胶红酵母产类胡萝卜素固态发酵工艺

doi:10.3864/j.issn.0578-1752.2018.10.017

摘要/Abstract

摘要：

目的优化胶红酵母固态发酵底物与发酵条件,提高类胡萝卜素产量,改善发酵产物营养价值,降低生产成本。方法选用胶红酵母TZR₂₀₁₄作为发酵菌种,采用Design-Expert软件的Mixture-Design设计固态发酵底物的配比,各底物原料的范围如下：麸皮50%—80%、豆粕6%—20%、玉米粉3%—15%、米糠2%—14%、玉米浆2%—10%、硫酸铵0.4%—2.5%、磷酸二氢钾0.05%—0.5%和硫酸镁0.03%—0.3%,通过固态发酵工艺生产类胡萝卜素,并根据类胡萝卜素的产量来确定最优的发酵底物。确定最适发酵底物配比后,利用L₁₆(4⁵)正交设计对发酵条件进行优化设计,各参数的范围如下：接种量5.0%—12.5%、发酵时间60.0—96.0 h、发酵温度26—32℃、pH 4.0—7.0、含水量60.0%—75.0%,根据试验结果确定胶红酵母产类胡萝卜素的最优发酵条件。研究优化的胶红酵母固态发酵工艺对发酵产物粗纤维、粗蛋白质、水分、粗脂肪、粗灰分、钙、磷和氨基酸等营养物质的影响。结果胶红酵母发酵产物中类胡萝卜素含量与固态发酵底物中麦麸的添加量呈显著负相关（r=-0.336,P=0.045）,与发酵底物中玉米浆的添加量呈显著正相关（r=0.344,P=0.040）,与发酵底物中米糠添加量为正相关（r=0.329,P=0.050）。发酵产物中胶红酵母活菌数与底物中豆粕含量呈显著正相关（r=0.510,P=0.001）。接种量、发酵温度、pH、底物含水量对胶红酵母活菌数均有极显著的影响（P<0.01）,但其中发酵温度对胶红酵母菌体数影响最大,其次是底物含水量,之后依次是接种量和pH。发酵时间、发酵温度和pH均显著影响发酵产物中类胡萝卜素含量（P<0.05）,其中发酵温度对发酵产物中类胡萝卜素含量影响最大,pH次之,发酵时间影响最小。经过发酵工艺的优化,发酵产物中类胡萝卜素产量提高到4 535 μg·kg^-1,发酵产物中的活菌数为3.79×10⁹CFU/g;发酵后粗纤维、粗蛋白质、粗灰分、苏氨酸、谷氨酸、脯氨酸含量均显著高于发酵前（P<0.05）,而组氨酸、水分、粗脂肪含量显著低于发酵前（P<0.05）。结论胶红酵母固态发酵产类胡萝卜素底物的最佳配比为麦麸52.5%、豆粕20.0%、玉米粉3.00%、米糠14.0%、玉米浆10.0%、硫酸铵0.40%、磷酸二氢钾0.05%和硫酸镁0.04%;最佳发酵条件为菌液接种量5.0%、发酵时间72.0 h、发酵温度28.0℃、pH 6.0、底物含水量60.0%。经过优化胶红酵母发酵工艺,类胡萝卜素产量得到显著提高,并且发酵产物营养价值得到明显改善。

关键词: 胶红酵母, 固态发酵, 类胡萝卜素, 发酵底物, 发酵条件

Abstract:

