Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa
© Waghmare et al. 2016
Received: 13 November 2015
Accepted: 14 March 2016
Published: 26 March 2016
Many methods are available for the concentration of proteins; however, most are not easily scalable due to costs, the need of specialized instruments and skilled workers or are very time-consuming. Three-phase partitioning (TPP) is a separation technique that has gained a lot of interest due to its rapid, simple and scalable use for concentration, isolation and decontamination of proteins from crude samples with high recovery yields. In the present work, the effect of various parameters of TPP was evaluated to optimize the concentration of proteins from Chlorella pyrenoidosa (CP), is green algae that increasingly being used as food supplements because of its positive impacts on human health.
Chlorella pyrenoidosa was cultivated in a closed system under controlled conditions. After reaching maximum growth, the microalgae was harvested, dried and powdered. Afterwards, TPP of CP cell lysate was done to concentrate protein content. To maximize protein concentration, various parameters were optimized such as solvent (t-butanol), ammonium sulphate concentration (40 % w/v), solid load (0.75 g/20 mL), pH (6), incubation time (20 min), slurry to butanol ratio (1:1.5) and enzymatic treatment (combination of Stargen™ and Carezyme™). Also, total starch, cellulose and carbohydrate content before and after the enzymatic treatment were determined to comprehend the impact of enzymatic treatment on protein concentration. Using these optimized parameters, 78.1 % w/w protein concentration was obtained in middle protein concentrate phase. This protein concentrate was characterizedfor proximate composition, colour analysis, water holding capacity, oil-holding capacity, foaming capacity, foam stability, amino acid composition, protein quality and thermal properties.
KeywordsMicroalgae Three-phase partitioning Protein concentrate Enzymatic treatment Protein quality
Microalgae proteins have a great potential to be an alternative protein source since they contain all essential amino acids (Becker 2007; Barbarino and Lourenço 2005; Lourenco et al. 2004; Safi et al. 2014a, b). Microalgae have created immense interest due to their unconventional growth requirements such as being able to grow in salt water in the presence of CO2 and sunlight on unfertile land. This makes microalgae a potentially sustainable source of feedstock for fuel, food, chemical, textile, polymer and even the pharmaceutical industry (Viegas et al. 2015). Microalgae contain lipids, protein and carbohydrate as their main components and their protein concentrates can be used in the food, feed and bulk chemical industry (Chacon-Lee and Gonzalez-Marino 2010).
Chlorella pyrenoidosa (CP) is a species of the freshwater green algae that is widely used as food supplements (Garcia et al. 2012; Wang and Zhang 2012). Each year, the United States, Japan, China, Taiwan and Indonesia produce over 2500 tons of dried Chlorella (Bishop and Zubeck 2012) because it is not only considered a good source of nutrients but also a functional food due to its positive health effects (Gao 1998; Richmond 1990). Various clinical and animal studies have shown that CP is a safe source of protein for consumption and dietary supplementation with Chlorella may reduce high blood pressure, lower serum cholesterol and glucose levels, accelerate wound healing, and enhance immune functions (Viegas et al. 2015; Cherng and Shih 2005; Halperin et al. 2003; Lee et al. 1967; Merchant and Andre 2001; Merchant et al. 2002; Mizoguchi et al. 2008; Lisboa et al. 2014). Microalgae proteins also have high techno-functional potential such as being usable as an emulsifier (Ursu et al. 2014). Since proteins from Chlorella can act as an animal protein replacement, its use can have a positive impact on the environment (Jones 2016).
