沼泽红假单胞菌产类胡萝卜素及抗氧化性

doi:10.16085/j.issn.1000-6613.2022-2242

摘要/Abstract

摘要：

类胡萝卜素是一种应用广泛的脂溶性天然色素和抗氧化剂。本文以一株典型的紫色非硫光合细菌——沼泽红假单胞菌（Rhodopseudomonas palustris）为实验对象，研究了光照厌氧体系下有机酸碳源种类、盐浓度和光照强度等培养条件对其生长和产类胡萝卜素量及组成的影响，并探究了类胡萝卜素的抗氧化性。研究结果表明：在实验的8种有机酸中，以乳酸为碳源可获得最高生物量以及类胡萝卜素含量；产类胡萝卜素最适光照强度（500lx）低于该菌生长所需（2000lx）；高盐浓度（7.5g/L）可促进该菌合成类胡萝卜素，是其生长所需最佳盐浓度的三倍。在优化后的培养条件下，该沼泽红假单胞菌类胡萝卜素含量可达15.35mg/L。所产类胡萝卜素由6种组成，培养条件对其组成有明显影响，高盐浓度和强光照会促进3,4-脱氢玫红品（3,4-didehydrorhodopin）的合成，可能是光合细菌应对环境压力的一种机制。抗氧化性分析和数据拟合表明不同组成的类胡萝卜素抗氧化能力差异显著，经基螺菌黄素和3,4-脱氢玫红品的抗氧化能力最强。

关键词: 光合细菌, 类胡萝卜素, 抗氧化活性, 有机酸

Abstract:

Carotenoids are widely used as fat-soluble natural pigments and antioxidants. Here we chose Rhodopseudomonas palustris, a typical purple nonsulfur photosynthetic bacteria, to produce carotenoids under photo-anaerobic conditions. The effects of culture conditions such as organic acid types, salt concentration and light intensity on the cell growth, carotenoid content and composition were studied, and the antioxidant activities of carotenoids were also explored. The results showed that lactic acid as carbon source produced the highest biomass and carotenoid content among the eight organic acids tested. The optimum light intensity (500lx) for carotenoid content was lower than that required for the growth of Rhodopseudomonas palustris (2000lx). High salt concentration promoted the synthesis of carotenoids, and the optimum concentration (7.5g/L) was three times that required for its growth. Under optimized conditions, the carotenoid content reached 15.35mg/L. Six kinds of carotenoids were detected, and the composition was greatly affected by the culture conditions. High salt concentration and strong light favored the synthesis of 3,4-didehydrorhodopin, which might be a mechanism for photosynthetic bacteria to adapt to environmental stress. The antioxidant activity test and data analysis showed that antioxidant activities of the produced carotenoids highly depended on their composition, and OH-spirilloxanthin and 3,4-didehydrorhodopin showed strong antioxidant activities.

Key words: photosynthetic bacteria, carotenoid, antioxidant activity, organic acids

中图分类号:

Q936

李南楠, 彭思雨, 闫海, 尹春华. 沼泽红假单胞菌产类胡萝卜素及抗氧化性[J]. 化工进展, 2023, 42(11): 5891-5899.

LI Nannan, PENG Siyu, YAN Hai, YIN Chunhua. Production and antioxidant activities of carotenoids from Rhodopseudomonas palustris[J]. Chemical Industry and Engineering Progress, 2023, 42(11): 5891-5899.

