| Sažetak | Siliranje visoko vlažnog zrna kukuruza način je da se očuva zrno kukuruza od berbe do
berbe. U klasičnoj teoriji siliranje je metoda konzerviranja uz pomoć anaerobnih uvjeta i
bakterija mliječne kiseline. Proizvodnja organskih kiselina, u najvećem dijelu mliječne
kiseline, snižava pH koji inhibira nepoželjne mikroorganizame i sprječava daljnje promjene
u siliranoj masi 1−2 mjeseca nakon početka siliranja. Suvremena istraživanja ukazuju na
to da silaža nastavlja fermentirati i nakon 1−2 mjeseca, što bi za posljedicu moglo imati i
kontinuirane promjene sastava i probavljivosti zrna kukuruza. Cilj istraživanja bio je utvrditi
kako dodatak inokulanta i mliječne kiseline prilikom siliranja vlažnog zrna u periodu od
godine dana utječe na intenzitet fermentacije i kvalitetu silaža te na razgradnju 16 kDa i
27 kDa γ-zeina hibrida različite tvrdoće zrna i udjela ukupnih zeina. Dodatno se ispitala i
buražna razgradivost suhe tvari i škroba ispitivanih silaža. Tri hibrida (Bc 513 pc, Bc 462 i
Bc 344) uzgojena u split-plot dizajnu u pet ponavljanja pri istim agroklimatskim uvjetima
razlikovala su se u tvrdoći zrna, što su pokazali rezultati Stenvertove analize tvrdoće zrna
i prava gustoća. Udio ukupnih zeina razlikovao se signifikantno između tri ispitivana
hibrida kukruza (Bc 513 pc: 59,96% SP; Bc 462: 55,13% SP; Bc 344: 48,75% SP). Silaže
sva tri hibrida silirane su s dodatkom inokulanta (Bio-Sil; 3x105 CFU/g), dodatkom
mliječne kiseline (linearni porast 5 ‒ 15 g/kg ST) i bez dodataka (kontrola) u periodu od
godine dana. Silaže su imale optimalne fermentacijske karakteristike tijekom godine dana
siliranja. Suha tvar (ST) silaža brojčano se minimalno mijenjala tijekom siliranja 69,11 ‒
71,04% i nije se razlikovala između tretmana i hibrida. Sadržaj mliječne kiseline
signifikantno je rastao do 182. dana stajanja silaža (1,01 ‒ 15,38 g/kg ST), iza čega je
opadao do zadnjeg uzorkovanja (9,10 g/kg ST). Nije utvrđena razlika između silaža
pojedinih hibrida u sadržaju mliječne kiseline. Skoro dvostruko viši sadržaj mliječne
kiseline imale su silaže s inokulantom, manje s mliječnom kiselinom i najmanje silaže bez
dodataka. Titracijska kiselost rasla je tijekom stajanja silaža (0,02 ‒ 0,74 meq/g ST) s
najvišim vrijednostima u silažama s dodatkom inokulanta. Vrijednost pH bilježila je
očekivan signifikantan pad tijekom siliranja (6,45 ‒ 4,18), s najnižim vrijednostima u
silažama s inokulantom i sukladno porastu mliječne kiseline te titracijske kiselosti u
silažama s dodatkom inokulanta. Tijekom godine dana stajanja silaža rastao je i sadržaj
produkata proteolize: topljivi protein (17,9 ‒ 47,0% SP) te amonijak (0,38 ‒ 3,73% SP).
Silaže hibrida Bc 513 pc imale su viši sadržaj sirovog proteina i ukupnih zeina od silaža
ostalih dvaju hibrida u svim vremenskim točkama. Na kraju siliranja zabilježena je
redukcija količine 16 kDa i 27 kDa γ-zeina, što ukazuje na to da siliranje uzrokuje
razgradnju ovih proteina. U svim silažama na kraju siliranja nije detektiran 27 kDa γ-zein.
