INTRODUCTIONThe ensiling process ma

INTRODUCTION
The ensiling process may occur either naturally, with
epiphytic microorganisms present on the plant material,
or with the addition of inoculants to improve the
process, thus resulting in better quality silage. Microbial
inoculants are commercially available for use in
silage, and lactic acid bacteria (LAB) are the main
microorganisms used for this purpose (Cai et al., 1999;
Driehuis et al., 2001; Filya, 2003).
In general, studies with LAB inoculants show that
inoculation before ensiling increases the fermentation
quality of the ensiled forage (Kleinschmit and Kung,
2006; Zopollatto et al., 2009). However, the results can
be inconsistent when forage crops are evaluated under
different conditions, such as silo size, climate, and packing
density. Factors related to the storage and application
of inoculants might influence their effects on silage
quality. Nevertheless, one of the determining factors
for the successful application of microbial inoculants
in silage is the compatibility between the plant and the
microorganisms used (Muck, 2008; Ávila et al., 2009).
This compatibility can be assessed by the ability of the
microorganisms to use carbohydrates present in the forage
and to produce metabolites of interest, primarily in
the preservation of silage (e.g., acetic and lactic acids).
Sugar cane (Saccharum spp.) is a forage crop widely
used in animal feed because of its high DM production
(25 to 40 t/ha) and high energy concentration, which is
due to the high concentration of sugars, mainly sucrose
(250 to 300 g/kg). The ensiling of sugar cane often results
in problems with the overgrowth of yeasts, which
leads to high losses of DM throughout the fermentative
process (Kung and Stanley, 1982). Chemical and microbiological
additives have been tested with the aim of
reducing yeast growth. However, microbial inoculants
have produced better results than chemical additives
(Carvalho et al., 2012).
Inoculants with LAB, which produce higher concentrations
of acetic or propionic acids, are more suitable
for yeast control because of the fungicidal effect of
these acids (Moon, 1983). The addition of microorganisms
that produce greater amounts of lactic acid are of
interest because of their rapid effect in reducing the pH
value. However, lactic acid is a potential substrate for
yeast during feeding-out, reducing the aerobic stability
of the silage. Inoculation with facultative heterofermentative
Lactobacillus plantarum and obligatory heterofermentative
Lactobacillus buchneri has been tested during
ensiling of sugar cane. The results are variable, but in
general, Lb. buchneri showed good results in reducing the DM losses and increased aerobic stability (Ávila
et al., 2009; Roth et al., 2010). Pedroso et al. (2008)
observed that Lb. buchneri improved fermentative and
aerobic stability in silages, whereas Lb. plantarum
strains interfered negatively in the fermentation and
preservation of sugar cane silages. Ávila et al. (2010b)
evaluated different LAB species (Lb. plantarum, Lactobacillus
paracasei, Lactobacillus brevis, and Lactobacillus
brevis buchneri) and observed that the effect of the
inoculant is more related to the strain used than to the
species. Inoculations with different strains that belong
to the same species have resulted in silages with different
characteristics, suggesting that studies should be
conducted not only at the species level but also at the
strain level (Saarisalo et al., 2007; Ávila et al., 2011).
The effects of microbial inoculants on the fermentation
process of silage are mainly due to the production
of metabolites of interest able to inhibit the growth
of undesired microorganisms. Therefore, the ability of
a strain to utilize different substrates present in the
forage plant and to produce different metabolites can
be an advantage in the competition with other microorganisms.
This ability can be used as a criterion for
selecting inoculants (Saarisalo et al., 2007). The present
study aimed to isolate, identify, and select LAB strains
for the ensiling of sugar cane by a rapid method based
on the production of metabolites that are relevant for
the silage process. In addition to strain performance,
we evaluated the improvement of chemical and microbiological
silage characteristics in experimental silos.
MATERIALS AND METHODS
Experiment 1: Isolation and Characterization
of LAB from Sugar Cane Silage
Silages were made with fresh-cut sugar cane from
plants that were approximately 12 mo old. The sugar
cane was manually harvested and chopped using a
laboratory-type chopper (PP-47, Pinheiro, Itapira, SP,
Brazil) at an approximate length of 30 mm. Approximately
10 kg of chopped material was immediately
conditioned in 15-L plastic buckets without valves for
gas release or effluent (mini-silos). The material was
compacted to a density of approximately 700 kg of
fresh matter/m3. The mini-silos were stored at room
temperature (22°C) and opened after 0, 2, 15, 60, and
90 d of storage. Samples were taken on each opening
day for pH analysis. Two replicates were prepared for
each date of sampling.
