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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.49 No.5 pp.157-167

Effects of L. plantarum Application on Chemical Composition, Fermentation Indices and Fatty Acid Profiles of Barley Silage

Dong-Hyeon Kim1, Sardar M. Amanullah1,3, Hyuk-Jun Lee1, Young-Ho Joo1, Hee Yun1, Seong-Shin Lee1, Young-Min Song2, Hoi-Yun Kim2, Sam-Churl Kim1*
1Division of Applied Life Science (BK21Plus, Insti. of Agri. & Life Sci.), Gyeongsang National University, Jinju 52828, South Korea
2Department of Animal Resource Technology, Gyeongnam National University of Science & Technology, Jinju 52725, South Korea
3Bangladesh Livestock Research Institute, Savar, Dhaka-1314, Bangladesh
Corresponding author : Sam-Churl Kim Tel: +82-55-772-1947 Fax: +82-55-772-1949
March 5, 2015 September 4, 2015 September 23, 2015


This study was conducted to know effects of forage cutting height and inoculants application on chemical composition, fermentation characteristics and fatty acid profile of barley silage. Barley forage(Yuyeon hybrid) was harvested at two different cutting heights(5 vs. 15cm) and applied with or without Lactobacillus plantarum, and ensiled for 0, 2, 7, 28, 49 and 100days. On 0 to 49-d of ensiling, higher cutting height resulted rapid drop (p<0.05) in pH caused by higher lactate content. Crude protein (p<0.01) content of 100-d silage was decreased by inoculation, but increased by higher cutting height. However, neutral detergent fiber and acid detergent fiber contents were decreased (p<0.05) by both inoculation and cutting height. In vitro dry matter digestibility was improved by higher cutting height (p=0.01), while yeast and mold counts were reduced (p<0.0001). The C18:3n-3 content in barley silage was decreased (p=0.001) by inoculation, but increased (p=0.034) by higher cutting height. The DNA analysis indicated L. plantarum dominating fermentation in inoculated silages. The results showed that higher cutting height can improve silage quality in terms of increasing crude protein content and digestibility as well as reducing yeast and mold counts in barley silage.


    Rural Development Administration


    Whole crop barley, as winter forage, is widely using to make silage for its high contents of rumen- fermentable carbohydrates(Eun et al., 2004). Yuyeon barley is a new hybrid developed by cross breeding at National Institute of Crop Science, South Korea having no silk, which can potentially improve feed intake and digestibility. Forage having higher palatability and digestibility is potential to achieve higher growth and milk production(Hunt et al., 1993; Oba & Allen, 1999). The lower part of the forage crop is generally considered to be poorly digestible as it contains fiber and lignin(Tolera & Sundstøl, 1999). Bernard et al.(2004) showed that increased cutting height decrease the concentration of acid detergent fiber(ADF) and in vitro true dry matter digestibility (IVDMD). Wu & Roth(2005) reported that higher cutting height of corn forage increased the concentrations of crude protein(CP) and net energy for lactation(NEL), and neutral detergent fiber(NDF) digestibility compared to the normal cutting height. In case of whole crop silage, increasing cutting height off or ages increases the proportion of grain and decrease fiber content(Weller et al., 1995). Sinclair et al.(2003) reported that starch content and metabolisable energy were improved by increased cutting height of wheat silage.

    Schnyder(1993) suggested that accumulated soluble sugar reserve in the stem and leaf sheath can be mobilized and transported to the developing grain to ensure a significant contribution to final grain yield. Leaving some more portion of the stalk at forage harvesting may be beneficial for silage fermentation especially when bacterial inoculation is intended to use. Earlier studies showed that homofermentative bacteria(Lactobacillus plantarum) efficiently improved fermentation characteristics of different sisilages( Zahiroddini et al., 2004; Baah et al., 2011).

    Therefore, this study was conducted to know the effects of forage cutting height and bacterial inoculation on nutritive value, fermentation characteristics and fatty acid profile of whole crop Yuyeon barley silage.

