Spoilage Of Silage Research Articles

PSIV-11 The inhibitory activity of phyto-phenolic compounds on ammonia-producing ruminal bacteria

Abstract Antibiotic resistance has progressively increased over time, thus necessitating the discovery of sustainable, novel antimicrobials. Ammonia producing bacteria (APB) are predominant organisms occupying many niches in the rumens of cattle and soil. The objective of this study was to discover target phyto-phenolic compounds (PPCs) that can act as replacements for existing antimicrobials, as well as at what concentration ranges may be optimal for inhibition of APB growth. In recognition of this discovery, producers may have alternative antimicrobial compounds for use in managing APB and common silage spoilage organisms. To shed light on this matter, inhibitory concentrations (IC) and ammonia assays were implemented. ICs analyzed the inhibitory action of six structurally similar PPCs: thymol, eugenol, carvacrol, hydro-cinnamic acid (H-CA), trans-cinnamic acid (T-CA), and phloroglucinol. Five APB (SR, MD1, F, BG1, and B14) were exposed to five concentrations of phytochemicals via a 10% v/v serial dilution: 10, 1, 0.1, 0.01, 0.001 mM. Following serial dilution, the bacteria were incubated ~24h at 39°C in a water bath, and visually inspected for growth. Ammonia assays utilized the least, fully inhibitory concentration of each phytochemical identified via the inhibitory concentration experiments. Ammonia concentrations were determined using the phenolic acid/hypochlorite method. All protocols utilized anaerobic sterile technique. All experiments were repeated in triplicate and significant results (P < 0.05) were determined using a one-way ANOVA with Tukey’s post hoc test utilizing OriginPro statistical software. Inhibition results revealed that eugenol, thymol, and carvacrol were successful in inhibiting bacterial growth at a concentration range of 10 mM to 1 mM. However, phloroglucinol was not fully inhibitory at any concentration tested against any bacteria. Eugenol, thymol, carvacrol were fully inhibitory at either 10 mM or 1 mM for all organisms tested except BG1, where it was only inhibitory at 10 mM. Ammonia analyses determined that eugenol and carvacrol greatly reduced ammonia production for all species tested except for B14, a generalist, which had no measurable changes in ammonia for any of the treatments including control. Thymol reduced ammonia production for MD1, BG1, and SR. Additionally, H-CA and T-CA decreased ammonia production of SR compared with control but not as much as eugenol or carvacrol. Results suggested that the growth of APB was generally inhibited by the greatest concentrations of PPCs (10 mM to 1 mM) tested here. Additionally, phloroglucinol did not inhibit bacterial growth therefore it was not included in the ammonia analyses. Eugenol and carvacrol significantly reduced ammonia production in all organisms, therefore indicating possible antimicrobial candidates to be further investigated. In comparison with carvacrol and eugenol, T-CA and H-CA made a modest to negligible reduction in ammonia. Phyto-phenolic compounds that were successful in inhibiting bacterial growth and reducing ammonia production demonstrate several promising target antimicrobials.

Isolation of Bacillus velezensis from Silage and Its Effect on Aerobic Stability and In Vitro Methane Production of Whole-Plant Corn Silage

