玉米粉生物修饰菌种的筛选与改良研究
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摘要
玉米是三大主食谷物(小麦、水稻、玉米)之一,在保障全球食物安全方面扮演重要角色。但由于自身成分的组成等原因,玉米并不适合用于制作主食品。如何将其修饰改性,使玉米粉能够像小麦粉一样,用于制做饺子、面条等主食品的生产,是当今玉米科学研究的重大科学技术问题。本论文选用重要微生物,对玉米粉进行生物修饰改性,期望改善玉米粉的食用及加工品质。主要结论如下：
1、选用了胞外酶分泌能力强,安全、常用的枯草芽孢杆菌B、米曲霉、黑曲霉、根毛霉、总状毛霉、微小毛霉等几种微生物的胞外酶对玉米粉进行了生物修饰改性研究,对比分析了玉米粉改性前后凝胶性质、糊化性质、面团的质构性质、玉米粉的X-射线衍射及玉米粉颗粒的电镜观察等指标的变化。试验结果表,在选用的几种生物酶中,枯草芽孢杆菌B胞外酶对玉米粉的修饰改良作用最明显,因此,选用枯草芽孢杆菌B对玉米粉进行修饰改性研究。
2、对比分析了枯草芽孢杆菌B胞外酶修饰前后玉米粉中主要成分的变化,玉米粉经过修饰后,其粗脂肪、可溶固形物、粗灰分等相对含量都有增加,而真蛋白的含量明显减少,总氨基酸含量比对照玉米粉增加了26.46%。玉米粉中总淀粉和支链淀粉含量下降明显,而直链淀粉的相对含量略有增加。
3、玉米粉中蛋白的主要成分为醇溶蛋白及球蛋白等,不含有面筋蛋白,所以制成品缺乏粘弹性,柔韧性也很差,严重影响玉米食品的食用品质及加工品质。所以,本论文以提高菌株分泌蛋白酶能力为目标,采用紫外诱变、亚硝基胍(NTG)诱变、硫酸二乙酯(DES)诱变、微波辐射诱变等四种方法分别对出发枯草芽孢杆菌B进行了遗传改造,筛选出7株遗传稳定性较好,蛋白酶分泌能力较出发枯草芽孢杆菌B(699.62U/m1)明显提高的菌株,即BU-18、BU-61、BN-32、BN-84、BD-5、BD-73及BM-74,它们的蛋白酶分泌能力分别达到1274.98U/ml、1102.56U/ml、1198.51U/ml、947.62U/ml、899.67U/ml、1201.11U/ml及1089.34U/ml。
4、为了能够采用全基因组改组的方法,进一步提高菌株的蛋白酶分泌能力,确定了菌株原生质体制备及融合处理的最佳条件,在酶浓度10mg/mL,菌龄10h,酶解温度38℃,酶解时间90min条件下,枯草芽孢杆菌B原生质体制备率及再生率最高。在融合温度35℃,聚乙二醇(PEG)4000浓度在35%,CaCl2浓度为0.02mol/L时,融合时间l0min条件下,枯草芽孢杆菌原生质体的融合率最高。通过两轮的全基因组改组,筛选出改良菌株BSGl,该菌分泌的蛋白酶酶活最高达到2175.81U/ml,为原始菌株B(699.62U/m1)的3.11倍,而淀粉酶的分泌量则较原始菌株没有明显变化。而且,蛋白酶及淀粉酶的分泌能力都具有较好的遗传稳定性。
5、采用改良前后的菌株对玉米粉进行了修饰改性的比较研究,菌株改良后对玉米粉中真蛋白的分解能力明显增强,而对淀粉的影响不明显。玉米粉的凝胶性质、糊化性质及玉米面团的质构性质等方面也都较原始菌株处理的有明显改善,其中玉米面团的凝聚力增幅达到18.98%,胶着性增幅达到52.6%,咀嚼性增幅达到60.95%,回弹增幅达到25.89%。凝聚力增大,说明玉米粉分子内部间结合力增大,即玉米淀粉形成凝胶的网络增强。弹性、回弹能力的增强表明玉米面团的延展性较好,同时,胶着性、咀嚼性增大表明玉米面团更有韧性。
6、优化了枯草芽孢杆菌BSG1产蛋白酶的条件。优化出的最佳培养基为：大豆粉2%,玉米粉1%,硫酸镁0.6%。最佳培养条件为：pH值7.0,摇瓶装液量150/500m1,接种量5%,摇床转速为280r/min,35℃发酵24h。在这一条件下,枯草芽孢杆菌BSG1分泌的蛋白酶酶活达到2469.12U/ml,为发酵条件优化前(2175.81U/m1)的1.13倍,通过优化,不但提高了菌株的蛋白酶产量,更优化出了价格低廉的农产品作为碳氮源,取代了比较昂贵的生化试剂,这将会极大的降低菌株蛋白酶生产及玉米粉生物修饰的成本。
7、以玉米粉面团的咀嚼性为标准,优化了生物酶修饰玉米粉的酶促反应条件,即在蛋白酶酶浓度为800U/m1,20%的玉米粉作为底物,反应体系pH值为5.501,反应温度保持在51.720℃,反应时间达到18.790h条件下,玉米面团的最大咀嚼性可达到511.977g,为原始菌株枯草芽孢杆菌B修饰玉米粉的2.32倍,具有制作主食品的潜力。并建立了模拟方程,可为玉米粉生物酶修饰的工业化生产提供理论依据,也为玉米主食工业化的继续研究奠定了坚实的基础。
Corn is one of the three main food grains (wheat, rice, corn) and plays an important role in global food security. But because of its own composition, corn is not suitable for used to make the main food. How to improve the food and processing quality of corn flour and to make it suitable for the production of noodles, dumplings and other foods, is the corn research major scientific technology. This paper choose important microorganisms to modify the corn flour, hopes to improve the food and processing characteristics of corn flour,and make it suitable for used to make noodles, dumplings main food products, The main conclusions are as follows:
1. We selected extracellular enzymes of microorganism, such as Bacillus subtilis B, Aspergillus oryzae, Aspergillus niger, Mucor pusillus, Rhizomucor and Mucor racemosus etc,to study the modification properties of biological enzyme of corn meal. The study investigated the solubility, water holding capacity,gel swelling, the physical properties, the pasting properties, corn powder X-ray diffraction (XRD) and microscopic observation of corn meal before and after modification. The results showed that corn dough modified by extracellular enzymes of Bacillus subtilisB was improved obviously in many aspects.So select the Bacillus subtilis B as the starting strain of corn flour modified.
