乳酸菌素Lacticin 518发酵条件的优化
摘要
乳酸菌素是一类安全无毒的生物防腐剂,在食品、饲料和医药等领域中具有广泛的应用价值。乳酸菌细菌素的积累不仅受到菌株本身遗传特性的影响,还受到发酵条件和培养基成分的影响,因而为了提高细菌素的产量,本研究利用生物学软件进行了乳酸菌产生细菌素的条件摸索。
建立了利用酶标仪快速测定乳酸菌素lacticin 518效价的方法。研究表明,以Nisin为参照,在指示菌接种量为10%、培养时间为8h的条件下,Nisin效价和指示菌吸光值的对数值之间有着很好的线性关系。在用于乳酸菌素lacticin 518效价检测时,相对标准偏差小于5%,该方法比传统的琼脂扩散法所需要的检测时间要短,适合本实验乳酸菌素lacticin 518效价的研究。
研究了种龄、接种量、培养方式、发酵液的初始pH以及发酵温度对乳酸菌素lacticin 518效价的影响。研究表明,种龄为12h的种子液、4%的接种量、静置培养、发酵培养基初始pH为7.5、28℃是乳酸菌素lacticin 518合成的最优环境因素,其中以发酵初始pH和发酵温度的影响最大。
在培养基成分单因素实验的基础上,利用响应面法对培养基各组成成分的浓度进行了优化。单因素实验表明乳酸菌素lacticin 518合成的最佳碳、氮源分别为蔗糖和胰蛋白胨,吐温80是乳酸菌素lacticin 518产生最适合的刺激因子。通过Plackett-Burman实验,探明了胰蛋白胨、磷酸二氢钾和硫酸镁是对乳酸菌素lacticin 518的合成影响最大的培养基成分。通过快速上升实验和中心复合实验,确定了最适培养基(g/L)为:蔗糖20,酵母粉5.0,胰蛋白胨8.18,牛肉膏10,硫酸镁0.82,磷酸氢二钾2.8,柠檬酸铵5.0,乙酸钠5.0,硫酸锰0.25,吐温80 5ml。
研究了乳酸菌Lactc518的生长、乳酸菌素lacticin 518的产生以及发酵产酸三者之间的耦联性。在最适的发酵条件和最适培养基成分条件下,乳酸菌Lactc518的生长延滞期为2h,然后迅速的进入对数生长期,在12h时菌株的生长进入稳定期。乳酸菌素lacticin 518在4h开始,在14h乳酸菌素lacticin 518的效价达到顶峰,此时乳酸菌素lacticin 518效价为180.13U/ml,较之最初始条件下乳酸菌素lacticin 518效价96.72 U/ml,效价增加了86.3%。乳酸菌Lactc518在延滞期略微产酸,然后进入酸的高产期,10h后产酸能力逐渐减弱。乳酸菌Lactc518的生长、lacticin 518的产生及发酵产酸三者之间为部分耦联型。
As a type of safety and innoxious substance, lacticin is important in the field of food, diet and medicine. The production of lacticin was effected by not only bacteria genetic character but also fermentation conditions. To improve the production of lacticin, the fermentation conditions of lacticin were studied with statistical software, in this paper.
A method of determination the titer of lacticin was established with enzyme micro-plate reader. The results show there was a linear relation between the absorbancy of bacteria and the titer of bacteriocin under the condition that the inoculation amount of indicative bacteria was 10% and the reaction time was 8 h, took nisin as referent. The RSD of samples was less than 5% when this method was used for determination the titer of sample. The detection time of this method was shorter than that of agar diffusion method, and the method could be used in the studies on this bacteriocin because of its rapidity and accuracy.
The fermentation conditions of lacticin 518 were studied, including seed age, initial pH, inoculation volume, fermentation temperature, culture method.The optimum fermentation conditions were as follows: seed age of 12h, 4% inoculation volume, stationary culture, initial pH of 7.5, at 28℃. Among these, pH and temperature had more significant effects than the others.
After single-factor experiment, the level of component was optimized with response surface method. The optimal carbon source and nitrogen source were sucrose and tryptone. Tween 80 was the optimal stimulator substance. Application of Plackett-Burman design, determined the key factors were tryptone, K_2HPO_4 and MgSO_4·7H_2O. After steepest ascent design and central composite design, the optimum medium composition is obtained as follow (g/L): sucrose 20, yeast extract 5, tryptone 8.18, beef extract 10, MgSO_4·7H_2O 0.82, K_2HPO_4 2.8, tri-ammonium citrate 5.0, CH_3COONa 5.0, MnSO_4 0.25, tween 80 5ml.
The coupled response was studied among the growth of strain, the production of Lacticin and acid in the optimum fermentation conditions. The lag phase was 2h, then it transformed to exponential phase rapidly, the stationary phase began at 12h. Lacticin began to produce at 4h, rose its summit at the 14h, the titer was 180.13U/ml, compared with 96.72 U/ml in the original condition, increased by 86.3%. In the lag phase, strain began to produce acid slightly, then it produced acid rapidly, it obviously weakened after 10h. The relation of the three was partly correlative.
