Selection and characterisation of lactic acid bacteria ... - ScienceDirect

Journal of Integrative Agriculture 2016, 15(10): 2353–2362 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Selection and characterisation of lactic acid bacteria isolated from different origins for ensiling Robinia pseudoacacia and Morus alba L. leaves NI Kui-kui1, YANG Hui-xiao1, HUA Wei2, WANG Yan-ping1, PANG Hui-li1 1 2

Henan Provincial Key Laboratory of Ion Beam Bio-Engineering, Zhengzhou University, Zhengzhou 450051, P.R.China Henan Sanoterre Bio-Tec Company, Zhengzhou 450000, P.R.China

Abstract The objective of this study was to isolate lactic acid bacteria (LAB) strains from different origins and to select the best strains for ensiling Robinia pseudoacacia (RB) and Morus alba L. (MB) leaves. The LAB strains were inoculated into the extracted liquid obtained from RB and MB leaves to evaluate the fermentation products. 11 LAB strains were selected for further experiments based on the highest products of lactic or acetic acid, including 1 strain of Weissella confusa, 2 of Lactobacillus reuteri and 8 of Lactobacillus plantarum. The API 50 CH fermentation experiment indicated that all of the selected 11 LAB strains utilised most of the carbohydrates. All the strains grew at temperatures between 10 and 45°C and at a pH of 3.5 to 4.5; however, L. reuteri F7 and F8 tolerated a pH as low as 3.0. All 11 LAB strains showed antibacterial activity against Listeria monocytogens, Escherichia coil, Salmonella sp. and Acetobacter pasteurianus; however, after excluding the effect of organic acids, only F7 and F8 still exhibited antibacterial activity. The present study indicated that the selected 11 LAB strains could be used to prepare silages of RB and MB leaves, especially L. reuteri F7 and F8. Keywords: lactic acid bacteria, Morus alba L., Robinia pseudoacacia, silage

1. Introduction The demand for animal products is increasing notably in China and other developing countries. Therefore, it is now necessary to develop larger quantities of feedstuff for animals. Robinia pseudoacacia (RB) and Morus alba L. (MB)

Received 16 September, 2015 Accepted 2 December, 2015 NI Kui-kui, E-mail: [email protected]; Correspondence WANG Yan-ping, Mobile: +86-13938583206, E-mail: [email protected]; PANG Hui-li, Tel: +86-371-67897722, E-mail: [email protected] © 2016, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(15)61251-5

trees are fast-growing, drought-tolerant and adaptable to different growing climates and are cultivated in many parts of the world. Due to their high biomass yield, protein sources and digestibility, studies have been performed to explore their potential for animal feedstuff, indicating that they can be used in animal diets without negatively affecting animal products (Zhang et al. 2010). In addition, RB and MB trees are seasonally available and may require accumulating larger stocks for immediate use. Therefore, a method for preserving these two trees needs to be developed. Ensiling has recently been considered the best way to preserve fresh forage crops and grasses with minimal losses. During the ensiling process, epiphytic lactic acid bacteria (LAB) ferments water-soluble carbohydrates under anaerobic conditions into organic acids, primarily lactic acid, which greatly and quickly reduces the pH (Cai et al.

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NI Kui-kui et al. Journal of Integrative Agriculture 2016, 15(10): 2353–2362

1998). To improve the silage quality, many LAB inoculants have been developed and are proven to be useful in forage and grass species (Ni et al. 2015). These could be used to solve problems in the variation in fermentation quality and microbial stability in the process of ensiling. Recently, some heterofermentative inoculants were also developed to inhibit aerobic spoilage, such as Lactobacillus buchneri and Lactobacillus brevis (Kleinschmit and Kung 2006). The characteristics of LAB are primarily associated with their physiological features, such as the utilisation of various substrates, and for their metabolic and antimicrobial capabilities (Wang et al. 2006). Several reports have demonstrated that inoculants with different strains belonging to the same species result in different effects on silage quality, which may indicate that the suitable inoculant candidates for ensiling should be selected at the strain level. Although numerous studies had been conducted on selecting LAB for inoculating corn, rice, alfalfa grass and other crops, limited information is available on what type of LAB strains are suitable for inoculating RB and MB trees. The objective of this study was to select potentially suitable LAB strains to be used as inoculants for ensiling RB and MB trees. A large number of LAB, isolated from different origins, were evaluated by analysing their organic acid production, utilising substrate ability, tolerance to inhibitory conditions and antimicrobial activity.

