Lactic Acid Bacteria in Tropical Grass Silages

1. Introduction

Lactic Acid Bacteria (LAB) have applications in many industrial areas and play an important role in the preservation process of moist forages for animal feeding (silage).

The basic principle behind silage is to store the surplus forage while maintaining its stability and nutritional value until it is required to feed the animals. This process takes place in anaerobic conditions, where the lactic acid produced by the LAB inhibits the proliferation of spoilage microorganisms, which are less tolerant to acidic conditions. Thus, as the pH values decline, the silage losses decline as well due to the greater conversion of plant soluble carbohydrates (the main substrate for LAB) in lactic acid, with a 96.9% rate of energy recovery (Mc Donald et al., 1991). The major soluble carbohydrates present in forage crops are fructose, glucose, sucrose and frutosans, according to Woolford, (1984); sucrose and frutosans are rapidly hydrolyzed in their monomers at forage harvest.

Lactic fermentation produces lactic acid as the main product. Therefore, homofermentative bacteria such as Lactobacillus plantarum are desirable in the silage fermentation process once 87% of their metabolites become lactic acid. On the other hand, in the heterofermentative process, additional substances like ethanol, acetate and CO₂ are formed. Microbial inoculants used as additives include homofermentative LAB, heterofermentative LAB or both combined. The specificity between the forage species and its epiphytic micro flora implicates the need for studies related to the isolation and identification of the main microorganism groups present in the forage used for silage.

In this chapter, we will discuss the characteristics of tropical grasses, the main LAB species found in these grasses and how the LABs are used to improve the quality of tropical grass silages.

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 for making 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 to ensilage at the right developmental stage or if appropriate additives are used (Zanine et al., 2010).

The decline in pH values inhibits the spoilage microorganisms' proliferation, which allows the silage nutritive values to be preserved. Thus, the best silage forages are those with high soluble carbohydrates contents, which should be sufficient to promote fermentation and produce enough acid to preserve the silage. According to Ferreira (2002), the minimum soluble carbohydrates contents recommended to ensure the 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 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) and organic salts (malate, citrate).

Several factors affect the fermentation pattern and - consequently - the silage quality, including the dry matter content, the amount of soluble carbohydrates readily available and the initial LAB population (Pereira et al., 2006). These inherent plant characteristics may vary according to the species and the maturity stage. Corn (Zea mays L.) and sorghum (Sorghum bicolour 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, the low buffering capacity, the satisfactory dry matter productivity and the quality of the silage produced. Although sorghum silage's nutritional value is considered to be lower than that of corn, it has played an important role in forage production in Brazil and in the world at large 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 re-growth 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 caught 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 must 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 the high production potential of the 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 it is desired to store the sugarcane during 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 the 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 the dry matter of ethanol) caused by yeast (Kung Jr. & Stanley, 1982), leading to losses of up to 30% of the dry matter (Ferreira et al., 2007), the accumulation of cell wall components and the reduction in the in vitro dry matter's 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's nutritional value and allows for better logistics for their manufacture and use, which implies the hand labour rationalization, concentrating the sugarcane harvest process in a particular time of year or time period, resulting in easier daily farm handling and maximizing machinery use.

Thus, a growing number of research projects, especially in Brazil, have sought 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 suitable additives are applied (Zanine et al., 2010).

Tropical weather grasses have high production in favourable seasons and show a sharp decline in the less favourable ones. In this context, the surplus silage can be an option to increase the dry matter supply to the animals during unfavourable times. Such examples of tropical forages with a potential for silage are: Brachiaria brizantha (cv. Marandu), Brachiaria decumbens (cv. Basilisk), Brachiaria humidicola, 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 studies because of their high productivity and their higher concentration of soluble carbohydrates.

According to Evangelista et al. (2004), tropical grasses present low dry matter contents, high buffering capacity and low soluble carbohydrates during growth stages whereby they present good nutritive values, endangering conservation through ensilage, once secondary fermentations are able to occur. Bacteria from the Clostridium genus are favoured by humid environments with high pH values and temperatures. 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 most of the cases, different species occur simultaneously in the same culture (Daeschel et al., 1987). According to Pahlow et al. (2003), in the 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.

