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CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 Hrvatski časopis za prehrambenu tehnologiju, biotehnologiju
98 i nutricionizam 12 (3-4), (2017)

HRVATSKI ČASOPIS ZA PREHRAMBENU

TEHNOLOGIJU, BIOTEHNOLOGIJU I NUTRICIONIZAM
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 Hrvatski časopis za prehrambenu tehnologiju, biotehnologiju
i nutricionizam 12 (3-4), (2017) 99

SADRŽAJ / CONTENT

Radovi / Papers

Odabrani biotehnološki čimbenici koji utječu na alkoholnu fermentaciju pri proizvodnji vina..........................................100
Vlatka Petravić-Tominac, Sven Mujadžić, Vesna Zechner-Krpan, Hrvoje August, Darko Velić, Natalija Velić

Monitoring the influence of high power ultrasound treatment and thermosonication

on inactivation of Brettanomyces bruxellensis in red wine........................................................................................................107
Leo Gracin, Stela Križanović, Anet Režek Jambrak, Marina Tomašević, Karla Kelšin,
Katarina Lukić, Karin Kovačević Ganić

Characteristics and selection of cultures of photosynthetic purple non-sulphur bacteria as a potential 5-aminolevulinic
acid producers............................................................................................................................................................................... 113
Mario Novak, Mladen Pavlečić, Baghish Harutyunyan, Vigen Goginyan, Predrag Horvat, Božidar Šantek

Preferencije potrošača i važnost intrinzičnih i ekstrinzičnih obilježja čipsa od jabuke .......................................................120

Željka Mesić, Velimir Hunjak, Marina Tomić

Water quality on cattle farms in the northwest Croatia............................................................................................................126

Marija Denžić Lugomer, Vesna Jaki Tkalec, Maja Kiš, Damir Pavliček, Sanja Furmeg, Jadranka Sokolović

Changes in the quality characteristics of carrot juice preserved with Aframomum danielli seed extract............................131
Abiodun Aderonke Amanyunose, Olufunmilola Adunni Abiodun, Gabriel Olaniyi Adegoke, Adegbola Oladele Dauda

Reološka svojstva mliječnog proizvoda na bazi svježeg sira i voća..........................................................................................137

Tijana Brčina, Milica Vilušić, Amel Selimović

Osiguranje kvalitete rezultata ispitivanja vina u analitičkom laboratoriju............................................................................146

Renata Leder, Valentina Ščitnik, Marija Vukoja, Anita Boras, Ivana Vladimira Petric, Neven Antunac, Mara Banović

Upute autorima.............................................................................................................................................................................155
Instructions to authors.................................................................................................................................................................157

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
100 and Nutrition 12 (3-4), 100-106 (2017)

PREGLEDNI RAD / REVIEW

Odabrani biotehnološki čimbenici koji utječu na

alkoholnu fermentaciju pri proizvodnji vina
Selected biotechnological factors affecting
alcoholic fermentation in winemaking
Vlatka Petravić-Tominac1, Sven Mujadžić2, Vesna Zechner-Krpan1*, Hrvoje August3, Darko Velić4,

Natalija Velić4

1
Laboratorij za biokemijsko inženjerstvo, industrijsku mikrobiologiju i tehnologiju slada i piva, Prehrambeno-biotehnološki
fakultet, Sveučilište u Zagrebu, Pierottijeva 6, 10000 Zagreb, Hrvatska
2
Xellia d.o.o (Xellia Ltd), Slavonska avenija 24/6, 10 000 Zagreb, Hrvatska
3
Ireks Aroma d.o.o., Radnička c. 37, 10000 Zagreb, Hrvatska
4
Prehrambeno-tehnološki fakultet Osijek, Sveučilište Josipa Jurja Strossmayera u Osijeku, Franje Kuhača 20, 31000 Osi-
jek, Hrvatska

* Corresponding author:
Tel. +385 1 46 05 142; Fax. +385 1 48 36 424; e-mail: vzkrpan@pbf.hr

Sažetak

Odstupanja od normalnog tijeka fermentacije predstavljaju veliki problem u proizvodnji vina. Važno je poznavati čimbenike koji utječu na
tijek fermentacije kako bi se izbjeglo usporavanje i/ili zastoj fermentacije te spriječilo ekonomske gubitke. Brojna istraživanja pokazala su da
odstupanja od normalnog tijeka fermentacije mogu imati više uzroka. U ovom radu su ukratko objašnjeni najvažniji čimbenici koji utječu na tijek
fermentacije u proizvodnji vina, a mogu se grupirati u tri skupine - nedostatak nutrijenata, prisutnost spojeva koji inhibiraju alkoholnu fermen-
taciju te utjecaj enoloških postupaka. Pored zasebnih učinaka pojedinih čimbenika, moguće je i smanjenje brzine fermentacije zbog njihovog
sinergističkog djelovanja, što fermentacijske probleme čini još složenijima.
Ključne riječi: proizvodnja vina, fermentacija, usporena fermentacija, zastoj fermentacije

Abstract
Deviations from the normal course of fermentation have been a major problem in wine production. It is important to know the factors affec-
ting the fermentation in order to avoid sluggish or stuck fermentation and prevent economic losses. Numerous studies have shown that deviations
from the normal course of fermentation can have multiple causes. This paper gives a brief overview of the most important factors affecting the
wine fermentation, that can be further grouped into three categories, namely – the absence or lack of nutrients, the presence of compounds that
inhibit the alcoholic fermentation and the impact of oenological practices. In addition to individual effects of certain factors, the fermentation
speed can decrease because of the synergistic action of various factors, which makes fermentation problems even more complex.
Keywords: winemaking, fermentation, sluggish fermentation, stuck fermentation

Uvod killer-toksini i pesticidi (Alexandre i Charpentier, 1998; Bisson

Istraživanja problema koji se zbivaju pri proizvodnji vina
započeo je Louis Pasteur u drugoj polovici 19. stoljeća (Bar-
nett, 2000). Istraživanja uzroka problema tijekom fermentacije
intenzivno su nastavljena i u novije vrijeme, pri čemu su ra-
zličiti čimbenici prepoznati kao odgovorni, kao što su velika
početna koncentracija šećera, nedostatak dušika, nedostatak
vitamina (posebice tiamina), nedostatak kisika, pretjerano bi-
strenje mošta, anaerobni uvjeti, velike koncentracije etanola,
inhibicija stanica kvasca nusproduktima fermentacije (pose-
bice masnim kiselinama i octenom kiselinom), pH-vrijednost,
i Butzke, 2000; Malherbe i sur., 2007). Osim zasebnih učinaka
svakog od navedenih čimbenika koji uzrokuju smanjenje brzi-
ne fermentacije, moguće je i njihovo sinergističko djelovanje,
što čini fermentacijske probleme još složenijima. S obzirom
na sve navedeno, iznimno je teško točno predvidjeti probleme
koji se mogu pojaviti tijekom fermentacije i utvrditi njihove
uzročnike (Alexandre i Charpentier, 1998; Malherbe i sur.,
2007).
Utjecaji navedenih čimbenika na fermentaciju su mnogo-
brojni te uključuju sniženje pH-vrijednosti, inhibiciju aktivno-
sti ključnih enzima i promjene u staničnoj membrani kvasca.

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
To nadalje može inducirati promjene metabolizma stanica
kvasca zbog čega dolazi do smanjenja proizvodnje biomase
kvasca, vijabilnosti stanica i brzine fermentacije. Kako bi si-
mulirali realne uvjete, mnoga istraživanja provedena su kori-
štenjem sintetskih podloga ili podloga koji oponašaju sastav
mošta, međutim u takvim podlogama se kvasac može ponašati
drugačije nego u realnim uvjetima (Alexandre i Charpentier,
1998).
Vijabilnost kvasca je postotak živih stanica u ukupnom
broju stanica kvasca, a vitalnost označava fiziološko stanje
kvaščevih stanica tj. njihovu sposobnost provođenja potpune
fermentacije. Tijek alkoholne fermentacije i karakteristike vina
ovise o broju živih stanica kvasca i njihovoj vitalnosti. Zado-
voljavajuća vijabilnost i vitalnost kvasca potrebne su za pro-
vođenje fermentacije do suhog vina. Također je važno da oda-
brani soj kvasca može fermentirati pri kombinaciji uvjeta koji
vladaju tijekom fermentacije (Iland i sur., 2007). Značaj kvas-
ca za tijek fermentacije opisan je u literaturi (Bisson, 1999;
Bisson i Butzke, 2000; Boulton, 1996; Fugelsang i Edwards,
2007; Krieger-Weber, 2009) i nije predmet interesa ovog rada.
Unatoč značajnim poboljšanjima u praćenju i kontroli
fermentacije, usporavanje (eng. sluggish fermentation) i za-
stoj fermentacije (eng. stuck fermentation) ostaju velik izazov
za vinsku industriju širom svijeta (Malherbe i sur., 2007). Pri
proizvodnji vina važno je postići potpunu alkoholnu fermen-
taciju uz malu koncentraciju neprevrelih šećera, što smanjuje
mogućnost kontaminacije nepoželjnim mikroorganizmima, pr-
venstveno bakterijama octene i mliječne kiseline. One mogu
metabolizirati neprevreli šećer te povećati koncentraciju hla-
pljivih kiselina i nastajanje nepoželjnih estera uz mogućnost
nastajanja diacetila (Alexandre i Charpentier, 1998).
Bisson (1999) je definirala nepotpunu fermentaciju (eng.
incomplete fermentation) ili zastoj fermentacije kao one sluča-
jeve u kojima je na kraju prisutna veća koncentracija zaostalog
šećera od željene. Usporene fermentacije su one tijekom kojih
kvasac sporo troši šećer. Važno je istražiti uzroke nepravil-
nih fermentacija u industrijskim vinskim podrumima, jer one
mogu dovesti do ekonomskih gubitaka. Kinetika fermentacije
može se pratiti određivanjem koncentracije utrošenog šećera,
određivanjem nastalog CO2 ili mjerenjem gustoće (Ribéreau-
Gayon i sur., 2006a).
Temperatura ima utjecaja ne samo na brzinu fermentacije
koju provode kvasci iz roda Saccharomyces, nego i na ravno-
težu između kvasaca iz roda Saccharomyces i divljih kvasaca.
U proizvodnji crnih vina (20 – 30 ºC) dominantan je kvasac
Saccharomyces cerevisiae, djelomično i zbog veće temperatu-
re fermentacije. Pri manjim temperaturama, poput onih koje se
primjenjuju pri proizvodnji bijelih vina, divlji kvasci se mogu
umnožiti do puno većih koncentracija (Fugelsang i Edwards,
2007).
U ovom radu opisan je utjecaj sastava mošta i fermenta-
cijskih uvjeta na tijek alkoholne fermentacije pri proizvodnji
vina. Objašnjeno je kako na tijek fermentacije utječu nedosta-
tak hranjivih tvari (dušika, kisika, sterola, minerala, vitamina),
prisutnost spojeva koji inhibiraju fermentaciju (etanol, organ-
ske kiseline) te utjecaj sumporenja i ostalih postupaka koji se
provode u tehnologiji vina. Rad se temelji na razmatranju ke-
mijskih i biokemijskih čimbenika, dok utjecaj mikroflore pri-
sutne u moštu, kao i agrokemijskih čimbenika nije detaljnije
razrađen u ovom radu, jer se radi o preopsežnim temama.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 100-106 (2017) 101
1. Nedostatak hranjivih tvari
1.1. Nedostatak asimilacijskog dušika
Umjesto izraza asimilacijski dušik, koji je uobičajen
među vinarima, inače se u biotehnologiji koristi naziv asimi-
labilni dušik, što znači onaj kojeg kvasac može asimilirati.
Asimilacijski dušik (eng. yeast assimilable nitrogen, YAN)
koji kvasac može koristiti u svom metabolizmu tijekom fer-
mentacije predstavlja zbroj koncentracija dušika prisutnog u
primarnim aminokiselinama i amonijevim ionima prisutnima u
moštu. Pored ovih izvora, dušik je u moštu prisutan i u sastavu
drugih spojeva koje kvasac ne može asimilirati. Koncentraci-
ja asimilacijskog dušika može biti varijabilna, a uobičajeno se
koristi kao pokazatelj nutritivnog statusa mošta (Butzke, 2010;
Waterhouse i sur., 2016).
Zbog nedovoljne koncentracije asimilacijskog dušika
može doći do usporavanja ili zastoja fermentacije (Lagunas
i sur., 1982; Salmon, 1989; Alexandre i Charpentier, 1998;
Butzke, 2010). Na potrebu kvasca za dušikom utječe prisut-
nost zraka, ali i početna koncentracija glukoze. Mala početna
koncentracija odgovarajućih izvora dušika limitira rast bio-
mase kvasca, što dovodi do usporavanja katabolizma šećera.
Ako kvasac tijekom fermentacije potroši ove izvore dušika iz
hranjive podloge, u njegovoj stanici dolazi do zastoja sinteze
proteina i drastičnog pada transporta šećera.
Koncentracije asimilacijskog dušika od 120 do 140 mg/L
omogućavaju potpunu fermentaciju do suhog vina (Alexandre
i Charpentier, 1998; Malherbe i sur., 2007; Butzke, 2010), dok
su najveće brzine fermentacije mošta zabilježene pri 500 mg
N/L (Butzke, 2010). Pri proizvodnji bijelih vina će 250 - 300
mg dušika/L omogućiti normalnu brzinu fermentacije i izražen
voćni karakter vina. Za crna vina je dostupno manje podata-
ka, ali se općenito smatra kako je za postizanje odgovarajuće
kvalitete gotovog vina potrebno osigurati slične koncentraci-
je asimilacijskog dušika kao i pri proizvodnji bijelih vina. Pri
tome, prisutnost čvrstih dijelova grozda koji polako otpušta-
ju aminokiseline i druge nutrijente tijekom proizvodnje crnih
vina predstavlja prednost u odnosu na proizvodnju bijelih vina.
1.2. Nedostatak kisika
Metabolizam kvasca ovisi o koncentraciji otopljenog kisi-
ka na početku fermentacije. Otopljeni kisik se tijekom fermen-
tacije brzo troši djelovanjem kvasca i oksidaza (enzima koji
su prirodno prisutni u moštu). Nedostatak kisika tijekom fer-
mentacije mošta dovodi do kvalitativnih i kvantitativnih pro-
mjena u sastavu lipida stanica kvasaca. U staničnoj membrani
uslijed nedostatka kisika dolazi do inhibicije biosinteze lipida
te se smanjuje koncentracija ergosterola i nezasićenih masnih
kiselina. Zbog toga se smanjuju brzina glikolize i brzina ra-
sta stanica kvasca, usporava rast biomase i opada vijabilnost
kvasca, što dovodi do usporavanja fermentacije. Promjena u
sastavu lipida stanične membrane dovodi do oslabljene otpor-
nosti stanica kvasca na etanol (Alexandre i Charpentier, 1998;
Ribéreau-Gayon i sur., 2006a ; Krieger-Weber, 2009).
Aeracijom ili mikrooksigenacijom mošta tijekom fermen-
tacije poboljšavaju se rast i vijabilnost kvasca, jer je u aerob-
nim uvjetima stanična membrana kvasaca bogatija nezasiće-
nim masnim kiselinama i ergosterolom, koje kvasac u takvim
uvjetima može sintetizirati. Ergosterol i nezasićene masne ki-
 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
102 and Nutrition 12 (3-4), 100-106 (2017)
seline inače se u literaturi nazivaju i faktorima preživljavanja
(eng. „survival factors“), što dovoljno govori o njihovoj važ-
nosti (Boulton i sur., 1996; Ribéreau-Gayon, 2006b; Malherbe
i sur., 2007).
Nedostatak kisika također utječe na nastajanje toksičnih
masnih kiselina, tj. oktanske (kaprilne) kiseline i dekanske
(kaprinske) kiseline, a koncentracije ovih kiselina smanjuju se
tijekom aeracije mošta. Proizvođači vina često aeriraju mošt
kako bi spriječili štetne učinke nedostatka kisika. Tako tole-
rancija stanica kvasca prema etanolu i njihova vijabilnost rastu
zbog odgovarajućeg sastava lipida u staničnoj membrani kvas-
ca i pada koncentracije toksičnih masnih kiselina (Alexandre i
Charpentier, 1998).
Kritična koncentracija otopljenog kisika za kvasac S. ce-
revisiae iznosi 0,6 mg/L (Marić i Šantek, 2009). Kisik je pri
optimalnim uvjetima za provođenje fermentacije slabo topljiv
te je stoga njegova koncentracija u moštu relativno mala, a do-
datno je smanjuju oksidaze koje troše većinu otopljenog kisika.
SO2 inhibira oksidaze koje su prirodno prisutne u moštu, pose-
bice polifenol-oksidaze. Ukoliko oksidaze nisu već inhibirane,
aeracija neće biti učinkovita (Boulton i sur., 1996; Alexandre i
Charpentier, 1998).
1.3. Nedostatak minerala
Neki od minerala potrebnih za optimalan rast kvasca i fer-
mentaciju neophodni su u milimolarnim koncentracijama (npr.
magnezij). Drugi su potrebni u mikromolarnim koncentraci-
jama (npr. kalcij) dok su neki toksični za kvasac već u mikro-
molarnim koncentracijama (teški metali) (Birch i sur., 2003).
Prema nekim autorima, mošt uglavnom kvalitativno i kvantita-
tivno pokriva potrebe kvasca za mineralima (Ribéreau-Gayon
i sur., 2006a), dok drugi navode cink i magnezij kao potencijal-
no litimirajuće (Malherbe i sur., 2007; Walker i Stewart, 2016).
Koncentracija cinka u grožđu može varirati ovisno o bio-
raspoloživosti cinkovih iona u tlu vinograda, kao i o primjeni
gnojiva i fungicida tijekom uzgoja grožđa. Stoga su moguće
suboptimalne koncentracije cinka u moštu, što negativno utje-
če na fermentacijsku učinkovitost kvasaca. Soj kvasca je tako-
đer čimbenik o kojem ovisi optimalna koncentracija cinka (De
Nicola i sur., 2009).
Magnezij je kvascima potreban za mnoge metaboličke i
fiziološke funkcije. On stabilizira nukleinske kiseline, protei-
ne, polisaharide i lipide, a također ima ključnu ulogu u kontroli
metabolizma, rastu i staničnoj proliferaciji. Nema dostupnih
istraživanja koja bi odredila ulogu iona Mg2+ tijekom alkohol-
ne fermentacije u proizvodnji vina, ali je poznata njegova važ-
nost tijekom fermentacije melase ili glukoze (u minimalnoj ili
kompleksnoj podlozi). Zbog toga se može pretpostaviti da ma-
gnezij ima važnu ulogu i u fermentaciji mošta. Postoji izravna
veza između dostupnosti magnezija i kinetike fermentacije.
Limitacija magnezijem odgovorna je za smanjenu brzinu rasta
kvasca i smanjenu fermentacijsku aktivnost. Dodatak magne-
zija u fermentacijsku podlogu omogućava poboljšanje proi-
zvodnje etanola, što se može objasniti činjenicom da magnezij
stabilizira strukturu membrane stanica. Nažalost, malo je po-
dataka o koncentraciji iona Mg2+ u moštu. U moštu dobivenom
iz bijelog grožđa, određene su koncentracije iona Mg2+ od 10
- 140 mg L-1. Neki autori navode da je optimalna koncentra-
cija iona Mg2+ za vijabilnost kvasca (5 mg L-1) manja od kon-
centracije koja je inače prirodno prisutna tijekom vinifikacije
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
(Alexandre i Charpentier, 1998), dok drugi govore o mogućem
nedostatku magnezija (Walker i Stewart, 2016).
Primijećeno je da transport Mn2+ u stanicu ovisi o unu-
tarstaničnoj koncentraciji Mg2+ te da toksičnost mangana ovi-
si o omjeru iona mangana i magnezija u stanici kvasca. Isto
tako visoka koncentracija mangana negativno utječe na ulazak
magnezija u stanicu, što može dovesti do deficita magnezija u
kvascu (Blackwell i sur., 1997; Blackwell i sur., 1998; Malher-
be i sur., 2007). Također se pokazalo kako je međusobni omjer
koncentracija iona Mg2+ i Ca2+ u moštu izuzetno važan za fer-
mentaciju i za organoleptički profil vina (Birch i sur., 2003).
Može se pretpostaviti da međusobna neravnoteža koncentraci-
ja različitih anorganskih iona, slično kao u navedenim primje-
rima, može dovesti do promjene fizioloških svojstava stanica
vinskog kvasca i posljedično do problema pri fermentaciji.
Funkcija svih minerala pri fermentaciji mošta nije još do-
voljno poznata te je vrlo malo istraživanja provedeno upravo u
moštu, dok je veći dio podataka dostupan na temelju eksperi-
menata provedenih u drugim hranjivim podlogama (Ribéreau-
Gayon i sur., 2006a; De Nicola i sur., 2009). Pored koncen-
tracija pojedinih minerala u moštu valjalo bi istražiti i utjecaj
njihovih međusobnih odnosa na fiziološka svojstva pojedinih
enološki zanimljivih sojeva kvasca. Pritom treba uzeti u obzir
da se u suvremenom vinarstvu koriste komercijalne starter-kul-
ture kvasca roda Saccharomyces, ali u novije vrijeme i kvasaca
drugih rodova koji su tijekom selekcije pokazali poželjna eno-
loška svojstva.
1.4. Nedostatak vitamina
Smatra se kako su u nekim slučajevima usporene fermen-
tacije povezane s nedostatkom vitamina, ali je malo istraživanja
koja potkrepljuju navedenu tezu (Ough i sur., 1989; Alexandre
i Charpentier, 1998). Mošt sadrži faktore rasta, uključujući i
vitamine, ali tijekom alkoholne fermentacije dolazi do promje-
ne njihove koncentracije (Ribéreau-Gayon i sur., 2006a). Ovo
u slučaju nedovoljne početne koncentracije vitamina u moštu
može dovesti do potrebe za njihovim naknadnim dodavanjem
tijekom fermentacije.
Dostupna istraživanja fokusirala su se uglavnom na kon-
centracije tiamina (vitamin B1), koje se u moštu kreću u raspo-
nu 150 - 750 µg L-1. Iako kvasac S. cerevisiae može sintetizirati
tiamin, nedostatak ovog vitamina može dovesti do usporene
fermentacije (Ough i sur., 1989). Kvasci mogu utrošiti 600
– 800 μg/L tiamina, što je više od koncentracija prisutnih u
moštu (Ribéreau-Gayon i sur., 2006a). Do smanjenja koncen-
tracije tiamina u moštu može doći jer ga divlji kvasci brzo asi-
miliraju i mogu ga potrošiti u roku od nekoliko sati. Ukoliko
se odgađa inokulacija mošta selekcioniranim sojevima kvasca
S. cerevisiae, tada može doći do negativnog utjecaja popula-
cije divljih kvasaca na fermentacijsku aktivnost. Koncentraci-
ja tiamina može se smanjiti i zbog sumporenja. Djelovanjem
bisulfitnih iona dolazi do cijepanja tiamina, što može izazva-
ti poteškoće u fermentaciji, pogotovo kada je SO2 prisutan u
velikim koncentracijama (Alexandre i Charpentier, 1998; Fu-
gelsang i Edwards, 2007). Dodatkom 0,5 mg tiamina po litri
mošta može se postići porast žive aktivne populacije stanica za
30 % te istovremeno ubrzati fermentaciju. Međutim, ovakvi re-
zultati se dobivaju u laboratorijskim uvjetima, ali u praksi nije
uvijek tako. Koncentracija tiamina prisutna u grožđu može, ali
i ne mora biti limitirajući faktor za kinetiku fermentacije, što
 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
ovisi o grožđu i o drugim čimbenicima (Ribéreau-Gayon i sur.,
2006a).
Koncentracije ostalih vitamina su različite u moštu i vi-
nima (Ribéreau-Gayon i sur., 2006a). Promjene koncentracija
vitamina tijekom fermentacije nisu jednake za sve vitamine.
Dok tiamin može skoro potpuno nestati, s ostalim vitaminima
je situacija drugačija. Tako su npr. koncentracije nikotinamida
slične u moštu i u crnim vinima, ali su oko 40 % niže u bijelim
vinima. S druge strane, riboflavin nastaje djelovanjem kvas-
ca. Nadalje, kvasci koriste, a potom i otpuštaju pantotensku
kiselinu, piridoksin i biotin te je njihova koncentracija skoro
identična u moštu i vinu.
2. Spojevi koji inhibiraju
alkoholnu fermentaciju
2.1. Etanol
Kvasac fermentira šećer u etanol koji se akumulira i može
inhibirati samu alkoholnu fermentaciju te uzrokovati druge
nepoželjne učinke na stanice kvasca. Iako se etanol tijekom
alkoholne fermentacije nakuplja unutar stanice, unutarstanič-
ne i izvanstanične koncentracije etanola su slične (Alexandre i
Charpentier, 1998).
Dobro je poznato kako etanol inhibira rast i djeluje tok-
sično na stanice kvasca (Bauer i Pretorius, 2000). Primjerice,
etanol inhibira aminokiselinsku permeazu i mehanizam tran-
sporta glukoze. Važno je napomenuti kako se brzina transporta
(ulaska) glukoze u stanicu niti njezina razgradnja glikolizom
ne mijenja do volumnog udjela etanola od 8,5 % (v/v), ali se
smanjuje brzina fermentacije za oko 50 % (Cartwright i sur.,
1986; Salmon, 1989).
Prisutnost etanola uzrokuje promjenu u propusnosti mem-
brane kvasca S. cerevisiae (Alexandre i Charpentier, 1998).
Etanol vjerojatno ulazi u hidrofobnu unutrašnjost membrane
i time povećava njenu polarnost te tako dopušta slobodnu iz-
mjenu polarnih molekula i slabi hidrofobne interakcije. Stres
uzrokovan etanolom izaziva promjene sastava lipida kvaščeve
stanične membrane, tako da se mijenja stupanj zasićenosti i
dužina lanaca nezasićenih masnih kiselina te stoga dolazi do
promjene u fluidnosti membrane. Povećanje indeksa neza-
sićenosti pogoduje povećanoj otpornosti prema etanolu i vi-
jabilnosti kvasca S. cerevisiae. Kod kvasaca koji u staničnoj
membrani imaju puno nezasićenih masnih kiselina manji je
negativan utjecaj etanola na potrošnju glukoze i aminokiseli-
na. Uvođenjem kisika u mošt, u stanicama dolazi do povećanja
sinteze nezasićenih masnih kiselina i sterola (tzv. „faktora pre-
življavanja“), koji doprinose većoj otpornosti stanica na etanol
i smanjuju štetne učinke etanola, što dovodi do povećanja vi-
jabilnosti stanica. Jasno je kako je smanjenje brzine fermenta-
cije povezano s nastajanjem etanola, pogotovo kada se u obzir
uzmu svi učinci etanola na fermentaciju. Ovisno o toleranciji
kvasca prema etanolu, veća koncentracija etanola može brzo
dovesti do usporavanja ili zastoja fermentacije.
2.2. Organske kiseline
Na metabolizam kvasca tijekom vinifikacije mogu nepo-
voljno djelovati srednjelančane masne kiseline (npr. oktanska i
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 100-106 (2017) 103
dekanska kiselina) te octena kiselina (Alexandre i Charpentier,
1998).
2.2.1. Srednjelančane masne kiseline
Tijekom fermentacije u bogatom mediju uz određene fi-
zikalno-kemijske uvjete, stanični ciklus kvasca ipak može biti
narušen te se javlja značajno smanjenje fermentacijske aktiv-
nosti, sve dok se ne dogodi potpuni zastoj procesa. U ovom
slučaju, inhibicija je rezultat spojeva nastalih uslijed metabo-
lizma kvasca. Osim etanola, srednjelančane masne kiseline
su također inhibitori fermentacije koje kvasac S. cerevisiae
sintetizira tijekom alkoholne fermentacije (Lafon-Lafourca-
de i sur. 1984; Jackson, 2014). Koncentracija masnih kiselina
oslobođenih u fermentacijski medij ovisi o soju kvasca, sasta-
vu medija i fermentacijskim uvjetima (temperatura, pH-vrijed-
nost, aeracija). Srednjelančane masne kiseline se u literaturi
spominju kao antimikrobni spojevi. Pri koncentracijama od
114 µmol L-1 oktanske kiseline, odnosno 46 µmol L-1 dekanske
kiseline, uočeno je smanjenje specifične brzine rasta kvasca S.
cerevisiae koje eksponencijalno ovisi o koncentraciji masnih
kiselina. Ove masne kiseline također djeluju na brzinu odumi-
ranja stanica kvasca Saccharomyces bayanus zbog povećane
temperature ili uslijed temperaturnog stresa u kombinaciji s
etanolom, dok u stanicama kvasca S. cerevisiae stimuliraju gu-
bitak aminokiselina i drugih komponenata (Alexandre i Char-
pentier, 1998).
Toksičnost masnih kiselina na kvasac povećava se sma-
njenjem pH-vrijednosti medija. To ukazuje na činjenicu da
su nedisocirane molekule najtoksičnije, pri čemu dekanska
kiselina ima veći inhibicijski učinak nego oktanska kiselina.
Dekanska kiselina izaziva promjenu u staničnoj membrani po-
većavajući njezinu fluidnost, uslijed zakiseljavanja citoplazme.
Također je moguće da srednjelančane masne kiseline djeluju
sinergistički s etanolom i tako dodatno usporavaju fermenta-
ciju. Dekanska i oktanska kiselina inhibiraju transport heksoza
(pri čemu dekanska kiselina ima jače djelovanje), što može do-
vesti do usporavanja ili zaustavljanja fermentacije (Alexandre
i Charpentier, 1998).
2.2.2. Octena kiselina
Octena kiselina je još jedan od krajnjih produkata koji
nastaju tijekom alkoholne fermentacije te i ona dodatno nega-
tivno utječe na rast, fermentaciju i vijabilnost vinskih kvasaca.
Način djelovanja octene kiseline podsjeća na djelovanje sred-
njelančanih masnih kiselina. Kako bi se objasnila uloga octene
kiseline u smanjenju brzine fermentacije, istraživana su dva
moguća mehanizma inhibicije. Prvi mehanizam je zakiselja-
vanje citoplazme (utjecaj octene kiseline na pH-vrijednost o
kojoj ovisi aktivnost enzima), a drugi mehanizam je djelovanje
octene kiseline izravno na transport ili na aktivnosti enzima.
Mnogi slučajevi usporene i zaustavljene fermentacije uzro-
kovani su nastajanjem velikih količina octene kiseline. To se
može dogoditi tijekom nepravilnog transporta pljesnivog grož-
đa iz vinograda do vinarija, što dovodi do preuranjene alkohol-
ne fermentacije oslobođenog soka, ali i zbog naknadnog rasta
bakterija octene kiseline. Slično se događa i kod nepravilne
obrade svježe dobivenog mošta (najčešće pri proizvodnji crnih
vina, kada je početna pH-vrijednost mošta veća od 3,5) ili kada
mošt nije inokuliran vinskim kvascem niti tretiran sumporo-
vim dioksidom, što omogućava ubrzani rast nepoželjnih (ali
 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
104 and Nutrition 12 (3-4), 100-106 (2017)
autohtonih) bakterija rodova Lactobacillus i Pediococcus. Ove
bakterije proizvode velike količine octene kiseline, umjesto
mliječne kiseline (Boulton i sur., 1996; Alexandre i Charpen-
tier, 1998; Jackson, 2014).
2.3. Utjecaj sumporenja
Sumporenje se stoljećima koristi u vinarstvu (Boulton i
sur., 1996; Ribéreau-Gayon i sur., 2006a; Butzke i sur, 2010).
Sulfit je vrlo toksičan za mikroorganizme i mikroflora grož-
đa je osjetljiva na njegovo djelovanje, dok su selekcionirani
kvasci koji se rutinski koriste u fermentaciji manje osjetljivi.
Antimikrobno djelovanje sulfita u vodenim otopinama ovisi o
pH-vrijednosti, temperaturi i vremenu izlaganja. Sulfiti postoje
u otopinama u tri oblika, odnosno kao molekularni oblik (SO2)
te kao HSO3-, SO32-, a međusobni omjer navedenih oblika ovisi
o pH-vrijednosti. Ravnotežna reakcija između različitih mo-
lekularnih oblika (Guzzo i Desroche, 2009) prikazane su jed-
nadžbom:
H2O + SO2   H+ + (HSO3)-     2H+ + SO32-
pH-vrijednosti različitih vrsta vina kreću se u području od
2,8 do 4,0 (Ribéreau-Gayon i sur., 2006b). Molekularni SO2
500 puta jače djeluje na kvasac nego ostali oblici u kojima
sulfit može biti prisutan (HSO3-, SO32-) što objašnjava zašto je
on osobito učinkovit prema kvascima koji su prisutni u moštu,
koji je pH-vrijednosti između 3,0 i 3,5 (Alexandre i Charpen-
tier, 1998).
Sulfit se primjenjuje u raznim fazama tijekom proizvod-
nje vina, ali uglavnom se dodaje moštu prije alkoholne fer-
mentacije radi kontrole rasta nepoželjnih vrsta. Dodatak SO2
u mošt mora biti strogo kontroliran. Primijenjena doza treba-
la bi inhibirati rast nepoželjnih vrsta, ali istodobno omogućiti
rast kvasca koji provodi fermentaciju. Opće je poznato kako
otpornost kvasca na SO2 ovisi o vrsti kvasca (Stratford i sur.,
1987). Stoga, velike koncentracije SO2 u moštu mogu biti od-
govorne za odgođenu ili zaustavljenu fermentaciju (Romano
i Suzzi, 1993), premda se to u današnje vrijeme rijetko doga-
đa. Dodatak SO2 važan je radi inhibicije polifenol-oksidaze i
sprječavanja potpune potrošnje kisika. U daljnjem tekstu su sa-
žeti molekularni mehanizmi utjecaja SO2 na mikrobne stanice
(Alexandre i Charpentier, 1998).
Štetno djelovanje sulfita prema kvascu u velikoj mjeri
ovisi o akumulaciji SO2 u stanici. Neki autori smatraju kako
SO2 ulazi u kvasac S. cerevisiae aktivnim transportom, dok
drugi (Stratford i Rose, 1986) tvrde da SO2 ulazi u stanice
jednostavnom difuzijom. Brzina transporta sulfita ima važnu
ulogu za njegovo štetno djelovanje, a manja fluidnost mem-
brane će olakšati difuziju kroz staničnu membranu. Kvasac S.
cerevisiae nakuplja SO2 vrlo brzo i kad se uspostavi ravnote-
ža, unutarstanična koncentracija sulfita je nekoliko puta veća
nego njegova koncentracija u otopini. Ovo se može objasniti
disocijacijom SO2 na bisulfitni anion HSO3- i H+ (zbog više pH-
vrijednosti u stanici), što omogućava daljnju difuziju u stanicu.
Kada se nađe u stanici, sulfit uzrokuje ubrzano smanjenje
koncentracije ATP-a u stanici. Nedostatak ATP-a je odlučuju-
ći događaj koji uzrokuje odumiranje stanice (Hinze i Holzer,
1986). Mehanizmi odumiranja stanica uzrokovanog sulfitom
su i dalje nepoznati, iako su in vitro dokazane reakcije sulfita s
određenim molekulama poput proteina, koenzima i metaboli-
ta. Poznato je kako sulfiti reagiraju s NAD+/NADP te cijepaju
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
tiamin i disulfidne mostove proteina. SO2 utječe i na mnoge
enzimske sustave kvasca. Inhibicija može proizaći iz promjena
u konformaciji, interakcije s aktivnim mjestima ili s kofakto-
rima. Već je spomenuta važnost cijepanja tiamina pri visokim
koncentracijama disulfita ili pri dugotrajnom čuvanju grožđa-
nog soka i koncentrata grožđanog soka (Alexandre i Charpen-
tier, 1998).
3. Utjecaj enološke prakse na fermentaciju
Mošt je glavna sirovina u procesu proizvodnje vina. U
proizvodnji bijelih vina provodi se vinifikacija izbistrenog mo-
šta. Uobičajeni postupci bistrenja su sedimentacija (taloženje),
bistrenje pomoću separatora, filtracija, centrifugiranje i doda-
tak enzima (Höhn i sur., 2005; Jackson, 2014).
U novije vrijeme, otprilike unazad 10-ak godina, sve je
prisutnija tehnologija flotacije koja se umjesto klasičnog bi-
strenja koristi za pripremu mošta iz bijelih sorata grožđa. To
se u praksi provodi tako da se prvo dodatkom odgovarajućih
enzima provede enzimska depektinizacija grožđa, masulja ili
mošta, a potom se moštu u flotacijskom tanku dodaje želati-
na te se uvodi plinoviti dušik. Time se postiže efekt flotacije,
odnosno na površinu mošta se uzdižu koloidne čestice zamu-
ćenja (Marchal i Jeandet, 2009; Mierczynska-Vasilev i Smith,
2015). Na ovaj način se značajno skraćuje postupak pripreme
mošta, uz još neke prednosti kao što je sprečavanje neželje-
nih oksidativnih procesa uzrokovanih oksidazama, neželjenih
mikrobioloških procesa (razmnožavanje mikroorganizama kao
što su kvasci roda Brettanomyces te bakterije rodova Lacto-
bacillus ili Pediococcus) i sprečavanje spontanog početka
fermentacije uzrokovanog prisutnim sojevima kvasaca roda
Saccharomyces. Promatrano s tehnološko-enološkog aspekta,
postupkom flotacije postiže se znatno veće iskorištenje mošta
uz značajno bolju kvalitetu bijelih vina, a pritom ne dolazi do
prekomjernog bistrenja mošta kao što to može biti slučaj kod
taloženja (Jackson, 2014).
Kada je proces bistrenja mošta preintenzivan, dolazi do
smanjenja brzine fermentacije i do smanjenja rasta biomase,
jer bistrenje mošta uzrokuje veliko smanjenje koncentracije
masnih kiselina C12 do C18 (40 – 100%) te sterola i makromo-
lekula (15 – 50%). Sve navedeno često je povezano s uspo-
ravanjem alkoholne fermentacije. Bistrenje također povećava
sintezu octene kiseline i srednjelančanih masnih kiselina za
koje je već navedeno kako inhibiraju aktivnost stanica kvasaca
(Alexandre i Charpentier, 1998).
Mošt kao kompleksna hranjiva podloga može sadržavati
dovoljnu količinu svih hranjivih tvari potrebnih za alkoholnu
fermentaciju. U slučajevima pomanjkanja nekog važnog sa-
stojka mošta, radi poboljšanja fermentacijske aktivnosti kvas-
ca, a također i radi postizanja željene arome vina ili rješavanja
određenih problema u raznim fazama proizvodnje i dorade vina
mogu se upotrijebiti komercijalno dostupni enološki pripravci
(Walker i Stewart, 2016) koji se međusobno razlikuju po sasta-
vu i namjeni. Za različite specifične uvjete fermentacije mošta
dostupni su enološki pripravci koji mogu sadržavati više kom-
binacija sastojaka. Enološki pripravci za poticanje alkohol-
ne fermentacije, koji se često popularno nazivaju “hrana” za
kvasce, mogu sadržavati derivate kvaščevih stanica (termički
inaktivirani kvasac, autolizat kvasca, stanične stijenke kvasca
 V. Petravić-Tominac et al: Croatian Journal of Food Technology, Biotechnology
ili kvaščev ekstrakt), amonijev fosfat i minerale (npr. magne-
zij i cink) (Pozo-Bayón i sur., 2009; Walker i Stewart, 2016).
Jedan od djelotvornih kemijskih spojeva koji se može nalaziti
među sastojcima navedenih pripravaka je i glutation, tripeptid
koji je po svojem sastavu zapravo γ-glutamil-cisteil-glicin, a
može značajno poboljšati i produžiti fermentacijsku aktivnost
kvasca (Novak i sur., 2013) te pored toga povoljno utjecati na
aromu vina (Dubourdieu i Lavigne-Cruege, 2003; Kritzinger i
sur., 2013) i zahvaljujući svom antioksidacijskom djelovanju
spriječiti posmeđivanje (Dubourdieu i Lavigne-Cruege, 2003;
Pozo-Bayón i sur., 2009; Kritzinger i sur., 2013). Prikladnim
odabirom enoloških pripravaka, kao i vremena i načina njiho-
ve primjene, moguće je spriječiti ili riješiti mnoge probleme
navedene u ovom radu te postići željene karakteristike proi-
zvedenog vina.
Pored ranije navedenih čimbenika, u praksi se susreću još
neki uzročnici problema tijekom fermentacije mošta, koji nisu
detaljnije diskutirani u ovom radu. Vezano uz to treba istaknu-
ti nepoštivanje karence ili propisane koncentracije apliciranih
zaštitnih sredstava. Ovaj problem uočen je prvenstveno veza-
no uz primjenu botricida. Također često nastaju fermentacijski
problemi ukoliko je u moštu prisutna povećana koncentracija
bakra, koji je porijeklom npr. iz modre galice, tradicionalno
korištene za pripravu tzv. bordoške juhe, ali i iz drugih pripra-
vaka bakra namijenjenih zaštiti vinove loze.
Zaključci
U vinarstvu postoji velik interes za rješavanje problema
usporavanja i zastoja fermentacije zbog mogućih ekonomskih
gubitaka. Predvidljivost fermentacije i kvaliteta vina prven-
stveno ovise o svojstvima korištenih vinskih kvasaca, čak i
ako veći broj čimbenika utječu na ponašanje kvasaca tijekom
fermentacije. Sposobnost kvasca da se prilagodi nutritivnom
deficitu i da se nosi s prisutnošću inhibicijskih tvari od vitalne
je važnosti za provedbu fermentacije. Fermentacijski problemi
obično nastaju zbog prisutnosti i utjecaja različitih čimbenika
stresa u okruženju kvasca i bakterija. Čimbenici koji dovode
do smanjenja pH-vrijednosti, inhibicije glavnih enzimskih ak-
tivnosti kvasca i promjene stanične membrane kvasca prou-
zročit će promjene kvaščevog metabolizma, što će posljedično
dovesti do smanjenja brzine rasta i preživljavanja stanica kvas-
ca, odnosno smanjenja brzine fermentacije. Nadalje, pojava
zastoja fermentacije ili usporene fermentacije može biti i re-
zultat interakcije navedenih čimbenika. Uzrok zastoja fermen-
tacije ili usporene fermentacije rijetko je rezultat samo jednog
čimbenika pa će različiti čimbenici imati sinergistički učinak.
Stoga, istodobni učinak dvaju ili više nepovoljnih parametara
može prouzročiti puno veće probleme nego kad bi jedan čim-
benik djelovao samostalno. Neki od navedenih čimbenika stre-
sa su neizbježni, dok su drugi posljedica neprikladnih odluka
pri vođenju fermentacije. Zato bolje razumijevanje fiziologije
mikroorganizama vina omogućava smanjenje navedenih pro-
blema na najmanju moguću mjeru.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 100-106 (2017) 105
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ORIGINAL SCIENTIFIC PAPER

