Open Li e Sci. 2016; 11: 280–286
ace ic acid (−46%), ammonium (−59%), ash (−13%)
and a (−24%). Popula ions o bac e ia associa ed wi h
lac ic acid e men a ion, moulds, yeas s, en e obac e ia
and o al mic oo ganisms coun we e also in luenced.
Bo h biological and chemical addi i es can be highly
ecommended o p oducing high-quali y silages mee ing
hygienic equi emen s. In luce ne silage, he chemical
p ese a i e showed a s onge e ec in achie ing
he heal h sa e y o silage compa ed o he biological
inoculan .
Keywo ds: heal h sa e y, silage addi i es, mic oo ganisms
1 In oduc ion
Biogenic amines comp ise a g oup o alipha ic,
he e ocyclic o a oma ic bases de i ed om amino acids.
Biogenic amines a e p esen in all eeds ha con ain
p o eins o ee amino acids, and hey also exis in
e men a i e eeds [1-3]. The concen a ion o biogenic
amines (mono-, di- and polyamines) in silage and in he
umen, body issues and body luids mainly depends
upon he c op a ha es , he ensiling p ocess, he silage
and he diges ion in he animal. Bo h he syn hesis and
chemical s uc u es o mono- and diamines a e well
documen ed. The basis o hei o ma ion is p o eolysis,
a na u ally occu ing p ocess in ensiling consis ing in he
enzyma ic deca boxyla ion o amino acids by he ac ion
o plan p o eases and pep idases along wi h enzymes o
a ious lac ic acid bac e ia (LAB), clos idia and o he
bac e ial gene a [4]. High-quali y eeds u s and p ope
nu i ion a e impo an o all ca ego ies o li es ock
[1-3]. High le els o biogenic amines a e equen ly
obse ed in silages p epa ed om high-p o ein o ages
(luce ne, clo e , ce ain g ass species). Some biogenic
amines ha e signi ican biological cha ac e is ics, as
hey a e, o example, issue ho mones (his amine),
p o oalkaloids (ho denine, g amine) and building
DOI 10.1515/biol-2016-0037
Recei ed Augus 17, 2016; accep ed Sep embe 21, 2016
Abs ac : This expe imen examined he in luence o
wo di e en silage addi i es o biological (Lac ococcus
lac is, Lac obacillus plan a um, En e ococcus aecium,
enzyme xylanase) and chemical (43% o mic acid, 30%
ammonium o ma e, 10% p opionic acid, 2% benzoic
acid) ypes on biogenic amines concen a ion, nu ien
con en , e men a ion p ocess, and mic obiologic
indica o s in luce ne (Medicago sa i a) silage a e 90
days o e men a ion. The biological addi i e signi ican ly
(P < 0.05) inc eased pu escine (+51%), lac ic acid
(+11%) and p o ein con en (+11%) in compa ison wi h
con ol silage. I signi ican ly dec eased cada e ine
(−29%), his amine (−57%), spe midine (−15%), spe mine
(−55%), ace ic acid (−40%), e hanol (−55%), ammonium
(−25%) and ash (−9%). A e he chemical-addi i e
ea men , g ea e amoun s o his amine and y amine
we e eco ded. Signi ican dec ease was obse ed in he
concen a ions o pu escine (−18%), cada e ine (−55%),
spe midine (−47%), spe mine (−45%), lac ic acid (−16%),
© 2016 Ve onika Mlejnko a e al., published by De G uy e Open.
This wo k is licensed unde he C ea i e Commons A ibu ion-NonComme cial-NoDe i s 3.0 License.
