Effect of Microbial Fermentation Technology on Chemical Constituents of Pueraria thomsonii
-
摘要: 目的：利用微生物发酵技术提高粉葛中活性成分的含量，以扩大其功能性产品的研究。方法：首先以粉葛饮片为原料，嗜酸乳杆菌为发酵菌种，葛根素含量为指标，接种量、料液比、发酵时间、发酵温度为影响因素，通过单因素结合响应面设计法优化粉葛的发酵工艺。其次，通过高效液相色谱法、紫外分光光度法、双波长法测定发酵前、后粉葛中葛根素、大豆苷、大豆苷元、总异黄酮、可溶性多糖及总淀粉的含量。结果：粉葛最优发酵条件为：接种量5%，料液比1:3 g/mL，发酵时间36 h，发酵温度29 ℃，葛根素实际含量为8.1854 mg/g与预测值8.2092 mg/g相比，相对误差仅为0.29%。发酵后粉葛中葛根素、大豆苷、大豆苷元、总异黄酮、可溶性多糖、支链淀粉含量均增加，直链淀粉和总淀粉含量减少，与发酵前相比差异均具有显著性（P<0.05）。结论：结果表明微生物发酵技术能够提升粉葛中活性成分的含量。Abstract: Objective: In order to expand the research of functional products, the content of active ingredients in Pueraria thomsonii was increased by microbial fermentation technology. Methods: Firstly, the fermentation process of Pueraria thomsonii was optimized by the combination of single factor test and response surface methodology with the decoction pieces of Pueraria thomsonii as raw material, L. acidophilus as fermentation strain, puerarin content as index, and inoculation amount, solid-liquid ratio, fermentation time and fermentation temperature as acting factors. Secondly, the contents of puerarin, daidzin, daidzein, total isoflavones, soluble polysaccharides and total starch in Pueraria thomsonii were determined by high performance liquid chromatography, ultraviolet spectrophotometry and dual wavelength method before and after fermentation. Results: The optimal fermentation conditions were as follows: Inoculation amount 5%, solid-liquid ratio 1:3 g/mL, fermentation time 36 h, fermentation temperature 29 ℃. The relative error was 0.29% between the actual content 8.1854 mg/g and predicted value 8.2092 mg/g of puerarin. After fermentation, the contents of puerarin, daidzin, daidzein, total isoflavones, soluble polysaccharides and amylopectin in Pueraria thomsonii increased, while the contents of amylose and total starch decreased, showing significant differences compared with those before fermentation (P<0.05). Conclusion: The results showed that microbial fermentation technology could improve the content of active ingredients in Pueraria thomsonii.
-
Key words:
- Puerariae thomsonii /
- content of puerarin /
- fermentation process /
- content determination
-
图 5 接种量、料液比、发酵时间对葛根素含量的响应曲面图及等高线图
Figure 5. Response surface and contour plots of inoculation amount, solid-liquid ratio and fermentation time on puerarin content
图 7 发酵前、后粉葛中异黄酮类成分HPLC图
Figure 7. HPLC diagrams of isoflavones in Puerariae thomsonii before and after fermentation
注：A.混合标准品；B.发酵前粉葛样品；C.发酵后粉葛样品；1.葛根素；2.大豆苷；3.大豆苷元。
表 1 响应面因素和水平设计
Table 1. Response surface factor and level design
水平 因素 A接种量（%） B料液比（g/mL） C发酵时间（h） −1 4 1:2 24 0 6 1:3 36 1 8 1:4 48 
下载: 导出CSV
表 2 响应面设计及结果
Table 2. Response surface design and result
