940J. Dairy Sci. 97 :940–951http://

940
J. Dairy Sci. 97 :940–951
http://dx.doi.org/10.3168/jds.2013-6987
© American Dairy Science Association®, 2014.
ABSTRACT
Sugar cane (Saccharum spp.) is a forage crop widely
used in animal feed because of its high dry matter (DM)
production (25 to 40 t/ha) and high energy concentration.
The ensiling of sugar cane often incurs problems
with the growth of yeasts, which leads to high losses of
DM throughout the fermentative process. The selection
of specific inoculants for sugar cane silage can improve
the quality of the silage. The present study aimed to
select strains of lactic acid bacteria (LAB) isolated
from sugar cane silage and to assess their effects when
used as additives on the same type of silage. The LAB
strains were inoculated into sugar cane broth to evaluate
their production of metabolites. The selected strains
produced higher concentrations of acetic and propionic
acids and resulted in better silage characteristics, such
as low yeast population, lower ethanol content, and
lesser DM loss. These data confirmed that facultative
heterofermentative strains are not good candidates for
sugar cane silage inoculation and may even worsen the
quality of the silage fermentation by increasing DM
losses throughout the process. Lactobacillus hilgardii
strains UFLA SIL51 and UFLA SIL52 resulted in silage
with the best characteristics in relation to DM loss, low
ethanol content, higher LAB population, and low butyric
acid content. Strains UFLA SIL51 and SIL52 are
recommended as starter cultures for sugar cane silage.
Key words: inoculant , Lactobacillus hilgardii , 1,2-propanediol
, yeast
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 use of Lactobacillus species as starter cultures
for enhancing the quality of sugar cane silage
C. L. S. Ávila,* B. F. Carvalho,* J. C. Pinto,† W. F. Duarte,* and R. F. Schwan*1
*Department of Biology, and
†Department of Animal Science, Federal University of Lavras, 37.200-000, Lavras, Minas Gerais, Brazil
Received May 1, 2013.
Accepted November 2, 2013.
1 Corresponding author: [e-mail]
Journal of Dairy Science Vol. 97 No. 2, 2014
LACTOBACILLUS SPECIES IN SUGAR CANE SILAGE 941
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

Hasil (Bahasa Indonesia) 1:

