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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.51 No.5 pp.81-89

Effect of Homo or Heterofermentative Inoculants on Fermentation Characteristics and Aerobic Stability of Rye Silage

Dong Hyeon Kim1, Seong Shin Lee1, Dimas H. V. Paradipta1, Young Ho Joo1, Hyuk Jun Lee1, Youn Sig Kwak2, Ouk Kyu Han3, Sam Churl Kim1*
1Division of Applied Life Science(BK21Plus, Insti. of Agri. & Life Sci.), Gyeongsang National University, Jinju, 52828, Korea
2Department of Plant Medicine(Insti. of Agri. & Life Sci.), Gyeongsang National University, Jinju, 52828, Korea
3Central Area Crop Breeding Division, National Institute of Crop Science, RDA, Suwon, 16429, Korea
Corresponding author: Sam Churl Kim
20170204 20170531 20170623


This study was conducted to estimate the effect of home or hetero fermentative lactic acid bacteria(LAB) on chemical composition, fermentation quality, and aerobic stability of rye silage. Rye forage was harvested at dough stage(28.9% of dry matter), chopped to 3-5 cm length, and divided into 4 piles for different inoculations as treatment, following 1) No additives(CON); 2) Lactobacillus plantarum at rate of 1.5 × 105 cfu/g of fresh forage(LP); 3) L. buchneri at rate of 1.2 × 105 cfu/g of fresh forage(LB); and 4) Mixture of LP and LB at 1:1 ratio(MIX). Rye silage was ensiled into 20 L bucket silo in quadruplicate for 0, 1, 4, 7, and 100 day periods. After 100 days of ensiling, the silage treated with LB had lower acid detergent fiber content(p<0.05), but higher in vitro dry matter digestibility(p<0.05). The LB and MIX reduced (p<0.05) pH more rapidly than CON and LP across the ensiling days, but had no difference on 100 days. Silage treated LP had lowest(p<0.05) acetic acid, but highest(p<0.05) propionic acid. In contrast, LB treated silage had highest(p<0.05) acetic acid, but lowest(p<0.05) propionic acid with the absence of butyric acid. On microbial count, LP treated silage had lowest(p<0.05) LAB, yeast, and aerobic stability, whereas LB and MIX treated silages had highest(p<0.05). Mold was not detected across all silages. Therefore, it could be concluded that heterofermentative LAB solely or combo with homofermentative LAB might improve in vitro dry matter digestibility, fermentation characteristics, and aerobic stability of rye silage harvested at dough stage.



    Forage production for ruminant in South Korea has been primarily cultivated in the winter season, except for the cultivation period for rice that is the staple food. Rye(Secale cereale L.) is an important silage forage in South Korea as winter forage. It has high adaptability and resistance in barren soil, and also can be grown by double-cropping with paddy(Briggle, 1959; Kim et al., 1986). In addition, rye has the advantage of producing larger quantities than barley, triticale, and wheat which are the winter forages(4 to 18.4 ton/ha; Song et al., 2014). However, rye harvested after heading or dough stages has higher pH and lower dry matter digestibility(DMD) compare to other winter forages(Kim et al., 2001).

    Bacterial inoculants have been used to improve silage fermentation, to prevent the growth or survival of pathogens and to conserve nutritional quality of forages(Oliveira et al., 2017). The principal function of inoculant is that lactic acid bacteria(LAB) convert water-soluble carbohydrates(WSC) into organic acids leading to a rapid decrease in pH and the improvement of silage conservation with minimal nutrient losses(Weinberg et al., 1993). Silage inoculants are usually classified into two groups, homofermentative and heterofermentative. Homofermentative LAB produces organic acids via lactic acid fermentation as 1 mole of glucose to 2 moles of lactic acid(McDonald et al., 1991), whereas heterofermentative LAB not only produces lactic acid, but also complex organic acids such as acetic acid and propionic acid, likely because this bacteria converts lactic acid to acetic acid under anaerobic conditions(Oude Elferink et al., 2001). Previous studies reported that inoculation with homofermentative and heterofermentative LAB improved silage quality; particularly fermentation losses and aerobic stability (Weinberg et al., 1993; Oude Elferink et al., 2001; Kung et al., 2003; Arriola et al., 2011). However, these effects varied depending on the forage cultivars. Kung et al.(2003) reported that the combo inoculant of heterofermentative LAB with homofermentative LAB improved the fermentation and aerobic stability of alfalfa silage, whereas inoculating bermudagrass with combo inoculant only improved fermentation characteristics, but did not affect aerobic stability (Adesogan et al., 2004). Several studies of rye silage using homofermentative LAB were conducted to estimate the inoculant effect(Kim et al., 1999; Kim et al., 2015; Choi et al., 2016), but limited studies were conducted with heterofermentative LAB.

    Therefore, the objective of this study was to examine the effect of homofermentative and heterofermentative LAB on nutrient digestibility, fermentation characteristics, and aerobic stability of rye silage harvested at dough stage.

