**6. Optimizing fiber and carbohydrate digestibility**

The main metabolic activity of LAB during the ensiling process consists of reducing soluble carbohydrates to organic acids to acidify and preserve the forage for long-term storage. It has been observed that animal performance has been increased following the use of microbial inoculants, even if no or small changes in silage fermentation parameters were observed [2]. Future research is needed to explain why these improvements are observed. Yet, past research has made several important advancements.

As discussed previously, inoculation with LAB contributes to important modifications of the silage microbiota, for both the bacterial and the fungal communities. Some of these modifications could partly explain the contribution of the inoculant to one or more nutritional characteristics of silage. This could also support the theory of an indirect positive impact of these nutritional characteristics to the rumen microbial population and functions.

The rumen environment may also be affected by LAB forage inoculants. Some strains of LAB used as inoculants were shown to survive in the rumen fluid [46] and shift gas production toward other products or microbial cells [47]. Weinberg et al. [48] observed that LAB inoculants applied at ensiling, or into the rumen, had the potential to increase DM and fiber digestibility.

Studies using different inoculants showed increases in animal performance and milk production [49]. Mohammed et al. were also able to quantify elevated levels of *L. plantarum* in the rumen of cows eating the treated silage [50].

To help explain this improved animal performance, results from the studies of LAB used as a human probiotic may offer some clues. In a review of the metabolism of oligosaccharides and starch by lactobacilli, Gänzle and Follador [51] described limitations of the conversion of oligosaccharides since most related enzymes in LAB are active intracellularly and their substrates must be transported inside the cells to hydrolyze (**Figure 3**). By studying the genome of several LAB species, they report that most lactobacilli could generally metabolize α-glucans. They would require contribution of a trans-membrane transporter in order to hydrolyze small

**163**

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

oligosaccharides. Like some other lactobacilli, *L. plantarum* genome includes a gene encoding for an extracellular amylase with endoamylases activity. The presence of this amylase in the genome is strain specific as reported by Hattingh [52] for strains

*Starch granules of corn after several months of ensiling. Rod shape bacteria, putatively LAB, were thriving on fiber particles surrounding the starch granule but not on the granules. Micrograph provided by Lallemand* 

Selecting strains with a functional trait, for example, fiber- or starch-degrading functions, represents the initial step in the development of a new inoculant. The strain has to cope with the different stresses of silage and also compete against epiphytic LAB and other microorganisms. The function has also to be expressed under the targeted microbial niche. The extracellular enzymes then have to be optimized for the acidic conditions and cope with the specific nature of polysac-

Access by fibrolytic enzymes to cellulose is difficult due to steric hindrance of the lignin-hemicellulose-homocellulose matrix. Improving cellulose degradation was targeted by selecting a LAB strain producing ferulate esterase [53]. This enzyme

releases ferulic acid from arabinoxylans, improving access to other fibrolytic

**7. Improving animal performance with LAB forage inoculants**

fermentation. However, these improvements are difficult to quantify.

More research is needed in this area. The complexity and dynamic of the microbial communities following the inoculation provide an important challenge in understanding the impact and role of the key players involved in this beneficial

The expected effects of using a LAB forage additive are improved fermentation and enhanced feedout stability, which in turn lead to better recovery of nutrients and DM. However, expectations from producers are often beyond better silage characteristics, such as improvements in feed efficiency and, subsequently, animal performance. Scientific evidence shows positive impact from the use of microbial inoculants on increases animal performance and production, in addition to enhancing the

enzymes of the lignin-cellulose layer within cell walls.

effect of microbial silage additive [54].

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

of *L. plantarum* isolated from barley.

charide substrates.

**Figure 3.**

*Specialties Inc.*

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

#### **Figure 3.**

*New Advances on Fermentation Processes*

also of lower nutritional quality.

important advancements.

rumen microbial population and functions.

potential to increase DM and fiber digestibility.

*L. plantarum* in the rumen of cows eating the treated silage [50].

these forage additives (personal communication).

smell and high palatability (personal communication).

