谷氨酸双结晶高效提取工艺关键技术的研究与集成
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摘要
谷氨酸(glutamic acid)是蛋白结构氨基酸之一,也是一种重要的游离氨基酸,广泛应用于食品、医药、日化及饲料等工业领域。2009年我国谷氨酸总产量达到160万吨,占全球总产量的75%以上,是我国在国际市场上具有一定竞争优势的大宗发酵产品之一。我国现有谷氨酸提取工艺以“低温等电离交工艺”为主,该工艺提取收率高,但原辅材料消耗大,环境污染严重。国际上针对糖蜜原料的谷氨酸“浓缩连续等电工艺”虽原辅材料消耗低,但提取收率低。因此,研究改造谷氨酸现有提取技术,实现“增产、降耗、减排”的综合目标,对促进我国氨基酸产业可持续发展、提升我国氨基酸产品在国际市场上的竞争力具有重要意义。本论文以我国普遍采用的生物素亚适量谷氨酸发酵液为研究对象,重点研究了“高粘度溶液环境中谷氨酸蒸发结晶”、“细晶消除型谷氨酸连续等电结晶”及“基于蛋白质热变性的絮凝除菌”等关键技术,通过技术集成形成了“谷氨酸双结晶高效提取工艺”的技术路线,并完成、完善了总容积为76 m3规模的中试研究工作。主要研究结果如下:
(1)以MSG水溶液模拟发酵液,采用pH-shift模式研究溶液过饱和度对谷氨酸结晶的影响。结果表明:过饱和度同时影响谷氨酸结晶的晶习和粒径分布。25℃时,当过饱和度S≤7.15时谷氨酸主要以α型晶习析出,粒径呈对数对称分布。随着过饱和度增大,β型结晶比例增大,中位径趋小且分布范围变宽。当S≥14.17以后,晶体全部是β型晶习。结晶成核诱导时间实验和理论计算结果表明,成核速率随过饱和度增加而增大是导致晶体粒径分布范围变宽的主要原因,不同过饱和度时α、β型晶核的竞争性成核速率是导致晶体晶习变化的主要原因。基于实验结果提出了谷氨酸C-pH相图模型,该模型解释了过饱和度影响谷氨酸结晶晶习的机理。控制等电调酸速率或添加晶种能有效降低过饱和度,改善谷氨酸结晶晶习及粒径分布。
(2)添加蔗糖改变谷氨酸溶液粘度,采用pH-shift模式研究了溶液粘度对谷氨酸结晶的影响。结果表明,在1.00-10.26 mP·s的粘度范围内,溶液粘度不影响谷氨酸结晶晶习,但粘度增加使得晶体粒径减小。高粘度溶液中谷氨酸结晶速率受溶质扩散速率控制,谷氨酸结晶成核速率、晶体生长速率随溶液粘度增加而下降是晶体粒径减小的主要原因。提高结晶温度能显著降低溶液的粘度,从而提高晶体的成核与生长速率。添加适量晶种能提高晶体平均粒径,并改善晶体均匀性。
(3)以除菌后等电母液为研究对象,考察了温度、浓缩倍数对浓缩母液粘度及谷氨酸溶解度的影响规律。结果表明,母液浓缩倍数越大粘度越大,提高温度可显著降低母液粘度,当温度大于60℃后不同倍数浓缩液的粘度接近。但提高温度同时促进了谷氨酸的焦化速率,为避免蒸发结晶过程中损失谷氨酸,蒸发结晶温度应控制在70℃以下。以控制谷氨酸结晶晶习及粒径分布为目标,通过实验研究了结晶温度、晶种添加策略、蒸发速率及浓缩倍数等工艺参数对谷氨酸蒸发结晶的影响。实验确定的谷氨酸蒸发结晶工艺为:结晶温度60℃,晶种目数为120目,添加量为母液中理论谷氨酸总量的30%,控制蒸发速率为207 L/m2·h,共浓缩6倍。在此条件下,蒸发结晶获得中位径约122.2μm的颗粒状谷氨酸α结晶,提取收率达到72.8%以上。
(4)实验确定了最大细晶消除循环速率R=1.3。进一步研究了不同细晶消除循环速率(R)、结晶停留时间等工艺参数对谷氨酸结晶的影响,结果表明,随着细晶消除循环速率的增加,细晶切割粒径从36.44μm提高到51.35μm,晶体粒径增大,粒径分布趋向集中。其原因是因为随着R值增加结晶过饱和度下降,晶体成核速率下降而晶体生长速率增加。结晶停留时间(τ)的长短同样影响结晶过饱和度,进而影响晶体粒径分布。分析对比等电离交、浓缩等电及细晶消除型连续等电
(5)以菌体细胞、COD及可溶性蛋白质去除率为指标,考察了絮凝剂种类、絮凝剂添加量、pH、温度及维持时间等因素对絮凝效果的影响,在此基础上,通过响应面分析法优化了絮凝除菌工艺条件。最优絮凝工艺:等电母液原始pH3.1、絮凝剂PAAS添加量60 mg/L、温度80℃、维持时间30 min时。此时菌体细胞、COD和可溶性蛋白去除率分别达到99%、47%和65%,以滤布为过滤介质的过滤速率达到280 L/m2·h。探讨了热变性絮凝除菌的机理,研究表明提高温度能降低菌体细胞表面的zeta电位,压缩颗粒表面扩散层厚度,增强架桥效应的絮凝作用。絮团中位径达到148μm以上,借助普通滤布即能实现高速过滤。
(6)在完成上述机理探索及关键技术研究的基础上,技术集成形成了基于谷氨酸二步结晶的“谷氨酸双结晶高效提取工艺”技术路线,并开展了总容积为76m3规模的中试验证。在中试研究过程中,对晶体分离、结晶质量提升等工序进行创新和优化,形成了更为完善的“谷氨酸双结晶高效提取工艺”路线。中试平均提取收率93.4%,比“浓缩等电工艺”(平均收率88%)高出5.4个百分点,仅比“等电离交工艺”(平均收率95%)低1.6个百分点。产品纯度、SO42-离子浓度、透光率等主要质量指标均高于“浓缩等电工艺”,更优于“等电离交工艺”。硫酸、液氨等辅料消耗比“等电离交工艺”分别下降了53%和100%,略低于“浓缩等电工艺”。技术经济性分析表明,在相同发酵水平、相同谷氨酸质量(转晶指标)、相同环境友好度的前提下,“双结晶工艺”吨谷氨酸生产成本分别比“等电离交工艺”和“浓缩等电工艺”下降了15.8%和14.6%。中试成果于2010年3月通过了中国发酵工业协会主持的技术鉴定,鉴定结论:本项目综合技术指标达到国际领先水平。
