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. 2023 Aug 29;7:100581. doi: 10.1016/j.crfs.2023.100581

Microwave treatment on structure and digestibility characteristics of Spirulina platensis protein

Jian Zhang a,c, Yingying Zou a,c, Bowen Yan a,c,∗, Nana Zhang a,c, Jianxin Zhao a,b,c, Hao Zhang a,b,c, Wei Chen a,b,c, Daming Fan a,b,c
PMCID: PMC10484979  PMID: 37691697

Abstract

As a novel protein resource, the low digestibility of Spirulina platensis protein (SPP) limits its large-scale application. From the perspective of food processing methods, different heating treatments were explored to improve the structure and digestibility of SPP. In this study, SPP was heated by water bath and microwave at the same heating rate and heating temperature. Microwave accelerated protein denaturation and structure unfolded as the heating intensity increases, causing more exposed hydrophobic residues and enhancing surface hydrophobicity. The data of free sulfhydryl group, particle size, and gel electrophoresis, showed that microwave treatment promoted the formation of protein aggregates. The structural changes can potentially improve the accessibility of digestive enzymes, promote the in vitro digestibility rate, and further accelerate the production of small molecular peptides and the release of free amino acids. This study provided an innovative approach to improve the digestibility and therefore the utilization efficiency of SPP.

Keywords: Spirulina platensis protein, Microwave, Water bath, Protein structure, Digestibility

Graphical abstract

Image 1

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Highlights

  • •

    Different heating had discrepant structure characteristics of SPP.

  • •

    Microwave accelerated protein denaturation and structure unfolded.

  • •

    Structural changes of SPP increased the digestion rate in vitro.

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    SPP heated by microwave at 90 °C was conducive to protein hydrolysis and utilization.

1. Introduction

In the past few years, for the sake of the environment, health, and animal welfare, using plant proteins as a substitute for animal proteins in food industry is a new development trend. Currently, abundant plant proteins have been widely developed and applied. Besides the conventional soy, wheat, pea, rice, abundant emerging plant-based proteins, such as bean, chickpea, fungal, and algae, are constantly adopted by consumers due to their comprehensive nutrients and characteristic functional characteristics (Singh et al., 2021). As widely distributed species in the world, microalgae show strong resource-based advantages with reproductive ability, high biomass yields per unit area, cultivability on non-cultivated lands with undrinkable water, and high nutritional profile have become an important field in the development of new plant-based protein resources (Minhas et al., 2016). Microalgae abundant functional protein have attracted increasing attention. Recently, the extraction of microalgae proteins and exploitation of functional protein commercial products have created a significant achievement (Soto-Sierra et al., 2018). As an extremely important branch of microalgae, Spirulina platensis is not only rich in active ingredients, such as pigments and polysaccharides, but also has a considerably high protein content, which has been the most widely explored and applied in the food field. On the other hand, the protein content of Spirulina platensis accounted for more than 60% of the total weight, which is much higher than that of common crops, such as soybean and wheat, is a huge protein reservoir (Spolaore et al., 2006; Zhujun and Xuewu, 2017). Moreover, Spirulina platensis protein (SPP) has a reasonable amino acid ratio, strong functional activity, and can better satisfy human needs (Ejike et al., 2017). Recently, many studies have confirmed antioxidant, antibacterial, anti-inflammatory, and osteoporosis treatment abilities, in Spirulina platensis and/or active ingredient (Bortolini et al., 2022).

SPP is diverse, containing more than 2000 protein molecules, and has many specific protein molecules that endow protein with diversified properties. In the field of food, a large number of studies on the functional properties of SPP have been reported. It has good solubility, and the maximum nitrogen solubility is reported as 59.6 ± 0.7% (w/w) at suitable conditions (Benelhadj et al., 2016). In addition, SPP shows excellent gelation, emulsification, and foaming properties, which will further expand its application in the food field. However, compared with other advantages, the digestibility has become a major limitation for its application. Studies have found that the real fecal nitrogen digestibility of Spirulina is approximately 60%, which is lower than common crops such as soybean and wheat (Muys et al., 2019). Protein digestibility is correlated with its amino acids composition, and the spatial structure and chemical bond interaction of the amino acids. In general, high content of L-type amino acids, α-helices, and sulfhydryl groups, can promote the improvement of protein digestibility (Yu et al., 2021). Protein modification is a common method to improve digestibility, including physical, chemical, and enzymatic modifications (Almeida Sa et al., 2020; Yu et al., 2023). Physical modification is widely conducted due to the high safety, low cost, and short time. Exploring new physical methods in further enhancing the digestibility of microalgae protein while extracting and processing is necessary for expanding the source of protein and improving its use characteristics (Lovedeep et al., 2022).

Physical modification is mainly conducted by changing the protein structure and intermolecular forces, increasing the efficiency and functional properties of protein extraction from different sources and expanding their application in various foods. Diversiform physical treatments, including water bath (WB), ohmic heating, radio frequency, and other heat treatments, showed the improvement of protein digestibility. However, most of these methods have low processing efficiency and limited influence on protein structure and properties (Rahman and Lamsal, 2021). In-depth molecular studies link the processing to specific protein modifications that occur during digestion are limited. Microwaves (MWs) are electromagnetic waves with frequencies ranging from 0.3 to 300 GHz (Yuan et al., 2020). MW heating is a dielectric process that has been used in the food industry for many years. The special heating method may have a significant influence on the activity and structural properties of proteins and polypeptides with a high dielectric constant (Sheikh et al., 2022). Recently, MW pretreatment has shown good structural modification and improved digestibility for egg, soybean, and other protein substances (Sun et al., 2020). At present, MW accelerated maturation and protein extraction of Spirulina have been reported in the literature, but molecular studies on the relationship between the structure modification and digestibility of SPP under MW treatment are still limited.

Therefore, the aim of this study is to investigate the relations between structural characteristics and digestibility of SPP in different heating treatment. In order to accomplish this objective, laboratory cultivation of Spirulina was carried out, followed by isolation and extraction the proteins to eliminate interference from the culture conditions and other components present in Spirulina. The same microwave and water bath heating condition was set to analyze the differences of protein structure, size and distribution before and after different heat treatments and further in vitro simulated gastrointestinal digestion model was used to explore degree of hydrolysis and final digestion products. Finally, the relationship between protein characteristics and hydrolysate indices of different samples was established through correlation analysis and principal component analysis (PCA).

2. Materials and methods

2.1. Materials

Spirulina platensis was obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology, Wuhan, China. Trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) were purchased from Macklin Biotechnology Co., Ltd (Shanghai, China). 5,5-dimercapto-2,2-dinitrobenzoic acid (DTNB) was purchased from Solarbio Life Sciences Co., Ltd (Beijing, China). Ammonium 8-anilino-1-naphthalenesulfonate (ANS) was purchased from Aladdin Reagent Co., Ltd (Shanghai, China). All the other reagents were purchased from Sigma-Aldrich Pty. Ltd. (Shanghai, China). All reagents were analytically pure grade.

2.2. Spirulina platensis culture and collection

The Zarrouk medium was used for seed preservation and culture. The initial cell density was 0.1 optical density and aerobic cultivated by using an air filtered pump. The growth temperature and pH were 30.0 ± 0.2 °C and 7.5 ± 0.02, respectively, the light (L)/dark (D) ratio was 12 L/12 D, and the light intensity was an illuminance of 81.3 μmol photons m−2 s−1, which was provided by a daylight-type fluorescent tubular lamps (Rosero-Chasoy et al., 2022). After seven days of continuous culture, the cell suspension was centrifuged, and then the precipitate was collected and lyophilized at −20 °C for further study.