Monitoring the influence of high power ultrasound

treatment and thermosonication on inactivation
of Brettanomyces bruxellensis in red wine
Leo Gracin1, Stela Križanović1, Anet Režek Jambrak1, Marina Tomašević1, Karla Kelšin1, Katarina Lukić1,
Karin Kovačević Ganić1*

1
University of Zagreb, Faculty of Food Technology and Biotechnology,
Pierottijeva 6, 10 000 Zagreb, Croatia

* Corresponding author: kkova@pbf.hr

Abstract

Brettanomyces bruxellensis is one of the most important spoilage microorganisms in winemaking. It is harmful for wine industry because
it produces volatile phenols, compounds primarily responsible for off-odours in wine. One of the possible solutions for preventing its growth is
using new non-thermal processing technologies. The aim of this study was to investigate the application of one non-thermal processing tech-
nology, high power ultrasound and its combination with heating (thermosonication) on the inactivation of B. bruxellensis in red wine in batch
systems. Various parameters, such as treatment duration (1, 2, 3, 6, 10 and 15 minutes), temperature (25, 35, 40 and 43 ºC) and probe diameter
(12.7 mm and 19.1 mm), were examined. The combination of high power ultrasound and heating (thermosonication) proved to be a better method
compared to solely using high power ultrasound. However, the production of volatile phenols by B. bruxellensis was also reduced after high
power ultrasound treatment. The optimal treatment of 3 min at 43 ºC with high power sonicator at ultrasound frequency of 20 kHz with 12.7 mm
diameter ultrasonic probe for complete inactivation of B. bruxellensis was determined.
Keywords: red wine, spoilage yeast, B. bruxellensis, high power ultrasound treatment, thermosonication

Sažetak
Brettanomyces bruxellensis jedan je od najvažnijih mikroorganizama koji uzrokuju kvarenje vina. Ovaj kvasac je štetan za industriju vina
jer ima sposobnost stvaranja hlapivih fenola, spojeva koji su odgovorni za neugodan miris vina. Za sprečavanje njegova rasta mogu se primi-
jeniti nove procesne tehnologije tzv. netermalne tehnologije. U ovom istraživanju ispitana je primjena ultrazvuka visoke snage kao i zajednička
primjena ultrazvuka visoke snage i zagrijavanja (termosonifikacija) za šaržnu inaktivaciju B. bruxellensis u crnom vinu. Tijekom istraživanja
ispitan je utjecaj različitih parametara kao što su trajanje tretiranja (1, 2, 3, 6, 10 i 15 minuta), temperatura (25, 35, 40 i 43 ºC) i promjer
sonde (12,7 i 19,1 mm). Utvrđeno je kako je zajednička primjena ultrazvuka visoke snage i zagrijavanja (termosonifikacija) bolja metoda za
inaktivaciju ovog kvasca u odnosu na samostalnu primjenu ultrazvuka visoke snage. Ipak primjenom ultrazvuka visoke snage postignuta je
smanjena proizvodnja hlapivih fenola pomoću kvasca B. bruxellensis. Potpuna inaktivacija kvasca B. bruxellensis postignuta je pri 43 ºC
primjenom ultrazvuka frekvencije 20 kHz i sonde promjera 12,7 mm kroz 3 minute.

Ključne riječi: crno vino, kvasac uzročnik kvarenja, B. bruxellensis, ultrazvuk visoke snage, termosonifikacija

Introduction sulphur dioxide). The growth of B. bruxellensis can be con-

Brettanomyces bruxellensis is a non-Saccharomyces ye-
ast that has been found in different stages of winemaking as
well as in bottled wine (Jolly et al., 2014). This yeast is ge-
nerally associated with spoilage, especially in red wines. It is
able to convert hydroxycinnamic acids into volatile phenols
(4-ethylphenol and 4-ethylguaiacol), providing off-odours
described as ‘horse sweat’, ‘animal leather’ and ‘medicinal‘
even at low concentrations at order of magnitude of 3 log CFU
mL-1 (Kheir et al., 2013). This effect causes a decrease in wine
quality, leading to economic losses. B. bruxellensis is a slow
growth yeast, but it can grow in adverse conditions (low pH,
in the presence of high levels of ethanol and in presence of
trolled by the use of chemical, physical and biological methods
(Mehlomakulu et al., 2015). Sulphur dioxide is the most used
chemical preservative in winemaking. It can be used to control
the growth of spoilage microorganisms such as B. bruxellensis,
but it can affect human health (e.g. allergic reaction, asthma,
diarrhea, headache or nausea) (Guerrero and Cantos-Villar,
2015). In recent years, there is a growing interest to replace
the use of sulphur dioxide by applying non-thermal process
technologies. Until now, several different non-thermal techno-
logies such as pulsed electric fields (PEF), low electric current
treatment (LEC), high hydrostatic pressure (HHP) and high
power ultrasound (HPU) have already been tested for inacti-
vation of B. bruxellensis in wine (Puértolas et al., 2009; Lu-

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 L.Gracin et al: Croatian Journal of Food Technology, Biotechnology
108 and Nutrition 12 (3-4), 107-112 (2017)
strato et al., 2015; Delsart et al., 2016; González-Arenzana et
al., 2016; van Wyk and Silva, 2017; Luo et al., 2012; Gracin
et al., 2016; Bermúdez-Aguirre and Barbosa-Cánovas, 2012).
Among them, HPU has physical (cavitation and micro-mecha-
nical shocks) and chemical (formation of free radicals) effects
on inactivation of microorganisms (Piyasena et al., 2003;
O’Brien, 2007; Chandrapala et al., 2012). HPU combined with
heat treatment (thermosonication), pressure treatment (mano-
sonication) or both (manothermosonication) has been shown to
increase the inactivation of microorganisms (Awad et al., 2012;
Abdullah and Chin, 2014; Huang et al., 2016). Additionally,
HPU treatment can be performed in batch and continuous flow
systems (Bermúdez-Aguirre and Barbosa-Cánovas, 2012; Mo-
hideen et al., 2015). The use of HPU can also reduce treatment
duration, which prevents the loss of organoleptic qualities of
wine such as flavour, colour or taste (Awad et al., 2012). HPU
can be applied in winery to minimize the influence of unde-
sirable microorganisms during processing of must, alcohol
fermentation, malolactic fermentation and sanitation of barrels
(Jiranek et al., 2008; Yap et al. 2008).
The aim of this work was to investigate the impact of high
power ultrasound (HPU) and the combination of HPU and he-
ating (thermosonication) on the inactivation of B. bruxellensis
in red wine during treatment in batch systems.
Materials and methods
Inoculum preparation
Brettanomyces bruxellensis CBS 2499 (from Westerdijk
Fungal Biodiversity Institute) was grown in YPD (10 gL-1 ye-
ast extract, 20 gL-1 peptone, 20 gL-1 glucose) liquid culture
medium at 28 ºC without shaking. The inoculum was prepared
as described by Delsart et al (2016). When the population re-
ached the stationary phase in this medium, another medium
was prepared and supplemented with ethanol (4 % v/v) to re-
ceive the previous culture (inoculated at 10 % v/v). Following
that, a new medium was prepared with ethanol (8 % v/v) and
inoculated via the second medium at 10 % v/v. The last medi-
um was prepared with ethanol (12 % v/v) and inoculated via
the third medium at 10 % v/v. Growth was followed by measu-
ring the absorbance at 600 nm. Also, samples were decimally
diluted and cellular culturability was determined by surface
plating 0.01 mL onto YPD medium (with 20 gL-1 agar), in
duplicate and incubating at 24 ºC for up to 7 days.
Cells of B. bruxellensis were harvested by centrifugation
at 4000 × g for 10 min and inoculated into red wine (Cabernet
Sauvignon from Croatia, 2016) at approximately 6 log CFU-
mL-1. The initial cell concentration in wine was then confir-
med by plate counts. Inoculation was performed 24 h before
HPU treatment. Bottles were incubated at 20 ºC ± 2 °C and
shaken prior to sampling.
High power ultrasound (HPU) treatments
Red wine samples (200 mL) inoculated with B. bruxellen-
sis, were placed in a round-bottom glass vessel (250 mL), which
served as a treatment chamber. An ultrasonic processor (S-
4000, Misonix Sonicators, Newtown, CT, USA), set at nomi-
nal power of 600 W and 20 kHz was used for HPU treatments.
Diameters of probes were 12.7 mm and 19.1 mm. Each probe
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
was immersed in red wine (2.5 cm) and placed at the center
of treatment chamber. Ultrasonication was carried out at the
amplitude of 100 % or 120 µm (for 12.7 mm probe) and 60 µm
(for 19.1 mm probe). The samples were isothermally treated
for 3, 6, 10 and 15 min on 25 °C. For the thermosonication
experiments, before the ultrasonic treatment, the samples were
heated in water bath (about 2 min) at temperatures of 35, 40
and 43 °C. The samples were then treated by ultrasound with
a 12.7 mm diameter probe for 1, 2 and 3 min. Isothermal con-
ditions during the ultrasound treatment were achieved by cold
water cooling of the treatment chamber. After HPU treatments
samples were analyzed, and placed in equal size sterile bottles
(100 mL) and stored for 90 days at 20 °C ± 2 °C. Samples were
analyzed right after the thermosonication treatment.
Wine parameters
The physico-chemical wine parameters (pH, total and
volatile acidity, reducing sugars and alcoholic strength) were
determined before inoculation with B. bruxellensis using Fou-
rier transform infrared spectroscopy (FTIR; Bacchus II, Micro-
dom) equipped with autosampler. Determination of sulphur
dioxide in wine was done by titration with iodine / iodine solu-
tion where the iodine was reduced and sulfur dioxide oxidized
by potentiometric determination of the titration point via the
LED indicator (LDS Sulfilyser, Laboratoires Dujardin-Salle-
ron, Noizay, France).
Analysis of B. bruxellensis cell number
Before and after the HPU treatment and thermosonication
treatment and during storage after 30, 60 and 90 days wine
samples were analysed for the colony forming unit (CFU) of B.
bruxellensis on commercial Brettanomyces agar plates (with
100 mgL-1 chloramphenicol, 10 mgL-1 cycloheximide and
100 mgL-1 coumaric acid; Conda, Spain) incubated for 7-10
days at 24 °C. Serial dilutions were carried out using sterile
saline solution. From each dilution, 0.01 mL was plated out in
duplicate. The number of colony forming units (CFU) can be
calculated according to the following formula:
GC/MS analysis of volatile compounds
Wine sample volatile compounds were analyzed by gas
chromatography coupled with mass spectrometry (GC/MS)
using an Agilent Gas Chromatography 6890 series equipped
with an Agilent 5973 Inert mass selective detector (Agi-
lent Technologies, Santa Clara, CA, USA). Prior to GC/MS
analysis, volatile compounds were extracted from wine by
headspace solid-phase microextraction (HS-SPME) using 100
µm PDMS fiber (Supelco, Bellefonte, USA). 10 mL of a wine
sample, containing internal standards (20 mgL-1 n-amyl alco-
hol and 0.5 mgL-1 p-cresol), were placed into a 20 mL head-
space vial containing NaCl p.a. (2 g) and capped with a crimp
cap and silicone-PTFE septum. The fiber was exposed to the
wine headspace for 30 minutes at 40 °C with constant stirring.
This was followed by thermal desorption for 5 minutes in the
injector (splitless mode) at 250 °C (Tomašević et al., 2016).
The target analytes were separated by gas chromatography
using a BP20 capillary column (SGE Analytical Science, Vic-
toria, Australia), dimensions 50 m x 220 μm with 0.25 μm film
thickness. The interface temperature of the detector was kept
at 250 °C and the ion source working in EI mode at 70 eV was
 L.Gracin et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 107-112 (2017) 109

held at 280 °C. Helium 5.0 was used as a vector gas (Messer
Croatia Plin d.o.o., Zagreb, Croatia). The initial temperature
was set to 40 °C and maintained for 5 minutes, then raised
to 200 °C for 3 °Cmin-1 and finally raised to 240 °C for 30
°Cmin-1 (Tomašević et al., 2016). Volatile compounds were
identified using the Enhanced Chemstation software (Agilent
Technologies, Santa Clara, CA, USA) and by comparing the
peak retention times against those of authentic standards and
matching the mass spectra against Nist05 mass library (Wiley
& Sons, Hoboken, NJ, USA). For quantification, calibration
curves for each compound were prepared and analyzed using
GC/MS and the same extraction and chromatographic methods
as for wine samples. Identified volatile compounds included
ethyl esters (ethyl hexanoate, ethyl octanoate and ethyl deca-
noate), acetate esters (i-amyl acetate) and volatile phenols (4
ethyl phenol and 4-ethyl guaiacol).

Data analysis
Significant differences among control and treated wine
samples for each of the constituents was determined by one-
way analysis of variance (ANOVA) using the Statistica V.10
software (StatSoft Inc., Tulsa, USA). Tukey’s honestly signifi-
cant difference (HSD) test (p<0.05) was used for comparison
when samples differed significantly after ANOVA was perfor-
med.
Figure 1. B. bruxellensis CBS 2499 population in red
wine before and after different HPU treatment duration with
12.7 mm diameter ultrasonic probe at 25 °C and during stor-
age (30, 60 and 90 days). All parameters are expressed as av-
erage value of two repetitions ± standard deviation (n=2).

Table 1. Physico-chemical parameters of red wine

Parameter Red wine
pH 3.46 ± 0.00
Total acidity (gL-1 tartaric acid) 5.85 ± 0.78
Volatile acidity (gL-1 acetic acid) 0.60 ± 0.01
Reducing sugars (gL-1) 3.90 ± 0.28
Alcoholic strength (% vol) 13.00 ± 0.14
Free SO2 (mgL-1) 11.5 ± 0.7
Total SO2 (mgL-1) 24.0 ± 1.4

Data are presented as average value of two repetitions ± stan-

dard deviation (n=2).

Results and discussion

To investigate the possibility of using HPU for batch inac-
tivation of B. bruxellensis in red wine, the experiments were
performed at 25 °C and heated to 35, 40 and 43 °C. During the
HPU treatment at 25 °C, two probes (12.7 and 19.1 mm diame-
ter) and four treatment durations (3, 6, 10 and 15 minutes) were
used (Figure 1 and Figure 2). A slightly better inactivation of
B. bruxellensis was achieved using a 12.7 mm diameter probe
in comparison to 19.1 mm diameter probe. After 15 minutes
treatment the maximum inactivation of 0.5 log CFUmL-1 was
achieved using the 12.7 mm diameter probe, while the maxi-
mum inactivation achieved using the 19.1 mm diameter probe
was 0.18 log CFUmL-1.
Figure 2. B. bruxellensis CBS 2499 population in red
wine before and after different HPU treatment duration with
19.1 mm diameter ultrasonic probe at 25 °C and during stor-
age (30, 60 and 90 days). All parameters are expressed as av-
erage value of two repetitions ± standard deviation (n=2).
Generally, for the HPU treatment ultrasonic intensity
depends on ultrasonic power and surface area of the probe
(Margulis and Margulis, 2003; Piyasena et al. 2003; Gao et
al., 2014; Kentish and Feng, 2014; Harvey et al., 2014; Režek
Jambrak et al., 2017;). Therefore, a different diameter probe is
used depending on the volume of the treated medium. For the
volume of 200 mL (used in our experiments), both 12.7 mm
and 19.1 mm probes could be used. Since slightly better inacti-
vation of B. bruxellensis was achieved using 12.7 mm diameter
probe, this probe was chosen for further research. Furthermore,

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 L.Gracin et al: Croatian Journal of Food Technology, Biotechnology
110 and Nutrition 12 (3-4), 107-112 (2017)

obtained data demonstrate that HPU treatment at 25 °C for 15
minutes is not effective enough to notably inactivate B. bruxel-
lensis population in red wine (Figure 1 and Figure 2). Contrary
to our research, Luo et al. (2012) reported significant inactiva-
tion in B. bruxellensis in wine (about 2.15 log CFUmL-1) after
20 min of HPU treatment at 23 - 25 °C. However, they used a
higher frequency (24 Hz) in their work.
During the 90 days monitoring period of B. bruxellensis
population, a constant trend of reduction in all samples treated
with both probes was observed (Figure 1 and Figure 2). How-
ever, the population in the untreated sample decreased for
about 1 log CFUmL-1 after 30 days, while no further inacti-
vation was observed during the following 60 days. After 90
days, the population in all treated samples decreased for about
3 log CFUmL-1, while the population in untreated samples
decreased for about 1 log CFUmL-1. The physico-chemical
parameters of wine used in this work (Table 1) are common
in winemaking and are not expected to have a significant ef-
fect on the survival of B. bruxellensis. The observed difference
in B. bruxellensis population between treated and untreated
samples was probably due to HPU treatment that may have
caused damage to cellular components and interfered with
cellular functions. This is supported by the previous research
where it was showed that ultrasonic treatment singly could not
break down a yeast cell, but could damage cell wall and cyto-
plasmic membrane (Chemat and Khan, 2011; Chandrapala et
al., 2012; Huang et al., 2014; Režek Jambrak et al., 2017; Wu
et al., 2015; Ferrario and Guerrero, 2017). However, further
study is required to confirm this hypothesis.
Also, the presence of volatile compounds (4-ethylphe-
nol, 4-ethyl guaiacol, ethyl hexanoate, ethyl octanoate, ethyl
decanoate, isoamyl acetate) in treated and untreated samples
after 90 days was determined (Table 2). The concentration of
4-ethylphenol in the untreated sample was 1742 µgL-1, while
in treated samples was determined in the range of 919 to 1595
µgL-1. The concentration of 4-ethyl guaiacol was 668 µgL-1
in the untreated sample and in the range of 186 to 398 µgL-1
in treated samples. The concentration of the analyzed esters
(ethyl decanoate, ethyl hexanoate, isoamyl acetate and ethyl
octanoate) were higher in the untreated sample (88 to 646 µgL-
1) compared to the treated samples (34 to 456 µgL-1). The
concentration of volatile phenols produced in wine depends
on many parameters such as hydroxycinnamic acid composi-
tion in wine, developed active Brettanomyces population and
ability of different B. bruxellensis strains to produce and ac-
cumulate volatile compounds (Kheir et al., 2013). In addition
to volatile phenols, B. bruxellenasis could produce acetic acid
and volatile esters (Steensels et al., 2015). In our research,
lower concentration of volatile phenols in treated wines com-
pared to untreated wine was the effect of reduced active Brett-
anomyces population as well as possible reduced ability of B.
bruxellensis to produced volatile compounds due to cell dam-
age (Table 2). Cells of B. bruxellensis with a damaged cell wall
or membrane are more sensitive to their environment, which
could affect enzyme activity (Chemat and Khan, 2011; Chan-
drapala et al., 2012; Huang et al., 2016). This effect observed
during our work is supported by observations conducted by
other authors (Lustrato et al., 2015), that reported lower pro-
duction of volatile phenols by non active B. bruxellensis popu-
lation treated with non-thermal technology, low electric cur-
rent (LEC). An interesting topic for further research would be
to more precisely determine how sonification affects the cell
wall or membrane, and consequently how it affects the volatile
phenols production.

Table 2. B. bruxellensis production of volatile compounds in red wine after 90 days under different experimental conditions

Concentration (µgL-1)
Treatment
4-ethyl phenol 4-ethyl guaiacol ethyl hexanoate ethyl octanoate ethyl decanoate isoamyl acetate
untreatment
(wine+cells) 1742±8c 668±1d 269±3c 646±40b 88±3b 543±57d

12.7 diameter probe

3 min 919±5a 183±11a 174±22a,b 280±3a 34±3a 456±50c,d
6 min 1474±41b,c 398±26b,c 165±28a,b 357±48a 42±2a 410±29b,c
10 min 1066±105a,b 313±16b 143±11a 451±73a 47±9a 124±4a
15 min 1191±52a,b,c 321±38b 147±8a,b 344±24a 45±6a 303±10b

19.1 diameter probe

3 min 1595±142c 511±49c 206±24b 396±84a 52±20a 337±27b,c
6 min 1137±34a,b,c 313±24b 159±12a,b 306±31a 40±6a 345±17b,c
10 min 1236±47a,b,c 339±18b 185±3a,b 366±23a 56±1a,b 380±4b,c
15 min 927±74a,b 187±7a 169±1a,b 367±35a 58±7a,b 384±28b,c

All parameters are expressed as average value of two repetitions ± standard deviation (n=2). ANOVA to compare data; different
letters indicate statistical differences between wines of all treatments at the same time (Turkey’s test,<0.05).

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 L.Gracin et al: Croatian Journal of Food Technology, Biotechnology
Figure 3. Influence of thermosonication parameters (tre-
atment temperature, treatment duration and 12.7
mm diameter ultrasonic probe) on B. bruxellensis
CBS 2499 population in red wine immediately after
thermosonication treatment. All parameters are
expressed as average value of two repetitions ±
standard deviation (n=2).
In order to increase inactivation of B. bruxellensis yeast,
HPU treatments with a 12.7 mm diameter probe were per-
formed at 35, 40 and 43 °C for 1, 2 and 3 minutes (Figure
3). The number of B. bruxellensis cells was determined imme-
diately after thermosonication treatment. The B. bruxellensis
population changed only slightly over all treatment durations
at 35 °C. At higher temperatures (40 and 43 °C), an increased
inactivation of B. bruxellensis population was observed. Af-
ter 3 -minute treatment, the population decreased for 1 log
CFUmL-1 at 40 °C and for 6 log CFUmL-1 at 43 °C. Since
the initial growth was 6 log CFUmL-1, a complete inactiva-
tion of B. bruxellensis was achieved at 43 °C after 3 minutes.
An increase in B. bruxellensis inactivation compared to solely
HPU, could be explained by the fact that HPU causes the cell
wall weakening which could facilitate thermal coagulation of
the intracellular proteins (Yap et al. 2008; Chandrapala et al.,
2012; Cruz-Cansino et al., 2016; Wu et al., 2015). Accord-
ing to previous research data for S. cerevisiae (Cacciola et al.,
2013), maximal inactivation of this yeast in wine occurred af-
ter 3 minutes and at similar temperature (47.6 °C) with simi-
lar diameter ultrasonic probe (13 mm). The ultrasound affects
combined physical and chemical mechanisms occurring during
cavitation and increases yeast sensitivity to heat (Piyasena et
al., 2003; Wu et al., 2015).
The influence of thermosonication treatment on inactiva-
tion of bacteria, yeasts and molds depends on many conditions
such as ultrasonic power and amplitude, temperature, treat-
ment duration as well as composition of the treated medium
(Patil et al., 2009;Wong et al., 2010; Bermúdez-Aguirre and
Barbosa-Cánovas, 2012; Gao et al., 2014; Ortuño et al., 2014;
Mohideen et al., 2015). Combination of HPU and heating
(thermosonication) can have a greater effect on B. bruxellensis
inactivation because it damages cells at the subcellular level
(Abdullah and Chin, 2014; Huang et al., 2016; Cruz-Cansino
et al., 2016; Režek Jambrak et al., 2017). Proportion between
sonification and heat influence on Brettanomyces cells is hard
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 107-112 (2017) 111
to estimate. Our experiments show that sonification without
heating is not very effective for inactivation of Brettanomyces
cells in wine environment. However, it is possible that sonifi-
cation accelerates yeast inactivation by heating.
Conclusions
In the present work the use of high power ultrasound
treatment (HPU) and combination of HPU and heating (ther-
mosonication) to control the spoilage yeast B. bruxellensis in
red wine during the treatment in batch systems has been tested.
The results clearly indicate improved inactivation by com-
bined treatment (thermosonication) compared to solely using
HPU. Parameters that appear to have the greatest effect are
temperature of the treatment and treatment duration. Complete
inactivation of B. bruxellensis was achieved by treatment with
12.7 mm diameter ultrasonic probe at temperature of 43 °C
during 3 min. After HPU treatment, B. bruxellensis population
has also shown lower ability to produce volatile phenols.
Acknowledgments
This paper has been funded by Croatian Science Founda-
tion project IP-09-2014-3796.
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ORIGINAL SCIENTIFIC PAPER

Characteristics and selection of cultures of

photosynthetic purple non-sulphur bacteria as a
potential 5-aminolevulinic acid producers
Mario Novak1*, Mladen Pavlečić1, Baghish Harutyunyan2, Vigen Goginyan2, Predrag Horvat1, Božidar Šantek1

1
University of Zagreb, Faculty of Food Technology and Biotechnology, Department of Biochemical Engineering, Labora-
tory of Biochemical Engineering, Industrial Microbiology, Malting and Brewing Technology, Pierottijeva 6/IV, HR-10000
Zagreb, Croatia
Center of Excellence Bio Pro Cro - Marine Bioprospecting
2
The Scientific and Production Center “Armbiotechnology” of the National Academy of Sciences of the Republic of Arme-
nia, Laboratory of Energy Alternative Sources, 14 Gyurjyan Str., Yerevan 0056, Republic of Armenia

*Corresponding author: mnovak@pbf.hr

Abstract

Wild strains of purple non-sulphur bacteria: Rhodospirillum rubrum B-6505, Rhodopseudomonas palustris B-6506, Rhodobacter capsu-
latus B-6508 and Rhodobacter spheroides B-6509 were studied as 5-ALA (5 aminolevulinic acid) producers. Selected strains were subjected
to mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine to obtain a strain with high 5-ALA producing capacity. After mutagenesis 19 stable
mutant strains were selected from Rhodobacter capsulatus B-6508 and Rhodobacter sphaeroides B-6509. On the basis of obtained results, mu-
tant strain of Rhodobacter capsulatus B-6508 has shown the highest potential for 5-ALA production. The most favorable conditions for growth
and 5-ALA production by mutant strain R. capsulatus B-6508 were observed in media composed of glutamate and malate, light at 2000 Lux,
microaerophilic conditions and temperature of 28 °C. In these conditions, the highest 5-ALA concentration (179 mg/L) was detected together
with the highest bacterial physiological activity. The prolongation of mutant strain R. capsulatus B-6508 cultivation time after glycine, succinate
and levulinic acid addition is related to the reduction of 5-ALA concentrations (e.g. 124.5 mg/L after 48 h and 89.5 mg/L after 72 hours). In
the light/aerobic conditions R. capsulatus B-6508 produced only 58.1 mg/L of 5 ALA. Furthermore, in dark conditions even lower biomass and
5-ALA concentrations were observed during R. capsulatus B-6508 cultivation.

