Microalgal Proteins and Bioactives for Food, Feed, and Other Applications
Round 1
Reviewer 1 Report
The Review contains significant and highly relevant data, and it is written in a clear way. Having in mind the importance of microalgae, I believe the topic is highly relevant for publication.
Paper contains some technical errors, typos, therefore, please check the whole paper carefully.
Also, the last section “Conclusion, future directions” contains information related to difficulties related to the production and use of microalgae. However, future directions are not so well explained; the focus is more on current problems and drawbacks. Therefore, I recommend reorganizing this section and placing the mentioned challenges and bottlenecks related to microalgae into separate section, while the section “Conclusion, future directions” should include more specific information related to future directions.
Author Response
Re: We thank reviewer one for their comments on our review paper and we have tried to take these comments on board in our revision. We have done a spellcheck on the entire article and corrected any indicated typos.
We have used American English – for example “recognized” instead of “recognised” and “labor” instead of “labour” – as these were indicated in the spell check as errors. We have also removed spaces where they were erroneous.
Furthermore, we have revised the “Conclusion, future directions section” as recommended by the reviewer – we have removed the “Challenges and bottlenecks section” and made this an independent section and have re-worded the “Conclusion and future directions” section. We have also included more detail on the future directions in this section. We thank reviewer 1 for their kind and constructive feedback.
This section now reads as follows:
- “Challenges and Bottlenecks
Several barriers need to be addressed to ensure the successful incorporation of bioactive components extracted from microalgal biomass including proteins into food, feed, pharmaceuticals and other products. The main bottleneck are (I) high production costs of microalgal biomass and its components (ii) lack of knowledge about the impact of consumption of microalgal biomass and the digestibility and safety of microalgae and (iii) insufficient research into the development of new food products. One of the main bottlenecks regarding development of microalgal protein as a food ingredient is the high cost of microalgal biomass production, recently estimated to be 3.4 EUR/kg for DW microalgal production in Spain [153] or 5.1 EUR/kg of DW for Tetraselmis suecica production in Italy [154]. Nowadays, selective separation of microalgal products is at an early stage and most commercial facilities focus on one product, which is either dried biomass or extracted and purified specific high-value component like omega-3 fatty acid (DHA, EPA) or pigments likeastaxanthin. Selective separation of different products using a biorefinery approach aims at optimal exploitation of various biomass components and their allocation to different markets. The separation of functional proteins requires mild conditions and the costs are still too high currently to be economically viable. For example, where cell disruption using the bead-milling process is implemented, there is a a huge amount of energy (1 kWh/kg) dissipated in the liquid fraction as heat, which correspond to additional costs of approximately 0.15 EUR/kg. However, it was estimated that a 90 % reduction of energy consumption for cell disruption can be achieved by the use of novel techniques like pulse electric field (PEF) and ultrasound [153]. However, these technologies are not widely available currently and initial set-up costs are high.