2.3. Preparation of SPP

The extraction of SPP concentrate was performed according to the method of Benelhadj et al. with some modifications (Benelhadj et al., 2016). After 15 min of mortar grinding, Spirulina freeze-dried powder was dissolved in distilled water with the ratio of 1:50 (m/v), and the pH was adjusted to pH 8 with NaOH (0.5 M). After magnetic stirring (3 h and 20 °C) and centrifugation (10,000 g, 30 min, and 20 °C), the supernatant was collected (supernatant A). The pellet was re-dissolved in deionized water, the pH was re-adjusted and centrifugated under the same conditions followed by the supernatant collection (supernatant B). By combining the supernatant, the pH was adjusted to pH 3 with 0.1 M HCl. Then, the precipitated protein was collected through centrifugation (18,500 g, 30 min, and 25 °C). The precipitated protein was resuspended into deionized water and adjusted to pH 7.0 and diafiltered through a 1 kDa membrane. Finally, the protein solution was freeze dried and stored at − 80 °C.

2.4. Preparation of heated SPP solution

The appropriate amount of freeze-dried powder was dissolved in deionized water to reach the concentration of 12 mg/mL. Then, each tube of 5 mL protein solution was heated in a WB at temperatures of 40, 50, 60, 70, 80, 90, or 100 °C, for 25 min, respectively. A thermocoupler was used to record the temperature throughout the heating process. Similarly, the protein solution was added to a quartz tube as previously above and placed in an all-in-one MW Synthesis Labstation of Milestone (Milestone Srl, Sorisole, Italy). The device was programmed based on the temperature curve of the WB to ensure an equivalent heating rate. Therefore, the solution was heated via MW at 40, 50, 60, 70, 80, 90, or 100 °C, for 25 min, respectively. After the completion of heating, the samples were quickly cooled and placed in −20 °C refrigerator.

2.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)

The protein composition of Spirulina after heating in different temperatures and times, as well as the disappearance of each sampling point after digestion, were analyzed by SDS-PAGE using a 12% polyacrylamide gel (Benelhadj et al., 2016).

The presence and absence of beta-mercaptoethanol as the reducing agent were conducted in this study. Gels were run with MOPS SDS running buffer at a constant 80 V for the spacer phase and 120 V for the separation process. Then, gels were stained with Coomassie brilliant blue R-250 for approximately 1 h, followed by destaining in washing buffer (distilled water with 7.5% acetic acid and 10% ethanol) overnight.

2.6. Surface hydrophobicity

The heat-treated and untreated protein solutions were diluted by gradient using deionized water to 0.015625, 0.03125, 0.0625, 0.125, and 0.25 mg/mL. Then, each 4 mL protein solution and 40 μL of 8 mM ANS were mixed and left to react in the dark for 15 min. The initial calculated data of surface hydrophobicity were captured by the fluorescence spectrophotometer. The excitation and emission wavelengths were 380 and 470 nm with a slit correction of 0.5 mm, respectively. Similar ultrapure water and ANS were used as the blank control. The protein surface hydrophobicity was determined using the initial slope of the relative fluorescence intensity vs. protein concentration (Wang et al., 2022).

2.7. Circular dichroism

The heat-treated and untreated protein solutions were adjusted to 0.2 mg/mL with deionized water. Placed in a 1-mm quartz optical path absorption cell, the samples were scanned using the circular dichromatic spectrometer over a wavelength range of 190–260 nm at a scanning speed of 100 nm/min and scanning interval time of 1 s. The mean residual ellipticity [θ] (°·cm2/dmol) was used as the characteristic index of circular dichroism in the protein structure and the protein secondary structure content of each sample was calculated by Dichroweb online software with Selcon3 Programme (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml).

2.8. Particle size distribution

The mean particle diameter of the untreated and heat treat samples with 0.5 mg/mL adjusted by deionized water were measured using a Zetasizer Nano ZS particle size analyzer (Malvern Instruments Ltd., Malvern, Worcestershire, UK). A 633-nm laser and a 90° scattering angle were used in 25 °C, and the measurement time was 2 min to detect the intensity of the scattered light. The particle distribution and size were determined through a cumulative analysis. The average value of sample triplicate measured value was calculated.

2.9. Free sulfhydryl content and total sulfhydryl content

The free sulfhydryl and total sulfhydryl content were determined using the method of Beveridge et al. with slightly modified (Beveridge et al., 1984). The protein solution heated by MW and WH was diluted to 8 mg/mL. For the free sulfhydryl measure, 1 mL protein solution was speedy mixed with 4 mL of Tris-glycine buffer (containing 8 mol/L urea, pH 8.0) and 0.05 mL of 4 mg/mL DTNB. The mixture reaction was performed for 30 min at room temperature without light, and then the absorbance value was measured at 412 nm. All measurements were repeated three times. The content of free sulfhydryl was calculated as follows:

freesulfhydrylgroups(μmol/g)=73.53ASH*D/CS,

where CS is the sample concentration (mg/mL), ASH is the difference value of the sample and the blank solution at 412 nm, and D is the dilution factor for free sulfhydryl is 5.02.

For the total sulfhydryl content measure, 1 mL protein-containing buffer was mixed with 0.05 mL β-mercaptoethanol and 4 mL Tris-glycine buffer. The mixed solution was performed for 1 h before centrifugation (5000 g and 10 min). Subsequently, 10 mL Tris-glycine buffer and 0.04 mL of dithio-nitrobenzoic acid were added and mixed. The mixture reaction was conducted for 30 min at room temperature without light, and then the absorbance value was measured at 412 nm.

The content of total sulfhydryl was calculated as follows:

totalsulfhydrylgroups(μmolg)=73.53ASH*DCS,

where CS is the sample concentration (mg/mL), ASH is the difference value of the sample and the blank solution at 412 nm, and D is the dilution factor for the total sulfhydryl is 10.

2.10. Imaging microstructures of proteins by scanning electron microscopy (SEM)

The microstructures of SPP were visualized by SU8010 field-emission scanning electron microscope (Hitachi, Japan). The protein solution under each heating treatment was diluted to a concentration of 2 mg/mL. Then, a small amount of solution was titrated in the silicon wafer and naturally dried. Then, samples were sprayed with gold and placed on a carbon disk stuck to an aluminum stub and on the “Charge Reduction Sample Holder”. SEM images were acquired using a high sensitivity multi-mode backscatter electron (BSE) detector at 3 kV and 45 k or 90 k magnification.

2.11. In vitro simulated gastrointestinal digestion

According to the study of M. Minekus et al. (2014), preparing stock solutions of simulated digestion for 1.25 times and conducting standardized static in vitro digestion for untreated and heated samples with protein concentration of all samples was 12 mg/mL. In gastric phase, 10 mL of protein solution was mixed with 8 mL of simulated gastric fluid and 2 mL of deionized water containing 40000 U porcine pepsin and 1.5 mM CaCl2. The pH was adjusted to pH 3 with 1 M HCl. Then, the mixture was placed in a bed temperature incubator at 37 °C for 2 h. During the digestion process, 5 mL of solution was collected in 1 and 2 h. Additionally, gastric digestion was stopped by adjusting the pH to 6.6 using 1 M NaOH and temporarily stored at −20 °C. After the gastric phase, the remaining 10 mL digestive juice of each sample was mixed with 5.5 mL simulated intestinal fluid. Then, 2.5 mL simulated intestinal fluid containing trypsin solution of 800 U/mL and 1.25 mL fresh bile (160 mM in fresh bile), and 20 μL of 0.3 M CaCl2 were added. After neutralizing the mixture to pH 7.0 by 1 M NaOH, the remaining volume was supplemented with pure water to reach 20 mL final volume. Similarly, samples were placed in a bed temperature incubator at 37 °C for 2 h and collected at 1 and 2 h. Moreover, 5 mM Pefabloc® SC PLUS was used to stop intestinal digestion in each collection and stored in −20 °C for further experiments. Digestions were independently performed in duplicate.

