基于豆渣为氮源的红法夫酵母产虾青素的发酵条件优化

官雪芳; 陈舒莹; 赖恭梯; 王琦; 赖呈纯; 黄菊青; 郑琪; 林斌

基于豆渣为氮源的红法夫酵母产虾青素的发酵条件优化

官雪芳^{1, 2,},
陈舒莹¹,
赖恭梯^{1, 2},
王琦^{1, 2},
赖呈纯^{1, 2},
黄菊青^{1, 2},
郑琪¹,
林斌^1, ,

1.
福建省农业科学院农产品加工研究所，福建　福州　350002
2.
农业农村部亚热带特色果蔬菌加工重点实验室，福建　福州　350002

基金项目: 福建省科技计划公益类专项（2022R1032006）

详细信息

作者简介:
官雪芳（1979 —），女，副研究员，主要从事微生物发酵工艺及代谢产物研究，E-mail：guan-619@163.com

通讯作者:
林斌（1964 —），男，副研究员，主要从事废弃物利用及功能食品，E-mail：linbin591@126.com

中图分类号: TQ46
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出版历程
- 收稿日期: 2024-05-11
- 修回日期: 2024-09-02
- 网络出版日期: 2024-10-24

Optimization of Astaxanthin-producing Fermentation by Phaffia rhodozyma using Okara as Nitrogen Source

GUAN Xuefang^{1, 2
,},
CHEN Shuying¹,
LAI Gongti^{1, 2},
WANG Qi^{1, 2},
LAI Chengchun^{1, 2},
HUANG Juqing^{1, 2},
ZHENG Qi¹,
LIN Bin^{1
, ,}

1.
Institute of Food Science and Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350002, China
2.
Key Laboratory of Processing of Subtropical Characteristic Fruits, Vegetables and Edible Fungi, Ministry of Agriculture and Rural Affairs of China, Fuzhou, Fujian　350002, China

摘要

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。
- 红法夫酵母 /
- 虾青素 /
- 豆渣 /
- 影响因子 /
- 响应面
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.
- 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.

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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.

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图 3 不同影响因子加入量对红法夫酵母生物量及虾青素产量的影响。

Figure 3. P. rhodozyma biomass and astaxanthin production in fermentation with additions of varied amounts of 4 key ingredients

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图 3 不同影响因子加入量对红法夫酵母生物量及虾青素产量的影响。

Figure 3. P. rhodozyma biomass and astaxanthin production in fermentation with additions of varied amounts of 4 key ingredients

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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
促进因子 Promoter factor	编码 Code	−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

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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)
试验号 No.	K₂SO₄	维生素E Vitamins E	葡萄糖 Glucose	蔗糖 Sucrose	虾青素含量 Content of Astaxanthin/ (mg·L⁻¹)	试验号 No.	K₂SO₄	维生素E Vitamins E	葡萄糖 Glucose	蔗糖 Sucrose	虾青素含量 Content of Astaxanthin/ (mg·L⁻¹)
1	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

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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 Square	F值 F value	P值 P value	显著性 Significance
模型 Model	228.71	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-Glucose	7.04	1	7.04	3.89	0.0687
D-蔗糖 D-Sucrose	7.47	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 Residual	25.36	14	1.81
失拟项 Lack of Fit	22.94	10	2.29	3.79	0.1055	ns
纯误差 Pure error	2.42	4	0.61
总离差 Cor total	254.07	28
R²	0.9002
R_adj²	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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参考文献(23)

