Claims:1. A process for the extraction of phycocyanin from a residual algal biomass; said process comprising the following steps:
a) preparing an aqueous slurry of said biomass by adding a fluid medium to obtain a first mixture;
b) optionally freezing or chilling said first mixture at a temperature in the range of -20 to 20 °C for a time period of 1 minute to 12 hours to obtain a frozen/chilled biomass followed by thawing said frozen biomass at a temperature in the range of 20 to 40 °C for 2 minutes to 30 minutes to obtain a thawed biomass; and
c) separating said fluid medium from first mixture or said thawed/chilled biomass to obtain a supernatant containing phycocyanin and a residue.
2. The process as claimed in claim 1, wherein said algal biomass is at least one selected from the group consisting of Rhodophyta, Cyanobacteria, Spirulina, Geitlerinema, Microcystis, Anabaena, Nodularia, Oscillatoria, Spirogyra and Phormidium.
3. The process as claimed in claim 1, further comprises washing said residual algal biomass at least once with water before preparing said aqueous slurry.
4. The process as claimed in claim 1, wherein said fluid medium is 3 – 8 times by weight of said algal biomass, and is at least one selected from the group consisting of water and/or buffer.
5. The process as claimed in claim 4, wherein said buffer is at least one selected from the group consisting of Phosphate buffer, Tris buffer, MES buffer [2-(N-morpholino)ethanesulfonic acid].
6. The process as claimed in claim 1, wherein said steps a, b and c are repeated at least once.
7. The process as claimed in claim 1, wherein said separation is carried out by centrifugation at 8000 – 12000 rpm for 10 – 20 minutes at a temperature in the range of 2 – 40 °C or by any solid liquid separation method.
, Description:This application is a patent of addition with respect to Indian Patent Application No. 201621028171 dated 18.08.2016, the entire contents of which, are specifically incorporated herein by reference.
FIELD
The present disclosure relates to the field of extraction of phycocyanin from an algal biomass.
BACKGROUND
Algae forms the life-supporting foundation of the natural food chain by providing essential vitamins, minerals, proteins, and nutrients required to support life. The health benefits of some algae have long been appreciated when used as a dietary supplement for promoting and sustaining human health.
Phycocyanin is a pigment-protein complex of the light-harvesting phycobiliprotein family, along with allophycocyanin and phycoerythrin. It is an accessory pigment to chlorophyll. All phycobiliproteins are water-soluble, so they cannot exist within the membrane, like carotenoids can. Instead, phycobiliproteins aggregate to form clusters that adhere to the membrane called phycobilisomes. Phycocyanin comprises a protein and a choromophore; furthermore it exhibits a strong red fluorescence. Phycocyanin has wide range of applications such as, fluorescent markers, antioxidants, immuno-modulant, neuroprotective and hepatoprotective, natural pigments in food and cosmetics and the like.
Conventional methods for isolating phycocyanin from algae/cyanobacteria involve using various cell disruption techniques like Freeze-Thaw, Sonication, Homogenization, etc., thereby releasing cytoplasmic contents, to produce a disrupted cell suspension; separating solid and liquid phases of the disrupted cell suspension; contacting the liquid phase of the disrupted cell suspension with a non-ionic polyaromatic macroreticular adsorbent resin; collecting the liquid phase from the resin to produce a phycocyanin extract; and optionally dehydrating the phycocyanin extract.
Some processes involve separation and purification of phycocyanin including precipitation, centrifugation, dialysis and chromatography process. These methods are difficult to carry out and are expensive.
Freeze-Thawing technique is recognized as a superior and versatile technique for cell disruption & downstream processing of algal biomass. Repeated freezing & thawing method is more effective in extracting phycobiliprotein from various cyanobacteria than the cell disruptive methods. The advantage of this technique is that it is mild and non-denaturing. Ice crystals formed during freezing, break the cell wall and the cell membrane and release the phycobiliprotein into the extracting medium. However freezing at extremely low temperatures and thawing at high temperature in freeze-thaw cycle is energy intensive and difficult to scale-up.
The recovery of phycocyanin is normally done from fresh algal biomass where the cake collected after 1st stage of phycocyanin extraction is disposed. Phycocyanin being a very high value product, enhanced recovery from the residual biomass is highly desirable.