    Materials and methods

    1.Silage making and sampling

    Rye hybrid(Gogu) was grown at National Institute of Crop Science, Suwon, South Korea, and harvested at 28.9% of DM. The harvested rye forage(600 kg) was chopped to a theoretical cut length between 3-5 cm, separated into four piles, and treated with bacterial additives, as follows: 1) distilled water (CON); 2) homofermentative LAB, applied to fresh forage to supply 1.5 x 105 cfu/g of L. plantarum (LP); 3) heterofermentative LAB, applied to fresh forage to supply 1.2 x 105 cfu/g of L. buchneri(LB); and 4) Mixture of LP and LB at 1:1 ratio(MIX). Inoculants were dissolved in distilled water and sprayed uniformly onto the forages under constant mixing. One kilogram of forage of each treatment was prompt sub-sampled and composited for the analysis of nutrient contents. After then, 5 kg of forage in each treatment was weighed into 10 L bucket silo in quadruplicate, sealed, and stored for 1, 4, 7, 48 and 100 days. At the end of each ensiling periods, silages were sub-sampled for the analyses, except the silages ensiled for 1, 4, 7, and 48 days only used for pH analysis.

    2.Chemical composition and in vitro digestibility

    The sub-sampled fresh forage was dried in a forced-air oven at 105°C for 24 h to measure the DM content. Approximately 500 g of each subsampled silage was collected, dried in a forced-air oven 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 chemical composition of samples from each replicate silo was measured in triplicate. The contents of crude protein(CP) and ether extract(EE) were analyzed by the procedures of Kjeldahl and the Soxhlet(AOAC 1990), respectively. Ash content was determined with a muffle furnace at 550°C for 5 h. The contents of neutral detergent fiber (NDF) and acid detergent fiber(ADF) were determined by using Ankom200 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. In vitro digestibility of DM(IVDMD) and NDF(IVNDFD) were measured following the method described by Tilley & Terry(1963) using ANKOM DAISYII Incubator(ANKOM Technology, Macedon, NY, USA).

    3.Fermentation indices

    Twenty grams of silage along with 200 ml of sterile ultrapure water was macerated for 30 seconds in a laboratory blender followed by filtering through two layers of cheesecloth to obtain silage extract. Silage extract was used to analyze pH, the contents of ammonia-N, lactic acid, and volatile fatty acids (VFA). The pH and ammonia-N content were measured by pH meter(SevenEasy, Mettler Toledo, Switzerland) and colorimetry assay(Chaney & Marbach, 1962), respectively. Silage extract was centrifuged at 5,645 × g for 15 min, and then the supernatant was used for lactic acid and VFA(acetic acid, propionic acid and butyric acid) contents using HPLC(L-2200; Hitachi, Tokyo, Japan) fitted with a UV detector (L-2400; Hitachi) and a column(Metacarb 87H; Varian, CA, USA) described by Adesogan et al(2004).

    4.Microbial enumeration and aerobic stability

    Silage samples(20 g) 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 onto a selective agar medium. Microbial analysis was counted LAB, yeast, and mold. 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 yeast and mold. The MRS agar plates were placed in a CO2 incubator (Thermo Scientific, USA) at 39°C for 24 h, while PDA plates were incubated at 39°C for 24 h in an 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. After silo were opened, each sub-sampled silage(1 kg) was placed loosely into a polystyrene box for aerobic stability. Temperature sensors were connected to data logger(MORGAN™ TR-60CH, Korea), placed at the geometric center of each silage, covered with 2 layers of cheesecloth to prevent dust and drying, and recorded every 30 min. Aerobic stability was measured by the time(h) before a 2°C increase in silage temperature above the ambient temperature(Arriola et al., 2011). Four additional sensors were placed in the room to record the ambient temperature.

    5.Statistical analysis

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


    The chemical composition and in vitro digestibility of the rye forage harvest at dough stage are shown in Table 1. The contents of CP, NDF, IVDMD, and IVNDFD were 10.6, 70.5, 57.9, and 41.8%, respectively.

    The chemical composition and in vitro digestibility of rye silage ensiled for 100 days are shown in Table 2. There were no inoculant effects on chemical compositions and in vitro digestibility of rye silage, except on ADF and IVDMD contents. Silage treated with LP had higher(p<0.05) ADF content than that of LB and MIX silages, while LB silage had lowest (p<0.05). The IVDMD of LB silage was higher (p<0.05) than that of CON and LP silages.

    The change of pH during the ensiling period is shown in Fig. 1. In the early stage of fermentation(1 and 4 days of ensiling), pH decreased(p<0.05) rapidly by applications of LB solely or MIX. The LB and MIX silages had lower(p<0.05) pH than CON and LP silages on 7 and 48 days of ensiling, while CON silage had highest(p<0.05) pH on 48 days of ensiling.

    Fermentation characteristics of rye silage after ensiled for 100 days are shown in Table 3. All of treatment did not affect pH and concentrations of ammonia-N and lactic acid(p>0.05). However, microbial inoculants had the effect on the concentrations of acetic acid, propionic acid, and butyric acid(p<0.05) which acetic acid was highest in LB silage and propionic acid was highest in LP silage, while butyric acid was lowest in LB silage.

