**4. Lactic acid bacteria and their effects on silage fermentation**

A suitable acidification is essential for the silage successful preservation, especially when the crop moisture is relatively high, condition which favors the proliferation of spoilage microorganisms. The acidity prevents the development of spoilage microorganisms because they are less tolerant to the acidic conditions than lactic acid bacteria (Woolford, 1984; McDonald et al., 1991).

Among the fermentation stages, aerobic remains during the filling and some hours after the silage closing. The growth of aerobic microorganisms such as yeasts, fungi and bacteria, favored by high concentrations of oxygen (O2) with the plant respiration process, promotes the O2 reduction, initiating the active fermentation process. Thus, occurs a sharp drop in silage pH due to the formation of organic acids from sugars, in which initially actuate the heterofermentative bacteria and enterobacteriaceae, that becomes, then, dominated by homofermentative until the pH falls to below 5.0.

In the stability phase, when only the lactic acid bacteria are active, the anaerobic and acidic pH conditions preserve the silage until the opening time. When the silo is opened, it typically happen the molds and yeasts growth. The inhibition of the fungi multiplication through the contact with O2 is called aerobic stability (Santos et al., 2006).

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 and Lactobacillus.

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

(*Brachiaria decumbens* cv. Basiliski). + Intense fermentation, -

homofermentative until the pH falls to below 5.0.

predominant specie for most plants.

McDonald et al., 1991).

(Santos et al., 2011).

Isolated strain Lactobacillus

no fermentation; (+) less intense fermentation

EB1 EB2 EB5 EB6 plantarum

L-lyxose - - - - - D-tagatose - - - - - D-fucose - - - - - L-fucose - - - - - D-arabitol (+) (+) (+) (+) - L-arabitol - - - - - Gluconate + + + + + 2 Cetogluconate - - - - - 5 Cetogluconate - - - - - **Table 7.** Carbohydrate fermentation pattern of the isolates EB1, EB2, EB5, and EB6, signal grass plants

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

A suitable acidification is essential for the silage successful preservation, especially when the crop moisture is relatively high, condition which favors the proliferation of spoilage microorganisms. The acidity prevents the development of spoilage microorganisms because they are less tolerant to the acidic conditions than lactic acid bacteria (Woolford, 1984;

Among the fermentation stages, aerobic remains during the filling and some hours after the silage closing. The growth of aerobic microorganisms such as yeasts, fungi and bacteria, favored by high concentrations of oxygen (O2) with the plant respiration process, promotes the O2 reduction, initiating the active fermentation process. Thus, occurs a sharp drop in silage pH due to the formation of organic acids from sugars, in which initially actuate the heterofermentative bacteria and enterobacteriaceae, that becomes, then, dominated by

In the stability phase, when only the lactic acid bacteria are active, the anaerobic and acidic pH conditions preserve the silage until the opening time. When the silo is opened, it typically happen the molds and yeasts growth. The inhibition of the fungi multiplication

through the contact with O2 is called aerobic stability (Santos et al., 2006).

**4. Lactic acid bacteria and their effects on silage fermentation** 

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

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 and microorganism studied, suggesting a specificity between these components.

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

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.

Thereby the silage inoculants can facilitate or accelerate the ensiling process, but they do not replace the fundamental factors (plant maturity, dry matter content, oxygen exclusion), which are essential for producing good quality silage. Among these factors the regrowth age is the one that influences all the silage characteristics, from fermentation to the nutritional value, considering the losses.

Lactic Acid Bacteria in Tropical Grass Silages 349

found in grass and small grains silages. It was observed correlation between acetic acid

Crop Microrganism pH1 NH32 LA3 AA4 PA5 BA6 ET7 AE8 DML9 DMR10

Grass LP -- -- ++ -- -- ns ns -- ++ Corn LB ++ ns -- ++ ns ns + ns Grass -- ns -- ++ ++ ++ + -- Corn PA/ LP -- -- -- -- -- ++

% total N % DM hours %

LB ++ ns -- ++ ++ ++ LP ns -- ++ ns ns -- LP/ LB ns -- ns ++ ++ --

LB ns ns -- ++ ++ ++ LP ns -- ns ns -- -- LP/ LB ns -- -- ++ ++ --

LP ns -- ++ + --

SF/ PA/ LP ns ns ns ns ns ns ns ns LP/ L. ns ns ns ns ns ns ns ns SF/ LP -- -- ns -- ++ ns ns ns