Glutamic acid (GA) is not only one of proteinogenic amino acids, but also an important free amino acid which is widely used in the fields of food industry, medicine industry, chemical industry for daily supplies and feed industry. Total output of the GA was 1.6 million tons in China in 2009, accounting for 75 per cent of global output. GA has now become one of the domestic staple fermentation products with strong competitive advantage in the global market. In our country, isoelectric crystallization with ion exchange (IEIE) is now the most common GA extracting process. Although the yield coefficient is high in IEIE, large amount of raw material is consumed and environmental pollution is serious. Instead, a concentrated continuous isoelectric process (CCIC) with low level of raw material consumption is applied to extract molasses-based GA. However the yield coefficient is not high enough in CCIC. Therefore modification of the current GA extraction technology to achieve the target of "energy-saving, raw material consumption and emission reduction" is essential for the promotion of the sustainable development and international competitiveness of Chinese GA industry. In this paper, key technologies of GA evaporative crystallization in the high viscosity solution, continuous isoelectric crystallization with eliminating fine crystal, and bacterium removal by combining thermal denaturation with flocculation were studied using Biotin-limited GA fermentation broth as the research object. A novel and cleaner GA extraction technique, called two-stage crystallization technology (TSC), was proposed and tested in pilot scale (76 m3). The main research work is summarized as follows:
(1) pH-shift model was adopted to investigate the effect of supersaturation on the GA crystallization using monosodium glutamate (MSG) solution instead of fermentation broth. Results showed that the supersaturation influenced both the crystal habit and size distribution. The main crystal habit wasα-form and the crystal size showed a logarithmic symmetric distribution when the supersaturation S≤7.15 at 25℃. With the increase of the supersaturation, the percentage ofβ-form crystal increased whereas the median diameter decreased with a wider size distribution. Almost all the crystal habit wereβ-form when the S≥14.17. According to the theoretical calculation and measurement of the induce time of nucleation, the increase of nucleation rate with the supersaturation is the primary reason for the widened crystal size distribution range. A GA C-pH Phase diagram model was proposed based on the results. This model explained how the supersaturation affected the GA crystal habit theoretically. The supersaturation could be efficiently reduced, and GA crystal habit as well as the crystal size distribution range could be improved by controlling the rate of acidity adjustment or by adding crystal seed.
