ORIGINAL ARTICLE. D.M. Xu 1,2, W.C. Ke 1,2, P. Zhang 1,2, F.H. Li 2,3 and X.S. Guo 1,2. Abstract

Transcription

1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Characteristics of Pediococcus pentosaceus Q6 isolated from Elymus nutans growing on the Tibetan Plateau and its application for silage preparation at low temperature D.M. Xu 1,2, W.C. Ke 1,2, P. Zhang 1,2, F.H. Li 2,3 and X.S. Guo 1,2 1 State Key Laboratory of Grassland and Agro-ecosystems, School of Life Sciences, Lanzhou University, Lanzhou, China 2 Probiotics and Biological Feed Research Centre, Lanzhou University, Lanzhou, China 3 Stay Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, China Keywords Elymus nutans, fermentation, low temperature, Pediococcus pentosaceus, silage. Correspondence Xusheng Guo, School of Life Sciences, Lanzhou University, No. 222 Tianshui South Road, Lanzhou , China. guoxsh07@lzu.edu.cn 2018/1188: received 13 June 2018, revised 17 August 2018 and accepted 11 September 2018 doi: /jam Abstract Aims: Characteristics of a strain Pediococcus pentosaceus Q6 isolated from Elymus nutans growing on the Tibetan plateau and its effects on E. nutans silage fermentation stored at low temperature were investigated. Methods and Results: Sugar fermentation pattern and growth profiles of the strain Q6 and its reference strain APP were characterized. The strain Q6 and APP were inoculated to E. nutans at ensiling respectively; and ensiled at different temperatures (10, 15 and 25 C) for 30, 60 and 90 days. The results indicated that Q6 could grow at ph 30 and at 4 C. In contrast to APP, Q6 could ferment mannitol, saccharose, sorbitol and rhamnose. Lower ph in Q6- treated silages fermented for 60 days at 10 and 15 C was found compared with the control and APP-treated groups. For the silages that were stored at 10 or 15 C, the greatest lactic acid content were detected in Q6-inoculated silages ensiled for 30 and 60 days respectively. There were no differences in ph and lactic acid content between Q6- and APP-treated silages ensiled at 10 and 15 C for 90 days respectively. Conclusions: Inoculation of the strain P. pentosaceus Q6 could improve fermentation quality of ensiled E. nutans at the early stage of ensiling stored at low temperature (10 or 15 C). Significance and Impact of the Study: The selection of P. pentosaceus inoculants for improving silage quality at low temperature, which provides a candidate strain to make high-quality silage in regions with frigid climate. Introduction The Tibetan plateau is regarded as the Earth s third pole, located in southwest China with an average altitude of over 4000 m (Duan et al. 2008). The main land use type on the Tibetan plateau is livestock farming. Yak (Bos grunniens), Tibetan sheep (Ovis aries) and Tibetan goats (Capra hircus) are the main domestic livestock, and the natural vegetation supports livestock for grazing yearround. The frigid climate, heavy ultraviolet rays and short growing period ( days) are considered to be the major constrains for forage yield on the Tibetan plateau (Zhang et al. 2003; Thomas et al. 2016; Shao et al. 2017). Even with these limitations recent statistics indicate that the number of animals raised in Tibetan grasslands has increased considerably (Gongbuzeren et al. 2016; Yao et al. 2016) leading to a considerable feed shortage, especially in winter. Therefore, forage preservation is an important farming strategy for livestock production on the plateau. Ensiling is a fermentation process of fresh forage dominated by lactic acid bacteria (LAB) under anaerobic conditions. The LAB can convert water-soluble carbohydrates (WSC) into lactic acid with antibacterial activity to promote the conservation forages during the 40

