纳豆芽孢杆菌发酵菜用大豆中总黄酮的提取及其降血脂效果评价

李秀凉; 倪庆圆; 杨宸; 宋永; 韩晓云; 孙庆申

doi:10.13386/j.issn1002-0306.2020070003

纳豆芽孢杆菌发酵菜用大豆中总黄酮的提取及其降血脂效果评价

doi: 10.13386/j.issn1002-0306.2020070003

李秀凉^{1, 3},
倪庆圆^{1, 2},
杨宸^{1, 2},
宋永^{1, 3},
韩晓云^{1, 2},
孙庆申^{1, 2, ,}

1.
黑龙江大学，农业微生物技术教育部工程研究中心，黑龙江哈尔滨 150500
2.
黑龙江大学生命科学学院，黑龙江省普通高等学校微生物重点实验室，黑龙江哈尔滨 150080
3.
黑龙江大学生命科学学院，黑龙江省普通高等学校分子生物学重点实验室，黑龙江哈尔滨 150080

基金项目: 黑龙江省自然科学基金面上项目（C2006049）；哈尔滨市应用技术研究与开发项目（青年后备人才 A类RC2017QN020010）资助

详细信息

通讯作者:
孙庆申（1977−），男，博士，教授，研究方向：食品与药品活性物质的挖掘，E-mail：sunqingshen@hlju.edu.cn

中图分类号: TS201.2
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出版历程
- 收稿日期: 2020-07-02

Extraction of Total Flavonoids in Vegetable Soybean Fermented by Bacillus natto and Evaluation of Its Effect on Lowering Blood Lipid

Xiuliang LI^{1, 3},
Qingyuan NI^{1, 2},
Chen YANG^{1, 2},
Yong SONG^{1, 3},
Xiaoyun HAN^{1, 2},
Qingshen SUN^{1, 2
, ,}

1.
Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China
2.
Key Laboratory of Microbiology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080, China
3.
Key Laboratory of Molecular Biology, College of Heilongjiang Province, School of Life Sciences, Heilongjiang University, Harbin 150080, China