Once a silo has been opened, the silage inside will face challenges such as aerobic deterioration, rot, and contamination. Biocontrol bacteria, as a kind of biological antiseptic, are highly effective and natural and are gaining increasing attention. This study aimed to screen a strain with anti-microbial activity against silage spoilage microorganisms and examine its effects on the fermentation quality, aerobic stability, in vitro digestion, and methane production of silage. Lactic acid bacteria, pathogenic and rot-causing microorganisms, were used as indicators to screen the strains for putrefactive silage. The bacteriostatic spectrum, growth performance, and tolerance to the silage environment of the strain were tested. A strain named D-2 was screened from rotten whole-plant corn silage and identified as Bacillus velezensis through physiological and biochemical tests as well as 16S rDNA sequencing. This study found that D-2 exhibits antibacterial effects on several microorganisms, including Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, Aspergillus niger, Saccharomyces cerevisiae, Fusarium oxysporum, and Fusarium graminearum. However, it has no adverse effect on Lactobacillus reuteri, Enterococcus faecium, or Lactobacillus casei. D-2 can attain a stable stage within 10 h and withstand temperatures of up to 70 °C. Moreover, this study found that D-2 had a high survival rate of over 97% after 48 h in a lactic acid environment with pH 4. Freshly chopped whole-plant corn was inoculated without or with D-2 and ensiled for 60 days. The results show that D-2 inoculations increase the content of water-soluble carbohydrates, acetic acid, and propionic acid in the silage and decrease the number of yeasts and molds, the NH4+-N/TN ratio, and the pH. We also found that fermenting whole-plant corn with D-2 significantly increased the in vitro digestibility and the propionic acid content, while also significantly inhibiting methane production. After being exposed to air for 10 days, D-2 can still effectively reduce the total number of yeasts and molds, prevent the decrease in lactic acid bacteria, and inhibit the increase in the pH and NH4+-N/TN ratio of silage products. Overall, D-2 is resistant to pathogenic and rot-causing microorganisms, allowing for easy adaptation to silage production conditions. D-2 can effectively improve aerobic stability and reduce losses in the nutritional value of silage, indicating possible applications for the prevention of silage rot and methane production.

Проблема аэробной устойчивости при заготовке и использовании кукурузного силоса

One of the most important problems of preserving corn silage is its aerobic stability in the process of harvesting, storage and using. It depends on the term and mowing height of plants, the milling degree, and the compaction of ramming, the hermetic nature of shelter, the duration of storage and the sampling rate during feeding. Spoilage of silage from aerobic instability leads to large losses of feed and the acquisition of properties dangerous to animal health. The best time for harvesting corn for silage is the phase of waxy ripeness of grain, in which plants contain the optimal amount of dry matter for silage and the maximum yield and concentration of nutrients in it. To increase the nutritional value of corn silage, the mowing height of plants is increased. However, the decrease in the yield of grass stand should be taken into account. The milling degree of plants varies depending on the moisture content of the raw material: the higher the moisture content, the larger particles size of silage mass should be. However, their largest size should not exceed 20 mm. To exclude aerobic instability, the silage mass must be compactly packed and sealed. Unsealing of the silage for feeding assumes such sampling rates that exclude aerobic spoilage. They are differentiated depending on the season of feed use: large when feeding silage in summer and smaller in winter.

Perilla frutescens as potential antimicrobial modifier to against forage oat silage spoilage.

The aim of this study was to investigate the influence of Perilla frutescens, alone or in combination with Lactobacillus plantarum a214 or citric acid, on forage oat silage quality, bacterial and fungal microbiological profile during ensiling and aerobic exposure. With the exception of Perilla frutescens, all additives could improve silage quality of forage oat based on lower ammonia-nitrogen content and higher residual of water soluble carbohydrates during anaerobic fermentation compared to control silage, especially in Perilla frutescens combined with citric acid (CAPF). Lactobacillus was the dominant bacteria in all silages, while CAPF group increased the relative abundance of Lactobacillus lindneri and Lactobacillus brevis compared to control silage. The application of Perilla frutescens suppressed the relative abundance of yeasts such as Pichia fermentans and Wickerhamomyces anomalus in response to aerobic exposure, especially in CAPF treatment, leading to high acetic acids and lower dry matter loss, as well as good aerobic stability. Therefore, Perilla frutescens, alone or in combination with citric acid, has potential to improve aerobic stability of forage oat silage by shifting bacterial and fungal community composition, and can be used as new additive to prepare high-quality silage for animal production.

