保拉迪酵母菌粉的制备及稳定性研究

发布时间：2018-03-01 04:19

本文关键词： 保拉迪酵母菌流加补料培养动力学模型冻干保护剂抗热保护剂　出处：《陕西科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：保拉迪酵母是一种益生菌,其在37℃(动物体温)下生长良好,而普通酵母菌生长的最适温度30℃左右,保拉迪酵母具有独特的生物活性,是适合用于人和动物的益生菌。目前,保拉迪酵母普遍用于治疗婴幼儿腹泻,国内保拉迪酵母制剂主要依赖进口,有关培养及制备研究的报道甚少。本研究以实验室保藏的保拉迪酵母为试验菌株,研究其培养基组成和发酵工艺条件,进而对比研究冷冻干燥和喷雾干燥法制备菌粉的技术工艺、菌粉活性及稳定性,获得以下研究结论。1.单因素试验和响应面法优化确定了保拉迪酵母菌最适培养条件为:装液量52 mL/250 mL、转速181 r/min、接种量3.1%、培养基初始pH值6.4。此条件下培养液中的活菌数达到1.86×108 CFU/mL,比优化前提高了14.1%。2.上述培养条件下,单因素和Plackett-Burman试验设计筛选出影响保拉迪酵母菌生长的主要因子为:葡萄糖、酵母浸粉、低聚果糖、磷酸氢二钠、氯化钙和蛋白胨,其中葡萄糖、酵母浸粉和低聚果糖为主要影响因素,爬坡试验和Box-Behnken试验设计获得优化后的培养基组成为:葡萄糖4.83%、酵母浸粉0.18%、低聚果糖0.18%、磷酸氢二钠0.45%、氯化钙0.2%、蛋白胨2%。该条件下培养保拉迪酵母菌,活菌数达到3.91×108 CFU/mL,菌体干重5.1911 g/L,比对照组分别提高了2.1×108 CFU/mL和2.0811 g/L。3.保拉迪酵母菌在5 L通风发酵罐中分批培养和流加补料培养结果表明,流加补料培养能够促进保拉迪酵母菌的生长,活菌数和菌体干重最高可达到7.3×108 CFU/mL和12.7644 g/L。较发酵罐中分批培养,活菌数(4.8×108 CFU/mL)提高了1.5倍,菌体干重(6.5612 g/L)提高了近2倍。采用Logistic和DoseResp模型分别建立了分批培养过程中菌体生长和底物消耗的非线性拟合动力学模型为:。所得模型与试验数据拟合良好。4.响应面法优化确定了保拉迪酵母菌复合冻干保护剂配方为:菊糖0.6%、低聚木糖0.4%、碳酸氢钠0.3%、硫酸镁0.75%、乳糖21.41%、海藻糖21.98%、谷氨酸钠4.02%、脱脂乳22%,磷酸缓冲液添加量与菌泥重之比为1.2:1。保拉迪酵母菌冻干存活率和单位菌粉活菌数分别为72.55%和1.32×1010 CFU/g。水浴模拟试验优化确定了保拉迪酵母菌抗热保护剂配方为:脱脂乳15.12%、明胶1.8%、海藻糖9.72%。经验证测得菌体存活率为17.82%,与预测值无显著性差异。该条件下喷雾干燥制备的保拉迪酵母菌粉,菌体存活率达到26.12%,单位菌粉活菌数达到1.018×109 CFU/g。5.加速试验结果表明,保拉迪酵母菌冻干菌粉和喷雾干燥菌粉于-18℃(冻藏温度)下贮存的失活速率常数分别为:k-18F=8.04×10-6和k-18S=1.04×10-5,冻干菌粉的稳定性优于喷干菌粉。加速试验测得冻干菌粉制备的保拉迪酵母菌羊奶粉在4℃和25℃时下失活速率常数分别为:k4=4.48×10-4和k25=9.72×10-3,稳定性较好。研究确定的保拉迪酵母菌优化培养基和流加补料培养条件能显著提高菌体浓度;研究确定的冻干保护剂和抗热保护剂用于制备保拉迪酵母冻干菌粉和喷雾干燥菌粉,能显著提高菌粉的存活率,为保拉迪酵母菌制剂的开发提供了参考依据及技术支持。
[Abstract]:Paradi is a probiotic yeast, in 37 degrees (animal body temperature) grow well, and common yeast optimum growth temperature of 30 degrees Celsius, Paradi yeast has a unique biological activity, is suitable for probiotics for human and animal. At present, Paradi is widely used in the treatment of infantile diarrhea in yeast, yeast preparation mainly domestic Paradi the study on Preparation of dependence on imports, culture and system are rarely reported. In this study, Paradi yeast preserved in our laboratory as the test strains, the medium composition and fermentation conditions, technology and comparative study of freeze drying and spray drying method for preparing bacterial powder, powder bacteria activity and stability, and obtain the following conclusions.1. response single factor test method optimized the optimum culture conditions for yeast Paradi: Volume 52 mL/250 mL, speed of 181 r/min, 3.1% inoculation quantity, initial pH value of medium 6.4 The number of live bacteria. Up to 1.86 * 108 CFU/mL cultured under these conditions, improve the cultivation conditions of 14.1%.2. than before optimization, the single factor experimental design and Plackett-Burman screening of the main factors affecting the growth of yeast Paradi: glucose, yeast extract powder, fructo oligosaccharides, two sodium hydrogen phosphate, calcium chloride and peptone. The protein of glucose, yeast