固定化酵母细胞制备甘油酯型鱼油
摘要
本文采用固定化柱状假丝酵母细胞对甘油酯型鱼油进行了酶解富集制备的研究,主要研究内容包括脂肪酶活力测定方法的改进、柱状假丝酵母生长及产酶最适条件的优化、采用聚氨酯泡沫对酵母细胞进行固定的方法及条件的摸索、采用固定化柱状假丝酵母对粗鱼油进行酶解等。
针对传统橄榄油乳化法存在重复性不佳、精确性难以使人信服的缺点,对该法反应体系的各参数和条件进行了优化:以20%橄榄油乳化液5mL代替25%橄榄油乳化液4mL,以3mL磷酸盐缓冲液代替5mL磷酸盐缓冲液,将均质处理的速度与时间由高速(>10000r/min)、6min降低为5000r/min、2min,将反应体系温度由40℃降低为35℃,改变静止的反应环境为在100r/min下振荡反应,选择在空白组反应体系中加入1mL 0.025 mol·L~(-1) pH7.5磷酸缓冲液代替1mL酶液。在该条件下,脂肪酶催化能力得到了最大的释放,与传统方法比较,优化的反应体系测得的脂肪酶活力提高了35%以上,且重复性好,精密度高,对相关的研究有一定的指导和借鉴意义。
分别采用单因素试验法和正交试验法对柱状假丝酵母的生长条件进行了摸索,发现该酵母菌最适生长温度为25℃,生长环境为酸性,且在pH5.2、摇床转速为150r/min时产酶能力最强,接种10h后进入对数生长期,25h后进入稳定期,45h后进入衰亡期;最佳培养基配方为橄榄油浓度6.0 g·L~(-1)、酵母膏浓度9.0 g·L~(-1)、吐温-80 1%、Ca~(2+)0.5g·L~(-1)、K_2HPO_4 3 g·L~(-1)、MgSO_4·7H_2O 1 g·L~(-1)。在最适产酶条件下,重复发酵5批后的平均酶活在15.00U·mL~(-1)左右,RSD为2.34%,重复性较好。该株柱状假丝酵母可以油脂为单一碳源,且酶活高于其它试验碳源的酶活,在油脂诱导下,脂肪酶伴随细胞的生长而产生,但在有其他碳源存在时,酵母优先利用其他碳源。该柱状假丝酵母生存环境为酸性,并在pH5.2时产酶效果最好,推测其产生的脂肪酶为酸性脂肪酶,对油脂水解产生的游离脂肪酸将会有一定程度的抵抗作用,研究意义明显。
以市售聚氨酯泡沫为载体,考察了五种载体预处理方式对细胞固定及产酶的影响,确定最有效的处理方式为先用1%的NaOH溶液处理24h,再用1%的HCl溶液处理24h;考察六种载体尺寸对细胞固定及产酶的影响,确定载体的最佳尺寸为2.0cm×2.0cm×0.5cm;摇床转速影响是培养基的溶氧量和细胞的固着,在本试验中,150r/min下获得的固着态细胞更多,发酵液酶活也更高;从发酵液酶活和试验成本两方面考虑,确定向150ml培养基中投放12块尺寸为2.0cm×2.0cm×0.5cm的载体为佳。固定化细胞经水洗6次后,载体内的固着态细胞干重为水洗前的42.1%,稍低于文献报道。固定化细胞发酵无论是在总生物量上,还是在发酵液酶活上,均保持一定的优势。
利用固定化酵母细胞对精制鱼油进行甘油酯型DHA/EPA的酶解富集,确定摇床转速为110r/min、油水比为3:40、酶解时间为30h时,DHA/EPA总含量可达到28.55%。
In this paper, glyceride fish oil was prepared by enzymatic studies with immobilized Candida Cylindracea cell. The main contents included the development of lipase activity determination, optimization of Candida Cylindracea growth and optimal conditions for enzyme production, the exploration of Candida Cylindracea cell fix with polyurethane foam and the hydrolysis of crude fish oil with immobilized Candida Cylindracea and so on.
The traditional olive oil emulsion method was with shortcomings of poor reproducibility and low accuracy, so the parameters and reaction conditions were optimized: the 4mL, 25% olive oil emulsion instead of 5mL, 20% olive oil emulsion was used and the 3ml phosphate buffer instead of 5ml phosphate buffer was used. Meanwhile, the homogenization speed and time from high-speed (> 10000r/min), 6min reduced to 5000r/min, 2min; the reaction temperature decreased from 40℃to 35℃; the static reaction environment was changed to 100r/min oscillation; 1ml, 0.025mol / L phosphate buffer at pH7.5 was added in the control group instead of 1ml enzyme solution. Under optimum conditions, lipase activity was increased by 35% than the activity of before optimization. The method is with high precision and good repeatability and can be a reference for related study.
