产番茄红素菌株的选育及代谢调控的初步研究
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摘要
番茄红素是由11个共轭双键和2个非共轭双键组成的直链型碳氢化合物,因此具有极强的淬灭单线态氧和清除自由基的能力,它的抗氧化能力是β-胡萝卜素的2倍,是维生素E的100倍,近年来备受世人关注。
本研究通过诱变三孢布拉霉负菌菌株,提高番茄红素产量,优化培养条件,并通过代谢调控,提高代谢途径中关键酶的活性,加大类胡萝卜素代谢流,提高番茄红素的产量。
以三孢布拉霉负菌(-)08-09为出发菌株,经过紫外线诱变得到一株番茄红素产量高于原始菌株22.54%的突变株UV-312;经过亚硝酸诱变得到一株番茄红素产量高于原始菌株31.52%的突变株NA-62;经过紫外和亚硝酸复合诱变得到一株番茄红素产量高于原始菌株59.72%的突变株UN-10。
通过正交实验得到三孢布拉霉液体发酵最佳培养基组成为:玉米粉3.0%,棉籽饼粉4.0%,KH2PO4 0.5%,棉籽油9.0%,接种量15%(V/V)。最佳液体摇瓶培养条件为:培养温度27℃,装液量20 mL/250 mL,初始pH 7.5,正负菌接种比例1:20,接种量15%(V/V),转速180 r/min,培养时间5 d。在此培养条件下三孢布拉霉菌体分散较好,其菌体最大生物量、番茄红素产量分别为73.46 g/L、1.36 g/L。
正负菌平板上形成的色带宽度大小反映异宗配合的强弱,这与番茄红素的产量有密切的关系。色带宽的正负菌配对接合能力较强,番茄红素产量也较高。大豆卵磷脂的添加有利于色带的形成,当大豆卵磷脂的添加量为0.3%时,番茄红素产量为1.61 g/L,提高了21.97%。乙酸钠和乳酸钠的浓度分别为0.15 g/L和0.20 g/L时,色素带较宽;添加乙酸钠0.10 g/L或添加乳酸钠0.15 g/L时,较有利于产物的合成,番茄红素产量分别为1.56 g/L、1.45 g/L。
考察了代谢促进剂和阻断剂对番茄红素合成的代谢调控。在各种代谢促进剂中选取了乙醇、氨苄青霉素和H2O2进行代谢调控,发酵36 h添加0.10%的乙醇时,番茄红素产量达1.64 g/L,比对照提高了22.38%;24 h添加0.075 g/L的氨苄青霉素时,番茄红素产量达1.65 g/L,比对照提高了24.06%;36 h添加0.10%的H2O2时,番茄红素产量达1.63 g/L,比对照提高了23.48%;在各种代谢阻断剂中,添加咪唑对β-胡萝卜素合成途径的阻断效果较佳,当40 h添加1.0 g/L的咪唑时,番茄红素产量达1.62 g/L,且一次添加要比分批添加效果好。24 h添加0.075 g/L的氨苄青霉素,36 h分别添加0.3%的大豆卵磷脂和1.0 g/L的亚油酸,40 h添加1.0 g/L的咪唑进行复合调控,番茄红素产量达1.72 g/L。
Lycopene is a straight-chain hydrocarbon which has 11 conjugated double bonds and two non-conjugated double bonds. As a result, it is capable of quenching singlet oxygen and removing free radicals. The antioxidant capacity of it is as much as two times that ofβ-carotene and 100 times that of vitamin E. That is why people pay more attention to lycopene.
This paper focused on the breeding of high-production strain Blakeslea trispora(-) as well as the speciality of the strain. The fermentation condition and the effect of activator of key enzymes on lycopene production of Blakeslea trispora strain(-) were also studied.
Higher production strains was selected by mutation breeding. A strain UV-312 with high production of lycopene was selected which was treated with UV. The lycopene production was 22.54% higher than that of the original strain. NA-62 was mutant strain treated with nitrous acid whose production was 31.52% higher than that of the original strain. UN-10 was mutant strain treated by ultraviolet and nitrous acid combination mutation whose production was 59.72% higher than the original strain.
The optimum fermentation medium was as follows: corn mea 3.0%, cottonseed meal 4.0%, cottonseed oil 9.0%, KH2PO4 0.3%. The optimum fermentation conditions were as follows: pH 7.5, inoculation size 15%(V/V), medium load amount 20 mL/250 mL shaking flask, temperature 27℃, best proportion of (+),(-) 1:20, culture time 5 days, rotary speeds of shaking flask 180 r/min. Under the optimum conditions, the mycelium of Blakeslea trispora dispersed well, and the yields of mycelium dry weight and lycopene were repectively 73.46 g/L, 1.36 g/L.
