Methods to protein and peptide extraction from microalgae: a systematic review

Abstract

Currently, there is a demand for protein sources that do not use soil management or animal breeding. Among these sources we highlight the microorganisms, such cyanobacteria and microalgae, which have a simple growth using light, CO₂, water and some mineral salts to generate high protein production. The extraction of these proteins depends on the method used. The most used methods for extracting bio-functional proteins are mechanical, chemical and enzymatic. The aim of this work is to analyze the protein extraction methods in microalgae using Scielo, ScienceDirect and NCBI (PubMed) electronic databases that made it possible to select original studies published in the last five years (2018-2023). A total of 2707 articles, 25 of which were selected for further analysis and subjected to risk of bias assessment. The genera Chlorella, Scenedesmus and Nannochloropsis were the most studied due to their high protein content. Mechanical methods and chemical hydrolysis are the most used methods, achieving an extraction yield of 46.0 % and 64.0 %, respectively. The best extraction results are obtained with a combination of methods, reaching up to 80.0 % yield. However, some aspects need to be observed to choose an ideal protein extraction method.

Key words Extraction methods; proteins; peptides; microalgae

INTRODUCTION

Proteins are abundant macromolecules present in living organisms with different functions and are important nutrients for human and animal welf. Due to the increasing population, nutritional demand (on average 0.75 g protein/kg/day), the growth in emerging sectors such as the vegan market, it’s necessary to find new protein sources without increasing the use of farming area (Gunerken et al. 2015, Grossmann et al. 2018, Washington DC 1989). One of the alternatives is the production of proteins from microorganisms, mainly those with more than 30 % of protein by g dry weight cells, which can provide a healthy balance of essential amino acids. Microbial protein is generally referred to as single cell protein (SCP), for example, yeasts, and more recently, photosynthetic microorganisms such as cyanobacteria and microalgae (Ritala et al. 2017). These microorganisms can accumulate about 40-50 % protein, values higher than traditional protein sources such as milk (26 %), soy (37 %), meat (43 %) (Soto-Sierra et al. 2018). Algae-based protein can be commercialized with suitable cost-effective growth medium, converting solar energy to chemical energy by fixing CO₂, and its efficiency is ten times higher than terrestrial plants (Sathasivam et al. 2019), can be grown regardless of the season, dispense with the use of pesticides; can be grown in wastewater/seawater water on non-arable land, minimizing environmental impacts, without compromising the production of food and other products derived from agricultural crops (Li et al. 2019).

At the same time, microalgae are capable of producing a wide variety of high-value bioactive compounds which can be used in the manufacturing of health foods, pharmaceutical products such as antiviral, antimicrobial, antitumor, anti-inflammatory, antidiabetic, antioxidant, and antihypertensive activities (Rizwan et al. 2018, Afify et al. 2018, Galarza 2019, Hu et al. 2019). However, the final product application should also be taken into consideration when selecting a suitable disruption technique. To extract the bio-functional proteins from microalgae while preserving their bioactivity and functionality, it is necessary to use a mild treatment. In this regard, A variety of mild cell disruption methods are currently available to rupture the cell wall of microalgae. These mild techniques are divided into three main groups, mechanic, chemical and enzymatic (Phong et al. 2018a). Currently, the most used methods for extracting bio-functional proteins are homogenization with glass beads or enzymatic hydrolysis (Bleakley & Hayes 2017, Safi et al. 2015, Jaeschke et al. 2019).

Mechanical methods are more general, have low selectivity and need more separation steps compared to chemical and enzymatic. On the other hand, chemical methods might change the protein structure and subsequently cause the loss of specific functionality or activity, formation of potentially toxic by-products and anticipate equipment maintenance by solubilize compounds, favoring the availability of organic molecules that can cause equipment oxidation, while that enzymatic are knowing by its high biological specificity, operate in gentle conditions, prevent in high electricity consumption, high shear stress or high pressures and no used toxic chemicals in comparison with as chemical and mechanics methods (Passos et al. 2014, Gunerken et al. 2015, Phong et al. 2018a). By the way, a good protein extraction technology should be easy to handle, require low energy demand, have high yield, low cost, use fewer toxic reagents environmentally safe and friendly, and residue-free techniques or residue nontoxic (Phong et al. 2018a).

Reviews about protein extraction from microalgae have been descriptors on various aspects comparing different traditional and advanced methods, challenges, and future trends for make possible an extraction and commercialization of bio-functional proteins, since it is extraction is one of the main problems industrial (Soto-Sierra et al. 2018, Bleakley & Hayes 2017, Phong et al. 2018a). It is the first systematic review on protein or peptide extraction methods from microalgae studies with different objectives and methodologies as their advantages and disadvantages.

