Development of a new lactic acid bacterial inoculant for fresh rice straw silage.
The annual roughage demands of herbivorous animals in Korea are estimated to be about 5.6 million tons. Roughage self-sufficiency is about 82% with half of them being rice straw. More than 2 million tons of rice straw is typically used to raise Korean native cattle . However, rice straw is of low quality and does not provide enough nutrients for most ruminant animals. More than half of the dry matter (DM) of rice straw consists of cellulose and hemicellulose, and the remainder is comprised of lignin, nitrogenous compounds, and ash . The feed value of rice straw as hay is very poor because of its structural characteristics and long drying period. Although rice straw contains sufficient cellulose to make it an excellent energy source for ruminants, it is a poor-quality feed in its natural state.
As a means of improving rice straw quality various agents were used in the past and one of them was ammonia, which increases crude protein (CP) content. The palatability of rice straw for ruminants is low but this low palatability can be increased by cubing or pelleting the straw . Wrapping large round bales with stretch-wrap plastic film to make silage is a relatively new method of preserving forage. However, it is difficult to make good silage from fresh rice straw due to low water soluble carbohydrate (WSC) content at harvest, hollow stems, and small amount of epiphytic lactic acid bacteria (LAB) .
Inoculants are the most common biological additives used to preserve silage worldwide. These products have selected strains of homofermentative LAB, such as Lactobacillus plantarum, Enterococcus faecium, and Pediococcus spp. . Adding LAB helps decrease pH faster, lower the final pH value, increase lactic acid content, and decrease N[H.sub.3]-N in silage . Many microbial inoculant products are available commercially but most of them were not developed for rice straw but rather for major foraging crops, such as corn, sorghum, alfalfa, and grasses. Therefore, we tried to isolate, identify and evaluate novel LAB strains suitable for low WSC content rice straw silage.
MATERIALS AND METHODS
Collection and screening of microbes
Silage samples without any additives were collected from around the country. The samples were plated on MRS agar and incubated at 35[degrees]C for 48 h. LAB were quantified by manually counting all yellow colonies, which were subcultured in MRS broth (Difco, Detroit, MI, USA) to test their growing ability. Among numerous bacteria, 10 microbes were tested for their acid-producing ability in MRS broth for 8 h at 25[degrees]C. Finally, two LAB were isolated from the samples (LAB 1821 and 1841).
Fresh rice straw (Chucheong) was directly ensiled after harvesting on an experimental field at the National Institute of Animal Science of RDA (Cheonan, Korea). The straw was chopped into 2 to 3 cm pieces using a rice straw cutter and mixed well. Then, it was ensiled in an experimental silo (20 L) with or without microbial additives (1821, 1841, HM/F, Agri-Lloyd International Ltd., Leominster, UK; and P1132, Pioneer Hi-Bred International Inc., Plymouth, IN, USA) and stored at ambient temperature (<25[degrees]C) for 60 days. The addition rate of inoculant was 1 x [10.sup.6] cfu/g fresh matter. The control received the same amount of distilled water. Three replications for each treatment were ensiled, and the experimental silo was sealed.
The silo was opened after 2 months, and the DM contents of the rice straw silages were oven-dried at 65[degrees]C for 72 h. The dried samples were ground to pass through a 1 mm screen and kept in double-plug-type plastic bottles for analysis. CP was determined by the Kjeldahl method , and acid detergent fiber (ADF) and neutral detergent fiber (NDF) were measured by the method of Goering and Van Soest . The in vitro dry matter digestibility (IVDMD) of the rice straw silages was determined using the two-stage technique of Tilley and Terry  over 72 h. Ten grams sample of each silage was macerated with 90 mL of distilled water for 30 min in a shaker and filtered, and the filtrates were used to measure pH with a pH meter (HI 9024; Hanna Instruments Ltd., Leighton Buzzard, UK). Ten grams sample of each silage was macerated with 90 mL of distilled water for 24 h and filtered through filter paper (#6). The filtrates were analyzed for volatile fatty acid and lactic acid contents. Volatile fatty acids were analyzed using gas chromatography (Model 3400; Varian Co., Harbor City, CA, USA), and lactic acid was analyzed by high performance liquid chromatography (HP-1100; Hewlett-Packard Co., Palo Alto, CA, USA). WSC was determined using the anthrone method of Thomas  and N[H.sub.3]-N concentrations were analyzed by the method of Chaney and Marbach  using a spectrophotometer (UVIDEC-610; Jasco Co., Tokyo, Japan). LAB counts in silage were estimated on MRS agar, and fungal counts were estimated on potato dextrose agar, as described by the American Public Health Association .