extract and fructose as the main factors, climbing test and Box-Behnken test design to obtain the optimized medium was: glucose 4.83%, yeast extract 0.18%, fructose 0.18%, two sodium hydrogen phosphate 0.45%, calcium chloride 0.2%, peptone yeast culture Paradi 2%. in the condition of live bacteria the number is up to 3.91 * 108 CFU/mL dry cell weight 5.1911 g/L, than in the control group were increased by 2.1 * 108 CFU/mL and 2.0811 g/L.3. Paradi 5 L yeast in the fermentation tank ventilation in batch culture and fed batch culture. The result showed that the feeding culture can promote Paradi yeast growth, the number of viable cells and cell dry weight can reach 7.3 * 108 CFU/mL and 12.7644 g/L. fermentor batch culture, the number of live bacteria (4.8 x 108 CFU/mL) was 1.5 times higher than that of dry cell weight (6.5612 g/L) increased nearly 2 times were established. The batch culture nonlinear fitting kinetic models of cell growth and substrate consumption in the process of using Logistic and DoseResp model. The optimized surface method: Paradi yeast formula of cryoprotectants for the response model and test data fitting good.4.: inulin 0.6%, xylo oligosaccharide 0.4%, sodium bicarbonate 0.3%, Magnesium Sulfate 0.75% 21.41%, lactose, trehalose 21.98%, monosodium glutamate 4.02%, skim milk 22%, phosphate buffer and adding bacterial mud weight ratio and survival rate of freeze-dried bacteria powder unit living bacteria number 1.2:1. Paradi yeast were 72.55% and 1.32 * 1010 CFU/g. water bath simulation test of the optimized Paradi yeast protective agents formula: 15.12% skim milk, 1.8% gelatin, trehalose 9.72%. verified the measured cell survival rate was 17.82%, no significant difference with the predicted value. The conditions of Paradi yeast powder prepared by spray drying, the cell survival rate reached 26.12%. The number of viable bacteria powder units up to 1.018 * 109 CFU/g.5. accelerated test results show that the Paradi yeast freeze-dried powder and spray drying powder at -18 DEG C (freezing temperature) under the deactivation rate constant storage were: k-18F=8.04 * 10-6 and k-18S=1.04 * 10-5, stability is better than spraying freeze-dried powder dry powder accelerated test. The measured freeze-dried powder preparation of Paradi yeast sheep milk in 4 degrees and 25 degrees the inactivation rate constants were k4=4.48 * 10-4 and k25=9.72 * 10-3, good stability. Study on yeast and determined Paradi Culture medium and feeding culture conditions can significantly improve the cell concentration; of cryoprotector and thermal protective agent for the preparation of Paradi yeast freeze-dried powder and spray drying powder, can significantly improve the survival rate of bacteria powder, to provide a reference and technical support for the development of Paradi yeast preparation.

【学位授予单位】：陕西科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ926

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