Keywords: purple non-sulphur bacteria, Rhodobacter capsulatus, 5-aminolevulinic acid, optimization of cultivation conditions.

Sažetak

Divlji sojevi ljubičastih ne-sumpornih bakterija Rhodospirillum rubrum B-6505, Rhodopseudomonas palustris B-6506, Rhodobacter cap-
sulatus B-6508 i Rhodobacter spheroides B-6509 proučavani su kao potencijalni proizvođači 5-ALA (5-aminolevulinska kiselina). Odabrani
sojevi podvrgnuti su postupku mutageneze s N-metil-N-nitro-N-nitrosoguanidinom s ciljem dobivanja sojeva s visokom sposobnošću sinteze
5-ALA. Nakon procesa mutanogeneze selekcionirano je 19 stabilnih sojeva iz vrsta Rhodobacter capsulatus B-6508 i Rhodobacter sphaeroides
B-6509. Na temelju rezultata istraživanja vidljivo je da genetički modificirani soj Rhodobacter capsulatus B-6508 ima najveći potencijal za
proizvodnju 5-ALA. Najpovoljniji uvjeti za rast R. capsulatus B-6508 i proizvodnju 5-ALA zabilježeni su na hranjivoj podlozi koja je sadržavala
malat i glutamat, imala intenzitet svjetla od 2000 Lux, mikroaerofilne uvjete i temperaturu od 28°C. U tim uvjetima registrirana je najveća 5-ALA
koncentracija (179 mg/L) zajedno s najvećom fiziološkom aktivnošću bakterijske biomase. Produljenje vremena uzgoja R. capsulatus B-6508
nakon dodataka glicina, sukcinata i levulinske kiseline povezano je sa smanjenjem 5-ALA proizvodnje (npr. 124.5 mg/L nakon 48 h odnosno 89.5
mg/L nakon 72 h). Uzgoj R. capsulatus B-6508 u aerobnim uvjetima uz prisustvo svjetla kroz 48 sati dao je samo 58.1 mg/L 5-ALA. Nadalje, kod
uzgoja R. capsulatus B-6508 bez prisustva svjetla zabilježene su još manje koncentracije bakterijske biomase i 5-ALA.

Ključne riječi: purpurne nesumporne bakterije, Rhodobacter capsulatus, 5- aminolevulinska kiselina, optimizacija uvjeta uzgoja.

Introduction purple photosynthetic bacteria. The purple bacteria are a small

The photosynthetic bacteria can be found in fresh, salt,
acidic and basic waters, and also in various wastewaters. These
bacteria play important roles in CO2 assimilation and nitrogen
fixation (Lascelles et al, 1978). One of the major groups are
group of Gram-negative eubacteria, consisting of only about
30 species. They are unicellular, reproduce by binary fission,
and in most cases are motile by flagella. All purple bacteria
are capable of growing anaerobically in the light with CO2 as
the carbon source and reduced inorganic compounds as the

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 M. Novak et al: Croatian Journal of Food Technology, Biotechnology
114 and Nutrition 12 (3-4), 113-119 (2017)
electron donor (Madigan and Jung 2009). The only pathway
for carbon fixation by purple bacteria is the Calvin cycle. There
are two divisions of photosynthetic purple bacteria, the purple
sulphur bacteria and the purple non-sulphur bacteria.
The purple sulphur bacteria use the inorganic sulphur
compounds such as hydrogen sulphide as the electron donor.
These organisms are predominantly aquatic, found in sulphate-
rich environments, and are mostly strictly anaerobes that requ-
ire vitamin B12 as the only growth factor.
The purple non-sulphur bacteria (PNSB) use various
organic compounds as electron donors including fatty acids,
other organic acids such as succinate or malate, primary and
secondary alcohols, carbohydrates and even aromatic com-
pounds. Under phototrophic (anoxic/light) conditions, typical
purple non-sulphur bacteria can grow photoautotrophic with
H2 or low levels of sulphide as electron donors; a few speci-
es can use S2O3 or Fe2+ as photosynthetic electron donors
(Ehrenreich and Widdel, 1994; Brune, 1995). However, most
purple non-sulphur bacteria grow best as photoheterotrophs in
media containing a readily useable organic compound, such as
malate or pyruvate, and ammonia as nitrogen source (Sojka,
1978). Most purple non-sulphur bacteria have a predominantly
photoheterotrophic mode of metabolism.
The PNSB have been classified as anoxygenic photo-
trophic bacteria, and are divided into 6 genera. Bacteria from
genus Rhodospirillum have spiralshaped cells and 0.5 to 1.5 µm
width. This genus uses polar flagella for motility. The bacteria
have intracytoplasmic photosynthetic membranes arranged in
vesicles, lamellae, or stacks, but not as finger-like intrusions
of cytoplasmic membrane. Rsp. rubrum, Rsp. fulvum Rsp. oxi-
gens, Rsp. photometricum and Rsp. meliscchianum belong to
this genus (Madigan and Jung, 2009). The Rhodopseudomo-
nas cells are rod-shaped and display polar growth, dividing
asymmetrically. They use flagella for motility. These bacteria
contain intracytoplasmic photosynthetic membranes. Rps. pa-
lustris, Rps. acidophila, Rps. rutica, Rps. viridis belong to this
genus. The bacteria of the Rhodomicrobium genus are ovoid.
Their peritrichously arranged flagella are used for motility. Re-
production of these organisms occurs by budding. Members of
this genus have staked intracytoplasmic membranes. Rhodo-
microbium vannielii is a member of the genus (Madigan and
Jung 2009). The cells in the Rhodopila genus are spherical to
ovoid. They use flagella for motility. The photosynthetic mem-
branes of these bacteria are vesicular. The Rhodopila can grow
at low pH and biotin and p-aminobenzoic acid are required as
growth factor. These cells are sensitive to oxygen. Rhodopila
globiformis is a member of this genus. Rhodocyclus bacteria
are slender, curved cells and are 0.3-0.7 µm in diameter. The
bacteria in this genus are non-motile or motile by polar flage-
lla. They have intracytoplasmic membranes which form small
finger-like intrusions of the cytoplasmic membrane (Madigan
and Jung 2009). The genus Rhodobacter was formerly inclu-
ded in the genus Rhodopseudomonas, but is now differentia-
ted by their invaginating intracytoplasmic membranes that can
appear vesicular in thin sections. The cells are ovoid to rod-
shaped and the bacteria multiply by binary fission. They can be
motile or nonmotile. All species require thiamine and most of
them require biotin. Additional vitamin requirements are varia-
ble among the species. The photosynthetic pigments in purple
bacteria are “Bchl a” (bacterial chlorophyll a) and carotenoides
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
of the spheroidene group. Rba. sphaeroides, Rba. sulfidophi-
lus, Rba. capsulatus, Rba. blasticus, and Rba. adriaticus are
species of this genus (Madigan and Jung, 2009).
5-aminolevulinic acid (5-ALA) is an intermediate in
synthesis of different tetrapyrrole molecules in all living orga-
nisms, i.e. chlorophyll, hem or vitamin B12 (Kang et al, 2012).
There are two different pathways in which 5-ALA can be
produced: C4 pathway (Shemin pathway) which is present in
purple bacteria, yeasts and mammalian cells and C5 pathway
which is present in many plants and some microorganisms
(Woodard and Dailey, 1995). Today, 5-ALA is mostly produ-
ced using microbial fermentation, namely by photosynthetic
bacteria because chemical synthesis of 5-ALA has lower yields
and is more complicated in comparison to microbial producti-
on (Liu et al, 2014). Production of 5-ALA has been reported
using both wild strains of bacteria and their mutants. Appli-
cation of mutant strains is far more suited for 5-ALA produc-
tion. So far many different strains of photosynthetic bacteria
together with their mutants have been tested for their 5-ALA
production capacities (Sasaki et al, 1991; Xiu-yan et al, 2005;
Liu et al, 2015; Meng et al, 2016). Both chemically defined and
complex media can be used (Heiko et al, 1993). 5-ALA can
be used as an effective herbicide, or as a plant stress tolerance
enhancer (Nunkaew et al, 2014). It is not harmful to crops,
animals or humans and it is biodegradable which makes it inte-
resting from ecological point of view. Much attention was also
dedicated to its great potential in the field of medicine, namely
tumor-localizing and photodynamic therapy (Seiji et al, 1999).
In this research, strains of purple non-sulphur bacteria
belonging to genera Rhodobacter, Rhodopseudomonas and
Rhodospirillum were isolated and genetically modified by
chemical mutagenesis. Obtained mutant strains were used in
further research which the main goal was to define the optimal
bioprocess conditions for purple non-sulphur bacteria growth
and 5-ALA production.
Materials and methods
Microorganisms
In this study, Rhodospirillum rubrum B-6505, Rhodopseu-
domonas palustris B-6506, Rhodobacter capsulatus B-6508
and Rhodobacter spheriodes B-6509 from the Culture Collec-
tion of the Laboratory of Energy Alternative Sources, Scientif-
ic and Production Center “Armbiotechnology” of the National
Academy of Sciences of the Republic of Armenia were used as
wild strains as well as mutants. Wild strains of microorganisms
were isolated from local mineral water sources around the city
of Yerevan (Republic of Armenia).
Cultivation media
In this research, following media were used for cultiva-
tion of purple non-sulphur bacteria:
a) Ormerod agar medium (Ormerod et al. 1961):
2.0 g/L Na- malate, 0.1 g/L yeast extract, 0.2 g/L MgSO4 x
7H2O, 0.08 g/L CaCl2 x H2O, 0.01 g/L FeSO4 x 7H2O, 0.9
g/L K2HPO4, 0.6 g/L KH2PO4, 1.25 g/L (NH4)2SO4, 0.02 g/L
EDTA, 0.028 g/L H3BO3, 0.021 g/L MnSO4 x 4H2O, 0.075
g/L Na2MoO4 x 2H2O, 0.0024 g/L ZnSO4 x 2H2O, 0.01 g/L
Cu(NO3)2 x 3H2O 20g/L agar pH = 6.8-7.3.
 M. Novak et al: Croatian Journal of Food Technology, Biotechnology
b) GA (glutamate / acetate) liquid medium (Suwansaard
2010):
1.5 g/L yeast extract, 1.64 g/L C2H3NaO2, 2 g/L C5H8NO4Na,
0.8 g/L (NH4)2SO4 x 7H2O, 0.5 g/L KH2PO4, 0.5 g/L K2HPO4,
0.2 g/L MgSO4 x 7H2O, 0.053 g/L CaCl2 x 2H2O, 1.2 x 10-3
g/L MnSO4 x 7H2O, 1.0 x 10-3 g/L thiamin-HCl, 1.0 x 10-3 g/L
nicotinic acid, 1.0 x 10-5 g/L biotin, pH = 6.8-7.0.
c) GG (glutamate / glucose) liquid medium (Nishikawa et al.
1999):
1.5 g/L yeast extract, 3.6 g/L C6H12O6, 2 g/L C5H8NO4Na, 0.8
g/L (NH4)2SO4 x 7H2O, 0.5 g/L KH2PO4. 0.5 g/L K2HPO4,
0.2 g/L MgSO4 x 7H2O, 0.053 g/L CaCl2 x 2H2O, 1.2 x 10-3
g/L MnSO4 x 7H2O, 1.0 x 10-3 g/L thiamin-HCl, 1.0 x 10-3 g/L
nicotinic acid, 1.0 x 10-5 g/L biotin, pH = 6.8-7.0.
d) GM (glutamate / malate) liquid medium (Nishikawa et al.
1999):
1.5g/L yeast extract, 2.7 g/L C4H4Na2O5, 2 g/L C5H8NO4Na,
0.8 g/L (NH4)2SO4 x 7H2O, 0.5 g/L KH2PO4. 0.5 g/L K2HPO4,
0.2 g/L MgSO4 x 7H2O, 0.053 g/L CaCl2 x 2H2O, 1.2 x 10-3
g/L MnSO4 x 7H2O, 1.0 x 10-3 g/L thiamin-HCl, 1.0 x 10-3 g/L
nicotinic acid, 1.0 x 10-5 g/L biotin, pH = 6.8-7.0.
Selection of media for purple non-
sulphur bacteria growth
From Petrie dishes, previously grown bacterial biomass of
Rhodospirillum rubrum B-6505, Rhodopseudomonas palustris
B-6506, Rhodobacter capsulatus B-6508 and Rhodobacter
spheriodes B-6509 on Ormerod agar medium, was taken with
microbiological inoculation loop into test tubes with 5 mL of
sterile water. This prepared inoculum was homogenized and 2
mL were taken for inoculation of 200 mL flasks containing 25
mL of liquid media (GM, GG and GM) with different carbon
sources (acetate, glutamate and malate). Total of 12 flasks were
used and they were kept on a rotary shaker in light conditions
(1500-1800 lux) at 28°C. After 5 days of cultivation, samples
were taken and analyzed. Based on optical density (520 nm)
and microscopical evaluation of bacterial activity, appropriate
cultivation media was chosen for further research.
Chemical mutagenesis
Selected strains of purple non-sulphur bacteria Rhodopse-
udomonas palustris, Rhodobacter capsulatus and Rhodbacter
spheriodes were genetically modified by nitrosoguanidine (N-
methyl-N-nitro-N-nitrosoguanidine). The process of mutage-
nesis was performed according to the Nishikawa et al (1999).
Obtained genetically modified strains were cultivated in GM
medium (flasks with 20 mL of medium) in light and dark con-
ditions. After 2 days of cultivation, 1 mL of bacterial cultivati-
on medium was taken from light and dark cultivation and used
for inoculation of Petri dishes containing GM medium with
added 20 g/L of agar. Inoculum contained approximately 1 g/L
dry weight of biomass. Inoculated Petri dishes were kept in
both dark and light (1800-2000 lux) conditions at 28˚C. After
2-3 days of cultivation on the GM-agar, mutant strains that had
shown the highest visible amount of grown biomass were se-
lected and used for further research.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 113-119 (2017) 115
Comparison of mutants and wild strains of non-sul-
phur purple bacteria for 5-ALA production
Selected mutant strains of non-sulphur purple bacteria,
cultivated on Petri dishes under different cultivation (light and
dark) conditions were inoculated on microtiter plates. Each
microtiter plate well contained 200 µL of GM medium and
it was inoculated with single-cell colony isolated from Petri
dishes. The mutant strains were evaluated for 5-ALA produc-
tion using the Ehrlich reaction in a 96-well microtiter plate.
Plates were incubated at 30˚C until visually detectable biomass
quantity was observed. Selection of the most promising 5-ALA
producing mutants was carried out by the addition of the mix-
ture of succinate (30mM), glycine (30mM) and levulinic acid
(15mM) into each microtiter plate well. Succinate and glycine
are precursors for 5-ALA production via 5-ALA synthase and
levulinic acid is an inhibitor of 5-ALA dehydratase (Suwansaa-
rd 2010). After 24 hours of cultivation, each microtiter plate
well was checked for accumulated 5-ALA according to the
Nishikawa et al. (1999). An indicator of 5-ALA concentration
in media is the intensity of the colorimetric reaction. Selected
mutants that have shown the highest 5-ALA production were
transferred into new test tubes containing fresh GM media for
new cultivation in order to obtain bacterial biomass for further
research.
Optimization of cultivation conditions for 5-ALA pro-
duction
In order to define optimal conditions for 5-ALA produc-
tion, cultivation of non-sulphur purple bacterial mutant strain
Rhodobacter capsulatus B-6508 was carried out in different
conditions (medium composition, light/dark regime, aerobic/
microaerophilic conditions). Cultivation of bacterial biomass
lasted 24 to 120 h depending on the experimental setup. Inocu-
lum for cultivation was grown in light conditions under 2000
Lux (28 °C) in Erlenmeyer flasks (without mixing) for 48 hours.
Erlenmeyer flasks with 50 mL of GM media were inoculated
with 10 % vol/vol of inoculum. The impact of the dark and light
regime, as well as aerobic and microaerophilic conditions, on the
bacterial growth and 5-ALA production was also studied. The
light regime was established by cultivation in luminostat with a
constant light source intensity of 2000 Lux and cultivation tem-
perature (28 oC). The dark regime was achieved by cultivation
in flasks that were enveloped in dark paper and aluminum foil
in the thermostat, without light. Aerobic cultivation was carried
out by shaker mixing intensity of 150 min-1 for surface aeration
and the free flow of air through the sterile porous filter. Bacterial
cultivation in microaerophilic conditions was carried out by low
shaker mixing intensity (<50 min-1) to prevent surface aeration
as well as without any free air flow in the flask. In these condi-
tions, oxygen present in the liquid free volume of flask can be
used for bacterial physiological activities. After defined biomass
cultivation period (24, 48, 72 h) a mixture of glycine, succinate
and levulinic acid (GSLA) was added to stimulate the 5-ALA
production. The quantity of added GSLA mixture was defined
to obtain the concentration in cultivation medium of 30 mmol/L
of glycine, 30 mmol/L of succinate and 15 mmol/L of levulinic
acid. After addition of GSLA, cultivation was carried out for an-
other 24 to 72 h, depending on the experimental setup. Samples
from each flask (2 mL) were taken for analytical purposes. After
centrifugation at 8000 min-1 for 20 min supernatant was used
 M. Novak et al: Croatian Journal of Food Technology, Biotechnology
116 and Nutrition 12 (3-4), 113-119 (2017)
for 5-ALA determination by using Suwansaard (2010) protocol.
Separated biomass was dried at 105°C (24 hours) for determina-
tion of dry biomass concentration. The growth rate of studied
bacterial strains was determined by the standard kinetic proce-
dure (Novak 2015).
Spectrophotometric determination of
5-ALA (modified Erlich reaction)
Samples taken from cultivation broth were centrifuged
and supernatant was used for further analysis. Sample of 0.2
mL was mixed together with 0.65 mL of acetaldehyde-acetone
buffer (acetate buffer with 1% of acetylacetone). Sample was
well mixed and then put at 100° C for 20 minutes. After de-
fined time (20 min) sample was cooled at 0 °C and then 0.65
mL Erlich reagents (acetic acid with 2% w/v polimethylami-
nobenz- aldehyde and perchloric acid; Sato et al. 1981) was
added. After 20 minutes, sample absorbance was measured on
a spectrophotometer at 553 nm and 5-ALA concentration was
determined from the calibration curve.
Table 1. Comparison of different chemically defined media for phototrophic cultivation of purple non-sulphur bacteria
Strains
GM (glutamate-malate)
Rhodospirillum rubrum B-6505 ++
Rhodopseudomonas palustris ++
B-6506
Rhodobacter capsulatus B-6508 ++
Rhodobacter spheroides B-6509 ++
Legend: «-» - no growth, «+» - negligible growth, «++» - good growth, «+++» - excellent growth.
On the basis of these results it is obvious that the GG
(glutamate-glucose) media is only suitable for cultivation of
Rhodobacter capsulatus B-6508 and Rhodobacter spheroides
B-6509. On the other hand, GM (glutamate-malate) media is
suitable for cultivation of all selected strains. Bacterial growth
on the GA (glutamate-acetate) medium was on the lowest level
compared to other two media. Therefore, for further research
GM media was selected. Willison (1988) reported that Rhodo-
bacter capsulatus B-6508 prefers organic acids as malate or
lactate as a carbon source, whereas slower growth was obser-
ved on media with sugars as a carbon source.
Mutagenesis and isolation of 5-ALA producing strains
As wild strains of purple non-sulphur bacteria have a very
low production rate of 5-ALA (Tangprasittipap and Prasertsan
2002), for example only11 mg/L in our research, strains of Rho-
dopseudomonas palustris, Rhodobacter capsulatus and Rhodo-
bacter spheroides were subjected to mutagens with N-methyl-
N-nitro-N-nitrosoguanidine in order to obtain mutants that have
a higher capacity for 5-ALA production. Total of 19 stable mu-
tant strains were obtained from Rhodobacter capsulatus B-6508
(strain marks: D-7, D-8, E-1, E-5, E-6, E-10, F-5, F-6, G-3,
G-4) and Rhodobacter sphaeroides B-6509 (strain marks: A-12,
B-10, C-8, D-7, E-10, F-7, F-10, G-9, G-10). Mutant strains of
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
Results and discussion
Selection of media for cultivation of
purple non-sulphur bacteria
Wild strains of purple non-sulphur bacteria: Rhodospirillum
rubrum B-6505, Rhodopseudomonas palustris B-6506, Rhodo-
bacter capsulatus B-6508 and Rhodobacter spheroides B-6509
were inoculated in different cultivation media in order to defi-
ne the most adequate carbon source for biomass growth. Three
different media were used, GA (glutamate-acetate), GG (glutama-
te-glucose) and GM media (glutamate-malate), where glutamate
serves as a nitrogen source. During purple non-sulphur bacteria
cultivations optical density of medium was monitored in order to
evaluate the intensity of bacterial growth. Furthermore, the bacte-
rial morphological stage was also examined under a microscope.
Results of these experiments are presented in Table 1.
Nutrient media / growth characteristics
GG (glutamate-glucose) GA (glutamate-acetate)
- ++
- +
+++ +
+++ +
Rhodopseudomonas palustris (B-6506) have shown that they
can grow under dark conditions, but they were not able to pro-
duce 5-ALA. Mutant strains of Rba. capsulatus B-6508 and Rps.
sphaeroides B-6509 did not grow in dark conditions. However,
in light conditions (1800 - 2000 Lux) only the growth of se-
lected mutants of Rba. capsulatus B-6508 and Rps. sphaeroides
B-6509 was observed. The photosynthetic bacterium accumu-
lates 5-ALA, which is a precursor in tetrapyrrole biosynthesis,
under light illumination and upon addition of levulinic acid as
an inhibitor of 5-ALA dehydratase (Suwansaard M., 2010). Ba-
sed on the colorimetric determination of 5-ALA ten mutants of
Rhodobacter capsulatus B-6508 have shown the approximately
similar intensity of color reaction (Fig. 1b) that was considerably
higher compared to the mutants of Rhodopseudomonas palustris
B-6506 (Fig, 1a). Therefore, for further research, mutant strain
Rhodobacter capsulatus B-6508 E-10 was selected due to its
higher potential for 5-ALA production.
 M. Novak et al: Croatian Journal of Food Technology, Biotechnology
Figure 1. Microtiter plate containing mutants of strain Rho-
dopseudomonas palustris B-6506 (a) and strain
Rhodobacter capsulatus B-6508 (b) (quantitative
reaction with Ehrlich reagent: purple colour indi-
cates positive reaction)
Optimization of cultivation conditions
for 5-ALA production
At the beginning of this research, growth in the light re-
gime under aerobic and microaerophilic conditions was studi-
ed. As it can be seen in Figure 2, the maximum biomass con-
centration in microaerophilic cultivation was 2.6 g/L and in
aerobic 2.5 g/L. The initial concentration of biomass in both
experiments was 0.7 g/L. After 48 h of cultivation, biomass
concentration in microaerophilic conditions was 2.5 g/L and
in aerobic conditions 2.2 g/L. At this time point, the mixture
of glycine, succinate and levulinic acid (GSLA) was added.
5-ALA concentration was determined after further 24 h of cul-
tivation (72 h of total cultivation time). In aerobic conditions
5-ALA concentration reached the value of 58.1 mg/L and in
microaerophilic conditions 86.49 mg/L, respectively. Based
on the obtained results it is obvious that light combines with
microaerophilic conditions are favorable for 5-ALA synthesis.
Therefore, the further optimization of mutant strain Rhodobac-
ter capsulatus B-6508 E-10 cultivation was performed in these
conditions. The activity of 5-ALA synthetase is considerably
depending on the oxygen concentration and its higher concen-
trations can cause the loss of pigmentation due to the impact
on the 5-ALA synthetase activator (glycine and succinate).
The shift of aerobic cultivation conditions into microaerophilic
conditions is related to the slight increase of 5-ALA production
(Tangprasittipap and Prasertsan 2002).
Figure 2. Changes of mutant strain Rhodobacter capsulatus
B-6508 E 10 biomass concentration during cultiva-
tion in microaerophilic and aerobic conditions at
27-29 °C and light intensity of 2000 Lux.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 113-119 (2017) 117
On the basis of results presented in Figure 2, the growth
rate of mutant strain of Rhodobacter capsulatus B-6508 E-10
was determined by the standard kinetic procedure. In microae-
rophilic conditions, Rhodobacter capsulatus B-6508 E-10
reached the growth rate of 0.05 h-1 and in aerobic conditions
0.035 h-1, respectively. The maximum biomass concentration
was observed in the time period of 22 - 28 h what is impor-
tant for the addition of glycine, succinate and levulinic acid
(GSLA) during 5-ALA production. It is known that the addi-
tion of GSLA has to be done in the high exponential bacterial
growth phase in order to obtain the highest 5-ALA yields (Liu
et al. 2014; Liu et al. 2015). It this research, selected mutant
strain of Rhodobacter capsulatus B-6508 E-10 shows also the
ability to produce relatively small amounts of 5-ALA (8 mg/L)
even without GSLA addition. The maximum biomass concen-
tration during Rhodobacter capsulatus B-6508 E-10 cultivation
in microaerophilic conditions was observed after approxima-
tely 24 h and it did not decrease significantly for another 24
hours. This observation confirms that the addition of GSLA has
to be performed after 24 hours or in a later cultivation stage.
In another experimental setup, prolonged cultivation af-
ter GSLA addition of Rhodobacter capsulatus B-6508 E-10 in
microaerophilic light conditions was tested. It was determined
that bacterial biomass concentrations were at approximately
similar levels (2.3 g/L after 24 hours, 2.5 g/L after 48 hours
and 2.25 g/L after 72 hours). However, it was observed that
5-ALA concentration was reduced with prolongation of cul-
tivation time due to the negative impact of light, biomass and
medium (e.g. pH alteration) on the 5-ALA stability. In these
experiments, the most physiologically active biomass and the
highest 5-ALA concentration (179 mg/L) were observed after
24 hours of cultivation after addition of GSLA. Prolongation
of cultivation time after GSLA addition on the 48 and 72 hours
was related to the reduction of 5-ALA concentration on the
124.5 mg/L and 89.5 mg/L, respectively.As a versatile group
of microorganisms purple non-sulphur bacteria can grow un-
der aerobic and anaerobic conditions as well as in dark and
light conditions (Suwansaard 2010; Nishikawa et. al 1999).
Selected strain Rhodobacter capsulatus B-6508 E-10 was
cultivated under light conditions and it was proven that light
conditions are favorable for 5-ALA production. During purple
non-sulphur bacterium cultivation light is used as energy sour-
ce and therefore it is necessary to optimize the light features in
order to reduce the operational bioprocess costs. However, the
cultivation of Rhodobacter capsulatus B-6508 E-10 has to be
examined in dark conditions in order to evaluate the potential
of these conditions for bacterial growth and 5-ALA synthesis.
In these experiments, Rhodobacter capsulatus B-6508 E-10
was cultivated in aerobic and microaerophilic conditions wit-
hout light (dark conditions) for 48 h and then GSLA mixture
was added (Figure 3).
 M. Novak et al: Croatian Journal of Food Technology, Biotechnology
118 and Nutrition 12 (3-4), 113-119 (2017)
Figure 3 Change of biomass (X) and 5-ALA concentrati-
ons during mutant strain Rhodobacter capsulatus
B-6508 E 10 cultivation in microaerophilic and
aerobic conditions without light (dark conditions)
As it can be seen in Figure 3, very low 5-ALA concentra-
tion (below 2 mg/L) was observed during Rhodobacter capsu-
latus B-6508 E-10 cultivation in dark microaerophilic conditi-
ons. On the contrary, during Rhodobacter capsulatus B-6508
E-10 cultivation in dark aerobic conditions considerably higher
5-ALA concentration 46.5 mg/L (31 mg/g dry weight biomass)
was detected. ALA synthase activity is the same in cultures
grown under high and low oxygen concentration (Biel 1992),
but ALA dehydratase activity is lower when aerobic conditions
are met (Nishikawa et al. 1999) meaning ALA accumulation
is possible during aerobic cultivation. Comparison between
Rhodobacter capsulatus B-6508 E-10 cultivations in light and
dark conditions shows considerably lower bacterial biomass
yield as well as 5-ALA yield. It is known that light intensity
is an important factor for 5-ALA production (Tangprasittipap
and Prasertsan 2002) and together with low oxygen concentra-
tion 5-aminolevulinate synthase is stabilized (Abu-Farha et al
2005) so that 5 ALA production can be enlarged. On the basis
of previous facts, it is obvious that higher 5-ALA production
efficiency can only be obtained during Rhodobacter capsula-
tus B-6508 cultivation in light microaerophilic conditions.
The impact of Fe2+ ion and vitamin B12
on the growth and 5-ALA production by
Rhodobacter capsulatus B-6508 E-10
Because of fact that Fe2+ ion and vitamin B12 have a
great role in the metabolism of purple bacteria (Liu et al 2015)
their impact on the growth and 5-ALA production was also
examined during Rhodobacter capsulatus B-6508 E-10 culti-
vations. In GM media Fe2+ and vitamin B12 were added so
that their concentrations were 30 µM and 2.5 mg/L, respecti-
vely. After 48 h of Rhodobacter capsulatus B-6508 E-10 culti-
vation in light microaerophilic conditions it was observed that
relatively low biomass concentration (≈ 1g/L) was obtained
together with the absence of purple pigment synthesis. Prolon-
gation of this cultivation (5 days) did not show any positive im-
pact on bacterial growth. It is known that addition of Fe2+ ion
to media can have a positive impact on the growth of purple
bacteria (Liu et al. 2005). However, the results of this study did
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
not support this claim. In this Rhodobacter capsulatus B-6508
E-10 cultivation, pH was relatively quickly increased up to
9.50 what considerably slowed down the bacterial growth, due
to the fact that purple bacteria prefer neutral to slightly aci-
dic (pH = 6.5 - 7.0) conditions. Furthermore, this relatively
high pH value was probably related to the absence of 5-ALA
production although GSLA mixture was added to the medium.
This observation is in accordance with literature where is po-
inted out that growth media should not contain iron or cobalt
ions if enlarged 5-ALA accumulation would like to happen du-
ring purple bacteria cultivation (Tangprasittipap and Prasertsan
2002). Although metal ions are important elements in regula-
tion of tetrapyrrole biosynthesis in Rhodobacter sphaeroides,
5-ALA synthetase is obviously regulated by hem compounds
through feedback inhibition or repression under iron sufficient
conditions (Sasikala and Ramana 1995).
Conclusions
The purple non-sulphur bacteria are able to use various
organic compounds for their growth and the medium compo-
sed of glutamate and malate (GM) shows the highest potential
for their cultivation. The most favorable conditions for Rhodo-
bacter capsulatus B-6508 E-10 growth and 5-ALA synthesis
are light (2000 Lux), microaerophilic conditions and tempera-
ture 28 °C. During Rhodobacter capsulatus B-6508 E-10 cul-
tivation in light aerobic conditions lower 5-ALA concentration
(58.1 mg/L) was obtained compared to the bacterial cultivation
in light microaerophilic conditions (86.59 mg/L). In the dark
cultivation of Rhodobacter capsulatus B-6508 E-10, aerati-
on is required for 5-ALA production, but considerably lower
5-ALA concentrations (31 mg/L) were detected. Cultivation of
Rhodobacter capsulatus B-6508 E-10 in light microaerophilic
conditions for 24 hours resulted in the most physiologically
active bacterial biomass for 5-ALA production (179 mg/L)
compared to the bacterial cultivation in the time period of 48
(124.5 mg/L) and 72 (89.5 mg/L) hours.
Acknowledgment
This research was financed from the Phoenix Grant Agree-
ment (NUMBER - 690925 - Phoenix), Project „The study of
features of 5-aminolevulinic acid biosynthesis by photosynthe-
tic bacteria “(NUMBER- 16A-1f37; Ministry of Education and
Science, Republic of Armenia) and project Bioprospecting of
Adriatic sea (KK.01.1.1.01.0002).
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Biel, A. J. (1992) Oxygen-regulated steps in the Rhodo-
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of Bacteriology, 174 (22) 5272-5274.
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120 and Nutrition 12 (3-4), 120-125 (2017)

IZVORNI ZNANSTVENI RAD / ORIGINAL SCIENTIFIC PAPER

Preferencije potrošača i važnost intrinzičnih i

ekstrinzičnih obilježja čipsa od jabuke
Consumer preferences and the importance of intrinsic
and extrinsic apple chips characteristics
Željka Mesić1*, Velimir Hunjak2, Marina Tomić2
1
Sveučilište u Zagrebu Agronomski fakultet, Zavod za marketing u poljoprivredi,
Svetošimunska c. 25, 10000 Zagreb
2
Zdrava navika d.o.o., Industrijski odvojak 6, 10431 Sv. Nedelja

* Corresponding author: zmesic@agr.hr

Sažetak

Cilj istraživanja je utvrditi, za potrošače relevantna intrinzična i ekstrinzična obilježja čipsa od jabuke, stavove potrošača o tom proizvo-
du, te utvrditi potrošačke preferencije i njihovo ponašanje u kupnji i potrošnji čipsa od jabuke. Anketno istraživanje je provedeno na uzorku od
100 potrošača čipsa od jabuke na području Grada Zagreba. Najviše ispitanika kupuje čips od jabuke barem jednom mjesečno i to najčešće u
drogerijama i supermarketima. Čips od jabuke se najčešće konzumira nekoliko puta mjesečno, uglavnom kod kuće i na poslu. Više od polovice
ispitanih potrošača je izjavilo da čips od jabuke jede kao međuobrok, dok oko petina ispitanika čips jede kada su gladni ili kada im je dosadno.
Osnovni motivi za kupnju čipsa od jabuka su zdravstveni aspekt, dobar okus te interes za isprobavanjem novih proizvoda. Ispitani potrošači sma-
traju intrinzična, odnosno senzorička svojstva čipsa od jabuke važnijima u odnosu na ekstrinzična obilježja. Od intrinzičnih obilježja najvažniji
su im okus, miris, izgled i boja, dok od ekstrinzičnih obilježja potrošači najveću pažnju pridaju cijeni, preporuci i marki proizvoda. Ispitanici
višeg stupnja obrazovanja smatraju važnijima prodajno mjesto, marku/brend i miris čipsa od jabuke dok ispitanici nižeg stupnja obrazovanja
veću važnost pridaju veličini pakiranja čipsa od jabuke. Rezultati istraživanja mogu pomoći proizvođačima čipsa od jabuke, kao i nutritivno
vrjednijih grickalica pri izradi komunikacijskih strategija za poboljšanje prodaje.

