ~n r-ach'd'' It i~ a furth-r ob~-ct o~ t~- pro--nt inv-ntion to ' ~, provid- a n-w and i~prov-d ti~ t-op-ratur- int-grating ~ -indicator which ~ay b~ u~-d with a wid- varioty of foodstuffs and in a wido vari-ty of packaging configuratlona 2~3~73~
, ,SUMMARY OF THE INVENTION
In accordancQ with th-sc and other ob~ects, the present invontion provides a new and improved time-temporature integrating indicator which comprises a first S , specific reaction pair including an activ~ reag~nt and a subatrat~ spQcific for that activ- r~ag-nt Th~ first ~p-cific r~action pair m-Dber~, i - , th- activ- r~agent and its specific ~ub~tr~t~, ar- r-activ- to form a first reaction product Thi~ first rcaction product t-nds to cause th- pH ~alu of th- indic tor to chang- or b~
shiftcd in an acidic or a basic first dir-ction away from noutrality. - -Th- indicator also comprise~ a s~cond spocific reaction pair ineluding an active reag-nt ~nd a substrate specific for t~at activ r~agent The ~-cond specific react~on pair ~b r~ are ~l-o r-activ to fo ~ a ~-cond r-action-product Th- -cond r-action produet t-nd~ to cau-- tha pH valu- of th indicator to chang or b shift d in a Joeond diroction oppo-it- th- fir-t diroction and pr-forably b yond n-utrality Th- indicator coupo-ition addition~lly co~prises at l-a-t on- pH,--n-itiv dy- whieh xhibit~ on- color for a pH in a ba~ie pH r~ng-,and a ~ cond diff-r-nt color for a pH in an aeidie pH rang In aeeord~ne- w$th thi- inv-ntion, th ub~trat- in th -fir-t p ei~c r-aetion pair i~ pr-~-nt in a ~toiehio~ trie xe ~ a ount o~-r th- ~,~ount of ~ubstrate in th eond p eifle reactlon pair Th--flr-t r-action produet and th -eond raaction product ar~ r-aetive to for~ a third r-aetion produet Th- third r-aetion product compri~-- a tabl- pH burr-r whieh maintain- th- pH of the indicator at a t bl- v~lu- on th- ~-eond dir-etion side of n-utrality until th- ~cond r-aetion produet i~ no long-r produe-d Th ~-cond r-action produet e-a--- to be produc-d in th- indieator wh-n th- sub-trat- ~tarting mat-rial for th- ~-eond specific rcaction pair is d-pl-ted or is u~-d up Wh-n this occurs, th- capaeity of th- pH

233~7~

buffer is rapidly exceeded by the first reaction product and the pH of th~ indicator changes very rapidly from the buffer pH value to a different pH on the first direction ~id~ of n~utràlity In the proce~s, the indicator exhibits a rapid and distinct color change from the first color to the second color or from tho s-cond color to the first color The aharp color chang- of th~ indicator composition is s-lQcted to occur after a prsd~termined amount of time ~ -has elaps-d upon exposure to a r-lativoly con~tant predetermined temperature or temp-rature range Moreover, beeau~ the rates of the first and s-eond specifie pair reaetions are temperature dependent, expo~ure of the indieator composition to el-v~tod t~peratures abov~ the predeter~ined temperature range will eause both reaetions to proceed at faster rat-~ Aecordingly, th~ third r-aetion produet will be produe-d at a fa-t~r rate and the substrat~ for the s-eond reaetion pair is depletQd in a short~r tim- p-riod AJ a re-ult, the burr-r eapaeity is exeeed-d and th- eolor ehange oeeur~ soon-r than is ob~-rved with low r t-mp~rature expo~ure ~ he sharp and di~tinet eolor ehang- aehi-vod by foroing a stabl- buff r fro~ two t-mp-ratur- rat-d-p-nd-nt but g-n r_lly ind-p-nd-nt sp-eifie r-aetion~ is mueh quiek-r than prior art indieator sy-t~ which start out, not fro~ n utral sub-~rat-~ a- with th- pr-~-n~
inv ntion, but rath-r with a highly aeidie or highly ba~ic st~rtlng olution In th-~- prior art indicator-, a r-aetion product i~ produced which ffectiv-ly neutralizes th- starting olution gradually, a- th- r~aetion between r-aetiv- compon-nt~ in the indieator proe--ds Any color ehang~ i~ slow to d v lop du- to th- lag ti~ assoeiated with the n-utr_lization portion of th- eurv In contradistinetion, the indieator of th- pre~ent invention b gins with a pH-neutral substrate solution and generates a stabl- pH buffer having a mod~rate pN valus on a given side of neutrality Both the fir~t and the seeond - 2 ~ 9 ~
_9_ specific roactions proceed simultaneously until the second specific roaction pair substrate is doplet-d At that point in time no new buffer is formed and th- influence on pH of the first reaction product is gonerally immediately ob erved In accordance with a preferred us-, the indicators of the invention ar- cmployed to indicat- tim--tomperature histories of a product subj~ct to biologically causod or influenced chang-~ in product quality A typical exa~ple ~ `~
lo includes a cook-d'''food prodùct int~nd-d for r~frigerated storago and sub~ect to biological degradation over timo, due to the harmfùl effects of bactQria or fungi The rate of deterioration in product guality for tho food product will vary with r-sp-ct to thc duration of it~ storag-, a~
woll as the temporatur~ history of th- stor-d timo period In accordance with a pr-f-rred ~mbodi~-nt, th- indicators of this invontion mploy fir~t and s-cond sp-cific reaction pair compon-nt~ s-locted fro~ biological sources and/or proe-s--- wnich'ar- cho~n to provid- an indicator ' ~
having an ov rall Qlo valu- similar to th- Qlo valu- of the - -product b ing ~onitor d Mor- part$cularly, in accordanc- with a pr-f-rred Q~bodim-nt, th- n~w and improv-d tim--t-Jp-ratur-integrating indleator'eompri--~ a first gp-Cif iC r-action pair ineluding~'an nzye or enzyme aetiv component a~ the activa r-ag nt and a -ub-trat- ~p-eific for th- nzym ~or r-acting to for~Van aeid a~ th- fir~t r-action product A
s-cond p-elfle r-aetion pair i- al-o provid-d ineluding a diff-r-nt, ~-eond nzym or nzyo activ- eompon-nt a~ the 30 act$v- r-ag-nt and a -eond diff-r-nt ub-trat- p-cific for th- s-cond nzy~- Th- s-cond r-ae'tion pair m-~b~rs ~ -roaet to form a ba-ic i-cond r-aetion produet Th- indieator~'in aeeordanc- with thi- pr-f-rred o~bodiment inelud-- a pH-~on~i~iv- eombination dya paekage including a eo~bination or mixtur- of m-nitroph-nol, p-nitrophenol and litou- This eombination of pH dy-~ i9 ~ffectiv~ to cau-- th- indicator to oxhibit a gro-n color -10-- ~ `3 J ~ 7 ~
at neutral to basic pH values (> 7 0) and to exhibit a distinct reddish-pink color in the acidic pH range (<

