Biogenic amines and hygienic quality of lucerne silage

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. Re e ences [1] Ho ky P., E ec o p o ein concen a e supplemen on he quali a i e and quan i a i e pa ame e s o milk om dai y cows in o ganic a ming, Ann. Anim. 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