Tanenler; Silajlarda ve Hayvan Besleme Uygulamalarında Kullanımı

Aim: This review was made to determine the effects of using tannin on the fermentation quality and animal nutrition as a different alternative to the additives used to prevent the spoilage of silages, which are widely used in today's livestock. Tannins are polyphenolic compounds and are natural compounds with a characteristic odor, acrid taste, and different colors from yellow to white, which can also be found in feeds such as gallnut, oak, chestnut and rapeseed, broad bean, sorghum. Since BC 1500 they have been used in various fields from medicine to food. Apart from its antidiarrheal, antiseptic and anthelmintic effects, its most well-known feature is that it forms compounds with proteins. The high protein/water presence of the feed to be ensiled makes ensiling difficult and affects fermentation negatively. High protein will reduce lactic acid formation in silages due to proteolysis and the desired pH value will not be reached. It is thought that tannins can form compounds with proteins and prevent proteolysis in silages and provide by-pass properties to proteins. Thus, the proteins that pass through the rumen will be digested in the small intestine and their utilization will increase. In addition, methane gas produced by ruminants causes loss of gross energy and contributes to global warming with greenhouse gas. Tannins are considered as an alternative to reduce methane gas and dispose of waste ammonia.
 Conclusion: The use of tannin in silages can facilitate the ensiling of high protein feeds, increase their quality, and meet the nutrient needs of animals better. It can improve the storage conditions of roughage. An innovative feeding method can be provided by reducing the methane production of animals that consume tannin-containing feeds. It can be a natural approach to the disposal of ammonia.

Farm Silage Facilities and Their Management for the Prevention of Anaerobic Bacteria Spore Contamination in Raw Milk

At feed-out, aerobic spoilage of silage enables an increase in anaerobic spore-forming bacteria (ANSB) that may enter the total mixed ration (TMR). The aim of our study was to understand whether in hot summers the silage structures and management may affect the level of ANSB in milk for long-ripening cheese production. A survey of silage facilities, management, and their relationships with silage, TMR, feces, and milk ANSB most probable number (MPN) content was conducted in the Po Valley during summer months. Silo type did not affect the mean ANSB, but only the wideness of their value distributions, with a narrow range for bags and a wider range for bunkers. The unloading equipment affected the ANSB count; the front-end loader with cutter was associated with a lower ANSB count—probably as a result of the reduced surface left after daily silage removal. Silo length and daily removed face width were the main factors affecting contamination of silage by spore-forming bacteria during summer, with longer silos and wider surface removal reducing ANSB contamination—probably as a consequence of reduced aerobic spoilage at the silage surface. The silage contamination by spore-forming bacteria within a log10 2 MPN g−1 allowed a low concentration of spore-forming bacteria at the farm bulk milk tank level. Fecal ANSB levels did not factor into the regression that explains the ANSB in farm milk. It has been found that silage facilities’ features and their management are an important first step to reduce the extent of ANSB contamination at the farm level.

Feed-out rate used as a tool to manage the aerobic deterioration of corn silages in tropical and temperate climates

There is an increasing recognition throughout the world that many of the feeding problems of dairy herds are linked to the presence of aerobically deteriorated parts on a silo face, causing farmers to pose questions on what amount of silage should be removed daily to feed their animals. Since an adequate feed-out rate helps to prevent silage spoilage, a simple tool is needed to manage the aerobic deterioration of corn silages during feed-out. The aims of this study were to develop an unloading rate index, which we have called the mass feed-out rate (MFR), expressed in kilograms of fresh matter silage unloaded daily per square meter of silo face, to better predict the aerobic deterioration of silage and to offer management solutions to help prevent spoilage, through a survey on 97 commercial dairy farms in Italy and Brazil. Silages were sampled and analyzed for their main microbial, fermentative, and nutritional characteristics, whereas silage temperatures were measured in the core and peripheral areas of the silo working face. Moreover, a detailed questionnaire on silo management and silage utilization was administered to the farmers during each farm visit. The size and silage density of the silos presented a wide variability in the 2 countries, thus indicating that different management practices were adopted during corn harvesting, silo filling, and silage compaction. The differences between pH and temperature in the peripheral areas and in the core of the silage (dpH and dT, respectively) were tested as a single indicator to identify any aerobic deteriorated areas on the silo face, associated with the yeast and mold counts. Both indicators correctly identified aerobic deterioration in 86.6% and 93.8% of the studied silos, respectively. The lactic acid and ethanol increased as the MFR increased, whereas the starch, dT, and the yeast and mold counts decreased with increasing MFR. A daily removal rate of over 250 kg of silage/m2 markedly reduced the risk of spoilage in corn silages at a farm level in both temperate and tropical environments. The new MFR index can substitute for the commonly used linear feed-out rate as it includes the silage density and can be obtained from 1 single recording.

Dynamics of Fermentation Parameters and Bacterial Community in High-Moisture Alfalfa Silage with or without Lactic Acid Bacteria.

