**3. Abatement of rumen methanogenesis by direct action of lactic acid bacteria as prebiotics producer**

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

 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 *Leuconostoc citreum* JCM9698 that produced almost the same size of clear zone on a Umamizyme G containing plate as that on a plate without Umamizyme G, were selected as PRA producers. *Lactobacillus plantarum* TUA1490L and *Leuconostoc citeum* JCM9698 were selected as PRA-1 and PRA-2 producers. GYEKP medium to prepare inoculants for PRA-1, PRA-2, nisin Z and control were used for the culture of lactic acid bacteria. Each strain of lactic acid bacteria was inoculated into a shaking flask containing GYEKP, and was cultivated for 20 h at 30C using SILIKOSEN (Shin-Etsu polymer, Tokyo), which was culture plug for aeration cultivation after confirmation of the stationary phase. The cells were removed by centrifugation at 8,000 g at 4C and filtration with 0.45 μm membrane filter. The supernatants were used as PRA inoculants in the in vitro gaseous quantification trials. Methane mitigating effects of PRA-1 from *Lactobacillus plantarum* TUA1490L and PRA-2 from *Leuconostoc citeum* JCM9698 isolated from foods were determined in comparison with *Lactococcus lactis* ATCC19435 which did not produce any antibacterial substances as a negative control and *Lactococcus lactis* NCIMB702954 which produced nisin-Z as a positive control using in vitro continuous incubation system equipped with automated infra-red quantification apparatus (Takahashi *et al*., 2005). Fig.2 shows effects of PRA-1 and PRA-2 produced by *Lactobacillus plantarum* and *Leuconostoc citreum* on cumulative methanogenesis extrapolated by nonlinear regression analyses. PRA-1 remarkably decreased cumulative methane production. For PRA-2, there were no effects on CH4 and CO2 production and fermentation characteristics in mixed rumen cultures. Fig. 3 shows the effect of PRA on potential methane production which estimated from non-linier regression analysis of cumulated methane production. It has been suggested that PRA-1 significantly decreases potential methane production by rumen methanogens (Asa *et al*., 2010). The PRA maintained their antimicrobial effects after incubation with proteases, while nisin lost its activity. Therefore, the PRA was hypothesized to be a more sustained agent than nisin for the mitigation of rumen methane emission. Fig. 4 shows DGGE band patterns of archaea and eubacteria. All fluorescence brightness of methanogens bands of PRA-1 were remarkably light in color compared with control. Band No. 1 to No.3 in archaea might be *Methanobrevibacter sp.* which is a Gram positive or parasitic methanogens sticking on protozoan surface (Fig.5). PRA-1 increased the fluorescence brightness of the band of the Gram positive bacteria and declined the fluorescence brightness of the band of the Gram negative bacteria. For Gram positive bacteria, *Streptococcus* sp*., Clostridium* sp., *Butyrivibrio*  sp. and *Clostridium aminophilum* were increased, whereas *Prevotella* sp., *Prevotella ruminicola, Pseudobutyrivibrio* sp, *Prevotella* sp, *Succinivibrio dextrinosolvens and Schwartzia succinivorans* in Gram negative bacteria were decreased by adding PRA-1.

Lactic Acid Bacteria and Mitigation of GHG Emission from Ruminant Livestock 461

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was the antibacterial substance produced from a strain of *Lactobacillus plantarum* TUA1490L that was isolated from tomato in Japan. However, methane suppressing activity of PRA-1 was not inactivated by treatments Umamizyme G and protease K. Moreover, aeration cultivation is an essential procedure for activation of PRA-1 to abate methanogenesis. Therefore, possible mechanism of PRA-1 produced by *Lactobacillus plantarum* TUA1490L on rumen methane production might be assumed as resulting from the direct involvement of low molecule substance such as hydrogen peroxide due to the requirement of aeration for

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**Figure 4.** DGGE band patterns

