A G R I C U LT U R A L A N D F O O D S C I E N C E Agricultural and Food Science (2022) 31: 187–197 187 https://doi.org/10.23986/afsci.115381 Effect of inoculants of different composition on the quality of rye silages harvested at different stages of maturity Jonas Jatkauskas1, Vilma Vrotniakiene1, Ivan Eisner2, Kristian K. Witt2 and Giuseppe Copani2 1Institute of Animal Science of the Lithuanian University of Health Sciences, Baisogala, Lithuania 2Chr. Hansen Animal Health and Nutrition, Hørsholm, Denmark e-mail: jonas.jatkauskas@lsmuni.lt Winter rye (Secale cereale L.), one of the small-grain winter annuals, can be used as a cover crop for protection against soil erosion for absorption of unused soil nitrogen, and for cattle feed by preserving as silage. The experi- ment was conducted with the objective to evaluate the potential of the blend of homofermentative and hetero- and homofermentative lactic acid bacteria (LAB) as a rye silage additive. Early-cut rye (at boot stage, wilted) and whole-crop rye (at milk and soft dough stages of grain) were ensiled in laboratory mini-silos with (1) a blend of ho- mofermentative LAB strains containing Lactobacillus plantarum (DSM26571), Enterococcus faecium (DSM22502), and Lactococcus lactis (NCIMB30117), (2) a blend of hetero- and homofermentative LAB strains containing Lacto- bacillus plantarum (DSM26571), Enterococcus faecium (DSM22502), and Lactobacillus buchneri (DSM22501), or (3) a blend of hetero- and homofermentative LAB strains containing Lactobacillus buchneri (DSM22501) and Lactococ- cus lactis (DSM11037). They were compared to ensiling without additive. After 60 days of fermentation at room temperature, mini-silos were opened, sampled for proximate analysis, forage hygiene, fermentation profile, and subjected to an aerobic stability (AS) test. Although the addition of homofermentative LAB strains was effective in reducing fermentation losses, it impaired the aerobic stability of rye silages. The combination of hetero- and ho- mofermentative LAB strains was effective in reducing the aerobic deterioration of the rye silages by supporting a low pH value and inhibiting the proliferation of yeast and moulds. Key words: aerobic spoilage, additives, bacteria strains, Secale cereale, dry matter loss Introduction Cover crops or double-cropping, used to protect against soil erosion, is becoming increasingly important. Imple- mentation of double-cropping is generally recognised as an accessible in-field method for reducing N leaching from fields by 40–70% compared to winter-bare fallow (Tonitto et al. 2006). Cover crops add organic matter to the soil, reduce the nitrate loss to groundwater and surface water, reduce soil erosion, suppress weeds, and keep valuable nutrients in the soil, hereby increasing the sustainability and reducing the environmental impact of the production system (Everett et al. 2019). Harvested at the boot (early-cut) or at the soft dough (whole-crop) stag- es of grain, double-cropped winter annuals can increase total dry matter (DM) yield per hectare up to 33% com- pared with a single crop of corn (Jemison et al. 2012). Moreover, this technique can decrease N leaching, reduce the phosphorus loss, and increase income over feed cost (Ranck et al. 2020). Like other small grain winter annuals (wheat, triticale, and barley), whole-crop rye can be a valuable forage for silage production; it was found to be a good forage source for many ruminants (Kennelly and Weinberg 2003) and could be fed to lactating dairy cows (Harper et al. 2017). Auerbach and Theobald (2020) showed poor fermentation quality in early-cut whole plant rye reflected by a high content of acetic acid, butyric acid, and ammonia-N despite the high content of water-sol- uble carbohydrates at harvest. According to Weissbach and Honig (1996), a lack of nitrate in whole-crop cereals (milk stage or later) may explain the fermentation of butyric acid despite high fermentability coefficients (FC). Because of the hollow nature of the stem and high porosity, whole-crop rye silages are prone to aerobic dete- rioration after silo opening. Application of the right silage inoculant can facilitate a successful fermentation pro- cess and inhibit aerobic deterioration during feed out (Wilkinson and Muck 2019). Generally, LAB in inoculants can be either homofermentative such as Lactobacillus plantarum, Enterococcus faecium, and various Pediococcus species that mainly convert crop sugars into lactic acid leading to a rapid decrease in silage pH, or heterofer- mentative such as Lactobacillus buchneri and other species capable of converting crop sugars and lactic acid into acetic acid and 1,2-propanediol during silo storage and increase the aerobic stability of the silages. Lactobacillus brevis is an obligate heterofermentative LAB