The effects of adding lacticacid bacteria and cellulase inoil palm (Elais guineensisJacq.) frond silages on fermentation quality, chemical composition and in vitro digestibilityMahdi Ebrahimi,1 Mohamed Ali Rajion,1
Yong Meng Goh,1
Abdoreza Soleimani Farjam,2
Awis Qurni Sazili,3
Jan Thomas Schonewille41Department of Veterinary PreclinicalSciences, Universiti Putra Malaysia,Serdang, Selangor, Malaysia2Institute of Tropical Agriculture,Universiti Putra Malaysia, Serdang,Selangor, Malaysia3Department of Animal Sciences,Universiti Putra Malaysia, Serdang,Selangor, Malaysia4Department of Farm Animal Health,Utrecht University, The Netherlands
Abstract
The main objective of the current study wasto evaluate whether oil palm frond (OPF) canbe successfully ensiled without or with theadditives cellulase or lactic acid bacteria(LAB). Thus, fresh OPF was ensiled eitherwithout additives or with cellulase or LAB ortheir combination. Ensiling was carried out bystoring 2 kg samples in airtight glass jars at 25-30°C for 12 weeks. Thereafter, the silage sam-ples were subjected to proximate analyses, anin vitro digestibility assay and measures onselected indices of fermentation. Fermentationof OPF without additives appeared to be unsuc-cessful as both pH and ammonia content weretoo high (4.9 and 9.9%, respectively). In con-trast, the use of cellulase or LAB resulted insilages with a pH<4.5 and ammonia fractions<8.4%, but the lowest values were found whenboth cellulase and LAB were used, i.e. pH=4.1and ammonia fraction=6.7%. In vitrodigestibility of dry matter was significantlyhigher in the cellulase treated silages. Theprocess of ensiling was associated with both asignificant decrease of the fat content of OPFand a significant change of the fatty acid pro-file. However, the proportions of major fatty
acids (C16:0 and C18:2n-6) were not affectedby the process of ensiling. In conclusion, theuse of cellulase additive appears a practicaltool to safeguard the process of fermentation.Using a cellulase enzyme or its combinationwith LAB improves the fermentation profileand increases the nutritional value of the OPFsilage.
Introduction
An adequate supply of roughage to rumi-nants is essential for optimum rumen functionand thus important in relation to the animals’health and production. However, the availabili-ty of roughage for ruminant nutrition may varyfrom day to day in Malaysia mainly becausefarmers usually own only a limited amount ofland to grow forages. However, the oil palmfrond (OPF), a byproduct of the oil palm tree(Sumathi et al., 2008), is widely available inMalaysia throughout the year (Goh et al.,2010). Thus, the use of OPF may provide a con-tinuous source of roughage for the Malaysianruminant livestock industry. In practice, theOPF is harvested periodically usually after har-vesting of the fruits. Consequently, the freshlyharvested OPF has to be preserved to ensurethe continuity of roughage supply to the ani-mals. For obvious reasons, sun drying can beused to preserve OPF but adequate drying offresh OPF (>85% DM) is not possible duringperiods of heavy rainfall. Wilting of grasses upto a dry matter (DM) content of least 35% iscommonly practiced to prevent high levels ofNH3 and butyric acid in silage (Kung andRanjit, 2001). However, fresh OPF usually hasa DM content of 45%. Thus, it can be speculat-ed that the DM content of fresh OPF is alreadyhigh enough to ensure an uncomplicatedprocess of fermentation but this is yet notknown. Furthermore, the concentration ofwater-soluble carbohydrates (WSC) in OPF(approximately 1% DM) may be too low forsuccessful ensiling (McDonald et al., 1991).Addition of cellulase, potentially increases theamount of substrate for lactic acid bacteria(LAB) and thus may be a practical tool toenhance the process of ensiling. It is also notknown whether the numbers of epiphytic LABin OPF are high enough to ensure sufficientconversion of sugars into lactate to attain a pH<4.5. Thus, the main objective of the currentstudy was to evaluate the effects of adding cel-lulase and LAB or the combination of the twoon the process of fermentation of OPF. Oil palmfrond is rich in both linoleic and a-linolenicacid and values are typically in the order of
12.8 and 24.5 g/100 g fatty acids, respectively(Ebrahimi et al., 2012). However, to theauthors’ knowledge, there is yet no informa-tion on the fate of linoleic and a-linolenic acidduring the process of ensiling. Therefore, thefatty acid composition of OPF both before andafter ensiling was assessed as well.