2 D. M. Xu et al. LAB for silage at low temperature ensiling process (McAllister et al. 1999). Thus, ensiling is deemed to be a desirable way to alleviate the seasonal unbalanced feed supply problem. For making silage, LAB inoculants are often inoculated to improve silage fermentation quality. However, the commercially available LAB strains are generally isolated from temperate environment and adapted to at higher temperatures such as 37 C (Zahar et al. 2002). On the Tibetan plateau, the average diurnal temperature at the stage of forage harvest is about C. Therefore, the low temperature is considered as a main limitation factor for commercial inoculants to work effectively in ensiling forage on the plateau. Several studies indicated that bacteria that survive under a hostile environment for a long time have stronger adaptive capacity. Thermo-tolerance and acid resistance of LAB vary with different origins (Chen et al. 2013; Huang et al. 2013). The diversity and physiological-biochemical characteristics of LAB from the Tibetan plateau are affected by low temperature. Numerous studies have been devoted to investigate enhancing silage by inoculation of LAB (Weinberg and Muck 1996; Kizilsimsek et al. 2007; Contreras-Govea et al. 2013). However, few studies have been conducted to investigate the effect of low temperature resistance LAB on fermentation quality of silage made in a lowtemperature environment. The purposes of the present study were to investigate the characteristics of a strain, Pediococcus pentosaceus Q6, isolated from Elymus nutans, and to evaluate its effects on fermentation quality of E. nutans silage stored at low temperatures. It is hypothesized that P. pentosaceus Q6 could grow well, as well as improve fermentation quality of E. nutans silage at low temperatures. inoculated to MRS culture medium at 3% (v/v) and then grown at 4, 10, 15, 25, 45 and 50 C for 72 h and at different initial ph values (30, 35, 40, 80, 85 and 90) at 37 C for 72 h. The growth performance of the tested strains at different temperatures and different initial ph values was indicated by turbidity (using ultraviolet spectrophotometer at 600 nm). Carbohydrate fermentation, arginine ammonia production, nitrate reduction and gas production from glucose were examined by the commercial biochemistry kits (Hangzhou Binhe Microorganism Reagent Co., Ltd, Hangzhou, China) according to the instructions of the manufacturer. Silage preparation Elymus nutans was mowed at heading period from a native pasture of Zhuaxixiulong village, Wuwei city, Gansu province, China. The harvested grass was chopped into 2 3-cm sections and wilted to the targeted moisture content of 70%. To apply the LAB to the chopped forage, strain Q6 and its reference strain APP were dissolved in 10 ml of sterile distilled water and sprayed evenly onto the forage for each treatment at an application rate of CFU per gram FW. For the control, the same amount of distilled water was sprayed. After homogeneous mixing, approximately 300 g of fresh forage were packed into vacuum-sealing polythene plastic bags (30 cm 9 23 cm) and vacuum sealed firmly, with three replicates for each treatment. The polythene bag silos were kept at 10, 15 and 25 C for 30, 60 and 90 days respectively. Initial fresh forage samples were taken before ensiling. Materials and methods LAB strains The tested strain P. pentosaceus Q6 was isolated from E. nutans silage made in the region of the Tibetan plateau, and screened in our lab based on its performance of growing better in MRS medium and distinguished acid production ability at 10 C. The sequence data of strain Q6 have been deposited in the GenBank database (Genbank accession no. MH348180). Physiological and biochemical tests The physiological and biochemical tests were conducted for the selected strain Q6 and its reference strain P. pentosaceus APP (isolated from a commercial silage inoculant produced by Vita Plus Co, Madison, WI). Bacterial suspension with 10 6 CFU per gram fresh weight (FW) was Chemical and microbial analyses of raw material and silage Triplicate mini-silos were opened at each sampling time for microbiological and biochemical analyses. The fermentation products of silages were analysed with cold water extracts. A 20-g FW sample from each bag was homogenized in 180 ml of sterile distilled water using a juice extractor (BA-828; Mannengda Plasthetics Co. Ltd, Guangzhou, China), then squeezed for 30 s at a high speed, and filtered through four layers of medical gauze. The filtrate ph was measured with a glass electrode ph meter immediately. A portion of the filtrate was stored at 20 C for determining WSC and the rest was acidulated with 714 mol l 1 H 2 SO 4 and filtered with a 045-lm dialyser. Lactic acid, acetic acid, propionic acid and butyric acid were analysed by high performance liquid chromatography (HPLC, KC-811 column; Shodex, Shimadzu, Japan; oven temperature 50 C; flow rate 1 ml min 1 ; 41