2.12. Degree of protein hydrolysis (DH)

The hydrolysis degree of the sample was determined by o-phthaldialdehyde (OPA) reagent reaction, which consisted of OPA, di-Na-tetraborate decahydrate, SDS, dithiothreitol (DTT), ethanol, and Milli-Q water. The two phases digestion samples were sequentially diluted to 1 mg/mL with Milli-Q water, which were then centrifuged (Centrifuge 5430 R, Eppendorf, Germany) at 10,000 g for 5 min at 23 °C. Then, the supernatant was collected for DH determination. Moreover, 225 μL of OPA reagent was mixed with 30 μL sample supernatant or 0.1 mg/mL serine standard or Milli-Q water in a 96-well microplate. The mixture was incubated at 25 °C for 2 min with shaking and incubated at 340 nm with a Spark multimode microplate reader and DH was calculated as follows:

DH(%)=hhTotal×100
h(meqv/gprotein)=Serine−NH2−βa
Serine−NH2=(Asample−AblankAstandard−Ablank)×0.9516meqv/L×0.1×100X×P

2.13. Molecular weight distribution in simulated intestinal fluid

Gel-filtration chromatography on a Superdex Peptide HR 10/300 column was used to separate peptides from the hydrolyzed products. The mobile phase consisted of 70% (v/v) Milli-Q purified water, 30% (v/v) acetonitrile with 0.1% TFA. The 2-h simulated intestinal fluid was eluted by the mobile phase at the flow rate of 0.5 mL/min, and compounds were detected by recording the absorbance at 214 nm. Bovine serum albumin (66.0 kDa), vitamin B (1355 kDa), and tyrosine (181 kDa), were used as the calibrator for the molecular weight.

2.14. Determination of free amino acids in simulated intestinal fluid

Here, 1 mL simulated intestinal fluid after 2 h digestion was mixed with an equal volume of 10% TCA and let stand for 1 h to remove large proteins. The mixture was centrifuged at 15000 rpm for 30 min. Then, the composition and content of free amino acids were determined by reversed-phase high performance liquid chromatography (Agilent 1260). Amino acid separation was achieved using Agilent Hypersil ODS column (5 μm，4.6 × 100 mm) and a binary gradient system. Solvent A was sodium acetate, triethylamine, and tetrahydroquantine (500/0.11/2.5, v/v/v), at pH 7.2, and Solvent B contained sodium diacetate and methanol-acetonitrile (1/2/2, v/v/v) at pH 7.2. The detection wavelength of UV detector (VWD) was 338 nm, and amino acid content was determined by the external standard method basing 17 amino acid standards.

2.15. Statistical analysis

Sample preparations were conducted in triplicates. The results were expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) or an unpaired t-test using IBM SPSS Statistics 19 (SPSS Inc., USA). Significant differences were defined at a 5% level (P < 0.05). Correlation analysis with protein structure characteristics and simulated intestinal fluid parameters was carried out by Pearson's test. Also, PCA was used to understand the impact of different heat treatment on different properties of SPP samples using R with packages factoextra and FactoMineR.

3. Results and discussion

3.1. SDS-PAGE analysis of heated and unheated protein

Protein gel electrophoresis is the most common method to determine the molecular weight of protein components. SDS-PAGE was conducted under both reducing and non-reducing conditions to understand the differences in the subunit composition before and after heating.

The electrophoretogram revealed that un-heated protein with a molecular weight of 100–10 kDa were strongly involved, showing several major bands at MW of 70, 40, 28 and 18 kDa (Fig. 1). Among them, the most prominent one was occupied around 15–20 kDa, which is composed of phycocyanin (Benelhadj et al., 2016). Phycocyanin is a brilliant blue water-soluble pigment protein consists of a monomer formed by two helix-shaped subunits, called alpha (15.4 kDa) and beta (17.3 kDa) subunits (Zheng et al., 2020). These subunits associate into (αβ) monomers, followed by the aggregation into (αβ)3 trimers and (αβ)6 hexamers (Padyana et al., 2001). The results of gel distribution showed that the monomer structure of phycocyanin was degraded to helix-shaped subunits after protein extraction. Compared with un-heated sample, the distribution of molecular weight was significantly changed under two heat treatments. The two heating methods did not cause large-scale aggregation of protein molecules when the heating temperature was below 70 °C. At this time, the protein molecules were relatively stable, and the structure of small molecules was not significantly damaged, which is consistent with the stability of phycocyanin (Zhang et al., 2023). With the increasing of temperature, the aggregation of protein molecules occurred at the top of the wells. Moreover, the aggregation degree gradually increased as the heating temperature increased, corresponding to the decrease of protein near 15 kDa (Fig. 1A). This showed that thermal treatment destroyed the protein subunits and had tremendous impact on the protein's primary structure. With the increase of the heating temperature, the small molecular chains of SPP unfold, which may produce the smallest net irreversible aggregates whose nuclei aggregate through covalent and/or non-covalent interactions and condensation (Nicolai and Durand, 2013). However, compared with traditional heating, MW treatment showed a greater degree of protein aggregation, which may be closely related to the high-frequency vibration of protein molecules under the MW effect (Fig. 1A). MW resulted in a fast exposure of protein groups and the dissociation and recombination through hydrophobic interaction and sulfhydryl/disulfide bond exchange reaction, resulting in the formation of random structural aggregates. Similarly, when MW heating pigeon pea protein, Sun et al. found that the degree of protein aggregation was clearer under the MW compared with soaking and ultrasound (Sun et al., 2020).

Fig. 1.

Fig. 1

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SDS-PAGE patterns of SPP composition under reducing (A) and non-reducing (B) conditions, at un-heated (NT) and heating treatment of 50–100 °C MW/WH. Lane M: molecular weight marker from 10 to 250 kDa.

As a reducing agent, mercaptoethanol can reduce the degree of protein aggregation by opening the disulfide bond between protein molecules. In our study, many aggregates at the top of the wells were lost and small molecular weight molecules increased in the reducing gel with mercaptoethanol under two heating methods, indicating that these aggregates were formed by disulfide rearrangement (Fig. 1B). However, when the temperature exceeded 90 °C, the MW treated protein still showed significant aggregation at the top of the wells, indicating that the structure of SPP molecules may be greatly destroyed by MW under overheating condition, resulted to a change in the way of intermolecular aggregation. Under MW treatment, protein-protein interaction, including electrostatic interaction, hydrophobic interaction, and hydrogen bonding can be disrupted, leading to alterations in protein binding patterns and protein aggregation. (Huang et al., 2022).

3.2. Particle size distribution

SDS-PAGE results show the aggregation of protein with heat treatment. The light scattering intensity was measured by dynamic light scattering technique to reveal the particle size distribution of protein and better illustrate the degree of protein aggregation. As previously described, the results of both thermal treatment increased protein aggregation, leading to the larger particles in solution. In the control sample, SPP exhibited three stable peaks with mainly particle sizes of 1, 30, and 2300 nm, among which the particle size of 1 nm was dominant, accounting for approximately 50% of the total volume of all particle sizes. Similarly, Ahmed & Kumar (2022) also found a unimodal distribution curve with two small shoulders in Spirulina, whereas, the peak of particle size had imparity between two studies, the inconsistent findings could be due to the differences in the Spirulina sources and protein extraction ways between their study and the present study. With the increasing of heating temperature, heat aggregation leads to the gradual increase of the particle size under the two heating treatments, and the distribution and width of the peak value render the differences (Fig. 2). With the increasing of bath temperature, the three protein peaks shifted to the right, and the peak value at 1 nm gradually decreased, while peak value of 1300 nm gradually increased (Fig. 2A). Although volume of the small size peak constantly decreased, the maximum size peak did not increase when the temperature raised to 90 °C, accompanied by the formation of a new peak near 600 nm (Fig. 2A). One factor may be that the small particle size no longer completely aggregated to the formed large particles, and some small and medium particles may aggregate together to form new peaks. Similar to WH heating, the main peaks at 1 and 2300 nm shift to the right under MW, while the small volume peaks at 30 nm did not regularly change (Fig. 2B). Conversely, although there were new peaks near 600 nm with rising temperature, large particles tend to form with a high temperature. At the temperature of 100 °C, the maximum volume peaks at 4800 nm, surpassing the size of WH heating significantly (Fig. 2B).