**6. Optimizing fiber and carbohydrate digestibility**

Producers may often choose not to purchase forage inoculants due to the cost of the products. Other top reasons that influence purchase decisions are (1) not believing inoculants work, (2) lack of knowledge, or (3) lack of specific equipment for inoculating the forage. With all these factors in mind, there is a strong need for proper education on the application and showing the cost-to-benefit calculation of

Even though some producers are nonusers, they believe that inoculants have the potential to improve consistency of silage quality, enhance ration quality, and increase feedout stability. In the same question, just 40% answered that improving ROI is one of the most important benefits of purchasing inoculants. Even though some producers do not associate inoculants with contributing to overall herd ROI and profitability, they positively associate the word "fresh" to silage having a good

During typical field and harvest management conditions, silage losses are easily reported between 15 and 20%. If inoculant use can reduce DM losses by 5 percentile points, there would be savings of \$2000 (US\$) per thousand tons of silage, assuming the silage is valued at \$40.00 (US\$) per ton FM. Moreover, silage with high degree of deterioration not only has less overall tonnage to be fed, but the feed is

The main metabolic activity of LAB during the ensiling process consists of reducing soluble carbohydrates to organic acids to acidify and preserve the forage for long-term storage. It has been observed that animal performance has been increased following the use of microbial inoculants, even if no or small changes in silage fermentation parameters were observed [2]. Future research is needed to explain why these improvements are observed. Yet, past research has made several

As discussed previously, inoculation with LAB contributes to important modifications of the silage microbiota, for both the bacterial and the fungal communities. Some of these modifications could partly explain the contribution of the inoculant to one or more nutritional characteristics of silage. This could also support the theory of an indirect positive impact of these nutritional characteristics to the

The rumen environment may also be affected by LAB forage inoculants. Some strains of LAB used as inoculants were shown to survive in the rumen fluid [46] and shift gas production toward other products or microbial cells [47]. Weinberg et al. [48] observed that LAB inoculants applied at ensiling, or into the rumen, had the

Studies using different inoculants showed increases in animal performance and milk production [49]. Mohammed et al. were also able to quantify elevated levels of

To help explain this improved animal performance, results from the studies of LAB used as a human probiotic may offer some clues. In a review of the metabolism of oligosaccharides and starch by lactobacilli, Gänzle and Follador [51] described limitations of the conversion of oligosaccharides since most related enzymes in LAB are active intracellularly and their substrates must be transported inside the cells to hydrolyze (**Figure 3**). By studying the genome of several LAB species, they report that most lactobacilli could generally metabolize α-glucans. They would require contribution of a trans-membrane transporter in order to hydrolyze small

**162**

*Starch granules of corn after several months of ensiling. Rod shape bacteria, putatively LAB, were thriving on fiber particles surrounding the starch granule but not on the granules. Micrograph provided by Lallemand Specialties Inc.*

oligosaccharides. Like some other lactobacilli, *L. plantarum* genome includes a gene encoding for an extracellular amylase with endoamylases activity. The presence of this amylase in the genome is strain specific as reported by Hattingh [52] for strains of *L. plantarum* isolated from barley.

Selecting strains with a functional trait, for example, fiber- or starch-degrading functions, represents the initial step in the development of a new inoculant. The strain has to cope with the different stresses of silage and also compete against epiphytic LAB and other microorganisms. The function has also to be expressed under the targeted microbial niche. The extracellular enzymes then have to be optimized for the acidic conditions and cope with the specific nature of polysaccharide substrates.

Access by fibrolytic enzymes to cellulose is difficult due to steric hindrance of the lignin-hemicellulose-homocellulose matrix. Improving cellulose degradation was targeted by selecting a LAB strain producing ferulate esterase [53]. This enzyme releases ferulic acid from arabinoxylans, improving access to other fibrolytic enzymes of the lignin-cellulose layer within cell walls.

More research is needed in this area. The complexity and dynamic of the microbial communities following the inoculation provide an important challenge in understanding the impact and role of the key players involved in this beneficial effect of microbial silage additive [54].

## **7. Improving animal performance with LAB forage inoculants**

The expected effects of using a LAB forage additive are improved fermentation and enhanced feedout stability, which in turn lead to better recovery of nutrients and DM. However, expectations from producers are often beyond better silage characteristics, such as improvements in feed efficiency and, subsequently, animal performance.

Scientific evidence shows positive impact from the use of microbial inoculants on increases animal performance and production, in addition to enhancing the fermentation. However, these improvements are difficult to quantify.

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 production [55].

Oliveira et al. [43] analyzed 31 studies—including animal performance results. This meta-analysis showed that microbial inoculation at a rate of at least 105 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 and diet, inoculant bacterial species and application rate (105 vs. 106 CFU/g of forage), and level of milk production.

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 environment, resulting in improved effects on animal performance.

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 needs to be adapted to target the correct ecological niche.

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

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 inconsistent.

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* environments needs additional research to be better understood.

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 generation of this type of additive.

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

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

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

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

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

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

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

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

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

agricultural sector [65].

preserved forages.

(freestall stocking—lower housing density).

observed by Daniel et al. [69].

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