Many methods have been used for fractionation/concentration of proteins from microalgae such as ultrafiltration, precipitation, chromatography, dialysis and centrifugation. Even though these methods are effective, they are not easily scalable due to the costs of the process, requirement of a specialized instrument, time-consuming and the need of skilled operators. The drawbacks of ultrafiltration include the susceptibility of rapid membrane clogging, prolonged processing time and the use of expensive membrane filters (Walsh 2007). Precipitation is an industrially suitable method for protein concentration and is performed by salting out, isoelectric focusing and solvent precipitation. However, each of these methods has their limitation such as the low recovery during salting out and irreversible denaturation of proteins after isoelectric focusing and solvent precipitation (Ustunol 2015). Chromatography, dialysis and centrifugation are preferable methods for final-stage purification of proteins but are very much time-consuming (Bio-Sciences 2010). Three-phase partitioning (TPP) is a separation technique useful for initial purification of proteins with high recovery yields. It has gained a lot of interest due to its rapid, simple and scalable use for concentration, isolation and decontamination of proteins from crude samples (Harde and Singhal 2012). TPP can efficiently fractionate non-polar components in solvent phase (upper phase), polar components in aqueous (lower phase) and proteins in the middle phase. This technique uses a combination of ammonium sulphate and t-butanol to precipitate proteins from crude extracts. Precipitated protein binds to t-butanol, thereby increasing their buoyancy and causing the precipitates to float above the denser aqueous salt phase. Optimum pH, temperature, ammonium sulphate and t-butanol concentrations can selectively precipitate proteins at the interface of the organic and aqueous phases. Kosmotropic, salting out, co-solvent precipitation, isoionic precipitation, osmotic electrostatic forces, conformation tightening and protein hydration shifts all contribute to protein precipitation at this interface (Vetal and Rathod 2014). It has been reported that TPP can even enhance enzyme activity compared to conventional methods (Garg and Thorat 2014; Ketnawa et al. 2014; Ozer et al. 2010; Şen et al. 2011). Furthermore, the protein recovery yield is almost perfect reaching values near 100 % of the total protein present (Phongthai and Rawdkuen 2015).
In the present work, the effect of various parameters of the TPP method was evaluated to optimize the concentration of proteins from CP. The effect of an enzymatic treatment on protein concentration efficiency was evaluated by quantifying initial and final starch, cellulose and total carbohydrate concentrations. Also, the protein concentrate obtained using optimized conditions was characterized to understand the influence of the TPP method.
All chemicals, salts, solvents and reagents used in this study were of analytical grade and were purchased from SD Fine, Mumbai, India. Stargen™ 002 was gifted from Genencor International, Danisco US and Carezyme™ was procured from Sigma-Aldrich, USA. Corn oil was gifted by Kamani oils, Mumbai, India.
Culture growth conditions and harvesting
The species Chlorella pyrenoidosa strain NCIM 2738 is a freshwater green alga from the genus Chlorella. This strain was obtained from National Centre for Industrial Microorganisms (NCIM), Pune, India. Batch size of 40 L (20 flasks) was cultivated in 3-L glass conical flasks containing 2 L modified Chlorella medium in each flask and sterilized in an autoclave at 120 °C, 15 bar pressure for 20 min. Modified chlorella medium, adjusted to pH 7.5, consisted of following components (g/l): KNO3 (4), NaNO3 (0.1), MgSO4·7H20 (0.1), CaCl2·H2O (0.05), H3BO3 (0.00286), MnCl2·4H2O (0.00181), ZnSO4·7H2O (0.00022), NaMoO4·2H2O (0.0004), CuSO4·5H2O (0.00008), FeSO4·7H2O (0.00557), and Na2EDTA (0.00745). The flasks were maintained in a plant growth chamber where the illumination (800–1000 lux) was provided by cool white fluorescent lamps (Philips) and light intensity was measured with an LUX meter. The temperature was maintained at 25 ± 2 °C and flasks were agitated twice daily for 2 min at 60 revolutions per min. Ten-day-old cultures were inoculated in the media-containing flasks and incubated during 45 days. The biomass was harvested by sedimentation and further centrifuged (Backman J2-MC, USA) at 5000×g for 10 min to collect the thick algal slurry. The algae slurry was washed with distilled water to remove salts. This slurry was dried in an oven at 50 °C for 24 h. Dried biomass was ground and passed through 36-mesh-size sieve to obtain uniform particle size then stored at −20 °C in an air-tight seal container until used. Afterwards, cell disruption of dried biomass was performed using ultrasonication (Branson Ultrasonifier 450, USA) and disruption was confirmed by microscopic (Motic BA310, with Moticam 480, China) observation at 40×.
TPP of cell lysed CP biomass was carried out to concentrate its proteins. In 100 mL glass beakers, 20 mL cell lysed biomass was mixed with 8 g of ammonium sulphate followed by addition of 20 mL t-butanol. This mixture was stirred for 20 min at 28 ± 2 °C. Immediately, three distinct phases were separated carefully using Pasteur pipette. Initially, the upper organic phase was pipetted then lower aqueous phase was pipetted by piercing middle phase. The middle phase, containing the concentrated proteins (see “Background” section), was used for further study. Salt was removed from protein concentrate using Hi-media dialysis membrane-50 for 12 h in 1 L distilled water (fresh distilled water was replaced after 6 h). Dialysed protein concentrate was precipitated by adjusting the pH to 5.0 followed by centrifugation at 8000×g for 10 min to remove water. This protein concentrate was dried at 50 °C for 12 h and used for analysis. Effect of various process parameters was evaluated such as solvent, ammonium sulphate, solid load, pH, incubation time, slurry to butanol ratio and enzymatic treatment. The protein concentration of middle phase proteins was represented as a concentrate in % w/w.
