γ-聚谷氨酸发酵及提取工艺研究
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摘要
γ-聚谷氨酸(Poly-γ-glutamic acid;γ-PGA)是以L-、D-型谷氨酸为单体通过γ-酰胺键聚合而成的一种均聚氨基酸聚合物,可以通过微生物发酵生产获得。由于其具有良好的生物相容性、生物降解性、水溶性、无毒害无污染等优点,可作为生物絮凝剂、重金属吸收剂、保水剂、高吸水性树脂、食品添加剂、药物载体、药物缓释剂等广泛应用于环境、食品、化妆品、医药领域中,具有广阔的应用前景。本课题对γ-PGA产生菌的发酵条件优化、γ-PGA提取纯化和结构表征以及γ-PGA的初步应用进行了研究。主要研究结果如下:
通过对培养基组分进行单因素实验和响应面实验分析,确定了菌株L536的最佳发酵培养基组成为(g/L):氯化钠15、豆粕53、柠檬酸钠16、谷氨酸钠40、氯化铵4.6、磷酸氢二钾0.625、硫酸镁1.25、硫酸锰0.35、氯化钙0.2。通过对接种量、发酵温度、初始pH值、溶氧量等培养条件的考察,获得了菌株L536的较优培养条件:接种量3%(V/V),发酵温度37℃,初始pH值pH7.0,摇瓶装液量35mL/250mL三角瓶,摇床转速200r/min,发酵时间48h左右。菌株L536在最佳培养基和较优培养条件下培养,发酵液的γ-PGA产量可达25.81g/L,比优化前提高了约1.5倍。
对γ-PGA不同的水解方法进行比较,确定采用盐酸水解法,其最适水解时间为24h。研究乙醇法提取γ-PGA,确定乙醇的最适添加量为2倍体积,考察pH值对发酵液的粘度和γ-PGA提取的影响,最终确定γ-PGA的最佳提取路线。
纯化样品经氨基酸分析仪分析纯度可达88.6%。纯化的γ-PGA样品经纸层析、紫外扫描和红外光谱扫描等方法进行初步分析,结果表明:γ-PGA样品是由谷氨酸单体聚合而成且其在215nm处吸收值达到最大,在260nm~280nm处没有明显的吸收峰,表明其没有典型的肽键结构。对照日本明治制药株式会社的γ-PGA标准品红外图谱,γ-PGA样品与标准品的特征吸收峰基本吻合,经图谱解析可以初步确定纯化样品为γ-聚谷氨酸。
以高岭土悬浊液为絮凝介质,研究了菌株L536产γ-PGA的絮凝特性。结果表明:在中性pH范围内、投加量为1.2g/L时,对高岭土的絮凝率达最大。Fe2+、Ca2+、Cu2+、Fe3+、Al3+均能不同程度地提高γ-PGA对高岭土的絮凝活性。其中, Ca2+的助凝效果最为显著,其最佳助凝浓度为0.09mol/L。另外,菌株L536产γ-PGA对于活性炭、Ca(OH)2也表现出较强的絮凝活性。另外,γ-PGA对于水果蔬菜的保鲜和土壤的保水具有一定的作用。
Poly-γ-glutamic acid was a compound of equal poly amino acid that consist of L- and D-glutamic acids throughγ-glutamyl bonds. Owing to its excellent characters including biocompatibility, biodegradability, non-toxicity and non-pollution,γ-PGA has been used in many areas such as environment, food, cosmetics and pharmaceutical.γ-PGA and its derivatives had a widely applications including being used as heavy metal absorber, bioflocculant, super absorbent polymers, food additive, drug carrier, sustained-release material and so on. The paper concentrated on the optimization of the strain L536’s culture medium and conditions, the separation and purification ofγ-PGA and the initial application ofγ-PGA. Main results were as follows:
By single-factor experiments and response surface methodology, the culture medium and conditions of the strain L536 outputingγ-PGA were optimized. The optimum culture conditions were as follows (g/L): NaCl 15, soybean flour 53, sodium citrate 16, sodium glutamate 40, NH4Cl 4.6, K2HPO4 0.625, MgSO4·7H2O 1.25, MnSO4·H2O 0.35, CaC12·2H2O 0.2, inoculation amount 3%(V/V), temperature 37℃, initial pH 7.0, broth’s volume in shake flask 35mL/250mL, rotation speed of rocking bed 200r/min and culture time 48h. Under these condition, the yield ofγ-PGA was 25.81g/L, increased for about 1.5 times more than before.
Compared the different methods of hydrolysis ofγ-PGA, the hydrochloric acid hydrolysis was adoptes and the optimum hydrolysis time was 24h. The optimum ethanol concentration was 2 times than the volume of fermentation broth. The effect of pH on the viscosity of fermentation broth and extraction ofγ-PGA was studied. Ultimately, the best route of extraction ofγ-PGA was determined.
The concentration of pureγ-PGA was 88.6% by amino acid analyzers. By paper chromatography, UV and IR, the characterization of'γ-PGA was analyzed. The results showed that the polymer was only composed of glutamic acid. There was an absorption peak at 215nm by the UV scanning. The purifiedγ-PGA basically coincidented with that of the standardγ-PGA prepared by Meiji Pharmaceutical Co., Ltd through the IR chromatograph. It indicated the polymer wasγ-PGA.
