Silage Carbon Sources Preferred by Epiphytic Lactic Acid Bacteria
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摘要:
目的 探究饲草源乳酸菌对碳源的选择性,为不同草种筛选适宜的青贮菌种。 方法 选取饲草中分离获得的16株代表性乳酸菌作为研究对象,在MRS液体培养基中以葡萄糖、蔗糖、果糖、木糖、棉籽糖以及5种糖混合为碳源,进行不同菌株发酵培养。每株乳酸菌设3个重复,每个糖处理设空白对照(不接菌)。采用高效液相色谱仪测定各处理发酵24 h后液体培养基中剩余碳源含量和乳酸产量。 结果 (1)蔗糖是大部分菌株发酵首选碳源,在棉籽糖、葡萄糖、果糖和木糖的选择上不同菌株差异较大,发酵乳杆菌(Lactobacillus fermentum, LbF-WM)首选棉籽糖和木糖,假肠膜明串珠菌(Leuconostioc pseudomesenteroides, LeP-IR)首选木糖。(2)罗伊氏乳杆菌(Lactobacillus reuteri, LbR-EG)和柠檬明串珠菌(Leuconostoc citreum, LeC-IR)对蔗糖,LbF-WM和LbR-EG对果糖, LbF-WM 、LbR-EG和LeP-IR对棉籽糖的利用率均达100%,屎肠球菌(Enterococcus faecium, EF-KG)、短乳杆菌(Lactobacillus brevis, LbB-KG)、戊糖乳杆菌(Lactobacillus pentosus, LbPe-EG)、乳酸乳球菌(Lactococcus lactis, LcL-SC)对葡萄糖的利用率高于其他碳源。(3)灰黄色肠球菌(Enterococcus durans, EG-IR)利用葡萄糖和混合糖,LbPe-EG利用果糖,肠膜明串珠菌(Leuconostoc mesenteroides, LeM-IR)利用棉籽糖和混合糖,LbPe-EG利用蔗糖,LbF-WM利用木糖,格氏乳球菌(Lactococcus garvieae, LcG-IR)利用混合糖发酵乳酸产量最高。(4)耐久肠球菌(Enterococcus durans, ED-IR)利用葡萄糖,植物乳杆菌(Lactobacillus plantarum, LbPl-IR)、LbPe-EG利用果糖, LbPl-IR和LeM-IR利用棉籽糖, LbPe-EG、LbPl-IR和ED-IR利用蔗糖, LeP- IR利用木糖,LcG-IR和EG-IR利用混合糖产乳酸效率较高。LbPl-IR在5种单糖中产酸效率均达80%以上。 结论 不同菌株对同一碳源的利用率、产酸量和产酸效率均不同;同一株菌株对不同碳源的利用率、产酸量和产酸效率也不相同。其中,蔗糖为大部分菌株的首选碳源,蔗糖和葡萄糖的利用率和产乳酸量较高,葡萄糖的产酸效率较高;青贮调制时,可针对饲草中分布乳酸菌种类选择最适碳源。 Abstract:Objective Preferential carbon sources of epiphytic lactic acid bacteria (LAB) were studied in laboratory to aid turning native grasses into silage. Method Sixteen representative LAB were isolated from forage and cultured on MRS broth media using glucose, sucrose, fructose, xylose, raffinose, or a mixture of the 5 sugars as carbon source. Three replicates for each LAB strain and a blank control without LAB inoculation for each sugar treatment were included. After incubation for 24 h, contents of residual sugars and generated lactic acid in the medium were determined by high-performance liquid chromatography. Results (1) Sucrose appeared to be the preferential carbon source by most LAB. Among the other sugars, raffinose and xylose were preferred by Lactobacillus fermentum (LbF-WM), and xylose by Leuconostoc pseudomesenteroides (LeP-IR). (2) An 100% utilization on sucrose by Lb. reuteri (LbR-EG) and Leuconostoc citreum (LeC-IR), on fructose by LbF-WM and LbR-EG, as well as on raffinose by LbF-WM, LbR-EG, and LeP-IR was observed. Enterococcus faecium (EF-KG), Lb. brevis (LbB-KG), Lactobacillus pentosus (LbPe-EG), and Lactococcus lactis (LcL-SC) could more efficiently utilize glucose than the other sugars. (3) The greatest amounts of lactic acid were produced from the