species that produces acetate from sugar as well as supports aero- bic stability. Using inoculants that combine a mixture of homo- and hetero LAB should contribute to fast reduc- tion of pH, control of the early active fermentation period by suppressing enterobacteria, clostridia, and other Received 14 March 2022 / Accepted 8 August 2022 The Scientific Agricultural Society of Finland ©This is an open access article under the CC BY 4.012 J. Jatkauskas et al. 188 microorganisms thus reducing proteolysis, the DM losses caused by fermentation, and improvement of the aerobic stability after the active silage fermentation period (Oliveira et al. 2017, Muck et al. 2018). Moreover, inhibiting the growth of undesirable microorganisms in silage (fungi, most of the yeasts, and some groups of bacteria) is an important aspect of using combined hetero- and homofermentative LAB (Auerbach and Theobald 2020). There are only few published studies (Lee et al. 2004, Auerbach et al. 2020, Auerbach and Theobald 2020, Paradhipta et al. 2020) on the fermentation pattern, DM losses during fermentation, and the aerobic stability of early-cut and whole-crop rye harvested at different stages of maturity. The experiment was motivated by the lack of informa- tion and evidence on the preservation of early-cut and whole-crop rye by fermentation as silage and the use of an additive containing a combination of LAB strains in the literature. Our hypothesis was that due to different behaviour of the strains used in combined LAB inoculants, treatment with an inoculant could differently affect the fermentation characteristics, dry matter loss, the microbial popula- tion, and the aerobic stability of early-cut or whole-crop rye silage ensiled in a mini-silo. Materials and methods The ensiling procedure Rye (Secale cereale L.) was harvested at three stages of maturity from the same field and location: (1) the ear- ly-cut harvest (slightly wilted, 24 h before ensiling) was reaped on 10 May 2019, when forage contained 273.6 g kg-1 DM, (2) the whole-crop harvest (milk stage of grain) was brought in on 19 June 2019, when forage contained 390 g kg-1 DM, and (3) the whole-crop harvest (soft dough stage of grain) was gathered on 26 June 2019, when forage contained 457 g kg-1 DM. To avoid contamination with soil, forages were harvested by hand at the height of 10 cm and chopped to about 2 cm using a laboratory-type chopper. Prior to ensiling, rye forage was separated into four piles, and four additive treatments for each maturity stage were applied: control (T0) with no additive (only tap water added) and three commercial (Chr. Hansen A/S, Denmark) LAB inoculants: (1) a homofermentative SiloSolve® MC (T1) containing Lactobacillus plantarum (DSM26571), Enterococcus faecium (DSM22502), and Lac- tococcus lactis (NCIMB30117), at the proportion 40:30:30, (2) a hetero- and homofermentative SiloSolve® AS200 (T2) containing Lactobacillus plantarum (DSM26571), Enterococcus faecium (DSM22502), and Lactobacillus buch- neri (DSM22501), at the proportion 20:30:50, and (3) a hetero- and homofermentative SiloSolve® FC (T3) contain- ing Lactobacillus buchneri (DSM22501) and Lactococcus lactis (DSM11037), at the proportion 50:50. The products were applied at the dose of 150 000 CFU g-1 forage. The application rate of the inoculant was calculated according to the target dose and the actual bacterial concentration in the products. Additive-treated carefully mixed forage was weighed for each mini-silo and packed tightly into 3-l glass jars by hand with periodic tamping. Each jar was filled with 2000 ± 52 g, 1600 ± 56 g, and 1500 ± 54 g, or at density 184 ± 4 kg/m3, 213 ± 7 kg/m3, and 239213 ± 9 kg/m3 of rye forage at early-cut (wilted) and whole-crop (milk and soft dough stages of grain), respectively. Ten experimental silages were prepared for each vegetation stage and for each treatment: five for the chemical and microbiological analyses and five for the aerobic stability test after the targeted fermentation period. Silages were stored for 60 days in a dark room at an ambient temperature (20–22 °C). The fermentation period of 60 days was chosen based on the study of Kleinschmit and Kung (2006). In this study, the positive effect of L. buchneri on the aerobic stability of silages was reported after 56 days of storage. Samples of the water and suspension used for inoculation were collected, and the total number of LAB was counted immediately (within an hour) using the ISO method 15214. The analysis was consistent with the expected number of LAB in the suspension (Table 1). Sampling and measurements For each vegetation stage, five samples representing the fresh material prior to ensiling were randomly obtained from the chopped rye herbage. Forage samples were subjected to chemical and microbiological