2. Materials and methods 2.1. Materials Corn (Zhengchaotian 2) at the dough-ripe stage, crop rice (Oryza sativa L.) at the milk ripe stage, alfalfa (Medicago sativa) at the flowering stage and pig feces were collected in Zhengzhou, Henan, China. RB and MB leaves were obtained at Luoyang, Henan, China. Dry matter (DM) and crude protein (CP) were analyzed by AOAC (1990) 934.01 and 967.05, respectively. Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed according to the methods of Van Soest et al (1991). For RB, the DM was 34.8%; CP, ADF and NDF were 19.6, 30.2 and 44.6% of DM, respectively. For MB, the DM was 37.2%; CP, ADF and NDF were 12.3, 16.2 and 31.7% of DM. For the number of isolated strains, please see Table 1.

2.2. Strains A total of 65 LAB strains were isolated from the employed materials and cultivated on lactobacilli de Man, Rogosa, Sharpe (MRS) agar incubated at 30°C for 2 days under anaerobic condition. Each colony was purified two times by streaking on the MRS agar, then transferred to nutrition broth

Table 1 The source of lactic acid bacteria (LAB) strains used in this study Sources Corn Rice Alfalfa Pig feces Morus alba L. (MB) Robinia pseudoacacia (RB)

Strains names C1, C2, C3, ..., C13 R1, R2, R3, ..., R10 A1, A2, A3, ..., A12 F1, F2, F3, ..., F15 MB1, MB2, MB3, ..., MB8 RB1, RB2, RB3, ..., RB7

(Difco Laboratories, USA) with dimethyl at a ratio 9:1 and stored as stock cultures at –80°C for further examination.

2.3. Inoculation experiment First, 65 LAB strains were cultivated in MRS broth for 24 h at 30°C. The broth of RB and MB was crushed out from 2 000×g of fresh materials mixed with 5 000 mL deionized water in a squeezer, separately, then filtered and sterilized (121°C, 15 min). After this period, the inoculum was standardized using a spectrophotometer (600 nm) at an optical density of 1.0. Subsequently, approximately 100 μL of each strain was inoculated into 30 mL of paddy rice broth, which was incubated at 30°C; two replicates were made for each treatment. After 48 h of fermentation, samples of cultures were taken to evaluate metabolite production by HPLC (1200 series; Agilent, USA). The HPLC system included column temperature 55°C, the speed of mobile solution 0.6 mL min–1 and UV detector 210 nm. Besides, 1 mL of fermented liquid were blended with 9 mL of sterilized water, and serially diluted from 10–1 to 10–5 in sterilized water. The number of LAB were measured by plate count on MRS agar incubated at 30°C for 48 h under anaerobic conditions (DG 250/min MACS; Don Whitley Science, England).

2.4. 16S rRNA gene sequencing Cells grown at 30°C for 24 h in MRS agar were used for 16S rRNA gene sequence. The 16S rRNA gene sequence coding region was amplified by PCR in a PCR thermal cycler as described by Suzuki et al. (1996). The sequences of the PCR products were determined directly with a sequencing kit using the prokaryotic 16S ribosomal DNA universal primers 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-GGTTACCTTGTTACGACTT-3´). Sequence similarity searches were performed using the DNA database of Japan (DDBJ) and the Basic Local Alignment Search Tool (BLAST).

2.5. Morphological, physiological and biochemical tests of LAB Morphological, physiological and biochemical tests of LAB


morphology and Gram-staining response were examined after 24 h of incubation on MRS agar. Catalase activity and gas production from glucose were determined using the methods of Kozaki et al. (1992). Growth at different temperatures was observed in MRS broth after incubation at 5, 10 and 15°C for 14 days, and at 45 and 50°C for 7 days. Growth at pH 3.0, 3.5, 4.0 and 4.5 was observed in MRS broth after incubation at 30°C for 7 days. Salt tolerance of LAB was tested in MRS broth containing 3.0 and 6.5% NaCl at 30°C for 2 days. Carbohydrate assimilation and fermentation of 49 compounds with one control were identified on API 50 CH strips (BioMerieux, Tokyo, Japan). The preliminary identification of the LAB isolates based on the phenotypic characteristics was performed according to the criteria of Bergey’s Manual of Determinative Bacteriology.

