**2. Tropical grass characteristics**

The forage characteristics that contribute to a good fermentation are: dry matter content, autochthonous plant microbiota and, most importantly, the quantity of soluble carbohydrates. Corn and sorghum are the most appropriate grasses to make silages due to their high soluble carbohydrate contents and dry matter production. However, some studies have shown that different grasses can be utilized if they are ensilage at the right developmental stage or if appropriate additives are used (Zanine et al., 2010).

Lactic Acid Bacteria in Tropical Grass Silages 337

Sugarcane (*Saccharum officinarum* L.) is an important grass due to its tolerance to drought periods and high production potential of dry matter and soluble carbohydrates per hectare. The sugarcane silage confection has been unusual, being used more for animal feeding in its natural form, after cutting and chopping, but it can be recommended when desires to store the sugarcane in its higher nutritional value stage (the dry season) for use throughout the year (Molina et al., 2002). However, according to Santos et al. (2006), sugar cane silage becomes justifiable only when there is a surplus or when accidental burning of sugar cane fields happen, always taking into account the difficulty of achieving a good fermentation pattern due to intense alcoholic fermentation (8% to 17% of dry matter of ethanol) caused by yeast (Kung Jr. & Stanley, 1982), leading to losses of up to 30% of dry matter (Ferreira et al., 2007), accumulation of cell wall components and reduction in the *in vitro* dry matter digestibility. Furthermore, sugar cane silage has low aerobic stability, as result of high residual carbohydrate and lactic acid contents (McDonald et al., 1991). On the other hand, the adoption of the silage method represents a chance to keep the sugarcane nutritional value and allows better logistics for their manufacture and use, what implies the hand labor rationalization, concentrating the sugar cane harvest process in a particular time of year or time period, resulting in easier daily farm handling and maximizing the machinery use.

Thus, there has been a growing number of research projects, especially in Brazil, seeking additives that inhibit yeast growth in sugar cane silages (Valeriano et al., 2009). Nevertheless, some studies have shown that grasses can also be stored if they are ensiled at the ideal stage of development, or if the suitable additives are applied (Zanine et al., 2010).

Tropical weather grasses have high production in favorable seasons and a sharp decline in the less favorable ones. In this context, the surplus silage can be an option to increase the dry matter supply to the animals in unfavorable times. Such examples of tropical forages with a potential for silage are: *Brachiaria brizantha* (cv. Marandu), *Brachiaria decumbens* (cv. Basilisk), *Brachiaria humidicula*, *Panicum maximum* Jacq. (Cv. Colonião, Tobiatã, Tanzânia, Mombaça, Vencedor, Centauro, Massai), *Pennisetum purpureum* Schum. (Cv. Napier, Taiwan, Merker, Porto Rico, Cameroon, Mott), *Cynodon dactylon* (Tifton) and the hybrid of *Cynodon dactylon* x *C. nlemfuensis* (Coastcross). (Patrizi et al., 2004; Santos et al., 2006; Ribeiro et al., 2008; Oliveira et al., 2007; Zopollatto et al., 2009; Lopes & Evangelista, 2010). When compared to the others, elephant grass stands out in silages researches because of present high

According to Evangelista et al. (2004), the tropical grasses present low dry matter contents, high buffering capacity and low soluble carbohydrates in growth stages in which they present good nutritive values, endangering the conservation through ensilage, once secondary fermentations are possible to occur. Bacteria from the *Clostridium* genus are favored by humid environments with high pH values and temperature. These bacteria are responsible for large losses because they produce CO2 and butyric acid instead of lactic acid. The grasses are colonized by a large number of LAB. In the most of the cases different species occur simultaneously in the same culture (Daeschel et al., 1987). According to Pahlow et al. (2003), in literature review studies, the species more commonly found in plants

productivity and higher soluble carbohydrates concentration.