analyses. After 60 days of ensiling, five mini-silos per treatment were opened for the analyses of the nutrient composition and Table 1. The number of lactic acid bacteria in the inoculum suspension Rye Date T0 T1 T2 T3 Early-cut (wilted) 10 May 2019 <1.0 × 10 1.5 × 108 1.5 × 108 1.5 × 108 Whole-crop (milk stage) 19 June 2019 <1.0 × 10 1.5 × 108 1.5 × 108 1.5 × 108 Whole-crop (soft dough stage) 26 June 2019 <1.0 × 10 1.5 × 108 1.5 × 108 1.5 × 108 Agricultural and Food Science (2022) 31: 187–197 189 fermentation quality. Five mini-silos represented five repetitions. For the aerobic stability test, the other five mini-silos per vegetation stage and per treatment were used, where five mini-silos represented five repetitions. The temperature was measured by using data loggers that recorded temperature readings every six hours from the thermocouple wires placed in five replicates, 1200 g silage representative samples aerated in open polysty- rene boxes. A thermocouple wire was inserted into the geometric centre of the silo. The boxes were kept in a con- stant room temperature (20 ± 0.5 °C). Aerobic stability was considered lost when the temperature of the ensiled material exceeded the temperature of the room by 3 °C. Therefore, the aerobic stability test lasted 30, 14, and 15 days for early-cut (boot stage) and whole-crop rye (milk and soft dough stages), respectively. At the end of the aerobic stability test, losses during exposure to air were calculated based on the silage weight before and after the aerobic stability test. The increase in temperature, the change in the pH value, the DM content, the fresh weight loss, and the numbers of yeasts and moulds during aerobic exposure were used as indicators of aerobic spoilage. Chemical and microbiological analyses Samples of fresh rye and rye silage were processed, and DM and the nutrient content (crude protein, crude fibre, water-soluble carbohydrates (WSC), crude ash, acid detergent fibre (ADF), neutral detergent fibre (NDF), and fer- mentation products (pH, lactic acid, acetic acid, propionic acid, butyric acid, ammonia nitrogen, and alcohols) were determined as described previously by Jatkauskas et al. (2013). The dry matter (DM) content measured by oven drying was consequently corrected for volatile compounds (DM c ) and was calculated according to the method de- scribed by Weissbach (2009): DM c = DM n + (1.05 − 0.059 pH) FA + 0.08 LA + 0.77 PD + 0.87 BD +1.00 OA (g kg-1 FM) for the early-cut of rye and DM c = DM n + 0.95 FA + 0.08 LA + 0.77 PD + 1.00 OA (g kg-1 FM) for the whole-crop rye (milk and soft dough stages) grain, where: FA – fatty acids (C 2 …C 6 ), PD – 1,2-propanediol, BD – 2,3-butanediol, LA – lactic acid, and OA – other alcohols (C 2 …C 4 ). Different equations for the DM content correction were used due to the differences between early-cut and whole-crop rye (either without or with grain, respectively). Fresh weight losses during the ensiling period were determined by weighing the silos after filling and after 60 days of storage. Given the DM content of the forage and silage, the DM loss was calculated using the following equation: Dry mat- ter loss = (DM at ensiling – DM c silage) / DM at ensiling. The buffer capacity of rye was determined according to Playne and McDonald (1966). A chemical analysis of fresh forages and silages was performed in two repetitions for each five replications (mini-silages) and presented on a DM basis. Samples of rye and silage were analysed for LAB (ISO 15214:2009), yeasts, and moulds (ISO 21527-1:2008). Statistical analysis Data from the microbial counts were transformed to log 10 . The statistical analysis of the results included a com- pletely randomised design using the general linear model (GLM) procedure (SAS, 9.4) with treatment as a fixed effect for each maturity stage separately. A pair-wise comparison between LSMEANS was performed by the Tuk- ey’s test, when a significant F-test (p < 0.05) was detected. Results Characteristics of rye forage The basic nutrient content and the microbiological characteristics of rye forage prior to ensiling and before addi- tive application are presented in Table 2. Wilted early-cut (boot stage) and whole-crop (milk and dough stages) rye had a DM content of 273.6, 390.2, and 457.1 g kg-1, respectively, and it contained 238.2 g, 132.4 g, and 117.6 g WSC kg-1 DM and 99.0 g, 85.3 g, and 78.9 g crude protein kg-1 DM, respectively. The buffering capacity of rye for- age at all three vegetation stages was low (25.2–21.2). The numbers of the initial yeasts and moulds in fresh ear- ly-cut (boot stage) and whole-crop (milk and soft dough stages) forages were high, 5.26 and 4.25; 6.20 and 5.84; and 6.36 and 6.03 log CFUg-1, respectively. The number of epiphytic LAB in rye forage was low at <4 log CFU g-1. J. Jatkauskas