图/表 13

参考文献 44

1	LIU H L, CHEN B H, KAO T H, et al. Carotenoids composition in Scutellaria barbata D. Don as detected by high performance liquid chromatography-diode array detection-mass spectrometry-atmospheric pressure chemical ionization[J]. Journal of Functional Foods, 2014, 8: 100-110.
2	SAEJUNG Chewapat, APAIWONG Pawittra. Enhancement of carotenoid production in the new carotenoid-producing photosynthetic bacterium Rhodopseudomonas faecalis PA2[J]. Biotechnology and Bioprocess Engineering, 2015, 20(4): 701-707.
3	PATTHAWARO Sirada, LOMTHAISONG Khomsorn, SAEJUNG Chewapat. Bioconversion of agro-industrial waste to value-added product lycopene by photosynthetic bacterium Rhodopseudomonas faecalis and its carotenoid composition[J]. Waste and Biomass Valorization, 2020, 11(6): 2375-2386.
4	MUSSAGY C U, WINTERBURN J, SANTOS-EBINUMA V C, et al. Production and extraction of carotenoids produced by microorganisms[J]. Applied Microbiology and Biotechnology, 2019, 103(3): 1095-1114.
5	CAPSON-TOJO G, BATSTONE D J, GRASSINO M, et al. Purple phototrophic bacteria for resource recovery: Challenges and opportunities[J]. Biotechnology Advances, 2020, 43: 107567.
6	LI Zuming, KONG Lina, HUI Bodi, et al. Identification and antioxidant activity of carotenoids from superfine powder of Rhodobacter sphaeroides [J]. Emirates Journal of Food and Agriculture, 2017, 29(11): 833-845.
7	LIU Chang, HU Bin, CHENG Yuliang, et al. Carotenoids from Fungi and microalgae: A review on their recent production, extraction, and developments[J]. Bioresource Technology, 2021, 337: 125398.
8	崔艺久, 王悦, 牛明芬, 等. 沼泽红假单胞菌产类胡萝卜素发酵条件优化[J]. 沈阳农业大学学报, 2012, 43(2): 225-228.
	CUI Yijiu, WANG Yue, NIU Mingfen, et al. Rhodopseudomonas palustris optimization of fermentation conditions for production of carotenoids[J]. Journal of Shenyang Agricultural University, 2012, 43(2): 225-228.
9	MENG Fan, PENG Meng, WANG Xintian, et al. Lactic acid wastewater treatment by photosynthetic bacteria and simultaneous production of protein and pigments[J]. Environmental Technology, 2022, 43(2): 163-170.
10	李美洁, 夏青青, HARWOOD C S, 等. 沼泽红假单胞菌作为微生物细胞工厂的应用[J]. 生物加工过程, 2020, 18(1): 27-34.
	LI M J, XIA Q Q, HARWOOD C S, et al. Application of Rhodopseudomonas palustris as microbial cell factories[J]. Chinese Journal of Bioprocess Engineering, 2020, 18(1): 27-34.
11	COOPER D A. Carotenoids in health and disease: Recent scientific evaluations, research recommendations and the consumer[J]. The Journal of Nutrition, 2004, 134(1): 221S-224S.
12	WOODALL A A, LEE S W, WEESIE R J, et al. Oxidation of carotenoids by free radicals: Relationship between structure and reactivity[J]. Biochimica et Biophysica Acta, 1997, 1336(1): 33-42.
13	CONN P F, LAMBERT C, LAND E J, et al. Carotene-oxygen radical interactions[J]. Free Radical Research Communications, 1992, 16(6): 401-408.
14	俞吉安, 叶永钧, 林志新, 等. 光合细菌中类胡萝卜素对脂质过氧化抑制作用研究[J]. 上海交通大学学报, 1998, 32(3): 107-110.
	YU Jian, YE Yongjun, LIN Zhixin, et al. On effect of carotenoids in photosynthetic bacteria on inhibiting lipid peroxiation[J]. Journal of Shanghai Jiao Tong University, 1998, 32(3): 107-110.
15	方立超, 魏泓. 光合细菌抗氧化特性的体外实验研究[J]. 解放军预防医学杂志, 2008, 26(3): 164-167.