Materials and methodsPreparation of the experimentalsilagesFresh OPF was harvested from the fields of
the Malaysian Agricultural Research andDevelopment Institute (MARDI), KualaLumpur, Malaysia. The OPF was chopped into2-3 cm and immediately transferred to the lab-oratory after harvesting. The freshly choppedOPF was either not treated with additives ortreated with LAB (Lactobacillus plantarumMTD1; Ecosyl, Stokesley, UK) or cellulase(Onozuka R-10; Yakult Ltd., Tokyo, Japan) or acombination of LAB and cellulase. Oil palmfrond treated with LAB contained at least1×106 colonies forming units (CFU) per gramfresh weight and cellulase was added at a levelof 2 g/kg fresh weight. The LAB and cellulasewere dissolved in sterile water and then
Corresponding author: Prof. Mohamed AliRajion, Department of Veterinary PreclinicalSciences, Faculty of Veterinary Medicine,Universiti Putra Malaysia, 43400 Serdang,Selangor, Malaysia. Tel. +603.8609.3411 - Fax: +603.8947.1973. E-mail: [email protected]
Acknowledgments: the authors are very gratefulto the Faculty of Veterinary Medicine, UniversitiPutra Malaysia, Serdang, Selangor, Malaysia.This research was supported by the MalaysianGovernment E-Science Grant No. 05-01-04-SF0200.
Received for publication: 3 March 2014.Accepted for publication: 13 June 2014.
This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).
Italian Journal of Animal Science 2014; volume 13:3358
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sprayed on the OPF by means of a watersprayer. The control treatment was sprayedwith sterile water alone to adjust the moisturecontent of the experimental forages.Thereafter, the experimental OPF were tightlypacked in three glass jars for each treatmentuntil the jars were completely filled. Each jarwas then sealed with a lid and the joint wascovered with parafilm to prevent the entry ofair. The jars were stored at ambient tempera-tures ranging from 25 to 30°C. The silages intriplicates were opened after 12 weeks forchemical analysis.
Collection and preparation of samplesAfter 12 weeks of ensiling, the top 5 cm of
the OPF silage was removed and from theremaining silage, a subsample of 20 g of silagewas taken and mixed for 2 min with 180 g ster-ile water by means of a laboratory blender(Waring, Torrington, CT, USA). Then, theextract was filtered through four layers ofgauze and No. 1 filter paper (Whatman Inc.,Maidstone, UK) and the pH of the filtrate wasrecorded (Mettler-Toledo Ltd., Leicester, UK).The filtrate was stored at -20°C until the analy-sis of lactic acid, NH3-N, WSC, volatile fattyacids (VFA), ethanol and LAB.Samples of fresh and ensiled OPF samples
were dried at 55°C for 48 h, ground to pass a 1mm screen and stored at -80°C until analysedfor analysis for ash, crude protein (CP), etherextract (EE), acid detergent fibre (ADF), neu-tral detergent fibre (NDF) and acid detergentlignin (ADL).A triplicate portion (0.25 g) of dried, ground
non-ensilaged and ensilaged OPF was taken todetermine the in vitro digestibility of DM. TheOPF was incubated in gas-tight 100 mL, plasticsyringes containing 20 mL of a phosphate-bicarbonate buffer adjusted to a pH of 6.8(Fievez et al., 2005) and 5 mL of rumen con-tent. Then, all air was expelled from thesyringes, after which their tips were closed.Syringes were placed in an incubator at 39°Cfor 24 h. The rumen contents were obtainedfrom four adult, rumen fistulated, Kacangcrossbred male goats that were fed a rationconsisting of 30% fresh OPF and 70% commer-cial concentrate (W/W). Rumen contents weretransferred into pre-warmed thermos flaskswhich were flushed with CO2 during transportto the laboratory. Prior to the incubations,rumen contents were filtered through four lay-ers of cheesecloth under continuous flushingwith CO2. After incubation, the content of thegas syringes for all treatments including theblank, was quantitatively transferred into pre-dried beakers and subjected to digestion as
described by Tilley and Terry (1963). Briefly, atthe end of 24 h incubation, rumen fluid sam-ples were centrifuged at 378 g for 10 min andthe precipitated sample was washed by dis-tilled water thrice. In the next step, washedsamples were mixed with 50 mL pepsin-HClsolution (containing 2 g/L pepsin and 17.8mL/L HCl) in 100 mL serum bottles and incu-bated at 39°C for 24 h. After incubation, thesamples were centrifuged and the precipitatedfeed was dried at 100°C for 48 h. The in vitrodry matter disappearance (IVDMD) was calcu-lated according to the following formula:
After the process of digestion, the content ofthe beakers was dried at 100°C until a constantweight.
