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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.1 pp.35-44

Effects of Microbial and Organic Additives on Fermentation Quality and Aerobic Stability of Barley Silage

Young-Ho Joo1, Dong-Hyeon Kim1,2, Hyuk-Jun Lee1, Seong-Shin Lee1, Dimas Hand Vidya Paradhipta1,3, In-Hag Choi4, Sam-Churl Kim1*
1Division of Applied Life Science (BK21Plus, Institute of Agriculture & Life Science), Gyeongsang National University, Jinju, 52828, South Korea
2National Institute of Animal Science, Rural Development Administration, Cheonan, 31000, South Korea
3Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
4Joongbu University, Department of Companion Animal & Animal Resources Sciences, Geumsan, 32713, South Korea
Corresponding author: Sam-Churl Kim Tel: +82-55-772-1947 Fax: +82-55-772-1949 E-mail:
February 13, 2020 February 13, 2020 February 14, 2020


The goal of this study was to evaluate effects of various microbial and organic additives on chemical compositions, fermentation indices, and aerobic stability of barley silage. Youngyang barley harvested at 31.5% dry matter (DM), and ensiled into 20 L bucket silo for 0, 1, 3, 7, 48, and 100 d in quadruplicates with four additives following: sterile destilled water at 1% of fresh forage (CON); Lactobacillus plantarum at 1.2 × 105 cfu/g of fresh forage (CL); Lactobacillus buchneri at rate of 1.2 × 105 cfu/g fresh forage (LB); Fermented Persimmon Extract at 1% of fresh forage (FPE); and Essential Oil at 1% of fresh forage (EO). On 100 d of ensiling, CL and FPE silages had lower (p<0.05) DM than CON silage. Additionally, FPE silage had higher (p<0.05) crude protein than CON silage. All silages with additives, except EO, had higher (p<0.05) neutral detergent fiber (NDF) than CON silage. Silage treated with CL, LB, and FPE had lower in vitro DM digestibility than CON silage, and silages treated with LB and FPE had higher in vitro NDF digestibility (IVNDFD) on 100 d of ensiling. The PFE silage produced the highest (p<0.05) lactate during ensiling period, while LB silage produced the highest (p<0.05) acetate. All inoculated silages had higher (p<0.05) LAB count than control silage. Only CL silage had higher (p<0.05) yeast count than control silage, while the other silages were not differ compared to CON silage. The aerobic stability was higher (p<0.05) in LB and FPE silages than in CON silage. In conclusion, FPE could be an alternative additive to increase IVNDFD, fermentation indices, and aerobic stability of barley silage.


    Rural Development Administration


    During a decade, the requirement of forage is supplied by preservation forage technology that is called silage. In silage production, ensiling is an important process, which is affected by natural epiphytic bacteria. This process involves lactic acid bacteria (LAB), which converts soluble carbohydrate of forage into lactate during anaerobic condition. However, the growth of harmful bacteria and fungi are susceptible to cause spoilage due to fungi during ensiling that reduce silage quality (Woolford, 1990). Thereby, high concentration of lactate by LAB reduces pH that inhibits spoilage microbes and preserves the nutrient of forages. Inoculation of LAB is capable of improving the ensiling process that enhances silage quality (McDonald et al., 1991). Usually, barley is known forage source for silage production. Barley forage contains a high amount of soluble carbohydrate (Hargreaves et al., 2009) that provides substrate for LAB to growth and produce lactate.

    Recently, microbial additives have been used to enhance silage quality. Microbial additives such as LAB, are classified into two groups based on their metabolic pathway. Homofermentative LAB produced lactate as main product during anaerobic condition, while heterofermentative LAB not only produce lactate but also another product such as acetate (McDonald et al., 1991). Previous studies reported that high concentration of lactate by homofermentative LAB during ensiling enhanced fermentation indices, while a high concentration of acetate by heterofermentative LAB improved aerobic stability (Adesogan et al., 2003; Filya, 2003; Kung et al., 2003). Some of organic compounds, such as fermented persimmon extract (FPE) and essential oil (EO) can be applied to to inhibit the growth of undesriable microbes and reduces the decaying nutrient of silage during the ensiling. These organic compounds have been used for fermenting food to reduced growth of spoiled microorganism. Fermented persimmon extract is a by-product of the persimmon industry in South Korea through aerobic fermentation for 1-2 year. As food additive, FPE produces high acetate concentration and low pH, which can reduces lipid oxidation, off-flavor, and inhibit microbial growth (Kittelmann et al., 1989; Woo et al., 2004;Sakanaka & Ishihara, 2008). The previous study showed that FPE increased acetate concentration, which has potential to improve aerobic stability (Kim et al., 2013). EO is the phytochemicals with antifungal activities (Chao & Young, 2000) that reduce the food spoiling yeast as additive (Souza et al., 2007). Essential oils have applications to reduce spoiling yeast in silage technology because feed and food fermentation are dependent on microbial activities. In terms of antifungal of function, Supplementation of EO inhibited species of Clostridium (Candan, 2003; Wannissorn, 2005) that growth during ensiling and lead to degrade nutrients. The objectives of this study were to compare the effect of microbial additives containing homo and heterofermentative LAB, and organic compounds or phytochemicals on the chemical compositions, fermentation indices, and aerobic stability.


