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Article

Studies on the Impact of Selected Pretreatments on Protein Solubility of Arthrospira platensis Microalga

by
Maria P. Spínola
1,2,†,
Mónica M. Costa
1,2,† and
José A. M. Prates
1,2,*
1
CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal
2
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2023, 13(1), 221; https://doi.org/10.3390/agriculture13010221
Submission received: 11 December 2022 / Revised: 10 January 2023 / Accepted: 13 January 2023 / Published: 16 January 2023
(This article belongs to the Special Issue Evaluation of Feed Additives in Animal Production)
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Abstract

:
Arthrospira platensis has emerged as a novel protein feed source since it contains high protein level and quality. However, this microalga presents a recalcitrant cell wall and its main proteins form protein-pigment complexes attached to the thylakoid membrane. The objective of the present study was to evaluate the influence of mechanical/physical pretreatments (bead milling, extrusion, freeze-drying, heating, microwave and sonication) on A. platensis protein solubility. Total protein content and solubility were assessed by Bradford method and SDS-PAGE quantification. Protein degradation was assessed through quantification of protein fractions (18–26 kDa, 40–48 kDa and others) in SDS-PAGE gels. Peptide formation was evaluated using the o-phthaldialdehyde assay. The results showed a decrease in total protein content in the supernatant with extrusion (0.07 to 1.42 mg/mL) and microwave pretreatments, and in the pellet with extrusion. Therefore, extrusion, followed by microwave, was the most effective pretreatment for A. platensis proteins denaturation and solubility. It is suggested that the extrusion process cause an irreversible denaturation and aggregation of the major microalga proteins (c-phycocyanin and allophycocyanin), with a strong decrease in their solubility. Therefore, extrusion could increase the bioaccessibility of A. platensis proteins and enable the incorporation of this microalga at higher levels in monogastric diets.

1. Introduction

Microalgae have increased their potential as novel nutrient sources because of their richness in proteins (values can vary from 8% in Porphyridium cruentum to 70% in Arthrospira platensis on a dry weight basis [1]), linolenic acid, fibers, vitamins, some minerals, pigments and several bioactive compounds [2].
Arthrospira platensis is a filamentous cyanobacterium that is known for its richness in proteins [3] and has been used as animal feed in aquaculture [4,5], for farm animals [6,7] and for pets [8,9]. Despite its great protein value, A. platensis has a recalcitrant cell wall that interferes with bioavailability and accessibility of nutrients and, therefore, the application of some pretreatments is necessary to increase protein availability [2]. As a cyanobacteria, A. platensis principal protein-pigment complex, phycocyanin, forms complexes with other phycobiliproteins, which constitutes the phycobilisomes. These complexes, which are linked to the thylakoid membrane, are composed by hexameric discs of c-phycocyanin attached to trimers of allophycocyanin, which are the core of phycobilisomes, together with hexameric discs of phycoerythrin. c-phycocyanin and allophycocyanin have, respectively, two or three chromophores per monomer and these monomers form higher aggregated structures that are linked dependently on solvent conditions [10,11]. This complex microalga structure influences bioaccessibility and digestibility of A. platensis nutrients [12], especially proteins with high biological value. So, it is important to find out alternative methods to overcome this issue [3] and understand the effect of pretreatments on degradation of A. platensis cell wall.
Although there are various types of methods to disrupt the microalga cell wall in an attempt to improve nutrient accessibility (mechanical, physical, chemical and enzymatic), the mechanical and physical have been the most used for this purpose [13,14,15], with various reports describing the effect of some of them on the degradation of microalga proteins [1,16,17,18,19].
According to Krishnamoorthy, et al. [20], bead milling is commonly used for lipid extraction because of its effectiveness in alga cell disruption and easy operating procedures. This method damages the cells by applying forces that promote the collision between the beads and the cells, but the efficiency of this is closely related on the load and diameter of the beads [19,21]. Bead milling is usually used for improving the digestibility of microalgae in aquaculture [22]. Extrusion conditions, which include extreme temperature with high pressure, mixing and shear forces, can cause some damages in cell wall structure because it leads to physical/mechanical modification and disruption of biomass through passage in the extruder [23]. Wang, et al. [23] tested the extrusion method in Nannochloropsis oceanica with good results in cell disruption. Moreover, freeze-drying is recognized as an easy technique and is one of the most used pretreatments for lipid extraction because it helps solvent extraction [24]. One advantage of this pretreatment is the production of high valuable compounds without adverse effects on cellular constituents. Heating can be a good choice to increase the accessibility of algal proteins because of partial protein denaturation and the breakdown of proteins into peptides and amino acids that become more accessible for enzymes [25]. For microwave pretreatment, microalga cells are disrupted by an electromagnetic field, induced by microwave irradiation and heat generated by the combination of dielectric medium and polar molecules [19]. Sonication includes continuous compression and decompression of ultrasonic waves which provoke cavitation in the cell forming microbubbles that compress and collapse according to the intensity of the ultrasound [19]. These waves can cause cell wall disruption because of their high shear force [20].
The objective of this work was to assess and compare the effects of the most used physical and mechanical pretreatments (bead milling, extrusion, freeze-drying, heating, microwave and sonication) on A. platensis protein content and solubility.

