**3. Characteristics of lactic acid bacteria (LAB) present in tropical grasses**

Lactic acid bacteria are gram-positive. They are negative catalase, do not present motility and do not produce spores. The final fermentation product is lactic acid, however, some groups produce considerable amount of CO2, ethanol and other metabolites, these being called heterofermentative. Particularly, *Lactobacillus plantarum* are the larger silage fermentative bacteria (Ohmomo et al., 2002). *Lactococcus, Streptococcus* and *Enterococcus* are very important in the fermentation initial stage, because they keep an acidic environment, which then becomes, predominantly colonized by Lactobacillus.

Fermentation can be considered the anaerobic decomposition of organic compounds to organic products, which may be metabolized by the cells without the oxygen intervention. Under anaerobiosis conditions, phosphorylation occurs at the substrate level in which an organic acid donates electrons to a NAD+, so that in microorganisms the NAD+ needs to be regenerated and it occurs through various oxidation-reduction pathways, involving pyruvate or its derivatives, like acetyl-CoA. Pyruvate is a key molecule of fermenting microorganisms, from that, it can be formed by several compounds such as: acetaldehyde (ethanol), acetyl-CoA, lactate, acetoacetate (butyrate, isopropanol), acetoin (2, 3-butanediol, diacetyl), acetate, oxaloacetate, succinate, and propionate.

The homofermentative LAB are characterized by a faster fermentation rate, reduced proteolysis, higher lactic acid concentrations, lower acetic and butyric acids contents, lower ethanol content, and higher energy and dry matter recovery. Heterofermentative bacteria utilize pentoses as substrate for acetic and propionic acids production, which are effective at controlling fungi, at low pH values. The facultative heterofermentative use the same hexoses pathway of homofermentative, but they are able to ferment pentoses, as they have aldolase and fosfocetolase enzymes. The facultative heterofermentative may produce lactic and acetic acids when the substrate is a pentose, or lactic acid, ethanol and CO2 when hexose is the substrate, due to the need of oxidation of two NAD molecules produced in the glycolytic pathway (White, 2000).

Table 3 summarizes the main lactic acid bacteria found in silages including some *Lactobacillus* with heterofermentative metabolism and some *Leuconostoc* species which have heterofermentative metabolism also.

For species of *Lactobacillus* genus were defined three groups based on the presence or absence of aldolase and fosfocetolase enzymes (Kandler and Weiss, 1986). These groups are as follows:


**Table 3.** Main lactic acid bacteria found in silages. (Woolford, 1984)

340 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

higher quality silage.

pathway (White, 2000).

as follows:

heterofermentative metabolism also.

It is observed that tropical grasses have characteristics influenced by several factors, ranging from species choice to maturity stage at harvest. These factors are primordial in silage confection, because if handled properly, they will favor the LAB development, resulting in

To understand how the factors related to the grass management will influence the LAB population dynamics consequently the fermentation, it is necessary to know the

**3. Characteristics of lactic acid bacteria (LAB) present in tropical grasses** 

Lactic acid bacteria are gram-positive. They are negative catalase, do not present motility and do not produce spores. The final fermentation product is lactic acid, however, some groups produce considerable amount of CO2, ethanol and other metabolites, these being called heterofermentative. Particularly, *Lactobacillus plantarum* are the larger silage fermentative bacteria (Ohmomo et al., 2002). *Lactococcus, Streptococcus* and *Enterococcus* are very important in the fermentation initial stage, because they keep an acidic environment,

Fermentation can be considered the anaerobic decomposition of organic compounds to organic products, which may be metabolized by the cells without the oxygen intervention. Under anaerobiosis conditions, phosphorylation occurs at the substrate level in which an organic acid donates electrons to a NAD+, so that in microorganisms the NAD+ needs to be regenerated and it occurs through various oxidation-reduction pathways, involving pyruvate or its derivatives, like acetyl-CoA. Pyruvate is a key molecule of fermenting microorganisms, from that, it can be formed by several compounds such as: acetaldehyde (ethanol), acetyl-CoA, lactate, acetoacetate (butyrate, isopropanol), acetoin (2, 3-butanediol,

The homofermentative LAB are characterized by a faster fermentation rate, reduced proteolysis, higher lactic acid concentrations, lower acetic and butyric acids contents, lower ethanol content, and higher energy and dry matter recovery. Heterofermentative bacteria utilize pentoses as substrate for acetic and propionic acids production, which are effective at controlling fungi, at low pH values. The facultative heterofermentative use the same hexoses pathway of homofermentative, but they are able to ferment pentoses, as they have aldolase and fosfocetolase enzymes. The facultative heterofermentative may produce lactic and acetic acids when the substrate is a pentose, or lactic acid, ethanol and CO2 when hexose is the substrate, due to the need of oxidation of two NAD molecules produced in the glycolytic