Fig. 2.

Fig. 2

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Structure characteristics of SPP at un-heated (NT) and heating temperature of 50–100 °C. Particle size distribution under WB (A) and MW (B). Surface hydrophobicity(C). Total sulfhydryl groups (D). Free sulfhydryl groups (E). Circular dichroism and secondary structure content under WB (F, H) and MW (G, I). Different letters indicate significantly different mean values (P < 0.05). Different superscript letters indicate significant differences between groups (p < 0.05); * indicates significant differences within groups.

When the temperature is mild, the particle size distribution under the two treatments present similar characteristics, and the thermal effect played a major role. However, with a high heating temperature, the large particle size under MW may be related to the special heating mode (Mondal et al., 2010). The structure was damaged fast under MW treatment due to the large number of polar groups in SPP, resulting in the exposure of polar molecules. Then, the rapid collision among polar molecules lead to the gradual aggregation of particles, forming the morphology of flocs. Moreover, the molecular clusters in the flocs were further polarized, leading to the change of the morphology and surface area of the flocs and increasing the binding sites and binding modes of small molecular particles, which also resulted in the enlargement of the flocs (Cheng et al., 2013). Different flocs may be further combined and twine to form further aggregation due to the difference of structure and charge, increasing the particle size.

3.3. Surface hydrophobicity

Heat treatment could destruct the protein tertiary structure, leading to the change of the state of the hydrophobic group. Adjusting the solution properties or temperature caused the change of conformation, and the exposure of hydrophobic amino acid residues increased surface hydrophobicity. Hydrophobicity, which is an important property, played an important role in the stability, conformation, and digestive characteristics of protein molecules (Wei et al., 2018). In Fig. 2C, under the two thermal treatment, the surface hydrophobicity increased as the temperature increased, which was consistent with other plant proteins such as soy and pea protein (Tang et al., 2022). However, at a temperature of 100 °C, the hydrophobicity of the MW treatment did not increase, whereas, the hydrophobicity of the WH treatment continued to increase. This phenomenon may be related to the strong aggregation of protein. The aggregation of protein caused the hydrophobic groups to be buried, reducing the surface hydrophobicity. Moreover, substantial accumulation of hydrophobic groups brought by hydrophobic interactions may cause “hydrophobic collapse”, which may bury binding sites again (Brylinski et al., 2006).

When the temperature is between 70 and 90 °C, compared with WH, MW caused more exposure of hydrophobic groups under the same temperature conditions, which may be related to the “limit pulling force” on protein molecules. With the electromagnetic oscillation, ionic and non-covalent bonds in protein structures are strongly damaged, rapidly exposing the hydrophobic groups (Pooventhiran et al., 2020).

In this study, the formation of large soluble aggregates during heat treatment may reduce the hydrophobicity. However, their contribution did not exceed the exposure of hydrophobic groups, as shown by the continuous increase in hydrophobicity.

3.4. Free sulfhydryl groups and total sulfhydryl groups

The sulfhydryl group is involved in the weak secondary and tertiary structures of the protein. During heat treatment, the denaturation of protein will destroy the covalent interaction, making the sulfhydryl group reacting easily. With the increase of the temperature, the content of total sulfhydryl group decreased under the two heating methods (Fig. 2D). At 50 °C, the number of sulfhydryl groups did not significantly change, which was related to low protein aggregation, in which the protein structure was hardly destroyed, and disulfide bonds were difficult to establish.

When the temperature exceeds 60 °C, the thermal effect leads to the destruction of the protein structure and promotes aggregation, in which disulfide bond formed by exposed sulfhydryl group is the main mode of protein aggregation. During MW heating, the polar substrate could strongly capture MW energy, increasing the chance of collision of some molecules and resulting in high destruction of the protein structure and less sulfhydryl groups. Furthermore, the total sulfhydryl content was reduced in 100 °C. Although with an apparent difference between the MW and WH treatment, the gap between them was relatively narrow compared with 80 and 90 °C (Fig. 2D). At 100 °C, the oxidation of sulfhydryl groups or the exchange of sulfhydryl disulfide bonds in the two ways triggered the semblable polymerization reaction and may lead to the same downward trend (Zhou et al., 2020). On the other side, MW may accelerate aggregation through the interaction between other protein groups. Although MW leads to more exposure of sulfhydryl groups, the other ways of aggregation made sulfhydryl groups buried again.

As reduced sulfhydryl groups, the exposure of free sulfhydryl groups did not result in the formation of disulfide bonds. Under the two heating methods, the content of free sulfhydryl group showed the same trend: first stable, then rising, and finally falling (Fig. 2E). The low temperature did not cause significant structure changes and exposure of internal free sulfhydryl groups. As the temperature increased, MW treatment speeded up the intense movement and friction inside the protein molecules and exacerbated structural damage, allowing the protein to completely unfold and exposing more active sites and sulfhydryl groups on the polar surface of the protein molecule. As the temperature continuously increased, the degree of protein aggregation was deepened, and some sulfhydryl groups were buried.

3.5. Effect of heating on the secondary structure

Under heating treatment, the decomposition and polymerization of non-covalent bonds, such as disulfide bonds and sulfhydryl groups, contribute to the unfolding of the protein structure and the constant change. The secondary structure refers to the spatial arrangement of atoms on the polypeptide main chain, whose variation is regularly evaluated by the content of α-helix, β-sheet, β-turn, and random coil conformations. In this study, the circular dichroism spectrum in the far ultraviolet region was used to evaluate the secondary structure changes under two heating methods, and the Selcon3 program was used to predict the secondary structure content of different samples.

Similar to most protein structures, CD spectra showed an apparent positive peak near 196 nm, which is unique to the β-sheet structure, and a negative peak near 208 and 222 nm, close to the main secondary structure of α helix in Spirulina, in which the peak sharpness constantly decreases as the temperature increases. Heating significantly altered the CD profile of the natural SPP, which is consistent with the study of L. Böcker (Boecker et al., 2020) who reported that different heat treatments affect the secondary structure. In our study, the protein secondary structure showed a similar transformation law under two treatments. The ratio of the helices and the strand decreased, and the protein state tends from order to disorder, which may be due to the degeneration caused by the depressed protein stability (Fig. 2F and G).

These results were enhanced by quantifying the protein secondary structure using an online software, which exhibited quantitative relationship between the transformation proportions of a typical secondary structure and ascending temperature. In Table 1, the content of α-helix reached 58% without heating, and the α helix content rapidly decreased as the temperature increased. The α helix content was transformed into β-turn and random coil conformations, and the effect of MW was more significant. Without heating, α-helix occupied the dominant position with content of 58%. With the increase of temperature, the helix content rapidly decreased and transformed into β-turn and irregular curling structure, and the effect of MW was more significant. MW energy induces the vibration of polar groups lead to the generation of free radicals, destroying multiple interactions between protein molecules, including van der Waals forces and hydrogen bonds, which result in the reduced ordered structure and frail stability of the protein. In addition, the alternating electric field can superimpose the dipole moment of the peptide bond, causing great damage to the α-helix.

Table 1.