2. Materials and Methods

2.1. Microalga Pretreatments

A. platensis dried powder obtained from Allmicroalgae Natural Products SA company (Pataias, Leiria, Portugal) was submitted to six different pretreatments following a specific protocol.
For bead milling, one bead was added per mL of microalga resuspension in 1 × phosphate buffered saline (PBS) buffer (BioWhittaker, Verviers, Belgium) at 20 mg/mL, and left under continuous rotation at 2000 rpm for 30 min in a shaker (Multi Reax Heidolph Instruments, Schwabach, Germany). Extrusion was done by Sparos company (Olhão, Algarve, Portugal) at 118 °C and 34 bars for 3 to 7 s with addition of water, 340 mL/min, and drying at 120 °C for 8–10 min. Then, microalga was resuspended with PBS buffer. The freeze-drying consisted in freezing A. platensis at −80 °C for 24 h and then freeze-drying it (Labogene, CoolSafe, Frilabo, Milheirós, Portugal) for another 24 h. In heating treatment, microalga stayed in dry heat (Melag, Geneststraße, Berlin, Germany) at 70 °C for 30 min. For microwave (Whirlpool, Household Microwave Oven, MI, USA) technique, alga suspension with PBS buffer was submitted to the keep warm mode until it boiled. Sonication (Bandelin ultrasonic homogenizer, Heinrichstraße, Berlin, Germany) conditions were 7 cycles at 70% power for 15 min with manual agitation of microalga suspension with PBS buffer at middle time. The chemical composition of non-treated A. platensis is presented in Table 1, which was conceded by Allmicroalgae company. The chemical composition was determined using routine and widespread methods [26]. Briefly, dry matter was analyzed by drying a sample at 105 °C to a constant weight. The nitrogen content for crude protein determination was obtained using the Kjeldahl method. Ash content was determined after burning the sample at 525 °C. Crude fat value was obtained with Soxhlet extraction using petroleum. Gross energy and carbohydrates were determined by standardized calculations. Phycocyanin was extracted following the procedures described by Ritchie [27] and were analyzed by high-performance liquid chromatography.

2.2. Incubation for A. platensis after Pretreatments

A. platensis resuspension in PBS buffer at 20 mg/mL was incubated overnight with each pretreatment, in triplicate, in an orbital shaker (Sanyo MIR-220RU Refrigerated, Shaking Incubator) at 37 °C and 160 rpm, in a 24-well plate (VWR Chemicals, West Chester, PA, USA) (Figure 1) [28]. After incubation, the plate was centrifuged at 1500× g, 15 min, 4 °C, followed by the recovery of 1 mL supernatant and the resuspension with 1 mL PBS buffer to recover the pellet to 1.5 mL microtubes.

2.3. Determination of Total Protein by Bradford Method

The Bradford method [29] was used to quantify solubilized total protein by spectrophotometry in the collected supernatant and pellet. Thus, 1.5 mL for Bradford solution (PanReac, AppliChem, ITW Reagents) was mixed with 30 μL of sample previously diluted in 1:3 factor with PBS buffer, and, after waiting 5 min at room temperature, the absorbance was read at 595 nm. A standard curve was done with bovine serum albumin at concentrations from 0.0125 to 1 mg/mL.

2.4. Electrophoretic Analysis of Proteins by SDS-PAGE

Microalga proteins, before and after pretreatments, were separated by 14% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). To evaluate protein fractions, 24 μL of each sample for supernatant or pellet (n = 5) was added to 6 μL of sample buffer, prepared according to Vizcaíno, et al. [30] but with some differences (prepared with 30% instead of 10% (v/v) β-mercaptoethanol). Then, the mixture was centrifuged, boiled for 5 min and added at 12 μL (9.00 µg of protein in each gel lane) into a 14% SDS-PAGE gel. The protein marker used was a low molecular weight marker (5 μL, about 1.95 µg of protein) with several protein fractions between 18.5 and 96 kDa (Nzytech, Lisbon, Portugal). Electrophoresis was done at a constant amperage of 12 mA per gel at 300 V (PowerPac Basic, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Then, the gels were colored with Coomassie brilliant blue and stayed overnight in distilled water before we processed the gel into an image using a software ChemiDoc XRS+ (Bio–Rad Laboratories, Inc., Hercules, CA, USA). The two most prominent protein fractions (18–26 kDa protein fraction 1, F1, and 40–48 kDa protein fraction 2, F2), other proteins and total protein, were quantified, in triplicate, by Image J software (NIH, Bethesda, MA, USA). The proportion of these fractions (F1, F2 and others) was also calculated relative to total protein.

2.5. Determination of Total Peptides by O-Phthaldialdehyde (OPA) Assay

The OPA spectrophotometric assay, used to measure free amino acids and peptides released from protein hydrolysis, was done according to Vizcaíno, et al. [30] and Sedighi, et al. [31], with some modifications. For each sample, 200 μL of each supernatant was mixed with 100 μL of 20% trichloroacetic acid for protein precipitation, followed by a centrifugation at 12,000× g for 15 min. Then, 200 μL of supernatant was added to 1 mL of OPA reagent, daily prepared according to Sedighi, et al. [31] and Church, et al. [32]. Blank was made with 200 μL distilled water, instead of supernatant sample. After incubation at room temperature for 5 min, the absorbance was read at 340 nm. A standard curve was done with peptone at ten concentrations between 5–500 μg/mL.