[1]	王宝贝, 凌雪萍, 郑宗宇, 等. 维生素对红法夫酵母产虾青素的影响 [J]. 厦门大学学报(自然科学版), 2011, 50(1):111−116. WANG B B, LING X P, ZHENG Z Y, et al. Effect of vitamins on astaxanthin production of Phaffia rhodozyma [J]. Journal of Xiamen University (Natural Science), 2011, 50(1): 111−116. (in Chinese)
[2]	周桂雄, 王闻, 谭雪松, 等. 利用农业废弃物碳源的红法夫酵母生产虾青素研究进展 [J]. 农业工程学报, 2016, 32(15):308−314. doi: 10.11975/j.issn.1002-6819.2016.15.043 ZHOU G X, WANG W, TAN X S, et al. Review on astaxanthin production from agricultural wastes by Phaffia rhodozyma [J]. Transactions of the Chinese Society of Agricultural Engineering, 2016, 32(15): 308−314. (in Chinese) doi: 10.11975/j.issn.1002-6819.2016.15.043
[3]	LAI J X, LIU W P, BU J, et al. Enhancement of astaxanthin production from food waste by Phaffia rhodozyma screened by flow cytometry and feed application potential [J]. Biotechnology and Applied Biochemistry, 2023, 70(6): 1817−1829. doi: 10.1002/bab.2484
[4]	张辰, 徐慧, 朱坤福, 等. 微生物法生产虾青素的研究进展 [J]. 中国酿造, 2021, 40(10):29−35. doi: 10.11882/j.issn.0254-5071.2021.10.005 ZHANG C, XU H, ZHU K F, et al. Research progress in the production of astaxanthin by microbial method [J]. China Brewing, 2021, 40(10): 29−35. (in Chinese) doi: 10.11882/j.issn.0254-5071.2021.10.005
[5]	HIGUERA-CIAPARA I, FÉLIX-VALENZUELA L, GOYCOOLEA F M. Astaxanthin: A review of its chemistry and applications [J]. Critical Reviews in Food Science and Nutrition, 2006, 46(2): 185−196. doi: 10.1080/10408690590957188
[6]	ELBAHNASWY S, ELSHOPAKEY G E. Recent progress in practical applications of a potential carotenoid astaxanthin in aquaculture industry: A review [J]. Fish Physiology and Biochemistry, 2024, 50(1): 97−126. doi: 10.1007/s10695-022-01167-0
[7]	NAIR A, AHIRWAR A, SINGH S, et al. Astaxanthin as a king of ketocarotenoids: Structure, synthesis, accumulation, bioavailability and antioxidant properties [J]. Marine Drugs, 2023, 21(3): 176. doi: 10.3390/md21030176
[8]	林嘉祺, 庄岩, 刘骁, 等. 一株海洋红法夫酵母Phaffia rhodozyma RP-306产虾青素的发酵条件优化 [J]. 海洋科学, 2024, 48(2):69−78. LIN J Q, ZHUANG Y, LIU X, et al. Optimization of fermentation conditions for astaxanthin production by marine Phaffia rhodozyma RP-306 [J]. Marine Sciences, 2024, 48(2): 69−78. (in Chinese)
[9]	潘雪珊, 凌雪萍, 叶驰名, 等. 红发夫酵母生产虾青素的氮源补加策略 [J]. 厦门大学学报(自然科学版), 2013, 52(4):545−552. PAN X S, LING X P, YE C M, et al. Nitrogen feeding strategies on astaxanthin production by Xanthophyllomyces dendrorhous [J]. Journal of Xiamen University (Natural Science), 2013, 52(4): 545−552. (in Chinese)
[10]	安君. 红法夫酵母发酵钝顶螺旋藻合成虾青素及载虾青素微粒功能性质研究[D]. 北京: 北京林业大学, 2020. AN J. Study on synthesis of astaxanthin from Spirulina platensis by Phaffia rhodozyma and functional properties of astaxanthin-loaded particles[D]. Beijing: Beijing Forestry University, 2020. (in Chinese)
[11]	江文涛, 彭立影, 马加军, 等. 产虾青素红法夫酵母的发酵工艺优化 [J]. 饲料博览, 2021(12):13−18. doi: 10.3969/j.issn.1001-0084.2021.12.003 JIANG W T, PENG L Y, MA J J, et al. Optimization of fermentation process of astaxanthin producing Phaffia rhodozyma [J]. Feed Review, 2021(12): 13−18. (in Chinese) doi: 10.3969/j.issn.1001-0084.2021.12.003
[12]	张晶. 红法夫酵母菌株筛选及TiO₂胁迫提高其虾青素产量的作用机制[D]. 长春: 吉林农业大学, 2022. ZHANG J. Screening of Phaffia rhodozyma strain and the mechanism of Increasing Its astaxanthin production under TiO₂ stress[D]. Changchun: Jilin Agricultural University, 2022. (in Chinese)