*Co esponding au ho s: Ji i Socho , Depa men o Vi icul u e
and Enology, Facul y o Ho icul u e, Mendel Uni e si y in B no,
Val ická 337, CZ-691 44 Lednice, Czech Republic;
E-mail:socho [email p o ec ed]
Ve onika Mlejnko a, Pa el Ho ky, Ji i Skladanka, Lucia Hodulíko á,
Depa men o Animal Nu i ion and Fo age P oduc ion, Facul y o
Ag onomy, Mendel Uni e si y in B no, Zemedelska 1, CZ-613 00
B no, Czech Republic
Ma ke a Kominko a, Voj ech Adam, Depa men o Chemis y and
Biochemis y, Facul y o Ag onomy, Mendel Uni e si y in B no,
Zemedelska 1, CZ-613 00 B no, Czech Republic
Ma ke a Kominko a, Cen al Eu opean Ins i u e o Technology, B no
Uni e si y o Technology, Technicka 3058/10, CZ-616 00 B no, Czech
Republic
Ji i Mlcek, Tomas Ba a Uni e si y in Zlin, Facul y o Technology,
Depa men o Food Analysis and Chemis y, T.G.Masa yk Sq. 275,
762 72 Zlin, Czech Republic
Tunde Ju iko a, Cons an ine he Philosophe Uni e si y in Ni a,
Facul y o Cen al Eu opean S udies, Ins i u e o Teache s T aining,
D azo ska 4, SK-949 74 Ni a, Slo ak Republic
Resea ch A icle Open Access
Ve onika Mlejnko a, Pa el Ho ky, Ma ke a Kominko a, Ji i Skladanka, Lucia Hoduliko a,
Voj ech Adam, Ji i Mlcek, Tunde Ju iko a, Ji i Socho *
Biogenic amines and hygienic quali y o luce ne
silage
Biogenic amines and hygienic quali y o luce ne silage  281
2 Ma e ial and Me hods
Expe imen al ma e ial was ob ained om Resea ch
Ins i u e o Fodde C ops, L d. T oubsko (Czech
Republic). In he model expe imen , luce ne (Medicago
sa i a, a . Pála a) was ob ained om he i s cu ing o
an expe imen al plo . The expe imen al plo was loca ed
in T oubsko. The luce ne was g own in a suga bee a ea a
an al i ude o 270 m. The p e alen soil ype he e consis s
o b own soil, and he soil ex u e is sil y o clayey. The
expe imen al pa cels had been e ilized wi h pul e ized
limes one (10 ha−1).
Luce ne was ha es ed in he bud s age while lea ing
s ubble heigh o 8 cm. The plan ma e ial was le o wil
o a sho ime and hen cu o leng h o 3–4 cm. I was
hen d ied o 6 h o 35% d y ma e con en . A model
expe imen was se up whe eby he obse ed ac o s
we e he addi ion o biological and chemical silage
addi i es and hei in luence on he silage mic o lo a. An
expe imen al con ol a ian (g oup K) was used (wi h
addi ion o an equi alen amoun o po able wa e in
o de o main ain he same con en o d y ma e ). The
biological silage addi i e (g oup B), applied a a a e o
20 g/ , consis ed o he lac ic acid bac e ia En e ococcus
aecium (DSM 22502/NCIMB 11181), Lac ococcus lac is
(NCIMB 30117), and Lac obacillus plan a um (DSM
16568), as well as he enzyme xylanase EC 3.2.1.8 in he
o al amoun o 1.25 × 1010 KTJ/g. A chemical p ese a i e
(g oup CH) was applied a he a e o 2 L −1. The chemical
addi i e consis ed o 43% o mic acid, 30% ammonium
o ma e, 10% p opionic acid and 2% benzoic acid. Each
a ian (K, B, CH) was done in h ee epe i ions, and each
expe imen al silage a ian was made in h ee epe i ions.
Mo eo e , he samples we e analysed in h ee epe i ions.
In o al, he e o e, 27 silage samples we e analysed.
T ea ed o age ma e ial weighing 8 kg was in each case
compac ed, weighed and anae obically sealed. The silage
densi y was 750 kg/m2. The model silages we e s o ed in
a labo a o y a a e age empe a u es wi hin he ange
20–25°C o a pe iod o 3 mon hs. A e he silage ubes
we e opened, hey we e weighed and om each a ian
a ep esen a i e sample was aken in o de o e alua e
mois u e con en , e men a ion p ocess, nu ien con en
and biogenic amines, as well as o mic obiological analysis.
2.1 Mic obiology
Twen y g ams o a gi en sample was shaken in a lask on
a P omax 1020 ecip oca ing pla o m shake (Heidolph,
Ge many) oge he wi h 180 mL o physiological solu ion.
blocks o biosyn hesis o o he ho mones in animals
(phenyle hylamine), phy oho mones, auxins, alkaloids
and o he seconda y me aboli es o plan s [5,6].
