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Article

Improved Efficiency of Lutein Extraction from Hens’ Feed Mixture and Food Samples Using Less Toxic Solvent Mixture

by
Olivera Galović
1,*,
Doris Prokopec
1,
Zlata Kralik
2,
Manuela Košević
2 and
Gordana Kralik
3
1
Department of Chemistry, Josip Juraj Strossmayer University of Osijek, Ulica Cara Hadrijana 8a, 31000 Osijek, Croatia
2
Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia
3
Nutricin j.d.o.o., Đure Đakovića 6, 31326 Darda, Croatia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(5), 2360; https://doi.org/10.3390/app15052360
Submission received: 17 January 2025 / Revised: 18 February 2025 / Accepted: 19 February 2025 / Published: 22 February 2025
(This article belongs to the Special Issue Food Chemistry, Analysis and Innovative Production Technologies)
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Abstract

:
Lutein is one of the nutrients necessary for the proper functioning of our organism. The majority of research focuses on the impact of lutein on eye health and its role as an antioxidant. Although fruits and vegetables are the most important sources of lutein, chicken eggs are considered an excellent and widely used source, necessitating the determination of lutein concentration in food. One of the methods for extracting lutein from various food samples is solvent extraction. Although it is one of the older methods and has disadvantages compared to instrumental methods, it is still widely used. In this investigation, the adapted AOAC method developed by Leeson et al. (used here as a standard method), was modified by adjusting the composition of the extraction solvent mixture in order to reduce the amount of toxic solvents used. The mixture of hexane:acetone:ethanol:toluene (10:7:6:7, v/v/v/v) was replaced with a mixture of methanol:acetone (1:1, v/v). The concentration of lutein extracted from the hens’ feed mixture was 52% higher compared to the method developed by Leeson et al. The suitability of the modified method was tested on two parallel samples, and the obtained recovery values were 95.68% and 98.38%, respectively. The influence of ultrasound on lutein extraction was examined, but the obtained concentration of lutein was 8.66% lower than the concentration determined by the modified standard method. The modified standard method was then used to determine lutein concentration in six hens’ feed mixture samples and eight food samples. The results obtained were in accordance with the data from the literature.

1. Introduction

Human health is a priority, and maintaining good health is challenging, especially in today’s fast-paced world. Oxidants, reactive oxygen species (ROS), and reactive nitrogen species (RNS) are continuously produced by our bodies, where they play significant roles in immune function and signal transmission. The presence of oxidants is balanced by our body’s antioxidant defense system. While endogenous enzymatic and non-enzymatic antioxidant systems provide excellent protection, we sometimes need help from the outside. Antioxidants present in food, particularly in vegetables and fruits, are an important supplement to these systems [1,2]. Oxidative stress is one of the major causes of various illnesses and can be described as an imbalance between antioxidants and prooxidants (prooxidants promote oxidation) [3]. A natural source of antioxidants is carotenoids, a group of pigments that are widely studied, especially for their impact on human health [4,5]. Good sources of carotenoids are marine organisms, mainly phytoplankton, but these pigments can also be found in organisms such as crabs, lobster, tuna, shrimp, and salmon, where they contribute to the coloration of these organisms [6]. Lutein belongs to the group of carotenoids known as xanthophylls, yellowish-orange pigments that are synthesized exclusively in plants (dark green vegetables, flowers, cereals) [7,8], as well as in some microalgae, fungi, and bacteria. Because humans cannot synthesize lutein, it must be ingested through food. It contributes to human health through its anti-cataract, anti-inflammatory, antioxidant, anti-arthritis, neuroprotective, bone-remodeling, hepatoprotective, cardioprotective, anticancer, and antidiabetic retinopathy activities [9], as well as through its positive effects on cognitive functions [10]. Lutein’s influence on eye health has also been extensively researched [11]. Fruits and vegetables are the most common source of lutein (80–90% of all carotenoids derive from these sources). Animal sources include eggs and, to some extent, raw or processed milk [12,13,14]. Although lutein is synthesized exclusively in plants, egg yolk is considered a better source of lutein for humans due to its high fat content, which enhances bioavailability [15,16]. Lutein also has a favorable effect on the health of laying hens by enhancing protection against respiratory infections [17]. Beyond its health benefits for humans, lutein contributes to customers’ perception of egg yolk color and hen skin color. In hens’ feed mixtures, the crops that make up the mixture are a source of lutein; however, lutein may also be added as an additive. According to EU legislation, between 50 and 80 mg of total carotenoids/kg of complete feed can be included [18]. Since eggs are a good source of lutein and other nutrients, and hen meat is widely used in nutrition, researchers aim to optimize feed composition for laying hens in order to increase the lutein content as well as other nutrients [19].
Many methods are used for the extraction of various bioactive compounds. Soxhlet, maceration, decoction, and liquid–liquid extraction are among the older methods. More recently, modern, instrumentally guided extraction processes have become more common: extraction with supercritical CO2, extraction with ionic solvents, microwave-assisted extraction, and extraction with ultrasound [20,21]. The most important advantages of these newer techniques are high extraction efficiency and time and chemical savings. The choice of solvent or solvent mixture has a significant impact on the success of extraction of bioactive compounds. Various organic solvents (hexane, acetonitrile, acetone, ethanol, methanol) and water are commonly used. The polarity of the solvent or solvent mixture affects the solubility of the target bioactive compound; therefore, a well-chosen solvent or solvent mixture enhances extraction efficiency [22]. In addition to the solvent mixture, the success of the extraction is influenced by particle size and temperature. While reducing the sample particle size makes extraction more efficient [21,23], some studies have found that further reduction below a certain size does not improve extraction [24]. Generally, increasing the temperature also improves extraction efficiency; however, excessively high temperatures may lead to analyte degradation.
Although numerous methods have been developed for carotenoid extraction from different samples, one of the methods still used is the classic solvent extraction method [25]. However, this method has several drawbacks, including a time-consuming, often multi-step extraction procedure and the use of large quantities of organic solvents, which leads to waste disposal issues and concerns about toxicity. Despite these drawbacks and its somewhat outdated nature, this method remains in use for analyzing small numbers of samples, primarily because purchasing expensive instruments is not cost-effective in such cases. This affordability is the most significant advantage of the solvent extraction method.
The aim of this study was to modify the solvent composition in an adapted AOAC method developed by Leeson et al. [26] and use the modified method for lutein extraction from samples of different origin (hens’ feed mixture, food samples). By modifying the solvent composition, lutein extraction efficiency would be improved. By reducing the volume of toxic organic solvents for extraction or by replacing them with solvents which are considered preferred solvents according to Pfizer’s recommendation list [27], the modified method would be less toxic.