The width of pigments band formed could predicted the heterothallic, which was closely related to lycopene production. The wider pigments band was, the higher lycopene produced. The addition of soybean lecithin stimulated the formation of pigment. When 0.3% soybean lecithin was added, lycopene yields reached 1.61 g/L which was 21.97% higher than the control. The addition concentrations of sodium acetate and sodium lactate were 0.10 g/L, 0.15 g/L respectively which were the most favorable to the lycopene yields, and the lycopene yields reached 1.56 g/L, 1.45 g/L respectively.
Metabolic regulations of lycopene synthesis with metabolic enhancers and blockers were investigated. Alcohol, ampicillin and H2O2 were selected as the metabolic enhancers. When 0.10% alcohol was added at 36 h, the lycopene yields reached 1.64 g/L which was 22.38% higher than the control; When 0.075 g/L ampicillin was added at 24 h, the lycopene yields reached 1.65 g/L which was 24.06% higher than the control. When 0.10% H2O2 was added at 36 h, the lycopene yields reached 1.63 g/L which was 23.48% higher than the control. Imidazole was the best metabolic blocker to theβ-carotene synthesis. When 1.0 g/L imidazole was added at 40 h, the lycopene yields reached 1.62 g/L. When 0.075 g/L ampicillin was added at 24 h, 0.3% soy lecithin and 1.0 g/L linoleic acid were added at 36 h, and 1.0 g/L imidazole was added at 40 h, the lycopene yields reached 1.72 g/L.
本研究通过诱变三孢布拉霉负菌菌株,提高番茄红素产量,优化培养条件,并通过代谢调控,提高代谢途径中关键酶的活性,加大类胡萝卜素代谢流,提高番茄红素的产量。
以三孢布拉霉负菌(-)08-09为出发菌株,经过紫外线诱变得到一株番茄红素产量高于原始菌株22.54%的突变株UV-312;经过亚硝酸诱变得到一株番茄红素产量高于原始菌株31.52%的突变株NA-62;经过紫外和亚硝酸复合诱变得到一株番茄红素产量高于原始菌株59.72%的突变株UN-10。
通过正交实验得到三孢布拉霉液体发酵最佳培养基组成为:玉米粉3.0%,棉籽饼粉4.0%,KH2PO4 0.5%,棉籽油9.0%,接种量15%(V/V)。最佳液体摇瓶培养条件为:培养温度27℃,装液量20 mL/250 mL,初始pH 7.5,正负菌接种比例1:20,接种量15%(V/V),转速180 r/min,培养时间5 d。在此培养条件下三孢布拉霉菌体分散较好,其菌体最大生物量、番茄红素产量分别为73.46 g/L、1.36 g/L。
正负菌平板上形成的色带宽度大小反映异宗配合的强弱,这与番茄红素的产量有密切的关系。色带宽的正负菌配对接合能力较强,番茄红素产量也较高。大豆卵磷脂的添加有利于色带的形成,当大豆卵磷脂的添加量为0.3%时,番茄红素产量为1.61 g/L,提高了21.97%。乙酸钠和乳酸钠的浓度分别为0.15 g/L和0.20 g/L时,色素带较宽;添加乙酸钠0.10 g/L或添加乳酸钠0.15 g/L时,较有利于产物的合成,番茄红素产量分别为1.56 g/L、1.45 g/L。
考察了代谢促进剂和阻断剂对番茄红素合成的代谢调控。在各种代谢促进剂中选取了乙醇、氨苄青霉素和H2O2进行代谢调控,发酵36 h添加0.10%的乙醇时,番茄红素产量达1.64 g/L,比对照提高了22.38%;24 h添加0.075 g/L的氨苄青霉素时,番茄红素产量达1.65 g/L,比对照提高了24.06%;36 h添加0.10%的H2O2时,番茄红素产量达1.63 g/L,比对照提高了23.48%;在各种代谢阻断剂中,添加咪唑对β-胡萝卜素合成途径的阻断效果较佳,当40 h添加1.0 g/L的咪唑时,番茄红素产量达1.62 g/L,且一次添加要比分批添加效果好。24 h添加0.075 g/L的氨苄青霉素,36 h分别添加0.3%的大豆卵磷脂和1.0 g/L的亚油酸,40 h添加1.0 g/L的咪唑进行复合调控,番茄红素产量达1.72 g/L。
Lycopene is a straight-chain hydrocarbon which has 11 conjugated double bonds and two non-conjugated double bonds. As a result, it is capable of quenching singlet oxygen and removing free radicals. The antioxidant capacity of it is as much as two times that ofβ-carotene and 100 times that of vitamin E. That is why people pay more attention to lycopene.
