Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Isolating and evaluating lactic acid bacteria strains for effectiveness of Leymus chinensis silage fermentation Q. Zhang, X.J. Li, M.M. Zhao and Z. Yu College of Animal Science and Technology, China Agricultural University, Beijing, China
Significance and Impact of the Study Leymus chinensis is an important grass in China and Russia, being the primary grass of the short grassland ‘steppe’ regions of central Asia. However, it has been difficult to make high-quality silage of this species because of low concentration of water-soluble carbohydrates (WSC). Isolating and evaluating lactic acid bacteria strains will be helpful for improving the silage quality of this extensively grown species.
Keywords isolation, identification, lactic acid bacteria, Leymus chinensis, silage quality. Correspondence Zhu Yu, College of Animal Science and Technology, China Agricultural University, West Road 2 Yuan Ming Yuan, Beijing 100193, China. E-mail: [email protected] 2014/0378: received 23 February 2014, revised 11 May 2014 and accepted 27 May 2014 doi:10.1111/lam.12291
Abstract Five LAB strains were evaluated using the acid production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All five strains (LP1, LP2, LP3, LC1 and LC2) grew at pH 40, and LP1 grew at 15°C. Strains LP1, LP2 and LP3 were identified as Lactobacillus plantarum, whereas LC1 and LC2 were classified as Lactobacillus casei by sequencing 16S rDNA. The five isolated strains and two commercial inoculants (PS and CL) were added to native grass and Leymus chinensis (Trin.) Tzvel. for ensiling. All five isolated strains decreased the pH and ammonia nitrogen content, increased the lactic acid content and LP1, LP2 and LP3 increased the acetic content and lactic/acetic acid ratio of L. chinensis silage significantly. The five isolated strains and two commercial inoculants decreased the butyric acid content of the native grass silage. LP2 treatment had lower butyric acid content and ammonia nitrogen content than the other treatments. The five isolated strains improved the quality of L. chinensis silage. The five isolated strains and the two commercial inoculants were not effective in improving the fermentation quality of the native grass silage, but LP2 performed better comparatively.
Introduction Leymus chinensis (Trin.) Tzvel., often known as Chinese leymus, is a native, cool-season, perennial grass in the Gramineae tribe that is widely distributed throughout the northern temperate Asia (Peng et al. 2013). Due to its high yield, protein content, palatability and ability to meet livestock nutritional needs, this species is cultivated as one of the principal forage grasses in northern China, the Mongolia plateau, and western and eastern Siberia (Shu et al. 2005; Yan et al. 2011). In these areas, almost all native grasses are preserved as hay. However, during July and August, the principal harvest season, the weather is unreliable for drying. In these areas, ensiling is preferable for conserving this forage resource. However,
successful ensiling of L. chinensis is difficult due to its low concentration of WSC (Sun et al. 2007). Lactic acid bacteria inoculation has been proven to be useful for promoting favourable fermentation in many forage species, with investigations from the 1970s onwards (Ohyama et al. 1975; Chen et al. 2013). The efficacy of LAB inoculants is highly influenced not only by their phenotypic characteristics and range of ecological niches, but also by several environmental factors, including chemical composition and indigenous microbial diversity of crops (Tohno et al. 2012). However, limited information is available on the characteristics of naturally occurring LAB on L. chinensis, and no effective LAB strains have been found and used for ensiling native rangeland forages. This study was conducted to isolate
Letters in Applied Microbiology 59, 391--397 © 2014 The Society for Applied Microbiology