2.6. Statistical Analysis

Data were analysed using General Linear Models of SAS to perform analysis of variance (ANOVA) and the Tukey–Kramer method (PDIFF option) for multiple comparisons of adjusted least square means. The homogeneity of variances was tested using Levene’s test. Values were considered significant when p < 0.05.

3. Results

Table 2 shows the effect of pretreatments on the degradation of A. platensis biomass proteins in the supernatant fraction.
Total protein content (determined by the Bradford method) and total protein solubility (evaluated by SDS-PAGE gel quantification) significantly decreased (p < 0.001) with extrusion and microwave conditions. Concerning the protein quantification in the gel, only extrusion and microwave conditions caused a significant decrease (p < 0.001) in proteins with 18–26 kDa and other proteins. In addition, extrusion led to a significant (p < 0.001) decrease in proteins with 40–48 kDa, whereas an increase in this protein fraction was found with heating. However, no significant differences (p > 0.050) were found between pretreatments for the percentage of protein fractions. There was a significant decrease in the proportion of total protein (p = 0.005) and protein fractions with 18–26 kDa (p < 0.001) and 40–48 kDa (p < 0.001) relative to control (protein with pretreatments/protein with control; PTRAT/PCON) with extrusion and microwave pretreatments. Only extrusion led to significant decrease (p < 0.001) in peptide formation quantified by OPA assay.
Figure 2 presents representative images of SDS-PAGE gels (n = 3) for the effect of the pretreatments on algal proteins in the supernatant. It is possible to confirm the effect of extrusion on degradation of A. platensis proteins since the lanes concerning the influence of extrusion are clearly lighter than those with other pretreatments, indicating the disappearance of proteins.
The effect of pretreatments on A. platensis proteins in the pellet fraction is shown in Table 3, whereas the most representative SDS-PAGE gels (n = 3) are depicted in Figure 3. Only total protein content quantified by Bradford method decreased significantly (p < 0.001) with extrusion conditions compared to control. All the other variables were not significantly (p > 0.050) affected by treatments. Extrusion and sonication caused a decrease and increase, respectively, of the proportion of protein fraction with 18–26 kDa relative to the control assay.
Figure 3 shows no clear difference between the lanes of each pretreatment in the pellet fraction.

4. Discussion

The present results showed that most of the mechanical/physical pretreatments studied here (bead milling, freeze-drying, heating and sonication) did not affect protein amount and solubility. However, both extrusion and microwave pretreatments were effective on the reduction of solubility of A. platensis proteins, with significant decreases of total protein and proteins fractions with 18–26 kDa and 40–48 kDa. Nevertheless, the lower content of proteins (assessed by the Bradford method) in the pellet fraction of extruded microalga relative to the microwave-treated indicate a stronger impact of extrusion on protein denaturation and aggregation.
Extrusion conditions seem to drastically change A. platensis protein’s structure and solubility when these were submitted to the severe conditions of temperature and pressure of this mechanical/physical pretreatment. Visualizing the gels (Figure 2 and Figure 3), it is possible to see that the lanes referred to extrusion conditions in the supernatant fraction disappeared and are, instead, present in the pellet fraction, which seems to indicate a strong protein aggregation after applying this pretreatment, although there were no significant differences in the total protein quantified in SDS-PAGE gel (Table 3). In fact, Böcker, et al. [10] reported that c-phycocyanin and allophycocyanin, which correspond to protein fraction with 18–26 kDa, are sensitive to thermal processing, suffering modification in their protein structure and properties. Indeed, high temperatures can lead to the denaturation of proteins and the loss of secondary and tertiary protein structures. Such high temperatures were achieved, in the present study, with extrusion and microwave pretreatments, which can explain the reduction of total protein in the algal supernatant with both pretreatments.
The aggregation of algal proteins, an irreversible structure, with extrusion conditions, and to a lesser extent by microwave conditions, seems to promote their accessibility to peptidases, thus improving digestibility [33]. Carbonaro, et al. [33] attested that the improved digestibility of chickpea proteins, after their heat-induced denaturation, was due to the enhanced accessibility of susceptible sites to proteolysis of denaturized/aggregated proteins. Moreover, according to Wang, et al. [23], extrusion is a good method for improving the extraction of bioactive compounds from Nannochloropsis oceanica, increasing 74.3% of polyunsaturated fatty acids and 20.5% of essential amino acids in comparison with control. The authors observed a minimum decrease in protein yield with extrusion conditions, probably influenced by shear and heat conditions, which affected protein structure or even caused protein degradation. It seems that lower water content in extrusion conditions is beneficial for microalga cell disruption and consequently increasing soluble protein formation. Moreover, extrusion conditions tend to increase algal essential amino acids with improvement of protein solubility due to the degradation of proteins into amino acids. However, it is important to consider the possible occurrence of Maillard reaction, involving free protein amino groups and reducing the sugar carbonyl groups, which reduces amino acid availability [23]. Moreover, Arthrospira maxima extruded pellets led to high in vivo protein digestibility (up to 82.5%) when fed to fish [22].
Microwave pretreatment also had a significant effect on protein amount and solubility in previous studies. This pretreatment has been tested in some microalgae (Chlorella sp. and Scenedesmus), but specifically for lipid extraction without any focus on proteins [19,34,35,36]. Piasecka, et al. [35] analyzed the fatty acid profile of Chlorella protothecoides and concluded that microwave pretreatment increased the content of palmitic acid and modified the unsaturated/saturated fatty acids ratio. Viner, et al. [36] tested microwave radiation with distilled water in Scenedesmus sp., finding very good results with respect to the release of neutral lipids and free fatty acids. Only one study, so far, reported the effect of a microwave-assisted technique in protein extraction, showing that microwave pretreatment was a good method for disruption of Chlorella vulgaris cell wall with significant results on proteins recovery (increase in 2.54 times in comparison with three phase partitioning extraction) [37].
Although bead milling, in theory, is a good method for cell disruption, and possible to apply in a large-scale [21], it was not significantly efficient in the present study, which can be attributed to the characteristics of the beads. Freeze-drying conditions also did not cause any differences on total protein amount or solubility, although they have been tested for improvement of soluble proteins extraction with good results for C. vulgaris [38]. A study concerning the effect of bead milling and freeze-drying applied to microalga Nannochloropsis gaditana on apparent digestibility coefficients in fish showed increased digestibility values with bead milling but no differences with freeze-drying (75.6% and 59.8%, respectively, in comparison with control, 59.3%) [12].
Concerning heating pretreatment, Abbassi, et al. [39] tested five different temperatures (20, 40, 60, 80 and 100 °C) with two pressure levels (10 and 50 bar) and the results confirmed that the temperature–pressure combination has an effect on cell wall disruption. These results did not occur in our work, probably because of the low heating temperature (70 °C) that was not sufficient for protein denaturation and to decrease in its solubility [10,11]. Although, in the present study, no significant differences were found with sonication, possibly due to the conditions used (combination of time, power and cycles), this method has been successfully applied to disrupt cells of various microalga species; however, it was not effective when microalgae were in very high concentrations [21]. Martínez-Sanz, et al. [40] tested the influence of ultrasound in the extraction of proteins, carbohydrates and lipids of three different species of microalga, such as A. platensis, with production of 27% of proteins. Moreover, the authors concluded that sonication conditions promoted the release of intracellular compounds without damaging the cell wall integrity.

