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High-p essu e p ocessing (HPP) o aw and d y-cu ed ham om
expe imen ally in ec ed pigs as a po en ial ool o he isk con ol
o Toxoplasma gondii
Ma ía Jesús G acia, Regina Láza o, Consuelo Pé ez-A quillué,
Ra ael Pagán, Se gio Ramos, João Luis Ga cia, Susana Baya i
PII: S1466-8564(19)31241-X
DOI: h ps://doi.o g/10.1016/j.i se .2020.102315
Re e ence: INNFOO 102315
To appea in: Inno a i e Food Science and Eme ging Technologies
Recei ed da e: 30 Sep embe 2019
Re ised da e: 5 Feb ua y 2020
Accep ed da e: 13 Feb ua y 2020
Please ci e his a icle as: M.J. G acia, R. Láza o, C. Pé ez-A quillué, e al., High-p essu e
p ocessing (HPP) o aw and d y-cu ed ham om expe imen ally in ec ed pigs as a
po en ial ool o he isk con ol o Toxoplasma gondii, Inno a i e Food Science and
Eme ging Technologies(2020), h ps://doi.o g/10.1016/j.i se .2020.102315
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TITLE:
High-P essu e P ocessing (HPP) o aw and d y-cu ed ham om expe imen ally
in ec ed pigs as a po en ial ool o he isk con ol o Toxoplasma gondii
Au ho names: Ma ía Jesús G acia, Regina Láza oa, Consuelo Pé ez-A quilluéa, Ra ael
Pagána, Se gio Ramosb, João Luis Ga ciac and Susana Baya ia*
A ilia ion:
a Ins i u o Ag oalimen a io de A agón - IA2 (Uni e sidad de Za agoza - CITA),
Ve e ina y Facul y, 50013 Za agoza (Spain)
b Cen o Nacional de Tecnología & Segu idad Alimen a ia CNTA, 31570 Na a a
(Spain)
c P o ozoology labo a o y, Depa amen o de Medicina Ve e iná ia P e en i a,
Uni e sidade Es adual de Lond ina - UEL, Pos al Box 6001, 86050-970 Lond ina, PR,
(B azil)
*Co esponding au ho : D a. Susana Baya i
Add ess: Dp o. P oducción Animal y Ciencia de los Alimen os. Facul ad de
Ve e ina ia. Uni e sidad de Za agoza. C/ Miguel Se e 177, 50013, Za agoza, Spain.
Phone numbe : +34-976-764135
Fax. numbe : +34-976-761612
E-mail: [email p o ec ed]
Abs ac
Raw and d y-cu ed mea s ha e been iden i ied as a po en ial sou ce o Toxoplasma
gondii in ec ion o humans. The p esen s udy e alua ed he e icacy o an al e na i e
non- he mal ood-p ocessing ea men , high hyd os a ic p essu e, on he iabili y o T.
gondii b adyzoi es in aw and d y-cu ed ham. Mea o pigs expe imen ally exposed o
4,000 oocys s o T. gondii VEG s ain was acuum-packaged and subjec ed o high
p essu e p ocessing (HPP). Tap wa e (6ºC±1ºC) was used as he p essu e- ansmi ing
luid, and i s empe a u e du ing HPP inc eased 2.7ºC pe 100 MPa. The e ec was
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e alua ed by bioassay in mice ollowed by qPCR. In aw ham, 100-400 MPa/1 min did
no inac i a e T. gondii, whe eas 600 MPa/20 min was e ec i e. In d y-cu ed ham, 600
MPa o 3 o 10 min we e no e ec i e and a 20-min ea men was equi ed o ende
he b adyzoi es non-in ec ious o mice. Ou esul s poin owa d he po en ial use o
HPP as a ool o isk con ol o T. gondii and as a ood sa e y gua an ee.
Key wo ds: Toxoplasma gondii; High-hyd os a ic p essu e p ocessing; HPP; Po k
mea ; D y-cu ed ham.
1. In oduc ion
Toxoplasma gondii is a ood-bo ne pa asi e ha causes human and animal
oxoplasmosis. Human oxoplasmosis can be li e- h ea ening in immunocomp omised
subjec s. In addi ion, ansplacen al ansmission du ing p ima y in ec ion in p egnancy
can esul in blindness, congeni al de ec s, men al e a da ion, o e en e al dea h
(Robe -Gangneux, Aube , & Villena, 2015). Fu he mo e, T. gondii in ec ion has been
implica ed in di e en o ms o beha iou al al e a ion and neu ological diso de s in
humans, sugges ing ha he in ec ion is po en ially mo e se ious han p e iously
supposed (Webs e , Kaushik, B is ow, Glenn, & McConkey, 2013).
