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Article

Additive Type Affects Fermentation, Aerobic Stability and Mycotoxin Formation during Air Exposure of Early-Cut Rye (Secale cereale L.) Silage

by
Horst Auerbach
1,* and
Peter Theobald
2
1
International Silage Consultancy, Thomas-Müntzer-Strasse 12, 06193 Wettin-Löbejün, Germany
2
Faculty of Agriculture, Economics and Management, HfWU Nürtingen-Geislingen University, Neckarsteige 6-10, 72622 Nürtingen, Germany
*
Author to whom correspondence should be addressed.
Agronomy 2020, 10(9), 1432; https://doi.org/10.3390/agronomy10091432
Submission received: 28 July 2020 / Revised: 5 September 2020 / Accepted: 15 September 2020 / Published: 21 September 2020
(This article belongs to the Special Issue Potential Roles of Forage Silage in Sustainable Agricultural Production)
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Abstract

:
Whole-crop rye harvested before maturity represents a valuable forage for silage production. Due to the scarcity of data on fermentation characteristics and aerobic stability (ASTA) and the lack of information on mycotoxin formation during aeration of early-cut rye (ECR) silage after silo opening, we evaluated the effects of different additive types and compositions. Wilted forage was treated with various biological and chemical additives, ensiled in 1.5-L glass jars and stored for 64 days. Fermentation pattern, yeast and mould counts and ASTA were determined at silo opening. In total 34 mycotoxins were analysed in wilted forage and in silage before and after 240 h of air exposure. Chemical additives caused the lowest dry matter (DM) losses during fermentation accompanied with the lowest ethanol production and the highest water-soluble carbohydrate concentration. Aerobic deterioration, which started within two days after silo opening in silage left untreated and inoculated with homofermentative lactic acid bacteria, was prevented by the combined use of hetero- and homofermentative lactic acid bacteria and the chemical additive containing sodium nitrite, hexamethylene tetramine and potassium sorbate. Moreover, these two additives largely restricted the formation of the mycotoxin roquefortine C to < 0.05 mg kg−1 DM after aeration, whereas untreated silage contained 85.2 mg kg−1 DM.

1. Introduction

Early-cut winter rye (ECR, Secale cereale L.) has been widely used for decades as a silage crop in Germany, especially in the eastern regions, and hybrids specifically bred for forage production have become available. Along with triticale, rye cut early before maturity has very recently attracted attention in other European countries, e.g., Hungary [1]. Although it is also grown as cover crop between cash crops [2], it is mainly used as forage for silage production in double-cropping systems with maize [3], but cultivation as a summer catch crop has been increasing [4]. The early harvest, which at the latest takes place just before flowering, enables farmers to grow two crops per year, which is usually maize after winter rye and summer rye after cereals or rape seed, thereby increasing land productivity [3,5]. Double-cropping increases nutrient cycling within the farm and reduces the amount of imported feed reducing negative environmental impacts [3].
With increasing stages of maturity, the dry matter (DM) yield increases [1], whereas a marked decline in the nutritive value is observed by rapid plant development [4]. The optimal stage of maturity is largely determined by the dietary requirements of the animals to which ECR silage is fed. Despite typically high sugar concentrations in ECR at harvest exceeding 100 g kg−1 DM [1,6,7], fermentation quality can be poor, as reflected by acetic acid concentrations of up to 84.2 g kg−1 DM, butyric acid concentrations of up to 25.2 g kg−1 DM, and by ammonia-N levels of up to 230 g kg−1 of the total N concentration (Table 1). Analytical results on the nutritive value of ECR silage, which were produced on commercial German dairy farms and analysed by the accredited feed analysis laboratory, LKS mbH, Niederwiesa, Germany, are presented in Table 1.
To our knowledge, data on fungal contamination of fresh and ensiled ECR is very limited [6,7]. The increase in temperature caused by aerobic yeast and mould metabolism is known to cause not only losses in DM, but also to lead to indirect losses due to lower nutritive value, reduced feed intake and animal performance [8,9]. Moreover, a range of mycotoxins produced by moulds have been detected in several silage types produced under laboratory and commercial farm conditions [10,11,12,13], but no data is available on ECR. These toxic fungal metabolites may negatively affect the health of farm animals [13,14,15]. Depending on storage conditions regarding the availability of air, a succession of the mycoflora takes place, which is also affected by climatic conditions, leading to significant differences in silage mould populations between regions [16]. In temperate climates, the most prominent field fungi, e.g., Fusarium and Alternaria, disappear quickly after anaerobiosis has been reached [17], whereas other moulds survive the fermentation process and can grow and produce mycotoxin after silo opening [18]. Penicillium roqueforti (sensu lato), which consists of three distinctive species—P. roqueforti, P. carneum and P. paneum [19,20], Monascus ruber and Aspergillus fumigatus—are most commonly found in several silage types in temperate climate [10,12,21,22].
In addition to the well-known management principles of producing good silage, silage additives have been used to improve the fermentation process and to alleviate the detrimental effects of air on silage quality during the feed-out phase. Biological and chemical additives differ in their mode of action and are used depending on the desired effect, either to improve fermentation quality or aerobic stability, or both [23,24]. Although there is an overwhelming number of articles on the effects of additives used in crops like lucerne, clover, grass, maize, whole-crop cereals and small grains, systematic studies on their effects in ECR are limited [6,7], comprising a total of only five published trials testing only a small range of potential silage additive types. Therefore, the aim of our study was to deepen the knowledge on fermentation characteristics, fungal populations, aerobic stability and mycotoxin formation in silage produced from ECR stored under challenging conditions with short fermentation length and air infiltration during storage. Moreover, the effects of biological and chemical additives of different composition were evaluated to determine potential candidates ensuring consistently high silage quality under farm conditions for further investigation. We hypothesised that fermentation pattern, fungal populations, and aerobic stability (ASTA) can be altered by silage additives in accordance with their expected mode of action, and that the prevention of aerobic deterioration will result in the suppression of mycotoxin formation by moulds during feed-out.