摘要

摘要: 本文以菜用大豆等外品为原料，通过纳豆枯草芽孢杆菌发酵来提高其中总黄酮的含量，为提高菜用大豆产品的附加值提供理论支持。实验利用纳豆芽孢杆菌对菜用大豆等外品进行发酵，以发酵产品中总黄酮含量为评价指标确定纳豆发酵的最优条件，通过D101型大孔树脂对提取的总黄酮进行纯化，采用芦丁比色法测定黄酮含量。体内实验分为肥胖预防实验和缓解实验：以C57BJ/6L小鼠为模型，预防实验在饲喂高脂饲料的同时，灌胃总黄酮提取物溶液（50 mg/kg·d）；缓解实验是利用高脂饲料建立起肥胖小鼠模型以后，灌胃低、中、高剂量的总黄酮提取物溶液（分别为20、50和100 mg/kg·d）。通过小鼠体重、血脂以及促炎因子等指标评价总黄酮溶液的降脂效果。结果表明，菜用大豆发酵生产黄酮的最佳条件为：枯草芽孢杆菌接菌量为1.15 × 10¹¹ CFU/100 g菜用大豆，发酵温度37 ℃，发酵时间24 h，后熟时间12 h，此时纳豆总黄酮得率为4.30 mg/g菜用大豆；测得该粗提物中黄酮含量为22.1%；预防实验组给予黄酮处理后，小鼠体重和Lee's指数与正常对照组相比均无显著差异。缓解实验中高、中、低剂量黄酮提取物处理组小鼠血清中甘油三酯（TG）含量分别为313.1 ± 10.12、310.39 ± 31.76和310.1 ± 10.20 nmol/L，与正常对照组（330.8 ± 34.36 nmol/L）无显著差异（P>0.05）；黄酮各组灌胃干预后，小鼠血清总胆固醇（TC）、白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）含量均较高脂对照组显著下降（P <0.05）。结论：利用纳豆芽孢杆菌发酵菜用大豆，提高了其中的总黄酮含量，该黄酮提取物具有预防和缓解高脂饮食小鼠血脂升高的效果。
- 菜用大豆 /
- 总黄酮 /
- 发酵条件优化 /
- 降血脂 /
- 纳豆芽孢杆菌
Abstract: This paper used vegetable soybeans which were not qualified for export as raw materials to increase total flavonoid yield through Bacillus natto fermentation, thus providing theoretical support for increasing the added value of vegetable soybeans. Bacillus natto was used to ferment vegetable soybeans, and the total flavonoid content in the fermented product was used as criteria to determine the optimal fermentation conditions. The extracted total flavonoids were purified using D101 macroporous resin. The content of total flavonoids was determined by Rutin colorimetric method. The in vivo experiments were divided into obesity preventive and alleviating tests using C57BJ/6L mice. Mice from the preventive experiment were fed high-fat diet while administered flavonoid extracts (50 mg/kg·d) through gavage; in alleviating groups, mice were administered total flavonoid extracts at low, medium and high doses (20, 50, and 100 mg/kg·d, respectively) through gavage after establishing obese mouse model with high-fat diet. The hypolipidemic effect of total flavonoids was evaluated through parameters such as mouse body weight, blood lipids and pro-inflammatory factors. Results showed that the optimal conditions for production of total flavonoids via fermentation of were as follows: Bacillus natto was inoculated at 1.15 × 10¹¹ CFU/100 g vegetable soybeans, then undergone fermentation at 37 ℃ for 24 h and post ripeness for another 12 h. The yield of total flavonoids was 4.30 mg/g vegetable soybean, with the content of flavonoids in the crude extract at 22.1%. In the prevention experiment, there was no significant difference in the weight and Lee's index of the high-fat diet mice after gavage with total flavonoids compared with the normal control group. In the alleviating experiment, the serum triglyceride (TG) content of the mice with high, medium, and low-dose flavonoid extract treatment were 313.1 ± 10.12, 310.39 ± 31.76 and 310.1 ± 10.20 nmol/L, respectively, with no significant difference compared with the normal control group (330.8 ± 34.36 nmol/L) (P>0.05); After intragastric intervention with flavonoids, serum total cholesterol (TC), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels in mice showed a significant decrease compared with that of the higher fat control group (P<0.05). In summary, fermentation using Bacillus natto helped to increase the total flavonoid content in vegetable soybeans. The flavonoid extract showed the effect of both preventing and alleviating mouse hyperlipidemia.
- vegetable soybean /
- total flavonoids /
- fermentation condition optimization /
- hypolipidemic effects /

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图 1 发酵时间(A)、发酵温度(B)、接菌量(C)和后熟时间(D)对纳豆中总黄酮含量的影响

注：图中标记小写字母不同者表示差异显著(p<0.05)。

Figure 1. Effect of fermentation time (A), fermentation temperature(B), inoculation dose(C) and ripening time(D) on the content of total flavonoids in natto

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图 2 缓解实验（A）和预防实验（B）各组小鼠体重变化

注：图中同一时间内标记小写字母不同者表示差异显著(p<0.05)。

Figure 2. Body weight change of mice in remission experiment（A）and prevention experiment（B）

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图 3 缓解实验（A）和预防实验（B）各组小鼠血清甘油三酯变化

注：图中标记不同小写字母表示差异显著(p<0.05)，图4～图6同。

Figure 3. Changes of triglycerides in mice in remission experiment（A）and prevention experiment（B）

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图 4 缓解实验（A）和预防实验（B）各组小鼠血清总胆固醇变化

Figure 4. Changes of total cholesterol in mice in remission experiment（A）and prevention experiment（B）

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图 5 缓解实验（A）和预防实验（B）各组小鼠高密度脂蛋白变化

Figure 5. Changes of High density lipoprotein in mice in remission experiment（A）and prevention experiment（B）

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图 6 缓解实验（A、C、E）和预防实验（B、D、F）各组小鼠血清炎性因子含量的变化

