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Development of a low-serum medium for the production of monoclonal antibody against congenital adrenal hyperplasia by hybridoma culture 用杂交瘤培养生产抗先天性肾上腺增生单克隆抗体的低血清培养基的研制
Pub Date : 2016-01-13 DOI: 10.1080/10826068.2015.1135450
G. K. Chua
ABSTRACT Statistically designed experiments were used in developing a low-serum medium for the production of a diagnostic monoclonal antibody against congenital adrenal hyperplasia using hybridoma 192. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate, and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco’s modified Eagle’s medium (DMEM; containing 4 mM L-glutamine, 1% antibiotic–antimycotic agent) supplemented with 0.4% by volume fetal bovine serum (FBS), 311.8 mM ferric citrate, 17.3 nM sodium selenite, and 4.5 mM zinc sulfate (LSD) was found to support the growth of the hybridoma. Specific cell growth rate in the LSD (0.033 ± 0.001/h) was slightly lower than in the control medium (i.e., basal DMEM supplemented with 2% FBS; 0.0045 ± 0.003/h). Nevertheless, the specific MAb production rate for LSD was higher (0.057 ± 0.015 pg/cell · h versus 0.004 ± 0.002 pg/cell · h in LSD and control, respectively). The antibody produced in the LSD showed high specificity and no cross-reactivity with the other structural resemblance’s steroid hormones, revealing no structural changes owing to the new medium formulation developed. The new medium formulation effectively reduced the medium cost by up to 64.6%.
采用统计学设计的实验,开发了一种低血清培养基,用于生产针对先天性肾上腺增生的诊断性单克隆抗体。采用双水平半分数析因设计筛选6种成分(Minimum Essential Medium Eagle氨基酸、2-巯基乙醇、乙醇胺、柠檬酸铁、硫酸锌和亚硒酸钠)。然后将实验设计扩展为中心复合设计。基底Dulbecco 's改良Eagle 's培养基(DMEM;结果表明,添加0.4%胎牛血清(FBS)、311.8 mM柠檬酸铁、17.3 nM亚硒酸钠和4.5 mM硫酸锌(LSD)的培养基有利于杂杂瘤的生长。LSD中的特定细胞生长速率(0.033±0.001/h)略低于对照培养基(即在基础DMEM中添加2% FBS;0.0045±0.003/h)。然而,LSD的特异性MAb产生率更高(0.057±0.015 pg/cell·h,而LSD和对照组分别为0.004±0.002 pg/cell·h)。在LSD中产生的抗体具有高特异性,且与其他结构相似的类固醇激素无交叉反应性,表明新培养基配方未引起结构变化。新培养基配方有效降低培养基成本达64.6%。
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引用次数: 1
Impact of microbial growth inhibition and proteolytic activity on the stability of a new formulation containing a phytate-degrading enzyme obtained from mushroom 微生物生长抑制和蛋白水解活性对蘑菇植酸降解酶新配方稳定性的影响
Pub Date : 2016-01-13 DOI: 10.1080/10826068.2015.1135456
M. R. Spier, F. B. Siepmann, Larissa Staack, P. Z. Souza, Vikas Kumar, A. Medeiros, C. R. Soccol
ABSTRACT The development of stable enzymes is a key issue in both the food and feed industries. Consequently, the aim of the current study is to evaluate the impact of various additives (sodium chloride, sodium citrate, mannitol, methylparaben, polyethylene glycol 3350, ethylenediaminetetraacetic acid disodium salt, and a serine protease inhibitor) on the stability of a mushroom phytase produced by solid-state cultivation and recovery. Also observed was the effect of the additives on microbial growth inhibition by monitoring both the change in optical density over 30 days of storage and proteolytic activity. Initially, eight experimental formulations were prepared along with a control. After screening, a 32 factorial design was applied to define suitable concentrations of the selected additives. Among the eight formulations tested, the formulation containing NaCl, PEG 3350, and methylparaben retained all of the initial phytase activity after 50 days of storage, with no detected interference from protease activity. Sodium citrate, a metal chelation agent, presented the unusual effect of reducing protease activity in the formulations. Although all formulations presented better phytase stability when compared to the control, NaCl and PEG were both able to prolong the stability of the enzyme activity and also to inhibit microbial growth during storage, making them favorable for application as food and feed additives.