2.6. Antimicrobial activity Strains of selected LAB were grown in the tubes containing 1.5 mL MRS broth at 30°C for 24 h and centrifuged at 10 000×g for 10 min. Then, 300 μL of the supernatants were used to examine the inhibition activity by agar diffusion assay method with Listeria monocytogens, Escherichia coil, Salmonella sp. and Acetobacter pasteurianus. After incubation at 30°C for 12 h, the diameters of inhibition zones were recorded by vernier caliper. In order to eliminate the effect of organic acid, the pH value of the obtained supernatants were adjusted to pH 7.0 with NaOH (1 N).

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MB broth were highly variable (Tables 2 and 3). For the RB broth, the LAB number and pH varied, respectively, from 6.2 to 8.9 colony form unit (cfu) per gram of fresh material and from 4.55 to 6.89; the contents of the lactic acid, acetic acid, propionic acid and ethanol ranged from 1.2 to 23.6, 0.8 to 10.8, 0.0 to 1.5 and 0.0 to 0.8 g kg–1, respectively. Compared with RB, the 65 LAB strains produced higher pH values and lower lactic acid in the MB broth. For the MB broth, the LAB number and pH varied, respectively, from 5.5 to 8.6 cfu g–1 and from 5.61 to 6.84; the contents of the lactic acid, acetic acid, propionic acid and ethanol ranged from 1.2 to 10.2, 0.9 to 11.8, 0.3 to 3.2 and 0.0 to 0.8 g kg–1, respectively. In general, lactic and acetic acid play an important role in inhibiting the undesirable bacteria and fungi. Based on the abovementioned results, the LAB strains producing the most lactic and acetic acid were selected separately for RB and MB. For RB, the 6 strains that produced the highest lactic acid contents (C13, 19.8; R2, 20.9; R3, 21.2; A3, 23.6; A4, 19.9; F8, 20.5) and the 3 strains that produced the highest acetic acid concentrations (C13, 10.8; R2, 9.8; A6, 9.5) were selected for further study. For MB, the 3 strains that produced the highest amounts of lactic acid (C5, 10.2; R9, 9.2; A4, 9.9) and the 3 strains that produced the highest acetic acid concentrations (C6, 9.6; F7, 10.8; F8, 11.8) were selected for further tests. Strain A4 produced high lactic acid in both RB and MB broth.

3.2. Biochemical, physiological properties and identification of selected LAB strains

3. Results 3.1. Preselection of LAB strains in RB and MB broth A total of 81 strains were isolated from the employed sources, of which 65 strains were considered to be LAB, as determined by their Gram-positive reaction and catalase-negative reaction (data not shown). The pH values and contents of the metabolites produced by the 65 LAB strains in RB and

Tables 4 and 5 show the physiological and biochemical properties of selected LAB strains. All LAB strains ferment a large number of carbohydrates (Table 4), but cannot utilise esculine, L-arabitol, 2-ceto-gluconate or 5-ceto-gluconate. Compared with the other LAB strains, Lactobacillus reuteri (F7 and F8) could not ferment inositol or gentiobiose. All the selected LAB strains were unable to grow at a low temperature (5°C) or high temperature (50°C) (Table 5).

