Effect of enzymes and bacterial inoculant and their combination on the fermentation parameter and aerobic stability of maize silage

Introduction

Silage is becoming increasingly popular in various regions of India due to its ability to provide a consistent supply of roughage throughout the year. The basis of ensiling phenomena is the naturally occurring anaerobic fermentation of fodder in the presence of lactic acid-producing bacteria, which convert readily fermentable carbohydrates into organic acids, primarily lactic acid (KocCoskuntuna and Ozduven, 2008) [26] . Ensiling is naturally a spontaneous process influenced by epiphytic microflora. However, the addition of silage additives can regulate and enhance fermentation, thereby improving silage quality and aerobic stability (Kung et al., 2003; Chauhan et al., 2021) [28,10] . Various additive combinations are used to enhance fermentation parameters and aerobic stability. Through analysis of multiple silage experiments, it has been observed that homofermentative lactic acid bacteria enhance dry matter recovery (DMR) during fodder fermentation, thereby improving the efficiency of the ensiling process. It has been observed that silage high in carbohydrates or well-preserved silages with high lactic acid concentrations and low quantities of volatile fatty acids (acetic acid) are particularly susceptible to aerobic degradation (McDonald et al., 1991) [30] . Aerobic deterioration of silage primarily occurs due to the proliferation of yeasts and moulds, which lie dormant under anaerobic conditions and rapidly multiply upon exposure to oxygen. Obligatory heterofermentative lactic acid bacteria enhance silage aerobic stability through the production of acetic acid and by suppressing the growth of undesirable fermentation bacteria (Kleinschmit and Kung, 2006) [25] . Homofermentative bacterial inoculants improve the fermentation profile by increasing the production of lactic acid.

Enzymes can partially break down plant cell walls (cellulose and hemicellulose), releasing soluble sugars that lactic acid bacteria (LAB) can ferment, thereby lowering the pH of the silage. This partial digestion of the plant cell wall may enhance the speed and/or efficiency of digestibility (Dean, 2005) [16] . In the present study, an attempt has been made to improve the fermentation parameters and aerobic stability of silage through the addition of bacterial inoculant and enzymatic additive.

Materials and Methods

Silage preparation and additive treatment

The research was conducted at the National Dairy Research Institute in Karnal, Haryana, situated at an altitude of 250 meters above sea level, with coordinates of 29° 42" N latitude and 79° 54" E longitude. Maize fodder was harvested from the NDRI fields at the one-third to half milk line stage, with a dry matter content of 30.62%. The fodder was chopped using an electric chaff cutter to an average length of 1.5-2 cm and ensiled in a plastic container equipped with a lid that allows gas release and creates a vacuum. For silage preparation following additives were used on a fresh basis. The treatments were control (no additive), cellulase, (6000 NCU/kg), xylanase (1500 IU/Kg 1 ), C+X, Lactiplantibacillus plantarum (LP @ 1*10 6 CFU g -1 ), LP+C, LP+X, LP+X+C, Limosilactobacillus fermentum, (LF @ 2*10 6 CFU g -1 ),), LF+C, LF+X, LF+X+C. Silage fermentation parameters and aerobic stability were evaluated after 30 days of ensiling. Each treatment involved filling three containers with maize fodder, thoroughly mixed with additives to assess their individual or combined effects. These containers were tightly sealed, weighed and stored at room temperature. After 30 days of ensiling, samples were analyzed in triplicate to evaluate fermentation and quality parameters, microbial composition and aerobic stability. Pre-ensiled samples were collected for chemical analysis prior to the ensiling process.

