EFSA Jou nal. 2025;23:e9160.
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h ps://doi.o g/10.2903/j.e sa.2025.9160
e sa.onlinelib a y.wiley.com/jou nal/1831-4732
SCIENTIFIC OPINION
Nu i ional sa e y and sui abili y o a speci ic p o ein
hyd olysa e manu ac u ed by Fon e a Co- ope a i e G oup L d
de i ed om a whey p o ein concen a e and used in in an
o mula and ollow- on o mula
EFSA Panel on Nu i ion, No el Foods and Food Alle gens (NDA) | To s en Bohn |
Mon aña Cáma a | Jacqueline Cas enmille | S e aan de Henauw | Ka en- Ildico Hi sch- E ns |
Angeles Jos | Alexand e Maciuk | Inge Mangelsdo | B eige McNul y | And oniki Naska |
K is ina Pen ie a | Al onso Siani | F ank Thies | Lau ence Cas le | Ma y Few ell |
Hildega d P zy embel | A iane Ti z | Dominique Tu ck
Adop ed: 28 No embe 2024
DOI: 10.2903/j.e sa.2025.9160
Abs ac
The Eu opean Commission asked EFSA o deli e an opinion on he nu i ional
sa e y and sui abili y o use in in an and ollow- on o mula o a speci ic p o ein hy-
d olysa e om whey p o ein concen a e manu ac u ed by Fon e a Co- ope a i e
G oup L d. Fon e a Co- ope a i e G oup L d. submi ed a dossie o he Eu opean
Commission o eques an amendmen o Regula ion (EU) 2016/127 wi h espec o
he p o ein sou ces ha may be used in he manu ac u e o in an and/o ollow-
on o mula. The p o ein hyd olysa e unde e alua ion is su icien ly cha ac e ised
wi h espec o he hyd olysed p o ein. In he pe inen in e en ion s udy p o-
ided, an in an o mula manu ac u ed om he p o ein hyd olysa e wi h a p o ein
con en o 2.0 g/100 kcal and consumed as he sole sou ce o nu i ion by in an s
o 3 mon hs led o a g ow h simila o a o mula manu ac u ed om in ac cow's
milk wi h he same p o ein con en . Da a on gas oin es inal ole ance o he o -
mula did no aise any conce ns. No expe imen al da a ha e been p o ided on he
nu i ional sa e y and sui abili y o his p o ein sou ce in ollow- on o mula. Gi en
ha ollow- on o mula is consumed wi h complemen a y oods and he p o ein
sou ce is nu i ionally sa e and sui able in an in an o mula ha is he sole sou ce
o nu i ion o young in an s, he Panel conside s ha he p o ein hyd olysa e is
also a nu i ionally sa e and sui able p o ein sou ce o use in ollow- on o mula.
The Panel concludes ha he p o ein hyd olysa e unde e alua ion is a nu i ion-
ally sa e and sui able p o ein sou ce o use in in an and ollow- on o mula, as
long as he o mula in which i is used con ains a minimum o 2.0 g/100 kcal p o-
ein and complies wi h he composi ional c i e ia o Regula ion (EU) 2016/127 and
he amino acid pa e n in i s Annex IIIA.
KEYWORDS
cha ac e isa ion, clinical ial, ollow- on o mula, in an o mula, nu i ional sa e y, p o ein
hyd olysa e, sui abili y
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion-NoDe i s License, which pe mi s use and dis ibu ion in any medium, p o ided he
o iginal wo k is p ope ly ci ed and no modi ica ions o adap a ions a e made.
© 2025 Eu opean Food Sa e y Au ho i y. EFSA Jou nal published by Wiley-VCH GmbH on behal o Eu opean Food Sa e y Au ho i y.
Co espondence: ni @e sa.eu opa.eu
The decla a ions o in e es o all scien i ic
expe s ac i e in EFSA's wo k a e a ailable
a h ps://open.e sa.eu opa.eu/expe s
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
CONTENTS
Abs ac ................................................................................................................................................................................................................................1
1. In oduc ion ..............................................................................................................................................................................................................3
1.1. Backg ound and Te ms o Re e ence as p o ided by he eques o .........................................................................................3
1.1.1. Backg ound ....................................................................................................................................................................................3
1.1.2. Te ms o Re e ence.......................................................................................................................................................................3
1.2. In e p e a ion o he Te ms o Re e ence ............................................................................................................................................4
2. Da a and Me hodologies ......................................................................................................................................................................................4
2.1. Da a ...................................................................................................................................................................................................................4
2.2. Me hodologies..............................................................................................................................................................................................4
3. Assessmen ................................................................................................................................................................................................................5
3.1. Cha ac e isa ion o he p o ein hyd olysa e ......................................................................................................................................5
3.2. Cha ac e isa ion o he o mula manu ac u ed om he p o ein hyd olysa e used in he clinical s udies
p o ided ..........................................................................................................................................................................................................6
3.3. Sa e y and sui abili y o he in an and ollow- on o mula ..........................................................................................................7
3.4. Unce ain ies ela ed o he nu i ional sa e y and sui abili y o he in an o mula ..........................................................9
4. Conclusions ................................................................................................................................................................................................................9
5. Documen a ion as p o ided o EFSA ...............................................................................................................................................................9
5.1. S eps aken by EFSA ....................................................................................................................................................................................9
Abb e ia ions ....................................................................................................................................................................................................................9
Acknowledgemen s ..................................................................................................................................................................................................... 10
Reques o ......................................................................................................................................................................................................................... 10
Ques ion numbe ..........................................................................................................................................................................................................10
Copy igh o non- EFSA con en .............................................................................................................................................................................. 10
Panel membe s .............................................................................................................................................................................................................. 10
Legal no ice .....................................................................................................................................................................................................................10
Re e ences........................................................................................................................................................................................................................ 10
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1 | INTRODUCTION
1.1 | Backg ound and Te ms o Re e ence as p o ided by he eques o
1.1.1 | Backg ound
Commission Di ec i e 2006/141/EC1 lays down ha monised ules applicable in he en i e EU o in an o mulae and ollow-
on o mulae. The Di ec i e allows he use o p o ein hyd olysa es as sou ce o p o ein in in an o mulae and ollow- on
o mulae unde ce ain condi ions (A icles 5–7; Annex I, poin 2.2; Annex II, poin 2.2 and Annex VI).