Chemical analysisLactic acid, VFA and ethanol were deter-
mined using gas-liquid chromatography(Quadrex Corporation Bethany, CT, USA)equipped with a flame ionisation detector. Inthe case of lactic acid and VFA, a silica capillarycolumn of 15 m�0.32 mm ID�0.25 µm film thick-ness (Agilent Technologies, Santa Clara, CA,USA) was used. The injector/detector tempera-ture was programmed at 220/230°C respective-ly. The column temperature was set in therange of 70 to 150°C with a temperature incre-ment at the rate of 7°C/min. Peaks for VFAwere identified by comparison with authenticstandards of acetic, propionic, butyric, isobu-tyric, valeric, isovaleric and 4-methyl-n-valericacids (Sigma Aldrich, St. Louis, MO, USA). Theinternal standard used for VFA was 4-methyl-n-valeric acids and the internal standard for lac-tic acid determination was fumaric acid(Sigma Aldrich). Peaks for lactic acid wereidentified by comparison with an authenticstandard of lactic acid (Sigma Aldrich). In thecase of ethanol a HP-1 capillary column of 30m�0.25 mm ID�0.25 µm film thickness (J&WScientific, Folsom, CA, USA) was used. Theflow rates of H2 and air were set at 30 and 300mL/min, respectively. The injector/detectortemperature was programmed at 225/285°Crespectively. The column temperature was setin the range of 45 to 245°C with a temperatureincrement at the rate of 45°C/min. The volumethat was injected was limited to 1 µL. Water-soluble carbohydrates were determined by themodified phenol sulfuric acid method asdescribed by Guiragossian et al. (1977) and theconcentrations of NH3-N were determined infresh silage with the use of colorimetricmethod as described by Solorzano (1969).
The total number of LAB in the silage wasdetermined on MRS Rogosa agar (Oxoid CM627; Oxoid Ltd., Basingstoke, UK). Agar plateswere incubated at 37°C for 72 h. The numbersof LAB were measured by the plate countmethod and the number of CFU was expressedas log10 per gram of DM OPF.The DM of silage samples was analysed by
drying at 55°C for 48 h. Ash was determined bycombustion at 525°C for 6 h (method 923.03;AOAC, 1990). Nitrogen was determined by theKjeldahl method (method 978.02; AOAC, 1990),and the CP content was calculated as N×6.25.Crude fat was extracted with petroleum ether(Soxtec 2050; Foss Analytical, Hillerød,Denmark). The contents of NDF, ADF and ADLwere determined according to Van Soest et al.(1991). Heat stable amylase and sodium sul-phite were used in the procedure of determin-ing NDF and the results of NDF and ADF wereexpressed on an ash-free basis. The total fatty acids were extracted from
fresh and ensiled OPF based on the method ofFolch et al. (1957) with some modifications byRajion et al. (1985). The methylated fatty acidswere separated by Agilent 7890A gas chro-matography (Agilent Technologies) asdescribed by Ebrahimi et al. (2013).
