Fortification of Chlorella vulgaris with citrus peel amino acid for improvement biomass and protein quality

Highlight • The major amino acids in citrus peels include proline, asparagine, aspartate, alanine, serine, and arginine.• Adding the amino acid extracts to the Chlorella medium enhanced microalgal biomass and protein content.• Citrus peel amino acids increase essential amino acids and decrease the non-protein amino acid of Chlorella.

potentially be used to extract flavonoids, favorable agents, and citric acid in the cosmetic and pharmaceutical sectors [9]. The macronutrient composition and profile of amino acids have been investigated to a lesser extent. Citrus peel waste with good nutritional quality offers enough nutrients for microalgae culture to enhance protein content in the microalgae. But the direct use of citrus peel has several limitations to microalgae culture due to antinutrient materials in the citrus peels. Partial extraction of amino acids and removing antinutrients could reduce these limitations [5].
Accordingly, in this research, the amino acid extract from different whole citrus peels was used as the organic nutrient source for cultivating Chlorella. The quality of extracted amino acids from bitter orange (Citrus aurantium), grapefruit (Citrus paradisi), sweet orange (Citrus sinensis), and mandarin (Citrus reticulata) peel waste and the stimulatory effects of their amino acid extracts on the manipulation of amino acid composition and protein quality of Chlorella are presented. The macronutrient composition, protein quality, and amino acid profile of citrus peel and Chlorella supplemented with amino acid extract from citrus peel have been explored.

Plant materials and biochemical analysis
The bitter orange, sweet orange, grapefruit, and mandarin were grown at Fars Research Center for Agriculture and Natural Resources (Jahrom, Fars, Iran). Fresh peels from ripped and harvested fruits were obtained from randomly chosen and healthy trees in December. Jahrom Agricultural and Natural Resources Research Station are allocated at an altitude of 1070 m with a longitude of 53 • , 37 ʹ, 33ʺ, and a latitude of 28 • , 30ʹ, 48ʺ. Jahrom Agricultural and Natural Resources Research Station confirmed all the source and batch number details. Plant field studies and experimental research, including collecting plant material, are conducted following relevant institutional, national, and international guidelines and legislation. The peel portion (albedo and flavedo) was separated from the fruit and dried out in the shade before being crushed using a household grinder. Citrus peel waste was evaluated for moisture content, ash content, total carbohydrate, total protein, total lipid, protein digestibility, and total energy using standardized techniques provided in the literature by following the Association of Official Analytical standard procedures Chemist's method (AOAC) [4,10]. The chemical components of citrus peels and Chlorella supplemented with citrus peel were characterized using Fourier transforms infrared (FT-IR) spectroscopy in the range of 4000-400 cm − 1 , performed with a Bruker FTIR spectrophotometer (Germany).

Amino acid extraction and profiling
Plant materials (10 g) were mixed with 100 mL of 6 M HCl and incubated for one day at 100 • C. Cheesecloth was used to separate the plant components. The amino acid then powdered by lyophilization. For amino acid analysis and microalgae supplementation amino acid powder were solubilized in NaCl (0.75%). The amino acids were analyzed using liquid chromatography-mass spectrometry (LC-MS/MS) on an Agilent mass spectrometer (G1313). To eliminate particles from the amino acids profile, 2.0 mL of the amino acids extract was centrifuged at 3000 × g for 20 min. At a temperature of 40 • C and a 0.01 mL injection volume, the amino acids were separated using an amino acid analyzer column (C18 reversed-phase, length=100 mm, inner diameter=3 mm, pore size=100, particle size= 2.7 m). The mobile phase solutions were 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (B). The elution protocol followed a linear gradient, starting at 5.0% solution B and increasing to 100% in 15 min. The flow rate of the mobile phase was 0.25 mL/min. The ion electrospray ionization (ESI) source obtained the mass spectrum in the m/z range of 30-3000 [11,12].

