**2. Possible control of indirect action of lactic acid bacteria as probiotics on rumen methanogenesis**

Rumen manipulation with ionophores such as monensin has been reported to abate rumen methanogenesis (Mwenya *et al*., 2005), However, there is an increasing interest in exploiting prebiotics and probiotics as natural feed additives to solve problems in animal nutrition and livestock production as alternatives of the antibiotics due to concerns about incidences of resistant bacteria and environmental pollution by the excreted active-antibacterial substances (Mwenya *et al*., 2006). Particular interest concerning bacteriocins which produced by lactic acid bacteria has increased recently.

Bacteriocins, antimicrobial proteinaceous polymeric material substances, are ubiquitous in nature being produced by a variety of Gram-negative and Gram-positive bacteria, and typically narrow spectrum antibacterial substances under the control of plasmid. Nisin is produced by *Lactococcus lactis* ssp. *lactis* which is an amphiphilic peptide composed by 34 amino acids with two structural domains that are connected by a flexible hinge (Breukink *et al*., 1998; Montville and Chen, 1998), and is classified into the group of lantibiotics. Nisin has a mode of action similar to ionophores, which show antimicrobial activity against a broad spectrum of Gram-positive bacteria and is widely used in the food industry as a safe and natural preservative (Delves-Broughton *et al*., 1996). It is generally recognized as safe (GRAS) and given international acceptance in 1969 by the joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food

Additives. Recent works have indicated that *Lactococcus lactis* subsp. *lactis* produce nisin Z, which has been identified from Korean traditional fermented food "Kimchi" besides nisin A (Park, 2003). They have similar antibacterial ability to mitigate methane emission (Mwenya *et al*., 2004; Santoso *et al*., 2004; Sar *et al*., 2006), to inhibit growth both of *Clostridium amoniphilum*, which is obligate amino-acid fermenting bacteria (Callaway *et al*., 1997) and lactic acid-producing ruminal Staphylococci and Enterococci (Lauková, 1995). *Leuconostoc mesenteroides* ssp. *mesenteroides, Leuconostoc lactis* and *Lactococcus lactis* ssp. *lactis* were isolated from "Laban" which was a traditional fermented milk product in Yemen and determined the mitigating effect on in vitro rumen methane production. These strains isolated from Laban enhanced propionate production and decreased acetate/propionate ratio. In consequence, they reduced methane production remarkably (Gamo *et al*., 2002). For *Leuconostoc mesenteroides* ssp. *mesenteroides*, in particular, the mitigating effect was amplified with GOS, which was degradable about 80% within 1 hour incubation in the artificial rumen fluid due to the stimulation of reduction reactions consuming metabolic hydrogen. However, direct involvement of bacteriocin or lower molecular substances produced by the strain on rumen methanogenesis remains to be elucidated.

Lactic Acid Bacteria and Mitigation of GHG Emission from Ruminant Livestock 459

(SEM = 20.802)

b

a

**Figure 3.** Effect of PRA on potential methane production. Control: *Lactococcus lactis* ATCC19435 (nonantibacterial substances), Nisin-A: *Lactococcus lactis* NCIMB702054, PRA-1: *Lactobacillus plantarum* TUA1490L, and PRA-2: *Leuconostoc citreum* JCM9698. Vertical bars represent standard deviation (n = 4).

a

Control Nisin Z PRA-1 PRA-2

**3. Abatement of rumen methanogenesis by direct action of lactic acid** 

For low molecular compounds, small amounts of volatile fatty acids (acetic acid, formic acid), hydrogen peroxide, β-hydroxy-propionaldehyde (reuterin) are produced by lactic acid bacteria as antibacterial substances in addition to lactic acid. Because lactic acid bacteria themselves don't have a group of catalase, considerable amount of hydrogen peroxide accumulates in the bacterial cells. Many strains of the genus *Lactobacillus* are commonly referred to as having high ability to produce hydrogen peroxide (Jaroni and Brashears, 2000;