The main ou e o T. gondii ansmission is ia consump ion o ood and wa e
con amina ed wi h spo ula ed oocys s, o mea con aining issue cys s. Inges ion o
unde cooked o cu ed mea con aining iable cys s has been sugges ed o be a majo
sou ce o T. gondii in ec ion (Bu olano e al., 1996; Cook e al., 2000; Jiang e al.,
2018; Kappe ud e al., 1996; Thalle , Tamma o, & Pen imalli, 2011).
D y-cu ed ham is a eady- o-ea p oduc (RTE) widely consumed in he Medi e anean
a ea. I is a nonsmoked po k mea p oduc cu ed wi h sal and ni i es, and s abilized
h ough dec eased aw. The en i e p ocess akes a leas 7 mon hs, al hough in some cases
he hams may be aged o mo e han 1 yea . Cu ing has adi ionally been ega ded as
an e ec i e echnology agains T. gondii (Genchi e al., 2017; F anssen e al., 2019).
Se e al s udies ha e poin ed ou ; howe e , ha cu ing may no be enough o inac i a e
T. gondii b adyzoi e cys s (Gómez-Samblás, Vilchez, Race o, Fuen es, & Osuna, 2015;
He e o e al., 2017; Wa nekulasu iya, Johnson, & Holliman, 1998).
Addi ional ea men o mea o elimina e iable T. gondii issue cys s would p o ide a
means o p o ec consume s. Ce ain ood-p ocessing echniques, such as hea ea men ,
eezing, high hyd os a ic p essu e p ocessing (HPP), and i adia ion ha e pa asi e-
inac i a ing po en ial (F anssen e al., 2019).
HPP is a echnology ha is ecei ing a g ea deal o a en ion wo ldwide. Fo he mea
sec o , HPP o e s a aluable al e na i e o he mal pas eu iza ion. Th ee main easons
make he HPP echnology bene icial o mea p oduc s: he inac i a ion o pa hogens
such as Lis e ia monocy ogenes o Salmonella, which allows he expo o mea
p oduc s o coun ies ha equi e he absence o L. monocy ogenes; an inc ease in shel -
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li e by inac i a ing spoilage mic oo ganisms; and he p ese a ion o nu i ional quali y
o aw and d y-cu ed mea wi hou hea ea men (Bajo ic, Boluma , & Heinz, 2012;
Hayman, Bax e , O’Rio dan, & S ewa , 2004; Jo é, Ayme ich, G èbol, & Ga iga,
2009; Rendueles e al., 2011).
The e ec i eness o HPP ea men depends p ima ily on he applied p essu e and on
holding ime. To pas eu ize mea and mea p oduc s, p essu e le els o 400-600 MPa
and holding imes o 3-10 min a e applied a low empe a u es, o 6-12ºC. In mos
cases, hese ea men s ha e led o an inac i a ion o mo e han ou log uni s o he
mos common ege a i e pa hogenic and spoilage mic oo ganisms, esul ing in
imp o ed sa e y and inc eased shel -li e (Bajo ic e al., 2012; Hugas e al., 2002;
Hyg ee a & Pandey, 2016; Rendueles e al., 2011). Al hough i is well known ha he
esis ance o mic oo ganisms is highly a iable, depending on he ype o o ganism and
he ood ma ix in ol ed (Rendueles e al., 2011), scan in o ma ion is a ailable on he
subjec o pa asi es in di e en ood p oduc s, pa icula ly in d y-cu ed ham.
S udies on he e ec i eness o HPP in elimina ing oodbo ne pa asi es ha e shown he
sensi i i y o T. gondii (Lindsay, Collins, Jo dan, Flick, & Dubey, 2005; Lindsay,
Collins, Holliman, Flick, & Dubey, 2006; Lindsay e al., 2008), C yp ospo idium
pa um (Collins e al., 2005), Anisakis la ae (B u i e al., 2010; Molina-Ga cía &
Sanz, 2002), T ichinella spi alis (Noeckle , Heinz, Lemkau, & Kno , 2001), Asca is
(Rosypal, Bowman, Holliman, Flick, & Lindsay, 2007), and T ichu is ulpis (Rosypal,
Zajac, Flick, Bowmand, & Lindsay, 2011) in low p essu e anges (100-400 MPa).