2. Materials and Methods

2.1. Ensiling

The leafy-type rye (Secale cereale L., cv. Protector, Saatenunion, Isernhagen, Germany), which was specifically bred for forage production, was sown at 3 × 106 seeds ha−1 on 11 September 2016, on a dairy farm in Klein Schulzendorf, Germany (52.1949552 N-13.250923 W). Prior to seeding, the field received N fertilizer exclusively from cattle slurry at a rate of 60 kg ha−1. In early March 2017, additional N (80 kg N ha−1) was applied in form of a mixture of ammonium carbonate and ammonium nitrate. Rye was cut on the morning of 10 May 2017 and wilted overnight before it was chopped by a precision chopper (Krone, Big X 650) set at 30 mm theoretical particle length. Equal quantities of forage (50 kg) from three different field locations with a distance between them of about 100 m were taken and thoroughly mixed to form a composite sample to minimize potential effects of sampling location on silage variables due to different forage composition [25], which may interfere with the effects of the tested additives.
Before manual packing of the forage into 1.5-L glass jars (Weck, Öfingen, Germany), the forage was divided into five piles. After suspending and diluting, respectively, of the products with tap water, the following additives (provided by KONSIL Europe GmbH, Wettin-Löbejün, Germany) were applied at a rate of 10 mL kg−1 wilted forage: no additive (tap water, CON), homofermentative lactic acid bacteria (LABho) composed of Lactobacillus plantarum DSM 16,627 and Lactobacillus paracasei NCIMB 30,151 (total inoculation rate: 1.5 × 105 cfu g−1), combination of hetero- and homofermentative lactic acid bacteria (LABheho) composed of Lactobacillus buchneri CNCM-I 4323 and Pediococcus acidilactici DSM 11,673 (total inoculation rate: 1.67 × 105 cfu g−1), liquid chemical mixture (SNHE) containing sodium nitrite (300 g L−1) and hexamethylene tetramine (200 g L−1), applied at 2 mL kg−1, or a liquid chemical blend (SNHEPS) containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), added at 2 mL kg−1. The jars had a 6-mm hole in the lid and in the body, which were closed by rubber stoppers. Along with the intentionally low packing density of 87 kg DM m−3, the removal of the rubber stoppers on day 28 and day 57 of storage for 24 h aimed at creating challenging test conditions by allowing air to penetrate and freely circulate through the silage mass, thereby stimulating fungal development. According to Jungbluth et al. (2017), packing density affected the gas exchange, reaching the oxygen concentration of air (21%, v/v) much faster in low density than in high density silage [26]. Experimental silages were prepared in triplicate for each treatment and were stored for 64 days in a dark room, whose temperature was set at 21 °C.

2.2. Dry Matter Determination

The DM content of forage and silage was determined by oven-drying at 60 °C until a constant weight was achieved, followed by 3 h of drying at 105 °C [27]. Silage DM content was corrected for the loss of volatiles during drying [27]. The losses of DM during fermentation were calculated as described by Weissbach [28].

2.3. Chemical and Microbiological Analysis

All samples for chemical analyses (pH, ammonia-N, acids and alcohols, water-soluble carbohydrates) were stored at −18 °C until use. Nutrient composition and energy concentration were determined by the accredited laboratory LKS mbH, Niederwiesa, Lichtenwalde on oven-dried (60 °C, 24 h) samples by using the official methods for feed evaluation [29] and energy estimation [30] in Germany. Buffering capacity (BC, given in g lactic acid kg−1 DM) was analysed by titration of dried forage samples (60 °C, 24 h) with lactic acid to pH 4.0. Finally, the fermentability coefficient (FC) was calculated using the equation by Schmidt et al. [31]: FC = DM [%] + 8 × WSC/BC, where WSC is the concentration of water soluble-carbohydrates (WSC) and BC is the buffering capacity.
Extracts of fresh silage were prepared by blending 50 g of sample with 200 mL of distilled water to which 1 mL toluene was added. After storage overnight at 4 °C, the extracts were filtered through a paper filter, followed by microfiltration (0.45 µm). Volatile fatty acids and alcohols were analysed by GC with flame-ionization detection according to Weiss et al. [32], whereas lactic acid was determined by HPLC coupled with a refractive index detector [33]. Silage pH was measured potentiometrically by using a pH electrode, and a colorimetric method based on the Berthelot-reaction (CFA, Scan++, Skalar Analytical, Breda, The Netherlands) was used to determine ammonia-N concentration. The concentration of WSC (mainly composed of glucose, fructose, disaccharides and fructosans) was determined after extraction of fresh silage samples in cold water for one hour, subsequent addition of anthrone reactant (composed of anthrone, thiourea and sulphuric acid) and subsequent spectro-photometrical analysis [34].
Serial dilutions of fresh forage and silage samples were prepared in 0.1% (w/w) peptone water broth. Lactic acid bacteria (LAB) were counted by pour-plating and incubation for four days on MRS (De Man, Rogosa and Sharpe) medium at 30 °C [35]. Yeasts and moulds were enumerated by spread-plating on yeast extract-dextrose-chloramphenicol agar after incubation for three to five days at 25 °C [36]. The total fungal count was calculated by summing up the number of individual yeast and mould colonies grown on the plates.

2.4. Aerobic Stability Measurement

As described by Honig [37], the ASTA of silage was evaluated based on the temperature development in the samples compared with that of the room, which was kept at 20.6 ± 0.2 °C. Data loggers (Tinytag Talk 2, Gemini, Chichester, UK) were placed in the geometric centre of a plastic container which was loosely filled with silage. The data loggers recorded silage and room temperature at 2-h intervals. Each plastic container was stored in an insulating polystyrene box, allowing free air circulation for 240 h. Silage was considered aerobically unstable once its temperature had increased by 2 °C above ambient temperature. To assess the extent of aerobic deterioration (AD), the cumulated temperature (TCUM) was calculated by summing up the temperature differences between silage and room [38]. The original procedure was modified by changing the interval of data recording from one hour to two hours.