(2) The pH-shift model was used to study how the viscosity of solution influenced the GA crystallization by adding sucrose to change the viscosity of solution. Results indicated that the viscosity did not affect the GA habit but reduce the nucleation rate in the range of 1.00-10.26 mP·s. The increase of viscosity led to smaller crystal size. The rate of crystallization was controlled by the solute diffusion rate in the high viscosity solution. Nucleation rate and crystal growth rate decreased with the increased viscosity which caused the smaller crystal size. The viscosity could be reduced by enhancing the temperature to increase the nucleation rate and crystal growth rate. Larger crystal size and better crystal habit could be obtained by adding crystal seed properly.
(3) The effects of temperature and condensed fold on the viscosity of condensed mother liquor and GA solubility were investigated using the isoelectric mother liquor in which the bacterium was removed. Results suggested that the viscosity increased as the folds of concentration enhanced. The viscosity could be reduced by rising the temperature. Besides, the viscosity under different concentration was similar when the temperature was over 60℃. On the other hand, high temperature could also promote the coking rate of GA. So the temperature should be controlled under 70℃to avoid the loss of GA in the evaporation crystallization process. To control the GA habit and size distribution, experiments were carried out to study the effects of crystallization temperature, strategy of seed addition, evaporation rate, folds of concentration and other factors during the evaporation crystallization process. The optimum operation conditions were: the optimum crystallization temperature at 60℃, seed crystal size of 120 mesh, the amount of seed addition of 30% of the theoretical GA quantity, evaporation rate of 207 L/m~2·h and 6 folds. Under these conditions, granularα-form crystal with median diameter of 122.2μm was obtained, and the yield coefficient of extraction reached to more than 72.8%.
(4) The maximum cycle rate of fine crystal elimination (R) was 1.3. Further research was done to investigate the effects of the technological parameters such as R, crystallization retention time and other parameters on the GA crystallization. As the increase of R, the cutting particle size of fine crystal was increased from 36.44μm to 51.35μm, and the crystal size was also increased. At the same time size distribution tended to be centralized. This phenomenon was due to the decrease of supersaturation and the nucleation rate, in contrast the increase of crystal growth rate with the increase of R value. Crystallization retention time could also influence the supersaturation and then crystal size distribution. This system was proved to efficiently eliminate the fine crystal and improve the crystal quality by comparing the size distribution of the three crystals in the processes of IEIE, evaporation isoelectric crystallization and continuous isoelectric crystallization respectively.
(5) In order to determine the optimum condition for the removal of bacterium, COD and soluble protein, the effects of flocculants, dosage, pH, temperature and time on flocculation were carried out using Response Surface Analysis. Highest bacterium, COD and soluble protein removal capacities (99%, 47% and 65%, respectively) were obtained when the PAAS was used under the condition of pH 3.1, dosage 60 mg/L, temperature 80℃and 30min. And the filtering rate was 280 L/m2·h when filter cloth was used. The mechanism of thermal denaturation with flocculation technology was studied. Results indicated that higher temperature could reduce the zeta potential on the surface of bacterium, compress the thickness of diffusion layer around the surface of particle, and enhance the bridge effect. The median diameter of the floccules reached to more than 148μm. In this condition, high filtering rate could be obtained using common filter cloth.