Secondary structure content (%) in SPP under different treatment samples.

secondary structure content (%) NT MW 50 °C MW 60 °C MW 70 °C MW 80 °C MW 90 °C MW 100 °C WH 50 °C WH 60 °C WH 70 °C WH 80 °C WH 90 °C WH 100 °C
Helix 57.7 56.7 52.7 42.8 29.6 27.2 21.5 56.6 56.7 51.8 38.2 29.9 26.7
Strand 4.3 5.2 5.2 9.3 8.3 7.3 6.1 5.4 5.4 5.8 6.6 3.5 2.5
Turns 18.7 18.5 19.6 23 25.6 28.2 31.9 18.5 18.5 18.9 22.1 27.1 29.6
Unordered 19.5 19.2 21.7 24.6 35.7 37.2 39.8 18.9 18.8 22.6 32.2 39.3 40.3
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Simultaneously, the β-sheet content increased first and then decreased with the rising temperature, and it reached the maximum content of 9.3% in MW treatment of 70 °C and 6.6% in 80 °C WH (Fig. 2H and I). This feature may be related to the denaturation degree of SPP at different temperatures. When the temperature was low, the partial helical structure was transformed into strand during the unfolding process, increasing the sheet content. However, the elevated temperature accelerated the exposure of the hydrophobic groups buried in the strands structure, reducing the content of sheet (Zhong et al., 2018). In addition, the sheet content reached the peak value at a low MW temperature, which may be due to the continuous high frequency movement of polar groups in the helical structure made the molecular structure bend, resulting in the appearance of sheet structure, and sheet molecule was rapidly destroyed with a high MW energy.

3.6. Imaging microstructures of Spirulina isolated proteins by SEM

The microscopic structure of SPP showed significant differences at 45-k magnification under different treatment samples (Fig. 3). In the unheated sample, the surface was regular and relatively smooth, with a major distribution form of clustered long strips (Fig. 3M and N). This may be related to the structural and functional phycobilisome, which contains more protein molecules, such as phycocyanin and allophycocyanin, to form a columnar or double cylindrical structure. In addition, although the protein molecules were stacked together without heating, the distribution was uniform with a low cross-linking degree (Fig. 3M and N). Heating treatment resulted in significant aggregation of proteins. With increasing temperature under two heat treatments, the smooth morphology of protein molecular surface was destroyed and became rough, and cracks and pits appeared (Fig. 3A–L). Protein molecules showed distinct cross-linking, and the degree was deep under MW treatment (Fig. 3K and L), which is consistent with the change of protein structure. Heat treatment, especially the special thermal method of MW, caused the destruction of protein secondary structure and the continuous stretching of protein molecules, leading to the aggregation of protein molecules. A large number of honeycomb structures appeared in the aggregates as the temperature increased, and the intergroup boundary became more distinct due to the change of protein structure and interaction forces.

Fig. 3.

Fig. 3

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SEM images of SPP by WH treatment at 50 °C (A), 60 °C (B), 70 °C (C), 80 °C (D), 90 °C (E), 100 °C (F), MW treatment at 50 °C (G), 60 °C (H), 70 °C (I), 80 °C (J), 90 °C (K), 100 °C (L) and un-heated treatment (M, N). The images were acquired using a Phenom ProX Desktop SEM system equipped with a backscatter electron detector at 3 kV and at45k × or 90k × magnification.

3.7. Degree of protein hydrolysis after thermal treatment

Protein digestion is a necessary step of absorption and use. As a rapid and reliable method, DH is used extensively to evaluate the efficiency of enzymatic hydrolysis reaction in vitro. The amino acid composition and sequence characteristic and the position of the peptide bond and its sensitivity to protease lead to the difference in the degree of hydrolysis. Under optimal environmental conditions, the hydrolysis of protein was captured by the sequential treatment of pepsin and trypsin and the reproducibility of the hydrolysate.

Pepsin in the protein solution simulated the process of human gastric digestion. As shown in Fig. 4A, the overall hydrolysis degree of protein during gastric juice digestion was low. At 1 h of digestion, although heating treatment improved the digestion degree, there was no significant difference among various treatment conditions, and the overall degree of hydrolysis was below 5%, which may be related to the short hydrolysis time. Simultaneously, OPA reagent may not effectively derive insoluble peptides or proteins, the formation of aggregates may lead to the initial weakness of OPA signal during gastric digestion. After 2 h of hydrolysis of pepsin, the enzyme molecule would strongly bind to the substrate and improve the protein digestibility on a wide range. The rise in temperature resulted in a high degree of hydrolysis (Fig. 4A), which could be attributed to the expanded protein structure, providing more accessible sites for enzymes and facilitating enzymatic hydrolysis.

Fig. 4.

Fig. 4

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The DH of SPP under WB (A) and MW (B) treatment at heating temperature of 50–100 °C. Different letters indicate significantly different mean values (P < 0.05). Different superscript letters indicate significant differences between groups (p < 0.05); * indicates significant differences within groups.

Pepsin has a certain amino acid sequence specificity in the cleavage of proteins, tending to split peptides with aromatic amino acids at the amino or carboxyl terminus. Although heating increases the degree of protein aggregation to some extent, heat treatment lead to molecular unfolding, exposing more sulfhydryl group and hydrophobic groups, changing the molecular conformation, enabling more enzymes to bind with characteristic peptide bonds, and improving the digestibility (Bhat et al., 2021). At 100 °C, the DH treated with WH decreased while the sample under MW increased, showing a significant difference. At high temperatures, the degree of protein aggregation was further deepened, and the mass of peptide bonds were folded and buried, decreasing the degree of hydrolysis. Meanwhile, although the aggregation degree was deepened, the vibrations of polar groups under the MW alternating electric field drove the movement of small molecules fragments and more peptide bonds could be bound by pepsin.

After 2 h of pepsin hydrolysis, trypsin and other substances were added to the protein solution for further hydrolysis at pH 7.0. Nevertheless, the DH sharply increased in the intestinal digestion (Fig. 4B). After 1 h of hydrolysis of trypsin enzyme, the digestibility of the unheated protein was 35%. The digestibility under two treatments increased as the pre-treatment increased. At 100 °C, the digestibility of WH sample was 55%, while that of MW was 58% (Fig. 4B). After 2 h, the digestibility was further improved and most proteins were hydrolyzed with digestibility reached 90% after MW treatment, which was higher than 5% of the WH sample. It indicated that the addition of simulated intestinal fluid rapidly hydrolyzed the relatively complete protein by trypsin and continued to hydrolyze into small peptides during intestinal digestion. The appearance of this result was related to the molecules unfolding and the apparent uneven structure under heating conditions. The restructure of the apparent protein structure and the increase of hydrophobic groups combines more hydrolysis sites with enzymes to accelerate the reaction process (Guijiang et al., 2020). On the other hand, protein digestibility is a factor related to the protease inhibitor. Many studies have shown that the activity of the protease inhibitor is restricted under high temperatures, and the inhibitory activity decreases as the target temperature increases, improving protein digestibility (Roy et al., 2010). Under the MW oscillating electric field, the movement and collision of polar molecules caused the destruction of secondary and tertiary structures, resulting in the destruction of trypsin inhibitor and trypsin binding sites and reducing the inhibitory activity. Meanwhile, the increase of MW temperature may accelerate the conformational change of trypsin inhibitor, leading to a more rapid sulfhydryl exchange reaction and sulfhydryl oxidation reaction and destructing the trypsin inhibitor. The decreased trypsin inhibitory activity increased the protein digestibility as the target temperature increased.

3.8. Molecular weight distribution after simulated gastrointestinal digestion

In GPC analysis, the molecular weights of SPP decreased over time. After 2 h of intestinal digestion, protein was sufficient hydrolyzed, and the peptides showed a tendency of multi-molecular weight distribution. The molecular weight of most peptides was small, and the peptides greater than 3000 Da accounted for less than 10% under each treatment condition (Fig. 5A and B). With the increase of the heat treatment temperature, the enzymatic hydrolysis was fast and the hydrolyzed product was more thorough. As Fig. 5C, in the untreated sample, the proportion of peptides below 1000 Da was 70.11%; under two heat treatments, the composition of small molecule peptides continuously increases, accounting for 77.94% of small molecule peptide in MW treatment and 75.65% in WB at 100 °C. This was consistent with the result of the degree of hydrolysis. Heating caused great damage to the structure of protein molecules, which was more conducive to the binding of enzyme and protein. Additionally, the special heating method of MW leads to the exposure of more active sites, prompting the hydrolysis more adequate.