Microalgal culture and treatment
Chlorella vulgaris (IBRC: M50026) was obtained from the Iranian Biological Resources Center (IBRC) (Tehran, Iran). Chlorella was isolated using the agar plate procedure, then purified in Bold's Basal Media (BBM) before being transferred to the same liquid medium. The Chlorella cells were transferred to BBM liquid medium, bubbled, and maintained at 28 • C with an initial pH of 6.8 and 2500 E.m − 2 . s − 1 light intensity to raise biomass. A mechanical pump aerated the culture after it passed through a 0.22 mm filter. The microalgal cells were supplied with amino acid (5.0 g/1000 mL) at the logarithmic phase, and the cultures were incubated for four days. The Chlorella biomass was collected at the end of the logarithmic phase by centrifugation, washed with deionized water to eliminate adhering residues, and dried by lyophilization [11,12]. Optical density (at 680 nm) and dry cell weight analyses were used to quantify cell proliferation [4]. Microalgae were evaluated for moisture content, ash content, total carbohydrate, total protein, total lipid, protein digestibility, and total energy using standardized techniques provided in the literature by following the Association of Official Analytical Standard Procedures Chemist's method (AOAC) [4,10].

Statistical analysis
The data is presented as a three-replicate average. Experimental data were evaluated using one-way analysis of variance (ANOVA) and Tukey post-hoc testing using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). The matrix of amino acid composition data in the samples was analyzed for major components to investigate the apparent difference in the distribution of the components between the samples. Minitab (version 20.1.2) was used for the principal component analysis (PCA).

Biochemical composition of citrus peel
The typical FTIR spectrum of bitter orange, sweet orange, grapefruit, or mandarin peels is shown in Fig. 1. The 3700-3100 cm − 1 bands are associated with stretching vibrations of OH in water or hydrogenbonded OH. Sharp peaks in the 2900-2700 cm − 1 region are associated with the stretching vibrations of lipid and fatty acid CH, CH2, and CH3 groups. The stretching of C = O amides, C = C aromatics, N-H, or carboxyl in proteins is represented by the bands in the 1600-1300 cm − 1 region. The bands at 1100-900cm − 1 are caused by polysaccharide vibrations, including symmetric stretching of OH and C -O-C. The vibration of terpenes and terpenoids causes the bands at 900-500 cm − 1 . According to FTIR analyses, citrus peels had a high concentration of carbohydrates and a lower concentration of protein, fatty acids, and terpenes [14].
The macronutrient composition of bitter orange, sweet orange, grapefruit, and mandarin is summarized in Table 1. Carbohydrates (56.10-58.83 g / 100 g dry matter), proteins (17.61-19.20 g / 100 g dry matter), and lipids (11.56-13.83 g / 100 g dry matter) are the main macronutrients in the analyzed citrus peels. The macronutrient composition of bitter orange, sweet orange, grapefruit, and mandarin is similar, although statistically significant differences exist (P ≤ 5%). Carbohydrates are the major macronutrient in citrus peels, but the most abundant carbohydrates are fibers (pectin) that cannot be digested [15]. Bitter orange and grapefruit have the similar dry matter, carbohydrate, and fat content. Sweet orange and mandarin have similar protein, moisture, ash contents, and digestibility (Table 1 and Figure S1 in the supplemental file). As reported in the previous research, Citrus maxima [6] and Citrus natsudaidai [16] peels have similar chemical compositions to our findings. Our experimental results and previous work confirmed that citrus peel waste contains macronutrients (carbohydrates, proteins, lipids) and micronutrients (vitamins and minerals), and volatile constituents (polyphenols and flavonoids) that make citrus peels as the raw and inexpensive materials for foodstuff, food ingredients, and food flavoring [17,18].

