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940J. Dairy Sci. 97 :940–951http://dx.doi.org/10.3168/jds.2013-6987© American Dairy Science Association®, 2014.ABSTRACTSugar cane (Saccharum spp.) is a forage crop widelyused 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 problemswith the growth of yeasts, which leads to high losses ofDM throughout the fermentative process. The selectionof specific inoculants for sugar cane silage can improvethe quality of the silage. The present study aimed toselect strains of lactic acid bacteria (LAB) isolatedfrom sugar cane silage and to assess their effects whenused as additives on the same type of silage. The LABstrains were inoculated into sugar cane broth to evaluatetheir production of metabolites. The selected strainsproduced higher concentrations of acetic and propionicacids and resulted in better silage characteristics, suchas low yeast population, lower ethanol content, andlesser DM loss. These data confirmed that facultativeheterofermentative strains are not good candidates forsugar cane silage inoculation and may even worsen thequality of the silage fermentation by increasing DMlosses throughout the process. Lactobacillus hilgardiistrains UFLA SIL51 and UFLA SIL52 resulted in silagewith the best characteristics in relation to DM loss, lowethanol content, higher LAB population, and low butyricacid content. Strains UFLA SIL51 and SIL52 arerecommended as starter cultures for sugar cane silage.Key words: inoculant , Lactobacillus hilgardii , 1,2-propanediol, yeastINTRODUCTIONThe ensiling process may occur either naturally, withepiphytic microorganisms present on the plant material,or with the addition of inoculants to improve theprocess, thus resulting in better quality silage. Microbialinoculants are commercially available for use insilage, and lactic acid bacteria (LAB) are the mainmicroorganisms used for this purpose (Cai et al., 1999;Driehuis et al., 2001; Filya, 2003).In general, studies with LAB inoculants show thatinoculation before ensiling increases the fermentationquality of the ensiled forage (Kleinschmit and Kung,2006; Zopollatto et al., 2009). However, the results canbe inconsistent when forage crops are evaluated underdifferent conditions, such as silo size, climate, and packingdensity. Factors related to the storage and applicationof inoculants might influence their effects on silagequality. Nevertheless, one of the determining factorsfor the successful application of microbial inoculantsin silage is the compatibility between the plant and themicroorganisms used (Muck, 2008; Ávila et al., 2009).This compatibility can be assessed by the ability of themicroorganisms to use carbohydrates present in the forageand to produce metabolites of interest, primarily inthe preservation of silage (e.g., acetic and lactic acids).Sugar cane (Saccharum spp.) is a forage crop widelyused in animal feed because of its high DM production(25 to 40 t/ha) and high energy concentration, which isdue to the high concentration of sugars, mainly sucrose(250 to 300 g/kg). The ensiling of sugar cane often resultsin problems with the overgrowth of yeasts, whichleads to high losses of DM throughout the fermentativeprocess (Kung and Stanley, 1982). Chemical and microbiologicaladditives have been tested with the aim ofreducing yeast growth. However, microbial inoculantshave produced better results than chemical additives(Carvalho et al., 2012).Inoculants with LAB, which produce higher concentrationsof acetic or propionic acids, are more suitablefor yeast control because of the fungicidal effect ofthese acids (Moon, 1983). The addition of microorganismsthat produce greater amounts of lactic acid are ofinterest because of their rapid effect in reducing the pHvalue. However, lactic acid is a potential substrate foryeast during feeding-out, reducing the aerobic stabilityof the silage. Inoculation with facultative heterofermentativeLactobacillus plantarum and obligatory heterofermentativeLactobacillus buchneri has been tested duringensiling of sugar cane. The results are variable, but ingeneral, Lb. buchneri showed good results in reducingThe use of Lactobacillus species as starter culturesfor enhancing the quality of sugar cane silageC. 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, BrazilReceived May 1, 2013.Accepted November 2, 2013.1 Corresponding author: rschwan@dbi.ufla.brJournal of Dairy Science Vol. 97 No. 2, 2014LACTOBACILLUS SPECIES IN SUGAR CANE SILAGE 941the DM losses and increased aerobic stability (Ávilaet al., 2009; Roth et al., 2010). Pedroso et al. (2008)observed that Lb. buchneri improved fermentative andaerobic stability in silages, whereas Lb. plantarumstrains interfered negatively in the fermentation andpreservation of sugar cane silages. Ávila et al. (2010b)evaluated different LAB species (Lb. plantarum, Lactobacillusparacasei, Lactobacillus brevis, and Lactobacillusbrevis buchneri) and observed that the effect of theinoculant is more related to the strain used than to thespecies. Inoculations with different strains that belongto the same species have resulted in silages with differentcharacteristics, suggesting that studies should beconducted not only at the species level but also at thestrain level (Saarisalo et al., 2007; Ávila et al., 2011).The effects of microbial inoculants on the fermentationprocess of silage are mainly due to the productionof metabolites of interest able to inhibit the growthof 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
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