The aim of this study was to gain deeper insights into the dynamics of fermentation parameters and the bacterial community during the ensiling of high-moisture alfalfa. A commercial lactic acid bacteria (YX) inoculant was used as an additive. After 15 and 30 days of ensiling, the control silage (CK) exhibited a high pH and a high concentration of ammoniacal nitrogen (NH3-N); Enterobacter and Hafnia-Obesumbacterium were the dominant genera. At 60 d, the pH value and the concentration of NH3-N in CK silage increased compared with 15 and 30 d, propionic acid and butyric acid (BA) were detected, and Garciella had the highest abundance in the bacterial community. Compared with CK silage, inoculation of YX significantly promoted lactic acid and acetic acid accumulation and reduced pH and BA formation, did not significantly reduce the concentration of NH3-N except at 60 d, and significantly promoted the abundance of Lactobacillus and decreased the abundance of Garciella and Anaerosporobacter, but did not significantly inhibit the growth of Enterobacter and Hafnia-Obesumbacterium. In conclusion, high-moisture alfalfa naturally ensiled is prone to rot. Adding YX can delay the process of silage spoilage by inhibiting the growth of undesirable microorganisms to a certain extent.

Application of monoclonal antibodies in quantifying fungal growth dynamics during aerobic spoilage of silage.

SummaryProliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony‐forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody‐based enzyme‐linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on‐farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post‐aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on‐farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health.

BIOCONVERSION OF CRUDE GLYCEROL INTO REUTERIN BY LACTOBACILLI ISOLATED FROM SILAGE

Heterofermentative lactobacilli were isolated from sorghum and maize silage, and their ability to produce the antimicrobial compound reuterin (3-hydroxypropionaldehyde, 3-HPA) was investigated. Additionally, reuterin production from crude and partially purified biodiesel-derived glycerol was evaluated as an alternative to the utilization of these by-products. Only two out of 148 lactobacilli isolated produced reuterin in the presence of glycerol. The gene encoding a subunit of glycerol dehydratase was detected by PCR in isolates SO8 and SO23, identified as Lactobacillus reuteri by 16S rRNA gene sequence analysis. Levels of reuterin produced by these two isolates and strain L. reuteri DSM 17938, used as control, were different. For each bacteria, the amount of reuterin detected from biodiesel-derived glycerol was comparable with that obtained from aqueous solution of pure glycerol. Among silage isolates, L. reuteri SO23 yielded the highest levels of reuterin production. In addition, cell-free supernatants revealed inhibitory activity against silage spoilage microorganisms like spore-forming bacteria, yeast and mould. L. reuteri SO23 proved to be a valuable candidate for use of crude glycerol and its bioconversion to reuterin. Addition of glycerol together with reuterin-producing lactobacilli strains could represent a good alternative as silage biopreservant and help optimize profit in biodiesel production.

ИНТЕНСИВНОСТЬ СПИРТОВОГО БРОЖЕНИЯ В ЗАВИСИМОСТИ ОТ СТЕПЕНИ ПРОВЯЛИВАНИЯ И ОБЕСПЕЧЕННОСТИ КОРМОВЫХ ТРАВ САХАРОМ И ЕГО РОЛЬ В ВОЗНИКНОВЕНИИ АЭРОБНОЙ ПОРЧИ СИЛОСА И СЕНАЖА

Aerobic spoilage in silage and haylage is affected by the intensity of alcoholic fermentation, which causes the amount of nutrient loss of the feed during excavation from the trench. The negative effect is not the alcohol content, as such, which in some cases even helps to improve the aerobic stability of the feed, but the amount of yeast found in the silo and hay at the time of opening the silos, which, in turn, depends on a number of factors. One of these factors is the low sugar-buffer ratio in plants, which should be ≤2.5. Under this condition, there is a slight accumulation of alcohol in the dry matter of the feed, which indicates a weak development of the yeast. The second factor that determines the aerobic stability of the feed and the amount of nutrient loss during storage in air is the dry matter content of plants. When stored in the air of haylage and silage from dried herbs, the loss of dry matter is reduced, reaching a minimum when preparing food from plants with a sugar-buffer ratio of ≤ 2.5. This is especially noticeable in the first 2–3 days of storing feed in the air. Subsequently, the loss of dry matter from aerobic spoilage begins to increase, causing the occurrence of secondary fermentation, which leads to its spoilage of feed. The third factor determining the aerobic stability of a silo is its shelf life under anaerobic conditions, which should not be less than 90 days. This follows from the biological features of the development of yeast, which are the main initiators of aerobic spoilage. However, haylage is predisposed to aerobic spoilage for any shelf life in trenches.