Protein concentration in middle phase was determined by the modified Kjeldahl method (Persson et al. 2008). This method mainly involved digestion, distillation and titration (Kel-Plus, Elite Ex 8L, Pelican equipments, India). For microalgae and cyanobacteria undergoing rapid growth, the recommended Kjeldahl nitrogen-to-protein conversion factor used was 5.95 instead of 6.25 (Lopez et al. 2010). In the case of enzymatically treated biomass, the amount of enzyme used was subtracted from total protein concentration found, to eliminate the contribution of enzymes in protein quantification. In every run, two digestion tubes were kept as blanks. All samples were analysed in triplicate and mean with standard deviation was reported.
Effect of enzymatic treatment on protein concentration
Effect of enzymatic treatment on protein concentration was studied using Stargen™ 002 (570 GAU/g from Genencor International) and Carezyme™ (1000 U/g from Sigma-Aldrich). Cell disrupted microalgae slurry was treated separately with each individual enzyme and also, both enzymes were used together to evaluate the combined effect. Simultaneously, non-enzymatic treated cell lysates were used as controls. The condition during Stargen™ 002 treatment was pH (4.5), temperature (50 °C) and enzyme loading (10 µg for 0.75 g of cell lysate in 20 mL distilled water) for 1 h with gentle stirring (300 rpm) using a magnetic stirrer. For, Carezyme™ treatment conditions were pH (5), temperature (37 °C) and enzyme loading (10 µg for 0.75 g of cell lysate in 20 mL distilled water) for 1 h with gentle stirring (300 rpm) using a magnetic stirrer. The combined effect of Stargen™ 002 and Carezyme™ was studied by first treating the cell lysate with Stargen™ 002 followed by Carezyme™ treatment both in the conditions described above for each individual enzyme. Effect of enzymatic treatment was assessed by determining protein concentration, starch, cellulose and total carbohydrate of TPP middle phase protein concentrate powder as described below.
Total starch quantification
Starch content was determined using a modified method of Takeshita et al. (2014). Briefly, 0.5 g of each sample was homogenized in 80 % ethanol at 50 °C to remove sugars. The residue was separated by centrifugation at 5000×g for 10 min then washed repeatedly with 80 % ethanol at 50 °C until the washings did not give a colour with anthrone reagent and removed all pigments. This residue was completely dried over a water bath then 2.5 mL of water and 2.5 mL of 60 % perchloric acid were added. These tubes were kept at 0 °C for 20 min for starch extraction. The supernatant was separated by centrifugation at 5000×g for 10 min and stored. The extraction was repeated using fresh 60 % perchloric acid and the supernatant was separated by centrifugation at 5000×g for 10 min. From the supernatant, 1 mL of extract was pipetted and 4 mL of anthrone reagent was added to each tube. Afterwards, the test tubes were heated in a boiling water bath for 10 min then cooled to room temperature, and the absorbance at 630 nm was measured using a spectrophotometer. Glucose was used as a standard for calibration graph to determine glucose concentration in samples. Starch content was calculated by multiplying the value by a factor 0.9.
Cellulose was quantified as described by Thayumanavan and Sadasivam (1984). Acetic/nitric reagent (3 mL of a solution consisting of 10 volumes of 80 % acetic acid and 1 volume of concentrated nitric acid) was added to 0.5 g of the sample in a test tube and mixed using a vortex mixer. The tubes were then placed in a water bath at 100 °C for 30 min, cooled and centrifuged at 5000×g for 15 min. The supernatant was discarded and cellulose-containing residue was washed with distilled water. To this residue, 10 mL of 67 % sulphuric acid was added and allowed to stand for 1 h. Further, 1 in 100 dilution was made and 1 mL of this diluted solution combined with 10 mL of anthrone reagent (200 mg anthrone in 100 mL chilled concentrated sulphuric acid) was added and mixed well. The tubes were heated in a boiling water bath for 10 min. Further, tubes were cooled and the absorbance at 630 nm was measured. Blank was prepared with anthrone reagent and distilled water and the calibration curve was prepared using cellulose in the range of 40–200 µg.