5. Conclusions and Future Perspectives

Data presented here indicate that the extrusion process, followed by the microwave methodology, was the pretreatment that mostly affected A. platensis proteins denaturation and solubility, especially for the main protein fractions (18–26 kDa and 40–48 kDa). Extrusion conditions seem to cause irreversible microalga protein denaturation and aggregation, with a strong decrease in their solubility, especially for c-phycocyanin and allophycocyanin. Therefore, it could be the most efficacious mechanical/physical pretreatment for improving A. platensis cell disruption and the bioaccessibility of proteins.
The other mechanical/physical pretreatments studied here (bead milling, freeze-drying, heating and sonication) did not affect A. platensis protein denaturation and solubility under these experimental conditions.
Further studies could elucidate the impact of other extrusion conditions, alone or in combination with other mechanical/physical methods, or even enzymatic pretreatments, on protein solubility and degradation. Moreover, considering the applicability of extrusion at an industrial scale, the effect of extrusion on the protein bioaccessibility of A. platensis in monogastric animals needs to be established. Therefore, in vivo trials with monogastric animals fed with high incorporation levels (up to 15–20% feed) of this extruded microalga are being performed by our team to test the effect of this pretreatment on A. platensis digestibility.

Author Contributions

Conceptualization, J.A.M.P.; data curation, M.P.S. and M.M.C.; formal analysis, M.P.S. and M.M.C.; investigation, M.P.S. and M.M.C.; writing—original draft preparation, M.P.S. and M.M.C.; writing—review and editing, J.A.M.P.; project administration, J.A.M.P.; funding acquisition, J.A.M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundação para a Ciência e a Tecnologia grants (Lisbon, Portugal; UI/BD/153071/2022 to M.P.S., UIDB/00276/2020 to CIISA and LA/P/0059/2020 to AL4AnimalS), and by Portugal2020 project (Lisbon, Portugal; P2020/17/SI/70114/2019 and associated researcher contract to M.M.C.).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Safi, C.; Charton, M.; Ursu, A.V.; Laroche, C.; Zebib, B.; Pontalier, P.-Y.; Vaca-Garcia, C. Release of hydro-soluble microalgal proteins using mechanical and chemical treatments. Algal Res. 2014, 3, 55–60. [Google Scholar] [CrossRef] [Green Version]
  2. Hayes, M.; Skomedal, H.; Skjånes, K.; Mazur-Marzec, H.; Toruńska-Sitarz, A.; Catala, M.; Isleten Hosoglu, M.; García-Vaquero, M. Microalgal proteins for feed, food and health. In Microalgae-Based Biofuels and Bioproducts; Gonzalez-Fernandez, C., Muñoz, R., Eds.; Woodhead Publishing: Sawston, UK, 2017; pp. 347–368. [Google Scholar]
  3. Spínola, M.P.; Costa, M.M.; Prates, J.A.M. Digestive Constraints of Arthrospira platensis in Poultry and Swine Feeding. Foods 2022, 11, 2984. [Google Scholar] [CrossRef] [PubMed]
  4. Palmegiano, G.B.; Agradi, E.; Forneris, G.; Gai, F.; Gasco, L.; Rigamonti, E.; Sicuro, B.; Zoccarato, I. Spirulina as a nutrient source in diets for growing sturgeon (Acipenser baeri). Aquac. Res. 2005, 36, 188–195. [Google Scholar] [CrossRef]
  5. Teimouri, M.; Amirkolaie, A.K.; Yeganeh, S. The effects of Spirulina platensis meal as a feed supplement on growth performance and pigmentation of rainbow trout (Oncorhynchus mykiss). Aquaculture 2013, 396-399, 14–19. [Google Scholar] [CrossRef]
  6. Martins, C.F.; Pestana Assunção, J.; Ribeiro Santos, D.M.; Madeira, M.S.M.d.S.; Alfaia, C.M.R.P.M.; Lopes, P.A.A.B.; Coelho, D.F.M.; Cardoso Lemos, J.P.; de Almeida, A.M.; Mestre Prates, J.A.; et al. Effect of dietary inclusion of Spirulina on production performance, nutrient digestibility and meat quality traits in post-weaning piglets. J. Anim. Physiol. Anim. Nutr. 2021, 105, 247–259. [Google Scholar] [CrossRef]
