Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors Carl Safi, Michael Charton, Olivier Pignolet, Françoise Silvestre, Carlos Vaca-Garcia, Pierre-Yves Pontalier To cite this version: Carl Safi, Michael Charton, Olivier Pignolet, Françoise Silvestre, Carlos Vaca-Garcia, et al..Influ- ence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to- protein conversion factors. Journal of Applied Phycology, Springer Verlag, 2013, 25 (2), pp.523-529. 10.1007/s10811-012-9886-1. hal-02064796 HAL Id: hal-02064796 https://hal.archives-ouvertes.fr/hal-02064796 Submitted on 12 Mar 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: http://oatao.univ-toulouse.fr/23265 Official URL: https://doi.org/10.1007/s10811-012-9886-1 To cite this version: Safi, Carl and Charton, Michael and Pignolet, Olivier and Silvestre, Françoise and Vaca-Garcia, Carlos and Pontalier, Pierre-Yves Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors. (2013) Journal of Applied Psychology, 25 (2). 523-529. ISSN 0921-8971 Any correspondence concerning this service should be sent to the repository administrator: [email protected] DOi 10.1007/s10811-012-9886-1 Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors Carl Safi • Michaël Charton • Olivier Pignolet • Françoise Silvestre • Carlos Vaca-Garcia • Pierre-Yves Pontalier Abstract Additional evidence about the influence of the time accurate protein quantification is required, and proved cell wall physical and chemical characteristics on protein that there is not a universal conversion factor that can be extractability was determined by calculating the conversion recommended. factors of five different microalgae known to have different cell wall composition, and their protein extracts. The con­ Keywords Amino acid profile • Cell wall • Conversion version factorsobtained forcrude rigid cell walled Chlore/la factor • Nitrogen • Protein extract vulgaris, Nannochloropsis oculata andHaematococcus plu­ vialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and Introduction 5.63. On the other band, conversion factor obtained for fragile cell walledmicroalgae Porphyridium cruentum and Microalgae have been consumed long time ago by the Athrospira platensis was 6.3 5 forthe formerand 6.27 forthe Aztecs and other Mesoamericans who used this biomass as latter, with no significantdifference fortheir protein extract an important food source (Vonshak 1997). Nowadays, in with 6.34 for the former and 6.21 for the latter. In addition, Japan for instance, Chlore/la vulgaris is added to food such the highest hydro-soluble protein percentage recovered from as noodles and pasta (Fradique et al. 2010) to improve the total protein was for P. cruentum 80.3 % and A. platensis nutritional quality of the meal. 69.5 % but lower for C. vulgaris with 43.3 %, N oculata Microalgae are gaining interest due to their capacity to with 33.3 % and H pluvialis with 27.5 %. The study spotted accumulate important amounts of multiple components the light on the influence of the cell wall on evaluating the (proteins, lipids, carbohydrates and pigments) compared to conversion factor and protein extractability. In addition, it any other sources, and therefore protein content is consid­ showed the necessity of finding the conversion factor every ered as one of the cardinal components determining their nutritional value. For instance, Arthrospira (Spirulina) max­ ima can accumulate proteins up to 71 % dry weight (Becker C. Safi(l81) · M. Charton· O. Pignolet · F. Silvestre· 1994). Thus, analysing and quantifying the protein content C. Vaca-Garcia· P.-Y. Pontalier are key factors that should be thoroughly investigated. A INP-ENSIACET, Laboratoire de Chimie Agro-industrielle (LCA), capital point is to calculate precisely the nitrogen-to-protein Université de Toulouse, 31030 Toulouse, France conversion factor (NTP). While the standard value of 6.25 is e-mail: [email protected] used, Kjeldahlor elemental analysismay lead to an overesti­ mation or underestimation of the protein quantity. Moreover, C. Safi· M. Charton· O. Pignolet· F. Silvestre· C. Vaca-Garcia· thesetwo methods takeinto account thetotality of thenitrogen P.