7 0) The substrate for the first specific reaction pair is present in the indicator in a stoichiometric excess amount over the amount of the substrate for the second specific reaction pair The tlm- temperatur~ integrating indicator~ in accordance with the pref-rred embodiment exhibit a distinct color change from gr--n to reddish-pink after a predet-rmined amount of tl~e has lapsed upon exposure to a relativ-ly constant t~mp~ratur~ range or, after a rel~tively short period of tim~ has elapsed, upon exposure to e}evated temperatures This combination dye package iQ preferred herein becaus- it employs or produces th- now universal symbols of green-rQd to indicate go-stop or use-do not use conditions Other pH dy-s and combinations or dye~ to produce other colors and color changes may also be us d if d-sired In tho pref-rr d ~bodi~ent, t~e -cond enzyme/sub~trat- sp cific r-action pair comprises a urease/urea reaction pair and the first enzymQ/~ubstrate specific reaction pair is a lipas-/triac-~in reaction pair Pr-ferably, the lipase nzyme pre--nt in the first enzy~/substrat- sp-cific react$on pair is provided in or from a ~icroorganism Qourc- xhibiting lipa-- nzym activity toward-th~ triacetin substrat- A pr-ferred ~icroorganl-m is y-a-t It ha- b~-n found that microbiological sourc-- of certain dig-~tivo nzymes may b- u~ d in th- indicator syst-ms, in accordanc- with th-pr-s-nt in~ention, to provid- a mor- accurat- t-mp-rature r--pon-iv profil- r---mbling th- d-t-rioration b ing measur-d ov-r th- temp-rature range~ of pri~ary concern The harm~ul effect of microbial action on product quality may b nitdr~d in accordan~n-vi~ t~is pr-ferred embodim-nt of the inv~ntion by monitoring the te~p-rature and tim dependent activity of an associated artificial syst-m containing similar microb--, as i~
provided in the presently preferred indicators Expressed - - . . .. .

:-` 2 ~ ~3 .~
--11-- .
differently, the indicators incorporating microorganisms as r-aqent in accordance with this invention mploy like to mea~ure like Moreover, the utilization of two sets of first and second enzyme/substrate specific reaction pairs results in a fa~ter, more reactive temp-ratur- sensitivity than may be achiev~d in prior art indicator systems containing only one enzyme/substrate reaction pair This is because, th- enzymes and/or microorganisms exhibiting enzym- activity mployed a- components Or th- indicators have higher Qlo valu-s as compared with th- inorqanic reagents used in the above mentloned US 3,942,467 In addition, the use of two neutral substrate~ together with two enzymatic reagents provides an indicator system which is a homogeneous solution which also contributes to faster indicator reaction ti~e If high temperatur- exposure of a food product exc--ds a safe level, then a rapid and distinct color chang- for the indicator within hours, pr-ferably within minut-- or ev-n s-cond~ is needed or d--ir~d to indicato a spoilag- condition In accordanc- with th- invention, t~- n-w and improvQd indicator in a preferrQd embodim nt reli~s on a ureas- enzym-lur-a ub-trate r-action pair to m asure or count out th- ti~ compon-nt of a pr-d-t-rmin-d tim-t-mperaturo int-gral ~h- rat- of reaction for th decompo-ition of uF-a in th- pr---nce of ur-a-- to form a~ooniu~ ion and carbonat- ion in agueous ~ dium is r-lati~-ly w ll Xnown ov-r th- t~op-rature ranges expected for f~--h-r-frig-rat d foods as compared to frozen foods, i - , b t!W--n about 25 F to 110F For a giv-n anount of ur-as- nzy~ , th- tim until conv-r~ion or d-pl-tion of the ur-a substrat- to the ammonia and carbon dioxide end product~ is compl-te and is dependent on th- r-action rate for thi- te~p-ratur- range and th- starting conc-ntration of th- urea sub-trat- By varying th- tarting concentration of urea sub~trate present in th- indicator, the time reguir-d to deplete the urea ~ubstrat- can be -- 2~J ~8 adju~ted to establish a desired maximum time to expiration or end point for the combined integrating indicator T~e temperature monitoring component or thermal hi~tory monitoring aspect of th- combined indicator is provided by the competitiv-, fir~t y-ast or lipase enzyme/triacetin substrate ~p-cifie react$on pair of the indicator The yeast/triacetin reaction produces one mole of glycerol and thre- moles of acetic acid for every mole of triaeetin digestQd In accordance with th- inv-ntion, th- indicator may be placed in a pouch and th- poueh may b- affixed to a packagQ intended to be maintainod at 40 F during ~torage and shipm-nt prior to sal- As ~oon as th- indicator component~ are mix-d, both of its enzyme/~ubstrat-conversion reactions proeeed at a first rate, respectively, d-ternined by the ~O F te~p-rature If the refrigeration-br~ak~ down, or if th~ product r-uains on a loading doek without r~frigation on a hot sumo r day, for example, th- t~ p-ratur- of that produet may inerease several d-gr~-s for a short p riod of tim- For very 10F increa~- in th t~p ratur~, th- y~ast/lip~s-triaeetin reaetion r~te iner-a--s at a tw-lv- fold rate as do-s th- rat- of th- ur-a~-/ur-a r-aetion A- a result, dramatieally ~ore of th f$r-t and ~eeond r-aetion produet~ ar- produe-d upon expo-ur~ to higher t-mp ratur---~ Th- r-aetion products r-act to for~ a stabl- pN buff-r until th- urea ~ub~trat- i- d-pl~t-d -Th-r-aft r, th pN of th- indieator solution drops very rapidly to th- aeid rang- Tho indieator r-aeheJ its end point ith-rr by b~ing xpos-d to a eon~tant 40 F
temperatur- for a giv n laps d p-riod of time or, upon expo~ur- to l-vat-d t-ep ratur~s in a relativ-ly acc-l-rated or short-n-d period tim-In accordanc- with an esp-eially pr-f-rr-d e~bodiment of th$s inv ntion, it ha~ now b-en surpr$singly discovered that the te~peratur- sensitivity of indieators pr-pared in accordanc- with the teachlngs of the pr-s-nt invention ... . ..