	FANG Lichao, WEI Hong. Anti-oxidative characteristics of photosynthetic bacteria in vitro [J]. Journal of Preventive Medicine of Chinese PLA, 2008, 26(3): 164-167.
16	孙震, 姚惠源. 类胡萝卜素的抗氧化活性与其量子化学参数的相关研究[J]. 食品与机械, 2007, 23(4): 56-59.
	SUN Zhen, YAO Huiyuan. Study on the correlation of antioxidation activity of carotenoids with quantum chemical parameters[J]. Food & Machinery, 2007, 23(4): 56-59.
17	穆青, 陈亚淑, 谢笔钧, 等. 北极海洋红球菌B7740(Rhodococcus sp.)产类胡萝卜素和类异戊二烯醌的抗氧化、抗增殖活性[J]. 食品科学, 2018, 39(11): 159-164.
	MU Qing, CHEN Yashu, XIE Bijun, et al. Antioxidant and antiproliferative activity of carotenoids and isoprenoid quinones from Rhodococcus sp. B7740[J]. Food Science, 2018, 39(11): 159-164.
18	董姗, 张光明. 不同条件对光合细菌沉降性的影响[J]. 工业安全与环保, 2013, 39(2): 5-7, 13.
	DONG Shan, ZHANG Guangming. Effects of different factors on PSB sedimentation[J]. Industrial Safety and Environmental Protection, 2013, 39(2): 5-7, 13.
19	WANG Hangyao, YANG Anqi, ZHANG Guangming, et al. Enhancement of carotenoid and bacteriochlorophyll by high salinity stress in photosynthetic bacteria[J]. International Biodeterioration & Biodegradation, 2017, 121: 91-96.
20	SERDYUK O P, SMOLYGINA L D, ASHIKHMIN A A. A new type of light-harvesting complex detected when growing Rhodopseudomonas palustris under low light intensity conditions[J]. Doklady Biochemistry and Biophysics, 2020, 491(1): 101-104.
21	SCHWERZMANN R U, BACHOFEN R. Carotenoid profiles in pigment-protein complexes of Rhodospirillum rubrum [J]. Plant and Cell Physiology, 1989, 30(4): 497-504.
22	赵江艳, 傅英楠, 赵春贵, 等. 一株高含玫红品的红树林海洋紫色硫细菌分离鉴定及特性[J]. 微生物学报, 2011, 51(10): 1318-1325.
	ZHAO Jiangyan, FU Yingnan, ZHAO Chungui, et al. Identification and characterization of a purple sulfur bacterium from mangrove with rhodopin as predominant carotenoid[J]. Acta Microbiologica Sinica, 2011, 51(10): 1318-1325.
23	张同, 朱瑞艳, 惠伯棣, 等. 沼泽红假单胞菌胞内番茄红素生物合成的研究[J]. 食品科学, 2013, 34(21): 283-291.
	ZHANG Tong, ZHU Ruiyan, HUI Bodi, et al. Lycopene biosynthesis in Rhodopseudomonas palustris [J]. Food Science, 2013, 34(21): 283-291.
24	赵春贵, 付乔明, 卓民权, 等. 薄层层析法(TLC)测定螺菌黄质系类胡萝卜素的相对极性和稳定性[J]. 微生物学通报, 2014, 41(7): 1448-1455.
	ZHAO Chungui, FU Qiaoming, ZHUO Minquan, et al. The relative polarity and stability of each carotenoid of spirilloxanthin biosynthesis pathway by thin-layer chromatography(TLC)[J]. Microbiology China, 2014, 41(7): 1448-1455.
25	MIZOGUCHI Tadashi, ISAJI Megumi, HARADA Jiro, et al. Identification of 3,4-didehydrorhodopin as major carotenoid in Rhodopseudomonas species[J]. Photochemical & Photobiological Sciences: Official Journal of the European Photochemistry Association and the European Society for Photobiology, 2008, 7(4): 492-497.
26	杨桥, 张晓玲, 张磊, 等. 类胡萝卜素在耐辐射奇球菌辐射抗性中的作用[J]. 生物化学与生物物理进展, 2009, 36(6): 715-721.
	YANG Qiao, ZHANG Xiaoling, ZHANG Lei, et al. Effects of carotenoids on the radioresistance of the extremely radioresistant bacterium deinococcus radiodurans[J]. Progress in Biochemistry and Biophysics, 2009, 36(6): 715-721.
27	邓永平, 车鑫, 艾瑞波, 等. 好食脉孢霉发酵产类胡萝卜素的鉴定、抗氧化性及稳定性研究[J]. 食品与发酵工业, 2021, 47(4): 15-20.
	DENG Yongping, CHE Xin, AI Ruibo, et al. Identification, antioxidant and stability of carotenoids from a GRAS fungus Neurospora sitophila [J]. Food and Fermentation Industries, 2021, 47(4): 15-20.