MATERIALS AND METHODS

Search and identification of articles

Pubmed, Science direct and Scielo electronic databases were used to search original articles that are in line with the scope of the study. The descriptors used in the review process were: “extraction”, “peptides”, “proteins” and “microalgae”. In this work, we considered the term “microalgae” to be only eukaryotic photosynthetic microorganisms, being prokaryotic cyanobacteria excluded in this study.

The search was carried out in three stages: Analysis of titles, abstracts and full text according to the eligibility criteria; any disagreements were resolved by peer discussion. In the first step, titles were evaluated, then the abstracts, and finally the full text was analyzed and the information that met the eligibility criteria was selected.

Eligibility and exclusion criteria

The eligibility criteria were English original articles published in the last five years (2018-2023) that evaluated protein or peptide extraction from eukaryotic microalgae. The information collected from each study were: species from eukaryotic microalgae, protein or peptide extraction methods and protein yield or protein recovery. The methods were classified on mechanical, chemical or enzymatic hydrolysis. Exclusion criteria were book chapters, review articles, scientific abstracts, short communication, technical notes, monographs, dissertations, thesis, the studies carried out in macroalgae, prokaryotic cyanobacteria, genetically modified strains of eukaryotic microalgae, yeasts, invertebrates, chicken proteins, rice proteins, swine waste or other waste, insoluble microalgae protein-rich fraction, and that showed the result in degree of hydrolysis.

Bias risk assessment

The quality criteria were synthesized in Table I, adapted from the Meta-Analysis of Statistics Assessment and Review Instrument (MASTARI) protocol (The Joanna Briggs Institute 2014). The reviewers evaluated eight quality criteria by using the following questions: 1) Include the yield of extracted protein; 2) Reports scale-up possibility of the extraction method; 3) Identify extraction of non-protein compounds; 4) Describe the protein extraction method; 5) Validates protein viability after extraction; 6) Check out biological activity with the extracted proteins; 7) Characterizes the extracted proteins or peptides; 8) Describes statistical methods. “Yes” was assigned to items reported in the text and “No” if the information is missing or incomplete. The risk of bias of articles was rated according to the sum of “yes” as follows: 1 to 3 = high, 4 to 5 = medium, 6 to 8 = low risk of bias (Wang et al. 2021).

Table I
Methods for protein extraction in different species of microalgae.