Lactic acid bacteria isolation
LAB 1821 was isolated from a good oat silage by plating on MRS agar containing 0.02% sodium azide and was identified based on its biochemical characteristics and a 16S rRNA analysis. The bacteria were screened by growing and acid-producing ability on minimal medium with a low carbohydrate content (Table 1). The 16S rRNA gene sequence analysis was performed using the method of Pavlova et al .
All data were analyzed using the general linear model procedure in SAS software . Differences among treatment means were determined using the least significant difference (LSD) test. A probability level of p < 0.05 was considered to be statistically significant.
Selection of microbes
The acid-producing ability (pH) of10 isolated bacteria is shown in Table 2. The LAB 1821 and 1841 microbial cultures showed the lowest pH values after an 8 h incubation and both were incubated in MRS broth at 25[degrees]C, 35[degrees]C, and 45[degrees]C for 12 h to evaluate their growth characteristics. The cultures grew well at 25[degrees]C and 35[degrees]C, but not at 45[degrees]C (Figure 1). Finally, strains 1821 and 1841 were selected as new silage inoculant candidates. The isolated strains were Gram-positive, rods, catalase-negative, and were identified to be the non-gas-forming Lactobacillus plantarum (L. plantarum) (Table 3), based on their biochemical characteristics and substrate utilization. The growing ability of L. plantarum 1821 on minimal medium with a low carbohydrate content was not different from that of L. plantarum (ATCC 10012).
Chemical composition of fresh rice straw
The chemical composition of fresh rice straw is shown in Table 4. The fresh rice straw had low WSC and CP contents, as well as low IVDMD. Insufficient WSC content could result in poor LAB fermentation during ensiling. The DM, CP, and WSC contents of fresh rice straw were 36.1%, 35.1 g/kg, and 43.3 g/kg, respectively. The ADF and NDF contents, as well as the IVDMD, were 418.9, 657.3, and 417.4 g/kg, respectively.
Effect of the new inoculant on rice straw silage quality
The rice straw silage quality is shown in Table 5. The fresh rice straw silages treated with additives (1821, 1841, HM/F, and P1132) were well-preserved at ambient temperature; the pH value and butyric acid content were lower, while the lactic acid and acetic acid contents, as well as the lactic/acetic acid ratio, were higher than those of the control. Acidity (pH) was lowest and lactic acid highest, in the 1821-treated silage (p < 0.05). All silages treated with inoculants had higher lactic/acetic acid ratios than the control, of 3.82-9.66. The N[H.sub.3]-N content decreased significantly in inoculant-treated silage (p < 0.05), while that of the 1821-treated silage was the lowest among the treatments.
The effect of inoculants on the microbial flora in rice straw silage is shown in Table 4. LAB counts increased in 1821-, 1841-, and P1132-inoculated silages (p < 0.05), and fungal counts decreased in response to the LAB 1821 treatment (p < 0.05).
Effect of the new inoculant on rice straw feed value
Table 6 shows the chemical composition of the fresh rice straw silage. The DM content of the control silage was lower compared with that of fresh rice straw (p < 0.05) and all inoculant-treated silages had a significantly higher DM content than that of the HM/F-treated silage. None of the microbial additives had any significant (p > 0.05) effect on ADF or NDF contents. The CP content and IVDMD increased after inoculation of the microbes (p < 0.05). The WSC content was lower in the inoculant-treated silages by an average of 7.6 g/kg compared to the control (9.4 g/kg).