The lactic acid bacteria from the autochthonous microbiota are essential for silage fermentation. However, no bacteria group varies as much as this as regards their 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 the LAB number of mombaça grass (Panicum maximum) and Brachiaria decumbens with different re-growth ages. It is observed that in none of the re-growth ages did grass show a dry matter content exceeding 30% and only those grasses cut more than 50 days after re-growth presented a LAB population of greater than 5 log CFU/g. On the other hand, there is a sharp drop in crude protein content with an increasing re-growth age.

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), soluble carbohydrates (SC) and number of lactic acid bacteria (LAB) in signal grass and mombaça grass silage with different re-growth ages (Sousa et al., 2006).

Santos et al. (2011), in studying the re-growth age's influence in the LAB population observed that silages made with older plants presented LAB populations higher than those silages made with younger plants. According to Knicky (2005), this can be attributed to the increase in soluble carbohydrates and dry matter content, as well as a decrease in the number of anionic substances, such as salts of organic acids, nitrates and sulphates, etc. Pereira et al. (2005) found an increase in the LAB population in elephant grass with an increase in the re-growth age.

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

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

	Corn	Sorghum	Pearl millet	Sugar Cane	Elephant grass	Buffel grass	Brachiaria brizantha	Brachiaria decumbens
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	-	-	-	-
BUFFERING CAPACITY	-	19.98	-	10.80	-	-	-	-
STARCH	21.31	-	-	5.50	-	-	-	-

Table 2.

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

(Pariz et al., 2011; Silva et al., 2011; Viana et al., 2011; Hu et al., 2009; Martinez et al., 2009; Valeriano, 2009; Benett et al., 2008; Reis et al., 2008; Ribeiro et al., 2008; Moreira et al., 2007; Pedroso et al., 2007; Velho et al., 2007; Valadares Filho et al., 2006; Velho et al., 2006; Kollet et al., 2006; Aroeira et al., 2005; Bernardino et al., 2005; Moraes et al., 2005; Santos et al., 2005; Silva et al., 2005; Patrizi et al., 2004; Dairy et al., 2003; Santos et al., 2003; Landell et al., 2002; Neumann et al., 2002; Rodrigues et al., 2002).

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

To understand how the factors related to grass management will influence the LAB population dynamics as a result of the fermentation, it is necessary to understand the characteristics related to metabolism and the main tropical grass species.

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 a considerable amount of CO₂, ethanol and other metabolites, these being called 'heterofermentative'. In particular, Lactobacillus plantarum are the larger silage fermentative bacteria (Ohmomo et al., 2002). Lactococcus, Streptococcus and Enterococcus are very important in the fermentation's initial stage because they maintain an acidic environment which then becomes - predominantly - colonized by Lactobacillus.

Fermentation can be considered as the anaerobic decomposition of organic compounds into organic products, which may be metabolized by the cells without any 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; this occurs through various oxidation-reduction pathways, involving pyruvate or its derivatives, like acetyl-CoA. Pyruvate is a key molecule of fermenting microorganisms. 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 a substrate for the production of acetic and propionic acids, 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 phosphoketolase 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 for 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 a heterofermentative metabolism and some Leuconostoc species which also have a heterofermentative metabolism.

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

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. cerevisiae
L. acidophilus	L. viridescens

Table 3.

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

Group 1: Homofermentative, which ferment hexoses almost exclusively to lactic acid (>85%), are unable to ferment pentoses due to the lack of the phosphoketolase enzyme;

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

Group 2: Facultative heterofermentative use the same hexoses pathway as that for group 1 but are able to ferment pentoses since they have aldolase and phosphoketolase enzymes;

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. coryniformis

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

Mandatory heterofermentative Lactobacillus
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

The presence of homofermentative LAB in silage is extremely important. CO₂ generation results in carbon loss, i.e., 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 related species or which have similar nutritional requirements. The bacteriocins action mechanism involves the interaction with specific receptors on the cell membrane upon its insertion, resulting in proton-motive force dissipation and the formation of pores, which may cause cell viability loss (Montville and Chen, 1998; Ennahar et al., 2000).

According to Lücke (2000), gram-negative bacteria are less susceptible to the action of bacteriocins from lactic acid bacteria due to the presence of an 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 as compared with the gram-positive bacteria (Ennahar et al., 2000).

Table 4 presents the lactic acid bacteria percentages isolated from the sorghum plant in a study conducted by Tjandraatmadja et al. (1991). Likewise, Lactobacillus plantarum was the predominant species and it kept for 100 days after ensiling. The presence of Lactobacillus fermentum and Lactobacillus brevis heterofermentative bacteria was observed in large quantities at the end of the ensiling process. This 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 plantarum was the predominant species in mombaça grass (Panicum maximum) and signal grass (Brachiaria decumbens).