et al. 190 Characteristics of ensiled early-cut rye (boot stage) The changes in the nutrient content and in the yeast and mould counts of silage on day 60 of storage are shown in Table 3. Corrected for volatiles, all three inoculant-treated silages (T1, T2, and T3) contained a significantly higher DM and crude protein content compared with control (T0) silage. Inoculants T1 and T2 preserved a larger amount of WSC compared with control (T0) or T3 treatment (p < 0.05). The number of LAB was significantly increased and the numbers of yeasts and moulds were significantly decreased by all three inoculant treatments. The strongest effect was observed in silages T2 and T3. Inoculant application had an effect on the silage fermentation profile (Table 4). During fermentation, the use of homofermentative LAB (T1) resulted in the highest concentration of lactic acid (p < 0.05) and the lowest concen- tration of acetic acid (p < 0.05), the lowest pH value (p < 0.05), and the lowest DM losses (p < 0.05). Application of hetero- and homofermentative LAB (T3) caused the highest concentration of acetic acid (p < 0.05) between the treatments. All three inoculant treatments suppressed the formation of ammonia-N, alcohols, and butyric acid. Table 2. Characteristics of rye forage at different stages of vegetation before ensiling Parameters Vegetation stage Early-cut (boot) Whole-crop (milk) Whole-crop (soft dough) Mean SD Mean SD Mean SD Ensiled forage density, kg/m3 184.7 4.24 213.1 7.52 238.8 8.85 DM, g kg-1 273.6 2.73 390.2 1.73 457.1 2.82 In DM, g kg-1 Crude protein 99.0 3.04 85.3 2.63 78.9 2.10 Crude fat 23.7 1.18 19.9 2.35 20.4 1.36 WSC 238.2 13.48 132.4 11.99 117.6 6.56 ADF 233.8 11.85 379.1 19.79 335.4 11.82 NDF 431.7 9.76 612.6 16.86 554.1 16.84 Starch – – 37.2 6.22 101.6 6.73 pH 5.73 0.06 5.47 0.07 5.44 0.10 BC, meq 100 g-1 DM 25.2 1.15 22.8 0.97 21.2 1.40 Yeast, log 10 CFU g-1 5.26 0.27 6.20 0.40 6.36 0.33 Mould, log 10 CFU g-1 4.25 0.34 5.84 0.27 6.03 0.14 LAB, log 10 CFU g-1 1.79 0.31 3.25 0.21 3.27 0.20 DM = dry matter; WSC = water soluble carbohydrates; ADF = acid detergent fibre; NDF = neutral detergent fibre; BC = buffer capacity; FC = fermentation coefficient; CFU = colony-forming units; LAB = lactic acid bacteria; SD = standard deviation TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; CP = crude protein; CF = crude fibre; WSC –=water-soluble carbohydrates; ADF = acid detergent fibre; NDF = neutral detergent fibre, CFU = colony- forming units; FF = fresh forage; LAB = lactic acid bacteria. Means with different superscripts within columns differed significantly at p< 0.05. Table 3. Nutrient composition and microbial characteristics of early-cut (boot stage) rye silage TR DM c 1, g kg-1 g kg-1 DM c Log 10 CFU g-1 of FF CP CF WSC ADF NDF LAB Yeast Mould T0 252.8c 96.3a 201.7 42.3c 260.5a 455.1a 5.38b 1.88a 2.19a T1 261.6b 99.9b 195.4 100.0b 255.5a 448.0a 7.13a 1.37b 1.78b T2 259.1b 99.9b 198.1 89.4b 257.5a 451.9a 7.31a 1.12c 1.55c T3 261.4b 98.8b 199.3 50.1c 254.4a 452.6a 7.46a 1.06c 1.48c SEM 1.320 0.973 3.882 4.536 2.072 3.251 0.22 0.06 0.06 Agricultural and Food Science (2022) 31: 187–197 191 The aerobic deterioration of silages was evaluated by observing temperature dynamics inside the silages, the pH value, and the number of yeasts and moulds at the end of the aerobic stability test (Table 5, Fig. 1). Untreated (T0) and homofermentative LAB-treated (T1) silages started deteriorating after 120 and 98 hours, respectively, of aerobic exposure and peaked on the pH value, fresh weight loss, and the number of yeasts and moulds at the end of the aerobic stability test when compared with T2 and T3. As expected, the application of hetero- and homofermentative LAB (T2) extended aerobic stability beyond T0 and T1 (approximately three times, 369 h) with a significantly reduced number of yeasts and moulds as well as the DM loss. The application of hetero- and homo- fermentative LAB (T3), however, extended aerobic stability approximately six times (688 h) and showed the lowest pH value, fresh weight loss, and the number of yeasts and moulds between all inoculant treatments. Table 4. Fermentation characteristics and dry matter losses of early-cut (boot stage) rye silage TR pH Ammonia-N, g kg-1 total N g kg-1 DM c LA AA Alcohols BA DM loss T0 3.91a 50.27a 52.37a 22.28a 25.94a 7.01a 93.7a T1 3.62b 32.63b 91.50b 8.23b 11.37b 0.70b 53.2b T2 3.69c 36.45c 79.91c 25.49a 12.66bc 0.74b 63.6c T3 3.74d 39.03c 73.97c 45.87c 13.74c 1.42b 57.8bc SEM 0.016 1.130 2.298 1.330 0.499 0.324 2.078 TR = treatment; T0 = control, T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; LA = lactic acid; AA = acetic acid; BA = butyric acid. Means with different superscripts within columns differed significantly at p< 0.05. Table 5. Characteristics of the aerobic stability