**8. Understanding the impact of ensiling on a global scale**

Silage represents an important part of animal diets. Challenges in production, reducing losses, and the impact on agricultural practices are often overlooked compared to other nutritive benefits provided. Microbial activity during fermentation produces several compounds besides the desirable organic acids. Some of those compounds were identified as negatively influencing air quality around farms. They are classified as alcohols, esters, and aldehydes [63, 64]. Production and volatilization of these compounds contribute to a reduction in quality of the stored feed, inducing ground-level ozone, and influence emission of greenhouse gases by the agricultural sector [65].

Forage characteristics and yield potential are influenced by several factors, including geographic and meteorological conditions. New analytical technologies and statistical methodologies now allow more comprehensive understanding of ensiling techniques and analyze productivity and nutritional quality on a broader scale.

Comparison between farms is always challenging, even between neighboring farms, since they could differ on animal husbandry, genetics of the herd, field management, harvesting periods, type and size of silos, management of the silos, and so on. On a broader geographic area, these differences will be minimized by the inclusion of higher numbers of farms, up to a point that patterns of variations could be analyzed. This type of analysis was performed by Gallo et al. in two recent studies [65, 66]. The team used a multivariate analysis technique, Principal Component Analysis (PCA), to evaluate ensiling of corn silage on 68 dairy farms [66] and generated a fermentation quality index to rank the silage [67]. Using 36 variables measured on every individual samples, they were able to group the silage according to quality parameters in relation to silo management techniques to discriminate between well-preserved and poorly preserved forages.

At the farm level, quality parameters from silage and feed analysis reports could be analyzed to identify trends in animal health and performance. Different types of data could be collected and analyzed to understand the main variations in milk quality and yield on a yearly or multi-year basis. Linking milk quality parameters to farm management practices was performed following the analysis of milk constituent using Fourier transformed mid-infrared spectroscopy results gathered from 33 farms [68]. The difference between observed high and low *de novo* fatty acid composition of milk allowed characterizing differences in feeding management (one or two feeding periods—fresher silage) and higher animal management scores (freestall stocking—lower housing density).

Up to now, few data analysis included data specific to silage fermentation beside the main fermentation acids. This is truer for other parameters related to silage production and management, including yield from the field, management of the silos, losses during fermentation, or type of silage additive used. This needs to be addressed considering important changes to the microbiota following the inoculation discussed previously and to differentiate in other fermentation chemicals or their relationship with the nature of the additives applied, as observed by Daniel et al. [69].

#### **9. Increasing the understanding of the fermentation process**

Compared to other research domains in agricultural and environmental sciences, using new sequencing technologies to understand the dynamics of the

*New Advances on Fermentation Processes*

age), and level of milk production.

production [55].

Some of the existing theories are that these bacteria may have a beneficial influence in the rumen environment, including altering the fermentation profile and interacting with the animal's existing digestive microbiota [48] and inhibiting undesirable microorganisms, which subsequently help reduce the potential for toxin

Oliveira et al. [43] analyzed 31 studies—including animal performance results.

colony-forming units (CFU) of LAB per gram of forage significantly increased milk production by 0.37 kg/d, increased DM intake, and had no effect on feed efficiency and total tract DM digestibility. Furthermore, the contents of milk fat and milk protein tended to be higher for cows fed inoculated silage. The effects on increased milk production due to LAB inoculation happened regardless of the type of forage

Among the animal performance trials, there are cases when the inoculant had no effect on the silage fermentation compared to untreated silage, although animal productivity was increased [56]. Therefore, this indicates that some LAB strains are positively affecting the rumen microbial community and the digestive tract envi-

Recent research has described these effects by evaluating the impact of inoculated silages in the populations of the rumen microbial community, but no significant changes were observed [51]. However, nitrogen efficiency seemed to be improved due to lower levels of milk urea nitrogen in cows fed inoculated silage and greater ruminal DM digestibility on the inoculated silage ration [57]. Since LAB were shown to attach to the fiber inside the rumen [58], isolation methodology

Changes in nitrogen compounds during ensiling are expected. For example, over half of the true protein in alfalfa is degraded to soluble nonprotein compounds initially by the plant's own proteases, and then later by microbial activity within the

Specifically, in the corn kernel or other cereal grain, a protein matrix (prolamins) around the starch granules partially prevents ruminal starch digestion. It has been reported that a slow and continuous breakdown of the prolamins during the storage phase makes the starch more digestible with longer storage time [60]. The authors explained that this effect is due to natural proteolytic mechanisms. This event, however, requires months of storage for the optimum level of starch digestibility in the rumen, in which it is not always feasible in commercial operations. One alternative solution would be to shorten the time necessary for storage to help enhance starch digestibility by inoculation with bacteria that possess high proteolytic activity, but, to date, limited research has been reported and results are

