Characteristics of isolated lactic acid bacteria and their effects on the silage quality

Siran Wang,Xianjun Yuan,Zhihao Dong,Junfeng Li,Gang Guo,Yunfeng Bai,Junyu Zhang,Tao Shao 아세아·태평양축산학회 2017 Animal Bioscience Vol.30 No.6

Objective: Four lactic acid bacteria (LAB) strains isolated from common vetch, tall fescue and perennial ryegrass on the Tibetan Plateau were characterized, and their effects on the fermentation quality of Italian ryegrass (Lolium multiflorum Lam.) silage were studied. Methods: The four isolated strains and one commercial inoculant (G, Lactobacillus plantarum MTD-1) were evaluated using the acid production ability test, morphological observation, Gram staining, physiological, biochemical and acid tolerance tests. The five LAB strains were added to Italian ryegrass for ensiling at three different temperatures (10°C, 15°C, and 25°C). Results: All isolated strains (LCG3, LTG7, I5, and LI3) could grow at 5°C to 20°C, pH 3.0 to 8.0 and NaCl (3.0%, 6.5%). Strains LCG3, LTG7, I5, and LI3 were identified as Lactobacillus plantarum, Pediococcus acidilactici, Lactobacillus paraplantarum, and Lactobacillus casei by sequencing 16S rDNA, respectively. All LAB inoculants significantly (p<0.05) increased lactic acid (LA) contents and ratios of lactic acid to acetic acid, and reduced pH and ammonia nitrogen/ total nitrogen (AN/TN) compared with uninoculated silages at various temperatures (10°C, 15°C, and 25°C). Compared to the commercial inoculant G, I5, and LI3 showed similar effects on improving the silage quality of Italian ryegrass at 10°C and 15°C, indicated by similar pH, LA content and AN/TN. Conclusion: All inoculants could improve the silage fermentation quality at various temperatures (10°C, 15°C, and 25°C). At the temperature of 10°C and 15°C, strain I5 and LI3 had similar effects with the commercial inoculant G on improving the silage quality of Italian ryegrass.

Fermentative products and bacterial community structure of C4 forage silage in response to epiphytic microbiota from C3 forages

Wang Siran,Shao Tao,Li Junfeng,Zhao Jie,Dong Zhihao 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.12

Objective: The observation that temperate C3 and tropical C4 forage silages easily produce large amounts of ethanol or acetic acid has puzzled researchers for many years. Hence, this study aimed to assess the effects of epiphytic microbiota from C3 forages (Italian ryegrass and oat) on fermentative products and bacterial community structure in C4 forage (sorghum) silage. Methods: Through microbiota transplantation and γ-ray irradiation sterilization, the irradiated sorghum was treated: i) sterile distilled water (STSG); ii) epiphytic microbiota from sorghum (SGSG); iii) epiphytic microbiota from Italian ryegrass (SGIR); iv) epiphytic microbiota from oat (SGOT). Results: After 60 days, all the treated groups had high lactic acid (>63.0 g/kg dry matter [DM]) contents and low pH values (<3.70), acetic acid (<14.0 g/kg DM) and ammonia nitrogen (<80.0 g/kg total nitrogen) contents. Notably, SGIR (59.8 g/kg DM) and SGOT (77.6 g/kg DM) had significantly (p<0.05) higher ethanol concentrations than SGSG (14.2 g/kg DM) on day 60. After 60 days, Lactobacillus were predominant genus in three treated groups. Higher proportions of Chishuiella (12.9%) and Chryseobacterium (7.33%) were first found in silages. The ethanol contents had a positive correlation (p<0.05) with the abundances of Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium. Conclusion: The epiphytic bacteria on raw materials played important roles in influencing the silage fermentation products between temperate C3 and tropical C4 forages. The quantity and activity of hetero-fermentative Lactobacillus, Chishuiella, Acinetobacter, Stenotrophomonas, Chryseobacterium, and Sphingobacterium may be the key factors for the higher ethanol contents and DM loss in silages. Objective: The observation that temperate C₃ and tropical C₄ forage silages easily produce large amounts of ethanol or acetic acid has puzzled researchers for many years. Hence, this study aimed to assess the effects of epiphytic microbiota from C₃ forages (Italian ryegrass and oat) on fermentative products and bacterial community structure in C₄ forage (sorghum) silage.Methods: Through microbiota transplantation and γ-ray irradiation sterilization, the irradiated sorghum was treated: i) sterile distilled water (STSG); ii) epiphytic microbiota from sorghum (SGSG); iii) epiphytic microbiota from Italian ryegrass (SGIR); iv) epiphytic microbiota from oat (SGOT).Results: After 60 days, all the treated groups had high lactic acid (>63.0 g/kg dry matter [DM]) contents and low pH values (<3.70), acetic acid (<14.0 g/kg DM) and ammonia nitrogen (<80.0 g/kg total nitrogen) contents. Notably, SGIR (59.8 g/kg DM) and SGOT (77.6 g/kg DM) had significantly (p<0.05) higher ethanol concentrations than SGSG (14.2 g/kg DM) on day 60. After 60 days, <i>Lactobacillus</i> were predominant genus in three treated groups. Higher proportions of <i>Chishuiella</i> (12.9%) and <i>Chryseobacterium</i> (7.33%) were first found in silages. The ethanol contents had a positive correlation (p<0.05) with the abundances of <i>Chishuiella</i>, <i>Acinetobacter</i>, <i>Stenotrophomonas</i>, <i>Chryseobacterium</i>, and <i>Sphingobacterium</i>.Conclusion: The epiphytic bacteria on raw materials played important roles in influencing the silage fermentation products between temperate C₃ and tropical C₄ forages. The quantity and activity of hetero-fermentative <i>Lactobacillus</i>, <i>Chishuiella</i>, <i>Acinetobacter</i>, <i>Stenotrophomonas</i>, <i>Chryseobacterium</i>, and <i>Sphingobacterium</i> may be the key factors for the higher ethanol contents and DM loss in silages.