Commission delega ed Regula ion (EU) 2016/1272 ans e s he exis ing ules o Di ec i e 2006/141/EC unde he new
amewo k o Regula ion (EU) No 609/2013 o he Eu opean Pa liamen and o he Council3 and e ises hem, based on he
opinion o he Eu opean Food Sa e y Au ho i y (EFSA) o 2014.4 In ha opinion, EFSA no ed ha ‘ he sa e y and sui abili y o
each speci ic o mula con aining p o ein hyd olysa es has o be es ablished by clinical s udies. In o ma ion on p o ein sou ces and
he echnological p ocesses applied should also be p o ided. In his con ex , he Panel no es ha one pa icula o mula con ain-
ing pa ially hyd olysed whey p o ein has been e alua ed o i s sa e y and sui abili y by he Panel (…) and has been au ho ised
o use by Di ec i e 2006/141/EC’. EFSA also no ed ha ‘ he c i e ia gi en in Di ec i e 2006/141/EC alone a e no su icien o
p edic he po en ial o a o mula o educe he isk o de eloping alle gy o milk p o eins. Clinical s udies a e necessa y o demon-
s a e i and o wha ex en a pa icula o mula educes he isk o de eloping sho - and long- e m clinical mani es a ions o
alle gy in a - isk in an s who a e no exclusi ely b eas ed’.
Taking in o accoun EFSA's opinion, he delega ed Regula ion es ablishes ha in an o mula and ollow- on o mula
manu ac u ed om p o ein hyd olysa es should only be allowed o be placed on he ma ke i hei composi ion co e-
sponds o he one posi i ely assessed by EFSA so a and p ohibi s he use o heal h claims desc ibing he ole o in an
o mula in educing he isk o de eloping alle gy o milk p o eins. The equi emen s o Commission delega ed Regula ion
(EU) 2016/127 shall apply o in an o mula and ollow- on o mula manu ac u ed om p o ein hyd olysa es om 22
Feb ua y 2021.
Pu suan o Reci al 21 o he Regula ion, hese equi emen s may be amended in he u u e in o de o allow he placing
on he ma ke o o mulae manu ac u ed om p o ein hyd olysa es wi h a composi ion di e en om he one al eady
posi i ely assessed, ollowing a case- by- case e alua ion o hei sa e y and sui abili y by EFSA. In addi ion, i , a e he as-
sessmen by EFSA, i is demons a ed ha a speci ic o mula manu ac u ed om p o ein hyd olysa es educes he isk o
de eloping alle gy o milk p o eins, u he conside a ion will be gi en o how o adequa ely in o m pa en s and ca egi e s
abou ha p ope y o he p oduc .
The equi emen s o Commission delega ed Regula ion (EU) 2016/127 shall apply o in an o mula and ollow- on o -
mula manu ac u ed om p o ein hyd olysa es om 22 Feb ua y 2021. I can be expec ed ha , be o e ha da e, dossie s
on o mulae manu ac u ed om p o ein hyd olysa es will be p esen ed by ood business ope a o s o assessmen by EFSA
wi h a iew o eques possible modi ica ions o he condi ions applicable o hese p oduc s in he delega ed Regula ion.
In his con ex , i is conside ed necessa y o ask EFSA o p o ide scien i ic ad ice o he Commission on dossie s on o mulae
manu ac u ed om p o ein hyd olysa es submi ed by ood business ope a o s o assessmen by EFSA in he u u e.
EFSA will be in o med by he Commission by le e when he applican has been asked by he Commission o ansmi
he dossie o EFSA o scien i ic assessmen .
1.1.2 | Te ms o Re e ence
In acco dance wi h A icle 29 o Regula ion (EC) No 178/2002,5 he Eu opean Commission eques s he Eu opean Food
Sa e y Au ho i y o issue scien i ic opinions on in an and ollow- on o mula manu ac u ed om p o ein hyd olysa es in
pa icula , depending on he na u e o he applica ion, on:
1. he sa e y and sui abili y o use by in an s o a speci ic o mula manu ac u ed om p o ein hyd olysa es;
I he o mula unde e alua ion is conside ed o be sa e and sui able o use by in an s, he Eu opean Food Sa e y
Au ho i y is also asked o ad ise on he minimum speci ic c i e ia on p o ein sou ce, p o ein p ocessing and p o ein
quali y o he o mula ha need o be sa is ied o he sa e y and sui abili y o such o mulae o be demons a ed.
1Commission Di ec i e 2006/141/EC o 22 Decembe 2006 on in an o mulae and ollow- on o mulae and amending Di ec i e 1999/21/EC, OJ L 401, 30.12.2006, p. 1.
2OJ L 25, 2.2.2016, p. 1.