Chemical composition, fermentation parameters and silage quality assessment

The chemical composition (DM, CP, OM and EE) of fresh maize fodder was determined as per the method described by AOAC (2005) [3] . The pH of fresh fodder and silage was determined using the Eutech pH meter from the aqueous extract. The water-soluble carbohydrate (WSC) content of fresh fodder was determined by a spectrophotometer after a reaction with an anthrone reagent. The fermentation coefficient (FC) of maize fodder was calculated using dry matter, water-soluble carbohydrates and buffering coefficient. The weight of the forage mass in the plastic container and its DM content at day 0 and 30 th day were used to calculate the DM recovery (DMR) (da Silva et al., 2020). Lactic acid estimation was done as per the method described by Barnett (1951) [6] . Acetic acid was estimated with the help of Nucon's gas-liquid chromatography. Flieg point was calculated from the pH value and DM of silage at the end of the fermentation period with the following equation. [15] obtained from Himedia Laboratories Pvt Ltd, Mumbai, India. The Petri plates were then incubated at 37°C for 36 hours to enumerate LAB. Total counts of yeasts and molds were assessed by pour-plating 10-fold serial dilutions on potato dextrose agar, which was acidified with 0.5% (vol/vol) of 85% lactic acid after autoclaving. These plates were incubated aerobically for 48 hours at 25°C. The concentration of Clostridia spores in fresh silage samples was determined using the most probable number (MPN) method. Fitness value (Davies et al., 2000) [14] were modified as follows for the present experiment conditions ) Weightage for different Parameters pH 4, DM Loss 3, Ammonia-N 3 Where wtg is weightage, cntl is control silage, DML is dry matter loss, and Amm-N is ammonia nitrogen content.

Aerobic stability

Aerobic stability was assessed by monitoring changes in pH and yeast and mould counts of silage during aerobic exposure. Approximately 2 kg of silage samples were placed in plastic buckets and stored at room temperature (25 °C). Samples were taken at intervals during aerobic exposure (0, 2, 4, 6 and 8 days) to measure pH and enumerate yeast and mould populations (Dolci et al., 2011) [17] . For yeast and mould enumeration, 11 grams of silage sample from each replicate were homogenized with 99 mL of sterile water. The homogenates were then plated on potato dextrose agar acidified with lactic acid. Yeasts and moulds were differentiated based on morphological characteristics. Plates with colony counts between 30 and 300 were used to determine colony-forming units (CFU).

Statistical design

The fermentation parameter data were analyzed using oneway analysis of variance (ANOVA) in SPSS (version 26.0) software. Aerobic stability data were subjected to a two-way analysis of variance, considering fixed effects of additives, ensilage period, and their interaction (additives × ensilage period). For LAB, yeast, and mould counts, logarithm base 10 transformations were applied to the data. Pairwise comparisons of mean values were conducted using Duncan's multiple range tests (Duncan, 1955) [18] with a significance level set at p<0.05.

Results

Chemical composition of maize green fodder

The composition of the maize fodder before ensiling (Table 1) shows the following values, dry matter (DM) 30.62%, crude protein (CP) 9.46%, ether extract (EE) 2.76% and total ash (TA) 6.05%. The pH of the maize fodder was 6.05 and its water-soluble carbohydrate content was 12.73%. The epiphytic LAB count in the maize fodder was 5.99 log10 CFU/g, while the yeast and mould count 3.20 log10 CFU/g.

Fermentation parameters of maize silage treated with varying additive combinations

The fermentation parameters of maize silage treated with various additive combinations were assessed after 30 days of ensiling (Table 2). The pH value showed a significant decrease (p<0.05) and lactic acid concentration increased in all treatment groups compared to the control. The control group exhibited the lowest lactic acid content (6.23%), whereas the highest (7.63%) was observed in maize silage treated with LP+C+X inoculation. Overall, the additives improved fermentation characteristics, notably pH and lactic acid levels. The acetic acid content was notably higher in silages treated with LF and their respective combinations than those treated with homofermentative bacterial inoculant and the control. Silages inoculated with heterofermentative bacteria and their combinations (LF, LF+C, LF+X, LF+C+X) exhibited elevated concentrations of acetic acid and a lower ratio of lactic acid to acetic acid compared to the homofermentative group and the control.