LB -- -- ++ ++ ns -- ++ LPa/ LL/ PA -- -- ++ -- -- -- -- **Table 8.** Effect of inoculants with lactic acid bacteria on the fermentation of the silage. \*Potato byproduct + 30% of wheat bran; 1lactic acid, 2acetic acid, 3propionic acid, 4butyric acid, 5ethanol, 6aerobic stability, 7dry matterlosses, 8dry matter recovery. ns = not significant, + = numerical increase, - = decreasing numbers; + + = significant increase (P <0.05) / - = significant decrease (P <0.05). (Filya et al., 2000; Rodrigues et al., 2001; Weinberg et al., 2002; Filya, 2003; Kleinschimit & Kung Jr., 2006; Rowghani & Zamiri, 2009; Ávila et al., 2009b; Nkosi et al., 2010; Santos et al., 2011). LP = *Lactobacillus plantarum,* EF = *Enterococcus faecium,* LPe = *Lactobacillus pentosus,* SF = *Streptococcus faecium,* PA = *Pediococcus acidilacti, L = Lactobacillus sp.,* LB = *Lactobacillus buchneri;* Pac = *Propionibacterium acidipropionici;* LPar *= Lactobacillus* 

In concluded studies, the inoculation with *Lactobacillus buchneri* changed silages fermentation pattern, decreasing the lactate/acetate ratio, without compromising the processes efficiency, because the dry matter values recovery remained above 90%, as the minimum value recommended for this variable in these plants. The authors also suggest the

Evaluating barley silage inoculated with *Lactobacillus buchneri*, Taylor et al. (2002) observed a decrease in yeasts and molds number, contrasting with an increase in aerobic stability.

Changes in dry matter consumption and milk production were not affected.

L.Pe -- ++ ns ns ns

Wheat LB ns ns ++ + ++

Wheat LP/EF -- ++ ns ns ns

Sugar cane LB ns ns ++ ++ ns -- +

concentration and fungi population reduction.

Wheat

Sorghum

Sunflowerl

Potato + WB\*

*paracasei paracasei* LL *= Lactococcus lactis.* 

existence of culture-specific effect.

Meeske & Basson (1998) evaluated the effect of inoculant containing *Lactobacillus acidophilus*, *Lactobacillus delbruekii ssp. bulgaricus* and *Lactobacillus plantarum* on corn silage and found no inoculants effect on pH values and the lactic acid production. According to the authors, the high LAB concentrations present in the plant before ensiling led to such results. Furthermore, the amount of bacteria from *Clostridium* genus present in greater numbers in the treatment without inoculants had no effect on the protein content decrease of the untreated silage. It was not detected the butyric acid formation.

The high residual soluble carbohydrates content in silage, mainly the ones made of corn, sorghum and sugarcane, favors the aerobic deterioration process by fungi and yeasts, causing losses after the silo opening. However, the organic acids produced by fermentation, mainly acetic acid, have fungicidal effect and can mitigate the deterioration, increasing silage aerobic stability (Ranjit & Kung Jr. 2000; Kung Jr. & Ranjit, 2001). Therefore, inoculants containing heterofermentative LAB (e.g. *Lactobacillus buchneri*) have been used to increase the silage aerobic stability.

Ávila et al. (2009a) evaluated the aerobic stability of mombaça grass silage (*Panicum maximum* Jacq. cv. Mombaça) inoculated with two *Lactobacillus buchneri* strains, one provinient from a commercial inoculant and another isolated from sugarcane (*Saccharum officinarum* L.) silage. It was observed an increase in dry matter content after silo opening, while the carbohydrate ratio did not change due to the low residual concentration, characteristic of grass silage. The ammonia (NH3) concentrations were above the 12% of the total-N recommended by Molina et al. (2002) for good quality silage, indicating high proteolysis during fermentation, due to low soluble carbohydrates supply, what makes possible a rapid decline of pH values.

Table 8 present few studies evaluating the effect of LAB on the silage fermentation. It is observed that there is a pattern of responses, as discussed previously, and its effect depends of the crop used, the microorganism strain and its concentration at the inoculation time. Although significant, the effects are of low magnitude, which leads to reflect about the use of inoculants without the microbiological principles and characteristics of forage plants knowledge.