3 LAB for silage at low temperature D. M. Xu et al. SPD 210 nm). The content of WSC was estimated using the method of Thomas (1977). Enumeration of LAB, yeasts and moulds in fresh and ensiled forage was detected according to the methods described by Reich and Kung (2010). Briefly, samples (10 g) were homogenized in 100 ml of sterile Ringer s solution (Oxide BR52) for 1 min and serially diluted (10-fold). The number of LAB was detected on spread plates using Rogosa agar (Oxoid CM627; Oxoid, Basingstoke, UK) and incubated at 37 C for h. Yeasts and moulds were determined by pour plating serial 10-fold dilutions of water extracts on malt extract agar (Oxoid CM0059) that had been acidified with lactic acid (concentration of 850 g kg 1 added at 50 g kg 1, v/v). Plates were incubated at 32 C for h. Colonies were counted from plates where appropriate dilutions yielded colonies. Silage samples from each bag were dried using a forced air oven at 65 C for 72 h and sieved through a mill with 10-mm screen for chemical analyses. Ground samples were analysed for Kjeldahl N (AOAC 1990, 95401). Crude protein (CP) was calculated as Kjeldahl N multiplied by 625. The content of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined according to the methods of Van-Soest et al. (1991) using an Ankom 200 fibre analyser (Ankom Technology, Fairport, NY). During the analysis procedure, heat-stable alpha amylase and sodium sulphite were added. The NDF and ADF were expressed in terms of residual ash. Statistical analyses The data for chemical composition, fermentation products and number of micro-organisms were compared using two-way analysis of variance to evaluate the effects of inoculation, temperature and their interaction. The mean comparisons were conducted using Duncan s multiple range tests (SPSS 17.0; SPSS Inc., Chicago, IL). The statistical significance was considered at P < 005. Results Characteristics and identification of strain Q6 The morphological, physiological and biochemical characteristics of Q6 and APP are shown in Table 1. The strain Q6 could grow at lowest initial ph 30 and at 4 C, but APP could only grow slightly at initial ph 35 and could not grow at 4 C. In contrast to the reference strain APP, Q6 could ferment mannitol, saccharose, sorbitol and rhamnose. The phylogenetic tree of partial 16S rrna sequences of Q6 is exhibited in Fig. 1. The sequence of Q6 with 100% similarity in line P. pentosaceus, indicating that Q6 belongs to P. pentosaceus. Table 1 Physiological and biochemical characteristics of Q6 and one strain from commercial addition Strains Q6 APP Carbohydrate fermentation Glucose Lactose Maltose Mannitol + Saccharose ++ L-Arabinose ++ Xylose + ++ Galactose Sorbitol + Amygdalin Rhamnose w Salicin Esculin Trehalose Raffinose Fructose Melibiose Cellobiose + ++ Mannose Starch Arginine ammonia production + Gluconate Nitrate reduction Growth at ph 30 w w 35 + w Growth at temperature ( C) 4 w w w PP, Pediococcus pentosaceus; APP, Pediococcus pentosaceus from a commercial addition; in carbohydrate fermentation, +++, biochemistry kits turning colour in h; ++, biochemistry kits turning colour in h; +, biochemistry kits turning colour in h; w, biochemistry kits turning colour from blue to colourless;, nondiscolouring. Growth at temperature and ph, ++, growing better (OD >05); +, growing (005 < OD < 01);, none growing (OD < 001); w, growing slightly (001 < OD < 005). Elymus nutans characteristics The chemical composition and epiphytic microflora of fresh E. nutans are shown in Table 2. Before ensiling, fresh E. nutans was wilted to a DM of 2856 gkg 1, and the WSC was 55 g kg 1 DM. The counts of epiphytic microbes in fresh forage such as LAB, yeasts and moulds, 42