Bacterial inoculants are known to improve silage fermentation and forage conservation. In present study inoculation with LAB significantly reduced pH, as well as the acetic acid, butyric acid and N[H.sub.3]-N contents, but increased the lactic acid content. Current results imply that LAB inoculation promoted rapid acidification, which inhibited the proteolytic activities of plant enzymes.
Most commercial inoculants consist of LAB. The LAB belong to a group of Gram-positive, low guanine-cytosine (G+C) containing, nonmotile, nonspore-forming, aerotolerant bacteria that ferment hexoses to lactic acid . Microorganisms can be categorized according to their ability to grow at low, moderate, or high temperatures (psychrophilic, mesophilic, and thermophilic microorganisms, respectively) . In general, the proper temperature for good silage fermentation is <32[degrees]C . The 1821 and 1841 microbes grew well at 25[degrees]C to 35[degrees]C in this study, so both were classified as mesophilic. McDonald  suggested that an ideal silage inoculant should grow at temperatures of up to 120[degrees]F (48[degrees]C).
Low WSC content of rice straw in the harvest stage restricts LAB growth during silage fermentation . Therefore, rice straw silage microbes must have good growth characteristics in low WSC content media. LAB 1821 showed better growth ability (pH and viable count) in sugar-restricted media compared with microbes in low WSC content media from the American Type Culture Collection (Table 7), and was identified as L. plantarum by the 16s rRNA gene sequence analysis. The 16S rRNA sequence analysis method is highly effective for identifying the genus and species of organisms. LAB 1821 was identified as L. plantarum by previous sequencing and phylogenetic tree analyses (Figure 2) .
Acidity (pH) is a crucial factor when evaluating silage fermentation quality . Compared with the control silage, the pH of LAB 1821-treated silage decreased significantly (p < 0.05). All inoculants significantly increased the lactic acid content of rice straw silage, except HM/F. The LAB 1821 and 1842 decreased the acetic acid content.
Adding the LAB decreased the N[H.sub.3]-N content of rice straw silage significantly (p < 0.05). McDonald  reported that a low pH inhibits protein degradation in silage. In this study, the low pH values in all LAB-treated silages prevented protein degradation. Whiter and Kung  stated that homofermentative LAB reduce proteolysis and deamination of silage through a more rapid fall in pH. The absolute value (5.86% to 6.81% of total N) indicated that all LAB-treated rice straw silage except the control could be considered as near acceptable silage according to Wilkinson , who reported that optimally preserved silage must have an N[H.sub.3]-N concentration <50 g/kg of total N. Haigh  stated that an N[H.sub.3]-N value <100 g/kg N usually indicates successful preservation.
The new LAB strains in this study (1821 and 1841) decreased the acetic acid, and increased the lactic acid content of silages significantly (p < 0.05). Some researchers have reported that use of a LAB inoculant increases lactic acid production and decreases silage pH, the acetic acid level, and butyric acid production [25,26]. The ratio of lactic acid to acetic acid is a good indicator of the type of fermentation undergone by the silage . Zhang et al  also reported that a ratio of at least 2:1 indicates strong homolactic fermentation. The ratios of lactic acid to acetic acid in LAB 1821 and 1841 were higher (9.66 and 9.58) than those of a commercial inoculant (p < 0.05). According to this result, LAB 1821 and 1841 are classified as homofermentative LAB.
Many studies have shown positive effects of bacterial inoculants on the digestibility of silage when fed to beef cattle , and dairy cattle . Aksu et al  also reported that the digestibility of DM and NDF in inoculated corn silages were higher than those of a control. Other researchers have reported that inoculating LAB does not have positive effects on digestibility . Although the average IVDMD of all rice straw silages was low in this study, all LAB significantly increased the IVDMD of silage (p < 0.05).
