纳豆菌液态发酵荞麦产纳豆激酶及其代谢特性分析
|
摘要
对纳豆枯草芽孢杆菌(Bacillus subtilis natto)液态发酵荞麦产纳豆激酶揺瓶、2.5 L发酵罐条件进行优化。对比自制大豆分离蛋白酶解物与商业大豆蛋白胨作为补充氮源时菌体生长规律以及代谢物质(酶、可溶性蛋白、还原糖、多酚及抗氧化物质)变化规律。纳豆菌液态发酵荞麦产纳豆激酶2.5 L发酵罐最优条件为荞麦浸泡6 h后,按料液比1∶10(g/mL)加水打浆,加入0.4%α-淀粉酶,90℃加热40 min,补充NS37071酶解12 h时所得酶解物,调节发酵培养基pH 7.0,接种量3%,通气量3.5 L/min,转速300 r/min,装液量1.2 L,发酵36 h。与商业大豆蛋白胨相比,补充大豆分离蛋白酶解物时,纳豆菌生长对数期较长,可溶性蛋白与还原糖的消耗量较大,在36 h趋于平稳,纳豆激酶活力持续上升至36 h达到最大值,酚类物质比溶出速率在12 h达到最大值,抗氧化物质比生成速率在6 h达到最大值。以荞麦为原料,补充自制大豆分离蛋白酶解物,通过优化纳豆菌液态发酵条件,可制备具有高纳豆激酶活力(152.5 FU/mL)、富含谷物多酚(0.109 mg/mL)且具有强抗氧化活性(27.43μmol/mL)的发酵产物。
The submerged fermentation conditions of buckwheat for nattokinase production in shake flasks and a 2.5 L fermentor using Bacillus subtilis natto were optimized. Changes in bacterial growth and the metabolism of nutrients(nattokinase, soluble protein, reducing sugar, phenolics and antioxidants) during fermentation using soybean protein isolate hydrolysate and commercial soybean peptone as a supplementary nitrogen source were comparatively studied. The optimized fermentation conditions in a 2.5 L fermentor were obtained as follows: 6 h of buckwheat soaking, pulping at a solid-towater ratio of 1:10(g/mL), addition of 0.4% α-amylase, heating for 40 min at 90 ℃, supplementation of 12 h hydrolyzed soybean protein isolate(SPI) with a commercial protease preparation(NS37071), pH adjustment to 7.0, inoculum size 3%,ventilation rate 3.5 L/min, stirring speed 300 r/min, medium volume 1.2 L, and 36 h fermentation. Supplementation of SPI hydrolysate prolonged the logarithmic growth phase and led to increased consumption of reducing sugar and soluble protein as compared with soybean peptone. Residual reducing sugar and soluble protein tended to level off at 36 h of fermentation with supplementation of SPI hydrolysate, and simultaneously the maximum nattokinase activity was obtained.In addition, the specific dissolution rate of phenolics and the specific formation rate of antioxidants reached a maximum at12 and 6 h, respectively. The nattokinase activity of the fermentation products obtained under the optimized conditions was152.5 FU/mL, which were rich in phenolics(0.109 mg/mL) and antioxidants(27.43 μmol/mL).
The submerged fermentation conditions of buckwheat for nattokinase production in shake flasks and a 2.5 L fermentor using Bacillus subtilis natto were optimized. Changes in bacterial growth and the metabolism of nutrients(nattokinase, soluble protein, reducing sugar, phenolics and antioxidants) during fermentation using soybean protein isolate hydrolysate and commercial soybean peptone as a supplementary nitrogen source were comparatively studied. The optimized fermentation conditions in a 2.5 L fermentor were obtained as follows: 6 h of buckwheat soaking, pulping at a solid-towater ratio of 1:10(g/mL), addition of 0.4% α-amylase, heating for 40 min at 90 ℃, supplementation of 12 h hydrolyzed soybean protein isolate(SPI) with a commercial protease preparation(NS37071), pH adjustment to 7.0, inoculum size 3%,ventilation rate 3.5 L/min, stirring speed 300 r/min, medium volume 1.2 L, and 36 h fermentation. Supplementation of SPI hydrolysate prolonged the logarithmic growth phase and led to increased consumption of reducing sugar and soluble protein as compared with soybean peptone. Residual reducing sugar and soluble protein tended to level off at 36 h of fermentation with supplementation of SPI hydrolysate, and simultaneously the maximum nattokinase activity was obtained.In addition, the specific dissolution rate of phenolics and the specific formation rate of antioxidants reached a maximum at12 and 6 h, respectively. The nattokinase activity of the fermentation products obtained under the optimized conditions was152.5 FU/mL, which were rich in phenolics(0.109 mg/mL) and antioxidants(27.43 μmol/mL).