The decline in pH values inhibit the spoilage microorganism proliferation, which allows the silage nutritive values to be preserved. Thus, the best silage forages are the ones with high soluble carbohydrates contents, which should be sufficient to promote the fermentation and produce enough acid to preserve the silage. According to Ferreira (2002), the minimum soluble carbohydrates contents recommended to ensure adequate fermentation of good silage, varies between 6% and 12% of the dry mass. McDonald et al. (1991) found that, since the soluble sugar level is adequate, dry mass contents higher than 25% are sufficient to ensure a good silage production. The buffering capacity is another factor affecting the silage final product. It reflects the capacity to resist change in the pH values, determined by buffering substances, represented in plants by inorganic bases such as potassium (K) and calcium (Ca), protein, ammonia (N-NH3), organic salts (malate, citrate).

Several factors affect the fermentation pattern and consequently the silage quality, including dry matter content, amount of soluble carbohydrates readily available and initial LAB population (Pereira et al., 2006). These inherent plant characteristics may vary according to species and maturity stage. Corn (*Zea mays* L.) and sorghum (*Sorghum bicolor* L. Moench), followed by millet (*Pennisetum glaucum*) and sunflower (*Helianthus annuus*) seems to be the most adapted species for silage due to the high soluble carbohydrates content, low buffering capacity, satisfactory dry matter productivity and quality of the silage produced. Although, sorghum silage nutritional value is considered lower than that of corn, it has shown an important role in forage production in Brazil and in the world as well, standing out as a resistant species to adverse environmental factors, such as drought stress (Miranda et al., 2010). This grass provides silage at low costs and the plant regrowth can be used (Rezende et al., 2011), because they keep the root system active.

As corn and sorghum have ideal characteristics for silage, a factor that drew the researcher's attention was the ideal harvest moment, considering the maturity stage and silage quality. Faria Júnior et al. (2011), working with the effect of seven grain maturity stages on the quality of sorghum BRS 610 silage, observed that the most appropriate stage for ensiling is the milk and soft dough stages, due to its higher silage fermentation quality and nutritional value.

Pearl millet silage presents high crude protein content as an intrinsic characteristic, when compared with corn and sorghum silage. Crude protein values varying from 8.51% to 10.68% were observed by Amaral et al. (2008). The storage system efficiency must not be defined only by the silage nutritional value, but also include the losses that occur from the plant harvest to the animal feeding (Neumann et al., 2007).

Sugarcane (*Saccharum officinarum* L.) is an important grass due to its tolerance to drought periods and high production potential of dry matter and soluble carbohydrates per hectare. The sugarcane silage confection has been unusual, being used more for animal feeding in its natural form, after cutting and chopping, but it can be recommended when desires to store the sugarcane in its higher nutritional value stage (the dry season) for use throughout the year (Molina et al., 2002). However, according to Santos et al. (2006), sugar cane silage becomes justifiable only when there is a surplus or when accidental burning of sugar cane fields happen, always taking into account the difficulty of achieving a good fermentation pattern due to intense alcoholic fermentation (8% to 17% of dry matter of ethanol) caused by yeast (Kung Jr. & Stanley, 1982), leading to losses of up to 30% of dry matter (Ferreira et al., 2007), accumulation of cell wall components and reduction in the *in vitro* dry matter digestibility. Furthermore, sugar cane silage has low aerobic stability, as result of high residual carbohydrate and lactic acid contents (McDonald et al., 1991). On the other hand, the adoption of the silage method represents a chance to keep the sugarcane nutritional value and allows better logistics for their manufacture and use, what implies the hand labor rationalization, concentrating the sugar cane harvest process in a particular time of year or time period, resulting in easier daily farm handling and maximizing the machinery use.