2 ~ 8 ineluding a microorganism source for at least one of the enzyme-aetive reagents of one of the speeifie reaetion pairs is improved by shoek treating the microorganism The preferred shoek-treated indicators provide a distinct visual color chang- in a shorter period of time upon exposure to abus- temp-ratures Moreovsr, the preferred shocked indieator~ exhibit extendad shelf lives under non-stress-d, refrigerat-d temperature storag-In accordane- with this asp-et of th- inv-ntion, unexp-ctedly improv-d time-temp-rature intcgrating indicators are provid~d by shoek treating a mieroorganism ~ ' containing indicator composition prior to assembli~g tho indieator onto a product package Mor- partieularly, the microorganism shoek treatment is generally a proe~ss which alters or partialiy alters the cellular structure of the microorgani~m~ but whieh do-s not alt-r or d-nature th-subcallular structur-- or proteins Spor-s from th-tr-at-d microorganism may b~ us-d direetly Illu~trativ m t~ods for diQrupting or altering th-cellular strueture of the microorgani~m referred to as shock tr-atment heroin may inelude heat treating, Pasteurization, sonifieation, radiation, eh-mical exposure to aeid or bas- or a eombination th-r-of Th- hock treatm-nt should not b- so xtrem- ~o as to d-stroy the biospeeifie aetlvity of th- nzym- b ing eontribut-d by the mieroorganisa or what-v-r oth-r prot-in ~truetur~ are b ing ube-llularly provid-d by th- mieroorganism and r-spon-ibl- for th- dlg--tion or oth-r r~action r-lied upon I~-'h-at-shoe~ed mieroorgani~ms t-nd to r-spond more rapidly at abu-- temperature~ allowing for th- use of high-r ur-a l-v l- on th- bas- sid- of th- indieator This ~urprising and un-xp-eted diseov-ry m ans that a tim -t-mperatur- s-nsitiv- indieator may now-b provided for monitoring longsr shelf lif- produet~ Without wishing to be bound by any partieular theory, th- improved performane- of heat shoeked microorganisms a~ compared 2~3~7~g with unshocked microorganisms may be due to the fact that the cell membranes of the microorganisms limit solution effect~ such as diffusion and may reduce the contact of the substrate molecules and the enzymes In the shocked microorganism indicators, the cellular membranes of the microorganisms have been disrupted, pos~ibly permitting relea6e of th- enzymes into solution and mor- complete diffusion of th- sub~trate molecules to and from the enzymes.
The microorganism reagent route for indicators of this type i5 also attractive from an economic standpoint, since very small quantities of microorganism~ are needed to provide satisfactory levels of enzyme_ Very large numbers of microorgani~ms can be produced r-latively lS inexpensively The ability to shock the microorganism~ to thereby provid- alt-r~d microorgani~ms increas-~ the '~
safety of using th-~- materials without dramatically increasing th- cost ,Moreovor, th- ability to mploy untreat~d microorgani~m~ to provid- th- r-quired enzy~es followed by shock treatmont is conomically attractive as compared with th- cost of i~olating and purifying the enzymes Th- n-w and improv-d indicators of tho pre~-nt invention m~y b plac-d on or in th- vicinity of a product to be monitor d Th- integrating indicator~ may be a~-ociat-d with packaging in any on- of a larg- number of way- Pr-~ r~bly~ an indicator pouch or env-lop- iQ
provid-d from a cl-ar film web approved for U5- with packages of this product A pioc- of filter pap-r may be placed in th- indicator pouch and minut- quantiti-- of the premixed indicator may be added dropwi~o onto th- filter paper to form an indicator patch Th~ pouch i~ s-aled and ;~
thQreaft~r iQ plac~d on th- packag- so that tho patch is vi~ually obs-rvabl- from tho outside of the product package ~h- pr-par-d indicator pouch-~ may b- ~tored at low temperatures, preferably at temperatures b low 0 until desired for us- Thereupon, the froz-n pouche~ may 2~97~,~