【Objective】 This study was performed to enhance carotenoid yield, to improve nutritional value of fermentation product, and to reduce the production cost of carotenoids through optimizing solid-state fermentation substrate and fermentation conditions of Rhodotorula mucilaginosa.【Method】 In this study, Rhodotorula mucilaginosa TZR₂₀₁₄ was used as a inoculant. First, the Mixture-Design of Design-Expert software was used to design the fermentation substrate, and the contents of ingredients as followed: 50%-80% wheat bran, 6%-20% soybean meal, 3%-15% maize flour, 2%-14% rice bran, 2%-10% maize syrup, 0.4%-2.5% ammonium sulfate, 0.05%-0.5% monopotassium phosphate, and 0.03%-0.3% magnesium sulfate. Then the optimal ratio of ingredients in substrate was determined according to the carotenoid yield. Based on this result, an L₁₆(4⁵) orthogonal design was used to optimize the fermentation conditions, including inoculum (5.0%-12.5%), fermentation time (60.0-96.0 h), fermentation temperature (26-32℃), and fermentation pH (60.0%-75.0%). Finally, the number of Rhodotorula mucilaginosa and contents of carotenoids, crude fiber, crude protein, water, crude fat, ash, calcium, phosphorus, and amino acids in fermentation product were determined to evaluate the effects of the optimized fermentation process on the nutritional values of fermentation product. 【Result】 The results showed that there was a positive correlation between maize starch content in substrate and carotenoid content in fermentation product (r=0.344, P=0.040) or between rice bran content in fermentation substrate and carotenoid content in fermentation product (r=0.329, P=0.050). There was a significantly negative correlation between carotenoid yield and the content of wheat bran in solid-state fermentation substrate (r=-0.336, P=0.045). There was a positive correlation between the number of live bacteria of Rhodotorula mucilaginosa in fermentation product and the content of soybean meal in fermentation substrate (r=0.510, P=0.001). Inoculum, fermentation temperature, pH, and moisture had extremely significant impacts on the number of Rhodotorula mucilaginosa (P<0.01), thereinto, fermentation temperature had the greatest effect on the number of Rhodotorula mucilaginosa, followed by moisture, inoculum, and pH. Fermentation time, fermentation temperature, and pH had extremely significant influences on the carotenoid content in the fermentention product (P<0.01), and fermentation temperature had the greatest influence on the carotenoid content in the fermented product, followed by pH and fermentation time. After the optimization of the fermentation process, the carotenoid yield by Rhodotorula mucilaginosa TZR₂₀₁₄ was increased to 4 535 μg·kg^-1; the bacteria number was increased to 3.79×10⁹ CFU/kg; the contents of crude fiber, crude protein, ash, threonine, glutamate, and proline in fermentation product were significantly increased (P<0.05), meanwhile, the contents of histidine, water, and crude fat was significantly decreased (P<0.05). 【Conclusion】 The optimal ratio of solid-state fermentation substrate for Rhodotorula mucilaginosa was as followed: 52.5% wheat bran, 20.0% soybean meal, 3.0% maize flour, 14.0% rice bran, 10.0% maize syrup, 0.4% ammonium sulfate, 0.05% monopotassium phosphate, and 0.04% magnesium sulfate. The optimal fermentation conditions were as followed: inoculum 5.0%, fermentation time 72 h, fermentation temperature 28.0℃, pH 6.0, and moisture 60.0%. The results suggested that the optimized fermentation process of Rhodotorula mucilaginosa enhanced the yield of carotenoids and improved the nutritional value of fermentation product.

Key words: Rhodotorula mucilaginosa, solid-state fermentation, carotenoids, fermentation substrates, fermentation conditions

孙佳静, 李貌, 孙志洪, 唐志如, 张相鑫, 陈进超. 胶红酵母产类胡萝卜素固态发酵工艺[J]. 中国农业科学, 2018, 51(10): 1982-1994.

JiaJing SUN, Mao LI, ZhiHong SUN, ZhiRu TANG, XiangXin ZHANG, JinChao CHEN. The Solid-State Fermentation Process of Rhodotorula Mucilaginosa for Producing Carotenoids[J]. Scientia Agricultura Sinica, 2018, 51(10): 1982-1994.

