细胞外细胞毒凝集素枯草芽孢杆菌IMV B-7724在实验室发酵条件下合成的特点

Q4 Biochemistry, Genetics and Molecular Biology
O. Kisten, K. Hetman, E. V. Koval, I. Hretskyi, L.F. Zyryanova, L. Tyshchenko, N. Fedosova, N. Cheremshenko, A. Chumak
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Batch antifoam-free fermentations were performed by culturing the strain in the modified Gause medium with galactose in two identical lab-scale fermenters with a working volume of 2.5 L at 37ºC for 48—72 h according to three fermentation variants. Variant 1: n — 400 rpm for the whole cultivation, the air supply to the CF — through a sparger at 0.1 vvm until the 39th h with further gradual decrease, KV — 4.2±0.3 g O2·L−1·h−1. Variant 2: n — 400 rpm for the first 24 h, then a gradual decrease to 200 rpm, air supply — through a sparger at 0.1 rpm for the first 12 h, followed by its switching into the fermenter free space, corresponding KV — from 4.2±0.3 to 0.3±0.1 g O2·L−1·h−1. Variant 3: n — 400 rpm and air supply to the fermenter free space during the whole cultivation, KV — 4.0±0.3 g O2·L−1·h−1. A number of biological properties of strain CF and isolated lectin samples were evaluated by biochemical, spectrophotometric, immunological, and culture methods. Statistical analysis was performed using Student’s t-test. Results. The maximum increase in the OD of CF relative to the initial values (28 and 21-fold) at the end of the period of the rapid growth of the strain (at 9th h), the μmax values of 0.33 and 0.41 h−1, and pH not lower than 6.7 and 6.3 units were observed for fermentation variants 1 and 2, respectively. In the case of variant 2, the HAA of CF reached 32 hemagglutinating units (HAU), and the samples isolated from it had a lectin activity of 512±64 HAU, whereas for variant 1 such values were lower:16 and 32±8 HAA, respectively; carbohydrate specificity of preparations to bovine submandibular gland mucin was the same, i.e. 0.2±0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μmax, and significant acid formation (15-fold at the 9th h, 0.25 h−1, and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2—4 HAU) and was absent in the corresponding isolated samples. The probable reason for such differences was the limited mass transfer in the CF due to the isolating effect of the foam layer on its surface formed as a result of intensive agitation. Conclusions. 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引用次数: 0

