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油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证
苏晓薇,唐庆九,张劲松,冯娜,王金艳,周帅,冯杰,俞苓
菌物学报 ›› 2021, Vol. 40 ›› Issue (9) : 2445-2460.
PDF(615 KB)
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油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证
Synthesis of triterpenes in liquid submerged fermentation of Ganoderma lingzhi promoted by oleic acid
, , ({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}灵芝三萜是灵芝中主要的活性成分之一,前期研究发现油酸可以促进灵芝三萜液态深层发酵下的发酵合成。本研究主要对油酸促进灵芝三萜液态深层发酵的工艺进行优化,并进行3L发酵罐规模的验证。通过单因素实验考察油酸的添加方式、添加时间和添加浓度对灵芝三萜的影响,结合响应面实验,获得最优工艺条件并进行验证：在发酵第32h添加1.21%高温灭菌油酸,最高灵芝三萜含量为42.69mg/g;在发酵第7h添加1.35%过滤除菌油酸,最高三萜含量为43.38mg/g,分别比对照提高2.04倍和2.08倍。在1 000mL摇瓶中添加高温灭菌油酸和过滤除菌油酸,灵芝三萜含量分别为32.18和32.48mg/g,为对照的1.96倍和1.95倍;在3L发酵罐规模下灵芝三萜含量分别为28.66和25.13mg/g,为对照的1.62倍和1.42倍。本研究系统优化了油酸促进灵芝三萜液态深层发酵的工艺条件,并在与工业生产相对应的3L发酵罐上进行验证。该研究可为灵芝三萜的规模化发酵提供重要参考和借鉴。
Triterpene is one of the most important active ingredients in Ganoderma lingzhi. Previous studies found that oleic acid could promote the biosynthesis of triterpenes in liquid submerged fermentation of Ganoderma lingzhi. In this study, the process of liquid submerged fermentation for synthesis of triterpenes by using oleic acid as promoter was optimized and verified on the scale of a 3L fermenter. The effects of the additive method, time for addition and concentration of oleic acid on triterpene synthesis were investigated by single factor experiment. The optimal process conditions were obtained and verified by response surface experiment. The highest triterpene content reaching 42.69mg/g was obtained by adding 1.21% moist heat sterilized oleic acid at 32h of fermentation, and by adding 1.35% filtered sterilized oleic acid at the 7h of fermentation, the highest triterpene content was 43.38mg/g, being 2.04 times and 2.08 times higher than triterpene content of the control, respectively. The triterpenes content was 32.18mg/g and 32.48mg/g respectively by adding moist heat sterilized and filterer sterilized oleic acid at 1 000mL scale of shake culture, being 1.96 times and 1.95 times higher as compared with the control. The triterpene content in 3L fermenter was 28.66mg/g and 25.13mg/g, 1.62 times and 1.42 times higher than that of the control. The technological conditions of liquid submerged fermentation of triterpenes promoted by oleic acid were successfully optimized and verified in a 3L fermenter, corresponding to industrial production. This study provide an important reference for large-scale fermentation of Ganoderma lingzhi for production of triterpenes.
灵芝三萜 / 工艺优化 / 响应面 / 油酸 / 规模化 {{custom_keyword}} /
Ganoderma triterpenes / process optimization / response surface / oleic acid / scaled {{custom_keyword}} /
表1 响应面实验因素水平表Table 1 Factors and levels of response surface design |
| 因素 Factors | 代码 Code | 水平Levels | ||||
|---|---|---|---|---|---|---|
| -a | -1 | 0 | +1 | +a | ||
| 时间 Fermentation time (h) | A | 0 | 7 | 24 | 41 | 48 |
| 浓度 Concentration of oleic acid (%, V/V) | B | 0.5 | 0.65 | 1 | 1.35 | 1.5 |
表2 添加高温灭菌油酸和过滤除菌两种方式下的响应面实验方案及结果Table 2 Design and results of adding oleic acid moist heat sterilized and filtered sterilized |
| 试验点 Run No. | 时间 Time (h) | 浓度 Concentration (%, V/V) | 高温灭菌油酸条件下三萜含量 Triterpene content under the condition of adding moist heat sterilized oleic acid (mg/g) | 过滤除菌油酸条件下三萜含量 Triterpene content under the condition of adding filtered sterilized oleic acid (mg/g) |