This paper focused on the breeding of high-production strain Blakeslea trispora(-) as well as the speciality of the strain. The fermentation condition and the effect of activator of key enzymes on lycopene production of Blakeslea trispora strain(-) were also studied.
Higher production strains was selected by mutation breeding. A strain UV-312 with high production of lycopene was selected which was treated with UV. The lycopene production was 22.54% higher than that of the original strain. NA-62 was mutant strain treated with nitrous acid whose production was 31.52% higher than that of the original strain. UN-10 was mutant strain treated by ultraviolet and nitrous acid combination mutation whose production was 59.72% higher than the original strain.
The optimum fermentation medium was as follows: corn mea 3.0%, cottonseed meal 4.0%, cottonseed oil 9.0%, KH2PO4 0.3%. The optimum fermentation conditions were as follows: pH 7.5, inoculation size 15%(V/V), medium load amount 20 mL/250 mL shaking flask, temperature 27℃, best proportion of (+),(-) 1:20, culture time 5 days, rotary speeds of shaking flask 180 r/min. Under the optimum conditions, the mycelium of Blakeslea trispora dispersed well, and the yields of mycelium dry weight and lycopene were repectively 73.46 g/L, 1.36 g/L.
The width of pigments band formed could predicted the heterothallic, which was closely related to lycopene production. The wider pigments band was, the higher lycopene produced. The addition of soybean lecithin stimulated the formation of pigment. When 0.3% soybean lecithin was added, lycopene yields reached 1.61 g/L which was 21.97% higher than the control. The addition concentrations of sodium acetate and sodium lactate were 0.10 g/L, 0.15 g/L respectively which were the most favorable to the lycopene yields, and the lycopene yields reached 1.56 g/L, 1.45 g/L respectively.
Metabolic regulations of lycopene synthesis with metabolic enhancers and blockers were investigated. Alcohol, ampicillin and H2O2 were selected as the metabolic enhancers. When 0.10% alcohol was added at 36 h, the lycopene yields reached 1.64 g/L which was 22.38% higher than the control; When 0.075 g/L ampicillin was added at 24 h, the lycopene yields reached 1.65 g/L which was 24.06% higher than the control. When 0.10% H2O2 was added at 36 h, the lycopene yields reached 1.63 g/L which was 23.48% higher than the control. Imidazole was the best metabolic blocker to theβ-carotene synthesis. When 1.0 g/L imidazole was added at 40 h, the lycopene yields reached 1.62 g/L. When 0.075 g/L ampicillin was added at 24 h, 0.3% soy lecithin and 1.0 g/L linoleic acid were added at 36 h, and 1.0 g/L imidazole was added at 40 h, the lycopene yields reached 1.72 g/L.
引文
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2.孙庆杰,丁霄霖.番茄红素的研究进展[J].中国食品添加剂,1998(2):1-6
3.徐伟,等.国外医学卫生学分册[M].1998,25(2):81-84
4. Nguyenm I,Schwartz S J.Lycopene: chemical and biological properties[J].Food Technology,1999,53(2):38-45
5.赵文恩,乔宪生,俞宏.番茄红素的生物学性质[J].生物学杂志,2000,17 (4):4-6
6. Boileau TW,Boileau AC Erdman JW Jr.Bioavailability of all-trans and cis-isomers of lycopene[J].Exp Biol Med(Maywood),2002,227(10):914-919
7. John S,Le Maguer M,Mike B,et.Kinetics of lycopene degradation in tomato puree by heat and light irradiation[J].Food Process Engineering,2003,25(6):485-489
8.尚德军,王军.番茄红素研究现状与展望[J].检验检疫科学,2004,14(2):59-61
9. L. De Franceschi,M. C. Arista, A. Callopoli,M.D.Flow Cytometric Study of Lymphocyte Subsets in Patients at Different Stages of Colorectal Carcinoma[J].Dis Colon Rectum,1994,37(Suppl):S30-S34
10.张艳春,万丽葵,潘洪志.番茄红素对小鼠移植性肿瘤的抑制作用研究[J].齐齐哈尔医学院学报,2006(3):265-266
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