    1. Silage production and sampling

    Youngyang barley was grown at Gyeongsang National University Research Farm, Jinju, South Korea, and harvested at 31.5% of DM. Youngyang barley forage was chopped to 3-5 cm length and treated with different additives as follows: Non additive, applied 1% distilled water in fresh forage (CON); Lactobacillus plantarum (Chungmilacto, CMbio, Anseong, South Korea) as homofermentative LAB at 1.2 x 105 cfu/g of fresh forage (CL); 2) Lactobacillus buchneri KACC12416 as heterofermentative LAB at 1.2 x 103 cfu/g of fresh forage (LB); 3) Fermented Persimmon Extract at 1% of fresh forage basis (FPE); and 4) Essential Oil at 1% of fresh forage (EO). Each treatment was ensiled into 20 L plastic bucket silo (5 kg) with four replication for 1, 3, 7, 48, and 100 d at room temperature. Fermented persimmon extract was obtained from Horticultural Primary Cooperatives, Jinju, South Korea, while essential oil was extracted wormwood silage according to protocol of Kim et al. (2005). Each silo was opened on the assigned day and sub-sampled for laboratory analyses. On 1, 3, 7, and 48 d of ensiling period, silages were sub-sampled for making silage extract (20 g) that was used to analyses fermentation indices. While on 100 d, silage was sub-sampled for analyses of chemical compositions (500 g), fermentation indices (20 g), and aerobic stability (1 kg).

    2. Chemical compositions

    The dry matter (DM) concentration was determined by drying silage sample (about 10 g) into the dry oven (OF- 22GW, JEIO TECH, Korea) at 105°C for 24 h. About 500 g of sample was dried separately at 60°C for 48 h and grinded by cutting mill (SHINMYUNG ELECTRIC Co., Ltd, South Korea) with 1 mm screen to determine chemical composition and in vitro digestibility. The procedures of Kjeldahl (B-324, 412, 435, and 719STitrino, BUCHI, Flawil, Switzerland) and Soxhlet (OB-25E, JeioTech, Seoul, South Korea) were used to determine crude protein (CP) and ether extract (EE), respectively (AOAC, 1995). The concentrations of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using Ankom200 fiber analyzer (Ankom Technology, Macedon, NY) following the method of Van Soest et al. (1991). The in vitro DM digestibility (IVDMD) and in vitro NDF digestibility (IVNDFD) were measured following the method described by Tilley & Terry (1963) using Ankom DaisyII Incubator (ANKOM Technology, Macedon, NY, USA).

    3. Fermentation indices

    Silage extract was prepared from fresh silage after opening the silo. Twenty gram of fresh sample along with 200 mL of sterile ultrapure water was macerated for 30 sec in a laboratory blender followed by filtering through two layers of cheesecloth to obtain silage extract (Arriola et al., 2012). A silage extract was used to determine pH, ammonia-N, volatile fatty acid (VFA), and microbial counts. The pH was measured by a pH meter (SevenEasy, Mettler Toledo, Switzerland). Ammonia-N concentration was determined by the colorimetric method described by Chaney & Marbach (1962). The silage extract was centrifuged at 5,645×g for 15 min then the supernatant was collected for determination of VFA. The concentration of VFA was 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).

    4. Microbial count analysis

    Microbial analysis was counted LAB, yeast, and mold. Considering the silage extract as the first dilution, serial dilutions were prepared and 100 μL 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 30°C for 24 h, while PDA plates were incubated at 30°C for 72 h in a normal incubator (Johnsam Corporation, Korea). Visible colonies were counted from the plates and the number of colonies forming units (cfu) was expressed per gram of silage.

    5. Aerobic stability analysis

    Aerobic stability was determined on all silage after silo opening. Silage was sub-sampled (1 kg) then placed loosely into a polystyrene box. Silage was exposed to air at room temperature. Temperature sensors (MORGANTM TR-60CH, Hong Kong, China) were placed at the geometric center of each silage sample and data were recorded every 30 min for the day. Four additional sensors were placed in the room temperature record. Aerobic stability was measured by the time (h) before a 2°C increase in silage temperature above the room temperature (Arriola et al., 2012).

