树脂吸附分离耦合固定化短乳杆菌生产GABA的研究
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摘要
γ-氨基丁酸(GABA)是哺乳动物中枢神经系统重要的抑制性神经递质,具有镇静安神、促进睡眠、增强记忆力、治疗癲痫、降低血压、控制哮喘、调节激素分泌、促进生殖、肾肝功能活化等多种生理活性。利用食品安全级微生物乳酸菌发酵生产GABA已成为近年来的研究热点,但细胞固定化技术在这一方面的应用很少。本文系统地研究了采用纤维素硫酸钠(NaCS)-聚二甲基二烯丙基氯化铵(PDMDAAC)微胶囊包埋短乳杆菌hjxj-01(Lactobacillus brevis hjxj-01)转化L-谷氨酸钠(L-MSG)生产GABA的过程,并探索了采用树脂吸附的原位分离技术与发酵相耦合生产GABA的新工艺,旨在提高发酵液中GABA的浓度、转化率及生产速率,降低产物对反应的抑制作用,简化产物的后续分离步骤,实现菌体的重复利用和连续化生产。
对短乳杆菌hjxj-01进行了游离培养,考察了pH值、底物浓度、葡萄糖浓度、温度等因素对发酵的影响,确定了最优发酵参数:培养温度30℃,发酵液pH恒定在4.0,底物L-MSG 73.3g/L,葡萄糖浓度20g/L,静置培养5d。在此条件下,短乳杆菌浓度最高达到2.48g/L,发酵50h葡萄糖基本耗尽,此后GABA被大量合成。GABA最高产量达到41.3g/L。
研究了用于包埋短乳杆菌hjxj-01的NaCS-PDMDAAC微胶囊的制备条件及其性能。制备得到的NaCS-PDMDAAC微胶囊为微白色半透明球体,平均直径2.82mm,机械强度良好,经过多批培养,胶囊仍保持完整。NaCS-PDMDAAC微胶囊与短乳杆菌hjxj-01有很好的生物相容性,L-MSG和葡萄糖等营养物质都可以顺利的从溶液中扩散进入NaCS-PDMDAAAC微胶囊。
在摇瓶中进行了短乳杆菌微囊化培养,确定了合适的发酵条件,并考察了微囊化培养对菌体生长、底物消耗及产物生成的影响。结果发现,与游离培养相比,微囊化培养促进了短乳杆菌对底物L-MSG和葡萄糖的利用速率,从而提高了囊内短乳杆菌浓度及GABA的产量。微胶囊内短乳杆菌最高浓度达到21.9g/L,约为游离培养菌体浓度最大值的8倍;GABA最高产量达到50.1g/L。微囊化短乳杆菌具有良好的重复利用性,发酵进行10批后对底物的转化率仍在60%以上。
以生物反应与分离耦合的集成化思路为指导,通过采用D001大孔强酸性阳离子交换树脂从反应体系中交换分离GABA,建立了原位分离技术耦合固定化短乳杆菌催化合成GABA的新方法。D001树脂对GABA有很高的静态交换容量,约为1.43mmol/g resin。在反应体系中原位添加10%(w/v)的D001树脂4h左右,底物L-MSG转化率达到100%,与之相比不添加树脂组5h时的转化率为81.4%,原位添加树脂的固定化细胞转化制备GABA不仅加快了底物的转化,还简化了GABA后续分离过程中的絮凝、脱色步骤。以氨水(2mol/L)对树脂进行静态解吸,产物GABA得到了初步的分离,回收率达到了93%。
Gamma-aminobutyric acid(GABA),serves as a major inhibitory neurotrans mitter in mammalian nervous systems.GABA has several physiological functio ns such as hypotensive activity,treatment of epilepsy,tranquilizing and allaying excitement,enhancing memory,controlling asthma,regulating hormone secretio n,promoting reproduction and activating liver and kidney function.Preparation of GABA by Lactobacillus brevis,which generally recognized as safe food add itive,has been among the most remarkable research focuses in the realm of bi ology.To extend the application of immobilization technique in this bioconversi on,the sodium cellulose sulfate / poly-dimethyl-diallyl-ammonium chloride micr ocapsule encapsulated cells of Lactobacillus brevis hjxj-01 for dissimilating sodi um glutamate(L-MSG) to GABA.In addition,a new technique,the integrated fermentation and separation technology based on the in situ product removal t echnique by adsorbent resin,was developed to produce GABA.By virtue of a dvantages of encapsulated cells and adsorbent resin,this immobilization ferment ation aimed at enhancing GABA concentration,yield and productivity,reducing the product inhibition,simplifying separation of product,achieving cell reutiliz ation and continuous fermentation.