The safety of microalgal biomass for consumption is another challenge. Van der Spiegel and colleagues warned that food safety of novel protein sources such as microalgae, seaweed or insects needs to be addressed. Potential hazards associated with their consumption include poisoning due to heavy metals, or mycotoxins, pesticide residues and pathogens. Other problems are the presence of anti-nutritional factors, allergens and modification of substances during processing that may increase allergenicity for example. In the future, research should focus on the safety of novel proteins from microalgae in food products and on the degradation and accumulation of bioactives and contaminants during processing [155]. Other issues include the digestibility of microalgal proteins, which has not been adequately explored to date. Although many studies have evaluated the digestibility of microalgae which is reported as 94 % for Arthrospira platensis in some studies, otherstudies found that microalgal proteins have lower digestibility compared to standard protein sources such as egg, soya and pea protein [156] and found digestibility values for Arthrospira platensis which were significantly lower at 78 % [157]. The importance of correct selection of methods to determine digestibility and bioavailability and standardization of these methods should not be underestimated. Furthermore, addition to the EU list of approved algae for use as novel foods is required beyond what is currently approved. At present there is a limited number of microalgal species approved and apart from omega-3-PUFA-rich oil extracted from certain heterotrophic microalgae, only Spirulina and Chlorella sp. exist on the market today and they are used primarily for their food colourant potential and not as a source of nutrients. Conclusion and future directions
The future for microalgae use looks promising despite the fore-mentioned bottlenecks and challenges. They are a noted source of PUFAs and proteins and plans to improve processing methods to make microalgae protein more acceptable to consumers should be pursued. These processing methods include cell disruption methods that can actually enhance uptake of key nutrients including amino acids by the consumer as well as methods to refine key ingredients from microalgae to generate acceptable powder formats with less sensory challenges compared to whole microalgae. Methods that can be applied to microalgal proteins include molecular weight cut off (MWCO) filtration and diafiltration that are used in the processing of proteins from the dairy and pea protein industries for example. According to Enzing, Europe has some important advances to make in this field – and this topic is of high priority in terms of R&D funding policies. Some bottlenecks in the European microalgae industry are obvious, including suboptimal climatic conditions (low levels of sun hours and intensity, low temperature, high level of rainfalls), high labor costs, lack of venture and seed capital for start-up companies, low entrepreneurial activity among researchers and engineers, low R&D investments by large companies, high cost of land and low domestic demand for microalgae based products [16]. The European Commission’s Green Deal targets numerous areas where microalgae production and processing can play an important role. For example, the goal of becoming climate neutral by 2050, protecting biodiversity, develo** a circular economy and contributing to the “farm to fork” strategy for sustainable food system development [112] could be a key driver of microalgal development for food, pharma and cosmetics in Europe and beyond.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments for author File: Comments.docx
Author Response
Reviewer 2: Response to reviewer comments – applsci-1658574-review
The authors have done extensive reviews of the usefulness of microalgae. Constituent and application as well as the process and legislation were presented. I have some comments and questions for the authors to edit and clarify.
Response: We thank reviewer two for their kind comments. We have tried to address these comments in the following edits.
Reviewer 2: 1. P.1 L.40: CO2, subscripted 2
Response: Item now subscripted
Reviewer 2: P1 L42: 160,000
Response: We have changed this to read 160,000
Reviewer 2: P2 L65: B2B should be declared first.
Response: We have declared this in the response along with B2C.
Response: We have edited this to read Business to Business (B2B)
Reviewer 2: 4. the use of microalga and microalgae should be consistent.
Response: We have changed text to read microalgae throughout
- P2 L76: Synechococcus sp., ….
Response: We have inserted a comma as requested
- Revise Table 1 title
Response: Table 1 title now reads “Compositional analysis of commercially available microalgae”
- Section 1.1.1: the meat and whey proteins have higher PDCAAS scores compared to the algal proteins. Should the authors address or discuss more on this point?
Response: Yes, we have included the following text on this point: “Meat and whey proteins are known to have PDCAAS values closer to 1 and are considered complete protein sources because of the amino acid content and digestibility (as measured using PDCAAS) values. The PDCAAS values found to date for selected microalgae (Table 2) are lower than 1 and this may result from the anti-nutritional factors present in microalgae including the constituents of the algal cell walls which may bind to available protein in microalgae when algae are consumed preventing their complete digestion.”
- P4 L127-129: do the authors mean peptides released after hydrolysis of the algal proteins or the native peptides encoded from the algal genome? This could be clarified, and research examples shall be provided.
Response: We believe the following text clarifies that microalgal peptides can be encoded from the algal genome or derived following hydrolysis using enzymes: “Peptides are short sequences of amino acids between two and thirty in length, with mass values less than 10-kDa [44]. They provide a health benefit that goes above and beyond basic, human nutrition to the consumer. Microalgae peptides can be generated using enzymes or are native and encoded from the algae genome. Both peptide types are associated with a wide range of hormone-like, biological activities [45]. The use of bioactive peptides in pharmacology was first described in 1950, when peptides of dairy origin were shown to enhance bone calcification in rachitic infants [46]. Table 3 lists the species of microalgae from which peptides with significant antimicrobial, antioxidant, anti-inflammatory, anti-hypertensive, and anti-atherosclerotic have been derived to date using enzymatic hydrolysis methods”. We have also edited table 3 to reflect that these peptides are derived using enzymatic hydrolysis – the enzymes are presented.
