植物乳杆菌R23耐受二氧化硫胁迫的应激机制

任香芸; 李维新; 林晓姿; 梁璋成; 何志刚

doi:10.19303/j.issn.1008-0384.2022.06.016

植物乳杆菌R23耐受二氧化硫胁迫的应激机制

doi: 10.19303/j.issn.1008-0384.2022.06.016

任香芸^{1, 2,},
李维新^{1, 2},
林晓姿^{1, 2},
梁璋成^{1, 2},
何志刚^{1, 2, ,}

1.
福建省农业科学院农业工程技术研究所，福建　福州　350003
2.
福建省农产品（食品）加工重点实验室，福建　福州　350013

基金项目: 福建省自然科学基金项目（2019J01114）；福建省农业科学院对外合作项目（DWHZ2021-11）；泉州市科技计划项目（2021N043）

详细信息

作者简介:
任香芸（1980−），女，硕士，副研究员，研究方向：食品微生物、食品发酵与酿造（E-mail：yuin09@163.com）

通讯作者:
何志刚（1964−），男，研究员，研究方向：食品科学（E-mail：njgzx@163.com）

中图分类号: Q 939.9
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- 被引次数: 0
出版历程
- 收稿日期: 2021-12-03
- 修回日期: 2022-05-23
- 网络出版日期: 2022-06-20
- 刊出日期: 2022-06-28

Response Mechanism of Lactobacillus plantarum R23 to Sulfur Dioxide Stress

REN Xiangyun^{1, 2
,},
LI Weixin^{1, 2},
LIN Xiaozi^{1, 2},
LIANG Zhangcheng^{1, 2},
HE Zhigang^{1, 2
, ,}

1.
Institute of Agricultural Engineering Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China
2.
Fujian Key Laboratory of Agricultural Products (Food) Processing, Fuzhou, Fujian　350013, China

摘要

摘要: 目的考察植物乳杆菌R23抗氧化酶与膜磷脂脂肪酸等在菌体应答二氧化硫胁迫中的作用机制。方法以植物乳杆菌R23为试验菌并对其进行梯度二氧化硫胁迫处理，借助扫描电子显微镜观察菌体超微形态，采用酶联免疫法与考马斯亮蓝法检测抗氧化酶活力及丙二醛含量，通过MIDI系统分析细胞膜磷脂脂肪酸结构。结果二氧化硫胁迫激发了植物乳杆菌R23胞内抗氧化酶（尤其是过氧化氢酶CAT）活力的显著提升，80 mg·L⁻¹二氧化硫胁迫下超氧化物歧化酶SOD、CAT和谷胱甘肽过氧化物酶GPX活力分别是对照处理的1.64、2.14、1.62倍，进而维持了丙二醛MDA较低的增长幅度和基本正常的菌体形态；然而过高二氧化硫（120 mg·L⁻¹）胁迫后，抗氧化酶活力趋于下降，膜脂质过氧化反应加剧，部分菌体细胞表面出现明显皱缩。细胞膜磷脂脂肪酸分析发现，二氧化硫胁迫促使植物乳杆菌R23中饱和、直链、长链和环丙烷脂肪酸总量发生不同程度提升，其中总直链脂肪酸高达52%，且与支链脂肪酸比例从7.15显著提升至9.72。结论植物乳杆菌R23通过提高抗氧化酶活力或增加饱和、直链、长链以及环丙烷脂肪酸比例，可以降低细胞膜对毒性物质的透过性或清除胞内过量的自由基，从而抵御二氧化硫对菌体细胞的伤害。
- 植物乳杆菌 /
- 二氧化硫胁迫 /
- 超微形态 /
- 抗氧化酶 /
- 磷脂脂肪酸
Abstract: Objective Roles of antioxidase and phospholipid fatty acid (PLFA) play in the response of Lactobacillus plantarum R23 to sulfur dioxide stress were studied. Methods L. plantarum R23 was exposed to a gradient of sulfur dioxide to observe the ultrastructural changes on the bacterium under scanning electron microscopy, determine the antioxidase activity and MDA content by ELISA and coomassie brilliant blue method, and analyze the PLFA composition using MIDI. Results The increasing sulfur dioxide stress induced in L. plantarum R23 a high antioxidase activity, especially CAT. Under 80 mg·L⁻¹ of sulfur dioxide exposure, 1.64-fold rise on SOD activity, 2.14-fold on CAT, and 1.62-fold on GPX were found in the bacteria that afforded a relatively low increasing rate on MDA and maintained a largely intact morphology. However, the imposition of 120 mg·L⁻¹ sulfur dioxide lowered the antioxidase activity and intensified the lipid peroxidation with appearance of wrinkles on the cellular surface. The PLFAs underwent varying degrees of increases on saturated, straight-chain, long-chain, and cyclopropane fatty acids under the stress. The straight-chain fatty acids accounted for 52% of all with a ratio to the branched-chain fatty acids significantly raised from 7.15 to 9.72. Conclusion When L. plantarum R23 encountered sulfur dioxide stress, by increasing the antioxidase activity and/or altering the PLFA composition (especially the saturated, straight-chain, long-chain, and cyclopropane fatty acids) it lowered the cell membrane permeability to deter the invasion of toxic substances or removed the excessive free radicals to prevent and mitigate possible damage.
- Lactobacillus plantarum /
- sulfur dioxide stress /
- ultrastructure /
- antioxidase /
- phospholipid fatty acid (PLFA)

