Optimization of Astaxanthin-producing Fermentation by Phaffia rhodozyma using Okara as Nitrogen Source
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摘要:
目的 研究以豆渣为氮源进行红法夫酵母产虾青素的可行性,并进行发酵工艺优化,为替代传统的红法夫酵母生产虾青素过程中通常使用的蛋白胨和酵母粉等高成本氮源、降低虾青素生产成本提供参考。 方法 以豆渣为有机氮源,分析碳源、前体物质、其他氮源、维生素和无机盐等对虾青素产量的影响,选取(NH4)2SO4、维生素E、葡萄糖、蔗糖4个影响因子进行发酵工艺的响应面优化。 结果 以湿豆渣为氮源时,葡萄糖是红法夫酵母产虾青素的最佳碳源,葡萄糖与蔗糖复合可促进虾青素增产,KCl、KNO3、K2HPO4等钾盐物质,(NH4)2SO4、VB2、VE、玉米黄素等均可显著促进虾青素的产量。对增产最优的4个因素(葡萄糖、蔗糖、K2SO4 和维生素E)进行响应面优化,获得产虾青素的最佳培养基配方为:湿豆渣 10%、K2SO4 0.22%、维生素E 0.6%、葡萄糖 1.08%、蔗糖1.50%,虾青素产量实测值为32.46 mg·L−1,是YM培养基产量的2.23倍。 结论 豆渣可作为唯一氮源进行红法夫酵母发酵产虾青素,经响应面试验进行工艺优化,红法夫酵母生产虾青素效率明显提升。研究可为虾青素生产中氮源成本控制及虾青素产量提升提供参考。 Abstract:Objective An astaxanthin-producing fermentation by Phaffia rhodozyma using okara for nitrogen was optimize. Methods On the conventional fermentation by P. rhodozyma to make astaxanthin, okara was used to replace the commonly applied peptone and yeast extract as the organic nitrogen source for cost reduction. Effects of carbon sources, precursor substances, other nitrogen supply, vitamins, and inorganic salts on yield of astaxanthin were analyzed with the amounts of (NH4)2SO4, vitamin E, glucose, and sucrose optimized by response surface methodology. Results When okara was used as a raw ingredient for the fermentation, glucose became the optimal carbon source. The yield of astaxanthin by the P. rhodozyma fermentation could be significantly increased by applying both glucose and sucrose, potassium salts such as KCl, KNO3, and K2HPO4 as well as (NH4)2SO4, VB2, VE, and zeaxanthin. Hence, the medium was optimized by response surface method on the 4 key ingredients of glucose, sucrose, K2SO4, and VE to arrive at a formulation consisting of 10% okara, 0.22% K2SO4, 0.6% VE, 1.08% glucose, and 1.50% sucrose to reach a yield of astaxanthin at 32.46 mg·L−1, which was 2.23 folds higher than what obtained by using the YM medium. Conclusion Okara could amply be used to replace peptone and yeast extract as the nitrogen source for the astaxanthin production by P. rhodozyma fermentation. -
Key words:
- Phaffia rhodozyma /
- astaxanthin /
- okara /
- impact factor /
- response surface
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图 1 红法夫酵母生长及虾青素积累基础培养条件分析
A:YM、C、Glc、Suc、Fru和Tre分别代表YM培养基、未加糖豆渣培养基、2.5%葡萄糖、2.5%蔗糖、2.5%果糖、2.5%海藻糖。不同字母代表样品间显著差异(P≤0.01),下同。
Figure 1. Required ingredients for P. rhodozyma growth and astaxanthin production
A: YM, C, Glc, Suc, Fru, and Tre represent YM medium, unsugar-added okara medium, 2.0% glucose, 2.0% sucrose, 2.0% fructose, and 2.0% trehalose treatment, respectively. Data with different letters represent significant differences between samples at p≤0.01. Same for below.
图 2 不同因子对红法夫酵母生物量及产虾青素含量的影响。
A:向对照组(CK组)中分别加入0.5%的蛋白胨、牛肉粉和酵母粉;B:Glc、Suc、Fru和Tre分别代表葡萄糖、蔗糖、果糖和海藻糖,+表示2种糖的等量混合物;C中,E:乙醇,Ace:乙酸,Lac:乳酸, Sul:磺基水杨酸,Ze:玉米黄素,Tom:番茄。D中,VA:维生素A,VB1:维生素B1,VB2:维生素B2,VC:维生素C, VE:维生素E。
Figure 2. Viable count and astaxanthin yield of P. rhodozyma under various treatments
A: 0.5% peptone, beef powder, and yeast powder added to CK; B: Glc, Suc, Fru, and Tre represent glucose, sucrose, fructose, and trehalose, respectively; "+" denotes an equal mixture of two sugars; C: in CK, 0.5% ethanol (E), acetic acid (Ace), lactic acid (Lac), sulfosalicylic acid (Sul), zeaxanthin (Ze), and tomato (Tom) were added; D: 0.5% vitamin A (VA), vitamin B1 (VB1), vitamin B2 (VB2), vitamin C (VC), and vitamin E (VE) were added.