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

The forage characteristics that contribute to a good fermentation are: dry matter content, autochthonous plant microbiota and, most importantly, the quantity of soluble carbohydrates. Corn and sorghum are the most appropriate grasses to make silages due to their high soluble carbohydrate contents and dry matter production. However, some studies have shown that different grasses can be utilized if they are ensilage at the right

The decline in pH values inhibit the spoilage microorganism proliferation, which allows the silage nutritive values to be preserved. Thus, the best silage forages are the ones with high soluble carbohydrates contents, which should be sufficient to promote the fermentation and produce enough acid to preserve the silage. According to Ferreira (2002), the minimum soluble carbohydrates contents recommended to ensure adequate fermentation of good silage, varies between 6% and 12% of the dry mass. McDonald et al. (1991) found that, since the soluble sugar level is adequate, dry mass contents higher than 25% are sufficient to ensure a good silage production. The buffering capacity is another factor affecting the silage final product. It reflects the capacity to resist change in the pH values, determined by buffering substances, represented in plants by inorganic bases such as potassium (K) and

Several factors affect the fermentation pattern and consequently the silage quality, including dry matter content, amount of soluble carbohydrates readily available and initial LAB population (Pereira et al., 2006). These inherent plant characteristics may vary according to species and maturity stage. Corn (*Zea mays* L.) and sorghum (*Sorghum bicolor* L. Moench), followed by millet (*Pennisetum glaucum*) and sunflower (*Helianthus annuus*) seems to be the most adapted species for silage due to the high soluble carbohydrates content, low buffering capacity, satisfactory dry matter productivity and quality of the silage produced. Although, sorghum silage nutritional value is considered lower than that of corn, it has shown an important role in forage production in Brazil and in the world as well, standing out as a resistant species to adverse environmental factors, such as drought stress (Miranda et al., 2010). This grass provides silage at low costs and the plant regrowth can be used (Rezende

As corn and sorghum have ideal characteristics for silage, a factor that drew the researcher's attention was the ideal harvest moment, considering the maturity stage and silage quality. Faria Júnior et al. (2011), working with the effect of seven grain maturity stages on the quality of sorghum BRS 610 silage, observed that the most appropriate stage for ensiling is the milk and soft dough stages, due to its higher silage fermentation quality and nutritional value.

Pearl millet silage presents high crude protein content as an intrinsic characteristic, when compared with corn and sorghum silage. Crude protein values varying from 8.51% to 10.68% were observed by Amaral et al. (2008). The storage system efficiency must not be defined only by the silage nutritional value, but also include the losses that occur from the

developmental stage or if appropriate additives are used (Zanine et al., 2010).

calcium (Ca), protein, ammonia (N-NH3), organic salts (malate, citrate).

et al., 2011), because they keep the root system active.

plant harvest to the animal feeding (Neumann et al., 2007).

**2. Tropical grass characteristics** 

Thus, there has been a growing number of research projects, especially in Brazil, seeking additives that inhibit yeast growth in sugar cane silages (Valeriano et al., 2009). Nevertheless, some studies have shown that grasses can also be stored if they are ensiled at the ideal stage of development, or if the suitable additives are applied (Zanine et al., 2010).

Tropical weather grasses have high production in favorable seasons and a sharp decline in the less favorable ones. In this context, the surplus silage can be an option to increase the dry matter supply to the animals in unfavorable times. Such examples of tropical forages with a potential for silage are: *Brachiaria brizantha* (cv. Marandu), *Brachiaria decumbens* (cv. Basilisk), *Brachiaria humidicula*, *Panicum maximum* Jacq. (Cv. Colonião, Tobiatã, Tanzânia, Mombaça, Vencedor, Centauro, Massai), *Pennisetum purpureum* Schum. (Cv. Napier, Taiwan, Merker, Porto Rico, Cameroon, Mott), *Cynodon dactylon* (Tifton) and the hybrid of *Cynodon dactylon* x *C. nlemfuensis* (Coastcross). (Patrizi et al., 2004; Santos et al., 2006; Ribeiro et al., 2008; Oliveira et al., 2007; Zopollatto et al., 2009; Lopes & Evangelista, 2010). When compared to the others, elephant grass stands out in silages researches because of present high productivity and higher soluble carbohydrates concentration.