Table 3 summarizes the main lactic acid bacteria found in silages including some *Lactobacillus* with heterofermentative metabolism and some *Leuconostoc* species which have

For species of *Lactobacillus* genus were defined three groups based on the presence or absence of aldolase and fosfocetolase enzymes (Kandler and Weiss, 1986). These groups are

characteristics related to metabolism and the main tropical grass species.

which then becomes, predominantly colonized by Lactobacillus.

diacetyl), acetate, oxaloacetate, succinate, and propionate.

**Group 1:** Homofermentative, which ferment hexoses homolacticly almost exclusively to lactic acid (>85%), however, they are unable to ferment pentoses, due to the fosfocetolase enzyme lack;


**Group 2**: Facultative heterofermentative that use the same hexoses pathway as the one of group 1, but are able to ferment pentoses, since they have aldolase and fosfocetolase enzymes;


**Group 3**: Obligately heterofermentative, which ferment hexoses, forming lactic acid, ethanol (or acetic acid) and CO2, being able to still ferment pentose to form lactic and acetic acids.

Lactic Acid Bacteria in Tropical Grass Silages 343

*Lactobacillus plantrum* was the predominant species in mombaça grass (*Panicum maximum*)

Species 0 4 8 100 *Lactobacillus plantarum* 35 84 87 44 *Leuconostoc spp.* 59 0 0 0 *Lactobacillus fermentum* 6 6 4 7 *Lactobacillus brevis* 0 10 9 49 **Table 4.** Percentage of lactic acid bacteria species isolated from sorghum silage (Tjandraatmadja et al.,

Species Days after ensiling

It is evident that *Lactobacillus plantarum* and the species from the *Pediococcus* genus are prevalent in forage plants. The species from *Leuconostoc* genus are present in plants. However, according to Chunjian et al. (1992) and Tjandraatmadja et al. (1991) they

According Lücke (2000), gram-negative bacteria are less susceptible to the action of bacteriocins from lactic acid bacteria due to the presence of outer membrane, which limits the access of peptides to the target site. In addition, the gram-negative bacteria are more sensitive to organic acid produced by LAB compared with the gram-positive bacteria

Santos et al. (2011) conducted a study aiming to characterize and quantify microbial populations in signal grass harvested at different regrowth ages. The six lactic acid bacteria strains isolated from signal grass were characterized according Gram staining, catalase enzyme reaction, and bacilli form, submitted to growth and identification tests. The microbial isolates identification was performed by carbohydrates fermentation in API 50 CH

*Lactobacillus plantarum* 21 39 47 *Lactobacillus coryneformis* 6 21 0 *Leuconostoc spp.* 27 12 0 *Enterococcus faeceium* 0 10 4 *Enterococcus faecalis* 3 0 3 *Pediococcus spp.* 30 12 31 *Lactobacillus brevis* 7 6 11 *Lactobacillus fermentum* 6 0 4 **Table 5.** Main lactic acid bacteria (%) isolated from grasses (*Panicum maximum* cv Hami; *Digitaria* 

*decumbens*; *Setaria sphacelata* cv Kazungula) (Tjandraatmadja et al., 1994).

disappear early in the ensiling process.

(Ennahar et al., 2000).

kit (BioMéurix - France).

Days after ensiling

*P. maximum D. decumbens S. sphacelata* 

and signal grass (*Brachiaria decumbens*).

1991).


The homofermentative LAB presence in silage is extremely necessary. CO2 generation results in carbon loss, ie, nutrient losses in plant materials. Therefore, homofermentative bacteria such as *Lactobacillus plantarum*, are desirable in the fermentation of silage.

Several lactic acid bacteria have antimicrobial peptides known as bacteriocins which are responsible for inhibiting the growth of or related species which have similar nutritional requirements. The bacteriocins action mechanism involves interaction with specific receptors on the cell membrane to its insertion resulting in proton-motive force dissipation and pores formation, which may cause cell viability loss (Montville and Chen, 1998; Ennahar et al., 2000).

According Lücke (2000), gram-negative bacteria are less susceptible to the action of bacteriocins from lactic acid bacteria due to the presence of outer membrane, which limits the access of peptides to the target site. In addition, the gram-negative bacteria are more sensitive to organic acid produced by LAB compared with the gram-positive bacteria (Ennahar et al., 2000).