3Regula ion (EU) No 609/2013 o he Eu opean Pa liamen and o he Council o 12 June 2013 on ood in ended o in an s and young child en, ood o special medical
pu poses, and o al die eplacemen o weigh con ol and epealing Council Di ec i e 92/52/EEC, Commission Di ec i es 96/8/EC, 1999/21/EC, 2006/125/EC and
2006/141/EC, Di ec i e 2009/39/EC o he Eu opean Pa liamen and o he Council and Commission Regula ions (EC) No 41/2009 and (EC) No 953/2009, OJ L 181, 29.6.2013,
p. 35.
4EFSA NDA Panel (EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies), 2014. Scien i ic Opinion on he essen ial composi ion o in an and ollow- on o mulae. EFSA
Jou nal, 12(7), 3760. h ps:// doi. o g/ 10. 2903/j. e sa. 2014. 3760.
5Regula ion (EC) No 178/2002 o he Eu opean Pa liamen and o he Council o 28 Janua y 2002 laying down he gene al p inciples and equi emen s o ood law,
es ablishing he Eu opean Food Sa e y Au ho i y and laying down p ocedu es in ma e s o ood sa e y, OJ L 31, 1.2.2002, p. 1.
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2. he p oduc 's e icacy in educing he isk o de eloping alle gy o milk p o eins;
3. he p oduc 's e icacy in educing he isk o de eloping alle gy/alle gic mani es a ions o alle gens in gene al.
1.2 | In e p e a ion o he Te ms o Re e ence
Fo he pa icula dossie which is he subjec o he p esen opinion, he eques om he Eu opean Commission was
ela ed o he assessmen o he nu i ional sa e y and sui abili y o a p o ein hyd olysa e o use in in an and ollow- on
o mula.
The in e p e a ion by he Panel on Nu i ion, No el Foods and Food Alle gens (NDA) is ha he sa e y o ood enzymes
o hei combina ion ha a e used in he manu ac u e o he p o ein hyd olysa e, is no o be assessed in his opinion.
The assessmen o he sa e y o he indi idual ood enzymes is pe o med by he he EFSA Panel on Food Enzymes (FEZ)
acco ding o he guidance and s a emen s o he CEF/CEP Panel (EFSA CEF Panel,2009, 2016; EFSA CEP Panel,2019). The
assessmen o ood enzymes is ongoing a he ime o he adop ion o he p esen opinion.
The e o e, he conclusions o he Panel a e ela ed o he nu i ional sa e y and sui abili y o he speci ic p o ein hyd oly-
sa e used o manu ac u e he in an and ollow- on o mula o which he dossie has been submi ed. They a e no ela ed
o he sa e y o he p o ein hyd olysa e in gene al, including he sa e y o he indi idual enzymes o hei combina ion.
Nei he a e hey ela ed o he sa e y o he inal o mula. This is jus i ied, as he composi ion o he o mula wi h espec o
subs ances o he han he p o ein ac ion should comply wi h he composi ional equi emen s laid down in Commission
Delega ed Regula ion (EU) 2016/127 in o de o ensu e he nu i ional sa e y and sui abili y o use by in an s. The conclu-
sions o he Panel also do no e e o he e icacy o he o mula in educing he isk o de eloping alle gic mani es a ions.
2 | DATA AND METHODOLOGIES
2.1 | Da a
The assessmen o he nu i ional sa e y and sui abili y o he speci ic p o ein hyd olysa e de i ed om whey p o ein con-
cen a e and used in in an o mula6 and ollow- on o mula7 is based on he da a supplied in he dossie submi ed o EFSA
and he addi ional in o ma ion p o ided by he ood business ope a o (FBO) upon eques om EFSA.
A common and s uc u ed o ma o he p esen a ion o dossie s ela ed o in an and ollow- on o mulae manu ac-
u ed om p o ein hyd olysa es is desc ibed in he EFSA scien i ic and echnical guidance o he p epa a ion and p esen-
a ion o an applica ion o au ho isa ion o an in an and/o ollow- on o mula manu ac u ed om p o ein hyd olysa es.8
As ou lined in his guidance, i is he du y o he FBO who submi ed he dossie o p o ide all a ailable scien i ic da a
which a e pe inen o he dossie . The p ocedu e ollowed by EFSA o handling dossie s on o mulae manu ac u ed om
p o ein hyd olysa es, he a ious s eps in he p ocedu e and es ima ed imelines a e desc ibed online.9
In acco dance wi h A icle 38 o Regula ion (EC) No 178/2002 and aking in o accoun he p o ec ion o con iden ial in-
o ma ion and o pe sonal da a in acco dance wi h A icles 39, 39a o 39e o he same Regula ion and o he Decision o he
EFSA's Execu i e Di ec o laying down p ac ical a angemen s conce ning anspa ency and con iden iali y,10 he non-
con iden ial e sions o he da a submi ed by Fon e a a e published in OpenEFSA.11
Acco ding o A . 32c (2) o Regula ion (EC) No 178/2002 and acco ding o he Decision o EFSA's Execu i e Di ec o lay-
ing down he p ac ical a angemen s on he p e- submission phase and public consul a ions, EFSA ca ied ou a public
consul a ion (PC- 1171)12 om 17 Oc obe o 7 No embe 2024 on he non- con iden ial e sion o he submission by Fon e a.
2.2 | Me hodologies
The assessmen ollows he me hodology se ou in he EFSA guidance o he p epa a ion and p esen a ion o an
applica ion o au ho isa ion o an in an and/o ollow- on o mula manu ac u ed om p o ein hyd olysa es,8 as well as in
he explana o y no e o he scien i ic and echnical guidance o he p epa a ion and p esen a ion o an applica ion o
au ho isa ion o an in an and/o ollow- on o mula manu ac u ed om p o ein hyd olysa es (wi h a iew o amend
6In an o mula means ood in ended o use by in an s du ing he i s mon hs o li e and sa is ying by i sel he nu i ional equi emen s o such in an s un il he
in oduc ion o app op ia e complemen a y eeding.