    6. Statistical analysis

    The data were analyzed as completely randomized design and subjected to analysis of variance by general linear models procedure of Statistical Analysis Software (SAS, 2002). Mean separation was performed by Tukey’s test of SAS. The significant differences were declared at p<0.05.


    1. Chemical composition

    The concentrations of DM, CP, EE, NDF, and ADF from fresh barley forage were 31.5, 7.71, 3.02, 63.5, and 37.2%, respectively (Table 1). Furthermore, IVDMD and IVNDFD were 45.2 and 25.4%, respectively. The CL and FPE silages had lower DM concentration than CON silage (p<0.05; 27.8 and 27.8 vs. 29.2%; Table 2). The FPE silage had higher CP and EE than CON silage (p<0.05; 7.23 vs. 6.86; P<0.05; 3.38 vs. 3.13%). The highest NDF was in LB silage, followed respectively by FPE silage and CL silage, while the lowest NDF was in EO and CON silages (p<0.05; 60.8 vs. 58.9 vs. 57.8 vs. 56.3 and 56.0%). The ADF concentration of barley silage was not affected by inoculant application in the present study. The CL, LB, and FPE silages had lower IVDMD than CON and EO silage (p<0.05; 58.8, 58.6 and 60.1 vs. 60.1 vs. 61.5%). On the other side, LB and FPE silages had higher IVNDFD than CON silage (p<0.05; 32.7 and 32.5 vs. 30.4%), while IVNDFD haw lower in CL silage had lower (p<0.05; 30.4 vs. 28.2%).

    2. Fermentation indices

    The pH silage decreased over day (Fig. 1A). On 3 d, LB and FPE silages had lower pH than CON silage. On 7 d, FPE and EO silages had higher pH compared to CON silage (p<0.05), but on 48 d these treatments had lower pH. The LB silage resulted in the lowest pH during 48 d of ensiling period, while the highest pH was CL silage (p<0.05). However on 100 d, all treatments had similar pH pattern. The concentration of ammonia -N increased over day (Fig. 1B). Additive treatments did not affect concentration of ammonia-N during 3 d of ensiling period (p<0.05). On 7 d, CON silage resulted in the lowest ammonia-N concentration, while the highest ammonia-N concentration was found in EO silage (p<0.05). The LB silage had the lowest ammonia-N concentration on 48 d, while CL and EO silages had the highest ammonia-N concentration (p<0.05).

    All silages increased lactate concentration as the days increased (Fig. 2A). However, a change trend of lactic acid concentration in all silages had shown various patterns during 7 d of ensiling. On 48 d, FPE silage produced the highest lactate concentration (p<0.05) and LB silage resulted in the lowest lactate concentration (p<0.05). In addition, lactate concentration among CON, CL and EO silages were not differ on this day. In general, LB, PFE, and EO silages seemed to present the stationary phase of lactate production after 48 d of ensiling.

    In general, all silages presented an increase of acetate concentration along with a longer ensiling day (Fig. 2B). The addition of microbial and chemical organic did not affect acetate concentration during the first 7 d of ensiling. On 48 d, LB silage produced the highest acetate concentration (p<0.05) and CL silage resulted in the lowest acetate concentration (p<0.05). In addition, FPE silage had higher acetate concentration than CON and EO silages (p<0.05) on this day. The LB and FPE silages seemed to present the stationary phase of acetate production after 48 d of ensiling, while CON and CL silages presented after 7 d of ensiling.

    At the end of 100 d of ensiling period, pH was not affect by all treatment (Table 3). The CL silage had the lowest ammonia-N concentration, while FPE and EO silages resulted in higher ammonia-N concentration than CON silage (p<0.05; 0.13 vs. 0.16 vs. 0.20 and 0.22%). The highest lactate concentration was in FPE silage, followed respectively by CL and CON silages, EO silage, and LB silage (p<0.05; 2.68 vs. 2.00 and 1.93 vs. 1.34 vs. 0.78%). Acetate concentration was highest in LB silage, while the lowest was in CL silage (p<0.05; 5.17 vs. 1.76%). In addition, EO silage had higher acetate concentration than FPE and CON silages (p<0.05; 3.50 vs. 2.55 and 2.22%). The LB silage also had the highest concentration of propionate, while CON and CL silages had lower concentration than FPE and EO silages (p<0.05; 0.42 vs. 0.28 and 0.29 vs. 0.17 and 0.13%). Besides lactate, FPE silage also had the highest butyrate than the other silages (p<0.05; 0.89 vs. 0.38, 0.18, 0.38, and 0.38%). The CL silage had the highest lactate to acetate ratio, while CON and FPE silages had higher ratio than LB and EO silages (p<0.05; 1.34 vs. 0.91 and 1.06 vs. 0.16 and 0.38%).