Statistical analysisAll data were checked for normality using
the UNIVARIATE procedure of the SAS rev. 9.1.,and LAB data were normalised using the log10-transformation. Then, all data were subjectedto ANOVA, using the MIXED procedure of theSAS software package, version 9.1 (SAS, 2007).The statistical model used the following equa-tion:
Yijk=µ+Ti+Fj+eijk
where µ is the overall mean, Ti is effect oftreatment (i=1 to 4), Fj is the random effect ofreplicate (j=1 to 3) and eij was the residualerror. When the influence of treatmentreached statistical significance, Tukey’s t testwas used to identify treatments with differenteffects on the variable involved. Throughout,the level of statistical significance was set atP<0.05.
Results and discussionFermentation qualityThe LAB count was raised after 12 weeks of
ensiling in all experimental silages but theLAB counts were only significantly higher inthe silages treated with additives (Table 1).
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The rise in LAB count was associated with asignificant decrease of the WSC content and aconcomitant increase of the lactic acid contentof the silages. The highest lactic acid contentwas found in the silage treated with both LABand cellulase (Table 1). The increase in thelactic acid was associated with a decrease ofthe pH and the lowest pH values were found inthe silages treated with additives. The NH3-Nfraction and the concentrations of ethanol,acetic-, propionic- and butyric acid were signif-icantly increased after 12 weeks of ensilingcompared to fresh OPF. The highest ethanolconcentrations were found in the silage treat-ed with both LAB and cellulase, while the high-est concentrations of acetic- and butyric acidand that of NH3-N were found in the silagewithout additives (Table 1). Parameters such as silage pH, short chain
fatty acids and ammonia content are common-ly used as indicators of silage quality. It is gen-erally accepted that in well preserved silages,pH values should be <4.5 (McDonald et al.,1991) and ammonia levels <100 g/kg totalnitrogen. Clearly, these criteria were not metby the OPF that was ensiled without additives.Thus, it seems that the osmotic pressure offresh OPF is not high enough to ensure anuncomplicated process of fermentation (Muck,1988). The relative low osmotic pressure canbe explained by the low WSC content (approx-imately 1% DM) in OPF which is consideredtoo low for successful ensiling (McDonald etal., 1991). This reasoning is in line with theobservation that the addition of cellulase sig-nificantly increased lactate concentrationswith a concomitant decrease in pH. Thus, itcan be suggested that the addition of cellulaseeffectively provided more substrate for fermen-tation by the LAB (Stokes, 1992; Ridla andUchida, 1993; Sheperd et al., 1995).Furthermore, the addition of LAB instead ofcellulase significantly increased the lactic acidcontent, suggesting that the numbers of epi-phytic LAB in OPF may limit the conversion ofsugars into lactate to attain a pH <4.5.Consequently, the addition of both cellulaseand LAB resulted in the highest lactate concen-trations (Table 1). However, the relevancy ofthis high lactate concentration is not exactlyclear because both silage pH and NH3-N con-centrations in the silages with additives werenot significantly different between silages andmet the criteria as indicated earlier. Interestingly, the highest lactate concentra-
tions found in the current study, were approxi-mately 33% lower than the threshold of suffi-cient preservation (McDonald et al., 1991).Thus, the required pH for well-preservedsilages was attained at relatively low lactate
concentrations. This result suggests that OPFhas a relatively low buffer capacity. It is wellknown that the buffer capacity of forage is pos-itively related to the CP content (McDonaldHenderson, 1962). Because OPF has a low CPcontent (4 to 5%, DM basis), it can be suggest-ed that OPF also has a low buffering capacity.Finally, in all silages the acetic acid/total fer-mentation acids ratio was found to be >0.57which is considerable higher than the recom-mended value, i.e. <0.20 (Lima et al., 2011).Therefore, it may be speculated that the aero-bic stability (Weinberg et al., 1993; Kung andRanjit, 2001; Danner et al., 2003) of all silagescan be disputed. The relative high proportionsof acetic acid are difficult to explain but theymay be related to a relative lack of rapid fer-mentable carbohydrates in OPF. Indeed, theuse of appropriate amounts of molasses as anadditive has been shown to produce silages
with low proportions of acetic acid in combina-tion with high proportions of lactic acid(Bureenok et al., 2012; Lima et al., 2011).