According to Evangelista et al. (2004), the tropical grasses present low dry matter contents, high buffering capacity and low soluble carbohydrates in growth stages in which they present good nutritive values, endangering the conservation through ensilage, once secondary fermentations are possible to occur. Bacteria from the *Clostridium* genus are favored by humid environments with high pH values and temperature. These bacteria are responsible for large losses because they produce CO2 and butyric acid instead of lactic acid.

The grasses are colonized by a large number of LAB. In the most of the cases different species occur simultaneously in the same culture (Daeschel et al., 1987). According to Pahlow et al. (2003), in literature review studies, the species more commonly found in plants are *Lactobacillus plantarum, Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecium.* Some heterofermentative lactic bacteria species can also be found in plants.

Lactic Acid Bacteria in Tropical Grass Silages 339

*Brachiaria brizantha* 

*Brachiaria decumbens* 

microbiota, found values lower than 3 log CFU/g of fresh forage. Pereira et al. (2007)

Table 2 presents a data compilation of chemical composition and other parameters considered determinants of tropical grass silages quality, such as buffering capacity, soluble

> Sugar Cane

CAPACITY - 19.98 - 10.80 - - - -

STARCH 21.31 - - 5.50 - - - - **Table 2.** Chemical characterization of tropical grass used for silage. \*Number of researches; DM = dry matter (%); OM = organic matter (%); CP = crude protein (%); EE = ether extract (%); NDF = neutral detergent fiber (%); NFC = non-fibrous carbohydrates (%); IVDMD = *in vitro* dry matter digestibility (%);

(Pariz, C.M. et al., 2011; Silva, T.C. et al., 2011; Viana, M.C.M. et al., 2011; Hu, W. et al., 2009; Martinez , J.C. et al., 2009; Valeriano, A.R, 2009; Benett, C.G.S. 2008; Reis, J.A.G. et al., 2008; Ribeiro, J.L. et al., 2008; Moreira, J.N. et al., 2007; Pedroso, A.F. et al., 2007; Velho, J.P. et al., 2007; Valadares Filho, S.C. et al., 2006; Velho, J.P. et al., 2006; Kollet, J.L. et al., 2006; Aroeira, L.J.M. et al., 2005; Bernardino, F.S. 2005; Moraes, E.H.B.K. et al., 2005; Santos, G.R.A. et al., 2005; Silva, A.V. et al., 2005; Patrizi, W.L. et al., 2004; Dairy, J. et al., 2003; Santos, M.V.F. et al., 2003; Landell, M.G.A. et al., 2002; Neumann, M. et al., 2002; Rodrigues, P.H.M. et al., 2002).

N-NH3= ammonia nitrogen (% TN); ADF = acid detergent fiber (%); MM = mineral matter (%).

n\* 6 6 6 7 5 4 6 6 DM 30.68 30.20 31.21 25.25 20.75 37.15 38.36 30.9 OM 96.91 92.79 90.9 97.45 90.91 90.60 92.89 92.25 CP 7.22 8.04 11.09 2.80 7.81 5.03 9.67 7.01 MM 5.81 4.45 9.1 2.68 9.53 9.92 5.29 7.53 EE 2.16 - - 0.82 3.33 1.8 1.16 2.51 NDF 50.32 61.36 60.64 46.88 72.44 73.94 70.05 75.47 ADF 26.57 37.27 35.68 28.24 44.11 50.60 38.64 38.26 NFC 32.49 - - 44.21 9.99 14.05 8.74 14.12 LIGNIN 4.72 6.2 4.24 4.72 6.24 8.4 4.67 5.9 IVDMD 59.19 52.87 - 53.87 60.90 37.4 58.77 51.61 pH 5.60 5.93 3.62 4.76 5.6 - - - N-NH3 0.785 - 1.28 1.20 - - - - ETHANOL - - - 2.12 - - - - YEASTS 5.30 - - 2.71 - - - -

Elephant grass

Buffel grass

reported initial LAB population of 4.92 log CFU/g in elephant grass plants.

millet

carbohydrates and pH values.