2. In this study, composition changes of corn flour were measured pre and post the modification of biological enzyme. After modified by enzyme, the fat, soluble solids, crude ash of corn meal were increased, with decreasing in true protein content of corn meal. the total amino acid content were increased by26.46%. The total starch and amylopectin starch content decreased significantly, meanwhile, amylose relative content increased.
3. To modify the original strain B, we use four methods as follows:UV mutagenesis, nitrosoguanidine (NTG) mutagenesis,mutagenesis of diethyl sulfate (DES),microwave radiation mutagenesis. We obtained7strains of bacteria were genetic stability is good, and protease ability were obviously improved than start bacillus B (699.62U/ml). That is BU-18, BU-61, BN-32, BN-84, BD-5, BD-73and BM-74, their protease ability is1274.98U/ml,1102.56U/ml,1198.51U/ml,947.62U/ml,899.67U/ml,1201.11U/ml and1089.34U/ml respectively.
4. We determine the optimum conditions of the Bacillus subtilis protoplast formation and fusion, the protoplast formation and regeneration rate is highest under following conditions: enzyme concentration of10mg/mL, bacteria age10h,hydrolysis temperature38℃, hydrolysis time was90min. The highest rate of Bacillus subtilis protoplast fusion happened under following conditions:temperature of35℃, PEG4000concentration35%, CaCl2concentration0.02mol/L, integration time10min. Through two rounds of genome shuffling, we get a genetic stability ideal mutant strain BSG1with protease activity2175.81U/ml,3.11times higher than the original strain,and the secretion of amylase secretion capacity than the original strain B did not change significantly.
5. By comparison of the modification on corn flour by Bacillus subtilis between pre and post, the result shows that the improvement strain enhanced significantly in decomposition of true protein in corn meal, and have no obviously change when it act on starch. There are markedly improvements in the gel nature、pasting properties and TPA properties of corn flour, which are all better than com flour treated by original strain of extracellular enzymes. The springiness, gumminess,chewing and resilience increased by18.98%,52.6%,60.95%,25.89%respectively.
6. The best culture condition for genetic transformation of Bacillus sutillus BSG1is as follows:2%soybean meal, corn flour1%,0.6%magnesium sulfate, medium pH7.0、bottle volume150/500ml、the initial inoculum5%、rotation speed280r/min and fermentation24hours at35℃. In this condition, bacillus subtilis BSG1secretion of protease enzyme reached2469.12U/ml, which1.13times compared with before optimization (2175.81U/ml). Not only increased production strains of protease, more optimized carbon and nitrogen sources, It will greatly reduce the production and com flour protease biological modified cost.
