**2. Materials and methods**

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

ability to dominate the fermentation.

silage ensiled with straw and molasses.

other types of fermentation because the theoretical recoveries of dry matter and energy are greatest. During this type of fermentation, LAB utilizes water-soluble carbohydrates to produce lactic acid, the primary acid responsible for decreasing the pH in silage. In contrast, other fermentations are less efficient. Natural populations of LAB on plant material are often low in number and heterofermentative. Thus, the concept of using a microbial inoculant to silage involves adding fast-growing homofermentative LAB in order to dominate the fermentation, thereby producing higher-quality silage. Some of the commonly used homofermentative LAB in silage inoculants include *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Pediococcus acidilactici*, and *Enterococcus faecium*. Commercially available microbial inoculants contain one or more of these bacteria that have been selected for their

Food by-product such as tofu cake is high in crude protein and fatty acids (Xu et al. 2001). Not only could the by-products be utilized as a source of nutrients for ruminants, but using them to replace imported commercial feedstuffs could save energy in transportation, and possibly reduce the environmental impact of burning them as waste of burying them landfills. Preparing total mixed ration (TMR) silage is one practice whereby food byproducts are stored and utilized as animal feeds in Japan (Imai, 2001). Our previous study (Cao et al., 2009b) showed that TMR silage with 30% dried tofu cake had the higher lactic acid content than that with rice bran or green tea waste. Alli et al. (1984) reported that as molasses can provide fermentable sugars for the production of organic acids, it has been used extensively as a fermentation aid, and that silage prepared with molasses may show a lower pH, higher residual water-soluble carbohydrates levels, greater quantities of lactic acid, lower levels of volatile basic nitrogen, and decreased dry matter (DM) loss compared to silage without molasses. Weinberg et al. (2003) also reported a high lactic acid content in

However, even if there is plenty of glucose as a substrate, if insufficient in lactic acid bacteria, to preparing good quality silage is difficult. Cai et al. (1999) reported that the factors involved in fermentation quality include chemical composition, particularly the water-soluble carbohydrates content of the silage material and the physiological properties of epiphytic bacteria. Conservation of forage crops by ensiling is based on natural fermentation in which epiphytic LAB convert sugars into lactic acid under anaerobic conditions. As a result, the pH decreases and the forage is preserved. The fermentation quality of silage is influenced by the size, diversity and activity of epiphytic LAB. The population density of LAB has been reported to range from 10–103 CFU/g fresh matter (FM) on standing forage crops to 103–107 CFU/g FM on chopped ones entering the silo. Generally, When LAB reaches at least 105 (CFU/g of FM), silage can be well preserved; if LAB values below 105 high-quality silage fermentation may need to be controlled using certain inoculants. *Lactobacillus plantarum* Chikuso-1 is shown to have great potential as an

Furthermore, Commercial processing of vegetables results in many residues, but most are burned and dumped into landfills or used as compost, which is a waste of resources and leads to possible environmental problems due to unsuitable disposal. Demand for efficient

inoculant for WCR silage (Cai et al., 1999, 2003; Cai, 2001; Cao et al., 2011).

Experiments were conducted with permission from the Committee of Animal Experimentation, and according to the animal care and use institutional guidelines for animal experiments at the Faculty of Agriculture, Yamagata University.

### **2.1. Silage preparation**

Whole crop rice (Haenuki) was cultivated using transplant cultivating methods in a paddy field on an experimental farm at Yamagata University, Japan, and harvested at the full-ripe stage with a length of 2 cm. As shown in Table 1, TMR was prepared using compound feed (Kitanihon-Kumiai Feed, Yamagata, Japan), WCR, dried beet pulp, a vitamin-mineral supplement (Snow Brand Seed, Iwate, Japan), dried tofu cake (Zenno, Tsuruoka, Japan), molasses (sugarcane; Dai-Nippon Meiji Sugar Co., Tokyo, Japan) and LAB (*Lactobacillus plantarum* Chikuso-1, Snow Brand Seed, Sapporo, Japan). Experimental treatments included control silage without additive, or with molasses, (4% FM basis), LAB (5 mg kg–1 FM basis), and molasses+LAB. Moisture was adjusted with water to approximately 65%. Silages were prepared using a small-scale system of silage fermentation. Approximately 1 kg TMR was packed into plastic film bags (Hiryu BN-12 type, 270 mm × 400 mm, Asahikasei, Tokyo, Japan), and the bags were sealed with a vacuum sealer (SQ303, Sharp, Osaka, Japan). Three silos per treatment were prepared and stored in a room at 20–25°C for 60 days.

Residues of white cabbage (*Brassica campestris* L. *var. capitata*), Chinese cabbage (*Brassica rapa* L. *var. glabra Regel*), red cabbage (*Brassica oleracea* var. *capitata F. rubra*), and lettuce (*Lactuca sativa* L.) (three samples of each) were collected from a local commercial vegetable factory (Fujiyama factory, Matsuya Foods Company, Limited, Fujinomiya, 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 chemical analysis and in vitro digestibility measurements.

Ruminal Digestibility and Quality of Silage Conserved via Fermentation by Lactobacilli 367

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

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

To terminate fermentation at the end of incubation, 25 µL of formaldehyde solution (35%) were injected into serum bottles, which were immediately sealed and cooled at room temperature. Gas samples were collected by air syringe from the serum bottles and injected into a gas chromatograph (GC323, GL Sciences, Tokyo, Japan) equipped with a thermal conductivity detector and a stainless steel column (WG-100 SUS, 1.8 m × 6.35 mm OD), and the methane production in each serum bottle was measured. The analytical conditions were as follows: column oven temperature at 50°C, injector temperature at 50°C, and detector

Separate sub-samples of the supernatant were taken to determine the pH and VFA concentration. The bottles were rinsed with warm water to remove all solid residues, which were then oven-dried at 60°C and stored for further analyses. In total, 2 g of dried residue

Analyses were performed using the general linear model procedure (SAS institute, Cary, NC, USA). Data on fermentative characteristics, in vitro DM digestibility, and ruminal methane and VFA production of TMR silages were subjected to one-way analysis of variance Tukey's test was used to identify differences (*P* < 0.05) between means. Data on the fermentative characteristics of each vegetable silage opened on d 30 were analyzed by one-

**2.5.** *In vitro* **DM digestibility, and methane and VFA production** 

were oven-dried at 135°C and stored to determine DM digestibility.

**2.3. Cultures and incubations** 

bath with a reciprocal shaker (100 strokes/min).

**2.4. Analysis of fermentation products** 

(Tsuruoka, Japan).

temperature at 50°C.

**2.6. Statistical analyses** 

### **2.2. Chemical analyses**

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 VFA contents in the silage.