(6) Based on the above results, a novel and cleaner GA extraction technique, called two-stage crystallization technology (TSC), was proposed and tested in pilot scale (76 m3). Crystal separation and crystal quality were optimized in the pilot scale test to form a more perfect two-stage crystallization technology. Average yield coefficient of extraction was 93.4% which is higher than that of evaporation isoelectric crystallization process by 5.4% and lower than that of IEIE process by only 1.6%. Purity, SO42- concentration and transmittance were superior to those of the other two processes. Compared with IEIE process, sulfuric acid and liquid ammonia consumptions were reduced by 53% and 100%, respectively. Technical analysis shows that production cost of TSC process would be lower than those of IEIE process and evaporation isoelectric crystallization process by 15.8% and 14.6%, respectively, provided that the fermentation level, the GA quality and effect on environment of the three processes were same.
(1)以MSG水溶液模拟发酵液,采用pH-shift模式研究溶液过饱和度对谷氨酸结晶的影响。结果表明:过饱和度同时影响谷氨酸结晶的晶习和粒径分布。25℃时,当过饱和度S≤7.15时谷氨酸主要以α型晶习析出,粒径呈对数对称分布。随着过饱和度增大,β型结晶比例增大,中位径趋小且分布范围变宽。当S≥14.17以后,晶体全部是β型晶习。结晶成核诱导时间实验和理论计算结果表明,成核速率随过饱和度增加而增大是导致晶体粒径分布范围变宽的主要原因,不同过饱和度时α、β型晶核的竞争性成核速率是导致晶体晶习变化的主要原因。基于实验结果提出了谷氨酸C-pH相图模型,该模型解释了过饱和度影响谷氨酸结晶晶习的机理。控制等电调酸速率或添加晶种能有效降低过饱和度,改善谷氨酸结晶晶习及粒径分布。
(2)添加蔗糖改变谷氨酸溶液粘度,采用pH-shift模式研究了溶液粘度对谷氨酸结晶的影响。结果表明,在1.00-10.26 mP·s的粘度范围内,溶液粘度不影响谷氨酸结晶晶习,但粘度增加使得晶体粒径减小。高粘度溶液中谷氨酸结晶速率受溶质扩散速率控制,谷氨酸结晶成核速率、晶体生长速率随溶液粘度增加而下降是晶体粒径减小的主要原因。提高结晶温度能显著降低溶液的粘度,从而提高晶体的成核与生长速率。添加适量晶种能提高晶体平均粒径,并改善晶体均匀性。
(3)以除菌后等电母液为研究对象,考察了温度、浓缩倍数对浓缩母液粘度及谷氨酸溶解度的影响规律。结果表明,母液浓缩倍数越大粘度越大,提高温度可显著降低母液粘度,当温度大于60℃后不同倍数浓缩液的粘度接近。但提高温度同时促进了谷氨酸的焦化速率,为避免蒸发结晶过程中损失谷氨酸,蒸发结晶温度应控制在70℃以下。以控制谷氨酸结晶晶习及粒径分布为目标,通过实验研究了结晶温度、晶种添加策略、蒸发速率及浓缩倍数等工艺参数对谷氨酸蒸发结晶的影响。实验确定的谷氨酸蒸发结晶工艺为:结晶温度60℃,晶种目数为120目,添加量为母液中理论谷氨酸总量的30%,控制蒸发速率为207 L/m2·h,共浓缩6倍。在此条件下,蒸发结晶获得中位径约122.2μm的颗粒状谷氨酸α结晶,提取收率达到72.8%以上。
(4)实验确定了最大细晶消除循环速率R=1.3。进一步研究了不同细晶消除循环速率(R)、结晶停留时间等工艺参数对谷氨酸结晶的影响,结果表明,随着细晶消除循环速率的增加,细晶切割粒径从36.44μm提高到51.35μm,晶体粒径增大,粒径分布趋向集中。其原因是因为随着R值增加结晶过饱和度下降,晶体成核速率下降而晶体生长速率增加。结晶停留时间(τ)的长短同样影响结晶过饱和度,进而影响晶体粒径分布。分析对比等电离交、浓缩等电及细晶消除型连续等电
(5)以菌体细胞、COD及可溶性蛋白质去除率为指标,考察了絮凝剂种类、絮凝剂添加量、pH、温度及维持时间等因素对絮凝效果的影响,在此基础上,通过响应面分析法优化了絮凝除菌工艺条件。最优絮凝工艺:等电母液原始pH3.1、絮凝剂PAAS添加量60 mg/L、温度80℃、维持时间30 min时。此时菌体细胞、COD和可溶性蛋白去除率分别达到99%、47%和65%,以滤布为过滤介质的过滤速率达到280 L/m2·h。探讨了热变性絮凝除菌的机理,研究表明提高温度能降低菌体细胞表面的zeta电位,压缩颗粒表面扩散层厚度,增强架桥效应的絮凝作用。絮团中位径达到148μm以上,借助普通滤布即能实现高速过滤。
(6)在完成上述机理探索及关键技术研究的基础上,技术集成形成了基于谷氨酸二步结晶的“谷氨酸双结晶高效提取工艺”技术路线,并开展了总容积为76m3规模的中试验证。在中试研究过程中,对晶体分离、结晶质量提升等工序进行创新和优化,形成了更为完善的“谷氨酸双结晶高效提取工艺”路线。中试平均提取收率93.4%,比“浓缩等电工艺”(平均收率88%)高出5.4个百分点,仅比“等电离交工艺”(平均收率95%)低1.6个百分点。产品纯度、SO42-离子浓度、透光率等主要质量指标均高于“浓缩等电工艺”,更优于“等电离交工艺”。硫酸、液氨等辅料消耗比“等电离交工艺”分别下降了53%和100%,略低于“浓缩等电工艺”。技术经济性分析表明,在相同发酵水平、相同谷氨酸质量(转晶指标)、相同环境友好度的前提下,“双结晶工艺”吨谷氨酸生产成本分别比“等电离交工艺”和“浓缩等电工艺”下降了15.8%和14.6%。中试成果于2010年3月通过了中国发酵工业协会主持的技术鉴定,鉴定结论:本项目综合技术指标达到国际领先水平。