4 D. M. Xu et al. LAB for silage at low temperature Figure 1 Phylogenetic tree of partial 16S rrna sequences of Q6 and sequences of identified bacteria in the nucleotide database of Gen- Bank. The tree was created by the neighbour-joining method with Knuc values; the numbers indicate bootstrap values for the branch point. Table 2 Chemical and microbial compositions of fresh Elymus nutans (means SD) Item DM, g kg 1 ph WSC, g kg 1 DM CP, g kg 1 DM NDF, g kg 1 DM, ADF, g kg 1 DM LAB, log 10 CFU per gram FW Yeasts, log 10 CFU per gram FW Moulds, log 10 CFU per gram FW were 414, 393 and 315 log 10 CFU per gram FW respectively. Silage fermentation quality Mean DM, dry matter; WSC, water-soluble carbohydrate; CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; LAB, lactic acid bacteria; FW, fresh weight. The fermentation characteristics of E. nutans silage ensiled for 30 days are shown in Table 3. Compared with the control silage, silages inoculated with the strains of Q6 and APP had lower ph (P < 005) and greater lactic acid especially in samples treated with Q6 which were stored at 10 C. In addition, silages inoculated with Q6 had the lowest ph when stored at 15 C. The concentration of acetic acid in inoculated silages was less than that in the control silage, and it was increased with the decrease of storage temperature in the control and the Q6-inoculated groups. After 60 days of fermentation (Table 4), silages treated with the strain Q6 and stored at 10 and 15 C obviously decreased the ph, and produced more lactic acid compared with control and APP groups (P < 005). The amount of LAB in the silages inoculated with Q6 or APP and stored at both 10 and 15 C were higher than in the control group, but there is no difference between groups Q6 and APP. The silages inoculated with the two strains and stored at 15 and 25 C had lower yeasts than the control group. As the fermentation time prolonged to 90 days, silages inoculated with strain Q6 and APP had lower ph value than the control group. But there were no differences in ph and lactic acid between Q6- and APP-treated silages ensiled at 10 and 15 C respectively. The yeasts in all samples and moulds in Q6-treated samples have not been detected (Table 5). The chemical composition of E. nutans silages ensiled for 90 days are shown in Table 6. The WSC in silage treated with Q6 at 10 C was remarkably lower than the control and the APP-treated groups. The lowest dry matter occurred in samples inoculated with Q6 and stored at 15 C, and there were no differences in other treatments. Strain Q6- and APP-treated silages had lower CP than the control group. Samples inoculated with the two strains and ensiled at 10 C had higher NDF than the control group (P < 005). The samples treated with Q6 and APP ensiled at 10 C had higher NDF and ADF than the control group, but there is no difference between groups Q6 and APP. Discussion Inoculation of LAB during silage production has been proved to be effective to improve fermentation quality and inhibit undesirable microbes (Cai et al. 2001; Parvin and Nishino 2009). The LAB characteristics and the stored temperature also had effects on fermentation quality (Weinberg et al. 1998). Low temperature-resistant LAB are proposed to enhance silage fermentation in the Tibetan plateau with frigid climate because normal strains could not grow well at a low temperature of below 15 C (Chen et al. 2013; Zhang et al. 2015). Micro-organisms have the ability to adapt to a harsh environment which 43

5 LAB for silage at low temperature D. M. Xu et al. Table 3 Fermentation characteristics and microbial composition of Elymus nutans silage ensiled for 30 days Strain Temperature ( C) ph DM (g kg 1 ) LA (g kg 1 DM) AA (g kg 1 DM) LAB (log 10 CFU Yeasts (log 10 CFU Moulds (log 10 CFU Control a 283ab 57e 631a 892b 374ab 288ab b 277bc 75e 378bc 910ab 286bcde 272abc b 272 cd 91de 222e 838c 423a 287ab Q c 282ab 276a 441b 897b 374ab 245abc d 285a 139cd 288cde 933a 253cde 245abc d 277bc 186bc 269de 861c 232de 230bc APP c 271cd 241ab 363bcd 918ab 327bc 312a c 265d 123cde 296cde 909ab 293bcd 258abc d 266d 172bc 336cd 851c 200e 200c SEM P value Strain *** *** *** ** NS ** NS Temperature *** ** ** *** *** ** NS S 9 T *** NS NS *** NS ** NS DM, dry matter; LA, lactic acid; AA, acetic acid; LAB, lactic acid bacteria; FW, fresh weight; SEM, standard error of means; S, strain; T, temperature; S 9 T, interaction of S and T. Means within the same line with different letters are significantly different (P < 005). NS P > 005; **0001 < P < 001; ***P < Table 4 Fermentation characteristics and microbial composition of Elymus nutans silage ensiled for 60 days Strain Temperature ( C) ph DM (g kg 1 ) LA (g kg 1 DM) AA (g kg 1 DM) LAB (log 10 CFU Yeasts (log 10 CFU Moulds (log 10 CFU Control a 285abc 230d 293ab 843bc 262b 200b b 273c 194d 134c 834bcd 392a 254a b 291ab 387cd 210bc 819cd 385a 248ab Q de 278bc 639b 280ab 882a 329ab 208ab e 293a 1011a 386a 883a 282b 225ab de 282abc 851ab 351ab 847b 000c 200b APP c 285abc 404cd 218bc 875a 312b 208ab c 274c 493cd 259abc 878a 300b 205ab cd 282abc 703abc 286ab 813d 000c 237ab SEM P value Strain *** NS *** ** *** *** NS Temperature ** NS NS NS *** *** NS S 9 T ** * NS NS *** *** NS DM, dry matter; LA, lactic acid; AA, acetic acid; LAB, lactic acid bacteria; FW, fresh weight; SEM, standard error of means; S, strain; T, temperature; S 9 T, interaction of S and T. Means within the same line with different letters are significantly different (P < 005). NS P > 005; *001 <P< 005; **0001 < P < 001; ***P < had been investigated. Based our previous studies, some epiphytic LAB strains on Kobresia littledalei from the Tibetan plateau had stronger temperature, acid and alkali resistance than the strains isolated under conventional conditions (Yang 2012; Gao et al. 2013). These results were consistent with the present study, in which the strain Q6 could grow at 4 C and also at lowest initial ph 30, but APP could only grow slightly at initial ph 35 and could not grow at 4 C. Based on the literature on P. pentosaceus isolated from silages, the strain of P. pentosaceus isolated from king grass silage could not grow at the ph of 40 (Liu et al. 2012), and another strain of P. pentosaceus isolated from corn stove silage did not grow at 5 C (Li et al. 2015). Therefore, the strain of Q6 isolated from the Tibetan plateau might adapt to the low temperature. In order to investigate the potential effect of the strain Q6 on fermentation of silage stored at a low temperature, we inoculated the strain onto the grass of E. nutans before ensiling. The silos were then stored at various temperatures and fermented for different times. The species of P. pentosaceus is commonly inoculated at ensiling in 44

6 D. M. Xu et al. LAB for silage at low temperature Table 5 Fermentation characteristics and microbial composition of Elymus nutans silage ensiled for 90 days Strain Temperature ( C) ph LA (g kg 1 DM) AA (g kg 1 DM) LAB (log 10 CFU Yeasts (log 10 CFU Moulds (log 10 CFU Control a 507c 264bc 797bc 000c b 566bc 269bc 799abc 230a bc 819ab 390ab 778cd 000c Q cd 429c 264bc 771d 000c cd 345c 261bc 797bc 000c e 390c 193c 772d 000c APP cd 429c 316bc 820a 200b cd 366c 250bc 818ab 224a de 907a 450a 795c 000c SEM P value Strain *** * * *** *** Temperature *** ** NS ** *** S 9 T *** NS * NS *** DM, dry matter; LA, lactic acid; AA, acetic acid; LAB, lactic acid bacteria; FW, fresh weight; SEM, standard error of means; S, strain; T, temperature; S 9 T, interaction of S and T. Means within the same line with different letters are significantly different (P < 005). NS P > 005; *001 < P < 005; **0001 < P < 001; ***P < Table 6 Chemical composition of Elymus nutans silages ensiled for 90 days Strain Temperature ( C) DM (g kg 1 ) WSC (g kg 1 DM) CP (g kg 1 DM) AN/TN (% TN) NDF (g kg 1 DM) ADF (g kg 1 DM) Control a 827a 1740b 69ab 5564cd 3105bcd a 629b 1803a 66b 5409cd 3038d a 527bc 1771ab 72ab 5476cd 3102cd Q a 347c 1573c 76ab 5961a 3300a b 558bc 1546c 70ab 5846ab 3249abc a 256d 1547c 75ab 5685bc 3081cd APP a 618b 1560c 59b 6016a 3259ab a 568bc 1554c 57b 5625bc 3129a a 431c 1537c 87ab 5602cd 3096bcd SEM P value Strain NS NS *** NS *** * Temperature * NS NS NS *** * S 9 T NS NS NS NS NS NS DM, dry matter; WSC, water-soluble carbohydrate; CP, crude protein; AN/TN, ammonia nitrogen/total nitrogen; NDF, neutral detergent fibre; ADF, acid detergent