of early-cut (boot stage) rye silage TR pH at the end of test DM, g kg-1 Weight loss, % AS1, h Highest temp., °C Log 10 CFU g-1 of FF yeast mould T0 8.09a 211.4b 10.10a 120.0c 31.5 6.86a 8.36a T1 8.48a 225.0a 9.88a 98.4c 29.7 6.75a 7.26b T2 8.05a 221.3a 8.83b 369.6b 25.6 5.73b 5.04c T3 5.56b 224.0a 3.51c 687.6a 23.0 3.95c 3.22d SEM 0.159 3.373 0.362 18.969 - 0.224 0.247 TR = treatment; T0 = control, T1 = 1.5 × 105 CFU g-1. L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1. L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SE = standard error; DM = dry matter; AS = aerobic stability; CFU = colony-forming units; FF = fresh forage; 1number of hours needed for the silage to reach a sustained temperature higher than 3 °C above ambient. Means with different superscripts within columns differed significantly at p < 0.05. 18 23 28 33 0 30 60 90 12 0 15 0 18 0 21 0 24 0 27 0 30 0 33 0 36 0 39 0 42 0 45 0 48 0 51 0 54 0 57 0 60 0 63 0 66 0 69 0 72 0 te m pe ra tu re , ° C Time of aerobic exposure, h Ambient Ambient +3 °C T0 T1 T2 T3 Fig. 1. Temperature changes in early-cut (boot stage, slightly wilted) rye silage during the aerobic exposure period (T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis) J. Jatkauskas et al. 192 Characteristics of ensiled whole-crop rye (milk stage of grain) The nutrient content and the microbial characteristics of silage at day 60 of storage are presented in Table 6. When compared with control (T0), all three inoculant-treated silages (T1, T2, and T3) contain a significantly higher DM corrected for volatiles. Inoculant T3 preserved a lower amount of WSC (p < 0.05) compared with control (T0) or T1 and T2 treatments. All three inoculant treatments significantly increased the number of LAB , and the num- ber of yeasts was significantly decreased (p < 0.05) in T2 and T3 inoculant-treated silages; the number of moulds decreased (p < 0.05) in all three inoculant treatments compared to control (T0). The strongest effect was observed in T3 silages. Inoculant application had an effect on the silage fermentation profile (Table 7). During fermentation, the use of homofermentative LAB (T1) resulted in the highest concentration of lactic acid (p < 0.05) and the lowest concen- tration of acetic acid (p < 0.05), the lowest pH value (p < 0.05), and the lowest DM losses (p < 0.05). Application of hetero- and homofermentative LAB (T3) caused the highest concentration of acetic acid (p < 0.05) between all treatments. All three inoculant treatments suppressed the formation of ammonia-N, alcohols, and butyric acid. The aerobic deterioration of silages was evaluated by observing temperature dynamics inside silage, the pH value, and the number of yeasts and moulds at the end of the aerobic stability test (Table 8, Fig. 2). Homofermenta- tive LAB-treated silage (T1) began to deteriorate only 52 h upon aerobic exposure, while it took control (T0) and hetero- and homofermentative LAB (T2) silages 168 h and 132 h, respectively, to reach a temperature of more than 3 °C above ambient. At the end of the aerobic stability test, T0 and T1 silages reached a high pH value, fresh weight loss, and the number of yeasts and moulds. Application of hetero- and homofermentative LAB (T3) maintained aerobic stability for 310 h and showed the lowest pH value, fresh weight loss, and the lowest number of yeasts and moulds between all inoculant treatments. Table 6. Nutrient composition and microbial characteristics of whole-crop (milk stage of grain) rye silage TR DM c 1, g kg-1 g kg-1 DM c Log 10 CFU g-1 of FF CP CF WSC ADF NDF LAB yeast mould T0 368.0c 75.6b 328.4b 39.3b 418.4b 576.3b 5.54a 3.07a 2.74a T1 378.5b 77.6b 317.3c 45.2b 410.2b 560.4c 6.71b 3.00a 2.03b T2 376.7b 77.2b 322.2bc 39.8b 407.2b 564.1c 7.78c 2.18b 1.70c T3 375.5b 77.8b 319.4c 21.5c 404.1b 560.1c 7.96c 1.16c 1.12d SEM 1.399 0.641 2.231 2.448 5.177 4.393 0.14 0.07 0.05 TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; CP = crude protein; CF = crude fibre; WSC = water-soluble carbohydrates; ADF = acid detergent fibre; NDF = neutral detergent fibre; CFU = colony-forming units; FF = fresh forage; LAB = lactic acid bacteria. Means with different superscripts within columns differed significantly at p < 0.05. Table 7. Fermentation characteristics and dry matter losses of whole-crop (milk stage of grain) rye silage TR pH Ammonia-N, g kg-1 total N g kg-1 DM c LA AA Alcohols BA DM loss T0 4.36a 68.68a 10.59a 9.16a 16.50a 10.58a 77.8a T1 3.66b 36.95b 54.66b 9.58a 10.27b 0.32b 39.8b T2 3.72b 35.60b 40.30c 15.32b 9.25bc 0.38b 44.9bc T3 3.86c 35.41b 33.09d 35.81c 6.99c 1.58b 49.8c SEM 4.36a 1.104 1.583 1.223 1.038 0.508 3.299 TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; LA = lactic acid; AA = acetic acid; BA = butyric acid. Means with different superscripts within columns