2.5. Mycotoxin Analysis

Silage mycotoxin analyses were performed in the fresh forage before ensiling and in the silage on the day of silo opening and after 240 h of air exposure in treatments CON, LABheho and SNHEPS only. These were selected based on the results from the test for aerobic stability ensuring large treatment differences. Silage samples were stored at −18 °C until analysis, which was carried out by RIKILT Wageningen University and Research, The Netherlands, using an accredited and validated multi-species (34 mycotoxins) LC/MS-MS method. This method can detect fungal metabolites produced by the major forage and silage moulds, including the genera Fusarium, Alternaria, Penicillium, Aspergillus. From each sample, 2.5 g were weighed in a 50 mL plastic tube and 7.5 mL of water, 10 mL of extraction solvent (acetonitrile/acetic acid, 99:1, v/v), and 25 µL of 13C-caffeine internal standard (IS) were added. The tubes were shaken manually to disperse the sediments on the bottom of the tube and put in a ‘head-over-head’ extracting device (Heidolph Reax 2, Schwabach, Germany) for 30 min. Subsequently, 4 g of magnesium sulphate were added and the tubes vortexed for 1 min, followed by centrifugation at 3000 rpm for 10 min and transfer of 200 µL of sample extract to polypropylene vials (Whatmann syringeless filter device). After the addition of 200 µL of water, each vial was capped and shaken in a vortex mixer (for approximately 3 s). After storage in the refrigerator (4 °C) for 30–60 min, the vials were closed with a pressing device (Six Position Compression; Whatman‘s-Hertogenbosch, The Netherlands) and stored at 4 °C until analysis. Mycotoxin determinations were performed using an LC-MS/MS (Waters Acquity, Etten-Leur, The Netherlands, AB SCIEX QTRAP® 6500, Applied Biosystems, Nieuwekerk aan de IJssel, The Netherlands) with a Restek Ultra Aqueous C18 3 µm 100 × 2.1 mm UPLC column (Restek Corporation, Bellefonte, USA) and a sample injection volume of 5 µL. The gradient was performed using two different eluents: eluent A consisted of 1 mM ammonium formate and 1% (v/v) formic acid in water; eluent B consisted of 1 mM ammonium formate and 1% (v/v) formic acid in a mix of methanol and water 95:5 (v/v). Eluent C consisted of 5 mM ammonium acetate and 0.1% (v/v) acetic acid in water; eluent D contained 5 mM ammonium acetate and 0.1% (v/v) acetic acid in a mix of methanol and water 95:5 (v/v). Eluent A and B were used when analysing mycotoxins in positive mode (ESI+), whereas eluent C and D were used when detecting mycotoxins in negative mode (ESI-). Each mycotoxin was identified by its retention time and the peak area ratio between two transitions. Quantification was performed by a single point standard addition protocol. Calibration curves were linear with coefficients of determination of R2 ≥ 0.994. The limit of quantification was equal to the lowest validated mycotoxin concentration and varied between mycotoxins. Average recoveries ranged between 80–120%, except those for DON-3-glucoside (73%), nivalenol (73%), citrinin (121%) and moniliformin (125%).

2.6. Statistical Analysis

All data were analysed as a completely randomized design by the Faculty of Life Sciences, Department of Biometry and Agricultural Experimentation, Humboldt Universität zu Berlin, Germany, using the software package of SAS, version 9.4, (Cary, NC, USA). Treatments were compared in a framework of a fixed effects model using silage additive as experimental factor with three replications: yij = μ + αi + εij, where yij is the observed value of the jth replication from silage additive i; µ the population mean; αi the fixed effect of silage additive i; and eij the random residual effect of the ith treatment and jth observation, ~N(0,σ2e). For silage mycotoxin analyses upon silo opening and after air exposure, a two-factorial fixed effects model with three replications was used: yijk = μ + αi + βj + (αβ)ij + εijk, where yijk is the observed value of the kth replication from silage additive i and aeration treatment j; µ the population mean; αi the fixed effect of silage additive i, βj the fixed effect of aeration treatment j, and eijk the random residual effect of the ijth treatment and jth observation, ~N(0,σ2e).
After checking the assumption of normally distributed residuals by Shapiro-Wilk-Test and graphical diagnostics, traits for which normality of observations could be assumed were subjected to ANOVA, whereas a non-parametric rank procedure using an ANOVA-type statistics was employed for non-normally distributed data (procedure MIXED). When significance was detected p < 0.05 in the global F test, pairwise comparisons among means were performed followed by Tukey’s test or by pair-wise rank tests. Pair-wise differences among means were considered significant at p < 0.05, and a tendency to significance was declared at 0.05 ≤ p ≤ 0.10. The procedure REG was used to characterise the relationships between silage variables. The best-fit regression model at p < 0.05 was selected based on the root mean square error (RMSE) and the coefficients of determination (R2), which was adjusted for degrees of freedom.

3. Results

3.1. Wilted Forage

The chemical and microbiological characteristics of the forage prior to ensiling before additive application are presented in Table 2. Wilted and chopped ECR had a DM concentration of 250 g kg−1. It contained 188 g WSC kg−1 DM and 326 g crude fibre kg−1 DM. The fermentability coefficient (FC) was calculated to be 60. Yeast and mould populations were present at ≥log10 5.0 cfu g−1. None of the 34 mycotoxins, which could be detected by the employed method, were found.

3.2. Silage

Additive application had an effect on all tested silage parameters (Table 3). When compared with untreated silage, both chemical additives and LABho reduced DM loss during fermentation whereas LABheho application resulted in an increase (p < 0.001). The lowest pH (p < 0.001) was associated with the highest lactic acid concentration in LABho treated silage (p < 0.001). The use of LABheho stimulated acetic acid production, whereas application of LABho caused the lowest concentration (p < 0.001). Ethanol formation was largely suppressed by both chemical additives (p < 0.001). Propionic acid and n-propanol were only detected in silage left untreated or inoculated with LABheho, with the latter treatment containing larger concentrations of both substances (p < 0.001). Except LABheho, additives had no influence on 1,2-propanediol production (p < 0.001). Both chemical additives preserved the largest concentrations of WSC of all treatments (p < 0.001). The counts of yeasts (p < 0.05) and moulds (p < 0.01) were highest in treatments CON and LABho, and the application of LABheho and SNHEPS resulted in fungal counts below the detection limit.
Untreated and LABho-inoculated silage spoiled rapidly upon air exposure after 65 h, which was accompanied by an increase in TCUM to 285 and 308 °C, respectively (Figure 1). Adding LABheho or SNHEPS maintained silage stability over the entire period of aeration (p < 0.01) and largely restricted the extent of aerobic deterioration, as reflected by lower TCUM values (p < 0.001). A tendency was observed for an improvement of ASTA by SNHE use compared with untreated silage (p = 0.059).
There was a strong linear, positive relationship between the concentration of ethanol and DM losses during fermentation (R2 = 0.89, RMSE = 0.44, p < 0.001). Regressing the DM losses on the total concentration of ethanol and n-propanol also showed a close linear, positive relationship (R2 = 0.96, RMSE = 0.27, p < 0.001), and the additional inclusion of 1,2-propanediol into the model further improved the coefficient of determination of the relationship to R2 = 0.99 (Supplementary Materials, Figure S1, RMSE = 0.11, p < 0.001). The counts of yeasts and moulds upon silo opening were strongly positively correlated (R2 = 0.80, RMSE = 0.46, p < 0.001), and the ASTA highly depended on the numbers of yeasts (linear relationship, R2 = 0.77, RSME = 0.31, p < 0.001) and moulds (linear relationship, R2 = 0.95, RSME = 0.14, p < 0.001). However, the best-fit was obtained by regressing aerobic stability on total fungal counts (Figure 2, R2 = 0.98, RMSE = 0.09, p < 0.001). A weak, negative logarithmic relationship existed between the concentration of acetic acid and the ASTA (R2 = 0.43, RMSE = 62.6, p < 0.01). As presented in Figure 3, aerobic stability and the extent of aerobic deterioration as expressed as TCUM were very closely correlated and best described by a polynomial function (R2 = 0.96, RMSE = 27.2, p < 0.001).

