Manipulation of fatty acid profile and nutritional quality of Chlorella vulgaris by supplementing with citrus peel fatty acid

Lipids from citrus peel powder (1.0 g) were hydrolyzed and esterified with 5.0 ml of acidic methanol (methanol: sulfuric acid 80:20, v/v) in a shaking water bath for 120 min at 80 °C. At that time, 4.0 mL of normal saline was added to the tubes and vortexed for 5.0 min. Then, 4.0 mL of hexane was added to the tubes and vortexed for 5.0 min, and centrifuged at 3000g for 20 min in the next step. The upper hexane phase was extracted and transferred to gas chromatography vials for fatty acid methyl ester analysis. GC–MS analysis was performed using Agilent (Agilent 7890B GC 7955A MSD) device equipped with a silica capillary column (HP-5MS (30 m × 0.25 mm id; thickness 0.25 µm)), coupled with a quadrupole mass spectrometer according to the standardized method that is described elsewhere in detail¹⁵. For the preparation of free fatty acid, we used acid hydrolysis (without methanol) with the same procedure, followed by the addition of normal saline for better extraction of fatty acid and then hexane extraction. After hexane evaporation, the free fatty acid extract was added to the microalgae culture medium.

Total saturated fatty acids (SFA), unsaturated fatty acids (UFA), polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), omega-6/omega-3 ratio, PUFA/SFA (P/S), hypocholesterolemic index (HI), atherogenicity index (AI), thrombogenicity index (TI), peroxidizability index (PI), and nutritive value index (NVI) were calculated according to the formula described in the literature¹⁶.

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 m filter. The microalgal cells were supplied with fatty acid (5.0 g/1000 ml) at the logarithmic phase (after 12 days), and the cultures were incubated for 4 days¹¹. The biomasses from supplemented microalgal cultures were collected by centrifugation at the end of the stationary phase (after 17–18 days). It was washed with deionized water and lyophilized to remove the associated residues. The dry biomass was used to analysis of macronutrients and fatty acid composition¹⁴. The standardized protocols proposed in the literature were used to extract and profile fatty acids¹⁵.

The results are presented as averages with standard deviations from three replicates. Through SPSS software version 16, experimental data were evaluated as a completely randomized design using one-way analysis of variance (ANOVA) followed by Tukey post-hoc testing (P ≤ 5%). (SPSS Inc., Chicago, IL, USA). To investigate the apparent difference in the distribution of the components between the samples, the matrix of fatty acid composition and nutritional value data in the samples was evaluated for principal component analysis (PCA) using Minitab software (version 20.1.2).

There were no human subjects or animal experiments in our study.

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 groups in water or hydrogen-bonded OH. 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 amines, or carboxyl groups in proteins is represented by the bands in the 1300–1100 cm⁻¹ region. Polysaccharide vibrations, including symmetric stretching of C–O–C and OH groups, cause the band at 1100–900 cm⁻¹. The bands at 900–500 cm⁻¹ are due to the vibration of terpenes and terpenoids¹⁷. According to FTIR analyses, citrus peels have a high concentration of carbohydrates (1100–900 cm⁻¹) and a lower concentration of protein, fatty acids, and terpenes (Fig. 1).

The macronutrient analysis also confirmed that the tested citrus peels have a large carbohydrate and lesser protein, lipid, and fatty acid (Table 1). The macronutrient composition of bitter orange, sweet orange, grapefruit, and mandarin is similar, although there are statistically significant differences (P ≤ 5%). Carbohydrates are the major macronutrient in citrus peels that make them to have high energy value, but the most abundant carbohydrates are fibers (pectin) that cannot be digested¹⁸. Bitter orange and grapefruit have the similar dry matter, carbohydrate, and fat content, while sweet orange and mandarin consider protein, moisture, ash contents, and digestibility (Table 1 and Fig. S1 in the supplemental file).