BUFFERING

Corn Sorghum Pearl

The lactic acid bacteria from the autochthonous microbiota are essential for the silage fermentation. However, no bacteria group varies as much as this one regarding number, with a detection limit of 101 to 105 CFU g-1 in alfalfa forage, 106 in perennial grasses and 107 in corn and sorghum (Pahlow et al., 2003).

The Table 1 shows contents of dry matter, crude protein, soluble carbohydrates and LAB number of mombaça grass (*Panicum maximum*) and *Brachiaria decumbens* with different regrowth ages. It is observed that in none of regrowth ages, neither grass showed dry matter content exceeding 30% and only the grasses cuted over 50 days after regrowth presented LAB population greater than 5 log CFU/g. On the other hand, there is a sharp drop in crude protein content with increasing regrowth age.


**Table 1.** Dry matter (DM), crude protein (CP) and soluble carbohydrates (SC) and number of lactic acid bacteria (LAB) in signal grass and mombaça grass silage with different regrowth ages (Sousa et al., 2006).

Santos et al. (2011) studying the regrowth age influence in the LAB population observed that silages made with older plants presented LAB populations higher than the silages made with younger plants. According to Knicky (2005), it can be attributed to the increase in soluble carbohydrates and dry matter content, as well as to the decrease of anionic substances such as salts of organic acids, nitrate, sulfates, and so on. Pereira et al. (2005) found an increase in LAB population in elephant grass with the increase in regrowth age.

Meeske et al. (1999) found population of approximately 1 log CFU/g of fresh forage in *Digitaria eriantha*. Cai et al. (1998), analyzing Guinea grass (*Panicum maximum*) indigenous microbiota, found values lower than 3 log CFU/g of fresh forage. Pereira et al. (2007) reported initial LAB population of 4.92 log CFU/g in elephant grass plants.

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

in corn and sorghum (Pahlow et al., 2003).

protein content with increasing regrowth age.

2006).

are *Lactobacillus plantarum, Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecium.*

The lactic acid bacteria from the autochthonous microbiota are essential for the silage fermentation. However, no bacteria group varies as much as this one regarding number, with a detection limit of 101 to 105 CFU g-1 in alfalfa forage, 106 in perennial grasses and 107

The Table 1 shows contents of dry matter, crude protein, soluble carbohydrates and LAB number of mombaça grass (*Panicum maximum*) and *Brachiaria decumbens* with different regrowth ages. It is observed that in none of regrowth ages, neither grass showed dry matter content exceeding 30% and only the grasses cuted over 50 days after regrowth presented LAB population greater than 5 log CFU/g. On the other hand, there is a sharp drop in crude

Signal grass (Brachiaria decumbens.) AGE (days) DM (%) CP (%) SC (%) LAB (log CFU/g) 30 20.99 9.65 2.62 3.93 40 21.23 6.97 2.92 4.81 50 21.94 5.86 3.13 5.37 60 22.35 5.30 2.73 5.32 70 23.67 4.37 2.53 5.51 Mombaça grass(Panicum maximum Jacq. cv. Mombaça) AGE (days) DM (%) CP (%) SC (%) LAB (log CFU/g) 30 17.75 7.43 3.34 4.35 40 19.63 7.30 4.12 4.56 50 21.50 6.47 4.18 5.16 60 23.38 4.94 5.43 5.55 **Table 1.** Dry matter (DM), crude protein (CP) and soluble carbohydrates (SC) and number of lactic acid bacteria (LAB) in signal grass and mombaça grass silage with different regrowth ages (Sousa et al.,

Santos et al. (2011) studying the regrowth age influence in the LAB population observed that silages made with older plants presented LAB populations higher than the silages made with younger plants. According to Knicky (2005), it can be attributed to the increase in soluble carbohydrates and dry matter content, as well as to the decrease of anionic substances such as salts of organic acids, nitrate, sulfates, and so on. Pereira et al. (2005) found an increase in LAB population in elephant grass with the increase in regrowth age.