Total carbohydrate quantification
A sample (100 mg) was taken in a test tube and 5 mL of 2.5 N HCl was added and kept in boiling water bath for 3 h for hydrolysis. This solution was allowed to cool and was neutralized using sodium carbonate until effervescence ceases. Volume was adjusted to 100 mL with distilled water and centrifuged at 8000×g for 20 min. The supernatant was collected and 1 mL aliquot was taken for analysis to which 4 mL of anthrone reagent was added followed by heating for 10 min in boiling water bath. After cooling to room temperature, absorbance was measured at 630 nm. Glucose was used as a standard for the preparation of calibration graph (Thayumanavan and Sadasivam 1984) and distilled water was used as blank in place of sample.
Characterization of protein concentrate
Proximate composition of dried biomass and protein concentrate
Samples were analysed for moisture and total ash contents following standard AOAC (2001) method. Total protein content was determined using the micro-Kjeldahl procedure with a nitrogen-to-protein conversion factor of 5.95 (Lopez et al. 2010). Fat content was determined by the Soxhlet method (using Instant Soxhlet apparatus-Socs Plus, Pelican Equipments, Chennai, India) using petroleum ether (B. P. 60–80 °C) as the solvent. Carbohydrate was estimated by difference [Percentage carbohydrate content = 100 − (% moisture + % ash + protein + % fat)] and also using the method with anthrone as described above.
Color of the dried biomass and protein concentrate were measured using Hunter Lab colorimeter model DP-9000 D25A (Hunter Associates Laboratory, Reston, VA, USA) in terms of Hunter L (lightness, ranging from 0 to 100 indicating black to white), a (+a, redness and –a, greenness) and b (+b, yellowness and –b, blueness). The determination of color was done on six different samples. Standardisation of the instrument was done using a black and white standard plates.
Water-holding capacity (WHC) and oil-holding capacity (OHC)
To determine WHC, 1 g protein concentrate was dispersed in 10 mL of distilled water and placed in centrifuge tubes. The dispersions were stirred for 5 min, held for 30 min, followed by centrifugation at 3000×g for 25 min. The supernatant was eliminated, the excess of water was removed by draining for 25 min at 50 °C and the sample was reweighed. For OHC, 0.5 g of sample was mixed with 6 mL of corn oil and allowed to soak for 30 min. Then, tubes were centrifuged at 3000×g for 25 min. The separated oil was then removed with a pipette and the tubes were inverted for 25 min to drain the oil prior to reweighing. The WHC and OHC were expressed as grams of water or oil bound per 100 g of the sample on dry weight basis (Ghribi et al. 2015).
Foaming capacity (FC) and foaming stability (FS)
Samples were dispersed in 50 mL distilled water at a concentration of 3 % (w/v) and homogenized at 8000×g for 3 min. The blend was immediately transferred into a graduated cylinder. The volume was recorded before and after whipping. FC was expressed as the volume (%) increase due to whipping.
Amino acid composition and protein quality
Amino acid composition was determined using high-pressure liquid chromatography (HPLC). The sample of protein concentrate was dissolved in 6 mL of 6 N HCl and was subjected to hydrolysis in boiling water bath for a period of 24 h. The tubes were vortex mixed after every 1 h for proper hydrolysis. After 24 h of hydrolysis, the tubes were centrifuged at 3500×g for 15 min. The supernatant was filtered and was neutralized with 1 N NaOH. Then, the filtered solution was diluted to 1:100 with HPLC grade water and was used for estimation of protein amino acids in HPLC (Huesgen 1999; Sonawane et al. 2015). Protein quality was evaluated by the essential amino acid index (EAAI), which is based on the content of all essential amino acids compared to a reference protein and human requirements as mentioned in FAO/WHO/UNU standard (2007). The EAAI is a rapid method to evaluate and optimize the amino acid content of food formulations (Smith and Nielsen 2010). CP protein concentrate by TPP was comparatively evaluated with literature data on best protein sources such as soy protein and casein. However, EAAI does not include any estimate of protein digestibility, which could be affected by processing method.
To study thermal properties, differential scanning calorimetry (DSC) analysis was done using DSC-60 (Shimadzu Scientific Instruments, Kyoto, Japan) instrument fitted with TA 60 WS detector and computer-aided data analysis. The empty sample and reference pans were taken of equal mass for analysis. 5.00 ± 0.25 mg of sample was weighed in the DSC pan, sealed hermetically and placed on the sample side. A hermetically sealed identical empty pan was placed on the reference side. Scans were performed between 30 and 200 °C at a heating rate of 5 °C/min with 70 mL/min nitrogen gas purging.