Ključne riječi: čips od jabuke, potrošači, intrinzična obilježja, ekstrinzična obilježja, stavovi

Abstract

The purpose of this paper is to examine intrinsic and extrinsic apple chips characteristics, consumer attitudes about this product and
consumer preferences and their behavior in purchase and consumption of apple chips. The survey was conducted on a sample of 100 consum-
ers of apple chips in the City of Zagreb. Most respondents buy apple chips at least once a month, mostly in drugstores and supermarkets. Apple
chips is mostly consumed several times a month, at home and at work. More than half of the respondents consume chips as a snack, while 1/5 of
respondents eat chips when they are hungry or bored. The basic motives for apple chips purchase are the health aspect, good taste and interest
in trying new product. Respondents consider intrinsic or sensory characteristics of apple chips more important than extrinsic. Most important
intrinsic characteristics are taste, smell, appearance and colour, while from the extrinsic or external characteristics consumers give the greatest
attention to price, recommendation and brand. Respondents with higher education consider selling place, brand and smell of the apple chips
more important, while those with lower education give more importance to the size of apple packaging. This study could help apple chips produc-
ers, as well as producers of more nutritive valuable snacks, in developing communication strategies to increase sales.

Key words: Apple chips, Consumer, Intrinsic characteristics, Extrinsic characteristics, Attitudes

Uvod pna proizvodnja jabuka u Hrvatskoj u 2015. godini iznosila

Jabuke imaju dobar okus, miris, teksturu i veliku nutri-
tivnu vrijednost. Osim u svježem stanju, jabuke se troše i u
prehrambenoj industriji u proizvodnji džemova, marmelada,
sokova i dehidriranih proizvoda (Woodroof i Luh, 1975). Uku-
je 101.750 tona (DZS, 2016), dok je potrošnja jabuke iznosi-
la 95.000 tona godišnje - kao konzumna i u obliku raznih
prerađevina (sokovi, osušena jabuka i dr.). Da bi se izbjegla pri-
nudna prodaja svježeg voća po vrlo niskim cijenama, potrebno
je preraditi jabuke u proizvode s dodanom vrijednošću, koji

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 Ž. Mesić et al: Croatian Journal of Food Technology, Biotechnology
bi ujedno donijeli i veću cijenu jabuci kao sirovini. Jedan od
takvih proizvoda s dodanom vrijednošću je čips od jabuke.
Čips od jabuke je proizvod koji se svrstava u skupinu sušenog
voća. Svjetski trend brige za zdravlje i bolji izgled doveo je
do povećane potrošnje suhog voća, a samim time i čipsa od
jabuke. Istraživanje autora Cerjak i Tomić (2014) pokazalo je
da potrošači smatraju da je suho voće, u koje pripada i čips od
jabuke, jako ukusno i da povoljno utječe na zdravlje. Takvo
suho voće sadrži male količine natrija te ne sadrži masti,
kolesterol ili dodatak šećera (Aksoy i sur., 2011).
Tehnologija proizvodnje čipsa od jabuke potpuno je
drugačija od tehnologije proizvodnje slanog čipsa koji se
dobije prženjem u vrlo slanom ulju (Sito i sur., 2013). Prema
Hunjak (2011) čips od jabuke se proizvodi na način da se
svježa jabuka, nakon pranja, nareže na ploške debljine 0,6
cm te se nakon izbacivanja usplođa slaže na police sušare i
suši toplim zrakom. Temperatura zraka za sušenje kreće se
od 60°C do 70 °C, a sam proces sušenja traje od 12 do 17
sati. Nakon sušenja čips se hladi te pakira u posebno uređenoj
prostoriji kontroliranih uvjeta - pakirnici. Nakon pakiranja u
za to predviđenu ambalažu, čips se stavlja u transportne kutije
te se plasira na tržište. Čips od jabuke je prirodni proizvod,
dobiven prirodnim putem bez konzervansa i aditiva pa prema
tome njegova kvaliteta prvenstveno ovisi o kvaliteti sirovine.
U samoj proizvodnji koristi se jabuka određenog kalibra sorte
Granny Smith, Golden Delicious te Jonagold. Čips bi trebao
biti jednolike boje, zadržati prirodni miris jabuke te biti
potpuno bez vlage, radi čega se u konačnici i naziva čipsom,
zbog svojih hruskavih svojstava.
Pri kupnji čipsa od jabuke, kao i ostalih poljoprivredno-
prehrambenih proizvoda, potrošači veliku važnost pridaju
kvaliteti proizvoda. Kvaliteta se može odnositi na različite
aspekte proizvoda: organoleptička svojstva, sigurnost,
podrijetlo, način proizvodnje (organski, tradicionalni). Kako
bi odredili kvalitetu proizvoda potrošači koriste intrinzična i
ekstrinzična obilježja (Lee i Lou, 2011). Ekstrinzična obilježja
kvalitete se odnose na proizvod, ali nisu fizički dio njega.
Ekstrinzična obilježja kvalitete uglavnom se odnose na cijenu,
ime marke, oznaku kvalitete, ime trgovine, pakiranje (Bernue´s
i sur., 2003; Rao i Monroe, 1989) ili općenitije pokazatelje,
kao što je zemlja podrijetla (Hoffmann, 2000). Prema Olson
i Jacoby (1972), intrinzična obilježja su dio fizičkog izgleda
proizvoda i ne mogu se mijenjati bez same fizičke promjene
proizvoda. Dobro poznata intrinzična obilježja kvalitete
prehrambenih proizvoda su boja, miris, okus i svježina. Ranija
istraživanja iz područja ponašanja potrošača su pokazala da
potrošači pridaju veliku važnost određenim ekstrinzičnim i
intrinzičnim obilježjima pri kupnji hrane (Steenkamp, 1997;
Espejel i sur., 2007) ili nakon donošenja kupovne odluke
(Enneking i sur., 2007). Fandos i Flavian (2006) su utvrdili da
intrinzična obilježja imaju pozitivan utjecaj na namjeru kupnje
dok su Enneking i sur. (2007) istraživali važnost intrinzičnih
i ekstrinzičnih obilježja pri kupnji proizvoda. Cerjak i Tomić
(2014) su utvrdili da su najvažnija ekstrinzična obilježja suhog
voća, prema mišljenju ispitanika, cijena i podrijetlo.
Poznavanje i pravilno predviđanje ponašanja potrošača
pretpostavka je za uspješno opsluživanje ciljnog potrošačkog
segmenta. Čips od jabuke je novost na domaćem tržištu i za
sada ne postoje podaci o tome u kojoj su mjeri potrošači upo-
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 120-125 (2017) 121
znati i zadovoljni s proizvodom i na koji način ga doživljavaju.
Tržište čipsa od jabuke u tom smislu je potpuno neistraženo.
Zbog toga je cilj ovog istraživanja utvrditi, za potrošače
relevantna ekstrinzična i intrinzična obilježja čipsa od jabu-
ke, stavove potrošača o tom proizvodu, te utvrditi potrošačke
preferencije i njihovo ponašanje u kupnji i potrošnji čipsa od
jabuke.
Materijali i metode
Primarni podaci prikupljeni su metodom anketnog is-
pitivanja s potrošačima čipsa od jabuke. Anketno ispitivanje
provedeno je na uzorku od 100 ispitanika na području grada
Zagreba u drogerijama, supermarketima i trgovačkim centri-
ma: DM-u, BIPA-i, Konzumu, Mülleru i Intersparu. Anketa je
trajala od 5 do 7 minuta. Prije provođenja anketnog ispitivanja
anketni upitnik je testiran na uzorku od pet ispitanika da bi
se ispravile eventualne nejasnoće u redoslijedu i formulaciji
pitanja.
Anketnim upitnikom obuhvaćeno je ponašanje potrošača
u kupnji i potrošnji čipsa od jabuke (učestalost kupnje, mjesto
kupnje, učestalost konzumacije, mjesto konzumacije, razlo-
zi konzumacije, zadovoljstvo ponudom). Zatim je uslijedila
grupa pitanja o važnosti ekstrinzičnih i intrinzičnih obilježja
čipsa od jabuke, koja je mjerena na skali od pet stupnjeva (1
= potpuno nevažno obilježje, 2= nevažno obilježje, 3= niti važ-
no, niti nevažno obilježje, 4= važno obilježje, 5= jako važno
obilježje). Mjerena su sljedeća ekstrinzična obilježja čipsa od
jabuke: cijena, preporuka, marka/brand, veličina pakiranja, di-
zajn pakiranja, certifikat (HACCP) i prodajno mjesto, dok je
od intrinzičnih obilježja mjerena važnost okusa, mirisa, tvrdo-
će odnosno hruskavosti, izgleda i boje.
Nadalje, anketnim ispitivanjem su se obuhvatila pitanja
koja su se odnosila na stavove ispitanika prema čipsu od ja-
buke. Ispitanici su na ljestvici od 5 stupnjeva (1=uopće se ne
slažem, 2=ne slažem se, 3=niti se slažem, niti ne slažem, 4=
slažem se, 5=potpuno se slažem) iskazali stupanj suglasnosti s
pet izjava kojima su se mjerili stavovi prema čipsu od jabuke.
Posljednja skupina pitanja u anketi odnosila se na soci-
odemografska obilježja; spol, dob, stupanj obrazovanja i do-
hodak.
Obrada prikupljenih podatka je provedena pomoću jed-
novarijatne (frekvencije, distribucija podataka) i dvovarijatne
analize podataka (ANOVA) za što je korišten statistički pro-
gram SPSS, verzija 21.
 Ž. Mesić et al: Croatian Journal of Food Technology, Biotechnology
122 and Nutrition 12 (3-4), 120-125 (2017)

Rezultati i rasprava
Više od polovice ispitanika je odgovorilo da kupuju čips
Struktura uzorka prema socio-demografskim obilježjima od jabuke u Drogerie Market-u (DM), zatim Konzumu, Inter-
prikazana je u Tablici 1. sparu, Mülleru i Billi, dok je kupnja u ostalim prodajnim mjes-
tima manje zastupljena.
Tablica 1. Socio-demografska obilježja uzorka Čips od jabuke se najčešće konzumira nekoliko puta
Table 1. Socio-demographic characteristics of the sample mjesečno (28,3%). Slijede ispitanici koji konzumiraju taj
% proizvod nekoliko puta tjedno (18,2%) i jednom tjedno
Sociodemografska obilježja (17,2%). Čak 9,1% ispitanika konzumira čips od jabuke svaki
N=100 dan (Slika 2).
Spol Muški 36,0
Ženski 64,0
Dob 16 - 35 50,0
36 - 45 25,0
Više od 46 25,0
Stupanj obrazovanja Osnovna škola 4,0
SSS 22,0
VSŠ/VSS 74,0
Mjesečni osobni do 2.500 kn 9,0
dohodak Slika 2. Učestalost konzumacije čipsa od jabuke
2.500 – 5.000 kn 18,0 Figure 2. Frequency of apple chips consumption
5.001 – 9.000 kn 56,0 Na pitanje o mjestu konzumacije čipsa od jabuke je posto-
jala mogućnost više odgovora. Čips se najčešće konzumira kod
više od 9.000 kn 17,0 kuće (83%) i na poslu (35%), u autu ili autobusu dok se na
ostalim mjestima konzumira u manjoj količini (tablica 2).
U istraživanju je sudjelovalo 36% muških i 64% ženskih
ispitanika. Prosječna dob ispitanika je 35 godina.  Više od S obzirom na prigodu u kojoj ispitanici najčešće
dvije trećine ispitanika, odnosno njih 74% ima visoku ili višu konzumiraju čips od jabuke, gotovo 3/4 njih je izjavilo da
stručnu spremu. Ukupna mjesečna primanja kod najvećeg navedeni proizvod jede kao međuobrok (74%), dok oko petina
broja ispitanika su od 5.001,00 – 9.000,00 kuna, dok najmanje ispitanika čips jede kada su gladni ili kada im je dosadno
ispitanika ima mjesečna primanja do 2.500,00 kuna - Tablica
1. Tablica 2. Mjesto konzumacije čipsa od jabuke
Table 2. Place of consumption of apple chips
Ponašanje potrošača prilikom kupnje Mjesto konzumacije N %
i potrošnje čipsa od jabuke
Prema učestalosti kupnje, najveći broj ispitanika, njih Kod kuće 83 83,0
36%, kupuje čips od jabuke barem jednom mjesečno. Gotovo
četvrtina ispitanika kupuje čips od jabuke više puta mjesečno, Na poslu 35 35,0
18% jednom tjedno, dok tek 3% ispitanika kupuje čips od ja- U autu/autobusu/vlaku 12 12,0
buke svaki dan (Slika 1).
U kinu 5 5,0
Na zabavama 4 4,0
U školi/na fakultetu 4 4,0
Na izletu 4 4,0

Najčešći razlozi konzumacije čipsa od jabuke su

Slika 1. Učestalost kupnje čipsa od jabuke
Figure 1. Purchasing behaviour of apple chips consumers
zdravstveni aspekt (n=69), dobar okus (n=39) i želja za
isprobavanjem novih proizvoda (n=20). 14 ispitanika ne
kupuje taj proizvod za sebe dok jedan ispitanik kupuje čips od
jabuke jer voli sušeno voće.
Po pitanju zadovoljstva ponudom čipsa od jabuke na
zagrebačkom tržištu, ispitanici su iskazali nezadovoljstvo
(srednja vrijednost 2,25).

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 Ž. Mesić et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 120-125 (2017) 123

Najveći udio ispitanika je spreman platiti 7-8 kn za 50g
čipsa od jabuke (46%), slijede oni koji su spremni platiti 5-6
kn za istu količinu čipsa od jabuke, dok je samo jedan ispitanik
spreman platiti preko 10 kn za čips od jabuke pakiranja 50g.
Važnost intrinzičnih i ekstrinzičnih
obilježja čipsa od jabuke
Rezultati istraživanja su pokazali da su ispitanicima važ-
nija intrinzična, odnosno senzorička svojstva u odnosu na ek-
strinzična (vanjska) obilježja čipsa od jabuke.
Rezultati istraživanja su pokazali da je ispitanicima pri
kupnji i potrošnji čipsa od jabuke od intrinzičnih obilježja naj-
važniji okus čipsa (srednja ocjena 4,79). Također, vrlo bitno
svojstvo prilikom kupnje čipsa od jabuke je miris čipsa (sred-
nja ocjena 4,43), dok su izgled (srednja ocjena 3,71) i boja
(srednja ocjena 3,65) najvećem broju ispitanika manje važna
intrinzična obilježja čipsa od jabuke – Tablica 3.
Ispitanicima su od ekstrinzičnih obilježja čipsa od jabu-
ke najvažniji cijena (srednja ocjena 4,01), preporuka (srednja
ocjena 3,82) i marka/brand čipsa od jabuke (srednja ocjena
3,74). Najmanje važno ekstrinzično obilježje za ispitane po-
trošače čipsa od jabuke je prodajno mjesto (srednja vrijednost
2,94).
Rezultati jednosmjerne analize varijance (ANOVA)
pokazali su da od ispitanih sociodemografskih obilježja (spol,
dob, mjesečni osobni dohodak, stupanj obrazovanja), jedino
stupanj obrazovanja potrošača utječe na važnost intrinzičnih
i ekstrinzičnih obilježja čipsa od jabuke (p<0,05). Kao što je
vidljivo iz rezultata prikazanih u tablici 3., ispitani potrošači
više i visoke stručne spreme pridaju veću važnost marki ili
brandu čipsa od jabuke u odnosu na ispitanike srednjoškolske
i osnovnoškolske stručne spreme. Nadalje, ispitanici sa višom
i visokom stručnom spremom smatraju prodajno mjesto
srednje važnim dok je za ispitanike sa završenom osnovnom
i/ili srednjom školom prodajno mjesto nevažno. Od ostalih
ekstrinzičnih obilježja čipsa od jabuke veličina pakiranja je
vrlo važna ispitanicima srednjoškolskog i osnovnoškolskog
obrazovanja dok je ispitanica sa završenom višom i visokom
školom veličina pakiranja srednje važna. Nije utvrđena
statistički značajna razlika u važnosti ostalih ekstrinzičnih
obilježja čipsa od jabuke (cijena, preporuka, dizajn pakiranja,
certifikat (HACCP) s obzirom na stupanj obrazovanja
ispitanika (p>0,05).
Što se tiče važnosti intrinzičnih obilježja, statistički
značajna razlika je utvrđena kod mirisa čipsa od jabuke
pri čemu ispitanici sa višom i visokom stručnom spremom
smatraju miris važnijim obilježjem u odnosu na ispitanike
sa završenom srednjom i osnovnom školom (p<0,05). Nije
utvrđena statistički značajna razlika među ispitanicima
različitog stupnja obrazovanja s obzirom na važnost ostalih
intrinzičnih obilježja čipsa od jabuke (okus, tvrdoća, boja i
izgled), p>0,05 – Tablica 3.

Tablica 3. Važnost intrinzičnih i ekstrinzičnih obilježja čipsa od jabuke prema stupnju obrazovanja
Table 3. The importance of intrinsic and extrinsic characteristics of apple chips according to level of education
Srednja Srednja SD p
Ekstrinzična obilježja SD Obrazovanje
ocjena ocjena
OŠ 3,25 1,26
Cijena 4,01 0,79 SSS 4,18 0,73 n.s.
VŠS/VSS 4,00 0,80
OŠ 3,75 1,26
Preporuka 3,82 1,03 SSS 3,45 1,22 n.s.
VŠS/VSS 3,95 0,95
OŠ 2,75 1,26
Marka/brand 3,74 0,94 SSS 3,55 0,74 <0,05
VŠS/VSS 3,86 0,96
OŠ 4,75 0,50
Veličina pakiranja 3,57 0,97 SSS 3,68 0,84 <0,05
VŠS/VSS 3,46 0,98
OŠ 3,75 0,96

Dizajn pakiranja 3,53 1,01 SSS 3,14 0,89 n.s.

VŠS/VSS 3,62 1,03
OŠ 2,75 2,06
Certifikat (HACCP) 3,43 1,21 SSS 3,00 0,98 n.s.
VŠS/VSS 3,58 1,20
OŠ 2,25 1,50
Prodajno mjesto 2,94 1,29 SSS 2,32 1,25 <0,05
VŠS/VSS 3,15 1,23

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124 and Nutrition 12 (3-4), 120-125 (2017)

Srednja Srednja
Intrinzična obilježja SD Obrazovanje SD p
ocjena ocjena
OŠ 4,75 0,50
Okus 4,79 0,41 SSS 4,82 0,39 n.s.
VŠS/VSS 4,78 0,41
OŠ 3,50 1,91
Miris 4,43 0,72 SSS 4,41 0,73 <0,05
VŠS/VSS 4,50 0,58
OŠ 4,25 0,50

Tvrdoća/hruskavost 4,52 0,66 SSS 4,50 0,86 n.s.

VŠS/VSS 4,54 0,60
OŠ 3,25 1,71
Izgled 3,71 1,05 SSS 3,45 1,01 >0,05
VŠS/VSS 3,82 1,02
OŠ 3,00 1,83
Boja 3,65 1,05 SSS 3,59 1,05 n.s.
VŠS/VSS 3,72 1,01

Stavovi potrošača prema čipsu od jabuke
Na temelju rezultata prikazanih u tablici 4., može se za-
ključiti da se ispitanici najviše slažu s izjavom da im je važno
da je čips proizveden prirodnim putem (srednja ocjena 4,46) te
da je čips od jabuke dobar za zdravlje (srednja ocjena 4,14). S
druge strane, ispitanici se ne slažu da bi cijena čipsa od jabu-
ke trebala biti slična cijeni čipsa od krumpira (srednja ocjena
2,54), kao niti da je čips od jabuke isti kao i svi ostali čipsevi
(srednja ocjena 1,81).
Rezultati istraživanja su pokazali da postoji statistički
značajna razlika u stavovima ispitanika o čipsu od jabuke s ob-
zirom na njihov stupanj obrazovanja (p<0,05). Ispitanicima sa
završenom višom i visokom školom je važnije da je čips proi-
zveden prirodnim putem u odnosu na ispitanike nižeg stupnja
obrazovanja. Nadalje, ispitanici višeg stupnja obrazovanja se
više slažu s izjavom da su potrošači čipsa od jabuke osobe koje
drže do svog zdravlja u usporedbi s ispitanicima nižeg stupnja
obrazovanja (p<0,05, ANOVA). Spol, dob i mjesečni osobni
dohodak ispitanika ne utječu signifikantno na njihove stavove
prema čipsu od jabuke.

Tablica 4. Stavovi ispitanika o čipsu od jabuke prema stupnju obrazovanja

Table 4. Consumers attitudes towards apple chips according to level of education
Srednja Obrazo- Srednja
IZJAVE SD SD p
ocjena vanje ocjena
OŠ 3,50 1,915
Važno mi je da je čips proizveden prirod-
4,46 0,86 SSS 4,05 1,133 <0,05
nim putem.
VŠS/VSS 4,64 ,587
OŠ 3,75 1,89
Čips od jabuke je dobar za zdravlje. 4,14 0,88 SSS 3,95 0,78 n.s.
VŠS/VSS 4,22 0,83
OŠ 3,25 2,06
Potrošači čipsa od jabuke su osobe koje
3,80 1,17 SSS 3,32 1,29 <0,05
drže do svog zdravlja.
VŠS/VSS 3,97 1,05
OŠ 2,00 1,41
Cijena čipsa od jabuke bi trebala biti
2,54 1,25 SSS 2,64 1,25 n.s.
slična cijeni čipsa od krumpira.
VŠS/VSS 2,54 1,25
OŠ 1,25 0,50
Čips od jabuke je isti kao i svi ostali
1,81 0,94 n.s.
čipsevi. SSS 2,14 0,99
VŠS/VSS 1,74 0,92

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 Ž. Mesić et al: Croatian Journal of Food Technology, Biotechnology
Zaključci
Istraživanje je pokazalo da u Hrvatskoj raste potražnja za
alternativnim grickalicama veće nutritivne vrijednosti. Trend
brige za zdravlje doveo je do povećane potrošnje sušenog
voća, a samim time i čipsa od jabuke.
Više od trećine ispitanika kupuje čips od jabuke barem
jednom mjesečno dok skoro četvrtina ispitanika kupuje čips
od jabuke nekoliko puta mjesečno, i to najčešće u drogerijama
i supermarketima. Najveći udio ispitanika konzumira čips
nekoliko puta mjesečno, uglavnom kod kuće i na poslu. Više
od polovice ispitanih potrošača je izjavilo da čips od jabuke
jede kao međuobrok, dok oko petine ispitanika čips jede kada
su gladni ili kada im je dosadno. Osnovni motivi za kupnju
čipsa od jabuka su zdravstveni aspekt, dobar okus te interes za
isprobavanjem novih proizvoda. Unatoč čestoj kupnji i kon-
zumaciji čipsa od jabuke, ispitanici su iskazali nezadovoljstvo
ponudom čipsa od jabuke na zagrebačkom tržištu, što upućuje
na zaključak da je potrebno prošiti ponudu čipsa od jabuke ali i
ostalih alternativa klasičnim grickalicama (čips od rogača, čips
od kelja, čips od povrća...).
Ispitani potrošači smatraju intrinzična, odnosno senzorička
svojstva čipsa od jabuke važnijima u odnosu na ekstrinzična
obilježja. Od intrinzičnih obilježja najvažniji su im okus, miris,
izgled i boja. Od ekstrinzičnih obilježja potrošači najveću
pažnju pridaju cijeni, preporuci i marki proizvoda. Ispitanici
višeg stupnja obrazovanja smatraju važnijima prodajno mjesto,
marku/brend i miris čipsa od jabuke dok ispitanici nižeg
stupnja obrazovanja veću važnost pridaju veličini pakiranja
čipsa od jabuke.
Ispitanicima je važno da je čips proizveden prirodnim
putem te smatraju da je čips od jabuke dobar za zdravlje. Što
se tiče cijene čipsa od jabuke, ispitanici se ne slažu da bi cijena
čipsa od jabuke trebala biti slična cijeni čipsa od krumpira,
kao niti da je čips od jabuke isti kao i svi ostali čipsevi. Iako
ispitanici ne smatraju da bi cijena čipsa od jabuke trebala biti
slična cijeni čipsa od krumpira, proizvođači čipsa od jabuke
trebaju imati na umu da je cijena najvažnije ekstrinzično
obilježje za potrošače.
Informacije o preferencijama i stavovima potrošača
te o važnosti ekstrinzičnih i intrinzičnih obilježja čipsa od
jabuke mogu pomoći proizvođačima čipsa od jabuke, kao
i proizvođačima drugih nutritivno vrijednijih grickalica pri
izradi komunikacijskih strategija za unapređenje prodaje.
S obzirom na dobivene rezultate, preporuča se u budućim
istraživanjima provesti segmentaciju tržišta čipsa od jabuke
kako bi se identificirali tržni segmenti prema kojima bi se mo-
gle kreirati precizne marketinške strategije.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 120-125 (2017) 125
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 M. Denžić Lugomer et al: Croatian Journal of Food Technology, Biotechnology
126 and Nutrition 12 (3-4), 126-130 (2017)

ORIGINAL SCIENTIFIC PAPER

Water quality on cattle farms in the northwest Croatia

Marija Denžić Lugomer1*, Vesna Jaki Tkalec2, Maja Kiš1, Damir Pavliček1, Sanja Furmeg2, Jadranka Sokolović2
1
Croatian Veterinary Institute, Veterinary Department Križevci, Laboratory for Analytical Chemistry and Residues, Ivana
Zakmardija Dijankovečkog 10, 48260 Križevci
2
Croatian Veterinary Institute, Veterinary Department Križevci, Laboratory for Microbiology of Food and Feed, Ivana Za-
kmardija Dijankovečkog 10, 48260 Križevci

* Corresponding author: denzic.vzk@veinst.hr

Abstract

In this study, quality of well water used for cattle watering on farms in the northwest Croatia was investigated. The following microbiolo-
gical parameters were analysed: total coliforms, Escherichia coli, intestinal enterococci, the number of microorganism colonies at 37 ° C and
22 ° C, Clostridium perfringens and Pseudomonas aeruginosa. Physico-chemical analysis included following parameters: colour, taste, odour,
pH, chlorides, sulphates, nitrites, nitrates, sodium, potassium, ammonium, free chlorine, iron and the consumption of KMnO4. Among 34 samples
analysed, 79.4% were microbiologically, 61.8% were chemically and 94.1% of the samples were in total unsuitable according to the Croatian
water quality regulations (OJ 125/2013, 141/2013, 128/2015).
Keywords: water quality, cattle, northwest Croatia, watering

Sažetak

U ovom radu ispitivana je kvaliteta bunarske vode koja se koristi za napajanje goveda na farmama sjeverozapadne Hrvatske. Analizirani
su slijedeći mikrobiološki parametri: ukupni koliformi, Escherichia coli, crijevni enterokoki, broj kolonija mikroorganizama na 37 °C i 22 °C,
Clostridium perfringens i Pseudomonas aeruginosa. Od fizikalno-kemijskih parametara analizirani su: boja, okus, miris, pH, kloridi, sulfati,
nitriti, nitrati, natrij, kalij, amonijak, slobodni klor, željezo i utrošak KMnO4. Od 34 pretražena uzorka, prema Pravilniku o parametrima suklad-
nosti i metodama analize vode za ljudsku potrošnju (NN 125/2013, 141/2013, 128/2015) mikrobiološki nije bilo sukladno 79,4%, kemijski 61,8%
uzoraka, odnosno ukupno 94,1% uzoraka.
Ključne riječi: kvaliteta vode, goveda, sjeverozapadna Hrvatska, napajanje

Introduction
The existence of water is certainly one of the conditions
of life on our planet because it is necessary for all vital pro-
cesses in the biosphere. The water accounts for about 70% of
the mass of all living organisms, including animals. It is a vital
body fluid essential for the transport of nutrients and removal
of waste, metabolic reactions, regulation of the body tempe-
rature, etc. In most cases animals satisfy the need for water
from watering and water quantity depends on their species and
category, nutrition, physiological condition, activities, produc-
tivity and environmental conditions. Domestic animals, such
as cows must have 4.5 L of water per 50 kg of body weight per
day and 3 L for each liter of milk. Yearling cattle require on an
average 20-30 L of water per day, and calves up to 1 month of
age 8-10 L of water (Naletilić et al., 2013).
In the Republic of Croatia, the quality of water for animals
must correspond to that for human consumption whose control
is carried out in accordance with the criteria of the Croatian
water quality regulation (OJ 125/2013, 141/2013, 128/2015).
However, the water quality for the animals may often be over-
looked. By investigating the quality of water used as drink-
ing water for turkeys in Dalmatian hinterland, Ostović et al.
(2011) found only water from the water supply system to be
safe, whereas water from all other sources investigated (well,
cistern and barrel) did not meet health safety criteria. Moreo-
ver, our previous study on water quality for broiler chickens
and laying hens on farms in the northwest Croatia showed that
25.0% of samples were chemically and/or microbiologically
unsuitable and all of the unsuitable samples come from wells
(Kiš et al., 2017). Therefore, more emphasis should be placed
on a systematic study of drinking water for animals with a goal
of achieving its health and ecological acceptability.
The aim of this study was to investigate the quality of
well water used for supplying cattle on Croatian farms in the
northwest since, according to our knowledge, no such studies
have been done yet.
Materials and methods
Water sampling
Water was sampled on dairy and beef cattle farms in the
northwest Croatia from the wells used for their watering. The
samples were collected by qualified employees, in clean, steri-
le bottles and transported in portable refrigerators to laborato-
ries within 6 hours of sampling. Immediately after coming to
the lab, they underwent microbiological and physical-chemical

