Screening of superior lactic acid bacteria from natural Lolium multiflorum silage and their effects on silage quality

doi:10.11686/cyxb2021064

Abstract

Abstract:

The aim of this study was to isolate superior lactic acid bacteria （LAB） strains suitable for Italian ryegrass silage fermentation. LAB were isolated from naturally fermenting Italian ryegrass and strains with rapid growth rate and high acid production efficiency were identified and selected. Subsequently the physiological and biochemical characteristics of these strains were analyzed and we then investigated the influence of these strains on the quality of Italian ryegrass silage. Using the traditional plate culture method， 180 strains of LAB were isolated from naturally fermenting Italian ryegrass silage in this study. Among them， five strains of LAB grew rapidly and had strong acid production ability. These five strains grew well in MRS liquid medium with a NaCl concentration of 3.0%， temperature of 15-35 ℃ and pH of 3.5-7.0. According to 16S rRNA gene sequence alignment and identification， four of the strains （designated PR_LAB_9， PR_LAB_34， PR_LAB_67 and PR_LAB_86） were Lactobacillus plantarum， and a fifth （PR_LAB_76） was Pediococcus pentosaceus. The growth curve and acid production ability of these five strains were determined. All these five LAB strains were able to reproduce and produce acid in 12 hours， and strain PR_LAB_76 had faster growth rate and better acid production efficiency in 24 h than the other four LAB strains. Fermentation quality， nutrient composition and microbial counts of Italian ryegrass ensiled without or with the five LAB strains were also investigated in the present study. All five LAB strains improved the quality of Italian ryegrass silage， including significant reduction in the pH， ammonia nitrogen content and dry matter loss， and increase in the water soluble carbohydrate concentration of ensiled forage. The dry matter content， crude protein content， lactic acid content and LAB counts in the group treated with PR_LAB_76 were significantly higher， and the pH value and yeast counts in the PR_LAB_76 treated silage were significantly lower than those of the four groups treated with other LAB strains. In conclusion， P. pentosaceus PR_LAB_76 was identified in this study as a superior inoculum for industry use in the production of Italian ryegrass silage.

Key words: Lolium multiflorum, silage, Lactobacillus plantarum, Pediococcus pentosaceus, acid production characteristics, silage quality

Jie YUAN, Ran-ran MA, Wen-jie ZHANG, Neng-xiang XU, Ran-ran ZHAO, Hong-ru GU, Cheng-long DING. Screening of superior lactic acid bacteria from natural Lolium multiflorum silage and their effects on silage quality[J]. Acta Prataculturae Sinica, 2021, 30(11): 132-143.

Figures/Tables 9

References 39

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项目 Item	值 Value
干物质 Dry matter （g·kg^-1 FM）	302.30±2.30
粗蛋白 Crude protein （g·kg^-1 DM）	81.75±0.06
可溶性碳水化合物 Water soluble carbohydrate （g·kg^-1 DM）	140.72±5.60
淀粉 Starch （g·kg^-1 DM）	91.23±5.60
中性洗涤纤维 Neutral detergent fiber （g·kg^-1 DM）	566.27±0.55
酸性洗涤纤维 Acid detergent fiber （g·kg^-1 DM）	313.04±8.17
酸性洗涤木质素 Acid detergent lignin （g·kg^-1 DM）	24.77±2.50
纤维素 Cellulose （g·kg^-1 DM）	287.02±5.67
半纤维素 Hemicellulose （g·kg^-1 DM）	253.24±8.72
乳酸菌 Lactic acid bacteria （Log₁₀ cfu·g^-1 FM）	4.38±0.10
好氧细菌 Aerobic bacteria （Log₁₀ cfu·g^-1 FM）	6.18±0.03
酵母 Yeast （Log₁₀ cfu·g^-1 FM）	3.37±0.10
霉菌 Mold （Log₁₀cfu·g^-1 FM）	3.65±0.05