Amino acid composition of citrus peel
Amino acids from citrus peels have a slightly similar FTIR spectrum, represented by bands in the 1700-500 cm − 1 area (Fig. 1). The amino The values are expressed as means ± SD for three replicates. Mean values with different letters within a row are significantly different by the Tukey test (p < 0.05). acid extract possesses a high concentration of amino acids and a low quantity of phenolic hydrocarbon and terpenes, according to FTIR analysis [14]. LC-MS/MS results show that proline, asparagine, aspartate, alanine, beta-aminoisobutyric acid, serine, arginine, gamma-aminobutyric acid, glutamate, glycine, and valine are the major amino acids found in citrus peel (Table 2). Mandarin and sweet orange had similar amounts of valine, alanine, asparagine, tyrosine, phenylalanine, leucine, isoleucine, methionine, citrulline, beta-aminobutyric acid, glycylproline, homocitruline, and cysteine (P ≤ 5%). Bitter orange and grapefruit had similar amounts of arginine, glutamic acid, alpha-aminobutyric acid, serine, aspartic acid, histidine, glutamine, proline, glycine, threonine, hydroxyproline, and gamma-aminobutyric acid (P ≤ 5%). The most abundant amino acids are leucine, threonine, lysine, phenylalanine, and histidine. Tryptophan and cysteine are the restricted amino acids ( Table 2 and Fig. 2). Citrus natsudaidai peel [16] contains similar amounts of amino acids as found in this study. Table 2 summarizes the protein quality of citrus peel in terms of amino acids. Sweet orange and mandarin are similar according to PER2, SuAA, EAA, AAA, PER1, and NPAA (P ≤ 5%). Grapefruit and bitter orange have the same amounts of FAA, NEAA, BAA, and SWAA (P ≤ 5%) ( Table 2 and Figure S2 in supplemental file). Citrus peels have a low EAA content; however, compared to the amino acid necessities of adults, they can be a good supply of EAA [16]. NPAA in citrus peels, such as aminobutyric acid, ornithine, and citrulline, are critical for plant physiology and development, carbon/nitrogen balance, energy dissipation, fruit growth, and immune response enhancement [19]. FAA produces umami flavors related to the palatability of food and its beneficial effects in regulating gastrointestinal function and treating clinical gastrointestinal diseases and stomach diseases [20]. SAA and BAA can make meals taste more natural. The amount of tasty amino acids found in citrus peel is much lower than the taste threshold. As a result, citrus peel amino acid does not affect the taste of food ingredients. Due to a lack of supply, SuAA has become the limited amino acid in plant proteins. SuAAs are involved in the formation of cellular antioxidants like n-acetylcysteine and glutathione. SuAA can also be used as a heavy metal chelating antidote to eliminate harmful metals from the body and prevent the accumulation of hazardous metal ions [21]. In addition, citrus peel usually contains other macronutrients (carbohydrates and lipids), micronutrients, natural antioxidants, and carotenoids, making it suitable for use as certain food additives.

Biochemical composition of Chlorella vulgaris
The FTIR patterns of Chlorella supplemented with citrus peels differ, indicating the various components in these products (Fig. 3). The 3700-3100 cm − 1 region is associated with the stretching vibrations of OH in water and hydrogen bonds. The peaks in the 2900-2700 cm − 1 region could be linked to the stretching vibration of lipid and fatty acids  at 1100-900 cm − 1 . The vibration of terpenes and terpenoids causes the bands at 900-500 cm − 1 . FTIR findings suggest that Chlorella includes mostly proteins, carbohydrates, fatty acids, and lower levels of hydrocarbon and terpenes [22].
For 14 days, optical density measurements and changes in dry mass levels were used to monitor Chlorella growth in the presence and absence of citrus peel protein hydrolysates (Fig. 4). Chlorella grows much faster in the citrus peel protein hydrolysates (amino acid) until 12 days reach to stationary phase. Chlorella supplementation with citrus peel amino acids increases total biomass (more than two folds p < 0.05) and protein content (more than 1.25 folds p < 0.05) while reducing carbohydrates (p < 0.05) and lipid (p < 0.05) to some extent (Table 3). Previous research also reported that protein hydrolysates and nitrate (amino acid as nitrogen source) could increase biomass and protein production in microalgae [4,12].
The macronutrient composition of Chlorella and Chlorella supplemented with citrus peel amino acids is stated in Table 3. Proteins (38.85 g / 100 g dry matter), carbohydrates (28.35 g / 100 g dry matter), and lipids (24.50 g / 100 g dry matter) are the main macronutrients in the Chlorella ( Table 3). The previously reported Chlorella biomass contains an average of 40 gs of protein, 12 gs of fiber, 18 gs of carbohydrates, and 10 gs of lipids per 100 gs of dry biomass, to some extent differing from our experimental results, especially in lipids and carbohydrates [3,13]. Proteins (47.50-49.72 g / 100 g dry matter), carbohydrates (22.75-24.55 g / 100 g dry matter), and lipids (18.56-21.83 g / 100 g dry matter) are the main macronutrients in the Chlorella supplemented with different citrus peels protein hydrolysate (amino acid). Chlorella and Chlorella/grapefruit samples are closely similar according to fat, ash, energy, dry matter, and carbohydrate (p < 0.05). On the other hand, Chlorella/bitter orange, Chlorella/mandarin, and Chlorella/sweet orange are similar according to protein, moisture, and digestibility (p < 0.05) ( Table 3 and Figure S3 in supplemental file). The carbohydrates (mainly starch) and dietary fiber (mainly cellulose) in the microalgae biomass could be possible raw materials for bioethanol synthesis and have increased interest due to their low concentration of lignin and hemicellulose. Compared to proteins and carbohydrates, the lipid content of Chlorella is relatively low because the algae culture is not affected by nutrients and other mechanisms to stimulate lipid production [3,4].

