N~+注入选育产ACE抑制的保加利亚乳杆菌
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摘要
生物体内的许多系统都与血压形成有关,而肾素-血管紧张素系统(RAS)和激肽-激肽释放酶系统(KKS)是两个重要的血压调节控制系统。在这两个系统中,血管紧张素转化酶(ACE)起着重要的调节作用。ACE抑制剂可使血管紧张素Ⅰ(AngⅠ)转化为血管紧张素Ⅱ(AngⅡ),从而起到降压的作用。目前,大多数用于治疗高血压的药的ACE抑制肽类药物,物都是人工合成的,虽然降压效果明显,但是副作用大,利用乳酸菌开发制备ACE抑制肽虽然效果不及人工合成,但其安全性较高,并对血压正常者无降压效果,现已成为研发的重点,具有安全降血压的作用。
实验一:本实验对实验室保存的9株保加利亚乳杆菌的基本生长特性及ACE抑制率大小进行比较,用于对出发菌株的生产性能充分了解。结果表明,不同菌株凝乳的致密性、滴定酸度、生物量等存在较大的差异,不同菌株个体间的ACE抑制活性差异也较大,通过对四株产ACE抑制肽活性较高的菌株研究发现,其共同点是具有较高的滴定酸度并且在发酵乳中生长良好。
实验二:利用N+注入技术对保加利亚乳杆菌进行诱变,以ACE抑制率为指标进行选育,以获得高产突变菌株。当N+注入能量在20 KeV,剂量设定为五个梯度,分别为0.5、1.0、1.5、2.0、2.5×1015 ions/cm2。结果表明,随着注入量的增加,其菌株存活率呈“马鞍型”曲线。通过试验,确立最佳离子诱变参数为:能量为20 KeV,剂量为1.5×1015 ions/cm2,在此条件下获得了四株高产菌株M601、M602、M603、M606,并通过稳定性试验,获得高产菌株M602-4-1。
实验三:通过L9(34)正交实验设计,研究不同的脱脂乳浓度、接种量、发酵时间和发酵温度对高产菌株M602-4-1产ACE抑制肽的影响,确立高产菌株最佳培养条件。在此基础上,设计五种不同氮源配比的培养基,根据高产菌的生长特性,确立最佳培养基命名为MRS-N3,在此培养基上菌株的生长优于MRS培养基。
实验四:高产菌株M602-4-1与出发菌株M602生长性能的比较。通过对生物量、滴定酸度、粘度以及形态方面进行对比,对诱变前后菌株经行对比。实验结果证明,诱变后的高产菌株无论在生物量、滴定酸度、粘度方面均有提高,在形态上有较大的改变。
In organism many systems affect the blood pressure in which renin-angiotensin system and bradykinin-Kallikrein system are two important modulators for blood pressure control system. Angiotensin converting enzyme (ACE) plays an important regulatory role in these two systems, and ACE inhibitors can transform the angiotensinⅠ(AngⅠ) into angiotensinⅡ(AngⅡ). Currently, most drugs used to treat high blood pressure are synthetic ACE inhibitory peptides. The antihypertensive effect was significant, while the side-effects were also large. Although the antihypertensive effect of ACE inhibitory peptides attained from lactic acid bacteria are weaker than synthetic ACE inhibitory peptides, but it was much safer and has no side-effects for the people with normal blood pressure. Therefore, ACE inhibitory peptides from lactic acid bacteria have become the focus of research and development. It can achieve safety effect to lower the blood pressure.
Experiment 1: This study compared the basic growth characteristics and ACE inhibition rates among nine strain lactobacillus Bulgaria in our laboratory. It was shown that the density, titratable acidity and biomass are very different of different strain curds. The ACE inhibitory activity among different strains was also different. It was also found that the four strains which can produce more ACE inhibitor show the common characteristic. That is they had a higher titratable acidity and good growth in the fermented milk.
Experiment 2: We selected and bred lactobacillus Bulgaria through N+ implantation technique based on the ACE inhibition rate. We set five dose gradient (0.5, 1.0, 1.5, 2.0, 2.5×1015 ions/cm2) when the N+ implantation energy is 20 KeV. It is shown that the surviral rate of strains shows a "saddle shape" curve along with the increase of injection. Through the experiment, we established the best ion mutation parameters: energy of 20 KeV, a dose of 1.5×1015 ions/cm2. Under this condition, we obtained four strains: M601, M602, M603 and M606. At last, we obtained the high-yield strains M602-4-1 through the stability experiments.
Experiment 3: We used the L9 (34) orthogonal experimental design to study high-yield strain M602-4-1. We studied the impact of different concentrations of skim milk, inoculums size, fermentation time and fermentation temperature upon the yield of the ACE inhibitory peptides, and we established the optimum culture conditions. We got an optimum culture medium MRS-N3 based on the growth characteristics of high-yield strains which grew in 5 different ratios of nitrogen culture-medium. Strains in this mediun grew better than in MRS medium.