2. Materials and Methods

2.1. Chemicals and Reagents

Toluene (Carlo Erba, Val-de-Reuil, France), n-hexane (Carlo Erba, Val-de-Reuil, France), acetone (Gram-mol, Zagreb, Croatia), ethanol (Carlo Erba, Val-de-Reuil, France), isopropanol (J. T. Baker, Gliwice, Poland), methanol (J. T. Baker, Gliwice, Poland), tetrahydrofuran (Fisher Scientific, Loughborough, UK), ethyl acetate (Fisher Scientific, Loughborough, UK) were all HPLC grade. Potassium hydroxide (Gram-mol, Zagreb, Croatia), sodium hydroxide (Gram-mol, Zagreb, Croatia), sodium sulfate anhydrous (Gram-mol, Zagreb, Croatia), butylated hydroxytoluene (Acros Organics, Steinheim, Germany), and lutein standard (Merck, Darmstadt, Germany) were also used.

2.2. Instrumentation

Chromatographic measurements were carried out using a Shimadzu (Japan) HPLC system with UV-VIS detector (system controller SCL-10A VP, degasser DGU-14A, pumps LC10AD VP, UV-VIS detector SPD-10AV VP, fluorescence detector RF-20A (Prominence), auto injector SIL-10AD VP, column oven CTO-10AS VP). For the preparation of standards and the sample solution, the following were employed: analytical balance (Sartorius), centrifuge (Hettich, Rotina 380R, Hettich, Kirchlengern, Germany), vortex (Lab dancer, IKA, Staufen, Germany), ultrasonic bath with temperature regulator (Sonorex, Bandelin, Berlin, Germany), homogenizer (BANDELIN SONOPULS HD 3100, Bandelin, Berlin, Germany) with probe vs. 70 T and noise protection box (Bandelin Sonoplus, Bandelin, Berlin, Germany), rotary evaporator (Büchi, Rotavapor® R-300, Büchi, Flawil, Switzerland), household grinder, ultrapure water system (Purelab Flex, Elga LabWater, High Wycombe, UK), and laboratory drying oven (ST-05, Instrumentaria, Zagreb, Croatia).

2.3. Chromatographic Procedure

For the separation, a Shimadzu (Japan) Shim_pack GIST, 4.6 × 250 nm, 5 µm, C18 column was used. The mobile phase consisted of methanol:THF (90:10, v/v) which, prior to use, was filtered through a 0.20 µm membrane filter (Whatman). Separation was carried out at room temperature with a flow rate of 1 mL/min at 450 nm, and the volume of the sample injected into the HPLC system was 20 µL. The total analysis time was 8 min.

2.4. Ultrasound-Assisted Procedure

Direct ultrasound-assisted extraction was performed with the following parameters: amplitude of 70%, with an interval of ultrasound of 3 s, interval of pause 2 s, total time 3 min. The ultrasonic homogenizer operated at a frequency of 20 kHz ± 500 Hz, with adjustable power ranging from 10 to 75 W.

2.5. Real Samples

The samples of hens’ feed mixtures were obtained from the Faculty of Agrobiotechnical Sciences Osijek, University of Osijek, Department of Animal Production and Biotechnology, as well as a few family farms. After collecting, samples were stored at 4 °C in the dark. Samples of food (fresh kale, parsley leaves, carrots, lettuce, butternut squash, paprika spice, cornmeal (polenta), wheat flour) were purchased from a local grocery store.