391
LAB strains for Leymus chinensis silage
Q. Zhang et al.
and identify lactic acid bacteria (LAB) from the shortgrass rangelands of China using phenotypic and molecular methods. Isolates were evaluated for their effects on fermentation quality measures of pure L. chinensis and a mixture of native forage species harvested from rangeland grazing areas. Results and discussion LAB strain characteristics All isolates were found to be Gram-positive, homofermentative and catalase-negative rod-shaped bacteria. Their morphological and physiological characteristics are presented in Table 1. Strain LP1 grew at 15°C, but others Table 1 The characteristics of lactic acid bacteria isolates Item
LP1
Source Corn Shape Rod Gram stain + Fermentation type Homo Catalase activity Growth at temperature 10°C 15°C + 45°C 50°C Growth at pH 300 350 400 + 450 ++ 550 ++ 750 + 850 + 950 Carbohydrate fermentation Xylose + Gluconate Glucose + Fructose + Galactose + Cellobiose + Lactose + Maltose + Sucrose + Mannitol + Sorbitol + Esculin + Salicin + Starch
grew slowly at temperatures below 15°C; only strain LC1 was able to grow at 45°C. All strains were able to grow at pH 40–85. All strains fermented glucose and sucrose, but none effectively utilized starch. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique (Clarridge 2004). Strains LP1, LP2 and LP3 were most closely related to Lactobacillus plantarum (Fig. 1), with 99, 97 and 98% similarity in their 16SrDNA gene sequences, respectively. Strains LC1 and LC2 were most closely related to Lactobacillus casei, supporting the 100% value from bootstrap analysis of the phylogenetic tree (Fig. 1), and showing 98% similarity in their 16S rDNA gene sequences. Therefore, strains LP1, LP2 and LP3 were identified as Lact. plantarum, and LC1 and LC2 were identified as Lact. casei. Forage characteristics
LP2
LP3
LC1
LC2
Alfalfa Rod + Homo
Native grass Rod + Homo
Alfalfa Rod + Homo
Alfalfa Rod + Homo
Lin et al. (1992) reported that among epiphytic LAB, Lactobacilli play an important role in promoting lactic Lactobacillus parabrevis (LMG 11984)
77 96
Lactobacillus hammesii (TMW 1.1236)
100
+ ++ ++ + +
+ ++ ++ + +
+ ++ ++ + +
+ ++ ++ + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + +
Lactobacillus senmaizukei (L13) Lactobacillus brevis (ATCC 367)
98
Lactobacillus namurensis (LMG 23583) 94
99
Lactobacillus zymae (LMG 22198) Lactobacillus farraginis (NRIC 0676) Lactobacillus buchneri (JCM1115)
100 67
100
100
Lactobacillus buchneri (NRRL B-30929)
Pediococcus damnosus (DSM 20331) Pediococcus inopinatus (DSM 20285) Lactobacillus plantarum (NRRL B-14768)
42
LP3
100
+ + + + + + + + + + +
LP1
100 98
LP2
Lactobacillus sakei (DSM 20017) Lactobacillus manihotivorans (OND 32)
69
Lactobacillus pantheris (LMG 21017) Lactobacillus camelliae (MCH3-1)
98
Lactobacillus rhamnosus (ATCC 53103) 84
LC1
100
100
LC2 100
Lactobacillus casei (ATCC 334)
0·01
, no growth; , weak growth; +, normal growth; ++, good growth; Native grass, a mixture of species harvested from the rangeland (comprised of 71% Leymus chinensis, 18% Elymus dahuricus Turcz, 6% Artemisia frigid and 3% Melilotus officinalis).
392
Figure 1 Phylogenetic tree of partial 16S rDNA sequences of isolated strains and sequences of identified bacteria in the nucleotide database of GenBank.
Letters in Applied Microbiology 59, 391--397 © 2014 The Society for Applied Microbiology
