648 Open Access Asian Australas. J. Anim. Sci. Vol. 27, No. 5 : 648-657 May 2014 http://dx.doi.org/10.5713/ajas.2013.13670 www.ajas.info pISSN 1011-2367 eISSN 1976-5517 Naturally Occurring Lactic Acid Bacteria Isolated from Tomato Pomace Silage Jing-jing Wu, Rui-ping Du1, Min Gao1, Yao-qiang Sui, Lei Xiu, and Xiao Wang* College of Life Science, Inner Mongolia University, Huhhot 010021, China ABSTRACT: Silage making has become a significant method of forage conservation worldwide. To determine how tomato pomace (TP) may be used effectively as animal feed, it was ensilaged for 90 days and microbiology counts, fermentation characteristics and chemical composition of tomato pomace silage (TPS) were evaluated at the 30th, 60th, and 90th days, respectively. In addition, 103 lactic acid bacteria were isolated from TPS. Based on the phenotypic and chemotaxonomic characteristics, 16S rDNA sequence and carbohydrate fermentation tests, the isolates were identified as 17 species namely: Lactobacillus coryniformis subsp. torquens (0.97%), Lactobacillus pontis (0.97%), Lactobacillus hilgardii (0.97%), Lactobacillus pantheris (0.97%), Lactobacillus amylovorus (1.9%), Lactobacillus panis (1.9%), Lactobacillus vaginalis (1.9%), Lactobacillus rapi (1.9%), Lactobacillus buchneri (2.9%), Lactobacillus parafarraginis (2.9%), Lactobacillus helveticus (3.9%), Lactobacillus camelliae (3.9%), Lactobacillus fermentum (5.8%), Lactobacillus manihotivorans (6.8%), Lactobacillus plantarum (10.7%), Lactobacillus harbinensis (16.5%) and Lactobacillus paracasei subsp. paracasei (35.0%). This study has shown that TP can be well preserved for 90 days by ensilaging and that TPS is not only rich in essential nutrients, but that physiological and biochemical properties of the isolates could provide a platform for future design of lactic acid bacteria (LAB) inoculants aimed at improving the fermentation quality of silage. (Key Words: Lactic Acid Bacteria, Silage, Tomato Pomace, 16S rRNA Gene) INTRODUCTION nutrients, such as vitamins, minerals, vegetable fiber and protein (Delvalle et al., 2006; Abdollahzadehi et al., 2010). Using byproducts of food processing as animal feed has TP is produced in vast amounts annually in the Bayannur proven economically viable, not only because it is a method region, the largest tomato production base in Inner of waste and residue disposal but also because it is a real Mongolia, generating 1.8 to 2.3 million tons of tomato and alternative for feeding livestock (Parvin et al., 2010). In 100,000 tons of tomato byproducts. The high water content China, it is increasingly important to produce highly (75%) of this byproduct limits its length of storage. Thus, digestible forage to support the expanding dairy industry. TP is often dried. Dried TP is fed to dairy cows and sheep The number of dairy cattle reached 12.6 million in 2010 (Belibasakis and Ambatzidiz, 1995; Zheng et al., 2011a), (Wang et al., 2013). Usually, food processing byproducts but artificial drying increases the price of TP substantially; contain high amounts of protein and fiber, rendering them thus, much of the pomace that is produced is discarded suitable for animal feed production (Council for Science (Weiss et al., 1997; Nemat Ziaei and Sadrollah Molaei, and Technology, 2005). Tomato pomace (TP), a byproduct 2010). In the light of the above, it is obvious that TP has a of the tomato juice industry, contains an abundance of high potential to become a highly nutritious and economic livestock feed if it can be economically preserved. * Corresponding Author: Xiao Wang. TP can be fed to ruminant animals for longer periods of E-mail: [email protected] time without spoilage, when it is ensiled with or without 1 Animal Nutrition Institute of Agriculture and Animal Husbandry Academy of Inner Mongolia, Huhhot 010031, China. additives (Denek and Can, 2006). Of the many factors that Submitted Oct. 21, 2013; Revised Dec. 24, 2013; Accepted Jan. 12, 2014 influence this process, the number and type of Copyright © 2014 by Asian-Australasian Journal of Animal Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 649 microorganisms