HPP could he e o e be an app op ia e ool o educing he isk o T. gondii. Al hough
p e ious s udies ha e indeed e alua ed he e ec o HPP on oocys s o T. gondii
(Lindsay e al., 2005, 2008), li le has been published on he e ec s o HPP on issues
in ec ed wi h T. gondii. Rega ding aw mea , only he s udy conduc ed by Lindsay e al.
(2006) has shown ha T. gondii issue cys s in g ound po k ha e been success ully
inac i a ed by applying 300-400 MPa o 30 s, whe eas 100 and 200 MPa ha e been
ine ec i e. The issues, howe e , did no come om in ec ed animals (na u ally o
expe imen ally), bu had been in en ionally spiked wi h pa asi es.
The p ope ies o he ood ma ix ha e been shown o ha e a g ea in luence on he
le hali y o HPP. D y-cu ed ham, a sal ed and d ied p oduc wi h low aw, has shown
lowe inac i a ion le els o some pa hogens (Salmonella, L. monocy ogenes, and S.
au eus) and spoilage (lac ic acid bac e ia) mic oo ganisms a e HPP han o he mea
p oduc s (Jo é e al., 2009; Rendueles e al., 2011).
To he bes o ou knowledge, no p e ious s udies e alua ing he e ec o high p essu e
on d y-cu ed mea pa asi ized wi h T. gondii ha e been ca ied ou o da e. The e o e,
he objec i e o he p esen s udy was o e alua e he po en ial use o he ac ual HPP
ea men s commonly employed by he mea indus y o isk con ol o T. gondii in
d y-cu ed ham. Fo his pu pose, we e alua ed he e ec s o HPP on he iabili y o T. gondii
b adyzoi es in ec ing aw and d y-cu ed ham made om expe imen ally in ec ed pigs.
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2. Ma e ial and me hods
2.1. Animal in ec ion, aw and d y-cu ed ham sampling, and sample p epa a ion o
analysis
Two T. gondii se onega i e whi e pigs we e used (~50 kg, 5 mon hs o age). One pig
was in ec ed o ally wi h 4,000 spo ula ed T. gondii oocys s (VEG s ain). The o he pig
was le unin ec ed and was used as he nega i e con ol. Animal in ec ion and slaugh e
was app o ed by he E hics Commi ee o he Complu ense Uni e si y o Mad id (Re .
PROEX 415/15). The pigs we e housed and main ained unde Biosa e y Class III
condi ions (in he VISAVET acili ies o he Ve e ina y Facul y o Complu ense
Uni e si y o Mad id) un il he s udy was comple ed. Six y days pos in ec ion, he
animals we e slaugh e ed and bo h haunches we e collec ed. One haunch was analysed
on day ze o, and he o he was cu ed o 12 mon hs. The echnological ea men o he
ham was ca ied ou in a cu ed ham p ocessing acili y as desc ibed by He e o e al.
(2017). Wa e ac i i y was de e mined in inal d y-cu ed hams by he Associa ion o
O icial Analy ical Chemis s O icial Me hod 978.18 (AOAC, 1998). Boneless aw and
d y-cu ed ham we e cu in o small pieces, minced, homogenized and e ige a ed.
2.2. High p essu e p ocessing
Fi y g ams o p epa ed samples we e placed in sealable plas ic bags and acuum-
packaged. The sealed bags we e hen placed in addi ional sealable bags and sealed
again. These addi ional bags we e used o p e en any po en ial con amina ion o he
HPP uni . Samples we e s o ed unde e ige a ion un il HPP ea men and analysed
he ea e .
HPP p ocess was pe o med in an indus ial Hipe ba ic 135 high p essu e p ocessing
uni (Hipe ba ic Bu gos, Spain), equipped wi h a 135 L essel, using ap wa e
(6ºC±1ºC) as he p essu e- ansmi ing luid. The wa e empe a u e du ing HPP
inc eased 2.7ºC pe 100 MPa.