引文
[1]刘振杰,郭伟鹏,张菊梅,等.纳豆的保健功效及开发应用[J].热带农业工程,2010(3):25-29.
[2]SUMI H,HAMADA H,TSUSHIMA H,et al.A novel fibrinolytic enzyme(nattokinase)in the vegetable cheese natto;a typical and popular soybean food in the Japanese diet[J].Experientia,1987,43(10):1110-1111.DOI:10.1007/BF01956052.
[3]HUANG S H,PAN S H,CHEN G G,et al.Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium,Bacillus subtilis HQS-3[J].International Journal of Biological Macromolecules,2013,62:124-130.DOI:1016/j.ijbiomac.2013.08.048.
[4]MURAKAMI K,YAMANAKA N,OHNISHI K,et al.Inhibition of angiotensin I converting enzyme by subtilisin nat(nattokinase)in natto,a Japanese traditional fermented food[J].Food&Function,2012,3(6):674-678.DOI:10.1039/c2fo10245e.
[5]葛春蕾.高效分泌表达纳豆激酶的重组枯草芽孢杆菌的构建[D].无锡:江南大学,2016.
[6]JING R,LI H Q,HU C L,et al.Phytochemical and pharmacological profiles of three fagopyrum buckwheats[J].International Journal of Molecular Sciences,2016,17(4):589.DOI:10.3390/ijms17040589.
[7]王世霞,李笑蕊,贠婷婷,等.不同品种苦荞麦营养及功能成分对比分析[J].食品与机械,2016,32(7):5-9.
[8]ZHAO G,PENG L X,WANG S,et al.HPLC Fingerprint-antioxidant properties study of buckwheat[J].Journal of Integrative Agriculture,2012,11(7):1111-1118.DOI:10.1016/S2095-3119(12)60104-X.
[9]赵谋明,邹颖,林恋竹,等.纳豆菌液态发酵谷物产纳豆激酶及发酵产物抗氧化活性研究[J].现代食品科技,2018,34(2):1-6.
[10]赵芳芳.大豆肽的生物学功能研究[D].北京:中国农业大学,2004.
[11]万春艳.大豆活性肽对酵母增殖代谢及啤酒发酵的影响研究[D].广州:华南理工大学,2012.
[12]ASTRUP T,MULLERTZ S.The fibrin plate method for estimating fibrinolytic activity[J].Archives of Biochemistry and Biophysics,1952,40:346-351.DOI:10.1016/0003-9861(52)90121-5.
[13]Japan Nattokinase Association.Degradation of artificial thrombus by nattokinase[EB/OL].2012-05-18[2018-01-20].http://jnattokinase.org/en/jnka_nattou_1.html.
[14]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(8):534-536.
[15]LIN L Z,ZHAO H F,DONG Y,et al.Macroporous resin purification behavior of phenolics and rosmarinic acid from Rabdosia serra(Maxim.)Hara leaf[J].Food Chemistry,2012,130(2):417-424.DOI:10.1016/j.foodchem.2011.07.069.
[16]林恋竹,焦铭.光果甘草叶中性多糖结构表征及抗氧化活性研究[J].现代食品科技,2016,32(1):106-110.DOI:10.13982/j.mfst.1673-9078.2016.1.017.