    Materials and Methods

    1.Silage making and sampling

    Barley(Yuyeon) was grown at Gyeongsang National University Research Farm, Jinju, South Korea(Fig. 1). Barley forage was harvested at about 26% dry matter( DM) with two different cutting heights (5 vs. 15cm). Forage was chopped to 5cm length and assigned into the following four treatments: 1) 5cm cutting height without inoculants (Applied distilled water at 2mL/kg of fresh forage), 2) 5cm cutting height with inoculant (L. plantarum; 1.5×107cfu/g of fresh forage; CMbio, South Korea); 3) 15cm cutting height without inoculant and 4) 15cm cutting height with inoculant. Then the forage was ensiled into mini bucket silos (10L capacity) in quadruplicate for 0, 2, 7, 28, 49 and 100days. At each silo opening, barley forage and silages were sub-sampled (500g) for the analyses of nutrient contents, fermentation indices, fatty acid profile and microbial counts.

    2.Silage extract preparation

    Twenty grams of fresh sample along with 200mL of sterile ultrapure water was macerated for 30seconds in a laboratory blender followed by filtering through two layers of cheesecloth to obtain silage extract( Kim & Adesogan, 2006). A part of the fresh extract was used to determine pH and the counts of lactic acid bacteria(LAB), yeast and mold at the day of silo opening, and the rest was stored at -20℃ for ammonia-N, volatile fatty acids(VFA), fatty acids(FA) analysis and extraction of microbial DNA.

    3.Laboratory analysis

    The DM content was determined by drying sample( about 10g) in a forced air dry oven(OF-22GW, JEIO TECH, Korea) at 105℃ for 24h. About 500g of sample was dried separately at 60℃ for 48h and ground by a cutting mill(SHINMYUNG ELECTRIC Co., Ltd, South Korea) with 1mm screen to use for chemical analysis. The procedures of Kjeldahl(B-324, 412, 435 and 719 S Titrino, BUCHI, Germany) and Soxhlet(OB-25E, JeioTech, South Korea) were used to determine CP and ether extract(EE), respectively following AOAC(1995). Crude ash was analyzed with a muffle furnace at 550℃ for 5h. The contents of NDF and ADF were determined by using Ankom 200 fiber analyzer(Ankom Technology, Macedon, NY) following the method of Van Soest et al.(1991). Hemicellulose content was calculated by the difference between NDF and ADF. The IVDMD was measured following the method described by Tilley & Terry(1963) using ANKOM DAISY II Incubator (ANKOM Technology, Macedon, NY, USA). The pH was measured by a pH meter(SevenEasy, Mettler Toledo, Switzerland) and ammonia-N content was determined by colorimetric method described by Chaney & Marbach(1962). For lactate and VFA analysis, extracted silage juice was centrifuged at 5,645×g for 15min, and then, the supernatant was collected. Contents of lactate and VFA were measured in HPLC(L-2200, Hitachi, Tokyo, Japan) fitted with a UV detector(L-2400, Hitachi, Tokyo, Japan) and a column(Metacarb 87H, Varian, CA, USA) according to the method described by Adesogan et al.(2004). The two steps methylation procedure of Jenkins et al.(2001) was used for the preparation of fatty acid(FA) methyl ester. The content of FA was determined using gas chromatograph(450-GC, Bruker) equipped with an autosampler(CP-8400, Varian), a flame ionization detector and a Varian capillary column( CP-Sil 88, 100m × 0.25mm × 0.2μm). Nitrogen was the carrier gas, and the temperatures of injector and detector were maintained at 230℃. The oven temperature was initially set at 120℃ for 1min, increased by 5℃/min up to 190℃, held at 190℃ for 30min, increased again by 2℃/min up to 220℃, and held at 220℃ for 40min. The peaks of the sample were identified and calculated based on the retention time and peak area of known standards.

    4.Microbial enumeration

    Extracted fresh silage juice was used to count LAB, yeast and mold in silage. Considering the extracted juice as first dilution, serial dilutions were prepared and 100μL aliquots of three consecutive dilutions( 10-5 to 10-7) were plated in triplicate onto a selective agar medium. The lactobacilli MRS agar media(MRS; Difco, Detroit, MI, USA) was used for culturing LAB, and potato dextrose agar (PDA; Difco, Detroit, MI, USA) for yeasts and molds. The MRS agar plates were placed in a CO2 incubator( Thermo Scientific, USA) at 39℃ for 24h, while PDA plates were incubated at 39℃ for 24h in normal incubator(Johnsam Corporation, Korea). Visible colonies were counted from the plates and the number of colony forming units(CFU) was expressed per gram of silage.