The significant difference between mean values was assessed by one-way analysis of variance (ANOVA). Tukey test was carried out using SPSS 16.0 software to determine whether there was any significant difference at the level of p < 0.05.
Results and discussion
TPP of Chlorella pyrenoidosa cell lysate for protein concentration
Effect of solvent on protein concentration
Effect of ammonium sulphate on protein concentration
Effect of solid load on protein concentration
Effect of pH on protein concentration
Effect of incubation time on protein concentration
Effect of slurry to butanol ratio on protein concentration
Effect of enzymatic treatment on protein concentration
Effect of enzymatic treatment on starch, cellulose and total carbohydrate content in protein concentrate (n = 3)
Starch (% w/w)
Cellulose (% w/w)
Total carbohydrate (% w/w)
5.30 ± 0.52
10.16 ± 1.11
22.43 ± 0.94
2.10 ± 0.25
10.13 ± 1.00
19.91 ± 0.80
5.03 ± 0.45
1.40 ± 0.41
16.06 ± 1.30
Stargen™ + carezyme™
2.20 ± 0.20
1.23 ± 0.40
12.80 ± 0.83
Characterization of protein concentrate
Proximate composition of dried biomass and protein concentrate
Physiochemical characterization of dried biomass and protein concentrate (n = 3)
Dried biomass (% w/w)
Protein concentrate (% w/w)
6.42 ± 0.34
4.43 ± 0.02
4.56 ± 0.26
4.10 ± 0.13
45.02 ± 2.73
78.30 ± 0.93
22.89 ± 1.78
4.12 ± 0.12
Carbohydrate (by difference)
Carbohydrate (by anthrone method)
23.32 ± 1.05
12.80 ± 0.83
Water-holding capacity (WHC)
3.09 ± 0.01
Oil-holding capacity (OHC)
2.02 ± 0.04
Foaming capacity (FC)
95.00 ± 1.14
Foaming stability (FS)
97.45 ± 0.46
T 0 (°C)
43.29 ± 1.91
T d (°C)
60.10 ± 2.43
Colour analysis of dried biomass and protein concentrate (n = 6)
28.26 ± 0.05
34.78 ± 0.02
−0.48 ± 0.38
0.07 ± 0.01
1.07 ± 0.03
4.72 ± 0.03
Water-holding capacity and oil-holding capacity
Water-holding capacity of protein concentrate can be explained by the presence of polar amino acid and lower amounts of water-soluble protein. WHC of the optimized protein concentrate was 3.1 g/mL. The presence of non-proteinaceous material in protein concentrate also contributes WHC (Kaur and Singh 2007). The OHC is of great importance from an industrial viewpoint since it reflects the emulsifying capacity, a highly desirable characteristic in products such as mayonnaise. The presence of several non-polar side chains may bind the hydrocarbon chains of fats, thereby resulting in higher absorption of oil (Kaur and Singh 2007). OHC of protein concentrate was 2.1 g/mL which is within the range previously published for various protein concentrate of 1.3–4.1 g/mL. High OHC is desirable for food formulation (Kaur and Singh 2007).
Foaming capacity (FC) and foaming stability (FS)
Foamability is linked with flexible protein molecules that reduce the surface tension which keeps air bubbles in suspension and slows down the rate of coalescence. Globular proteins are relatively stable to surface denaturation which gives low foam. Previously, foaming capacity of winged bean protein concentrate (36 %), soya isolates (235 %) and mucuna bean protein concentrate (58 %) were reported. In this study, the microalgae protein concentrate had an FC of 95 %. FS is important since the usefulness of whipping agents depends on their ability to maintain the whip as long as possible. In the case of microalgae protein concentrate, foaming stability was 97 % after 180 min of storage. This excellent FS suggests that the native proteins that were soluble in the continuous phase (water) were very surface-active in microalgae proteins (Kaur and Singh 2007).