Citrus maxima¹⁹ and citrus natsudaidai²⁰ peels have chemical compositions similar to our findings. Citrus peel waste contains polysaccharides (cellulose and pectin), monosaccharides (sucrose, fructose, glucose, galactose), organic acid (citric, succinic, malic, tartaric, oxalic), fatty acid (palmitic, oleic, linoleic, linolenic, and stearic), and minerals (nitrogen, calcium, magnesium, potassium)²¹. Citrus peel waste also contains volatile constituents (alcohols, ester, aldehydes, ketone, hydrocarbon), flavonoids (flavanones, anthocyanins, flavones), essential oil (limonene and linalool), limonoids (limonin), enzymes (pectin esterase, peroxidase, phosphatase), carotenoids (carotene, lutein), polyphenolic constituents, nitrogen compounds (nitrate, ammonia), and vitamin (B groups vitamins, ascorbic acid)²². All of them make citrus peels, the raw and inexpensive materials for foodstuff, ingredients, and flavoring.

The typical FTIR patterns of fatty acid extracts of bitter orange, sweet orange, grapefruit, and mandarin peels are shown in Fig. 1. The strong 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 C=C stretching of unsaturated fatty acids and C=O of carboxyl groups can be related to the variations in FTIR patterns observed in the 1900–1500 cm⁻¹ region. The weak bands in the 1200–1000 cm⁻¹ might be related to lipophilic volatile hydrocarbon and terpenes¹⁷. There were no proteins and carbohydrates in prepared extracts of fatty acids.

The fatty acid composition of bitter orange, sweet orange, grapefruit, and mandarin peels are stated in Table 2. The most predominant components in the citrus oil were linoleic acid, palmitic acid, oleic acid, alpha-linolenic acid, stearic acid, palmitoleic acid, and myristic acid. The fatty acid composition of bitter orange, sweet orange, grapefruit, and mandarin remains similar, as there are some statistically significant differences, especially in the oleic acid and alpha-linolenic acid content (P ≤ 5%). Grapefruit had the highest oleic acid and alpha-linolenic acid and sweet orange the lowest, while mandarin had significantly the lowest oleic acid compared to all other citrus peels (P ≤ 5%). The contents of arachidic acid, heptadecanoic acid, docosadienoic acid, palmitic acid, tridecanoic acid, stearic acid, myristic acid, decanoic acid, and pentadecanoic acid were similar in sweet orange and mandarin (P ≤ 5%). At the same time, bitter orange and grapefruit peel fatty acid compositions were similar considering percentages of oleic acid, hexadecadienoic acid (C16:2n−6), gamma-linolenic acid, palmitoleic acid, and dodecanoic acid (Table 2 and Fig. S2 in the supplemental file).

Generally, the major fatty acids in bitter orange, sweet orange, grapefruit, and mandarin peels were linoleic, palmitic, oleic, linolenic, stearic, palmitoleic, and myristic acids. Fatty acid composition from Citrus maxima¹⁹ and C. natsudaidai peel²⁰ display comparable results to those in our study. Citrus peel oils have a low omega-6/omega-3 ratio, high omega-9 fatty acid (mainly oleic acid), high omega-6 fatty acids, and high omega-3 fatty acids (especial grapefruit and mandating peels) could be a better dietary source of fatty acids than most of the vegetable oils by authors opinion. Animals and humans cannot synthesize linoleic and linolenic acids, which are essential and provided exclusively from dietary sources. Citrus peel fatty acids could be a promising candidate for food supplements and ingredients to provide linoleic and linolenic acids²³.

The medium-chain SFA (8–12 carbon atoms) is linked to lower blood pressure, better cardiac function, lower cancer risk, lower atherosclerosis risk, and lower low-density lipoprotein (LDL) cholesterol^24,25. Moderate myristic acid consumption improves long-chain omega-3 fatty acid levels in plasma phospholipids, positively impacts cardiovascular health, and regulates key metabolic processes²⁶. Palmitic acid (16:0, PA) is the most common saturated fatty acid found in the human body and can be provided in the diet or synthesized endogenously from other fatty acids. PA is a precursor for palmitoleic acid (16:1n-7, POA) synthesis by delta 9 desaturase. The disruption of PA homeostatic balance, implicated in different physiopathological conditions such as atherosclerosis, neurodegenerative diseases, and cancer, is often related to an uncontrolled PA endogenous biosynthesis, irrespective of its dietary contribution²⁷. On the other hand, although it belongs to saturated fatty acids, stearic acid is not atherogenic and reduces cardiovascular and cancer risk²⁸.