A se ies o di e en HPP ea men s we e ca ied ou . HPP was applied o aw mea a
100, 200, 300, and 400 MPa wi h a holding ime o 1 min a each p essu e es ed. In
addi ion, a HPP ea men o 600 MPa was applied du ing a holding ime o 20 min.
High hyd os a ic p essu e was applied o d y-cu ed ham a 600 MPa wi h holding ime
o 3, 10, and 20 min. Each HPP ea men was eplica ed h ee imes. P essu e buildup
was 240 MPa/min, and decomp ession was ins an aneous.
A nega i e con ol and a posi i e con ol we e included o each assay ( aw and d y-
cu ed ham). The nega i e con ol did no con ain T. gondii cys s, while he posi i e
con ol did, bu was no subjec ed o any HPP ea men .
2.3. Mouse bioassay o issues o T. gondii
To de e mine iabili y, we pe o med bioassay on mice, ollowed by eal ime PCR
(qPCR). A e he inocula ion o mice o he sample o be analysed, se ocon e sion
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occu ed i he pa asi e was con ained in i . I he pa asi e was iable in he po k sample,
he issue cys s could o m in he b ain o exposed mice (Figu e 1) and issue cys
o ma ion could be con i med by PCR. Fo each HPP- ea ed o con ol sample, a
concen a ion bioassay echnique wi h an acid pepsin diges ion p ocedu e was applied
o aw and d y-cu ed ham, as desc ibed elsewhe e (Baya i e al., 2010; Dubey, 1998).
A 0.5 ml aliquo o diges ion ex ac was inocula ed in ape i oneally in o each o eigh
20-25 g CD1 Swiss emale mice pe sample (Jan ie Labs, Le Genes -Sain -Isle,
F ance). All expe imen s included nega i e con ol mice. The mice came wi h a heal h
ce i ica e a es ing ha hey we e ee om pa hogens. They we e kep a he Cen o de
In es igación Biomédica de A agón (CIBA) in Za agoza (Spain). The inocula ion,
main enance, and eu hanasia o he mice we e pe o med unde he s anda ds o he
E hics Ad iso y Commission o Animal Expe imen a ion and he Biosecu i y
Commission o he Uni e si y o Za agoza, as g an ed by Judgmen No PI55/14. These
guidelines a e in acco dance wi h he P o ocol o In e na ional Guiding P inciples o
Biomedical Resea ch In ol ing Animals (Di ec i e 2010/63/EU).
2.3.1. IFA o mouse se a
Blood samples we e d awn om mice ha su i ed 60 days a e inocula ion. Se a
samples o mice we e analysed by IFA o de ec an ibodies agains T. gondii wi h
polyclonal abbi an imouse immunoglobulins (DakoCy oma ion). Se um om each
mouse was dilu ed 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320. A posi i e and a nega i e
con ol se um om p e ious s udies in ou labo a o y we e included in each es . Final
p epa a ions we e examined wi h an Eclipse 80i luo escence mic oscope (Eclipse 80i,
Nikon ins umen s INC, Ne he lands). Se a samples wi h a i e o 1:10 we e conside ed
posi i e.