Amino acid composition and protein quality
Composition of amino acids in protein concentrate
Protein concentrate (g/100 g of protein)
8.12 ± 0.16
7.87 ± 0.23
2.79 ± 0.03
1.64 ± 0.01
9.73 ± 0.42
3.45 ± 0.04
5.91 ± 0.07
5.08 ± 0.19
1.22 ± 0.01
3.83 ± 0.11
5.17 ± 0.05
3.30 ± 0.02
2.82 ± 0.06
6.20 ± 0.14
3.44 ± 0.06
8.14 ± 0.37
Comparative study of amino acid pattern in CP protein concentrate, soya protein and Casein protein with FAO/WHO/UNU standard and calculation of EAAI
Essential amino acids (EAA)
CP protein concentrate
(Bertol et al. 2001)
(Han et al. 2015)
FAO/WHO/UNU standard (2007)
Microalgae have attracted the attention of the food industry mainly because of the need for valuable, sustainable, unconventional, non-climate-dependent protein source for food and feed. Chlorella species have a great potential to be used as an alternative valuable protein source for humans, especially CP. TPP is an interesting method because of its ability to separate proteins from lipids simultaneously and because it is scalable and an economical process to obtain high protein yields. In this study, it was shown that process parameters such as solvent, ammonium sulphate, solid load, pH, incubation time and slurry to butanol ratio were important in TPP for protein concentration. Enzymatically treated cell lysate of CP increased protein concentration of protein concentrate. Using optimized TPP parameters and enzymatic treatment, the protein concentration was 78.1 % w/w. Characterization of this protein concentrate revealed that it was a high-quality protein for human consumption because of its high essential amino acid content. TPP concentrated protein was less intense in colour and also foaming stability was excellent. Thus, TPP concentrated proteins could be easily used in techno-functional applications in pharmaceutical, food and chemical industries. Animal trials and other in vivo studies of the protein concentrate of CP obtained by TPP concentration must be performed to ensure safety and digestibility before proposing its introduction in the food chain of humans and other animals.
high-pressure liquid chromatography
essential amino acid index
differential scanning calorimetry
AGW have conducted an experiment, analysed results and prepared the manuscript. MKS have analysed samples for protein quantification by Kjeldahl method. JGL have provided technical support and assisted in manuscript drafting. SSA have supervised and guided in experimental work as well as in writing manuscript. All authors read and approved the final manuscript.
The authors would like to gratefully acknowledge the UGC-SAP, Government of India for their financial support.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- AOAC (Association of Official Analytical Chemists) (2001) Official methods of analysis, 16th edn. Washington
- Barbarino E, Lourenço SO (2005) An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. J Appl Phycol 17:447–460View ArticleGoogle Scholar
- Becker EW (2007) Micro-algae as a source of protein. Biotechnol Adv 25(2):207–210View ArticleGoogle Scholar
- Bertol TM, Moraes N, Franke MR (2001) Partial substitution of soybean meal by texturized soybean protein in diets for weaned piglets. J Anim Sci 30:141–149Google Scholar
- Bishop WM, Zubeck HM (2012) Evaluation of microalgae for use as nutraceuticals and nutritional supplements. J Nutr Food Sci 2:5View ArticleGoogle Scholar
- Cano-Medina A, Jimenez-Islas H, Dendooven L, Herrera RP, Gonzalez-Alatorre G, Escamilla-Silva EM (2011) Emulsifying and foaming capacity and emulsion and foam stability of sesame protein concentrates. Food Res Int 44(3):684–692View ArticleGoogle Scholar
- Chacon-Lee TL, Gonzalez-Marino GE (2010) Microalgae for “healthy” foods-possibilities and challenges. Compr Rev Food Sci F 9:655–675View ArticleGoogle Scholar
- Cherng JY, Shih MF (2005) Potential hypoglycemic effects of Chlorella in streptozotocin-induced diabetic mice. Life Sci 77:980–990View ArticleGoogle Scholar
- Dhananjay SK, Mulimani VH (2009) Three-phase partitioning of α-galactosidase from fermented media of Aspergillus oryzae and comparison with conventional purification techniques. J Ind Microbiol Biotechnol 36:123–128View ArticleGoogle Scholar