Figure 6. Changes of proinflammatory factors in mice serum in alleviating experiment（A, C, E）and prevention experiment（B, D, F）

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表 1 纳豆发酵工艺优化正交实验因素与水平

Table 1. Factors and levels of orthogonal experiment for optimization of natto fermentation process

水平	A发酵时间(h)	B接菌量(CFU /100 g)	C后熟时间(h)
1	24	5.75 × 10¹⁰	6
2	36	1.15 × 10¹¹	12
3	48	1.73 × 10¹¹	18

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表 2 纳豆发酵工艺优化正交实验结果

Table 2. Orthogonal experiment result for optimization of natto fermentation process

实验号	A	B	C	Y:总黄酮含量（mg/g）
1	1	1	1	3.34 ± 0.17
2	1	2	2	4.30 ± 0.12
3	1	3	3	3.70 ± 0.24
4	2	1	2	3.66 ± 0.27
5	2	2	3	3.79 ± 0.21
6	2	3	1	2.99 ± 0.21
7	3	1	3	3.30 ± 0.26
8	3	2	1	3.23 ± 0.28
9	3	3	2	2.40 ± 0.26
k₁	3.78	3.43	3.19
k₂	3.48	3.77	3.45
k₃	2.98	3.03	3.59
R	0.80	0.74	0.41

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表 3 正交实验方差分析

Table 3. Analysis of orthogonal experimental variance

因素	SS	自由度	F值	Fa
A	0.9893	2	3.874	F_0.05（2,4） = 6.944
B	0.8294	2	3.248
C	0.2604	2
误差e	0.2503	2
总变异	2.329

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表 4 各组小鼠体长及Lee’s指数

Table 4. Body length and Lee’s index of mice in different groups

组别	缓解实验		组别	预防实验
组别	体长（cm）	Lee’s指数	组别	体长（cm）	Lee’s指数
正常对照组（A）	8.98 ± 0.33^a	344.94 ± 5.82^b	正常对照组	8.63 ± 0.24^a	356.58 ± 3.79^b
阳性对照组（B）	8.90 ± 0.23^a	349.73 ± 10.19^b	阳性对照组	8.31 ± 0.39^a	363.58 ± 11.18^b
模型组（C）	8.98 ± 0.36^a	363.56 ± 18.18^a	模型组	8.57 ± 0.62^a	382.03 ± 11.15^a
总黄酮高剂量组（D）	8.73 ± 0.15^a	350.20 ± 7.17^b	总黄酮组	8.34 ± 0.38^a	362.54 ± 6.76^b
总黄酮中剂量组	8.81 ± 0.08^a	351.15 ± 4.96^b
总黄酮低剂量组（F）	8.84 ± 0.28^a	348.65 ± 12.22^b
注：表中同一列标记小写字母不同者表示差异显著(p<0.05)，表5同。

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表 5 缓解实验及预防实验各组小鼠肝脏及脾脏系数

Table 5. Liver and spleen coefficients of mice in alleviating experiment and prevention experiment

组别	缓解试验		组别	预防实验
组别	肝脏指数	脾脏指数	组别	肝脏指数	脾脏指数
正常对照组	3.55 ± 0.13^a	0.24 ± 0.03^a	正常对照组	3.57 ± 0.39^a	2.57 ± 0.25^a
阳性对照组	3.73 ± 0.33^a	0.27 ± 0.04^a	阳性对照组	3.65 ± 0.20^a	2.58 ± 0.43^a
模型组	3.25 ± 0.37^b	0.23 ± 0.05^b	模型组	3.38 ± 0.33^b	2.46 ± 0.42^a
总黄酮高剂量	3.53 ± 0.20^a	0.26 ± 0.05^a	异黄酮组	3.62 ± 0.11^a	2.63 ± 0.19^a
总黄酮中剂量组	3.51 ± 0.63^a	0.27 ± 0.04^a
总黄酮低剂量组	3.58 ± 0.33^a	0.26 ± 0.05^a

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