    5.DNA extraction, primer and PCR condition

    The DNA of L. plantarum was extracted by using QIAamp DNA mini kit(Qiagen, USA) following the manufacturer’s protocol. The DNA concentrations were measured by using a NanoDrop Spectrophotometer (ND-1000, USA). Amplification of DNA was performed using Bio-Rad C1000 TouchTM Thermal cycler real-time PCR detection system(CFX96TM Real-Time system, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers and PCR conditions was followed as described by Amanullah et al.(2014). The amplified fragments were subjected to electrophoresis on 1.5% agarose gel and visualized after stained with ethidium bromide under UV illumination.

    6.Statistical analysis

    A 2(no inoculant vs. inoculant) × 2(5cm vs. 15cm cutting height) factorial design with 4 replications per treatment was used in this study. The data from silage ensiled for 100days except the gel electrophoresis measurements were analyzed using GLM procedure of SAS(2002). The model included main effects of inoculant, cutting height and the interactions of these factors. The Tukey test was used to differentiate the treatment means of pH and the contents of ammonia-N, lactate and acetate at 0, 2, 7, 28, 49 and 100days of ensiling. The significance level was declared at p<0.05.

    Results and Discussion

    The chemical composition and FA profile of barley forage(Yuyeon hybrid) before ensiling is illustrated in Table 1. The DM, CP and NDF contents in fresh forages cut at 5 and 15cm height were 26.1 vs. 24.9%, 7.03 vs. 8.92% and 58.4 vs. 60.0%, respectively. The main FAs in fresh forage were C18:3n-3(40.2%), C18:2n-6(24.9%), C16:0(22.9%) and C18:1n-9(6.62%).

    The changes in pH and contents of ammonia-N, lactate and acetate over ensiling periods are showed in Fig. 1. During the ensiling period, the pH in all silages dropped by day 7, and then maintained under 4.70. The rapid pH drop was shown by inoculation, and inoculated silage had lower pH (4.13 vs. 4.61; p<0.05) than uninoculated silage on day 7. However, inoculated silage with 15 cm cutting height had the highest pH (p<0.05) on 100 days. Ammonia-N content in all silages were increased with ensiling time, and the increasing rate was higher in the silage of higher cutting height (15cm) irrespective of inoculation and the difference was significant (p<0.05) throughout the entire fermentation periods. Lactate contents were highest (p<0.05) in inoculated silage with 5cm cutting height on day 2 (10.9%), and in inoculated silage with 15cm cutting height on day 7, 28 and 49 (10.6, 8.67 and 9.60%, respectively). However, at 100days, it was highest (p<0.05) in uninoculated silage of 15cm cutting height. Acetate content was highest (p<0.05) in inoculated silage with 15cm cutting height on day 7, 28 and 49(1.21, 1.23 and 1.09%, respectively), and in uninoculated silage with 5 cm cutting height on day 49 and 100(1.38 and 4.26%).

    The chemical composition of barley silage ensiled for 100days are stated in Table 2. Inoculant application increased (p<0.05) DM(20.2 vs. 19.2%) content, but decreased CP(8.13 vs. 8.84%), crude ash(7.69 vs. 8.17%), NDF(55.1 vs. 58.4%) and ADF(25.8 vs. 38.9%) contents. As increased cutting height, CP(9.23 vs. 7.74%) content was increased (p<0.05) while DM(19.4 vs. 20.0%), crude ash(7.70 vs. 8.16%), NDF(54.6 vs. 58.9%), ADF(35.9 vs. 38.8%) and hemicellulose(18.8 vs. 20.1%) contents were decreased. There were no interaction effects(inoculant × cutting height) in chemical compositions.