fermentation of glucose or the mixed sugars by E. durans (EG-IR), from that of fructose by LbPe-EG, from that of raffinose or the sugar mixture by Leuconostoc mesenteroides (LeM-IR), from that of sucrose by LbPe-EG, from that of xylose by LbF-WM, and from that of the mixed sugars by Lactococcus garvieae (LcG-IR). (4) The lactic acid production was more efficient by E. durans (ED-IR) on glucose, by Lb. plantarum (LbPl-IR), or LbPe-EG on fructose, by LbPl-IR or LeM-IR on raffinose, by LbPe-EG, LbPl-IR, or ED-IR on sucrose, Le. pseudomesenteroides (LeP-IR) on xylose, and LcG-IR or EG-IR on mixed sugars. A lactic acid production efficiency greater than 80% was reached by LbPl-IR grown on the medium with the mixed sugars. Conclusion The sugar utilization and lactic acid production of the LAB fermentations on native grasses varied significantly. Sucrose appeared to be the preferential carbon source by most LAB, sucrose and glucose could obtain the higher utilization rate and lactic acid amount, and glucose could obtain higher lactic acid efficiency. For efficient and effective application of LAB to obtain extended shelf-life silage, it was imperative that careful selection of microbe strains and carbon source be exercised. -
Key words:
- forage grass /
- epiphytic lactic acid bacteria /
- carbon source /
- glucose /
- sucrose /
- selectivity
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图 1 16株乳酸菌培养24 h后在5种糖混合培养基中各种糖的剩余含量
1)图中柱状数据表示混合糖培养基中剩余各糖的百分含量。2)CK,无接菌的混合糖培养基。
Figure 1. Contents of residual sugars in mixed sugar media of 16 different LAB cultured for 24 h
1) Datas on the column are contents of residual individual sugars in medium of mixed sugars; 2) CK, without inoculation on medium of mixed sugars.
表 1 16株乳酸菌的名称及来源
Table 1. Names and sources of 16 LAB strains
序号
No.编号
Code种名
Species来源
Source序号
No.编号
Code种名
Species来源
Source1 EF-KG 屎肠球菌 E. faecium 热研四号王草 9 LcG-IR 格氏乳球菌 Lc. garvieae 意大利黑麦草 2 ED-IR 耐久肠球菌 E. durans 意大利黑麦草 10 LcL-SC 乳酸乳球菌 Lc. lactis 甜玉米 3 EG-IR 灰黄色肠球菌 E. gilvus 意大利黑麦草 11 LeC-IR 柠檬明串珠菌 Le. citreum 意大利黑麦草 4 LbB-KG 短乳杆菌 Lb. brevis 热研四号王草 12 LeM-IR 肠膜明串珠菌 Le. mesenteroides 意大利黑麦草 5 LbF-WM 发酵乳杆菌 Lb. fermentum 糯玉米 13 LeP-IR 假肠膜明串珠菌 Le. pseudomesenteroides 意大利黑麦草 6 LbPe-EG 戊糖乳杆菌 Lb. pentosus 摩特矮象草 14 WCo-KG 融合乳杆菌 W. confusa 热研四号王草 7 LbPl-IR 植物乳杆菌 Lb. plantarum 意大利黑麦草 15 WCi-MT 魏斯特菌 W. cibaria MT-1杂交象草 8 LbR-EG 罗伊氏乳杆菌 Lb. reuteri 摩特矮象草 16 WP-IR 类肠膜魏斯氏菌 W. paramesenteroides 意大利黑麦草 表 2 牧草原料中糖分组成
Table 2. Sugars in native grass material
(单位:%) 项目
Item葡萄糖
Glucose蔗糖
Sucrose果糖
Fructose棉籽糖
Raffinose木糖
