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Comparison of Extraction Assays and Quantification of Protein from Ulva anandii (Cholorophycota)
Abstract
Seaweeds contain many macronutrients including protein, therefore they can be utilized to fulfil the protein requirements of human beings. This research focused on extracting total protein in green seaweed Ulva anandii (Amjad et Shameel 1993), from the crude extracts, by using the trichloroacetic acid (TCA) and acetone precipitation methods, and the estimation of crude extract (water-soluble proteins), and those obtained from the two above-mentioned methods. The results indicate that the water-soluble proteins had the highest quantity (949.75µg/mL) followed by the TCA precipitation method (831µg/mL), while the acetone precipitation method had the least concentration of total protein (100 µg/mL). The study concludes that treatment with organic solvents lowers the quantity of protein extracted from U. anandii.
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Seaweed is considered a potentially sustainable source of protein for human consumption, and rapid, accurate methods for determining seaweed protein contents are needed. Seaweeds contain substances which interfere with common protein estimation methods however. The present study compares the Lowry and BCA protein assays and protein determination by N-ratios to more novel spectroscopic methods. Linear regression of the height or the integrated area under the Amide II band of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) was used to predict seaweed protein with good prediction performance. Partial least squares regression (PLSR) was performed on both DRIFTS and near-infrared (NIR) spectra, with even higher prediction accuracy. Spectroscopy performed similar to or better than the calculated N-ratio of 4.14 for protein prediction. These spectral prediction methods require minimal sample preparation and chemical use, and are easy to perform, making them environmentally sustainable and economically viable for rapid estimation of seaweed protein.
The global population is expected to reach 11 billion people by the year 2100 and will require sustainable sources of dietary protein. Most dietary protein originates from animal and terrestrial plant agriculture which lead to deforestation, water pollution, and greenhouse gas emissions. Discovering alternative protein sources that are nutritionally adequate for the human diet without harmful environmental effects is imperative. Seaweeds are a promising option as they produce abundant protein with a low carbon-footprint. Experimental evidence shows that seaweeds contain high concentrations of the essential amino acids (EAAs) necessary for human consumption, but seaweeds have yet to be evaluated with standardized metrics to compare their nutritional value to other protein sources. In this technical note, independent literature describing the EAA content and protein digestibility of three commonly consumed species of seaweeds were evaluated alongside traditional protein sources using a novel hybrid protein quality (HPQ) metric. HPQ is derived from the protein digestibility-corrected amino acid score (PDCAAS) and digestibility indispensable amino acid score (DIAAS) but includes modifications to address the lack of in vivo digestibility data for seaweeds. Seaweed proteins are similar in quality to common plant protein sources such as peas, soy, and tree nuts. Furthermore, seaweed proteins from different species have complementary EAA profiles and can be mixed to form protein blends that are nutritionally on-par with animal products such as milk and whey. Thus, seaweeds may be viable protein sources with a reduced footprint that provide beneficial ecosystem services.
Protein is one of the major macronutrients essential in human nutrition. Protein sources especially animal sourced proteins are expensive, thus much work has been carried out to explore alternative protein sources. Seaweeds, or macroalgae, are emerging as one of the alternative protein sources. They are rich in protein with an excellent amino acid profile comparable to the other conventional protein sources. Seaweed protein contains bioactive components, such as free amino acids, peptides, lectins, and phycobiliproteins, including phycoerythrin and phycocyanin, among others. Seaweed proteins have been proved for their antihypertensive, antidiabetic, antioxidant, anti-inflammatory, antitumoral, antiviral, antimicrobial, and many other beneficial functional properties. Therefore, seaweed proteins can be a natural alternative source for functional food development. This paper discusses the compositional and nutritional aspects of seaweed protein, protein extraction techniques, functional properties of various seaweed proteins, as well as their safety for new product development and functional food applications.
Seaweeds, as a dietary protein source originated in Asian countries and later expanded towards France, Chile, etc. Food applications have been narrowed down due to complications in extraction. Therefore, products are not yet available in the market. Extraction of other phytochemicals along with seaweed proteins provides value addition in food products. Therefore, a trend has emerged to extract protein from edible seaweeds with many health beneficial applications. Also, consumption of many animal proteins like meat are now becoming a threat on humans due to infectious viral diseases. Hence, seaweed proteins are emerging as a global alternative source of protein.