fibre. SEM, standard error of means; S, strain; T, temperature; S 9 T, interaction of S and T. Means within the same line with different letters are significantly different (P < 005). NS P > 005; *001 < P < 005; ***P < order to promote fermentation process of the ensiled forage in the early stages of fermentation (Majumdar et al. 2011; Contreras-Govea et al. 2013). Filya et al. (2007) also reported that inoculation of the two isolated P. pentosaceus strains produced the highest lactate : acetate ratio of the first-cut alfalfa silages than commercial inoculant silages. As expected, inoculation of the strain Q6 at ensiling of E. nutans enhanced fermentation quality of the E. nutans silage under low temperatures. When the silos were stored at 10 or 15 C, samples treated with the strain Q6 had higher concentrations of lactic acid after 30 and 60 days of preservation compared with the control silage or silage inoculated with the commercial strain APP. Furthermore, increases in acetic acid were observed in samples fermented for 30 days under low temperatures regardless of treatments. A previous study also indicated that a higher acetate concentration was detected in corn silage stored at a low temperature of 20 C (Kim and Adesogan 2006). However, contrasting results were found in ensiled stylo (Stylosanthes guianensis Sw.) growing in 45

7 LAB for silage at low temperature D. M. Xu et al. the southern region of China with a humid and hightemperature climate (Liu et al. 2011). The study results indicated that a lower concentration of acetic acid was observed in samples fermented at high (40 C) and low (10 C) temperatures than at middle temperatures (20 and 30 C). Moreover, Muck and Dickerson (1988) reported that the content of acetic acid increased with the decreased temperatures in 55% DM alfalfa silages, but not in 40% DM silages. The differences between epiphytic bacteria communities and their activity at different temperatures might account for the inconstancies. The microbial composition of the ensiled forage is also important for estimating the silage quality. The LAB counts of silages inoculated with Q6 or APP and stored at 15 C for 60 days were higher and the yeasts were lower than the control group, which was consistent with previous reports (Daniel et al. 2015; Zielinska et al. 2015). The count of LAB was decreased from 30 to 90 days regardless of treatments, which due to the low ph inhibited the growth of LAB as reported by Denoncourt et al. (2007). After 90 days of fermentation, comparable results on ph and lactic acid concentration were obtained in Q6- and APP-inoculated silages preserved under low temperatures. It is generally considered that Pediococci, Lactococci, Enterococci and Leuconostocs are starters of initial silage fermentation and then replaced by more acid-tolerant lactobacilli, such as Lactobacillus plantarum and Lactobacillus brevis (Mcdonald et al. 1981; Lin et al. 2010). Therefore, it might because that the most competitive LAB species, especially lactobacilli dominated the bacteria community at the late stage of fermentation (Lin et al. 2010), and consequently similar results on fermentation quality were observed in Q6 and APP inoculated silages. The content of lactic acid in the Q6-inoculated silage decreased obviously from 60 to 90 days. Similar results were reported by Kleinschmit and Kung (2006), and their results indicated that the concentrations of lactic acid decreased in corn silages stored from 56 to 282 days. Herrmann et al. (2011) also reported similar consequences in sorghum, forage rye and triticale silages added with L. plantarum and L. buchneri, the lactic acid content of the samples stored for 90 days was lower than those silages stored for 10 days. The decrease in lactic acid from 60 to 90 days might be because of the restricted content of the available WSC with prolonged fermentation, and subsequently a part of the lactic acid was used as a substrate by microbes (Herrmann et al. 2011). However, a contrary result occurred in the control group. The increased lactic acid in the control silage after 90 days of fermentation suggested that the lactic acid fermentation