Experiment 4: The comparison of growth performance between mutagenesis high-yield strains M602-4-1 and starting strains M602 in biomass, titratable acidity, viscosity, and morphology. It was shown that high-yield strains have increased in biomass, titratable acidity and viscosity, and the morphous greatly changed after mutated.
实验一:本实验对实验室保存的9株保加利亚乳杆菌的基本生长特性及ACE抑制率大小进行比较,用于对出发菌株的生产性能充分了解。结果表明,不同菌株凝乳的致密性、滴定酸度、生物量等存在较大的差异,不同菌株个体间的ACE抑制活性差异也较大,通过对四株产ACE抑制肽活性较高的菌株研究发现,其共同点是具有较高的滴定酸度并且在发酵乳中生长良好。
实验二:利用N+注入技术对保加利亚乳杆菌进行诱变,以ACE抑制率为指标进行选育,以获得高产突变菌株。当N+注入能量在20 KeV,剂量设定为五个梯度,分别为0.5、1.0、1.5、2.0、2.5×1015 ions/cm2。结果表明,随着注入量的增加,其菌株存活率呈“马鞍型”曲线。通过试验,确立最佳离子诱变参数为:能量为20 KeV,剂量为1.5×1015 ions/cm2,在此条件下获得了四株高产菌株M601、M602、M603、M606,并通过稳定性试验,获得高产菌株M602-4-1。
实验三:通过L9(34)正交实验设计,研究不同的脱脂乳浓度、接种量、发酵时间和发酵温度对高产菌株M602-4-1产ACE抑制肽的影响,确立高产菌株最佳培养条件。在此基础上,设计五种不同氮源配比的培养基,根据高产菌的生长特性,确立最佳培养基命名为MRS-N3,在此培养基上菌株的生长优于MRS培养基。
实验四:高产菌株M602-4-1与出发菌株M602生长性能的比较。通过对生物量、滴定酸度、粘度以及形态方面进行对比,对诱变前后菌株经行对比。实验结果证明,诱变后的高产菌株无论在生物量、滴定酸度、粘度方面均有提高,在形态上有较大的改变。
In organism many systems affect the blood pressure in which renin-angiotensin system and bradykinin-Kallikrein system are two important modulators for blood pressure control system. Angiotensin converting enzyme (ACE) plays an important regulatory role in these two systems, and ACE inhibitors can transform the angiotensinⅠ(AngⅠ) into angiotensinⅡ(AngⅡ). Currently, most drugs used to treat high blood pressure are synthetic ACE inhibitory peptides. The antihypertensive effect was significant, while the side-effects were also large. Although the antihypertensive effect of ACE inhibitory peptides attained from lactic acid bacteria are weaker than synthetic ACE inhibitory peptides, but it was much safer and has no side-effects for the people with normal blood pressure. Therefore, ACE inhibitory peptides from lactic acid bacteria have become the focus of research and development. It can achieve safety effect to lower the blood pressure.
Experiment 1: This study compared the basic growth characteristics and ACE inhibition rates among nine strain lactobacillus Bulgaria in our laboratory. It was shown that the density, titratable acidity and biomass are very different of different strain curds. The ACE inhibitory activity among different strains was also different. It was also found that the four strains which can produce more ACE inhibitor show the common characteristic. That is they had a higher titratable acidity and good growth in the fermented milk.
Experiment 2: We selected and bred lactobacillus Bulgaria through N+ implantation technique based on the ACE inhibition rate. We set five dose gradient (0.5, 1.0, 1.5, 2.0, 2.5×1015 ions/cm2) when the N+ implantation energy is 20 KeV. It is shown that the surviral rate of strains shows a "saddle shape" curve along with the increase of injection. Through the experiment, we established the best ion mutation parameters: energy of 20 KeV, a dose of 1.5×1015 ions/cm2. Under this condition, we obtained four strains: M601, M602, M603 and M606. At last, we obtained the high-yield strains M602-4-1 through the stability experiments.
Experiment 3: We used the L9 (34) orthogonal experimental design to study high-yield strain M602-4-1. We studied the impact of different concentrations of skim milk, inoculums size, fermentation time and fermentation temperature upon the yield of the ACE inhibitory peptides, and we established the optimum culture conditions. We got an optimum culture medium MRS-N3 based on the growth characteristics of high-yield strains which grew in 5 different ratios of nitrogen culture-medium. Strains in this mediun grew better than in MRS medium.
Experiment 4: The comparison of growth performance between mutagenesis high-yield strains M602-4-1 and starting strains M602 in biomass, titratable acidity, viscosity, and morphology. It was shown that high-yield strains have increased in biomass, titratable acidity and viscosity, and the morphous greatly changed after mutated.
引文
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