be removed from low temperature storage an~ affixed to the outside of th- product package by means of a pr~ssure sensitive adh-sive, or other suitable attachment means Alternatively, the indicator system may be provided in the form of a 2-part composition which is placed in a 2-compartment barrier pouch affixed to the product packaging The individual parts are segregated from each other by mean~ of a rupturable film barri-r which may be ruptured to mix th- indicator compon~nt- to start the functioning of the indicator at an appropriate starting point Moreover, a smaller seaied envelope or see-through pouch of packaging film material containing the indicator may be placed in an obsQrvabl~ location within th- product packagQ or on the exterior thereof to provide a visual lS indication of frQshness As has been m-ntion-d above, the indicator pouch can b- affixed to th- product p~ckaging by any suitabl- m ans including by th- us- of adh-sive~
and/or simple and doubl- sid-d adhesiv tap~ strips may be used Wh-r- the packag d product is a foodstuff such as a packaged meat, it is pr-ferred to place the indicator pouch on the outside surface of th~ p~ckag- to monitor t-mperatures exp-rienc-d by the product at the surface of the meat or oth-r food product In this mann-r, th-indicator ~olution will b- expos-d to gen-rally the same ~l-vated t~op ratur-s as the out-r surfac- of th~ food product~ which is usually wh r- spoilaq- b gins on the product Other ob~-cts and advantages of th~ present invention will b co~- appar-nt fro~ the following Detailed D-~cription of th- Inv ntion and th- illustrativ-Examples .

Dr~LI~L~l~cRIpTSO~ OJ~ s~y5~
In accordanc~ with a preferred ~bodi~ nt, the new and i~proved time-te~p-rature integrating indicator of the present invention co~prises a first sp-cific reaction pair including an active reagent and a sub~trate sp-cific to 7~ ~

said active reagent In the preferred embodiment, the first specific reaction pair is reactive to form a first reaction product which causes or tends to cause the pH
value Or the indicator composition to change in an acidic first direction on the acid side of neutrality The first speci~ic reaction pair is for generating acid in the indicator 3y "specific reaction pair" h-rein is meant an observable bio-specific tendency for the active reagent and its substrate to react and form a given reaction product, regardle~e of the pre~ce of-oth~r ~actors which normally hinder or advers-ly influence non-bio specific interactions such ~g pH (within limits~, solution effects, relativ concentratlon of reactants, the pr~sence or absence of other materials in solution or the like Illustrativ- specific pair reactions includ- thos-reactions betw -n enzy~-~ and th~ir substrat~s, enzyme-co-factor interactlon~, antibody-an~igen reactions, immunologica} r-actions and other types of bio-specific interactions to nam- a few In th- pr f-rr-d first specific pair, the active reagent reactiv- with th- first sub~trate to produce an acidic first r-action product pref-rably is an enzyme or a microbiological organism or entity which ~xhibits enzyme activity with r-sp-ct to the substrat~ An espccially pref-rr-d first specific reaction pair includes y-ast a~
th- activc r-ag-nt, alon- or in combination with add-d lipa~- nzyo , and a triacetin substratQ Other acid-generating microb-s which may b- used heroin as the fir~t activ- r-ag-nt includ- lactobacilli which r-act with gluco~a to produc- lactic acid Other lactic acid-producing organisms which digest various sugars or carbohydrates to produce bio-acids or organic acids such as lactic acid including the lactobacilli, streptococci and p~diococci, may also be employed with th-ir respective carbohydrate sub trat-s to provide the first specific reaction pair 2 3 ~

'The active reagents and their specific substrates for use herein as the first specific reaction pair are generally commercially available and may be obtained from a number of sources For example, a suitable yeast is sold under the tradename RED STARo Lactobacilli may be obtained under the tradename LACTACEL from Microlife Technologies, Sarasota, Florida Triacetin, a triacetic acid ester of glycerol also known as glycerol triacetate, and various glucose and carbohydrate solutions may be obtained from sQveral commercial sourc-s The~ second specific reaction pair in the indicator is for generàting a base which changes or tends to change the pH of th- indicator to the basic ~ide of neutrality and to maintain the pH in the basic range until th- second pair sub~trate is depleted The second spacific reaction pair al-o compriseQ an active reagent and a second sub~trate with which or upon which the activ- reagent acts to generate a basic second reaction product Pr-f-rably, th- activ reag-nt'in the second Qpecific r-action p~'ir is''also an anzyme or a biological organism or entity exhibiting enzyme activity toward~ a s-l-ct d'sub~trata for g-n-rating ba~- In the preferr-d e~b~dim-nt, the sacond sp-cific raaction pair is reactiv- to form a s-cond r-action product including ammonia Accordingly, enzyma-~ubstrata pairQ known to generat- a~oonia ar- pr-~-rred E~p-cially praferred s-cond speci~$c'r-action pair m-mbQrs ar- urease and urea, as w-ll a~, micro-orgarism~ exhibiting signi'ficant urease actlvity and a ur-a substrate Other enzymes known to gen-rato ammonia in connection with th- dig-stion, or metabolism or catabolis~ of proteins, amino acids or nitrog-n-containing bases might also be su~ce~fully employed Urea~ commercially available in many forms One commercially available form iQ JacX b-an meal powder with documanted ureale activity from Sigma Cb-mical Company Th- urea substrate for the urea~e/ur-a 2~ $