[17]雷芬芬.深海菌株Exiguobacterium sp.SWJS2发酵产蛋白酶、酶学性质及酶解特异性研究[D].广州:华南理工大学,2014.
[18]李小丽.纳豆芽孢杆菌分离及纳豆激酶活性的研究[D].合肥:安徽农业大学,2009.
[19]谢秋玲.纳豆激酶液体发酵的条件优化及其调控的研究[D].广州:华南理工大学,1999.
[20]史一一.荞麦淀粉酶水解特性研究[D].杨凌:西北农林科技大学,2007.
[21]谢秋玲,郭勇,林剑,等.纳豆激酶的合成机制[J].暨南大学学报(自然科学与医学版),2001(3):110-114.DOI:10.3969/j.issn.1000-9965.2001.03.023.
[22]莫芬.小麦面筋蛋白水解物对酵母增殖代谢及啤酒发酵的影响研究[D].广州:华南理工大学,2014.
[23]毕静.糖化酶发酵生产中溶解氧的研究[J].中国酿造,2015,34(8):97-100.DOI:10.11882/j.issn.0254-5071.2015.08.020.
[24]SINGH P,SHERA S S,BANIK J,et al.Optimization of cultural conditions using response surface methodology versus artificial neural network and modeling of L-glutaminase production by Bacillus cereus MTCC 1305[J].Bioresource Technology,2013,137:261-269.DOI:10.1016/j.biortech.2013.03.086.
[25]SUWANMANON K,HSIEH P C.Isolating Bacillus subtilis and optimizing its fermentative medium for GABA and nattokinase production[J].CyTA,2014,12(3):282-290.DOI:10.1080/19476337.2013.848472.
[26]祝亚娇,宋嘉宾,陈杨阳,等.地衣芽胞杆菌工程菌高产纳豆激酶的发酵罐工艺优化及中试放大[J].食品与发酵工业,2016,42(1):37-41.DOI:10.13995/j.cnki.11-1802/ts.201601007.
[27]WEI X,ZHOU Y,CHEN J,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology&Biotechnology,2015,42(2):287-295.DOI:10.1007/s10295-014-1559-4.
[28]YIN L J,LIN H H,JIANG S T.Bioproperties of potent nattokinase from Bacillus subtilis YJ1[J].Journal of Agricultural&Food Chemistry,2010,58(9):5737-5742.DOI:10.1021/jf100290h.
[2]SUMI H,HAMADA H,TSUSHIMA H,et al.A novel fibrinolytic enzyme(nattokinase)in the vegetable cheese natto;a typical and popular soybean food in the Japanese diet[J].Experientia,1987,43(10):1110-1111.DOI:10.1007/BF01956052.
[3]HUANG S H,PAN S H,CHEN G G,et al.Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium,Bacillus subtilis HQS-3[J].International Journal of Biological Macromolecules,2013,62:124-130.DOI:1016/j.ijbiomac.2013.08.048.
[4]MURAKAMI K,YAMANAKA N,OHNISHI K,et al.Inhibition of angiotensin I converting enzyme by subtilisin nat(nattokinase)in natto,a Japanese traditional fermented food[J].Food&Function,2012,3(6):674-678.DOI:10.1039/c2fo10245e.
[5]葛春蕾.高效分泌表达纳豆激酶的重组枯草芽孢杆菌的构建[D].无锡:江南大学,2016.
[6]JING R,LI H Q,HU C L,et al.Phytochemical and pharmacological profiles of three fagopyrum buckwheats[J].International Journal of Molecular Sciences,2016,17(4):589.DOI:10.3390/ijms17040589.
[7]王世霞,李笑蕊,贠婷婷,等.不同品种苦荞麦营养及功能成分对比分析[J].食品与机械,2016,32(7):5-9.
[8]ZHAO G,PENG L X,WANG S,et al.HPLC Fingerprint-antioxidant properties study of buckwheat[J].Journal of Integrative Agriculture,2012,11(7):1111-1118.DOI:10.1016/S2095-3119(12)60104-X.