An investigation on fermentative profile, microbial numbers, bacterial community diversity and their predicted metabolic characteristics of Sudangrass (Sorghum sudanense Stapf.) silages

Wang Siran,Li Junfeng,Zhao Jie,Dong Zhihao,Shao Tao 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.8

Objective: This study aimed to investigate the fermentation profiles, bacterial community and predicted metabolic characteristics of Sudangrass (Sorghum sudanense Stapf.) during ensiling. Methods: First-cutting Sudangrass was harvested at the vegetative stage and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30, and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through high-throughput sequencing technology, and 16S rRNA-gene predicted functional profiles were analyzed according to the Kyoto encyclopedia of genes and genomes using Tax4Fun. Results: The Sudangrass silages showed good fermentation quality, indicated by higher lactic acid contents, and lower pH, butyric acid and ammonia nitrogen contents. The dominant genus Lactococcus on day 3 was replaced by Lactobacillus on day 60. The metabolism of amino acid, energy, cofactors and vitamins was restricted, and metabolism of nucleotide and carbohydrate was promoted after ensiling. The 1-phosphofructokinase and pyruvate kinase of bacterial community seemed to play important roles in stimulating the lactic acid fermentation, and the promotion of arginine deiminase could help lactic acid bacteria to tolerate the acidic environment. Conclusion: High-throughput sequencing technology combined with 16S rRNA genepredicted functional analyses revealed the differences during the early and late stages of Sudangrass ensiling not only for distinct bacterial community but also for specific functional metabolites. The results could provide a comprehensive insight into bacterial community and metabolic characteristics to further improve the silage quality. Objective: This study aimed to investigate the fermentation profiles, bacterial community and predicted metabolic characteristics of Sudangrass (Sorghum sudanense Stapf.) during ensiling.Methods: First-cutting Sudangrass was harvested at the vegetative stage and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30, and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through high-throughput sequencing technology, and 16S rRNA-gene predicted functional profiles were analyzed according to the Kyoto encyclopedia of genes and genomes using Tax4Fun.Results: The Sudangrass silages showed good fermentation quality, indicated by higher lactic acid contents, and lower pH, butyric acid and ammonia nitrogen contents. The dominant genus <i>Lactococcus</i> on day 3 was replaced by <i>Lactobacillus</i> on day 60. The metabolism of amino acid, energy, cofactors and vitamins was restricted, and metabolism of nucleotide and carbohydrate was promoted after ensiling. The 1-phosphofructokinase and pyruvate kinase of bacterial community seemed to play important roles in stimulating the lactic acid fermentation, and the promotion of arginine deiminase could help lactic acid bacteria to tolerate the acidic environment. Conclusion: High-throughput sequencing technology combined with 16S rRNA genepredicted functional analyses revealed the differences during the early and late stages ofSudangrass ensiling not only for distinct bacterial community but also for specific functional metabolites. The results could provide a comprehensive insight into bacterial community and metabolic characteristics to further improve the silage quality.