-Y. Pontalier INRA, UMR 1010 CAi, present in the biomass from which 59-98 % (Lourenço et al. 31030 Toulouse, France 1998; Fujihara et al. 2001; Lourenço et al. 2004; Gonzalez Lopez et al. 2010) oft otal nitrogen belongs to protein and the recorded by a pH/temperature probe and pH was regulated rest cornes from pigments, nucleic acids and other inorganic at 7 .5 with CO2 • The algae were harvested during the components. It is true that the colorimetric method of exponential growth phase and concentrated by centrifuga­ Lowry (Lowry et al. 1951) is an accurate method for tion, and thensupplied as a frozenpaste fromAlpha Biotech protein quantification (Peterson 1979; Gonzalez Lopez (Asserac, France). The biomass contained 20 % dry weight. et al. 2010) and it does not require a conversion factor. The frozen paste of crude microalgae was freeze-dried in a Nevertheless, this method determines only the hydro­ Fisher Bioblock Scientific Alpha 2-4 LD Plus device soluble proteins (Crossman et al. 2000; Diniz et al. (Illkirch, France). 2011) and not the total protein content. In addition, the extraction of proteins can be dimin­ Protein extraction ished by the cell wall barrier, which can prevent the solubilisation of all the intracellular proteins affecting Stock solutions were prepared with approximately 500 mL of thus the value of the nitrogen-to-protein conversion ultrapure water and some drops of 2 N NaOH to adjust the factor. Therefore, the impact of the cell wall character­ solution to pH 12. A sample of 1 g of freeze-dried biomass istics on protein extractability should be taken into was added to 50 mL ofstock solution. The mixture was heated account and analysed in order to prevent an incorrect to 40 °C with stirring for 1 h followed by centrifugation at estimation of the protein content. 5,000xg for 10 min. Samples were taken for analysis by the Multiple studies have focused on findinga methodto rec­ colorimetricmethod of Lowry et al. ( 1951 ), elemental analysis ommend the right conversion factor; for instance, Gonzalez and amino acid analysis. Lopez et al. (2010) focused on obtaining the conversion factor offive microalgae a:fterbreaking the cell wall, andthen finding Lowry method a correlation between protein contentand total nitrogencontent (elemental analysis or Kjeldahl). As a result, amongfive micro­ A calibration curve was prepared using a concentration 1 algae a new mean conversion factor was estimated to 4.44 range of bovine serum albumin from O to 1,500 µg mL- . (elemental analysis) and 5.95 (Kjeldahl). Another study In order to measure the protein content, 0.2 mL of each (Lourenço et al. 2002) determined the conversion factor for standard or samples containing the crude protein extract 19 tropical seaweeds ha:rvesteddirectly from theb each; and in a were withdrawn and then 1 mL of modified Lowry reagent second study (Lourenço et al. 1998), 12 marine microalgae was added to each sample. Each sample was then vortexed were analysedu nder differentg rowthphases anda meanv alue and incubated for exactly 10 min. After incubation, 100 µL of 4.58 was found. of Folin-Ciocalteu reagent (1 N) were added and again The following study assesses the impact of the cell wall vortexed and incubated for exactly 30 min. The absorbance on the protein extractability and the evaluation of the NTP was then measured at 750 nm. for five microalgae intensively grown worldwide and having wide taxonomie diversity. Elemental analysis Total nitrogen of the freeze-dried biomass was evaluated Materials and methods using a PerkinElmer 2400 series II elemental analyser. Samples of2 mg were placed in tin capsules and then heated The microalgae used a:re: the cyanobacterium Arthrospira at 925 °C using pure oxygen as the combustion gas and pure platensis (strain PCC 8005), two Chlorophyceae C. vulgaris helium as the carrier gas, then evaluating the nitrogen per­ (strain SAG 211-19), Haematococcus pluvialis (unknown centage and converting it into protein percentage by using strain), one Rhodophyta Porphyridium cruentum (strain the conversion factors calculated for each microalga in this UTEX 161) and the Eustigmatophyceae Nannochloropsis study. oculata (unknown stra:in). Each alga was cultivated in a different culture medium: Amino acid analysis and NTP calculation Hemerick medium for P. cruentum, Sueoka medium for C. vulgaris, Basal medium for H pluvialis, Conway medium The determination of the amino acid composition of the for N oculata, and Zarrouk medium for A. platensis. All biomass was performedaccording to a widely used standard were grown in batch mode in an indoor tubula:r air-lift method (Moore and Stein 1948). The samples were hydro­ photobioreactor (PBR, 10 L) at 25 °C (Loubiere et al. lysed with 6 N hydrochloric acid at 103 °C for 24 h. Then, 2011) after inoculation from a prior culture in a fiat panel the hydrolysed material was adjusted to pH 2.2 with 6 N air-liftPB R (1 L).