LAB strains for Leymus chinensis silage
Q. Zhang et al.
acid fermentation over the course of ensiling, whereas the cocci, e.g. Streptococci, Leuconostocs, Lactococci and Enterococci, grow vigorously only in the early stage of ensiling processes. A well-preserved silage requires that lactobacilli reach a level of at least 105 CFU g1 fresh weight (FW) at ensiling (Lin et al. 1992). However, the number of epiphytic lactic acid bacteria on fresh crops is often very low, and this was true in the present study. As shown in Table 2, LAB numbers on the two materials were relatively low (
ORIGINAL ARTICLE
Isolating and evaluating lactic acid bacteria strains for effectiveness of Leymus chinensis silage fermentation Q. Zhang, X.J. Li, M.M. Zhao and Z. Yu College of Animal Science and Technology, China Agricultural University, Beijing, China
Significance and Impact of the Study Leymus chinensis is an important grass in China and Russia, being the primary grass of the short grassland ‘steppe’ regions of central Asia. However, it has been difficult to make high-quality silage of this species because of low concentration of water-soluble carbohydrates (WSC). Isolating and evaluating lactic acid bacteria strains will be helpful for improving the silage quality of this extensively grown species.
Keywords isolation, identification, lactic acid bacteria, Leymus chinensis, silage quality. Correspondence Zhu Yu, College of Animal Science and Technology, China Agricultural University, West Road 2 Yuan Ming Yuan, Beijing 100193, China. E-mail: [email protected] 2014/0378: received 23 February 2014, revised 11 May 2014 and accepted 27 May 2014 doi:10.1111/lam.12291
Abstract Five LAB strains were evaluated using the acid production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. All five strains (LP1, LP2, LP3, LC1 and LC2) grew at pH 40, and LP1 grew at 15°C. Strains LP1, LP2 and LP3 were identified as Lactobacillus plantarum, whereas LC1 and LC2 were classified as Lactobacillus casei by sequencing 16S rDNA. The five isolated strains and two commercial inoculants (PS and CL) were added to native grass and Leymus chinensis (Trin.) Tzvel. for ensiling. All five isolated strains decreased the pH and ammonia nitrogen content, increased the lactic acid content and LP1, LP2 and LP3 increased the acetic content and lactic/acetic acid ratio of L. chinensis silage significantly. The five isolated strains and two commercial inoculants decreased the butyric acid content of the native grass silage. LP2 treatment had lower butyric acid content and ammonia nitrogen content than the other treatments. The five isolated strains improved the quality of L. chinensis silage. The five isolated strains and the two commercial inoculants were not effective in improving the fermentation quality of the native grass silage, but LP2 performed better comparatively.
Introduction Leymus chinensis (Trin.) Tzvel., often known as Chinese leymus, is a native, cool-season, perennial grass in the Gramineae tribe that is widely distributed throughout the northern temperate Asia (Peng et al. 2013). Due to its high yield, protein content, palatability and ability to meet livestock nutritional needs, this species is cultivated as one of the principal forage grasses in northern China, the Mongolia plateau, and western and eastern Siberia (Shu et al. 2005; Yan et al. 2011). In these areas, almost all native grasses are preserved as hay. However, during July and August, the principal harvest season, the weather is unreliable for drying. In these areas, ensiling is preferable for conserving this forage resource. However,
successful ensiling of L. chinensis is difficult due to its low concentration of WSC (Sun et al. 2007). Lactic acid bacteria inoculation has been proven to be useful for promoting favourable fermentation in many forage species, with investigations from the 1970s onwards (Ohyama et al. 1975; Chen et al. 2013). The efficacy of LAB inoculants is highly influenced not only by their phenotypic characteristics and range of ecological niches, but also by several environmental factors, including chemical composition and indigenous microbial diversity of crops (Tohno et al. 2012). However, limited information is available on the characteristics of naturally occurring LAB on L. chinensis, and no effective LAB strains have been found and used for ensiling native rangeland forages. This study was conducted to isolate
Letters in Applied Microbiology 59, 391--397 © 2014 The Society for Applied Microbiology