Impacts of some factors that effect spoilage of silage at the periphery of the exposed face of corn silage piles

Preserving silage in large piles is popular due to its flexibility and affordability that allows storage of large amounts of silage with low facility investment. Management of silage piles during feed-out impacts silage spoilage since, once the plastic covers are removed and silage near the exposed silage ‘face’ is exposed to air, undesirable microbes can proliferate to negatively impact its hygienic quality and nutritional value. Pile silage also has characteristics, such as a long feed-out phase and a large exposed face peripheral area, which makes it prone to spoilage during feed-out, especially in the exposed face area. Silage samples were collected from nine corn silage piles in the San Joaquin Valley of California (USA) in order to evaluate impacts of feed-out speed, days after opening and surface silage density on degree of silage spoilage at the periphery of the exposed face. Faster feed-out speed (i.e., 0.59 versus 0.29 m/day) suppressed the degree of spoilage, as indicated by lower temperature and pH, and higher lactic levels, as well as higher abundance of lactic acid bacteria (LAB). However, because surface spoilage in both fast- and slow-speed piles was judged to be in the spoilage initiation stage, the feed-out speed of 0.29 m/day was judged sufficient to prevent substantive spoilage. Fewer days after opening (i.e., 69 versus 110 days) impacted degree of spoilage as indicated by lower mold counts and higher lactic and acetic acid levels. As spoilage in long-open piles had reached mid-stage, while spoilage in short-open piles was still in the initiation stage, days after opening of 110 was judged too long to prevent surface spoilage. Higher surface density (i.e., 360 versus 312 kg/m3 wet weight) had little impact on spoilage, due to its low impact on all response parameters. Overall, faster feed-out speed and fewer days after pile opening had the most positive impacts on suppressing spoilage in corn silage at the periphery of the exposed face, while increasing surface silage density was less impactful.

Bacterial and fungal core microbiomes associated with small grain silages during ensiling and aerobic spoilage

BackgroundDescribing the microbial populations present in small grain silage and understanding their changes during ensiling is of interest for improving the nutrient value of these important forage crops. Barley, oat and triticale forages as well as an intercropped mixture of the 3 crops were harvested and ensiled in mini silos for a period of 90 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics and nutritive value were assessed in terminal silages and bacterial and fungal communities during ensiling and aerobic exposure were described using 16S and 18S rDNA sequencing, respectively.ResultsAll small grain silages exhibited chemical traits that were associated with well ensiled forages, such as low pH value (4.09 ± 0.28) and high levels of lactic acid (59.8 ± 14.59 mg/g DM). The number of microbial core genome operational taxonomic units (OTUs) decreased with time of ensiling. Taxonomic bacterial community profiles were dominated by the Lactobacillales after fermentation, with a notable increase in Bacillales as a result of aerobic exposure. Diversity of the fungal core microbiome was shown to also be reduced during ensiling. Operational taxonomic units assigned to filamentous fungi were found in the core microbiome at ensiling and after aerobic exposure, whereas the Saccharomycetales were the dominate yeast population after 90 days of ensiling and aerobic exposure. Bacterial and fungal orders typically associated with silage spoilage were identified in the core microbiome after aerobic exposure.ConclusionNext Generation Sequencing was successfully used to describe bacterial communities and the first record of fungal communities throughout the process of ensiling and utilization. Adequately describing the microbial ecology of silages could lead to improved ensiling practices and the selection of silage inoculants that act synergistically with the natural forage microbiome.

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomic Analysis of Silage.