The results for the nutritional quality of fatty acids from bitter orange, sweet orange, grapefruit, and mandarin peels are presented in Table 2. The citrus peel contains PUFA, SFA, and MUFA, respectively. PUFA mainly contains omega-6 and omega-3, respectively. MUFA mainly contains omega-9 and omega-7, respectively. Considering the fatty acid nutritional quality, bitter orange, sweet orange, grapefruit, and mandarin are similar, although there are statistically significant differences in the total MUFA and omega-9 and omega-3 fatty acid content (P ≤ 5%). Grapefruit had the highest MUFA, omega-9, and omega-3 fatty acid. Mandarin had the lowest MUFA and omega-9 fatty acids (P ≤ 5%). Bitter orange and grapefruit are similar according to UFA/SFA, HI, UFA, PUFA/SFA, NVI, MUFA/SFA, MUFA, omega-9, PUFA, omega-3, PI, and omega-7. Sweet orange and mandarin are similar according to SFA, AI, TI, PUFA/MUFA, and omega-6/omega-3 (Table 2 and Fig. S3 in the supplemental file). Dietary variables are closely linked to the development of diet-related disease, and the consumption of fatty acids is thought to play a unique role. Because of their relevance to health, an adequate supply of UFA plays an important role in disease treatment. The ratio of SFA to UFA in biological membranes is an important feature. Reduced levels of UFA in membranes have been linked to various pathophysiological conditions, including cardiovascular disease, diabetes, and cancer²⁹. MUFA are beneficial to biological membranes because they are liquid at body temperature but not quickly oxidized, allowing them to retain proper membrane fluidity. UFA also functions as a lipid hormone or lipokine, coordinating metabolic responses between tissues³⁰. Accordingly, citrus peels fatty acids with a good balance of PUFA, SFA, MUFA, omega-6, omega-3, omega-9, and omega-7 are beneficial for human health and microalgae culture and biomass production.

The FTIR patterns of Chlorella supplemented with fatty acid from bitter orange, sweet orange, grapefruit, and mandarin peels differ, indicating the various components in these products (Fig. 2). The 3700–3100 cm⁻¹ bands are associated with the stretching vibrations of OH groups in water or aromatic molecules. Peaks in the 2900–2700 cm⁻¹ region could be linked to the stretching vibration of lipid and fatty acid CH, CH2, and CH3 groups. The majority of the variations in FTIR patterns observed in the 2900–2700 cm⁻¹ region can be attributed to a variation in fatty acid composition caused by citrus peel fatty acids. The C=C stretching of unsaturated fatty acids and the formation of MUFA and PUFA in Chlorella after supplementing with citrus peel can be related to the variations in FTIR patterns observed in the 1900–1500 cm⁻¹ region. The stretching of C=O of amides, C=C of aromatics, N–H of amines, or carboxyl groups in proteins is represented by the bands in the region 1300–1000 cm⁻¹. The bands at 1100–900 cm⁻¹ are created by polysaccharide vibrations, including symmetric stretching of the C–O–C and OH groups. The vibration of terpenes and terpenoids causes the bands at 900–500 cm⁻¹³¹.

The macronutrient composition of Chlorella and Chlorella supplemented with citrus peel fatty acids is stated in Table 3. Protein, carbohydrates, and lipids are the major components of Chlorella, respectively. Chlorella supplementation with citrus peel fatty acids increases total biomass and fat content while reducing carbohydrates and protein to some extent (P ≤ 5%). In this research, microalgae were separated by centrifugation, washed with distilled water, and then dried by lyophilization after culture. Wet biomass washing and lyophilization reduce the moisture content and salt (ash) of the dry matter of microalgae. Chlorella/bitter orange, Chlorella/grapefruit, Chlorella/sweet orange, and Chlorella/mandarin are similar according to energy, fat, moisture, and digestibility. Chlorella is different from other samples based on the ash, protein, dry matter, and carbohydrate (Table 3, Fig. S4 in supplemental file). Accordingly, supplementation with fatty acids from tested citrus peels modulates the macronutrient composition of Chlorella, especially fatty acids. Previous reports suggested that Chlorella biomass included an average of 40 g of protein, 18 g of carbohydrates, 12 g of fiber, and 10 g of lipids per 100 g of dry biomass^32–34.