Evaluating fermentation quality, in vitro digestibility and aerobic stability of a total mixed ration ensiled with different additives on Tibet plateau

Dong, Zhihao,Wang, Siran,Zhao, Jie,Li, Junfeng,Liu, Qinhua,Bao, Yuhong,Shao, Tao Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.2

Objective: To investigate the improvement in utilization efficiency of total mixed ration (TMR) on Tibetan plateau, TMR were ensiled with different additives. Methods: A total of 150 experimental silos were prepared in a completely randomized design to evaluate the six treatments: i) control (without additive), ii) Lactobacillus buchneri (L. buchneri), iii) acetic acid, iv) propionic acid, v) 1,2-propanediol; and vi) 1-propanol. After 90 days of ensiling, silos were opened for fermentation quality and in vitro analysis, and then subjected to an aerobic stability test for 14 days. Results: Treating with L. buchneri, acetic acid, 1,2-propanediol and 1-propanol decreased propionic acid contents and yeast number, whereas increased (p<0.05) pH, acetic acid and ethanol contents in the fermented TMR. Despite increased dry matter (DM) loss in the TMRs treated with 1,2-propanediol and 1-pronanol, additives did not affect (p>0.05) all in vitro parameters including gas production at 24 h (GP24), GP rate constant, potential GP, in vitro DM digestibility and in vitro neutral detergent fibre digestibility. All additives improved the aerobic stability of ensiled TMR to different extents. Specially, aerobic stability of the ensiled TMR were substantially improved by L. buchneri, acetic acid, 1,2-propanediol, and 1-propanol, indicated by stable pH and lactic acid content during the aerobic stability test. Conclusion: L. buchneri, acetic acid, 1,2-propanediol, and 1-propanol had no adverse effect on in vitro digestibility, while ensiling TMR with the additives produced more acetic acid and ethanol, subsequently resulting in improvement of aerobic stability. There is a potential for some fermentation boosting additives to enhance aerobic stability of fermented TMR on Tibetan plateau.

Effects of additives on the fermentation quality, in vitro digestibility and aerobic stability of mulberry (Morus alba L.) leaves silage

Dong Zhihao,Wang Siran,Zhao Jie,Li Junfeng,Shao Tao 아세아·태평양축산학회 2020 Animal Bioscience Vol.33 No.8

Objective: To explore feed resources capable of replacing regular poor-quality fodder, this study was conducted to evaluate the effects of additives on the fermentation quality, in vitro digestibility and aerobic stability of mulberry leaves silage. Methods: The mulberry leaves were ensiled either untreated (control) or treated with 1×106 cfu/g fresh matter Lactobacillus plantarum (L), 1% glucose (G), 3% molasses (M), a combination of 1% glucose and Lactobacillus plantarum (L+G), and a combination of 3% molasses and Lactobacillus plantarum (L+M). The fermentation quality and chemical composition were analyzed after 7, 14, 30, and 60 d, respectively. The 60-d silages were subjected to an aerobic stability test and fermented with buffered rumen fluid to measure the digestibility. Results: Inoculating lactic acid bacteria (LAB) resulted in more rapid increase in lactic acid concentrations and decline in pH of mulberry leaves silage as compared control. Higher acetic acid and lower ethanol and ammonia nitrogen concentrations (p<0.05) were observed in the LAB-inoculated silages as opposed to control during ensiling. The LAB-inoculated silages contained lower water-soluble carbohydrates compared with control during the first 14 d of ensiling, and lower neutral detergent fibre (p<0.05) concentrations as compared with non-LAB inoculated silages. Adding molasses alone increased (p<0.05) the digestibility of dry matter (DM). The aerobic stability of mulberry leaves silage was increased by LAB inoculation, whereas decreased by adding glucose or molasses. Conclusion: The LAB inoculation improved fermentation quality and aerobic stability of mulberry leaves silage, while adding glucose or molasses failed to affect the fermentation and impaired the aerobic stability. Inoculating LAB alone is recommended for mulberry leaves especially when ensiled at a relatively high DM.