2.3.2. DNA ex ac ion and iden i ica ion o T. gondii
Analysis o T. gondii DNA om b ains o se ologically posi i e mice was pe o med by
qPCR o de e mine iabili y o he pa asi e. Fi een mg piece o each b ain we e
homogena ed h ough hand pis el plas ic o a ing plunge s in an eppedo . The DNA
ex ac ion was pe o med using Ul aClean® Tissue & Cells DNA Isola ion Ki Sample
Ca alog No. 12334-S (Mobio Labo a o ies, Inc.) acco ding o he manu ac u e ’s
ins uc ions. Two se s o p ime s o DNA ampli ica ion a ge ing speci ic sequence o
529 epea elemen we e used (ToxoRoc F TAGACGAGACGACGCTTTCC, ToxoRoc
R TCGCCCTCTTCTCCACTCT, and ToxoRepea 500 F
CGCTGCAGGGAGGAAGACGAAAGTTG, ToxoRepea 500 R
CGCTGCAGACACAGTGCATCTGGATT) and ano he se o p ime s o he su ace
an igen ma ke genes (SAG) (ToxoSG1 F TCATCGGTCGTCAATAA, ToxoSG1 R
CTTTGACTCCATCTTTCC). A CFX Connec eal ime PCR ins umen (Bio-Rad
Labo a o ies) was used o he ampli ica ion and de ec ion o T. gondii using Go aq
Sybe g een Mas e Mix (ca alogue # A6002) om P omega. The eac ion olume was
20 ml, and samples we e un in iplica es. The p o ocol consis ed o 7 min a 94 ºC o
enzyme ac i a ion (ho s a ), and 40 cycles o dena u a ion a 94 ºC o 5 s, annealing a
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55 ºC o 30 s, and ex ension a 72 ºC o 10 s. The p og am ended wi h a dissocia ion
cu e om 60 o 94 ºC wi h a 0.5 ºC inc ease in e al. Each PCR un included a
nega i e con ol, a posi i e con ol, and a sepa a e eac ion o Ac in DNA copies as
in e nal con ol (IC). A sample was conside ed posi i e i a leas wo o he iplica es
we e posi i e wi h bo h ma ke s. The h eshold cycle (C ) alue used was indica ed by
he ma ke 529 epea elemen (C mus be lowe han 38), and he SAG ma ke se ed
o con i m he esul . Calib a ion cu es we e p epa ed using 15 mg o homogena e
nega i e issues spiked wi h a known numbe o achyzoi es (104, 103, 102, 101, and
100). The samples we e subsequen ly homogenized and p ocessed wi h he comme cial
DNA ex ac ion ki ollowing he manu ac u e ’s ins uc ions in he same way as he
es o he samples.
3. Resul s and Discussion
The esul s o he e ec o high-p essu e p ocessing on T. gondii issue cys s in aw
ham a e p esen ed in Table 1. High-p essu e p ocessing o aw mea a p essu es o 100,
200, 300, and 400 MPa wi h holding ime o 1 min was no e ec i e in educing
in ec i i y o T. gondii. T ea men wi h HPP a 600 MPa o 20 min comple ely
elimina ed in ec i i y o mice. None o he mice inocula ed wi h non-in ec ed aw mea
became in ec ed wi h T. gondii. In ec ion was obse ed in mice inocula ed wi h non-
p essu e- ea ed T. gondii issue cys con aining g ound po k.
Ou esul s di e om hose desc ibed by he only s udy ha has p e iously e alua ed
he applica ion o HPP ea men s agains T. gondii inocula ed in aw mea (Lindsay e
al., 2006). Acco ding o hose au ho s, he HPP ea men has achie ed he inac i a ion
o he pa asi e wi h p essu es a 300-400 MPa and a holding ime o 30 s. These esul s,
di e ing om ou s, could be due o he ac ha he au ho s ha e wo ked wi h g ound
po k in en ionally spiked wi h mouse b ains con aining iable issue cys s; la e samples
ha e been diges ed in acid pepsin solu ion and bioassayed in mice. Since he pa asi e is
con ained in he mouse b ain issue and no in pig muscle, i is possible ha he
diges ion p ocess may ha e a ec ed he iabili y o b adyzoi es o a g ea e ex en
(Dubey e al., 1995; Dubey, 2010; Mendoça, Domingues, da Sil a, Peze ico, &
Langoni, 2004). Ou s udy, on he con a y, used expe imen ally in ec ed animals,
which implies ha T. gondii issue cys s we e loca ed in he muscle issue, which is
mo e esis an o he diges i e p ocess. As he i s in es iga ion o e alua e HPP in
mea om animals expe imen ally in ec ed wi h T. gondii, his s udy holds special
ele ance, since i mo e ai h ully ep oduced he condi ions in which he pa asi e is
ound wi hin he muscula issues.
Ano he possible eason o ou disc epancy wi h he esul s o he esea ch ca ied ou
by Lindsay e al. (2006) is he echnique we used o con i m iabili y. Bioassay is
ega ded as he gold s anda d o he e alua ion o ea men e icacy o pa asi es in
ood (F anssen e al., 2019). Bo h Lindsay e al. (2006) and ou own s udy ha e used
bioassay; howe e , we con i med iabili y by analysing he mouse b ain by q-PCR,
unlike Lindsay e al. (2006), who ha e used his ology. PCR has mo e speci ici y and
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sensi i i y in de ec ing he p esence o T. gondii han his ological de ec ion (Es eban-
Redondo e al., 1999; Ops eegh, Scha es, & an de Giessen, 2016). Resea ch on
oodbo ne pa asi es should be imp o ed wi h he goal o s anda dizing expe imen al
app oaches o he e alua ion o inac i a ion me hods, bu also in o de o s anda dize
me hods designed o moni o inac i a ion (F anssen e al., 2019).