    3. Microbial counts and aerobic stability

    The EO silage had higher LAB count than LB and FPE silages (p<0.05; 6.45 vs. 5.97 and 5.91 log10 cfu/g; Table 4). In addition, CL silage had higher this count than CON silage (5.30 vs. 4.97 log10 cfu/g; Table 4). The LB and EO silages had lower yeast count than FPE and CL silages (p<0.05; 3.00 and 3.00 vs. 4.14 vs. 5.24 log10 cfu/g), while the CON silage only had lower yeast count than CL silage (p<0.05; 3.45 vs. 5.24 log10 cfu/g). The mold was not detected in all silages. The highest aerobic stability was in LB silage, followed respectively by FPE silage and CL silage (p<0.05; 348 vs. 307 vs. 254 h). In addition, the aerobic stability in CON silage was not differ with in CL and EO silages.


    The chemical composition of fresh Youngyang barley is in agreement with the previous studies (Park et al., 2008; Lee et al., 2014). In the present study, CL and FPE silages had lower DM concentration than CON silage. Weinberg et al. (2007) reported that some strains of LAB such as Pediococcus pentosaceus (Agri-King), Entrococcus faecium (Agri-King), L. pentosus (Agri-King), and L. plantarum (Agri-King) were capable of lowering DM concentration on wheat silage, but the other strains of LAB consisting of L. buchneri (Biotal) and L. buchneri 11A44 (Pioneer) resulted in a higher DM concentration after ensiling. This indicated that application of LAB in the silage is not always promising to reduce DM loss. In addition, application of FPE in barley silage up to 1.6% of fresh weight had no effect on DM concentration (Kim et al., 2013). The low DM concentration of FPE silage in the present study could be occurred depending on the fermentation process (McDonald et al., 1991), which might be differ with other study. An increase in NDF concentration after ensiled could be affected by DM losses during ensiling, which increased the proportion of NDF. Numerically, all silages had higher DM loss than CON silage, which might increase NDF concentration in the present study. It could be a reason of no IVDMD improvement in the present study. Silage treated with FPE and LB produced the highest IVNDFD compared to all of treatments. Weinberg et al. (2007) also indicated that inoculation of some strain of LAB, such as L. buchneri 11A44 (Pioneer) on the corn silage could increase IVNDFD after 24 h of in vitro incubation. The L. buchneri might enhance the activity of ruminal cellulolytic, thereby the further study is necessary to explain this result. According to Kim et al. (2013), FPE had xylanase and endoglucanase enzyme that capable of enhancing fiber digestibility. Furthermore, supplementation of FPE at 0.4% of fresh weight increased NDF and IVNDFD concentrations, but high supplementation of FPE at 1.6% was reported to decrease these concentrations.

    All of treatments reduced the silage pH along with longer ensiling day. Interestingly, the CL silage had the highest pH, while the LB silage had the lowest pH on 48 d. These result could be influenced by the presence of ammonia-N (Fig. 1B) and organic acid such as lactate and acetate (Fig. 2). However, on 100 d of the ensiling period, all treatments had no difference on silage pH compared to control. Similar to this present study, Ranjit & Kung (2000) reported that inoculation of L. plantarum and L. buchneri did not affect pH of corn silage after ensiled 100 d. Furthermore, Kim et al. (2013) and Kung et al. (2008), supplementation PFE or EO also did not affect the pH of silage.

    Ammonia-N concentration reflect proteolysis during ensiling period (McDonald et al., 1991; Huisden et al., 2009). Low pH condition decrease ammonia-N concentration (McDonald et al., 1991). This was a reason of low ammonia-N concentration in CL silage of the present study. The concentration of ammonia-N on 100 d indicated the similar result with previous studies, which inoculation of L. plantarum reduced the ammonia-N concentration, but inoculation of L. buhcneri increased it (Filya, 2003; Kung et al., 2003). Furthermore, supplementation FPE increased concentration of ammonia-N on the barley silage (Kim et al., 2013). However, Kung et al. (2008) explained that essential oil did not affect concentration of ammonia-N on the corn silage.