28	BENZIE I F, STRAIN J J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay[J]. Analytical Biochemistry, 1996, 239(1): 70-76.
29	Virginia MONTIEL-CORONA, Germán BUITRÓN. Polyhydroxyalkanoates from organic waste streams using purple non-sulfur bacteria[J]. Bioresource Technology, 2021, 323: 124610.
30	LAGUNA R, TABITA F R, ALBER B E. Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin-Benson-Bassham reductive pentose phosphate cycle in Rhodobacter sphaeroides and Rhodopseudomonas palustris [J]. Archives of Microbiology, 2011, 193(2): 151-154.
31	DANIAL A W, ABDEL W A, ARAFAT H H, et al. Bioenergetics of lactate vs. acetate outside TCA enhanced the hydrogen evolution levels in two newly isolated strains of the photosynthetic bacterium Rhodopseudomonas [J]. Zeitschrift Fur Naturforschung C, Journal of Biosciences, 2017, 72(3/4): 99-105.
32	WANG Zhaobao, WEN Qifeng, HARWOOD Caroline S, et al. A disjointed pathway for malonate degradation by Rhodopseudomonas palustris [J]. Applied and Environmental Microbiology, 2020, 86(11): e00631-e00620.
33	BOLSHAKOV M A, ASHIKHMIN A A, MAKHNEVA Z K, et al. Distribution of rhodopin and spirilloxanthin between LH1 and LH2 complexes when incorporating carotenoid mixture into the membrane of purple sulfur bacterium Allochromatium minutissimum in vitro [J]. Doklady Biochemistry and Biophysics, 2016, 471(1): 383-386.
34	GARCIA-CORTES A, GARCIA-VÁSQUEZ J A, ARANGUREN Y, et al. Pigment production improvement in Rhodotorula mucilaginosa AJB01 using design of experiments[J]. Microorganisms, 2021, 9(2): 387.
35	PALIWAL Chetan, PANCHA Imran, GHOSH Tonmoy, et al. Selective carotenoid accumulation by varying nutrient media and salinity in Synechocystis sp. CCNM 2501[J]. Bioresource Technology, 2015, 197: 363-368.
36	KUMAR M, KULSHRESHTHA J, SINGH G P. Growth and biopigment accumulation of cyanobacterium Spirulina platensis at different light intensities and temperature[J]. Publication of the Brazilian Society for Microbiology, 2011, 42(3): 1128-1135.
37	EGGERSDORFER M, WYSS A. Carotenoids in human nutrition and health[J]. Archives of Biochemistry and Biophysics, 2018, 652: 18-26.
38	Sladjana STAJČIĆ, Gordana ĆETKOVIĆ, Jasna ČANADANOVIĆ-BRUNET, et al. Tomato waste: Carotenoids content, antioxidant and cell growth activities[J]. Food Chemistry, 2015, 172: 225-232.
39	XIA Yichen, BAMDAD Fatemeh, Michael GÄNZLE, et al. Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis[J]. Food Chemistry, 2012, 134(3): 1509-1518.
40	KANG Wenyi, WANG Jinmei. In vitro antioxidant properties and in vivo lowering blood lipid of Forsythia [J]. Medicinal Chemistry Research, 2010, 19(7): 617-628.
41	胡珂, 李娜. 倍受青睐的抗氧化家族——类胡萝卜素[J]. 大学化学, 2010, 25(S1): 94-98.
	HU Ke, LI Na. Carotenoids: A favored antioxidant family[J]. University Chemistry, 2010, 25(S1): 94-98.
42	KUMARAN A, KARUNAKARAN R J. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus [J]. Food Chemistry, 2006, 97(1): 109-114.
43	TERAO J. Antioxidant activity of β-carotene-related carotenoids in solution[J]. Lipids, 1989, 24(7): 659-661.
44	TAN Si, KE Zunli, CHAI Dan, et al. Lycopene, polyphenols and antioxidant activities of three characteristic tomato cultivars subjected to two drying methods[J]. Food Chemistry, 2021, 338: 128062.