|---|---|---|---|---|
| 1 | 0 | 1 | 33.35±0.88 | 40.41±1.75 |
| 2 | 7 | 1.35 | 40.91±1.38 | 45.74±0.09 |
| 3 | 7 | 0.65 | 26.21±2.28 | 31.64±1.16 |
| 4 | 24 | 1 | 42.28±1.50 | 40.35±1.50 |
| 5 | 24 | 0.5 | 31.25±1.31 | 35.31±0.69 |
| 6 | 24 | 1 | 41.49±1.35 | 43.36±0.75 |
| 7 | 24 | 1.5 | 43.70±1.19 | 47.21±1.37 |
| 8 | 24 | 1 | 47.80±0.50 | 42.08±1.85 |
| 9 | 41 | 1.35 | 45.53±0.47 | 44.59±0.34 |
| 10 | 41 | 0.65 | 42.23±1.63 | 37.12±0.22 |
| 11 | 48 | 1 | 36.56±1.79 | 43.39±1.09 |
表3 响应面方差分析结果Table 3 Analyses of variance for CCD |
| 来源 Source | 平方和 Sum of squares | 自由度 Df | 均方 Mean square | F value | P value |
|---|---|---|---|---|---|
| 模型Model | 365.01 | 7 | 52.14 | 123.94 | 0.0011 |
| A | 17.53 | 1 | 17.53 | 41.67 | 0.0075 |
| B | 66.26 | 1 | 66.26 | 157.49 | 0.0011 |
| AB | 1.44 | 1 | 1.44 | 3.43 | 0.1609 |
| A² | 58.47 | 1 | 58.47 | 138.98 | 0.0013 |
| B² | 21.62 | 1 | 21.62 | 51.40 | 0.0056 |
| A3 | 6.28 | 1 | 6.28 | 14.93 | 0.0306 |
| B3 | 11.05 | 1 | 11.05 | 26.25 | 0.0144 |
| 残差Residual | 1.26 | 3 | 0.4207 | ||
| 失拟项 Lack of fit | 0.1294 | 1 | 0.1294 | 0.2285 | 0.6798 |
| 误差 Pure error | 1.13 | 2 | 0.5664 | ||
| 总误差 Cor total | 366.27 | 10 | |||
| R2 | 0.9966 | R2Adj | 0.9885 |
表4 响应面方差分析结果Table 4 A analysis of variance for CCD |
| 来源 Source | 平方和 Sum of squares | 自由度 Df | 均方 Mean square | F value | P value |
|---|---|---|---|---|---|
| 模型Model | 276.33 | 5 | 55.27 | 46.19 | 0.0003 |
| A | 9.14 | 1 | 9.14 | 7.64 | 0.0397 |
| B | 242.50 | 1 | 242.50 | 202.67 | < 0.0001 |
| AB | 11.01 | 1 | 11.01 | 9.20 | 0.0290 |
| A² | 0.2672 | 1 | 0.2672 | 0.2233 | 0.6564 |
| B² | 13.35 | 1 | 13.35 | 11.16 | 0.0205 |
| 残差Residual | 5.98 | 5 | 1.20 | ||
| 失拟项 Lack of fit | 1.42 | 3 | 0.4747 | 0.2083 | 0.8839 |
| 误差 Pure error | 4.56 | 2 | 2.28 | ||
| 总误差 Cor total | 282.31 | 10 | |||
| R2 | 0.9788 | R2Adj | 0.9576 |
表5 250mL摇瓶规模的响应面摇瓶验证Table 5 Response surface validation at the scale of 250mL shaking flask |
| 类别 Category | 高温灭菌 Moist heat sterilization | 过滤除菌 Filtration sterilization | |||
|---|---|---|---|---|---|
| 三萜含量 Triterpene content (mg/g) | 菌丝体生物量 Mycelial biomass (g/L) | 三萜含量 Triterpene content (mg/g) | 菌丝体生物量 Mycelial biomass (g/L) | ||
| 对照组 Control | 20.89±0.30 | 10.12±0.78 | 20.89±0.30 | 10.12±0.78 | |
| 响应面优化组 Optimal response surface group | 42.69±0.19 | 14.42±0.06 | 43.38±0.30 | 20.79±0.35 | |
| 响应面中心组 Response surface center group | 39.38±0.10 | 13.50±0.59 | 38.78±0.04 | 14.30±0.33 | |
| 单因素优化组 Optimal single factor group | 浓度 Concentration of oleic acid | 37.65±1.62 | 13.89±0.19 | 37.96±0.61 | 19.03±0.16 |
| 时间 Time of fermentation | 30.08±0.18 | 13.69±0.75 | 28.27±0.58 | 14.19±0.97 | |
表6 放大规模的响应面验证Table 6 Response surface validation at large scales of fermentation |
| 规模 Scale | 类别 Category | 高温灭菌 Moist heat sterilization | 过滤除菌 Filtration sterilization | ||
|---|---|---|---|---|---|
| 三萜含量 Triterpene content (mg/g) | 菌丝体干重 Mycelial dry weight (g/L) | 三萜含量 Triterpene content (mg/g) | 菌丝体干重 Mycelial dry weight (g/L) | ||
| 1 000mL摇瓶 1 000mL shaking flask | 对照组 Control | 16.62±0.22 | 7.22±0.12 | 16.62±0.22 | 7.22±0.12 |
| 响应面优化组 Optimal response surface group | 32.18±0.89 | 13.63±0.14 | 32.43±0.17 | 14.01±0.01 | |