- Table 3: is it one peptide per reference or multiple peptide sequences per reference? The authors should format a clearer representation.
Response: The table references refer to peptides presented adjacent to the reference – more than one peptide sequence shown per reference.
- Table 3: what does “-” mean?
Response: We have deleted this as we agree with the reviewer that it makes no sense and we have inserted n/d = not determined in its place
- P6 L150-151: define the essential amount for human consumption.
Response: PUFAs in sufficient amounts (males and females 0.25 g of EPA and DHA daily) in the human diet [64].
- Table 4: describe GLA, ARA, and AEA.
Response: arachidonic acid (ARA); gamma-linolenic acid (GLA); anandamide (AEA)
- Table 5: could the authors provide specific carbohydrate names as examples?
Response: We have provided exopolysaccharides and glycogen as examples in this table as this is what is referenced in the papers cited.
- Table 6: full name of RDA
Response: Recommended Daily Allowance (RDA) added to the table
- Despite thousands of algal species, only a limited number of species have been used. Could the authors discuss this point?
Response: yes, we thank the reviewer for this point and we have discussed this point in relation to those approved for use in EU for example and found on the novel foods list of EFSA. Please see text as follows in revised paper: “Furthermore, addition to the EU list of approved algae for use as novel foods is required beyond what is currently approved. At present there is a limited number of microalgae species approved and apart from omega-3-PUFA-rich oil extracted from certain heterotrophic microalgae, only Spirulina and Chlorella sp. exist on the market today and they are used primarily for their food colourant potential and not as a source of nutrients.”
- Table 7: the second column uses a mix of common names and scientific names. Could the authors use the same pattern?
Response: We agree with the reviewer and we have edited column two and have inserted the common names for all animals or fish used in the study
- Table 7: ADG and TAG with their full names
Response: We have included the full name for average daily gain (ADG) and triglycerides (TAG) in the table
- Table 7: sp. Non-italic
Response: We have removed the italics as suggested by the reviewer.
- P9 L246: This area of biotechnology is growing and thanks …
Response; We agree that this is a strange sentence – in the revision we have deleted it.
- Please revise this sentence: Moreover, lipid extract from Nannochloropsis gaditana reduced blood glucose and LDL cholesterol, while the concentration of blood insulin and HDL cholesterol increased. Does it conflict with the previous sentence?
Response: Many thanks for the reviewer for bringing this to our attention and yes, they are correct, the results conflict so we have indicated this in the revised manuscript as follows;
“. In a study by Ramos-Romero and colleagues lipid extracts from Nannochloropsis sp. reduced plasma and liver cholesterol in rats significantly. In contrast, a lipid extract derived from Nannochloropsis gaditana was found to reduce blood glucose and LDL cholesterol, while the concentration of blood insulin and HDL cholesterol increased [75].”
- P10 L287: … , low accumulation in tissues and low toxicity. This phrase is unclear. How the recombinant algal-produced proteins will be low toxic?
Response: We agree with the reviewer that the low toxicity part does not make sense – we have revised the manuscript and removed low toxicity from it.
- P10 L291-293: yeast is also a eukaryote and has a modification system.
Response: We have edited this and removed yeasts – this was an error on our part, we meant to only mention bacteria here. The sentence now reads: “However, all of these host organisms have some drawbacks. In the case of bacteria they do not make the same post-translation modifications of proteins as higher eukaryotes, so they are not appropriate for the production of eukaryotic proteins. Plant cells have different glycosylation patterns and mammalian tissues are costly and instable. Microalgae cells may be effective hosts for the expression of recombinant therapeutic proteins [112].”
- P11 L304: revise this “may find use”. Similar style was used in other part of the manuscript.
Response: We have changed this “may find use” in the text – there is none in the revised manuscript.
- P11 L315-316: please change to “a peptide sequence LVMH derived from Chlorella sp.”