Effect of additives on microbial count

Figure 1 illustrates the variations in lactobacilli and yeastmould counts in maize silage. The count of lactic acid bacteria was significantly higher (p<0.05) in additive-treated maize silage compared to the control. Yeast and mould counts ranged from 2.86 to 4.97 log10CFU/g. The lowest count was found in LF+C+X treated silage (2.86 log10CFU/g). 3 presents the modified fitness value, flieg point and dry matter recovery (DMR) of maize silage. Compared to the control, all additive-treated silages showed significantly higher values (p<0.05) for modified fitness, flieg point and DMR. The highest modified fitness value was observed in LP+X+C (0.1072), while the lowest was in the control (0.0909). The modified fitness value is influenced by pH, dry matter loss and ammonia nitrogen content of the silage. The modified fitness value is indicative of the efficacy of silage additives. Flieg points of all silages were significantly higher (p<0.05) than the control, although there were no significant differences among the treatments. The flieg point is determined by the pH and dry matter content of the silage. The dry matter recovery of maize silage ranged from 85.85% (control) to 88.79% (LP+C+X). This indicates that the additives used in this study effectively increased dry matter recovery. Treatment with inoculants containing LF led to lower dry matter recovery (DMR). This could be attributed to more extensive hetero-lactic fermentation and increased CO2 production. Figure 2 illustrates the yeast counts in additive-treated maize silage during aerobic exposure. Silage inoculated with LF and respective combinations exhibited the lowest yeast counts, correlating with higher aerobic stability. Yeast growth was significantly higher (p<0.05) in LP-treated silage, followed by the control and LF-inoculated silage. Figure 3 presents the mould counts (log10CFU/g) of additive-treated maize silage over different aerobic exposure days. (2017). Johansson (2011) [23] suggests that a dry matter content of less than 30% increases the risk of bacterial and fungal spoilage. The chemical composition of the maize fodder aligns with findings from previous studies (Arriola et al., 2011) [4] . The pH and water-soluble carbohydrate levels observed were consistent with reported ranges for maize fodder.

The epiphytic LAB count in the maize fodder (5.99 log10 CFU/g) aligns with findings by Contreras-Govea et al. (2013). In contrast to our results, Hrafner et al. (2015) reported higher counts of epiphytic lactic acid bacteria, specifically 7.04 log10 CFU/g and yeast counts of 5.77 log10 CFU/g. Addah et al. (2011) [2] observed 8.57 log10 CFU/g of Lactobacillus counts in corn silage. According to Lin et al. (1992), the abundance of epiphytic LABs on fresh plants varies widely, ranging from less than 10 CFU/g to 10 4 CFU/g, influenced by factors such as crop species, climate, maturity stage and chopping method.

The results showing lower pH and increased lactic acid concentration in inoculated silages are consistent with previous studies, investigating bacterial inoculants and cellulase enzyme in sorghum forage silage, observed similar effects with reduced pH and higher lactic acid content compared to the control. Similarly, Sucu and Filya (2006) [46] noted elevated lactic acid levels and lower pH in corn silage treated with additives. Acosta et al. (2012) [1] found that inoculating whole maize fodder at ensiling with a commercial additive containing both homo and heterofermentative lactic acid bacteria resulted in significantly improved fermentation quality, characterized by lower pH and increased lactic acid concentration compared to untreated silage. The finding of higher concentrations of acetic acid and a lower ratio of lactic acid to acetic acid in silage inoculated with the homofermentative group heterofermentative bacteria. This observation aligns with previous studies. Hashemzadeh-Cigari et al. (2013) [12] found a higher lactic acid to acetic acid ratio in alfalfa silage treated with a combination of homofermentative and propionate-producing bacterial inoculants. Similarly, Kleinschmit and Kung (2006) [25] reported that silage treated with L. buchneri had increased acetic acid concentrations and a decreased lactic acid to acetic acid ratio compared to untreated silage. This effect is likely due to the conversion of lactic acid to acetic acid, along with the production of 1, 2-propanediol and small amounts of ethanol by heterofermentative bacteria.

According to Kung and Shaver (2001), an ideal lactic acid to acetic acid ratio should not be less than 3:1, with a higher ratio being preferable. These finding are consistent with Cai et al. (1999) [7] and Kumari et al. (2023) [27] , who concluded that inoculating silage with homofermentative lactic acid bacteria (LAB) has beneficial effects by promoting LAB growth. In the present study, LF inoculants were more effective in reducing yeast growth compared to LP, likely due to their higher acetate production. Acetic acid is known to inhibit yeasts responsible for aerobic spoilage, possibly due to its antifungal properties. Additionally, Danner et al. (2003) [13] identified that acetate exhibits antimicrobial properties against undesirable microbes. The reduction in yeast population by chemical additives may occur through mechanisms such as altering membrane functions or inducing cytosolic acidification.