Table 2 The fermentation quality by LAB in Robinia pseudoacacia (RB) broth LAB strians C1 C2 C3 C4 C5 C6 C7 C8

pH 4.75 5.14 5.36 6.21 5.84 5.55 6.07 5.24

Lactic acid 17.8 10.2 8.8 5.5 7.4 6.8 2.5 5.6

Acetic acid 8.9 7.2 5.4 6.8 5.4 1.8 0.8 4.5

Propionic acid 0.2 0.8 0.4 0 0.5 0.4 0 0

Ethanol 0.1 0.2 0.2 0.1 0.1 0.3 0.2 0.2

Lactic acid bacteria 7.8 8.5 6.2 8.5 8.2 7.7 8.1 8.9

(Continued on next page)

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Table 2 (Continued from preceding page) LAB strians C9 C10 C11 C12 C13 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 MB1 MB2 MB3 MB4 MB5 MB6 MB7 MB8 RB1 RB2 RB3 RB4 RB5 RB6 RB7

pH 4.69 5.71 6.01 4.79 4.55 6.21 4.80 4.62 5.89 6.23 4.78 5.87 5.23 4.81 4.68 4.71 5.65 4.60 4.60 4.72 5.86 6.23 4.66 6.37 5.93 5.55 4.98 6.23 6.13 6.06 6.71 6.54 4.68 6.30 4.65 5.89 5.95 5.67 6.35 6.23 5.77 5.45 4.85 6.15 6.75 6.32 5.69 5.41 5.25 5.47 6.89 6.41 6.25 4.95 5.12 5.68 6.30

Lactic acid 15.6 6.8 2.4 9.5 19.8 2.4 20.9 21.2 5.8 1.2 16.9 5.4 9.6 15.4 18.9 15.6 8.2 23.6 19.9 16.8 7.7 2.1 17.5 3.5 4.2 6.5 12.4 2.1 3.2 3.1 1.2 2.1 18.5 2.2 20.5 4.7 5.4 8.5 2.5 3.4 7.5 7.8 11.2 2.7 2.2 3.5 7.1 6.9 10.8 7.6 2.4 3.2 4.2 10.9 9.6 5.6 3.2

Acetic acid 6.1 3.6 3.5 5.6 10.8 3.5 9.8 2.3 2.7 2.2 3.6 4.4 6.5 5.4 4.2 7.4 7.8 6.5 6.8 8.7 9.5 1.7 8.6 2.1 3.2 6.8 6.8 3.2 2.5 3.6 2.2 1.2 6.9 2.5 4.8 5.8 6.5 8.1 4.5 5.5 2.1 2.3 2.4 3.5 1.5 2.1 3.6 5.5 6.4 7.6 1.8 1.5 1.9 8.1 5.4 3.6 0.8

Propionic acid 0.5 0.4 0 0.5 1.2 0 0.3 0.5 0.8 0 0.6 0.2 0.3 0.8 0.7 1.5 0 0.6 1.1 1.2 0.5 0 0 0 0.5 0.4 0.4 0 0 0 0 0 0.4 0 0 0 0.5 0.1 0 0 0.5 0.4 0 0 0 0 0.7 0 0.2 0.1 0 0 0 0.2 0 0 0

Ethanol 0.2 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.1 0.2 0.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.0 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.0 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Lactic acid bacteria 8.6 6.8 6.7 7.7 8.7 6.8 8.5 7.4 8.2 7.2 7.9 6.9 7.5 8.2 6.5 8.6 6.7 8.5 8.1 6.8 7.6 7.2 8.1 8.6 7.9 7.5 6.8 6.5 7.2 6.4 8.3 8.4 7.9 6.5 7.5 6.5 7.6 6.8 8.3 6.8 6.8 7.6 8.6 6.9 7.6 6.5 7.2 6.5 7.5 6.5 6.2 7.3 7.6 8.3 7.2 5.3 7.3


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Table 3 The fermentation quality by LAB in Morus alba L. (MB) broth LAB strians C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 MB1 MB2 MB3 MB4 MB5 MB6 MB7

pH 6.84 6.19 5.98 5.95 5.66 5.74 5.79 5.86 5.96 5.71 6.01 6.35 5.96 5.97 5.94 5.77 5.82 5.82 6.61 5.87 5.93 5.62 6.81 5.91 5.65 5.91 5.71 5.92 6.03 6.23 5.88 6.29 5.93 6.32 5.89 6.23 6.13 6.06 6.71 6.54 5.67 5.73 5.61 5.89 5.98 6.45 6.35 6.23 5.77 5.98 6.35 6.15 6.75 6.32 6.38 6.45 6.25