enzyme/substrate pair is a standard chemical reagent available from a number of sources Th~ amount of urease enzyme and the relative amount of urea substrate used may be varied between reasonable S limits For example, tho concentration of urease enzyme -provided should be high enough so that a sufficiently strong green coloration of the indicator characteristic of the pH buffer initially appears An upper limit for the amount of ureaso may be determined by reaching a point of ~0 diminishing return~, namely, that adding more urease enzyme does not significantly increase the rate of production of urea d-composition products in the indicator and unnecessarily increases the expense The relative proportion of urea a-dded to thQ
-indicator may change the time component of the indicator function For a given amount of urease enzyme, for example, varying th- concentration of urea added to the indicator will vary~~b~ ti~ req~ired-to reach an end point signal in a non-t~operature stressed indicator ~ample For ex~ople, at a fir~t starting conc-ntration of urea, the indicator may not reach its color chang- time end point signal until two day~ have pa~s-d at exposure to 80F, whereas at a high-r starting concentration, the same indicator systam may take 3 5 days to reach its time end point signal color change Th- n-w and-i~prov~d tim~ t~mperatur~ int-grating indicator ~y~t-~ of th- present inv~ntion include at least on- pH is-nsitive dy- In the preferred embodiment, a pH s-nsitiu- combination dye package including m-nitroph-nol, p-nitroph-nol and litmus is includ-d for the indicator Th- meta- and para-nitrophenols exhibit a yellow coloration in the basic pH ranges abov- 7 0 with a sharp change to colorl-~- at 7 0 and less Th- litmus dye exhibits a dark blu- color in the ba~ic pH ranges above 7 0 and a distinct reddish pink color at a pH in the acidic rang- b low 7 0 The preferred combination of dyes therefore provide the indicator solution with a green ., ,.
color until such time as the urea substrate and the third reaction product pH buffer has been completely depleted and the pH of th~ indicator system has shifted to the acid range, whereupon a vivid and rapid reddish pink color develops The pH sensitive dye package should provide a distinct color change near neutrality, pH - 7 0 and preferably relies on an unmistakable signal value, such as a chango from green to red, to indicat- an expiration warning Although the above-packag- is preferred, other co~bin~tions may be used, such as a combination of bromothymol blue and neutral red If this lat-r combination is employed, the indicator is initially green and changes to an orange red color in th- acid range The indicator solution is preferably an agueous lS solution or a mixture of all of the ingredients, and may optionally include other ingredients, such as a monosodium phosphate buffer and/or a carbohydrate energy source for the microorganism, such as, preferably glucose may be provid~d The indicators may also includ- a wat-r-miscible co-solv-nt such as, any polar organic solvent, including, for example, lower alkanols such as ethanol, liquid~ketones, such a~ ac-ton- or m-thylethylk-tone, to nam--but a f-w In accordanc- with th- pre~ent inv-ntion, th- various compon-nts of th- indicator solution are imm-diately preoix-d prior to packaging with th- food products This comm nc s~th- ch-oi~try of th- indicating reactions and accordingly, a~nonia will b- st-adily produced from the s-cond nzym-/sub~trat- pair imm-diat-ly after mixing, at a reaction rat- d-t-rmined by the ambient t-mp-rature The total amount of the individual components comprising the indicator of th- pres-nt invention are d-t-roined by such consid-ration- a- the manufacturing proc--- costs, material compatibilities, application and th~ like and these factor~ are, in turn, largely determined by matters of convenienc- or deQign choice since the indicator system begins reacting a~ soon as the components are mixed r~

or brought into contact, it is apparent that these compononts may be kept separated until such time as activation of the indicator is desired Therefore, the components comprising the indicator may be enclosed in a rupturable container which is associated with a food package, or with a simple one part pouch or envelope container In a 2-part packago indicator may bo provided so that ono portion of a rupturabl- barrier package contains the enzym- and the microbes responsible for providing at least th- econd enzyme componont an illustrative 2-part package is aescribod in US 3,977,945 The dyes and substrates may be placed in the other chamber of the barrier package In use, the blister-pack is squeez~d to rupture the barrier between the two parts to mix and form the indicator The product packaging materials are preferably transparent and fabricated from chemically inert re~ilient materials such as polyethylene Moreover, the packaging for the indicator pouch or pocket i~ pr-ferably also mad- from a polyethylen- film The pouches or envelopes may be provided with an adhesive backing or may be affixed in some othor manner to the food packaging Alternativ ly, it is Or cours- pos~ible to mix th-components o~ th- indicator together in th- cours- of manufactur- and to fill and ~-al a singl- compartment contain-rs In a pr-f-rr-d embodiment, tho indicator is added onto a w-tt-d pi-ce of filter paper which is placed in a pock-t or nv-lop- which is adh~siv-ly bonded to tho outsid- surface of a food containor In accordance with this single compartm-nt approach, a r-eled tap- of an absorbent substrate may be un-rolled and pa~sed through a tr-ating station Th- mixed indicator composition~ may be applied to the substrato tape at thil tr-ating station to form a w~tted indicator tape The w-tted tap- may be re-rollod onto a reel and frozen until it i~ ready to be cutinto small piece~ for insertion in a pouch envelope The wetted tape may be cut and assembled into a pouch or strip 7 ~ 3 of continuous pouches which are thereafter rrozen if desired Freezing of the indicator solution may help to prevent the reactions from occurring, or may dramatically reduce the rates of reactions, so that the indicators are effectively "on hold" or "turned off" while frozen or chilled until they are ready for use Other objects and advantages of the present invention will become apparent from the following working Examples as follows ~CR~PrlON 0~ T~J PR~F~aR~ ODlM~NT8 A new and improved time-temperature integrating indicator was prepared as follows A 2% cold filtered sterilized solution of urease reagent was prepared by admixing 2 grams of Jack bean meal powder with ur-ase activity (SIGHA) Chemical Co in 100 mls of de-ionized water to which a diatomaceous earth filter aid was add~d Th- admixtur~ was cold-filtered through a 2~ Millipore filter to provide about 100 mls of a clear, substantially bacteria-free aqueous urease solution Part A o~ th- indicator was prepared by making a solution of th- fo~lowing 2 0 grams of a 0 4 M NaH2P04 6 0 grams of triac-tin (glycerol --triacet~te) 1 0 gram of glucos-O 4 gr~s of urea S O gr~a~ of the 2~ cold filtered urease solution prepared abov- and O l gr~m~ of dry yeast A Part B solution for th- indicator composition was prepared a- follow-In a 100 ml volum tric flask containing about 50 mls of deionized water 0 01 grams of m-nitroph-nol, 0 01 grams of p-nitrophenol and O OS grams of litmus were added and the mixture wa- agitat-d until each of th- pH-s-n itive dyes w-re completely dis olved to form a-cl-ar solution 2~
.