Fig. 5.

Fig. 5

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SPP hydrolysate characteristics at un-heated (NT) and heating temperature of 50–100 °C. Molecular weight distribution of peptides in WH (A), MW (B), variation trend of total small molecular weight peptides (<1000 Da) (C). Total free amino acids (D), essential amino acids (E) and umami amino acids (F) distribution. Different letters indicate significantly different mean values (P < 0.05). Different superscript letters indicate significant differences between groups (p < 0.05); * indicates significant differences within groups.

3.9. Composition and distribution of free amino acids in simulated intestinal fluid

The composition and content of free amino acids in the hydrolysate after intestinal digestion of 2 h were determined, which are primary evaluating means of protein bioavailability, absorption rate, and metabolic pathway, to further understand the protein hydrolysis process of SPP. HPLC showed that the contents of Asp, Glu, and Arg, had high amino acid content, while Thr, His, and Met, were relatively low (Table S1), which was consistent with the study of Campanella et al. (1999).

Compared with the untreated sample, after protein hydrolysis, the content of free amino acids in the solution increased as the pretreatment temperature increased (Fig. 5D). Similarly, the content of free amino acids in MW pre-treatment samples was significantly higher than that in WH samples when the temperature exceeds 80 °C, which was consistent with the result of hydrolysis degree. This may be attributed to the fact that MW protein samples have more cleavage sites, leading to more peptide chain breaks, and the small molecule peptides are hydrolyzed to release more free amino acids.

The content and proportion of amino acids are important means to evaluate the quality of amino acid. As a representative microalgae, the amino acid quality of Spirulina has long been confirmed and amino acid pattern compares favorably with that of the criterion recommended by WHO/FAO (Joint and Organization, 1973) and most plant proteins. The transformation of total content of essential amino acids, including Thr, Val, Met, Phe, Ile, Leu, and Lys, showed a similar pattern as total free amino acids with rising temperature.

In the unheated sample, the content of essential amino acids was 6.12 mg/g, accounting for 16% of the free amino acids. The content of essential amino acids gradually increased and reached 15.89 mg/g as the pretreatment temperature increased, accounting for 27% in 100 °C for water bathing, and 21.34 mg/g, accounting for 32.8% in 100 °C MW (Fig. 5E). The increase of the MW temperature leads to the change of protein structure, such as glycosylation, oxidation, deamidation and other modifications in local regions of protein, which will inevitably change the restriction site and the type and content of peptides, causing the difference of the content and type of free amino acids and improving the use rate of amino acids ulteriorly (Stadtman and Levine, 2003).

Different amino acids cause special foods have various tastes, such as sweet, bitter, and umami. Umami is central feature of Spirulina, which is composed of Asp, Glu, Gly, and Ala. The increase of its content contributes to the flavor related characteristics of seafood and is responsible for the special taste of seaweed. In our study, the umami amino acids were 22 mg/g in the un-heating sample. Umami amino acids increased; however, no significant differences among different temperatures and treatments (Fig. 5F). This may be related to the cleavage sites and hydrolysis rates of different amino acids. Umami amino acids may be more exposed to the protein surface, which is conducive to the rapid cutting and release of protease cleavage. In addition, hydrolysis for 2 h may be sufficient for the complete hydrolysis of the umami related peptide in the protein, and the final free amino acid content remains unchanged. Umami amino acids play a crucial role in the aroma of spirulina. Understanding the content t variation of umami-related amino acids is essential for elucidating the mechanisms underlying its flavor characteristics and assessing the quality of associated products. Additionally, it can offer valuable insights for future studies focusing on enhancing the taste and flavor of spirulina-based food products through the regulation of amino acid content.

3.10. Comparative analysis of protein characteristics and hydrolysate indices with different samples

Correlation analysis of different indicators can provide clear and accurate information and reveal the correlation and trend of variables (Granato et al., 2014). To better understand the interdependence between the intrinsic characteristics of heat treated proteins and hydrolysate indicators, this study investigated the relationship between structure and digestion by correlating quantifiable structural indicators such as protein hydrophobicity, secondary structure, and sulfhydryl content with DH and the properties of hydrolysate after simulated digestion. From Fig. 6A, it can be visualized that each structural parameter had a consistent correlation with all the hydrolysate properties, and the largest correlation was with small molecular peptides, which indicated that the structural changes were more conducive for the production of small molecules in the proteolytic solution. Similarly, a significant positive correlation was observed between protein surface hydrophobicity and the hydrolysis process, indicating that the increase in protein hydrophobicity was the main factor driving the hydrolysis. Additionally, a negative correlation was observed between sulfhydryl content and hydrolysis, with a higher negative correlation for total sulfhydryl groups, suggesting that free sulfhydryl groups may play a secondary role in the detection and evaluation of the bioaccessibility of SPP products. Furthermore, the relationship between protein secondary structure and digestive characteristics was clearly, with higher correlation between helix, turns and unordered compared to strand structure, which reinforces that the transformation of helix rather than strand structure may promote the digestive efficiency. The results of the correlation analysis demonstrated that the increase of protein hydrolysis degree and hydrolysis products were directly related to the observed directional changes of surface hydrophobicity, secondary structure and tertiary structure. This implies that future studies could establish a mathematical quantitative relationship between heating mode, energy output, and protein structure to enhance protein digestibility with greater precision and efficiency (Dobson, 2019).

Fig. 6.

Fig. 6

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Comparative analysis of association patterns in protein property and hydrolysate indices under different heating treatment sample. (A). Correlation heatmap between structure parameters and digestibility characteristics. The proportional color of the circles represents the strength of the linear pearson correlation coefficient and whether it is positive (blue) or negative (light blue),asterisks (*) show significant correlations: * P-value <0.05; (B). Biplot of objects (samples) and component loadings (protein and hydrolysate characteristics). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The calculation of the interrelationships between different indicators of the sample is valuable for providing an overview of the changes in the sample under different conditions and identifying distinct patterns of behavior. Moreover, PCA method could effectively reduce the initial variables of the sample to a smaller number of principal components which allows for a visual exploration of the differential relationships between variables and factors. Fig. 6B showed the correspondence between different feature indicators and scores, specifically if the loading map and the score vector are unidirectional, it indicates a positive correlation, the opposite is a negative correlation, and the orthogonal position is not correlated. And the correlation between different heat treatment conditions (scores) and structural and digestive characteristics of the proteins (loading plots) was 93.2%. Protein surface hydrophobicity, amino acid content, small molecule peptide, secondary structure of unordered and turns exhibited a positive correlation with higher treatment temperatures. Furthermore, this correlation was stronger under microwave conditions at the same temperature, suggesting that higher-intensity microwave treatment resulted in more significant alterations in the target structure of SPP. Conversely, total sulfhydryl and helix exhibited a substantial negative correlation with high temperature, indicating that high temperature adversely affects their stability. In contrast, the correlation between free sulfhydryl and strand and high temperature was weaker. On the other hand, for the same temperature and heating time, the score and position matrices of the microwave and water bath samples exhibit significant differences. It was evident as the temperature gradually increases, the distance between the positions of the microwave and water bath apparatus becomes more significant and microwave has a higher score, visually demonstrating the distinct heating patterns of the two ways and higher microwave efficiency. Moreover, when the microwave heating temperature reached 90 °C, the sample score exhibited a similar trend as the loading graph, indicating that this condition was conducive to protein hydrolysis and utilization.