试验号 A接种量 B料液比 C发酵时间 含量（mg/g） 1 0 1 1 6.21 2 1 1 0 5.85 3 1 0 1 5.64 4 1 −1 0 6.41 5 0 −1 1 7.00 6 0 0 0 8.04 7 0 0 0 7.78 8 1 0 −1 6.57 9 −1 0 1 7.59 10 0 1 −1 6.04 11 0 −1 −1 6.10 12 −1 −1 0 5.90 13 0 0 0 8.40 14 −1 1 0 7.44 15 0 0 0 8.26 16 −1 0 −1 7.91 17 0 0 0 7.71
下载: 导出CSV
表 3 回归模型方差分析表
Table 3. Regression model variance analysis table
方差来源 平方和 自由度 均方 F值 P值 显著性 模型 12.71 9 1.41 4.83 0.0250 显著 A 2.69 1 2.69 9.19 0.0191 显著 B 0.020 1 0.020 0.068 0.8013 C 4.050×10−3 1 4.050×10−3 0.014 0.9097 AB 0.84 1 0.84 2.86 0.1346 AC 0.093 1 0.093 0.32 0.5905 BC 0.13 1 0.13 0.46 0.5216 A2 1.31 1 1.31 4.47 0.0722 B2 5.55 1 5.55 18.95 0.0033 显著 C2 1.29 1 1.29 4.39 0.0743 残差 2.05 7 0.29 失拟项 1.69 3 0.56 6.37 0.0528 不显著 纯误差 0.35 4 0.089 总离差 14.76 16 注：P<0.05表示具有显著性，P˃0.05表示不具有显著性。
下载: 导出CSV
表 4 发酵前、后粉葛中水分、灰分测定结果（n=3）
Table 4. Determination results of moisture and ash in Pueraria thomsonii before and after fermentation (n=3)
测定项 水分（%） 灰分（%） 发酵前粉葛 9.69±0.01 3.89±0.05 发酵后粉葛 8.19±0.08 4.19±0.03
下载: 导出CSV
表 5 发酵前、后粉葛中异黄酮类成分含量测定结果（n=3）
Table 5. Determination results of isoflavone content in Puerariae thomsonii before and after fermentation (n=3)
测定项 测定成分（mg/g） 大豆苷元 大豆苷 葛根素 总异黄酮 发酵前粉葛 0.22±0.02 0.84±0.02 4.85±0.06 25.68±0.15 发酵后粉葛 0.38±0.02* 1.45±0.03** 8.17±0.02** 33.26±0.34** 注：*表示P<0.05，**表示P<0.01；表6同。
下载: 导出CSV
表 6 粉葛发酵前、后可溶性多糖及淀粉含量测定结果（n=3）
Table 6. Determination results of soluble polysaccharides and starch contents in Puerariae thomsonii before and after fermentation (n=3)
测定项 可溶性多糖（%） 淀粉（%） 直链淀粉 支链淀粉 总淀粉 发酵前粉葛 2.47±0.05 8.28±0.07 − 8.28±0.07 发酵后粉葛 12.6±0.06** 6.44±0.11** 0.98±0.04 7.42±0.07** 注：−表示未检出。
下载: 导出CSV
-
[1] 国家药典委员会. 中华人民共和国药典. 一部[S]. 北京: 中国医药科技出版社, 2020.National Pharmacopoeia Commission. Chinese Pharmacopoeia. Part I [S]. Beijing: China Pharmaceutical Science and Technology Press, 2020. [2] AHMAD B, KHAN S, LIU Y, et al. Molecular mechanisms of anticancer activities of puerarin[J]. Cancer Manag Res,2020,12:79. doi: 10.2147/CMAR.S233567 [3] 钟凌云, 邓小燕, 黄艺, 等. 葛(葛根、粉葛)不同炮制品的药效与肠道菌群研究[J]. 中国中药志,2021,46(17):4403−4409. [ZHONG L Y, DENG X Y, HUANG Y, et al. Study on the efficacy and intestinal flora of different processing products of Radix Puera-riae and Radix Puerariae[J]. Chinese Materia Medica,2021,46(17):4403−4409. [4] JEON Y D, LEE J H, LEE Y M, et al. Puerarin inhibits inflammation and oxidative stress in dextran sulfate sodium-induced colitis mice model[J]. Biomed Pharmacother,2020,124:109847. doi: 10.1016/j.biopha.2020.109847 [5] CHEN Y G, SONG Y L, WANG Y, et al. Metabolic differentiations of Pueraria lobata and Pueraria thomsonii using ¹H NMR spectroscopy and multivariate statistical analysis[J]. J Pharm Biomed Anal,2014,93:51−58. doi: 10.1016/j.jpba.2013.05.017 [6] 于钦辉, 杜以晴, 孙启慧, 等. 基于功效和物质基础的野葛、粉葛在解热和抗病毒作用研究进展[J]. 中华中医药学刊,2021,39(9):89−94. [YU Q H, DU Y Q, SUN Q H, et al. Research progress on antipyretic and antiviral effects of Pueraria lobata and Puerariae thomsonii based on efficacy and material basis[J]. Chinese Journal of Traditional Chinese Medicine,2021,39(9):89−94. [7] 中国科学院中国植物志编制委员会. 