His amine, y amine and 2-phenyle hylamine ha e been
ound o ha e nega i e e ec s, in luencing he ne ous
sys em, a ec ing blood p essu e, and p o oking skin
mani es a ions. Polyamines (spe midine, pu escine,
cada e ine) a e essen ial o cell di ision and g ow h,
o main aining high me abolic a e, o immune sys em
unc ion, and o o he p ocesses. They also suppo he
g ow h o umou s, despi e hei no being ca cinogenic
on hei own [7,8]. The oxic e ec o biogenic amines
is highly in luenced by he ac i i y o hese enzymes,
which may di e by indi idual and is dependen on
se e al ac o s, such as he p esence o inhibi o s (some
medicines, alcohol). When e alua ing oxic e ec , i is
necessa y o conside no only he p esence o a speci ic
biogenic amine bu also o he ac o s such as he amoun
o a oods u consumed and p esence o o he oxic
subs ances. I is he e o e di icul o s a e he bounds o
hei oxici y [9]. The use o chemical subs ances based
on o ganic acids, and pa icula ly hose con aining
la ge quan i ies o o mic acid, in luence in a posi i e
way he con en o ni ogenous subs ances in luce ne
silages. Fo mic acid posi i ely a ec s p ese a ion and
main enance o p o ein con en , and especially o hose
p o eins composed om la ge numbe s o amino acid
componen s [10,11]. The addi ion o he e o e men a i e
bac e ia o p omo e lac ic acid e men a ion has been
shown o ha e no e ec on nu ien indica o s o silages
[12,13]. He e o e men a i e species o lac obacilli a e
cha ac e ized as ha ing an imic obial e ec s, and
Lac obacillus plan a um has high an imic obial e ec s.
This inding inc eases he possibili y ha a highe densi y
o he e o e men a i e LAB species wi h po en ially
p obio ic cha ac e is ics would ha e a posi i e in luence
on he esul ing quali y o p ese ed eeds [14,15]. High-
quali y silage depends on he de elopmen o a ou able
mic o lo a unde anae obic condi ions. Silage ea men s
wi h homo e men a i e and he e o e men a i e
lac obacilli can achie e conside ably be e ae obic
s abili y. Addi i e applica ion o lac obacilli species
dec eases pH, as well as le els o bu y ic acid, alcohol
and ammonium [16]. The aim o he p esen s udy was
o de e mine he in luence o wo ypes o ea men
(biological and chemical) on he occu ence o biogenic
amines, nu ien alue, and hygienic quali y o luce ne
silages. The esea ch hypo hesis was ha chemical and
biological addi i es would dec ease he con en s o
biogenic amines and un a ou able mic o-o ganisms,
he eby imp o ing he hygienic quali y o silages.
282   V. Mlejnko a, e al.
2.4 Biogenic amines analysis
2.4.1 P epa a ion o samples
Luce ne samples weighing 300 mg we e ans e ed o a
g inding mo a whe e hey we e e oded by liquid ni ogen
(pou ing liquid ni ogen in o he g inding mo a wi h he
luce ne and g inding wi h a pes le o abou 2 min). These
samples we e homogenized wi h 2 mL o deionised Milli-Q
wa e , hen incuba ed a 25°C and 300 pm o 60 min on a
The momixe com o (Eppendo ). A e incuba ion, he
samples we e cen i uged a 4°C and 25000 g o 10 min
(Cen i uga 5417R, Eppendo ). A e cen i uga ion, he
supe na an was emo ed and he sample was cen i uged
again (4°C, 25 000 g, 10 min). Supe na an was once again
emo ed and he sample p epa ed in his manne hen
analysed using liquid ch oma og aphy.