7Follow- on o mula means ood in ended o use by in an s when app op ia e complemen a y eeding is in oduced and which cons i u es he p incipal liquid elemen in
a p og essi ely di e si ied die o such in an s.
8EFSA NDA Panel (EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies). (2021). Scien i ic and echnical guidance o he p epa a ion and p esen a ion o a dossie o
e alua ion o an in an and/o ollow- on o mula manu ac u ed om p o ein hyd olysa es (Re ision 1). EFSA Jou nal, 19(3), 6556. h ps:// doi. o g/ 10. 2903/j. e sa. 2021. 6556.
9h ps:// www. e sa. eu opa. eu/ si es/ de au l / iles/ appli ca io ns/ apdes kappl wo k lownu iin an . pd .
10P ac ical a angemen s conce ning he T anspa ency Regula ion a e accessible unde : h ps:// www. e sa. eu opa. eu/ en/ co po a e- pubs/ ans pa en cy- egul a ion- p ac
ical- a an gemen s.
11h ps:// open. e sa. eu opa. eu/ ques ions/ EFSA-Q- 2021- 00339 .
12h ps:// conne c . e sa. eu opa. eu/ RM/s/ consu l a i ons/ publi ccons ul a ion2/ a0lTk 00000 2dKqX/ pc1171.
18314732, 2025, 1, Downloaded om h ps://e sa.onlinelib a y.wiley.com/doi/10.2903/j.e sa.2025.9160 by Readcube (Lab i a Inc.), Wiley Online Lib a y on [09/06/2025]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
Regula ion (EU) 2016/127).13 A p e ious EFSA opinion o 201414 and he egula o y amewo k15 we e also aken in o
accoun .
As he in an o mula in which he p o ein hyd olysa e unde e alua ion is used is ma ke ed only in powde o m,
s abili y da a we e no e alua ed o he o mula (e en hough eques ed in he scien i ic and echnical guidance8) as i is
no expec ed ha hyd olysis con inues in powde ed o mulae.
3 | ASSESSMENT
3.1 | Cha ac e isa ion o he p o ein hyd olysa e
P o ein sou ce
The p o ein hyd olysa e unde e alua ion is p oduced om whey p o ein concen a e (WPC) om cow's milk wi h an
a e age p o ein con en o 16 an a e age lac ose con en o and an a e age a con en o . Raw ma e ial
speci ica ions and ce i ica es o analysis we e p o ided. Indi idual in ac p o eins in he sou ce ma e ial ha e been
iden i ied by e e sed phase- high pe o mance liquid ch oma og aphy (RP- HPLC) based on he me hod by Elga
e al.(2000).
P o ein p ocessing
The p o ein hyd olysa e is p oduced unde Haza d Analysis and C i ical Con ol Poin s (HACCP) and addi ional Food Sa e y
Sys em Ce i ica ion (FSSC) 22000 equi emen s. Ce i ica es o FSSC 22000 ce i ica ions we e p o ided.
In o de o p oduce he hyd olysa e, he sou ce ma e ial (WPC) is hyd a ed o a solids con en o and hea ed o
o he enzyma ic hyd olysis s ep ha is pe o med . This s ep is ollowed by a hea ea men o
. The enzyma ic hyd olysis s ep is ca ied ou a and ollowed by a hea ea men o
.
The ood enzymes used o hyd olysis ha e been iden i ied by he FBO. he ood enzymes employed in he p o-
cess a e cu en ly unde sa e y assessmen by he EFSA CEP Panel, while o he assessmen has been inalised
In he hyd olysis s ep, a se ine endopep idases
is used. The hyd olysis
s ep is pe o med wi h
The FBO s a es ha he inal hea ea men s ep educes he enzyme ac i i y by .
Deg ee o hyd olysis and molecula weigh dis ibu ion o pep ides, con en o ee amino acids and esidual p o eins
Desc ip ions o all analy ical me hods used we e p o ided oge he wi h documen a ion on me hod alida ion and
acc edi a ion o he labo a o ies pe o ming he analyses, excep o he analyses o ee amino acids which we e based
on he me hod desc ibed by Mondino e al.(1972), o which he alida ion documen a ion was no a ailable om he
labo a o y pe o ming he measu emen s.
The deg ee o hyd olysis (DH) was app oxima ed by di iding he ee amino ni ogen (AN) in he p o ein hyd olysa e,
analysed by a alida ed o- ph haldialdehyde (OPA) me hod, by he o al numbe o pep ide bonds in he sou ce
ma e ial, h o , as epo ed by Adle - Nissen(1986) o whey p o ein. The FBO p esen s his a io as DH. The Panel no es ha
he da a p esen ed by he FBO a e no equi alen o DH and a e only conside ed o app oxima e DH (as explained in
13h ps:// www. e sa. eu opa. eu/ en/ suppo ing/ pub/ en- 1858.
14EFSA NDA Panel (EFSA Panel on Die e ic P oduc s, Nu i ion and Alle gies), 2014. Scien i ic Opinion on he essen ial composi ion o in an and ollow- on o mulae. EFSA
Jou nal, 2(7), 3760. h ps:// doi. o g/ 10. 2903/j. e sa. 2014. 3760.
15h ps:// ec. eu opa. eu/ ood/ sa e y/ label ling_ nu i ion/ speci al_ g oups_ ood/ child en_ en.
16Calcula ed as Nx6.38.
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
EFSA(2020)). The a e age o his a io, exp essed as a pe cen age, based on 18 samples om i e independen ly p oduced
ba ches was wi h a s anda d de ia ion (SD) o . The manu ac u ing speci ica ions o he FBO o his a io is
. Ce i ica es o analyses we e p esen ed.