    Inoculation of L. plantarum on silage increased the lactate concentration, whereas L. buchneri increased acetate concentration (Kung et al., 2003). Homofermentative LAB uses soluble carbohydrate to produced high concentration of lactate as a main product. Another side, heterofermentative LAB is able to produce not only lactic acid but also acetic acid, ethanol, and carbon dioxide (McDonald et al., 1991). Therefore, LB silage produced the highest production of acetate than the other treatment during ensiling. However, this present study showed interesting result that FPE silage had the highest lactate production during ensiling. Contrast with the previous study, Kim et al. (2013) reported that supplementation of FPE on barley silage reduced concentration of lactate, but increased the concentration of acetate. The result of high lactate concentration in FPE silage could be occurred because fermented persimmon extract could still contain high water soluble carbohydrate, which could converted mainly into lactate by epiphytic LAB. In this present study, silage treated with EO decreased lactate concentration, but increased acetate concentration of barley silage. However, previous study reported that supplementation blend essential oil did not affect lactate, acetate, and propionate concentrations of corn silage after ensiled 256 d (Kung et al., 2008). The different type source of EO could affected different results of organic acid production. The LB, FPE, and EO silages increased propioante concentration, which could indicate the presence of hetefermentative LAB or propionibacterium in those silage (McDonald et al., 1991). On the other side, CL silage decreased butyrate concentration. This result was in agreement with previous studies, which homofermentative LAB could decrease the butyrate concentration due to the presence of lactate that caused rapid acidification (McDonald et al., 1991; Filya, 2003; Kung et al., 2003).

    All treated silages, including CL and LB silages had higher LAB count than CON silage. Similar to this present study, Filya (2003) reported that inoculation homo and heterofermentative LAB bacteria increase LAB count. In addition, the present study indicated that organic compound additive and heterofermentative LAB had higher LAB count compared to homofermentative LAB. Furthermore, acetate production by these additives reduced the yeast and mold count more effectively compared to homofermentative LAB. These results suggested that treatment with a low ratio of lactate to acetate had the low count of yeast and mold. According to Ranjit & Kung (2000), inoculation of L. buchneri more inhibited yeast growth compared to L. plantarum at 1 x 106 cfu/g, but there was not difference at a concentration 1 x 105 cfu/g. Inoculation LB as the highest production of acetate increased the aerobic stability of barley silage, which was supported by previous studies (Adesogan et al., 2003; Kung et al., 2003; Filya, 2003). Acetic acid has a role as an antimicrobial effect that inhibited the growth of fungi (Palmqvist et al., 1998). On the other side, FPE silage was capable to increase aerobic stability in the present study. The increase of acetate concentration through FPE silage caused greater inhibitory effects on yeast and mold growth, therefore increasing aerobic stability (Danner et al., 2003). According to Kim et al. (2013), FPE silage increased the acetate concentration, which had a potential to increase aerobic stability. Pathogenic and spoiled microorganisms have also been inhibited by the some essential oil (Kim et al., 1995; Candan, 2003; Wannissorn, 2005; Souza et al., 2007). However, EO silage in the present study had no different with CON silage. Kung et al., (2008) reported similar result with the present study that application of essential oil with different level had no effect on aerobic stability.

    The IVNDFD increased in LB and FPE silages. The lactate improved in FPE silage and the acetate improved in LB silage. Nevertheless, acetate concentration in FPE silage was still higher than CL silage. Silages treated with LB, FPE, and EO had no effect on yeast growth, while silage treated with CL increased it. Additionally, aerobic stability was increased by LB and FPE silages. The present study concluded that application of fermented persimmon extract could be an alternative additive to improve nutrient digestibility, fermentation indices, and aerobic stability of barley silage.


    This research was performed with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ013869022019)” Rural Development Administration South Korea.



    Changes of pH (A) and ammonia-N (B) concentrations in barley silage (Youngyang) by additives during ensiling period. CON (●), control; CL (□); L. plantarum; LB (■), L. buchneri; FPE (Δ), fermented persimmon extract; EO (▲), essential oil. Bar indicated standard error. Values differ between groups within same hour * p<0.05 and ** p<0.01.


    Changes of lactate (A) and acetate (B) concentrations in barley silage (Youngyang) by additives during ensiling period. CON (●), control; CL (□); L. plantarum; LB (■), L. buchneri; FPE (Δ), fermented persimmon extract; EO (▲), essential oil. Bar indicated standard error. Values differ between groups within same hour * p<0.05 and ** p<0.01.


    Chemical compositions of barley forage (Youngyang) before ensiling (%, DM)

    Effect of additives on chemical compositions and in vitro digestibility of barley silage (Youngyang) ensiled for 100 d (% of DM)

    Effect of additives on fermentation indices of barley silage (Youngyang) ensiled for 100 d

    Effect of additives on microbial count and aerobic stability of barley silage (Youngyang) ensiled for 100 d


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