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 M. Denžić Lugomer et al: Croatian Journal of Food Technology, Biotechnology
analysis. A total of 34 samples of well water from different
farms collected during the autumn of 2016 were analysed.
Physico-chemical analysis
Colour
Colour of samples was determined according to the norm
HRN EN ISO 7887:2012. Principle of method is based on
using optical apparatus for comparison with hexachloroplati-
nate concentration at wavelength, λ=410 nm.
Odour and taste
Determination of odour and taste were carried out accor-
ding norm HRN EN 1622:2008 using short method which
is applicable when either a sample has no odour and taste or
for compliance of odour and taste with specified level. Only
microbiologically suitable samples were subjected to analysis.
pH value
The concentration of hydrogen ions or pH value of wa-
ter was determined potentiometrically, i.e. by measuring the
pH value using the Meter Toledo Meter SevenCompact S220
instrument. The procedure with the pH meter and the pH me-
asurement procedure itself were carried out according to the
norm HRN ISO 10523:2012.
Dissolved anions
Dissolved anions (chlorides, nitrites, nitrates, suphates)
were determined by ion-liquid chromatography according to
the norm HRN EN ISO 10304-1:2009. The method was set up
on the DIONEX ion chromatography assay and the detection
was performed with a conductometric detector with suppressi-
on. As eluent, a solution of 4.5 mmol L-1 Na2CO3 and 1.4 mmol
L-1 NaHCO3 was used. Dionex IonPac AS22 column was used,
4 × 250 mm, thermostated at 30 ° C. The flow rate was 1.2 mL
min-1 The sample was filtered through a membrane filter Ø 45
μm prior to injection.
Dissolved cations
Dissolved cations (sodium, potassium and ammonium)
were determined by ion-liquid chromatography according to
the norm HRN EN ISO 14911:2001. The method was set up on
the DIONEX ion chromatography assay and the detection was
performed with a conductometric detector with a suppression.
A solution of 20 mM methanesulfonic acid was used as eluent
at a flow rate of 1 mL min-1.. Dionex IonPac CS12 column, 4
× 250 mm, thermostated at 22 ° C was used. The sample was
filtered through a membrane filter Ø 45 μm prior to injection.
Free chlorine
The free chlorine determination method is based on a di-
rect reaction with N, N-diethyl-1,4-phenylenediamine (DPD)
and the formation of a pink coloured compound at pH 6.2 to
6.5. The measurement of colour intensity is achieved by a chlo-
rine pocket meter, HACH colorimeter.
Iron
The method for determining iron in water was carried out
according to the norm HRN ISO 6332:1998 and is based on the
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 126-130 (2017) 127
measurement of absorbance at 510 nm of an orange-red com-
plex formed by the reaction of iron (II) ions with a solution of
1,10-phenanthroline. Iron (III) ions are reduced to iron (II) ions
by adding ascorbic acid before forming the complex.
Consumption of KMnO4
The method for determining consumption of KMnO4, i.e.
the permanganate index, is based on determining the amount
of oxygen required for oxidation of dissolved organic matter
in water with some strong oxidizing agent (KMnO4). The con-
sumption of KMnO4 was determined in accordance with the
norm HRN EN ISO 8467:2001. The method is based on hea-
ting the sample in a boiling water bath with a known amount
of potassium permanganate and sulfuric acid at a fixed time
period (10 min). Reduction of part of the permanganate by oxi-
dizing substances in the sample and the determination of the
consumed permanganate by addition of an excess of oxalate
solution, followed by titration with permanganate.
Microbiological analysis
For the purpose of confirming and determining the total
number of coliform bacteria and Escherichia coli bacteria in
water, the membrane filtration method was used according to
the standard procedure HRN EN ISO 9308-1:2014, while the
procedure HRN EN ISO 7899-2:2000 was used to confirm and
determine the number of intestinal enterococci. For the detecti-
on of bacterial species of Pseudomonas aeruginosa, a procedu-
re was used according to the norm HRN EN ISO 16266:2008.
The detection and number of Clostridium perfringens spores
after membrane filtration and anaerobic incubation at 44 °C
and 37 °C for 24 hours was determined on m-CP agar (Clo-
stridium Perfringens agar base, Merck, Germany) and TSC
agar (Tripton sulfit cycloser agar, Biokar, France). The method
HRN EN ISO 6222:2000 was used to determine the number of
microorganism colonies colonizing on a nutrient agar.
The sample search procedure was carried out in accor-
dance with the requirements of the above mentioned standards
by membrane filtration of 100 mL of water sample through
0.45 μm pore size filters and incubated on solid selective agar
(Lactose TTC agar with Tergitol 7, Merck, Germany) for total
coliforms, TTC and TBX (Tryptone Bille X-gluconide, Merck,
Germany) agar for Escherichia coli and Slanetz and Bartley
agar for intestinal enterococci. Morphological and biochemi-
cal properties were used to confirm and identify the grown
microorganisms. Coliform bacteria were confirmed by the pro-
duction of indole from tryptophane and by negative oxidase.
The bacterial species of Escherichia coli grows as blue-green
colonies on TBX agar, indole is positive and oxidase negative.
The intestinal enterococci hydrolyse aesculin on bile-aesculin-
azide agar. The end-product, 6,7-dihydroxycumarin, combines
with iron(III) ions to give a tan coloured to black compound
which diffuses into the medium. Pseudomonas aeruginosa is a
microorganism that grows on a selective medium with cetrimi-
de and produces pyocyanin, it is the oxidase positive, fluores-
ces under UV light (360±20 nm) and is capable of producing
ammonia from acetamide.
To determine the number of microorganism colonies in a
1 mL of water sample, yeast extract agar was applied and the
 M. Denžić Lugomer et al: Croatian Journal of Food Technology, Biotechnology
128 and Nutrition 12 (3-4), 126-130 (2017)

plates were incubated at 22 °C for 68 hours and 37 °C for 44 Apart from housing and nutrition, cattle breeding also
hours. depends on water supply. Unsatisfactory, poor quality of wa-
ter, either organoleptic, physico-chemical or microbiological,
affects the animal health, welfare and productivity. Problems
Results and discussion related to the quality of water for livestock watering, in terms
of impaired health and productivity, are caused by the qualita-
The percentage of microbiologically and chemically un- tive composition of water but may also be the result of water
suitable samples among 34 analysed samples regarding the disinfection due to the resulting harmful by-products (Marja-
Maximum Permissible Concentration (MPC) set out by the nović and Tofant, 2008).
Croatian water quality regulations (OJ 125/2013, 141/2013, Water for animals should be colourless, odourless and ta-
128/2015) are presented in Table 1 and the results of unsuit- steless. The colour of water comes from dissolved and suspen-
able samples according to a individually analysed parameter ded matter. The water colour does not have a hygienic meaning,
are shown in Table 2. but it gives to water an unappealing look. In this study, the co-
lour of all water samples was within the MPC of 20 mg L-1 PtCo
Table 1. Microbiologically and chemically unsuitable sam- scale. Odour and taste have sanitary significance because they
ples pursuant to the exposure levels set out by the are often the first obvious indicators of water contamination. The
Croatian water quality regulations (OJ 125/2013, present study showed that all of the analysed microbiologically
141/2013, 128/2015) suitable samples were odourless and tasteless.
Type of analysis Analysed Unsuitable % Determining the pH value is very important because of
the influence on the chemical and biological properties of wa-
samples (n) samples (n) ter. Water with low pH is corrosive to equipment and has a
Microbiological 27 79.4 negative impact on animal acceptability, because it is acidic,
34 while water with high pH is also unacceptable because it is
Chemical 21 61.8 unsavoury. No sample was found to be unsuitable with this
parameter and the average pH value was 7.4.
Table 2. Unsuitable samples pursuant to the exposure The consumption of KMnO4 is a parameter that points
levels set out by the Croatian water quality regulations (OJ to content of dissolved organic substances in water. In natural
125/2013, 141/2013, 128/2015) per individually analysed pa- waters, increased amounts of organic matter are usually due to
rameter with minimum and maximum obtained values among secondary sources of pollution (e.g. the rupture of agricultural
34 analyzed samples surfaces, human or animal waste substances or industrial waste
Unsuitable Min-max products) and that is why the determination of this parameter
Parameter samples (n) % values among in water is an important indicator of a potential water sour-
34 samples
ce contamination with an organic matter (Asaj, 1974). All the
samples tested were suitable with this parameter, respecting
Total coliforms 25 73.5 0-890 cfu/100
the MPC of 5.0 mg O2 L-1.
mL
The concentration of chlorides in water has no major he-
Microorganism count at 24 70.6 0-2000 cfu/
alth significance, but it is important because of their corrosive
37 °C mL action on metal tubes of the water supply systems and because
Microorganism count at 23 67.6 0-990 cfu/mL of the taste of water (Naletilić et al., 2013). Dissolved chlorides
22 °C are the most dissolved anions in water. Their concentration in
Intestinal enterococci 23 67.6 0-300 cfu/100 water sources is largely constant and any significant change
mL may indicate to secondary pollution of the water source with
Escherichia coli 21 61.8 0-400 cfu/100 faecal matter and/or wastewaters (Asaj, 1974). MPC for chlo-
mL rides is 250 mg L-1. All analysed water samples showed com-
Nitrates 13 38.2 1.2-212.7 mg pliance with this parameter. In addition, sulphate concentration
L-1
was also monitored, as their increased concentrations in water
(>150 ppm) cause salty taste, diarrhoea, and in some cases co-
Clostridium perfringens 9 26.5 0-47 cfu/100
pper deficiency (Higgins et al., 2008). Increased sulphate con-
mL
centrations stimulate the development of polioencephaloma, a
Ammonium 8 23.5 <0.03-1.31
neurological disorder characterised by weakness, muscle tre-
mg L-1 mors, lethargy and even paralysis and death in cattle (Higgins
Pseudomonas aeruginosa 3 8.8 0-61 cfu/100 et al., 2008).The highest measured sulphate concentration was
mL 105 mg L-1, well below prescribed 250 mg L-1.
Iron 2 5.9 <20 -819 µg For the purpose of testing and assessing the water qua-
L-1 lity, determining the presence of substances resulting from the
Potassium 2 5.9 0.21- 101.9 decomposition of waste from plants and/or animals is of great
mg L-1 importance. Cattle breeding affects the deterioration of water
Free chlorine 1 2.9 <0.02-1.03 quality through different sources of contamination (e.g. farms,
mg L-1 fertilizers on agricultural surfaces) (Nemčić-Jurec and Valda,
2010). Nitrogen compounds, such as ammonia, nitrite and ni-

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 M. Denžić Lugomer et al: Croatian Journal of Food Technology, Biotechnology
trate, are products of the breakdown of the substances of biolo-
gical origin. All these forms of nitrogen can be discharged into
the water by polluting water sources with wastewaters. Presen-
ce of nitrites in the drinking water points to partially decom-
posed organic waste substances. Nitrites are very toxic. Nitrite
concentrations in all water samples were satisfactory, below
MPC, which is 0.5 mg L-1.
The concentration of nitrates as nitrogen compounds,
which are the highest degree of the nitrogen oxidation in its
natural cycle, is small in the surface waters (usually 1 to 30
mg L-1), but can be found in larger quantities in deep waters.
Increased concentration of nitrate in drinking water can po-
tentially cause health problems in humans and animals. It is
known that when nitrite in the blood binds to hemoglobin, it
turns into methemoglobin, reducing the oxygen transfer capa-
city, which can be very dangerous. Nitrate toxicity causes poor
growth, slimness and poor coordination in poultry (Ostović et
al., 2011). It is also known that nitrates can produce nitrosa-
mines which can modify certain DNA components and cause
tumours (Nemčić-Jurec et al., 2009). Signs of lower level of
nitrate poisoning in cattle include inferior growth, infertility,
abortion and vitamin A deficiency (Higgins et al., 2008). In
acute nitrate poisoning there is breathing difficulty, rapid pul-
se, foaming, convulsion, blue muzzle and dark circles around
the eyes. Signs of chronic nitrate poisoning include reduced
weight gain, decreased appetite, reduced milk production and
increased exposure to infection (Higgins et al., 2008). In this
study, nitrate concentrations were satisfactory (below MPC
which is 50 mg L-1) in 61.8% of the analysed water samples.
Ostović et al. (2011) found that nitrate concentrations in water
supply containers for turkeys, though within the permitted li-
mits, were almost twice as high when compared to water sour-
ces. The mean nitrate concentration in the suitable samples was
14.3 mg L-1, with the highest measured value of 36.6 mg L-1.
From all dissolved cations, ammonium, potassium and so-
dium concentrations were monitored. The presence of ammo-
nium in water indicates a “fresh” pollution with organic matter
and presents a danger to users of such water. Sometimes rain-
fall and very often the water of deep wells can contain traces
of ammonia that is of geological origin and does not pose a
threat to users. In this study 76.5% of the samples were found
to be suitable with this parameter taking into account MPC of
0.5 mg L-1.Monitoring the concentration of the other two cati-
ons was decided because of their role in the functioning of the
organism. Studies have shown that animals can tolerate large
doses of sodium if they are given sufficient amounts of water.
Cattle given water containing 975 mg L-1 of sodium ions for 28
days increased the water intake, reduced milk production and
had diarrhoea. Excessive intake of potassium salts suppresses
absorption of magnesium in ruminants (Olkowski, 2009). All
analysed samples were compliant with sodium ion MPC (200
mg L-1), while 94.1% of samples were found to be compliant
with potassium ion MPC (12 mg L-1).
Iron is an important microelement, but in excessive doses
it can also be harmful. Water with an increased concentration of
iron has a negative influence on the organoleptic properties of
the water, causes bitter, oily and sour taste, so during the proce-
ssing and preparation of water for consumption it is necessary to
remove iron ions. Iron in the drinking water causes cloudiness
and at higher concentrations gives it a taste of ink. In the wa-
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
and Nutrition 12 (3-4), 126-130 (2017) 129
ter supply system, the occurrence of iron is the most common
consequence of corrosion of pipes and reservoirs. Although high
levels of iron in drinking water do not have toxicological signi-
ficance, secondary metabolic effects should be considered for
at least two reasons: 1) iron may affect the taste of water, and
consequently reduce water intake; and 2) excessive iron intake
may have harmful effects on the metabolism of several essential
micronutrients including copper, zinc, magnesium, manganese
and calcium (Olkowski, 2009). Of the 34 examined samples, 32
(94.1%) were found to be suitable with MPC of 200 µg L-1.
The most common and most noticeable problems with
drinking water are due to its microbiological composition. Not
only are microorganisms considered dangerous but also are their
toxins which often remain in the water when microorganisms are
no longer present. As a sanitary indicator, the number of bacteria
in a milliliter of water is most often determined, and as the most
common causes of infection, especially of the digestive system,
the total number of coliforms and intestinal enterococci is deter-
mined. Microbial contamination, besides the negative impact on
animal health, is also reflected in their productivity, usually by
reduced weight gain. Our previously study of health safety of
drinking water which are used in the milk collection points in the
Bjelovar-bilogora’s district showed that very high percentage of
samples (46.3%) were microbiologically unsuitable (Denžić et
al., 2016). The results of this study showed that only 7 samples
(20.6%) were microbiologically suitable. In 11 (32.4%) samples,
the number of colonies at 22 ° C in 1 mL of water was within
the permissible level which for aerobic mesophiles is 100, while
in 10 (29.4%) samples the number of colonies at 37 ° C in 1
mL of water was below the permitted 20 colonies. According to
the Croatian water quality regulations (OJ 125/2013, 141/2013,
128/2015), coliform bacteria must not be present in the drin-
king water, and only 9 (26.5%) of the samples were found to
be suitable. Water contaminated with Escherichia coli bacteria
and intestinal enterococci is the most common evidence of fae-
cal contamination because they are part of the normal intestinal
microflora of humans and warm-blooded animals. These bac-
teria can survive in the environment for a long period of time
and their concentration is so high that they can be easily proven
even in very diluted samples. In the present study, Escherichia
coli was detected in 21 (61.8%) and enterococci in 23 samples
(67.6%). Clostridium perfrigens can survive for an indefinite pe-
riod because it creates spores in unfavourable conditions. There-
fore, the presence of Clostridium perfrigens and Pseudomonas
aeruginosa indicates contamination with faecal or wastewater
for a longer period of time. Clostridium perfrigens was detected
in 9 (26.5%) samples, while Pseudomonas aeruginosa was de-
tected in only 3 (8.8%) samples.. In the study of Marjanović and
Tofant (2008) who investigated the quality of water for cattle
watering, the water from all analysed wells was also unsuitable,
and only water from water supply system was safe.
In order to destroy pathogenic microorganims, disinfecti-
on of water is necessary. However, irregular dosage of disin-
fectant can affect the taste of water to the extent that animals do
not want to drink it; the concentrations of residual disinfectants,
i.e. free chlorine, are also monitored. Residues of disinfectants
with other substances present in the water can produce by-pro-
ducts with a consequent impact on animal health and produc-
tivity. An example being the dissolved organic carbon which,
when heated with chlorine, produces high-dose trihalomethane
 M. Denžić Lugomer et al: Croatian Journal of Food Technology, Biotechnology
130 and Nutrition 12 (3-4), 126-130 (2017)
carcinogens. Some studies show that over-chlorinated drinking
water was the cause of reproducitive failure, increased number
of spontaneous abortion, return to oestrus number and percen-
tage of stillbirths, and reduced farrowing and total number of
piglets in gilts and sows (Tofant et al., 2010). Also, adverse
effects manifested as an increased percentage of death losses
in all production categories, i.e., suckling, nursery and fatte-
ning pigs (Tofant et al., 2011). Almost all investigated samples
(97.1%) were within MPC for free chlorine (0.5 mg L-1), with a
large number of samples with the free chlorine concentrations
below or very close to the detection limit of 0.02 mg L-1.
Conclusions
The analysis of water supply for cattle on the farms in
northwest Croatia reveals high percentage of chemically and
microbiologically unsuitable water samples. The study showed
that cattle are given unsuitable water on as many as 94.1% of
farms. Since only water supply sources were investigated in the
study, the situation in drinkers and in water containers could be
even worse. These results dictate that farmers should be infor-
med about the importance of the drinking water hygiene. The
quality of the well water is variable, as it may contain contami-
nants due to inadequate drainage from its own or nearby facili-
ties (stables, septic tanks) or after rainfall when surface water
flows into wells. Laboratory test results show water status only
at the time when the sample was taken, so the true state of well
water health safety and its changes can only be determined by
more frequent repetition of the analysis and by appropriate su-
pervision. As one of the solutions, it is recommended to use pu-
blic water supply systems for livestock watering because basic
microbiological, physico-chemical and organoleptic properties
of water are controlled regularly thus making the water safer.
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and Nutrition 12 (3-4), 131-136 (2017) 131

SCIENTIFIC NOTE

Changes in the quality characteristics of carrot juice

preserved with Aframomum danielli seed extract
Abiodun Aderonke Amanyunose1, Olufunmilola Adunni Abiodun2*, Gabriel Olaniyi Adegoke3,
Adegbola Oladele Dauda2

1
Department of Food Science and Technology, Iree, Osun State, Nigeria
2
Department of Home Economics and Food Science, University of Ilorin, Kwara State, Nigeria
3
Department of Food Technology, University of Ibadan, Oyo State, Nigeria

*
Corresponding author: funmiabiodun2003@yahoo.com, abiodun.oa@unilorin.edu.ng

Abstract

Effect of using different concentrations of Aframomum danielli on the physicochemical, microbiological and sensory characteristics of
carrot juice stored under refrigerated and ambient temperatures were examined. This was done to ascertain the concentration and storage
conditions that will enhance the quality of the juice. Carrot juice was produced and treated with different concentrations of Aframomum danielli
extract (5, 10 and 15 %). The juice was pasteurized and stored at 4 oC and 27 oC. Control sample was prepared for each group and the quality
attributes of the samples were monitored for five days. Sensory properties of the juice were conducted on the first day of production. A decrease
in pH was observed during the five days storage with the control samples showing lower pH when compared to the treated samples. The pH of
treated samples (10 and 15 %) was slightly maintained at 4 oC. Titratable acidity increased while the total soluble solids decreased with days of
storage. Vitamin A loss was more pronounced in the control samples than the treated samples which showed more vitamin A retention at higher
concentration of Aframomum danielli at 4 oC. The microbial population was higher in the control juice stored at ambient temperature but was
retarded by low temperature coupled with higher concentrations of A. danielli extract. Sensory evaluation showed that the control and up to 10%
inclusion of A. danielli extract were acceptable

Keywords: Aframomum danielli, carrot juice, physicochemical properties, spices, temperature

Introduction been reduced due to their suspected contribution to the growth

Carrots (Daucus carota) are an excellent source of anti-
oxidant compounds and the richestvegetable sources of the pro-
vitamin A (carotene) which can be used in our daily diet (Bao
and Chang, 1994). They are of good nutritional quality and
could be used to make carrot juice and fiber products which can
be supplied all year round (Bao and Chang, 1994). Fresh carrot
juice has a short shelf life, which makes centralized industrial
production and distribution difficult, and there is also high de-
gree of spoilage in the retail outlet (Alkint, 2003). Reiter et al.
(2003) observed that enzymatic mash maceration, acidification
and thermal treatment of carrot juices prior to juice extraction
was found to be an important step in the production of cloud
stable juices. Reiter et al. (2003) and Demir et al. (2004) pro-
duced carrot juices using different methods and additives but,
however, thermal processing had been reported to alter food
qualities. Thus suitable procedure for maintaining quality of
carrot juice is important (Huang and Bourne, 1983). The use of
artificial additives on food had been reported to cause harmful
effect on human health (Al-Shammari et al., 2014; Inetianbor
et al., 2015). According to Kennedy et al. (1992), additives
have been found to reduce the quality loss usually associated
with juice processing. Synthetic antioxidants are very effec-
tive with high stability and low cost but their use in food has
of carcinogenic tissues in humans (Ho, 1997, Anbudhasan et
al., 2014). This invariably led to general rejection of synthetic
food additives, thus increasing the trend among consumers to-
ward natural ingredients. Several compounds with antioxidant
properties have been isolated from spices, herbs, oil seeds, fer-
mented foods and vegetables (Taghvaei and Jafari, 2015).
Aframomum danielli, an aromatic spice belonging to the
Zingiberaceae family is an effective spice that has been of
commercial value and of wide application in the country. Both
the seeds and leaf extracts do not cause tissue damage and it
lowers significantly the effect of some enzymes that lead to
liver toxicity (Adegoke et al., 2002). It possesses broad-spec-
trum antimicrobial properties as it has been found to inhibit the
growth of some microorganisms such as Salmonella enteridi-
tis, Streptococcus aureus, Aspergillus niger and Pseudomonas
fragii (Adegoke and Skura, 1994, Ashaye et al., 2006). With
the existing information on the application of Aframomum
danielli (Adegoke et al., 2002), this spice could be used as a
preservative on the carrot juice. However, the objective of this
work was to ascertain the changes in the quality characteristics
of carrot juice preserved with Aframomum danielli extract.

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 A. A. Amanyunose et al: Croatian Journal of Food Technology, Biotechnology
132 and Nutrition 12 (3-4), 131-136 (2017)
Materials and methods
Materials
Fresh carrots and Aframomum danielli seeds were pur-
chased from a local market in Ibadan, Oyo State, Nigeria.
MethodsProduction of carrot juice
Matured carrot (500 g) were sorted, washed, trimmed
and sliced with a stainless steel knife. The carrot slices were
weighed and blended with distilled water (125mL) in a warring
blender (Howard et al., 1996). The blended carrots roots were
filtered using a muslin cloth to obtain the juice.
Preparation of Aframomum danielli extract
Aframomum danielli seeds were obtained and cleaned to
remove extraneous matter and adhering particles, the seeds
were then sorted, dried and milled in a hammer mill to separate
the endosperm from the seed coat. The powder (89 % dry mat-
ter) obtained was packed in polyethylene bag to prevent mois-
ture absorption. Fifty grams of Aframomum danielli powder
was dissolved in 1000 mL of distilled water. The suspension
was kept in the refrigerator at 4 oC for 5 days followed by cen-
trifuging at 1000 rpm to obtain supernatant solution and was
then stored at 4 oC until required (Adegoke et al., 2000).
Treatment of carrot juice with
Aframomum danielli extract
Different concentrations of Aframomum danielli extracts
(5, 10 and 15 mL) were added to 95, 90 and 85 mL of the car-
rot juice respectively. The juice was sieved, filled into sterile
bottles and then pasteurized at 71 oC for 15 s. After pasteuriza-
tion the juice was allowed to cool before storage at both refrig-
eration temperature (4 oC) and ambient temperature (27 oC).
The samples were then monitored for their physicochemical
properties for a period of five days.
Physico-chemical composition of carrot juice
The pH of the samples was determined using a pH meter
standardized with buffer solution of pH 7.0 and 4.0 respectively
according to AOAC’s method (1990). The pH of the juice was
read directly after dipping the electrode into the juice. Titrat-
able acidity was determined according to the method of AOAC
(1990) and was expressed in citric acid equivalent. Soluble
solids was expressed as ºBrix and determined by refractometer
(Abbe refractometer).
Vitamin A determination
Carrot juice (2 mL) was added to 10 mL of chloroform
and homogenized properly for 1 hour. The mixture was then
filtered into a 30 mL test tube. Standard solution of β-carotene
was prepared by dissolving 30 µg/mL β-carotene stock solu-
tions in 100 mL of chloroform. Absorbance of sample and
standards were read on the spectrophotometer (Jenway Analog
Colorimeter) at a wavelength of 449 nm and absorption coeffi-
cients of 2592 (Cardoso et al., 2009). Conversion of β-carotene
to vitamin A was done according to Barr (2003) as follows;
Retinol Equivalents (RE) and International Units (IU).
1 RE = 1 μg retinol or 6 μg beta-carotene
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
Sensory analysis
Sensory evaluation was carried out on the carrot juice im-
mediately after processing using 20 panelists chosen from stu-
dents of Food Technology Department, University of Ibadan.
Evaluation was on the following sensory parameters: colour,
taste, aroma and overall acceptability. Panelist evaluated the
quality attributes using 9 point hedonic scale, with 9-liked ex-
tremely and 1-disliked extremely. The data obtained were sub-
jected to analysis of Variance.
Statistical analysis
The analyses were carried out in triplicate. The mean and
standard deviation of the data obtained were calculated. The
data were evaluated for significant differences in their means
using One-Way Analysis of Variance at p ≤ 0.05. Differences
between the means were separated using Tukey’s test with
SPSS (17.0).
Results and Discussion
pH of the control and A. danielli treated carrot juices
ranged from 5.1-6.4. The pH value decreased in juices kept at
27±2 oC than at refrigeration temperature. The control samples
had lower pH values than the treated samples. There was slight
decrease in the control juice pH from 6.3 to 6.08 while at 27±2
ºC, the pH dropped drastically to 5.1 in the juice at day 5. The
pH values of the juice samples dropped during the five days
storage period. pH drop was pronounced in the control samples
than in the samples treated with varying concentrations of A.
danielli (Fig. 1). The pH drop was accompanied with cloud
formation for the samples stored at room temperature similar
to observations of Alkint (2003). Carrot juice stored at 27±2 ºC
had the lowest range of pH values after five days and this could
be as a result of the onset of fermentation process. The pH drop
was inhibited by the addition of A. danielli extract above 10 %
at 4 ºC. However, pH drop was suppressed in dose-dependent
manner at 27±2 ºC. This drop in pH was more prevalent at
27±2 oC due to greater rate of fermentation at this tempera-
ture according to Ashaye et al. (2013). The A. danielli treated
samples had higher pH values compared to the control samples
at refrigerated temperature showing that the spice extract had a
stabilizing effect on the juice.
 A. A. Amanyunose et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 131-136 (2017) 133

A A

B B

Fig. 1: Effect of Aframomum danielli on the pH of carrot Fig. 2: Effect of Aframomum danielli on the titratable acidity
juice stored at 4 oC (A) and 29 oC (B) of carrot juice stored at 4 oC (A) and 27±2 oC (B)

The total titratable acidity changes are shown in Fig. 2.
Titratable acidity of control and Aframomum danielli treated
carrot juices ranged from 0.63-0.70 %. Titratable acidity in-
creased with days of storage (Fig. 2). These values were sta-
bilized at higher concentration (15 %) till the third day but in-
creased slightly at 4 and 5 days of storage at both refrigeration
and ambient temperatures. Total titratable acidity of the sam-
ples increased with days of storage, with the sample stored at
ambient temperature recording higher range of total titratable
acidity. The control samples had higher values compared to
the samples treated with A. danielli extract. A. danielli extract
inhibited the increase in titratable acidity at 4 oC and 27±2 ºC.
Increase in titratable acidity of the carrot juice may possibly
be due to reactions between the carrot juice components and
A. danielli extracts at low temperature. The high pH value 6.4
in the juice treated with 15 % Aframomum danielli extract at
first day result in low acidity and characterizes the carrot juice
as a low-acid food. Rise in total titratable acidity and decrease
in pH indicates increase in the acid concentrations of the carrot
juice with increase in storage time. Jatto and Adegoke (2010)
reported stabilized pH for cashew juice with Aframomum dan-
ielli for one week but addition of additives was observed to
have differential effect on the pH of tomato juice with length
of storage (Hossain et al., 2011).
Total soluble solid are shown in Fig. 3. The values of the
control and Aframomum danielli treated carrot juice ranged
from 6.5-8.5 oBrix and decreased with days of storage. At 4
o
C, the values were constant at 0-2 days and the 4-5 days in the
treated samples. Total soluble solids followed the same trend
in the samples treated with different concentrations of Afra-
momum danielli at 4 and 27±2 oC. The total soluble solids of
A. danielli treated carrot juices were higher than the control
carrot juice and may be as a result of dissolved compounds in
the A. danielli extract. Total soluble solid obtained was with-
in the value (6.9 oBrix) reported by Sharma et al. (2006) for
carrot juice. The values decreased at both temperatures with
storage periods. This corroborates the report of Hossain et al
(2011) that the soluble solids decreased rapidly in tomato juice
with storage time. This could be due to fermentation process
in which the components are broken down into alcohol, wa-
ter and carbon dioxide. Sugars and acids can be considered as
parameters of composition of the juices and hence indices of
adulteration Maireva et al. (2013).

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 A. A. Amanyunose et al: Croatian Journal of Food Technology, Biotechnology
134 and Nutrition 12 (3-4), 131-136 (2017)
A control and carrot juices treated with A. danielli extract at
B vitamin A in carrot juice was possibly due to degradation of
Fig. 3: Effect of Aframomum danielli on the total soluble
solid of carrot juice stored at 4 oC (A) and 27±2 oC (B)
The control sample had the lowest vitamin A value 520.20
µg/100 g at 4 oC in the fifth day while the highest value was
Table 1: Effect of Aframomum danielli on vitamin A (µg/100g) content of carrot juice
Sample
0 1
Control 3150±0.41a 2077±0.21a
5% 3016±0.63b 2900±0.10a
10 % 2961±0.38c 2742±0.19a
15 % 2827±0.22d 2689±0.27a
Value with the same letter down the column are not significantly (p<0.05) different
Microbial load was noticed in the control and 5 % A dani-
elli at 4oC from 3 -5 days (Table 2a and b). The value increased
in the control with days of storage but was constant at 5 % A.
danielli concentration. There was increase in microbial load
of the juice stored at 27±2 oC with higher value (15.0x104cfu/
mLmL) in the control juice at the fifth day. The total viable
bacterial count of the carrot juice increased rapidly with in-
crease in storage periods in the samples kept at 27±2 oC. The
carrot juice stored at ambient temperature had the highest num-
ber of microbial population which may be due to the favorable
temperature with the higher levels of A. danielli concentration
showing a reduction in growth of microorganisms when com-
pared to the control. Retardation of growth was recorded for
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
3150.10 µg/100g in the control juice after production (Table 1).
The values decreased with increased concentration of Aframo-
mum danielli in the first day but later increased with increase
in Aframomum danielli concentration in the fifth day. Storage
at 4 oC retained appreciable level of vitamin A than storing
at ambient temperature. The vitamin A content of the juice
which was calculated as β-carotene equivalent decreased af-
ter the five days of storage for the samples stored 4 and 27±2
o
C. The values for the carrot juices were higher after prepa-
ration but reduced greatly with storage days. There were no
significant differences (p>0.05) in the vitamin A contents of
day 1 in the samples kept at 4 oC. There were significant dif-
ferences (p<0.05) in the juices stored at ambient temperature
at all periods. The vitamin A contents of the juice decreased
with increase in concentration of A. danielli extract. This may
be due to the reduction in carrot juice with increased concen-
tration of A. danielli extract. It has been stated that only about
25 % of total carrot β-carotene is extracted by juicing and
retention during processing has been found to be reasonably
high (Bates et al., 2001). Chen et al. (1995) observed highest
destruction of carotenoids in carrot juice using convectional
canning (121oC for 30min) processing method. Reduction in
carotenoids and was reported to vary according to preserva-
tion techniques and processing conditions (Bao and Chang,
1994). Carotenoids are generally stable in their natural envi-
ronments but when food is heated or when they are extracted
into solution, in oils or organic solvents, they become much
more labile (Coultate, 2002).
4 oC 27±2 oC
Days of storage
5 1 5
520±0.15d 1931±0.23c 782±0.33d
1907±0.12c 1986±0.16a 989±0.19c
1927±0.24b 1966±0.10b 1012±0.09b
1934±0.11a 1902±0.12d 1361±0.18a
refrigerated samples at increasing A. danielli concentrations.
According to Akhtar et al. (2012) spoilage can be controlled
by keeping the juice in refrigerator and using preservatives.
Quality changes in strawberry were also observed to be due
to microbiological and physiological processes. This was in
agreement with Adegoke and Skura (1994) that A. danielli pos-
sesses broad-spectrum antimicrobial activity and the results in
study showed that the spice antimicrobial activity is enhanced
with low-temperature storage. Moreover, it was concluded that
beyond one week, the preservative potential of Aframomum
danielli at 15% concentration becomes very low (Adegoke et
al., 2000).
 A. A. Amanyunose et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 131-136 (2017) 135

Table 2a: Microbial counts (cfu/mL) of carrot juice at 4 oC difference (p<0.05) in the color, taste, aroma and overall ac-
ceptability of all the juice produced. The sensory properties
Sample Days revealed no significant differences (p>0.05) in the colour and
aroma of carrot juice with 10 and 15 % A. danielli extract.
1 3 5 The control carrot juice was significantly different (p<0.05)
Control Nil 1.8 x10 1
2.5 x 102 from A. danielli treated carrot juices in the taste and overall
acceptability. The taste and acceptability of the carrot juices
5% A. danielli Nil 1.0 x102 1.0 x 102 were affected by increased concentration of A. danielli extract
10% A. danielli Nil Nil Nil in the carrot juice. The taste of the carrot juice was acceptable
at 5 % A. danielli extract concentration but at 10 % concentra-
15% A. danielli Nil Nil Nil tion, slight change in the taste of the carrot juice was observed.
There was an objectionable taste at 15 % A. danielli extract
concentration. The control carrot juice was more acceptable
Table 2b: Microbial counts (cfu/mL) of carrot juice at 27±2 oC than the A. danielli treated juices. Addition of up to 10 % A.
danielli concentration to carrot juice is desirable as higher con-
Sample Days centrations (15%) of A. danielli impact undesirable effect on
the taste of the product. Ashaye et al. (2013) reported accept-
1 3 5 ability of roselle grape juice preserved with A. danielli at zero
Control Nil 10.8 x10 2
15.0 x 104 and one week of storage. Likewise, Ishola et al. (2015) pre-
served zobo drink with Aframomum danielli and black pepper
5% A. danielli Nil 6.6 x102 8.1 x 103 extracts. But it was observed that 12% and 4% Aframomum
10% A. danielli Nil 4.0 x 102 5.0 x 102 danielli and black pepper extracts were the most preferred in
the zobo drink. Adegoke et al. (2013) reported acceptability
15% A. danielli Nil 2.1 x 102 3.0 x 102 of yoghurt with A.danielli, strawberry and vanilla. Therefo-
re, addition of A. danielli to food had impact on the taste and
The sensory properties carried out on the freshly prepared aroma of the product.
carrot juice were shown in Table 3. There were significant

Table 3: Sensory characteristics of carrot juice treated with A. danielli

Sample Colour Taste Aroma Overall Acceptability
Control 6.2±0.16c 6.6±0.12a 6.5±0.10a 7.1±0.15a
5% A. danielli 6.4±0.20b 6.4±0.15b 6.4±0.08b 6.7±0.14b
10% A. danielli 6.5±0.11a 6.0±0.09c 6.0±0.17c 6.5±0.11c
15% A. danielli 6.5±0.19a 5.2±0.14d 6.0±0.13c 6.4±0.07d

Value with the same letter down the column are not significantly (p<0.05) different

Conclusions Adegoke, G.O. and Skura, B.J. (1994). Nutritional profile

The results of this study showed that A. danielli can be
used effectively as a preservative for carrot juice. This was
reflected in the total soluble solids, acidity and vitamin A re-
tention of the juice when compared to the control samples. Al-
though there were significant differences in the taste and over-
all acceptability of the control and carrot juice treated with A.
danielli but the sensory analysis conducted on the fresh juice
revealed that incorporation of up to 10 % A. danielli extract
into carrot juice were desirable.
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 T. Brčina et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 137-145 (2017) 137

ZNANSTVENA BILJEŠKA / SCIENTIFIC NOTE

Reološka svojstva mliječnog proizvoda

na bazi svježeg sira i voća
Rheological properties of dairy products
based on fresh cheese and fruit
Tijana Brčina*, Milica Vilušić, Amel Selimović

Odsjek za Prehrambenu tehnologiju, Tehnološki fakultet, Univerzitet u Tuzli, Univerzitetska 8, 75 000 Tuzla, Bosna i Her-
cegovina

* Corresponding author:
Tel: 00 387 35 320 806; Fax: 00 387 35 320 741; E-mail: tijana.brcina@untz.ba

Sažetak

Cilj rada je bio ispitati reološka svojstva mliječnog proizvoda na bazi svježeg sira i voća, u ovisnosti o udjelu mliječne masti i upotrje-
bljenih starter kultura. Reološka svojstva su ispitana na Physica MCR 301 reometru (Anton Paar, Austria). Rezultati reoloških ispitivanja su
pokazali razlike u modulima elastičnosti G’ i viskoznosti G’’ u ovisnosti o sadržaju suhe tvari, masti i mikrobnoj starter kulturi. Mliječni proizvod
je proizveden upotrebom mikrobnih starter kultura CHN 22, XT - 303 i mješavina CHN-22 i Lyofasf SAB 440 B u omjeru 2:1, a svježi sir koji
je korišten za dobivanje mliječnog proizvoda je proizveden od obranog (0.9% m.m.) i djelomično obranog mlijeka (1.5% m.m.). Produkcija eg-
zopolisagarida od strane mikrobne sarter kulture Lyofast SAB 440 B je utjecala na manje vrijednosti G ‘, G “i G * u uzorcima koji su dobiveni
inokulacijom sa ovom kulturom u kombinaciji sa CHN 22.
Ključne riječi: mliječni proizvod, modul elastičnosti G’, modul viskoznosti G’’, mikrobne sarter kulture, egzopolisaharidi.