4. Conclusions

As an emerging protein resource, the processing method of SPP is significant for the efficient and large-scale utilization. In this study, the changes of structural properties and in vitro digestibility of SPP under two heating treatments at different temperatures were investigated by setting the same heating conditions of WH and MW. The results showed that heat treatment significantly promoted the protein aggregation and changed the secondary and tertiary structures. Moreover, the structural changes increased the digestion rate in vitro. Compared with the WH treatment, MW demonstrated a more pronounced impact, which could be attributed to the unique heating pattern and the positive structural response of SPP. As a new efficient processing method, MW treatment has a broad space in the field of microalgae processing.

CRediT authorship contribution statement

Jian Zhang: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing – original draft, Visualization. Yingying Zou: Methodology, Investigation. Bowen Yan: Conceptualization, Methodology, Investigation, Writing – review & editing, Supervision, Project administration. Nana Zhang: Conceptualization, Writing – review & editing, Project administration. Jianxin Zhao: Supervision, Project administration. Hao Zhang: Supervision, Project administration. Wei Chen: Supervision, Project administration. Daming Fan: Conceptualization, Supervision, Project administration.

Declaration of competing interest

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my coauthors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32225040), the Fundamental Research Funds for the Central Universities, the National First-class Discipline Program of Food Science and Technology (JUFSTR20180102), and the “Collaborative innovation center of food safety and quality control in Jiangsu Province” program.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crfs.2023.100581.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (15.7KB, docx)

Data availability

Data will be made available on request.