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参考文献 32

[1]	BANZATTO D, FREITA L A D,MUTTON M J R. Carotenoid production by Rhodotorula rubra cultivated in sugarcane juice, molasses, and syrup. Ciência e Tecnologia de Alimentos, 2013, 33(33): 14-18.
[2]	TANG F Y.The silver bullet for cancer prevention: Chemopreventive effects of carotenoids.Biomedicine, 2012, 2(3): 117-121. doi: 10.1016/j.biomed.2012.06.004
[3]	BHAGAVATHY S, SUMATHI P.Evaluation of antigenotoxic effects of carotenoids from green algae Chlorococcum humicola using human lymphocytes. Asian Pacific Journal of Tropical Biomedicine, 2012, 2(2):109-117. doi: 10.1016/S2221-1691(11)60203-7 pmid: 3609264
[4]	NELIS H J, DELEENHEER A P.Microbial sources of carotenoid- pigments used in foods and feeds. Journal of Applied Bacteriology, 1991, 70(3): 181-191.
[5]	PERRIER V, DUBREUCQ E, GALZY P.Fatty-acid and carotenoid composition of rhodotorula strains. Archives of Microbiology, 1995, 164(3): 173-179.
[6]	董娟, 郑晓吉, 孙静涛, 史学伟. 红酵母产类胡萝卜素影响因素及检测方法研究进展. 粮食与油脂, 2013, 26(3): 49-51.
	DONG J, ZHENG X J, SUN J T, SHI X W.Research advance in influencing factors of carotenoid producted by red yeasts and detection methods.Cereals & Oils, 2013, 26(3): 49-51. (in Chinese)
[7]	FERRAO M, GARG S.Studies on effect of media components on growth and α-carotene production by Rhodotorula graminis RC04. Journal of Cell Tissue Reseach, 2011, 11(1): 2551-2556.
[8]	HERNANDEZ-ALMANZA A, CESAR MONTANEZ J, AGUILAR- GONZALEZ M A, MARTINEZ-AVILA C, RODRIGUEZ- HERRERA R, N.AGUILAR C.Rhodotorula glutinis as source of pigments and metabolites for food industry. Food Bioscience, 2014, 5:64-72. doi: 10.1016/j.fbio.2013.11.007
[9]	KOT A M, BLAZEJAK S, KURCZ A, GIENTKA I, KIELISZEK M.Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 2016, 100(14): 6103-6117. doi: 10.1007/s00253-016-7611-8 pmid: 4916194
[10]	ROADJANAKAMOLSON M, SUNTORNSUK W.Production of beta-carotene-enriched rice bran using solid-state fermentation of Rhodotorula glutinis. Journal of Microbiology and Biotechnology, 2010, 20(3): 525-531.
[11]	HERNANDEZ-ALMANZA A, MONTANEZ-SAENZ J, MARTINEZ- AVILA C, RODRIGUEZ-HERRERA R, N.AGUILAR C. Carotenoid production byRhodotorula glutinis YB-252 in solid-state fermentation. Food Bioscience, 2014(7): 31-36. doi: 10.1016/j.fbio.2014.04.001
[12]	杨文, 吉春明. 一种简单的胞壁破碎方法. 微生物学通报, 1995, 22(1): 58-59.
	YANG W, JI C M.A simple method of crushing cell wall.Microbiology, 1995, 22(1): 58-59. (in Chinese)
[13]	王岁楼, 张鑫, 张平之. 红酵母类胡萝卜素提取方法研究.食品与机械, 2000(6): 14-16.
	WANG S L, ZHANG X, ZHANG P Z.The extraction of carotenoids fromRhodotorula. Food & Machinery, 2000(6): 14-16. (in Chinese)
[14]	张玉诚, 薛白, 达勒措, 李秋瑾, 何宇. 混菌固态发酵白酒糟开发为蛋白质饲料的条件优化及营养价值评定. 动物营养学报, 2016, 28(11): 3711-3720. doi: 10.3969/j.issn.1006-267x.2016.11.042
	ZHANG Y C, XUE B, DA L C, LI Q J, HE Y.Distillers Grains: Optimization of mixed bacterial solid-state fermentation conditions to produce protein feed and nutrient value analysis.Chinese Journal of Animal Nutrition, 2016, 28(11): 3711-3720. (in Chinese) doi: 10.3969/j.issn.1006-267x.2016.11.042
[15]	GABERT V M, SAUER W C, SCHMITZ M, AHRENS F, MOSENTHIN R.The effect of formic acid and buffering capacity on the ileal digestibilities of amino acids and bacterial populations and metabolites in the small intestine of weanling pigs fed semipurified fish meal diets.Canadian Journal of Animal Science, 1995, 75(4): 615-623. doi: 10.4141/cjas95-091
[16]	王岁楼, 章银良, 王平诸. 红酵母RY-98产类胡萝卜素培养基的优选及其发酵生理学研究. 生物技术, 2000, 10(4): 24-27.
	WANG S L, ZHANG Y L, WANG P Z.Studies on the selection of carotenoids fermentation medium for RY- 98 strain of Rhodotorula and its fermentative physiology. Biotechnology, 2000, 10(4): 24-27. (in Chinese)
[17]	唐棠. 红酵母Y-5菌株产类胡萝卜素发酵条件的研究[D]. 雅安: 四川农业大学, 2011.