progress in control silage was slower than the inoculated silages. In summary, inoculation of the strain P. pentosaceus Q6 could improve fermentation quality of ensiled E. nutans at the early stage of ensiling stored at 10 or 15 C, as indicated by lower ph value and higher lactic acid concentration in Q6-inoculated silage in comparison with the control silage or silage inoculated by a commercial strain of APP under low temperatures. Therefore, the strain Q6 can be used as a candidate strain for improving the fermentation quality of ensiled forages in the areas with low temperature climate, such as the Tibetan plateau. Acknowledgements The financial supports from the National Key R&D Program of China (2017YFE ) and the Double first class Funding-International Cooperation and Exchange Program ( ) are gratefully acknowledged. Conflict of Interest No conflict of interest declared. References AOAC. Association of Official Agricultural Chemists (1990) Official Methods of Analysis of the Association of Official Analytical Chemists. 1, 15th edn. Washington, DC: AOAC. Cai, Y., Uegaki, R. and Fujita, Y. (2001) Lactic acid bacteria isolated from forage crops and silage fermentation. In Proceedings of the XIX International Grassland Congressgrassland Ecosystems: An Outlook into the 21st Century ed. Gomide, J.A., da Silva, S.C. and Mattos, W.R.S. pp Sao Paolo: FEALQ. Chen, M.M., Liu, Q.H., Xin, G.R. and Zhang, J.G. (2013) Characteristics of lactic acid bacteria isolates and their inoculating effects on the silage fermentation at high temperature. Lett Appl Microbiol 56, 71. Contreras-Govea, F.E., Richard, E., Broderick, G.A. and Weimer, P.J. (2013) Lactobacillus plantarum effects on silage fermentation and in vitro;microbial yield. Anim Feed Sci Technol 179, Daniel, J.L.P., Checolli, M., Zwielehner, J., Junges, D., Fernandes, J. and Nussio, L.G. (2015) The effects of Lactobacillus kefiri and L. brevis on the fermentation and aerobic stability of sugarcane silage. Anim Feed Sci Technol 205, Denoncourt, P., Caillet, S. and Lacroix, M. (2007) Bacteriological and chemical changes occurring in Bunkerstored silage covered with biodegradable coating. J Appl Microbiol 103, Duan, Y., Tan, Z., Wang, Y., Li, Z., Li, Z., Qin, G., Huo, Y. and Cai, Y. (2008) Identification and characterization of 46

8 D. M. Xu et al. LAB for silage at low temperature lactic acid bacteria isolated from Tibetan Qula cheese. J Gen Appl Microbiol 54, Filya, I., Muck, R.E. and Contrerasgovea, F.E. (2007) Inoculant effects on alfalfa silage: fermentation products and nutritive value. J Dairy Sci 90, Gao, J., Guo, X.S. and Wang, G.C. (2013) Physiological and biochemical characteristics of lactic acid bacteria isolated from Kobresia littledalei in Tibet. Pratacultural Sci 30, Gongbuzeren, Zhuang, M. and Li, W. (2016) Market-based grazing land transfers and customary institutions in the management of rangelands: two case studies on the Qinghai-Tibetan Plateau. Land Use Policy 57, Herrmann, C., Heiermann, M. and Idler, C. (2011) Effects of ensiling, silage additives and storage period on methane formation of biogas crops. Bioresour Technol 102, Huang, Y., Wang, X., Wang, J., Wu, F., Sui, Y., Yang, L. and Wang, Z. (2013) Lactobacillus plantarum strains as potential probiotic cultures with cholesterol-lowering activity. J Dairy Sci 96, Kim, S.C. and Adesogan, A.T. (2006) Influence of ensiling temperature, simulated rainfall, and delayed sealing on fermentation characteristics and aerobic stability of corn silage. J Dairy Sci 89, Kizilsimsek, M., Schmidt, R.J. and Kung, L. Jr (2007) Effects of a mixture of lactic acid bacteria applied as a freezedried or fresh culture on the fermentation of alfalfa silage. J Dairy Sci 90, Kleinschmit, D.H. and Kung, L. Jr (2006) The effects of Lactobacillus buchneri and Pediococcus pentosaceus R1094 on the fermentation of corn silage. J Dairy Sci 89, Li, D., Ni, K., Pang, H., Wang, Y., Cai, Y. and Jin, Q. (2015) Identification and antimicrobial activity detection of lactic acid bacteria isolated from corn stover silage. Asian- Australas J Anim Sci 28, 620. Lin, C., Bolsen, K.K., Brent, B.E. and Fung, D.Y.C. (2010) Epiphytic