Photosynthetic Microorganisms	Method	Protein concentration/ Yield/ Degree of hydrolysis	Reference
Chlorella vulgaris	Pulsed electric field + Homogenization	69.2 ± 1.4 %( g/L)	Buchmann et al. (2019).
Chlorella vulgaris	Sonication + chemical hydrolysis in alkaline pH	68.1 %	Phusunti & Cheirsilp (2020).
Chlorella vulgaris	1M NaOH Basic hydrolysis	47.3 ± 3.41 % (dry weight)	Gonzalez et al. (2020).
Chlorella vulgaris	Lyophilization + Sonication	~17.0 % (mg/dry weight)	Stirk et al. (2020).
Chlorella vulgaris	Lyophilization + bead milling	~19.0 % (mg/dry weight)	Stirk et al. (2020).
Chlorella vulgaris	Bead milling + acid-basic hydrolysis	78.3 % (aa content)	Zhao et al. (2019).
Chlorella vulgaris	Bead milling	40.0 % (g/g dry weight)	Alavijeh et al. (2020).
Chlorella vulgaris	Bead milling + Enzymatic Hydrolysis	68.0 % (g/g dry weight)	Alavijeh et al. (2020).
Chlorella vulgaris	Bead milling	0.12 ± 0.02 g/g	Mchardy et al. (2021).
Chlorella vulgaris	Ultrassound	0.20 g/g	Mchardy et al. (2021).
Chlorella pyrenoidosa	Organic solvent (ethanol)	27.8 ± 0.70 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Immersion in distilled water	28.5 ± 0.74 % ((mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Ultrasound	19.8 ± 0.79 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Homogenization	12.1 ± 0.68 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Enzymatic hydrolysis	17.0 ± 0.59 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Freeze-thaw + ultrasonication	23.5 ± 0.80 % (mg/g ptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Enzymatic Hydrolysis + Ultrasonication	29.6 ± 0.74 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Enzymatic Hydrolysis + Homogenization	26.5 ± 0.74 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Enzymatic hydrolysis + Ultrasonication + Homogenization	39.5 ± 1.17 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Immersion in distilled water + Enzymatic hydrolysis + Ultrasonication + Homogenization	66.1 ± 1.02 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Organic solvent + enzymatic hydrolysis + ultrasonication + homogenization	72.4 ± 1.4 % (mg/gptn total)	Zhang et al. (2018).
Chlorella pyrenoidosa	Microfluidization	44.0 %	He et al. (2023).
Chlorella pyrenoidosa	Hydrothermal pretreatment	265.2 mg/g	Xiao et al. (2019).
Chlorella sorokiniana	Bead Miling + membrane filtration	12.0 %	Gifuni et al. (2020).
Chlorella sp.	Saponification (Basic hydrolysis + heating + addition of solvents)	44.0 %	Karan et al. (2023).
Coelastrella sp.	Thermal extraction+ Basic hydrolysis	20.4 %	Sousa et al. (2022).
Haematococcus pluvialis	Pulsed electric	46.0 %	Gateau et al. (2021).
Scenedesmus acutus	Lyophilization + sonication	~19.0 % (mg/dry weight)	Stirk et al. (2020).
Scenedesmus acutus	Freeze-drying + bead milling	~20.0 % (mg/dry weight)	Stirk et al. (2020).
Scenedesmus almeriensis	Ultrasound	30.2 ± 0.30 %	Martínez-Sanz et al. (2020).
Scenedesmus almeriensis	Pulsed electric field + Enzymatic hydrolysis	60.4 % (cleaved ppt/total peptide bonds)	Guo et al. (2019).
Scenedesmus almeriensis	Pulsed electric field + Enzymatic hydrolysis (Alkalase)	50.6 % degree of hydrolysis (cleaved peptide bonds (aa)/total peptide bond (protein)	Akaberi et al. (2019).
Scenedesmus almeriensis	High pressure homogenization + Enzymatic hydrolysis (Alkalase)	48.50 % degree of hydrolysis (cleaved peptide bonds (aa)/total peptide bond (protein)	Akaberi et al. (2019).
Scenedesmus almeriensis + Scenedesmus acuminatus	Enzymatic hydrolysis	5.30 - 34.2 %	Rojo et al. (2021).
Scenedesmus obliquus	Basic hydrolysis (NaOH) 2 M pH 12 (neutralized) + enzymatic hydrolysis	34.5 ± 0.01 % (mg/mg ptn gross)	Afify et al. (2018).
Scenedesmus obliquus	Basic hydrolysis-acid (NaOH) 2 M pH 12 + HCl 1 M + enzymatic hydrolysis	39.6 ± 0.02 % (mg/mg ptn gross)	Afify et al. (2018).
Scenedesmus obliquus	Bead milling	24.9 % (mg/L)	Amorim et al. (2020).
Scenedesmus obliquus	Bead milling+ SDS	49.6 % (mg/L)	Amorim et al. (2020).
Scenedesmus obliquus	Basic hydrolysis 1 M NaOH	63.9 ± 0.02 % (dry weight)	Gonzalez et al. (2020).
Nannochloropsis sp. Chlorella vulgaris, Scenedesmus obliquus (microalgal consortium)	Alkaline extraction + enzymatic hydrolysis	81.2 %	Callejo-López et al. (2020).
Nannochloropsis gaditana	Ultrasound	22.8 ± 0.2 %	Martínez-Sanz et al. (2020).
Nannochloropsis gaditana	Alkaline extraction + acid hydrolysis	2.20 %	Niu et al. (2023).
Nannochloropsis oculata	Basic hydrolysis	54.0 %	Obeid et al. (2022).
Nannochloropsis oculata	Soxhlet extraction + Basic hydrolysis	73.0 %	Obeid et al. (2022).
Nannochloropsis sp.	Alkaline extraction + acid hydrolysis + High pressure homogenization + Microfluidization	75.0 - 80.0 %	Soto-Sierra et al. (2021).
Tetraselmis chui	Bead milling	11.0 - 32.0 %	Mear et al. (2023).

Quality criteria

A systematic review is a formal process to gather and evaluate literature to answer a defined question. The systematic review process is transparent. The steps: the posing of the question, continuing with the plans for describing the inclusion criteria of trials, searching for pertinent articles, extracting the necessary data from each one, deciding if and how to combine the data, and concluding with the analyses of the information obtained are laid out in a methods section (Koretz & Lipman 2017).

Each item was evaluated as: yes or no. The frequency of yes for each criterion was used to evaluate the quality of methods extraction of protein or peptides. When the frequency was higher than 70.0 % the quality was considered to be good, when the frequency was between 50 and 69.0 % the quality was considered to be moderate, and when the frequency was lower than 50.0 % the quality was considered to be bad (Gadioli et al. 2018).