Lactic acid 4.3 4.5 8.1 5.5 10.2 6.8 7.6 5.6 6.2 7.2 2.4 1.2 7.5 7.6 7.6 8.1 5.8 6.3 2.3 7.9 6.6 9.2 1.9 6.9 8.3 8.6 9.9 6.8 4.7 2.3 7.8 3.5 4.2 6.5 2.4 2.1 2.4 3.1 1.2 2.1 8.5 7.5 6.4 4.7 7.5 2.6 2.5 3.4 7.5 7.7 1.2 2.7 2.2 3.5 7.1 5.3 1.5

Acetic acid 2.1 2.3 5.4 3.6 7.5 9.6 4.2 4.5 6.1 3.6 3.3 1.2 5.3 3.5 4.2 6.2 6.3 6.2 2.6 4.4 6.5 7.5 3.2 7.4 7.8 6.5 6.8 5.3 3.9 1.8 8.6 2.1 3.4 6.8 3.2 3.5 2.5 3.6 2.2 2.8 8.9 10.8 11.8 4.6 6.5 3.2 3.6 5.5 2.1 7.5 1.4 2.5 1.5 2.1 3.6 5.5 6.4

Propionic acid 1.8 0.8 0.6 0.5 0.6 0.4 0.7 0.7 1.1 0.8 0.6 0.5 0.9 0.7 0.4 0.5 0.8 0.5 0.4 0.3 1.2 0.8 3.2 1.5 1.8 0.9 1.1 1.3 0.5 1.2 1.6 1.2 0.5 1.1 0.4 0.4 0.6 0.3 1.6 1.2 2.1 1.2 2.3 1.8 1.2 0.6 1.5 1.4 0.5 2.6 0.9 0.4 0.6 0.6 0.7 1.2 1.5

Ethanol 0.4 0.1 0.2 0.5 0.1 0.0 0.2 0.5 0.2 0.1 0.4 0.6 0.1 0.1 0.1 0.1 0.1 0.2 0.5 0.1 0.1 0.2 0.5 0.1 0.1 0.1 0.2 0.1 0.8 0.1 0.1 0.2 0.1 0.1 0.5 0.2 0.1 0.2 0.1 0.1 0.0 0.0 0.5 0.1 0.2 0.1 0.1 0.2 0.4 0.2 0.5 0.2 0.6 0.1 0.1 0.2 0.1

Lactic acid bacteria 6.5 7.2 6.5 5.9 7.9 6.3 6.5 6.5 7.8 6.6 7.8 7.7 8.5 7.4 5.8 7.5 7.5 6.9 7.2 6.2 7.8 8.2 6.9 8.5 7.2 8.6 8.3 6.9 7.1 6.6 7.6 8.5 6.3 5.5 6.7 6.5 7.5 6.4 8.1 6.8 8.5 7.5 6.8 7.5 6.6 7.2 7.5 6.5 7.1 7.6 7.5 6.8 8.2 7.5 6.6 6.5 7.5


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Table 3 (Continued from preceding page) LAB strians MB8 RB1 RB2 RB3 RB4 RB5 RB6 RB7

pH 6.47 6.89 6.41 6.25 6.35 6.12 6.14 6.30

Lactic acid 1.8 2.4 3.2 2.2 2.5 5.5 2.3 3.2

Acetic acid 2.6 1.5 1.5 1.9 1.8 4.2 3.6 0.9

Propionic acid 1.3 0.8 1.2 0.6 0.2 1.5 1.2 0.9

Ethanol 0.1 0.5 0.4 0.4 0.1 0.2 0.1 0.3

Lactic acid bacteria 6.5 6.8 7.3 7.6 7.5 7.2 6.3 6.5

Table 4 API 50 CH fermentation patterns of isolated LAB strains Item Control Glycerol Erythritol D-Arabinose L-Arabinose D-Ribose D-Xylose L-Xylose D-Adonitol Methyl-D-xylopyraniside D-Galactose D-Glucose D-Fructose D-Mannose L-Sorbose L-Rhamnose Dulcitol Inositol D-Mannitol D-Sorbitol Methyl-D-mannopyranoside Methyl-D-glucopyranoside N-acetylglucosamine Amygdaline Arbutine Esculine Salicine D-Celiobiose D-Maltose D-Lactose D-Melibiose D-Saccharose D-Trehalose Inuline D-Melezitose D-Raffinose Amidon Glycogen Xylitol Gentiobiose D-Turanose D-Lyxose D-Tagatose D-Fucose