Natural antimicrobial substances can be used alone or in combination with other novel preservation technologies to facilitate the replacement of traditional approaches (Brijesh, 2009). *Lactobacillus plantarum* produces bacteriocin from many foods including meat and meat products (Garriga *et al*.,1993; Enan *et al*.,1996; Aymerich *et al*., 2000), milk (Rekhif *et al*., 1995), cheese (Gonzalez *et al*.,1994), fermented cucumber (Daeschel *et a*l.,1994), olives (Jimenez-Diaz *et al*., 1993; Leal *et al*., 1998), grapefruit juice (Kelly *et al*.,1996), Turkish fermented dairy products (Aslim *et al*., 2005), and sourdough (Todorov *et al*., 1999). PRA-1 was the antibacterial substance produced from a strain of *Lactobacillus plantarum* TUA1490L that was isolated from tomato in Japan. However, methane suppressing activity of PRA-1 was not inactivated by treatments Umamizyme G and protease K. Moreover, aeration cultivation is an essential procedure for activation of PRA-1 to abate methanogenesis. Therefore, possible mechanism of PRA-1 produced by *Lactobacillus plantarum* TUA1490L on rumen methane production might be assumed as resulting from the direct involvement of low molecule substance such as hydrogen peroxide due to the requirement of aeration for the preparation.

**Figure 4.** DGGE band patterns

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

Gram negative bacteria were decreased by adding PRA-1.

Natural antimicrobial substances can be used alone or in combination with other novel preservation technologies to facilitate the replacement of traditional approaches (Brijesh, 2009). *Lactobacillus plantarum* produces bacteriocin from many foods including meat and meat products (Garriga *et al*.,1993; Enan *et al*.,1996; Aymerich *et al*., 2000), milk (Rekhif *et al*., 1995), cheese (Gonzalez *et al*.,1994), fermented cucumber (Daeschel *et a*l.,1994), olives (Jimenez-Diaz *et al*., 1993; Leal *et al*., 1998), grapefruit juice (Kelly *et al*.,1996), Turkish fermented dairy products (Aslim *et al*., 2005), and sourdough (Todorov *et al*., 1999). PRA-1

*Leuconostoc citreum* JCM9698 that produced almost the same size of clear zone on a Umamizyme G containing plate as that on a plate without Umamizyme G, were selected as PRA producers. *Lactobacillus plantarum* TUA1490L and *Leuconostoc citeum* JCM9698 were selected as PRA-1 and PRA-2 producers. GYEKP medium to prepare inoculants for PRA-1, PRA-2, nisin Z and control were used for the culture of lactic acid bacteria. Each strain of lactic acid bacteria was inoculated into a shaking flask containing GYEKP, and was cultivated for 20 h at 30C using SILIKOSEN (Shin-Etsu polymer, Tokyo), which was culture plug for aeration cultivation after confirmation of the stationary phase. The cells were removed by centrifugation at 8,000 g at 4C and filtration with 0.45 μm membrane filter. The supernatants were used as PRA inoculants in the in vitro gaseous quantification trials. Methane mitigating effects of PRA-1 from *Lactobacillus plantarum* TUA1490L and PRA-2 from *Leuconostoc citeum* JCM9698 isolated from foods were determined in comparison with *Lactococcus lactis* ATCC19435 which did not produce any antibacterial substances as a negative control and *Lactococcus lactis* NCIMB702954 which produced nisin-Z as a positive control using in vitro continuous incubation system equipped with automated infra-red quantification apparatus (Takahashi *et al*., 2005). Fig.2 shows effects of PRA-1 and PRA-2 produced by *Lactobacillus plantarum* and *Leuconostoc citreum* on cumulative methanogenesis extrapolated by nonlinear regression analyses. PRA-1 remarkably decreased cumulative methane production. For PRA-2, there were no effects on CH4 and CO2 production and fermentation characteristics in mixed rumen cultures. Fig. 3 shows the effect of PRA on potential methane production which estimated from non-linier regression analysis of cumulated methane production. It has been suggested that PRA-1 significantly decreases potential methane production by rumen methanogens (Asa *et al*., 2010). The PRA maintained their antimicrobial effects after incubation with proteases, while nisin lost its activity. Therefore, the PRA was hypothesized to be a more sustained agent than nisin for the mitigation of rumen methane emission. Fig. 4 shows DGGE band patterns of archaea and eubacteria. All fluorescence brightness of methanogens bands of PRA-1 were remarkably light in color compared with control. Band No. 1 to No.3 in archaea might be *Methanobrevibacter sp.* which is a Gram positive or parasitic methanogens sticking on protozoan surface (Fig.5). PRA-1 increased the fluorescence brightness of the band of the Gram positive bacteria and declined the fluorescence brightness of the band of the Gram negative bacteria. For Gram positive bacteria, *Streptococcus* sp*., Clostridium* sp., *Butyrivibrio*  sp. and *Clostridium aminophilum* were increased, whereas *Prevotella* sp., *Prevotella ruminicola, Pseudobutyrivibrio* sp, *Prevotella* sp, *Succinivibrio dextrinosolvens and Schwartzia succinivorans* in

