选育高脂肪酶活性菌株,考察其发酵制备豆豉过程中磷脂酰胆碱(phosphatidylcholine,PC)、磷脂酰肌醇(phosphatidylinositol,PI)和磷脂酰乙醇胺(phosphatidylethanolamine,PE)的含量变化。采用维多利亚蓝和中性红油脂平板从淡豆豉饮片中初筛高脂肪酶活性菌株,运用对硝基苯酚法进行复筛,通过常压室温等离子体(atmospheric and room temperature plasma, ARTP)技术对筛选的菌株进行诱变以提高其酶活力,分别以出发菌株和诱变菌株发酵制备豆豉,采用HPLC测定豆豉不同发酵阶段PC、PI和PE的含量变化。筛选出1株高脂肪酶活性菌株,经ARTP诱变后,酶活性提高了25.98%,经Biolog微生物鉴定系统鉴定为伞枝梨头霉;出发菌株和突变菌株发酵7 d制备的豆豉PC、PI和PE的含量没有明显差异,但显著高于市售淡豆豉相应成分的含量,分别约为后者的5倍、8~11倍和30倍。该研究优选了高脂肪酶活性的菌株,显著提高了豆豉中磷脂类成分的含量,确定了富含功能性磷脂的豆豉制备工艺,为后续研究奠定基础。
Screening and breeding the strain with high lipase activity, and the content changes of phospholipids, i.e.phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) in Douchi were investigated. The Victoria blue and neutral red lipase plates were used to preliminary screen the strains with high lipase activity from Semen Sojae Praeparatum, the P-nitrophenol method was used to re-screen the strains, and atmospheric and room temperature plasma (ARTP) was adopted to mutagenize the selected strain to improve its enzyme activity. The Douchi was prepared using black soybean with different starters (original strain and mutant), the contents of PC, PI and PE in Douchi at different fermentation stages were detected by HPLC. A strain with high lipase activity was screened out, and the enzyme activity of the strain after treated by ARTP increased by 25.98%, and it was identified as Absidia corymbifera by Biolog microbial identification system. There is no significant difference of the contents of PC, PI and PE in Douchi fermented 7 d by the original and mutated strains, however, the contents of PC, PI and PE in Douchi fermented by the selected strains were 5 times, 8-11 times and 30 times than that of Semen Sojae Praeparatum. The strain with high lipase activity was selected, the content of phospholipid in Douchi improved significantly, and the preparation process of Douchi with rich phospholipid was determined, which lays a foundation for subsequent research.
[1] 王思齐, 王满元, 关怀, 等.淡豆豉的本草考证[J].中国现代中药, 2018, 20(4):473-477;488.
WANG S Q, WANG M Y, GUAN H, et al.Herbalogical textual research of sojae semen praeparatum[J].Modern Chinese Medicine, 2018, 20(4):473-477;488.
[2] 赵九永. 传统大豆发酵食品的营养价值与保健功能[J].粮食科技与经济, 2017, 42(2):71-73;76.
ZHAO J Y.Nutritional value and health care function of traditional soybean fermented food[J].Grain Science and Technology and Economy, 2017, 42(2):71-73,76.
[3] 曾艳, 朱玥明, 张建刚, 等.大豆发酵食品中的活性肽及其生理功能研究进展[J].大豆科学, 2019, 38(1):159-166.
ZENG Y, ZHU Y M, ZHANG J G, et al.Process in bioactive peptides during soybean fermentation and their potential health benefits[J].Soybean Science, 2019, 38(1):159-166.
[4] 沈畅萱, 王修俊, 黄珊.豆豉纤溶酶等微生物源纤溶酶的研究与应用进展[J].中国酿造, 2017, 36(8):6-10.
SHEN C X, WANG X J, HUANG S.Research and application progress of fibrinolytic enzyme from Douchi and other microbial source[J].China Brewing, 2017, 36(8):6-10.
[5] YEU E, SILVERSTEIN S, GUILLON M, et al.Efficacy and safety of phospholipid nanoemulsion-based ocular lubricant for the management of various subtypes of dry eye disease:A phase IV, multicenter trial[J].Clinical Ophthalmology (Auckland, N.Z.), 2020, 14:2 561-2 570.
[6] LI J, WANG X L, ZHANG T, et al.A review on phospholipids and their main applications in drug delivery systems[J].Asian Journal of Pharmaceutical Sciences, 2015, 10(2):81-98.
[7] KÜLLENBERG D, TAYLOR L A, SCHNEIDER M, et al.Health effects of dietary phospholipids[J].Lipids in Health and Disease, 2012, 11:3.
[8] 肖志刚, 杨国强, 杨舒, 等.功能性磷脂的酶法合成及其生理功能研究进展[J].沈阳师范大学学报(自然科学版), 2020, 38(3):238-244.