- Eskilsson CS, Bjorklund E, Karlsson L, Torstensson A (1999) Microwave-assisted extraction of felodipine tablets. J Chromatogr A 1:59–70View ArticleGoogle Scholar
- FAO/WHO/UNU (2007) Protein and amino acid requirements in human nutrition. WHO, pp 150 http://whqlibdoc.who.int/trs/WHO_TRS_935_eng.pdf
- Gao K (1998) Chinese studies on the edible blue-green alga, Nostoc flagelliforme—A review. J Appl Phycol 10:37–49View ArticleGoogle Scholar
- Garcia JMR, Fernandez FGA, Sevilla JMF (2012) Development of a process for the production of l-amino-acids concentrates from microalgae by enzymatic hydrolysis. Bioresource Technol 112:164–170View ArticleGoogle Scholar
- Garg R, Thorat BN (2014) Nattokinase purification by three phase partitioning and impact of t-butanol on freeze drying. Sep Purif Technol 131:19–26View ArticleGoogle Scholar
- GE Healthcare Bio-Sciences (2010) Strategies for protein purification. Amersham Biosciences, Uppsala. http://proteins.gelifesciences.com
- Ghribi AM, Gafsi IM, Blecker C, Danthine S, Attia H, Besbes S (2015) Effect of drying methods on physicochemical and functional properties of chickpea protein concentrates. J Food Eng 165:179–188View ArticleGoogle Scholar
- Halperin SA, Smith B, Nolan C, Shay J, Kralovec J (2003) Safety and immunoenhancing effect of a Chlorella-derived dietary supplement in healthy adults undergoing influenza vaccination: randomized, double-blind, placebo-controlled trial. Can Med Assoc J 169:111–117Google Scholar
- Han SW, Chee KM, Cho SJ (2015) Nutritional quality of rice bran protein in comparison to animal and vegetable protein. Food Chem 172:766–769View ArticleGoogle Scholar
- Harde SM, Singhal RS (2012) Extraction of forskolin from Coleus forskohlii roots using three phase partitioning. Sep Purif Technol 96:20–25View ArticleGoogle Scholar
- Huddleston J, Veide A, Kohlez K, Flanagan J, Enfors SO, Lyddiatt A (1991) The molecular basis of partitioning in ATPS: review. Trends Biotechnol 9:381–388View ArticleGoogle Scholar
- Huesgen AG (1999) Sensitive and reliable amino acid analysis in protein hydrolysates using the HP 1100 Series HPLC, Hewlett-Packard, Technical Note
- Ibanoglu E (2005) Effect of hydrocolloids on the thermal denaturation of proteins. Food Chem 90:621–626View ArticleGoogle Scholar
- Jones OG (2016) Recent advances in the functionality of non-animal-sourced proteins contributing to their use in meat analogs. Curr Opin Food Sci 7:7–13View ArticleGoogle Scholar
- Kaur M, Singh N (2007) Characterization of protein isolates from different Indian chickpea (Cicer arietinum L.) cultivars. Food Chem 102(1):366–374View ArticleGoogle Scholar
- Ketnawa S, Benjakul S, Martínez-Alvarez O, Rawdkuen S (2014) Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera. Sep Purif Technol 132:174–181View ArticleGoogle Scholar
- Lee SK, Fox HM, Kies C, Dam R (1967) The supplementary value of algae protein in human diets. J Nutr 92:281–285Google Scholar
- Lisboa CR, Pereira AM, Ferreira SP, Costa JA (2014) Utilisation of Spirulina sp. and Chlorella pyrenoidosa biomass for the production of enzymatic protein hydrolysates. Int J Eng Res Appl 4(5):29–38Google Scholar
- Lopez CVG, Garcia MCC, Fernandez FGA, Bustos CS, Chisti Y, Sevilla JMF (2010) Protein measurements of micro-algal and cyanobacterial biomass. Bioresoure Technol 101:7587–7591View ArticleGoogle Scholar
- Lourenco SO, Barbarino E, Lavin PL, Lanfer Marquez UM, Aidar E (2004) Distribution of intracellular nitrogen in marine microalgae: calculation of new nitrogen-to-protein conversion factors. Eur J Phycol 39(1):17–32View ArticleGoogle Scholar
- Merchant RE, Andre CA (2001) A review of recent clinical trials of the nutritional supplement Chlorella pyrenoidosa in the treatment of fibromyalgia, hypertension, and ulcerative colitis. Altern Ther Health Med 7:79–91Google Scholar
- Merchant RE, Andre CA, Sica DA (2002) Nutritional supplementation with Chlorella pyrenoidosa for mild to moderate hypertension. J Med Food 5:141–152View ArticleGoogle Scholar
- Mizoguchi T, Takehara I, Masuzawa T, Saito T, Naoki Y (2008) Nutrigenomic studies of effects of Chlorella on subjects with high-risk factors for lifestyle-related disease. J Med Food 3:395–404View ArticleGoogle Scholar