RESULTS

Literature search

As shown in Figure 1a, we identified potentially relevant articles in three different databases, 2.707 articles, and after removing duplicates and non-original articles reviews, letters, book chapters, conference abstracts, 1.163 were selected. After screening the article titles and abstracts based on the eligibility criteria, we identified 42 articles. After initial screening, potentially eligible studies were retrieved in full text and evaluated following the criteria: Quantify proteins, extracted protein yield, describe the extraction method. After reviewing the full text, 14 papers were excluded for at least one of five reasons: a) it did not meet inclusion criteria; b) the studies carried out in macroalgae; c) Study reported extraction of fractions rich in insoluble proteins; d) Study did not perform or report protein dosage extracted; e) Study does not make clear the species of microalgae used. Overall, 25 articles were identified and included in the final analysis.

Figure 1
a) Flow chart of included articles. (n) indicates the number of studies; b) Number of articles selected in study.

Studies description

Microalgae species of selected articles

Table I shows that microalgae genus the most studied are Chlorella (50.0 %, n=12), following by Scenedesmus (36.0 %, n=9) and Nannochloropsis (20.0 %, n=5), while only three articles used Coelastrella, Haematococcus, Tetraselmis (Sousa et al. 2022 , Gateau et al. 2021, Mear et al. 2023).

Chlorella vulgaris was the most studied species of the Chlorella genus (32.0 %; n=8) (Buchmann et al. 2019, Phusunti & Cheirsilp 2020, Gonzalez et al. 2020, Stirk et al. 2020, Zhao et al. 2019, Alavijeh et al. 2020, Mchardy et al. 2021), following by C. pyrenoidosa with three articles (12.0 %; n=3) (Zhang et al. 2018, Ke et al. 2023, Xiao et al. 2019).

Scenedesmus were the second genus most reported, being represented by S. obliquus and S. almeriensis with 16.0 % of studies (n=4) each species (Afify et al. 2018, Amorim et al. 2020, Gonzalez et al. 2020, Callejo-López et al. 2020, Martínez-Sanz et al. 2020, Guo et al. 2019, Akaberi et al. 2019, Rojo et al. 2021), while only one reported S. acutus (4.0 %; n=1) (Stirk et al. 2020) and one study of S. acuminatus in co-culture with S. almeriensis (Rojo et al. 2021).

Cell disruption techniques used in microalgae biorefinery

Mechanical methods are most used method with 72.0 % (n=18) articles distributed in: Bead milling (28%; n=7) (Stirk et al. 2020, Zhao et al. 2019, Alavijeh et al. 2020, Mchardy et al. 2021, Gifuni et al. 2020, Amorim et al. 2020, Mear et al. 2023), sonication or ultrassonication (20.0 % ; n= 5) (Stirk et al. 2020, Phusunti & Cheirsilp 2020, Mchardy et al. 2021, Zhang et al. 2018, Martínez-Sanz et al. 2020), pulsed electric (16.0 %; n= 4) (Akaberi et al. 2019, Buchmann et al. 2019, Guo et al. 2019, Gateau et al. 2021), Homogenization (12.0 %; n=3) (Zhang et al. 2018, Buchmann et al. 2019, Soto-Sierra et al. 2021), thermal extraction (Xiao et al. 2019, Sousa et al. 2022), high pressure homogenization (Akaberi et al. 2019, Soto-Sierra et al. 2021) and microfluidization (Soto-Sierra et al. 2021, Ke et al. 2023) where it is reported in 8.0 % in articles each (n=2).

In general, mechanical methods used alone achieve yields lower than 50.0 %, as observed in pulsed electric, microfluidization, bead milling which obtained protein extraction values of 46.0 %, 44.0 %, 40.0 %, respectively (Gateau et al. 2021, He et al. 2023, Alavijeh et al. 2020). However, when a mechanical method was associated with another, this yield increases to up to 80.0 % (Zhao et al. 2019, Soto-Sierra et al. 2021).

Chemical extraction is the second used method with 48.0 % (n=12) of the articles (Phusunti & Cheirsilp 2020, Gonzalez et al. 2020, Zhao et al. 2019, Zhang et al. 2018, Karan et al. 2023, Sousa et al. 2022, Afify et al. 2018, Gonzalez et al. 2020, Niu et al. 2023, Obeid et al. 2022, Soto-Sierra et al. 2021, Amorim et al. 2020), achieving protein yields 63.9 ± 0.02% (Gonzalez et al. 2020).