Xylose热研四号王草
Pennisetum purpureum × P. americanum cv. Reyan No.40.07±0.01c 1.91±0.37a 1.34±0.23b 1.86±0.06a 0.00±0.00c MT-1象草
P. purpureum cv. MT-10.10±0.01c 1.82±0.08a 1.41±0.25b 1.28±0.27b 0.03±0.00c 摩特矮象草
P. purputeum cv. Mott0.12±0.01c 1.75±0.29ab 2.11±0.13a 1.58±0.31b 0.09±0.09c 甜玉米
Zea mays L. saccharata Sturt.1.84±0.26b 6.00±0.96a 1.12±0.11b 0.00±0.00c 0.00±0.00c 糯玉米
Zea mays L. Sinensisa Kulesh1.81±0.36b 5.06±0.37a 0.93±0.02c 0.00±0.00d 0.00±0.00d 意大利黑麦草
Lolium multiflorum0.70±0.39ab 0.94±0.36a 0.34±0.07b 0.08±0.08c 0.10±0.04c 同行数据后不同小写字母表示差异显著(P<0.05),相同或无字母表示差异不显著(P>0.05);下表同。
Data with different lowercase letters on same row represent significant difference(P<0.05); those with same or no letter, no significant difference(P>0.05). Same for below.表 3 16株乳酸菌对6种糖源培养液发酵后的碳源利用率
Table 3. Sugar utilization by 16 different LAB in 6 sugar treatments
(单位:%) 菌株编号
Strain code葡萄糖MRS培养基
MRS-P果糖MRS培养基
MRS-G棉籽糖MRS培养基
MRS-MZ蔗糖MRS培养基
MRS-Z木糖MRS培养基
MRS-M混合糖MRS培养基
MRS- MIXEF-KG 94.68±1.01a 29.25±3.43e 0.73±0.53f 89.99±1.72b 59.95±1.48c 50.27±0.54d ED-IR 44.76±2.10b 60.48±1.52a 63.67±0.91a 69.87±1.16a 5.22±0.18c 39.83±2.34b EG-IR 71.44±3.35ab 58.97±3.27bc 49.20±1.22cd 81.23±1.54a 0.00±0.00e 39.56±0.89d LbB-KG 72.13±1.05a 18.10±0.53c 7.80±4.49c 50.41±0.10b 46.74±0.10b 38.82±2.37b LbF-WM 87.50±1.43b 100.00±0.00a 100.00±0.00a 89.79±3.33b 79.15±0.84b 33.33±4.13c LbPe-EG 82.97±0.73a 60.31±0.62b 37.42±3.02c 76.75±1.05ab 1.10±0.19d 38.56±2.88c LbPl-IR 69.56±1.06a 58.45±1.72a 60.34±1.81a 72.80±2.56a 0c 39.57±2.86b LbR-EG 82.07±2.63b 100.00±0.00a 100.00±0.00a 100.00±0.00a 0d 31.01±5.99c LcG-IR 78.09±0.84ab 60.59±1.02b 74.69±0.71ab 81.48±1.64a 1.92±0.42d 38.11±3.78c LcL-SC 93.58±1.01a 0e 18.52±0.52d 90.29±0.82a 37.25±0.12c 52.81±5.62b LeC-IR 83.17±3.13b 79.15±0.61b 10.49±0.03d 100.00±0.00a 15.54±0.16d 44.51±10.98c LeM-IR 73.89±1.05a 55.36±4.22b 70.94±0.01a 74.02±0.65a 0d 24.70±4.80c LeP-IR 80.19±1.64b 90.59±0.10a 100.00±0.00a 52.23±6.10c 24.86±20.14c 33.81±2.39c WCo-KG 76.66±1.64b 9.93±5.08e 17.11±1.42d 96.40±1.00a 48.27±0.10c 53.27±6.55c WCi-MT 67.93±3.46a 56.89±0.82a 68.83±1.55a 71.00±0.86a 8.80±0.18b 27.70±7.52b WP-IR 79.14±1.24a 73.84±0.01a 79.16±2.21a 79.09±1.13a 0c 41.12±7.76b 表 4 16株乳酸菌在不同碳源培养基发酵中的乳酸产量
Table 4. Lactic acid production by 16 different LAB on different carbon media
(单位:%) 菌株编号
Strain code葡萄糖MRS培养基
MRS-P果糖MRS培养基
MRS-G棉籽糖MRS培养基
MRS-MZ蔗糖MRS培养基
MRS-Z木糖MRS培养基
MRS-M混合糖MRS培养基