	Days after ensiling
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., 1991).

Species	Days after ensiling
P. maximum	D. decumbens	S. sphacelata
Lactobacillus plantarum	21	39	47
Lactobacillus coryniformis	6	21	0
Leuconostoc spp.	27	12	0
Enterococcus faecium	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).

It is evident that Lactobacillus plantarum and the species from the Pediococcus genus are prevalent in forage plants. The species from the 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.

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

With regard to the predominant bacteria identification in signal grass plants, it is observed in Table 6 that all of the isolates had the form of short bacilli with rounded ends, arranged in pairs or in short chains (3-4 cells). All of them showed a 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 they all grew at pH 7.2 and 4% NaCl at 45°C.

	Isolated strain
	EB1	EB2	EB3	EB4	EB5	EB6	Lactobacillus plantarum
Test
form	bacillus	bacillus	bacillus	bacillus	bacillus	bacillus	bacillus
Arrangement	DB*	DB	DB	DB	DB	DB	DB
Gram	+	+	+	+	+	+	+
Catalase	-	-	-	-	-	-	-
	Growth at different pH
7,2	+	+	+	+	+	+	+
9,6	-	-	-	-	-	-	-
	Growth at different salt concentration (NaCl)
NaCl 4%	+	+	+	+	+	+	+
NaCl 6,5%	-	-	-	-	-	-	-
	Growth at different temperatures (T ^oC)
15^oC	+	+	+	+	+	+	+
45^oC	+	+	+	+	+	+	+

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 and EB6 were identified as Lactobacillus plantarum with 99.9% similarity.

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

	Isolated strain				Lactobacillus plantarum
EB1	EB2	EB5	EB6
Glycerol	-	-	-	-	-
Erythritol	(+)	(+)	(+)	(+)	-
D-arabinose	-	-	-	-	-
L-arabinose	+	+	+	+	+
Ribose	+	+	+	+	+
D-xylose	-	-	-	-	-
L-xylose	-	-	-	-	-
Adonitol	-	-	-	-	-
β-methyl D-xyloside	-	-	-	-	-
Galactose	+	+	+	+	+
D-glucose	+	+	+	+	+
D-fructose	+	+	+	+	+
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	-	-	-	-	-
Glycogen	-	-	-	-	-
Xylitol	-	-	-	-	-
β-gentiobiose	+	+	+	+	+
D-turanose	+	+	+	+	+
L-lyxose	-	-	-	-	-
D-tagatose	-	-	-	-	-
D-fucose	-	-	-	-	-
L-fucose	-	-	-	-	-
D-arabitol	(+)	(+)	(+)	(+)	-
L-arabitol	-	-	-	-	-
Gluconate	+	+	+	+	+
2 Ketogluconate	-	-	-	-	-
5 Ketogluconate	-	-	-	-	-

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 the elephant grass plants cv. Cameroon (Pennisetum purpureum Schum), identified the isolates as being Lactobacillus casei ssp. Pseudoplantarum, using the carbohydrate fermentation profile as an identification criterion. Santos et al. (2011) observed the Lactobacillus plantarum as a LAB predominant species in signal grass (Brachiaria decumbens Stapf). Based on the details 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 species for most plants.

4. Lactic acid bacteria and their effects on silage fermentation

A suitable acidification is essential for the silage's successful preservation, especially when the crop moisture is relatively high (a condition which favours 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, the aerobic stage remains during filling and for some hours after the silage closing. The growth of aerobic microorganisms such as yeasts, fungi and bacteria favoured by high concentrations of oxygen (O₂) in the plant respiration process, promotes O₂ reduction, initiating the active fermentation process. Thus, there occurs a sharp drop in silage pH due to the formation of organic acids from sugars, in which they initially actuate the heterofermentative bacteria and enterobacteriaceae which become, then, dominated by homofermentative until the pH falls below 5.0.

During 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 sees the growth of moulds and yeasts. The inhibition of the fungi's multiplication through contact with O₂ is called 'aerobic stability' (Santos et al., 2006).