- Narayan AV, Madhusudhan MC, Raghavarao KS (2008) Extraction and purification of ipomoea peroxidase employing three phase partitioning. Appl Biochem Biotechnol 151:263–272View ArticleGoogle Scholar
- Ozer B, Akardere E, Çelem EB, Onal S (2010) Three-phase partitioning as a rapid and efficient method for purification of invertase from tomato. Biochem Eng J 50:110–115View ArticleGoogle Scholar
- Persson JA, Wennerholm M, O’Halloran S (2008) Handbook for kjeldahl digestion, FOSS, DK-3400 Hilleroed, Denmark
- Phongthai S, Rawdkuen S (2015) Preparation of rice bran protein isolates using three-phase partitioning and its properties. Food Appl Biosci J 3(2):137–149Google Scholar
- Rawdkuen S, Vanabun A, Benjakul S (2012) Recovery of proteases from the viscera of farmed giant catfish (Pangasianodon gigas) by three-phase partitioning. Process Biochem 47:2566–2569View ArticleGoogle Scholar
- Richmond A (1990) Handbook of micro-algal mass culture. CRC Press, Boca RatonGoogle Scholar
- Roe S (2000) Protein purification techniques: a practical approach, 2nd edn. Oxford University Press, OxfordGoogle Scholar
- Safi C, Liu DZ, Yap BHJ, Martin GJO, Vaca-Garcia C, Pontalier PY (2014a) A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption. J Appl Phycol 26(6):2379–2387View ArticleGoogle Scholar
- Safi C, Ursu AV, Laroche C, Zebib B, Merah O, Pontalier PY, Vaca-Garcia C (2014b) Aqueous extraction of proteins from microalgae: effect of different cell disruption methods. Algal Res 3:61–65View ArticleGoogle Scholar
- Şen A, Eryılmaz M, Bayraktar H, Onal S (2011) Purification of α-galactosidase from pepino (Solanum muricatum) by three-phase partitioning. Sep Purif Technol 83:130–136View ArticleGoogle Scholar
- Smith DM, Nielsen SS (2010) Protein separation and characterization procedures. In: Heldman DR (ed) Food analysis. Springer, Berlin, pp 261–281View ArticleGoogle Scholar
- Sonawane SK, Bagul MB, LeBlanc JG, Arya SS (2015) Nutritional, functional, thermal and structural characteristics of Citrullus lanatus and Limonia acidissima seed flours. J Food Meas Ch. doi:10.1007/s11694-015-9278-8 Google Scholar
- Takeshita T, Ota S, Yamazaki T, Hirata A, Zachleder V, Kawano S (2014) Starch and lipid accumulation in eight strains of six Chlorella species under comparatively high light intensity and aeration culture conditions. Bioresoure Technol 158:127–134View ArticleGoogle Scholar
- Tana ZJ, Wanga CY, Yi YJ, Wanga HY, Zhoua WL, Tana SY, Li FF (2015) Three phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-step extraction. Process Biochem 50:482–486View ArticleGoogle Scholar
- Thayumanavan B, Sadasivam S (1984) Physicochemical basis for the preferential uses of certain rice varieties. Plant Food Hum Nutr 34:253–259View ArticleGoogle Scholar
- Ursu AV, Marcati A, Sayd T, Sante-Lhoutellier V, Djelveh G, Michaud P (2014) Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresource Technol 157:134–139View ArticleGoogle Scholar
- Ustunol Z (2015) Applied food protein chemistry. John Wiley & Sons Ltd, ChichesterGoogle Scholar
- Vetal MD, Rathod VK (2014) Three phase partitioning a novel technique for purification of peroxidase from orange peels (Citrus sinenses). Food Bioprod Process 487:6Google Scholar
- Viegas CV, Hachemi I, Maki-Arvela P, Smeds A, Aho A, Murzin DY (2015) Algal products beyond lipids: comprehensive characterization of different products in direct saponification of green alga Chlorella sp. Algal Res 11:156–164View ArticleGoogle Scholar
- Vinoth Kumar V, Premkumar MP, Sathyaselvabala VK, Dineshkirupha S, Nandagopal J, Sivanesan S (2011) Aspergillus nigerexo-inulinase purification by three phase partitioning. Eng Life Sci 11:607–614View ArticleGoogle Scholar
- Walsh G (2007) Pharmaceutical biotechnology, Concept and applications. Wiley, ChichesterGoogle Scholar
- Wang X, Zhang X (2012) Optimal extraction and hydrolysis of Chlorella pyrenoidosa proteins. Bioresource Technol 126:307–313View ArticleGoogle Scholar
- Yi L, Lakemond CMM, Sagis LMC, Eisner-Schadler V, van Huis A, van Boekel MAJS (2013) Extraction and characterisation of protein fractions from five insect species. Food Chem 141(4):3341–3348View ArticleGoogle Scholar