that dominate the fermentation process lactobacilli MRS agar (Huankai Microbial SCI. and Tech, often dictate the final quality of the silage (Cai, 1999a; Co., Ltd. Guangdong, China) under anaerobic conditions Weinberg et al., 2004). The preservation of silage depends (anaerobic jar, AnaeroPack-Anaero, Tokyo, Japan) and on the production of sufficient organic acid mainly measured by plate count. Coliform bacteria were incubated produced by lactic acid bacteria (LAB) to inhibit the at 30C for 24 h and counted on blue light broth agar activity of undesirable microorganisms, such as clostridia (Huankai Microbial SCI. and Tech, Co., Ltd.). Molds and and molds, under anaerobic conditions (Cai et al., 1997). yeast were incubated for 24 h at 30C and counted on The aim of this study was to determine whether TP potato dextrose agar (Huankai Microbial SCI. and Tech, Co., could be preserved for a long time as a livestock diet by Ltd.). Yeast was distinguished from mold and bacteria by ensilaging and to examine the LAB in tomato pomace colony appearance and cell morphology. Bacilli and aerobic silage (TPS) that are most likely to have significant bacteria were distinguished by colony shape and counted on functions in the fermentation process. In this study, the nutrient agar (Huankai Microbial SCI. and Tech, Co., Ltd.), chemical composition, silage fermentation characteristics incubated for 24 h at 30C under aerobic conditions. and microbiology counts of TPS were measured. Further, Colonies were counted as viable numbers of LAB isolates from tomato silage were identified based on microorganisms in colony-forming units per gram fresh phenotypic and chemotaxonomic characteristics, 16S rDNA matter (FM). Each LAB colony was purified twice by being sequence analysis and carbohydrate fermentation tests. streaked on MRS agar. The purified strains were stored at 80C in nutrient broth (Difco) and dimethyl sulfoxide at a MATERIALS AND METHODS 9:1 ratio for further analysis (Cai et al., 1999d,e). Tomato pomace sampling and silage preparation Morphological, physiological and biochemical tests TP was collected from local tomato juice industries Gram-staining and catalase activity were examined after (Inner Mongolia, China) on October 7, 2012. TP is a residue 48 h of incubation on MRS agar. Gram-positive and that remains after juice is extracted, consisting of remainder catalase-negative bacteria were identified as LAB. flesh, seeds and peels without the addition of bacteria. TPS Morphological characteristics and gas production from was prepared in a small-scale fermentation system using a glucose were measured as described (Camu et al., 2007). slightly modified method of Tanaka and Ohmomo (1994). Growth at various temperatures was measured in MRS agar Approximately 100 g of tomato pomace, chopped into after incubation at 5C and 10C for 10 d and at 45C and about 10 mm lengths, was packed into plastic film bags and 50C for 7 d. Growth at pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 sealed. The film bag silos were stored outdoors. Three was monitored in MRS broth after incubation at 37C for 7 parallel experiments were used for silage treatment. d. Salt tolerance of LAB was tested in MRS agar that Samples were collected at the 30th, 60th, and 90th days of containing 3.0% and 6.5% NaCl. the ensiling process. Carbohydrate fermentation tests were performed using API 50 CH strips (BioMérieux, France) for 49 compounds Chemical analysis and 1 control per the manufacturer’s instructions; reactions TPS and the fresh TP were ground to pass a 1-mm screen and analyzed in 3 parallel experiments. Dry matter were measured after incubation at 37C for 48 h. The (DM), crude protein (CP), and ash content were determined organisms were identified using API LAB Plus, version by standard methods (AOAC, 2000) (930.15, 976.05, and 3.3.3 from the BioMérieux and Analytab Products database 942.05, respectively). Neutral detergent fiber (NDF) was for comparison of assimilation and fermentation patterns. analyzed with thermostable amylase and sodium sulfite and acid detergent fiber (ADF) was analyzed nonsequentially; PCR