C5 – w w + + + + w w w + + + + + + + + + + + + + + + – + + + + + + + + + + + + + + + + w w

C6 – + w + + + + w w w + + + + + + + + + + + + + + + – + + + + + + + + + + + + w + + + + w

C13 – w w w + + w w w w + + + + + + + + + + + + + + + – + + + + + + + + + + + w w + + + + w

R2 – + w + + + + w w w + + + + + + w w + + + + + + + – + + + + + + + + + + + w w + + w + w

R3 – + w w + + w w – – + + + + w w w w + + + + + + + – + + + + + + + + + + + w w + + w + w

R9 – + w + + + + w – – + + + + + + w w + + + w + + + – + + + + + + + w + + + w w + + w + w

A3 – + w – w + w – – – + + + + w w w w + + w + + + + – + + + + w + + w + + + – w + w w + w

A4 – + w w + + w w – – + + + + + + w w + + w + + + + – + + + + + + + + + + + w w + + w + w

A6 – + w + + + + – – – + + + + + + w w + + + + + + + – + + + + + + + + + + + w w + + w + w

F7 – + w + + + + w – w + + + + + + w – + + + + + + + – + + + + + + + w + + + + + – + w w w

F8 – – w w + + + w – w + + + + – + + – + + + + + + + – + + + + + + + – + + w w w – w w w w


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Table 4 (Continued from preceding page) Item L-Fucose D-Arabitol L-Arabitol Potassium gluconate 2-Ceto-gluconate 5-Ceto-gluconate

C5 w w – + – –

C6 w – – + – –

C13 w w – + – –

R2 w w – + – –

R3 w w – + – –

R9 – – – + – –

A3 – – – + – –

A4 w w – + – –

A6 – – – + – –

F7 – w – – – –

F8 – w – w – –

+, positive; w, weakly positive; –, negative. The same as in Table 5.

Table 5 Tolerance to inhibitory conditions by LAB isolates 　 Growth at temperature (°C) 5 10 15 45 50 Growth at pH 3.0 3.5 4.0 4.5 Growth in NaCl (w/v, %) 3.0 6.5

C5

C6

C13

R2

R3

R9

A3

A4

A6

F7

F8

– + + + –

– w + + –

– w + + w

– w + + –

– w + + –

– w + + –

– w + + –

– w + + –

– w + + –

– w + + w

– w + + –

– w + +

– + + +

– + + +

– + + +

– + + +

– + + +

– + + +

– + + +

– + + +

+ + + +

+ + + +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

+ +

Strains F7 and F8 were able to grow at a low pH value (3.0), whereas the other strains could not. All the isolated LAB strains showed the same survival ability at pH 3.0 and 6.5% NaCl solution. Following the 16S rRNA gene sequence analysis, Figs. 1 and 2 showed that strain C6 had an identity similar to (100%) Weissella confusa. C5, C13, R2, R3, R9, A3, A4 and A6 were assigned to Lactobacillus plantarum. F7 and F8 were identified as being similar to (100%) L. reuteri.

L. monocytogens, E. coil and Salmonella sp. It is possibly because of strain-specific inhibition which occurs via production of ribosomally encoded bacteriocins. For example, L. reuteri can produce reuterin, which has an ability inhibiting the growth of L. monocytogens, E. coil and Salmonella enterica (Zhao et al. 2012).

3.3. Antibacterial activity

In the present study, 65 LAB strains were isolated from 5 origins and used to perform a fermentation test on the substrates of RB and MB broth. The results (Tables 2 and 3) showed that great variance existed among the different strains in terms of their pH and metabolites. The major metabolite variations of these LAB strains were found in their production of lactic and acetic acids. Generally, LAB produces lactic acid as a major by-product of carbohydrate fermentation, but some of the isolated strains produced more acetic acid than lactic acid. That is because LAB can be classified as homo- or hetero-fermentative based on the fermentation products. Homo-fermentative LAB promotes rapid fermentation, primarily producing lactic acid and rapidly reducing the pH, preventing the growth of other undesirable spoilage organisms such as coliform and clostridium (Cai et al. 1998). Hetero-fermentative LAB, however, produces a mix of lactic acid and acetic acid, which results in a slower