Improvement of fiber digestibility has to be considered in relation to the activity of silage inoculants. Some strains of LAB have been reported to produce the enzyme ferulic acid esterease, which breaks the esterease bond between the lignin and the hemicellulose fraction, leading to more digestible fiber portions for the rumen microorganisms [61]. However, data from animal performance or production studies did not show consistencies in the improvements [61, 62]. While *in vitro* and *in situ* effects may be conceivable, the expression of this phenomenon within *in vivo*

There is still a need to better understand how the microbial additives for ensiling positively affect animal performance, so this should be used as criteria for a new

vs. 106

CFU/g of for-

This meta-analysis showed that microbial inoculation at a rate of at least 105

and diet, inoculant bacterial species and application rate (105

ronment, resulting in improved effects on animal performance.

needs to be adapted to target the correct ecological niche.

cow, resulting in inefficient nitrogen use to the cow [59].

environments needs additional research to be better understood.

generation of this type of additive.

**164**

inconsistent.

microbial communities in silage is recent. McAllister et al. [12] published a review providing a technological and methodological overview. Currently, the number of trials performed using this technique is small enough that repetitions between geographical regions and over time are nonexistent.

Amplicon-based metasequencing represents the entry level of the -omic techniques. For silage research, the industry could also consider metagenomic, proteomic, transcriptomic, or epigenomic as a potential area of study. A review of the possibilities offered by metabolomics in agriculture was recently published [70].

Since ensiling is based on the fermentation of forage crops, knowledge of the metabolic activity of the forage prior to ensiling would be useful. A review by Rasmussen et al. [71] provides an insight into how plants are coping with physiological changes due to breeding strategies, associations with endophytes or rhizobia, responses to nutrients, and, more interestingly, on the metabolic responses to the osmotic stress. Harvesting and wilting will directly influence plant cell activities and nutrient cycling. The authors reported that amino acids, fatty acids, and phytosterols generally decrease following the water stress, while sugars and organic acids increased. Since the fermentation process requires fermentable sugars for optimal acidification of the forage, wilted plants may respond positively toward ensiling. We need to consider the speed of those changes in concentration of metabolites during wilting compared in order to propose a model of the response to an osmotic stress. Ould-Ahmed et al. [72] provided some knowledge on this response to wilting while studying changes in fructan, sucrose, and some associated hydrolytic enzymes, concluding there is a positive effect toward ensiling requirements from the different metabolites.

Metabolomic profiling of silage was performed in a study aiming to understand the role of inoculation with *L. plantarum* or *L. buchneri* in alfalfa silage against a noninoculated control [13]. The authors were able to distinguish all three inoculation treatments by a PCA of the 102 metabolites surveyed. The major metabolites observed were related to amino acids, organic acids, polyhydric alcohols, and some derivatives. One of the main observations was an increase in free amino acids and 4-aminobutyric acid following the inoculation with *L. buchneri* and a decrease in cadaverine and succinic acid following the inoculation with *L. plantarum.*

Testing the same two LAB strains on whole plant corn silage instead of alfalfa, Xu et al. [32] observed a total of 979 chemical substances, from which 316 were identified and quantified. The PCA allowed separating the three inoculation treatments along the first axis, representing nearly 80% of the variations between samples. The second axis was able to further distinguish how inoculation with *L. buchneri* influenced the fermentation. Inoculation with either *L. plantarum* or *L. buchneri* contributes to increase the concentration of amino acids and phenolic acids, 4-hydroxycinnamic acid, 3,4-dihydroxycinnamic acid, glycolic acids, and other organic acids. Inoculation with *L. buchneri* also induces higher concentration of 2-hydroxybutanoic acid, saccharic acid, mannose, and alpha-d-glucosamine-1-phosphate, among others. Other substances were increased by ensiling without specific impact of the inoculants, such as catechol and ferulic acid that could have antioxidant functions.

Metabolomic studies can also be used in defining a metabolomic signature specific of different forage and silage on feed efficiency of ruminants. With the aim of identifying feed efficiency traits in beef cattle, Novais et al. [73] investigated how serum metabolomic profiles could be used to predict feed intake and catabolism. They identified different molecules having feed efficiency role. Two molecules from the retinol pathway, vitamin A synthesis, were significantly associated with feed efficiency (higher concentration of retinal and lower concentration of retinoate).