3.3. Mycotoxin Formation

Roquefortine C was the only mycotoxin detected in ECR silage at silo opening. The results given in Figure 4 demonstrate that untreated silage contained this mycotoxin already before air exposure (0.20 mg kg−1 DM), whereas the tested additives completely inhibited mycotoxin formation during storage (p < 0.001). During subsequent exposure to air, roquefortine C concentrations generally increased (p < 0.001) but they were much larger in untreated (85.2 mg kg−1 DM) than in treated (0.04 mg kg−1 DM) ECR silage (p < 0.001) at the end of aeration.

4. Discussion

4.1. Wilted Forage

The DM content at ensiling was similar to that shown by Auerbach et al. [7] and corresponded well with the average DM content of 281 g kg−1 found in ECR silage from commercial dairy farms in Germany (Table 1). The concentration of crude fibre was higher and crude protein content was lower than reported earlier [1,7], highlighting the influence of the stage of maturity at harvest [4]. Concomitantly, energy concentrations were much lower than previously observed [7].
The forage contained large quantities of sugar and water-soluble carbohydrates, respectively, which agrees with previous findings [6,7]. On this account, and due to low buffering capacity, the ECR could be classified as easy-to-ensile [39], reflecting a low risk of poor fermentation quality in the silage produced from it. However, Bader [6] reported lower FC values ranging between 29 and 35, which was mainly due to using direct-cut ECR of low DM content. The yeast count represented a typical value of above 105 cfu g−1 for ECR [7] or for whole-crop wheat cut at flowering [40].
None of the 34 detectable mycotoxins were found in ECR at ensiling. This may be explained by the early harvest, giving moulds not enough time to infest the plant, and produce mycotoxins. It was shown for maize and wheat that the concentrations of mycotoxins produced by Fusarium species in the field increased with progressing length of vegetation [41,42].