References

  1. 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 doi: 10.1016/j.algal.2021.102617. [DOI] [Google Scholar]
  2. Almeida Sa A.G., Franco Moreno Y.M., Mattar Carciofi B.A. Food processing for the improvement of plant proteins digestibility. Crit. Rev. Food Sci. Nutr. 2020;60(20):3367–3386. doi: 10.1080/10408398.2019.1688249. [DOI] [PubMed] [Google Scholar]
  3. Benelhadj S., Gharsallaoui A., Degraeve P., Attia H., Ghorbel D. Effect of pH on the functional properties of Arthrospira (Spirulina) platensis protein isolate. Food Chem. 2016;194:1056–1063. doi: 10.1016/j.foodchem.2015.08.133. [DOI] [PubMed] [Google Scholar]
  4. Beveridge T., Jones L., Tung M.A. Progel and gel formation and reversibility of gelation of whey, soybean, and albumen protein gels. J. Agric. Food Chem. 1984;32(2):307–313. doi: 10.1021/jf00122a033. [DOI] [Google Scholar]
  5. Bhat Z.F., Morton J.D., Bekhit A.E.-D.A., Kumar S., Bhat H.F. Processing technologies for improved digestibility of milk proteins. Trends Food Sci. Technol. 2021;118:1–16. doi: 10.1016/j.tifs.2021.09.017. [DOI] [Google Scholar]
  6. Boecker L., Hostettler T., Diener M., Eder S., Demuth T., Adamcik J., Reineke K., Leeb E., Nystroem L., Mathys A. Time-temperature-resolved functional and structural changes of phycocyanin extracted from Arthrospira platensis/Spirulina. Food Chem. 2020;316 doi: 10.1016/j.foodchem.2020.126374. [DOI] [PubMed] [Google Scholar]
  7. Bortolini D.G., Maciel G.M., Fernandes I.d.A.A., Pedro A.C., Rubio F.T.V., Branco I.G., Haminiuk C.W.I. Functional properties of bioactive compounds from Spirulina spp.: current status and future trends. Food Chem. Mol. Sci. 2022;5 doi: 10.1016/j.fochms.2022.100134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brylinski M., Konieczny L., Roterman I. Hydrophobic collapse in (in silico) protein folding. Comput. Biol. Chem. 2006;30(4):255–267. doi: 10.1016/j.compbiolchem.2006.04.007. [DOI] [PubMed] [Google Scholar]
  9. Campanella L., Crescentini G., Avino P. Chemical composition and nutritional evaluation of some natural and commercial food products based on Spirulina. Analusis. 1999;27(6):533–540. doi: 10.1051/analusis:1999130. [DOI] [Google Scholar]
  10. Cheng J., Sun J., Huang Y., Feng J., Zhou J., Cen K. Dynamic microstructures and fractal characterization of cell wall disruption for microwave irradiation-assisted lipid extraction from wet microalgae. Bioresour. Technol. 2013;150:67–72. doi: 10.1016/j.biortech.2013.09.126. [DOI] [PubMed] [Google Scholar]
  11. Dobson C.M. Biophysical techniques in structural biology. Annual Rev. Biochem. 2019;88:25–33. doi: 10.1146/annurev-biochem-013118-111947. [DOI] [PubMed] [Google Scholar]
  12. Ejike C.E.C.C., Collins S.A., Balasuriya N., Swanson A.K., Mason B., Udenigwe C.C. Prospects of microalgae proteins in producing peptide-based functional foods for promoting cardiovascular health. Trends Food Sci. Technol. 2017;59:30–36. doi: 10.1016/j.tifs.2016.10.026. [DOI] [Google Scholar]
  13. Granato D., de AraúraCalado V.M., Jarvis B. Observations on the use of statistical methods in food science and technology. Food Res. Int. 2014;55:137–149. doi: 10.1016/j.foodres.2013.10.024. [DOI] [Google Scholar]
  14. Guijiang L., Wenpu C., Xuejiao Q., Maomao Z., Fang Q., Zhiyong H., Jie C. Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties. Food Hydrocolloids. 2020;105:105764. doi: 10.1016/j.foodhyd.2020.105764. 105764. [DOI] [Google Scholar]
  15. Huang K., Shi J., Li M., Sun R., Guan W., Cao H., Guan X., Zhang Y. Intervention of microwave irradiation on structure and quality characteristics of quinoa protein aggregates. Food Hydrocolloids. 2022;130 doi: 10.1016/j.foodhyd.2022.107677. [DOI] [Google Scholar]
  16. Joint F., Organization W.H. World Health Organization; 1973. Energy and Protein Requirements: Report of a Joint FAO. [Google Scholar]
  17. Lovedeep K., Boning M., Singh Beniwal A., Null A., Ramandeep K., Ming Chian F., Jaspreet S. Alternative proteins vs animal proteins: the influence of structure and processing on their gastro-small intestinal digestion. Trends Food Sci. Technol. 2022;122:275–286. doi: 10.1016/j.tifs.2022.02.021. [DOI] [Google Scholar]
  18. Minekus M., Alminger M., Alvito P., Ballance S., Bohn T., Bourlieu C., Carriere F., Boutrou R., Corredig M., Dupont D., Dufour C., Egger L., Golding M., Karakaya S., Kirkhus B., Le Feunteun S., Lesmes U., Macierzanka A., Mackie A., Marze S., McClements D.J., Menard O., Recio I., Santos C.N., Singh R.P., Vegarud G.E., Wickham M.S.J., Weitschies W., Brodkorb A. A standardised static in vitro digestion method suitable for food - an international consensus. Food Funct. 2014;5(6):1113–1124. doi: 10.1039/c3fo60702j. [DOI] [PubMed] [Google Scholar]
  19. Minhas A.K., Hodgson P., Barrow C.J., Adholeya A. A review on the assessment of stress conditions for simultaneous production of microalgal lipids and carotenoids. Front. Microbiol. 2016;7 doi: 10.3389/fmicb.2016.00546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mondal A., Shukla A., Upadhyaya A., Agrawal D. Effect of porosity and particle size on microwave heating of copper. Soc. Sinter. 2010;42(2):169–182. doi: 10.2298/sos1002169m. [DOI] [Google Scholar]
  21. Muys M., Sui Y., Schwaiger B., Lesueur C., Vandenheuvel D., Vermeir P., Vlaeminck S.E. High variability in nutritional value and safety of commercially available Chlorella and Spirulina biomass indicates the need for smart production strategies. Bioresour. Technol. 2019;275:247–257. doi: 10.1016/j.biortech.2018.12.059. [DOI] [PubMed] [Google Scholar]
  22. Nicolai T., Durand D. Controlled food protein aggregation for new functionality. Curr. Opin. Colloid Interface Sci. 2013;18(4):249–256. doi: 10.1016/j.cocis.2013.03.001. [DOI] [Google Scholar]
  23. Padyana A.K., Bhat V.B., Madyastha K.M., Rajashankar K.R., Ramakumar S. Crystal structure of a light-harvesting protein C-phycocyanin from Spirulina platensis. Biochem. Biophys. Res. Commun. 2001;282(4):893–898. doi: 10.1006/bbrc.2001.4663. [DOI] [PubMed] [Google Scholar]
  24. Pooventhiran T., Bhattacharyya U., Rao D.J., Chandramohan V., Karunakar P., Irfan A., Mary Y.S., Thomas R. Detailed spectra, electronic properties, qualitative non-covalent interaction analysis, solvatochromism, docking and molecular dynamics simulations in different solvent atmosphere of cenobamate. Struct. Chem. 2020;31(6):2475–2485. doi: 10.1007/s11224-020-01607-8. [DOI] [Google Scholar]
  25. Rahman M.M., Lamsal B. Ultrasound-assisted extraction and modification of plant-based proteins: impact on physicochemical, functional, and nutritional properties. J. Am. Oil Chem. Soc. 2021;98:177–178. doi: 10.1111/1541-4337.12709. [DOI] [PubMed] [Google Scholar]
  26. Rosero-Chasoy G., Rodriguez-Jasso R.M., Aguilar C.N., Buitron G., Chairez I., Ruiz H.A. Growth kinetics and quantification of carbohydrate, protein, lipids, and chlorophyll of Spirulina platensis under aqueous conditions using different carbon and nitrogen sources. Bioresour. Technol. 2022;346 doi: 10.1016/j.biortech.2021.126456. [DOI] [PubMed] [Google Scholar]
  27. Roy F., Boye J.I., Simpson B.K. Bioactive proteins and peptides in pulse crops: pea, chickpea and lentil. Food Res. Int. 2010;43(2):432–442. doi: 10.1016/j.foodres.2009.09.002. [DOI] [Google Scholar]
  28. Sheikh M.A., Saini C.S., Sharma H.K. Synergistic effect of microwave heating and hydrothermal treatment on cyanogenic glycosides and bioactive compounds of plum (Prunus domestica L.) kernels: an analytical approach. Curr. Res. Food Sci. 2022;5:65–72. doi: 10.1016/j.crfs.2021.12.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Singh M., Trivedi N., Enamala M.K., Kuppam C., Parikh P., Nikolova M.P., Chavali M. Plant-based meat analogue (PBMA) as a sustainable food: a concise review. Eur. Food Res. Technol. 2021;247(10):2499–2526. doi: 10.1007/s00217-021-03810-1. [DOI] [Google Scholar]
  30. Soto-Sierra L., Stoykova P., Nikolov Z.L. Extraction and fractionation of microalgae-based protein products. Algal Res. 2018;36:175–192. doi: 10.1016/j.algal.2018.10.023. [DOI] [Google Scholar]
  31. Spolaore P., Joannis-Cassan C., Duran E., Isambert A. Commercial applications of microalgae. J. Biosci. Bioeng. 2006;101(2):87–96. doi: 10.1263/jbb.101.87. [DOI] [PubMed] [Google Scholar]
  32. Stadtman E.R., Levine R.L. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids. 2003;25(3–4):207–218. doi: 10.1007/s00726-003-0011-2. [DOI] [PubMed] [Google Scholar]
  33. Sun X., Ohanenye I.C., Ahmed T., Udenigwe C.C. Microwave treatment increased protein digestibility of pigeon pea (Cajanus cajan) flour: elucidation of underlying mechanisms. Food Chem. 2020;329:127196. doi: 10.1016/j.foodchem.2020.127196. 127196. [DOI] [PubMed] [Google Scholar]
  34. Tang J., Wichers H.J., Hettinga K.A. Heat-induced unfolding facilitates plant protein digestibility during in vitro static infant digestion. Food Chem. 2022;375:131878. doi: 10.1016/j.foodchem.2021.131878. 131878. [DOI] [PubMed] [Google Scholar]
  35. Wang X., Cheng L., Wang H., Yang Z. Limited Alcalase hydrolysis improves the thermally-induced gelation of quinoa protein isolate (QPI) dispersions. Curr. Res. Food Sci. 2022;5:2061–2069. doi: 10.1016/j.crfs.2022.10.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wei Y., Sun C., Dai L., Zhan X., Gao Y. Structure, physicochemical stability and in vitro simulated gastrointestinal digestion properties of beta-carotene loaded zein-propylene glycol alginate composite nanoparticles fabricated by emulsification-evaporation method. Food Hydrocolloids. 2018;81:149–158. doi: 10.1016/j.foodhyd.2018.02.042. [DOI] [Google Scholar]
  37. Yu E., Chen D., Yu B., Huang Z., Mao X., Zheng P., Luo Y., Yin H., Yu J., Luo J., Yan H., He J. Manno-oligosaccharide attenuates inflammation and intestinal epithelium injury in weaned pigs upon enterotoxigenic Escherichia coli K88 challenge. Br. J. Nutr. 2021;126(7):993–1002. doi: 10.1017/s0007114520004948. [DOI] [PubMed] [Google Scholar]
  38. Yu N., Wang Y., Shao S., Li J., Li M., Zhu L., Ye Q., Huan W., Meng X. Functional properties of glutelin from Camellia oleifera seed cake: improvement by alkali-assisted phosphorylation through changes in protein structure. Curr. Res. Food Sci. 2023;6 doi: 10.1016/j.crfs.2023.100438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yuan T., Bowen Y., Daming F., Nana Z., Shenyan M., Liyun W., Yejun W., Mingfu W., Jianxin Z., Hao Z. Structural changes of starch subjected to microwave heating: a review from the perspective of dielectric properties. Trends Food Sci. Technol. 2020;99:593–607. doi: 10.1016/j.tifs.2020.02.020. [DOI] [Google Scholar]
  40. Zhang Z., Holden G., Wang B., Adhikari B. Maillard reaction-based conjugation of Spirulina protein with maltodextrin using wet-heating route and characterisation of conjugates. Food Chem. 2023;406 doi: 10.1016/j.foodchem.2022.134931. [DOI] [PubMed] [Google Scholar]
  41. Zheng J.-X., Yin H., Shen C.-C., Zhang L., Ren D.-F., Lu J. Functional and structural properties of spirulina phycocyanin modified by ultra-high-pressure composite glycation. Food Chem. 2020;306 doi: 10.1016/j.foodchem.2019.125615. [DOI] [PubMed] [Google Scholar]
  42. Zhong H., Meng-Jie C., Jun-Hu C., Da-Wen S. Effects of electric fields and electromagnetic wave on food protein structure and functionality: a review. Trends Food Sci. Technol. 2018;75:1–9. doi: 10.1016/j.tifs.2018.02.017. [DOI] [Google Scholar]
  43. Zhou L.-Y., Zhu Y.-H., Wang X.-Y., Shen C., Wei X.-W., Xu T., He Z.-Y. Novel zwitterionic vectors: multi-functional delivery systems for therapeutic genes and drugs. Comput. Struct. Biotechnol. J. 2020;18:1980–1999. doi: 10.1016/j.csbj.2020.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhujun W., Xuewu Z. Isolation and identification of anti-proliferative peptides from Spirulina platensis using three-step hydrolysis. J. Sci. Food Agric. 2017;97(3):918–922. doi: 10.1002/jsfa.7815. [DOI] [PubMed] [Google Scholar]

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