Glutamic acid (GA) is not only one of proteinogenic amino acids, but also an important free amino acid which is widely used in the fields of food industry, medicine industry, chemical industry for daily supplies and feed industry. Total output of the GA was 1.6 million tons in China in 2009, accounting for 75 per cent of global output. GA has now become one of the domestic staple fermentation products with strong competitive advantage in the global market. In our country, isoelectric crystallization with ion exchange (IEIE) is now the most common GA extracting process. Although the yield coefficient is high in IEIE, large amount of raw material is consumed and environmental pollution is serious. Instead, a concentrated continuous isoelectric process (CCIC) with low level of raw material consumption is applied to extract molasses-based GA. However the yield coefficient is not high enough in CCIC. Therefore modification of the current GA extraction technology to achieve the target of "energy-saving, raw material consumption and emission reduction" is essential for the promotion of the sustainable development and international competitiveness of Chinese GA industry. In this paper, key technologies of GA evaporative crystallization in the high viscosity solution, continuous isoelectric crystallization with eliminating fine crystal, and bacterium removal by combining thermal denaturation with flocculation were studied using Biotin-limited GA fermentation broth as the research object. A novel and cleaner GA extraction technique, called two-stage crystallization technology (TSC), was proposed and tested in pilot scale (76 m3). The main research work is summarized as follows:
(1) pH-shift model was adopted to investigate the effect of supersaturation on the GA crystallization using monosodium glutamate (MSG) solution instead of fermentation broth. Results showed that the supersaturation influenced both the crystal habit and size distribution. The main crystal habit wasα-form and the crystal size showed a logarithmic symmetric distribution when the supersaturation S≤7.15 at 25℃. With the increase of the supersaturation, the percentage ofβ-form crystal increased whereas the median diameter decreased with a wider size distribution. Almost all the crystal habit wereβ-form when the S≥14.17. According to the theoretical calculation and measurement of the induce time of nucleation, the increase of nucleation rate with the supersaturation is the primary reason for the widened crystal size distribution range. A GA C-pH Phase diagram model was proposed based on the results. This model explained how the supersaturation affected the GA crystal habit theoretically. The supersaturation could be efficiently reduced, and GA crystal habit as well as the crystal size distribution range could be improved by controlling the rate of acidity adjustment or by adding crystal seed.