    The IVDMD, fermentation characteristics and microbial counts are presented in Table 3. The IVDMD improved by not only inoculation (51.8 vs. 49.4%, p=0.045), but also increased cutting height (52.2 vs. 49.0%, p=0.01). There were interaction effects (inoculant × cutting height, p<0.05) on pH, the contents of ammonia-N, ammonia-N per total N, lactate and acetate, lactate to acetate ratio and mold count. However, LAB count was affected neither by cutting height nor by inoculation (p>0.05).

    The FA profile of barley silage ensiled for 100days are described in Table 4. Inoculant application increased (p<0.05) C16:0(2.59 vs. 2.45%), C18:0(1.80 vs. 1.70%), C18:1n-9(9.14 vs. 7.40%), monounsaturated FA(MUFA, 10.3 vs. 8.67%) contents, but decreased C18:3n-3(28.2 vs. 36.6%) content. The contents of C14:0(2.42 vs. 2.89%), C16:0(23.8 vs. 26.6%) and C18:0(16.3 vs. 18.7%) decreased (p<0.05) by increased cutting height, while C18:3n-3(32.7 vs. 29.4%) content increased (p=0.034). There were interaction effects(inoculant × cutting height, p<0.05) on the contents of C15:0, C17:0, saturated FA(SFA) and polyunsaturated FA(PUFA), and the ratio of PUFA to SFA.

    The Polymerase chain reaction(PCR) amplification of DNA of barley silage ensiled for 100days is shown in Fig. 2. The DNA band was more prominent in inoculated silages, and the band mass was also numerically higher (239.2 vs. 48.9ng) than uninoculated silages.

    Chemical composition of barley forage and silage are in agreement with our previous studies(Kim et al., 2013; Amanullah et al., 2014). The DM of L. plantarum inoculated silage was well preserved than of uninoculated silages. In the present study, we observed an increase in CP content due to increasing cutting height irrespective of inoculation. However, variable results were observed with increase of cutting height on corn forage in earlier studies, such as decrease of CP concentration(Neylon & Kung, 2003), but increase of CP(Kung et al., 2008). On the other hand, Song et al.(2009) reported no change in CP due to increasing cutting height of barley and some other winter crop silages(Oat, wheat and triticale). The increased CP content of silages with higher cutting height in this study might be due to the increase of CP content in those barley forage(Table 1), which means the lower part of Yuyeon barley forage has lower CP content than the upper part. Usually inoculant application helps to preserve the CP content in silage(McDonald et al., 1991). The reduced CP content with L. plantarum application in this study is unusual, but partially explained by the increased pH and ammonia-N concentration in inoculated silage of 15 cm cutting height at 100 days of ensiling(Fig. 2). In agreement with the present result, the reduction of CP content of inoculated barley silage was reported by Amanullah et al.(2014) and Taylor et al.(2002). The reduced fiber concentration as NDF, ADF and hemicelluloses in higher cut silages are in agreement with most other previous studies with corn or other silages(Sinclair et al., 2003; Neylon & Kung, 2003; Kennington et al., 2005; Kung et al., 2008). The reduced fiber content in higher cut silages is straightly due to the fact that more fibrous and less digestible part of the plant was left in the field (Tolera & Sundstøl, 1999; Kung et al., 2008). This further might contributed to increase the IVDMD in silage of higher cutting height in this study. In agreement with the result of present study, Kennington et al.(2005) observed greater DM degradability in higher cut silage, and Kang et al.(2009) reported inoculant application increased IVDMD. However, the inoculant effects on IVDMD of silage are variable. This might be due to the difference in forage hybrid and bacterial strain.

    The rapid reduction of pH in inoculated silages at early stages of fermentation(2 - 49 days) in this study suggests that inoculation with L. plantarum was beneficial to reduce pH at the beginning of ensiling( Fig. 2). Rapid reduction at early stage of fermentation is important to ensure good preservation of forage nutrients(Adesogan et al., 2004; Kim & Adesogan, 2006). The silage pH is associated with organic acid concentration in silage, especially lactic acid(Shaver, 2003; Zahiroddini et al., 2004). In this study, lactate concentration in inoculated silages was increased from day 2 - 49 of ensiling, but not persisted at 100day(Fig. 2). After 49days, in most of the silages(except uninoculated higher cut silage) lactate concentration was dropped but acetate was increased at variable rate. This might be the underlying reason for increasing pH in inoculated silage at 100days. The reason for decreasing lactate and increasing acetate concentration in silage of later stage needs to be identified. It may happen that Yuyeon barley hybrid have contain such heterolactic bacteria that activates at later stage of fermentation and/or can convert lactic acid to acetic acid under anaerobic condition. Generally, increased ammonia-N concentration in silage is considered as loss of protein due to fermentation. However, in this study, higher ammonia- N concentration in higher cut silages may be originated from increased CP content in corresponding forages. Sinclair et al.(2003) also reported higher ammonia- N in higher cut whole crop wheat silage.