The FTIR patterns of fatty acid from Chlorella supplemented with bitter orange, sweet orange, grapefruit, and mandarin peel fatty acids are sated in Fig. 2. Peaks in the 2900–2700 cm⁻¹ region could be linked to the stretching vibration of lipid and fatty acid CH, CH2, and CH3 groups. The majority of the variations in FTIR patterns observed in the 2900–2700 cm⁻¹ region can be attributed to a variation in fatty acid composition caused by citrus peel fatty acids. The C=C stretching of unsaturated fatty acids and the formation of MUFA and PUFA in Chlorella after supplementing with citrus peel can be related to the variations in FTIR patterns observed in the 1900–1500 cm⁻¹ region. The vibration of terpenes and terpenoids causes the bands at 1000–500 cm⁻¹³¹. According to FTIR findings, Chlorella includes mostly proteins, carbohydrates, fatty acids, and lower hydrocarbons and terpenes. Because of the high level of essential fatty acid, high level of omega-3, and high levels of omega-6, microalgae such as Chlorella are preferred over bacteria and fungi as the single-cell oil for human eating. Carbohydrates (starch) and dietary fibers (cellulose) in microalgae biomass vary on average 20% and 12%, respectively. Because of the lack of lignin and hemicellulose, microalgae carbohydrates have been potential raw materials for bioethanol production. If the characteristics of Chlorella fatty acids are nutritionally attractive, Chlorella with citrus peel fatty acids can be marketed as a single-celled oil³³.

Fatty acid composition of Chlorella and Chlorella supplemented with bitter orange, sweet orange, grapefruit, and mandarin peel fatty acids stated in Table 4. The primary fatty acids of Chlorella are palmitic, linoleic, oleic, alpha-linolenic, gamma-linolenic, hexadecatetraenoic, palmitoleic, hexadecadienoic, hexadecatrienoic, lauric and eicosapentaenoic acids (Table 4). To some extent, the fatty acid composition reported in Chlorella is similar to the previous results^32,34. The fatty acid composition of Chlorella remains almost unchanged after supplementation with the fatty acids, as there are some statistically significant differences (Table 4). Chlorella/sweet orange, and Chlorella/mandarin according to docosahexaenoic acid, eicosanoic acid, heptadecanoic acid, myristic acid, tridecanoic acid, 4,7,10-hexadecatrienoic acid, pentadecanoic acid, 7,10,13-hexadecatrienoic acid, linoleic acid, palmitic acid, eicosapentaenoic acid, and lauric acid are correlated to each other (P ≤ 5%). Chlorella, Chlorella/bitter orange, and Chlorella/grapefruit samples are correlated according to 4,7,10,13-hexadecatetraenoic acid, gamma-linolenic acid, 6,9,12,15-octadecatetraenoic acid, oleic acid, palmitoleic acid, and alpha-linolenic acid (Table 4, Fig. S5 in supplemental file).

Fatty acid nutritional quality of Chlorella and Chlorella supplemented with bitter orange, sweet orange, grapefruit, and mandarin peel fatty acids stated in Table 4. Chlorella manly contains PUFA, SFA, and MUFA, respectively. PUFA mainly contains omega-6 and omega-3, respectively. MUFA contains omega-9 and omega-7, respectively (Table 4). Chlorella had nutritionally acceptable AI, TI, omega-6/omega-3, HI, PI, and NVI (Table 4). Supplementation of Chlorella with citrus peel fatty acids increases the total biomass and lipid content, while carbohydrate and protein, to some extent, decrease (P ≤ 5%). Chlorella and supplemented Chlorella's nutritional quality and fatty acid composition are similar, but total fatty acid increased. Chlorella, Chlorella/bitter orange, and Chlorella/grapefruit are correlated according to MUFA/SFA, MUFA, omega-9, NVI, UFA, UFA/SFA, HI, and PUFA/SFA. Chlorella/sweet orange and Chlorella/mandarin are similar according to PUFA/MUFA, AI, SFA, PUFA, omega-3, and omega-6 (Table 4, Fig. S6 in supplemental file).