S druge strane razgradnja 16 kDa γ-zeina bila je različita u silažama ispitivanih hibrida
siliranim s različitim dodacima. Dodatak inokulanta imao je veći utjecaj na razgradnju γzeina
silaža tvrđih hibrida (Bc 513 pc i Bc 462), a dodatak mliječne kiseline na silaže
mekšeg hibrida (Bc 344). Silaže s dodacima imale su veću razgradnju 16 kDa γ-zeina od
kontrolnih silaža kod svih ispitivanih hibrida. Na kraju siliranja zabilježen je signifikantan
porast buražne razgradivosti suhe tvari (od 0,59 na početku do 0,77 na kraju) i škroba (od
0,53 do 0,78), što je potvrda da siliranje (i razgradnja γ-zeina) povisuju razgradnju škroba i
suhe tvari. Istraživanjem je potvrđena hipoteza o većoj razgradnji 16 kDa i 27 kDa γ-zeina
u starijim silažama te hibridima tvrđeg, nego mekšeg endosperma te u silažama s
dodacima, nego bez dodataka. Dodatno, istraživanje ukazuje da uz slične vrijednosti
razgradivosti suhe tvari i škroba na kraju siliranja, kao i hibridi mekšeg zrna, hibridi tvrđeg
zrna (Bc 462) imaju bolje proizvodne karakteristike. |
| Sažetak (engleski) | Influence of corn hybrids and type of silage additives on total and γ- zein content
during the high moisture corn silage production
Silage production is a conservation method used for high moisture crop
preservation. It is based on natural fermentation that is under control of epiphytic
microflora, optimally-mostly by lactic acid bacteria (McDonald et al., 1991). Protein
fractions are important indicators of silage quality (Der Bedrosian et al., 2012; Ferraretto
et al., 2014). In particular, corn silage soluble crude protein (sCP) fraction and ammonium
(NH3) content are positively associated with proteolysis (Winters et al., 2000; Johnson et
al., 2002; Slottnerr and Bertilsson, 2006; Ferraretto et al., 2014) and correlate positively
with ruminal in vitro starch digestibility (Ferraretto et al., 2015b). Zein proteins, the most
abundant protein fraction in corn grain, surround starch granules and their hydrophobic
nature acts as a barrier to starch digestion by rumen microbes (Giuberti et al., 2014).
Besides their influence on starch digestibility, zeins also appear to influence the texture
and hardness of the grain (Holding and Larkins, 2009). Below et al. (2004) reported that
total zein content of corn grain depends primarily on genotypic factors; with environmental
factors such as N supply or plant maturity at harvest both causing smaller or larger
phenotypic variations.
In the corn endosperm zein proteins are located within protein aggregates called
protein bodies and are comprised of four distinctive zein classes: alpha (α), beta (β), delta
(δ) and gamma (γ) zein. Location of distinctive zein class differs within protein bodies.
Gamma zein is thought to be the most important zein fraction. This zein class is located
on the surface of protein bodies and in small spots within where they together with beta
zein stabilize alpha zein core. Gamma zeins are comprised from three distinctive proteins:
16-kDa gamma zein, 27-kDa gamma zein and 50-kDa gamma zein (Woo et al., 2001;
Holding and Larkins, 2006; Boston and Larkins, 2009; Holding and Larkins, 2009).
In silage production, lactic acid bacteria (LAB) additives are widely used to optimize
fermentation and thereby maximize lactic acid production, quickly reduce pH, and
preserve nutrients (McDonald et al., 1991). Lactic acid production during ensiling of
forages is well established (McDonald et al., 1991); this most abundant and the strongest
acid in silages dissolves zein proteins, leading to a reduction in grain total zein content
(Lawton, 2002). In addition to lactic acid, enzymes from plants or microorganisms degrade
zein proteins (McDonald et al., 1991; Lawton, 2002; Hoffman et al., 2011).
The purpose of the present study was to investigate the additive effect of lactic acid
bacteria and lactic acid application on the gamma zeins content, total zein content and
grain rumen degradability during high moisture corn fermentation process of corn hybrids
that differ in endosperm texture and total zein content. In commercial production ensiling
is conducted on yearly basis e.g. between two corn harvests. Main silage conservation
characteristics where monitored at different time points during one year ensiling period.
The gamma zein proteome analysis e.g. differences between gamma zeins content in
silages were detected using 2-D gel electrophoresis followed by densitometry
quantification and MALDI-TOF/TOF protein identification.
Hybrids (Bc 513 pc, Bc 462 and Bc 344) were grown in 2013 under the same
production conditions in split-plot experimental fields at the Faculty of Agriculture of the
University of Zagreb. Each hybrid was grown in five replicates. Chopped material from
each replicate was divided in three parts. Silages where ensiled at the black layer stage of
growth (67.61% – 72.42% DM) in laboratory scale bag silos with one part ensiled with BioSil
LAB inoculant (Lactobacillus plantarum DSM 8862 and DSM 8866) in a concentration
3x105 CFU/g of fresh material, second one was left untreated while the third was treated
with lactic acid (linear application 5 ‒ 15 g/kg DM). Silages were maintained at 25 ± 2 °C
and sampled at the beginning and on 15th, 48th, 98th, 182nd, 274th and 364th day of ensiling.