Rice straw has low WSC and epiphytic LAB contents. A high-performance LAB must be inoculated to produce high-quality silage, and must grow well in a low sugar content environment. In this study, LAB 1821 met these requirements considering all criteria we assessed
CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
This research was supported by Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01177902), Rural Development Administration, Republic Korea.
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Jong Geun Kim (1) *, Jun Sang Ham (2), Yu Wei Li (1), Hyung Soo Park (3), Chul-Sung Huh (1), Byung-Chul Park (1) *
* Corresponding Authors: Jong Geun Kim
Tel: +82-33-339-5728, Fax: +82-33-339-5763, E-mail: email@example.com Byung-Chul Park Tel: +82-33-339-5792, Fax: +82-33-339-5763, E-mail: firstname.lastname@example.org
(1) Graduate School of International Agricultural Technology and Institute of Green Bio Science & Technology, Seoul National University, Pyeongchang 25354, Korea
(2) Animal Products Research and Development Division, National Institute of Animal Science, Wanju 55365, Korea
(3) Grassland and Forage Division, National Institute of Animal Science, Cheonan 31000, Korea
Submitted Apr 14, 2017; Revised May 16, 2017;
Accepted May 18, 2017
Caption: Figure 1. Growth characteristics of selected two microbes (1821; upper, 1841; low) at different temperature.
Caption: Figure 2. Phylogenetic tree of partial 16S rRNA sequence of lactic acid bacteria (LAB) 1821 and identified bacteria in the nucleotide database of GeneBank.
Table 1. Low carbohydrate minimal medium used for the screening Ingredients Amount [K.sub.2]HP[O.sub.4] (g) 1.0 KHT[O.sub.4] (g) 1.0 Yeast extract (g) 5.0 FeS[O.sub.4] (g) 0.001 MgS[O.sub.4] (g) 0.25 NaCl (g) 0.005 Glucose (g) 1 Water (mL) 1,000 pH 7.0 Table 2. Change of acid producing ability (pH) for the selected cultures during the incubation Incubation 1808 1819 1821 1823 1827 1828 1830 time (h) 0 6.0 6.0 6.0 6.0 6.0 6.0 6.0 8 4.41 4.38 4.36 4.42 4.46 4.43 4.41 (bc) (cde) (def) (bc) (a) (abc) (bcd) Incubation 1834 1840 1841 Mean time (h) 0 6.0 6.0 6.0 -- 8 4.44 4.43 4.35 4.41 (ab) (abc) (df) (a-f) Means within rows followed different superscript letters are statistically different (p < 0.05). Table 3. The biochemical characteristics of selected lactic acid bacteria, 1821 Item Characteristics Major +/- carbohydrate fermentation Shape Rod L-Arabinose + Fermentation type Homo Galactose + Gram stain + Fructose + Catalase activity - Dulcitol - Gas from glucose - Sorbitol + Growth potential temp. 13-43 Lactose + ([degrees]C) Growth potential pH 3.5-7.5 Mannitol + +, Active; -, Inactive. Table 4. Nutrient contents and in vitro dry matter digestibility of rice straw at ensiling Composition Fresh rice straw DM (%) 36.1 WSC (g/kg DM) 43.3 CP (g/kg DM) 35.1 ADF (g/kg DM) 418.4 NDF (g/kg DM) 657.3 IVDMD (g/kg DM) 417.4 