The modified fitness value is influenced by pH, dry matter loss and ammonia nitrogen content of the silage. The modified fitness value is indicative of the efficacy of silage additives. The flieg point is determined by the pH and dry matter content of the silage. Kilic (1986) [24] introduced the rapid assessment method (flieg points) for quality evaluation based on dry matter and pH. According to this method, silage is categorized as very high quality if it scores between 81 and 100 on the flieg point scale. Based on flieg point assessment, all the silages in this study were classified as very good quality silage. Additives used in this study effectively increased dry matter recovery. Treatment with inoculants containing LF led to lower dry matter recovery (DMR). This could be attributed to more extensive heterolactic fermentation and increased CO2 production. Heterolactic bacteria convert lactic acid and carbohydrates into acetic and propionic acids, which contributes to dry matter losses characterized by CO2 production (Filya, 2003) [19] . Similarly, studies by Arriola et al. (2021) [5] have reported that inoculants containing L. buchneri resulted in lower DMR compared to untreated silage. Typically, these losses range between 2% and 4% the predominant bacterial species and fermentable substrates play crucial roles in fermentation-related dry matter losses. [30] . Silage treated with homofermentative bacterial inoculants exhibited lower aerobic stability compared to both the control and silage treated with heterofermentative bacteria. These findings are consistent with those of Filya and Succu (2007) [20] , which could be attributed to homofermentative bacteria-treated silage containing higher levels of lactic acid and lower levels of acetic acid compared to silage treated with heterofermentative bacterial inoculants. Yeasts and moulds utilize lactic acid as a substrate, converting it into CO2 and other byproducts, which accelerates the increase in silage pH. Yeasts are commonly recognized as the primary initiators of aerobic deterioration in silage. The reduced aerobic stability observed in silages inoculated with LP may be attributed to higher yeast numbers. Additionally, LP inoculation often leads to lower levels of acetic acid production, which can accelerate yeast proliferation and diminish aerobic stability. According to Carvalho et al. (2015) [8] , yeasts such as Candida spp., Hansenula spp., Pichia spp., Issatchenkia spp., and Saccharomyces spp. are primarily responsible for aerobic deterioration of silage by assimilating lactate. The aerobic stability of silage can be compromised if yeast counts exceed 6 log10 CFU/g of silage. In contrast, the use of heterofermentative lactic acid bacteria (LAB) during ensiling enhances acetate concentration, thereby improving aerobic stability (Danner et al., 2003; Filya & Sucu, 2007) [13, 20] . Consistent with previous studies, higher acetate levels in LF-inoculated silages resulted in lower yeast counts compared to LP-inoculated silages (Ranjit & Kung, 2000) [

. The increased rate of deterioration observed in silages treated solely with homolactic acid-producing LAB is attributed to the fermentation process that limits the accumulation of compounds such as acetic acid, which possesses antifungal properties. Undissociated acetic acid, along with other short-chain fatty acids, is effective in suppressing the growth of yeasts and moulds, whereas lactic acid alone is less effective against these organisms that initiate aerobic degradation (Danner et al., 2003 [13] . According to Chauhan et al. (2022), if the mould count reaches 7 log10 CFU/g of silage, it indicates reduced nutritional quality and signifies aerobic deterioration of the silage.

Conclusion

The additives effectively improved the maize silage fermentation parameters and quality attributes after aerobic exposure. Among the treatments, combination of LP+C+X was effective in improving the fermentation quality of silage, while the combination of LF and their respective has shown potential in maintaining quality of maize silage in terms of pH and yeast and mould count after aerobic exposure.

Acknowledgement

AcknowledgmentsThe authors thank the Director, National Dairy Research Institute, Karnal, India, for all the necessary facilities provided during the study.