Response: We have revised the text to read: “Microalgae are found in several products for personal skin care. For example, the company Soliance uses whole Arthrospira sp. and a peptide sequence LVMH derived from Chlorella sp is used in personal skin care products. Furthermore, the company Solazyme use alguronic acid in its anti-aging skin products”.
- P11 L332: species
Response: This typo was corrected and is species in the revised manuscript
- No explanation of Tables 9 and 10 in the text body of the manuscript. The authors should discuss or contrast to point out the key difference and methods that others might be easily select and follow.
Response: We agree with reviewer 2 and we have edited the text accordingly. This section now reads:
“In general, microalgae cells disruption techniques can be divided into mechanical and non-mechanical techniques. The main advantages and disadvantages of selected techniques suitable for microalgae protein extraction are listed in Table 9. Mechanical methods are suitable for cell disruption where the bioactive in question is not heat sensitive a fast process method is required (Table 9). For heat sensitive actives like proteins and peptides, enzymatic methods are preferred. In addition, enzymatic methods are considered to be more environmentally friendly. Enzymatic methods and the physical methods are both scalable. The yields of proteins obtained following use of different disruption methods are shown in Table 10. Ultrasound and high pressure homogenisation combined with enzymatic treatment have resulted in protein yields of between 74-90% when applied to microalgae previously.”
- P15 L373: external?
Response: We have deleted “external” and replaced with “skin or outer membranes”
- Section 2.2: which method will be the most suitable for algal protein purification. Which was commonly used?
Response; We have included the following text to address the reviewers concerns:
“Proteins can be separated from other compounds using molecular weight cut off filtration also. However, proteins less than the membrane size are only recovered along with salts which can pose problems for later applications in food or feeds for example. Proteins can be salted out using ammonium sulphate precipitation also and this method is the most commonly described in the literature to date. Purification of protein extracts is usually achieved using charcoal filtration or TiO2 clean-up methods, especially if proteins are to be characterised for their peptide content using mass spectrometry.”
- P15: “Furthermore, some of the compounds are attached to the cell wall by hydrogen or hydrophobic bonds so they are not accessible for chemical solvents [134].” Is this correct?
Response: We have edited this section as follows: Chemical hydrolysis of proteins is performed at higher temperatures (over 40 °C) and extreme pH. The advantages of this method are low costs, simplicity and short processing time, but on the other hand, the process lacks sensitivity and specificity and some amino-acids can be destroyed [134].
- Section 2.3: the authors could add more advantages to the use of microbial fermentation.
Response: We agree with the reviewer here. We have added additional text and a new reference (Sharma – 134) to overcome this. The text now reads as follows:
“Microbial fermentation was evaluated as an eco-friendly method suitable for protein hydrolysis on a large scale. Other advantages include elimination of hyper-allergic and anti-nutritional factors. For the purpose of protein hydrolysis by fermentation, lactic acid bacteria such as Lactobacillus brevis, Bacillus subtilis, Enterococcus gallinarum or Pediococcus acidilactii are frequently used [133]. As discussed by Sharma and colleagues recently [134] “fermented foods comprise very complex ecosystems consisting of enzymes from raw ingredients that interact with the fermenting microorganisms’ metabolic activities. Fermenting microorganisms provide a unique approach towards food stability via physical and biochemical changes in fermented foods. These fermented foods can benefit consumers compared to simple foods in terms of antioxidants, production of peptides, organoleptic and probiotic properties, and antimicrobial activity.”
- Table 11: the content of this table shall be highlighted or contrasted in the body text. Were the emulsifying properties of the algal proteins better than those of animals?
Response: We have added the following text to address this concern: “Proteins are widely recognized as the major component influencing the rheological properties of food products and the stability during storage [42]. Due to high content of surface active proteins, various microalgae species were proved to have great ability to stabilize proteins and foams and exhibited comparable stabilization properties to commonly used synthetic surfactants or animal based proteins. In the future, microalgae proteins have the potential to replace surfactant and animal proteins in the food industry [143]. Techno-functional properties of selected microalgae species and their protein fractions are listed in Table 11, together with the effect of their incorporation to the food. As outlined in Table 11, the emulsifying properties of selected proteins from different Chlorella sp. were excellent and comparable to egg protein in many instances. Microalgae proteins exhibit comparable to superior interfacial stabilization compared to animal or plant based proteins [143]. Their emulsions and foams exhibit minor pH-dependency due to a characteristically low isoelectric point and extraordinary resistance towards increased ionic strength.”