  7. Pestana, J.M.; Puerta, B.; Santos, H.; Madeira, M.S.; Alfaia, C.M.; Lopes, P.A.; Pinto, R.M.A.; Lemos, J.P.C.; Fontes, C.M.G.A.; Lordelo, M.M.; et al. Impact of dietary incorporation of Spirulina (Arthrospira platensis) and exogenous enzymes on broiler performance, carcass traits, and meat quality. Poult. Sci. 2020, 99, 2519–2532. [Google Scholar] [CrossRef]
  8. Beynen, A.C. Microalgae in petfood. Creat. Companion 2019, 40, 42. [Google Scholar]
  9. Satyaraj, E.; Reynolds, A.; Engler, R.; Labuda, J.; Sun, P. Supplementation of Diets With Spirulina Influences Immune and Gut Function in Dogs. Front. Nutr. 2021, 8, 667072. [Google Scholar] [CrossRef]
  10. Böcker, L.; Hostettler, T.; Diener, M.; Eder, S.; Demuth, T.; Adamcik, J.; Reineke, K.; Leeb, E.; Nyström, L.; Mathys, A. Time-temperature-resolved functional and structural changes of phycocyanin extracted from Arthrospira platensis/Spirulina. Food Chem. 2020, 316, 126374. [Google Scholar] [CrossRef]
  11. Buecker, S.; Grossmann, L.; Loeffler, M.; Leeb, E.; Weiss, J. Thermal and acidic denaturation of phycocyanin from Arthrospira platensis: Effects of complexation with λ-carrageenan on blue color stability. Food Chem. 2022, 380, 132157. [Google Scholar] [CrossRef]
  12. Agboola, J.O.; Teuling, E.; Wierenga, P.A.; Gruppen, H.; Schrama, J.W. Cell wall disruption: An effective strategy to improve the nutritive quality of microalgae in African catfish (Clarias gariepinus). Aquac. Nutr. 2019, 25, 783–797. [Google Scholar] [CrossRef]
  13. Middelberg, A.P.J. Process-scale disruption of microorganisms. Biotechnol. Adv. 1995, 13, 491–551. [Google Scholar] [CrossRef] [PubMed]
  14. Lee, A.K.; Lewis, D.M.; Ashman, P.J. Disruption of microalgal cells for the extraction of lipids for biofuels: Processes and specific energy requirements. Biomass Bioenergy 2012, 46, 89–101. [Google Scholar] [CrossRef]
  15. Sousa, C.V.d. Microalgas: Do Tratamento de Efluentes Para a Biorrefinaria. Master’s Thesis, Universidade Nova de Lisboa, Lisbon, Portugal, 2014. [Google Scholar]
  16. Ahmed, J.; Kumar, V. Effect of high-pressure treatment on oscillatory rheology, particle size distribution and microstructure of microalgae Chlorella vulgaris and Arthrospira platensis. Algal Res. 2022, 62, 102617. [Google Scholar] [CrossRef]
  17. Safi, C.; Ursu, A.V.; Laroche, C.; Zebib, B.; Merah, O.; Pontalier, P.-Y.; Vaca-Garcia, C. Aqueous extraction of proteins from microalgae: Effect of different cell disruption methods. Algal Res. 2014, 3, 61–65. [Google Scholar] [CrossRef] [Green Version]
  18. Soto-Sierra, L.; Stoykova, P.; Nikolov, Z.L. Extraction and fractionation of microalgae-based protein products. Algal Res. 2018, 36, 175–192. [Google Scholar] [CrossRef]
  19. Rahman, M.M.; Hosano, N.; Hosano, H. Recovering Microalgal Bioresources: A Review of Cell Disruption Methods and Extraction Technologies. Molecules 2022, 27, 2786. [Google Scholar] [CrossRef]
  20. Krishnamoorthy, A.; Rodriguez, C.; Durrant, A. Sustainable Approaches to Microalgal Pre-Treatment Techniques for Biodiesel Production: A Review. Sustainability 2022, 14, 9953. [Google Scholar] [CrossRef]
  21. Wang, M.; Chen, S.; Zhou, W.; Yuan, W.; Wang, D. Algal cell lysis by bacteria: A review and comparison to conventional methods. Algal Res. 2020, 46, 101794. [Google Scholar] [CrossRef]
  22. Annamalai, S.N.; Das, P.; Thaher, M.I.A.; Abdul Quadir, M.; Khan, S.; Mahata, C.; Al Jabri, H. Nutrients and Energy Digestibility of Microalgal Biomass for Fish Feed Applications. Sustainability 2021, 13, 13211. [Google Scholar] [CrossRef]
  23. Wang, M.; Cheng, H.; Chen, S.; Wen, S.; Wu, X.; Zhang, D.; Yuan, Q.; Cong, W. Microalgal cell disruption via extrusion for the production of intracellular valuables. Energy 2018, 142, 339–345. [Google Scholar] [CrossRef]