4.2. Fermentation Characteristics and Aerobic Deterioration

The DM losses during fermentation in untreated silage agree with those previously reported [6,7]. According to Borreani et al. [8], DM losses of as low as 5% DM caused by in-silo respiration, fermentation and effluent production can be expected under good management conditions on farms. However, the available data on DM losses in ECR silage produced in laboratory silos have consistently been higher than this threshold value (7.0 to 9.1%) [6,7], highlighting the prominent role of specific fermentation pathways involved and substrates used. Production of butyric acid by clostridia and ethanol formation by yeasts result in the highest DM losses [43]. In our study, no butyric acid was found, which can be attributed to the high FC of 60, and a sufficiently large epiphytic LAB population (>105 cfu g−1) on the forage at ensiling. According to Weissbach and Honig [39], silage was particularly prone to clostridial fermentation when forages contained less than 105 LAB g−1 and 0.5 g nitrate kg−1 DM. In their study on different forages (n = 244), including ECR, 78% of the produced silages contained butyric acid, despite a rather high FC of >35. The ECR used by Auerbach et al. [7] contained 0.4 g nitrate g−1 DM, leading to the formation of butyric acid (1.5 g kg−1 DM), whereas in three out of four trials by Bader [6], ECR silages contained no butyric acid despite low FC. This can be attributed to high nitrate levels above 2.5 g kg−1 DM. On the contrary, high butyric acid concentrations were detected in the fourth study (>10 g kg−1 DM), which used forage comparably low in nitrate (0.7 g kg−1 DM) and epiphytic LAB (<104 cfu g−1).
Lactic and acetic acids and ethanol concentration were in the range typical for grass silage at 25 to 35% DM [44] and confirmed earlier results [7]. However, ECR silage may contain much higher concentrations of these fermentation end-products, especially when DM at ensiling is low resulting in excessive fermentation intensity [6]. The comparison of WSC concentrations determined in the fresh forage at ensiling and in the silage revealed that >90% was utilized during fermentation, which agrees with data by Auerbach et al. [7], reporting a decline in WSC from 173 g kg−1 DM at ensiling to 24 g kg−1 DM after 91 days of fermentation.
An improvement in the efficiency of the fermentation process as reflected by lower DM losses was found by all additives, except for LABheho. The largest restriction was observed for chemical additives regardless of their composition. This substantiates earlier findings showing a reduction in DM loss by 0.8 percentage units by the use of homofermentative LAB, and by 2.1 percentage units by application of a chemical blend having the same composition as treatment SNHE in our study [6]. Moreover, inoculation with homofermentative LAB also decreased DM loss by 2.9 percentage units in the study by Auerbach et al. [7], in which chemical additives were not tested. Our data on ECR silage are in general agreement with the results of a meta-analysis on the use of homofermentative and facultative heterofermentative LAB in various silage types [45]. These authors detected a significant improvement in DM recovery in grass and legume silages by homofermentative LAB. The combined use of LABheho increased DM loss in ECR silage, as was previously reported by Auerbach et al. [7] for the sole use of heterofermentative LAB of the L. buchneri-type. This is in line with observations by Kleinschmit and Kung [46] stating lower DM recovery in grass and small grain silage inoculated with this additive type.
Obviously, the additives used in our study differently affected microbial populations and their production of CO2, which is the main source of DM losses during fermentation [47,48]. Inoculation with LABho resulted in a more homolactic fermentation, leading to the lowest pH of all treatments [7] but, in general, the differences between treatments were of no biological and practical relevance. The most significant change in fermentation pattern was observed in LABheho silage, as reflected by very high acetic acid concentrations and the lowest content of lactic acid [7]. The low DM at ensiling may have caused intensive metabolic activity of L. buchneri, supporting recent data by Gomes et al. [49], who determined higher acetic acid contents in direct-cut than in wilted whole-crop oat silage. Obviously, L. buchneri, which can anaerobically degrade lactic acid to acetic acid, and 1,2-propanediol [50] dominated the fermentation in our trial. The concentration of 1,2-propanediol in LABheho silage was highest compared with all other treatments but still only about 60% of the quantity determined earlier [7]. Thus, large variations regarding this parameter between trials can be expected. The presence of 1,2-propanediol in all treatments other than LABheho, including CON, may be explained by the presence of wild, epiphytic L. buchneri strains on the forage at ensiling, but their numbers and metabolic activity, respectively, must have been lower because this compound was detected at small quantities only. In agreement with Bader [6], the chemical additives proved their potential to largely restrict ethanol production with no differences between them. Moreover, LABho application reduced ethanol production when compared with untreated silage at similar magnitude as reported by Bader [6] and Oliveira et al. [45]. On the contrary, LABheho stimulated ethanol formation, which is attributable to the capacity of obligately and facultatively heterofermentative LAB also to produce alcohol, depending on sugar type and metabolic pathway [43]. Our results agree with other reports on substantially increased ethanol level in grass and small grain silage by L. buchneri treatment [46]. The lack of response to L. buchneri-inoculation in ECR regarding ethanol production described by Auerbach et al. [7] may be associated with the strictly anaerobic storage, whereas air infiltration was allowed in our study. It is known that air can alter sugar utilization by LAB leading to different metabolic end-products [51]. The proportion of ethanol originating from yeast metabolism can be considered insignificant as in treatments LABheho, SNHE and SNHEPS yeast numbers were below the detection limit upon silo opening, and no differences were observed between untreated and LABho-inoculated silage. However, data on the accumulation pattern during storage were not recorded. The synthesis of the n-propanol and propionic acid was totally suppressed in treatments LABho, SNHE and SNHEPS, but most likely by different mechanisms. Although it can also be produced by yeasts [43], its presence in silage is most commonly associated with the activity of L. diolivorans, which utilizes 1,2-propandiol to form equimolar quantities of propionic acid and n-propanol under anoxic conditions [52]. Recently, Zielinska et al. [53] also demonstrated 1,2-propanediol-degrading capabilities of L. reuteri and L. buchneri strains but the conversion efficiency was much poorer than that of L. diolivorans, and there is no data yet available on the occurrence of L. reuteri in a silage environment. The added LABho may have outcompeted the epiphytic L. diolivorans population, whereas we assume that the chemical additives suppressed this species directly. Additionally, storage length may affect 1,2-propanediol conversion, as described by Auerbach and Nadeau [54] using the same LABheho product and the same chemical additive SNHEPS in grass-clover silage of 35% DM. More so, Gomes et al. [49] showed an effect of DM at ensiling on L. diolivorans activity demonstrating limited osmotolerance of this species. Despite the same total concentration of the three compounds involved in its unique metabolic pathway in untreated and L. buchneri-inoculated silage, direct-cut oat silage had much higher concentrations of n-propanol and propionic acid and contained less 1,2-propanediol than detected in silage from wilted forage.
Both chemical additives preserved more WSC during fermentation when compared with untreated or LABheho silage. This strongly suggests a direct inhibitory effect on sugar-utilizing microbial populations during storage.
The differences in the counts of yeasts between untreated and LABho-inoculated silage and LABhohe application can be ascribed to different acetic acid concentrations. This compound has been repeatedly shown to reduce silage yeast numbers [7,46], and the magnitude of the effect strongly depended on the acetic acid content. However, this observation cannot explain the lower yeast counts in silages treated with chemical additives due to similarly high acetic acid concentrations detected in untreated silage. According to Woolford [55], sodium nitrite has very limited activity against yeasts, leading us to conclude that hexamethylene tetramine, and even more so the inclusion of the potent antimycotic agent potassium sorbate [56,57], caused their complete elimination. Untreated silage and that inoculated with LABho had the highest mould count whereas this population was not detected in treatments LABheho and SNHEPS. Typically, moulds die off soon after anaerobiosis has been attained [58], and only a few species can survive in an anaerobic silage environment [17,18]. The number of moulds on forages can vary significantly depending on location, but they are frequently near or below the detection limit in silage after extended storage length [57,59]. In our study, however, air was allowed to penetrate twice during storage, which likely caused the survival and growth of moulds, leading to visible signs of mould infestation already upon silo opening in untreated silage and those inoculated with LABho. The reason for the lack of response in ASTA to homolactic inoculation, which frequently reduced aerobic stability in ECR silage [6,7,60] may be attributable to similar fungal counts, comparable concentrations of WSC and acetic acid, and the generally fast onset of aerobic deterioration when compared with untreated silage. When L. buchneri, applied alone or in combination with homofermentative LAB, dominated the fermentation process resulting in higher acetic acid concentrations, aerobic stability was improved [46,47,61]. In agreement with observations by Auerbach and Nadeau [9] on increased stability by using the chemical additive SNHEPS, this effect should mainly be attributed to the antimycotic action of potassium sorbate contained in the additive. This also explains why this product outperformed treatment SNHE without this substance. The tendency towards better aerobic stability by SNHE than that of untreated silage was likely caused by the antifungal properties of hexamethylene tetramine [55].