2.4.2 De e mina ion o biogenic amines
An AAA 400 ion exchange liquid ch oma og aphy
appa a us (Ingos, P ague, Czech Republic) was used
o de e mina ion o biogenic amines. The sys em
consis ed o a glass illing ch oma og aphic column
and s eel p e-column, wo ch oma og aphic pumps o
anspo o elu ion bu e s and de i a iza ion eagen ,
a cooled ca ousel o 25 Eppendo ubes, dosing
al e, hea eac o , Vis de ec o , and cooled chambe
o de i a iza ion eagen . The olume o he injec ed
sample was 100 µL, and he applica ion’s p ecision was
wi hin 1% ela i e s anda d de ia ion. We used a wo-
channel Vis de ec o wi h a 5 µL low olume cu e e
ope a ed a wa eleng hs o 440 and 570 nm. A solu ion o
ninhyd in was p epa ed wi h 75% me hyl cellosol e ( / )
and 25% 4M ace ic bu e ( / ). Tin chlo ide was used as
a educing eagen . The p epa ed solu ion o ninhyd in
was s o ed unde an ine a mosphe e (N2) wi h cooling
a 4°C. Flow a e was 0.25 mL min−1 unde a p essu e
anging om 4.5 o 6.0 MPa. Reac o empe a u e was se
o 120°C. Fo elu ion, wo bu e s we e employed: bu e
A was composed o 5.5 mM C6H8O7, 81 mM Na3C6H5O7,
257mM NaCl, 350 mM KB , and 250 mL o C3H8O pe 1 L o
Milli-Q wa e , wi h a inal pH o 5.78. Bu e B consis ed
o 73 mM C6H8O7, 3 M NaCl, and 10.0 mL o 50% solu ion
o KOH (w/w) pe 1 L o MilliQ wa e , wi h a inal pH o
3.27. A WTW inoLab pH me e (Weilheim, Ge many) was
used o pH measu emen s.
Nex , a en- old se ial dilu ion was done and 1 mL om he
co esponding dilu ion was inocula ed on o a Pe i dish
and laye ed wi h cul i a ing medium.
Pla e coun aga (Bioka Diagnos ics, F ance) was used
as cul i a ion medium o de e mining he o al mic o-
o ganisms coun . Inocula ion was a 30°C o 72 h. Fo
LAB de e mina ion, an MRS aga (i.e. Lac obacillus aga
in acco dance wi h DeMan, Rogosa and Sha pe; Bioka
Diagnos ics, F ance) was used. Inocula ion was again
o 72 h a 30°C. Chlo amphenicol glucose aga (Bioka
Diagnos ics, F ance) was used in de e mining he o al
coun o yeas s and moulds, wi h incuba ion las ing 120h
a 25°C. To de e mine bac e ia o he En e obac e iaceae
amily, iole ed bile glucose (VRBG) aga (Bioka
Diagnos ics, F ance) was used, wi h incuba ion o 24 h a
37°C. A e incuba ion, he g own colonies we e ead om
he Pe i dishes and he esul s we e exp essed in colony-
o ming uni s (CFU) pe g am o silage.
2.2 P epa a ion o wa e leaching o
assessmen o e men a ion indica o s
A sample o silage weighing 50 g was su used wi h 450
mL o dis illed wa e . A e 24 h he leacha e was il e ed
h ough il a ing pape (FILTRAK 388) and analysed.
2.3 Nu ien analysis
Ash was de e mined by weighing he ma e ial emaining
a e bu ning a ixed-weigh sample a 550°C in speci ied
condi ions. Fa was de e mined by weighing he di ec
ex ac aken om a sample using pe oleum e he on
an ex ac ion de ice acco ding o Soxhle . Ni ogenous
subs ances we e measu ed by Kjeldal me hod (N * 6.25
coe icien ) using a Kjel ec 2300 de ice (Foss, Hille ød,
Denma k).
Acid de e gen ib e (ADF), neu al de e gen ib e
(NDF), and o al ib e we e measu ed on an ANKOM 220
Fibe Analyze (ANKOM Technology, Macedon, NY, USA)
by wo-s ep hyd olysis in a boiling solu ion o sulphu ic
acid. Po assium hyd oxide was used in analysing he
emaining ib e con en , including speci ica ion o ash
con en om he es o he sample. NDF and ADF con en s
we e de e mined using de e gen s sodium lau yl sulpha e
in he case o NDF and ce yl ime hylammonium b omide
in he case o ADF [17].
Biogenic amines and hygienic quali y o luce ne silage  283
he con ol g oup. The highes pu escine concen a ion
was in g oup B, whe e in compa ison wi h he con ol
g oup he inc ease was 51% (P < 0.05). In he CH g oup,
a signi ican 18% dec ease o pu escine was obse ed.