The esidual amoun o p o eins was analysed in h ee ways:
Fi s , he FBO quan i ied he amoun o esidual in he p o ein hyd olysa e by using an enzyme-
linked immunoso ben assay (ELISA, i e samples, ba ch- o- ba ch a iabili y and ce i ica es o analyses p o ided). The
de ec ed by ELISA was on a e age pe kg powde . The FBO explained ha he
ELISA me hod also de ec s epi opes p esen ing pep ides oge he wi h in ac and he e o e likely o e es-
ima es he con en o in ac . The FBO used his me hod o es ima e he con en o esidual p o eins in he
p o ein hyd olysa e and o se a limi o o p oduc elease.
Then, sodium dodecyl sulpha e polyac ylamide gel elec opho esis (SDS- PAGE) was used o quali a i ely assess he
p esence o esidual p o eins, bu no p o eins we e de ec ed.
Finally, esidual in ac and we e quan i ied using e e se phase- high pe o -
mance liquid ch oma og aphy–mass spec ome y (RP- HPLC- MS; alida ed). The h ee p o eins we e below he limi o
de ec ion o he me hod o p o ein hyd olysa e, co esponding o a educ ion o o he
o iginal p o ein con en and in ag eemen wi h he ELISA esul s.
The a e age amoun o ee amino acids in he p o ein hyd olysa e (powde o m) is ( ). Da a on ba ch- o-
ba ch a iabili y o 18 samples o i e independen ly p oduced ba ches ha e been p esen ed oge he wi h he ce i ica es
o analyses.
A e age alues o he molecula weigh dis ibu ion o pep ides and da a on he ba ch- o- ba ch a iabili y o 18 sam-
ples om i e ba ches we e p esen ed oge he wi h he ce i ica es o analyses. Manu ac u ing speci ica ion limi s as
minimum and maximum pe cen age o each molecula weigh ange a e gi en in b acke s.
< 1000 Da:
1000–5000 Da:
5000–20,000 Da:
> 20,000 Da:
The molecula weigh dis ibu ion o pep ides was measu ed by size exclusion- HPLC (SE- HPLC) wi h UV de ec ion
. In o ma ion on he column ( ) used was p o ided. In o ma ion on he calib a ion o he
sys em wi h ega ds o molecula weigh , including de ails on he calib a o s used and da a indica ing he ep oducibili y
o he me hod we e p o ided.
Concen a ions o u osine in 18 samples o i e ba ches o he p o ein hyd olysa e (ce i ica es o analyses p o ided)
we e analysed using a alida ed me hod based on he Ag icul u e Indus y S anda d o he People's Republic o China NY/
IT 939- 2016. Fu osine concen a ions anged om
which was in he ange o u osine concen a ions, o example, in an
ul a- high- empe a u e (UHT) milk analysed as a con ol es sample.
The Panel conside s ha he p o ein hyd olysa e ha is used in he manu ac u e o he in an and ollow- on o mulae o
which he dossie has been submi ed is su icien ly cha ac e ised wi h espec o he hyd olysed p o ein.
3.2 | Cha ac e isa ion o he o mula manu ac u ed om he p o ein hyd olysa e used in he
clinical s udies p o ided
The in an o mula manu ac u ed wi h he p o ein hyd olysa e ha is used in he clinical s udies submi ed complies
wi h he composi ional c i e ia o Regula ion (EU) 2016/127, excep o i s con en o i amin D, which is 0.62 μg/100 kJ
(2.6 μg/100 kcal) and highe han he maximum con en o 0.60 μg/100 kJ (2.5 μg/100 kcal) se up in Regula ion (EU)
2016/127.
The p o ein con en o his o mula is 0.48 g/100 kJ (2.0 g/100 kcal) and is based on a calcula ion o o al ni ogen × 6.25.
a e added as ee amino acids o he o mula. The amino acid p o ile mee s he one laid down in
Annex IIIA o Regula ion (EU) 2016/127, excep o he concen a ions o which is
and lowe han he minimum con en o laid down in Regula ion (EU) 2016/127 and
he concen a ion o which is and lowe han he minimum o
) laid down in Regula ion (EU) 2016/127. Ce i ica es o analyses o wo ba ches o he in an o mula we e
p o ided by he FBO. The FBO s a es ha
. Analyses we e ca ied ou in he labo a o y o
he manu ac u e o in an ISO 17025 acc edi ed labo a o y. The in an o mula, p oduced in powde o m, is manu ac u ed
ollowing FSSC 22000 s anda ds, acco ding o ce i ica es p o ided.
The wo ba ches o he o mula we e analysed o hei u osine and ca boxyme hyllysine (CML) concen a ions (ce i -
ica es o analyses p o ided). The me hods used o analyses we e alida ed and desc ip ions we e p o ided. Fu osine was
analysed using , and CML analyses we e done using
. In he wo ba ches, concen a ions o u osine we e ;
CML concen a ions we e . The con ol o mula used in he s udy desc ibed in Sec ion3.3
had a u osine concen a ion o and a CML concen a ion o in bo h
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
ba ches. The concen a ions we e wi hin he ange o alues epo ed o in an o mula based on a da abase main ained
by he Technical Uni e si y D esden as epo ed by he FBO.
The Panel conside s ha he in an o mula ha is used in he pe inen human in e en ion s udy is su icien ly
cha ac e ised.