2.6. Standard Solutions

Lutein standard was used to prepare stock solutions for calibrating the curve and suitability of the chosen extraction procedure. A 5 mg/mL stock solution for the calibration curve was prepared in hexane:ethyl acetate (65:35, v/v). A series of solutions with concentrations of 0.25, 0.50, 1.25, 2.50, and 5.00 mg/L were then prepared by diluting the stock solution. All standard solutions were prepared in amber volumetric flasks and stored at 4 °C.

2.7. Sample Preparation

Prior to analysis, samples of hens’ feed mixture were ground and sieved. Samples of food (fresh kale, parsley leaves, carrots, lettuce, butternut squash) were dried at 55 °C for 24–48 h and, after drying, samples were ground and sieved. Paprika spice, cornmeal (polenta), and wheat flour were used in their native form.

2.7.1. Influence of Sample Particle Size

To investigate the influence of sample particle size on determination of lutein concentration, the collected hens’ feed mixture sample was used either in its native form or was ground using a household grinder and then sieved.

2.7.2. Lutein Extraction Method

One of the first and widely used AOAC (Association of Official Analytical Chemists) methods [28] required extensive time and a large volume of chemicals. Chen et al. [29] improved the AOAC method by simultaneously carrying out extraction and saponification under an inert atmosphere with the addition of a BHT or BHA (butylated hydroxyanisole) solution. Cold saponification was found to be better for extraction of xanthophylls. For separation of xanthophylls, the composition of the ternary solvent mixture was changed to hexane:acetone:methanol = 85:15:1 and the composition of adsorbent was adjusted to an activated MgO:diatomaceous earth = 1:1. Further improvement of the AOAC method was conducted by Leeson et al. [26]. The adapted AOAC method developed by Leeson et al. used hot saponification, and extraction and saponification was also conducted simultaneously but over a significantly shorter time. After extraction and saponification aliquot of the hexane layer was evaporated, diluted with a mixture of hexane:ethyl acetate = 65:35 and analyzed by HPLC. The method developed by Leeson et al. was adopted as the standard method for this investigation. A detailed description of the Leeson et al. method is as follows: 1 g of sample was weighed into 50 mL glass vials, then 15 mL of the extraction mixture of hexane:acetone:ethanol:toluene (10:7:6:7, v/v/v/v) and 1 mL of 0.2% methanolic BHT solution were added. The content was vortexed for 30 s, then 2 mL of ultrapure water and 2 mL of 40% methanolic KOH solution were added, and the mixture was vortexed again for 30 s. The vials were covered with aluminum foil to reduce the effect of light on the samples and heated in a water bath at 56 °C for 20 min. After heating, the vials were left in the dark for 1 h to cool down. Next, 15 mL of n-hexane and 5 mL of 10% aqueous Na2SO4 solution were added to the mixture, after which the mixture was vigorously stirred. The mixture was again left in the dark for 1 h to allow the layers to separate. An aliquot of the upper hexane layer was filtered through a membrane filter with a 0.20 µm pore size and 1 mL of the sample was evaporated on a rotary evaporator at 55 °C until dry. After evaporation, 1 mL of a hexane:ethyl acetate = 65:35 mixture was added to each sample. 1 mL of the prepared sample was transferred to an amber vial and the sample was analyzed by HPLC. The sample-to-extraction mixture ratio was 1:40. From this point forward, the method developed by Leeson et al. will be abbreviated as SM.
To reduce the use of toxic solvents, guidelines from Pfzier’s recommendation list [27] and relative solubility list published by Neal E. Craft and Joseph H. Soares, Jr. [30] were followed. The procedure used in this study was as follows: 1 g of sample was weighed, then 15 mL of the solvent or solvent mixture, 2 mL of 0.2% BHT solution, and 3 mL of 40% KOH solution were added. The extraction was assisted by direct ultrasound for 3 min. After sonication, samples were left in the dark for 1 h, after which 1 mL of the upper layer was evaporated on a rotary evaporator at 55 °C until dry. Following evaporation, 1 mL of hexane:ethyl acetate (65:35, v/v) mixture was added to each sample, and the resulting sample was transferred to the amber vial. The sample was analyzed using HPLC. The ratio of the sample to the solvent or solvent mixture was 1:20. From this point forward, the ultrasound-assisted extraction method for solvent selection will be abbreviated as UAE-SOL.
Modification of SM refers to changing the solvent composition in the extraction mixture. The mixture of hexane:acetone:ethanol:toluene (10:7:6:7, v/v) was replaced with a chosen solvent mixture. All other SM parameters remained unchanged. From this point forward, the modified SM will be abbreviated as MSM.
All samples were analyzed the same day they were prepared, and measurements were performed on five parallel samples.