amplification, sequencing and analysis of 16S results were expressed without residual ash (Van Soest et al., rRNA genes 1991). The pH was measured with a glass electrode pH Cells were grown at 37°C for 8 h in MRS broth and meter (MP230, Mettler Toledo, Greifensee, Switzerland) used for DNA extraction and purification (Meroth et al., and ammonia-N was measured by steam distillation of the 2003). The 16S rRNA coding region was amplified by PCR filtrates (Xu et al., 2007). The organic acid content was in a thermal cycler (Analytikjena, Flexcycler Block, measured per Cai et al. (1999b). Germany) using reagents from the Takara Taq PCR Kit (Takara Biotechnology Co., Ltd, Dalian, China).The Microbiological analysis and lactic acid bacteria isolates primers were prokaryotic 16S ribosomal DNA universal Each of the samples (1 g) were blended with 9 mL primers 27F (5’-GAGTTTGAT CCTGGCTCA-3’) sterilized water and serially diluted (101 to 105) in and1492R (5’-TACCTTGTTACGACTT-3’). sterilized water. LAB were incubated at 37C for 48 h on The PCR performed was as follows: 50 L PCR 650 Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 reactions contained 0.25 L Takara Taq (5 U/L), 1 L 27F and alignment (Thompson et al., 1994). The 16S rDNA (10 mol/L) and 1492R (10 mol/L) primer, 5 L 10PCR sequences of the isolated LAB strains were compared with buffer (Mg2+-free), 3 L MgCl (25 mol/L), 4 L dNTP sequences of LAB strains in NCBI (Figure 1). Nucleotide 2 mixture, 2 L template DNA and ddH O up to 50 L. The substitution rates (Knuc values) were calculated (Pang et al., 2 2012) and phylogenetic tree was constructed using the PCR program was 94C for 5 min, 30 cycles at 94C for 1 neighbor-joining method (Ennahar et al., 2003). Bacillus min, 53C for 1 min and 72C for 2 min and 72C for 10 subtilis NCDO 1769 was used as an outgroup organism min. (Duan et al., 2008). The topologies of the tree was 16S rRNA gene sequences were compared using evaluated by bootstrap analysis of the sequence data using GenBank and the BLAST program Molecular Evolutionary Genetics Analysis (MEGA) 5 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence (Tamura et al., 2007), based on 1,000 random resamplings information was introduced into CLUSTALW for assembly Figure1. Phylogenetic tree showing the relative positions of the representative isolates isolated from tomato pomace silage as inferred by the neighbor-joining method of complete 16S rDNA sequences. Bootstrap values for 1,000 replicates are shown at the nodes of the tree. Bacillus subtilis is used as an outgroup. The bar indicates 1% sequence divergence. Knuc = Nucleotide substitution rate. Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 651 (Eitan et al., 2006). The sequences were aligned with when probability was less than 0.05. All reported values published sequences of type strains in GenBank. were the average of three replicates. Nucleotide sequence accession number RESULTS AND DISCUSSION The nucleotide sequences for the 16S rRNA genes were deposited into GenBank under the following accession Microorganism counts, chemical composition and numbers: KF312693, KF312677, KF312678, KF312679, fermentation characteristics of TPS KF312680, KF312681, KF312682, KF312683, KF312684, The counts of viable microorganisms, chemical KF312685, KF312686, KF312687, KF312688, KF312689, composition and silage fermentation characteristics of TPS KF312690, KF312691, and KF312692 for the are shown in Table 1. Overall, 107 to 108 (cfu/g FM) LAB, representative strains TCP001, TCP007, TCP004, TCP008, 102 molds, 103 to 105 aerobic bacteria, 102 yeasts and 105 TCP015, TCP016, TCP017, TCP050, TCP029, TCP024, bacilli were found in TPS samples. It is notable that TCP045, TCP071, TCP073, TCP102, TCP080, TCP063 and coliform bacteria were not present and that LAB was the TCP037, respectively. dominant group among the microorganisms in the TPS. Due to the anaerobic fermentation process, the number Statistical analyses of aerobic bacteria in the TPS was less than that of fresh TP Data on chemical composition and silage quality of TPS and continued to reduce with time during the