Balanced fatty acid in the Chlorella leads to an acceptable AI, TI, HI, PUFA/SFA, omega-6/omega-3 ratio, PI, and NVI, making Chlorella suitable for food supplements. Because the ratio of UFA/SFA in the Chlorella is above 1.5, it can recommend a promising candidate for raising high-density lipoprotein (HDL) cholesterol and depressing LDL cholesterol. Lowering triglycerides, preventing inflammation, increasing mitochondrial biogenesis, restoring insulin sensitivity, reducing central body fat, and suppressing thrombosis and inflammation are benefits of an omega-3-rich diet³⁵. Increased blood viscosity, vasoconstriction, and promoting prothrombotic, proinflammatory, and proaggregatory factors are linked to a diet with high omega-6 fatty acids. Maintaining good health necessitates a sensible omega-6/omega-3 ratio³⁶. Chlorella supplemented with citrus peel is beneficial as a rich source of essential fatty acids and the excellent omega-6/omega-3 ratio, which is crucial in dietary supplements to prevent and manage chronic illnesses and obesity problems³⁴.

A PUFA/SFA ratio of over 0.45 is advised to avoid cardiovascular disease and several chronic illnesses in human diets, including cancer. The recommended PUFA/SFA ratio, which indicates the quality of lipid nutrition in a given diet, is 1–2, decreasing blood cholesterol and lowering the risk of heart disease³⁴. Low AI and TI values indicate that foodstuffs have a greater preventive impact in avoiding heart and coronary illnesses, decreasing overall and abdominal obesity, and reducing diabetes in pregnant women. High PUFA levels are closely connected to high HI fatty acid ratios, which are more favorable for human health since they are regarded as the optimal quantity of cholesterol. The PI is a measure of PUFA sensitivity to oxidation. The range of 70–90 is a favorable PI ratio representing the lipid nutritional quality of a certain meal and lowers blood cholesterol and cardiovascular disease. A higher amount of fatty acid oxidation is associated with higher PI levels. However, high PI levels due to high omega-3 PUFA and omega-6 PUFA lead to greater antioxidant and anti-inflammatory actions³⁶.

Citrus peel oil produces a rise in the nutritional quality of Chlorella due to the findings, which is a valuable gain for Chlorella applications such as food supplements, medicinal benefits, and biodiesel generation. Citrus peel oil can be converted to acetyl-CoA, then converted to palmitic acid, and subsequently to an unsaturated fatty acid by elongases and desaturases. (1) Fatty acids may be incorporated into the phospholipids and glycolipids of the plasma membrane. (2) Fatty acids may be incorporated into the triglyceride and used as a storage resource. (3) Fatty acids may be incorporated into the metabolic pathway of fatty acids and converted to omega-3 and omega-6 fatty acids. Citrus peel oil enters directly into the synthesis pathway of unsaturated fatty acids like linoleic acid and linolenic acid³⁵. In the omega-3 pathway, 15-desaturase converts linoleic acid to alpha-linolenic acid. In the omega-6 pathway, 6-desaturase transforms linoleic acid into gamma-linolenic acid²⁴. When the diet contains high linoleic acid (citrus peel oil) levels, placed in the Chlorella culture medium, ∆15-desaturase is involved in the biosynthesis of alpha-linolenic acid from linoleic acid¹¹. In contrast, ∆6-desaturase is involved in the biosynthesis of gamma-linolenic acid from linoleic acid¹¹. Adding citrus peel oil to the culture medium of Chlorella leads to an acceptable production of omega-9, omega-6, omega-3, and nutritionally suitable omega-6/omega-3 ratio²⁶. Accordingly, humans whose diet is rich in these fatty acids have lower inflammation and better insulin sensitivity than those with saturated fatty acids in their diet. So, these diets are beneficial for human health²⁶. The use of fatty acids by microalgae is complex, and explanation in this case is very difficult and need more studies.

The data that support the findings of this study are available from the corresponding author, upon reasonable request.