Amino acid composition of Chlorella vulgaris
Amino acids from Chlorella and supplemented Chlorella have a slightly comparable FTIR spectrum, mainly indicated by bands in the 1700-500 cm − 1 (Fig. 3). The amino acid extract included a high concentration of amino acids and a small proportion of phenolic hydrocarbon and terpenes, according to FTIR studies [22]. Table 4 shows the amino acid composition of Chlorella supplemented with amino acids from bitter orange, grapefruit, sweet orange, and mandarin peel. The main amino acid components in Chlorella protein were alanine, serine, aspartic acid, glutamic acid, glycine, leucine, threonine, phenylalanine, proline, lysine, arginine, isoleucine, valine, tyrosine, citrulline, methionine, histidine, and aspartic acid, to some extent similar to other results [3,23]. Because there are some statistically significant alterations, the amino acid composition of Chlorella remains essentially constant after supplementation with citrus peel amino acids (p < 0.05). The amino acid profile of Chlorella and supplemented Chlorella are similar, but the total amino acids increased.
Liang et al. [24] reported lipid accumulation in Chlamydomonas reinhardtii by branched-chain amino acid catabolism through an increase in the acetyl CoA level. The amino acids could influence the contents of ω− 6 and ω− 3 fatty acids in C. vulgaris culture and improve the quality of microalgal fatty acids as a food supplement [25]. No comprehensive study has so far been reported on the techniques for promoting microalgal amino acid productivity by supplementing their culture media with citrus peel amino acids. Citrus peel amino acids can easily enter the catalytic pathway of the amino acid in microalgae and affect their levels. Amino acids are broken down into precursors or mediators of the citric acid cycle, most of which occur in the mitochondria ( Figure S5 in the supplemental file). Citrus peel amino acids can enter the catabolism pathway of Chlorella through the glycine or any other amino acid and affects biomass, amino acid profile, and nutritional quality of microalgae [12]. Generally, adding amino acids as carbon and nitrogen sources resulted in better growth and increased total amino acid production. In general, the main amino acids of the citrus peels were proline, serine, aspartic acid, glycine, alanine, ornithine, glutamic acid, lysine, and valine, most of which are glucogenic. Ketogenic amino acids (leucine, lysine, tryptophan, phenylalanine, and tyrosine) are metabolized to acetyl CoA. Glucogenic amino acids (glutamic acid, glutamine, aspartic acid, asparagine, arginine, histidine, proline, valine, methionine, and threonine) are metabolized to glucose precursor and glucose metabolism pathway intermediates. Glucose is metabolized to acetyl-CoA by the glycolysis pathway. Acetyl-CoA produced in the mitochondria is the main building block for ATP production and synthesizing other amino acids under high-energy conditions. Acetyl-CoA produced from the catabolism of branched-chain amino acids can be used for the biosynthesis of some amino acids, causing an increase in amino acid accumulation [26].
Neutral non-polar and acidic polar amino acids are catabolized via extracellular amino acid oxidases. At the same time, the basic polar amino acids are transported to the cell. Inside the cell, amino acids are converted to nitrogen supply and alpha ketoacids by the activity of enzymes amino acid oxidase and dehydrogenase. The carbon skeletons of amino acids are generally converted to precursors or intermediates of the tricarboxylic acid cycle, which can contribute to ATP production and amino acid synthesis. Intermediates of the tricarboxylic acid cycle, including α-ketoglutarate, can convert to glutamic acid, the precursor for proline and arginine synthesis [26]. Succinyl-CoA and fumarate are precursors for synthesizing threonine, methionine, valine, isoleucine, tyrosine, and phenylalanine. Oxaloacetate can be converted to aspartic acid, asparagine, lysine, methionine, and threonine. Pyruvate and phosphoenolpyruvate can also be converted to valine, leucine, alanine, tyrosine, and phenylalanine. Aspartic acid converted to oxaloacetic acid, entered the tricarboxylic acid cycle, and produced energy and acetyl-CoA. Under amino acid supplementations, aspartic acid is transferred to the cytosol by the malate/aspartate shuttle and converted to threonine, methionine, and lysine. As can be seen, the levels of these amino acids have increased in Chlorella supplemented with citrus peel amino acids [26]. Table 4 summarizes the amino acid nutritional quality of Chlorella cultured in the presence of amino acids from bitter orange, grapefruit, sweet orange, and mandarin. Chlorella/bitter orange, Chlorella/grapefruit, and Chlorella/sweet orange were similar according to EAA, SwAA, HAA, PER2, AAA, BAA, and PER1. While, Chlorella and Chlorella/mandarin were similar according to FAA, NPAA, and NEAA (Table 4, Figure S4 in supplemental file). Due to its high EAAs, and high biological value, Chlorella is more popular than bacteria and fungi as a single-cell protein source for human feeding. Microalgae have a greater protein nutritional quality than conventional plants. Other nutritional components in microalgae include carbohydrates, polysaccharides, lipids, vitamins, and minerals, which can be used as functional foods, supplements, and nutrients. Compared to WHO/FAO recommendations for adults and newborns, Chlorella may be an excellent source of EAA. Chlorella can improve the amino acid quality of foodstuffs as a food additive. Based on the interaction between proline-leucine (PER1) and tyrosine-leucine (PER2), the protein efficiency ratio (PER) evaluates the nutritional quality of food protein. Low-quality protein has a PER value of less than 1.5, whereas high-quality protein has a PER value of more than 2.0. The PER values for Chlorella are greater than 2, indicating that the protein from this microalga is of good quality [20,27].