fermentation were subjected to ANOVA and differences between means process. The populations of LAB in TPS (107 to 108 cfu/g were assessed by Tukey test using the statistical packages FM) increased significantly compared to that of fresh TP for the social sciences (SPSS 13.0 for Windows; SPSS Inc., (103 cfu/g FM) and kept increasing in the ensiling process, Chicago, IL, USA). The effect was considered significant which was good for the conversation of TPS. This great Table 1. Colony counting of microorganisms, feed value analysis and fermentation end-production in TPS1 stored for 0, 30, 60, and 90 days Item 0 d 30 d 60 d 90 d Counts (cfu/g of FM) Lactic acid bacteria 5.0103 2.8107 9.2107 1.4108 Bacilli 1.5103 1.5105 4.8105 3.3105 Coliform bacteria 6.0104 ND2 ND ND Aerobic bacteria 3.7107 8.1105 9.1104 2.0103 Molds ND 6.1102 2. 8102 1.1102 Yeasts 1.5103 6.4102 6.1102 1.0102 Chemical composition (g/kg DM) CP 164.1c 172.1cd 188.5d 192.7d NDF 694.1b 693.2b 692.0b 662.5a ADF 534.3d 536.2d 506.9c 475.6a OM 813.4b 805.6b 822.4b 815.4b Ether extract 272.5a 275.2a 284.3a 274.5a Silage quality (g/kg DM) pH 4.6b 4.60b 4.56ab 4.40a Ammonia-N 5.3b 6.2c 7.2c 11.7d DM 345.4a 347.5a 346.4 a 343.4 a Calcium 3.1a 3.8b 12.6c 16.6 d Phosphorus 3.1c 3.5b 3.8d 3.9d Ash 34.3a 34.2a 35.0a 34.6a Lactic acid ND 202.3b 245.6c 354.5d Acetic acid ND 48.1b 69.3ab 83.4a Propionic acid ND 54.5b 56.7b 66.81c Butyric acid ND 6.3b 5.9b 5.0c Color of TPS Light red Light red Red Reddish yellow TPS, tomato pomace silage; FM, fresh matter; ND, not detected; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; OM, Organic Matter; DM, dry matter. Values with different superscripts differ significantly (p<0.05), values with same superscripts means no significant difference (p>0.05). 1 Silage was stored for 30 to 90 d; data of cfu/g of FM are the average of three parallel experiments. 652 Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 increase was mainly related with the anaerobic environment. indicated that the indigestible fibrous material hydrolyzed 104 coliform bacteria were found in fresh TP, while in the to cause more energy value of silage. Lower ADF and NDF TPS they were totally inhibited. Although the count of are good for digestion and metabolism in ruminants. In molds in the TPS increased compared with that of fresh TP, addition, too much ADF and NDF would reduce the dry the number of moulds was still low. Moreover, the count of matter intake of ruminants. It was observed that the color of molds kept reducing with time. The inhibition of TP was virtually preserved in the TPS during the ensiling undesirable microorganisms, such as coliform bacteria and period. For instance the red color of the TP was still molds was attributable to the increase of LAB which maintained in the TPS at the 30th day while its light red and produce lactic acid that inhibits the growth of these reddish yellow colors at the 60th and 90th days respectively undesirable microorganisms. From a microbiology point of were only slightly and thus inconsequentially, different view, therefore, ensilaging TP in this study has been from the original red color of the TP. The original color of successful as it witnessed a simultaneous time-dependent the TP was therefore largely preserved in the TPS. Given increase and decrease in desirable and undesirable the chemical composition and fermentation characteristics microbial populations respectively. of TPS, therefore, ensilaging TP to preserve it would be an The levels of CP, lactic acid, acetic acid, phosphorus, economic way of producing a highly nutritive livestock calcium and ammonia-N increased compared with those of forage alternative. the fresh TP and continued to significantly increase during the whole storage period (p<0.05). The ADF content and Morphological, physiological and biochemical pH of TPS declined (p<0.05) during storage. The levels of properties ash, organic matter, ether extract and DM in the TPS did The morphology, characteristics and API 50 CH not have significant change (p>0.05) over time. The level of fermentation patterns of representative strains from TPS are NDF