The LAB were isolated from 80 g of sugar cane silage
that was mixed with 720 mL of 0.1% sterile peptone
water and homogenized in an orbital mixer for 20
min. Subsequently, 10-fold dilutions were prepared to
quantify the LAB using de Man, Rogosa, and Sharpe
agar (MRS, Difco, Detroit, MI) containing 0.1%
cysteine-HCl and cycloheximide (0.4%). The plates
were incubated at 30°C for 48 h under anaerobic conditions
(Gas Pack Anaerobic System, BBL, Cockeysville,
MD). Colonies were counted on plates with 30 to 300
well-isolated cfu, and a number of colony-forming units
corresponding to the square root of the total was taken
at random for identification (Holt et al., 1994). The
isolates were further purified by streaking individual
colonies onto MRS agar. The purified isolates were
maintained at −80°C in MRS broth containing 20%
(vol/vol) glycerol.
The size, shape, color, height, and edge morphology
of each colony were noted. The presumptive lactobacilli
were counted on MRS agar. The isolates were examined
by Gram stain and for colony and cell appearance,
catalase activity, motility and production of CO2 from
glucose, and gluconate in MRS broth with a Durham
tube. The lactobacilli were recognized as gram-positive,
catalase-negative, oxidase-negative, regular fermentative
rods, and were classified as homofermentative or
heterofermentative lactobacilli by their ability to produce
CO2 from glucose and gluconate.
Preselection of Bacterial Strains Based on
Metabolite Production in Sugar Cane Broth. Fifty-
seven isolates classified as LAB were isolated from
sugar cane silage and evaluated for metabolite production.
The LAB were evaluated in a 5° Brix sugar cane
broth medium supplemented with 0.1% yeast extract.
The Brix degree (soluble solids) was determined according
to AOAC (1990) using a digital refractometer
Atago PR-32 (Atago USA Inc., Bellevue, WA), with
automatic temperature compensation. The broth was
filtered (gauze) and sterilized (120°C, 15 min). First,
the 57 strains were cultivated in MRS broth for 24 h
at 35°C. After this period, the inoculum was standardized
using a spectrophotometer (600 nm) at an optical
density of 1.0. Subsequently, approximately 400 μL of
each strain was inoculated into 300 mL of sugar cane
broth, which was incubated at 35°C and 120 rpm. After
24 h of fermentation, samples of the cultures were taken
to evaluate metabolite production by HPLC.
Data regarding the production of metabolites by
strains were analyzed using principal component
analysis (PCA). Sugar cane has a high concentration
of soluble carbohydrates, a low buffering capacity, and
DM content suitable for ensiling. Therefore, a decrease
in pH occurs quickly (Kung and Stanley, 1982; Ávila et
al., 2009). However, the overgrowth of yeast in sugar
cane silage throughout the process causes DM losses
(Kung and Stanley, 1982). Moreover, problems with
deterioration after silo opening are common because of
the high concentration of lactic acid,

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PENDAHULUANProses ensiling dapat terjadi baik secara alami, denganepifit mikroorganisme hadir pada bahan tanaman,atau dengan penambahan inokulan untuk meningkatkanproses, sehingga mengakibatkan lebih baik kualitas silase. Mikrobainokulan komersial tersedia untuk digunakan disilase, dan bakteri asam laktat (LAB) yang utamamikroorganisme yang digunakan untuk tujuan ini (Cai et al., 1999;Driehuis et al., 2001; Filya, 2003).Secara umum, studi dengan inokulan laboratorium menunjukkan bahwaInokulasi sebelum ensiling meningkatkan fermentasikualitas