3.3 | Sa e y and sui abili y o he in an and ollow- on o mula
The FBO p esen ed an unpublished ull s udy epo (Gunn e al.,2023) o one pe inen double- blind andomised con olled
ial (RCT) wi h 249 in an s who we e andomised in blocks o 10, s a i ied by si e and sex, o consume exclusi ely ei he
he in an o mula manu ac u ed om hyd olysed whey p o ein o which he dossie has been submi ed (HF) o an in an
o mula manu ac u ed om in ac cow's milk p o ein (con ol o mula, CF). An exclusi ely b eas - ed e e ence g oup o 50
in an s was also included. This g oup was en olled in a a io o 4:1 in each s udy si e and had simila baseline cha ac e is ics,
including age a en olmen , as he o mula- ed g oups. The s udy was designed as an equi alence s udy o he p ima y
ou come o weigh gain and an equi alence ma gin o ±3 g/day was se a p io i.
The HF and he CF had p o ein con en s o 0.48 g/100 kJ (2.0 g/100 kcal). The composi ion o bo h o mulae complied
wi h Commission Delega ed Regula ion (EU) 2016/127, excep o he p o ein sou ce o he HF.
The s udy was ca ied ou in Bulga ia and Ge many.
Heal hy e m exclusi ely o mula- ed in an s up o 28 days o age wi h a bi hweigh be ween 2500 and 4500 g and a
5- min Apga sco e o ≥ 7 we e included in he s udy. The andomisa ion schedule was compu e - gene a ed and p o ided
o s udy si es in sealed opaque consecu i ely numbe ed en elopes wi h he indica ion whe he i was o a male o emale.
The Panel no es he
limi a ions o his app oach in e ms o success ul blinding o he s udy.
Fo mulae powde s wi h iden ical smell and appea ance we e p o ided in me allic ins labelled wi h he unique code.
The p ima y ou come o he s udy was he a e age weigh gain om en olmen o day 90 o he s udy.
Seconda y ou comes included body weigh , leng h, head ci cum e ence (HC), weigh - o - age, weigh - o - leng h,
leng h- o age z- sco es, ad e se e en s and se ious ad e se e en s, s ool consis ency assessed by he B is ol s ool o m
scale (Chumpi azi e al.,2010), s ool equency, o mula in ake, spi ing up and egu gi a ion, la ulence and bloa ing, om-
i ing, c ying/sc eaming episodes, colic, ussiness and sleep ime.
All an h opome ic measu emen s a baseline we e pe o med a he s udy si es (17 si es in Ge many and h ee in
Bulga ia had ac i e pa icipan s) bu du ing he COVID- 19 pandemic, some pa en s ca ied ou measu emen s a home. A
he inal isi , pa en al home measu emen s we e comple ed o ou o in an s (including all h ee eeding g oups,
including he b eas - ed e e ence g oup) in Bulga ia. In Ge many, in an s we e measu ed by pa en s, h ee by heal h
p o essionals in hei o ices and a he s udy si es ( o al numbe o in an s measu ed ). Fo home measu emen s,
pa en s we e supplied wi h medical g ade scales, measu ing boa ds and ape measu es o HC measu emen s. Pa en s
ecei ed w i en ins uc ions. In Bulga ia, elephone assis ance was also a ailable.
The Panel assumes ha home measu emen s we e equally dis ibu ed among g oups, because o he andomised na-
u e o he s udy, and no es ha in Ge many 20% o all measu emen s a he inal ime poin we e done by pa en s, while
i was 6% in Bulga ia.
In e en ion o mula eeding began he e ening a e he ini ial en olmen isi (V1) o in an s aged 0 o 28 days. The
ollowing measu emen s ook place a days 30 (V2), 60 (V3) and 90 (V4) a e he s a o eeding he in e en ion o mulae.
Ano he s udy isi ook place a he age 6 mon hs (V5), du ing which also an h opome ic pa ame e s and ole ance o he
p oduc we e assessed.
Th ee days be o e each isi , pa en s we e asked o eco d on dia y ca ds he equency and amoun o o mula ed pe
day and whe he any complemen a y ood had been consumed.
Compliance was assessed by collec ing he emp y ins and by checking he dia y ca ds.
Analyses we e pe o med in he in en ion- o- ea popula ion (ITT, i.e. all indi iduals andomised) and in he pe p o o-
col popula ion (PP, all indi iduals a ending all isi s wi h > 66.6% compliance and no majo p o ocol iola ions, no ha ing
been gi en complemen a y oods and no ha ing been ed mo e han one bo le o a o mula o he han he in e en ion
o mula du ing he whole s udy pe iod). The ITT popula ion was he popula ion used o he p ima y analysis.
Fo analysing he di e ence in weigh gain o e 90 days be ween in e en ion and con ol g oup an analysis o co a-
iance (ANCOVA) was used wi h in e en ion and coun y and hei in e ac ion as ixed ac o s, gende as ixed blocking
ac o and ges a ional age a bi h and baseline body weigh as co a ia es. The Panel no es ha , o he analysis o his
p ima y endpoin , i is unclea how missing da a we e handled in he ITT analysis.
The place a which measu emen s ook place (s udy si es s. a home) was ini ially included in he s a is ical model, bu
as he e was no in e ac ion wi h he o mula g oups i was emo ed om he inal model.
Con inuous a iables o seconda y endpoin s we e analysed using a mixed model o epea ed measu es including all
i e measu emen ime poin s. An au o eg essi e co a iance pa e n was chosen. Pos - hoc compa isons we e pe o med
using he Tukey es o pai wise compa isons. Fo seconda y endpoin s, which we e only assessed once a e baseline, he
same model as o he p ima y analysis was used. Fo ca ego ical a iables he chi- squa ed es was applied. A Poisson
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
model was used o assess incidence a es. z- Sco es we e calcula ed using WHO e e ence s anda ds.17 The Benjamini-
Hochbe g p ocedu e was used o con ol o he alse disco e y a e and he p- alue o s a is ical signi icance o second-
a y ou comes was se a ≤ 0.0025.