References

1. Acosta Aragón Y, Jatkauskas J, Vrotniakienė V. The effect of a silage inoculant on silage quality, aerobic stability, and meat production on farm scale. International Scholarly Research Notices. 2012;2012(1):345927. https://doi.org/10.5402/2012/345927
2. Addah W, Baah J, Groenewegen P, Okine EK, McAllister TA. Comparison of the fermentation characteristics, aerobic stability and nutritive value of barley and corn silages ensiled with or without a mixed bacterial inoculant. Canadian Journal of Animal Science. 2011;91(1):133-46. https://doi.org/10.4141/CJAS10071
3. Arriola KG, Kim SC, Adesogan AT. Effect of applying inoculants with heterolactic or homolactic and heterolactic bacteria on the fermentation and quality of corn silage. Journal of Dairy Science. 2011 Mar 1;94(3):1511-1516. https://doi.org/10.3168/jds.2010-3807
4. Arriola KG, Oliveira AS, Jiang Y, Kim D, Silva HM, Kim SC, et al, Cervantes AA, Ferraretto LF. Metaanalysis of effects of inoculation with Lactobacillus buchneri, with or without other bacteria, on silage fermentation, aerobic stability, and performance of dairy cows. Journal of Dairy Science. 2021 Jul 1;104(7):7653-7670. https://doi.org/10.3168/jds.2020-19647
5. Barnett AJ. The colorimetric determination of lactic acid in silage. Biochemical Journal. 1951 Sep;49(4):527. https://doi.org/10.1042/bj0490527
6. Cai Y, Benno Y, Ogawa M, Kumai S. Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics and aerobic deterioration of silage. Journal of Dairy Science. 1999 Mar 1;82(3):520-526. https://doi.org/10.3168/jds.S0022-0302(99)75263-X
7. Carvalho BF, Ávila CL, Miguel MG, Pinto JC, Santos MC, et al. Aerobic stability of sugar-cane silage inoculated with tropical strains of lactic acid bacteria. Grass and Forage Science. 2015 Jun;70(2):308-323. https://doi.org/10.1111/gfs.12117
8. Chauhan N, Kumari N, Mishra DB, Mani V, Tyagi N. Dynamic changes in microbial succession and fermentation profiles of sugarcane tops silage treated with exogenous enzymes and lactic acid bacteria following various duration of ensiling. Sugar Tech. 2023 Jun;25(3):592-602. https://doi.org/10.1007/s12355-022-01205-4
9. Chauhan N, Singh D, Kumari N, Tyagi N. Impact of lactic acid bacteria and enzymes on the fermentation processes of sugarcane tops silage at a particular time interval. The Pharma Innovation Journal. 2021;10(2):332-336. https://doi.org/10.22271/tpi.2021.v10.i2e.5684
10. Contreras-Govea FE, Muck RE, Broderick GA, Weimer PJ. Lactobacillus plantarum effects on silage fermentation and in vitro microbial yield. Animal Feed Science and Technology. 2013 Jan 31;179(1-4):61-68. https://doi.org/10.1016/j.anifeedsci.2012.11.008
11. da Silva ÉB, Savage RM, Biddle AS, Polukis SA, Smith ML, Kung Jr L. Effects of a chemical additive on the fermentation, microbial communities, and aerobic stability of corn silage with or without air stress during storage. Journal of Animal Science. 2020 Aug;98(8). https://doi.org/10.1093/jas/skaa246
12. Danner H, Holzer M, Mayrhuber E, Braun R. Acetic acid increases stability of silage under aerobic conditions. Applied and Environmental Microbiology. 2003 Jan;69(1):562-7. https://doi.org/10.1128/AEM.69.1.562-567.2003
13. Davies ZS, Gilbert RJ, Merry RJ, Kell DB, Theodorou MK, Griffith GW. Efficient improvement of silage additives by using genetic algorithms. Applied and Environmental Microbiology. 2000 Apr 1;66(4):143543. https://doi.org/10.1128/AEM.66.4.1435-1443.2000
14. de Man JD, Rogosa D, Sharpe ME. A medium for the cultivation of lactobacilli. Journal of Applied Microbiology. 1960 Apr 1;23(1):130-135.
15. Dean DB, Adesogan AT, Krueger N, Littell RC. Effect of fibrolytic enzymes on the fermentation characteristics, aerobic stability and digestibility of bermudagrass silage. Journal of Dairy Science. 2005 Mar 1;88(3):994-1003.
