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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.52 No.5 pp.71-79

Effects of New Inoculants on In vitro Digestibility and Fermentation Indices of High Moisture Rye Silage

Seong Shin Lee1,Dimas H. V. Paradhipta1,Young Ho Joo1,Hyuk Jun Lee1,Hyun Son1,Ouk Kyu Han2,Dong Hyeon Kim3,Sam Churl Kim1,3,*
1Division of Applied Life Science(BK21Plus, Insti. of Agri. & Life Sci.), Gyeongsang National University, Jinju, 52828, Korea
2Central Area Crop Breeding Division, National Institute of Crop Science, RDA, Suwon, 16429, Korea
3Department of Animal Sciences, IFAS, University of Florida, Gainesville, FL 32608, USA
*Corresponding author: Sam Churl Kim
Tel: +82-55-772-1947
Fax: +82-55-772-1949
July 27, 2018 August 13, 2018 August 13, 2018


This study was conducted to examine the effect of new inoculants on in vitro digestibility and fermentation characteristics of high moisture rye silage. Rye was harvested at heading stage and divided into 5 treatments, following: No additives(CON); L. plantarum R48-27(NI1); L. buchneri R4-26(NI2); mixture of NI1 and NI2 at 1:1 ratio(MIX); and L. buchneri(LB). The rye forage was ensiled into 10 L bucket silo for 100 days. in vitro digestibility of dry matter and neutral detergent fiber were highest(p<0.05) in NI2 silage. The pH in NI2 and LB silages were lower(p<0.05) than CON silage. Lactate concentration was highest(p<0.05) in NI1 silage. While concentrations of acetate and propionate were highest(p<0.05) in MIX silage. Lactates : acetate ratio was highest(p<0.05) in NI1 silage, but lowest in LB silage. Butyrate concentrations of NI2 and LB silages were lower(p<0.05) than that in CON and NI1 silages. Lactic acid bacteria (LAB) count in all inoculated silages was higher(p<0.05) than that in CON silage, while yeast count in LB silage was lower than in CON, NI1, and MIX silages. In conclusion, application of NI2 inoculant could improve potentially fermentation quality and digestibility of high moisture rye silage.



    Rye is one of main winter crop in South Korea, which is applied after harvesting rice or before farming corn(Heo et al., 2004). The characteristics of rye have high dry matter(DM) yield, adaptability, and resistance(Briggle, 1959; Kim et al., 1986). Especially, DM yield of rye increase dramatically by delaying harvest period(Morris & Gardner, 1985). However, total digestible nutrients of rye were lower than other winter crops such as Italian ryegrass and barley, due to rye had high neutral detergent fiber(NDF) and acid detergent fiber(ADF) concentrations(Yang et al., 2012).

    Bacterial inoculants are wildly used to improve fermentation quality and nutrient digestibility of silage. Various studies reported that application of lactic acid bacteria(LAB) improved silage preservation by increasing organic acids concentration(Seale, 1986; Filya et al., 2000; Baah et al., 2011). Homofermentative LAB such as Lactobacillus plantarum produce lactate mainly by using water soluble carbohydrates, which can lead the rapid decrease of silage pH(Zahiroddini et al., 2004) and inhibit the growth of undesirable microbes during ensiling(Oliveira et al., 2017). However, high production of lactate could decrease aerobic stability of silage because lactate is an energy source of yeast (Wang et al., 2014). In the other side, heterofermentative LAB such as L. buchneri could produce not only lactate, but also acetate and propionate(Elferink et al., 2001). These acetate and propionate have strong antimicrobial activity, which can increase aerobic stability of silage by inhibition of undesirable microbes such as yeast and mold during aerobic exposure (Courtin & Spoelstra, 1990). In our previous study(Kim et al., 2017), new strains of homo and heterofermentative LAB were isolated from rye silage and identified as L. plantarum R48-27 and L. buchneri R4-26, respectively. Based on this plate assays, L. plantarum R48-27 had reported to produce fibrinolytic enzymes, while L. buchneri R4-26 had shown high acidification ability and antifungal activities. Fibrinolytic and antifungalproducing inoculant, respectively, are expected to increase the fiber digestibility and inhibit the undesirable microbes in rye silage.

    In South Korea, it usually has a lot of rain during the harvesting period of rye forage, which is difficult to wilt properly. In the practical of industry, the abnormal fermentation of silage can be occurred by high moisture silage, which leads the growth of clostridia. The clostridia increase pH by increasing concentrations of butyrate and ammonia-N(Leibensperger & Pitt, 1987). Consequently, abnormal fermentation in silage could lead decreases of nutritional value and fermentation quality of silage(McDonald et al., 1991). Application of bacterial inoculant is one of solution to increase silage quality with high moisture content, which can stimulate the growth of LAB and reduce the clostridia effects. However, none of the studies have tested the effect of new inoculants on rye silage with high moisture content.