2.7.3. Suitability of MSM Extraction Procedure

After selecting a suitable solvent mixture, the suitability of the MSM extraction procedure was evaluated by analyzing spiked samples of hens’ feed mixture. Two samples of hens’ feed mixture were spiked with standard lutein solution and prepared according to the MSM. The values of extraction recovery were calculated by comparing the results obtained from the standard samples and the hens’ feed mixture samples spiked with standard.
To examine the influence of ultrasound on lutein extraction using the MSM, the following sample preparation method was used: 1 g of sample was weighed, then 15 mL of the chosen solvent mixture, 1 mL 0.2% methanol solution of BHT, 2 mL of 40% methanolic solution of KOH, and 2 mL of ultrapure water were added. The contents were vortexed for 30 s, and 15 mL of n-hexane and 5 mL of 10% aqueous Na2SO4 solution were added to the mixture. The extraction was assisted by direct ultrasound using an ultrasound probe for 3 min. The samples were left in the dark for 10 min, after which they were centrifuged for 10 min at 6000 rpm. The supernatant was filtered through a syringe filter with a pore size of 0.20 µm and 1 mL of the filtered supernatant was evaporated on a rotavapor until dry, after which 1 mL of a mixture of hexane:ethyl acetate (65:35, v/v) was added. Finally, 1 mL of the prepared sample was transferred to the amber vial and the sample was analyzed using HPLC. The sample-to-extraction mixture ratio was 1:40. Henceforth, the abbreviation MSM-UAE will be used for this modified standard method with ultrasound-assisted extraction.

2.8. Statistical Analysis

The results obtained from this study are presented in tables and were processed using the statistical packages Microsoft 365 Excel and TIBCO Statistica® 14.0.0. (TIBCO Software Inc. 2020, Palo Alto, CA, USA) [31]. Differences determined among the studied groups were tested with Fisher’s LSD test at significance levels of p < 0.05, p < 0.01, and p < 0.001. In the tables, values above the mean are marked with exponents a,b,c,d,e.

3. Results and Discussion

All analyses conducted prior to analyzing real samples obtained from the Faculty of Agrobiotechnical Sciences Osijek, University of Osijek, Department of Animal Production and Biotechnology and a few family farms, as well as before analyzing the food samples, were conducted on the same sample of hens’ feed mixture obtained from a family farm.
All measurements were performed on five parallel samples (n = 5).

3.1. Calibration Curve

Prior to HPLC analysis of the samples, a calibration curve was constructed using standard solutions prepared according to the protocol described in Section 2.6 and HPLC analysis performed according to the previously described protocol (Section 2.3). The analysis time was 8 min and retention time for lutein was 4.2 min. The constructed calibration curve is shown in Figure 1 and obtained R2 = 0.99989.
The time of analysis was shortened from 20 min, as recorded in previous research [14,20,32], to 8 min. The retention time of lutein was also reduced to 4.2 min. Shortening analysis time and shorter retention time represent significant savings of time and chemicals.

3.2. Comparison of Unground and Ground Samples

Since the hens’ feed mixture consists of several components with different particle sizes, the influence of particle size on lutein extraction was investigated. The hens’ feed sample was ground until no residue remained on a sieve mesh with a 300 μm pore size. A comparison of unground (a) and ground and sieved samples (b) is shown in Figure 2. Extraction of lutein was carried out using the SM, and the resulting chromatogram is presented in Figure 3. Analysis of the ground samples yielded a 25% higher lutein concentration (in unground samples, the lutein concentration was 10.145 mg of lutein/kg of feed mixture vs. 12.753 mg of lutein/kg of feed mixture in ground samples). Hojnik et al. investigated the influence of particle size on the efficiency of lutein extraction from marigold flower petals. They concluded that particle size does not significantly affect the extraction efficiency of lutein, but does reduce the extraction time [22]. In our study, better results were obtained with ground samples; therefore, all further analyses were performed on ground and sieved samples.

3.3. Selection of the Solvent

The solvents typically used for lutein extraction are methanol, dichloromethane, acetone, and ethanol [33]. In this investigation, for selection of the solvent in the extraction mixture, the previously mentioned guidelines (Section 2.7.2) and extraction protocol UAE-SOL (described in Section 2.7.2) were used. Methanol, ethanol, acetone, 2-propanol, and a mixture of methanol:acetone (1:1, v/v) were used. Results are shown in Table 1. The significantly higher lutein content in the hens’ feed mixture was determined when using the solvent mixture methanol:acetone (1:1, v/v), and the lowest when using 2-propanol (4.572 mg/kg and 0.543 mg/kg, respectively; p < 0.01). Similar amounts of lutein in the hens’ feed mixture were determined when using methanol and ethanol as solvents (3.499 mg/kg and 3.201 mg/kg, respectively; p > 0.05). Chandra-Hioe et al. investigated the influence of isopropanol, acetone, ethyl acetate, and hexane on the extraction of lutein from Australian Sweet Lupin Flour [34], and Liu et al. [35] determined that isopropanol is the appropiate solvent for lutein extraction. Islam et al. used a mixture of n-hexane:isopropanol (3:2, v/v) to extract lutein from hens’ feed mixture [36], and Hojnik et al. concluded that hexane was the best solvent for lutein extraction under the conditions they investigated [22]. The mixture of acetone and magnesium sulfate was used by Pérez-Vendrell et al. [37], while Koutsos et al. used butanol:acetonitrile (1:1, v/v) and hexane:chloroform (2:1, v/v) [38]. De Oliveira et al. used acetone followed by petroleum ether [39], and Breithaupt et al. used methanol:ethyl acetate:light petroleum (1:1:1, v/v/v) [40]. Meanwhile, methanol and dichloromethane (1:1, v/v) was used by Dansou et al. [41], acetone:methanol 8:2 (v/v) was used by Xiao et al. [42], Šivel et al. used an acetone:methanol solvent mixture (1:1, v/v), and samples were subsequently treated in an ultrasound bath [32]. A mixture of chloroform:methanol (1:1, v/v) was used by Casella et al. [43].
For the analysis of real samples, we chose methanol:acetone (1:1, v/v) to use in the composition of the extraction mixture.