-Thereafter 10 gra~s of the Part A solution were added to the Part ~ dye solution in the flask which turned a medium green color 10 grams of reagent grade ethanol was also added and the combined solution in the flask was diluted with de-ionized water to 100 ml volume The indicator solution was incorporated into an indicator label suitable for us- with food packaging by first cutting 1/4 inch diameter discs from white filter paper The paper di~ks were plac~d in anvQlope~ or pouch pock-ts made from 2 ml thicknes~ lin~ar low density polyethylene film measuring about 1/2 of an inch square Threè sides of th~ pouch w-re h-at seamed in a DYN0 packaging machine - The filter paper was in~erted in the interior cavity of the pouch thus formed Thereafter, 30~1 of the green-co}ored indicator solution was transferred by an auto-pipettor onto th* paper disk and the open t-op of the pouch was imm-diately sealed using the Dyno equipment Thè pouch pocket containing the indicator was transferred to a controlled t-mp-ratur- ov-n and maintained at 80 ~ Under thes- conditions, the indicator compo~ition Or Exampl- 1 changed from a gre-n-colored disk to a vivid r dd$~h-pink disk within 2 0 day--' ~ el~_Z~
Anoth r indlcator solution was prepared by the same m thod mploy d ln Example 1, ~xc~pt tbat th- amount of ur-a~dd d to th- Part A solution wa- increasod from 0 04 gra to 0 1 gr~ o~ ur-a ~`~
This indicator was placed in an indicator pouch label and tested at 80-F ln accordanc~ with the m-thod of Exampl- ~- Th- indicator of Example 2 changed color from gr--n to reddi~h pink within 3 5 days 2~ 3-5 -- 21~79~

The following examples illustrate the improvement i~
performance obtained by shocking microorganisms incorporated into the indicator composition An aqueous solution of 0 0011% sodium phosphate, 6%
triacetin, 1% gl-ucose, 0 4% urea, 0 1% Jack Bean Meal and 0 1% yeast was diluted l to 10 with a solution containing 0 05% litmus, 0 01% p-nitrophenol, 0 01% m-nitrophenol and 10% reagent alcohol Dried yeast was used directly or mixed with water and heated in a water bath for l hour at 180 , 190F or 200 F This was followod by chilling to `37F - ' One-half $nch paper disc (SchleIcher and Schnell) were dipped into th- solution, placed into polyethylene pouches, frozen at -65, v~`uum s-alQd and stored at 80F
The results obtained are se~ forth in Table I, as follows TAB~E 1- EEE$ÇI~ OF HEAT-S~OCXING TREATMEN~
ON INDICATOR P W ORMAN OE
- - - Days to Reach Examole - Treatment~n~9i~ at 80F
A No H-~t 3 34 3 180 F for 1 hour 3 09 4 l90 F for 1 hour 2 71 ~' -' 5 20~ P for 1 hour 2 22 . . - . ~ :
''' IY~el~ 6-13 ~ ~;
'TX- sa~ iolution preparation m-thod of Examples 3-5 was r-p-at-d with various amounts of urea Th-~y-a~t for th- te~t ~ampl-s was mix-d with water and heated on a'steam table for 1/2 hour (t-mp-raturQ~
reaching 165 F) She indicator~ were held at 80 and' Th- results obtained are set forth in Table 2, as follow~

2 ~
TABLE 2: EFFECT OF VARYING UREA
SUBSTRATE CONCENTRATION
IN HEAT SHOCXED INDIC~TOR~
. . .

Exam~le: 6 ~ 7 C
U~ Coa~ent, 0.19- Ql% 0.2% 02% 03* 03% Q4~ 0.4%
% b~ Wd~l Time to End- ~~~
~Q~, Daya t P. 0.41.0~ 1.21 1~3 1.7~ 234 2.63 334 ~ 40 P. ~

TABLE 2 (Continued) Q E ~ g U~ Co~t. Ql~ Q~% 02% 02% 03~ Q39~ Q4~ 0.~%
%b~W~

; : :' Ti~ to End-Doint. Day~, ~a~ ~ ~. -- -- _ _ _ _ _ 6i~ 4~ 1~. 16 S0 ~6,5~ U.01 42.03 54.90 42~3 II~Q0 67.14 2 ~ ~ 3 ~ ~ ~

Average Qlo~S (reaction rate change with every 10C
change) of the test samples were approximately 13.5 as compared to 9.0 for the control samples.
Increasing levels of urea extends the shelf-life of S the indicator at refrigerated temperatures of 40or less.
However, limited concentrations of urea are used in this system because untreated microorganisms respond slowly at abuse temperatures of 80F or higher to produce visual changes in the indicator. "Heat shocked" microorganisms respond more rapidly at abuse temperatur-s , allowing for the application of higher urea levels. Thus, a temperature sensitive indicator has been developed which produces a visual change in a shorter time at abuse temperatures and a longer time at refrigerated temperatures.
The microorqanisms treatment may not be limited to "heat shoak." Other examples may possibly inslude: -pasteurization, sonification, radiation, chemical exposur~, acidity, alkalinity or any combination thereof.
Generally, any process which will alter or partially alter microorganisms cells (but not the spores) is considered suitable. Th- direct use of pure sporas may be employed.