Seaweeds are a rich source of protein and can contain up to 47% on the dry weight basis. It is challenging to extract proteins from the raw biomass of seaweed due to resilient cell-wall complexes. Four species of macroalgae were used in this study-two brown, Fucus vesiculosus and Alaria esculenta, and two red, Palmaria palmata and Chondrus crispus. Three treatments were applied individually to the macroalgal species: (I) high-pressure processing (HPP); (II) laboratory autoclave processing and (III) a classical sonication and salting out method. The protein, ash and lipid contents of the resulting extracts were estimated. Yields of protein recovered ranged from 3.2% for Fucus vesiculosus pre-treated with high pressure processing to 28.9% protein recovered for Chondrus crispus treated with the classical method. The yields of protein recovered using the classical, HPP and autoclave pre-treatments applied to Fucus vesiculosus were 35.1, 23.7% and 24.3%, respectively; yields from Alaria esculenta were 18.2%, 15.0% and 17.1% respectively; yields from Palmaria palmata were 12.5%, 14.9% and 21.5% respectively, and finally, yields from Chondrus crispus were 35.2%, 16.1% and 21.9%, respectively. These results demonstrate that while macroalgal proteins may be extracted using either physical or enzymatic methods, the specific extraction procedure should be tailored to individual species.
The demand for vegetable proteins increases globally and seaweeds are considered novel and promising protein sources. However, the tough polysaccharide-rich cell walls and the abundance of polyphenols reduce the extractability and digestibility of seaweed proteins. Therefore, food grade, scalable, and environmentally friendly protein extraction techniques are required. To date, little work has been carried out on developing such methods taking into consideration the structural differences between seaweed species. In this work, three different protein extraction methods were applied to three Swedish seaweeds (Porphyra umbilicalis, Ulva lactuca, and Saccharina latissima). These methods included (I) a traditional method using sonication in water and subsequent ammonium sulfate-induced protein precipitation, (II) the pH-shift protein extraction method using alkaline protein solubilization followed by isoelectric precipitation, and (III) the accelerated solvent extraction (ASE®) method where proteins are extracted after pre-removal of lipids and phlorotannins. The highest protein yields were achieved using the pH-shift method applied to P. umbilicalis (22.6 ± 7.3%) and S. latissima (25.1 ± 0.9%). The traditional method resulted in the greatest protein yield when applied to U. lactuca (19.6 ± 0.8%). However, the protein concentration in the produced extracts was highest for all three species using the pH-shift method (71.0 ± 3.7%, 51.2 ± 2.1%, and 40.7 ± 0.5% for P. umbilicalis, U. lactuca, and S. latissima, respectively). In addition, the pH-shift method was found to concentrate the fatty acids in U. lactuca and S. latissima by 2.2 and 1.6 times, respectively. The pH-shift method can therefore be considered a promising strategy for producing seaweed protein ingredients for use in food and feed.
Seaweeds are well recognized as a potential protein source. The edible green seaweed, Ulva sp., is abundant in the Brittany Coast, France. This study examined the extraction of proteins and glycoproteins from this seaweed. Four different extraction procedures (Procedure 1: deionized water, DW; Procedure 2: lysis solution 1 (LS1) containing 8 M urea, 2% Tween 20, 2% Triton X-100, 30 mM dithiothreitol, and 1% polyvinylpyrolidine; Procedure 3: lysis solution 2 (LS2) containing 50 mM Tris-HCl buffer pH 8, 10 mM EDTA, 2 mM Na2S2O5, and 1% Triton X-100; Procedure 4: 50 mM Tris-HCl buffer, pH 8) were applied to extract proteins from Ulva sp. in Brittany. The protein contents (%, dry basis) in the above extracts from Procedures 1–4 were 4.36 ± 0.21, 11.88 ± 0.23, 10.34 ± 0.35, and 3.58 ± 0.48, respectively. Moreover, electrophoresis (SDS-PAGE) revealed that the protein profile varies with season. Three glycoprotein-rich fractions, namely, GP-1 (from Procedure GP1), GP-2-DA, and GP-2-DS (from Procedure GP2), were extracted from Ulva sp. GP-1 and GP-2-DA fractions have a higher protein content than neutral sugars, while GP-2-DS contains a higher amount of neutral sugars than proteins. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) technique was applied to further proteomic analysis of each glycoprotein-rich fraction. GP-2-DS was hydrolyzed with protease enzyme to confirm the availability of proteins, and interestingly, the particular hydrolysate shows no original peaks in the MALDI-TOF/MS analysis. All three glycoprotein-rich fractions show no cytotoxicity in Vero cells at the tested concentration (500 mg dw mL⁻¹). Collectively, these results revealed that the extractable protein content and protein profile of Ulva sp. differ according to the extraction liquid system and the season. The utilization and value addition of proliferative Ulva sp. in Brittany as a protein source is promising but needs to consider the seasonal change of the protein profile.