中国植物志(第四十一卷)[M]. 北京: 中国科学出版社, 1995: 219.Chinese Flora Compilation Committee, Chinese Academy of Sciences. Flora of China (Volume 41)[M]. Beijing: Science Press, 1995: 219. [8] 吴文杰, 刘良红, 黄莺, 等. LC-MS/MS法同时测定葛根药材中14种异黄酮类成分的含量[J]. 中国药房,2018,29(10):1320−1324. [WU W J, LIU L H, HUANG Y, et al. LC-MS/MS method was used to simultaneously determine the contents of 14 isoflavones in Radix Puerariae[J]. Chinese Pharmacy,2018,29(10):1320−1324. doi: 10.6039/j.issn.1001-0408.2018.10.06 [9] 赵婧文, 张建逵, 魏巍, 等. 粉葛中淀粉含量与葛根素含量的相关性研究[J]. 中国中医药现代远程教育,2018,16(15):95−97. [ZHAO W J, ZHANG J K, WEI W, et al. Study on the correlation between starch content and puerarin content in Puerariae thomsonii[J]. Modern Distance Education of Chinese Traditional Medicine,2018,16(15):95−97. doi: 10.3969/j.issn.1672-2779.2018.15.040 [10] 董凡, 李浩然, 王少平, 等. 中药发酵的现代研究进展与展望[J]. 中华中医药杂志,2021,36(2):628−633. [DONG F, LI H R, WANG S P, et al. Modern research progress and prospect of traditional Chinese medicine fermentation[J]. Chinese Journal of Traditional Chinese Medicine,2021,36(2):628−633. [11] LI L, WANG L, FAN W, et al. The application of fermentation technology in traditional Chinese medicine: A review[J]. Am J Chin Med,2020,48(4):899−921. doi: 10.1142/S0192415X20500433 [12] CAO G, MA F, XU J, et al. Microbial community succession and toxic alkaloids change during fermentation of Huafeng Dan Yaomu[J]. Lett Appl Microbiol,2020,70(4):318−325. doi: 10.1111/lam.13276 [13] 张红艳, 冉淦侨, 韩姗姗, 等. 微生物多级发酵对中药方剂功能活性及有效组分的影响[J]. 食品与生物技术学报,2021,40(11):90−96. [ZHANG H Y, RAN J Q, HAN S S, et al. Effect of microbial multi-stage fermentation on functional activity and effective components of Chinese medicinal formulae[J]. Journal of Food and Biotechnology,2021,40(11):90−96. [14] WANG T, WANG Z, YANG Z, et al. Effect of the fermentation broth of the mixture of Pueraria lobata, Lonicera japonica, and Crataegus pinnatifida by Lactobacillus rhamnosus 217-1 on liver health and intestinal flora in mice with alcoholic liver disease induced by liquor[J]. Front Microbiol,2021,12:722171−722171. doi: 10.3389/fmicb.2021.722171 [15] 陈艳艳. 葛根发酵工艺优化及解酒活性评价研究[D]. 长春: 长春中医药大学, 2021.CHEN Y Y. Optimization of fermentation process and evaluation of alcoholysis activity of Pueraria lobata[D]. Changchun: Changchun University of Traditional Chinese Medicine, 2021. [16] 朱盼, 谢娟平. 不同产地粉葛不同部位中7种化学成分的含量测定与比较[J]. 化学与生物工程,2019,36(7):59−64. [ZHU P, XIE J P. Determination and comparison of seven chemical components in different parts of Pueraria lobata from different producing areas[J]. Chemistry and Bioengineering,2019,36(7):59−64. doi: 10.3969/j.issn.1672-5425.2019.07.014 [17] 蒙秋艳, 黄靖洲, 梁艳玲, 等. 广西不同产地粉葛、野葛的多糖和异黄酮含量比较[J]. 