391
LAB strains for Leymus chinensis silage
Q. Zhang et al.
and identify lactic acid bacteria (LAB) from the shortgrass rangelands of China using phenotypic and molecular methods. Isolates were evaluated for their effects on fermentation quality measures of pure L. chinensis and a mixture of native forage species harvested from rangeland grazing areas. Results and discussion LAB strain characteristics All isolates were found to be Gram-positive, homofermentative and catalase-negative rod-shaped bacteria. Their morphological and physiological characteristics are presented in Table 1. Strain LP1 grew at 15°C, but others Table 1 The characteristics of lactic acid bacteria isolates Item
LP1
Source Corn Shape Rod Gram stain + Fermentation type Homo Catalase activity Growth at temperature 10°C 15°C + 45°C 50°C Growth at pH 300 350 400 + 450 ++ 550 ++ 750 + 850 + 950 Carbohydrate fermentation Xylose + Gluconate Glucose + Fructose + Galactose + Cellobiose + Lactose + Maltose + Sucrose + Mannitol + Sorbitol + Esculin + Salicin + Starch
grew slowly at temperatures below 15°C; only strain LC1 was able to grow at 45°C. All strains were able to grow at pH 40–85. All strains fermented glucose and sucrose, but none effectively utilized starch. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique (Clarridge 2004). Strains LP1, LP2 and LP3 were most closely related to Lactobacillus plantarum (Fig. 1), with 99, 97 and 98% similarity in their 16SrDNA gene sequences, respectively. Strains LC1 and LC2 were most closely related to Lactobacillus casei, supporting the 100% value from bootstrap analysis of the phylogenetic tree (Fig. 1), and showing 98% similarity in their 16S rDNA gene sequences. Therefore, strains LP1, LP2 and LP3 were identified as Lact. plantarum, and LC1 and LC2 were identified as Lact. casei. Forage characteristics
LP2
LP3
LC1
LC2
Alfalfa Rod + Homo
Native grass Rod + Homo
Alfalfa Rod + Homo
Alfalfa Rod + Homo
Lin et al. (1992) reported that among epiphytic LAB, Lactobacilli play an important role in promoting lactic Lactobacillus parabrevis (LMG 11984)
77 96
Lactobacillus hammesii (TMW 1.1236)
100
+ ++ ++ + +
+ ++ ++ + +
+ ++ ++ + +
+ ++ ++ + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + +
Lactobacillus senmaizukei (L13) Lactobacillus brevis (ATCC 367)
98
Lactobacillus namurensis (LMG 23583) 94
99
Lactobacillus zymae (LMG 22198) Lactobacillus farraginis (NRIC 0676) Lactobacillus buchneri (JCM1115)
100 67
100
100
Lactobacillus buchneri (NRRL B-30929)
Pediococcus damnosus (DSM 20331) Pediococcus inopinatus (DSM 20285) Lactobacillus plantarum (NRRL B-14768)
42
LP3
100
+ + + + + + + + + + +
LP1
100 98
LP2
Lactobacillus sakei (DSM 20017) Lactobacillus manihotivorans (OND 32)
69
Lactobacillus pantheris (LMG 21017) Lactobacillus camelliae (MCH3-1)
98
Lactobacillus rhamnosus (ATCC 53103) 84
LC1
100
100
LC2 100
Lactobacillus casei (ATCC 334)
0·01
, no growth; , weak growth; +, normal growth; ++, good growth; Native grass, a mixture of species harvested from the rangeland (comprised of 71% Leymus chinensis, 18% Elymus dahuricus Turcz, 6% Artemisia frigid and 3% Melilotus officinalis).
392
Figure 1 Phylogenetic tree of partial 16S rDNA sequences of isolated strains and sequences of identified bacteria in the nucleotide database of GenBank.
Letters in Applied Microbiology 59, 391--397 © 2014 The Society for Applied Microbiology
LAB strains for Leymus chinensis silage
Q. Zhang et al.
acid fermentation over the course of ensiling, whereas the cocci, e.g. Streptococci, Leuconostocs, Lactococci and Enterococci, grow vigorously only in the early stage of ensiling processes. A well-preserved silage requires that lactobacilli reach a level of at least 105 CFU g1 fresh weight (FW) at ensiling (Lin et al. 1992). However, the number of epiphytic lactic acid bacteria on fresh crops is often very low, and this was true in the present study. As shown in Table 2, LAB numbers on the two materials were relatively low (