Algae are a diverse group of aquatic autotrophs that represent important models for studying morphology, physiology, and molecular biology. Many, because of their adaptation to the marine environment, cell wall composition, and secondary metabolites, present difficulties when trying to extract significant amounts of protein for analyses. In this study, we compared protein extraction methods for 39 different species of marine algae and cyanobacteria. Since successful protein detection requires the liberation of protein from a cell, the goal of the current investigation was to establish a high yield protein extraction method that would allow for protein extraction from different organisms ranging from cyanobacteria, unicellular, multicellular, and coenocytic algae. Six protein extraction assays were tested (three from literature citations and three from commercially available kits). A modification of the technique originally described by Barbarino and Lourenço (J Appl Phycol 17:447–460, 2005) displayed significantly higher yields (p > 0.001, average of 6.7–30.87% dry weight) in all algal and cyanobacterial species examined. Protein yields using this modified procedure were especially successful in the Trebouxiophyceae, Bangiophyceae, Phaeophyceae, and Cyanobacteria.
It is crucial to determine several protein-related parameters at the initial stages of proteomic analysis of any biological samples. In this study, crude protein content, total soluble protein, total phenolic content and the SDS-PAGE profile of fifteen varieties of seaweed from Semporna, Sabah, Malaysia were analysed. The crude protein, total soluble protein and total phenolic content of all seaweed samples were in the range of 3.99 to 13.18 % of dry weight, 0.52 to 1.45 mg/mL in acetone dried powder samples and 8.59 to 48.98 mg PGE/g dry weight, respectively. In general, the differences (crude protein, total soluble protein and total phenolic content) among all fifteen varieties of seaweeds were significant (p<0.05). There was also a strong positive correlation between crude protein and total soluble protein concentration (Pearson's Correlation Coefficient (r)=0.923; p=0.01) in these fifteen varieties of seaweed. A distinctive protein pattern was observed in the SDS-PAGE gels between three different seaweed classes of green, red and brown colours. All of these results are important in sample preparations (extractions) before furthering proteomic analysis in order to identify and characterize seaweed proteomes.
A global drive to source additional and sustainable biomass for the production of protein has resulted in a renewed interest in the protein content of seaweeds. However, to determine accurately the potential of seaweeds as a source of protein requires reliable quantitative methods. This article systematically analysed the literature to assess the approaches and methods of protein determination and to provide an evidence-based conversion factor for nitrogen to protein that is specific to seaweeds. Almost 95 % of studies on seaweeds determined protein either by direct extraction procedures (42 % of all studies) or by applying an indirect nitrogen-to-protein conversion factor of 6.25 (52 % of all studies), with the latter as the most widely used method in the last 6 years. Meta-analysis of the true protein content, defined as the sum of the proteomic amino acids, demonstrated that direct extraction procedures underestimated protein content by 33 %, while the most commonly used indirect nitrogen-to-protein conversion factor of 6.25 over-estimated protein content by 43 %. We therefore determined whether a single nitrogen-to-protein conversion factor could be used for seaweeds and evaluated how robust this would be by analysing the variation in this factor for 103 species across 44 studies that span three phyla, multiple geographic regions and a range of nitrogen contents. An overall median nitrogen-to-protein conversion factor of 4.97 was established and an overall mean nitrogen-to-protein conversion factor of 4.76. We propose that the overall median value of 5 be used as the most accurate universal seaweed nitrogen-to-protein (SNP) conversion factor.