Effect of storage time and the level of formic acid on fermentation characteristics, epiphytic microflora, carbohydrate components and in vitro digestibility of rice straw silage

Zhao Jie,Wang Siran,Dong Zhihao,Li Junfeng,Jia Yushan,Shao Tao 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.6

Objective: The study aimed to evaluate the effect of storage time and formic acid (FA) on fermentation characteristics, epiphytic microflora, carbohydrate components and in vitro digestibility of rice straw silage. Methods: Fresh rice straw was ensiled with four levels of FA (0%, 0.2%, 0.4%, and 0.6% of fresh weight) for 3, 6, 9, 15, 30, and 60 d. At each time point, the silos were opened and sampled for chemical and microbial analyses. Meanwhile, the fresh and 60-d ensiled rice straw were further subjected to in vitro analyses. Results: The results showed that 0.2% and 0.6% FA both produced well-preserved silages with low pH value and undetected butyric acid, whereas it was converse for 0.4% FA. The populations of enterobacteria, yeasts, moulds and aerobic bacteria were suppressed by 0.2% and 0.6% FA, resulting in lower dry matter loss, ammonia nitrogen and ethanol content (p<0.05). The increase of FA linearly (p<0.001) decreased neutral detergent fibre and hemicellulose, linearly (p<0.001) increased residual water soluble carbohydrate, glucose, fructose and xylose. The in vitro gas production of rice straw was decreased by ensilage but the initial gas production rate was increased, and further improved by FA application (p<0.05). No obvious difference of FA application on in vitro digestibility of dry matter, neutral detergent fibre, and acid detergent fibre was observed (p>0.05). Conclusion: The 0.2% FA application level promoted lactic acid fermentation while 0.6% FA restricted all microbial fermentation of rice straw silages. Rice straw ensiled with 0.2% FA or 0.6% FA improved its nutrient preservation without affecting digestion, with the 0.6% FA level best. Objective: The study aimed to evaluate the effect of storage time and formic acid (FA) on fermentation characteristics, epiphytic microflora, carbohydrate components and <i>in vitro</i> digestibility of rice straw silage.Methods: Fresh rice straw was ensiled with four levels of FA (0%, 0.2%, 0.4%, and 0.6% of fresh weight) for 3, 6, 9, 15, 30, and 60 d. At each time point, the silos were opened and sampled for chemical and microbial analyses. Meanwhile, the fresh and 60-d ensiled rice straw were further subjected to <i>in vitro</i> analyses.Results: The results showed that 0.2% and 0.6% FA both produced well-preserved silages with low pH value and undetected butyric acid, whereas it was converse for 0.4% FA. The populations of enterobacteria, yeasts, moulds and aerobic bacteria were suppressed by 0.2% and 0.6% FA, resulting in lower dry matter loss, ammonia nitrogen and ethanol content (p<0.05). The increase of FA linearly (p<0.001) decreased neutral detergent fibre and hemicellulose, linearly (p<0.001) increased residual water soluble carbohydrate, glucose, fructose and xylose. The <i>in vitro</i> gas production of rice straw was decreased by ensilage but the initial gas production rate was increased, and further improved by FA application (p<0.05). No obvious difference of FA application on <i>in vitro</i> digestibility of dry matter, neutral detergent fibre, and acid detergent fibre was observed (p>0.05).Conclusion: The 0.2% FA application level promoted lactic acid fermentation while 0.6% FA restricted all microbial fermentation of rice straw silages. Rice straw ensiled with 0.2% FA or 0.6% FA improved its nutrient preservation without affecting digestion, with the 0.6% FA level best.