Table 4 presents the lactic acid bacteria percentages isolated from sorghum plant in a study conducted by Tjandraatmadja et al. (1991). Likewise, *Lactobacillus plantarum* was the predominant specie and it kept 100 days after ensiling. It was observed the presence of *Lactobacillus fermentum* and *Lactobacillus brevis* heterofermentative bacteria in large quantities at the end of the ensiling process. It demonstrates that these bacteria are active during the fermentation process.

Evaluating the microbiological composition of silages obtained from three different grass species, Tjandraatmadja et al. (1994) found that *Lactobacillus plantarum* and *Pediococcus spp.* are the predominant species, observing one more time the presence of significant amounts of *Lactobacillus brevis* and *Lactobacillus fermentum* (Table 5). Santos et al. (2006) observed that *Lactobacillus plantrum* was the predominant species in mombaça grass (*Panicum maximum*) and signal grass (*Brachiaria decumbens*).

342 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

27. *L. bifermentans* 36. *L. halotolerans 28. L. brevis* 37. *L. hilgardii 29. L. buchneri* 38. *L. kandleri* 30. *L. collinoides 39. L. kefir* 31. *L. confusus* 40. *L. minor* 32. *L. divergens* 41. *L. reuteri 33. L. fermentum* 42. *L. sanfrancisco* 34. *L. fructivorans* 43. *L. vaccinostercus*  35. *L. fructosus* 44. *L viridescens*

acids.

Ennahar et al., 2000).

(Ennahar et al., 2000).

fermentation process.

**Group 3**: Obligately heterofermentative, which ferment hexoses, forming lactic acid, ethanol (or acetic acid) and CO2, being able to still ferment pentose to form lactic and acetic

Mandatory heterofermentative *Lactobacillus*

The homofermentative LAB presence in silage is extremely necessary. CO2 generation results in carbon loss, ie, nutrient losses in plant materials. Therefore, homofermentative

Several lactic acid bacteria have antimicrobial peptides known as bacteriocins which are responsible for inhibiting the growth of or related species which have similar nutritional requirements. The bacteriocins action mechanism involves interaction with specific receptors on the cell membrane to its insertion resulting in proton-motive force dissipation and pores formation, which may cause cell viability loss (Montville and Chen, 1998;

According Lücke (2000), gram-negative bacteria are less susceptible to the action of bacteriocins from lactic acid bacteria due to the presence of outer membrane, which limits the access of peptides to the target site. In addition, the gram-negative bacteria are more sensitive to organic acid produced by LAB compared with the gram-positive bacteria

Table 4 presents the lactic acid bacteria percentages isolated from sorghum plant in a study conducted by Tjandraatmadja et al. (1991). Likewise, *Lactobacillus plantarum* was the predominant specie and it kept 100 days after ensiling. It was observed the presence of *Lactobacillus fermentum* and *Lactobacillus brevis* heterofermentative bacteria in large quantities at the end of the ensiling process. It demonstrates that these bacteria are active during the

Evaluating the microbiological composition of silages obtained from three different grass species, Tjandraatmadja et al. (1994) found that *Lactobacillus plantarum* and *Pediococcus spp.* are the predominant species, observing one more time the presence of significant amounts of *Lactobacillus brevis* and *Lactobacillus fermentum* (Table 5). Santos et al. (2006) observed that

bacteria such as *Lactobacillus plantarum*, are desirable in the fermentation of silage.


**Table 4.** Percentage of lactic acid bacteria species isolated from sorghum silage (Tjandraatmadja et al., 1991).


**Table 5.** Main lactic acid bacteria (%) isolated from grasses (*Panicum maximum* cv Hami; *Digitaria decumbens*; *Setaria sphacelata* cv Kazungula) (Tjandraatmadja et al., 1994).

It is evident that *Lactobacillus plantarum* and the species from the *Pediococcus* genus are prevalent in forage plants. The species from *Leuconostoc* genus are present in plants. However, according to Chunjian et al. (1992) and Tjandraatmadja et al. (1991) they disappear early in the ensiling process.

According Lücke (2000), gram-negative bacteria are less susceptible to the action of bacteriocins from lactic acid bacteria due to the presence of outer membrane, which limits the access of peptides to the target site. In addition, the gram-negative bacteria are more sensitive to organic acid produced by LAB compared with the gram-positive bacteria (Ennahar et al., 2000).