Kleinschimit and Kung Jr. (2006), in a meta-analysis study (43 experiments), evaluated the *Lactobacillus buchneri* effect on fermentation and aerobic stability of corn, grasses and small grains silages. In general, the inoculation reduced pH, lactic acid concentration and mold counts. At the same time increases in acetic acid concentrations and aerobic stability were detected in all silage types. The increase in aerobic stability was more pronounced in corn silage. Furthermore, it was observed an increase in the propionic acid and ethanol concentrations, on the other hand decreases in soluble carbohydrates concentrations were


found in grass and small grains silages. It was observed correlation between acetic acid concentration and fungi population reduction.

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

untreated silage. It was not detected the butyric acid formation.

value, considering the losses.

increase the silage aerobic stability.

possible a rapid decline of pH values.

knowledge.

Thereby the silage inoculants can facilitate or accelerate the ensiling process, but they do not replace the fundamental factors (plant maturity, dry matter content, oxygen exclusion), which are essential for producing good quality silage. Among these factors the regrowth age is the one that influences all the silage characteristics, from fermentation to the nutritional

Meeske & Basson (1998) evaluated the effect of inoculant containing *Lactobacillus acidophilus*, *Lactobacillus delbruekii ssp. bulgaricus* and *Lactobacillus plantarum* on corn silage and found no inoculants effect on pH values and the lactic acid production. According to the authors, the high LAB concentrations present in the plant before ensiling led to such results. Furthermore, the amount of bacteria from *Clostridium* genus present in greater numbers in the treatment without inoculants had no effect on the protein content decrease of the

The high residual soluble carbohydrates content in silage, mainly the ones made of corn, sorghum and sugarcane, favors the aerobic deterioration process by fungi and yeasts, causing losses after the silo opening. However, the organic acids produced by fermentation, mainly acetic acid, have fungicidal effect and can mitigate the deterioration, increasing silage aerobic stability (Ranjit & Kung Jr. 2000; Kung Jr. & Ranjit, 2001). Therefore, inoculants containing heterofermentative LAB (e.g. *Lactobacillus buchneri*) have been used to

Ávila et al. (2009a) evaluated the aerobic stability of mombaça grass silage (*Panicum maximum* Jacq. cv. Mombaça) inoculated with two *Lactobacillus buchneri* strains, one provinient from a commercial inoculant and another isolated from sugarcane (*Saccharum officinarum* L.) silage. It was observed an increase in dry matter content after silo opening, while the carbohydrate ratio did not change due to the low residual concentration, characteristic of grass silage. The ammonia (NH3) concentrations were above the 12% of the total-N recommended by Molina et al. (2002) for good quality silage, indicating high proteolysis during fermentation, due to low soluble carbohydrates supply, what makes

Table 8 present few studies evaluating the effect of LAB on the silage fermentation. It is observed that there is a pattern of responses, as discussed previously, and its effect depends of the crop used, the microorganism strain and its concentration at the inoculation time. Although significant, the effects are of low magnitude, which leads to reflect about the use of inoculants without the microbiological principles and characteristics of forage plants

Kleinschimit and Kung Jr. (2006), in a meta-analysis study (43 experiments), evaluated the *Lactobacillus buchneri* effect on fermentation and aerobic stability of corn, grasses and small grains silages. In general, the inoculation reduced pH, lactic acid concentration and mold counts. At the same time increases in acetic acid concentrations and aerobic stability were detected in all silage types. The increase in aerobic stability was more pronounced in corn silage. Furthermore, it was observed an increase in the propionic acid and ethanol concentrations, on the other hand decreases in soluble carbohydrates concentrations were **Table 8.** Effect of inoculants with lactic acid bacteria on the fermentation of the silage. \*Potato byproduct + 30% of wheat bran; 1lactic acid, 2acetic acid, 3propionic acid, 4butyric acid, 5ethanol, 6aerobic stability, 7dry matterlosses, 8dry matter recovery. ns = not significant, + = numerical increase, - = decreasing numbers; + + = significant increase (P <0.05) / - = significant decrease (P <0.05). (Filya et al., 2000; Rodrigues et al., 2001; Weinberg et al., 2002; Filya, 2003; Kleinschimit & Kung Jr., 2006; Rowghani & Zamiri, 2009; Ávila et al., 2009b; Nkosi et al., 2010; Santos et al., 2011). LP = *Lactobacillus plantarum,* EF = *Enterococcus faecium,* LPe = *Lactobacillus pentosus,* SF = *Streptococcus faecium,* PA = *Pediococcus acidilacti, L = Lactobacillus sp.,* LB = *Lactobacillus buchneri;* Pac = *Propionibacterium acidipropionici;* LPar *= Lactobacillus paracasei paracasei* LL *= Lactococcus lactis.* 

In concluded studies, the inoculation with *Lactobacillus buchneri* changed silages fermentation pattern, decreasing the lactate/acetate ratio, without compromising the processes efficiency, because the dry matter values recovery remained above 90%, as the minimum value recommended for this variable in these plants. The authors also suggest the existence of culture-specific effect.