Enzymatic hydrolysis is the third method most used with 28.0 % (n=7) of the studies each which obtained protein yields of 5.30% - 34.2% (Rojo et al. 2021). Combined with enzymatic and mechanical methods this yield increases, reaching between 68.0 and 78.0 %, as reported in the species Chlorella vulgaris (Alavijeh et al. 2020, Zhao et al. 2019).

Combination of methods enzymatic and acid-basic hydrolysis increases protein extraction yield, reaching more than 80.0 %, as reported in the species Chlorella vulgaris, Nannochloropsis sp. and Scenedesmus obliquus (Callejo-López et al. 2020).

The higher yield of extraction is achieved by combining more than one extraction method, reaching yields of up to 81.0 % in the microalgae Nannochloropsis sp (Callejo-López et al. 2020). The combination of methods was used by 60.0 % of the studies (n=15) (Buchmann et al. 2019, Phusunti & Cheirsilp 2020, Stirk et al. 2020, Zhao et al. 2019, Alavijeh et al. 2020, Zhang et al. 2018, Gifuni et al. 2020, Karan et al. 2023, Sousa et al. 2022, Guo et al. 2019, Akaberi et al. 2019, Afify et al. 2018, Amorim et al. 2020, Callejo-López et al. 2020, Soto-Sierra et al. 2021).

Risk of bias and quality of the included studies

As shown in Figure 2, most of the criteria were rated as good quality (80.0-100.0%), while only criterion 5 was rated as moderate, because 50% (n=13) of studies evaluated protein viability after extraction. Two criteria were identified as bad quality because only 16.0 % (n=4) of the studies checked out biological activity after protein extraction and 48.0 % (n=12) characterized the proteins or peptides extracted. The quality of articles was defined on the basis of the specific criteria used in this review and do not refer at all to their scientific level. Therefore, included studies that did not respond positively to them, being considered too bad or moderate because may have had other targets not considered in this analysis.

Figure 2
Risk of bias according as percentages of all included studies response to the sum of “yes”. () = Good, () = Moderate, () = Bad.

As shown in Table II most of the articles responded positively to the criteria, being considered low (44.0 %; n=11) or moderate (60.0 %, n=15) risk of bias.

Table II
Description of tools to assess risk of bias and quality criteria in protein extraction studies in microalgae.

Article	1. Include the yield of extracted protein	2. Reports the scale-up possibility of the extraction method	3. Identify extraction of non-protein compounds	4. Describe the protein extraction method	5. Evaluated protein viability after extraction	6. Check out biological activity with the extracted proteins	7. Characterises the extracted proteins or peptides	8. Describes statistical analysis	Number of criteria in each study/%	Risk of Bias
Afify et al. (2018)	Yes	No	No	Yes	Yes	Yes	Yes	Yes	6/ 75.0 %	Low
Akaberi et al. (2019)	No	Yes	Yes	Yes	Yes	No	Yes	No	5/62.5 %	Medium
Alavijeh et al. (2020)	Yes	Yes	Yes	Yes	Yes	No	No	No	5/62.5%	Medium
Amorin et al. (2020)	Yes	Yes	Yes	Yes	No	No	No	Yes	5/ 62.5%	Medium
Buchmann et al. (2019)	Yes	Yes	No	Yes	Yes	No	Yes	Yes	6/75.0 %	Low
Callejo-López et al. (2020)	Yes	Yes	Yes	Yes	No	No	Yes	Yes	6/75.0 %	Low
Gateau et al. (2021)	Yes	Yes	No	Yes	Yes	No	Yes	Yes	6/75.0 %	Low
Gifuni et al. (2020)	Yes	Yes	Yes	Yes	No	No	No	Yes	5/62.5 %	Medium
Gonzalez et al. (2020)	Yes	Yes	Yes	Yes	No	No	No	Yes	5/ 62.5 %	Medium
Guo et al. (2019)	No	Yes	Yes	Yes	Yes	No	No	Yes	5/ 62.5 %	Medium
Karan et al. (2023)	Yes	Yes	Yes	Yes	No	No	No	No	4/ 50.0 %	Medium
Ke et al. (2023)	Yes	Yes	Yes	Yes	No	No	No	Yes	5/62.5 %	Medium
Martínez-Sanz et al. (2020)	No	No	Yes	Yes	No	Yes	No	Yes	4/ 50.0 %	Medium
Mchardy et al. (2021)	Yes	Yes	No	Yes	No	No	No	Yes	4/ 50.0 %	Medium
Mear et al. (2023)	Yes	Yes	Yes	Yes	No	No	Yes	Yes	6/ 75.0 %	Low
Niu et al. (2023)	Yes	Yes	Yes	Yes	Yes	No	No	No	5/ 62.5 %	Medium
Obeid et al. (2022)	Yes	Yes	Yes	Yes	No	No	Yes	Yes	6/ 75.0 %	Low
Phusunti & Cheirsilp (2020)	Yes	Yes	Yes	Yes	Yes	No	Yes	Yes	7/ 87.5 %	Low
Rojo et al. (2021)	Yes	No	Yes	Yes	No	No	Yes	Yes	5/ 62.5%	Medium
Soto-Sierra et al. (2021)	Yes	Yes	Yes	Yes	Yes	No	Yes	Yes	7/ 87.5%	Low
Sousa et al. (2022)	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	8/ 100.0 %	Low
Stirk et al. (2020)	Yes	Yes	Yes	Yes	Yes	Yes	No	Yes	7/ 87.5 %	Low
Xiao et al. (2019)	Yes	Yes	Yes	Yes	Yes	No	No	No	5/ 62.5 %	Medium
Zhang et al. (2018)	Yes	Yes	No	Yes	No	No	No	Yes	4/ 50.0 %	Medium
Zhao et al. (2019).	Yes	Yes	Yes	Yes	Yes	No	No	No	5/ 62.5 %	Medium
Studies meet the criterion	n= 23 (92%) Good	n= 23 (92%) Good	n=21 (84%) Good	n=25 (100%) Good	n= 13 (50%) Moderate	n= 4 (16%) Bad	n=12 (48%) Bad	n=20 (80%) Good	-