Though various processes for the extraction of phycocyanin are known, the extraction rate is not high, resulting in an increased production costs. Also the processes are tedious, time consuming and energy intensive.
It is therefore, desired to provide a simple, economic and rapid phycocyanin extraction process which can be easily scaled up and can be an alternative to the known process and which can also overcome the drawbacks associated with the known process such as complex process steps, time consuming and high cost.

OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a process for the extraction of phycocyanin from residual algal biomass.
Another object of the present disclosure is to provide a process for the extraction of phycocyanin which is simple, rapid and economic.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure envisages a process for the extraction of phycocyanin from a residual algal biomass. Initially, an aqueous slurry is prepared by adding a predetermined amount of a fluid medium to a predetermined quantity of the residual algal biomass to obtain a first mixture. The first mixture is then transferred into a container, followed by freezing/chilling at a temperature in the range of -20 to 20 °C for a time period of 1 minute to 12 hours to obtain a frozen/chilled biomass. The frozen biomass is then thawed by keeping the container containing the frozen biomass at a temperature in the range of 20 to 40 °C for 1 to 30 minutes to obtain a thawed biomass. The fluid medium is then separated from the thawed/chilled biomass to obtain a supernatant containing phycocyanin and a residue.
The algal biomass can be at least one selected from the group consisting of Rhodophyta, Cyanobacteria, Spirulina, Geitlerinema, Microcystis, Anabaena, Nodularia, Oscillatoria, Spirogyra and Phormidium. The residual algal biomass can optionally be washed at least once with water before preparing the aqueous slurry. Typically, the fluid medium can be water and/or buffer. The buffer is selected from the group consisting of Phosphate buffer, Tris buffer, MES buffer [2-(N-morpholino)ethanesulfonic acid] or any other buffer. In an embodiment of the present disclosure, the buffer is selected from the group consisting of biologically similar buffer, preferably buffers having a neutral pH. The process is repeated at least once for extracting the remaining phycocyanin. The separation is carried out by centrifugation at 8000 – 12000 rpm for 10 – 20 minutes at a temperature in the range of 2 – 40 °C or by any other solid liquid separation process as appropriate.
DETAILED DESCRIPTION
Phycocyanin is a water soluble protein and is one of the main pigments of cyanobacteria. It has a blue colour and a red fluorescence. It has a maximum absorption at 620 nm and an emission radiation at 635 nm. This quality makes it a natural fluorescent product which is a favoured label in biomedical diagnostics. At the physiological level, phycocyanin is an important antioxidant particularly in protecting plasma proteins against oxidative modifications. Phycocyanin is a molecule of great interest, because of its beneficial properties for human and animal health. Phycocyanin is consumed particularly for its antioxidant properties and also for its ability to promote the production of stem cells.
Conventional methods for isolating phycocyanin from algae/cyanobacteria involve using various cell disruption techniques like Freeze-Thaw, Sonication, Homogenization, etc., thereby releasing cytoplasmic contents, to produce a disrupted cell suspension; separating solid and liquid phases of the disrupted cell suspension; contacting the liquid phase of the disrupted cell suspension with a non-ionic polyaromatic macroreticular adsorbent resin; collecting the liquid phase from the resin to produce a phycocyanin extract; and optionally dehydrating the phycocyanin extract.
Some processes involve separation and purification of phycocyanin including precipitation, centrifugation, dialysis and chromatography process. These methods are difficult to carry out and are expensive.
Freeze-Thawing technique is recognized as a superior and versatile technique for cell disruption and downstream processing of algal biomass. Repeated freezing and thawing method is more effective in extracting phycobiliprotein from various cyanobacteria than the cell disruptive methods. The advantage of this technique is that it is mild and non-denaturing. Ice crystals formed during freezing, break the cell wall and the cell membrane and release the phycobiliprotein into the extracting medium. However, freezing at extremely low temperatures and thawing at high temperature in freeze-thaw cycle is energy intensive and difficult to scale-up.
Though various processes for the extraction of phycocyanin are known, the extraction rate is not high, resulting in increased production costs. Also, the processes are tedious, time consuming and energy intensive.
It is therefore, desired to provide a simple and economic phycocyanin extraction process which can be easily scaled up and can be an alternative to the known processes and which can also overcome the drawbacks associated with the known processes such as complex process steps, time consuming and high cost.