4.3. Relationships between Silage Variables

In our study, DM losses during fermentation were largely affected by the concentrations of ethanol alone, or the sum of ethanol and n-propanol substantiating previous results by Auerbach et al. [47] on whole-crop rye silage harvested at dough stage (R2 = 0.86, p < 0.001), by Weiss et al. [62] on maize silage (R2 = 0.70, p < 0.001), and data from a meta-analysis by Rabelo et al. [47] on sugarcane silage (R2 not given, p < 0.01). This highlights the prominent role of ethanol and other silage alcohols regarding the release of CO2, which always results in significant DM losses, regardless of the metabolic pathway and the microbial population producing it.
The power of the linear relationship between the counts of yeasts and moulds upon silo opening confirmed data on maize silage (R2 = 0.75) by Teller et al. [56]. On the contrary, Schmidt and Kung [59] detected a much weaker degree of correlation (R2 = 0.12). This highlights the role other factors may have on the degree of correlation between yeast and mould counts, e.g., forage, location, storage length, number and composition of the epiphytic mycoflora, and the succession pattern during storage.
The strong negative correlations between acetic acid content and ASTA reported by Kleinschmit and Kung [46] (R2 = 0.66) and by Auerbach et al. [7] (R2 = 0.85) could not be confirmed by our results. Their evaluations exclusively considered untreated silage, or silage inoculated with heterofermentative LAB alone or in combination with homofermentative LAB. However, in our study, the reduction of yeast numbers in the chemical treatments was mainly caused by the used chemicals with antifungal properties, including hexamethylene tetramine, and potassium sorbate because only minor effects on acetic acid content existed. This also explains why the detected relationship between acetic acid concentration and aerobic stability was weaker than that previously found in ECR silage (R2 = 0.88) by Auerbach et al. [7], or by Schmidt and Kung [59] in maize silage (R2 = 0.95). Omission of the values from silage treated with the potassium sorbate-containing additive SNHEPS improved the coefficient of determination to R2 = 0.86 (p < 0.001) in our study.
We found similar characteristics of the function describing the relationship between yeast count and aerobic stability to previous findings (R2 = 0.85, p < 0.01) [7]. However, as a better-fit model was found when aerobic stability was regressed on mould count, we assume mould activity had played a more substantial role in the spoilage process than yeasts, which were typically considered the main causative fungal population to initiate aerobic deterioration [63].
The increase in aerobic stability was associated with a lower extent of aerobic deterioration as reflected by TCUM and substantiate data by Auerbach and Nadeau [9] on grass and grass-clover silage. However, the type and power of the relationship can differ widely between studies because silage may rapidly heat-up upon air exposure but at different rates depending on additive use and application rate and amount and type of utilizable substrate to support and sustain fungal growth [57,64,65]. This leads us to suggest the parameter TCUM be more suitable to characterise and predict potential changes in nutritive value during aeration [9,38]. However, type and slope of the regression function may vary widely between studies because of different test conditions. Frequently, like in our study, the tests for ASTA are terminated before silages of all treatments become aerobically unstable, thereby exerting a significant impact on the regression function.

4.4. Mycotoxin Formation

The low concentration of roquefortine C found in untreated silage before air exposure confirmed results from laboratory ensiling experiments by Auerbach [18] using maize silage. Like in our trial, air ingress was enabled to stimulate mould growth, and mycotoxin formation, but the aeration protocol was different. Roquefortine C, which has been detected in numerous studies in temperate climates [10,22,66], was very likely produced by two representatives of the P. roqueforti-group frequently found in silage, P. roqueforti and P. paneum [10,20,67] although mould identification was not performed in our study. In addition to other species, e.g., P. expansum [67], these two representatives were shown to synthesise roquefortine C, whereas the third species, P. carneum, lacked this capacity [19]. The high incidence of species of the P. roqueforti-group in silage can be explained by its good adaptation to acidic environments and to anaerobic or micro-aerophilic conditions [68]. The detected roquefortine C quantities in untreated ECR silage (>85 mg kg−1 DM) were much higher than those reported in the literature for other silages, e.g., grass and maize silage [10,69]. This may be explained by the extent of mould growth in the samples because differences were frequently observed between visibly moulded and unmoulded silage [10,70]. In our study, untreated silage was completely decomposed after 240 h of aeration.
Our hypothesis was not confirmed to detect metabolites other than roquefortine C, which can be produced by the P. roqueforti-group, e.g., mycophenolic acid, patulin, penicillic acid, PR toxin. These have been found in grass and maize silage [22,69,70], and co-occurrence with roquefortine C has been observed [69,71]. Even if PR toxin had been formed, it could not have been detected by the employed method. Penicillic acid may be produced by P. carneum whose incidence in silage seems to be low [19,20]. However, other factors may have played an important role, too. According to Müller and Amend [70], the length of exposure to air affected mycotoxin synthesis, and the formed quantities. An extended period of aeration of >13 days had elapsed to the first detection of mycophenolic and penicillic acids, which was longer than the length of air exposure in our study. Moreover, these authors found the concentrations of all mycotoxins, including PR toxin, to peak and thereafter decline to below the detection limit. This was explained by the low stability of certain mycotoxins at high pH values that are typical in deteriorated silage, and by possible interactions with ammonia or amino acids, rendering the parent compound undetectable. These reactions have not been described for roquefortine C. Moreover, depletion of carbon sources for mould growth and mycotoxin production rates lower than that of their disappearance may have contributed to our results. Thus, mycotoxin formation and accumulation pattern are affected by numerous factors, including the presence and the metabolic activity of the producing mould species, silage type, available carbon sources and length of exposure to air. As expected, the tested silage additives largely restricted the formation of roquefortine C in ECR silage. This supports observations by Auerbach [18] on roquefortine C formation in maize silage by benzoic acid, and by Cavallarin et al. on aflatoxin production in corn silage exposed to air for 7 days by a heterofermentative inoculant [72]. Whenever mould growth is restricted or inhibited upon exposure to air, mycotoxin biosynthesis is delayed and completely prevented, respectively.

5. Conclusions

Low DM rye harvested before maturation was well fermented but deteriorated rapidly upon exposure to air. The dual-purpose inoculant composed of L. buchneri and P. acidilactici and the chemical additive containing sodium nitrite, hexamethylene tetramine and potassium sorbate, ensured long-lasting protection against aerobic instability caused by fungal microorganisms. The production of the mycotoxin roquefortine C during air exposure was largely restricted by the use of these additives. In conclusion, the use of biological and chemical additives preventing changes in silage quality upon exposure to air is encouraged but further studies are warranted to determine the additive type which is best suited to consistently ensure high quality of silage made from early-cut rye.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4395/10/9/1432/s1, Figure S1: Relationship between the total concentration of ethanol + n-propanol + 1,2-propanediol (x) at silo opening and the dry matter (DM) losses during fermentation (y) in early-cut rye silage treated with additives of different types and compositions after 64 days of storage. R2 = 0.99, RMSE = 0.11, p < 0.001, n = 15.