E~a~pl- 14 Another n-w and improved time-temperature integrating ~-indicator was prepared as follows:
An acid free biological enzyme active reagent was prepar-d in the following manner: a frozen slurry sample of ~YgiQ9SQs9~ bactoria available from Microlife Technologies, Sarasota, Florida and sold under the trade designation LACTACEL~ 115 was obtain-d. LACTACEL 115 is sold as a starter culture for sausage making. A 0.5 gram sample of the frozen slurry was thawed. The thawed bacterial solution had a pH of about 5Ø 2.5 ounces (about 75ml~) of thawed LACTACEL were placed in a flask and diluted with deionized water to 450 mls. The diluted sample was centrifuged in an International Centrifuge apparatus at a setting of 2,000 rpm for a period of 30 minutes. The supernatant water was carefully decanted~
The concentrat-d bacterium was again diluted to 450 mls with deionized water, and was re-centrifuged. Again, the ' supernatant liquid was carefully decanted. The remaining bacterial residue was re-suspended in deionized water to 75 mls of volume. The washed LACTACEL 1150 product had a pH of 7Ø The 75 ml washed sample was refrozen and maintained at -60F until ready for use.
Another indicator composition in accordance with this invention was prepared by making a 100 ml solution of the following ingrèdiënts:
5.O mls of thawed, rinsed LACTACEL~ 115;
4.62 grams of APT broth;
5.0 grams of 2% Jack'Bean meal solution -- 'taS prepared in Ex~mple l);
5.0 grams of glucose (lactose may be substituted);- -0.09 grams of urea;
0.04 grams m-nitrophenol;
0'.004 gra~s Neutral Red;
0.01 grams'bromothymol blue.
The abov ingr~dients were agitated briefly until a clear, gre-n c~lor-d'solution was obtain-d.
Indicator*labels including a 1/4 inch diamater filter papar disks and''~/2-~nch square polyethylene film pouches w-re prepared in accordance with the method of Example 1, 30 ~1 of thQ ina$caeor solution of Example 14 was added to each o~ pouchas and the pouche~ were sealed on Dyno equipm-n~. '-' ' One portion of the pouches were transferred to a controll-d temp-rature oven maintained at 80F and another group of pouch-s were placed in a 40F refrigerator.
Undar thesa conditions, the Example 14 indicators after continuou~ expo~ure at 80F changed from a green colored to a reddi~h orange colored label after 2.0 days. The pouches maintained at 40F remained unchanged after at least 95 days.

r7 9 ~

~su~pl- ~5 According to this Example 1,000 lbs. of fresh turkey ~' breast are in~cted with a brine solution comprising 90%
water. The turkey breasts each ranged in weight from 2.5 to about 3.75 lbs. The turkey breasts ar placed on a rack in an oven and cooked at a high humidity at 160F dry bulb for two hours and then at 170F dry bulb until the internal temperature of the turkey breast~ is about 160F.
After cooking, the turkey brea~t~ are removed from the oven, cut into ~arters and then coated with a preservative solution. The turkey breasts were then cooled to approximately 32F and then vacuum packed in moisture-oxygen barrier films of ethylene vinyl acetate and saran laminated material.
An indicator label pouch is prepared in accordance ~ ~, with the method of Example 14 and is affixed by means of a '~
pressure-sensitive adhesive onto the outer surface of the va~uun packag,ed ,breaQts. Prior testing had revealed that when these vacuum packaged turkey breasts are carefully maintained under refrigerated storage at 40-F or less, they have an aver,ag- shelf life of about 65 days. The indicator w$1i not change color if the s~mple~ are, scrupulousl~ ~a,i~tained under controlled 40-F conditions.
However, $f th- ~a~ple~ are re~oved fro~ 40-F refrigerated storag-, and expo-ed to an abuse cond$tion at 80-F, the indicators w;ill change color with one day upon being Qxpos-d to *~ 80-F abuse te~perature.

l-- 16 30 ' In accord,ance with this example, a number of fresh turkey breasts~are injected with a brine solution comprising 90% water. The turkey brea~ts ,a,ch weigh about 9.0 lb~. The turkey breasts are vacuum packaged in a commercial ethylene vinyl acetate/saran laminate film.
Thereafter, the vacuum packaged turkoy brea~ts are cooked in a high humidity oven until the internal temperature of 2~7c~

the br-asts reaches 160F The turkey breasts are then remov-d from th~ oven and are permitted to cool to room temperature Th-r~after, the cool-d package~ ar- placed in refrigerated storage at 40F In accordance with th~
present invention and in accordance with the method of Exampl- 15, indicator lab l pouche~ containing th-indicator solution a- pr-par-d in Exampl- 13 are placed on the outsid- ~rapp~r by m-an~ of adhesive The indicator lab l~ are effectiv- to monitor th- tim- and temperature historieJ of th- product-, exhibiting distinct color change- at times in days which vari-~-d-pending on the storag- treat~ent for that sample test product :