The Candida Cylindracea growth conditions were explored by single factor test and orthogonal test. The results showed that the optimum temperature for Candida Cylindracea growth was 25℃, the living environment was acidic, and when pH was 5.2 and rotation speed was 150r/min, it had the strongest enzyme production ability. 10h after inoculation, it was in logarithmic growth phase and 25h after inoculation was the stable phase and after 45h was the decline phase; The best medium was olive oil concentration of 6.0 g·L~(-1), yeast extract concentration of 9.0 g·L~(-1), Tween-80 1%, Ca~(2 +)0.5 g·L~(-1), K_2HPO_4 3.0 g·L~(-1), MgSO_4·7H_2O 1.0 g·L~(-1). Under the optimum conditions for enzyme production, after 5 times of repeated experiments, the average enzyme activity was around 15.00U/ml and RSD was 2.34% with good reproducibility. The Candida Cylindracea can use cylindrical oil as single carbon source and the enzyme activity was better than other carbon source tests. With oil induction, lipase was produced with cell growth, but in the presence of other carbon sources, yeast uses other carbon sources first. The living environment for the Candida was acid and enzyme production was the best when pH was 5.2, suggesting that it produced acid lipase. Fat hydrolysis on free fatty acids will be resistant to a certain extent and it has clear research meaning.
With commercial polyurethane foam as a carrier, the impact of five kinds of pretreatment patterns on cell fixation and enzyme production were investigated and the most effective treatment was first with 1% NaOH solution treatment for 24h, and then 1% HCl solution treatment for 24h; the impact of six different carrier sizes on cells fixation and enzyme production was also investigated and the best carrier size was 2.0cm×2.0cm×0.5cm; rotation speed influenced the dissolved oxygen amount and cell fixation and in the test, fixed cells were more and broth activity was also higher under150r/min; considered from the cost of test and fermentation activity, the best carrier was to put 12 carriers with size 2.0cm×2.0cm×0.5cm in 150ml .after Immobilized cells were washed 6 times, the dry cell weight in the carrier was of 42.1% of the pre-washed state, slightly lower than reported in the literature. Immobilized cell fermentation maintains a certain advantage either in total biomass, or in the activity of the fermentation broth.
Immobilized yeast cells was used in refined fish oil to enrich glyceride DHA/EPA .when the rotation speed was 110r/min, ratio of oil to water was 3:40 and reaction time was 30h, the total DHA / EPA content can reach 28.55%.
针对传统橄榄油乳化法存在重复性不佳、精确性难以使人信服的缺点,对该法反应体系的各参数和条件进行了优化:以20%橄榄油乳化液5mL代替25%橄榄油乳化液4mL,以3mL磷酸盐缓冲液代替5mL磷酸盐缓冲液,将均质处理的速度与时间由高速(>10000r/min)、6min降低为5000r/min、2min,将反应体系温度由40℃降低为35℃,改变静止的反应环境为在100r/min下振荡反应,选择在空白组反应体系中加入1mL 0.025 mol·L~(-1) pH7.5磷酸缓冲液代替1mL酶液。在该条件下,脂肪酶催化能力得到了最大的释放,与传统方法比较,优化的反应体系测得的脂肪酶活力提高了35%以上,且重复性好,精密度高,对相关的研究有一定的指导和借鉴意义。
分别采用单因素试验法和正交试验法对柱状假丝酵母的生长条件进行了摸索,发现该酵母菌最适生长温度为25℃,生长环境为酸性,且在pH5.2、摇床转速为150r/min时产酶能力最强,接种10h后进入对数生长期,25h后进入稳定期,45h后进入衰亡期;最佳培养基配方为橄榄油浓度6.0 g·L~(-1)、酵母膏浓度9.0 g·L~(-1)、吐温-80 1%、Ca~(2+)0.5g·L~(-1)、K_2HPO_4 3 g·L~(-1)、MgSO_4·7H_2O 1 g·L~(-1)。在最适产酶条件下,重复发酵5批后的平均酶活在15.00U·mL~(-1)左右,RSD为2.34%,重复性较好。该株柱状假丝酵母可以油脂为单一碳源,且酶活高于其它试验碳源的酶活,在油脂诱导下,脂肪酶伴随细胞的生长而产生,但在有其他碳源存在时,酵母优先利用其他碳源。该柱状假丝酵母生存环境为酸性,并在pH5.2时产酶效果最好,推测其产生的脂肪酶为酸性脂肪酶,对油脂水解产生的游离脂肪酸将会有一定程度的抵抗作用,研究意义明显。
以市售聚氨酯泡沫为载体,考察了五种载体预处理方式对细胞固定及产酶的影响,确定最有效的处理方式为先用1%的NaOH溶液处理24h,再用1%的HCl溶液处理24h;考察六种载体尺寸对细胞固定及产酶的影响,确定载体的最佳尺寸为2.0cm×2.0cm×0.5cm;摇床转速影响是培养基的溶氧量和细胞的固着,在本试验中,150r/min下获得的固着态细胞更多,发酵液酶活也更高;从发酵液酶活和试验成本两方面考虑,确定向150ml培养基中投放12块尺寸为2.0cm×2.0cm×0.5cm的载体为佳。固定化细胞经水洗6次后,载体内的固着态细胞干重为水洗前的42.1%,稍低于文献报道。固定化细胞发酵无论是在总生物量上,还是在发酵液酶活上,均保持一定的优势。
利用固定化酵母细胞对精制鱼油进行甘油酯型DHA/EPA的酶解富集,确定摇床转速为110r/min、油水比为3:40、酶解时间为30h时,DHA/EPA总含量可达到28.55%。
