کشت سلولی میکروجلبک اسپیرولینا (Spirulina platensis) و مقایسه راندمان روش‌های آنزیمی، اولتراسوند، انجماد-انجمادزدایی و حلال معدنی در استخراج رنگدانه فیکوسیانین

نوع مقاله : مقاله پژوهشی

نویسندگان

• رضا صفری ¹
• سهیل ریحانی پول ²

¹ پژوهشکده اکولوژی دریای خزر، موسسه تحقیقات علوم شیلاتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، ساری، ایران

² گروه فراوری محصولات شیلاتی، دانشکده شیلات و محیط‌زیست، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران

https://doi.org/10.22067/ifstrj.2023.78800.1204

چکیده

جهت استخراج رنگدانه­ها از جلبک­های دریایی از تکنیک­های مختلفی استفاده می­شود که هر تکنیک علاوه بر داشتن معایب و مزایایی، راندمان متفاوتی دارد. یکی از این رنگدانه­ها، فیکوسیانین است که از روش­های مختلفی جهت استخراج آن استفاده می­گردد. هدف پژوهش حاضر کشت میکروجلبک اسپیرولینا پلاتنسیس و استخراج فیکوسیانین از آن با استفاده از چهار روش اولتراسوند، انجماد-انجمادزدایی، آنزیمی و حلال معدنی بود. در مراحل بعد، میزان راندمان هر روش از طریق اندازه­گیری غلظت و خلوص فیکوسیانین ارزیابی شد. ضمن اینکه اثر اعمال فرایند خالص­سازی با سولفات آمونیوم نیز بر غلظت و خلوص رنگدانه مستخرج مورد سنجش قرار گرفت. نتایج نشان داد رشد میکروجلبک از زمان صفر تا 14 روز، دارای روند صعودی و تغییرات حاصله نیز در تمامی زمان­ها، بجز روزهای 14 و 16، معنی­دار بوده است (05/0>p). همچنین جلبک بعد از سپری­کردن فاز سکون کوتاه (2 روز)، وارد مرحله رشد لگاریتمی شد و تا روز 14 به رشد خود ادامه داد اما بین روزهای 14 و 16، رشد تقریبا روند ثابتی بخود گرفت. در ادامه مشخص شد که میزان توده تولیدشده پس از 16 روز، 1120 میلی­گرم بر لیتر می­باشد. غلظت فیکوسیانین استخراج­شده در روش­های آنزیمی و اولتراسوند (به ترتیب 815/1 و 786/1 میلی­گرم در میلی­لیتر) فاقد اختلاف معنی­دار (05/0<p) و در سطح بالاتری از دو روش دیگر قرار داشت (05/0>p)؛ ضمن اینکه غلظت رنگدانه در تکنیک انجماد-انجمادزدایی (535/1 میلی­گرم در میلی­لیتر) بیشتر از روش حلال معدنی (121/1 میلی­گرم در میلی­لیتر) بود (05/0>p). پس از خالص­سازی رنگدانه با استفاده از سولفات آمونیوم، غلظت و خلوص رنگدانه به صورت معنی­داری در هر روش افزایش یافت (05/0>p). نتایج تحقیق حاضر نشان داد که با انتخاب روش بهینه و همچنین اعمال فرایند خالص­سازی با سولفات آمونیوم، می­توان راندمان استخراج فیکوسیانین از میکروجلبک اسپیرولینا پلاتنسیس را افزایش داد.

کلیدواژه‌ها

• میکروجلبک اسپیرولینا
• استخراج فیکوسیانین
• روش اولتراسوند
• تکنیک آنزیمی
• آنزیم لیزوزیم

موضوعات

• فناوری مواد غذایی

عنوان مقاله [English]

Cell Culture of Spirulina Microalgae (Spirulina platensis) and Comparison the Efficiency of Enzymatic, Ultrasound, Freeze-defrosting and Mineral Solvent Methods in Extraction of Phycocyanin Pigment

نویسندگان [English]

• Reza Safari ¹
• Soheil Reyhani Poul ²

¹ Caspian Sea Ecology Research Institute, Fisheries Science Research Institute, Agricultural Research Education And Extention Organization, Sari, Iran