食品研究与开发,2020,41(21):43−50. [MENG Q Y, HUANG J Z, LIANG Y L, et al. Comparison of polysaccharide and isoflavone contents of Pueraria lobata from different habitats in Guangxi[J]. Food Research and Development,2020,41(21):43−50. [18] 黄再强, 张燕飞, 陈玲, 等. 川产葛根、粉葛总黄酮和多糖含量的对比分析[J]. 中药与临床,2017,8(3):11−14. [HUANG Z Q, ZHANG Y F, CHEN L, et al. Comparative analysis of the content of total flavonoids and polysaccharides in Pueraria lobata and Puerariae thomsonii from Sichuan[J]. Traditional Chinese Medicine and Clinical,2017,8(3):11−14. [19] 崔晋, 李建军, 马艳弘, 等. 双波长法测定山药中直链和支链淀粉含量[J]. 食品研究与开发,2017,38(13):150−154. [CUI J, LI J J, MA Y H, et al. Determination of amylose and amylopectin content in yam by dual-wavelength method[J]. Food Research and Development,2017,38(13):150−154. doi: 10.3969/j.issn.1005-6521.2017.13.031 [20] 赵璇, 李瑞颖, 马彦江, 等. 清炒法炮制粉葛过程中主要化学成分与工艺、物性及颜色变化的相关性分析[J]. 中国医院药学杂志,2020,40(23):2414−2418. [ZHAO X, LI R Y, MA Y J, et al. Correlation analysis of main chemical components and process, physical properties and color changes in the process of stir-frying Puerariae thomsonii[J]. Chinese Journal of Hospital Pharmacy,2020,40(23):2414−2418. doi: 10.13286/j.1001-5213.2020.23.04 [21] 陈旋, 杨国琴, 高书彦, 等. 响应曲面优化苦荞多肽固态发酵工艺及其抗菌活性研究[J]. 食品与发酵科技,2018,54(1):30−38. [CHEN X, YANG G Q, GAO S Y, et al. Optimization of solid state fermentation process and antibacterial activity of tartary buckwheat peptides by response surface methodology[J]. Food and Fermentation Technology,2018,54(1):30−38. [22] 冯颖, 陶亮, 肖学爱, 等. 人参叶中高产皂苷菌种的筛选及培养条件优化[J]. 中国酿造,2021,40(8):117−122. [FENG Y, TAO L, XIAO X, et al. Selection of high-yield saponins strains in ginseng leaves and optimization of culture conditions[J]. Chinese Brewing,2021,40(8):117−122. doi: 10.11882/j.issn.0254-5071.2021.08.021 [23] ZHANG H, WANG L, WANG H, et al. Effects of initial temperature on microbial community succession rate and volatile flavors during Baijiu fermentation process[J]. Food Res Int,2021,141:109887. doi: 10.1016/j.foodres.2020.109887 [24] 宋艳秋, 陈有为. 红曲霉转化中药葛根固体发酵条件研究[J]. 安徽农业科学,2010,38(4):1707−1708. [SONG Y Q, CHEN Y W. Study on solid fermentation conditions of Monascus transformed Pueraria lobata[J]. Anhui Agricultural Science,2010,38(4):1707−1708. doi: 10.3969/j.issn.0517-6611.2010.04.020 [25] 倪以宇, 赵大庆, 倪伟锋, 等. 葛根-枳椇子药对发酵工艺及解酒功效评价研究[J/OL]. 食品与发酵工业: 1−102022-04-30]. NI Y Y, ZHAO D Q, NI W F, et al. Evaluation of the fermentation process and anti-alcoholism effect of Pueraria lobata-Hovenia dulcis Thunb seed medicine[J/OL]. Food and Fermentation Industry: 1−10[2022-04-30]. [26] 孙双, 张婷, 方琰, 等. 雨生红球藻高产虾青素的培养条件优化[J]. 现代食品科技,2021,37(6):98−107. [SUN S, ZHANG T, FANG Y, et al. Optimization of culture conditions for high astaxanthin production by Haematococcus pluvialis[J]. Modern Food Technology,2021,37(6):98−107. doi: 10.13982/j.mfst.1673-9078.2021.6.1000 [27] 罗盟錡, 张惠捷, 王金龙, 等. 