Chemical composition anddigestibility Both the DM and EE contents were signifi-
cantly decreased due to the process of ensilingbut there was no significant differencebetween DM in the ensiled OPF. The CP con-tent of the OPF silages was significantly higherafter 12 weeks of ensiling (Table 2). In allsilages, the NDF content was numericallylower than in the fresh OPF, but the differenceonly reached statistical significance in the twosilages treated with cellulase. Likewise, theADF and cellulose contents of the silages werelower compared to the fresh OPF and the dif-ferences were most pronounced when thesilages were treated with both cellulase and
Oil palm frond silage
Table 1. Selected indexes of fermentation of oil palm frond, before and after an ensilingperiod of 12 weeks.
LAB, lactic acid bacteria; WSC, water soluble carbohydrates; nd, not detected (zero value was used in statistical analysis); DM, drymatter. a-dMeans within the same rows with different superscripts are significantly different at P<0.05.
Table 2. Chemical composition and in vitro dry matter digestibility of oil palm frondbefore and after an ensiling period of 12 weeks.
LAB, lactic acid bacteria; DM, dry matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fibre; ADF, acid detergentfibre; cellulose, ADF-ADL; hemicellulose, NDF-ADF; ADL, acid detergent lignin. a,bMeans within the same rows with different super-scripts are significantly different at P<0.05.
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LAB. The process of ensiling did not signifi-cantly affect the hemicellulose, ADL and ashcontent of OPF (Table 2). Ensiling withoutadditives influence the digestibility of OPF,especially when cellulase was used as an addi-tive, the in vitro digestibility of the ensiledOPF increased by 8 to 10% compared to thefresh OPF (Table 2).The fermentation of OPF was associated
with an overall loss of 15% of DM (Table 2).The underlying reason is not exactly clear butit may be, at least partly, related to the forma-tion of volatile end-products of fermentation.This reasoning is corroborated by Pedroso etal. (2008) who showed a similar loss of DMdue to the evaporation of volatile end-productsduring drying. Next to the loss of DM an 8%loss of EE also occurred during the process ofensiling. The loss of EE during the ensilingprocess is corroborated by several other stud-ies (Dewhurst and King, 1998; Elgersma et al.,2003; Van Ranst et al., 2009) and can beexplained by the oxidation of fatty acids(Dewhurst and King, 1998). In the current study, an increase of 12% of
the CP content was observed in the ensiledOPF with additives. This observation is in linewith that of Zahiroddini et al. (2004) andBureenok et al. (2012) who also observed anincrease in the CP content of silages fromwhole-crop barley and Napier grass, respec-tively. The observed increases of the CP con-tent of the ensiled OPF is most likely related tothe observed decrease of the NDF and to a less-er extent of the EE content of the silages.Clearly, the loss of NDF and EE increases therelative proportion of CP, expressed in g/kgDM.Compared to fresh OPF, the addition of cel-
lulase alone or in combination with LAB,caused a significant decrease of the NDF andADF content of the ensiled OPF. It appears thatcellulase effectively degraded at least partly,structural carbohydrates such as cellulose(Ren et al., 2007). Indeed, the lowest cellulosecontents were found in the OPF silages treatedwith cellulase. This result is corroborated byRidla and Uchida (1993) who also showed thatthe addition of cellulase decreases the contentof cell wall carbohydrates associated with theNDF and ADF content. It appears that the addi-tional cellulase stimulated the conversion ofcellulose to WSC thereby rendering glucoseavailable for LAB and potential subsequentconversion to lactate (Stokes, 1992; Ridla andUchida, 1993; Sheperd et al., 1995). The effec-tive degradation of NDF and ADF by cellulase isalso reflected by the improvement of DMdigestibility. It appeared that the in vitrodigestibility of OPF ensiled with cellulase was
at least 6.9% greater than the silage withoutadditives.