Four species were used as targeted strains, including E. coil, A. pasteurianus, Salmonella sp. and L. monocytogens. Because these 4 strains not only can decrease the nutritional value of the silage, but also present a risk to animal health (Driehuis et al. 2000). All the selected LAB strains exhibited the ability to inhibit the activity of the 4 indicators. Furthermore, it was determined that the antagonistic activity toward L. monocytogens and Salmonella sp. were higher than the capacity against E. coil and A. pasteurianus (Table 6). The pH value of natural fermentation products of the selected LAB strains ranged from 3.8–4.2. When the pH value was adjusted to 7.0, no strains exhibited an inhibitory activity toward A. pasteurianus. However, the 3 remaining strains (C13, F7 and F8) exhibited inhibiting activity toward

4. Discussion

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C5 C13 R2 Knuc 0.02

R3 R9 A3

100

A4 A6

84 L. plantarum

L.farciminis L. alimentarius

100

L. paralimentarius 99 100 L. kimchii L. fermentum L. mucosae

100

L. colehominis 45

F7 100

79

F8 L. reuteri L. pontis

91

L. vaginalis L. frumenti L. panis 99

L. oris B. subtilis

Fig. 1 Phylogenetic tree showing the relative positions of isolates C5, C13, R2, R3, R9, A3, A4, A6, F7 and F8 as referred by the neighbor-joining method of complete 16S rDNA sequences. Bootstrap values for 100 replicates are shown at the nodes of the tree. Bacillus subtilis is used as an outgroup. The bar indicates 1% sequence divergence. L., Lactobacillus; Knuc, nucleotide substitution rate.

C6

100 99

W. confusa W. kimchii

67

100 W. cibaria W. hellenica

92

Knuc 0.02

W. thailandensis

100 71

53

W. paramesenteroides W. soli

62

W. kandleri W. viridescens

100

W. minor W. halotolerans B. subtilis

Fig. 2 Phylogenetic tree showing the relative positions of isolate C6 as referred by the neighbor-joining method of complete 16S rDNA sequences. Bootstrap values for 100 replicates are shown at the nodes of the tree. B. subtilis is used as an outgroup. The bar indicates 1% sequence divergence. W., Weissella.


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Table 6 Antimicrobial ability of isolated LAB 　

C5

Listeria monocytogens +++ Escherichia coil + Salmonella sp. +++ Acetobacter pasteurianus ++

C6 C13 R2

R3

+ +++ +++ +++ + ++ + + + +++ +++ +++ + ++ ++ ++

R9

A3

A4

A6

F7

F8

+++ +++ +++ +++ +++ +++ ++ ++ ++ + ++ ++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++

C5 C6 C13 – – ++ – – + – – + – – –

pH=7.0 R2 R3 R9 A3 A4 A6 F7 F8 – – – – – – + ++ – – – – – – + + – – – – – – + + – – – – – – – -

–, no visible inhibition; +, weak suppression (diameter of inhibition zone: 0.1–5.0 mm); ++, good supression (5.0–10.0 mm); +++, strong supression (more than 10.0).