    Therefore, this study was performed to examine the effect of new inoculants on fermentation indices and In vitro digestibility of rye silage with high moisture content.

    Materials and methods

    1 Silage production

    The rye(Gogu, Secale cereale L.) forage was grown at the animal research unit, Gyeongsang National University, Jinju, South Korea and harvested at the heading stage(25.4% of DM). The fresh rye was chopped by conventional forage harvester(BHC-90, BUHEUNG Machinery Ltd., Jinju, South Korea) into 3~5 cm length, and divided into 5 treatments: 1) No additives (CON); 2) L. plantarum R48-27 at 4.0×104 cfu/g of fresh forage(NI1); 3) L. buchneri R4-26 at 4.0×104 cfu/g of fresh forage(NI2); 4) Mixture of NI1 and NI2 at 1:1 ratio (MIX); and 5) L. buchneri KACC 12416 at 4.0×104 cfu/g of fresh forage(LB). The inoculants, L. plantarum R48-27 and L. buchneri R4-26, in this study were isolated from Kim et al. (2017) and confirmed the fibrinolytic enzyme and the antifungal substance, respectively. Rye forages from each treatments were sub-sampled(1 kg) just after inoculant application, for the chemical compositions. After then, rye forage (5 kg) was ensiled into 10 L bucket silo with 4 replications for 100 days.

    2 Chemical compositions and In vitro digestibility

    The sub-sampled rye forage and silage(10 g) were dried at 105°C for 24 h to measure DM content. Approximately 500 g of sub-sampled each silage was collected, dried at 60°C for 48 h and ground to pass a 1 mm screen using a cutting mill(SHINMYUNG ELECTRIC Co., Ltd, South Korea). The concentrations of crude protein(CP) and ether extract(EE) were analyzed by the Kjeldahl method and the Soxhlet method (AOAC, 1990), respectively. Crude ash concentration was determined with a muffle furnace at 550°C for 5 h. The method of Van Soest et al.(1991) was used to measure NDF and ADF concentrations using an Ankom200 fiber analyzer(Ankom Technology, Macedon, USA). In vitro digestibility of DM(IVDMD) and NDF (IVNDFD) were determined using the method of Tilley & Terry(1963) using Ankom DaisyII Incubators (Ankom Technology, Macedon, USA).

    3 Fermentation indices

    About 20 g of fresh silage were homogenized with 200 mL of distilled water by blending for 30 s, and then filtered through two layers of cheese cloth to make silage juice used for pH, ammonia-N, lactate and volatile fatty acid(VFA) analysis. The pH and ammonia-N concentration were measured with a pH meter(SevenEasy, Mettler Toledo, Switzerland) and colorimetry(Chaney & Marbach, 1962), respectively. Silage juice was centrifuged at 5645 × g for 15 min, and then, the supernatant was used to measure the concentrations of lactate and VFA using HPLC (L-2200; Hitachi, Tokyo, Japan) fitted with a UV detector(L-2400; Hitachi, Tokyo, Japan) and a column (Metacarb 87H; Varian, CA, USA) described by Adesogan et al.(2004).

    4 Microbial enumeration

    About 20 g of fresh silage samples from each treatment were diluted with 180 mL of distilled water and macerated in a blender to obtain silage extract for enumeration of 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 into 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 72 h, while PDA plates were incubated at 30°C for 72 h in a aerobic 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 Statistical analysis

    The data were analyzed using ANOVA procedure of SAS(2002). Mean separation was performed by Tukey test and the significant differences were declared at p<0.05.


    The chemical compositions and In vitro digestibility of the rye foragebefore ensiling were shown in Table 1. The mean concentrations of CP, NDF, ADF, IVDMD, and IVNDFD across treatments were 12.1, 55.7, 30.8, 82.9, and 77.1%, respectively.

    The chemical compositions and In vitro digestibility of rye silage ensiled for 100 days were shown in Table 2. Inoculant applications were not affected(p>0.05) on chemical compositions of rye silages, except on In vitro digestibility of rye silage. The NI2 silage had highest(p<0.05) IVDMD and IVNDFD.

    The fermentation indices of rye silage ensiled for 100 days were shown in Table 3. The NI2 and LB silages had lower pH than CON silage(p<0.05; 4.28 and 4.40 vs. 4.59). Ammonia-N concentration was not affected(p>0.05) by inoculant applications. Lactate concertation was highest(p<0.05) in NI1 silage. MIX silage had highest acetate concentration, which had significant difference compared to CON and NI1 silages (p<0.05; 4.97 vs. 1.47 and 2.45%). The concentration of propionate was detected in NI2, MIX, and LB silages, but not detected in CON and NI1 silages. The concentration of butyrate was highest in NI1, followed by CON, and the lowest was in NI2 and LB silages(p<0.05; 2.29 vs. 1.54 vs. 0.79 and 0.49%). Furthermore, concentration of butyrate in MIX silage was not different(p>0.05) with CON, NI2, and LB silages. Lactate : acetate ratio was highest in NI1, followed by LB, and the lowest was in MIX silages(p<0.05; 0.45 vs. 0.12 vs. 0.07%).