表 1 响应面优化设计因素及水平编码表
Table 1. Factors and codes applied in response surface experiment
促进因子
Promoter factor编码
Code水平 level −1 0 1 硫酸钾 K2SO4/% A 0.2 0.4 0.6 维生素E Vitamin E/% B 0.6 0.8 1.0 葡萄糖 Glucose/% C 1.00 1.25 1.50 蔗糖 Sucrose/% D 1.00 1.25 1.50 表 2 响应面优化试验结果
Table 2. Response surface optimization results
试验号
No.影响因子加入量
Dosage of influence factors/%虾青素含量
Content of
Astaxanthin/
(mg·L−1)试验号
No.影响因子加入量
Dosage of influence factors/%虾青素含量
Content of
Astaxanthin/
(mg·L−1)K2SO4 维生素E
Vitamins E葡萄糖
Glucose蔗糖
SucroseK2SO4 维生素E
Vitamins E葡萄糖
Glucose蔗糖
Sucrose1 0.4 0.6 1.5 1.25 24.14±0.94 16 0.4 0.6 1.25 1 20.92±1.68 2 0.4 0.8 1.25 1.25 29.13±0.32 17 0.6 0.8 1.25 1.5 20.90±1.58 3 0.4 0.6 1 1.25 27.38±0.48 18 0.4 0.8 1 1 20.14±1.03 4 0.2 1 1.25 1.25 22.27±0.94 19 0.2 0.8 1.25 1 19.28±2.90 5 0.6 0.6 1.25 1.25 20.42±0.77 20 0.2 0.6 1.25 1.25 24.02±0.06 6 0.4 0.8 1.5 1.5 22.43±1.16 21 0.4 1 1.5 1.25 23.27±0.68 7 0.4 0.8 1.25 1.25 27.19±0.23 22 0.6 0.8 1 1.25 26.60±0.94 8 0.4 0.8 1.5 1 25.51±1.26 23 0.2 0.8 1 1.25 24.25±2.00 9 0.4 1 1.25 1.5 20.83±0.19 24 0.4 0.8 1.25 1.25 28.63±0.19 10 0.4 0.8 1 1.5 26.65±0.10 25 0.6 0.8 1.5 1.25 21.42±0.39 11 0.4 0.6 1.25 1.5 28.01±0.35 26 0.4 0.8 1.25 1.25 27.60±1.65 12 0.6 1 1.25 1.25 28.13±1.48 27 0.6 0.8 1.25 1 24.55±1.32 13 0.4 1 1.25 1 26.94±0.45 28 0.2 0.8 1.25 1.5 27.99±1.23 14 0.2 0.8 1.5 1.25 24.57±0.32 29 0.4 1 1 1.25 25.51±1.06 15 0.4 0.8 1.25 1.25 28.27±2.26 表 3 回归模型的方差分析
Table 3. ANOVA of quadratic model
方差来源
Source平方和
Sum of
Squares自由
度
df均方差
Mean
SquareF值
F valueP值
P value显著性
Significance模型
Model228.71 14 16.34 9.02 < 0.0001 ** A-K2SO4 0.011 1 0.011 6.13×10−3 0.9387 B-VE 0.35 1 0.35 1.90×10−1 0.6664 C-葡萄糖
C-Glucose7.04 1 7.04 3.89 0.0687 D-蔗糖
D-Sucrose7.47 1 7.47 4.12 0.0617 AB 22.41 1 22.41 12.37 0.0034 ** AC 7.56 1 7.56 4.17 0.0604 AD 38.22 1 38.22 21.1 0.0004 ** BC 0.25 1 0.25 0.14 0.7148 BD 43.62 1 43.62 24.08 0.0002 ** CD 22.95 1 22.95 12.67 0.0031 ** A2 41.79 1 41.79 23.07 0.0003 ** B2 16.78 1 16.78 9.26 0.0088 C2 16.72 1 16.72 9.23 0.0089 D2 42.83 1 42.83 23.64 0.0003 ** 残差
Residual25.36 14 1.81 失拟项
Lack of Fit22.94 10 2.29 3.79 0.1055 ns 纯误差
Pure error2.42 4 0.61 总离差
Cor total254.07 28 R2 0.9002 Radj2 0.8003 CV/% 5.44 *表示 P <0. 05, 差异显著;**表示 P <0. 01;差异极显著;ns表示P≥0.05,差异不显著。*: Significant difference at P<0.05; **: extremely significant difference at P<0.01; ns: no significant difference at P≥0.05. -
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