Santos et al. (2011) conducted a study aiming to characterize and quantify microbial populations in signal grass harvested at different regrowth ages. The six lactic acid bacteria strains isolated from signal grass were characterized according Gram staining, catalase enzyme reaction, and bacilli form, submitted to growth and identification tests. The microbial isolates identification was performed by carbohydrates fermentation in API 50 CH kit (BioMéurix - France).

Regarding the predominant bacteria identification in signal grass plants, it is observed in Table 6 that all isolates had the form of short bacilli with rounded ends, arranged in pairs or in short chains (3-4 cells). All of them showed negative reaction to the catalase enzyme test and were gram-positive. None of the strains grew at pH 9.6 and 6.5% NaCl, but all grew at pH 7.2 and 4% NaCl at 45°C.

Lactic Acid Bacteria in Tropical Grass Silages 345

Isolated strain Lactobacillus

EB1 EB2 EB5 EB6 plantarum

Glycerol - - - - - Erythritol (+) (+) (+) (+) - D-arabinose - - - - - L-arabinose + + + + + Ribose + + + + + D-xylose - - - - - L-xylose - - - - - Adonitol - - - - β-methyl D-xyloside - - - - - Galactose + + + + + D-glucose + + + + + D-frutose + + + + + D-mannose + + + + + L-sorbose - - - + - Rhamnose (+) (+) (+) (+) - Dulcitol - - - - - Inositol - - - - - Mannitol + + + + + Sorbitol + + + + + α-methyl D-mannose - - - - + α-methyl D-glycoside - - - - - N-acetyl-glucosamine + + + + + Amygdaline + + + + + Arbulin + + + + + Esculin + + + + + Salicin + + + + + Cellobiose + + + + + Maltose + + + + + Lactose + + + + + Melibiose + + + + + Saccharose + + + + + Trehalose + + + + + Inulin - - - - - Melezitose + + + + + D-raffinose + + + + + Amidon - - - - - Glycogene - - - - - Xylitol - - - - β-gentibiose + + + + + D-turanose + + + + +


**Table 6.** Morphology and biochemical characteristics of the isolates EB1, EB2, EB3, EB4, EB5, EB6, signal grass plant (*Brachiaria decumbens* cv. Basiliski). \*DB: diplobacillus. (Santos et al., 2011).

According with the carbohydrate fermentation pattern (Table 7), the isolates EB1, EB2, EB5 e EB6 were identified as *Lactobacillus plantarum* with 99.9% of similarity.

The *Lactobacillus plantarum* specie, identified as dominant in signal grass plants (*Brachiaria decumbens* cv. Basiliski) (Santos et al., 2011) has been isolated and characterized as major species in several cultures. Lin et al. (1992) evaluated the corn and alfalfa autochthonous microbiota and found that from the total lactic acid bacteria isolated, over 90% were homofermentative lactic bacteria, being *Lactobacillus plantarum* the predominant specie. Tjandraatmadja et al. (1994), in studies on tropical grasses silage, found *Lactobacillus plantarum* and *Pediococcus spp*. as the predominant species.


pH 7.2 and 4% NaCl at 45°C.

Test

Regarding the predominant bacteria identification in signal grass plants, it is observed in Table 6 that all isolates had the form of short bacilli with rounded ends, arranged in pairs or in short chains (3-4 cells). All of them showed negative reaction to the catalase enzyme test and were gram-positive. None of the strains grew at pH 9.6 and 6.5% NaCl, but all grew at

Isolated strain

Growth at different pH

Growth at different salt concentartion (NaCl)

Growth at different temperatures (T oC)

*plantarum* 

EB1 EB2 EB3 EB4 EB5 EB6 *Lactobacillus* 

form bacillus bacillus bacillus bacillus bacillus bacillus bacillus

Arranjement DB\* DB DB DB DB DB DB Gram + + + + + + + Catalasis - - - - - - -

7,2 + + + + + + + 9,6 - - - - - - -

NaCl 4% + + + + + + + NaCl 6,5% - - - - - - -

15 oC + + + + + + + 45 oC + + + + + + +

According with the carbohydrate fermentation pattern (Table 7), the isolates EB1, EB2, EB5 e

The *Lactobacillus plantarum* specie, identified as dominant in signal grass plants (*Brachiaria decumbens* cv. Basiliski) (Santos et al., 2011) has been isolated and characterized as major species in several cultures. Lin et al. (1992) evaluated the corn and alfalfa autochthonous microbiota and found that from the total lactic acid bacteria isolated, over 90% were homofermentative lactic bacteria, being *Lactobacillus plantarum* the predominant specie. Tjandraatmadja et al. (1994), in studies on tropical grasses silage, found *Lactobacillus* 

**Table 6.** Morphology and biochemical characteristics of the isolates EB1, EB2, EB3, EB4, EB5, EB6, signal grass plant (*Brachiaria decumbens* cv. Basiliski). \*DB: diplobacillus. (Santos et al., 2011).