7. Using corn dough chewing as the standard, the enzymatic reaction conditions of the modified cornmeal by biological enzyme was optimized, which is protease enzyme concentration800U/ml,20%of the com flour as a substrate, pH5.501, temperature51.720℃, reaction time18.790h. Under above condition, com dough chewing could get up to511.977g, which2.32times compared with com flour.It has the potential to make main food.
1、选用了胞外酶分泌能力强,安全、常用的枯草芽孢杆菌B、米曲霉、黑曲霉、根毛霉、总状毛霉、微小毛霉等几种微生物的胞外酶对玉米粉进行了生物修饰改性研究,对比分析了玉米粉改性前后凝胶性质、糊化性质、面团的质构性质、玉米粉的X-射线衍射及玉米粉颗粒的电镜观察等指标的变化。试验结果表,在选用的几种生物酶中,枯草芽孢杆菌B胞外酶对玉米粉的修饰改良作用最明显,因此,选用枯草芽孢杆菌B对玉米粉进行修饰改性研究。
2、对比分析了枯草芽孢杆菌B胞外酶修饰前后玉米粉中主要成分的变化,玉米粉经过修饰后,其粗脂肪、可溶固形物、粗灰分等相对含量都有增加,而真蛋白的含量明显减少,总氨基酸含量比对照玉米粉增加了26.46%。玉米粉中总淀粉和支链淀粉含量下降明显,而直链淀粉的相对含量略有增加。
3、玉米粉中蛋白的主要成分为醇溶蛋白及球蛋白等,不含有面筋蛋白,所以制成品缺乏粘弹性,柔韧性也很差,严重影响玉米食品的食用品质及加工品质。所以,本论文以提高菌株分泌蛋白酶能力为目标,采用紫外诱变、亚硝基胍(NTG)诱变、硫酸二乙酯(DES)诱变、微波辐射诱变等四种方法分别对出发枯草芽孢杆菌B进行了遗传改造,筛选出7株遗传稳定性较好,蛋白酶分泌能力较出发枯草芽孢杆菌B(699.62U/m1)明显提高的菌株,即BU-18、BU-61、BN-32、BN-84、BD-5、BD-73及BM-74,它们的蛋白酶分泌能力分别达到1274.98U/ml、1102.56U/ml、1198.51U/ml、947.62U/ml、899.67U/ml、1201.11U/ml及1089.34U/ml。
4、为了能够采用全基因组改组的方法,进一步提高菌株的蛋白酶分泌能力,确定了菌株原生质体制备及融合处理的最佳条件,在酶浓度10mg/mL,菌龄10h,酶解温度38℃,酶解时间90min条件下,枯草芽孢杆菌B原生质体制备率及再生率最高。在融合温度35℃,聚乙二醇(PEG)4000浓度在35%,CaCl2浓度为0.02mol/L时,融合时间l0min条件下,枯草芽孢杆菌原生质体的融合率最高。通过两轮的全基因组改组,筛选出改良菌株BSGl,该菌分泌的蛋白酶酶活最高达到2175.81U/ml,为原始菌株B(699.62U/m1)的3.11倍,而淀粉酶的分泌量则较原始菌株没有明显变化。而且,蛋白酶及淀粉酶的分泌能力都具有较好的遗传稳定性。
5、采用改良前后的菌株对玉米粉进行了修饰改性的比较研究,菌株改良后对玉米粉中真蛋白的分解能力明显增强,而对淀粉的影响不明显。玉米粉的凝胶性质、糊化性质及玉米面团的质构性质等方面也都较原始菌株处理的有明显改善,其中玉米面团的凝聚力增幅达到18.98%,胶着性增幅达到52.6%,咀嚼性增幅达到60.95%,回弹增幅达到25.89%。凝聚力增大,说明玉米粉分子内部间结合力增大,即玉米淀粉形成凝胶的网络增强。弹性、回弹能力的增强表明玉米面团的延展性较好,同时,胶着性、咀嚼性增大表明玉米面团更有韧性。
6、优化了枯草芽孢杆菌BSG1产蛋白酶的条件。优化出的最佳培养基为：大豆粉2%,玉米粉1%,硫酸镁0.6%。最佳培养条件为：pH值7.0,摇瓶装液量150/500m1,接种量5%,摇床转速为280r/min,35℃发酵24h。在这一条件下,枯草芽孢杆菌BSG1分泌的蛋白酶酶活达到2469.12U/ml,为发酵条件优化前(2175.81U/m1)的1.13倍,通过优化,不但提高了菌株的蛋白酶产量,更优化出了价格低廉的农产品作为碳氮源,取代了比较昂贵的生化试剂,这将会极大的降低菌株蛋白酶生产及玉米粉生物修饰的成本。
7、以玉米粉面团的咀嚼性为标准,优化了生物酶修饰玉米粉的酶促反应条件,即在蛋白酶酶浓度为800U/m1,20%的玉米粉作为底物,反应体系pH值为5.501,反应温度保持在51.720℃,反应时间达到18.790h条件下,玉米面团的最大咀嚼性可达到511.977g,为原始菌株枯草芽孢杆菌B修饰玉米粉的2.32倍,具有制作主食品的潜力。并建立了模拟方程,可为玉米粉生物酶修饰的工业化生产提供理论依据,也为玉米主食工业化的继续研究奠定了坚实的基础。
Corn is one of the three main food grains (wheat, rice, corn) and plays an important role in global food security. But because of its own composition, corn is not suitable for used to make the main food. How to improve the food and processing quality of corn flour and to make it suitable for the production of noodles, dumplings and other foods, is the corn research major scientific technology. This paper choose important microorganisms to modify the corn flour, hopes to improve the food and processing characteristics of corn flour,and make it suitable for used to make noodles, dumplings main food products, The main conclusions are as follows:
1. We selected extracellular enzymes of microorganism, such as Bacillus subtilis B, Aspergillus oryzae, Aspergillus niger, Mucor pusillus, Rhizomucor and Mucor racemosus etc,to study the modification properties of biological enzyme of corn meal. The study investigated the solubility, water holding capacity,gel swelling, the physical properties, the pasting properties, corn powder X-ray diffraction (XRD) and microscopic observation of corn meal before and after modification. The results showed that corn dough modified by extracellular enzymes of Bacillus subtilisB was improved obviously in many aspects.So select the Bacillus subtilis B as the starting strain of corn flour modified.
