Identification, optimization of culture conditions and activity analysis of fermentation products of Xylaria escharoidea
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摘要:目的
炭角菌属具有珍贵的药用价值和较高的经济价值。本研究对采自白蚁废弃巢穴上的炭角菌菌株进行鉴定，对其液体发酵和固体培养条件进行优化，并对液体发酵产物抗菌、抗氧化活性进行测定，以期为痂状炭角菌的开发利用提供参考。
方法通过形态观察及ITS序列测序对炭角菌进行鉴定；探讨液体发酵中添加不同碳源、氮源和金属离子对痂状炭角菌菌丝体生长的影响；分析固体培养基中添加氨基酸对无性子座生长的影响；利用平板抑菌法和DPPH法对液体发酵产物抗菌和抗氧化活性进行测定。
结果采集菌株经形态鉴定和分子鉴定，为痂状炭角菌。液体发酵最佳碳源为可溶性淀粉，最佳氮源为蚕蛹粉，最佳无机盐为MgSO4。正交试验筛选的最佳液体培养基组成为可溶性淀粉4%(w)+蚕蛹粉0.6%(w)+MgSO40.06%(w)。固体培养基中添加缬氨酸、异亮氨酸和苏氨酸均能显著促进无性子座的生长。液体发酵产物抗菌效果显著优于山梨酸钾，对DPPH的抗氧化活性为(75.19±2.08)%，显著优于维生素E。
结论优化的液体发酵和固体培养条件可以显著提高痂状炭角菌菌丝体产量，促进无性子座生长。痂状炭角菌发酵产物具有较好的抗菌和抗氧化作用，具有较高的开发利用价值。
Abstract:ObjectiveXylaria has valuable medicinal value and high economic value. Xylaria species collected from abandoned nest of termites were identified, and its liquid fermentation and solid culture conditions were optimized. The antibacterial and antioxidant activities of liquid fermentation product were determined. The study aimed at providing reference for exploitation and application of Xylaria escharoidea.
MethodThe morphology observation and ITS sequencing were used to identify Xylaria species. The effects of adding different carbon sources, nitrogen sources and metal ions in liquid fermentation on mycelial growth were investigated. The effects of adding amino acids in solid medium on stromata growth were also analyzed. Furthermore, the antibacterial and antioxidant activities of liquid fermentation product were determined through plate bacteriostatic method and DPPH method.
ResultThe collected strains were identified as X. escharoidea based on morphology and molecular data. The optimum carbon source in liquid fermentation was soluble starch, the optimum nitrogen source was silkworm pupa powder, and the optimum inorganic salt was MgSO4. The optimum liquid medium component screened by orthogonal test was soluble starch 4% (w), silkworm pupa powder 0.6% (w) and MgSO4 0.06% (w). All valine, isoleucine and threonine could significantly promote the growth of stromata in solid medium. The antibacterial effect of liquid fermentation product was significantly better than that of potassium sorbate, and the antioxidant activity to DPPH was (75.19±2.08)%, significantly superior than that of vitamin E.
ConclusionThe optimized liquid fermentation and solid culture conditions can increase mycelial yield and promote stromata growth. The liquid fermentation product of X. escharoidea has excellent antibacterial and antioxidant activities, and possesses high exploitation and utilization value.