  24. Al Hattab, M.; Ghaly, A. Microalgae Oil Extraction Pre-treatment Methods: Critical Review and Comparative Analysis. J. Fundam. Renew. Energy Appl. 2015, 5, 4. [Google Scholar] [CrossRef] [Green Version]
  25. Meade, S.J.; Reid, E.A.; Gerrard, J.A. The impact of processing on the nutritional quality of food proteins. J. AOAC Int. 2005, 88, 904–922. [Google Scholar] [CrossRef] [PubMed]
  26. AOAC. Official Methods of Analysis, 17th ed.; Association of Official Analytical Chemists: Washington, DC, USA, 2000. [Google Scholar]
  27. Ritchie, R.J. Universal chlorophyll equations for estimating chlorophylls a, b, c, and d and total chlorophylls in natural assemblages of photosynthetic organisms using acetone, methanol, or ethanol solvents. Photosynthetica 2008, 46, 115–126. [Google Scholar] [CrossRef]
  28. Lopes, P.A.; Coelho, D.; Prates, J.A.M. Testimony on a successful lab protocol to disrupt Chlorella vulgaris microalga cell wall. PLoS ONE 2022, 17, e0268565. [Google Scholar] [CrossRef]
  29. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
  30. Vizcaíno, A.J.; Sáez, M.I.; Martínez, T.F.; Acién, F.G.; Alarcón, F.J. Differential hydrolysis of proteins of four microalgae by the digestive enzymes of gilthead sea bream and Senegalese sole. Algal Res. 2019, 37, 145–153. [Google Scholar] [CrossRef]
  31. Sedighi, M.; Jalili, H.; Darvish, M.; Sadeghi, S.; Ranaei-Siadat, S.-O. Enzymatic hydrolysis of microalgae proteins using serine proteases: A study to characterize kinetic parameters. Food Chem. 2019, 284, 334–339. [Google Scholar] [CrossRef]
  32. Church, F.C.; Swaisgood, H.E.; Porter, D.H.; Catignani, G.L. Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J. Dairy Sci. 1983, 66, 1219–1227. [Google Scholar] [CrossRef]
  33. Carbonaro, M.; Cappelloni, M.; Nicoli, S.; Lucarini, M.; Carnovale, E. Solubility−Digestibility Relationship of Legume Proteins. J. Agric. Food Chem. 1997, 45, 3387–3394. [Google Scholar] [CrossRef]
  34. Zheng, H.; Yin, J.; Gao, Z.; Huang, H.; Ji, X.; Dou, C. Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: A comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Appl. Biochem. Biotechnol. 2011, 164, 1215–1224. [Google Scholar] [CrossRef] [PubMed]
  35. Piasecka, A.; Krzemiñska, I.; Tys, J. Physical methods of microalgal biomass pretreatment. Int. Agrophys 2014, 28, 341–348. [Google Scholar] [CrossRef] [Green Version]
  36. Viner, K.J.; Champagne, P.; Jessop, P.G. Comparison of cell disruption techniques prior to lipid extraction from Scenedesmus sp. slurries for biodiesel production using liquid CO2. Green Chem. 2018, 20, 4330–4338. [Google Scholar] [CrossRef]
  37. Chew, K.W.; Chia, S.R.; Lee, S.Y.; Zhu, L.; Show, P.L. Enhanced microalgal protein extraction and purification using sustainable microwave-assisted multiphase partitioning technique. Chem. Eng. J. 2019, 367, 1–8. [Google Scholar] [CrossRef]
  38. Unterlander, N.; Champagne, P.; Plaxton, W.C. Lyophilization pretreatment facilitates extraction of soluble proteins and active enzymes from the oil-accumulating microalga Chlorella vulgaris. Algal Res. 2017, 25, 439–444. [Google Scholar] [CrossRef]
  39. Abbassi, A.; Ali, M.; Watson, I.A. Temperature dependency of cell wall destruction of microalgae with liquid nitrogen pretreatment and hydraulic pressing. Algal Res. 2014, 5, 190–194. [Google Scholar] [CrossRef]
  40. Martínez-Sanz, M.; Garrido-Fernández, A.; Mijlkovic, A.; Krona, A.; Martínez-Abad, A.; Coll-Marqués, J.M.; López-Rubio, A.; Lopez-Sanchez, P. Composition and rheological properties of microalgae suspensions: Impact of ultrasound processing. Algal Res. 2020, 49, 101960. [Google Scholar] [CrossRef]
Agriculture 13 00221 g001 550
Figure 1. Typical aspect of the in vitro incubation of Arthrospira platensis with pretreatments in a 24-well plate.