稳定酶的开发是食品和饲料工业的关键问题。因此,本研究的目的是评估各种添加剂(氯化钠、柠檬酸钠、甘露醇、对羟基苯甲酸甲酯、聚乙二醇3350、乙二胺四乙酸二钠盐和丝氨酸蛋白酶抑制剂)对通过固态培养和回收产生的蘑菇植酸酶稳定性的影响。通过监测贮藏30 d光密度变化和蛋白水解活性,观察了添加剂对微生物生长的抑制作用。最初,制备了8种实验配方并进行对照。筛选后,采用32因子设计来确定所选添加剂的合适浓度。在8个试验配方中,含有NaCl、PEG 3350和对羟基苯甲酸甲酯的配方在保存50天后保留了所有初始植酸酶活性,没有检测到蛋白酶活性的干扰。金属螯合剂柠檬酸钠在配方中表现出非同寻常的降低蛋白酶活性的作用。虽然与对照相比,所有配方均表现出更好的植酸酶稳定性,但NaCl和PEG均能延长酶活性的稳定性,并在储存过程中抑制微生物生长,有利于作为食品和饲料添加剂的应用。
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引用次数: 3
Development of a semidefined growth medium for Pedobacter cryoconitis BG5 using statistical experimental design 采用统计实验设计开发一种半确定的冷冻结肠炎杆菌BG5生长培养基
Pub Date : 2016-01-13 DOI: 10.1080/10826068.2015.1135447
M. Ong, Clarence M. Ongkudon, C. Wong
ABSTRACT Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K2HPO4 along with three additional nutrients (yeast extract, MgSO4, and NH4Cl) were identified to form the basal medium which was further analyzed by Plackett–Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH4Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600 nm) cell density of 7.5 using 23.7 g/L of tryptone, 8.8 g/L of yeast extract, and 0.7 g/L of NH4Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.
低温嗜冷杆菌BG5是从寒冷环境中分离出来的嗜冷菌,能够在低温条件下增殖和生长。他们的细胞产品已经发现了广泛的应用,包括食品,医药和生物修复。因此,开发高细胞密度的培养策略和优化的生长培养基对冻结菌BG5的培养具有重要意义。到目前为止,还没有关于冷冻结膜假单胞菌生长培养基设计和优化的报道,因此本研究项目的目的是。初步筛选了四种市售培养基,即色氨酸大豆肉汤、R2A、Luria Bertani肉汤和营养肉汤,以制定基础培养基。在初步筛选的基础上,确定了色氨酸、葡萄糖、NaCl和K2HPO4以及另外三种营养物质(酵母提取物、MgSO4和NH4Cl)构成基础培养基,并通过Plackett-Burman实验设计进一步分析。采用响应面法进行中心复合实验设计,优化培养基中色氨酸、酵母提取物和NH4Cl的浓度。统计数据分析表明,色氨酸浓度为23.7 g/L,酵母浸膏浓度为8.8 g/L, NH4Cl浓度为0.7 g/L,预测最佳光学(600 nm)细胞密度为7.5,回归系数为0.84。对优化后的冷冻结缔组织BG5培养基进行实验,观察到的光密度为7.8。优化培养基的成本效益以每克细胞在250毫升Erlenmeyer烧瓶中生产6.25单位价格确定。
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引用次数: 2
Editorial Board EOV 编辑委员会EOV
Pub Date : 2015-11-17 DOI: 10.1080/10826068.2015.1018044
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引用次数: 0
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Preparative Biochemistry and Biotechnology
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