- Table 11: what is LAB?
Response: Lactic acid bacteria (LAB) – we have added this to the table
- P18: “there were 22 algae listed. (https://ec.europa.eu/food/safety/novel-food/novel-food-catalogue_en) 6 microalgae – Arthrospira platensis, Chlorella luteoviridis, Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas reinhardtii, Spirulina sp. (accessed 11/01/2022).” Please revise this part. Do the last phrases belong to the sentence?
Response: We have edited this sentence – it now reads as follows:
“The EU through Regulation (EU) 2017/2470 maintains an online list - the novel food catalogue - that contains the unions list of all authorized novel foods. This legislation applies to microalgae intended to be used as food. This catalogue contains both European and imported algae and to the current date there were 22 algae listed. The list is accessible at https://ec.europa.eu/food/safety/novel-food/novel-food-catalogue_en and includes six microalgae includingArthrospira platensis, Chlorella luteoviridis, Chlorella pyrenoidosa, Chlorella vulgaris, Chlamydomonas reinhardtii, and Spirulina sp. when the list was accessed on 11/01/2022.”
- Section 4: Could the authors propose the improvement of the legislation process? Is there any scientific innovation that could help the safety ascertainment and thus speed the process? The comparison of the legislation and regulation between EU and US would be more useful.
Response: I’m afraid not – we are not in a position to do this. We have presented the relevant legislation in the paper and it is up to the reader to interpret the data. Recommending improvements to the legislation is beyond the scope of the article in my opinion.
- P20: “For example, the goal of becoming climate neutral by 590 2050, protecting biodiversity, develo** a circular economy and contributing to the 591 “farm to fork” strategy for sustainable food system development [112].” This is not a sentence.
Response: We have checked this sentence and revised as follows:
….”Green Deal targets numerous areas where microalgae production and processing can play an important role. For example, the goal of becoming climate neutral by 2050, protecting biodiversity, develo** a circular economy and contributing to the “farm to fork” strategy for sustainable food system development [112] could be a key driver of microalgae development for food, pharma and cosmetics in Europe and beyond.”
- Please check ref format such as 7, 65, 67, 82, 132, 135, and 148
Response: We have checked these references and made sure in revision that they are in line with the journal instruction for authors.
Author Response File: Author Response.docx
Reviewer 3 Report
This review article provide beneficial information concerning microalgal application for foof, feed, cosmatics, and pharmaceuticals for researchers. I recommend to revise several minor points before publication.
- In the article, many components of micaroalgae (proteins, lipids, carbohydates, vitamins , and etc) were described. Thersore, I suggest to revise a title of 'Microalgal proteins and bioactives for food, feed, and pharma-2 ceuticals application'.
- Keywords are too many. Please reduce number of keywords. Especially, astaxanthin is not proper.
- (L 50) Annual production of microalgae in 2011 was described. More recent data is required to understand situation up-to-date.
- I can't find essential difference between subsections of 1.1/1.1 and a section of 3. You have to consider to merge those. Subsections of 1.3 and 1.4 are better to make independent sections. I recommend to revise the structure of writing.
- Contents matching between columns in some tables is not clear.
Author Response
Response to reviewer three comments
- Response: Many thanks to reviewer three for their valuable review. We agree with the reviewer and we have edited the title of the article to “Microalgal proteins and bioactives for food, feed, and other applications” as suggested.
- Response: We have reduced the number of keywords and removed astaxanthin from the list as requested. Keywords: microalgae; protein; bioactive peptides, lipids, prebiotics, , feeds
- Response; We agree with the reviewer and in this paper have decided to leave out figures regarding quantity of microalgae produced globally as there are few figures for 2020 and 2021 on this.
- Response: We have edited tables where possible as requested by reviewer 3.
Author Response File: Author Response.docx