Silage conservation characteristics (DM, lactic acid (LA), titratable acidity (TA), pH, crude
protein (CP), sCP, NH3, total zein) where monitored. At the beginning and the end of
ensiling period gamma zein content and silage ruminal degradability was evaluated. At the
end of ensiling total bacterial count, lactic acid and yeast count was determined. At the
beginning of ensiling the corn grain hardness was evaluated in green material of hybrid
tested with Stenvert hardness test and true density. Effects of time, silage additive and
hybrid genotype together with their interactions on silages were tested as a split-plot
design using the PROC MIXED procedure in SAS 9.4 (SAS Institute, Cary, NC, USA).
Means with different superscript letter groups were obtained with postfitting statistical
PROC PLM. The differences of green material endosperm texture and microbial count
were performed separately as a split-plot completely randomized design and differences
between means were assessed for significance using the Tukey test. The correlation
parameters where determined using PROC CORR and linear regression between the dry
matter and starch degradability rate with PROC REG in SAS 9.4. Differences and
interactions were considered significant when P < 0.05.
Grain hardness is important corn characteristic that plays a role in the final product
quality and nutritional value of corn. It is highly connected with the ratio of horny and floury
endosperm in corn grain and with total zein content (Dombrink-Kurtzman and Bietz, 1993;
Pratt et al., 1995). Stenvert hardnes test parameters (time of grinding, volume of grinded
sample and coarse to fine particle ratio, Wt C/F) showed the highest hardness in Bc 513
pc and lowest in Bc 344. Hybrids differed significantly in time of grinding and Wt C/F. Bc
513 pc had longer time of grinding (3.94 sec) in contrast with Bc 344 (3.50 sec) and Bc
462 (3.75 sec) and higher Wt C/F than other two hybrids (Bc 513 pc: 1.96; Bc 344: 1.46;
Bc 462: 1.48). True density analysis showed similar results to Stenvert hardness test; Bc
513 pc had highest values (1.36 g/ml) in contrast to Bc 344 (1.26 g/ml) and Bc 462 (1.31
g/ml) (P < 0.001). Harder corn grains have higher true density than soft corn grain (Wu,
1992; Radosavljević et al., 2000). Results proved the highest hardness in Bc 513 pc and
lowest in Bc 344. Bc 462 is more similar to Bc 513 pc (corn type popcorn) than Bc 344,
and is regarded as somewhat harder corn than Bc 344. The total zein content was in
accordance with observed differences in hardness between hybrids tested, Bc 513 pc had
highest total zein content (59.96 % CP) and Bc 344 lowest (48.75 % CP) total zein content
(Bc 462: 55.13 % CP) (P < 0.001).
The silage fermentation profiles obtained with these 3 hybrids are in accordance
with other high moisture corn silages ensiled at the black layer (Ferraretto et al., 2014).
Dry matter value showed significant but numerically low change during ensiling (69.11 ‒
71.04 %). The hybrid tested had no influence on lactic acid content, pH value or titratable
acidity in silages. However, the use of inoculant was associated with higher lactic acid
content (12.63 g/kg DM; P < 0.001), faster pH reduction (6.45 ‒ 4.03; P < 0.001) and
higher titratable acidity (0.57 meq/g DM; P < 0.05) in tested silages, consistent with the
ability of LAB to promote fermentation. Application of lactic acid resulted with similar trend
in silages (lactic acid, LA: 8.55 g/kg DM; control, LA: 7.14 g/kg DM; lactic acid, pH: 6.45 ‒
4.17; control, pH: 6.45 ‒ 4.35; lactic acid, TK: 0.51 meq/g DM; control, TK: 0.51 meq/g
DM). Both types of silage additives used in this study improved silage fermentation
characteristics when compared to control silages. In all silages regardless of the hybrid or
additive tested increase in lactic acid content to 182nd day of ensiling was observed (1.01
‒ 15.89 g/kg DM; P < 0.001), after which small reduction in LA content up to 364th day
was observed (274th: 11.60 g/kg DM; 364th: 9.10 g/kg DM). In substrate deficiency,
primarily glucose, lactic acid bacteria (including Lactobacillus plantarum) metabolize lactic
acid to acetic acid (Lindgren, 1990). In the case of negative yeast or clostridia activity,
parallel to lactic acid reduction the increase in pH should be observed (McDonald et al.,
1991; Weinberg and Muck, 1996). However that was not the case in this study. Crude
protein content in silages was significantly different between hybrid tested, with highest
values observed in Bc 513 pc and lowest in Bc 344 (Bc 513 pc: 133 g/kg DM; Bc 344:
105.6 g/kg DM; Bc 462: 109.8 g/kg DM) and is in accordance with total zein content
observed for hybrids tested, with highest values observed for Bc 513. Although the triple
interaction influence (corn hybrid×silage additive×ensiling period) observed for ammonium
(P < 0.001) and sCP (P < 0.05) content in silages implies different hybrid responses to
additive application during ensiling, the same trend was observed. The sCP and
ammonium content showed increase during ensiling (sCP: 17.9 ‒ 47.0 % CP; ammonium:
0.38 ‒ 3.73 % CP; P < 0.001) and are evidence of proteolysis in silages (Winters et al.,
2000; Johnson et al., 2002; Slottnerr and Bertilsson, 2006).