3.4. Comparison of SM and MSM

The results (Table 2) obtained by the MSM were significantly higher than the results obtained by the SM (19.410 mg/kg and 12.753 mg/kg of feed mixture; p < 0.001). Considering all of the above, lutein from real samples was extracted using the MSM. During the investigation, it was determined that the volume of used solvents could be reduced further; however, both the volume of each solvent and the sample mass must be reduced by the same percentage to maintain the solvent-to-sample ratio of 40:1.

3.5. Suitability of MSM Extraction Procedure

Measurements were performed on two parallel hens’ feed mixture samples (S1 and S2) by using the MSM. The obtained results are shown in Table 3. For both samples, the obtained values of recovery were acceptable (sample 1 (S1) 95.68% and sample 2 (S2) 98.38%). Therefore, we concluded that the MSM is suitable for determining the concentration of lutein in real samples.

3.6. Influence of Ultrasound on Extraction

Ultrasound is frequently used to extract bioactive substances from various types of samples or to improve the extraction efficiency. Using the MSM-UAE (described in Section 2.7.3, the influence of ultrasound on total extraction time and lutein extraction was examined. Using direct ultrasound, the extraction and saponification time was reduced from 2.5 h (MSM) to 25 min (MSM-UAE). In addition to reducing the time of extraction, the effect of ultrasound on lutein extraction efficiency was examined. During direct ultrasound extraction, the average temperature reached was 50 °C, and the determined concentration of lutein was 17.728 mg/kg feed mixture, which is 8.66% lower than the concentration determined using the MSM. The resulting chromatograms are presented in Figure 4. The lower lutein concentration obtained with the MSM-UAE method suggests that the shorter waiting time after sonication may be a contributing factor. The waiting time after extraction and saponification is 2 h for MSM, whereas it is only 10 min for the MSM-UAE. Nevertheless, the results obtained in our research leave ample room for further investigation of the method. Chandra-Hioe et al. investigated the influence of indirect and direct ultrasound on the extraction of lutein from Australian Sweet Lupin Flour, and they determined that the use of indirect ultrasound gave better results than direct ultrasound [34]. Saini et al. used direct ultrasound for the extraction of lutein from the peel of Citrus reticulate. They observed that the lutein yield increased up to 44 °C, then decreased with further increases in temperature [44]. Ye et al. also used direct ultrasound for extraction of lutein from corn with ethanol as the solvent. In their study, a longer period of sonication yielded a higher concentration of lutein. The temperature during sonication was 38–40 °C [45]. Wang et al. used indirect ultrasound and ethanol as the solvent for extraction of lutein from corn gluten meal. Extraction was conducted for 40 min at a temperature of 60 °C [20]. Ahmadi et al. extracted lutein from pistachio waste with indirect ultrasound during 45 min, using ethyl acetate as the solvent at a temperature of 50 °C [46]. Mahneshwari et al. used a 1% surfactant aqueous solution as the solvent and ultrasound-assisted extraction to obtain lutein from marigold flowers, with an optimal temperature of 35 °C [47].

3.7. HPLC Analysis of Real Samples

3.7.1. Analysis of Hens’ Feed Mixtures

HPLC analysis of hens’ feed mixture samples was performed according to the previously described protocol for Chromatographic Procedure (Section 2.3) and MSM protocol for extraction of lutein. Composition of standard hens’ feed mixture is shown in Table S1 in Supplementary Materials. The obtained results and composition of analyzed samples are shown in Table 4. The data show that there was a significant difference in the lutein content among the analyzed hens’ feed mixtures (p < 0.001). The standard feed mixture had the highest lutein content, while the lowest content was recorded in the standard hens’ feed mixture mixed with ground corn, oats, and wheat. The Premix (vitamin and mineral supplement) had significantly less lutein than all tested feed mixtures (p < 0.001). In all samples, the lutein concentration ranged from 31.487 to 41.732 mg of lutein/kg of feed mixture, which is consistent with the results in the literature [48]. The reported range of lutein concentrations in feed for laying hens is from 5.5 mg of lutein/kg of feed mixture to 50 mg of lutein/kg of feed mixture [49]. The difference of about 10 mg of lutein/kg feed mixture which is analyzed is probably due to variations in the composition and storage conditions of the hens’ feed mixture (e.g., longer storage time, variation in temperature, exposure to light).