and butyric acid did not change significantly before 60 shown in Table 2 and 3. A total of 103 (TCP001 to TCP103) days (p>0.05) but declined afterwards (p<0.05). Lactic acid LAB strains were isolated from silage, on the basis of their ensures long-stem storage of silage by creating a low pH gram-positive reaction, negative catalase reaction and environment to inhibit the survival of undesirable microbes. production of lactic acid as the chief fermentation product. Phosphorus and calcium are needed for good nutritional Ultimately, they were identified as 17 species and all value of silage. The decrease of ADF and NDF was belonged to genus Lactobacillus, in contrast to previous attributed to the degradation of fibrous material and this research. For example, Cai et al. (1998; 1999a,b) noted that Table 2. Characterristics of representative strains (neighbor-joining method or NJ designations) isolated from TPS1 TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP Characteristic 001 007 004 008 015 016 017 050 029 024 045 071 073 102 080 063 037 No. of isolates 17 6 36 11 1 3 7 4 4 3 1 2 1 1 2 2 2 Shape Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Gram stain + + + + + + + + + + + + + + + + + Catalase                  Gas from glucose      +    +    +    Fermentation from type Homo Homo Homo Homo Homo Hetero Homo Homo Homo Hetero Homo Homo Homo Hetero Homo Homo Homo Growth at temperature (C) 5.0                  10.0 + w + + +  + +  +        45.0         +  + +  + + + w 50.0            +  +    Growth in NaCl 3.0% + + + +  + + +  + + w + + + + 6.5%    +             Growth at pH 3.0 + + + + +  + +  + + + + + + +  4.0 + + + + + + + + + + + + + + + + + 5.0 + + + + + + + + + + + + + + + + + 6.0 + + + + + + + + + + + + + + + + + 7.0 + + + + + + + + + + + + + + + + + 8.0 + + + + + + + + + + + + + + + +  16Sr DNA 99.86 99.59 99.59 99.38 99.52 99.67 98.00 99.23 99.00 99.93 97.30 99.72 98.33 99.79 99.65 99.40 100.00 similarity2 (%) TPS, tomato pomace silage. 1 +, positive; w, weakly positive; , negative. 2 16S rDNA sequence similarity between isolate and each type strain was analyzed by BLAST search program. Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 653 Table 3. Fermentation patterns (evaluated using API 50 CH strips; BioMérieux France) of lactic acid bacteria strains isolated from TPS1 TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP TCP Item 001 007 004 008 015 016 017 050 029 024 045 071 073 102 080 063 037 Glycerol                 w Erythritol                  D-Arabinose w                 l-Arabinose + +  +  +    +  + +    + Ribose  + + +  +    +   + +   + d-Xylose + +    +   + +   +    + l-Xylose                  Adonitol   ＋               -Methyl-xylopyranoside                 + Galactose +  + + + + + + + +  + +  w w + d-Glucose + + + + + + + + + +  + + + +  + d-Fructose + + + + + － + + + +  + + +   + d-Mannose +  + + +  + + + w  +     + l-Sorbose                  Rhamnose       w ＋          Dulcitol   +               Inositol                  Mannitol   + + +     w  +   w  + Sorbitol   + + +            + -Methyl-d-mannoside    +             + -Methyl-d-glucoside +  +   +           w N-Acetyl glucosamine + － + + + － + + + － － + － － －  + Amygdalin +  + +     +   +     + Arbutin +  + +     +   w     + Esculin + － + + + － － w + － － w － － －  + Salicin +  + + +    +   w     + Cellobiose +  + +   + + +   +     + Maltose + + + + + + + + + ＋  + + + w  + Lactose   + +  + w +    +     + Melibiose + +  +  +    ＋   +  +  + Saccharose + + + + + +   w ＋  + + + + + + Trehalose +  + +     + － － + － － － － + Inulin   +               Melezitose +  + +  ＋    +  w +    + d-Raffinose + +  +      +  w +  w  + Starch w        w   +      Glycogen            +      Xylitol            w      -Gentiobiose +  + +     +   +     + d-Turanose w  + +  +           + d-Lyxose     +             d-Tagatose w  +               d-Fucose                  l-Fucose w                 d-Arabitol    w      +        l-Arabitol                  Gluconate + w + +  +    +   + w   + 2-Keto-gluconate      w    w        5-Keto-gluconate  w    +    +   +    w 1 +, positive; w, weakly positive; , negative. the