Lactic Acid Bacteria and Mitigation of GHG Emission from Ruminant Livestock 463

Breukink, E., C. Van Kraaij, A. Van Dalen, R.A. Demel, R.J. Siezen, B. De Kruijff, and O.P. Kuipers., 1998. The orientation of nisin in membranes. Biochem., 37: 8153–8162. Brijesh, K. T., P. V. Vasilis, P. O. D. Colm, M. Kasiviswanathan B. Paula and P. J. Cullen., 2009. Applivation of natural antimicrobials for food preservation. J. Agric. Food Chem.,

Callaway, T. R., Alexandra M. S. Carneiro De Melo and J. B. Russell., 1997. The effect of nisin

Cadieux, P., J. Burton, G. Gardiner, J. Braunstein, A.W. Bruce, G.Y. Kang and G. Reid., 2002.

Chalupa, W., 1984. Manipulation of rumen fermentation. In: Recent Advances in Animal Nutrition. W. Haresign and D. Cole, (eds). Butterworths, London, England,

Chen, M. and M.J. Wolin, M.J., 1979. Effect of monensin and lasalocid-sodium on the growth of methanogenic and rumen saccharolytic bacteria. Appl. Environ. Microbiol., 38: 72-77. Daeschel, M. A., M. C. Mckenny and L. C. McDonald., 1990. Bacteriocidal activity of

Delves-Broughton, J., P. Blackburn., R. Evans and J. hugenholtz., 1996. Applications of the

Enan, G., A. A. El-Essawy., M. Uyttendaele and J. Debevere., 1996. Antibacterialactivity of Lactobacillus plantarum UG1 isolated from dry sausage: characterization, production

Garriga, M., M. Hugas., T. Aymerich and J. M. monfort., 1993. Bacteriocinogenic activity of

González, B., P. Arca., B. Mayo and J. E. Suárez., 1994. Detection, purification and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum

Hopgood, M. F. and D. J. Walker., 1967. Succinic acid production by rumen bacteria. II. Radioisotope studies on succinateproduction by *Ruminococcus flavefaciens.* Aust. J. Biol.

IPCC (Intergovermental Panel on Climate Change), 1994. Houghton, J.H., Meria filho, J. Bruce, L.Hoesung, B.A.Callander, H. Haites, N.Harris and K.Maskell, (eds), Cambridge

Immig I., D. Demeyer, D. Fiedler, C. Van Nevel and L. Mbanzamihigo., 1996. Attempts to induce reductive acetogenesis into a sheep rumen. Arch. Tierernahr. 49:363-370.

lactobacilli from fermented sausages. J. Appl. Bacteriol., 75: 142-148.

Houghton, J., 1994. Global warming. Lion Publishing plc. Oxford, UK, pp: 29-45.

strain of dairy origin. Appl. Environ. Microbiol., 6: 2158-2163.

and bactericidal action of plantarcin UG1. Int. J. Food. Microbiol., 30: 189-215. Gamo, Y., M. Mii, X.G. Zhou, C. Sar, B. Santoso, I. Arai, K. Kimura and J. Takahashi., 2002. Effects of lactic acid bacteria, yeasts and galactooligosaccharides supplementation on in vitro rumen methane production. In: J. Takahashi and B.A. Young, (eds), Greenhouse Gases and Animal Agriculture. ELSEVIER, Amsterdam,

and monensin on ruminal fermentations in vitro. Curr Microbiol., 35:90-96.

Lactobacilli strains and vaginal ecology. J. Am. Med. Assoc., 287: 1940-1941.

Lactobacillus plantarum C11. Food Microbiol. 7: 91-99.

bacteriocin, nisin. Antonie van Leeuwenhoek. 69: 193-202.

57: 5987-6000.

pp.143-160.

Netherland. pp201-204.

Sci., 20: 183-192.

University Press. New York. pp25-27.

**Figure 5.** Electric scanning microscopy of symbioses of methanogens on the surface of Ciliate Protozoa.