3.7.2. Analysis of Food Samples

Food samples were prepared according to the previously described protocol (Section 2.7) and MSM protocol for lutein extraction. HPLC analysis of food samples was performed according to the previously described protocol for Chromatographic Procedure (Section 2.3). The percentages of moisture in food samples calculated after drying are shown in Table 5. The concentration of lutein in fresh samples (FW) in our study was calculated based on the percentage of moisture in the sample. Concentrations of lutein obtained from food samples in our investigation and previously conducted investigations are shown in Table 6. The highest lutein concentration was determined in parsley leaves (1581.489 mg/kg), and the lowest was found in wheat flour (4.737 mg/kg). Significantly higher lutein concentrations were found in samples of parsley leaves, lettuce, and kale (p < 0.001) compared to other food samples.
The concentration of lutein in various samples depends on the cultivar and the production system used, which is the reason for the wide range of determined concentrations. Table 6 presents data obtained from previously conducted investigations.
Lettuce is a vegetable known for its benefits to human health, largely due to the various bioactive compounds it contains. One of the primary carotenoids in lettuce is lutein [50]. Kim et al. reported lutein concentrations ranging from 16.4 mg/kg FW in cultivar Abata to 38.2 mg/kg FW in cultivar Super Caesar Red [51], while El-Nakhel et al. determined concentrations from 5.58 mg/kg FW in Green Salanova to 13.38 mg/kg FW in Red Salanova, depending on the variety [52]. Rouphael et al. found high lutein concentrations of 754.5 and 746.0 mg/kg DW in the cultivars Red Salanova and Baby Romaine, respectively, while the lowest lutein concentration was determined in cultivar Lollo Verde (176.9 mg/kg DW) [53]. Massa et al. reported that the ‘Outredgeous Red Romaine’ cultivar contained 3160 mg/kg DW [54]. In our investigation, the concentration of lutein in lettuce was 1179.246 mg/kg DW. Becerra-Moreno et al. [55] determined lutein concentrations of 100.05 mg/kg FW in the Winterbor kale cultivar and 71.58 mg/kg FW in the Maribor kale cultivar. de Azevedo et al. [56] reported an average of 50.6 mg/kg FW, while 16.57 mg/kg FW was determined by Lefsurd et al. and the same authors determined 15.1 mg/kg FW in later research [57]. Piyarach et al. reported a lutein concentration of 619.37 mg/kg FW in a sample of Chinese kale [58]. Zhang et al. investigated lutein concentrations in varieties of Chinese kale and determined concentrations ranging from 660 mg/kg DW to 2530 mg/kg DW. In our investigation, a kale sample contained a lutein concentration of 966.543 mg/kg DW [59]. Hajare et al., using TLC, reported lutein concentrations in carrot between 0.830 and 1.665 mg/kg FW, depending on standing time in acetone [60]. Piyarach et al. determined a lutein concentration of 22.30 mg/kg FW in a sample of carrot [58]. Yoo et al. analyzed lutein concentrations in selected carrot lines and determined lutein concentrations between 4.9 and 15.2 mg/kg FW [61]. Blando et al. found a lutein concentration of 57.58 mg/kg DW in black carrot [62]. Our investigation determined a lutein concentration of 15.734 mg/kg DW in a carrot sample. In butternut pumpkin, Bohoyo-Gil et al. [63] determined a lutein concentration of 16.8 mg/kg FW. Piyarach et al. determined lutein in a higher concentration of 383.98 mg/kg FW in a sample of pumpkin [58]. In our investigation, the concentration of lutein in butternut squash was 3.592 mg/kg FW. Dadan et al. investigated the influence of ultrasound, steaming, and dipping on the lutein concentration in parsley and reported a lutein concentration of 413.0 mg/kg DW [64]. The same authors, in a previously conducted study, determined lutein concentrations in the range of 811.0–1306.0 mg/kg DW [65]. de los Ángeles Proz et al. investigated the influence of cultivation systems on lutein concentration in parsley. The obtained results showed the highest concentration in parsley cultivated indoors (780.52 mg/kg DW) [66]. In our investigation, parsley leaves showed a lutein concentration of 1581.489 mg/kg DW. The paprika spice sample in our investigation showed a lutein concentration of 522.734 mg/kg DW. Chili pepper had the highest lutein concentration (61.4 mg/kg DW under an organic production system and 55.0 mg/kg DW under a conventional production system) in research conducted by Ponder et al. [67]. Arrizabalaga-Larrañaga et al. used methanol:acetone (1:1, v/v) as a solvent mixture for carotenoid extraction from paprika samples, but without the saponification step, and they did not confirm the presence of lutein in their research samples [68]. Research conducted by Kim et al. demonstrated that the concentration of lutein in paprika depends on the cultivation method and color. The highest concentration was obtained in orange papers grown using a soil-based cultivation method (376.9 mg/kg DW), while in yellow peppers with a soilless cultivation method, the highest concentration was 291.0 mg/kg DW [69]. Carniero et al. determined that the lutein concentration in heirloom varieties of corn grains ranged from 8.53 mg/kg DW to 10.08 mg/kg DW [70], while Kurilich et al. found 27.59 mg/kg DW [71]. Chai et al. determined a lutein concentration of 2438.18 mg/kg DW in the corn grain of one Chinese cultivar [72]. The lutein concentration of 27.185 mg/kg DW determined in our sample is consistent with those reported in the literature. The content of lutein in Chinese common wheat was the subject of investigation by Li et al. The concentration range was between 0.87 to 8.90 mg/kg DW [73]. Abdel-Aal et al. found that wheat species Durum, Kamut, and Khorasan have levels of lutein in the range of 5.41 to 5.77 mg/kg DW, while common bread or pastry wheat had concentrations ranging from 2.01 to 2.11 mg/kg DW [74]. The result obtained in our investigation (4.737 mg/kg DW) is in accordance with previous investigations.