² Department of Processing of Fishery Products, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

چکیده [English]

Introduction
 Phycocyanin is one of the pigments used in the food industry due to its antioxidant and antibacterial as well as coloring properties. This pigment is commercially produced from Spirulina platensis microalgae, in the form of photoautotrophic cultures and in open environments in large ponds or pools in tropical or subtropical areas at the edges of oceans. Different techniques are used in order to extract phycocyanin from spirulina microalgae.. Each technique has its own advantages and disadvantages besides different efficiency. These methods include freezing-defrosting, enzymatic, ultrasound, high hydrostatic pressure, ultracentrifuge, ultra homogenization, extraction using water and various solvents. Of course recently, the production of recombinant phycocyanin has been considered as a suitable option for the production of heterotrophic phycocyanin. The purpose of the current research was to cultivate Spirulina platensis, evaluation of the microalgae growth process, and comparison of the efficiency of different methods in the extraction of phycocyanin pigment.
 
Materials and Methods
 The pure sample of Spirulina platensis microalgae was prepared from Algaeology Laboratory, Biology Department of Tarbiat Modares University. For the cultivation of spirulina, Zarrouk culture medium with different compositions was used, and after cultivation in smaller scales (100 and 500 ml), the final cultivation was carried out in volumes of 5 and 50 liters. After cultivating the microalgae and exposing them to fluorescent light with appropriate light lux intensity (3500 to 8000) and a period of 12 hours of darkness and 12 hours of light, the samples were placed at 29 °C for 16 days. In order to evaluate the growth process of the algal mass, the absorbance of the solution containing the algal cells was read at a wavelength of 540 nm. After preparing the dry mass of spirulina microalgae, four methods of ultrasound, freezing-defrosting, enzymatic and mineral solvent technique were used to extract phycocyanin. In the next steps, the efficiency of each method was evaluated by measuring the concentration and purity of phycocyanin. In addition, the effect of applying the purification process by ammonium sulfate on the concentration and purity of the extracted pigment was also evaluated. This research was conducted in a completely randomized design and SPSS and EXCEL softwares were used for statistical analysis and drawing of diagram, respectively. Data were analyzed using one-way analysis of variance and the difference between the means was evaluated by Duncan's test at 95% confidence level.
 
Results and Discussion
 The results showed that microalgae growth from day 0 to 14 had an upward trend and the resulting changes were significant at all times, except days 14 and 16 (p<0.05). Also, after passing the short resting phase (2 days), the microalgae entered the logarithmic growth phase and continued to grow until the 14th day, but between the 14th and 16th days, the growth was almost constant. In the following, it was found that the mass produced after 16 days is 1120 mg/l. The concentration of phycocyanin extracted in enzymatic and ultrasound methods (1.815 and 1.786 mg/ml, respectively) had no significant difference (p>0.05) and was at a higher level than the other two methods (p<0.05); In addition, the pigment concentration was higher in the freezing-defrosting technique (1.535 mg/ml) than in the mineral solvent method (1.121 mg/ml). After purification of the pigment using ammonium sulfate, the pigment concentration and purity increased significantly in each method (p<0.05). The results of this research showed that by choosing the optimal method and applying the purification process using ammonium sulfate, the extraction efficiency of phycocyanin from Spirulina microalgae (Spirulina platensis) could be increased.
 
Conclusion
 Based on the results of this research, the growth trend of Spirulina platensis in Zarrouk culture medium was  ascending first and then constant (during 16 days). Ultrasound technique and enzymatic method (lysozyme enzyme) to extract phycocyanin pigment from Spirulina platensis microalgae have more efficiency than freezing-defrosting and inorganic solvent (hydrochloric acid) methods. Also, purification of the extracted pigment using 40% ammonium sulfate increases the concentration and purity of phycocyanin in each method.

کلیدواژه‌ها [English]

• Enzymatic technique
• Lysozyme enzyme
• Phycocyanin extraction
• Spirulina microalgae
• Ultrasound method

©2023 The author(s). This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source.

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