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

New techniques

Feeding systems need to be monitored and evaluated just like milking systems. A TMR Audit® is a systematic approach designed to evaluate the feeding program. The objectives are to look for ways to minimize variation between the formulated and consumed ration, minimize shrink, and to improve the efficiency of the feeding system. Areas evaluated include silage and feedstuff management, TMR load preparation, TMR consistency within and between loads, and feedbunk management. Management procedures should be in place to minimize top spoilage of silage stored in bunker silos and with other feedstuffs, and feeding personnel should be trained that spoiled feed needs to be discarded, not fed. Due to variation in DM and nutrient content across the face of the silo and across bales of hay or balage, it is best to premix an individual forage prior to using that forage in preparing a load of TMR. The Penn State Particle Separator can be used to evaluate TMR consistency. We have identified 10 primary factors that can lead to variation within and between loads of TMR. These factors include the following: worn equipment, mixing time, load size, levelness of mixer during mixing, loading position on the mixer box, hay or straw quality and processing, loading sequence, liquid distribution, vertical mixer auger speeds, and hay restrictor plate settings in vertical mixers. Feed efficiency can be improved by feeding multiple times per day as opposed to once daily. Feed push-ups need to be done frequently enough to ensure easy access to feed along the entire feed bunk. TMR Audits can help to improve performance and health on the dairy.

Aerobic deterioration stimulates outgrowth of spore-forming Paenibacillus in corn silage stored under oxygen-barrier or polyethylene films

The occurrence of Bacillus and Paenibacillus spores in silage is of great concern to dairy producers because their spores can survive pasteurization and some strains are capable of subsequently germinating and growing under refrigerated conditions in pasteurized milk. The objectives of this study were to verify the role of aerobic deterioration of corn silage on the proliferation of Paenibacillus spores and to evaluate the efficacy of oxygen-barrier films used to cover silage during fermentation and storage to mitigate these undesirable bacterial outbreaks. The trial was carried out on whole-crop maize (Zea mays L.) inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium. A standard polyethylene film and a polyethylene-polyamide film with an enhanced oxygen barrier were used to produce the silage bags for this experiment. The silos were stored indoors at ambient temperature (18 to 22°C) and opened after 110 d. The silage was sampled after 0, 2, 5, 7, 9, and 14 d of aerobic exposure to quantify the growth of endospore-forming bacteria during the exposure of silages to air. Paenibacillus macerans (gram-positive, facultatively anaerobic bacteria) was able to develop during the aerobic exposure of corn silage. This species was present in the herbage at harvesting, together with clostridial spores, and survived ensiling fermentation; it constituted more than 60% of the anaerobic spore formers at silage opening. During silage spoilage, the spore concentration of P. macerans increased to values greater than 7.0 log10 cfu/g of silage. The use of different plastic films to seal silages affected the growth of P. macerans and the number of spores during aerobic exposure of silages. These results indicate that the number of Paenibacillus spores could greatly increase in silage after exposure to air, and that oxygen-barrier films could help to reduce the potential for silage contamination of this important group of milk spoilage microorganisms by delaying the onset of aerobic deterioration.

The effect of Lactobacillus buchneri inoculation on the aerobic stability and fermentation characteristics of alfalfa-ryegrass, red clover and maize silage

Aerobic spoilage of silages occurs frequently and is undesirable because it reduces both its nutritive and hygienic quality. Silage inoculants containing heterofermentative lactic acid bacteria, like Lactobacillus buchneri, have already been proven to improve aerobic stability by augmented production of acetic acid, which inhibits yeasts. In this study, the effect of L. buchneri on fermentation characteristics and aerobic stability of alfalfa-ryegrass silage, red clover silage and maize silage was assessed using microsilos. Two dosages, 1×105 and 3×105 cfu g-1 of fresh matter, were compared to untreated control silage. Inoculation with L. buchneri clearly altered the fermentation characteristics of alfalfa-ryegrass and red clover silage, resulting in a significantly higher aerobic stability at both dosages. The effects of L. buchneri inoculation on maize silage were less clear, but nevertheless the aerobic stability of maize silage inoculated with 1×105 cfu g-1 of fresh matter was significantly higher compared to the untreated silage.