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图 1 采集的炭角菌子座外观形态及子囊横切面
Figure 1. Appearance and ascus cross-section of collected Xylaria stromata
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图 2 添加不同碳源、氮源、金属离子的菌丝体干质量
各小图中柱子上的不同小写字母表示处理间差异显著（P<0.05，Duncan’s法)
Figure 2. Dry weight of mycelia with different carbon and nitrogen sources, and metal ions
Different lowercase letters on the columns in each figure indicate significant differences among treatments (P<0.05, Duncan’s method)
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图 3 添加不同质量分数可溶性淀粉、蚕蛹粉和MgSO4的菌丝体干质量变化
各小图中柱子上的不同小写字母表示处理间差异显著（P<0.05，Duncan’s法)
Figure 3. Dry weight of mycelia with different mass fractions of soluble starch, silkworm pupa powder and MgSO4
Different lowercase letters on the columns in each figure indicate significant differences among treatments (P<0.05, Duncan’s method)
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图 5 平板抑菌法测定痂状炭角菌液体发酵产物抗菌活性
A: 金黄色葡萄球菌, B: 大肠埃希菌; 1: 液体发酵液, 2: 山梨酸钾, 3: 水
Figure 5. Antibacterial activity of liquid fermentation product of Xylaria escharoidea by plate bacteriostatic method
A: Staphylococcus aureus, B: Escherichia coli; 1: Liquid fermentation product, 2: Potassium sorbate, 3: Water
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图 6 痂状炭角菌液体发酵产物的抗氧化活性
柱子上的不同小写字母表示处理间差异显著（P<0.05，Duncan’s法)
Figure 6. Antioxidant activity of liquid fermentation product of Xylaria escharoidea
Different lowercase letters on the columns indicate significant differences among treatments (P<0.05, Duncan’s method)
表 1 痂状炭角菌液体培养优化正交试验结果
Table 1 Results of Xylaria escharoidea liquid culture optimization using orthogonal experiment
序号
Numberw/% 菌丝干质量/g
Dry weight
of mycelia可溶性
淀粉
Soluble
starch蚕蛹粉
Silkworm pupa
powderMgSO4 1 3 0.2 0.04 2.946 2 3 0.4 0.06 2.495 3 3 0.6 0.08 2.879 4 4 0.2 0.06 2.626 5 4 0.4 0.08 3.318 6 4 0.6 0.04 3.247 7 5 0.2 0.08 2.040 8 5 0.4 0.04 1.659 9 5 0.6 0.06 3.335 K1 8.320 7.611 7.852 K2 9.191 7.473 8.456 K3 7.034 9.461 8.237 k1 2.773 2.537 2.617 k2 3.064 2.491 2.819 k3 2.345 3.154 2.746 R 0.719 0.663 0.201 
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表 2 添加不同氨基酸对无性子座生长的影响1)
Table 2 Effects of different amino acid additions on the growth of asexual stromata
氨基酸
Amino acid数量
Number粗细
Thickness长度
Length缬氨酸 Valine 8.67±1.53a 粗 Thick 较长 Longer 精氨酸 Arginine 5.33±0.58de 粗 Thick 较短 Shorter 苯丙氨酸 Phenylalanine 8.33±2.08ab 细 Thin 短 Short 组氨酸 Histidine 7.67±1.53abc 细 Thin 短 Short 甲硫氨酸 Methionine — — — 甘氨酸 Glycine 6.33±2.08cd 细 Thin 长 Long 丝氨酸 Serine 6.67±0.58bcd 细 Thin 短 Short 苏氨酸 Threonine 8.33±0.58ab 细 Thin 长 Long 亮氨酸 Leucine 6.00±1.00cde 粗 Thick 较短 Shorter 异亮氨酸 Isoleucine 8.33±2.52ab 粗 Thick 长 Long CK 4.33±0.58e 细 Thin 较短 Shorter 1)“—”表示未见无性子座
1)“—”indicates no asexual stromata
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表 3 痂状炭角菌液体发酵产物对细菌的抗菌活性1)
Table 3 Antibacterial activity of liquid fermentation product of Xylaria escharoidea against bacteria
样品
Sample大肠埃希菌
Escherichia coli金黄色葡萄球菌
Staphylococcus aureus液体发酵液
Liquid fermentation product++ +++ 山梨酸钾
Potassium sorbate+ + 1)“+++”：抑菌圈直径≥20 mm；“++”：10 mm<抑菌圈直径<20 mm；“+”：抑菌圈直径≤10 mm
1)“+++”: Diameter of bacterial inhibition zone≥20 mm; “++”: 10 mm<diameter of bacterial inhibition zone<20 mm; “+”: Diameter of bacterial inhibition zone≤10 mm
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