Cada e ine con en s we e signi ican ly lowe in g oups B
and CH by 29% and 55%, espec i ely. Simila esul s we e
obse ed in le els o spe midine, which we e 15% lowe
(P<0.05) in g oup B and 47% lowe in g oup CH (P <0.05).
The e also occu ed signi ican dec ease o spe mine in
bo h g oups B and CH by 55% and 45%, espec i ely.
Fe men a ion p oduc s measu emen s a e epo ed
in Table 2. pH alues anged om 4.37 o 4.96. Wi h he
addi ion o silage addi i es, pH dec eased by a signi ican
11.7% in he B g oup and 11.9% in he CH g oup. In silages
ea ed wi h biological addi i e, he con en s o lac ic
acid, which is bene icial o success ul e men a ion,
inc eased by 11% (P < 0.05) in he B g oup. A e ea men
wi h he chemical addi i e, lac ic acid con en dec eased
by 16% (P < 0.05) in compa ison wi h he con ol g oup.
Ace ic acid con en was also signi ican ly lowe in bo h
expe imen al g oups: by 40% in he B g oup and 46%
in he CH g oup. E hanol dec eased by 55% (P < 0.05) in
he B g oup as compa ed o he con ol g oup. E hanol
concen a ion in he CH g oup was unde he de ec ion
h eshold. Ammonia alues dec eased signi ican ly in
compa ison wi h he con ol g oup: by 59% in he B g oup
and 25% in he CH g oup.
Table 3 epo s mean nu ien con en alues. Ash was
obse ed o dec ease signi ican ly in he B g oup by 9%
and in he CH g oup by 13% as compa ed o he con ol
g oup. The amoun o p o ein in he B g oup inc eased
by a signi ican 11%. The CH g oup had p o ein le els
2.5 S a is ics
The da a we e p ocessed s a is ically using STATISTICA.
CZ, e sion 10.0 (Czech Republic). Resul s a e exp essed as
means ± s anda d de ia ion. Pa ame ic dis ibu ion was
analysed by Kolmogo o -Smi no es . Fo nepa ame ic
da a in mic obiological analysis he K uskal-Wallis es
was used and o pa ame ic da a s a is ical signi icance
was de e mined by examining he di e ences among
g oups using ANOVA and Sche é’s es (one-way analysis)
o he ollowing pa ame e s: his amine, y amine,
pu escin, cada e ine, spe mine, spe midine, o mic acid,
ace ic acid, e hanol, ammonium, ash, p o ein, a , ib e,
ADF, NDF, yeas s, moulds, LAB, and o al coun o mic o-
o ganisms. Di e ences wi h P < 0.05 we e conside ed
signi ican .
3 Resul s
Le els o biogenic amines, nu ien composi ion, amoun s
o e men ing acids, and mic obiological ma ke s in
luce ne silage we e measu ed and compa ed among
ea men s o he ensiled ma e ial using biological o
chemical silage addi i es.
Among biogenic amines, we measu ed he con en s o
his amine, y amine, pu escine, cada e ine, spe midine
and spe mine (Table 1). His amine was obse ed in he
B g oup o be dec eased by 57% (P < 0.05) in compa ison
wi h he con ol g oup while in he CH g oup i s con en
inc eased by 63% (P < 0.05). Ty amine was highe in
g oups B and CH by 37% and 34%, espec i ely, ela i e o
Table 1. In luence o biological and chemical ea men s on le els o biogenic amines.
Biogenic amines (µg/kg)
G oups His amine Ty amine Pu escine Cada e ine Spe midine Spe mine
Con ol 13.7 ± 1.0 a 10.4 ± 1.2 a 33.4 ± 1.2 a 9.1 ± 0.3 a 15.2 ± 0.5 a50.9 ± 2.2 a
Biological (B) 5.9 ± 0.6 b 14.2 ± 3.8 b 50.9 ± 1.7 b 6.4 ± 0.3 b12.9 ± 1.1 b22.7 ± 3.3 b
Chemical (CH) 22.3 ± 1.6 a 13.9 ± 0.6 b 27.6 ± 0.4 c 4.1 ± 0.8 b 8.1 ± 1.1 b28.0 ± 1.4 b
Means ± s anda d de ia ion wi h di e ing supe sc ip s di e signi ican ly (P < 0.05).