MRS- MIXEF-KG 0.69±0.01a 0.32±0.02b 0. 01±0.00c 0.62±0.01a 0.58±0.03a 0.55±0.02a ED-IR 0.89±0.02a 0.85±0.05a 0.21±0.03b 1.14±0.01a 0.02±0.00c 0.86±0.06a EG-IR 1.25±0.05a 0.73±0.05b 0.66±0.03b 0.87±0.00b 0.01±0.00c 1.00±0.03a LbB-KG 0.71±0.00a 0.29±0.03c 0.23±0.00c 0.56±0.06b 0.63±0.04b 0.61±0.04b LbF-WM 0.67±0.01b 0.36±0.04c 0.71±0.01b 0.73±0.00b 0.94±0.00a 0.67±0.05b LbPe-EG 1.08±0.04b 1.10±0.06a 0.43±0.04c 1.31±0.00a 0.01±0.00c 0.98±0.02b LbPl-IR 1.14±0.01a 1.07±0.05a 1.01±0.02a 1.23±0.03a 0.61±0.02b 0.77±0.00b LbR-EG 0.73±0.02ab 0.69±0.04ab 0.86±0.02a 0.55±0.03b 0.63±0.04ab 0.67±0.00 ab LcG-IR 1.18±0.04a 0.88±0.00b 1.02±0.04a 1.15±0.01a 0.31±0.00c 1.08±0.05a LcL-SC 0.69±0.02a 0.45±0.06b 0.15±0.00c 0.52±0.05ab 0.50±0.02ab 0.58±0.00a LeC-IR 0.71±0.02a 0.67±0.02a 0.14±0.05c 0.42±0.02b 0.15±0.00c 0.45±0.01b LeM-IR 1.13±0.04a 1.01±0.05a 1.16±0.00a 1.13±0.00a 0.71±0.01b 1.06±0.02a LeP-IR 0.79±0.00a 0.54±0.02c 0.76±0.02a 0.65±0.01bc 0.78±0.00a 0.61±0.01bc WCo-KG 0.60±0.02a 0.43±0.00b 0.13±0.00c 0.62±0.06a 0.55±0.02a 0.50±0.02ab WCi-MT 1.11±0.05a 0.54±0.00b 0.36±0.02c 0.67±0.05b 0.10±0.01d 0.69±0.02b WP-IR 0.58±0.00a 0.52±0.00a 0.08±0.01c 0.32±0.00b 0.64±0.02a 0.31±0.00b 表 5 16株乳酸菌的产酸效率
Table 5. Acid production efficiency of 16 different LAB
(单位:%) 菌株编号
Strain code葡萄糖MRS培养基
MRS-P果糖MRS培养基
MRS-G棉籽糖MRS培养基
MRS-MZ蔗糖MRS培养基
MRS-Z木糖MRS培养基
MRS-M混合糖MRS培养基
MRS- MIXEF-KG 36.44±0.01c 54.69±0.06a 34.26±1.17d 34.45±0.01d 48.38±0.06b 54.71±0.59a ED-IR 99.41±0.22a 70.27±0.02d 16.49±0.00f 81.58±0.07b 19.16±0.66e 71.47±0.00c EG-IR 87.49±0.06a 61.90±0.02d 67.07±0.02c 53.55±0.03e 50.00±0.00f 82.72±0.00b LbB-KG 49.21±0.04b 80.12±0.15ab 81.69±0.20a 55.55±0.11b 67.40±0.15ab 49.85±0.00b LbF-WM 38.29±0.01d 18.00±0.00f 35.50±0.00e 40.65±0.01c 59.38±0.03a 49.12±0.00b LbPe-EG 65.08±0.02d 91.20±0.03a 57.46±0.03e 85.34±0.06b 23.10±3.98f 79.76±0.00c LbPl-IR 81.94±0.06d 91.53±0.03a 83.69±0.01c 84.70±0.45b 81.33±0.00e 63.71±0.00f LbR-EG 44.48±0.02c 34.50±0.00e 43.00±0.00d 27.50±0.00f 45.99±0.00b 48.56±0.00a LcG-IR 75.55±0.04b 72.61±0.02c 68.28±0.01e 70.57±0.03d 41.77±0.00f 87.25±0.00a LcL-SC 36.87±0.01d 66.18±0.00b 40.49±0.05e 28.80±0.01f 67.11±0.22a 61.45±0.00c LeC-IR 42.69±0.02c 42.32±0.01c 66.75±0.14a 21.00±0.00e 48.28±0.50b 40.55±0.00d LeM-IR 76.47±0.05d 91.22±0.03a 81.76±0.01b 76.33±0.05e 79.78±0.00c 70.38±0.00f LeP-IR 49.26±0.03c 29.80±0.01f 38.00±0.00e 62.23±0.12b 86.66±0.00a 46.08±0.00d WCo-KG 39.13±0.02d 43.10±0.00c 37.98±0.05e 32.16±0.00f 56.97±0.12a 53.50±0.00b WCi-MT 81.70±0.07a 47.46±0.02c 26.15±0.00d 47.18±0.04c 56.85±1.12b 47.72±0.00c WP-IR 36.65±0.02a 35.21±0.01a 5.05±0.00b 20.23±0.01ab 15.80±27.37ab 26.33±0.00ab -
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