Electrophoretic separation and mass spectrometry identification of gamma zein
extract of tested silages showed thirteen distinctive spots of zein proteins. Of three
gamma zein types, 16-kDa and 27-kDa gamma zeins were detected. 50-kDa gamma zein
is expressed usually in undetectable quantities on molecular level (Woo et al., 2001). At
the end of ensiling, densitometry of electrophoretically separated proteins showed
reduction of both gamma zein proteins. 27-kDa zein was reduced at undetectable level in
all silages. Inoculant application resulted with higher degradability of 16-kDa gamma zein
proteins in silages of corn hybrids with harder endosperm type. Lactic acid application
resulted with higher degradability of 16-kDa gamma zein proteins in softer corn hybrid (Bc
344) silages. In all silages degradation of 16-kDa zein was lowest in control silages
regardless of the hybrid used. Other zein detected were alpha zeins, which showed
reduction at the end of ensiling. However the reduction was less expressive than in the
case of gamma zeins. Gamma zeins are located on the surface of protein bodies and in
small spots at the core of protein bodies (Holding and Larkins, 2006) which can explain
higher degradability of gamma zeins compared to alpha zeins. Interestingly, increase in
total zein content was observed during ensiling regardless of the hybrid tested or additive
used and is opposite to proteolytic activity observed in silages (increase in ammonium and
sCP contents) and observed reduction in gamma zein and alpha zein. Analytical method
used for zein quantification proved to be efficient for defining difference between hybrids
tested, but inefficient for defining zein degradation rate during ensiling (Hoffman et al.,
2011).
High moisture corn is highly used feed in ruminant and monogastic animals nutrition
as it reduces costs of drying during corn grain storage and it allows earlier harvest of corn
grain during unfavourable weather. Additionally, high moisture corn silage preparation
allows corn utilization from harvest to harvest, during whole year and it has positive effect
on corn grain nutritional value. Ensiling boosts ruminal starch degradability (Philippeau et
Michalet-Doreau, 1998; Firkins, 2001; Ferraretto et al., 2013). High moisture corn silages
had higher starch and dry matter degradation parameters at the end of ensiling when
compared with green material (effective degradability of dry matter: from 0.59 to 0.77;
effective degradability of starch: from 0.53 to 0.78; P < 0.001). All the starch degradation
parameters showed improvement at the end of ensiling; Lag phase was shorter (from 7.37
to 5.43 hours; P < 0.001), rapidly degradation fraction was higher (from 0.28 to 0.54; P <
0.001) as well as the rate of starch degradation (from 0.051 h-1 to 0.098 h-1; P < 0.001).
Similar trend was observed in dry matter degradation. Strong linear regression of rate of
dry matter degradation and starch degradation observed by us implies that dry matter
degradation is highly related to starch degradation in high moisture corn silages
(Philippeau et al., 2000; Correa et al., 2002; Ngonyamo-Majee et al., 2008). Inoculant
application had positive effect on degradation of dry matter and starch, but overall this
effect was minimal. The hybrid effect on dry matter degradability or starch degradability
showed somewhat better parameters for softer corn hybrid (Bc 344) although the
difference was minimal compared to harder corn hybrid (Bc 462).
The study confirmed the hypothesis of a greater degradation of 16-kDa gamma zein
in silages at the end of one year ensiling and in hybrids with harder than softer grain, as
well in silages with additives. Inoculant application had more significant influence on
harder grain hybrids; and lactic acid on softer hybrid. The greater 16-kDa gamma zein
degradation was connected with higher ruminal degradation of starch and dry matter.
Results showed that harder hybrid (Bc 462) had similar ruminal degradability as softer
hybrid (Bc 344) with the somewhat better fermentation characteristics and better 16-kDa
gamma zein degradation thus implying better production characteristics for more vitreous
corn. |