Figure 1. Typical aspect of the in vitro incubation of Arthrospira platensis with pretreatments in a 24-well plate.
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Agriculture 13 00221 g002 550
Figure 2. Images of SDS-PAGE gels of the supernatant fraction showing the effect of pretreatments on Arthrospira platensis protein amount and solubility after 24 h incubation (n = 3); LMW, low molecular eeight protein marker; BM, bead milling; ET, extrusion; HT, heating; FD, freeze-drying; MW, microwave; NoP, no pretreatment; SO, sonication.
Figure 2. Images of SDS-PAGE gels of the supernatant fraction showing the effect of pretreatments on Arthrospira platensis protein amount and solubility after 24 h incubation (n = 3); LMW, low molecular eeight protein marker; BM, bead milling; ET, extrusion; HT, heating; FD, freeze-drying; MW, microwave; NoP, no pretreatment; SO, sonication.
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Agriculture 13 00221 g003 550
Figure 3. Images of SDS-PAGE gels of the pellet fraction showing the effect of pretreatments on Arthrospira platensis protein amount and solubility after 24 h incubation (n = 3); LMW, low molecular weight protein marker; BM, bead milling; ET, extrusion; HT, heating; FD, freeze-drying; MW, microwave; NoP, no pretreatment; SO, sonication.
Figure 3. Images of SDS-PAGE gels of the pellet fraction showing the effect of pretreatments on Arthrospira platensis protein amount and solubility after 24 h incubation (n = 3); LMW, low molecular weight protein marker; BM, bead milling; ET, extrusion; HT, heating; FD, freeze-drying; MW, microwave; NoP, no pretreatment; SO, sonication.
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Table
Table 1. Chemical composition of Arthrospira platensis.
Table 1. Chemical composition of Arthrospira platensis.
Nutritional Composition
Energy (MJ/kg)13.9
Crude protein (% dry matter)62.6
Ash (% dry matter)14.9
Crude carbohydrates (% dry matter)6.06
Crude fibre (% dry matter)9.78
Crude fat (% dry matter)6.70
Pigment composition
Phycocyanin (% dry matter)11.2
Table
Table 2. Effect of pretreatments on the solubility of Arthrospira platensis biomass proteins in the supernatant fraction (n = 5) (Values are presented as mean ± standard deviation).
Table 2. Effect of pretreatments on the solubility of Arthrospira platensis biomass proteins in the supernatant fraction (n = 5) (Values are presented as mean ± standard deviation).
ItemPretreatments 1p-Value
NoPBMETFDHTMWSO
Total protein (mg/mL)
Bradford method1.42 ± 0.112 ab1.30 ± 0.061 b0.07 ± 0.060 c1.20 ± 0.105 b1.29 ± 0.125 b0.41 ± 0.054 c1.72 ± 0.418 a<0.001
   SDS-PAGE gel9.9 ± 1.58 a10.1 ± 0.20 a6.50 ± 1.035 b9.6 ± 0.99 a10.5 ± 1.61 a7.0 ± 0.32 b10.7 ± 0.54 a<0.001
Proteins (mg/mL) in SDS-PAGE gel
Proteins 18–26 kDa2.41 ± 0.066 ab2.35 ± 0.107 ab1.44 ± 0.176 c2.25 ± 0.030 b2.74 ± 0.315 a1.73 ± 0.256 c2.65 ± 0.244 a<0.001
Proteins 40–48 kDa1.71 ± 0.094 bc2.02 ± 0.205 ab1.19 ± 0.188 d1.94 ± 0.113 ab2.10± 0.273 a1.41 ± 0.057 cd2.00 ± 0.056 ab<0.001
   Other proteins5.74 ± 1.553 a5.76 ± 0.152 a3.87 ± 0.755 bc5.39 ± 0.909 abc5.61 ± 1.027 ab3.81 ± 0.327 c6.09 ± 0.588 a0.001
Proteins (% total protein) in SDS-PAGE gel
Proteins 18–26 kDa22.3 ± 5.22 ab23.2 ± 1.30 ab18.7 ± 2.07 b23.7 ± 2.49 ab26.4 ± 1.17 a24.9 ± 3.58 a24.7 ± 2.72 a0.011
Proteins 40–48 kDa17.7 ± 3.1519.9 ± 1.8518.3 ± 0.9520.3 ± 1.2520.2 ± 0.5620.3 ± 0.3618.6 ± 0.520.047
    Other proteins60.0 ± 7.46 ab56.8 ± 0.79 ab63.0 ± 1.48 a56.0 ± 3.64 ab53.5 ± 1.70 b54.9 ± 3.89 b56.6 ± 3.13 ab0.008
Proteins (PTRAT/PCON) in SDS-PAGE gel
    Total proteinnd1.05 ± 0.196 abc0.68 ± 0.207 c1.00 ± 0.206 abc1.07 ± 0.206 ab0.72 ± 0.135 b c1.12 ± 0.223 a0.005
Proteins 18–26 kDand1.18 ± 0.110 a0.69 ± 0.105 b1.13 ± 0.046 a1.23 ± 0.190 a0.82 ± 0.038 b1.17 ± 0.054 a<0.001