predominant LAB in many forage crops and grasses This difference may be the one of the reasons for the were lactic acid-producing cocci and that the least frequent contrasting results. According to the literature, in the early were lactobacilli (primarily homofermentative). In our study, fermentation, lactic acid-producing cocci reproduced the LAB strains were isolated from tomato pomace ensiled massively and pH fell rapidly. During fermentation, lactic at least 30 days, whereas in the research of Cai et al. (1998; acid-producing cocci were replaced by lactobacilli 1999a,b), the strains were isolated from fresh materials. gradually until lactobacilli became dominant. In addition, 654 Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 the distinct materials in tomatos may also contributed the fermentum produced a large amount of lactic acid to reduce difference in LAB. However, this reason is only speculation. the pH rapidly to below 4.5 which would inhibit Clostridia Determining the true cause will require future research. and enterobacteria. Winters et al. (2000) examined the Generally, the lactobacilli play a more important role in effect of inoculation with Lactobacillus plantarum and fermentation processes and effectively promoted lactic acid Lactobacillus paracasei subsp. paracasei during ensilage of fermentation for a longer time than lactic acid-producing sterile and nonsterile ryegrass and found that they had a cocci, eg. enterococci, streptococci, leuconostocs, Weissella significant effect on silage amino acid profiles. Tabacco et and pediococci. When the lactobacilli reach a level of 105 al. (2011) noted that silage inoculated with Lactobacillus cfu/g FM, silage can be well preserved (Cai et al., 1999c). buchneri had a lower concentration of lactic acid, lactic-to- In this study, the isolated LAB were all lactobacilli and the acetic acid ratio, and yeast count and higher aerobic amount of lactobacilli reached as high as 107 to 108 cfu/g stability compared with untreated silage. Inoculation with FM. This indicated that TP can be well preserved by Lactobacillus buchneri reduced the yeast count to <2 log ensilaging. cfu/g of silage in 16 of 21 farm silage samples, confirming No LAB isolate grew at 5C. TCP001, TCP004, the ability of this inoculum to enhance the aerobic stability TCP008, TCP015, TCP017, TCP050, and TCP024 grew of corn silage in farm bunker silos. well at 10C. The results will be valuable for the future Summarily, therefore, some isolates from this study design of appropriate inoculants for silage fermentation in (including Lactobacillus fermentum, Lactobacillus cold areas. TCP071 and TCP102 grew at 45C and 50C. plantarum, Lactobacillus paracasei subsp. paracasei and TCP029, TCP045, TCP080, and TCP063 grew well at 45C. Lactobacillus buchneri) have already been used as good These strains were thus more heat tolerant and could be silage inoculation in the past, producing significant different added to silage at high temperature. TCP037 grew weakly effects in improving fermentation and preservation. A comprehensive knowledge of the biochemical and at 45C and failed to grow at 50C. physiological properties of both these low and high All isolates, expect TCP015 and TCP029, grew well in temperature-tolerant and other isolates should hopefully 3.0% NaCl and only TCP008 grew well in 6.5% NaCl. All provide a platform for the discovery and design of isolates, except TCP016, TCP029, and TCP037, grew well inoculants aimed at improving silage fermentation and from pH 3.0 to 8.0, indicating that most isolated strains quality under various prevailing conditions. were resistant to acid and could grow well during the production of silage. 