Abstract

The aim of the paper was to examine the rheological properties of dairy products based on fresh cheese and fruit, depending on the pro-
portion of dairy fat and the use of starter cultures. Rheological properties were tested on the Physica MCR 301 rheometer (Anton Paar, Austria).
The results of rheological tests have shown differences in elasticity modulus G ‘and viscosity G’ depending on the content of dry matter, fat and
microbial starter culture. The dairy product was produced using CHN 22, XT - 303 microbial starter cultures and CHN - 22 and Lyofasf SAB
440 B mixes in 2:1 ratio, and the fresh cheese used to obtain dairy products was manufactured from molded (0.9% mm) and partially skimmed
milk (1.5% mm). The production of exopolysaccharide by microbial sarter culture Lyofast SAB 440 B has affected the reduction of both modules
in the samples obtained by inoculation with this culture in combination with CHN 22.
Key words: dairy product, modulus of elasticity G ‘, viscosity modulus G’’, microbial culture starter, exopolysaccharides.

Uvod jali se smatraju viskoelastičnima, ako je za vrijeme i poslije

Uloga fermentiranih mliječnih proizvoda u ljudskoj pre-
hrani je poznata od davnina. Ovi proizvodi odavno predstav-
ljaju sastavni dio prehrane. Ljekovita i prehrambena svojstva
raznih fermentiranih mliječnih proizvoda iskusile su mnoge
generacije (Panesar, 2011). Reološka i mikroskopska istraži-
vanja su pokazala da se svježi sir može opisati kao disperzija
ili pasta hidratiziranog kiselog gela kazeinskih čestica u sirutki
(Korolczuk, 1993; Tscheuschner i Nimbs, 1993; Ozer i sur.,
1998; Senge i sur., 1998; Senge, 2002). Inače, svježi sirevi pos-
jeduju viskoelastična svojstva.
Sir je viskoelastični materijal i sve teksturalne karakter-
istike su kombinacija mjerljivih reoloških i mehaničkih svo-
jstava. Svježi sirevi posjeduju viskoelastična svojstva. Materi-
deformacije dio dobivene mehaničke energije pohranjen u ma-
terijalu (elastični dio), a dio raspršen (viskozni dio).
Dinamičke metode prate proces geliranja proteina (ko-
agulacije) i određuju reološka svojstva finalnog proizvoda
(gruša). Kod ove metode, materijal koji se ispituje je podrvrg-
nut kontroliranim oscilacijama, tj. frekvenciji koja određuje
brzinu deformacije uzorka.
Ispitivanje proteinske mreže hrane, kao što je sir, obav-
lja se sa malim amplitudama oscilatornog smicanja, koje
nisu destruktivnog karaktera, i pri tome daju informacije o
modulima elastičnosti (G’) i viskoznosti (G’’) (Tunick i Hek-
ken, 2012). Viskoelastične karakteristike, kao što su modul
elastičnosti i viskoznosti pomažu da se bolje razumije struktura
supstrata, utjecaj temperature, interakcije komponenata i fazna

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 T. Brčina et al: Croatian Journal of Food Technology, Biotechnology
138 and Nutrition 12 (3-4), 137-145 (2017)
transformacija (Velez-Ruiz, 2008). Reološka svojstva sira su ta
koja daju odgovor na stres i naprezanje koji se primjenjuju ti-
jekom kompresije, sječenja ili rezanja (Andronoiu i sur, 2010).
Viskoelastične gelove karakterizira određivanje modula
G’ i G’’ u linearno viskoelastičnoj regiji (LVR). U linearno
viskoelastičnoj regiji, moduli G’ i G’’ ostaju konstantni pri
promjeni amplitude deformacije. Ova regija se uočava pri
nižim vrijednostima frekvencije ili deformacije. Kako se vri-
jednost deformacije povećava, tako će vrijednosti modula G’
i G’’ početi smanjivati ili povećavati te to predstavlja kraj lin-
erno viskoelastične regije.
Važno je razlikovati elastični i viskozni parametar u
viskoelastičnim mliječnim proizvodima kako bi se mogao
bolje procijeniti sam tijek razvoja varijabli proizvoda (Pechak
i Smith, 2007).
Faktor gubitka (faktor prigušenja) tan δ se računa iz odno-
sa G’’ i G’ (). ″Gel točka″ (točka križanja) se može odrediti
određivanjem mjesta na kojem je tan δ neovisan o frekvenciji
ili temperaturi i gdje vrijednosti modula G’ i G’’ postaju jed-
nake (G’ = G’’). Vrijednost tan δ u ovoj točki je 1. Pojam tan
δ se spominje kao tangens gubitka i mjera je relativne vrijed-
nosti viskozne i elastične komponente ili kao mjera energije
koja se izgubila i energija koja se pohranila pod djelovanjem
deformacije.
Kazeinski gelovi su odgovorni za strukturu i reološka
svojstva proizvoda koji imaju gelastu strukturu, rastezljivi
su i podložni frakturama. Reološka ispitivanja se koriste kao
metode određivanja kvalitete proizvoda mliječne industrije i
kao metode istraživanja strukture proizvoda (Tunick, 2000).
Kiseli kazeinski gel se ponaša kao viskoelastični materijal.
Sa povećanjem viskoznih svojstava povećava se tan δ,
dok se povećanjem elastičnih svojstava smanjuje tan δ. Ta-
kođer, može se zaključiti da je tan δ idealan parametar za
praćenje promjene iz viskoznog tečnog (mlijeko) u čvrsto (gel)
stanje. Primjena malog napona na kiseli kazeinski gel, rezul-
tira izduživanjem proteinskog matriksa (koje ima povratnu
reakciju). Čvrstoća gela se povećava sa povećanjem sadržaja
proteina, toplinskim tretmanom mlijeka, povećanjem tempera-
ture pri kojoj se dodaju kiseline ili sredstava za acidifikaciju, i
sniženjem pH vrijednosti (Maćej i sur., 2004).
Dinamička mjerenja na kazeinskim gelovima daju in-
formacije o kratkim interakcijama, posebno o konformaciji i
strukturi kazeinskih čestica (Roefs i sur., 1990). Eksperimenti
moraju biti provedeni pri linearnom viskoelastičnom rasponu
kako bi struktura sira ostala netaknuta. Na taj način, moduli su
funkcije vremena, a ne samo veličine naprezanja ili deformaci-
je (Rao i Skinner, 1986).
Kelly i O’Donnell (1998) su ispitivali Quark, koji sadrži
80 % vode, utvrdivši da proteoliza i način proizvodnje sman-
juju G’ vrijednost. Schulz i sur. (1999) su razvili metodu za
in-line (unutarnje) praćenje formacija kazeinskih gelova ti-
jekom proizvodnje quark sira i jogurta upotrebom oscilacijske
reometrije.
Bakterije mliječne kiseline koje su sposobne producirati
egzopolisaharide (EPS) imaju važnu ulogu u mliječnoj indu-
striji zbog njihovog doprinosa konzistenciji i reologiji fermen-
tiranih mlijeka (Florenca, 2013). Također je utvrđeno da je niži
modul elastičnosti i viskoznosti pri oscilatornom mjerenju kod
jogurta sa egzopolisaharidima (EPS) nego kod jogurta bez eg-
zopolisaharida zbog indikacija inicijalne neelastičnosti koja je
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
niža kod uzoraka koji sadrže egzopolisaharide (Hassan i sur.,
2003). Do istih rezultata je došla i Florenca (2013) istraživan-
jem reologije kozjih sireva za mazanje koji su proizvedeni sa
autohtonim starter kulturama sa i bez produkcije egzopolisa-
harida. Oba uzorka sira su okarakterizirana kao slabo visko-
elastični gelovi i pseudoplastični proizvodi, gdje su sirevi sa
EPS pokazivali manje vrijednosti G’, G’’ i η*, u istom raspo-
nu frekvencije, od sireva bez EPS. Svrha rada je bila ispitati
promjene modula G’ i G” ovisno o udjelu suhe tvari i masti u
mliječnom proizvodu.
Također je praćen utjecaj EPS mikrobne starter kulture na
navedene module.
Materijali i metode
Proizvodnja prototipa miječnog proizvoda na bazi svje-
žeg sira i voća provedena je u laboratoriju za Prehrambenu teh-
nologiju Tehnološkog fakulteta Univerziteta u Tuzli. Za proi-
zvodnju svježeg sira korišteno je komercijalno UHT mlijeko sa
0.9% (M1) i 1.5% (M2) mliječne masti. Za izravnu inokulaciju
u mlijeko za sir korištene su FD-DVS kulture (proizvođača
Chr. Hansen, Danska i Sacco, Italija):
1. CHN-22 u Lactococcus lactis ssp. lactis
sastavu:
Lactococcus lactis ssp. cremoris
Lactococcus lactis ssp. lactis biovar diacety-
lactis
Leuconostoc mesenteorides ssp. cremoris
Streptococcus thermophilus
2. XT–303 u Lactococcus lactis ssp. lactis
sastavu:
Lactococcus lactis ssp. cremoris
Lactococcus lactis ssp. lactis biovar diacety-
lactis
Leuconostoc mesenteorides ssp. cremoris
Leuconostoc pseudomesenteorides
3. CHN – 22 : Lyofast SAB 440B = 2:1
Lyofast SAB
440 B (pro-
ducira EPS) Streptococcus thermophilus
u sastavu:
Lactobacillus acidophilus
Bifidobacterium animals ssp.lactis
Kao voćni dodatak korišteno je voće iz pasteriziranih
kompota ananasa (V1), breskve (V2) i kruške (V3), u laganom
sirupu (Iska Qualität, I. Schroeder KG, Njemačka). Dodava-
nje je provedeno po odabranoj formulaciji (25% voća na masu
sira). Mlijeko je termički obrađeno na temperaturi 75°C/30
sekundi, zbog homogenizacije i bolje denaturacije proteina si-
rutke i njihova uklapanja u strukturu gruša, ohlađeno na tem-
peraturu inokulacije (25°C), nacjepljeno sa mikrobnim starter
kulturama.
 T. Brčina et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 137-145 (2017) 139

Fermentacija je trajala između 11 i 12 sati na 25°C, do po- provedena su na temperaturi 20°C. Test amplitude proveden
stizanja vrijednosti pH od 4.6 – 4.7. Potom je izvršeno rezanje je sa utvrđenim parametrima: deformacija γ = 0.01-100% i
gruša, hlađenje i cijeđenje sirutke pomoću pamučnih tkanina, kutne frekvencije ω = 10 rad/s. Kod ove oscilacijske probe
u trajanju od oko 8 sati, na 20 °C. Nakon završenog cijeđenja varira amplituda od γ = 0.01-100%, a frekvencija se održava
gruša, svježi sir je dobro homogeniziran u mikseru i razdijeljen konstantnom. Ovim testom se ne ispituje stanje mirovanja
na 3 dijela. U svaki dio je dodana jedna vrsta sjeckanog voća. uzorka, uz kutnu frekvenciju ω = 10 rad/s gdje opterećenje
U jedan dio ananas, u drugi breskva i u treći je dodana kruška. smicanja ne odgovara mirovanju.
Dobiveni proizvodi su skladišteni u hladnjaku u plastič- Ako bi se ispitivalo stanje mirovanja uzorka onda bi
nim posudama i čuvani 10 dana na temperaturi +4°C. Oznake trebalo smanjiti ω. Sve vrijednosti reoloških parametara su u
uzoraka, udjel mliječne masti, vrste mikrobnih kultura i vrste logaritamskom obliku.
voća korištene pri laboratorskoj proizvodnji sirnog deserta pri- Dinamički test je definiran slijedećim karakteristikama:
kazane su u tablici 1. modula elastičnosti - G›, modula viskoznosti - G» i dinamičkim
Tablica 1. Oznake uzoraka sirnog deserta viskozitetom (η) u obliku kompleksnog modula - G*, gdje je
Table 1. Samples of dairy desserts
G* = G'+G''
Oznaka Mliječna Mikrobna kultura Voće
uzorka mast u G* mjera ukupnog otpora gruša na deformaciju (Mezger,
mlijeku (%) 2000; Vilušić, 2002).
M1K1A Ananas
CHN -22
M1K1B Breskva Statistička obrada
M1K1K Kruška
0.9 Analiza varijance (ANOVA) je provedena upotrebom
M1K2A Ananas SPSS softvera (verzija 22) gdje je određen utjecaj kulture,
XT - 303 voća i udjela mliječne masti u mlijeku od kojeg je proizveden
M1K2B Breskva
mliječni proizvod. Tukey test je korišten da bi se odredilo koji
M1K2K Kruška uzorci su statistički značajni (p< 0.05).
M1K3A Ananas
CHN – 22 : Lyofast
M1K3B SAB 440 B = 2:1 Breskva Rezultati i rasprava
M1K3K Kruška
Sadržaj mliječne masti u uzorcima mliječnog proizvoda
M2K1A Ananas na bazi svježeg sira i voća je varirao od 1.40 do 2.48%, a sa-
CHN -22 držaj suhe tvari od 15.13 do 20.62%. Dinamičke metode često
M2K1B Breskva
se koriste za praćenje procesa koagulacije, odnosno formiranja
M2K1K Kruška gruša, i određuju reološka svojstva finalnog proizvoda.
1.5
M2K2A Ananas Pri upotrebi dinamičkih metoda za reološka mjerenja
XT - 303 sam proces koagulacije ne utječe na mjerenje. Izlazni signal
M2K2B Breskva je predstavljen kao dvije krivulje od kojih jedna predstavlja
M2K2K Kruška elastična svojstva, a druga viskozna svojstva reološko-defor-
macijskog ponašanja. Ovakva metoda se može primjenjivati
M2K3A Ananas
za mjerenje elastičnih i viskoznih svojstava proizvoda tijekom
CHN – 22 : Lyofast
M2K3B SAB 440 B = 2:1 Breskva cijelog procesa koagulacije ili praćenje svojstava gotovog pro-
izvoda. Kada se provode mjerenja pri konstantnoj frekvenci-
M2K3K Kruška
ji, ova metoda omogućava snimanje viskoelastičnog svojstva
sustava kao funkcija vremena (Bohlin i sur., 1984; Ikeda i
Mliječna mast i suha tvar uzoraka mliječnog proizvoda Foegeding, 2003). Kod dinamičkih metoda materijal, koji se
na bazi svježeg sira i voća je utvrđena standardnim analitičkim ispituje je podvrgnut kontroliranim oscilacijama, tj. frekvenci-
metodama: mliječna mast Gerber metodom, a suha tvar suše- ji koja određuje brzinu deformacije uzorka. Nastanak gruša je
njem na 105°C do konstantne mase. osnova svakog fermentiranog mliječnog proizvoda, koji prema
Kudryashov i sur. (2001), prati prvo povećanje energije gubi-
taka uslijed agregiranja kazeinskih čestica u nakupine i dalj-
Reološka mjerenja njeg međusobnog povećanja modula elastičnosti i viskoznosti,
obzirom da je gruš već formiran. Dinamički test je definiran
Reološka svojstva mliječnog proizvoda na bazi svježeg sa modulom elastičnosti (G›), modulom viskoznosti (G››), di-
sira i voća ispitana su na Physica MCR 301 reometru namičkim viskozitetom (η-Pas) i kompleksnim modulom G*.
(Anton Paar, Austria). MCR reometri temelje se na konceptu Vrlo niska temperatura inkubacije (23°C), sporo zakiseljavanje
tehnologije na granici rezanja (rezni rub). Sva reološka (fermentacija duže traje) i procesi formiranja gruša rezultiraju
mjerenja mliječnog proizvoda na bazi svježeg sira i voća manjim vrijednostim tan δ (krivulje su ravnije) u odnosu na

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gruš nastao na višoj temperaturi (30°C) (Lucey, 2004). Tempe-
ratura fermentacije svih proizvedenih uzoraka mliječnog proi-
zvoda na bazi svježeg sira je bila 25°C i bila je relativno spora
sa trajanjem od 11.5 i 12 sati. Na osnovu rezultata reoloških is-
pitivanja vrijednost G››, koja opisuje viskozna svojstva mliječ-
nog proizvoda na bazi svježeg sira i voća, niža je od G›, koja
opisuje elastični dio reološko-deformacijskog ponašanja. Na
osnovu toga, proizlaze elastična svojstva uzoraka mliječnog
proizvoda na bazi svježeg sira i voća. Sumiranjem realne kom-
ponente G› i imaginarne komponente G›› dobije se kompleksni
modul smicanja G* koji predstavlja čvrstoću gruša. Paralelne
krivulje G› i G›› pokazuju tipično viskoelastično ponašanje,
što i odgovara tvrdnji da uzorci mliječnog proizvoda na bazi
svježeg sira i voća posjeduju viskoelastična svojstva. Odnos
G› i G›› nije puno odstupao kod uzoraka proizvoda na bazi
svježeg sira i voća i iznosio je G›/G›› ≈ 4. Odgovor gruša na
oscilacijske deformacije i postojanje relativno veće vrijednosti
modula elastičnosti, prikazane u obliku veće energije čuvanja
na skali kraćeg frekvencijskog perioda ili skali deformacije,
mogu biti posljedica preniskog ili visokog napona smicanja iz
linearnog viskoelastičnog područja dinamičkog testa napona
smicanja. Kiseli kazeinski gel ima viskoelastična svojstva, a
kao takav pokazuje karakteristike i čvrstih tijela (elastičnosti)
i tekućina (viskozitet) (Lucey i sur., 1997b), (vrijednosti koja
ovisi od fine ravnoteže spontane dezintegracije strukture i br-
zine deformacije primjenjene preko instrumenta). Analiza va-
rijance (tablica 2) pokazala je statistički signifikantnu razliku
za vrijednosti G›, G›› i G*, jer je dobivena F-vrijednost (tes-
tna vrijednost) veća od granične tablične vrijednosti. Ovome
u prilog govori i činjenica da je nivo signifikatnosti p < 0.05.
Analiza varijance (tablica 3) pokazala je da postoji statistički
signifikantna razlika unutar grupa podataka za G›, G›› i G* (p
< 0.05). S obzirom na statistički signifikantnu razliku, prove-
den je Tukey test (tablica 4). Radi uspoređivanja vrijednosti
unutar grupa G›, G›› i G*, proizvoljno su izabrane tri točke
amplituda 0.0316, 0.1 i 1%, pri konstantnoj frekvenciji kako je
ranije i navedeno.

Tablica 2. Analiza varijance podataka modula G›, G›› i G*

Table 2. Analysis of variance G›, G›› i G* moduls

Izvor Sume kvadrata Stupnjevi Prosjeci F (testna p-nivo sig.

slobode vrijed.)
Varijance kvadrata
Između proizvoda 1.135 · 10 7
8 1418828.294 5.406 0,000
moduli

Analitička greška 4.016· 10 7

153 262459.734

Ukupno 5.151· 107 161

Tablica 3. Analiza varijance modula G›, G›› i G* unutar grupe podataka

Table 3. Analysis of variance G›, G›› i G* moduls in side group
Izvor Sume Stupnjevi Prosjeci F (testna p-nivo
slobode vrijed.)
Varijance kvadrata kvadrata sig.
G’ Između proizvoda 51003315,716 17 3000195,042 32,089 0,000
Analitička greška 25524232,910 273 93495,359
Ukupno
76527548,626 290

G’’ Između proizvoda 3646394,239 17 214493,779 125,977 0,000

Analitička greška 464821,719 273 1702,644

Ukupno
4111215,958 290

G* Između proizvoda 81926367,846 17 4819198,109 40,949 0,000

Analitička greška 32128733,320 273 117687,668

Ukupno
114055101,166 290

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Tablica 4. Statistička obrada rezultata reoloških mjerenja (a)

(Tukey test)
Table 4. Statistical analysis of rheological measurement
results (Tukey test)

G’ G’’ G*
M1K1A5,6 M1K1A8 M1K1A6

M1K1B2,3 M1K1B3 M1K1B3,4

M1K1K7 M1K1K9 M1K1K7

M1K2A10 M1K2A13 M1K2A10

M1K2B8 M1K2B11 M1K2B8

(c)
M1K2K9 M1K2K12 M1K2K9

M1K3A4 M1K3A6 M1K3A5

M1K3B1,2,3 M1K3B2 M1K3B2,3,4

M1K3K4 M1K3K5 M1K3K5

M2K1A1,2 M2K1A2 M2K1A1,2,3

M2K1B1 M2K1B1 M2K1B1

M2K1K4 M2K1K4 M2K1K4

M2K2A1 M2K2A2 M2K2A1,2

M2K2B4,5 M2K2B5 M2K2B5

(d)
M2K2K6 M2K2K7 M2K2K6

M2K3A7 M2K3A10 M2K3A7

M2K3B1,2,3 M2K3B2 M2K3B1,2,3

M2K3K1 M2K3K1 M2K3K1,2

1,2,3,4,5,6,7,8,9,10,11,12,13 - srednje vrijednosti u istoj ko-

loni, sa različitim eksponentom su signifikantno različite
(p<0,05)
U reološkom dinamičkom spektru, modul elastičnosti
(G›) i modul viskoznosti (G››) se smanjuju pri zagrijavanju i
povećavaju pri hlađenju. Svježi sirevi (Queso Blanco, Queso
Blanco con Frutas, Queso Para Freir and Parela) imaju veći
elastični modul G› 2.64-3.63 x 104 Pa) i viskozni modul G›› (f)
(0.79 -1.12 x 104Pa) u odnosu na sireve za topljenje. Sa po-
većanjem temperature i dužine čuvanja, G› i G›› kod svježih
sireva značajno se smanjuju (Park, 2006). Na osnovu rezultata
reoloških mjerenja vrijednosti modula G› i G›› bile su veće kod
uzoraka proizvedenih od mlijeka sa 0.9 % mliječne masti, pa se
može zaključiti da se vrijednosti modula G› i G›› smanjuju sa
povećanjem sadržaja suhe tvari i masti u uzorcima mliječnog
proizvoda na bazi svježeg sira i voća što je vidljivo na slikama
1(a), 1(c)¸ 1(f), 2 (a) i 2(e).

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(g) mlijeka sa 0.9 % m.m., sadržaj suhe tvari iznosio je od 15.13

do 16.25 %, a sadržaj masti od 1.40 do 1.76% dok su uzorci
proizvedeni od mlijeka sa 1.5 % m.m. sadržavali od 20.32 do
21.40% suhe tvari te od 1.96 do 2.48% mliječne masti.

(a)

(h)

(b)

(i)

(d)

Slika 1. Promjene modula čuvanja G› i modula gubitka

G›› u ovisnosti o deformaciji uzorka proizvedenog
od mlijeka M1: M1K1A (a), M1K1K (c), M1K2A
(d), M1K2K (f), M1K3A (g), M1K3B (h) i M1K3K
(i)
Figure 1. Changes of G ‹storage module and loss modu-
lus G› depending on deformation of the sample
produced from M1 milk: M1K1A (a), M1K1K (c),
M1K2A (d), M1K2K (f), M1K3A (g), M1K3B (h)
and M1K3K (i)

Uzorci koji su dobiveni od obranog mlijeka sa 0.9% mli-

ječne masti imali su veće vrijednosti modula G› i G›› od uzo-
raka proizvedenih od djelomično obranog mlijeka s 1.5 % mli-
ječne masti. U uzorcima mliječnog proizvoda proizvedenih od

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(e) (i)

(f)
Slika 2. Promjene modula čuvanja G› i modula gubitka
G›› u ovisnosti o deformaciji uzorka proizvedenog
od mlijeka M2K1A (a), M2K1B (b), M2K2A (d),
M2K2B (e), M2K2K (f), M2K3A (g), M2K3B (h) i
M2K3K (i)
Figure 2. Changes of G ‹storage module and loss modu-
lus G› depending on deformation of the sample
produced from M2 milk: M2K1A (a), M2K1B (b),
M2K2A (d), M2K2B (e), M2K2K (f), M2K3A (g),
M2K3B (h) and M2K3K (i)

Mliječna mast pozitivno utječe na teksturu, okus i boju

mliječnih proizvoda (Haque i Ji, 2003). Smanjen sadržaj masti
u jogurtu uzrokuje formiranje krhke gel strukture jogurta,
(g) što ukazuje na lošija reološka svojstva, teksturu, okus i miris
(Lobato-Calleros i sur, 2014). Iz tablice 4 vidljivo je i da
uzorci sa većim udjelom mliječne masti imaju niže vrijednosti
modula G› i G›› kod uzoraka nacijepljenih sa K1 i K2 starter
kulturom (p<0.05). Uzorci nacijepljeni s mješavinom starter
kultura (K3) ne pokazuju takve rezultate.
Kod uzoraka s ananasom i breskvom, vrijednosti modula
G’’ su manje od vrijednosti modula G’, što znači da prevla-
dava karakter elastičnog dijela uzorka svježeg sira. Budući da
u svim proizvedenim uzorcima mliječnog proizvoda na bazi
svježeg sira i voća dominira G’ > G’’ znači da spomenuti uzorci
zadržavaju tan δ < 1. Svi uzorci mliječnog proizvoda na bazi
svježeg sira i voća su imali omjer viskoznih i elastičnih karak-
teristika između 0 i 1 (tan δ). To potvrđuje vrijednost tan δ koja
se nalazila u intervalu od 0.22 do 0.3 što dokazuje da mliječni
(h) proizvod na bazi svježeg sira i voća posjeduje elastična svoj-
stva. U slučaju idealno elastičnog tijela tan δ je jednak 0, a za

idealno viskoznu tekućinu tan . Tan δ je idealan para-

metar za praćenje promjene iz viskoznog tekućeg (mlijeko) u
čvrsto (gel) stanje (Mezger, 2000; Maćej i sur., 2004). Pojam
tan δ se spominje kao tangens gubitka i mjera je relativne vri-
jednosti viskozne i elastične komponente ili kao mjera energije
koja se izgubila i energija koja se pohranila pod djelovanjem
deformacije. U studiji koju su proveli El-Garawany i Abd El
Salam (2005) modul čuvanja G’ je bio veći od modula gubitaka
G’’ na različim temperaturama. Općenito, log (G′) i log (G″)
rastu sa povećanjem primjenjene frekvencije, čime se ukazuje
na slabo umreženu strukturu gela.