名称	化学式	特征吸收峰/nm		保留时间/min	分子量[MH⁺]
名称	化学式	实测值	文献值^[24]	保留时间/min	实测值	理论值^[25]
番茄红素^①	C₄₀H₅₆	446 472 503	446 472 502	23.01	537.45	537.44
玫红品	C₄₀H₅₈O	445 471 502	442 471 502	11.96	555.46	555.45
3,4-脱氢玫红品	C₄₀H₅₆O	457 483 516	454 482 515	11.20	553.44	553.43
脱水紫菌红醇	C₄₁H₅₈O	457 482 516	457 482 514	16.20	567.46	567.45
紫菌红醇	C₄₁H₆₀O₂	455 483 515	457 483 515	8.47	585.47	585.46
经基螺菌黄素	C₄₁H₅₈O₂	466 493 527	461 493 525	7.93	583.45	583.44

名称	化学式	特征吸收峰/nm		保留时间/min	分子量[MH⁺]
名称	化学式	实测值	文献值^[24]	保留时间/min	实测值	理论值^[25]
番茄红素^①	C₄₀H₅₆	446 472 503	446 472 502	23.01	537.45	537.44
玫红品	C₄₀H₅₈O	445 471 502	442 471 502	11.96	555.46	555.45
3,4-脱氢玫红品	C₄₀H₅₆O	457 483 516	454 482 515	11.20	553.44	553.43
脱水紫菌红醇	C₄₁H₅₈O	457 482 516	457 482 514	16.20	567.46	567.45
紫菌红醇	C₄₁H₆₀O₂	455 483 515	457 483 515	8.47	585.47	585.46
经基螺菌黄素	C₄₁H₅₈O₂	466 493 527	461 493 525	7.93	583.45	583.44

实验组样品	羟基螺菌黄素	紫菌红醇	3,4-脱氢玫红品	玫红品	脱水紫菌红醇	番茄红素	DPPH自由基清除率/%	还原铁离子能力/μmol	ABTS自由基清除率/%	菌体总类胡萝卜素含量/mg·L^-1
1	3.5	0	76.54	16.99	1.86	1.11	38.85	46.64	43.68	5.49
2	4.65	8.43	47.12	36.27	0.94	2.59	38.9	41.18	42.28	7.19
3	8.22	4.14	61.25	24.08	1	1.31	39.16	47.04	42.48	2.36
4	1.4	4.76	35.96	45.37	5.24	7.27	37.16	38.73	35.35	12.02
5	3.54	8.69	38.91	44.35	2.43	2.08	35.86	42.05	37.85	4.55
6	4.93	4.74	50.36	33.77	2.53	3.67	38.1	42.29	41.3	10.14
7	6.18	5.85	54.1	30.16	1.98	1.74	39.66	46.75	41.93	1.17
8	4.63	5.28	44.77	37.82	3.8	3.69	40.43	40.81	41.73	1.93
9	4.22	8.07	29.36	49.56	5.73	3.06	34.65	34.61	38.62	7.36
10	3.53	6.63	30.21	47.75	6.32	5.55	35.76	42.43	36.47	7.26
11	4.35	4.67	34.53	43.61	4.62	8.23	35.7	39.21	40.48	1.29
12	4.02	5.76	42.43	35.32	5.07	7.4	35.44	40.52	38.86	1.09
13	3.88	3.65	52.1	27.64	4.87	7.86	39.98	41.69	40.91	0.52
14	1.4	4.76	35.96	45.37	5.24	7.26	33.17	36.73	36.35	12.92
15	3.12	6.63	32.21	45.75	6.32	5.97	33.44	37.1	37.07	15.35
16	2.56	6.48	32.7	43.66	5.93	8.66	33.52	37.94	37.07	14.58
17	3.11	6.3	36.41	42.55	4.21	7.42	34.55	38.98	37.65	13.94
18	2.8	6.29	36.49	41.07	5.14	8.21	34.46	38.77	37.9	12.92
19	5.05	8.07	36.33	38.86	5.16	6.54	35.83	40.12	39.67	9.78
20	3.16	6.48	38.15	38.83	5.6	7.78	36.08	41.24	40.45	9.30

