**2.3. Cultures and incubations**

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

chemical analysis and in vitro digestibility measurements.

**2.2. Chemical analyses** 

VFA contents in the silage.

Shizuoka, Japan). The vegetable residue comprised the outside leaf part of white cabbage, Chinese cabbage, red cabbage, and lettuce with no added bacteria. Experimental treatments included control silage without additive or with LAB, beet pulp (DM, 90.7%; organic matter, 94.9%; crude protein, 8.4%; ether extract, 0.7%; acid detergent fiber, 25.6%; neutral detergent fiber, 52.1%; WSC, 2.1% of DM), and beet pulp+LAB. The strain FG1 (Lactobacillus plantarum Chikuso-1; Snow Brand Seed, Sapporo, Japan) isolated from a commercial inoculant was used. The de Man Rogosa Sharpe agar broth was inoculated with strain FG1 and incubated overnight. After incubation, the optical density of the suspension at 700 nm was adjusted to 0.42 using sterile 0.85% NaCl solution. The inoculum size of LAB was 1 mL of suspension per kilogram of FM. The inoculated LAB number was 1.0 × 105 CFU/g of FM. The addition ratio of beet pulp was 300 g per kg of FM. Silages were prepared using a small-scale system of silage fermentation (Cai et al., 1999). Approximately 100-g portions of forage material, chopped into about 20-mm length, were mixed well and packed into plastic bags (Hiryu KN type, 180 × 260 cm; Asahikasei, Tokyo, Japan). The bags were sealed with a vacuum sealer (BH 950; Matsushita, Tokyo, Japan). The plastic-bag silos were stored at a room at 25°C. There were ten bag silos per treatment. One bag of silo per treatment was opened on d 60. Samples were dried in a forced-air oven at 60°C for 48 h, ground to pass through a 1-mm screen with a Wiley mill (ZM200, Retch GmbH & Co. KG, Haan, Germany), and used for

The TMR silages, vegetable and its silages were dried in a forced draft oven at 60°C for 48 h and ground into a 2-mm powder with a sample mill (Foss Tecator; Akutalstuku, Tokyo, Japan). Moisture, ash, crude protein, ether extract, and crude fiber contents were determined by general methods. Analyses of neutral detergent fiber and acid detergent fiber contents were made following Van Soest et al. (1991). Heat-stable amylase and sodium sulfite were used in the neutral detergent fiber procedure, and the results were expressed without residual ash. Nonfibous carbohydrate was calculated by the formula as: Nonfibous carbohydrate = organic matter − crude protein − nonfibous carbohydrate − ether extract. The fermentation products of silages were determined using cold-water extracts. Wet silage (50 g) was homogenized with 200 ml sterilized distilled water and stored at 4°C overnight (Cai et al. 1999). The pH of the silages was determined using a glass electrode pH meter (D-21, Horiba, Kyoto, Japan). Lactic acid was analyzed using the methods of Cai et al. (1999). Ammonia- N was determined as described by (Cai et al. 2003). To measure total VFA, silage and ruminal fluid were steam-distilled and titrated using sodium hydroxide. Dried VFA salt was separated and quantified using gas chromatography (G-5000A, Hitachi, Tokyo, Japan) equipped with a thermal conductivity detector and a stainless column (Unisole F-200, 3.2 mm × 2.1 m). The analytical conditions were as follows: column oven temperature, 140°C; injector temperature, 210°C; detector temperature, 250°C. V-score, which was used to assess silage quality, was determined from the proportion ammonia-N in the total nitrogen and Two adult wethers (average initial body weight, 78.5 kg) fitted with rumen cannulae were used as donors of ruminal fluid. The wethers were fed a basal diet of 50% reed canary grass (Phalaris arundinacea L.) hay and 50% commercial feed concentrate (Koushi-Ikusei-Special, Kitanihon-Kumiai-Feed, Miyagi, Japan) at maintenance energy level (2.0% DM of their body weight) and had free access to clean drinking water. They were fed once daily at 09:00 h. Wethers were cared for in accordance with the animal care and use institutional guidelines for animal experiments at the Faculty of Agriculture, Yamagata University (Tsuruoka, Japan).

Rumen fluid was collected through the rumen cannulae 2 h after feeding and diverted to plastic bottles. The fluid was filtered through four layers of cheesecloth and combines on an equal volume basis. The combined filtrate was mixed with CO2-bubbled McDougall's artificial saliva (pH 6.8) at a ratio of 1:4 (vol/vol). Then 50 mL buffered rumen fluid was transferred to 128-mL serum bottles containing 0.5 g sample, and flushed with O2-free CO2. Tubes were capped with a butyl rubber stopper and sealed with an aluminum cap. Incubations were performed in triplicate at 39°C for 6 h (Mohammed et al., 2004) in a water bath with a reciprocal shaker (100 strokes/min).