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Uzorci mliječnog proizvoda na bazi svježeg sira i ananasa
proizvedenih od obranog mlijeka sa 0.9% mliječne masti i
djelomično obranog mlijeka sa 1.5% mliječne masti inokulirani
sa mikrobnim starter kulturama XT-303 i mješavinom starter
kultura CHN-22 i Lyofast SAB 440 B u omjeru 2:1 imali su
najveće vrijednosti modula G› (M1K2A i M2K3A) (slike
1(d) i 2(g)). Također, Tukey testom pokazalo se da ovi uzorci
posjeduju veće vrijednosti. Najmanje vrijednosti pokazali su
uzorci mliječnog proizvoda i breskve proizvedeni od obranog
mlijeka s 0.9% mliječne masti i djelomično obranog mlijeka
sa 1.5% mliječne masti inokulirani sa mikrobnim starter
kulturama CHN-22 i mješavinom starter kultura CHN-22 i
Lyofast SAB 440 B u omjeru 2:1 (M1K3B – G››, M1K3K–
G›, M2K1B – G››, M2K2A – G›) (slike 1(h), 1(i), 2(b) i 2(d))
(tablica 4). Najveće vrijednosti modula G›› su imali uzorci
mliječnog proizvoda na bazi svježeg sira i ananasa proizvedeni
od obranog mlijeka sa 0.9% mliječne masti i djelomično
obranog mlijeka sa 1.5% mliječne masti inokulirani sa
mikrobnim starter kulturama XT - 303 i mješavinom starter
kultura CHN-22 i Lyofast SAB 440 B u omjeru 2:1 (M1K2A
i M2K3A) (slike 1(d) i 2(g)), a najmanje vrijednosti pokazali
su uzorci mliječnog proizvoda na bazi svježeg sira i breskve
proizvedeni od obranog mlijeka sa 0.9% mliječne masti i
djelomično obranog mlijeka sa 1.5% mliječne masti inokulirani
sa XT – 303 i mješavinom starter kultura CHN-22 i Lyofast
SAB 440 B u omjeru 2:1 (M1K3B i M2K1B) (slike 1 (h) i
2(b) Starter kulture također značajno mogu poboljšati reološke
karakteristike proizvoda. Primjena i inkorporiranje probiotika
u starteru također može utjecati na tijek fermentacije, proces
formiranja gela i reološke karakteristike finalnog proizvoda.
Poznato je da su egzopolisaharidi (EPS) produkti fermentacije
koji poboljšavaju viskozitet proizvoda i smanjuju sinerezu
u usporedbi sa proizvodima bez EPS kultura (Iličić, 2010).
Utvrđeno je da je niži modul elastičnosti i viskoznosti pri
oscilatornom mjerenju kod EPS jogurta nego kod jogurta bez
EPS zbog indikacija inicijalne neelastičnosti koja je niža kod
uzoraka koji sadrže egzopolisaharide (Hassan i sur., 2003).
Mikrobna starter kultura Lyofast SAB 440 B u svom sastavu
pored dvije probiotičke bakterije sastoji se i od posebno
odabranih sojeva Streptococcus thermophilus koji produciraju
egzopolisaharide. Pet od šest uzoraka mliječnog proizvoda na
bazi svježeg sira i voća koji su bili inokulirani ovom starter
kulturom su imali niže vrijednosti modula G› i G›› (slike 1 (g-i),
2(h) i 2 (i)) jer je produkcija egzopolisaharida imala utjecaj na
sniženje vrijednosti oba modula. Analiza varijance je pokazala
statistički signifikantnu razliku unutar grupa podataka G› i G››
između uzoraka (p<0.05). Tukey test (tablica 4) je pokazao da
su egzopolisaharidi imali utjecaja na niže vrijednosti modula
G’ i G’’ samo kod uzoraka koji su proizvedeni od mlijeka s 0.9
% m.m. To nije bio slučaj kod uzoraka proizvedenih od mlijeka
sa 1.5% m.m. uz EPS kulture, gdje je došlo do porasta vrijed-
nosti modula G’ i G’’ u odnosu na uzorke bez EPS kulture.
Kod uzorka mliječnog proizvoda na bazi svježeg sira i voća
proizvedenog od mlijeka s 1.5% m.m. dva od tri uzorka s EPS
imaju više vrijednosti modula G’ i G’’ (M2K3A i M2K3K).
Hook-ov zakon navodi da je proporcionalan odnos između
naprezanja i deformacije za viskoelastične materijale kada
je primijenjen mali napor. Ovaj fenomen se naziva linearna
viskoelastičnost (LVE). Unutar ove linearno viskoznoelastične
regije, viskoelastični parametri G’ i G’’ ostaju konstantni pri
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
promjeni deformacije. Iz rezultata reoloških ispitivanja, LVE
regija se uočava na niskim frekvencijama u kojoj su moduli
G’ i G’’ ostali konstantni pri promjeni frekvencije. Kako se
frekvencija počela povećavati uočava se pad vrijednosti oba
modula. Taj pad predstavlja kraj LVE regije. Kod nekih ma-
terijala, kraj LVE regije može predstavljati i porast ovih mod-
ula. Kako se vidi sa slika (1(a), 1(c)¸ 1(f), 2 (a), 2(d), 2(e) i 2
(g)), dužina LVE regija je različita od uzorka do uzorka. Kod
uzoraka mliječnog proizvoda na bazi svježeg sira i ananasa
proizvedenih od djelomično obranog mlijeka s 1.5% mliječne
masti i inokuliranih s mikrobnom starter kulturom XT-303 i
mješavinom mikrobnih starter kultura CHN-22 i Lyofast SAB
440 B u omjeru 2:1 (M2K2V1 i M2K3V1) (slike 2 (d) i (g))
ova regija je najkraća. Kod ostalih uzoraka kraj LVE regije
nastupio je u intervalu deformacije od 1 do 10%. Promatrajući
rezultate reoloških ispitivanja, primjetno je da je kod uzoraka
mliječnog proizvoda na bazi svježeg sira i voća proizvedenih
od obranog mlijeka s 0.9% mliječne masti i djelomično obra-
nog mlijeka s 1.5% mliječne masti inokuliranih s mikrobnim
starter kulturama CHN–22, XT–303 i mješavinom mikrobnih
starter kultura CHN-22 i Lyofast SAB 440 B u omjeru 2:1,
od kojih su tri uzorka bila sa ananasom (M1K1A, M1K2A i
M2K3A) (slike 1(a), 1(d) i 2(g)), a jedan sa kruškom (M2K2K)
(slika 2(f)), na kraju mjerenja došlo do križanja krivih G’ i G’’.
Ova točka se često naziva ″gel točka″ u kojoj se vrijednosti
modula G’ i G’’ izjednačavaju i tan δ = 1 (δ = 45°). U ovoj
točki, dolazi do promjene viskoznog elastičnog ponašanja iz
dominatnog elastičnog (G’ > G’’) u viskozno ponašanje (G’’
> G’). To se vidi najbolje kod uzorka mliječnog proizvoda na
bazi svježeg sira i kruške proizvedenog od djelomično obra-
nog mlijeka s 1.5% mliječne masti i mikrobne starter kulture
XT-303 (M2K2K) koja je nastupila prije završetka mjerenog
područja deformacije, tj. na nešto nižoj deformaciji od kra-
jnje (slika 2(f)) za razliku od ostalih uzoraka kod kojih je ta
točka nastupila točno na kraju mjernog područja. Budući da su
moduli elastičnosti i viskoznosti ovisni o frekvenciji, i vrijeme
postizanja ″gel točke″ je također ovisno o frekvenciji. Ova
točka se koristi u reologiji emulzija za definiranje promjene
ponašanja iz tekućeg u čvrsto i obratno. Ako su formirane
idealne točke križanja, tada su moduli potpuno neovisni o
frekvenciji.
Prema Allmere i sur. (1999) nađene su vrijednosti G›
oscilacijskih mjerenja značajno promijenjene udjelom ukupnih
proteina mliječnih uzoraka. Za razliku od vrijednosti G››, gdje
ni ukupni proteini, ni udio kazeina, nemaju značajan uzajamni
odnos sa modulom gubitaka. Činjenica je da veći udio ukupnih
proteina kreira mrežu sa većom gustoćom strukture matriksa,
koja onda rezultira većim elasticitetom, bez utjecaja na
viskozni modul.
Zaključci
Na osnovu reološke procjene, vrijednosti modula čuvanja
G› i modula gubitaka G›› variraju kod svih uzoraka s obzirom
na različit sadržaj suhe tvari i masti.
Svi uzorci mliječnog proizvoda na bazi svježeg sira i voća
su imali G› > G›› odakle proizilazi da uzorci posjeduju ela-
stična svojstva sa omjerom viskoznih i elastičnih karakteristika
između 0 i 1 (tan δ).
 T. Brčina et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 137-145 (2017) 145

Uzorci dobiveni od obranog mlijeka imali su veće vrijed-
nosti modula G› i G›› od uzoraka proizvedenih od djelomično
obranog mlijeka.
Niže vrijednosti modula G› i G›› kod pet uzoraka mliječ-
nog proizvoda na bazi svježeg sira i voća (M1K3A, M1K3B,
M1K3K, M2K3B i M2K3K) dobivenih inokulacijom sa mi-
krobnom starter kulturom Lyofast 440 SAB B, rezultat su
utjecaja produkcije egzopolisaharida od strane sojeva Strepto-
coccus thermophilus.
Pechak, D. G., Smith, A. K. (2007): Instrumental Tech-
niques for Sample Preparation, Chapter 2, u Structure of dairy
foods, Blackwell Publishing Ltd., ur. A. Tamime, 17-50.
Rao, V.N.M., Skinner, G. E. (1986): Rheological proper-
ties of solid foods, u Engineering Properties of Foods, Marcel
Dekker, New York, NY, ur. M. A. Rao, S. S. H. Rizvi, 215–254.
Robinson, R. K. (1991) Therapeutic Properties of Fer-
mented Milks, Elsevier Applied Science Publishers, London.

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properties od acidified gels of skim milk from cows selected
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Korolczuk, J. (1993) Flow behaviour of low solids fresh
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centz Verlag, Hannover
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(1998) Rheological properties of concentrated yogurt (Lab-
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CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 R. Leder et al: Croatian Journal of Food Technology, Biotechnology
146 and Nutrition 12 (3-4), 146-154 (2017)

STRUČNI RAD / PROFESSIONAL PAPER

Osiguranje kvalitete rezultata ispitivanja 2

vina u analitičkom laboratoriju

Quality assurance of wine testing results in analytical laboratory

Renata Leder1*, Valentina Ščitnik2, Marija Vukoja2, Anita Boras2, Ivana Vladimira Petric1, Neven Antunac2,
Mara Banović3
1
Hrvatski centar za poljoprivredu, hranu i selo, Zavod za vinogradarstvo i vinarstvo, Jandrićeva 42, 10000 Zagreb, Hrvat-
ska
2
Sveučilište u Zagrebu, Agronomski fakultet, Svetošimunska cesta 25, 10000 Zagreb, Hrvatska
3
Sveučilište u Zagrebu, Prehrambeno-biotehnološki fakultet, Pierrottieva 6, 10000 Zagreb, Hrvatska
*
Corresponding author: renata.leder@hcphs.hr

Sažetak

Osiguranje kvalitete temeljni je čimbenik u dobivanju pouzdanih, vjerodostojnih i ponovljivih rezultata analiza. Cilj rada je prikazati na-
čin kontrole kvalitete rezultata ispitivanja vina, a time i osposobljenost laboratorija, provedbom unutarnje i vanjske kontrole kvalitete za četiri
odabrana parametra: relativna gustoća, alkoholna jakost, hlapljiva i ukupna kiselost. Unutarnja kontrola kvalitete provedena je primjenom
kontrolnih karata za interni referentni materijal, a vanjska kontrola kvalitete sudjelovanjem u međulaboratorijskim usporedbenim ispitivanjima.
Prikazane su vrijednosti statističke obrade rezultata laboratorija na osnovu kojih se procjenjuje uspješnost sudjelovanja laboratorija u
međulaboratorijskim usporedbenim ispitivanjima. Na osnovu prikazanih rezultata može se zaključiti da je odabrani laboratorij u promatranom
razdoblju uspješno provodio kontrolu kvalitete rezultata analitičkih metoda za sva četiri ispitivana parametra, budući da dobiveni rezultati
referentnog materijala nisu prelazili zadane granične vrijednosti. Laboratorij je uspješno sudjelovao i u međulaboratorijskim usporedbenim
ispitivanjima, što je potvrđeno dobivenim Z-vrijednostima koje su bile između -2,00 i +2,00 za sve ispitivane parametre. Primjenom predstavljenih
postupaka kontrole postignuta je visoka kvaliteta rezultata ispitivanja.
Ključne riječi: analize vina, kontrolne karte, kvaliteta, međulaboratorijska usporedbena ispitivanja, osiguranje kvalitete, referentne metode

Abstract

Quality assurance is the key factor in producing reliable, trustworthy and reproducible analyses results. The goal of this research is to
demonstrate the modality of results quality control in the scope of wine analyses and thus the qualification of laboratory, by implementation of
internal and external quality control for four selected parameters:relative density, alcoholic strength, volatile and total acidity. Internal qua-
lity control is carried out by applying control charts for in-house reference material and external quality is carried out by the participation in
interlaboratory comparison examinations. Here are presented the results of laboratories data statistical processing from which the success of
participation in interlaboratory comparisons is being estimated. Based on the presented results we can conclude that laboratory successfully
implemented quality control of testing results during controlled period for four analyzed parameters, because the reference material results did
not exceed the specified limit values. Laboratory has successfully participated in interlaboratory comparisons which was confirmed by obtained
Z-values that were between -2.00 and +2.00 for all tested parameters. By application of presented control procedures, high quality of testing
results is achieved.
Key words: wine analyses, control charts, quality, interlaboratory comparison, quality assurance, reference methods

Uvod a za rad u laboratoriju u primjeni je norma HRN EN ISO/

U uvjetima sve jače konkurencije i sve većih zahtjeva
potrošača, kvaliteta je postala temeljni čimbenik opstanka na
tržištu. Principi tzv. dobre laboratorijske prakse (DLP) primje-
njuju se od 1970. g., da bi u današnje vrijeme bili zamijenje-
ni sa sustavom upravljanja kvalitetom, koji se primjenjuju u
svim djelatnostima (Peterlić, 2008). Raditi kvalitetno znači
raditi prema najvišim svjetskim standardima (normama),
IEC 17025:2007 „Opći zahtjevi za osposobljenost ispitnih
i umjernih laboratorija“. Primjer organizacije koja je akre-
ditirana prema ovim zahtjevima je Hrvatski centar za poljo-
privredu, hranu i selo (HCPHS), Zavod za vinogradarstvo i
vinarstvo (ZVV), Odjel za kontrolu kvalitete proizvoda (u
daljnjem tekstu Odjel) u kojem se provode fizikalno-kemij-
ska ispitivanja kakvoće grožđa, mošta, vina i voćnih vina te
jakih alkoholnih pića.

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 R. Leder et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 146-154 (2017) 147

Kvaliteta rezultata ispitivanja ili umjeravanja može se i učestalosti sudjelovanja u ispitivanjima sposobnosti (HAA-
pratiti unutarnjim ili vanjskim mjerama kontrole kvalitete. Pr-2/6, 2015). Cilj rada bio je prikazati način kontrole kvalitete
Unutarnja kontrola kvalitete sastoji se od postupaka koje pro- rezultata ispitivanja vina, provedbom unutarnje i vanjske kon-
vodi osoblje laboratorija u svrhu kontinuirane procjene kva- trole kvalitete.
litete rezultata ispitivanja, dobivenih određenim analitičkim
postupkom. Unutarnje mjere osiguravanja kvalitete rezultata
ispitivanja imaju za cilj svakodnevno dobivanje valjanih i kva- Materijali i metode
litetnih rezultata analiza. Unutarnja kontrola kvalitete rezulta-
ta može se osigurati uporabom referentnog materijala (RM) Materijali
(Gašljević i sur., 2007), te kontrolnim kartama (KK), kojima Sve analize vina provedene su u Odjelu za kontrolu kva-
se vrijednosti dobivene jednoga dana uspoređuju s vrijednosti- litete proizvoda, ZVV, HCPHS, tijekom 2015. i 2016. godine.
ma dobivenih kroz više prethodnih dana (Hovind i sur., 2011). Unutarnja kontrola kvalitete provedena je primjenom KK za
Prikazivanje rezultata analiza na KK omogućuje analitičaru interni RM, a vanjska je kontrola kvalitete provedena sudjelo-
utvrđivanje raspodjele analiziranih vrijednosti, prikaz podata- vanjem u MUI.
ka na sažeti način, utvrđivanje trendova i poduzimanje odgo-
varajućih popravnih radnji (Runje, 2015; Runje i Ančić, 2015; Priprema internog referentnog materijala
Štajdohar-Pađen, 2015). U praktičnoj svakodnevnoj primjeni i Interni RM odgovarajuće matrice (vino) u Odjelu se pri-
tumačenju kontrolnih karata moguća su tri slučaja: prema sukladno normama i propisima za validacijske postupke
• metoda je pod kontrolom ukoliko je rezultat između „gra- te statističku obradu dobivenih rezultata (ISO Guide 80:2014;
nica upozorenja“ (± 2 standardne devijacije, SD), (engl. Leder i sur., 2014). RM-u se najprije utvrđuje homogenost i
warning limits) te ukoliko je rezultat između „granica stabilnost, nakon čega slijedi provjera ekstremnih vrijednosti
upozorenja“ i „granica akcije“ (± 3 SD), (engl. action li- (engl. outliera), određivanje referentne vrijednosti te izrada
mits) a dva prethodna rezultata su unutar „granica upozo- kontrolnih karata s dozvoljenim granicama odstupanja, odno-
renja“ (± 2 SD) može se nastaviti s analizama redovnih sno „granicama upozorenja“ ( ± 2 SD) i „granicama akcije“ (
uzoraka, ± 3 SD). Ovdje prikazane referentne vrijednosti za sve para-
• metoda je pod kontrolom, ali se može smatrati statistički metre, odnosno ispitne metode, dobivene su mjerenjima prove-
izvan kontrole ukoliko su rezultati između „granica upo- denim u 25 jedinica proizvoda izuzetih iz ukupne nabavljene
zorenja“, s najviše jednim rezultatom između „granica količine (500 boca volumena 0,187 L) vina sorte Graševina,
upozorenja“ i „granica akcije“. U slučajevima kada se se- berbe 2013., proizvođača Kutjevo d.d. Sva mjerenja određi-
dam uzastopnih rezultata postepeno povećava, odnosno vanih značajki RM-a provodili su analitičari Odjela koji su se
smanjuje ili se deset od jedanaest nalazi ispod ili iznad svaki tjedan izmjenjivali u provođenju navedenih ispitivanja.
centralne linije, može se pristupiti analizama redovnih Primijenjene metode i uređaji navedeni su u tablici 1.
uzoraka. Ovakvi trendovi kontrolnih uzoraka ukazuju na
moguće probleme s primjenom metode. Tablica 1. Parametri, metode i uređaji za pripremu internog
• metoda je izvan kontrole kad je rezultat izvan „granica referentnog materijala u Odjelu
akcije“ te kad je rezultat između „granica upozorenja“ i Table 1. Parametres, methods and instruments of in-house
„granica akcije“, a najmanje jedan od prethodna dva re- reference material preparation in Department
zultata također između tih granica (pravilo dva od tri). U
ovakvoj situaciji ne smiju se analizirati redovni uzorci, parametar /
već se moraju poduzeti odgovarajući koraci za otklanja- metoda / method uređaj / instrument
parameter
nje uzroka dobivanja rezultata izvan dozvoljenih granica
(Hovind i sur., 2011). OIV-MA-AS2- tri uređaja DMA
relativna gustoća
01A:R2012, 4500
Vanjska kontrola kvalitete temelji se na ispitivanju ospo- (20/20°C) relative
(Compedium OIV,
sobljenosti laboratorija (eng. Proficiency Testing) u različitim density (20/20°C)
2016.) (Anton Paar)
shemama ispitivanja sposobnosti ili drugim međulaboratorij-
skim usporedbama i najbolji je alat u procijeni sposobnosti NIR spektrometrija
i kvalitete mjerenja. Unutarnje i vanjske mjere osiguravanja alkoholna jakost tri uređaja Alcolyzer
Patent Anton Paar
kvalitete međusobno se ne isključuju. Redovito sudjelovanje (% vol) alcoholic
laboratorija u međulaboratorijskim usporedbenim ispitivanji- strength (% vol) (Anton Paar)
(US 6,690,015; AT
ma (MUI) nije dovoljno za praćenje svakodnevnih rezultata. 406711).
Organizator programa MUI odgovoran je za oblikovanje she-
me programa, pomoć laboratorijima u analizi neprihvatljivih OIV-MA-
ukupna kiselost (g/L)
AS313-01:R2015
rezultata te izvještavanje o dobivenim rezultatima koje mora -
(Compedium OIV,
uključiti: tumačenje ciljnih vrijednosti, način vrednovanja re- total acidity (g/L)
2016.)
zultata, grafički prikaz dobivenih rezultata te kontinuirano pra-
ćenje rezultata svakog pojedinog laboratorija sudionika (Ančić, hlapljiva kiselost OIV-MA-
destilator
2014). Iako HRN EN ISO/IEC 17025 ne propisuje učestalost (g/L) AS313-02:R2015
sudjelovanja laboratorija u MUI, prema preporukama Hrvatske (Compedium OIV,
(Leo Kuebler)
volatile acidity (g/L) 2016.)
akreditacijske agencije (HAA), laboratorij mora razviti strate-
giju kontrole kvalitete koja uključuje strategiju sudjelovanja
u ispitivanjima sposobnosti koja uključuje određivanje razine

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 R. Leder et al: Croatian Journal of Food Technology, Biotechnology
148 and Nutrition 12 (3-4), 146-154 (2017)
Primjena internog referentnog materijala
Interni RM primjenjuje se u Odjelu svakodnevno prije po-
četka analiza uzoraka vina koji se taj dan ispituju i to za svaki
navedeni parametar, odnosno metodu. Dobiveni rezultati uno-
se se u kontrolne karte, te se provodi svakodnevna i dugoroč-
na procjena rezultata. Za potrebe dnevnih analiza pristupa se
analizama ukoliko je dobiveni rezultat unutar zadanih granica,
a ukoliko nije, provodi se analiza uzroka nesukladnosti i od-
govarajuće popravne radnje. Za dugoročne analize KK procje-
njuje se pojava pozitivnih ili negativnih trendova, te se također
po potrebi poduzimaju popravne radnje (Hovind i sur., 2011).
U razdoblju pripreme internog RM, kontrola kvalitete rezulta-
ta ispitivanja provedena je pomoću prethodne šarže internog
RM-a i pomoću certificiranog RM (TITRIVIN BTB), koji su-
kladno ISO 17034:2016 proizvodi Chambre d`agriculture de la
Gironde – Service Vigne et Vin, Francuska.
Uzorci za međulaboratorijska usporedbena ispitivanja
U uzorcima vina za MUI koje organizator kontinuirano
tijekom godine isporučuje u Odjel, provedena su ispitivanja
relativne gustoće u 20 uzoraka, alkoholne jakosti u 12 uzo-
raka, hlapljive kiselosti u 14 uzoraka te ukupne kiselosti u 19
uzoraka.
Metode
Sva ispitivanja u promatranom razdoblju provedena su
metodama akreditiranim sukladno normi HR EN ISO/IEC
17025:2007.
Relativna gustoća. Određivanje relativne gustoće pro-
vedeno je metodom elektronske denzimetrije OIV-MA-AS2-
01A:R2012 (Compendium OIV, 2016) s frekventnim oscilato-
rom, na uređaju DMA 4500 (Anton Paar, Austrija).
Alkoholna jakost. Alkoholna jakost određena je NIR
spektrometrijom, uređajem Alcolyzer, Anton Paar, Austrija
(US 6,690,015; AT 406711).
Hlapljiva kiselost. Određivanje hlapljive kiselosti vina
provedeno je destilacijom uzorka vina vodenom parom te titra-
cijama destilata otopinom natrijevog hidroksida te otopinom
joda radi korekcije kiselosti uzrokovane slobodnim i vezanim
sumporovim dioksidom u dobivenom destilatu (OIV-MA-
AS313-02:R2015), (Compendium OIV, 2016).
Ukupna kiselost. Određivanje ukupne kiselosti se prove-
deno je titracijom s 0,1 mol/L otopinom natrijevog hidroksida
uz indikator bromtimol modro (OIV-MA-AS313-01:R2015),
(Compendium OIV, 2016).
Statistička obrada podataka. Statistička obrada poda-
taka izvršena je u programu Microsoft Office Excel®. Od
mjera centralne tendencije izračunata je aritmetička srednja
vrijednost ( ), a od mjera varijabilnosti: standardna devijacija
(SD) i Z-vrijednost kao mjera relativnog položaja. Provjera
ekstremnih vrijednosti, odnosno outliera provedena je
Grubbsovim testom (Microsoft Office Excel®, Real Statistics
Resource Pack).
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
Rezultati i rasprava
U domeni unutarnje kontrole kvalitete jedan je od cilje-
va bio i primijeniti interni RM odgovarajuće matrice - vino,
pripremljen u laboratoriju Odjela, te na taj način uštedjeti zna-
čajna financijska sredstva, budući je time u znatnoj mjeri sma-
njena potreba za uvozom skupog certificiranog RM-a iz ino-
zemnih laboratorija. Ovakav RM mora imati zadovoljavajuću
homogenost i stabilnost (HAA-Pr-2/8:2015), što je provjereno
sukladno ISO Guide 80:2014.
U svrhu vanjske kontrole kvalitete Odjel sudjeluje u MUI
za vino (program 17- Wines), koje organizira BIPEA (Bureau
Interprofessionnel d›Etudes Analytiques, Francuska), tvrtka
akreditirana za organiziranje MUI. Organizator MUI dostavlja
svim prijavljenim laboratorijima identične uzorke vina i pro-
vodi statističku obradu rezultata analiza. Kriterij uspješnosti
sudjelovanja u MUI je Z-vrijednost, koja označava odstupanje
od prosjeka izraženo kao standardna devijacija. Ako je |Z| = 0,
rezultat je izvrstan, odnosno vrijednost koju je dobio labora-
torij identična je referentnoj vrijednosti za taj parametar. Pri-
hvatljiv i zadovoljavajući rezultat je ako je |Z| ≤ 2 te se nalazi
unutar „granica upozorenja“ ( ± 2 SD), dok je rezultat upitan
i nalazi se između „granica upozorenja“ i „granica akcije“ (
± 3 SD) ako je 2 < |Z| ≤ 3. Rezultat ne zadovoljava i nalazi
se izvan „granica akcije“ ako je |Z| > 3, što zahtijeva pokreta-
nje popravne ili korektivne radnje, radi uspostave nadzora nad
rezultatima ispitivanja (HRN ISO 13528:2017; HAA-Pr-2/6,
2015). Na temelju referentnih vrijednosti laboratorij provodi i
procjenu sistematske pogreške mjerenja (eng. bias). Vrijednost
biasa (%) je veća ako je veće odstupanje vrijednosti rezulta-
ta mjerenja od referentne vrijednosti (IUPAC, 1995; Hovind i
sur., 2011).
Rezultati dobiveni analizom vina RM-a testirani su na
outliere te primijenjeni za kreiranje kontrolnih karata s refe-
rentnim i graničnim vrijednostima (tablica 2).
U području unutarnje kontrole kvalitete za svaki analizi-
rani parametar odabrana je po jedna KK (slike 1-4). Rezultati
vanjske kontrole kvalitete za pojedine parametre prikazani su
u tablicama 3-6. To su vrijednosti koje laboratoriju šalje orga-
nizator MUI nakon provedene statističke obrade rezultata svih
laboratorija koji sudjeluju u ispitivanjima.
 R. Leder et al: Croatian Journal of Food Technology, Biotechnology
and Nutrition 12 (3-4), 146-154 (2017) 149

Tablica 2. Rezultati pripreme internog referentnog materijala u Odjelu

Table 2. Results of in-house reference material preparation in Department

Parametar / parameter n – 3 SD – 2 SD + 2 SD + 3 SD
relativna gustoća (20/20°C) relative density
153 0,99266 0,99250 0,99256 0,99267 0,99282
(20/20°C)

alkoholna jakost (% vol) alcoholic strength (% vol) 153 12,200 12,105 12,137 12,263 12,295

ukupna kiselost (g/L)

63 6,16 5,939 6,013 6,308 6,381
total acidity (g/L)
hlapljiva kiselost (g/L)
63 0,271 0,194 0,219 0,323 0,348
volatile acidity (g/L)

n: broj mjerenja / number of measurements; : referentna vrijednost / reference value; SD:standardna devijacija / standard de-
viation; - 3 SD: donja „granica akcije“ / lower action limit; - 2 SD: donja „granica upozorenja“ / lower warrning limit; +
2 SD: gornja „granica upozorenja“ / upper warning limit; - 3 SD:.gornja „granica akcije“ / upper action limit

Relativna gustoća Relativna gustoća je omjer izražen kao
decimalni broj gustoće nekog određenog volumena vina pri
20°C prema gustoći istog volumena vode pri istoj temperaturi.
Obično se kreće u rasponu od 0,9850 do 0,9970, dok u vini-
ma koja imaju visok udio šećera, može biti i veća od 1,0000
(Panda, 2011). Primjer kontrolne karte za relativnu gustoću
internog RM-a u promatranom razdoblju prikazan je na slici 1.
Na osnovu vrijednosti relativne gustoće internog RM-a
prikazanih na slici 1, vidljivo je da se u promatranom razdo-
blju dobivena vrijednost relativne gustoće vina RM-a dobro
podudarala s referentnom vrijednosti, što potvrđuje veliku pre-
ciznost i točnost izvođenja analiza i dobivenih rezultata. Budu-
ći da su vrijednosti bile unutar „granica upozorenja“ ( ± 2 SD)
metoda određivanja gustoće vina bila je uspješno primjenjena
(Hovind i sur., 2011).
Usporedbom rezultata analiza vina u MUI za relativnu
gustoću u promatranom razdoblju (tablica 3) vidljiva je izvr-
sna podudarnost rezultata s referentnim vrijednostima, što se
može zaključiti na osnovu vrlo niske vrijednosti biasa (između
–0,009 % i 0,011 %) i Z-vrijednosti, koja je varirala od -0,60 do
1,75, te je bila manja od 2, odnosno veća od -2, što se smatra
prihvatljivim i zadovoljavajućim rezultatom (ISO 13528:2015;
HAA-Pr-2/6, 2015).

Slika 1. Kontrolna karta za relativnu gustoću vina

Figure 1. The control chart for the relative density of wine

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Tablica 3. Rezultati sudjelovanja Odjela u međulaboratorijskim usporedbenim ispitivanjima za relativnu gustoću vina
Table 3. Results of participation of Department in interlaboratory comparison for relative density of wine

1. 2. 3. 4. (2.-3.) 5. 6. 7. 8. 9. 10. 11.

RB LAB RV RAZLIKA min max BIAS (%) nLab s(MUI) s(MUI) (%) Z-vrijednost

BIAS S(MUI)
DIFFE-
No LAB RV min max nLab S(MUI) Z-value
RENCE
(%) (%)

1. 0,99165 0,99158 0,00007 0,99128 0,99188 0,007 74 0,00006 0,0061 0,47

2. 1,03086 1,03078 0,00008 1,03048 1,03108 0,008 63 0,00011 0,0107 0,53

3.
0,99131
0,99133
-0,00002 74 0,0001 0,0101 -0,13
0,99103
0,99163
-0,002

4. 0,99313 0,99278 0,00035 0,99238 0,99318 0,035 69 0,0002 0,0201 1,75

5. 1,01858 1,01867 -0,00009 1,01837 1,01897 -0,009 67 0,0001 0,0098 -0,60

6. 0,99227 0,99226 0,00001 0,99196 0,99256 0,001 73 0,00007 0,0071 0,07

7. 0,99142 0,99144 -0,00002 0,99114 0,99174 -0,002 83 0,00007 0,0071 -0,13

8. 1,04245 1,04254 -0,00009 1,04224 1,04284 -0,009 70 0,0002 0,0192 -0,60

9. 1,00548 1,00542 0,00006 1,00502 1,00582 0,006 69 0,0003 0,0298 0,30

10. 1,00548 1,00542 0,00006 1,00502 1,00582 0,006 69 0,0003 0,0298 0,30

11. 0,99281 0,99279 0,00002 0,99249 0,99309 0,002 78 0,0001 0,0101 0,13

12. 0,99281 0,99279 0,00002 0,99249 0,99309 0,002 78 0,0001 0,0101 0,13

13. 0,99147 0,99151 -0,00004 0,99121 0,99181 -0,004 71 0,00007 0,0071 -0,27

14. 0,99147 0,99151 -0,00004 0,99121 0,99181 -0,004 70 0,00007 0,0071 -0,27

15. 0,99068 0,99057 0,00011 0,99027 0,99087 0,011 73 0,00007 0,0071 0,73

16. 1,01141 1,01137 0,00004 1,01097 1,01177 0,004 70 0,00007 0,0069 0,20

17. 0,99068 0,99057 0,00011 0,99027 0,99087 0,011 73 0,00007 0,0071 0,73

18. 1,01141 1,01137 0,00004 1,01097 1,01177 0,004 70 0,00007 0,0069 0,20

19. 0,99040 0,99033 0,00007 0,99003 0,99063 0,007 81 0,00015 0,0151 0,40

20. 0,99040 0,99033 0,00007 0,99003 0,99063 0,007 81 0,00015 0,0151 0,40

1. redni broj uzoraka / ordinal number of samples, 2. vrijednosti dobivene u laboratoriju / values obtained in the laboratory, 3.
referentne vrijednosti / reference values, 4. razlika između (2) i (3) / difference between (2) and (3); 5. i 6. minimalni i maksimalni
dozvoljeni interval / minimum and maximun allowed interval; 7. vrijednosti biasa / bias values, 8. broj laboratorija uključenih u
pojedini krug MUI / number of laboratories included in a single cycle of ILC, 9. i 10. standardne devijacije prihvatljivih rezultata
izražene u apsolutnim i relativnim vrijednostima / standard deviations of acceptable results expressed in absolute and relative
values, 11. Z-vrijednosti / Z-values.

U svakom je krugu MUI za parametar relativne gustoće Alkoholna jakost

sudjelovalo od 63 do 83 europska laboratorija, dok je Odjel
sudjelovao u ukupno 20 MUI. Vrijednosti minimalne i mak-
simalne prihvatljive vrijednosti izračunava organizator MUI i
označavaju granice prihvatljivosti rezultata, a vrijednosti Odje-
la su u svakom sudjelovanju bile unutar tih granica.
Alkoholna jakost (% vol.) izražena volumenom je broj li-
tara etanola sadržanog u 100 litara vina, a oba volumena mjere
se pri temperaturi od 20°C (Compendium OIV, 2016). U vinu
su uz etanol prisutni jednovalentni i viševalentni alkoholi koji
dolaze ili iz grožđa ili kao produkt fermentacije. Udio etanola,
ovisi o udjelu fermentabilnih šećera, vrsti kvasaca, temperaturi
i uvjetima fermentacije. U standardnim uvjetima fermentacije,

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može nastati 14-15 % etanola. Veća koncentracije upućuju na
došećeravanje tijekom fermentacije, dok su vina s manje od
10 % etanola podložnija mikrobiološkim kvarenjima. Osim na
stabilnost, etanol značajno utječe na sposobnost starenja i sen-
zorske karakteristike vina (Ribéreau-Gayon i sur., 2006; Jack-
son, 2014). Primjer kontrolne karte alkoholne jakosti internog
RM-a za promatrano razdoblje prikazan je na slici 2.
Vrijednosti alkoholne jakosti internog RM-a (slika 2) na-
lazile su se uglavnom unutar „granica upozorenja“ ( ± 2 SD).
Tijekom 2015. g. vrijednosti su se nalazile na samoj „granici
upozorenja“ i u blizini „granice akcije“ ( ± 3 SD), što još
uvijek predstavlja prihvatljiv rezultat, ali prisutnost negativnog
trenda ukazuje na potrebu kalibracije uređaja. Nakon provede-
ne kalibracije sve vrijednosti se ponovo nalaze unutar „granica
upozorenja“.
U promatranom razdoblju u MUI je za alkoholnu jakost
sudjelovalo od 85 do 101 europskih laboratorija. Odjel je bio
vrlo uspješan u ukupno 12 MUI (tablica 4) što je vidljivo iz do-
bre podudarnosti vrijednosti dobivenih u Odjelu s referentnim
vrijednostima, odnosno niskih relativnih vrijednosti biasa (od
–0,92 % do 2,31 %). Z-vrijednosti su u svim sudjelovanjima
bile manje od +2, odnosno veće od –2, a u dva sudjelovanja je
bilo Z=0,00.

Slika 2. Kontrolna karta za alkoholnu jakost vina

Figure 2. The control chart for the alcoholic strength of wine

Tablica 4. Rezultati sudjelovanja Odjela u međulaboratorijskim usporedbenim ispitivanjima za alkoholnu jakost vina
Table 4. Results of participation of Department in interlaboratory comparison for alcoholic strength of wine

1. 2. 3. 4. (2-3) 5. 6. 7. 8. 9. 10. 11.

RAZLI- BIAS
s(MUI) Z-vrijed-
RB LAB RV min max nLab s(MUI)
(%) nost
KA (%)

DIFFE- BIAS S(MUI)

No. LAB RV min max nLab S(MUI) Z-value
RENCE (%) %

1. 10,65 10,56 0,09 10,37 10,75 0,85 87 0,07 0,66 0,95

2. 12,25 12,17 0,08 11,98 12,36 0,66 91 0,07 0,58 0,84

3. 12,30 12,24 0,06 12,05 12,43 0,49 86 0,11 0,90 0,63

4. 12,00 11,99 0,01 11,80 12,18 0,08 85 0,08 0,67 0,11

5. 13,70 13,75 -0,05 13,56 13,94 -0,36 97 0,09 0,65 -0,53

6. 14,59 14,26 0,33 14,26 14,64 2,31 97 0,1 0,70 1,47

7. 11,84 11,95 -0,11 11,76 12,14 -0,92 90 0,12 1,00 -1,16

8. 10,89 10,84 0,05 10,65 11,03 0,46 101 0,08 0,74 0,53

9. 12,83 12,80 0,03 12,61 12,99 0,23 97 0,09 0,70 0,32

10. 12,07 12,07 0,00 11,88 12,26 0,00 91 0,08 0,66 0,00

11. 9,91 9,91 0,00 9,72 10,10 0,00 97 0,08 0,81 0,00

12. 12,16 12,13 0,03 11,94 12,32 0,25 95 0,1 0,82 0,32

1. redni broj uzoraka / ordinal number of samples, 2. vrijednosti dobivene u laboratoriju / values obtained in the laboratory, 3.
referentne vrijednosti / reference values, 4. razlika između (2) i (3) / difference between (2) and (3); 5. i 6. minimalni i maksimalni
dozvoljeni interval / minimum and maximun allowed interval; 7. vrijednosti biasa / bias values, 8. broj laboratorija uključenih u
pojedini krug MUI / number of laboratories included in a single cycle of ILC, 9. i 10. standardne devijacije prihvatljivih rezultata
izražene u apsolutnim i relativnim vrijednostima / standard deviations of acceptable results expressed in absolute and relative
values, 11. Z-vrijednosti / Z-values.