HTML全文

图 1 梯度二氧化硫胁迫下植物乳杆菌R23胞内抗氧化酶活力

小写字母表示梯度二氧化硫质量浓度胁迫后菌体抗氧化酶活性差异显著（P＜0.05）。

Figure 1. Intracellular antioxidase activity of L. plantarum R23 under gradient of SO₂ stress

Data with different lowercase letters represent significant differences on antioxidase activity of L. plantarum R23 treated by gradient of SO₂ at P＜0.05

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图 2 梯度二氧化硫胁迫下植物乳杆菌R23细胞膜丙二醛含量

小写字母表示梯度二氧化硫质量浓度胁迫后菌体丙二醛含量间差异显著（P＜0.05）。

Figure 2. MDA content inside cell membrane of L. plantarum R23 under gradient of SO₂ stress

Data with different lowercase letters represent significant differences on MDA content after SO₂ treatments at P＜0.05.

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图 3 梯度二氧化硫胁迫下植物乳杆菌R23菌体细胞电镜扫描结果 (SEM, ×10 000)

植物乳杆菌R23分别用0（a）、40（b）、80（c）、120（d） mg·L⁻¹ 二氧化硫胁迫处理。

Figure 3. Effects of SO₂ treatments on morphology of L. plantarum R23 shown under SEM (×10 000)

L. plantarum R23 treated by 0 (a), 40 (b), 80 (c) or 120 mg·L⁻¹ (d) of SO₂.

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图 4 二氧化硫对植物乳杆菌R23细胞膜磷脂脂肪酸相对含量的影响

a、b、c、d分别表示二氧化硫胁迫导致的总饱和脂肪酸与总不饱和脂肪酸、总长链脂肪酸与总短链脂肪酸、总直链脂肪酸与总支链脂肪酸、总顺式脂肪酸与总反式脂肪酸的变化；“*”表示两者间的差异显著（P ＜ 0.05）。

Figure 4. Effect of SO₂ on relative contents of PLFA in L. plantarum R23

a, b, c, and d: changes caused by SO₂ stress on saturated (Sa-), unsaturated (Us-), long-chain (L-), short-chain (S-), straight-chain (Sc-), branched-chain (Bc-), cis (Cis-), and trans fatty acids (Trans-) PLFAs. “*” indicates significant difference at P＜0.05.

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表 1 蛋白质含量测定标准曲线

Table 1. Standard curve for protein determination

试剂 Reagent	管号 Tube No.
试剂 Reagent	1	2	3	4	5	6
蛋白质标准液 Standard solution of protein/mL	0	0.2	0.4	0.6	0.8	1.0
蒸馏水 Distilled water/mL	1.0	0.8	0.6	0.4	0.2	0
考马斯亮蓝G-250试剂 Coomassie brilliant blue G-250 reagent/mL	5	5	5	5	5	5

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表 2 不同二氧化硫胁迫下植物乳杆菌R23细胞膜磷脂脂肪酸相对含量

Table 2. Relative contents of PLFA in L. plantarum R23 under SO₂ stress

磷脂脂肪酸 PLFA	磷脂脂肪酸相对含量 Relative amount of PLFA/%
磷脂脂肪酸 PLFA	胁迫前 Before stress	胁迫后 After stress
十二碳异脂肪酸 12:0 iso	1.36±0.08	2.45±0.11
十四烷酸 14:0	7.34±0.21	3.36±0.31
十四碳异脂肪酸 14:0 iso	0	1.92±0.18
十五碳异脂肪酸 15:0 iso	0	0.98±0.04
十六碳异脂肪酸 16:1 iso	3.99±0.18	0
十六碳顺式单不饱和脂肪酸 16:1 cis	3.19±0.55	2.87±0.65
十六烷酸 16:0	27.77±1.29	30.10±1.20
十七碳前异脂肪酸 17:0 anteiso	1.55±0.10	0
Sum In Feature 8	7.35±0.23	12.05±0.92
Sum In Feature 9	18.67±0.11	8.14±0.96
十八烷酸 18:0	8.65±0.59	10.92±0.17
十八碳顺式单不饱和脂肪酸 18:1 cis	0	2.46±0.20
十九碳反式脂肪酸 19:1 trans	2.41±0.16	2.29±0.40
Sum In Feature 11	4.28±0.03	7.80±0.08
环丙烷脂肪酸 19:0 cyclo	13.44±0.31	14.66±0.34

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