A larg- nu~b r of ti~- temp~ratur~ indicators in accordanc- with th- pr---nt inv ntion may b- prepared in an auto~at~ ~Rnner a~ s-t forth h einaft-r An indicator pouch as~-~bly apparatus is provided includlng a-longitu~inally xtending f- d axis along which are arrang-d a re-l d filter papor ~upply, a pair of spaced upper and low-r packaging film web supplies, low t-mp-ratur- indicator bath olution, a pap r sta~ping station, ~ s-a~ w ld~ng staticn for fu~ing th oppo~ing w bs around a ~ tt-d indicator patch to for~ discrete spac-d-apart indlc~tor pouch-s, a p-rforating station wh-r-~t t-ar lin-~ b tw en ad~ac-nt lab-l pouches are i~part d to th- strip, and an adh-~ive strip applying ~tation, wh-r-at a line of adh-siv- and contact/release sh--t ar- appli-d to th- out-r surfac- of on- Or the web face~ forming th- pouches A tak- up r--l is al~o provid-d at th- downstrea~ end of th~ apparatus to provide a r--l-d carri-r Th- r~ d strip of t-ar-off pouches may b- placed in a ~r-ezer until r-ady for us-Th-reafter, when ready for us- tho re-l-d carrier may be r-mov-d from th- fr-ez-r Individual indicator pouches may b removed from th- r-mainder of the pouch-s on the re-led strip by tearing along tho p-rforations ~he 2 ~ 7 ~ ~

indicator labels may then be placed on the exterior of the product packages by p--ling the releas- sh--t away ~rom the adh~sive strip and pressing the adh-sivo strip and pouch against the product packaqlng - ~lternatively, S individual indicator labels without th- adhesivQ strip and release sheet combination, may simply b- placed on the exterior of the product package-in pIàin view, or if the product is doubl- wrapp~d,' in between tha wrap layers Although th- pres-nt inv-ntion h~s b en--d~'cri~ed with ref-r-nc~ to certain pref-rred mbodiments', modificati~ns~~r-changes may be made- herein b-y those skilled"in tni~ art without departing ~ro~ the scope a'nd ~
spirit of this invention as defined by the appended claims ''-'~ '' ' '' ~; - --.; . - . - ~

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A time-temperature integrating indicator comprising:
a first specific reaction pair including an active reagent and a substrate specific to said active reagent, said first specific reaction pair being reactive to form a first reaction product said first reaction product tending to cause the pH value of the indicator to change in an acidic or a basic first direction away from neutrality;
a second specific reaction pair including an active reagent and a substrate specific to said active reagent, said second specific reaction pair being reacted to form a second reaction product, said second reaction product tending to causing the pH
value of the indicator to change in a second direction opposite said first direction and beyond neutrality;
said first reaction product and said second reaction product in turn being reacted to form a third reaction product, said third reaction product including a stable pH buffer which maintains the pH
of the indicator at a stable value on the second direction side of neutrality until said second reaction product is no longer produced, said substrate in said first specific reaction pair being present in a stoichiometric excess amount over the amount of the substrate in said second specific reaction pair; and at least one pH-sensitive dye exhibiting one color for a pH in a basic pH range and a second different color for a pH in an acidic pH range, whereby said time-temperature integrating indicator exhibits a rapid and distinct color change from one to the other of said first and second colors after a predetermined amount of time has elapsed upon exposure to relatively constant temperatures, or after a relatively shorter period of time has elapsed, upon exposure to elevated temperatures.

2. A time-temperature integrating indicator as defined in Claim 1, wherein said first reaction product is acidic.

3. A time-temperature integrating indicator as recited in Claim 2, wherein said second reaction product is basic.

4. A time-temperature integrating indicator as defined in Claim 1, wherein said third reaction product comprises a stable basic pH buffer.

5. A time-temperature integrating indicator as defined in Claim 1, wherein said at least one pH-sensitive dye exhibits a green first color and a red second color.

6. A time-temperature integrating indicator as defined in Claim 1, wherein said first specific reaction pair and said second specific reaction pair each include an enzyme active reagent as said active reagent and an enzyme substrate as said substrate specific to said active reagent.

7. A time-temperature integrating indicator as defined in Claim 6, wherein the enzyme in said first specific reaction pair is provided by a microorganism source.

8. An indicator as defined in claim 6, wherein the second specific reaction pair is urease/urea.

9. An indicator as defined in Claim 6, wherein said first specific reaction pair is provided by yeast/triacetin.

10. An indicator as defined in Claim 9, wherein in said first enzyme/substrate specific reaction pair, the first enzyme further includes lipase.

11. An indicator as defined in Claim 7, wherein said microorganism is yeast.

12. An indicator as defined in Claim 7, wherein said microorganism is a shock-treated microorganism.

13. A time-temperature integrating indicator as defined in Claim 6, wherein said first specific reaction pair includes a microorganism exhibiting lactic acid-generating enzymatic activity as the active reagent and at least one carbohydrate as the substrate.

14. A time-temperature integrating indicator as defined in Claim 1, wherein said at least one pH-sensitive dye comprises a mixture of m-nitrophenol, p-nitrophenol and litmus.

15. A time-temperature integrating indicator as defined in claim 1, wherein said at least one pH-sensitive dye comprises a mixture of m-nitrophenol, bromothymol blue and Neutral Red.

16. A method for making an indicator for indicating time-temperature histories in conjunction with the storage of sensitive products and compositions, comprising admixing a first specific reaction pair for generating acid, a second specific reaction pair for generating base and at least one pH sensitive dye to form an aqueous solution, said first specific reaction pair having a first substrate present in a stoichiometric excess amount over the amount of a specific substrate provided in said second specific reaction pair.

17. A method as recited in Claim 16, wherein the first specific reaction pair for generating acid includes an enzyme component contributed by a microorganism source.

18. A method as recited in Claim 17, wherein said microorganism is shock-treated prior to addition to the indicator solution.

19. An improved packaging for room temperature and refrigerated storage of a foodstuff including a label incorporating an indicator as defined in Claim 1.

20. An indicator pouch for use in monitoring the time and temperature histories of an adjacent packaged food product to which said pouch ? affixed, said pouch comprising a thermoplastic pouch envelope having an indicator solution receiving cavity with a visually observable amount of the integrating indicator composition as defined in Claim 1 in said cavity.

21. An indicator pouch as defined in Claim 13 further including a substrate sheet material disposed in said pouch cavity with the indicator solution.