实验组样品	羟基螺菌黄素	紫菌红醇	3,4-脱氢玫红品	玫红品	脱水紫菌红醇	番茄红素	DPPH自由基清除率/%	还原铁离子能力/μmol	ABTS自由基清除率/%	菌体总类胡萝卜素含量/mg·L^-1
1	3.5	0	76.54	16.99	1.86	1.11	38.85	46.64	43.68	5.49
2	4.65	8.43	47.12	36.27	0.94	2.59	38.9	41.18	42.28	7.19
3	8.22	4.14	61.25	24.08	1	1.31	39.16	47.04	42.48	2.36
4	1.4	4.76	35.96	45.37	5.24	7.27	37.16	38.73	35.35	12.02
5	3.54	8.69	38.91	44.35	2.43	2.08	35.86	42.05	37.85	4.55
6	4.93	4.74	50.36	33.77	2.53	3.67	38.1	42.29	41.3	10.14
7	6.18	5.85	54.1	30.16	1.98	1.74	39.66	46.75	41.93	1.17
8	4.63	5.28	44.77	37.82	3.8	3.69	40.43	40.81	41.73	1.93
9	4.22	8.07	29.36	49.56	5.73	3.06	34.65	34.61	38.62	7.36
10	3.53	6.63	30.21	47.75	6.32	5.55	35.76	42.43	36.47	7.26
11	4.35	4.67	34.53	43.61	4.62	8.23	35.7	39.21	40.48	1.29
12	4.02	5.76	42.43	35.32	5.07	7.4	35.44	40.52	38.86	1.09
13	3.88	3.65	52.1	27.64	4.87	7.86	39.98	41.69	40.91	0.52
14	1.4	4.76	35.96	45.37	5.24	7.26	33.17	36.73	36.35	12.92
15	3.12	6.63	32.21	45.75	6.32	5.97	33.44	37.1	37.07	15.35
16	2.56	6.48	32.7	43.66	5.93	8.66	33.52	37.94	37.07	14.58
17	3.11	6.3	36.41	42.55	4.21	7.42	34.55	38.98	37.65	13.94
18	2.8	6.29	36.49	41.07	5.14	8.21	34.46	38.77	37.9	12.92
19	5.05	8.07	36.33	38.86	5.16	6.54	35.83	40.12	39.67	9.78
20	3.16	6.48	38.15	38.83	5.6	7.78	36.08	41.24	40.45	9.30

抗氧化性测定方法	R²	类胡萝卜素组分抗氧化系数
抗氧化性测定方法	R²	羟基螺菌黄素	3,4-脱氢玫红品	紫菌红醇	番茄红素	玫红品	脱水紫菌红醇
DPPH	0.998	0.696	0.416	0.330	0.323	0.311	0.143
FRAP	0.998	0.861	0.498	0.432	0.333	0.289	0.219
ABTS	0.999	0.913	0.445	0.491	0.425	0.282	0.243