 Its antimicrobial activity is effective against numerous Gram-positive bacteria. Although it has been reported that nisin suppress rumen methanogenesis, the suppressing efficacy of nisin on rumen methanogenesis may not be sustained, because proteinaceous nisin is degradable in the rumen due to bacterial protease (Sang *et al*., 2002). Several strains of lactic acid bacteria produce different types of protease resistant antimicrobial substance (PRA). In our research, the strain of lactic acid bacteria that produce PRA were screened on MRS agar plates containing Umamizyme G (protease mixture from *Aspergillus oryzae*, amino Enzyme Inc, Nagoya, Japan) as follows: candidates were inoculated onto MRS agar with or without 1,000 IU ml-1 of Umamizyme G and incubated for 24 h at 30 C. the plates were then overlaid with Bacto Lactobacilli agar AOAC (Becton, Dickinson and Company, NJ. USA) containing an indicator strain, *Lactobacilli sakei* JCM1157T. The agar overlays were incubated for 24 h at 30C and examined for zones of clearing. Protease degradable anti-microbial substances were decomposed by Umamizyme G, thus a clear zone did not form on the plate with Umamizyme G. Two strains of lactic acid bacteria, *Lactobacillus plantarum* TUA1490L and

Means with different letters differ significantly (p<0.01).

**bacteria as prebiotics producer** 

0

50

100

150

200

250

a

300

Potential methane production (ml)

Aroucheva *et al,* 2001; Gardiner *et al*., 2002).

Where, y(ml) =gas produced at time *t* (min), a=first gas production, b=second gas production and c=frctional rate gas production, using Kaleida Graph (Version 3.6, Synergy Software, Reading, PA, USA).

**Figure 2.** Effect of PRA on the cumulative methane production extrapolated by nonlinear regression analysis; y=a+b (1-e-ct)3.

strain on rumen methanogenesis remains to be elucidated.

0

analysis; y=a+b (1-e-ct)3.

50

100

150

Methane production (ml)

200

250

Additives. Recent works have indicated that *Lactococcus lactis* subsp. *lactis* produce nisin Z, which has been identified from Korean traditional fermented food "Kimchi" besides nisin A (Park, 2003). They have similar antibacterial ability to mitigate methane emission (Mwenya *et al*., 2004; Santoso *et al*., 2004; Sar *et al*., 2006), to inhibit growth both of *Clostridium amoniphilum*, which is obligate amino-acid fermenting bacteria (Callaway *et al*., 1997) and lactic acid-producing ruminal Staphylococci and Enterococci (Lauková, 1995). *Leuconostoc mesenteroides* ssp. *mesenteroides, Leuconostoc lactis* and *Lactococcus lactis* ssp. *lactis* were isolated from "Laban" which was a traditional fermented milk product in Yemen and determined the mitigating effect on in vitro rumen methane production. These strains isolated from Laban enhanced propionate production and decreased acetate/propionate ratio. In consequence, they reduced methane production remarkably (Gamo *et al*., 2002). For *Leuconostoc mesenteroides* ssp. *mesenteroides*, in particular, the mitigating effect was amplified with GOS, which was degradable about 80% within 1 hour incubation in the artificial rumen fluid due to the stimulation of reduction reactions consuming metabolic hydrogen. However, direct involvement of bacteriocin or lower molecular substances produced by the

0 1000 2000 3000 4000 5000 6000

time(min) Where, y(ml) =gas produced at time *t* (min), a=first gas production, b=second gas production and c=frctional rate gas

**Figure 2.** Effect of PRA on the cumulative methane production extrapolated by nonlinear regression

production, using Kaleida Graph (Version 3.6, Synergy Software, Reading, PA, USA).

Control(actual measurement) NisinZ(actual measurement) PRA-1(actual measurement) PRA-2(actual measurement)

Control NisinZ PRA-1 PRA-2

**Figure 3.** Effect of PRA on potential methane production. Control: *Lactococcus lactis* ATCC19435 (nonantibacterial substances), Nisin-A: *Lactococcus lactis* NCIMB702054, PRA-1: *Lactobacillus plantarum* TUA1490L, and PRA-2: *Leuconostoc citreum* JCM9698. Vertical bars represent standard deviation (n = 4). Means with different letters differ significantly (p<0.01).