differed significantly at p< 0.05. Agricultural and Food Science (2022) 31: 187–197 193 Characteristics of ensiled whole-crop rye (soft dough stage of grain) The nutrient content and the microbial characteristics of silage at day 60 of storage are presented in Table 9. When compared with control silage (T0), all three inoculant-treated silages (T1, T2, and T3) contain significantly high- er DM content corrected for volatiles. Inoculant T3 preserved a lower amount of WSC (p < 0.05) compared with control (T0) or T1 and T2 treatments. The number of LAB was significantly increased, and the numbers of yeasts and moulds were significantly decreased by all three inoculant treatments. The strongest effect was observed in silages T2 and T3. Table 8. Characteristics of the aerobic stability of whole-crop (milk stage of grain) rye silage TR pH DM, g kg-1 Weight loss, % AS1 h Highest temp., °C Log 10 CFU g-1 of FF yeast mould T0 6.10a 328.2a 4.77a 168.0a 29.7 6.24a 7.68a T1 7.76b 338.5b 5.35a 51.60b 32.2 6.82b 7.00b T2 7.97b 340.5b 5.18a 132.0c 30.0 5.46c 5.92c T3 5.09c 343.0b 1.71b 310.0d 23.8 3.51d 2.97d SE 0.135 2.733 0.372 8.325 - 0.165 0.080 TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM = dry matter; AS = aerobic stability; CFU = colony-forming units; FF = fresh forage; 1number of hours needed for the silage to reach a sustained temperature higher than 3 °C above ambient. Means with different superscripts within columns differed significantly at p < 0.05. 18 20 22 24 26 28 30 32 34 0 12 24 36 48 60 72 84 96 10 8 12 0 13 2 14 4 15 6 16 8 18 0 19 2 20 4 21 6 22 8 24 0 25 2 26 4 27 6 28 8 30 0 31 2 32 4 33 6 te m pe ra tu re , ° C Time of aerobic exposure, h Ambient Ambient +3 °C T0 T1 T2 T3 Fig. 2. Temperature changes in whole-crop (milk stage of grain) rye silage during aerobic exposure period (T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis) TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; CFU = colony-forming units; FF = fresh forage; CP = crude protein; CF = crude fibre; WSC = water- soluble carbohydrates; ADF = acid detergent fibre; NDF = neutral detergent fibre; LAB = lactic acid bacteria. Means with different superscripts within columns differed significantly at p < 0.05. Table 9. Nutrient composition and microbial characteristics of whole-crop (soft dough stage of grain) rye silage TR DM c 1, g kg-1 g kg-1 DM c Log 10 CFU g-1 of FF CP CF WSC ADF NDF LAB yeast mould T0 432.9b 70.3b 291.3b 23.2b 352.3b 504.7b 5.67a 3.45a 2.90a T1 446.7c 73.5c 285.9b 42.5c 346.4a 493.3b 7.16b 3.02b 2.07b T2 444.1d 71.8d 287.7b 24.9b 346.9a 502.3b 8.21c 2.89b 1.70c T3 445.9cd 70.0bd 286.3b 17.6d 344.4a 501.4b 8.58c 1.18c 1.12d SEM 0.806 1.011 3.096 1.587 5.173 8.333 0.18 0.06 0.04 J. Jatkauskas et al. 194 Inoculant application had an effect on the silage fermentation profile (Table 10). The use of homofermentative LAB (T1) and hetero- and homofermentative LAB (T2) resulted in the highest concentration of lactic acid (p < 0.05), and T1 silage showed the lowest DM loss (p < 0.05). The lowest concentration of acetic acid (p < 0.05) was detected in control (T0) and homofermentative LAB (T1) silages. Hetero- and homofermentative LAB silage (T3) caused the highest concentration of acetic acid (p < 0.05) between all treatments. All three inoculant treatments suppressed the formation of ammonia-N, alcohols, and butyric acid. The aerobic deterioration of silages was evaluated by observing temperature dynamics inside the silages, the pH value, and the number of yeasts and moulds at the end of the aerobic stability test (Table 11, Fig. 3). Homofer- mentative LAB-treated silage (T1) began to deteriorate 65 h after aerobic exposure, and it took control (T0) and hetero- and homofermentative LAB (T2) silages 176 h and 116 h, respectively, to reach a temperature of more than 3 °C above ambient. At the end of the aerobic stability test, T1 and T2 silages reached the highest pH value, fresh weight loss, and the largest number of yeasts and moulds. Application of hetero- and homofermentative LAB (T3) supported aerobic stability for almost 340 h and showed the lowest pH value, fresh weight loss, and the lowest number of yeasts and moulds between all inoculant treatments. Table 10. Fermentation characteristics and dry matter losses of whole-crop (soft dough stage of grain) rye silage TR pH Ammonia-N, g kg-1 total N g kg-1 DM c LA AA Alcohols BA DM loss T0 4.41a 51.32a 12.58a 5.89a 12.52a 6.93a 75.2a T1 3.70b 32.73b 34.57b 5.65a 8.32b 0.65b 29.7b T2 3.77c 34.97b 32.74b 10.69b 5.25c 0.71b 36.1c T3 3.96d 39.06c 7.78c 29.26c 6.78d 0.73b 36.8c SEM 0.014 1.106 1.030 0.997 0.482 0.265 1.555 TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM c = dry matter corrected for volatiles; LA = lactic acid; AA = acetic acid; BA = butyric acid. Means with different