(2) The pH-shift model was used to study how the viscosity of solution influenced the GA crystallization by adding sucrose to change the viscosity of solution. Results indicated that the viscosity did not affect the GA habit but reduce the nucleation rate in the range of 1.00-10.26 mP·s. The increase of viscosity led to smaller crystal size. The rate of crystallization was controlled by the solute diffusion rate in the high viscosity solution. Nucleation rate and crystal growth rate decreased with the increased viscosity which caused the smaller crystal size. The viscosity could be reduced by enhancing the temperature to increase the nucleation rate and crystal growth rate. Larger crystal size and better crystal habit could be obtained by adding crystal seed properly.
(3) The effects of temperature and condensed fold on the viscosity of condensed mother liquor and GA solubility were investigated using the isoelectric mother liquor in which the bacterium was removed. Results suggested that the viscosity increased as the folds of concentration enhanced. The viscosity could be reduced by rising the temperature. Besides, the viscosity under different concentration was similar when the temperature was over 60℃. On the other hand, high temperature could also promote the coking rate of GA. So the temperature should be controlled under 70℃to avoid the loss of GA in the evaporation crystallization process. To control the GA habit and size distribution, experiments were carried out to study the effects of crystallization temperature, strategy of seed addition, evaporation rate, folds of concentration and other factors during the evaporation crystallization process. The optimum operation conditions were: the optimum crystallization temperature at 60℃, seed crystal size of 120 mesh, the amount of seed addition of 30% of the theoretical GA quantity, evaporation rate of 207 L/m~2·h and 6 folds. Under these conditions, granularα-form crystal with median diameter of 122.2μm was obtained, and the yield coefficient of extraction reached to more than 72.8%.
(4) The maximum cycle rate of fine crystal elimination (R) was 1.3. Further research was done to investigate the effects of the technological parameters such as R, crystallization retention time and other parameters on the GA crystallization. As the increase of R, the cutting particle size of fine crystal was increased from 36.44μm to 51.35μm, and the crystal size was also increased. At the same time size distribution tended to be centralized. This phenomenon was due to the decrease of supersaturation and the nucleation rate, in contrast the increase of crystal growth rate with the increase of R value. Crystallization retention time could also influence the supersaturation and then crystal size distribution. This system was proved to efficiently eliminate the fine crystal and improve the crystal quality by comparing the size distribution of the three crystals in the processes of IEIE, evaporation isoelectric crystallization and continuous isoelectric crystallization respectively.
(5) In order to determine the optimum condition for the removal of bacterium, COD and soluble protein, the effects of flocculants, dosage, pH, temperature and time on flocculation were carried out using Response Surface Analysis. Highest bacterium, COD and soluble protein removal capacities (99%, 47% and 65%, respectively) were obtained when the PAAS was used under the condition of pH 3.1, dosage 60 mg/L, temperature 80℃and 30min. And the filtering rate was 280 L/m2·h when filter cloth was used. The mechanism of thermal denaturation with flocculation technology was studied. Results indicated that higher temperature could reduce the zeta potential on the surface of bacterium, compress the thickness of diffusion layer around the surface of particle, and enhance the bridge effect. The median diameter of the floccules reached to more than 148μm. In this condition, high filtering rate could be obtained using common filter cloth.
(6) Based on the above results, a novel and cleaner GA extraction technique, called two-stage crystallization technology (TSC), was proposed and tested in pilot scale (76 m3). Crystal separation and crystal quality were optimized in the pilot scale test to form a more perfect two-stage crystallization technology. Average yield coefficient of extraction was 93.4% which is higher than that of evaporation isoelectric crystallization process by 5.4% and lower than that of IEIE process by only 1.6%. Purity, SO42- concentration and transmittance were superior to those of the other two processes. Compared with IEIE process, sulfuric acid and liquid ammonia consumptions were reduced by 53% and 100%, respectively. Technical analysis shows that production cost of TSC process would be lower than those of IEIE process and evaporation isoelectric crystallization process by 15.8% and 14.6%, respectively, provided that the fermentation level, the GA quality and effect on environment of the three processes were same.
引文
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