4. Conclusions

Our investigation demonstrated that by replacing the solvents used in SM (the adapted AOAC method developed by Leeson et al.), hexane:acetone:ethanol:toluene (10:7:6:7, v/v/v/v), with a mixture of methanol:acetone (1:1, v/v), the use of the toxic solvent n-hexane was reduced and toluene was completely eliminated. This new solvent mixture contributed to a 52% higher extraction of lutein from the hens’ feed mixture. It was also found that using the modified standard method (MSM) and the application of direct ultrasound (MSM-UAE) did not lead to a higher concentration of extracted lutein, likely due to the shorter waiting time following ultrasound treatment. The volume of solvents used can be further reduced, but the volumes of each component in the solvent mixture must be reduced by the same percentage, and the ratio of total volume of solvent to sample used must be 40:1. The MSM was successfully used for determination of lutein concentration in hens’ feed mixture samples. The concentration of lutein in food is influenced by numerous factors, such as cultivar, soil quality, light exposure, fertilizer use, etc. By obtaining lutein concentrations in food samples that are consistent with the literature, our investigation confirmed that the MSM is suitable for determining lutein concentrations in food samples. Although solvent extraction is somewhat outdated, it is still successfully used for the extraction of lutein from hens’ feed mixtures and food samples, especially if the number of samples to be analyzed is relatively small. The simplicity of the method and the reduced amount of toxic solvents make this method readily available. Moreover, the potential for further investigation of the influence of ultrasound and modification of solvent composition on improving lutein extraction from feed and food samples offer opportunities to develop methods that are effective but simple.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15052360/s1, Table S1: Composition of standard hens feed mixture.