Sample size calcula ions we e based on a wo- sided, wo- sample - es assuming a s anda d de ia ion (SD) o 5–6 g/day
and a di e ence in weigh gain o 3 g/day. Fo ha ing a 90% powe a a 5% signi icance le el, 86 in an s pe g oup we e o
be included. Assuming a 40% d op- ou a e, 143 in an s pe g oup we e needed.
In o al, 249 in an s we e andomised, 123 o he HF g oup and 126 o he CF g oup. In he HF g oup, 56 discon inued
he in e en ion and/o did no comply wi h he p o ocol up o V4 (45.5% o andomised in an s): 11 we e los o ollow
up, 4 wi hd ew consen , 17 le he s udy because o in ole ance and 24 did no comply o had a p o ocol iola ion. In he
CF g oup, 50 s opped pa icipa ion and/o did no comply wi h he p o ocol up o V4 (39.7%): 1 in an did no s a he
in e en ion, 12 we e los o ollow up, 1 wi hd ew consen , 20 s opped because o in ole ance and 16 did no comply o
had a p o ocol iola ion. Hence, a V4, 67 in an s in he HF and 76 in an s in he CF g oup we e in he PP popula ion. A V5,
he numbe s we e 59 and 68, espec i ely. F om he da a p o ided in esponse o a eques o addi ional in o ma ion om
EFSA, he Panel no es ha in an s in he HF g oup ( o all andomised in an s) and in an s in he CF g oup (
o all andomised in an s) had ou come da a bo h a V1 and V4. The Panel no es he high d op- ou a e which was simila
be ween bo h g oups.
Baseline cha ac e is ics o in an s in he HF and CF g oups we e simila in bo h he ITT and PP popula ions. In he PP
popula ion, age and weigh a inclusion in he HF g oup we e mean ± SD 9.1 ± 8.3 days and 3304 ± 457 g (unadjus ed) and
in he CF g oup 9.3 ± 9.2 days and 3340 ± 502 g, espec i ely. Howe e , age a baseline was conside ably di e en be ween
in an s ec ui ed in Bulga ia and hose ec ui ed in Ge many. In Bulga ia, in an s in he HF g oup had a mean (SD) age a
baseline o 14.8 (8.21) days and hose in he CF g oup 16.0 (9.14) days. In Ge many, hey we e 3.5 (2.60) and 3.4 (3.18) days
old, espec i ely. Including age ins ead o weigh a baseline as co a ia e did no change he esul s o he analysis (analysis
p o ided by he FBO upon eques om EFSA).
The mean di e ence in weigh gain be ween V1 and V4 be ween he HF and he CF g oup was 1.9 g/day (95% CI 0.0 o
3.9 g/day) in he ITT and 2.4 g/day (0.3 o 4.5 g/day) in he PP popula ion. The Panel no es ha he uppe bound o he 95%
CI exceeded he p ede ined ma gin o equi alence. Howe e , he Panel also no es ha his likely e lec ed he lowe weigh
gain in in an s in he Bulga ian CF g oup compa ed o Ge man in an s in bo h eeding g oups and Bulga ian in an s in he
HF g oup, a di e ence o which no explana ion could be ound. In addi ion, he Panel no es ha bo h o mula- ed g oups
gained weigh wi hin he ange expec ed acco ding o WHO s anda ds.
Resul s o leng h, HC and an h opome ic measu es gi en as z- sco es we e consis en wi h he conclusions o weigh
gain.
Fo mula eeding olume was simila be ween g oups. Fo mula in ake was on a e age (95% CI) 828 mL (786 o 871 mL)
in he HF g oup and 798 mL (758 o 837 mL) in he CF g oup a V4. In a epea ed measu es analysis, also including V5, he e
was no s a is ically signi ican ea men pe ime in e ac ion.
The e we e 111 ad e se e en s epo ed in 123 in an s (ITT popula ion) in he HF g oup and 152 e en s in 125 in an s in
he CF g oup. A leas one ad e se e en occu ed in 53% o in an s in he HF g oup and 60% in he CF g oup. They we e
judged by he in es iga o s o be p obably ela ed o he s udy o mula in 7 cases o he HF g oup and 12 cases o he
CF g oup. All ad e se e en s classi ied as se ious we e conside ed by he in es iga o s as un ela ed o he s udy o mula.
S ool equency was lowe in he HF g oup han he CF g oup [mean (95% CI) HF s. CF: 1.42 (1.21–1.53) s. 1.60 (1.40–1.80)
bowel mo emen s pe day, which showed a s a is ically signi ican ea men pe ime in e ac ion (p = 0.04) in a epea ed-
measu es analysis]. Ha d s ools occu ed in one in an in he HF and wo in an s in he CF g oup a V2 bu no a subsequen
isi s. None o he o he seconda y ole ance ou comes we e signi ican ly di e en be ween g oups, excep o he num-
be o sc eaming episodes which was highe in he CF (1%–2% o in an s, depending on he isi ) han he HF g oup (0%
o in an s). The Panel conside s ha he abo e- men ioned di e ences in s ool equency and sc eaming episodes a e no
clinically signi ican .