The free cell culture was carried out with Lactobacillus brevis hjxj-01 and effects of pH,L-MSG,glucose,temperature on the formation of GABA were investigated and the fermentation information about Lactobacillus brevis hjxj-0 1 was obtained.The fermentation should be performed for 5 days at 30℃,pH 4.0,L-MSG 73.3g/L,glucose 20g/L and no aeration.After 50h fermentation,gl ucose was consumed.Then,GABA was synthesized rapidly.The GABA final c oncentration was 41.3 g/L,with maximum biomass of 2.48g/L.
We studied the preparation and effects of the sodium cellulose sulfate / po ly-dimetbyl-diallyl-ammonium chloride microcapsule encapsulated cells of Lacto bacillus brevis hjxj-O1.The capsules were white and transparent,with an avera ge diameter 2.82mm.It's compression intensity was strong enough to keep the capsules in good condition after several cultivation.As nutrients like L-MSG and glucose can easily diffuse into the capsules,the sodium cellulose sulfate / poly-dimethyl-diallyl-ammonium chloride microcapsule showed a good biocompa tibility to the Lactobacillus brevis hjxj-01
A serious of shake flask cultures with encapsulated Lactobacillus brevis hj xj-01 were performed to obtain the optimized fermentation conditions.Then the cell growth,L-MSG consumption and GABA production of encapsulated cells were investigated.Compared with free cell culture in the same condition,the encapsulated cells could endure higher concentration of L-MSG and glucose,th us got a higher productivity of GABA and biomass.GABA final concentration reached 50.1g/L.The biomass of encapsulated cell culture reached 21.9g/L,8-fold higher than that of free cell culture.After ten-time reutilization of the enc apsulated cells,the L-MSG conversion ratio still reached more than 60%and c apsules kept integrity.
Taking the integrated bioreaction and separation process as guide,a new t echnique,the technique of in situ product removal based on the adsorption of product by macro-reticular resin D001 directly from the fermentation broth,wa s developed for GABA production.4h after 10%(w/v) resin D001 was added i nto the reaction solution,the conversion ratio of L-MSG reached 100%,while the conversion ratio of L-MSG was only 81.4%after 5h without addition of re sin.The adsorption capacity of resin D001 on GABA was about 1.43 mmol/g resin.GABA was separated initially without flocculation and decolorization afte r elution of the resin by 2mol/L ammonia,the recovery of GABA reached 9 3%.
对短乳杆菌hjxj-01进行了游离培养,考察了pH值、底物浓度、葡萄糖浓度、温度等因素对发酵的影响,确定了最优发酵参数:培养温度30℃,发酵液pH恒定在4.0,底物L-MSG 73.3g/L,葡萄糖浓度20g/L,静置培养5d。在此条件下,短乳杆菌浓度最高达到2.48g/L,发酵50h葡萄糖基本耗尽,此后GABA被大量合成。GABA最高产量达到41.3g/L。
研究了用于包埋短乳杆菌hjxj-01的NaCS-PDMDAAC微胶囊的制备条件及其性能。制备得到的NaCS-PDMDAAC微胶囊为微白色半透明球体,平均直径2.82mm,机械强度良好,经过多批培养,胶囊仍保持完整。NaCS-PDMDAAC微胶囊与短乳杆菌hjxj-01有很好的生物相容性,L-MSG和葡萄糖等营养物质都可以顺利的从溶液中扩散进入NaCS-PDMDAAAC微胶囊。
在摇瓶中进行了短乳杆菌微囊化培养,确定了合适的发酵条件,并考察了微囊化培养对菌体生长、底物消耗及产物生成的影响。结果发现,与游离培养相比,微囊化培养促进了短乳杆菌对底物L-MSG和葡萄糖的利用速率,从而提高了囊内短乳杆菌浓度及GABA的产量。微胶囊内短乳杆菌最高浓度达到21.9g/L,约为游离培养菌体浓度最大值的8倍;GABA最高产量达到50.1g/L。微囊化短乳杆菌具有良好的重复利用性,发酵进行10批后对底物的转化率仍在60%以上。
以生物反应与分离耦合的集成化思路为指导,通过采用D001大孔强酸性阳离子交换树脂从反应体系中交换分离GABA,建立了原位分离技术耦合固定化短乳杆菌催化合成GABA的新方法。D001树脂对GABA有很高的静态交换容量,约为1.43mmol/g resin。在反应体系中原位添加10%(w/v)的D001树脂4h左右,底物L-MSG转化率达到100%,与之相比不添加树脂组5h时的转化率为81.4%,原位添加树脂的固定化细胞转化制备GABA不仅加快了底物的转化,还简化了GABA后续分离过程中的絮凝、脱色步骤。以氨水(2mol/L)对树脂进行静态解吸,产物GABA得到了初步的分离,回收率达到了93%。