EB6 were identified as *Lactobacillus plantarum* with 99.9% of similarity.

*plantarum* and *Pediococcus spp*. as the predominant species.

#### Lactic Acid Bacteria in Tropical Grass Silages 345


Lactic Acid Bacteria in Tropical Grass Silages 347

According to Ohmomo et al. (2002) in the early fermentation stage, Lactococus species, such as Lactococcus lactis, Enterococcus faecalis, Pediococcus acidilactici, Leuconostoc mesenteroides, and Lactobacillus species such as Lactobacillus plantarum, Lactobacillus cellobioses grow together with aerobic microorganisms like yeasts, molds and aerobic bacteria, due to the presence of air between the plant particles. At the same time, it is the plant respiration process. To promote the fermentation, an anaerobic environment is formed making the population to become predominantly composed by LAB, basically Lactococcus

At the final fermentation stage, *Lactobacillus* becomes prevalent, due to their tolerance to the acidity. However, the silage LAB is pretty well diversified, depending on plant material properties, silage technology and silo type. The LAB predominance change from *Lactococcus* to *Lactobacillus* usually occurs in the final fermentation stage. According to Langston et.al (1960), these chemical changes is resulted from bacterial or plant enzymes action making the conversion of carbohydrates into other components such as gas and organic acids, as well as

The LAB use as microbial inoculants have been widely documented in research (Penteado et al., 2007; Ávila et al., 2009a; Ávila et al., 2009b; Jalč et al., 2009; Reich & Kung Jr., 2010).

Zopollatto et al. (2009) in a meta-analysis study (1999-2009) found a data limitation on the effect of microbial additives in silage quality. They observed that the number of conduced studies is not enough to provide conclusive positions regarding the effects of additives, emphasizing also the data scarcity in certain areas, such as dairy cattle performance. The results documented by these authors show that the magnitude of the response, especially on animal performance, is low. Thus, the justification for the use of additives should be evaluated considering the losses reduction in silage and the higher plant nutritional value preservation. Furthermore, they found that the response intensity varies with plant species

However, studies conducted in the 1980s and 1990s had already shown that the fermentation responses differ between strains of the same species (Wooflford & Sawczyc 1984, Hill, 1989; Fitzsimons et al., 1992). Hill (1989) found that inoculating corn silage with two *Lactobacillus plantarum* strains isolated from corn and grass, the dominant strain after ensiling was the isolated from corn. The same was observed for the grass silage, where the

Many inconclusive results observed in silage fermentation studies may be related to this principle, which must have been overlooked. The specificity between the forage specie and its epiphytic microflora implicates in the need for studies related with isolation and identification of the main microorganism groups present in the forage used for silage. Ávila et al. (2009b) isolated *Lactobacillus buchneri* strains from sugar cane (*Saccharum officinarum* L.) and found that *L. buchneri* UFLA SIL 72 addition reduced the fungi population and the ethanol concentration in silages. Santos et al. (2007) observed reduction in ammonia concentration and enterobacteria population in mombaça grass silage (*Panicum maximum*) inoculated with *Lactobacillus plantarum*, which were isolated from the epiphytic microflora.

the partial protein breakage resulting in formation of non-protein structures.

and microorganism studied, suggesting a specificity between these components.

dominant lactic bacteria strain of were the one isolated from grass.

and Lactobacillus.

**Table 7.** Carbohydrate fermentation pattern of the isolates EB1, EB2, EB5, and EB6, signal grass plants (*Brachiaria decumbens* cv. Basiliski). + Intense fermentation, no fermentation; (+) less intense fermentation (Santos et al., 2011).

In another study, Rocha (2003), evaluating the lactic acid bacteria populations in elephant grass plants cv. Cameroon (*Pennisetum purpureum* Schum) identified the isolates as *Lactobacillus casei ssp. Pseudoplantarum,* using the carbohydrate fermentation profile as an identification criterion. Santos et al. (2011) observed the *Lactobacillus plantarum* as LAB predominant specie in signal grass (*Brachiaria decumbens* Stapf). Based on the reported above, it is observed that there were differences between the LAB dominant species among the cultures evaluated, however *Lactobacillus plantarum* has been identified as the predominant specie for most plants.