[9]赵谋明,邹颖,林恋竹,等.纳豆菌液态发酵谷物产纳豆激酶及发酵产物抗氧化活性研究[J].现代食品科技,2018,34(2):1-6.
[10]赵芳芳.大豆肽的生物学功能研究[D].北京:中国农业大学,2004.
[11]万春艳.大豆活性肽对酵母增殖代谢及啤酒发酵的影响研究[D].广州:华南理工大学,2012.
[12]ASTRUP T,MULLERTZ S.The fibrin plate method for estimating fibrinolytic activity[J].Archives of Biochemistry and Biophysics,1952,40:346-351.DOI:10.1016/0003-9861(52)90121-5.
[13]Japan Nattokinase Association.Degradation of artificial thrombus by nattokinase[EB/OL].2012-05-18[2018-01-20].http://jnattokinase.org/en/jnka_nattou_1.html.
[14]赵凯,许鹏举,谷广烨.3,5-二硝基水杨酸比色法测定还原糖含量的研究[J].食品科学,2008,29(8):534-536.
[15]LIN L Z,ZHAO H F,DONG Y,et al.Macroporous resin purification behavior of phenolics and rosmarinic acid from Rabdosia serra(Maxim.)Hara leaf[J].Food Chemistry,2012,130(2):417-424.DOI:10.1016/j.foodchem.2011.07.069.
[16]林恋竹,焦铭.光果甘草叶中性多糖结构表征及抗氧化活性研究[J].现代食品科技,2016,32(1):106-110.DOI:10.13982/j.mfst.1673-9078.2016.1.017.
[17]雷芬芬.深海菌株Exiguobacterium sp.SWJS2发酵产蛋白酶、酶学性质及酶解特异性研究[D].广州:华南理工大学,2014.
[18]李小丽.纳豆芽孢杆菌分离及纳豆激酶活性的研究[D].合肥:安徽农业大学,2009.
[19]谢秋玲.纳豆激酶液体发酵的条件优化及其调控的研究[D].广州:华南理工大学,1999.
[20]史一一.荞麦淀粉酶水解特性研究[D].杨凌:西北农林科技大学,2007.
[21]谢秋玲,郭勇,林剑,等.纳豆激酶的合成机制[J].暨南大学学报(自然科学与医学版),2001(3):110-114.DOI:10.3969/j.issn.1000-9965.2001.03.023.
[22]莫芬.小麦面筋蛋白水解物对酵母增殖代谢及啤酒发酵的影响研究[D].广州:华南理工大学,2014.
[23]毕静.糖化酶发酵生产中溶解氧的研究[J].中国酿造,2015,34(8):97-100.DOI:10.11882/j.issn.0254-5071.2015.08.020.
[24]SINGH P,SHERA S S,BANIK J,et al.Optimization of cultural conditions using response surface methodology versus artificial neural network and modeling of L-glutaminase production by Bacillus cereus MTCC 1305[J].Bioresource Technology,2013,137:261-269.DOI:10.1016/j.biortech.2013.03.086.
[25]SUWANMANON K,HSIEH P C.Isolating Bacillus subtilis and optimizing its fermentative medium for GABA and nattokinase production[J].CyTA,2014,12(3):282-290.DOI:10.1080/19476337.2013.848472.
[26]祝亚娇,宋嘉宾,陈杨阳,等.地衣芽胞杆菌工程菌高产纳豆激酶的发酵罐工艺优化及中试放大[J].食品与发酵工业,2016,42(1):37-41.DOI:10.13995/j.cnki.11-1802/ts.201601007.
[27]WEI X,ZHOU Y,CHEN J,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology&Biotechnology,2015,42(2):287-295.DOI:10.1007/s10295-014-1559-4.
[28]YIN L J,LIN H H,JIANG S T.Bioproperties of potent nattokinase from Bacillus subtilis YJ1[J].Journal of Agricultural&Food Chemistry,2010,58(9):5737-5742.DOI:10.1021/jf100290h.