superscripts within columns differed significantly at p < 0.05. TR = treatment; T0 = control; T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis; SEM = standard error of means; DM = dry matter; AS = aerobic stability; CFU = colony-forming units; FF = fresh forage; 1number of hours needed for the silage to reach a sustained temperature higher than 3 °C above ambient. Means with different superscripts within columns differed significantly at p < 0.05. Table 11. Characteristics of the aerobic stability of whole-crop (soft dough stage of grain) rye silage TR pH DM, g kg-1 Weight loss, % AS1, h Highest temp., °C Log 10 CFU g-1 of FF yeast mould T0 6.23a 396.5a 5.29a 176.4a 29.8 7.60a 8.06a T1 7.17b 407.4b 6.44 b 64.8b 29.9 8.34a 7.26b T2 7.63c 409.7b 5.96ab 116.4c 28.2 7.71a 7.05b T3 4.32d 411.5b 2.51c 339.6d 23.3 4.78b 2.24c SEM 0.149 3.303 0.289 11.392 - 0.255 0.214 18 20 22 24 26 28 30 32 0 18 36 54 72 90 10 8 12 6 14 4 16 2 18 0 19 8 21 6 23 4 25 2 27 0 28 8 30 6 32 4 34 2 36 0 te m pe ra tu re , ° C Time of aerobic exposure, h Ambient Ambient +3 °C T0 T1 T2 T3 Fig. 3. Temperature changes in whole-crop (soft dough stage of grain) rye silage during aerobic exposure period (T0 – control, T1 = 1.5 × 105 CFU g-1 L. plantarum, E. faecium, L. lactis; T2 = 1.5 × 105 CFU g-1 L. buchneri, L. plantarum, E. faecium; T3 = 1.5 × 105 CFU g-1 L. buchneri, L. lactis) Agricultural and Food Science (2022) 31: 187–197 195 Discussion At the time of ensiling, early-cut (boot stage, slightly wilted) rye forage contained 274 g kg-1 DM and 99 g kg-1 DM crude protein and was similar to that studied by Kim et al. (2001) and Auerbach and Theobald (2020). The DM content increased with increasing maturation of rye up to 390 and 457 g kg-1 for whole-crop rye at milk and soft dough stages, respectively. The increase in the DM content during maturation was accompa- nied by a decrease in crude protein and WSC content. Micek et al. (2001) reported the same tendencies in re- lation to the stage of maturity of rye forage. However, as the DM content increased with maturation of whole- crop rye, the buffering capacity slightly decreased. On this account and regarding Auerbach and Theobald (2020), early-cut and whole-crop rye forage was considered to be a moderately easy to easy to ensile crop. The number of epiphytic LAB was low and reached only 1.79–3.27 log10 CFU g-1. The number of yeasts and moulds above 5 log10 CFU g-1 and above 4.5 log10 CFU g-1, respectively, represent typical values for the Lithuanian climatic conditions. Untreated (T0) silage underwent butyric fermentation or contained a considerable amount of butyric acid, had a high pH value, a high ammonia-N concentration (high proteolysis), and a high DM loss. This can be attributed to a slow acidification rate caused by a low number of epiphytic LAB (1.79–3.27 log10 CFU g-1) and a low nitrate level of the crop, although this parameter was not measured. These findings agree with the observations by Auerbach et al. (2013), who indicated that under these ensiling conditions, clostridia could thrive during the initial stage of fermentation. Compared to control (T0), homofermentative (T1), and hetero- and homofermentative LAB (T2) treatments resulted in a significantly higher DM content (corrected for volatiles) for early-cut and whole-crop rye silages. As expected, the WSC content in all silages was reduced during fermentation. The residual WSC content was the highest for the T1 treatment among all vegetation stages of rye indicating more effective WSC utilisation by homofermentative LAB. Inoculation with L. buchneri in combination with homofermentative LAB (T3) resulted in a lower content of residual WSC in silages. Similar results were reported by Kleinschmit and Kung (2006). The homofermentative LAB treatment (T1) produced a typical effect on silage quality at all three vegetation stages of rye: more lactic acid, less acetic acid (for early-cut rye), less ethanol, and lower proteolysis (lower ammonium-N content) compared to control (T0). A positive effect of T1 treatment was also manifested by a lower pH value of this silage and a lower content of butyric acid in it. The homofermentative LAB treatment (T1) resulted in the lowest DM losses among all inoculant treatments during the storage period. Our previous observations (Jatkauskas et al. 2018) and results reported by other authors (Basso et al. 2014, Borreani et al. 2018, Muck et al. 2018) confirm the findings of this study. According to Schmidt and Kung (2010) and Muck et al. (2018), homofermentative LAB usually promote lac- tic acid fermentation by shifting fermentation products towards lactic acid and away from ethanol and acetic acid fermentation products with a decreased DM loss as the main benefit. Homofermentative LAB-based silage additives sometimes