[13]	祝义伟, 龙勃, 龙勇, 等. 豆渣中营养成分的检测及其含量声称 [J]. 食品研究与开发, 2017, 38(8):117−120. doi: 10.3969/j.issn.1005-6521.2017.08.027 ZHU Y W, LONG B, LONG Y, et al. Determination and content claim of nutrients in okara [J]. Food Research and Development, 2017, 38(8): 117−120. (in Chinese) doi: 10.3969/j.issn.1005-6521.2017.08.027
[14]	韩扬, 何聪芬, 董银卯, 等. 响应面法优化超声波辅助酶法制备燕麦ACE抑制肽的工艺研究 [J]. 食品科学, 2009, 30(22):44−49. doi: 10.3321/j.issn:1002-6630.2009.22.006 HAN Y, HE C F, DONG Y M, et al. Response surface optimization of ultrasonic-assisted enzymatic preparation of ACE inhibitory peptides from oat [J]. Food Science, 2009, 30(22): 44−49. (in Chinese) doi: 10.3321/j.issn:1002-6630.2009.22.006
[15]	倪辉, 何国庆, 杨远帆, 等. 法夫酵母虾青素提取工艺的优化研究 [J]. 农业工程学报, 2004, 20(2):204−208. doi: 10.3321/j.issn:1002-6819.2004.02.049 NI H, HE G Q, YANG Y F, et al. Optimization of condition for extracting astaxanthin from Phaffia rhodozyma [J]. Transactions of the Chinese Society of Agricultural Engineering, 2004, 20(2): 204−208. (in Chinese) doi: 10.3321/j.issn:1002-6819.2004.02.049
[16]	STOKLOSA R J, JOHNSTON D B, NGHIEM N P. Phaffia rhodozyma cultivation on structural and non-structural sugars from sweet Sorghum for astaxanthin generation [J]. Process Biochemistry, 2019, 83: 9−17. doi: 10.1016/j.procbio.2019.04.005
[17]	MIAO L L, CHI S, WU M R, et al. Deregulation of phytoene-β-carotene synthase results in derepression of astaxanthin synthesis at high glucose concentration in Phaffia rhodozyma astaxanthin-overproducing strain MK19 [J]. BMC Microbiology, 2019, 19(1): 133. doi: 10.1186/s12866-019-1507-6
[18]	王严飞, 刘燕青, 陶正国, 等. 玉米黄素转化为虾青素的工艺参数研究 [J]. 天然产物研究与开发, 2014, 26(2):278−282. WANG Y F, LIU Y Q, TAO Z G, et al. Study on technical parameters of converting Zeaxanthin into astaxanthin [J]. Natural Product Research and Development, 2014, 26(2): 278−282. (in Chinese)
[19]	刘春利, 沈宁燕, 倪辉, 等. 乙醇对法夫酵母发酵合成虾青素的影响 [J]. 食品与发酵工业, 2018, 44(3):1−7. LIU C L, SHEN N Y, NI H, et al. Effect of ethanol on synthesis of astaxanthin by Phaffia rhodozyma [J]. Food and Fermentation Industries, 2018, 44(3): 1−7. (in Chinese)
[20]	YAMANE Y, HIGASHIDA K, NAKASHIMADA Y, et al. Astaxanthin production by Phaffia rhodozyma enhanced in fed-batch culture with glucose and ethanol feeding [J]. Biotechnology Letters, 1997, 19(11): 1109−1111. doi: 10.1023/A:1018492611011
[21]	黎丽, 窦光鹏, 霍文严, 等. 高产虾青素红法夫酵母的选育和工艺优化 [J]. 中国食品添加剂, 2014, 25(9):140−146. doi: 10.3969/j.issn.1006-2513.2014.09.014 LI L, DOU G P, HUO W Y, et al. Screening and optimization of a high-yield astaxanthin-producing Phaffia rhodozyma [J]. China Food Additives, 2014, 25(9): 140−146. (in Chinese) doi: 10.3969/j.issn.1006-2513.2014.09.014
[22]	王雪, 孙美娟, 刘军贤, 等. 激光镊子拉曼光谱法优化红法夫酵母合成虾青素条件 [J]. 光散射学报, 2013, 25(2):152−157. doi: 10.3969/j.issn.1004-5929.2013.02.009 WANG X, SUN M J, LIU J X, et al. Condition optimization of astaxanthin production in Phaffia rhodozyma using laser tweezers Raman spectroscopy [J]. The Journal of Light Scattering, 2013, 25(2): 152−157. (in Chinese) doi: 10.3969/j.issn.1004-5929.2013.02.009
[23]	徐建春, 孙翰, 张睿钦, 等. 响应面分析法优化雨生红球藻产虾青素培养基 [J]. 中国酿造, 2014, 33(12):72−75. doi: 10.11882/j.issn.0254-5071.2014.12.014 XU J C, SUN H, ZHANG R Q, et al. Optimization of Haematococcus Pluvialis medium producing astaxanthin by response surface methodology [J]. China Brewing, 2014, 33(12): 72−75. (in Chinese) doi: 10.11882/j.issn.0254-5071.2014.12.014