Number of papers over time

Figure 1b shows that there was an increase in the amount of papers seeking proteins extracted from microalgae, especially in the last three years (2020-2023).

DISCUSSION

The microalgae in the studies

Chlorella sp., Scenedesmus sp. with approximately 50.0 % protein by dry weight (Galarza 2019) and Nannochloropsis sp. with 36.0 % protein by dry weight (Zhang et al. 2019) were the most reported genus, probably due to high protein content in their biomass cell. In addition to being the target of most studies, there is also a higher number of protein extraction techniques (Sathasivam et al. 2019). In addition to high protein content, the genera Chlorella, Scenedesmus and Nannochloropsis are widely used to obtain other biomolecules with high interest biotechnology, as pigments (carotenoids and chlorophyll and lipids which are used to biofuel production, being able to achieve 58.0 %, 42.0 %, 56.0 % to Chlorella, Scenedesmus and Nannochloropsis, respectively (Coronado-Reyes et al. 2022, Guimarães & França 2021, Shokravi et al. 2020). Lipids need to be extracted from the interior of their cells, and for this are widely used in studies to biomolecules extraction (Obeid et al. 2022, Niu et al. 2023, Kose & Oncel 2015). Chlorella vulgaris and Scenedesmus sp. are the most utilized in biotechnology, mainly as food and biofuel, and for this reason is more widely studied (Andreeva et al. 2021, Abdulsamad & Varghese 2017).

Other microalgae such as Coelastrella sp., Haematococcus pluvialis and Tetraselmis accumulate a high content of carotenoids, chlorophylls and proteins which have wide biotechnological application, as for example, astaxanthin from H. pluvialis that are used in aquaculture and human nutrition (Sousa et al. 2022, Muniz Souza et al. 2020, Mear et al. 2023).

Selection of appropriate cell disruption methods is dependent on cellular matrix and cell-wall characteristics. For instance, the robust structure of trilayered cell walls in Chlorella vulgaris or Tetradesmus obliquus commonly requires intense methods for efficient disruption (Stirk et al. 2020, Montone et al. 2018).

Effect of the extraction method on protein

To promote cell rupture, extraction methods need to disrupt hydrogen bonds and electrostatic forces between membrane-associated polar lipids and proteins and make them porous. This allows the nonpolar or polar solvent (e.g., chloroform, hexane, acid, hydroxide) to enter the cell and interact with the hydrophobic or hydrophilic contents (Du et al. 2015). In this review, the methods were classified as mechanical, chemical or enzymatic hydrolysis.

Bead milling is mechanical method more used, consisting of the collision of high-speed steel, zirconium, glass or ceramic beads against the microalgae cells obtaining high cell lysis efficiency, so it is widely used in the extraction process (Soto-Sierra et al. 2018). The size of the beads influences the cell wall disruption process and consequently the extraction of biocompounds, studies indicate that smaller beads are more efficient in extracting metabolites, due to the increased contact surface with the cell, for microalgae such as Chlorella vulgaris and Neochloris oleoabundans (Postma et al. 2017).