Proteins 40–48 kDand0.98 ± 0.028 ab0.60 ± 0.076 c0.93 ± 0.021 b1.13 ± 0.108 a0.72 ± 0.102 c1.10 ± 0.127 ab<0.001
    Other proteinsnd1.08 ± 0.3580.74 ± 0.3201.01 ± 0.3351.03 ± 0.3360.71 ± 0.2021.14 ± 0.4260.237
Total peptides (µg/mL)
   o-phthaldialdehyde assay42.7 ± 2.20 a50.4 ± 9.20 a24.6 ± 4.81 b44.4 ± 9.84 a38.5 ± 4.39 a39.0 ± 5.02 a43.4 ± 3.69 a<0.001
1 NoP: no pretreatment; BM, bead milling; ET, extrusion; FD, freeze-drying; HT, heating; MW, microwave; SO, sonication; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; PTRAT, protein obtained with pretreatments; PCON, protein obtained with control; a,b,c, different letters in the same line indicate statistically significant differences; no letters within lines indicate no statistically significant differences; nd, not detected.
Table
Table 3. Effect of pretreatments on the solubility of Arthrospira platensis biomass proteins in the pellet fraction (n = 5) (Values are presented as mean ± SD).
Table 3. Effect of pretreatments on the solubility of Arthrospira platensis biomass proteins in the pellet fraction (n = 5) (Values are presented as mean ± SD).
ItemPretreatments 1p-Value
NoPBMETFDHTMWSO
Total protein (mg/mL)
    Bradford method0.83 ± 0.094 bc1.13 ± 0.312 b0.03 ± 0.039 d0.71 ± 0.091 c0.67 ± 0.101 c1.13 ± 0.076 b2.36 ± 0.246 a<0.001
    SDS-PAGE gel11.5 ± 0.5912.9 ± 0.7913.8 ± 5.0711.5 ± 1.8511.3 ± 0.3113.6 ± 0.6410.2 ± 1.390.115
Protein quantification (mg/mL) by SDS-PAGE gel
    Proteins 18–26 kDa2.23 ± 0.0262.40 ± 0.0572.14 ± 0.2372.29 ± 0.1322.16 ± 0.0672.38 ± 0.0802.40 ± 0.3370.075
    Proteins 40–48 kDa1.95 ± 0.0322.14 ± 0.0881.97 ± 0.2551.98 ± 0.2641.96 ± 0.0452.20 ± 0.0902.19 ±0.4400.279
    Other proteins7.36 ± 0.5918.33 ± 0.6739.66 ± 5.0417.20 ± 1.4567.16 ± 0.2148.99 ± 0.6985.60 ± 2.0540.110
Protein proportion (% total) by SDS-PAGE gel
    Proteins 18–26 kDa19.3 ± 0.87 ab18.7 ± 0.80 ab16.7 ± 4.01 b20.3 ± 2.51 ab19.1 ± 0.15 ab17.6 ± 1.14 b24.2 ± 6.39 a0.020
    Proteins 40–48 kDa16.9 ± 0.6316.6 ± 0.2915.9 ± 0.5917.4 ± 1.2017.4 ± 5.5516.3 ± 1.0622.2 ± 7.310.129
    Other proteins63.7 ± 1.9164.7 ± 1.3767.3 ± 9.5362.4 ± 3.0963.5 ± 0.2266.2 ± 2.3053.6 ± 13.690.056
Protein proportion (PTRAT/PCON) by SDS-PAGE gel
    Total proteinnd1.11 ± 0.0371.21 ± 0.5050.99 ± 0.1260.98 ± 0.0711.18 ± 0.0830.88 ± 0.0930.169
    Proteins 18–26 kDand1.08 ± 0.0140.96 ± 0.1171.03 ± 0.0490.97 ± 0.0401.07 ± 0.0371.08 ± 0.1620.132
    Proteins 40–48 kDand1.10 ± 0.0511.01 ± 0.1401.02 ± 0.1441.00 ± 0.0211.13 ± 0.0521.12 ± 0.2140.385
    Other proteinsnd1.13 ± 0.0441.36 ± 0.8120.97 ± 0.1500.98 ± 0.1021.23 ± 0.1340.75 ± 0.2360.145
1 NoP: no pretreatment; BM, bead milling; ET, extrusion; FD, freeze-drying; HT, heating; MW, microwave; SO, sonication. SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; PTRAT, protein obtained with pretreatments; PCON, protein obtained with control; a,b,c, different letters in the same line represent means with statistically significant differences; no letters within lines indicate no statistically significant differences; nd, not detected.
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Spínola, M.P.; Costa, M.M.; Prates, J.A.M. Studies on the Impact of Selected Pretreatments on Protein Solubility of Arthrospira platensis Microalga. Agriculture 2023, 13, 221. https://doi.org/10.3390/agriculture13010221

AMA Style

Spínola MP, Costa MM, Prates JAM. Studies on the Impact of Selected Pretreatments on Protein Solubility of Arthrospira platensis Microalga. Agriculture. 2023; 13(1):221. https://doi.org/10.3390/agriculture13010221

Chicago/Turabian Style

Spínola, Maria P., Mónica M. Costa, and José A. M. Prates. 2023. "Studies on the Impact of Selected Pretreatments on Protein Solubility of Arthrospira platensis Microalga" Agriculture 13, no. 1: 221. https://doi.org/10.3390/agriculture13010221

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