The Panel conside s ha his s udy shows ha an in an o mula manu ac u ed om he p o ein hyd olysa e desc ibed
in Sec ion3.1 wi h a p o ein con en o 0.48 g/100 kJ (2.0 g/100 kcal) and consumed as he sole sou ce o nu i ion o 3
mon hs leads o g ow h ha is simila o an in an o mula manu ac u ed om in ac cow's milk p o ein wi h he same p o-
ein con en . The Panel concludes ha he p o ein hyd olysa e unde e alua ion is a nu i ionally sa e and sui able p o ein
sou ce o use in in an o mula, as long as he in an o mula in which i is used con ains a minimum o 0.48 g/100 kJ (2.0
g/100 kcal) p o ein and complies wi h he composi ional c i e ia o Commission Delega ed Regula ion (EU) 2016/127 and
he amino acid pa e n in Annex IIIA o he Regula ion.
No expe imen al da a ha e been p o ided on he nu i ional sa e y and sui abili y o his p o ein sou ce in ollow- on
o mula. Howe e , gi en he ac ha ollow- on o mula is consumed in conjunc ion wi h complemen a y oods and he
p o ein sou ce is conside ed nu i ionally sa e and sui able in an in an o mula ha is he sole sou ce o nu i ion o young
in an s, he Panel conside s ha he p o ein hyd olysa e unde e alua ion is also a nu i ionally sa e and sui able p o ein
sou ce o use in ollow- on o mula, as long as he ollow- on o mula in which i is used con ains a minimum o 0.48 g/100
kJ (2.0 g/100 kcal) p o ein and complies wi h he composi ional c i e ia o Commission Delega ed Regula ion (EU) 2016/127
and he amino acid pa e n in Annex IIIA o he Regula ion.
17Using he SAS mac os published by WHO (h ps:// www. who. in / ools/ child- g ow h- s and a ds/ so wa e).
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NUTRITIONAL SAFETY AND SUITABILITY OF A PROTEIN HYDROLYSATE
3.4 | Unce ain ies ela ed o he nu i ional sa e y and sui abili y o he in an o mula
The Panel no es ha weigh gain in Bulga ian in an s in he CF g oup was lowe han in he HF g oup and also lowe han in
Ge man in an s in he CF and HF g oups and he b eas - ed e e ence g oup, e en hough hei weigh gain was wi hin he
ange expec ed acco ding o WHO e e ence s anda ds. The e we e no ele an di e ences in baseline cha ac e is ics o
in an s o o mula in akes be ween g oups o o mula- ed in an s. The same wo ba ches o he con ol o mula we e used
bo h in Bulga ia and Ge many and ba ches did no di e in hei composi ion as con i med by ce i ica es o analyses, so
ha di e ences in o mula composi ion could no explain he di e ences in g ow h.
4 | CONCLUSIONS
The Panel concludes ha :
• he p o ein hyd olysa e o which he dossie has been submi ed and ha is o be used in he manu ac u e o in an and
ollow- on o mula is su icien ly cha ac e ised wi h espec o i s ac ion o hyd olysed p o ein;
• he minimum speci ic c i e ia o cha ac e isa ion o he p o ein hyd olysa e wi h espec o he p o ein sou ce, p o ein
p ocessing and p o ein quali y, as eques ed in he e ms o e e ence, a e hose gi en in Sec ion3.1;
• he p o ein hyd olysa e o which he dossie has been submi ed is a nu i ionally sa e and a sui able p o ein sou ce o
use in in an and ollow- on o mula, as long as he o mula in which i is used con ains a minimum o 0.48 g/100 kJ (2.0
g/100 kcal) p o ein and complies wi h he composi ional c i e ia o Commission Delega ed Regula ion (EU) 2016/127 and
he amino acid pa e n in Annex IIIA o he Regula ion.
5 | DOCUMENTATION AS PROVIDED TO EFSA
Dossie on he sa e y and sui abili y o use by in an s o an in an o mula manu ac u ed om p o ein hyd olysa es.
Sep embe 2023. Submi ed by Fon e a Co- ope a i e G oup Limi ed.
5.1 | S eps aken by EFSA
1. The echnical dossie was ecei ed by EFSA on 15/06/2021.
2. A le e om he Eu opean Commission wi h he eques o a scien i ic opinion on he sa e y and sui abili y o use by
in an s o an in an and ollow- on o mula manu ac u ed om p o ein hyd olysa e was ecei ed by EFSA on 06/09/2021.
3. The scien i ic e alua ion p ocedu e s a ed on 15/09/2023.
4. On 13/11/2023, he Wo king G oup on p o ein hyd olysa e- based o mula o he NDA Panel ag eed on a lis o ques ions
o he ood business ope a o o p o ide addi ional in o ma ion o accompany he dossie . The scien i ic e alua ion was
suspended on 21/11/2023 and was es a ed on 27/03/2024.
5. On 31/05/2024, he Wo king G oup on p o ein hyd olysa e- based o mula o he NDA Panel ag eed on a lis o ques ions
o he ood business ope a o o p o ide addi ional in o ma ion o accompany he dossie . The scien i ic e alua ion was
suspended on 04/06/2024 and was es a ed on 04/07/2024.
6. Du ing i s mee ing on 28/11/2024, he NDA Panel, ha ing e alua ed he da a, adop ed an opinion on 28/11/2024.
ABBREVIATIONS
AN amino ni ogen
ANCOVA analysis o co a iance
CEP Panel on Food Con ac Ma e ials, Enzymes and P ocessing Aids
CEF Panel on Food Con ac Ma e ials, Enzymes, Fla ou ings and P ocessing Aids
CF con ol o mula
CI con idence in e al
CML ca boxyme hyl- lysine
COVID- 19 Co ona i us disease o 2019
Da dal on
DH deg ee o hyd olysis
EC Enzyme Commission
ELISA enzyme- linked immunoso ben assay
FBO Food business ope a o
FEZ Panel on Food Enzymes
FSSC Food Sa e y Sys em Ce i ica ion
GMO gene ically modi ied o ganism
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