hijauan ensiled (Kleinschmit dan Kung,2006; Zopollatto et al., 2009). Namun, hasilnya bisamenjadi tidak konsisten ketika tanaman hijauan dievaluasi di bawahkondisi yang berbeda, seperti ukuran silo, iklim dan Kemasankepadatan. Faktor-faktor yang terkait dengan penyimpanan dan aplikasipola dapat mempengaruhi mereka efek pada silasekualitas. Namun demikian, salah satu faktor penentuuntuk aplikasi sukses inokulan mikrobasilase adalah kompatibilitas antara tanaman danmikroorganisme digunakan (kotoran, 2008; Ávila et al., 2009).Kompatibilitas ini dapat dinilai oleh kemampuanmikroorganisme menggunakan karbohidrat hadir dalam hijauandan untuk menghasilkan metabolit menarik, terutama dipelestarian silase (misalnya, asam asetat dan laktat).Tebu (Saccharum spp.) adalah tanaman hijauan yang luasdigunakan dalam pakan karena DM tinggi produksi ternak(25 sampai 40 t/ha) dan energi tinggi konsentrasi, yangkarena konsentrasi tinggi dari gula, terutama Sukrosa(250-300 g/kg). Ensiling tebu sering hasilmasalah dengan pertumbuhan berlebih dari ragi, yangmenyebabkan kerugian yang tinggi DM seluruh fermentasiproses (Kung dan Stanley, 1982). Kimia dan Mikrobiologiaditif telah diuji dengan tujuanmengurangi pertumbuhan ragi. Namun, mikroba inokulantelah menghasilkan hasil yang lebih baik daripada bahan kimia tambahan(Carvalho et al., 2012).Inokulan dengan LAB, yang menghasilkan konsentrasi yang lebih tinggidari asetat atau asam propionat, lebih cocokpengendalian ragi karena efek fungisidaasam (bulan, 1983). Penambahan mikroorganismemenghasilkan jumlah yang lebih besar dari asam laktat daribunga karena efek cepat dalam mengurangi pHnilai. Namun, asam laktat adalah substrat yang potensial untukragi selama makan-keluar, mengurangi stabilitas aerobikdari silase. Inokulasi dengan Heterofermentatif fakultatifLactobacillus plantarum dan wajib HeterofermentatifLactobacillus buchneri telah diuji selamaensiling tebu. Hasilnya adalah variabel, tapi diumum, Lb. buchneri menunjukkan hasil yang baik dalam mengurangi kerugian DM dan peningkatan stabilitas aerobik (Ávilaet al., 2009; Roth et al., 2010). Pedroso et al. (2008)mengamati bahwa Lb. buchneri ditingkatkan fermentasi danaerobik stabilitas di silages, sedangkan Lb. plantarumstrain mengganggu negatif di fermentasi danpelestarian tebu silages. Ávila et al. (2010b)dievaluasi spesies LAB yang berbeda (plantarum lb, Lactobacillusparacasei, Lactobacillus brevis dan Lactobacillusbrevis buchneri) dan mengamati bahwa efekinokulan lebih berkaitan dengan strain digunakan daripadaspesies. Inokulasi dengan strain yang berbeda yang termasukuntuk spesies yang sama telah mengakibatkan silages dengan berbedaKarakteristik, menyarankan bahwa studi harusdilakukan tidak hanya di tingkat spesies tetapi juga dilevel strain (Saarisalo et al., 2007; Ávila et al., 2011).Efek dari mikroba inokulan pada fermentasiproses silase adalah terutama karena produksimetabolit menarik dapat menghambat pertumbuhanmikroorganisme yang tidak diinginkan. Oleh karena itu, kemampuanregangan untuk memanfaatkan substrat yang berbeda hadir diHijauan tanaman dan menghasilkan berbeda metabolit dapatmenjadi keuntungan dalam kompetisi dengan mikroorganisme lainnya.Kemampuan ini dapat digunakan sebagai kriteria untukmemilih inokulan (Saarisalo et al., 2007). Masa kinistudi ini bertujuan untuk mengisolasi, mengidentifikasi dan pilih strain LABuntuk ensiling tebu oleh metode cepat yang didasarkanpada produksi metabolit yang relevan untukproses silase. Selain kinerja ketegangan,dievaluasi kami peningkatan kimia dan Mikrobiologisilase