16. Dolci P, Tabacco E, Cocolin L, Borreani G. Microbial dynamics during aerobic exposure of corn silage stored under oxygen barrier or polyethylene films. Applied and Environmental Microbiology. 2011 Nov 1;77(21):7499-507. https://doi.org/10.1128/AEM.05050-11
17. Duncan DB. Multiple range and multiple F tests. Biometrics. 1955 Mar 1;11(1):1-42.
18. Filya I. The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability and ruminal degradability of low dry matter corn and sorghum silages. Journal of Dairy Science. 2003 Nov 1;86(11):3575-81. https://doi.org/10.3168/jds.S0022-0302(03)73963-0
19. Filya I, Sucu E. The effect of bacterial inoculants and a chemical preservative on the fermentation and aerobic stability of whole-crop cereal silages. AsianAustralasian Journal of Animal Sciences. 2007;20(3):378-84. https://doi.org/10.5713/ajas.2007.378
20. Hafner SD, Windle M, Merrill C, Smith ML, Franco RB, Kung Jr L. Effects of potassium sorbate and Lactobacillus plantarum MTD1 on production of ethanol and other volatile organic compounds in corn silage. Animal Feed Science and Technology. 2015 Oct 1;208:79-85. https://doi.org/10.1016/j.anifeedsci.2015.07.007
21. Hashemzadeh-Cigari F, Khorvash M, Ghorbani GR, Ghasemi E, Taghizadeh A, Kargar S, et al. Interactive effects of molasses by homofermentative and heterofermentative inoculants on fermentation quality, nitrogen fractionation, nutritive value and aerobic stability of wilted alfalfa (Medicago sativa L) silage. Journal of Animal Physiology and Animal Nutrition. 2014 Apr;98(2):290-299. https://doi.org/10.1111/jpn.12079
22. Kilic A. Silo feed (instruction, education and application proposals). Bilgehan Pres. 1986;327.
23. Kleinschmit DH, Kung Jr L. A meta-analysis of the effects of Lactobacillus buchneri on the fermentation and aerobic stability of corn and grass and small-grain silages. Journal of Dairy Science. 2006 Oct 1;89(10):4005-4013. https://doi.org/10.3168/jds.S00220302(06)72444-4
24. Koc F, Coskuntuna L, Ozduven L. The effect of bacteria+ enzyme mixture silage inoculant on the fermentation characteristic, cell wall contents and aerobic stabilities of maize silage. Pakistan Journal of Animal Science. 2008 Jun 18;7(2):222-226.
25. Kumari N, Chauhan N, Mishra DB, Tyagi N. Effect of bacterial inoculants and their combination with enzymes and chemical additives on fermentation characteristics and ensiling period of maize silage. Range Management and Agroforestry. 2023;44(1):167174.
26. Kung Jr L, Taylor CC, Lynch MP, Neylon JM. The effect of treating alfalfa with Lactobacillus buchneri 40788 on silage fermentation, aerobic stability and nutritive value for lactating dairy cows. Journal of Dairy Science. 2003 Jan 1;86(1):336-343. https://doi.org/10.3168/jds.S0022-0302(03)73613-0
27. Li Y, Nishino N. Effects of inoculating Lactobacillus buchneri at ensiling with or without subsequent exposure to air on the fermentation products, aerobic stability, and microbial communities of total mixed ration silage. Journal of Dairy Science. 2011 Oct 1;94(10):5328-41. https://doi.org/10.3168/jds.2011-4424
28. McDonald P, Henderson AR, Heron SJ. The biochemistry of silage. Chalcombe publications; 1991. p. 340.
29. Nishino N, Harada H, Sakaguchi E. Evaluation of fermentation and aerobic stability of wet brewers' grains ensiled alone or in combination with various feeds as a total mixed ration. Journal of the Science of Food and Agriculture. 2003 Jan 15;83(6):557-563. https://doi.org/10.1002/jsfa.1376
30. Nishino N, Yoshida M, Shiota H, Sakaguchi E. Accumulation of 1, 2-propanediol and 1-propanol in alfalfa silage inoculated with Lactobacillus buchneri. Applied and Environmental Microbiology. 2003 Feb;69(2):1342-1347. https://doi.org/10.1128/AEM.69.2.1342-1347.2003