In he cou se o he HPP p ocess, p essu e is ansmi ed uni o mly and ins an ly
(Hugas, Ga iga, & Mon o , 2002) wi h li le a ia ion in empe a u e, independen o
ood shape o size (Rendueles e al., 2011). This aspec is o pa icula in e es when
in es iga ing pa hogens such as T. gondii, o which he dis ibu ion o cys s in issues is
ho oughly andom. I has been es ima ed ha he numbe o T. gondii cys s pe g am o
issue om ood animals such as pigs may be less han 1 cys /50 g o issue, and one
cys may con ain a ew o 1000 indi iduals (Es eban-Redondo e al., 1999; Ops eegh e
al., 2016).
High hyd os a ic p essu e usually exe s a s onge des uc i e e ec on o ganisms wi h
a highe deg ee o o ganiza ion and s uc u al complexi y. P oka yo es a e usually mo e
esis an han euka yo es (Yus e, Capellas, Fung, & Mo -Mu , 2001), and i is assumed
ha he des uc ion o pa asi es is achie ed wi h ela i ely low p essu es (Rendueles e
al., 2011). Howe e , he p essu es and imes equi ed o inac i a e T. gondii a e highe
han hose used in p e ious s udies agains issue cys (Lindsay e al., 2006) o oocys s
(Lindsay e al., 2008). They a e also supe io o hose used agains o he pa asi es
(B u i e al., 2010; Collins e al., 2005; Molina-Ga cía & Sanz, 2002; Noeckle e al.,
2001; Rosypal e al., 2007, 2011).
The applica ion o HPP o mea modi ies quali y pa ame e s such as colou , ex u e, and
wa e holding capaci y (Bajo ic e al., 2012). Mea colou is one o he mos impo an
quali y cha ac e is ics o consume s in a pu chase si ua ion. HPP a ec s quali y
pa ame e s o aw mea , pa icula ly depending on he p essu e le el applied, and hus
ypical cha ac e is ics associa ed wi h aw mea such as ex u e and, especially, colou
can be modi ied o a ema kable deg ee. The mea becomes mo e gel-s uc u ed and
pale , losing he ypical appea ance o aw mea (Bajo ic e al., 2012). Thus hampe s
he comme cializa ion o HPP- ea ed aw mea s due o he loss o he ypical aw mea
colou om a consume pe spec i e (Che el & Culioli, 1997). These changes a e
indeed ele an i he p oduc s a e u he p ocessed o e en di ec ly consumed; hey
would, howe e , be sa e and mo e nu i ious han cooked mea , since HPP has no
nega i e impac on nu i ional alue (Bajo ic e al., 2012). Rega ding he
physicochemical e ec o HPP echnology on ood, i is so e and p ese es la ou
be e han a he mal ea men (Schindle , K ings, Be ge , & O lien, 2010). Such
changes in colou and ex u e may e en be desi able in some cases, when he pu pose is
o elabo a e new p oduc s and new o ms o p esen a ion.
The chemical composi ion o ood has been iden i ied as an impo an ac o ha can
in luence he g ea e o lesse esis ance o mic oo ganisms o HPP. The e icacy o
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HPP dec eases wi h educed aw (Jo é e al., 2009; Rendueles e al., 2011), as is he case
o he d y-cu ed ham samples e alua ed in his s udy (aw ~0.85).
Fo ou s udy o he e ec o HPP on T. gondii issue cys s con amina ing d y-cu ed
ham, we assayed h ee di e en HPP ea men s. We i s applied wo ea men
condi ions (600 MPa/3 min and 600 MPa/10 min) commonly used by he ood indus y
o con ol L. monocy ogenes and ul il he equi emen s o USDA and Heal h Canada
(Bo e -Cid, Belle i, Ga iga, & Ayme ich, 2011; Hayman e al., 2004; Hugas e al.,
2002). These holding imes a e di ec ly ela ed wi h he wa e ac i i y and a con en o
he d y-cu ed p oduc s and, depending on hese wo cha ac e is ics, HPP is modula ed
be ween 3 and 10 min o ea men (habi ually 5 min) (He eu e al., 2012). We hen
applied a longe ea men , o 600 MPa/20 min. As desc ibed in Table 2, while HPP a
600 MPa o 3 and 10 min did no elimina e T. gondii in ec i i y o mice, 600 MPa o
20 min gua an eed i s inac i a ion. None o he mice inocula ed wi h non-in ec ed d y-
cu ed mea became in ec ed wi h T. gondii. Con e sely, in ec ion was obse ed in mice
inocula ed wi h non-p essu e- ea ed T. gondii issue cys con aining g ound po k.