Author Contributions

Conceptualization, funding acquisition, supervision, writing, resource supervision, O.G., Z.K., M.K. and G.K.; writing (review and revision), O.G.; methodology, visualization, O.G.; analysis and data curation, formal analysis, investigation, validation, writing (original draft), O.G. and D.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research is supported by the European Structural and Investment Fund’s grant for the Croatian National Scientific Center of Excellence for Personalized Health Care (grant #KK.01.1.1.01.0010) and by the Ministry of Science and Education of the Republic of Croatia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable, as there are no humans involved in this study.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Calibration curve for lutein (lutein concentrations γ = 0.25, 0.50, 1.25, 2.50, and 5.00 mg/L).
Figure 1. Calibration curve for lutein (lutein concentrations γ = 0.25, 0.50, 1.25, 2.50, and 5.00 mg/L).
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Figure 2. Comparison of unground (a) and ground and sieved (b) hens’ feed mixture.
Figure 2. Comparison of unground (a) and ground and sieved (b) hens’ feed mixture.
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Figure 3. Comparison of lutein content in unground (—) and ground (—) samples obtained with the standard method (SM).
Figure 3. Comparison of lutein content in unground (—) and ground (—) samples obtained with the standard method (SM).
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Figure 4. Comparison of lutein content obtained by MSM-UAE (—) and MSM (—).
Figure 4. Comparison of lutein content obtained by MSM-UAE (—) and MSM (—).
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Table 1. Comparison of lutein content extracted from samples of hens’ feed mixture obtained by using chosen solvents ( x ¯ ± sd).
Table 1. Comparison of lutein content extracted from samples of hens’ feed mixture obtained by using chosen solvents ( x ¯ ± sd).
Solvent/Solvent Mixturemg of Lutein/kg of Feed Mixture
methanol:acetone (1:1, v/v)4.572 ± 0.28 a
methanol3.499 ± 0.17 b
ethanol3.201 ± 0.13 b
acetone1.214 ± 0.04 c
2-propanol0.543 ± 0.09 d
p-value<0.01
x ¯ = mean; sd = standard deviation; a,b,c,d values within a column with different letters differ significantly at p < 0.001.
Table 2. Comparison of lutein content obtained by standard method and modified standard method ( x ¯ ± sd).
Table 2. Comparison of lutein content obtained by standard method and modified standard method ( x ¯ ± sd).
Methodmg of Lutein/kg of Feed Mixture
SM12.753 ± 0.715 b
MSM19.410 ± 1.337 a
p-value<0.001
x ¯ = mean; sd = standard deviation; a,b values within a column with different letters differ significantly at p < 0.001.
Table 3. Concentrations of lutein obtained from samples used for determination of suitability of MSM extraction procedure.
Table 3. Concentrations of lutein obtained from samples used for determination of suitability of MSM extraction procedure.
SampleLutein (mg/L)
S10.37727
S20.39106
standard0.08794
measuredS1 + standard0.48620
S2 + standard0.48688
theoreticallyS1 + standard0.46521
S2 + standard0.47900
recovery (%)S1 + standard95.68
S2 + standard98.38
Table 4. Composition of analyzed hens’ feed mixtures and obtained concentrations of lutein ( x ¯ ± sd).
Table 4. Composition of analyzed hens’ feed mixtures and obtained concentrations of lutein ( x ¯ ± sd).
SampleCompositionmg of Lutein/kg of Feed Mixture
1standard hens’ feed mixture41.732 ± 0.63 a
2Premix *8.482 ± 0.56 e
3standard hens’ feed mixture with the addition of corn40.181 ± 1.16 b
4ground corn and Premix * mixed in a ratio of 4:135.828 ± 0.67 c
5standard hens’ feed mixture mixed with ground corn, barley, and soy35.161 ± 0.61 c
6standard hens’ feed mixture mixed with ground corn, oats, and wheat3.483 ± 1.70 d
p-value<0.001
Samples 1 and 2 are provided by the Faculty of Agrobiotechnical Sciences Osijek, University of Osijek; Samples 3–6 are provided by family farms; x ¯ = mean; sd = standard deviation; a,b,c,d,e values within a column with different letters differ significantly at p < 0.001. * Premix composition is given in Supplementary Materials Table S1.
Table 5. Percentage of moisture in food samples.
Table 5. Percentage of moisture in food samples.
SampleMoisture (%)* DW (%)
kale82.03617.964
parsley leaves81.84618.154
carrot89.83510.165
lettuce94.2995.701
butternut squash92.7047.296
* DW—dry weight.
Table 6. Concentrations of lutein obtained from food samples in our investigation and previously conducted investigations ( x ¯ ± sd).
Table 6. Concentrations of lutein obtained from food samples in our investigation and previously conducted investigations ( x ¯ ± sd).
SamplePreviously Conducted InvestigationsOur Investigation
mg/kg of * DWmg/kg of ** FWReferencemg/kg of * DWmg/kg of ** FW
lettuce 16.4–38.2451179.246 ± 98.40 b67.229
5.58–13.3846
176.9–754.5 47
3160 48
kale 71.58–100.0549966.543 ± 74.43 c173.62
50.650
15.151
441.44619.3752
660–2530 53
carrot 0.830–1.6655415.734 ± 0.64 e1.599
22.352
4.9–15.255
57.58 56
butternut squash 16.85749.234 ± 2.95 e3.592
383.9852
parsley leaves413.0 581581.489 ± 124.82 a287.125
811.0–1306.0 59
780.52 60
paprika spice55.0–61.4 61522.734 ± 25.42 d-
n.d. ***n.d. ***62
291.0–376.9 63
cornmeal (polenta)8.53–10.08 6427.185 ± 0.84 e-
27.59 65
2438.18 66
wheat flour0.87–8.90 674.737 ± 0.17 e-
5.41–5.77 68
2.01–2.11
p-value <0.001-
* DW—dry weight, ** FW—fresh weight, *** n.d.—not determined; x ¯ = mean; sd = standard deviation; a,b,c,d,e values within a column with different letters differ significantly at p < 0.001.
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Galović, O.; Prokopec, D.; Kralik, Z.; Košević, M.; Kralik, G. Improved Efficiency of Lutein Extraction from Hens’ Feed Mixture and Food Samples Using Less Toxic Solvent Mixture. Appl. Sci. 2025, 15, 2360. https://doi.org/10.3390/app15052360

AMA Style

Galović O, Prokopec D, Kralik Z, Košević M, Kralik G. Improved Efficiency of Lutein Extraction from Hens’ Feed Mixture and Food Samples Using Less Toxic Solvent Mixture. Applied Sciences. 2025; 15(5):2360. https://doi.org/10.3390/app15052360

Chicago/Turabian Style

Galović, Olivera, Doris Prokopec, Zlata Kralik, Manuela Košević, and Gordana Kralik. 2025. "Improved Efficiency of Lutein Extraction from Hens’ Feed Mixture and Food Samples Using Less Toxic Solvent Mixture" Applied Sciences 15, no. 5: 2360. https://doi.org/10.3390/app15052360

APA Style

Galović, O., Prokopec, D., Kralik, Z., Košević, M., & Kralik, G. (2025). Improved Efficiency of Lutein Extraction from Hens’ Feed Mixture and Food Samples Using Less Toxic Solvent Mixture. Applied Sciences, 15(5), 2360. https://doi.org/10.3390/app15052360

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