| 响应面中心组 Response surface center group | 29.84±0.85 | 11.64±0.11 | 32.35±0.87 | 12.78±0.06 | |
| 3L发酵罐 3L fermenter | 对照组 Control | 17.67±0.29 | 7.76±0.15 | 17.67±0.29 | 7.76±0.15 |
| 响应面优化组 Optimal response surface group | 28.66±0.44 | 11.67±0.29 | 25.13±0.47 | 13.20±0.04 | |
图7 250mL摇瓶（左）、1 000mL摇瓶（中）和3L发酵罐（右）规模的灵芝三萜HPLC图Fig. 7 HPLC fingerprint of fermented Ganoderma triterpenes in the scales of 250mL shaking flask (left), 1 000mL shaking flask (medium) and 3L fermenters (right) under the condition of adding moist heat sterilized and filtered sterilized oleic acid. |
表7 不同发酵规模下的灵芝三萜HPLC图的峰面积Table 7 Peak area of fermentation parameters in different fermentation scales |
| 规模 Scale | 250mL摇瓶 250mL shaking flask | 1 000mL摇瓶 1 000mL shaking flask | 3L发酵罐 3L fermenter | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 名称 Name | 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid | 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid | 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid |
| 主要出峰数 The number of main peaks | 4 | 6 | 6 | 4 | 5 | 5 | 6 | 7 | 8 |
| 总面积 Total area (mAU·min) | 40 841 | 184 009 | 223 336 | 83 725 | 687 430 | 236 126 | 352 088 | 545 365 | 792 093 |
表8 不同培养规模下的灵芝菌丝体发酵参数比较Table 8 Fermentation parameters in different fermentation scales |
| 参数名称 Parameters | 发酵规模 Fermentation scales | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| 250mL摇瓶 250mL shaking flask | 1 000mL摇瓶 1 000mL shaking flask | 3L发酵罐 3L fermenter | |||||||
| 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid | 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid | 对照 Control | 高温灭菌 Moist heat sterilized oleic acid | 过滤除菌 Filtered sterilized oleic acid | |
| 初始还原糖浓度 Initial reducing sugar concentration (g/L) | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
| 最终还原糖浓度 Final reducing sugar concentration (g/L) | 5.4 | 11.2 | 11.5 | 3.2 | 12.25 | 13.65 | 15 | 15.4 | 15.5 |
| 发酵时间 Fermentation time (h) | 168 | 168 | 168 | 168 | 168 | 168 | 168 | 168 | 168 |
| 最大菌丝体生物量 Maximum mycelial biomass (g/L) | 10.12 | 14.42 | 20.79 | 7.22 | 13.63 | 14.01 | 7.76 | 11.67 | 13.20 |
| 灵芝三萜得率 Triterpene yield (g/L) | 0.212 | 0.610 | 0.886 | 0.120 | 0.463 | 0.461 | 0.137 | 0.334 | 0.332 |
| 还原糖消耗速率 Reducing sugar consumption rate [g/(L·h)] | 0.087 | 0.052 | 0.051 | 0.100 | 0.046 | 0.038 | 0.030 | 0.027 | 0.027 |
| 菌丝体对还原糖得率 Mycelial yield on reducing sugar (g/g) | 0.693 | 1.639 | 2.446 | 0.430 | 1.759 | 2.206 | 1.552 | 2.537 | 2.933 |
| 灵芝三萜对还原糖得率 Triterpene yield on reducing sugar (×10-2g/g) | 1.449 | 6.933 | 10.421 | 0.715 | 5.971 | 7.254 | 2.740 | 7.261 | 7.378 |
| 菌丝体生产强度 Mycelial productivity [g/(L·h)] | 0.060 | 0.086 | 0.124 | 0.043 | 0.081 | 0.083 | 0.046 | 0.069 | 0.079 |
| 灵芝三萜生产强度 Triterpene productivity [10-3g/(L·h)] | 1.259 | 3.632 | 5.273 | 0.715 | 2.754 | 2.742 | 0.816 | 1.991 | 1.975 |
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PDF(615 KB)
表1 响应面实验因素水平表
图1 不同浓度的高温灭菌油酸（左）和过滤除菌油酸（右）对三萜含量和菌丝体生物量的影响图2 不同时间添加高温灭菌油酸（左）和过滤除菌油酸（右）对三萜含量和菌丝体生物量的影响表2 添加高温灭菌油酸和过滤除菌两种方式下的响应面实验方案及结果表3 响应面方差分析结果图3 主要因素交互作用的响应曲面图表4 响应面方差分析结果图4 主要因素交互作用的响应曲面图表5 250mL摇瓶规模的响应面摇瓶验证表6 放大规模的响应面验证图5 不同添加方式的油酸的菌丝体生物量（左）和三萜含量（右）的变化图6 两种添加方式油酸发酵液DO（左）和pH（右）的变化图7 250mL摇瓶（左）、1 000mL摇瓶（中）和3L发酵罐（右）规模的灵芝三萜HPLC图表7 不同发酵规模下的灵芝三萜HPLC图的峰面积表8 不同培养规模下的灵芝菌丝体发酵参数比较
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