    In general, it was reported that unsaturated FA contents in silage decrease by hydrogenation during ensiling(McDonald et al., 1991). Bacterial inoculation reduced the C18:3n-3 FA content in silage by 20% compare to uninoculated silage. Similarly, Boufaïed et al.(2003) used L. plantarum as silage inoculant which results a declined concentration of C18:3n-3 in grass silage. The declined C18:3n-3 content further resulted in decreased PUFA content by inoculation in this study. On the other hand, C18:3n-3 and PUFA contents of silage increased by higher cutting height. The reason of this result is unclear, but might be due to the less hydrogentation of unsaturated FA in these silages. Dietary PUFA such as C18:2n-6 and C18:3n-3 is important not only for energy sources, but also for numerous biological functions(Raes et al., 2004). Again, higher contents of dietary PUFA could increase FA quality of animal products, which can help to improve human health(Connor, 2000). Therefore, increased cutting height of Yuyeon barley forage has potential benefit for animal performances.

    Inoculant applications were resulted the abundant DNA of applied inoculants(Arriola et al., 2011; Amanullah et al., 2014). In agreement with these previous studies, PCR analysis of DNA in the presented study shown that DNA band mass was more prominent in inoculated silages.

    Results showed that the concentration of CP and C18:3n-3 fatty acid was increased in barley(Yuyeon) silage by increasing cutting height at harvest. It also reduced the fiber concentration(ADF and NDF) in silage with the concomitant increase in in vitro DM digestibility. Use of L. plantarum inoculant did affect the silage chemical composition and fermentation characteristics, but in a non-specific manner. It is concluded that barley forage can be harvested at 15 cm height from the ground than at 5cm height to achieve increased CP concentration, reduced fiber and improved digestibility of whole crop barley silage.



    Effect of cutting height of forage and bacterial inoculation on pH and the concentrations of ammonia-N, lactate and acetate at different fermentation periods. (□), silage without bacterial inoculants and 5cm forage cutting height; (■), silage without bacterial inoculants and 15cm forage cutting height; (○), silage with L. plantarum and 5cm forage cutting height; (●), silage with L. plantarum and 15cm forage cutting height. At the same day of ensiling, symbols with different superscripts differ (p<0.05).


    Gel electrophoresis analyses after PCR amplification of DNA from barley silages(Yuyeon) ensiled for 100days with or without inoculants. Lane M, DNA marker; lane L.P, pure culture of L. plantanum; lanes 1 to 4, 5 and 15cm cutting height without or with inoculant; lane N, negative control.


    Chemical compositions of barley forage (Yuyeon) before ensiling

    SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.

    Chemical compositions of barley silage(Yuyeon) ensiled for 100 d(%, DM)

    †Forages were harvested at 5 and 15cm height from root, respectively.
    INO, inoculant effect; CUT, cutting height effect; INO*CUT, interaction between inoculant and cutting height. SEM, standard error of the mean.

    Fermentation indices and microbial growth of barley silage(Yuyeon) ensiled for 100 d

    †Forages were harvested at 5 and 15cm height from root, respectively.
    INO, inoculant effect; CUT, cutting height effect; INO*CUT, interaction between inoculant and cutting height. SEM, standard error of the mean.

    Fatty acid profiles of barley silage(Yuyeon) ensiled for 100 d(% of total FA or as stated)

    †Forages were harvested at 5 and 15cm height from root, respectively.
    INO, inoculant effect; CUT, cutting height effect; INO*CUT, interaction between inoculant and cutting height; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.
    SEM, standard error of the mean.


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