	TANG T.Optimization of fermentation condition for production of carotenoid by Rhotlotorula Y-5 [D]. Ya’an: Sichuan Agricultural University, 2011. (in Chinese)
[18]	张坤生, 连喜军, 李红, 任云霞. 红酵母高产β-胡萝卜素营养因子的选择. 食品工业科技, 2004, 25(7): 60-62. doi: 10.3969/j.issn.1002-0306.2004.07.024
	ZHANG K S, LIAN X J, LI H, REN Y X.Nutrition factors selection for β-carotene of red yeast.Science and Technology of Food Industry, 2004, 25(7): 60-62. (in Chinese) doi: 10.3969/j.issn.1002-0306.2004.07.024
[19]	梁晓华, 杨莺莺, 杨铿, 陈永青. 海洋红酵母Y2发酵产类胡萝卜素条件的研究. 中国微生态学杂志, 2011, 23(12): 1068-1073.
	LIANG X H, YANG Y Y, YANG K, CHENG Y Q.Production of carotenoid by marine red yeast Y2.Chinese Journal of Microecology, 2011, 23(12): 1068-1073. (in Chinese)
[20]	刘卉琳. 红酵母合成类胡萝卜素及体外转化维生素A的研究[D]. 长沙: 湖南农业大学, 2012.
	LIU H L.Studies on production of carotenoid by Rhodotorulla mucilaginosa and its conversion to vitamin A in vitro [D]. Changsha: Hunan Agricultural University, 2012. (in Chinese)
[21]	丛莉. 海洋红酵母产类胡萝卜素的研究[D]. 青岛: 中国海洋大学, 2006.
	CONG L.Investigation on carotenoids from Marine Rhodotorula sp. [D]. Qingdao: Ocean University of China, 2006. (in Chinese)
[22]	TINOI J, RAKARIYATHAM N, DEMING R L.Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochemistry, 2005, 40(7): 2551-2557.
[23]	王增煌, 王文策, 翟双双, 谢强, 左鑫, 杨琳. 香蕉茎叶粉固态发酵条件优化及鹅对其养分利用率的研究. 动物营养学报, 2017, 29(4): 1283-1293.
	WANG Z H, WANG W C, ZHAI S S, XIE Q, ZUO X, YANG L.Study on optimal solid state fermentation conditions of banana stems and leaves powder and its nutrient utilization for geese.Chinese Journal of Animal Nutrition, 2017, 29(4): 1283-1293. (in Chinese)
[24]	赵紫华, 韩雪, 王丽丽, 郭晶晶, 张率, 韩萌, 荀静娜. 高产β-胡萝卜素的红酵母培养条件的优化. 食品与发酵科技, 2014, 50(4): 34-38. doi: 10.3969/j.issn.1674-506X.2014.04-009
	ZHAO Z H, HAN X, WANG L L, GUO J J, ZHANG L, HAN M, XUN J N.The fermentation technology optimization with high β-carotene by red yeast.Food and Fermentation Technology, 2014, 50(4): 34-38. (in Chinese) doi: 10.3969/j.issn.1674-506X.2014.04-009
[25]	KAISER P, SURMANN P, VALLENTIN G, FUHRMANN H.A small-scale method for quantitation of carotenoids in bacteria and yeasts.Journal of Microbiological Methods, 2007, 70(1): 142-149. doi: 10.1016/j.mimet.2007.04.004 pmid: 17509707
[26]	DOMYOUNG K, EUNJI C, JIWON K, YONGWOOG L, HWAJEE C.Production of cellulases by Penicillium sp in a solid-state fermentation of oil palm empty fruit bunch. African Journal of Biotechnology, 2014, 13(1): 145-155. doi: 10.5897/AJB12.2970
[27]	MAURYA D P, SINGH D, PRATAP D, MAURYA J P.Optimization of solid state fermentation conditions for the production of cellulase by Trichoderma reesei. Journal of Environmental Biology, 2012, 33(1): 5-8. doi: 10.1038/emboj.2010.40
[28]	AKSU Z, EREN A T.Carotenoids production by the yeast Rhodotorula mucilaginosa: Use of agricultural wastes as a carbon source. Process Biochemistry, 2005, 40(9): 2985-2991.
[29]	张闯. 红酵母发酵生产类胡萝卜素的研究[D]. 大连: 大连工业大学, 2011.
	ZHANG C.Preparation of carotenoids by fermention of Rhodotorula [D]. Dalian: Dalian Polytechnic University, 2011. (in Chinese)
[30]	ZHANG X Y, YANG Z H, LIANG J, TANG L, CHEN F.Detoxification of Jatropha curcas seed cake in solid-state fermentation of newly isolated endophytic strain and nutrition assessment for its potential utilizations.International Biodeterioration & Biodegradation, 2016, 109: 202-210. doi: 10.1016/j.ibiod.2016.02.001
[31]	王建军. 混菌固态发酵黄酒糟生产蛋白饲料的研究[D]. 杭州: 浙江大学, 2007.
	WANG J J.Production and application of protein feed from yellow rice wine lees using mixed strains combinations [D]. Hangzhou: Zhejiang University, 2007. (in Chinese)
[32]	孙展英. 马铃薯渣固态发酵工艺研究及生物学价值评定[D]. 保定: 河北农业大学, 2014.
	SUN Z Y.Study on technology of solid-State fermentation and evaluation of biological value of potato residue [D]. Baoding: Hebei Agricultural University, 2014. (in Chinese)