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

During the final fermentation stage, Lactobacillus becomes prevalent due to their tolerance to acidity. However, the silage LAB is pretty well diversified, depending upon plant material properties, silage technology and silo type. The LAB predominance changes from Lactococcus to Lactobacillus, usually occurring during the final fermentation stage. According to Langston et al. (1960), these chemical changes resulted from bacterial or plant enzymes' action, causing the conversion of carbohydrates into other components such as gas and organic acids, as well as the partial protein breakage resulting in the formation of non-protein structures.

The use of LAB as microbial inoculants has been widely documented in the 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, also emphasizing a scarcity of data 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 loss reduction in silage and the higher degree of preservation of the plant's nutritional value. Furthermore, they found that the response intensity varies with the plant species or microorganism studied, suggesting a specificity between these components.

However, studies conducted in the 1980s and 1990s have 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 in inoculating corn silage with two Lactobacillus plantarum strains isolated from corn and grass, the dominant strain after ensiling was that isolated from corn. The same was observed for the grass silage, where the dominant lactic bacteria strains were those 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 species and its epiphytic microflora implicates the need for studies related to the 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 the addition of L. buchneri UFLA SIL 72 reduced the fungi population and the ethanol concentration in silages. Santos et al. (2007) observed a reduction in ammonia concentration and the enterobacteria population in mombaça grass silage (Panicum maximum) inoculated with Lactobacillus plantarum, which were isolated from the epiphytic microflora.

As such, 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 re-growth age is that which influences all the silage characteristics - from fermentation to the nutritional value - considering the losses.

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

The high residual soluble carbohydrates content in silage - mainly those made of corn, sorghum and sugarcane - favour the aerobic deterioration process by fungi and yeasts, causing losses after silo opening. However, the organic acids produced by fermentation - mainly acetic acid - have a fungicidal effect and can mitigate the deterioration, increasing the silage's 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's 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 from a commercial inoculant and another isolated from sugarcane (Saccharum officinarum L.) silage. It was observed that there was 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 the low level of supply of soluble carbohydrates, what makes possible a rapid decline of pH values.

Table 8 presents a 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 upon the crop used, the microorganism strain and its concentration at the time of inoculation. Although significant, the effects are of a low magnitude, which leads to reflections as to our knowledge 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 effect of Lactobacillus buchneri on the fermentation and aerobic stability of corn, grasses and small grains silages. In general, the inoculation reduced the pH, lactic acid concentration and mould 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 that there was 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 that there was a correlation between acetic acid concentration and fungi population reduction.

Crop	Micro-organism	pH^1	NH3^2	LA^3	AA^4	PA^5	BA^6	ET^7	AE^8	DML^9	DMR^10
			% total N	% DM					hours	%
Grass	LP	--	--	++	--		--	ns	ns	--	++
Corn	LB	++	ns	--	++	ns		ns	+		ns
Grass		--	ns	--	++	++		++	+		--
Corn	PA/ LP	--		--	--	--	--				++
Wheat	LB	++	ns	--	++				++	++
LP	ns	--	++	ns				ns	--
LP/ LB	ns	--	ns	++				++	--
Sorghum	LB	ns	ns	--	++				++	++
LP	ns	--	ns	ns				--	--
LP/ LB	ns	--	--	++				++	--
Wheat	LP/EF	--		++	ns		ns		ns
LPe	--		++	ns		ns		ns
Wheat	LB	ns		ns	++				+	++
LP	ns		--	++				+	--
Sugar cane	LB	ns		ns	++	++	ns	--	+
Sunflower	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
Potato + WB*	LB	--	--	++	++	ns	--		++
LPa/ LL/ PA	--	--	++	--	--	--		--

Table 8.

Effect of inoculants with lactic acid bacteria on the fermentation of the silage. *Potato by-product + 30% of wheat bran; ¹lactic acid, ²acetic acid, ³propionic acid, ⁴butyric acid, ⁵ethanol, ⁶aerobic stability, ⁷dry matter losses, 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 acidilactici, L = Lactobacillus sp., LB = Lactobacillus buchneri; Pac = Propionibacterium acidipropionici; LPar = Lactobacillus paracasei paracasei LL = Lactococcus lactis.

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

Evaluating barley silage inoculated with Lactobacillus buchneri, Taylor et al. (2002) observed a decrease in the number of yeasts and moulds, contrasting this 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 part, promote improvements, especially after the opening of the silo, increasing the aerobic stability of silage by inhibiting the growth of moulds and yeasts. Thus, many research papers have recommended the use of inoculants combining the above two groups of LAB due to their greater efficiency compared to the isolated use.