karakteristik di eksperimental silos.BAHAN DAN METODEPercobaan 1: Isolasi dan karakterisasiLab dari tebu silaseSilages dibuat dengan segar-memotong tebu daritanaman yang kira-kira 12 mo tua. Gulatebu secara manual dipanen dan cincang menggunakanLaboratorium-jenis helikopter (PP-47 Pinheiro, Itapira, SP,Brasil) pada perkiraan panjang 30 mm. sekitar10 kg bahan cincang itu segeradikondisikan dalam 15-L plastik ember tanpa katup untukrilis gas atau limbah (mini-Silo). Bahandipadatkan untuk kepadatan kira-kira 700 kgsegar masalah/m3. Mini-Silo disimpan di ruangsuhu (22° C) dan dibuka setelah 0, 2, 15, 60, dand 90 penyimpanan. Sampel yang diambil pada setiap pembukaanhari untuk analisis pH. Dua Ulangan digembleng selamasetiap tanggal pengambilan sampel.Laboratorium diisolasi dari 80 g gula tebu silaseyang dicampur dengan 720 mL peptone steril 0,1%air dan homogen di mixer orbit untuk 20min. Selanjutnya, pengenceran 10-kali lipat bersediamengukur laboratorium menggunakan de Man, Rogosa, dan Sharpeagar-agar (MRS, Difco, Detroit, MI) mengandung 0.1%Sistein-HCl dan cycloheximide (0,4%). Piringyang diinkubasi di 30° C selama 48 jam kondisi anaerobic(Gas sistem anaerobik Pack, BBL, Cockeysville,MD). koloni dihitung pada piring dengan 30 sampai 300cfu terisolasi dengan baik, dan sejumlah koloni membentuk unitsesuai dengan akar kuadrat dari total diambilsecara acak untuk identifikasi (Holt et al., 1994). Theisolat lebih lanjut ditahirkan oleh melesat individukoloni-koloni ke MRS agar-agar. Isolat murnidipertahankan pada −80 ° C dalam MRS kaldu yang berisi 20%gliserol (vol/vol).Ukuran, bentuk, warna, tinggi, dan tepi morfologisetiap koloni diperhatikan. Laktobasili anggapandihitung pada MRS agar. Isolat tersebut Diperiksapewarnaan Gram dan penampilan koloni dan sel,catalase kegiatan, motilitas dan produksi CO2 dariglukosa, dan glukonat dalam MRS kaldu dengan Durhamtabung. Laktobasili yang diakui sebagai gram-positif,catalase-negatif, oksidase-negatif, biasa fermentasibatang, dan diklasifikasikan sebagai homofermentatif ataulaktobasili Heterofermentatif oleh kemampuan mereka untuk menghasilkanCO2 dari glukosa dan glukonat.Yang dinginkan bakteri strain berdasarkanMetabolit produksi dalam kaldu tebu. Lima puluh-tujuh isolat diklasifikasikan sebagai laboratorium diisolasi darisilase tebu dan dievaluasi untuk metabolit produksi.Laboratorium dievaluasi dalam 5° Brix gula tebuekstrak kaldu menengah dilengkapi dengan ragi 0,1%.Tingkat Brix (padatan terlarut) ditentukan menurutuntuk AOAC (1990) menggunakan refraktometer digitalAtago PR-32 (Atago USA Inc, Bellevue, WA), denganotomatis suhu kompensasi. Kaldu itudisaring (kasa) dan disterilkan (120° C, 15 menit). Pertama,57 strain yang dibudidayakan di MRS kaldu untuk 24 hpada 35° C. Setelah masa ini, inoculum standarmenggunakan spectrophotometer (600 nm) di optikkepadatan 1.0. Selanjutnya, sekitar 400 μL dariketegangan setiap diinokulasi ke 300 mL tebukaldu, yang diinkubasi di 35° C dan 120 rpm. Setelah24 h fermentasi, diambil sampel dari budayaEvaluasi metabolit produksi oleh HPLC.Data mengenai produksi metabolit olehstrain dianalisis menggunakan komponen utamaanalisis (PCA). Gula tebu memiliki konsentrasi tinggikarbohidrat larut, kapasitas buffer yang rendah, danKonten DM cocok untuk ensiling. Oleh karena itu, penurunanpH terjadi cepat (Kung dan Stanley, 1982; Ávila etAl., 2009). Namun, pertumbuhan berlebih ragi gulatebu silase seluruh proses menyebabkan kerugian DM(Kung dan Stanley, 1982). Selain itu, masalah dengankerusakan setelah silo buka umum karenakonsentrasi tinggi asam laktat,