Fatty acid compositionThe process of ensiling was associated with
a significant decrease of the total fatty acidcontent of OPF and a significant effect on thefatty acid profile of the fat fraction. The propor-tion of C18:1trans-11 was significantly higherin the experimental silages with cellulaseadditives (Table 3). There were no significanteffects on the proportions of C15:0, C15:1 andC18:3n-3 compared to no additive OPF. Theproportions of the two major fatty acids, i.e.C16:0 and C18:2n-6, were not significantlyaffected by process of ensiling. The ratio of n-6: n-3 fatty acids remained unchanged afterensiling (Table 3).The loss of EE is in line with that of fatty
acids (Table 3). Some studies report adecrease in the total fatty acid content ofsilages compared to that of fresh products(Dewhurst and King, 1998; Elgersma et al.,2003; Van Ranst et al., 2009; Han and Zhou,2013). The decrease in proportions of C15:0,C15:1 and C18:3n-3 in ensiled OPF comparedto fresh OPF are probably related to the oxida-tion of these fatty acids by bacteria. Indeed, it
has been reported by Elgersma et al. (2003)that ensiling of grass decreased the concentra-tions of most fatty acids, especially C18:3n-3.This observation is corroborated by Lee et al.(2006), who also reported a reduced proportionof C18:3n-3 when grass was ensiled in combi-nation with Lactobacillus plantarum. Clearly,the current study did not provide clues withrespect to the underlying mechanism for thereduced proportions of C18:3n-3, but it wasshown by Lee et al. (2008) that lipoxygenasesfrom plants play a role in the oxidation ofC18:3n-3 during the process of ensiling.In the current study, the proportions
C18:1trans-11 was significantly higher in OPFsilage. This outcome is in line with the obser-vations of Lough and Anderson (1973) andVanhatalo et al. (2007) who reported increasedproportions of C18:1trans-11 in grass and redclover silages. The exact origin of C18:1trans-11 in silage is not clear but it was shown byOgawa et al. (2005) and Kishino et al. (2009)that LAB can produce this fatty acid. Kishino etal. (2009) demonstrated that LAB can iso-merise C18:2n-6 into conjugated C18:2 andsubsequently hydrogenate this fatty acid intoC18:1trans-11. However, this explanation isnot in line with the results of the current study
Ebrahimi et al.
Table 3. Total fatty acid content (mg/g DM) and fatty acid composition (g/100 g of totalidentified fatty acids) of oil palm frond before and after ensiling for 12 weeks.
LAB, lactic acid bacteria; TFA, total fatty acids; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturatedfatty acids; UFA, unsaturated fatty acids. °Total SFA, sum of C12:0, C14:0, C15:0, C16:0, C17:0, C18:0; #total MUFA, sum of C15:1, C16:1,C17:1, C18:1n-9; §total n-3PUFA, C18:3n-3; ^total n-6PUFA, C18:2n-6; $n-6: n-3, total PUFA n-6 (C18:2n-6):total PUFA n-3 (C18:3n-3).a,bMeans within the same rows with different superscripts are significantly different at P<0.05.
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because the proportion of C18:2n-6 was notaffected by the process of ensiling (Table 3).Likewise, it can be suggested that C18:3n-3acted as a precursor of C18:1trans-11 duringthe process of ensiling but, to the authors’knowledge, this is currently not known. Next to C18:1trans-11, the proportion of
C18:1n-9 also was increased in the experimen-tal silages. Theoretically, this result can beexplained by the conversion of C18:0 intoC18:1n-9 mediated by a D9 desaturase, whichis in line the observed decrease in the propor-tion of C18:0. Unfortunately, to the authors’knowledge, the activity of D9 desaturase insilage is not yet reported in the literature.Furthermore, when C18:1n-9 was expressed asg/kg DM (data not shown), the contents of theensiled OPF was similar to that of the freshOPF indicating that the observed proportionalincrease of C18:1n-9 after ensiling was, atleast partly, due to the loss of other fatty acidssuch as C15:0, C15:1 and C18:3n-3. Clearly, theissue on the alteration of the fatty acids profileassociated with ensiling is not yet settled(Glasser et al., 2013).
Conclusions
The quality of silage derived from fresh OPFis below standards. Addition with LAB or cellu-lase or the combination of LAB and cellulasesignificantly improves the quality of the OPFsilage. Cellulase appeared to be the most effec-tive additive in relation to silage quality. Theresults of the current study implicate that OPFcan be well preserved by ensiling and therebyrepresents a continuous source of roughagefor ruminant livestock diets in Malaysia.
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