5. Use of additives and management practices aimed at the development of lactic bacteria in tropical grass silages

For an appropriate fermentation process with a predominance of lactic acid, it is necessary to provide ideal conditions for the LAB to develop and predominate in the silage environment. In order to attend to these conditions, some additives are used which can absorb moisture or provide soluble carbohydrates, making a more propitious environment to the LAB growth. Some management practices may also be employed with the same purpose.

The key point in the management of grass for silage is undoubtedly the harvest time. Grass harvested during an advanced maturity stage presented a large LAB population; however, the high levels of the lignification of tissues is an intrinsic characteristic as well, which reduces their nutritional value. In contrast, young grasses have good nutritional value; however, they also have unfavourable characteristics in relation to the fermentation process, such as high humidity, low LAB population and a high buffering capacity. In the case of young grasses, various additives can be used. In the case of mature grasses, they can be settled to a point whereby the dry matter content and the LAB populations are suitable and the nutritive value is not compromised.

Research conducted with tropical grasses - evaluating the addition of a wide variety of additives - shows that an increase in the forage dry matter content or supply of soluble carbohydrates favours lactic fermentation and, in most cases, reduces the silage losses. Among others, it has been used with wheat bran, corn, fruit pulp and biodiesel industry by-products, sugar cane molasses and even tropical fruits, such as jackfruit (Zanine et al., 2006; Pardo et al., 2008; Santos et al., 2008; Rêgo et al., 2010; Andrade & Melotti, 2004; Zanine et al., 2010; Silva et al., 2011). It is important to remember that these additives should be used while respecting the level recommended by the authors, otherwise the effects can endanger the fermentative process.

Andrade and Melotti (2004) evaluated the effect of 20 additives on the quality of silage made of elephant grass over 80 days (Tables 9 and 10).

In this study, it is observed that cotton fibre, sweeping residue, corn meal, elephant grass hay and guandu hay were used as additives, absorbing moisture (90.91% of dry matter).The sweeping residue and molasses were used to supply carbohydrates (97.65%).

Looking at the N-NH₃ results, it seems that the use of urea, cotton fibre, elephant grass hay, guandu hay, corn meal and molasses with urea, resulted in increased protein degradation during the fermentation process. However, no changes were observed in the lactic acid concentration.

Treatment	DM	pH	N-NH3	Lactic; acid	Acetic; acid	Butyric acid
%		% total N	%DM
Control (without any additive)	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 fibre (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
Corn Meal (2%) /; Urea (0.5%)	17.96de	4.19b	36.67ab	5.31a	0.53b	0.04b
Corn Meal (4%)/; Urea (0.5%)	20.26c	4.29b	49.36a	1.96a	0.85b	0.05b
Corn Meal (6%) /; 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
Dried Molasses (1%); Urea (0.5%)	17.20de	4.18b	34.93abc	1.25a	0.46b	0.04b
Dried Molasses (2%); Urea (0.5%)	18.20de	4.09b	32.43abc	5.24a	0.44b	0.04b
Dried Molasses (3%); 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 DM. Equal means in the column do not differ (P>0.05): CV = coefficient of variation.

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 fibre had a higher dry matter loss (11.0 and 10.5%, respectively).

According to the authors, it is not recommended to make the inclusion of urea, hay and cotton fibre with elephant grass silage. Additives rich in non-structural carbohydrates, such as corn meal and molasses, can be used; however, further studies are required to establish suitable levels for better fermentation. The microbial inoculant 'BioSilo' does not benefit the elephant grass silage.

Treatment	IVDMD (%DM)	DML (%)
Control (without any additive)	41.62abcde	6.80b
Urea 0.5 %	34.47abcde	11.00a
Cotton fibre (10%)	27.62de	10.50a
Elephant grass hay (10%)	34.12abcde	9.80b
Guandu hay(10%)	26.36e	7.00b
Drying for 6 hours	41.71abcde	6.70b
Sugar waste (2%)	42.89abcd	6.85b
Corn Meal (2%)	41.36abcde	6.70b
Corn Meal (4%)	45.68abc	7.20b
Corn Meal (6%)	41.81abcde	5.70b
Corn Meal (2%) /Urea (0.5%)	50.30ab	6.60b
Corn Meal (4%)/ Urea (0.5%)	51.31a	7.10b
Corn Meal (6%) /Urea (0.5%)	41.82abcde	7.10b
Dried Molasses (1%)	40.03abcde	6.80b
Dried Molasses (2%)	46.84abc	6.65b
Dried Molasses (3%)	45.25abc	6.80b
Dried Molasses (1%) Urea (0.5%)	43.73abc	6.90b
Dried Molasses (2%) Urea (0.5%)	47.15bc	7.10b
Dried Molasses (3%) Urea (0.5%)	49.65ab	6.85b
BioSilo inoculant	32.52de	7.00b
CV (%)	13.70	18.5

Table 10.