2. In this study, composition changes of corn flour were measured pre and post the modification of biological enzyme. After modified by enzyme, the fat, soluble solids, crude ash of corn meal were increased, with decreasing in true protein content of corn meal. the total amino acid content were increased by26.46%. The total starch and amylopectin starch content decreased significantly, meanwhile, amylose relative content increased.
3. To modify the original strain B, we use four methods as follows:UV mutagenesis, nitrosoguanidine (NTG) mutagenesis,mutagenesis of diethyl sulfate (DES),microwave radiation mutagenesis. We obtained7strains of bacteria were genetic stability is good, and protease ability were obviously improved than start bacillus B (699.62U/ml). That is BU-18, BU-61, BN-32, BN-84, BD-5, BD-73and BM-74, their protease ability is1274.98U/ml,1102.56U/ml,1198.51U/ml,947.62U/ml,899.67U/ml,1201.11U/ml and1089.34U/ml respectively.
4. We determine the optimum conditions of the Bacillus subtilis protoplast formation and fusion, the protoplast formation and regeneration rate is highest under following conditions: enzyme concentration of10mg/mL, bacteria age10h,hydrolysis temperature38℃, hydrolysis time was90min. The highest rate of Bacillus subtilis protoplast fusion happened under following conditions:temperature of35℃, PEG4000concentration35%, CaCl2concentration0.02mol/L, integration time10min. Through two rounds of genome shuffling, we get a genetic stability ideal mutant strain BSG1with protease activity2175.81U/ml,3.11times higher than the original strain,and the secretion of amylase secretion capacity than the original strain B did not change significantly.
5. By comparison of the modification on corn flour by Bacillus subtilis between pre and post, the result shows that the improvement strain enhanced significantly in decomposition of true protein in corn meal, and have no obviously change when it act on starch. There are markedly improvements in the gel nature、pasting properties and TPA properties of corn flour, which are all better than com flour treated by original strain of extracellular enzymes. The springiness, gumminess,chewing and resilience increased by18.98%,52.6%,60.95%,25.89%respectively.
6. The best culture condition for genetic transformation of Bacillus sutillus BSG1is as follows:2%soybean meal, corn flour1%,0.6%magnesium sulfate, medium pH7.0、bottle volume150/500ml、the initial inoculum5%、rotation speed280r/min and fermentation24hours at35℃. In this condition, bacillus subtilis BSG1secretion of protease enzyme reached2469.12U/ml, which1.13times compared with before optimization (2175.81U/ml). Not only increased production strains of protease, more optimized carbon and nitrogen sources, It will greatly reduce the production and com flour protease biological modified cost.
7. Using corn dough chewing as the standard, the enzymatic reaction conditions of the modified cornmeal by biological enzyme was optimized, which is protease enzyme concentration800U/ml,20%of the com flour as a substrate, pH5.501, temperature51.720℃, reaction time18.790h. Under above condition, com dough chewing could get up to511.977g, which2.32times compared with com flour.It has the potential to make main food.
引文
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