Flocculating conditions ofγ-PGA produced by Bacillus amyloliquefaciens L536 were investigated. The flocculating activity got highest at theγ-PGA concentration of 1.2g/ L and in the neutral pH. The flocculating activity was synergistically simulated by the addition of cations, Ca2+ was the optimum cation that remarkably promoted the flocculating activity. The results showed that the optimum flocculation conditions were CaCl2 of 0.09mol/L.γ-PGA had a high flocculating activity to some organic and inorganic suspension. In addition,γ-PGA can be used to keeping the fruits and vegetables fresh and increased the soil moisture.
通过对培养基组分进行单因素实验和响应面实验分析,确定了菌株L536的最佳发酵培养基组成为(g/L):氯化钠15、豆粕53、柠檬酸钠16、谷氨酸钠40、氯化铵4.6、磷酸氢二钾0.625、硫酸镁1.25、硫酸锰0.35、氯化钙0.2。通过对接种量、发酵温度、初始pH值、溶氧量等培养条件的考察,获得了菌株L536的较优培养条件:接种量3%(V/V),发酵温度37℃,初始pH值pH7.0,摇瓶装液量35mL/250mL三角瓶,摇床转速200r/min,发酵时间48h左右。菌株L536在最佳培养基和较优培养条件下培养,发酵液的γ-PGA产量可达25.81g/L,比优化前提高了约1.5倍。
对γ-PGA不同的水解方法进行比较,确定采用盐酸水解法,其最适水解时间为24h。研究乙醇法提取γ-PGA,确定乙醇的最适添加量为2倍体积,考察pH值对发酵液的粘度和γ-PGA提取的影响,最终确定γ-PGA的最佳提取路线。
纯化样品经氨基酸分析仪分析纯度可达88.6%。纯化的γ-PGA样品经纸层析、紫外扫描和红外光谱扫描等方法进行初步分析,结果表明:γ-PGA样品是由谷氨酸单体聚合而成且其在215nm处吸收值达到最大,在260nm~280nm处没有明显的吸收峰,表明其没有典型的肽键结构。对照日本明治制药株式会社的γ-PGA标准品红外图谱,γ-PGA样品与标准品的特征吸收峰基本吻合,经图谱解析可以初步确定纯化样品为γ-聚谷氨酸。
以高岭土悬浊液为絮凝介质,研究了菌株L536产γ-PGA的絮凝特性。结果表明:在中性pH范围内、投加量为1.2g/L时,对高岭土的絮凝率达最大。Fe2+、Ca2+、Cu2+、Fe3+、Al3+均能不同程度地提高γ-PGA对高岭土的絮凝活性。其中, Ca2+的助凝效果最为显著,其最佳助凝浓度为0.09mol/L。另外,菌株L536产γ-PGA对于活性炭、Ca(OH)2也表现出较强的絮凝活性。另外,γ-PGA对于水果蔬菜的保鲜和土壤的保水具有一定的作用。
Poly-γ-glutamic acid was a compound of equal poly amino acid that consist of L- and D-glutamic acids throughγ-glutamyl bonds. Owing to its excellent characters including biocompatibility, biodegradability, non-toxicity and non-pollution,γ-PGA has been used in many areas such as environment, food, cosmetics and pharmaceutical.γ-PGA and its derivatives had a widely applications including being used as heavy metal absorber, bioflocculant, super absorbent polymers, food additive, drug carrier, sustained-release material and so on. The paper concentrated on the optimization of the strain L536’s culture medium and conditions, the separation and purification ofγ-PGA and the initial application ofγ-PGA. Main results were as follows:
By single-factor experiments and response surface methodology, the culture medium and conditions of the strain L536 outputingγ-PGA were optimized. The optimum culture conditions were as follows (g/L): NaCl 15, soybean flour 53, sodium citrate 16, sodium glutamate 40, NH4Cl 4.6, K2HPO4 0.625, MgSO4·7H2O 1.25, MnSO4·H2O 0.35, CaC12·2H2O 0.2, inoculation amount 3%(V/V), temperature 37℃, initial pH 7.0, broth’s volume in shake flask 35mL/250mL, rotation speed of rocking bed 200r/min and culture time 48h. Under these condition, the yield ofγ-PGA was 25.81g/L, increased for about 1.5 times more than before.
Compared the different methods of hydrolysis ofγ-PGA, the hydrochloric acid hydrolysis was adoptes and the optimum hydrolysis time was 24h. The optimum ethanol concentration was 2 times than the volume of fermentation broth. The effect of pH on the viscosity of fermentation broth and extraction ofγ-PGA was studied. Ultimately, the best route of extraction ofγ-PGA was determined.
The concentration of pureγ-PGA was 88.6% by amino acid analyzers. By paper chromatography, UV and IR, the characterization of'γ-PGA was analyzed. The results showed that the polymer was only composed of glutamic acid. There was an absorption peak at 215nm by the UV scanning. The purifiedγ-PGA basically coincidented with that of the standardγ-PGA prepared by Meiji Pharmaceutical Co., Ltd through the IR chromatograph. It indicated the polymer wasγ-PGA.
Flocculating conditions ofγ-PGA produced by Bacillus amyloliquefaciens L536 were investigated. The flocculating activity got highest at theγ-PGA concentration of 1.2g/ L and in the neutral pH. The flocculating activity was synergistically simulated by the addition of cations, Ca2+ was the optimum cation that remarkably promoted the flocculating activity. The results showed that the optimum flocculation conditions were CaCl2 of 0.09mol/L.γ-PGA had a high flocculating activity to some organic and inorganic suspension. In addition,γ-PGA can be used to keeping the fruits and vegetables fresh and increased the soil moisture.
引文
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