XIAO Z G, YANG G Q, YANG S, et al.Research progress of enzymatic synthesis and physiological function[J].Journal of Shenyang Normal University (Natural Science Edition), 2020, 38(3):238-244.
[9] KRETZA E, PAPANEOPHYTOU C P, PAPI R M, et al.Lipase activity in Thermus thermophilus HB8:Purification and characterization of the extracellular enzyme[J].Biotechnology and Bioprocess Engineering, 2012, 17(3):512-525.
[10] 陈雪, 冯莉, 秦义, 等.常压室温等离子体诱变选育低产挥发酸酿酒酵母[J].中国酿造, 2019, 38(11):43-48.
CHEN X, FENG L, QIN Y, et al.Mutation breeding of low-yield volatile acid Saccharomyces cerevisiae by atmospheric and room temperature plasma[J].China Brewing, 2019, 38(11):43-48.
[11] 杨佩斯. ARTP诱变黑曲霉絮凝菌株及应用研究[D].贵阳:贵州大学, 2020.
YANG P S.Screening of microbial flocculant high-producing Aspergillus niger strains by atmospherric and room temperature plasma and its application[D].Guiyang:Guizhou University, 2020.
[12] 贺秋红, 巩志金, 颜梅.产脂肪酶菌株的筛选、鉴定及发酵培养基优化[J].中国酿造, 2019, 38(10):84-88.
HE Q H, GONG Z J, YAN M.Screening and identification of lipase producing strain and optimization of the fermentation medium[J].China Brewing, 2019, 38(10):84-88.
[13] 郑小梅. 脂肪酶产生菌的筛选及其脂肪酶基因的克隆[D].北京:中国农业科学院, 2009.
ZHENG X M.Screening of lipase producing strains and cloning of lipase genes[D].Beijing:Chinese Academy of Agricultural Sciences, 2009.
[14] 姚泽, 李云晶, 马越, 等.高效液相色谱法测定多烯磷脂酰胆碱软胶囊中磷脂酰胆碱和溶血磷脂酰胆碱的含量[J].中南药学, 2017, 15(9):1 304-1 306.
YAO Z, LI Y J, MA Y, et al.Determination of phosphatidyl choline and lysophosphatidyl choline in polyene phosphatidyl choline soft capsules by HPLC[J].Central South Pharmacy, 2017, 15(9):1 304-1 306.
[15] 陈丽艳, 刘青, 孙银玲, 等.不同产地淡豆豉优势发酵菌群的筛选及酶活性分析[J].中国药房, 2017, 28(31):4 359-4 361.
CHEN L Y, LIU Q, SUN Y L, et al.Screening and enzymatic activity analysis of dominant fermentive bacteria of Sojae Semen Praeparatum from different production places[J].China Pharmacy, 2017, 28(31):4 359-4 361.
[16] 宓月光, 王伟明, 孙银玲, 等.淡豆豉和豆豉纯种发酵过程中游离氨基酸的含量变化[J].食品与发酵工业, 2019, 45(23):257-261.
MI Y G, WANG W M, SUN Y L, et al.Dynamic quantity changes of free amino acids during Semen Sojae Praeparatum and black beans fermentation using pure strain[J].Food and Fermentation Industries, 2019, 45(23):257-261.
[17] 刘德海, 郝益民, 岳丹丹, 等.一株产β-葡萄糖苷酶菌株的筛选及酶学性质研究[J].中国酿造, 2013, 32(6):47-51;60.
LIU D H, HAO Y M, YUE D D, et al.Isolation of a strain with β-glucosidase and its enzymatic properties[J].China Brewing, 2013, 32(6):47-51;60.
[18] 王涵欣. 传统豆糁的工艺优化与风味品质特征研究[D].郑州:河南工业大学, 2015.
WANG H X.Research on processing optimization and flavor quality of traditional Doushen[D].Zhengzhou:Henan University of Technology, 2015.
[19] 沈怡方. 白酒生产技术全书[M].北京:中国轻工业出版社, 2013.
SHEN Y F.White spirit production technology book[M].Beijing:China Light Industry Press, 2013.
[20] 陈嘉序, 陈如扬, 连媛, 等.大豆异黄酮的生物转化及功能活性研究进展[J].食品研究与开发, 2021, 42(9):176-182.
CHEN J X, CHEN R Y, LIAN Y, et al.Progress in microbial conversion and functional activity of soy isoflavones[J].Food Research and Development, 2021, 42(9):176-182.
[21] 刘莉, 张金兰, 孙勇, 等.发酵豆制品生物活性肽功能特性研究进展[J].中国酿造, 2017, 36(7):1-4.
LIU L, ZHANG J L, SUN Y, et al.Research progress of functional properties of bioactive peptides from fermented soybean products[J].China Brewing, 2017, 36(7):1-4.