Author Contributions

Conceptualization, H.A. and P.T.; methodology, H.A. and P.T.; software, P.T. and H.A.; validation H.A. and P.T.; formal analysis, B.K.; investigation, P.T. and H.A.; resources, P.T. and H.A.; data curation, H.A. and P.T.; writing—original draft preparation, H.A.; writing—review and editing, P.T. and H.A.; visualization, P.T. and H.A.; supervision, P.T.; project administration, P.T. and H.A.; funding acquisition, H.A. and P.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was primarily funded by the University Nürtingen-Geislingen, Germany and the International Silage Consultancy (ISC), Germany. Additional minor funding was provided by the KONSIL Europe GmbH, Germany.

Acknowledgments

We are deeply indebted to B. Kroschewski, Humboldt Universität zu Berlin, Germany for data processing and running statistical analyses of this trial. We greatly acknowledge the support by Kirsten Weiss, Humboldt University, Berlin, Germany, for analysing fermentation pattern, and W. Richardt, LKS mbH, Lichtenwalde, Germany, for providing data on nutritional and fermentation quality of ECR silage produced on commercial farms in Germany. We would also like to thank H. van Egmont and T. de Rijk, RIKILT Wageningen University and Research, The Netherlands, for mycotoxin analysis.

Conflicts of Interest

Horst Auerbach and Peter Theobald are holding shares in the company KONSIL Europe GmbH, which provided the tested additives.

Abbreviations

ASTA, aerobic stability; AD, aerobic deterioration; DM, dry matter; ECR, early-cut rye; LAB, lactic acid bacteria; RMSE, root mean square error; TCUM, cumulated temperature; WSC, water-soluble carbohydrates.