lactic acid bacteria succession during the preensiling and ensiling periods of alfalfa and maize. J Appl Microbiol 73, Liu, Q., Zhang, J., Shi, S. and Sun, Q. (2011) The effects of wilting and storage temperatures on the fermentation quality and aerobic stability of stylo silage. Anim Sci J 82, Liu, Q., Chen, M., Zhang, J., Shi, S. and Cai, Y. (2012) Characteristics of isolated lactic acid bacteria and their effectiveness to improve stylo (Stylosanthes guianensis Sw.) silage quality at various temperatures. Anim Sci J 83, Majumdar, K., Razdan, M., Aggarwal, N., Murali, K.K., Bhattacharya, R.C. and Dureja, P. (2011) Isolation and characterization of a potential biocontrol agent Bacillus KM5 from rhizosphere soil of a rice plant. Archives of Phytopathology & Plant 44, McAllister, T., Inglis, G., Mir, Z. and Hristov, A. (1999) Effect of inoculants on fermentation and performance of feedlot cattle fed barley and corn silage. Final Report, Project A Agriculture and Agri-Food Canada Research Centre/Pioneer Hi-Bred International, Marlborough, Wiltshire, UK. Mcdonald, P., Henderson, A.R. and Heron, S.J.E. (1981) The Biochemistry of Silage. Marlow: Chalcombe Publications. Muck, R.E. and Dickerson, J.T. (1988) Storage temperature effects on proteolysis in alfalfa silage. Trans Am Soc Agric Eng 31, Parvin, S. and Nishino, N. (2009) Bacterial community associated with ensilage process of wilted guinea grass. J Appl Microbiol 107, Reich, L.J. and Kung, L. Jr (2010) Effects of combining Lactobacillus buchneri with various lactic acid bacteria on the fermentation and aerobic stability of corn silage. Anim Feed Sci Technol 159, Shao, J.J., Liu, C.B., Zhang, Q.H., Fu, J.J., Yang, R.Q., Shi, J.B., Cai, Y. and Jiang, G.B. (2017) Characterization and speciation of mercury in mosses and lichens from the high-altitude Tibetan Plateau. Environ Geochem Health 39, Thomas, T.A. (1977) An automated procedure for the determination of soluble carbohydrates in herbage. J Sci Food Agric 28, Thomas, E.K., Huang, Y., Clemens, S.C., Colman, S.M., Morrill, C., Wegener, P. and Zhao, J. (2016) Changes in dominant moisture sources and the consequences for hydroclimate on the northeastern Tibetan Plateau during the past 32 kyr. Quaternary Sci Rev 131, Van-Soest, P.J., Robertson, J.B. and Lewis, B.A. (1991) Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 74, Weinberg, Z.G. and Muck, R.E. (1996) New trends and opportunities in the development and use of inoculants for silage. FEMS Microbiol Rev 19, Weinberg, Z.G., Szakacs, G., Ashbell, G. and Hen, Y. (1998) The effect of temperature and Lactobacillus amylovorus and Lact. plantarum, applied at ensiling, on wheat silage. J Appl Microbiol 84, Yang, Y. (2012) Characterization and Identification of Lactic Acid Bacteria Isolated from Kobresia littledalei in Tibet. Lanzhou: Lanzhou University. Yao, Z., Zhao, C., Yang, K., Liu, W., Li, Y., You, J. and Xiao, J. (2016) Alpine grassland degradation in the Qilian Mountains, China a case study in Damaying Grassland. Catena 137, Zahar, M., Benkerroum, N., Guerouali, A., Laraki, Y. and Yakoubi, K.E. (2002) Effect of temperature, anaerobiosis, stirring and salt addition on natural fermentation silage of sardine and sardine wastes in sugarcane molasses. Bioresour Technol 82, Zhang, Q.B., Cheng, G., Yao, T., Kang, X. and Huang, J. (2003) A 2,326-year tree-ring record of climate variability on the northeastern Qinghai-Tibetan Plateau. Geophy Res Lett 30,

9 LAB for silage at low temperature D. M. Xu et al. Zhang, Q., Yu, Z. and Wang, X. (2015) Isolating and evaluating lactic acid bacteria strains with or without sucrose for effectiveness of silage fermentation. Grassl Sci 61, Zielinska, K., Fabiszewska, A. and Stefanska, I. (2015) Different aspects of Lactobacillus inoculants on the improvement of quality and safety of alfalfa silage. Chilean J Agric Res 75, Supporting Information Additional supporting information may be found online in the Supporting Information section at the end of the article. Figure S1. Phylogenetic tree of partial 16S rrna sequences of Q6 and sequences of identified bacteria in the nucleotide database of GenBank. 48