Seaweed is more than the wrap that keeps rice together in sushi. Seaweed biomass is already used for a wide range of other
products in food, including stabilising agents. Biorefineries with seaweed as feedstock are attracting worldwide interest
and include low-volume, high value-added products and vice versa. Scientific research on bioactive compounds in seaweed usually
takes place on just a few species and compounds. This paper reviews worldwide research on bioactive compounds, mainly of nine
genera or species of seaweed, which are also available in European temperate Atlantic waters, i.e. Laminaria sp., Fucus sp., Ascophyllum nodosum, Chondrus crispus, Porphyra sp., Ulva sp., Sargassum sp., Gracilaria sp. and Palmaria palmata. In addition, Undaria pinnatifida is included in this review as this is globally one of the most commonly produced, investigated and available species. Fewer
examples of other species abundant worldwide have also been included. This review will supply fundamental information for
biorefineries in Atlantic Europe using seaweed as feedstock. Preliminary selection of one or several candidate seaweed species
will be possible based on the summary tables and previous research described in this review. This applies either to the choice
of high value-added bioactive products to be exploited in an available species or to the choice of seaweed species when a
bioactive compound is desired. Data are presented in tables with species, effect and test organism (if present) with examples
of uses to enhance comparisons. In addition, scientific experiments performed on seaweed used as animal feed are presented,
and EU, US and Japanese legislation on functional foods is reviewed.
KeywordsHigh value-added products–Health promotion–Biorefinery–Nutraceutical–Pharmaceutical–Feed supplement
Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical methods employed.
The different extraction procedures (exposure to water, grinding, etc.), protein precipitation using different amounts of
25% trichloroacetic acid and quantification of protein by two different methods and using two protein standards were evaluated.
All procedures were tested using freeze-dried samples of three macroalgae: Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocol for protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure
water for 12 h, followed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should
be done with 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in this study
was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima, Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae (Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Comparison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry's method
should be used instead of Bradford's using bovine serum albumin (BSA) as protein standard instead of casein. This may be related
to the reactivity of the protein standards and the greater similarity in the amino acid composition of BSA and algae. The
current results should contribute to more accurate protein determinations in marine algae.
Two spectrophotometric assays for protein commonly used in marine research (Coomassie stain, Bradford; alkaline copper, Lowry) and a more recent assay which has not been applied in this field (bicinchoninic acid, Smith) were compared for homogenates of the marine diatom Thalassiosira pseudonona using bovine serum albumin (BSA) as a standard. When homogenates were prepared by precipitating protein with trichloroacetic acid (TCA) and redissolving in 1 N NaOH, the protein content estimated by the Lowry and Smith assays agreed closely, but was consistently 20% higher than that indicated by the Bradford assay. To determine if this difference was due to the choice of a protein standard, protein from T. pseudonana was purified and compared to BSA, bovine gamma-globulin (BGG), and casein. The reactivity of the purified protein (expressed as the slope of the absorbance vs protein concentration curve) did not differ between cultures grown at high or low irradiance. For the Smith and Bradford assays the reactivity of BSA was not significantly different from algal protein, but for the Lowry assay, algal protein was significantly higher in reactivity than BSA. BGG was not significantly different in reactivity from algal protein for the Lowry and Smith assays, but BGG gave significantly lower absorbances than algal protein in the Bradford assay. These results suggest that BSA is a suitable standard for algal protein in the Bradford assays, while BGG is preferable for the Lowry assay. Either protein standard could be used for the Smith assay. Differences in purified algal protein reactivity compared to BSA could not account for the differences among the assays, nor could interference by chlorophyll a. Precipitating protein with TCA prior to analyses gave lower protein than direct analyses of homogenates for the Lowry and Smith assays, but no differences were found for the Bradford assay. As a result, the Lowry and Smith assays indicated up to 60% greater protein than the Bradford if TCA precipitation was not performed. This may be due to removal of free amino acids and small peptides which are less reactive in the Bradford assay. The 20% higher protein found in the Lowry or Smith vs Bradford assays may be due to different assay sensitivity to small peptides or other compounds which are precipitated along with proteins by TCA. Although the Smith assay is substantially simpler to perform than the Lowry, there appear to be no quantitative differences in the results. It remains unclear which spectrophotometric assay is most accurate, but the Bradford assay is faster and simpler, and is less likely to be affected by non-protein compounds found in marine phytoplankton.