Meeske et al. (1999) found population of approximately 1 log CFU/g of fresh forage in *Digitaria eriantha*. Cai et al. (1998), analyzing Guinea grass (*Panicum maximum*) indigenous

Some heterofermentative lactic bacteria species can also be found in plants.

Table 2 presents a data compilation of chemical composition and other parameters considered determinants of tropical grass silages quality, such as buffering capacity, soluble carbohydrates and pH values.


**Table 2.** Chemical characterization of tropical grass used for silage. \*Number of researches; DM = dry matter (%); OM = organic matter (%); CP = crude protein (%); EE = ether extract (%); NDF = neutral detergent fiber (%); NFC = non-fibrous carbohydrates (%); IVDMD = *in vitro* dry matter digestibility (%); N-NH3= ammonia nitrogen (% TN); ADF = acid detergent fiber (%); MM = mineral matter (%).

(Pariz, C.M. et al., 2011; Silva, T.C. et al., 2011; Viana, M.C.M. et al., 2011; Hu, W. et al., 2009; Martinez , J.C. et al., 2009; Valeriano, A.R, 2009; Benett, C.G.S. 2008; Reis, J.A.G. et al., 2008; Ribeiro, J.L. et al., 2008; Moreira, J.N. et al., 2007; Pedroso, A.F. et al., 2007; Velho, J.P. et al., 2007; Valadares Filho, S.C. et al., 2006; Velho, J.P. et al., 2006; Kollet, J.L. et al., 2006; Aroeira, L.J.M. et al., 2005; Bernardino, F.S. 2005; Moraes, E.H.B.K. et al., 2005; Santos, G.R.A. et al., 2005; Silva, A.V. et al., 2005; Patrizi, W.L. et al., 2004; Dairy, J. et al., 2003; Santos, M.V.F. et al., 2003; Landell, M.G.A. et al., 2002; Neumann, M. et al., 2002; Rodrigues, P.H.M. et al., 2002).

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 higher quality silage.

Lactic Acid Bacteria in Tropical Grass Silages 341

*Lactobacillus Enterococcus Leuconostoc Pediococcus* 

*L. plantarum L. brevis E. faecalis L. dextranicum P. acidilactici L. casei L. buchneri E. faecium L. citrovorum P. pentosaceus L. curvatus L. fermentum E. lactis L. mesenteroides P. cerevisae* 

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

Homofermentative *Lactobacillus*

1A. *Lactobacillus delbrueckii* subsp. *Delbrueckii* 9. *L. helveticus* 1B. *Lactobacillus delbrueckii* subsp. *lactis* 10. *L. jensenii* 1C. *Lactobacillus delbrueckii* subsp. *bulgaricus* 11. *L. ruminis* 2. *L. acidophilus* 12. *L. salivarius* 3. *L. amylophilus* 13. *L. sharpeae* 4. *L. amylovorus* 14. *L. vitulinus*

5. *L. animalis* 15. *L. yamanashiensis*

**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

Facultative heterofermentative *Lactobacillus*

16. *L. agilis* 20b. *L. coryniformis* subsp. *Torquens*

17. *L. alimentarius* 21. *L. curvatus* 18. *L. bavaricus 22. L. homohiochii* 19a. *L. casei* subsp. *Casei 23. L. maltaromicus*

19b. *L. casei* subsp. *pseudo-plantarum 24. L. murinus* 19c. *L. casei* subsp. *rhamnosus 25. L. plantarum*

19d. *L. casei* subsp. *tolerans 26. L. sake*

20a. *L. coryniformis* subsp. *coryniforms*

*L. acidophilus L. viridescens* 

fosfocetolase enzyme lack;

6. *L. crispatus* 7. *L. farciminis* 8. *L. gasseri*

enzymes;

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

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 characteristics related to metabolism and the main tropical grass species.