Gamma-aminobutyric acid(GABA),serves as a major inhibitory neurotrans mitter in mammalian nervous systems.GABA has several physiological functio ns such as hypotensive activity,treatment of epilepsy,tranquilizing and allaying excitement,enhancing memory,controlling asthma,regulating hormone secretio n,promoting reproduction and activating liver and kidney function.Preparation of GABA by Lactobacillus brevis,which generally recognized as safe food add itive,has been among the most remarkable research focuses in the realm of bi ology.To extend the application of immobilization technique in this bioconversi on,the sodium cellulose sulfate / poly-dimethyl-diallyl-ammonium chloride micr ocapsule encapsulated cells of Lactobacillus brevis hjxj-01 for dissimilating sodi um glutamate(L-MSG) to GABA.In addition,a new technique,the integrated fermentation and separation technology based on the in situ product removal t echnique by adsorbent resin,was developed to produce GABA.By virtue of a dvantages of encapsulated cells and adsorbent resin,this immobilization ferment ation aimed at enhancing GABA concentration,yield and productivity,reducing the product inhibition,simplifying separation of product,achieving cell reutiliz ation and continuous fermentation.
The free cell culture was carried out with Lactobacillus brevis hjxj-01 and effects of pH,L-MSG,glucose,temperature on the formation of GABA were investigated and the fermentation information about Lactobacillus brevis hjxj-0 1 was obtained.The fermentation should be performed for 5 days at 30℃,pH 4.0,L-MSG 73.3g/L,glucose 20g/L and no aeration.After 50h fermentation,gl ucose was consumed.Then,GABA was synthesized rapidly.The GABA final c oncentration was 41.3 g/L,with maximum biomass of 2.48g/L.
We studied the preparation and effects of the sodium cellulose sulfate / po ly-dimetbyl-diallyl-ammonium chloride microcapsule encapsulated cells of Lacto bacillus brevis hjxj-O1.The capsules were white and transparent,with an avera ge diameter 2.82mm.It's compression intensity was strong enough to keep the capsules in good condition after several cultivation.As nutrients like L-MSG and glucose can easily diffuse into the capsules,the sodium cellulose sulfate / poly-dimethyl-diallyl-ammonium chloride microcapsule showed a good biocompa tibility to the Lactobacillus brevis hjxj-01
A serious of shake flask cultures with encapsulated Lactobacillus brevis hj xj-01 were performed to obtain the optimized fermentation conditions.Then the cell growth,L-MSG consumption and GABA production of encapsulated cells were investigated.Compared with free cell culture in the same condition,the encapsulated cells could endure higher concentration of L-MSG and glucose,th us got a higher productivity of GABA and biomass.GABA final concentration reached 50.1g/L.The biomass of encapsulated cell culture reached 21.9g/L,8-fold higher than that of free cell culture.After ten-time reutilization of the enc apsulated cells,the L-MSG conversion ratio still reached more than 60%and c apsules kept integrity.
Taking the integrated bioreaction and separation process as guide,a new t echnique,the technique of in situ product removal based on the adsorption of product by macro-reticular resin D001 directly from the fermentation broth,wa s developed for GABA production.4h after 10%(w/v) resin D001 was added i nto the reaction solution,the conversion ratio of L-MSG reached 100%,while the conversion ratio of L-MSG was only 81.4%after 5h without addition of re sin.The adsorption capacity of resin D001 on GABA was about 1.43 mmol/g resin.GABA was separated initially without flocculation and decolorization afte r elution of the resin by 2mol/L ammonia,the recovery of GABA reached 9 3%.
引文
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