16S rRNA gene sequence analysis All isolates, except TCP016, TCP024, and TCP102, A phylogenetic tree of the isolated strains was were homofermentative rods that did not produce gas from constructed from evolutionary distances using the neighbor- glucose. In contrast to lactic acid-producing cocci, joining method, as shown in Figure 1. All strains were lactobacilli (Rodhe, 1990) are important promoters of lactic clustered in the genus Lactobacillus (Figure 1). acid fermentation for longer fermentation periods. Many Based on phylogenetic analysis, strains TCP102, studies (Cai et al., 1998; 1999b) have reported that the TCP029, TCP017, TCP001, TCP073, TCP024, TCP037, inoculation of forage with homofermentative lactobacilli, TCP016, TCP045, TCP063, TCP080, TCP007, and TCP071 such as Lactobacillus casei and Lactobacillus plantarum, were unambiguously identified as Lactobacillus pantheris, promotes lactic acid fermentation and improves silage Lactobacillus camelliae, Lactobacillus manihotivorans, quality. However, the heterofermentative weissellas and Lactobacillus harbinensis, Lactobacillus hilgardii, leuconostocs do not improve fermentation and might cause Lactobacillus parafarraginis, Lactobacillus rapi, fermentation loss (Cai et al., 1998). Lactobacillus buchneri, Lactobacillus pontis, Lactobacillus The species and characteristics of epiphytic LAB can vaginalis, Lactobacillus panis, Lactobacillus fermentum change and influence fermentation losses and silage quality and Lactobacillus amylovorus, respectively (bootstrap (Li et al., 2011), but the populations of epiphytic LAB are between 83%-100%). not always sufficiently large or of suitable composition to TCP015 was clustered with Lactobacillus coryniformis promote efficient fermentation under farm conditions subsp. torquens and Lactobacillus coryniformis, with a (Fenlon et al., 1995; McAllister et al., 2002). Thus, our bootstrap value of 100% and a similarity of 16S rDNA studies aimed at obtaining high-quality silage through the sequence of 99.52% with Lactobacillus coryniformis subsp. development of additives that stimulate and direct the torquens and 98% with Lactobacillus coryniformis; thus, fermentation process. TCP015 was identified as Lactobacillus coryniformis subsp. In addition, many isolated strains in this study have torquens. been demonstrated to be good inoculators of forage. For TCP004 formed a well-defined cluster with 3 strains example, Zheng et al. (2011b) reported that Lactobacillus Wu et al. (2014) Asian Australas. J. Anim. Sci. 27:648-657 655 (Lactobacillus paracasei subsp. tolerans, Lactobacillus strain and differed from that of the Lactobacillus paracasei subsp. paracasei and Lactobacillus casei), and gallinarum-type strain. 97% bootstrap values confirmed monophyly. Further, This study has demonstrated that TP could be well TCP004 appeared to be more similar to Lactobacillus casei preserved by ensilaging as evident in the virtual and Lactobacillus paracasei subsp. paracasei than maintenance of its original color and the minimal content of Lactobacillus paracasei subsp. tolerans, based on its 16S unwanted microbes (such as Clostridia and molds) during rDNA sequence, which shared 99.59% similarity with those and at the end of the storage. This coupled with the of Lactobacillus casei and Lactobacillus paracasei subsp. chemical composition and fermentation characteristics of paracasei. Based on carbohydrate fermentation patterns, the TPS could make TPS an economic highly nutritious forage pattern of strain TCP004 was an exact match to that of the alternative. Furthermore, to the best of our knowledge, this Lactobacillus paracasei subsp. paracasei-type strain and is the first isolation of Lactobacillus harbinensis, differed from that of the Lactobacillus casei-type strain. Lactobacillus manihotivorans, Lactobacillus helveticus, Unlike Lactobacillus paracasei subsp. paracasei, Lactobacillus camelliae, Lactobacillus pontis, Lactobacillus casei grew with glycerol as a carbon source Lactobacillus amylovorus, Lactobacillus hilgardii, (Table 3). All other isolates that were the same as TCP004 Lactobacillus pantheris, Lactobacillus panis, Lactobacillus were identified as Lactobacillus paracasei subsp. paracasei. vaginalis and Lactobacillus rapi from silage. Both these TCP008 was placed on the phylogenetic tree together and the remaining isolates could form a platform for future with Lactobacillus plantarum subsp. argentoratensis, design of appropriate inoculants for silage fermentation. 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