Ultrasound or Ultrasonication is the second mechanical method that stand out in protein extraction from microalgae (Zhang et al. 2018, Martínez-Sanz et al. 2020) and can be amplified when in association with other methods, such as enzymatic hydrolysis, homogenization and bead milling (Table I). Cell disruption occurs by the cavitation effect which occurs when vapor bubbles within the liquid form in an area where the pressure of the liquid is lower than its vapor pressure, then, these bubbles grow when the pressure is negative and compress under positive pressure, which causes a violent collapse of the bubbles (Safi et al. 2015). If it occurs close to cell walls, it causes stress on the cell wall in order to destroy it, thus releasing its compounds (Phong et al. 2018a, b). The procedure has high energy consumption by producing heat and requires a refrigeration system to avoid overheating. On the other hand, it does not cause damage to the environment because there is no waste, so it can be applied on a large scale, since it does not require further separation steps (Dixon & Wilken 2018).

The pulsed electric field is the third most used mechanical method, mainly in cells with thick cell walls, such as Chlorella sp. and Scenedesmus sp. This method uses an intense external electric field by a short period (nanoseconds to milliseconds) on cells. The electric pulses cause an electro propagation effect that increases the cell membrane permeability. One of the advantages of using this technique is the ease in separating the metabolites of interest, since it dispenses with the use of solvents and harmful products, not generating toxic waste for disposal (Coustets et al. 2014). This method is common to use as pre- treatments to metabolites extraction (Guo et al. 2019, Papachristou et al. 2020). The pulsed electric field generally is used in association with other treatments and achieves high extraction results to Chlorella sp. and Scenedesmus sp. cells (Buchmann et al. 2019, Guo et al. 2019).

Hydrothermal pretreatment was used by only two studies showing low yield protein (Table I). This method usually occurs at high temperatures, approximately 180 °C, and pressures below 2 MPa which damage the surface structure of microalgae cells by hydrolysis of carbohydrates and proteins (Sousa et al. 2022, Fu et al. 2021).

The homogenization or immersion technique was used in only one study and obtained low protein yields (Table I), being recommended to use it in combination with other methods to optimize extraction (Buchmann et al. 2019, Zhang et al. 2018). This technique consists of subjecting a cell suspension to a high pressure homogenizer (Zhang et al. 2018). To prevent overheating of the cell suspension, a cooling system is integrated into the homogenizer keeping the temperature below 30 °C during the process (Safi et al. 2017).

Another technique used in just two studies was microfluidization which it subjects cells to high pressure to force the fluid into microchannels with a specific configuration and initiates the effects of process via a cavitation, powerful shear, high-velocity impaction, turbulence and instantaneous pressure drop (He et al. 2021). In our search, only one study using this technique isolated and one combined with another were found to extract proteins because this emerging technology is more efficient at removing lipids (Ke et al. 2023, Ansari et al. 2018).

To promote cell rupture, extraction methods need to disrupt hydrogen bonds and electrostatic forces between membrane-associated polar lipids and proteins and make them porous. This allows the nonpolar or polar solvent (e.g., chloroform, hexane, acid, hydroxide) to enter the cell and interact with the hydrophobic or hydrophilic contents (Du et al. 2015).

Chemical hydrolysis is widely used because it generally breaks cell-wall through changing the pH. This method is frequently used to extract targeted molecules with a higher polarity, showing high yield to intracellular components but with a high level of impurities, such as pigments which require additional steps for purification (Obeid et al. 2022, Gonzalez et al. 2020, Zhang et al. 2018).

Another method very much used is enzymatic hydrolysis which uses enzymes to lyse microalgal cell walls. This method is more gentle and specific when compared to the other methods (Phong et al. 2018b). Enzymatic lysis is a very studied cell disruption method due to presenting mild operating conditions, low energy requirements, low capital investment, and the prevention of aggressive physical conditions such as high shear stress which can cause loss of activity of the compounds (Gunerken et al. 2015).

Cell disruption technology, whether by physical, chemical or mechanical methods, is a prerequisite for efficient extraction of intracellular proteins. The extraction efficiency using single methods is not satisfactory (Table I), however, the combination of some methods can significantly increase the extraction yield in some species such as Scenedesmus almeriensis, Chlorella pyrenoidosa and Chlorella vulgaris ( Zhang et al. 2018, Akaberi et al. 2019). Besides the combination of methods, other variants can influence extraction yields such as temperature, extraction time, and pH (Guo et al. 2019, Zhang et al. 2018, Amorin et al. 2020, Phusunti & Cheirsilp 2020). Among the methods that stand out in association are ultrasound, enzymatic and chemical hydrolysis (Akaberi et al. 2019, Zhang et al. 2018, Phusunti & Cheirsilp 2020). The advantages and disadvantages of each method are summarized in Table III.