Table 2. In luence o biological and chemical ea men s on con en o e men a ion p oduc s.
Fe men a ion p oduc s (g/kg)
G oups Lac ic acid Ace ic acid E hanol Ammonia
Con ol 113.2 ± 6.0 a27.8 ± 1.3 a9.4 ± 0.3 a2.6 ± 0.1 a
Biological (B) 125.9 ± 7.0 b16.6 ± 1.7 b4.3 ± 0.4 b0.4 ± 0.1 b
Chemical (CH) 94.9 ± 2.1 c14.9 ± 0.4 b0.0 ± 0.0 c1.1 ± 0.1 b
Means ± s anda d de ia ion wi h di e ing supe sc ip s di e signi ican ly (P < 0.05).
284   V. Mlejnko a, e al.
and impac s on animals’ heal h a e only a ely e alua ed.
P e iously s udies ha e di e ed in hei in e p e a ions as
o e ec s and doses o biogenic amines in a m animals.
Fo his amine, y amine, pu escine and cada e ine, and
depending on he animal ca ego y, sa e doses o 2–40 g o
amine/kg o d y ma e ha e been s a ed [4]. When using
he chemical silage addi i e Chemisile (2.5 kg/ ), Selwe
e al. obse ed a dec ease o biogenic amines in luce ne
silages [18]. His amine le els in chemically ea ed e sus
un ea ed con ol silages ha e been epo ed as dec easing
by 50% while hese alues we e −17% o pu escine, −33%
o cada e ine, and −21% o y amine [19]. A simila e ec
o chemical ea men was obse ed in ou expe imen ,
whe ein he con en s o pu escine and cada e ine
signi ican ly dec eased. The use o wo biological silage
inoculan s (L. casei a 106 CFU/g and L. buchne i a
106 CFU/g) in ensiling maize a e epo ed o ha e had a
ma ked e ec on dec easing biogenic amines in maize
silage. L. casei dec eased he occu ence o his amine,
y amine, pu escine and cada e ine. L. buchen i educed
only pu escine and his amine [20]. In con as o hese
esul s, we obse ed he occu ence o his amine o be
highes o he biological ea men . Ou esul s sugges
he chemical silage addi i e o be mo e e ec i e om he
pe spec i e o elimina ing biogenic amines, leading as
i did a he onse o e men a ion o a apid dec ease o
pH o 4.37 while a e ea men wi h a biological silage
addi i e he pH was 4.96. In ano he expe imen in which
inoculan doses we e selec ed simila o hose used in
ou case, a dec ease o biogenic amines was obse ed.
A he same ime, VanOs e al. con i m ha unless he
p ac ically iden ical o hose o he un ea ed con ol
g oup. Fa amoun was no signi ican ly a ec ed in he
B g oup, whe eas in he CH g oup a signi ican 24%
dec ease in a was obse ed. No signi ican di e ences
we e obse ed among any o he expe imen al g oups in
e ms o o al ib e con en , ADF o NDF.
Resul s om he mic obiological analyses a e
epo ed in Table 4. The numbe s o bac e ia o he
En e obac e iaceae amily we e wi hin he o de o
101 CFU/g o all silage g oups. The highes yeas and
mould con en s eached maximum alues on he o de
o 102 CFU/g in all expe imen al a ian s. Moulds we e
dec eased in he B g oup by a signi ican 73% as compa ed
o he con ol g oup. LAB numbe s eached alues in he
o de o 107 CFU/g o silage in g oups K and B. Va ian s
ea ed wi h he chemical addi i e had LAB alues one
o de o magni ude lowe . The o al numbe o mic o-
o ganisms in expe imen al silage ac oss all a ian s was
in he ange o 106–107 CFU/g. The o al numbe o mic o-
o ganisms includes bo h desi able and undesi able mic o-
o ganisms.
4 Discussion
Because luce ne has a highe p o ein con en in
compa ison o maize, i is mo e suscep ible o c ea ion o
e ec i e biogenic amines. These amines cons i u e one o
he mos impo an isk ac o s in dai y ca le nu i ion.
The Eu opean Union has no o da e es ablished any limi s
o he amoun s o biogenic amines wi hin luce ne silages,
Table 3. In luence o biological and chemical ea men s on nu ien con en in luce ne silages.