Such ex ended imes do no end o be employed a he indus y (Bajo ic e al., 2012;
Hyg ee a & Pandey, 2016; Rendueles e al., 2011); ins ead, imes o 3-10 min a e mo e
common (Bo e -Cid e al., 2011; Hayman e al., 2004; Jo é e al., 2009). Six hund ed
MPa o 10 min o holding ime ha e been necessa y o each 3 log-inac i a ion o
p essu e- esis an L. monocy ogenes in Spanish d y-cu ed ham (aw 0.880; pH 5.84)
(Bo e -Cid e al., 2011). This inac i a ion le el is ele an o ul illing he
equi emen s o USDA and Heal h Canada ega ding he con ol o L. monocy ogenes in
RTE ood p oduc s. To he bes o ou knowledge, ou s is he i s s udy o ha e
e alua ed he e ec o HPP on he inac i a ion o T. gondii in d y-cu ed ham om
expe imen ally in ec ed animals.
I is gene ally accep ed ha cu ed mea colou is pa icula ly esis an o HPP. The
majo i y o s udies ha e epo ed no change in edness, he eby suppo ing he
p o ec i e ole o ni i ica ion on mea colou (Bak, Boluma , Ka lsson, Lindahl, &
O lien, 2019). The colou o mea is s ongly ela ed o i s wa e con en (Fe ini,
Comaposada, A nau, & Gou, 2012), and HPP applied o up o 10 min ha e had a
negligible e ec on d y-cu ed ham. Howe e , wi h mo e ex ended ea men imes,
isible changes could appea in he p oduc s (Bajo ic e al., 2012; Bak e al., 2019).
These changes migh a y acco ding o he mea ’s ini ial ex u al cha ac e is ics and
depending on i s p o ein sys em, i s empe a u e, he p essu e applied, and he du a ion
he eo . Juiciness, sp inginess, and chewiness a e inc eased by HPP depending on
ea men ime (Coll-B asas e al., 2019). Longe ea men s inc ease p oduc ion cos s,
since hey limi he numbe o ea men cycles pe hou and educe he kilo p oduc ion
a e pe hou . In o de no o exceed hose imes, one o he solu ions applied bo h in he
mea sec o and in o he sec o s (juices and be e ages, p epa ed dishes, e c.), consis s in
applying mul i-pulsed p essu e cycles (comp ession and decomp ession a es). Mul i-
pulsed HPP could be used o inac i a e spoiling and pa hogenic agen s, he eby helping
o imp o e ood sa e y and quali y. An op imized balance be ween ideal p essu e,
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5 h Decembe 2019
Au ho s a emen
Dea edi o s,
Please, below you can ind he indi idual con ibu ion o au ho s o he pape
IFSET_2019_1177, using CRediT oles:
Susana Baya i, as coo dina o o he esea ch p ojec leading o his publica ion,has p oposed
he s udy, has deal wi h he acquisi ion o he inancial suppo , as well as he planning,
execu ion and supe ision o he esea ch ac i i y. She has coo dina ed he mul idisciplina y
eam in eg a ed by he au ho s o his wo k. (CRediT oles: Concep ualiza ion; Da a cu a ion;
Fo mal analysis; Funding acquisi ion; In es iga ion; Me hodology; P ojec adminis a ion;
Resou ces; Supe ision; Visualiza ion; W i ing - e iew & edi ing).
Ma ía Jesús G acia, pa asi ology expe , has p oposed he s udy and has pa icipa ed on he
planning o he ials. She has also con ibu ed o expe imen al asks ela ed o pig in ec ion
and sample analysis (mouse bioassay). She has been esponsible o w i ing he ini ial d a .
(CRediT oles: Concep ualiza ion; Da a cu a ion; Fo mal analysis; In es iga ion; Me hodology;
Visualiza ion; W i ing - o iginal d a ; W i ing - e iew & edi ing).