    The microbial counts of rye silage ensiled for 100 days were shown in Table 4. The LAB count was higher in NI2, MIX, and LB silages than that in CON and NI1 silages(p<0.05; 8.03, 8.24, and 8.04 vs. 6.15 and 6.65 log10 cfu/g). The yeast count was lower in LB silage than CON, NI1, and MIX silages (p<0.05; 5.05 vs. 6.27, 6.58, and 6.14 log10 cfu/g). The mold count was not detected in all silages.


    The previous studies had reported that DM, CP, ADF, and NDF concentrations of rye harvested at heading stage were about 23.5, 9.15, 37.7, and 63.2%, respectively(Kim et al., 2001; Han et al., 2015; Choi et al., 2016), which had slightly higher CP and fiber contents compare to the present study. This might be due to the different region, environmental, climatic factors and so on(Gonzalez et al., 2012).

    In the present study, new species of LAB(L. plantarum R48-27 and L. buchneri R4-26) were used, which have been identified before by Kim et al.(2017). The L. plantarum R48-27 produced fibrinolytic enzymes, while buchneri R4-26 had acidification ability and antifungal activity(Kim et al., 2017). According to Kang et al.(2009) and Nadeau et al.(2000), fibrinolyticproducing LAB could increase the digestibility of DM and NDF by break down the structure carbohydrate of forage. In the present study, NI1 silage did not increase the IVDMD and IVNDFD. And in the other side, NI2 silage instead showed the improvement on IVDMD and IVNDFD. The reason of these results were not clear. However, it might be affected partially by the high moisture content of rye forage in the present study. The moisture content of silage directly influenced the LAB growth and performance(McDonald et al., 1991), which also affected in production of organic acids and other metabolite produce such fibrinolytic enzymes and antifungal substances. The ideal DM content for silage production was approximately at 35%(Kaiser et al., 2004), which has water activity 0.93-0.94 for optimal growth of LAB(Pitt et al., 1985).

    Bacterial inoculants were used to increase fer mentation quality through producing organic acids such as lactate, acetate, and propionate(Borreani et al., 2018). In general, applied homofermentative LAB increase lactate concentration(Demirci et al., 2011). While, hetero-fermentative LAB increase acetate and propionate concentrations(Cho et al., 2014). In the present study, application of CON, NI1, and MIX silages had higher pH than NI2 silage due to higher butyrate concentration. Similar with this result, previous study also reported that clostridial bacteria increased the silage pH by increasing butyrate concentration (Leibensperger & Pitt, 1987). The lactate concentration was higher in NI1 silage because it was inoculated with L. plantarum R48-27 as homofermentative LAB. While NI2, MIX, and LB had higher acetate concentration because those silages were inoculated with L. buchneri strain as heterofermentative LAB. Even though NI1 silage had high lactate concentration, it was still produce high butyrate concentration. According to Weinberg & Muck(1996), lactate could be changed into butyrate during fermentation in high moisture forage, and this process was known as ‘secondary or clostridial fermentation’. The application of NI2 and LB as heterofermentative LAB had lower butyrate concentration than other silage treatments due to the high concentrations of acetate and propionate, which had antimicrobial activity to inhibit clostridia. Tabacco et al.(2009) also reported that applied heterofermentative LAB in silage could inhibit the growth of clostridia. In the other side, L. buchneri R4-26 in NI2 silage produced the antifungal activity that might inhibit clostridia growth effectively, and then produced the lowest butyrate concentration in the present study. The MIX silage also showed lower butyrate concentration than CON and NI1 silages because its mixture inoculant contained L. buchneri R4-26.

    In the microbial count, all inoculant applications had higher LAB count than CON. It indicated that applied bacterial inoculant stimulated the growth of LAB(McDonald et al., 1991). In the other side, among NI2 and LB silages were not different on yeast count. It indicated that antifungal substance by L. buchneri R4-26 was not enough effective to inhibit yeast growth compare to common heterofermentative LAB. In high moisture of silage, undesirable microbes such as yeast grow faster than in normal moisture (McDonald et al., 1991). Therefore, it was difficult the control yeast growth in the present study. In the other side, NI2, MIX, and LB silages might increase aerobic stability due to high acetate concentration as antimicrobial. Increases of acetate concentration will increase exponentially aerobic stability of silage (Danner et al., 2003).


    This research was performed with the support of “Cooperative Research Program for Agriculture Science & Technology Development(Project No. PJ01101203 2017)” Rural Development Administration, South Korea.



    Chemical compositions of rye before ensiling(% DM)

    Effects of selected inoculants on chemical compositions of rye silage ensiled for 100 days(% DM)

    Effects of selected inoculants on fermentation indices of rye silage ensiled for 100 days

    Effects of selected inoculants on microbial counts of rye silage ensiled for 100 days


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