31. Nocek JE, Kautz WP. Direct-fed microbial supplementation on ruminal digestion, health, and performance of pre and postpartum dairy cattle. Journal of Dairy Science. 2006 Jan 1;89(1):260-266. https://doi.org/10.3168/jds.S0022-0302(06)72090-2
32. Nsereko VL, Smiley BK, Rutherford WM, Spielbauer A, Forrester KJ, Hettinger GH, et al. Influence of inoculating forage with lactic acid bacteria at ensiling on ensilage process and ensilage quality. Animal Feed Science and Technology. 2008 Apr 14;145(1-4):122135. https://doi.org/10.1016/j.anifeedsci.2007.05.011
33. Oude Elferink SJ, Driehuis F, Gottschal JC, Spoelstra SF. Silage fermentation processes and their manipulation. FAO Plant Production and Protection Papers. 2000 Jan 1;170:17-30.
34. Pauly T, Sørensen J. Bacterial inoculants for improving aerobic stability of whole-crop cereal silage. Grass and Forage Science. 2008 Mar;63(1):1-21. https://doi.org/10.1111/j.1365-2494.2007.00611.x
35. Pires AJ, Carvalho GG, Silva FF, Santos EM, Sampaio CB, Souza DR, Tosto MS. Performance of dairy heifers fed sugarcane silage and concentrate. Revista Brasileira de Zootecnia. 2010 Sep;39:2072-2077. https://doi.org/10.1590/S1516-35982010000900021
36. Ranjit NK, Kung Jr L. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage. Journal of Dairy Science. 2000 Mar 1;83(3):526-35. https://doi.org/10.3168/jds.S0022-0302(00)74911-X
37. Ren H, Guo X, Zhang T, Kang J, Zhang Y, Yang F, et al. Research on the nutritive value and fermentation quality of alfalfa silage treated with lactic acid bacteria and cellulase. Pakistan Journal of Botany. 2014 Dec 1;46(6):2197-2201.
38. Santos MC, Nussio LG, Mari LJ, Rotger A, Ipharraguerre IR. Production, composition, and ruminal degradability of volatile fatty acids in dairy cows fed silages treated with microbial inoculants or chemical additives. Journal of Dairy Science. 2010 Nov 1;93(11):5079-89. https://doi.org/10.3168/jds.2010-3133
39. Saricicek BZ, Kilic U. Effect of homolactic and heterolactic inoculants on fermentation characteristics and aerobic stability of whole-plant corn silage. Cuban Journal of Agricultural Science. 2011;45(4):391-398.
40. Schmidt RJ, Hu W, Mills JA, Kung Jr L. The development of lactic acid bacteria and Lactobacillus buchneri during the ensiling of corn silage treated with and without a silage inoculant. Journal of Applied Microbiology. 2009 Feb;107(2):363-71. https://doi.org/10.1111/j.1365-2672.2009.04214.x
41. Sucu E, Filya I. Effect of lactic acid bacteria and chemical additive on fermentation characteristics and aerobic stability of sorghum silage. Journal of Animal Science. 2006 Oct 1;84(10):3354-62. https://doi.org/10.2527/jas.2006-057
42. Tager LR, Krause KM, Grant RJ, Beckett JL. Effect of molasses-based liquid feed supplements on ruminal fermentation and diet digestibility. Journal of Dairy Science. 2010 Apr 1;93(4):1592-1601. https://doi.org/10.3168/jds.2009-2738
43. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science. 1991 Oct 1;74(10):3583-97. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
44. Vissers MM, Driehuis F, Te Giffel MC, De Jong P, Lankveld JM. Concentrations of butyric acid bacteria spores in silage and relationships with aerobic deterioration. Journal of Dairy Science. 2007 Mar 1;90(2):928-36. https://doi.org/10.3168/jds.S0022-0302(07)71576-9
45. Weinberg ZG, Chen Y, Gamburg M, Yosef E, Nikbahat M, Miron J, et al. The effect of a lactic acid bacterium inoculant applied at ensiling, on the fermentation and aerobic stability of wheat and sorghum silages. Journal of Applied Microbiology. 2017 May;106(2):395-401. https://doi.org/10.1111/j.1365-2672.2008.04008.x