João Luis Ga cia, pa asi ology expe , has p o ided he Toxoplasma VEG s ain o he s udy
and has pa icipa ed in he expe imen al pig in ec ion design. (CRediT oles: Concep ualiza ion;
Fo mal analysis; In es iga ion; Me hodology).
Ra ael Pagán and Se gio Ramos ha e p o ided expe ise in ood echnology and
ins umen a ion o HPP ea men , and ha e pe o med HPP expe imen s. (CRediT oles:
Concep ualiza ion; Fo mal analysis; In es iga ion; Me hodology; Visualiza ion; W i ing - e iew
&edi ing).
Regina Láza o and Consuelo Pé ez-A quillué, ood sa e y expe s, ha e pa icipa ed in sample
p epa a ion o HPP ea men s and ha e con ibu ed o da a analysis ela ed wi h isk
assessmen . (CRediT oles: Concep ualiza ion; In es iga ion; Me hodology; Visualiza ion;
W i ing - e iew &edi ing).
Since ely you s,
Susana Baya i (Co esponding au ho )
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Figu e 1. Toxoplasma gondii cys in mouse b ain
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Table 1. E ec o di e en HPP ea men condi ions on Toxoplasma gondii issue cys s
in aw ham
T ea men
Exposu e ime
(min)
Se oposi i e
mice a
Viabili y o
T. gondii b
In ec i i y
o he
sample
Nega i e
con ol
0
0/8
-
-
Posi i e
con ol
0
4/8
4/4
35.61
33.83
34.23
35.02
+
100MPa
1
2/8
1/2
35.29
+
8/8
2/8
31.01
31.43
5/8
1/5
35.75
200MPa
1
2/8
0/2
+
2/8
0/2
3/8
2/3
33.20
31.45
300MPa
1
4/8
2/4
32.46
32.42
+
4/8
0/4
5/8
2/5
34.78
31.51
400MPa
1
2/8
2/2
33.88
32.35
+
3/8
0/3
2/8
0/2
600MPa
20
2/8
0/2
-
3/8
0/3
3/8
0/3
aSe oposi i e mice by IFA/To al o mice.
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bPosi i e mice b ain by PCR/To al o se oposi i e mice by IFA; C alues o posi i e
mice b ains (< 38).
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Table 2. E ec o di e en HPP ea men condi ions on Toxoplasma gondii issue cys s
in d y-cu ed ham
T ea men
Exposu e
ime
(min)
Se oposi i e
mice a
Viabili y o
T. gondii b
In ec i i y o
he sample
Nega i e
con ol
0
0/8
-
-
Posi i e
con ol
0
5/8
3/5
28.77
29.77
30.58
+
600MPa
3
5/8
0/5
+
3/8
0/3
3/8
1/3
34.84
600MPa
10
4/8
1/4
34.67
+
2/8
0/2
1/8
0/1
600MPa
20
1/8
0/1
-
1/8
0/1
4/8
0/4
aSe oposi i e mice by IFA/To al o mice.
bPosi i e mice b ain by PCR/To al o se oposi i e mice by IFA; C alues o posi i e
mice b ains (< 38).
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Highligh s
• Fi s HPP s udy on T. gondii in aw and cu ed ham om expe imen ally
in ec ed pigs
• Viabili y o T. gondii assessed by bioassay in mice ollowed by PCR
• 600 MPa/10 min does no inac i a e T. gondii in d y-cu ed ham while 600
MPa/20 min does
• HPP o aw and d y-cu ed ham is a po en ial ool o he isk con ol o T.
gondii
Indus ial ele ance ex
Unde eal p oduc ion condi ions, he usual HPP ea men s applied by he ood
indus y o con ol L. monocy ogenes and o he pa hogenic bac e ia a e 600 MPa o p essu e
and holding imes o 3-10 min. Ou s udy demons a ed, howe e , ha longe ea men imes
a e equi ed o inac i a e he pa asi e and, hus, o gua an ee he sa e y o aw and d y-cu ed
mea s in o de o educe he public heal h isk o oxoplasmosis. Fu he esea ch is needed o
e alua e o he HPP condi ions, including pulsed cycles, o he inac i a ion o T. gondii in oods
o animal o igin.
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Figu e 1