项目 Item	值 Value
干物质 Dry matter （g·kg^-1 FM）	302.30±2.30
粗蛋白 Crude protein （g·kg^-1 DM）	81.75±0.06
可溶性碳水化合物 Water soluble carbohydrate （g·kg^-1 DM）	140.72±5.60
淀粉 Starch （g·kg^-1 DM）	91.23±5.60
中性洗涤纤维 Neutral detergent fiber （g·kg^-1 DM）	566.27±0.55
酸性洗涤纤维 Acid detergent fiber （g·kg^-1 DM）	313.04±8.17
酸性洗涤木质素 Acid detergent lignin （g·kg^-1 DM）	24.77±2.50
纤维素 Cellulose （g·kg^-1 DM）	287.02±5.67
半纤维素 Hemicellulose （g·kg^-1 DM）	253.24±8.72
乳酸菌 Lactic acid bacteria （Log₁₀ cfu·g^-1 FM）	4.38±0.10
好氧细菌 Aerobic bacteria （Log₁₀ cfu·g^-1 FM）	6.18±0.03
酵母 Yeast （Log₁₀ cfu·g^-1 FM）	3.37±0.10
霉菌 Mold （Log₁₀cfu·g^-1 FM）	3.65±0.05

特性 Characteristics	菌株 Strain
特性 Characteristics	PR_LAB_9	PR_LAB_34	PR_LAB_67	PR_LAB_76	PR_LAB_86
菌株形态Shape	杆菌Bacillus	杆菌Bacillus	杆菌Bacillus	球菌Coccus	杆菌Bacillus
革兰氏染色Gram strain	阳性Positive	阳性Positive	阳性Positive	阳性Positive	阳性Positive
过氧化氢酶反应Catalase reaction	阴性Negative	阴性Negative	阴性Negative	阴性Negative	阴性Negative
葡萄糖产气Gas from glucose	阴性Negative	阴性Negative	阴性Negative	阴性Negative	阴性Negative
发酵类型Fermentation type	同型Homo	同型Homo	同型Homo	同型Homo	同型Homo
耐酸性 Aciduric ability
pH 2.5	-	-	-	-	-
pH 3.0	＋＋	＋＋	＋＋	＋＋	＋＋
pH 3.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 4.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 4.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 5.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 5.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 6.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 6.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
PH 7.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
耐盐性Salt tolerance ability
0.0% NaCl	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
3.0% NaCl	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
6.5% NaCl	＋＋	＋＋	＋＋	＋＋	＋＋
10.0% NaCl	＋	＋	＋	＋	＋
20.0% NaCl	-	-	-	-	-
耐温性Thermotolerant ability
5 ℃	＋	＋	＋	＋	＋
15 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
25 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
35 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
45 ℃	＋	＋	＋	＋	＋

特性 Characteristics	菌株 Strain
特性 Characteristics	PR_LAB_9	PR_LAB_34	PR_LAB_67	PR_LAB_76	PR_LAB_86
菌株形态Shape	杆菌Bacillus	杆菌Bacillus	杆菌Bacillus	球菌Coccus	杆菌Bacillus
革兰氏染色Gram strain	阳性Positive	阳性Positive	阳性Positive	阳性Positive	阳性Positive
过氧化氢酶反应Catalase reaction	阴性Negative	阴性Negative	阴性Negative	阴性Negative	阴性Negative
葡萄糖产气Gas from glucose	阴性Negative	阴性Negative	阴性Negative	阴性Negative	阴性Negative
发酵类型Fermentation type	同型Homo	同型Homo	同型Homo	同型Homo	同型Homo
耐酸性 Aciduric ability
pH 2.5	-	-	-	-	-
pH 3.0	＋＋	＋＋	＋＋	＋＋	＋＋
pH 3.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 4.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 4.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 5.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 5.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 6.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
pH 6.5	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
PH 7.0	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
耐盐性Salt tolerance ability
0.0% NaCl	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
3.0% NaCl	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
6.5% NaCl	＋＋	＋＋	＋＋	＋＋	＋＋
10.0% NaCl	＋	＋	＋	＋	＋
20.0% NaCl	-	-	-	-	-
耐温性Thermotolerant ability
5 ℃	＋	＋	＋	＋	＋
15 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
25 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
35 ℃	＋＋＋	＋＋＋	＋＋＋	＋＋＋	＋＋＋
45 ℃	＋	＋	＋	＋	＋

菌株 Strain	相似菌株 Similar strain	同源性 Homology
PR_LAB_9	Lactobacillus plantarum MT613628.1	99.93%
PR_LAB_34	Lactobacillus plantarum MT645503.1	100.00%
PR_LAB_67	Lactobacillus plantarum MT645503.1	100.00%
PR_LAB_76	Pediococcus pentosaceus LC119127.1	99.86%
PR_LAB_86	Lactobacillus plantarum AB830324.1	100.00%