Table III
Summary of advantages and disadvantages of the evaluated protein extraction methods.

Method	Advantages	Disadvantages	Reference
Bead milling	Low energy consumption Scalable Biocomposites with activity	Many executions parameters Uses high concentration of biomass Difficulty in separating the compounds of interest from insoluble fragments Requires cooling system Expensive Maintenance	Soto-Sierra et al. (2018)Postma et al. (2017), Montalescot et al. (2015), Zinkoné et al. (2018), Günerken et al. (2019).
Ultrasound and ultrasonication	Applicable to several species Combined with other methods Scalable Does not produce toxic residues Eliminates separation steps	Cooling required Energy consumption	Phong et al. (2018a, b).
Pulsed electric field	Pre-treatment Associated with other methods Scalable Select metabolites Does not produce waste	High power consumption Low efficiency in microalgae cell wall rupture	Coustets et al. (2014), Parniakov et al. (2015), Papachristou et al. (2020).
High pressure homogenization	Low energy consumption Scalable High extraction yield Does not produce toxic residues Low-cost reagents	Requires cooling system	Safi et al. (2017).
Microwave or ultrasound	Scalable Active biomolecules Short runtime Low energy consumption	Cooling Maintenance Cost Limited to polar solvents Infeasible for volatile compounds	Dixon & Wilken (2018), Gunerken et al. (2015).
Microfluidization	Short processing time Little nutritional component loss Scalable	Causes protein denaturation High temperature Emergent technology	He et al. (2021), Timira et al. (2021).
Hydrothermal pretreatment	Efficient removal of bioproducts Scalable	High operating temperatures Inevitable degradation of compounds	Karan et al. (2019), Fu et al. (2021).
Enzymatic treatment	Low energy cost Easily separable	Loss of activity Enzyme costs	Safi et al. (2017).
Chemical Treatment	Low energy consumption Scalable High Throughput Selects extracted compound Reusable	Continuous demand for solvents Decreases the equipment maintenance interval Requires proper disposal Can destabilise compounds	Phong et al. (2018a), Shankar et al. (2019), Dixon & Wilken (2018).

Number of articles selected in recent years

It is believed that the increase in the number of articles in recent years is a result of the demand for protein sources, as it is increasingly reported that microalgae are rich in essential amino acids, a protein supplement for human and animal nutrition, as well as rich in important metabolites. economic and nutraceutical character (Sathasivam et al. 2019, Andreeva et al. 2021, Williamson et al. 2023).

CONCLUSIONS

Microalgae are a promising source of organic metabolites from renewable sources, but current low-yield extraction methods limit their applicability, so it is imperative to find methods that meet economic and ecological needs. Knowing the benefits and limitations of different methods together with emerging technologies allows us to adapt execution techniques and optimize extraction procedures.

The species of microalgae is an important factor when choosing the extraction process, as variables such as cell shape and wall thickness will directly interfere with protein extraction. Mechanical methods such as ball milling are the most efficient, especially when combined with other methods. Based on the evaluated criteria, it is possible to understand the need to use more than one method for viable protein extraction.

Different cell wall disruption and protein and peptides extraction methods from the microalgae are available. Although many critical issues, such as energy consumption, toxicity, metabolite stabilities and scale-up, still need to be investigated for optimized extraction processes which can help in the cost-effective production of natural protein and peptides.

The choice of the ideal protein extraction method must take into account the type of cell, the application of the protein, the execution time, the separation treatments after extraction, the products or waste generated and the costs involved in the entire process. Therefore, protein extraction methodologies have been studied and developed in recent years. However, research is needed to ensure progress in this segment.

ACKNOWLEDGMENTS

This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – [Finance Code 001] and by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco [FACEPE, (FACEPE, APQ-0486-9.26 22; APQ-1480-2.08/22] and by the Fundação de Amparo à Ciência e Tecnologia de Pernambuco [FACEPE, (FACEPE, APQ-0486-9.26 22; APQ-1480-2.08/22].

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Publication Dates

• Publication in this collection
  21 Oct 2024
• Date of issue
  2024

History

• Received
  3 Feb 2024
• Accepted
  19 June 2024