    The microbial counts and aerobic stability of rye silage ensiled for 100 days are shown in Table 4. The counts of LAB and yeast were lowest in LP(p<0.05), whereas CON, LB, and MIX had no difference. The mold counts were not detected in all of the treatments. Aerobic stability of LB and MIX silages were higher than CON and LP silages.


    The chemical composition of rye forage before ensiling indicated a normal variation due to the differences such as the seed quantity and harvest time(Table 1). Previous studies also reported similar variations in CP(6.4 to 10.6 %), ADF(37.7 to 40.4%), and NDF(61.2 to 68.2%) contents(Han et al., 2015; Kim et al., 2015; Choi et al., 2016).

    The inoculation of LAB is known to improve the quality by directly or indirectly affecting silage due to the metabolic products produced through silage fermentation, and homofermentative LAB is known to produce mainly lactic acid, and does not produce fibrinolytic enzymes(Dewar et al., 1963; McDonald et al., 1991). Several previous studies reported that inoculation of L. plantarum(i. e. homofermentative LAB) had no effect on the chemical composition after ensiling(Kim et al., 2015; Choi et al., 2016), and it is consistent with our results. In case of the L. buchneri (i. e. herterofermentative LAB), activity of esterase was reported to have the potential to increase digestibility of fiber by separating hemicellulose from lignin(Kang et al., 2009). This could support partially the reason of increased IVDMD by LB application in this study. The ADF reduction in this study might have potential fiber degrading the ability of L. buchneri. However, it is unknown due to the limited studies which were tested the effects of LB on rye silage.

    After 100-d ensiling, inoculation of microbial additives did not affect the pH(Table 3). However, there was a pH reduction in LB and MIX after 7 and 48-d ensiling(Fig. 1). The reason for these results can be found in the content of organic acids. The heterofermentative LAB can converts lactic acid to acetic acid under anaerobic conditions(Oude Elferink et al., 2001). Our results showed that acetic acid increased in LB than LP, but there was no difference between CON, LB, and MIX. It implies that acetic acid was produced during that period. This situation might be due to the high concentration of epiphytic bacteria that naturally occurred in the untreated rye forage(Pravin et al., 2010).

    The antifungal characteristics of acetic and propionic acids are well known(Danner et al., 2003); they are effective at reducing the growth of yeasts and molds, which are responsible for causing and maintaining or enhancing aerobic deterioration in silages, respectively (Kleinschmit et al., 2005). In our study, there was no effect of aerobic stability in LP despite the content of high acetic acid and propionic acid. This may be due to the high content of pH in the early ensiling stage.

    The low pH in the early ensiling stage might contribute to silage quality. The lower pH can prevent undesirable bacteria such as clostridia, and enterobacteria by sufficient organic acids(Adesogan et al., 2014). However, the content of butyric acid in CON and LP was higher than LB in the current study. As a result, aerobic stability improved in LB and MIX that had lower pH. The butyric acid in MIX might be due to the numerically increased pH that affect the growth of undesirable bacteria.


    Application of LB improved IVDMD, acetic acid and aerobic stability in rye silage compare with LP. In addition, as a result of pH trend during ensiling, LB has been found to be more suitable for rye silage than LP. Inoculating rye forage harvested at dough stage with LB may reduce the financial losses from aerobic spoilage.


    This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development(Project No. PJ011012032 017)” Rural Development Administration, Republic of Korea.



    The change of pH of rye silage fermented 100 days with different inoculants. CON(□), control; LP(■), L. plantarum; LB(▲), L. buchneri; MIX(●), mixture of LP and LB at 1:1 ratio. a~cMeans at the same day with different superscripts differ significantly(p<0.05).


    Chemical compositions of rye before ensiling (% DM)

    1IVDMD, In vitro dry matter digestibility; IVNDFD, In vitro neutral detergent fiber digestibility.
    2Means°standard deviation.

    Chemical compositions of rye silage ensiled for 100 days(% DM)

    1CON, No additive; LP, L. plantarum at 1.5 x 107 cfu/g of fresh forage; LB, L. bucheneri at 1.2 x 107 cfu/g of fresh forage; MIX, mixture of LP and LB at 1:1 ratio.
    IVDMD, In vitro dry matter digestibility; IVNDFD, In vitro neutral detergent fiber digestibility.
    a~cMeans in the same row with different superscripts differ significantly(p<0.05).

    Effect of microbial additives on fermentation characteristics of rye silage ensiled for 100 days

    1CON, No additive; LP, L. plantarum at 1.5 x 107 cfu/g of fresh forage; LB, L. bucheneri at 1.2 x 107 cfu/g of fresh forage; MIX, mixture of LP and LB at 1:1 ratio.
    a~bMeans in the same row with different superscripts differ significantly(p<0.05).

    Effects of microbial additives on microbial counts and aerobic stability of rye silage ensiled for 100 days

    1CON, No additive; LP, L. plantarum at 1.5 x 107 cfu/g of fresh forage; LB, L. bucheneri at 1.2 x 107 cfu/g of fresh forage; MIX, mixture of LP and LB at 1:1 ratio.
    *ND: not detected
    a~bMeans in the same row with different superscripts differ significantly(p<0.05).


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