fermentation than homo-fermentative LAB. This type of LAB is designed to inhibit the yeast and mould that initiates the process of aerobic deterioration during feed-out. Based on the production of the most lactic and acetic acid content, 11 LAB strains isolated from corn, rice, alfalfa and faeces were selected for further study. Some studies have shown the best isolates for a specific crop come from the crop itself. Hill et al. (1989) found that inoculants isolated from corn were not efficient in lucerne and sorghum. Wang et al. (2006) found that adding selected LAB strains from alfalfa to a corn medium does not improve the ratio of lactate/acetate. It may be assumed that the bacteria originally present in a plant or crop has a good relationship with its host and can colonise the plant easily. However, in the present study, none of the best strains were selected from the RB and MB plants, which might indicate that the selection criterion should depend on the characteristics of the strains rather than their origin. The amount of sugars in different plant species was directly correlated with the maximum bacterial population sizes and suggested that microorganisms growing on plant surfaces might be competing for a limited amount of sugars which, in turn, would determine the microbial carrying capacity of the plant. Therefore, the LAB strains isolated from RB and MB do not possess a strong ability for making use of the sugars available in RB and MB. All of the selected 11 LAB were further characterized using sugar fermentation assays with API 50 CH strips. F7 and F8 displayed a distinct carbohydrate fermentation pattern with others. However, the phenotypic procedure used to assign isolates to known species is challenging because it can be difficult to differentiate between species. To identify LAB isolates at the species level, a molecular analysis was performed, and phylogenetic trees were constructed based on the evolutionary distances of their 16S rDNA sequences using the neighbour-joining method. The strains of C5, C13, R2, R3, R9, A3, A4 and A6 were identified as L. plantarum; C6 had an identity similar to Weissella confuse; F7 and F8 were identified as being similar to L. reuteri. All the 11 selected LAB strains, except C6, belong to the genus Lactobacillus and are considered to be safe for both humans and animals. Lactobacillus species are

among those most commonly used as silage inoculants, due to their high acid tolerance and crucial role in the final fermentation steps (Cao et al. 2011). In contrast to cocci, Lactobacillus are important promoters of lactic acid or acetic acid fermentation for longer lengths of time. Many studies have reported the beneficial effects to silage quality that has been inoculated with Lactobacillus strains. Of the 11 selected LAB strains, strain A4 produces the most lactic acid, and no strain produces the most acetic acid in both plant broths, which may indicate that the effect of LAB should be crop- or plant-specific. Avila et al. (2014) reported that no correlation exists between the best producer of lactic acid and the best producer of acetic acid, but strains C13 and R2 can produce a high concentration of lactic acid as well as a high concentration of acetic acid in RB. This is likely because some homo-fermentative lactobacilli (e.g., L. plantarum) can undergo an oxidative pyruvate and/or lactate dissimilation to acetate by stereospecific NAD-independent flavin-containing lactate dehydrogenase oxidases (Bobillo and Marshall 1991). The antibacterial activity of the LAB is tested by an in vitro assay, which is commonly used to select the potentially beneficial stains from various origins (Gollop et al. 2005). It was observed that all of the selected LAB strains, except C13, F7 and F8, did not exhibit an inhibition zone with a pH adjustment to 7.0, indicating the inhibitory effect is primarily due to the organic acids. Lactic and acetic acids are the primary products fermented in silage by LAB and are generally regarded as safe for humans and animals. Their potential mechanism lies in the reduction of pH, as well as the undissociated form of molecules (Gourama and Bullerman 1995; Carr et al. 2002). It is hypothesised that the low pH may result in the acidification of the cell cytoplasm, whereas acetic acid, a high undissociated acid, can diffuse passively across the membrane and is described as a better inhibitor of mould growth than lactic acid (Batish et al. 1997). Interestingly, F7 and F8 (both are L. reuteri) still exhibited inhibiting activity toward L. monocytogens, E. coil and Salmonella sp. when the fermentation product was adjusted to pH 7.0. However, the antibacterial effect decreased. These results indicated that the inhibitory ef-

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fects of F7 and F8 are likely due to the joint contribution of organic acids and other low-molecular-weight compounds, which may offer a completive advantage as an inoculant of silage. Previous reports have shown that L. reuteri can produce reuterin, which increases the antifungal activity (Dobrogosz et al. 1989). In this regard, it is worthwhile to investigate their effect on fermentation quality and the aerobic stability of silage.

5. Conclusion In this study, a total of 11 LAB strains were considered to be potential inoculants for ensiling RB and MB leaves based on the highest products of lactic or acetic acids. Only L. plantarum A4 produced a high amount of lactic acid in the extracted liquids of both RB and MB leaves. Of the 11 LAB strains, L. reuteri F7 and F8 isolated from pig faeces exhibited attractive properties, such as a tolerance to a low pH (3.0) and production of other low-molecular-weight antibacterial compounds. However, further research is warranted to investigate their effect on the silage quality of RB and MB leaves under small-scale fermentation conditions.

Acknowledgements This work was supported by the Key Technique Project from Henan Province, China (152102110045 and 152102310064).

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