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Hlapljiva kiselost
Hlapljive kiseline vina čine skupinu organskih kiselina
koje se uglavnom javljaju kao sekundarni proizvodi alkoholne
fermentacije ili se u vinu mogu pojaviti kao rezultat kvarenja
vina. Octena kiselina je najzastupljenija hlapljiva kiselina u
vinu (95–99 %), ali uz nju su prisutne i druge karboksilne kise-
line, kao što su mravlja, maslačna i propionska kiselina koje su
u zdravima vinima obično ispod senzorskog praga osjetljivosti
(Jackson, 2014). U zdravom grožđu sadržaj hlapljivih kiselina
je zanemariv (70–250 mg/L), a u vinima nenarušene kvalite-
te uglavnom se kreće između 0,4 i 0,8 g/L (Zoecklein i sur.,
1995).
Slika 3. Kontrolna karta za hlapljivu kiselost vina
Figure 3. The control chart for the volatile acidity of wine

Vrijednosti hlapljive kiselosti RM-a (slika 3) tijekom

promatranog razdoblja nalazile su se unutar „granica upozo-
renja“ ( ± 2 SD). U studenom i prosincu 2015. g., vrijednosti
su u dva navrata bile na „granici upozorenja“, što upućuje na
upitan ali još uvijek prihvatljiv rezultat, te je i iz ravnomjerne
distribucije rezultata oko referentne vrijednosti vidljivo da je
metoda uspješno primjenjena.

Tablica 5. Rezultati sudjelovanja Odjela međulaboratorijskim usporedbenim ispitivanjima za hlapljivu kiselost vina
Table 5. Results of participation of Department in interlaboratory comparison for volatile acidity of wine
1. 2. 3. 4.(2.-3.) 5. 6. 7. 8. 9. 10. 11.

LAB RV BIAS s(MUI)

RAZLI- Z-vrijed-
RB min max nLab s(MUI)
KA nost
(mEq) (mEq) (%) (%)

BIAS S(MUI)
DIFFE-
No. LAB RV min max nLab S(MUI) Z-value
RENCE
(%) %

1. 11,0 9,3 1,7 7,6 11,0 18,3 78 0,9 10 2,00

2. 8,2 6,8 1,4 5,1 8,5 20,6 86 0,6 9 1,65

3. 4,8 3,9 0,9 2,2 5,6 23,1 74 0,6 15 1,06

4. 4,7 3,8 0,9 2,1 5,5 23,7 95 0,5 13 1,06

5. 1,1 1,5 -0,4 0,0 3,2 -26,7 69 0,6 40 -0,47

6. 8,2 7,6 0,6 5,9 9,3 7,9 102 0,7 9 0,71

7. 6,5 6,5 0,0 4,8 8,2 0,0 91 0,8 12 0,00

8. 3,0 3,0 0,0 1,3 4,7 0,0 82 0,5 17 0,00

9. 7,2 6,4 0,8 4,7 8,1 12,5 97 0,7 11 0,94

10. 9,7 8,4 1,3 6,7 10,1 15,5 103 0,7 8 1,53

11. 7,4 6,9 0,5 5,2 8,6 7,2 84 1,0 14 0,59

12. 7,2 6,7 0,5 5,0 8,4 7,5 91 0,9 13 0,59

13. 7,1 6,6 0,5 4,9 8,3 7,6 109 1,1 17 0,59

14. 5,1 5,1 0,0 3,4 6,8 0,0 97 0,9 18 0,00

1. redni broj uzoraka / ordinal number of samples, 2. vrijednosti dobivene u laboratoriju / values obtained in the laboratory, 3.
referentne vrijednosti / reference values, 4. razlika između (2) i (3) / difference between (2) and (3); 5. i 6. minimalni i maksimalni
dozvoljeni interval / minimum and maximun allowed interval; 7. vrijednosti biasa / bias values, 8. broj laboratorija uključenih u
pojedini krug MUI / number of laboratories included in a single cycle of ILC, 9. i 10. standardne devijacije prihvatljivih rezultata
izražene u apsolutnim i relativnim vrijednostima / standard deviations of acceptable results expressed in absolute and relative
values, 11. Z-vrijednosti / Z-value

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Tijekom promatranog razdoblja u MUI za hlapljivu kise-

lost je sudjelovalo 69 do 109 laboratorija, dok je Odjel sudje-
lovao u ukupno 14 MUI (tablica 5). Iako se kod ovog parame-
tra mogu uočiti nešto veće relativne vrijednosti biasa (između
-26,7 % i 23,7 %), na osnovu dobivenih Z-vrijednosti (-0,47
do 2,00) vidljivo je da je Odjel bio uspješan u svih 14 MUI, sa
čak tri rezultata identična s referentnom vrijednosti (Z = 0,00).

Ukupna kiselost
Ukupna kiselost vina predstavlja sumu kiselina koje se
neutraliziraju do pH 7, sa standardnom otopinom lužine uz in-
dikator brom-timol modro, i kreće se u rasponu od 4 do 14
g/L. Ovisno o stupnju disocijacije ukupnoj kiselosti doprinose
organske i anorganske kiseline. Od organskih kiselina, najviše
su zastupljene vinska, limunska, jabučna i mliječna a od anor-
ganskih fosfatna, sulfatna i ugljična kiselina (Ribéreau-Gayon
i sur., 2006).
Slika 4. Kontrolna karta za ukupnu kiselost vina
Figure 4. The control chart for total acidity of wine
Vrijednosti ukupne kiselosti vina (slika 4) nalazile su se
unutar „granica upozorenja“, tj. ± 2 SD. U lipnju 2015. g.
vrijednost se samo jednom nalazila na „granici upozorenja“ i
„granici akcije“ što se još uvijek smatra prihvatljivim rezulta-
tom.
U MUI je za ukupnu kiselost u promatranom razdoblju
sudjelovalo od 61 do 126 laboratorija, dok je Odjel sudjelovao
u ukupno 19 MUI (tablica 6). Dobivena su dobra slaganja s
referentnim vrijednostima, odnosno male relativne vrijedno-
sti biasa (-0,5 % i 9,2 %). Na osnovu dobivenih Z-vrijednosti
(-0,14 i 1,89) vidljivo je da su analize provedene u Odjelu bile
uspješne u svih 19 MUI.

Tablica 6. Rezultati sudjelovanja Odjela u međulaboratorijskim usporedbenim ispitivanjima za ukupnu kiselost vina
Table 6. Results of participation of Department in interlaboratory comparison for total acidity of wine
1. 2. 3. 4. (2.-3.) 5. 6. 7. 8. 9. 10. 11.
BIAS s(MUI)
RAZLI- Z-vrijed-
RB LAB RV min max nLab s(MUI)
KA nost
(%) (%)
BIAS S(MUI)
DIFFE-
No. LAB RV min max nLab S(MUI) Z-value
RENCE
(%) %
1. 95,0 92,8 2,2 85,8 99,8 2,4 113 2,5 2,7 0,63
2. 74,5 70,6 3,9 63,6 77,6 5,5 95 2,1 3,0 1,11
3. 75,5 73,2 2,3 66,2 80,2 3,1 122 2,5 3,4 0,66
4. 99,0 99,5 -0,5 92,5 106,5 -0,5 110 3 3,0 -0,14
5. 50,8 47,9 2,9 40,9 54,9 6,1 100 1,8 3,8 0,83
6. 105,0 101,6 3,4 94,6 108,6 3,3 112 2,8 2,8 0,97
7. 78,0 71,4 6,6 64,4 78,4 9,2 122 2,4 3,4 1,89
8. 63,5 58,3 5,2 51,3 65,3 8,9 108 2,2 3,8 1,49
9. 101,0 99,9 1,1 92,9 106,9 1,1 113 3,2 3,2 0,31
10. 63,5 58,3 5,2 21,3 65,3 8,9 108 2,2 3,8 1,49
11. 101,0 99,9 1,1 92,9 106,9 1,1 113 3,2 3,2 0,31
12. 84,5 83,3 1,2 76,3 90,3 1,4 126 2,5 3,0 0,34
13. 84,5 83,3 1,2 76,3 90,3 1,4 126 2,5 3,0 0,34
14. 63,0 59,7 3,3 52,7 66,7 5,5 61 2,6 4,4 0,94
15. 84,5 82,0 2,5 75,0 89,0 3,0 120 3,1 3,8 0,71
16. 70,0 67,9 2,1 60,9 74,9 3,1 107 2,2 3,2 0,60
17. 84,5 82,0 2,5 75,0 89,0 3,0 120 3,1 3,8 0,71
18. 70,0 67,9 2,1 60,9 74,9 3,1 107 2,2 3,2 0,60
19. 87,0 84,6 2,4 77,6 91,6 2,8 115 3,5 4,1 0,69

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

 R. Leder et al: Croatian Journal of Food Technology, Biotechnology
154 and Nutrition 12 (3-4), 146-154 (2017)
1. redni broj uzoraka / ordinal number of samples, 2. vrijednosti dobivene u laboratoriju / values obtained in the laboratory, 3.
referentne vrijednosti / reference values, 4. razlika između (2) i (3) / difference between (2) and (3); 5. i 6. minimalni i maksimalni
dozvoljeni interval / minimum and maximun allowed interval; 7. vrijednosti biasa / bias values, 8. broj laboratorija uključenih u
pojedini krug MUI / number of laboratories included in a single cycle of ILC, 9. i 10. standardne devijacije prihvatljivih rezultata
izražene u apsolutnim i relativnim vrijednostima / standard deviations of acceptable results expressed in absolute and relative
values, 11. Z-vrijednosti / Z-values.
Zaključci
Osiguranje kvalitete u analitičkom laboratoriju podrazu-
mijeva sve one mjere koje se primjenjuju da bi se osigurao
kvalitetan rad, koji osigurava pouzdane i vjerodostojne re-
zultate analiza. U cilju osiguranja kvalitete rezultata analiza,
Odjel za kontrolu kvalitete proizvoda zadovoljava propisane
standarde kvalitete rada analitičkog laboratorija, koji predstav-
ljaju osnovu za akreditaciju sukladno zahtjevima norme HRN
EN ISO/IEC 17025. Tijekom prikazanog jednogodišnjeg raz-
doblja, Odjel je potvrdio da ima uspostavljen sustav osiguranja
kvalitete ispitnih rezultata, što dokazuje uspješnom kontinuira-
nom provedbom unutarnjih i vanjskih mjera kontrole kvalitete.
Referentni materijal – vino, pripremljen u Odjelu, prikladan je
za primjenu u kontroli kvalitete rezultata ispitivanja, sukladno
normama i propisima za validacijske postupke te statističku
obradu dobivenih rezultata. Zamjenom skupog certificiranog
referentnog materijala sa referentnim materijalom pripremlje-
nim u Odjelu, značajno su smanjeni troškovi unutarnje kontro-
le kvalitete rezultata ispitivanja.
Literatura
Ančić M. (2014) Međulaboratorijske usporedbe. II. blok.
U: Međulaboratorijska usporedbena ispitivanja. CROLAB se-
minar. Hrvatski inženjerski savez, Zagreb.
Anton Paar® (2005) Method for the spectroscopic deter-
mination of ethanol concentration in an aqueous sample, Eu-
ropski patent br. 1073896 B1.
Compendium OIV(2016) Compendium of International
Methods of Analysis of Wines and Musts. International Orga-
nization of vine and wine - OIV. Paris, France.
Gašljević V., Košiček M., Marinčić S. (2007) Planiranje,
primjena i ocjenjivanje unutrašnjih mjera osiguravanja kvali-
tete rezultata. Seminar. Hrvatsko mjeriteljsko društvo, Zagreb.
HAA-Pr-2/8 (2015) Pravila za izbor i uporabu referencij-
skih tvari, Hrvatska akreditacijska agencija, Zagreb.
HAA-Pr-2/6 (2015) Pravila za međulaboratorijske uspo-
rebe, Hrvatska akreditacijska agencija, Zagreb.
Hovind H., Magnusson B., Krysell M., Lund U., Mäkinen
I. (2011) Internal Quality Controll –Handbook for Chemical
Laboratories. Nordtest. NT Technical Report 569. Norway.
HRN EN ISO/IEC 17025:2007. Opći zahtjevi za ospo-
sobljenost ispitnih i umjernih laboratorija. Hrvatski zavod za
norme, Zagreb.
HRN ISO 13528:2017. Statističke metode pri ispitivanju
sposobnosti putem međulaboratorijskih usporedbi. Hrvatski
zavod za norme, Zagreb.
ISO 17034:2016. General requirements for the competen-
ce of reference material producers. International Organization
for Standardization.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
ISO Guide 80:2014. Guidance for the in-house preparati-
on of quality control materials (QCMs). International Organi-
zation for Standardization.
IUPAC (1995) International Union of Pure and Appli-
ed Chemistry. Analytical Chemistry Division Commission
on Analytical Nomenclature. Nomenclature in evaluation of
analytical methods, including detection and quantification ca-
pabilties. Lloyd A. Currie, Chemical Science and Technology
Laboratory, National Institute of Standards and Technology,
Gaithersburg, MD 20899, USA.
Jackson R.S. (2014) Wine science – principles and appli-
cation. 4. izdanje, Academic Press, San Diego, USA.
Leder R., Peran S., Petric I. V., Kubanović V. (2014) Isku-
stva u pripremi referentnih materijala kao alata interne kontrole
kvalitete. U: Zbornik sažetaka: 10 Međunarodna konferencija
“Kompetentnost laboratorija i Regional Conformity assesse-
ment Workshop“, CROLAB, 15.10- 18. 10, Šibenik.
Panda H. (2011) The Complete Book on Wine Production.
Niir Project Cosultancy Services (NPCS), New Delhi, India.
Peterlić S. (2008) Dobra profesionalna praksa u analitič-
kom laboratoriju. Hrvatsko mjeriteljsko društvo, Zagreb.
Ribéreau-Gayon P., Glories Y., Maujean A., Dubourdi-
eu D. (2006) Handbook of Enology: The Chemistry of Wine
Stabilization and Treatments, Volume 2, 2 Ed., John Wiley &
Sons, Ltd, Chichester, UK.
Runje B. (2015) Razumjeti kontrolne karte. Što su, čemu
služe, kako ih izraditi i kako ih tumačiti. II. Blok. CROLAB
seminar. Hrvatski inženjerski savez. 12. ožujka. Zagreb.
Runje B., Ančić M. (2015) Razumjeti kontrolne karte. Što
su, čemu služe, kako ih izraditi i kako ih tumačiti. III. Blok.
CROLAB seminar. Hrvatski inženjerski savez. 12. ožujka. Za-
greb.
Štajdohar-Pađen O. (2015) Razumjeti kontrolne karte.
Što su, čemu služe, kako ih izraditi i kako ih tumačiti. I. Blok.
CROLAB seminar. Hrvatski inženjerski savez. 12. ožujka. Za-
greb.
Zoecklein B.W, Fugelsang K.C., Gump B.H., Nury F.S.
(1995) Wine analysis and production. Chapman & Hall, New
York, USA.
 155

Zahvala
Hrvatski časopis za prehrambenu tehnologiju, biotehnologiju i nutricionizam/Croatian Journal of Food Technology, Bio-
technology and Nutrition zahvaljuje svim recenzentima koji su tijekom 2017. godine pomogli u uređivanju znanstvenih i stručnih
radova.

Marija Badanjak Sabolović

Sandra Balbino
Ines Banjari
Irena Barukčić
Danijela Bursać Kovačević
Irena Colić Barić
Mato Drenjančević
Ivana Generalić Mekinić
Tibor Janči
Ana Jeromel
Marko Jukić
Irena Keser
Kristina Kljak
Draženka Komes
Klara Kraljić
Marina Krpan
Nina Kudumija
Ružica Lončarić
Ksenija Markov
Ibrahim Mujić
Jasna Novak
Dubravka Novotni
Mario Ostović
Jelka Pleadin
Ljiljana Primorac
Ivana Radojčić Redovniković
Darja Sokolić
Zvonimir Šatalić
Jana Šic Žlabur
Marina Tišma
Nina Toth
Milna Tudor Kalit
Natalija Velić
Sanja Vidaček Filipec
Sandra Voća
Tomislava Vukušić
Ana Vulić
Manuela Zadravec

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION

156

Upute autorima
Mole se autori radova za objavljivanje u znanstveno-struč-
nom časopisu “HRVATSKI ČASOPIS ZA PREHRAMBENU
TEHNOLOGIJU BIOTEHNOLOGIJU I NUTRICIONIZAM”
da se prilikom pisanja drže slijedećih naputaka. Časopis objav-
ljuje radove na hrvatskom ili engleskom jeziku, koji spadaju u
slijedeće kategorije:
1. Izvorni znanstveni radovi
Sadrže neobjavljene rezultate vlastitih izvornih istraživa-
nja. Rezultati moraju biti popraćeni i objašnjeni raspravom, te
po potrebi statistički obrađeni. Eksperiment treba biti ponov-
ljiv, te tako dobiveni rezultati unutar granica predviđene ekspe-
rimentalne pogreške. Rad je podijeljen na slijedeća poglavlja:
- Sažetak (na hrvatskom i engleskom jeziku)
- Uvod
- Materijali i metode rada
- Rezultati i rasprava
- Zaključci
- Literatura
koji se podnosi uredništvu mora biti napisan bez tipografskih po-
grešaka, u trećem licu, uz izbjegavanje pasivnih glagolskih oblika.
RADOVI ZAPRIMLJENI U ČASOPIS ZA
MOGUĆU OBJAVU ŠALJU SE NA RECENZIJU
BAREM DVOJICI UGLEDNIH STRUČNJAKA
Opseg rada treba biti maksimalno 20 A4 stranica (21,0 x
29,7 cm), što uključuje tablice i slike (otprilike dvije tablice ili
slike po stranici).
UPUTE ZA PRIPREMU TABLICA I SLIKA:
SLIKE:
• Slike se trebaju poslati kao posebni JPEG ili TIFF dokumenti
(visoka rezolucija) i trebaju biti prilikom slanja označeni kao:
Slika 1., Slika 2., Slika XY. Na samoj slici ne smije biti naziv.
• Za radove koji se pišu na Hrvatskom jeziku naziv slike tre-
ba biti prvo na Hrvatskom pa zatim na Engleskom jeziku:

2. Prethodna priopćenja Slika 1. Ocjena prilagodbe (fitanja) izlaznih i ciljanih po-

Kraće obavijesti o novim znanstvenim spoznajama, čija dataka dobivenih preko UNM, za različite amplitu-
narav zahtijeva hitno objavljivanje. Ne moraju omogućavati po- de.
navljanje ni provjeru iznesenih rezultata, ali obveza autora je da Figure 1. Goodness of fit of output and experimental data
nakon završetka istraživanja objavi izvorni znanstveni rad. obtained from ANN, for different amplitudes.

3. Znanstvene bilješke • Nazivi slika se trebaju staviti u rad točno na mjesto gdje
Kratka priopćenja ili znanstvene bilješke sadrže rezulta- slika treba biti.
te završenih kraćih istraživanja, opise izvornih laboratorijskih
tehnika, metoda, aparata i dr. TABLICE:
• Tablice trebaju biti izrađene u Word formatu i stavljene u
4. Pregledni radovi rad s nazivom iznad tablice
U kategoriju preglednih radova spadaju cjeloviti prikazi • Za radove koji se pišu na Hrvatskom jeziku nazivi tablica
novih znanstvenih spoznaja, područja ili problema. Izrađeni trebaju biti prvo na Hrvatskom pa na Engleskom jeziku.
su na temelju rezultata izvornih znanstvenih istraživanja većeg Tablice trebaju biti pripremljene u jednostavnom obliku
broja autora, rasprave na temelju literaturnih znanstvenih či- - vidi ispod:
njenica i vlastitih pretpostavki.
Tablica 1. Utjecaj ultrazvuka (1000W) na kemijski sastav [%]
5. Autorski pregledi kravljeg mlijeka
Radovi u kojima se prikazuju cjeloviti pregledi nekog Table 1. Influence of ultrasound on chemical composition
problema ili područja u kojem je autor objavio veći broj izvor- [%] of cow milk.
nih znanstvenih radova.
Otopine/Solutions Koncentracija/Concentration (g/L)
6. Stručni radovi
Urea 0,4
Problematikom nisu vezani uz izvorna istraživanja, a u
njima se iznose mogućnosti razvoja struke na područjima pre- R, L - asparagine acid 2,0
hrambene tehnologije, biotehnologije i nutricionizma. Kod Glycine 2,5
stručnih radova bitna je primjena poznatih metoda i činjenica,
te se koriste već stečena znanja primijenjena na cilj ispitivanja. Na-succinate 0,1
Razlučivanje znanstvenog i stručnog rada vrši sam autor, a te- CaCO3 0,14
melji se na originalnosti rezultata istraživanja i zaključaka, te
MgCO3 0,4
prikazanih znanstvenih metoda.

Uvjeti za prihvaćanje rada na recenziju: Ako sadržaj i kvaliteta nekog rada to opravdavaju, u izu-
Rukopisi sa prilozima se šalju Uredništvu u dva primjerka zetnim slučajevima prihvatiti će se i duži radovi. Za pravilno
s CD-om, ili putem e-mail adrese na E-mail: jfrece@pbf.hr. Rad formatiranje dokumenta, potrebno je uključiti numeriranje

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION


redova, podesiti 1,5 prored, te rubne bjeline (margine) na 2.5
cm.
Font korišten za izradu rada treba biti Times New Roman,
s veličinom slova od 12 pt. Izuzetak je naslov rada, za kojeg se
koriste podebljana slova u fontu veličine 14 pt. Početak odlom-
ka ne smije se uvlačiti TAB tipkom, dok se pojedini odlomci
razdjeljuju tipkom ENTER. Za numeraciju stranica koristi se
automatska numeracija ugrađena u program, s time da se broj
stranice postavlja na poziciju dolje-desno. Slijedeći formati su
prihvatljivi: MS Word (do verzije 2010, ekstenzije.doc i.docx),
te WordPerfect (do verzije 6). OpenOffice ODT format nije
prihvatljiv.
Naslov rada treba biti kratak. Ispod naslova navode se
imena i prezimena autora. Na posebnom listu papira prilože-
nom uz rad navode se titule i adrese autora, s punim imenima
i prezimenima. U radu na hrvatskom jeziku se za pisanje deci-
malnih brojeva koriste isključivo zarezi, odnosno u engleskoj
verziji isključivo točke.
Sažetak ne smije biti duži od jedne stranice, te treba sa-
državati jezgrovit prikaz, metodologiju, glavne rezultate i za-
ključak. Piše se na hrvatskom i engleskom jeziku u kurzivu. U
samom sažetku ne koriste se skraćenice, niti literaturni navodi.
Ispod sažetka navode se ključne riječi.
Uvod sadrži ukratko opisane rezultate prethodnih istraži-
vanja, kao i svrhu vlastitog istraživanja. Materijale i metode
treba kratko izložiti. Za poznate metode i tehniku istraživa-
nja navodi se samo autor i literatura, dok se detaljnije opisuju
samo ako odstupaju od već objavljenih u literaturi.
Rezultati i rasprava služe za isticanje i objašnjavanje naj-
važnijih rezultata, te se ne ponavljaju podaci izneseni u tabli-
cama i slikama. Zaključci sadržavaju kratko i jasno iznesen
značaj rezultata istraživanja.
Literatura mora biti selektivna, osim ako je riječ o pre-
glednim člancima, kada može biti i opširna. Ako originalna
literatura nije dostupna, autor mora navesti izvor koji je kori-
stio. Autori odabrane literature navedeni u tekstu moraju biti
na popisu literature. Autorima citiranima u tekstu navodi se u
zagradama prezime i godina objavljivanja. U slučaju da je rad
napisalo dvoje autora, navode se oba, dok se za radove sa tri
ili više autora navodi samo prezime prvog autora uz oznaku “i
sur.”, te godina objavljivanja.
U popisu literature potrebno je navesti potpune podatke o
svim autorima (prezime i inicijali imena autora), naslov djela,
naziv izdavača, mjesto i godina izdavanja, te stranice (od - do).
Literaturni navodi navode se abecednim redom, a radovi istog
autora i kronološkim redom. U slučaju zajedničkog rada više
autora, u popisu se ne koristi oblik “i sur.”, nego se navode svi
autori. Naslovi knjiga i časopisa pišu se u kurzivu.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
157
Primjeri:
Znanstveni ili stručni radovi:
Brnčić M., Karlović S. Rimac Brnčić S., Penava A., Bo-
siljkov T., Ježek D., Tripalo B. (2010) Textural properties of
infra red dried apple slices as affected by high power ultraso-
und pre-treatment. African Journal of Biotechnology, 9 (41)
6907-6915.
Knjige:
Sun D-W (ed) (2005) Emerging Technologies for Food
Processing. Academic Press, Elsevier Science, London, UK.
Edwards M. (2004) Detecting Foreign Bodies in Food.
Woodhead Publishing, Boca Raton, USA
Heinz W. (2007) Pressure and Heat Resistance of Clostri-
dium botulinum and Other Endospores. U: Doona C.J., Fee-
herry F.E. (ed): High Pressure Processing of Foods, str. 95-115.
Blackwell Publishing, Ames, USA.
Kongresni radovi:
Bosiljkov T., Brnčić M., Tripalo B., Karlović S., Ukra-
inczyk M., Ježek D., Rimac-Brnčić S. (2009) Impact of ul-
trasound-enhanced homogenization on physical properties of
soybean milk. U: Chemical Engineering Transactions - volume
17, Proceedings of the 9th International Conference on Chemi-
cal and Process Engineering. Rim, Italija.
Patenti:
Haydock D. (1993) Ultrasonically driven cutting knife
and method and apparatus for cutting a soft yielding bakery
product. Europski patent br. 3817141.
WWW stranice:
Hielscher N. (2007) Ultrasonic Homogenizing and Blen-
ding. Dostupno na: http://www.hielscher.com/ultrasonics/. Pri-
stupljeno: 01.01.2010.
Tablice i dijagrami moraju biti razumljivi i bez detaljnog
opisa u radu. Isti podaci ne mogu se koristiti na tablicama i
dijagramima. Naslovi tablica i slika moraju biti pisani na hr-
vatskom i engleskom jeziku u kurzivu.
Opisi i legende tablica i slika moraju biti pisani na hrvat-
skom i engleskom jeziku, kao i sav tekst istih.
Radi sigurnije i kvalitetnije izvedbe tiskanja rada, potreb-
no ih je također dostaviti i u jednom od grafičkih ili slikovnih
formata (JPEG, TIFF, XLS i sl.).
Separati
Prvom autoru rada dostaviti će se 5 primjeraka časopisa
“HRVATSKI ČASOPIS ZA PREHRAMBENU TEHNOLO-
GIJU BIOTEHNOLOGIJU I NUTRICIONIZAM”.
Na zahtjev autora dostaviti će se i veći broj primjeraka.
Adresa Uredništva:
Kačićeva 21, 10000 Zagreb
HRVATSKA
E-mail: jfrece@pbf.hr
158

Instructions to authors
Authors of papers should adhere to the following rules Manuscript preparation:
before submission in scientific-professional paper “CROATIAN
JOURNAL OF FOOD TECHNOLOGY BIOTECHNOLOGY Manuscripts to be considered for publication should be
AND NUTRITION”. submitted to Editorial office in two copies with CD, or by
Journal publishes papers in Croatian or English language, e-mail.
which belong to one of the following categories:
ARTICLES SUBMITTED FOR POSSIBLE
1. Original scientific papers PUBLISHING IN JOURNAL UNDERGO
Full-length papers consisted of author’s own original REVIEW PROCESS BY AT LEAST TWO
research. Results have to be accompanied and thoroughly DISTINGUISHED REVIEWERS.
explained in discussion. Statistical analysis of results should
be conducted. Experiments must be reproducible, with results
inside margins for experimental errors. Manuscript has to be written without spelling errors.
Paper should be divided in the following chapters: Passive tenses should be avoided. Maximum number of pages
- Abstract should be 15 A4 pages (21.0 × 29.7 cm), including figures and
- Introduction tables (approximately 2 figures of tables per page).
- Materials and methods
- Results and discussion FIGURES:
- Conclusions • Figures should be sent as separate JPEG or TIFF files
- Literature (high resolution) and marked as Figure 1., Figure 2….Fi-
gure XY.
2. Preliminary communications • Title of the Figure should be in English:
Short notifications about new scientific findings, which
require immediate publication. Result do not necessary have to Figure 1. Goodness of fit of output and experimental data
be reproducible or verified, but authors has to publish original obtained from ANN, for different amplitudes.
scientific paper after completion of research.
Titles of the figures should be placed in the article at the
3. Scientific notes place where authors consider to be.
Scientific notes include results of finished shorter
research, descriptions of new laboratory techniques, methods, TABLES:
devices, etc. • Tables should be presented in Word format and placed in
the article with the title above the table.
4. Reviews • Title of the table should be in English.
In this category belongs integral review of new scientific • Tables should be prepared in simple format - see below:
knowledge, area or problems. Manuscript is based on results
from original scientific papers from larger number of authors, Table 1. Influence of ultrasound on chemical composition
as well as discussion on the basis of literature references and [%] of cow milk.
own hypothesis. Solutions Concentration (g/L)
5. Author reviews Urea 0,4
Papers that analyze and integrate knowledge in specific R, L - asparagine acid 2,0
field, in which author himself has larger number of original
scientific papers. Glycine 2,5
Na-succinate 0,1
6. Professional papers
CaCO3 0,14
Papers which are not linked to original research. In
manuscripts development possibilities of specific fields MgCO3 0,4
(food technology, biotechnology and nutrition) are presented.
Application of known methods and facts, as well as application Larger manuscripts will be accepted if quality and content
of already acquired knowledge on research objective is justify it.
presented. Differentiation between original scientific and For proper document formatting, line numbering as well
professional paper rest on author’s opinion and it is based as automatic page numbering (position down - right) has to be
on originality of research results and conclusions, as well as included. Line spacing should be set at 1.5; margins at 2.5 cm.
presented scientific methods. Times New Roman font with size 12 pt should be used. Title
of manuscript is exception with font size 14 pt. Indentation at
start of new paragraph is not allowed.

CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION


Manuscripts may be submitted in any standard format,
including Word (up to version 2010,.doc and.docx extensions),
Wordperfect (up to version 6). OpenOffice ODT format is not
acceptable.
Title of the manuscript should be short, with names and
surnames of authors below. Full names of authors and their
addresses should be written on separate page attached to the
paper.
Summary must be under one page long, and it has to in-
clude concise review, methodology, main results and conclu-
sion. Please do not use abbreviations and references in summa-
ry. After summary authors have to write up to five keywords.
Introduction has to present summary of previous research
results, as well as purpose of manuscript.
Materials and methods should be presented briefly. For
known methods, authors and references are sufficient. Detailed
description should be given in case of new or modified methods.
Results and Discussion chapter should be used for
emphasis and description of most important results. Data in
tables must not be repeated in figures.
Conclusions contain brief presentation of importance of
research results.
References should be selective, except in case of review
articles. Authors should quote source, if original literature is
not available. Authors of chosen references which are quoted
in text must be in reference list. For quoting in text, surname of
author and year of publication of paper should be used, such as
(Knorr, 2010). In case of two authors of paper, surnames of both
authors should be included. Only first author should be quoted
in case of three or more authors, with “et al” abbreviation, i.e.
(Knorr et al, 2010).
In reference list it is necessary to include full data about
all authors (surname, initials of name (s)), title of paper, pub-
lisher, place and year of publishing, as well as page numbers
in journal. Reference list has to be in alphabetical order, with
papers of the same author in chronological order.
CROATIAN JOURNAL OF FOOD TECHNOLOGY, BIOTECHNOLOGY AND NUTRITION
159
Original scientific or professional papers:
Brnčić M., Karlović S. Rimac Brnčić S., Penava A.,
Bosiljkov T., Ježek D., Tripalo B. (2010) Textural properties
of infra red dried apple slices as affected by high power
ultrasound pre-treatment. African Journal of Biotechnology, 9
(41) 6907-6915.
Books:
Sun D-W (ed) (2005) Emerging Technologies for Food
Processing. Academic Press, Elsevier Science, London, UK.
Edwards M. (2004) Detecting Foreign Bodies in Food.
Woodhead Publishing, Boca Raton, USA
Heinz W. (2007) Pressure and Heat Resistance of
Clostridium botulinum and Other Endospores. In: Doona C.J.,
Feeherry F.E. (ed) : High Pressure Processing of Foods, str. 95-
115. Blackwell Publishing, Ames, USA.
Congress papers:
Bosiljkov T., Brnčić M., Tripalo B., Karlović S.,
Ukrainczyk M., Ježek D., Rimac-Brnčić S. (2009) Impact of
ultrasound-enhanced homogenization on physical properties
of soybean milk. In: Chemical Engineering Transactions -
volume 17, Proceedings of the 9th International Conference on
Chemical and Process Engineering. Rim, Italija.
Patents:
Haydock D. (1993) Ultrasonically driven cutting knife
and method and apparatus for cutting a soft yielding bakery
product. European patent Nr. 3817141.
Web pages:
Hielscher N. (2007) Ultrasonic Homogenizing and
Blending. Available at: http://www.hielscher.com/ultrasonics/.
Accessed: 01.01.2010.
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