**167**

*Lactic Acid Bacteria as Microbial Silage Additives: Current Status and Future Outlook*

Besides the studies of Guo et al. [13] and Xu et al. [32], one other study combined different -omic techniques in understanding the ensiling process. The first glimpse of that study was presented at the International Silage Conference in 2018 [8] with data on microbiota dynamic between 1 and 64 days of fermentation of corn silage. Analysis of the amplicon-based metasequences, metagenomic, and metabo-

The potential of transcriptomic was also shortly covered by the *in vitro* trial of Eikmeyer et al. [34], which aimed to understand induction of genes in *L. buchneri* CD034 under different incubation settings. It is expected that additional studies performed directly under ensiling conditions may be published in the next few

Metabolomic data have shown how inoculation of LAB strains induces changes to the ensiled forage that goes beyond the simple production of lactic and acetic acids from the fermentation of sugars under anaerobic conditions. Increases in a whole array of molecules were observed, but the change also extends to the fibers and is either a direct or an indirect effect of the inoculant. Inoculation of alfalfa by *L. plantarum* or *Pediococcus pentosaceus* strains increased the release of different hemicellulose polysaccharides, including homogalacturonan, rhamnogalacturonan,

These new technologies will allow greater understanding of the impact of bacterial inoculants on improvements of the silage and their contribution in the induction of specific genes and proteins by other members of the microbial community at

Food processing residues represent high-energy organic material already used in some way that could include either food-processing residues from food industries or distiller's grains from the ethanol production. These residues could easily be used by farms closely located to the production site, but their relatively high humidity content renders them prone to a rapid deterioration. New ensiling techniques allow mixing them with low moisture forage or grain in order to perform a fermentation

Aiming to use a bakery co-product waste, Rezende et al. [75] tested possibilities of re-hydration, treating it with acid whey or water and levels of urea. The authors found that the resulting silages had reduced populations of molds and yeast by acidification process. However, the initial population of these microorganisms was high, mainly accounting of *Penicillium* and *Aspergillus* spp. Inoculating with a bacteria that could produce antifungal chemicals, including acetic and propionic

TMR silage is an important source of ruminant feed. This practice has been more common in some places, where companies or producers mix wet co-products with dry feeds to prepare TMR that is then preserved as silage. Based on conventional criteria, aerobic deterioration could occur easily in TMR silage, because lactic acid prevails during fermentation and any sugars remaining unfermented can serve as substrates for the growth of yeasts. However, some trials [76, 77] have been shown that when added concentrate, the brewer's grains or soybean curd residue, the main co-products used in TMR preserved do not show heating in the TMR. For the trial with brewers' grain-based TMR, the main bacteria found in the stable silages were *L. buchneri*, but for the soybean curd-based TMR, the main LAB found were *P. acidilactici* and *L. brevis* [78], showing potential association of those bacteria

**10. Co-ensiling forage with food processing waste and TMR** 

that is enclosed in a kind of total mixed ration (TMR) acidic conservation.

acids, might be considered for this kind of co-product.

*DOI: http://dx.doi.org/10.5772/intechopen.89326*

lomic data set is currently underway.

and arabinogalactan from the cell walls [74].

different stages of the ensiling process.

**conservation**

years.

#### *Lactic Acid Bacteria as Microbial Silage Additives: Current Status and Future Outlook DOI: http://dx.doi.org/10.5772/intechopen.89326*

Besides the studies of Guo et al. [13] and Xu et al. [32], one other study combined different -omic techniques in understanding the ensiling process. The first glimpse of that study was presented at the International Silage Conference in 2018 [8] with data on microbiota dynamic between 1 and 64 days of fermentation of corn silage. Analysis of the amplicon-based metasequences, metagenomic, and metabolomic data set is currently underway.

The potential of transcriptomic was also shortly covered by the *in vitro* trial of Eikmeyer et al. [34], which aimed to understand induction of genes in *L. buchneri* CD034 under different incubation settings. It is expected that additional studies performed directly under ensiling conditions may be published in the next few years.

Metabolomic data have shown how inoculation of LAB strains induces changes to the ensiled forage that goes beyond the simple production of lactic and acetic acids from the fermentation of sugars under anaerobic conditions. Increases in a whole array of molecules were observed, but the change also extends to the fibers and is either a direct or an indirect effect of the inoculant. Inoculation of alfalfa by *L. plantarum* or *Pediococcus pentosaceus* strains increased the release of different hemicellulose polysaccharides, including homogalacturonan, rhamnogalacturonan, and arabinogalactan from the cell walls [74].

These new technologies will allow greater understanding of the impact of bacterial inoculants on improvements of the silage and their contribution in the induction of specific genes and proteins by other members of the microbial community at different stages of the ensiling process.