响应面法优化混合益生菌发酵甘草的工艺[J]. 饲料研究,2022,45(3):80−84. [LUO M Q, ZHANG H J, WANG J L, et al. Response surface methodology was used to optimize the fermentation process of licorice by mixed probiotics[J]. Feed Research,2022,45(3):80−84. doi: 10.13557/j.cnki.issn1002-2813.2022.03.016 [28] 杜静, 王琪琪, 王云胜, 等. 冠突散囊菌发酵对葛根的活性物质和抗氧化活性的影响[J]. 食品工业科技,2021,42(1):121−125. [DU J, WANG Q Q, WANG Y S, et al. Effect of fermentation of P. coronarium on active substances and antioxidant activity of Pueraria lobata[J]. Food Industry Technology,2021,42(1):121−125. doi: 10.13386/j.issn1002-0306.2019120314 [29] 张群. 益生菌发酵富含黄酮农产品关键技术研究[J]. 食品与生物技术学报,2021,40(11):1673−1689. [ZHANG Q. Key technology of flavone-rich agricultural products fermentation by probiotics[J]. Journal of Food and Biotechnology,2021,40(11):1673−1689. [30] PERVEZ S, NAWAZ M A, SHAHID F, et al. Characterization of cross-linked amyloglucosidase aggregates from Aspergillus fumigatus KIBGE-IB33 for continuous production of glucose[J]. Int J Biol Macromol,2019,135(C):1252−1260. [31] 柴小涛. 金耳-葛根双向液体发酵条件优化及抗氧化的研究[J]. 轻工科技,2020,36(5):23−25. [CHAI X T. Optimization of fermentation conditions and antioxidant activity of Jiner-Gegen bidirectional liquid fermentation[J]. Light Industry Technology,2020,36(5):23−25. [32] 任佳楠. Massilia sp. UMI-21 PHA合成途径中相关酶鉴定及其对PHA合成的影响[D]. 长春: 长春理工大学, 2021.REN J N. Massilia sp. UMI-21 PHA synthesis pathway related enzyme identification and its effect on PHA synthesis[D]. Changchun: Changchun University of Technology, 2021. [33] 黄群, 肖文军, 孙术国, 等. α-淀粉酶和糖化酶协同酶解葛根淀粉动力学研究[J]. 食品科学,2012,33(21):187−191. [HUANG Q, XIAO W J, SUN S G, et al. Kinetics of α-amylase and glucoamylase synergistic enzymatic hydrolysis of Pueraria starch[J]. Food Science,2012,33(21):187−191. [34] 刘襄河, 郑丽璇, 郑丽勉, 等. 双波长法测定常用淀粉原料中直链淀粉、支链淀粉及总淀粉含量[J]. 广东农业科学,2013,40(18):97−100. [LIU X H, ZHENG L X, ZHEN L M, et al. Determination of amylose, amylopectin and total starch content in common starch raw materials by dual wavelength method[J]. Guangdong Agricultural Science,2013,40(18):97−100. doi: 10.3969/j.issn.1004-874X.2013.18.034 [35] 岳世彦, 周荣荣, 南铁贵, 等. 粉葛与葛根中主要化学成分的含量比较[J/OL]. 中国中药杂志: 1−10[2022-03-02].YUE S Y, ZHOU R R, NAN T G, et al. Comparison of the contents of main chemical constituents in Radix Puerariae and Radix Puerariae[J/OL]. Chinese Journal of Traditional Chinese Medicine: 1−10[2022-03-02]. [36] 张应, 李隆云, 舒抒, 等. 不同产地、品种及采收期粉葛可溶性糖和淀粉的含量测定[J]. 中药材,2013,36(11):1751−1754. [ZHANG Y, LI L Y, SHU S, et al. Determination of soluble sugar and starch content in Pueraria lobata from different habitats, varieties and harvest period[J]. Medicinal Materials,2013,36(11):1751−1754. doi: 10.13863/j.issn1001-4454.2013.11.021 -
点击查看大图
图(8) / 表(6)
计量
- 文章访问数: 109
- HTML全文浏览量: 28
- PDF下载量: 11
- 被引次数: 0