摘要

氧传质水平(KV)是影响好氧微生物生长速度和代谢产物合成的重要参数。这主要取决于发酵箱内的搅拌和曝气率。的目标。研究产生细胞外毒性凝集素(ECL)的枯草芽孢杆菌(Bacillus subtilis)菌株IMV B-7724培养液(CF)在不同搅拌和通风率下的pH、光密度(OD)和血凝(凝集素)活性(HAA)的变化,并测定和比较从枯草芽孢杆菌中分离的相应样品的HAA、碳水化合物特异性和细胞毒性。根据三种发酵方式,将菌株在含半乳糖的改良Gause培养基中,在两个相同的实验室规模发酵罐中,工作体积为2.5 L, 37ºC下培养48-72 h,进行批量无泡沫发酵。变异1:在整个培养过程中,以0.1 vvm的速度通过分散器给CF供气,直至第39 h, KV - 4.2±0.3 g O2·L−1·h−1。变体2:前24小时n - 400转/分,然后逐渐降低到200转/分,供气-在前12小时以0.1转/分的速度通过一个分散器,然后将其切换到发酵罐自由空间,相应的KV -从4.2±0.3到0.3±0.1 g O2·L−1·h−1。变体3:在整个培养过程中,n - 400rpm并向发酵罐自由空间供气,KV - 4.0±0.3 g O2·L−1·h−1。通过生化、分光光度、免疫学和培养等方法,对菌株CF和分离的凝集素样品的生物学特性进行了评价。统计学分析采用Student’s t检验。结果。菌株快速生长结束时(第9 h), CF的OD值较初始值(28倍和21倍)最大,μmax值分别为0.33和0.41 h−1,pH值分别不低于6.7和6.3个单位。在变异2中,CF的HAA达到32个血凝素单位(HAU),其凝集素活性为512±64个HAU,而变异1的这一数值较低,分别为16和32±8个HAA;各制剂对牛颌下腺黏液的糖特异性相同,均为0.2±0.1 mg/mL。与此相反,变异3的CF OD增加较慢,μmax降低,酸生成显著(第9 h 15倍,0.25 h−1,pH降至5.8单位);在这种情况下,CF的HAA水平极低(2-4 HAU),并且在相应的分离样品中不存在。产生这种差异的可能原因是CF中的传质有限,这是由于剧烈搅拌形成的泡沫层在其表面上的隔离作用。结论。在实验室规模的发酵罐培养过程中,通过分散器向CF供气,保持最大的氧传质水平,直到达到最大OD,然后在进一步培养过程中逐渐降低到规定的水平,观察到菌株的快速生长和CF的HAA的增加。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Features of the Synthesis of Extracellular Cytotoxic Lectin Bacillus subtilis IMV B-7724, Depending on the Cultivation Conditions in the Laboratory Fermenter
The level of oxygen mass transfer (KV) is an important parameter influencing the growth rate of aerobic microorganisms and the synthesis of metabolites. It is mainly determined by the agitation and the aeration rates in the fermenter. Aim. To study changes in pH, optical density (OD), and hemagglutinating (lectin) activity (HAA) of culture fluid (CF) of Bacillus subtilis strain IMV B-7724, a producer of extracellular cytotoxic lectin (ECL), during its cultivation in a laboratory fermenter at different agitation and aeration rates as well as to determine and compare the HAA, carbohydrate specifi city, and cytotoxic properties of the corresponding samples of the preparation isolated from CF. Methods. Batch antifoam-free fermentations were performed by culturing the strain in the modified Gause medium with galactose in two identical lab-scale fermenters with a working volume of 2.5 L at 37ºC for 48—72 h according to three fermentation variants. Variant 1: n — 400 rpm for the whole cultivation, the air supply to the CF — through a sparger at 0.1 vvm until the 39th h with further gradual decrease, KV — 4.2±0.3 g O2·L−1·h−1. Variant 2: n — 400 rpm for the first 24 h, then a gradual decrease to 200 rpm, air supply — through a sparger at 0.1 rpm for the first 12 h, followed by its switching into the fermenter free space, corresponding KV — from 4.2±0.3 to 0.3±0.1 g O2·L−1·h−1. Variant 3: n — 400 rpm and air supply to the fermenter free space during the whole cultivation, KV — 4.0±0.3 g O2·L−1·h−1. A number of biological properties of strain CF and isolated lectin samples were evaluated by biochemical, spectrophotometric, immunological, and culture methods. Statistical analysis was performed using Student’s t-test. Results. The maximum increase in the OD of CF relative to the initial values (28 and 21-fold) at the end of the period of the rapid growth of the strain (at 9th h), the μmax values of 0.33 and 0.41 h−1, and pH not lower than 6.7 and 6.3 units were observed for fermentation variants 1 and 2, respectively. In the case of variant 2, the HAA of CF reached 32 hemagglutinating units (HAU), and the samples isolated from it had a lectin activity of 512±64 HAU, whereas for variant 1 such values were lower:16 and 32±8 HAA, respectively; carbohydrate specificity of preparations to bovine submandibular gland mucin was the same, i.e. 0.2±0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μmax, and significant acid formation (15-fold at the 9th h, 0.25 h−1, and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2—4 HAU) and was absent in the corresponding isolated samples. The probable reason for such differences was the limited mass transfer in the CF due to the isolating effect of the foam layer on its surface formed as a result of intensive agitation. Conclusions. The rapid growth of the strain and an increase in the HAA of CF were observed during cultivation in a lab-scale fermenter by maintaining the maximum level of oxygen mass transfer with air supply into the CF through a sparger until the maximum OD was reached and the subsequent gradual decrease in the specifi ed level during further cultivation started.
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Mikrobiolohichnyi zhurnal
Mikrobiolohichnyi zhurnal Medicine-Microbiology (medical)
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