The present disclosure envisages a process for the extraction of phycocyanin from a residual algal biomass. Initially, an aqueous slurry is prepared by mixing predetermined amount of a fluid medium with predetermined amount of the residual algal biomass to obtain a first mixture. Typically, the fluid medium can be 3 to 8 times the weight of the residual algal biomass.
Further, the first mixture is transferred into a container, followed by freezing/chilling at a temperature in the range of -20 to 20 °C for a time period of 1 minute to 12 hours to obtain a frozen/chilled biomass. The frozen biomass is then thawed by keeping the container containing the frozen biomass at a temperature in the range of 20 to 40 °C for 1 to 30 minutes to obtain a thawed biomass. The fluid medium is then separated from the thawed biomass to obtain a supernatant containing phycocyanin and a residue. In an embodiment of the present disclosure, the separation can be carried out by centrifugation at 8000 – 12000 rpm for 10 – 20 minutes at a temperature in the range of 2 – 40 °C or by any appropriate solid liquid separation process.
In accordance with the embodiments of the present disclosure, the algal biomass can be at least one selected from the group consisting of Rhodophyta, Cyanobacteria, Spirulina, Geitlerinema, Microcystis, Anabaena, Nodularia, Oscillatoria, Spirogyra and Phormidium. Optionally, the residual algal biomass can be washed at least once with water before preparing the aqueous slurry.
In an embodiment of the present disclosure, Geitlerinema species is obtained from Gagva, Jamnagar, Gujarat, India.
The fluid medium can be water and/or buffer. The buffer is selected from the group consisting of Phosphate buffer, Tris buffer, MES buffer [2-(N-morpholino) ethanesulfonic acid] or any other buffer. In an embodiment of the present disclosure, the buffer is selected from the group consisting of biologically similar buffer, preferably buffers having a neutral pH.
In another embodiment of the present disclosure, the process is repeated at least once for extracting the remaining phycocyanin.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial scale.

Experiment 1: Phycocyanin extraction (from the residual algal biomass)
Residual algal biomass (residual Geitlerinema culture biomass post phycocyanin extraction) was used for additional phycocyanin recovery. The residual biomass was mixed with four times fresh wash water (by weight). This mixture was then kept in the freezer at -10 °C for 1 hour and then thawed at 30 °C for 5 minutes to obtain a thawed biomass. Post thawing, the thawed biomass was centrifuged using REMI PR-24 lab centrifuge at 10000 rpm for 15 minutes to obtain a supernatant containing phycocyanin and a residue. This residue was again mixed with four times fresh wash water (by weight) and the entire process is repeated to improve and further extract more phycocyanin.
The phycocyanin (PC) concentration in crude was estimated based on Equation 1.
Equation 1:
PC concentration [PC], mg/ml = (A620*D.F.-0.7A655*D.F.)/7.38 (1)
where A620 & A655 represent absorbance at 620 & 655 nm and D.F. represents dilution factor.
Table 1 summarizes the amount of phycocyanin extracted after each cycle of phycocyanin extraction from the residual biomass. It is observed that among all PC recovered in the four cycles, cycle 2-4 lead to 25.6% of the total cumulative recovery obtained until stage 4. Hence, enhanced recovery over first cycle is 34% that otherwise would be lost as waste, in absence of enhanced recovery process.
Table 1: Enhanced recovery of phycocyanin
Cycle No. PC conc.
(mg/ml) Stage wise PC recovery %, (recovery at any stage/total recovery) Enhanced cumulative recovery over 1st stage
(recovery at any stage/1st stage recovery)
1 6.04 74.4 1
2 1.26 16.7 1.22
3 0.49 6.7 1.31
4 0.17 2.2 1.34

TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of:
? a simple, efficient & rapid process to extract phycocyanin from the residual algal biomass, that is easy to scale up; and
? a chemical/enzyme-free extraction of phycocyanin.
The embodiments as described herein above, and various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the description. Descriptions of well-known aspects, components and molecular biology techniques are omitted so as to not unnecessarily obscure the embodiments herein.
The foregoing description of specific embodiments so fully reveal the general nature of the embodiments herein, that others can, by applying current knowledge, readily modify and/or adapt for various applications of such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Further, it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
Having described and illustrated the principles of the present disclosure with reference to the described embodiments, it will be recognized that the described embodiments can be modified in arrangement and detail without departing from the scope of such principles.
While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.