940J. susu Sci. 97:940-951http://DX.Doi.org/10.3168/JDS.2013-6987© Amerika susu Sains Asosiasi ®, 2014.ABSTRAKTebu (Saccharum spp.) adalah tanaman hijauan yang luasdigunakan dalam hewan feed karena bahan kering yang tinggi (DM)produksi (25 sampai 40 t/ha) dan energi tinggi konsentrasi.Ensiling tebu sering incurs masalahdengan pertumbuhan ragi, yang menyebabkan kerugian yang tinggi dariDM seluruh proses fermentasi. Pemilihanpola tertentu untuk tebu silase dapat meningkatkankualitas silase. Penelitian ini bertujuan untukPilih strain bakteri asam laktat (LAB) terisolasidari gula tebu silase dan untuk menilai efek mereka ketikadigunakan sebagai aditif pada jenis yang sama silase. Laboratoriumstrain yang diinokulasi ke dalam kaldu tebu untuk mengevaluasiproduksi mereka metabolit. Strain dipilihdiproduksi konsentrasi yang lebih tinggi dari asetat dan propionatasam dan mengakibatkan lebih baik silase karakteristik, sepertisebagai penduduk rendah ragi, menurunkan kandungan etanol, danlebih rendah DM kerugian. Data ini menegaskan bahwa fakultatifHeterofermentatif strain yang tidak baik untuk calongula tebu silase inokulasi dan bahkan dapat memperburukkualitas silase fermentasi dengan meningkatkan DMkerugian seluruh proses. Lactobacillus hilgardiistrain UFLA SIL51 dan UFLA SIL52 membuahkan silasedengan karakteristik yang terbaik dalam hubungannya dengan DM kerugian, rendahetanol konten, populasi LAB yang lebih tinggi dan rendah butiratasam konten. Strain UFLA SIL51 dan SIL52direkomendasikan sebagai starter budaya untuk tebu silase.Kata kunci: inokulan, Lactobacillus hilgardii, 1,2-propanediol, ragiPENDAHULUANProses 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 mengurangiPenggunaan spesies Lactobacillus sebagai starter budayauntuk meningkatkan kualitas silase tebuC. L. S. Ávila, * B. F. Carvalho, * J. C. Pinto, † W. F. Duarte, * dan R. F. Schwan * 1* Departemen Biologi, dan†Department ilmu hewan, Federal University of Lavras, 37.200-000, Lavras, Minas Gerais, BrasilMenerima 1 Mei 2013.Diterima pada 2 November 2013.1 Corresponding penulis: [e-mail] of Dairy Science Vol. 97 No. 2 tahun 2014SPESIES LACTOBACILLUS DI TEBU SILASE 941DM kerugian 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 pertumbuhanof undesired microorganisms. Therefore, the ability ofa strain to utilize different substrates present in theforage plant and to produce different metabolites canbe an advantage in the competition with other microorganisms.This ability can be used as a criterion forselecting inoculants (Saarisalo et al., 2007). The presentstudy aimed to isolate, identify, and select LAB strainsfor the ensiling of sugar cane by a rapid method basedon the production of metabolites that are relevant forthe silage process. In addition to strain performance,we evaluated the improvement of chemical and microbiologicalsilage characteristics in experimental silos.MATERIALS AND METHODSExperiment 1: Isolation and Characterizationof LAB from Sugar Cane SilageSilages were made with fresh-cut sugar cane fromplants that were approximately 12 mo old. The sugarcane was manually harvested and chopped using alaboratory-type chopper (PP-47, Pinheiro, Itapira, SP,Brazil) at an approximate length of 30 mm. Approximately10 kg of chopped material was immediatelyconditioned in 15-L plastic buckets without valves forgas release or effluent (mini-silos). The material wascompacted to a density of approximately 700 kg offresh matter/m3. The mini-silos were stored at roomtemperature (22°C) and opened after 0, 2, 15, 60, and90 d of storage. Samples were taken on each openingday for pH analysis. Two replicates were prepared foreach date of sampling.The LAB were isolated from 80 g of sugar cane silagethat was mixed with 720 mL of 0.1% sterile peptonewater and homogenized in an orbital mixer for 20min. Subsequently, 10-fold dilutions were prepared toquantify the LAB using de Man, Rogosa, and Sharpeagar (MRS, Difco, Detroit, MI) containing 0.1%cysteine-HCl and cycloheximide (0.4%). The plateswere 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 300well-isolated cfu, and a number of colony-forming unitscorresponding to the square root of the total was takenat random for identification (Holt et al., 1994). Theisolates were further purified by streaking individualcolonies onto MRS agar. The purified isolates weremaintained at −80°C in MRS broth containing 20%(vol/vol) glycerol.The size, shape, color, height, and edge morphologyof each colony were noted. The presumptive lactobacilliwere counted on MRS agar. The isolates were examinedby Gram stain and for colony and cell appearance,catalase activity, motility and production of CO2 fromglucose, and gluconate in MRS broth with a Durhamtube. The lactobacilli were recognized as gram-positive,catalase-negative, oxidase-negative, regular fermentativerods, and were classified as homofermentative orheterofermentative lactobacilli by their ability to produceCO2 from glucose and gluconate.Preselection of Bacterial Strains Based onMetabolite Production in Sugar Cane Broth. Fifty-seven isolates classified as LAB were isolated fromsugar cane silage and evaluated for metabolite production.The LAB were evaluated in a 5° Brix sugar canebroth medium supplemented with 0.1% yeast extract.The Brix degree (soluble solids) was determined accordingto AOAC (1990) using a digital refractometerAtago PR-32 (Atago USA Inc., Bellevue, WA), withautomatic temperature compensation. The broth wasfiltered (gauze) and sterilized (120°C, 15 min). First,the 57 strains were cultivated in MRS broth for 24 hat 35°C. After this period, the inoculum was standardizedusing a spectrophotometer (600 nm) at an opticaldensity of 1.0. Subsequently, approximately 400 μL ofeach strain was inoculated into 300 mL of sugar canebroth, which was incubated at 35°C and 120 rpm. After24 h of fermentation, samples of the cultures were takento evaluate metabolite production by HPLC.Data regarding the production of metabolites bystrains were analyzed using principal componentanalysis (PCA). Sugar cane has a high concentrationof soluble carbohydrates, a low buffering capacity, andDM content suitable for ensiling. Therefore, a decreasein pH occurs quickly (Kung and Stanley, 1982; Ávila etal., 2009). However, the overgrowth of yeast in sugarcane silage