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Figure 1. Effects of additive type and composition on the aerobic stability (ASTA) and the cumulated temperature (TCUM) in early-cut rye silage after 64 days of storage. CON, no additive; LABho, homofermentative inoculant composed of L. plantarum DSM 16,627 and L. paracasei NCIMB, total inoculation rate: 1.5 × 105 cfu g−1; LABheho, inoculant composed of L. buchneri CNCM-I 4323 and P. acidilactici DSM, total inoculation rate: 1.67 × 105 cfu g−1; SNHE, liquid chemical additive containing sodium nitrite (300 g L−1) and hexamethylene tetramine (200 g L−1), 2 mL kg−1; SNHEPS, liquid chemical additive containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), 2 mL kg−1; >denotes that ASTA was greater than 240 h; a–b open bars for TCUM bearing different superscripts differ, Tukey´s test, p < 0.001, SEM = 17.2; x–z solid bars for ASTA with unlike superscripts differ, non-parameteric rank test of ANOVA-type statistics, p < 0.01, SEM = 0–13.7, calculated separately for each treatment due to non-normal data distribution, n = 15.
Figure 1. Effects of additive type and composition on the aerobic stability (ASTA) and the cumulated temperature (TCUM) in early-cut rye silage after 64 days of storage. CON, no additive; LABho, homofermentative inoculant composed of L. plantarum DSM 16,627 and L. paracasei NCIMB, total inoculation rate: 1.5 × 105 cfu g−1; LABheho, inoculant composed of L. buchneri CNCM-I 4323 and P. acidilactici DSM, total inoculation rate: 1.67 × 105 cfu g−1; SNHE, liquid chemical additive containing sodium nitrite (300 g L−1) and hexamethylene tetramine (200 g L−1), 2 mL kg−1; SNHEPS, liquid chemical additive containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), 2 mL kg−1; >denotes that ASTA was greater than 240 h; a–b open bars for TCUM bearing different superscripts differ, Tukey´s test, p < 0.001, SEM = 17.2; x–z solid bars for ASTA with unlike superscripts differ, non-parameteric rank test of ANOVA-type statistics, p < 0.01, SEM = 0–13.7, calculated separately for each treatment due to non-normal data distribution, n = 15.
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Figure 2. Relationship between the total fungal count at silo opening (x) and the aerobic stability (y) in early-cut rye silage treated with additives of different type and composition and stored for 64 days upon subsequent air exposure for 240 h. R2 = 0.98, root mean square error (RMSE) = 0.09, p < 0.001, n = 15.
Figure 2. Relationship between the total fungal count at silo opening (x) and the aerobic stability (y) in early-cut rye silage treated with additives of different type and composition and stored for 64 days upon subsequent air exposure for 240 h. R2 = 0.98, root mean square error (RMSE) = 0.09, p < 0.001, n = 15.
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Figure 3. Relationship between the aerobic stability (x) and the cumulated temperature (y) in early-cut rye silage treated with additives of different type and composition and stored for 64 days upon subsequent air exposure for 240 h. R2 = 0.96, RMSE = 27.2, p < 0.001, n = 15.
Figure 3. Relationship between the aerobic stability (x) and the cumulated temperature (y) in early-cut rye silage treated with additives of different type and composition and stored for 64 days upon subsequent air exposure for 240 h. R2 = 0.96, RMSE = 27.2, p < 0.001, n = 15.
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Figure 4. Effects of additive type on the concentration of the mycotoxin roquefortine C in early-cut rye silage stored for 64 days before (black bar) and after (grey bar) subsequent air exposure for 240 h. LABheho, inoculant composed of L. buchneri CNCM-I 4323 and P. acidilactici DSM, total inoculation rate: 1.67 × 105 cfu g−1; SNHEPS, liquid chemical additive containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), 2 mL kg−1; Unlike letters in brackets denote significance of effect of aeration (X,Y, p < 0.001) and of effect of additive (x,y, p < 0.001), aeration × additive interaction not significant, ANOVA-type statistics, SEM = 0-8.79, calculated separately for each treatment due to non-normality of data.
Figure 4. Effects of additive type on the concentration of the mycotoxin roquefortine C in early-cut rye silage stored for 64 days before (black bar) and after (grey bar) subsequent air exposure for 240 h. LABheho, inoculant composed of L. buchneri CNCM-I 4323 and P. acidilactici DSM, total inoculation rate: 1.67 × 105 cfu g−1; SNHEPS, liquid chemical additive containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), 2 mL kg−1; Unlike letters in brackets denote significance of effect of aeration (X,Y, p < 0.001) and of effect of additive (x,y, p < 0.001), aeration × additive interaction not significant, ANOVA-type statistics, SEM = 0-8.79, calculated separately for each treatment due to non-normality of data.
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Table
Table 1. Nutritive value and fermentation characteristics (g kg−1 dry matter (DM), unless stated otherwise) of early-cut rye silages analysed in 2016–2018 by LKS mbH, Niederwiesa, Germany.
Table 1. Nutritive value and fermentation characteristics (g kg−1 dry matter (DM), unless stated otherwise) of early-cut rye silages analysed in 2016–2018 by LKS mbH, Niederwiesa, Germany.
ParameternMeanSDMinimumMaximum
Dry matter, g kg−165728161172584
Crude ash657751929166
Crude protein6571292753225
Crude fibre65728737186425
ADL6573010370
Sugar *65736380230
Metabolisable energy, MJ kg−1 DM65710.60.77.912.0
Net-Energy-Lactation, MJ NEL kg−1 DM6576.40.54.57.5
pH5594.20.43.35.3
Ammonia-N, g kg−1 total N)657862919230
Lactic acid5275.945.84.1152.2
Acetic acid ‡5225.921.92.684.2
Butyric acid †521.45.1025.2
SD, standard deviation; ADL, acid-detergent lignin; DM, dry matter; * sum of glucose, fructose and saccharose; ‡ sum of acetic and propionic acids, † sum of n-butyric, iso-butyric, n-valeric, iso-valeric and n-caproic acids; 0 denotes values below detection limit.
Table
Table 2. Composition of wilted forage before additive application (n = 3, data given in g kg−1 DM unless stated otherwise).
Table 2. Composition of wilted forage before additive application (n = 3, data given in g kg−1 DM unless stated otherwise).
ParameterMeanStandard Deviation
Dry matter, g/kg2501.4
Crude ash560.4
Crude protein1012.4
Crude fibre3269.4
Sugar *1661.6
Water-soluble carbohydrates † 1881.3
Buffering capacity, g lactic acid kg−1 DM430.7
Fermentability coefficient600.4
Metabolizable energy, MJ kg−1 DM9.60.1
Net-Energy-Lactation, MJ NEL kg−1 DM5.70
Lactic acid bacteria, log10 cfu g−15.90.2
Yeasts, log10 cfu g−15.30.1
Moulds, log10 cfu g−15.00.6
Total fungi, log10 cfu g−15.50.3
* sum of glucose, fructose and saccharose; † sum of sugar and fructans.
Table
Table 3. Effects of additive type and composition on dry matter (DM) losses, fermentation characteristics and fungal populations of early-cut rye silage stored for 64 days (data presented as LSmeans in g kg−1 DM unless stated otherwise, n = 3).
Table 3. Effects of additive type and composition on dry matter (DM) losses, fermentation characteristics and fungal populations of early-cut rye silage stored for 64 days (data presented as LSmeans in g kg−1 DM unless stated otherwise, n = 3).
ParameterCONLABhoLABhehoSNHESNHEPSSEMp
DM loss, %7.0 c6.4 b9.4 d5.9 a5.9 a0.03<0.001
pH4.08 b3.96 a4.33 d4.31 d4.12 c0.007<0.001
Lactic acid78.4 d89.0 e39.5 a57.0 b66.4 c0.60<0.001
Acetic acid19.5 b15.5 a52.7 c18.8 ab20.6 b0.81<0.001
Propionic acid0.3 y0 x4.1 z0 x0 x0–0.31<0.001
Ethanol8.6 c6.9 b13.9 d2.2 a2.3 a0.24<0.001
n-propanol0.5 y0 x3.9 z0 x0 x0–0.42<0.001
1,2-propanediol2.9 a2.2 a24.0 b1.3 a1.2 a0.93<0.001
Ammonia-N, gkg−1 total N26 ab24 a33 c32 c30 bc0.9<0.001
WSC *15.2 a20.1 ab11.2 a59.2 c36.9 b4.12<0.001
Yeast count, log10 cfu g−13.6 y3.6 y<2.0 x<2.0 x<2.0 x0–0.39<0.05
Mould count, log10 cfu g−14.0 z3.6 yz<2.0 x2.5 y<2.0 x0–0.25<0.01
CON, no additive; LABho, homofermentative inoculant composed of L. plantarum DSM 16,627 and L. paracasei NCIMB, total inoculation rate: 1.5 × 105 cfu g−1; LABheho, inoculant composed of L. buchneri CNCM-I 4323 and P. acidilactici DSM 11,673, total inoculation rate: 1.67 × 105 cfu g−1; SNHE, liquid chemical additive containing sodium nitrite (300 g L−1) and hexamethylene tetramine (200 g L−1), 2 mL kg−1; SNHEPS, liquid chemical additive containing sodium nitrite (195 g L−1), hexamethylene tetramine (71 g L−1) and potassium sorbate (106 g L−1), 2 mL kg−1; * WSC, water-soluble carbohydrates; a–e values in rows bearing unlike superscripts differ at p < 0.05, Tukey´s test; x–z values in rows bearing unlike superscripts differ at p < 0.05, non-parameteric rank test of ANOVA-type statistics, SEM of non-normally distributed data was calculated separately for each treatment.

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Auerbach, H.; Theobald, P. Additive Type Affects Fermentation, Aerobic Stability and Mycotoxin Formation during Air Exposure of Early-Cut Rye (Secale cereale L.) Silage. Agronomy 2020, 10, 1432. https://doi.org/10.3390/agronomy10091432

AMA Style

Auerbach H, Theobald P. Additive Type Affects Fermentation, Aerobic Stability and Mycotoxin Formation during Air Exposure of Early-Cut Rye (Secale cereale L.) Silage. Agronomy. 2020; 10(9):1432. https://doi.org/10.3390/agronomy10091432

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Auerbach, Horst, and Peter Theobald. 2020. "Additive Type Affects Fermentation, Aerobic Stability and Mycotoxin Formation during Air Exposure of Early-Cut Rye (Secale cereale L.) Silage" Agronomy 10, no. 9: 1432. https://doi.org/10.3390/agronomy10091432

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