Amino acid nutritional quality of Chlorella vulgaris
Because of population growth and a health-conscious society concerned with overconsumption of fats and carbohydrates, dietary protein intake is rising. Agriculture, aquaculture, and the food industry have been working actively in recent years to increase protein product output from both production and processing aspects. Dietary proteins derived from animal sources are of the highest quality, containing well-balanced profiles of essential amino acids that generally exceed those of other food sources. However, due to low production efficiency and significant environmental impacts, together with the negative health impacts associated with the dietary intake of some animal products, the consumption of animal proteins has been declining over the past few decades. Researchers and product development specialists have been working closely to discover new protein sources, such as microalga-based ingredients. Microalgae have been recognized as strategic crops. Due to their vast biological diversity, they have distinctive phenotypic traits and interactions with the environment in the production of  biomass and protein, offering possibilities for the production of large quantities of microalgal protein through manipulating growing systems and conditions and bioengineering technologies. Several microalgal species are currently exploited for various biological and industrial applications, including human foods, functional ingredients, cosmeceuticals, pharmaceuticals, animal and aquaculture feeds, fatty acids, alginates, carotenoids, wastewater treatment, and biofuels. Despite this, microalgae remain underexploited crops, and research into their nutritional values and health benefits is in its infancy. Only a small handful of microalgal species are being produced at a commercial scale for use as human food or protein supplements [13,27]. As our experimental results show, manipulating Chlorella with amino acids from citrus peel increases total biomass and protein content.

Conclusion
Citrus peel is rich in carbohydrates, proteins, and lipids. Given amino acid content, citrus peels are rich sours of proline, asparagine, aspartate, alanine, beta-aminoisobutyric acid, serine, arginine, gammaaminobutyric acid, glutamine, glutamic acid, glycine, essential amino acid, and non-essential oil with good nutritional quality. With this nutritional quality, citrus peels could be used as an inexpensive nutrient for microalgae growth to improve microalgae biomass. But the direct use of citrus peel has several limitations due to antinutrient materials in the citrus peels. Partial extraction of amino acids and removing antinutrients could reduce these limitations. Supplementing Chlorella with amino acids from citrus peel increases total biomass and protein content. Although further research is needed to increase Chlorella's protein and amino acid content, recent findings suggest that citrus peel may be utilized to grow microalgae and provide microalgal biomass for nutritional supplements at a low cost.

Author contributions
Gholamreza Kavoosi helped to conceptualize and design the study. Kourosh Ghodrat Jahromi and Zhila Heydari Koochi were responsible for material preparation, data collecting, and analysis. Zhila Heydari Koochi wrote the original draft of the work. The draft manuscript was reviewed and edited by Gholamreza Kavoosi and Asghar Ramezanian. Gholamreza Kavoosi was involved in the final manuscript's funding, validation, supervision, review, and submission. The final manuscript has been read and approved by all writers.

Funding
The fund for this research was provided by Shiraz University (grant No. 88-GR-AGRST-108).

Availability of data and materials
On reasonable request, the corresponding author can provide the data supporting this study's findings.

Ethics approval
In our research, no human subjects or animal studies were used.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.