In this paper, glyceride fish oil was prepared by enzymatic studies with immobilized Candida Cylindracea cell. The main contents included the development of lipase activity determination, optimization of Candida Cylindracea growth and optimal conditions for enzyme production, the exploration of Candida Cylindracea cell fix with polyurethane foam and the hydrolysis of crude fish oil with immobilized Candida Cylindracea and so on.
The traditional olive oil emulsion method was with shortcomings of poor reproducibility and low accuracy, so the parameters and reaction conditions were optimized: the 4mL, 25% olive oil emulsion instead of 5mL, 20% olive oil emulsion was used and the 3ml phosphate buffer instead of 5ml phosphate buffer was used. Meanwhile, the homogenization speed and time from high-speed (> 10000r/min), 6min reduced to 5000r/min, 2min; the reaction temperature decreased from 40℃to 35℃; the static reaction environment was changed to 100r/min oscillation; 1ml, 0.025mol / L phosphate buffer at pH7.5 was added in the control group instead of 1ml enzyme solution. Under optimum conditions, lipase activity was increased by 35% than the activity of before optimization. The method is with high precision and good repeatability and can be a reference for related study.
The Candida Cylindracea growth conditions were explored by single factor test and orthogonal test. The results showed that the optimum temperature for Candida Cylindracea growth was 25℃, the living environment was acidic, and when pH was 5.2 and rotation speed was 150r/min, it had the strongest enzyme production ability. 10h after inoculation, it was in logarithmic growth phase and 25h after inoculation was the stable phase and after 45h was the decline phase; The best medium was olive oil concentration of 6.0 g·L~(-1), yeast extract concentration of 9.0 g·L~(-1), Tween-80 1%, Ca~(2 +)0.5 g·L~(-1), K_2HPO_4 3.0 g·L~(-1), MgSO_4·7H_2O 1.0 g·L~(-1). Under the optimum conditions for enzyme production, after 5 times of repeated experiments, the average enzyme activity was around 15.00U/ml and RSD was 2.34% with good reproducibility. The Candida Cylindracea can use cylindrical oil as single carbon source and the enzyme activity was better than other carbon source tests. With oil induction, lipase was produced with cell growth, but in the presence of other carbon sources, yeast uses other carbon sources first. The living environment for the Candida was acid and enzyme production was the best when pH was 5.2, suggesting that it produced acid lipase. Fat hydrolysis on free fatty acids will be resistant to a certain extent and it has clear research meaning.
With commercial polyurethane foam as a carrier, the impact of five kinds of pretreatment patterns on cell fixation and enzyme production were investigated and the most effective treatment was first with 1% NaOH solution treatment for 24h, and then 1% HCl solution treatment for 24h; the impact of six different carrier sizes on cells fixation and enzyme production was also investigated and the best carrier size was 2.0cm×2.0cm×0.5cm; rotation speed influenced the dissolved oxygen amount and cell fixation and in the test, fixed cells were more and broth activity was also higher under150r/min; considered from the cost of test and fermentation activity, the best carrier was to put 12 carriers with size 2.0cm×2.0cm×0.5cm in 150ml .after Immobilized cells were washed 6 times, the dry cell weight in the carrier was of 42.1% of the pre-washed state, slightly lower than reported in the literature. Immobilized cell fermentation maintains a certain advantage either in total biomass, or in the activity of the fermentation broth.
Immobilized yeast cells was used in refined fish oil to enrich glyceride DHA/EPA .when the rotation speed was 110r/min, ratio of oil to water was 3:40 and reaction time was 30h, the total DHA / EPA content can reach 28.55%.
引文
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