Nu ien s (g/kg)
G oups Ash P o ein Fa Fib e ADF NDF
Con ol 102.0 ± 4.5 a194.6 ± 5.9 a49.7 ± 5.4 a 263.6 ± 11.2 a288.9 ± 10.8 a349.7 ± 8.1 a
Biological (B) 92.5 ± 1.6 b215.1 ± 4.9 b51.8 ± 3.1 a 270.0 ± 6.5 a293.4 ± 11.5 a374.9 ± 6.1 a
Chemical (CH) 88.6 ± 3.8 b194.3 ± 2.5 a 38.0 ± 3.4 b254.0 ± 12.7 a283.9 ± 13.3 a353.8 ± 14.3 a
ADF: acid de e gen ib e, NDF: neu al de e gen ib e. Means ± s anda d de ia ion wi h di e ing supe sc ip s di e signi ican ly (P < 0.05).
Table 4. In luence o biological and chemical ea men s on mic obiological ma ke s.
Mic obiological ma ke s (CFU/g)
G oups En e obac e Moulds Yeas s LAB TMC
Con ol 18.2 ± 4.5 ab 142.4 ± 10.5 a19.7 ± 6.4 a 1.6 × 107 ± 45 × 105 a 1.7 × 107 ± 65 × 105 a
Biological (B) 12.1 ± 1.0 a 37.9 ± 12.1 b57.6 ± 8.3 b 1.2 × 107 ± 54 × 105 a4.1 × 107 ± 56 × 105 a
Chemical (CH) 36.4 ± 3.2 b 240.9 ± 45.2 a531.8 ± 105.9 b 0.5 × 107 ± 0.7 × 105 a0.4 × 107 ± 0.3 × 105 a
En e obac e .: he En e obac e iaceae amily, LAB: lac ic acid bac e ia, TMC o al mic o-o ganisms coun . Means ± s anda d de ia ion wi h
di e ing supe sc ip s di e signi ican ly (P < 0.05).

Biogenic amines and hygienic quali y o luce ne silage  285
e men a ion p ocess. A he same ime, hey obse ed a
posi i e in luence o biological silage addi i es on lac ic
acid alues, which we e he highes in compa ison wi h
he con ol g oup. Simila indings we e epo ed by Filya
e al. [28] and Holze e al. [24]. Hassana e al. [29] epo
om hei expe imen al wo k alues o ADF, NDF, lac ic
acid and ace ic acid simila o hose de e mined in ou
expe imen al luce ne silages, as well as simila absence o
p opionic acid and bu y ic acid.
5 Conclusion
This expe imen compa ed wo ea men s (one biological,
one chemical) when ensiling luce ne. The biological
ea men dec eased he occu ence o some biogenic
amines (cada e ine, his amine, spe midine, spe mine),
e men a ion p oduc s (ace ic acid, e hanol, ammonium)
and ash. Mic obiological analysis showed he e was a
dec ease in he popula ion o moulds and bac e ia o
he En e obac e iaceae amily. Le els o pu escine and
lac ic acid inc eased. Meanwhile, he chemical addi i e
dec eased he le els o biogenic amines (pu escine,
cada e ine, spe midine, spe mine), e men a ion p oduc s
(lac ic acid, ace ic acid, ammonium), pH, ash and a .
This ea men also educed o al mic o-o ganism coun s.
Le els o his amine and y amine we e inc eased. The
biological addi i e suppo ed he e men a ion p ocess,
and he luce ne silage me he hygienic equi emen s.
Ou esul s indica e ha biological and chemical
addi i es can be ecommended o p omo ing he
e men a ion p ocess and mee ing hygienic s anda ds in
p oducing luce ne silages. Chemical addi i es a e mo e
e ec i e compa ed o biological silage ones.
Acknowledgemen s: This esea ch was suppo ed by
p ojec NAZV QJ1310100 - De elopmen and op imiza ion
me hods o he de e mina ion o biogenic amines in
esponse o inc easing heal h secu i y o silage and IGA
TP1/2016 New end in he cul i a ion and use o milk
his le (Silybum Ma ianum L.) in ag icul u e.
Con lic o in e es : Au ho s decl e no hing o disclose.
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