建立了利用酶标仪快速测定乳酸菌素lacticin 518效价的方法。研究表明,以Nisin为参照,在指示菌接种量为10%、培养时间为8h的条件下,Nisin效价和指示菌吸光值的对数值之间有着很好的线性关系。在用于乳酸菌素lacticin 518效价检测时,相对标准偏差小于5%,该方法比传统的琼脂扩散法所需要的检测时间要短,适合本实验乳酸菌素lacticin 518效价的研究。
研究了种龄、接种量、培养方式、发酵液的初始pH以及发酵温度对乳酸菌素lacticin 518效价的影响。研究表明,种龄为12h的种子液、4%的接种量、静置培养、发酵培养基初始pH为7.5、28℃是乳酸菌素lacticin 518合成的最优环境因素,其中以发酵初始pH和发酵温度的影响最大。
在培养基成分单因素实验的基础上,利用响应面法对培养基各组成成分的浓度进行了优化。单因素实验表明乳酸菌素lacticin 518合成的最佳碳、氮源分别为蔗糖和胰蛋白胨,吐温80是乳酸菌素lacticin 518产生最适合的刺激因子。通过Plackett-Burman实验,探明了胰蛋白胨、磷酸二氢钾和硫酸镁是对乳酸菌素lacticin 518的合成影响最大的培养基成分。通过快速上升实验和中心复合实验,确定了最适培养基(g/L)为:蔗糖20,酵母粉5.0,胰蛋白胨8.18,牛肉膏10,硫酸镁0.82,磷酸氢二钾2.8,柠檬酸铵5.0,乙酸钠5.0,硫酸锰0.25,吐温80 5ml。
研究了乳酸菌Lactc518的生长、乳酸菌素lacticin 518的产生以及发酵产酸三者之间的耦联性。在最适的发酵条件和最适培养基成分条件下,乳酸菌Lactc518的生长延滞期为2h,然后迅速的进入对数生长期,在12h时菌株的生长进入稳定期。乳酸菌素lacticin 518在4h开始,在14h乳酸菌素lacticin 518的效价达到顶峰,此时乳酸菌素lacticin 518效价为180.13U/ml,较之最初始条件下乳酸菌素lacticin 518效价96.72 U/ml,效价增加了86.3%。乳酸菌Lactc518在延滞期略微产酸,然后进入酸的高产期,10h后产酸能力逐渐减弱。乳酸菌Lactc518的生长、lacticin 518的产生及发酵产酸三者之间为部分耦联型。
As a type of safety and innoxious substance, lacticin is important in the field of food, diet and medicine. The production of lacticin was effected by not only bacteria genetic character but also fermentation conditions. To improve the production of lacticin, the fermentation conditions of lacticin were studied with statistical software, in this paper.
A method of determination the titer of lacticin was established with enzyme micro-plate reader. The results show there was a linear relation between the absorbancy of bacteria and the titer of bacteriocin under the condition that the inoculation amount of indicative bacteria was 10% and the reaction time was 8 h, took nisin as referent. The RSD of samples was less than 5% when this method was used for determination the titer of sample. The detection time of this method was shorter than that of agar diffusion method, and the method could be used in the studies on this bacteriocin because of its rapidity and accuracy.
The fermentation conditions of lacticin 518 were studied, including seed age, initial pH, inoculation volume, fermentation temperature, culture method.The optimum fermentation conditions were as follows: seed age of 12h, 4% inoculation volume, stationary culture, initial pH of 7.5, at 28℃. Among these, pH and temperature had more significant effects than the others.
After single-factor experiment, the level of component was optimized with response surface method. The optimal carbon source and nitrogen source were sucrose and tryptone. Tween 80 was the optimal stimulator substance. Application of Plackett-Burman design, determined the key factors were tryptone, K_2HPO_4 and MgSO_4·7H_2O. After steepest ascent design and central composite design, the optimum medium composition is obtained as follow (g/L): sucrose 20, yeast extract 5, tryptone 8.18, beef extract 10, MgSO_4·7H_2O 0.82, K_2HPO_4 2.8, tri-ammonium citrate 5.0, CH_3COONa 5.0, MnSO_4 0.25, tween 80 5ml.
The coupled response was studied among the growth of strain, the production of Lacticin and acid in the optimum fermentation conditions. The lag phase was 2h, then it transformed to exponential phase rapidly, the stationary phase began at 12h. Lacticin began to produce at 4h, rose its summit at the 14h, the titer was 180.13U/ml, compared with 96.72 U/ml in the original condition, increased by 86.3%. In the lag phase, strain began to produce acid slightly, then it produced acid rapidly, it obviously weakened after 10h. The relation of the three was partly correlative.
引文
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