Seaweed is gaining attention as a sustainable source of protein. However, the nutritional protein quality needs further investigation to explore the potential of seaweed as a protein source. This study evaluates in vivo digestibility of six different seaweed protein products, four from Ulva sp. and two from Saccharina latissima. Ulva sp. was either preserved in a preservation liquid and spin flash-dried, freeze-dried without any pre-treatment or protein was extracted from the crude biomass with a screw press and isoelectrically precipitated (pH 2.5) with or without sulfite addition during extraction. Saccharina latissima was either freeze-dried or fermented with lactic acid bacteria prior to freeze-drying. The digestibility of the seaweed products was tested in a rat trial, where seaweed protein products constituted 50% of the nitrogen (N) in diets, the other 50% of N coming from whey protein concentrate. The protein concentration and proportion of essential amino acids (EAA) were increased upon protein extraction from Ulva sp. and the ash content was reduced. This led to an increased N digestibility of 55.5 ± 2.7% for the protein extract without sulfite, being 10–13%-points higher than for the other Ulva sp. products. The sulfite addition seemed to counteract the effect of extraction regarding the N digestibility. Fermentation of S. latissima increased the total amino acid content along with increased EAA proportion and lowered the ash content. However, the fermentation did not improve the organic matter, ash or N digestibility.
Digestibility was in general low, but this study demonstrates the potential to increase seaweed N digestibility by more than 20% through protein extraction.
To elucidate the properties of protein in seaweeds, protoplasts were used as a starting material. Protoplasts were isolated from 4 green seaweeds Ulva pertusa, U. fasciata, Enteromorpha linza, and Monostroma nitidum; from 2 browns Laminaria japonica and Undaria pinnatifida; and from 1 red Callymenia perforata. Proteins in protoplasts were then extracted with low (I=0.05) and high (I=0.5) ionic strength phosphate buffers (pH 7.5) and 0.1N sodium hydroxide, and analyzed for protein composition, SDS-polyacrylamide gel electrophoretic pattern, and amino acid composition.
Non-protein nitrogen was a major fraction (59.5% of the total nitrogen) in L. japonica. While water-soluble proteins were major fractions in green and red seaweeds, and these amounted to 30.5-69.5% of the total nitrogen. Salt-soluble protein was a predominant fraction (26.4% of the total nitrogen) only in U. pinnatifida. Electrophoretic patterns of the protein fractions consisted of many bands, and most of the patterns differed one another among species or protein fractions. Amino acid compositions were similar in all protein fractions except arginine in some protein fractions from U. fasciata, M. nitidwn, and C. perforata. Protein compositions and electrophoretic patterns differed much between protoplasts and thalli in all seaweeds.
Solvent precipitation is commonly used to purify protein samples, as seen with the removal of sodium dodecyl sulfate through acetone precipitation. However, in its current practice, protein loss is believed to be an inevitable consequence of acetone precipitation. We herein provide an in depth characterization of protein recovery through acetone precipitation. In 80% acetone, the precipitation efficiency for six of 10 protein standards was poor (ca. ≤15%). Poor recovery was also observed for proteome extracts, including bacterial and mammalian cells. As shown in this work, increasing the ionic strength of the solution dramatically improves the precipitation efficiency of individual proteins, and proteome mixtures (ca. 80-100% yield). This is obtained by including 1-30mM NaCl, together with acetone (50-80%) which maximizes protein precipitation efficiency. The amount of salt required to restore the recovery correlates with the amount of protein in the sample, as well as the intrinsic protein charge, and the dielectric strength of the solution. This synergistic approach to protein precipitation in acetone with salt is consistent with a model of ion pairing in organic solvent, and establishes an improved method to recover proteins and proteome mixtures in high yield.
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