Effect of applying inoculants with heterolactic or homolactic and heterolactic bacteria on the fermentation and quality of corn silage

This study examined the effect of applying different bacterial inoculants on the fermentation and quality of corn silage. Corn plants were harvested at 35% DM, chopped, and ensiled in 20-L mini silos after application of (1) deionized water (CON) or inoculants containing (2) 1 × 105 cfu/g of Pediococcus pentosaceus 12455 and Propionibacteria freudenreichii (B2); (3) 4 × 105 cfu/g of Lactobacillus buchneri 40788 (BUC); or (4) 1 × 105 cfu/g of Pediococcus pentosaceus 12455 and 4 × 105 cfu/g of L. buchneri 40788 (B500). Four replicates of each treatment were weighed into polyethylene bags within 20-L mini silos. Silos were stored for 575 d at ambient temperature (25°C) in a covered barn. After silos were opened, aerobic stability, chemical composition, and yeast and mold counts were determined. The DNA in treated and untreated silages was extracted using lysozyme/sodium dodecyl sulfate lysis and phenol/chloroform and used as a template for a conventional PCR with primers designed on the 16S rRNA gene to detect the presence of L. buchneri in all silage samples. Acetic acid concentration was greater in B2 silages versus others (6.46 vs. 4.23% DM). Silages treated with BUC and B500 had lower pH and propionic acid concentration and greater lactic acid concentration than others. The B500 silage had the greatest lactic:acetic acid ratio (1.54 vs. 0.41), and only treatment with BUC reduced DM losses (5.0 vs. 14.3%). Yeast and mold counts were less than the threshold (105) typically associated with silage spoilage and did not differ among treatments. Consequently, all silages were very stable (>250 h). Aerobic stability was not improved by any inoculant but was lower in B500 silages versus others (276 vs. 386 h). The conventional PCR confirmed the presence of similar populations of L. buchneri in all silages. This may have contributed to the prolonged aerobic stability of all silages.

Low Permeability to Oxygen of a New Barrier Film Prevents Butyric Acid Bacteria Spore Formation in Farm Corn Silage

The outgrowth of Clostridium spore-forming bacteria causes late blowing in cheeses. Recently, the role of air diffusion during storage and feed-out and the role of aerobic deterioration has been shown to indirectly favor butyric acid bacteria (BAB) growth and to determine the presence of high concentrations of BAB spores in farm tank milk. A new oxygen barrier (OB) film was tested and compared with conventional polyethylene (ST). The objective was to verify whether the OB film could prevent BAB spore formation in whole-crop corn silage during storage on 2 commercial farms with different potential silage spoilage risks. Two bunkers (farms 1 and 2) were divided into 2 parts along the length so that half the feed-out face would be covered with ST film and the other half with OB film. Plastic net bags with freshly chopped corn were buried in the upper layer and in the central part (CORE) of the bunkers. The silos were opened in summer and fed out at different removal rates (19 vs. 33 cm/d). Herbage at ensiling, silage at unloading, and silage after air exposure (6 and 15 d) were analyzed for pH, nitrate, BAB spores, yeasts, and molds. The BAB spores in herbages at ensiling were 2.84 log10 most probable number (MPN)/g, with no differences between treatments or farms. Nitrate was below the detection limit on farm 1 and exceeded 2,300mg/kg of fresh matter on farm 2. At unloading, the BAB spores in the ST silage on farm 1 were greater than 5 log10 MPN/g, whereas in the CORE and the OB silages, they were approximately 2 log10 MPN/g. The ST silage had the greatest pH (5.89), the greatest mold count (5.07 log10 cfu/g), and the greatest difference between silage temperature and ambient temperature (dTsection–ambient). On farm 2, the ST silage had the greatest concentration of BAB spores (2.19 log10 MPN/g), the greatest pH (4.05), and the least nitrate concentration compared with the CORE and the OB silages. Pooled data on BAB spores collected from aerobically deteriorated samples showed a positive relationship with pH, mold count, and dTsection–ambient and a negative relationship with nitrate concentration. A high concentration of BAB spores (>5 log MPN/g) was associated with visible spoilage, high pH values (>5.00), high mold counts (>5 log cfu/g), high dTsection–ambient, and nitrate below 1,000mg/kg of fresh matter. We concluded that the use of a film with reduced oxygen permeability prevented the outgrowth of BAB spores during conservation and feed-out, and it could improve the microbiological quality of corn silage by eliminating the fractions of silage with high BAB spore concentrations.