In vitro dry matter digestibility (IVDMD) and dry matter losses (DML) of elephant grass, Napier, ensiled with different additives (Andrade & Melotti, 2004). Equal means in the column do not differ (P>0.05): CV = coefficient of variation.

In more recent studies, evaluating the effect of four additives in sugar cane silage (sugarcane with 1.5% urea; 0.5% urea + 4% corn; 0.5% urea + 4% dried cassava; 1.5% of starea and sugar cane control), Lopes and Evangelista (2010) concluded that the additive 0.5% urea + 4% corn provides better results than the sugar cane silage.

Ávila et al. (2006), using combinations of different additives types (citrus pulp, wheat bran and corn meal) with various doses (3%, 6%, 9% and 12%), found that Tanzania grass has a low soluble carbohydrates content while citrus pulp was the additive which contributed to increasing the forage carbohydrate concentration and reducing the buffering capacity. It provides an increase in the relation of the soluble carbohydrate and buffering capacity and better conditions for the fermentation process, resulting in better quality silages.

Besides the additives, some management practices from the harvest time to silo sealing can influence the LAB development. When the grass is chopped at harvest time, the LAB population tends to increase due to the reactivation of dormant and non-culturable cells. Thus, the shorter the time between cutting the grass and sealing the silo, the better the fermentation conditions.

A good compaction and sealing is one of the secrets for good silage. They serve to expel the air from inside the forage mass (considering that air presence affects the fermentation process, resulting in losses caused by undesirable microorganisms). According to Senger et al. (2005), the original material must exhibit a compression level exceeding 650 kg/m³ of green matter, reducing the quality losses of the ensiled material.

Furthermore, the particle size influences the compression and - consequently - the silo density. Igarasi (2002) observed an inverse relationship between particle size and silage density, suggesting that the smaller the particle size, the greater the density; thus, there will be more oxygen remaining among the plant particles.

Neumann et al. (2007) in evaluating the effect of particle size (small: 0.2 to 0.6 cm or large: 1.0 to 2.0 cm) and cutting height of corn plants (low: 15 cm or higher: 39 cm) on silage fermentation dynamics and the opening period found that small-sized particles provide greater compression efficiency and, consequently, reduces the temperature and pH gradients during the silo opening time. The temperature differential between silage and the environment is greater on the top, which is related to the time that the silo remains opened and is exposed to the external environment as well as a lower compression efficiency. It causes an increase in ammoniac nitrogen content and an elevation of silage pH values, indicating changes in the silage's nutritional value.

The plant moisture content and the particle size after chopping are directly related to the compression. Excessively wet forage provides favourable conditions for butyric fermentation and favours nutrient losses through leaching and the degradation of proteins. On the other hand, forage with a high dry matter content hinders compaction and air expulsion during the ensiling process. Amaral et al. (2007) found that an increase in compression of 100 to 160 kg MS/m³ increased effluent production from 2.2 to 9.8 kg/t of green matter.

In summary, the faster and more efficient the process of harvesting, chopping, compaction and sealing, the greater the amount of LAB present in silage and, thus, the lower the losses.

6. Conclusions

The increase in lactic acid fermentation is a big challenge for the confection of tropical grass silages, determining the success of this technology. It is extremely important to understand the species of lactic acid bacteria prevalent in tropical grasses, as well as their metabolism, in order to obtain maximum use with its utilization.

The use of lactic acid bacteria as microbial inoculants in tropical grass silages continues to show some inconsistency in the results obtained in research works. More research evaluating their effects on the fermentation parameters, dry matter losses and - mainly - on the quality in relation to nutrient intake and animal performance is required.

However, tropical grass silages represent a promising technology for livestock in areas threatened by periodic droughts. Furthermore, in tropical countries like Brazil, this practice has been taken up by producers.