序号 Order number	麸皮 Wheat bran	豆粕 Soybean meal	玉米 Maize	米糠 Rice bran	玉米浆 Maize syrup	硫酸铵 Ammonium sulphate	磷酸二氢钾 Monopotassium phosphate	硫酸镁 Magnesium sulphate
1	73.4	9.51	5.00	6.64	2.43	2.50	0.33	0.17
2	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
3	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
4	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
5	71.4	7.89	15.0	2.00	2.04	1.30	0.18	0.22
6	67.7	10.0	4.60	14.0	2.00	1.27	0.23	0.24
7	63.4	14.4	13.6	3.70	2.44	1.57	0.50	0.28
8	52.3	19.8	13.6	10.6	2.00	1.57	0.15	0.03
9	77.0	7.53	9.97	2.98	2.05	0.40	0.05	0.03
10	58.1	15.5	9.15	12.2	4.03	0.40	0.50	0.06
11	58.1	18.2	12.9	5.01	3.10	2.50	0.11	0.15
12	53.8	16.6	7.79	11.3	8.59	1.16	0.50	0.28
13	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
14	69.4	6.37	3.00	11.9	6.39	2.44	0.25	0.30
15	56.0	18.3	5.81	7.81	10.0	1.74	0.13	0.26
16	76.1	8.99	3.00	4.93	4.38	2.48	0.06	0.03
17	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
18	62.3	16.4	10.1	7.38	2.94	0.61	0.05	0.22
19	57.7	15.6	6.41	10.9	8.82	0.40	0.19	0.03
20	56.4	17.7	14.0	3.66	7.32	0.85	0.10	0.04
21	70.2	20.0	3.06	3.21	2.63	0.68	0.21	0.05
22	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
23	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
24	65.9	7.38	13.8	7.01	4.86	0.40	0.42	0.27
25	57.6	12.2	13.8	4.76	8.84	2.35	0.20	0.30
26	63.6	6.00	14.0	4.73	9.90	1.54	0.12	0.10
27	73.4	9.51	5.00	6.64	2.43	2.50	0.33	0.17
28	77.7	6.00	3.00	8.10	4.16	0.74	0.08	0.19
29	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
30	79.8	7.93	3.00	2.00	6.59	0.46	0.05	0.18
31	59.5	19.3	4.44	10.2	4.45	1.89	0.05	0.08
32	67.7	10.0	4.60	14.0	2.00	1.27	0.23	0.24
33	62.1	13.3	8.73	8.05	6.00	1.53	0.28	0.09
34	69.4	6.37	3.00	11.9	6.39	2.44	0.25	0.30
35	76.1	8.99	3.00	4.93	4.38	2.48	0.06	0.03
36	62.3	16.4	10.1	7.38	2.94	0.61	0.05	0.22