DM, dry matter; WSC, water soluble carbohydrate; CP, crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber; IVDMD, in vitro dry matter digestibility. Table 5. Effect of inoculant on the acidity (pH), ammonia-nitrogen, organic acid composition and microbial flora in rice straw silage Items Control 1821 1841 pH 4.93 4.40 4.51 N[H.sub.3]-N (% TN) 16.83 5.86 6.26 Acetic acid (% DM) 0.67 0.29 0.26 Butyric acid (% DM) 0.35 0.18 0.77 Lactic acid (% DM) 1.80 2.80 2.49 Lactic/acetic acid 2.69 9.66 9.58 LAB (cfu/g) 2.9 x 2.1 x 1.7 x [10.sup.7] [10.sup.8] [10.sup.8] Fungi (cfu/g) 100 20 150 Items P1132 HM/F LSD (p<0.05) pH 4.73 4.75 0.28 N[H.sub.3]-N (% TN) 6.07 6.81 0.82 Acetic acid (% DM) 0.50 0.50 0.15 Butyric acid (% DM) 0.14 0.11 0.12 Lactic acid (% DM) 2.65 1.91 0.24 Lactic/acetic acid 5.30 3.82 1.62 LAB (cfu/g) 1.9 x 9.1 x 4.0 x [10.sup.8] [10.sup.7] [10.sup.7] Fungi (cfu/g) 100 120 30 LSD, least significant difference; TN, total nitrogen; DM, dry matter; LAB, lactic acid bacteria. Table 6. Effects of inoculant on nutrient contents and in vitro dry matter digestibility in rice straw silage Items Control 1821 1841 P1132 HM/F LSD (p<0.05) DM (%) 30.5 37.6 39.7 40.7 35.0 3.3 WSC (g/kg DM) 9.4 7.2 7.6 7.3 8.2 1.3 CP (g/kg DM) 46.1. 56.0 50.8 52.2 47.5 4.4 ADF (g/kg DM) 491.3 481.7 493.7 484.6 472.8 NS NDF (g/kg DM) 703.7 712.4 694.2 703.9 696.1 NS IVDMD (g/kg DM) 333.1 357.6 345.7 367.2 357.4 14.2 LSD, least significant difference; DM, dry matter; WSC, water soluble carbohydrate; CP, crude protein; ADF, acid detergent fiber; NS, not significant; NDF, neutral detergent fiber; IVDMD, in vitro dry matter digestibility. Table 7. Comparison of growth ability of selected LAB in sugar restricted media Strains Concentration of sugar(%) 0 h Viable count pH (cfu/mL) 1821 0.1 5.2 x [10.sup.7] 6.69 0.5 6.43 Lactobacillus 0.1 4.0 x [10.sup.7] 6.70 plantarum ATCC 10012 0.5 6.43 Lactobacillus 0.1 4.9 x [10.sup.7] 6.71 bulgaricus ATCC 33409 0.5 6.44 Strains Concentration Incubation time of sugar(%) 6 h Viable count pH (cfu/mL) 1821 0.1 5.1 x [10.sup.8] 4.95 0.5 6.3 x [10.sup.8] 4.34 Lactobacillus 0.1 6.2 x [10.sup.7] 6.16 plantarum ATCC 10012 0.5 1.2 x [10.sup.8] 5.00 Lactobacillus 0.1 2.0 x [10.sup.8] 6.14 bulgaricus ATCC 33409 0.5 2.4 x [10.sup.8] 5.23 Strains Concentration of sugar(%) 24 h Viable count pH (cfu/mL) 1821 0.1 4.2 x [10.sup.8] 4.62 0.5 7.9 x [10.sup.8] 3.60 Lactobacillus 0.1 1.2 x [10.sup.8] 4.65 plantarum ATCC 10012 0.5 3.7 x [10.sup.8] 3.87 Lactobacillus 0.1 4.0 x [10.sup.8] 4.62 bulgaricus ATCC 33409 0.5 3.9 x [10.sup.8] 3.62 LAB, lactic acid bacteria.
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|Author:||Geun Kim, Jong; Sang Ham, Jun; Wei Li, Yu; Soo Park, Hyung; Huh, Chul-Sung; Park, Byung-Chul|
|Publication:||Asian - Australasian Journal of Animal Sciences|
|Date:||Jul 1, 2017|
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