render silages less stable when they are exposed to air, because lactic acid is not a strong antifungal agent and the production of antifungal compounds (acetic acid) in the silages is limited (Danner et al. 2003, Kung 2009). This effect was also observed in the current experiment: the aerobic stability of T1 silages treated with homofermentative LAB were clearly reduced compared to the control (T0) and the other (T2 and T3) treatments. Moreover, at the end of the aerobic stability test, T1 silages manifested the fresh weight loss and the number of yeasts and moulds close to the control (T0) silage at all three maturity stages of rye. Auerbach et al. (2013) indicated that the high residual sugar content may stimulate the extent and the rate of yeast survival and growth in the silages exposed to air and reduce aerobic stability of these silages. Irrespective of the rye crop maturity stage, hetero- and homofermentative LAB-treated silages (T2 and T3) had a higher content of lactic acid than non-inoculated (T0) silages, but lower than T1-treated silage. Muck et al. (2018) and da Silva et al. (2019) observed that heterofermentative LAB produce more acetic acid and increase the DM loss when compared with homofermentative LAB. The results of the present study are in line with these observa- tions, where T2 and T3 silages showed higher DM losses, when compared with T1 silages. The heterofermenta- tive pathway, however, is usually connected with increased fermentation losses compared to homofermentative pathway (McDonald et al. 1991). The highest content of acetic acid was detected in T3 silages. A capability of a LAB strain to increase the aerobic stability of silage is directly related to its ability to inhibit the growth of micro- organisms that deteriorate the silage and causes silage heating when it is exposed to air. Research by Driehuis et al. (2001) showed that addition of L. buchneri reduced the survival of yeasts during the anaerobic phase of silage fermentation and inhibited the growth of yeasts during exposure of silage to air. Tabacco et al. (2011) indicated the power of L. buchneri to inhibit the aerobic deterioration of silages through the fermentation of lactic acid to acetic acid and the inhibition of yeast and clostridial growth. J. Jatkauskas et al. 196 The results of our experiment show that rye silages inoculated with a blend of hetero- and homofermentative LAB (L. buchneri and L. lactis, T3) had the highest values of aerobic stability and LAB counts, the lowest fresh weight loss, and the lowest number of yeasts and moulds at mini-silo opening and at the end of aerobic exposure at all three vegetations stages. The significant decrease in the number of yeasts and moulds in T3 silage can be ascribed to the significantly highest acetic acid content in T3 silage among all inoculant treatments. Auerbach et al. (2013) and Kleinschmit and Kung (2006) reported that acetic acid can reduce the number of yeasts in silages, and the extent of this effect depends on the content of acetic acid. The growth-inhibiting effect of yeasts and moulds re- mained during the aerobic exposure phase and consequently resulted in a significantly decreased aerobic deterio- ration. Under aerobic conditions, T3-inoculated rye silages had a markedly lower pH value, fresh weight loss, and heat production than control and other inoculant treatments used at all three stages of maturity of rye. Conclusions Inoculation of early-cut and whole-crop rye harvested at milk or soft dough stages of grain with LAB blends af- fected silage fermentation properties and aerobic stability traits depending on their intended behaviour. The ho- mofermentative inoculant treatment improved the fermentation profile and reduced DM losses but impaired aerobic stability and increased fresh weight loss during the period of exposure to air. The product containing ho- moferementative Enterococcus faecium, L. plantarum, and heterofermentative L. buchneri improved the fermen- tative profile and reduced DM losses at all stages of maturity, but aerobic stability was improved only in early-cut rye silage. 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Ensiling in 2050: some challenges and opportunities. Grass and Forage Science 74: 178–187. https://doi.org/10.1111/gfs.12418 Effect of inoculants of different composition on the quality of ryesilages harvested at different stages of maturity Introduction Materials and methods The ensiling procedure Sampling and measurements Chemical and microbiological analyses Statistical analysis Results Characteristics of rye forage Characteristics of ensiled early-cut rye (boot stage) Characteristics of ensiled whole-crop rye (milk stage of grain) Characteristics of ensiled whole-crop rye (soft dough stage of grain) Discussion Conclusions References