序号 Order number	类胡萝卜素 Carotenoid (μg·kg^-1)	发酵料含菌数 Number of bacteria (10⁹CFU/g)
1	1 673±28.8	2.22±0.40
2	1 329±81.6	2.42±0.38
3	1 715±125	3.08±0.41
4	1 767±47.2	3.28±0.41
5	1 293±76.6	2.85±0.30
6	1 503±94.2	2.82±0.35
7	1 332±24.2	2.85±0.33
8	1 699±70.4	2.94±0.50
9	1 404±73.9	2.70±0.23
10	1 751±159	2.59±0.29
11	1 673±77.7	2.42±0.23
12	1 767±56.8	2.82±0.27
13	1 757±135	2.61±0.39
14	1 785±64.1	2.42±0.22
15	1 902±22.1	3.43±0.45
16	1 470±38.7	2.45±0.33
17	1 831±79.4	3.52±0.23
18	1 923±109	3.13±0.39
19	1 695±100	2.87±0.26
20	1 383±69.6	2.61±0.33
21	1 296±133	3.27±0.36
22	1 329±78.8	2.80±0.28
23	1 758±49.3	2.77±0.43
24	1 776±182	2.85±0.19
25	1 630±51.9	2.67±0.31
26	1 744±79.3	2.91±0.35
27	1 695±32.3	2.14±0.26
28	1 372±53.0	2.32±0.26
29	1 656±63.1	3.01±0.27
30	1 685±73.9	3.07±0.41
31	1 785±88.5	3.22±0.30
32	1 479±72.4	2.79±0.23
33	1 732±103	2.59±0.23
34	1 787±61.3	2.56±0.27
35	1 502±30.9	2.51±0.23
36	1 848±98.7	2.87±0.25

项目 Items	类胡萝卜素 Carotenoids	胶红酵母菌数 Number of Rhodototula mucilaginosa
模型的信噪比 Adeq Precision	6.23	5.32
R²预测值 Pred R-Squared	-0.095	-0.031

因素 Factor	类胡萝卜素 Carotenoids	P值 P-value	胶红酵母数 Number of Rhodototula mucilaginosa	P值 P-value
麦麸 Wheat bran	-0.336	0.045	-0.370	0.067
豆粕 Soybean meal	0.200	0.242	0.510	0.001
玉米粉 Maize	-0.049	0.777	0.005	0.978
米糠 Rice bran	0.329	0.050	-0.027	0.874
玉米浆 Maize syrup	0.344	0.040	0.183	0.284
硫酸铵 Ammonium sulphate	0.094	0.586	-0.296	0.080
磷酸二氢钾 Monopotassium phosphate	-0.009	0.960	-0.276	0.104
硫酸镁 Magnesium sulphate	0.196	0.253	-0.069	0.687

指标 Index	模型预测值 Model predictive value	实测值 Measured data	SEM	P值 P-value
类胡萝卜素 Carotenoids (μg·kg^-1)	1 923	1 937	22.6	0.65
活菌数 Number of bacteria (10⁹CFU/g)	3.2	3.18	0.38	0.31