Evaluating barley silage inoculated with *Lactobacillus buchneri*, Taylor et al. (2002) observed a decrease in yeasts and molds number, contrasting with an increase in aerobic stability. Changes in dry matter consumption and milk production were not affected.

The homofermentative LAB are used in order to improve the fermentation of the silage by increasing the concentration of lactic acid, which reduces the ammonia and the loss of dry matter. The heterofermentative LAB, for its turn, promote improvements, especially after the opening of the silo, increasing the aerobic stability of silage by inhibiting the growth of molds and yeasts. Thus, many research papers have recommended the use of inoculant combining the above two groups of LAB, due to its greater efficiency compared to the isolated use.

Lactic Acid Bacteria in Tropical Grass Silages 351

acid Acetic acid Butyric

acid

Lactic

% % total N %DM

Control (without aditive) 15.58f 4.15b 12.39d 2.40a 0.30b 0.00b Urea 0.5 % 15.49f 5.36a 35.76abc 1.05a 1.81a 0.57a Cotton fiber (10%) 23.25b 5.33a 36.07ab 1.8a 0.66b 1.73a Elephant grass hay (10%) 25.88a 4.26b 25.63bcd 2.48a 0.46b 0.12b Guandu hay(10%) 25.78a 4.21b 8.33d 1.38a 0.58b 0.14b Drying for 6 hours 19.84cd 4.08b 15.17d 1.81a 0.30b 0.02b Sugar waste (2%) 16.50de 4.09b 13.68d 4.69a 0.66b 0.00b Corn Meal (2%) 16.90de 4.00b 13.68d 2.47a 0.28b 0.00b Corn Meal (4%) 20.39c 4.00b 12.94d 4.96a 1.15a 0.08b Corn Meal (6%) 21.60c 4.04b 12.01d 4.41a 0.33b 0.00b

Urea (0.5%) 17.96de 4.19b 36.67ab 5.31a 0.53b 0.04b

Urea (0.5%) 20.26c 4.29b 49.36a 1.96a 0.85b 0.05b

Urea (0.5%) 20.43c 4.20b 46.86a 2.25a 0.38b 0.01b Dried Molasses (1%) 16.95de 4.04b 10.52d 3.60a 0.22b 0.00b Dried Molasses (2%) 17.58de 3.92b 10.27d 3.29a 0.23b 0.00b Dried Molasses (3%) 16.67de 3.89b 9.43d 3.98a 0.35b 0.00b

Urea (0.5%) 17.20de 4.18b 34.93abc 1.25a 0.46b 0.04b

Urea (0.5%) 18.20de 4.09b 32.43abc 5.24a 0.44b 0.04b

Urea (0.5%) 17.55ed 3.97b 11.50d 4.84a 0.36b 0.00b Biosilo inoculant 15.88f 4.06b 15.24d 2.61a 0.50b 0.03b CV (%) 7.04 5.55 34.87 50.62 62.54 137.65 **Table 9.** Dry matter (DM) content and fermentation pattern of elephant grass, Napier, ensiled with different additives (Andrade & Melotti, 2004). DM = dry matter (%), CP = crude protein (% DM), N-NH3 = ammonia nitrogen/total nitrogen (%), lactic acids, acetic and butyric acids: values in % of the silage

The lowest in vitro dry matter digestibility was obtained with the use of guandu hay. On the other hand the highest one was obtained using corn meal and urea (Table 10). Compared to the control treatment, only the urea and cotton fiber had higher dry matter loss (11.0 and

According to the authors, it is not recommended the inclusion of urea, hay and cotton fiber in elephant grass silage. Additives rich in nonstructural carbohydrates, such as corn meal and molasses can be used, however, further studies are required to establish suitable levels

DM. Equal means in column do not differ (P>0.05): CV = coefficient of variation.

Treatment DM pH N-NH3

Corn Meal (2%) /

Corn Meal (4%)/

Corn Meal (6%) /

Dried Molasses (1%)

Dried Molasses (2%)

Dried Molasses (3%)

10.5%, respectively).
