Stability and shelf life modeling of natural colorant from bee pollen

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Food Packaging and Shelf Life on December 2023, available at: https://doi.org/10.1016/j.fpsl.2023.101169 .” 1 Stability and shelf life modeling of natural colorant from bee pollen 1 Claudia Salazar-González1, Carlos A. Fuenmayor2, Consuelo Díaz- Moreno2*, Carla M. Stinco3, 2 Francisco J. Heredia3 3 1Facultad de Ingeniería – Departamento de Ingeniería Química y Ambiental, Universidad Nacional 4 de Colombia, Carrera 30 # 45-03, Bogotá, Colombia. 5 2Instituto de Ciencia y Tecnología de Alimentos (ICTA), Universidad Nacional de Colombia, 6 Carrera 30 # 45-03, Edificio 500, Bogotá D.C., Colombia. 7 3Laboratorio de Color y Calidad de Alimentos, Facultad de Farmacia, Universidad de Sevilla, 8 41012, Sevilla, Spain 9 *Correspondence to: Consuelo Díaz-Moreno, Instituto de Ciencia y Tecnología de Alimentos, 10 Universidad Nacional de Colombia. Carrera 30 # 45-03, Edificio 500, Bogotá D.C., Colombia. 11 Email: amcdiazm[email protected] Phone: +57 311 4348360. 12 13 ABSTRACT 14 One of the main limitations for natural food colorants as potential replacements for synthetic 15 alternatives is their stability and shelf life, which are determinant to their successful 16 marketing. Bee pollen can be used as a source of natural pigments to substitute artificial 17 colorants in the food, medicine, and cosmetics industries, showing advantages due to their 18 bioactivity. This work studied the stability and shelf life of an oil-based carotenoid-rich 19 extract (CRE) obtained from bee pollen via microwave-assisted extraction. Total carotenoid 20 content (TCC), lipid oxidation indices, and color in the CIELAB space were evaluated during 21 90 days at three different temperatures: 30°C, 45°C, and 60°C. The results showed that while 22 the color remains stable, the TCC decreases and the peroxide index increases as a function 23 of temperature. The overall CRE degradation kinetics was better modeled as a zero-order 24 2 reaction and, with the Arrhenius equation, the shelf life of the colorant was determined as 37 25 months (at 20°C) with a hue (hab) critical value of 70. These results demonstrate the high 26 stability of a new type of natural colorant as well as the efficacy of bee pollen carotenoids in 27 combination with oil lipids in color preservation. 28 Keywords: antioxidants, carotenoids, natural and functional ingredients, pigments 29 30 1. Introduction 31 Food secondary ingredients, such as colorants, are expected to have extended shelf life to 32 comply with industrial requirements. According to their composition and type of processing, 33 food additives have a maximum time in which they retain the physicochemical, 34 microbiological, or sensory characteristics. The stability and shelf life studies provide 35 information to developers on their behavior and the changes in the desired functionality (e.g., 36 pigmentation capacity) under storage conditions, which is critical for the use in standardized 37 and high-quality food (Bora et al., 2019). This is particularly important for natural additives, 38 which often contain molecules that are intrinsically less stable than their synthetic 39 counterparts. Indeed, the lower stability of natural colorants compared with artificial ones is 40 one of the main drawbacks for its industrial use (de Mejia et al., 2020). Carotenoids are an 41 important class of natural pigments that can be found in several plant tissues, able to confer 42 color, nutritional value, and bioactive properties as ingredients in processed foods 43 (Meléndez-Martínez, 2018). Bee pollen is one of the richest sources of carotenoids for the 44 food industry (Salazar-González et al., 2022), which can be extracted by different 45 technological strategies (Elik et al., 2020; Rahimi & Mikani, 2019; Salazar-Gonzalez et al., 46 2019). 47 48 3 One of those is microwave-assisted extraction (MAE), which uses waves that interact with 49 molecules, absorb energy and turn it into heat. In the case of carotenoids, the energy is 50 absorbed through temporary dipole moments generated by the uneven distribution of 51 electrons (Richter y Pirola, 2017). The absorption of this energy causes the internal 52 temperature of the biological structures, as well as their pressure, to increase drastically, 53 causing the structures to break. This destruction facilitates the transfer of the solvent to the 54 matrix and the secondary metabolites to the solvent. The protection is removed and 55 compounds are released, thus, the extraction is enhanced and continues until equilibrium is 56 reached (Chan et al., 2017; Chizoba-Ekezie et al., 2017; Vernès et al., 2020; Vinatoru et al., 57 2017). However, carotenoids are unstable molecules and suffer different degradation 58 mechanisms including temperature-induced and photooxidative-reactions (Meléndez- 59 Martínez, 2018). 60 61 The shelf life of natural colorants, such as carotenoids extracted from plant materials, 62 depends on factors related to their molecular structure, interaction with other extracted 63 compounds and solvents (intrinsic properties), and to environmental variables in their 64 processing and storage (external factors) (Hough, 2010). In oils and oil-based natural 65 colorants, these factors influence both sensory quality and physicochemical quality indexes 66 including oxidation values and composition (Farhoosh & Hoseini-Yazdi, 2013). 67 68 The extraction of natural hydrophobic pigments, such as carotenoids, from plant sources, 69 using vegetable oils is advantageous for the use as natural colorants in processed foods in 70 terms of safety and environmental impact, in comparison to the use of organic volatile 71 solvents (Che Musa et al., 2018; Golshany, 2019; Ho et al., 2015). However, due to the 72 4 impossibility of separating the vegetable oils from the extracted pigments by downstream 73 processing, the quality of the colorant not only depends on the properties of the extracted 74 compounds, but it is also strongly influenced by the physicochemical stability of the oil. 75 Studies with oils have evaluated different factors affecting their stability, such as high and 76 low temperatures, oleoresins blends, and preliminary raw material treatments (Andreou et 77 al., 2017; Farhoosh & Hoseini-Yazdi, 2013; Upadhyay & Mishra, 2015). 78 79 To elucidate the shelf life of oils, indirect or accelerated methods are usually preferred, due 80 to their long shelf life. In accelerated conditions, the test time is reduced by increasing the 81 rate of deterioration by modifying extrinsic factors such as temperature, exposure to light, or 82 relative humidity. With the results obtained, the shelf life under normal conditions is 83 calculated using mathematical models. Different predictive models can be selected 84 depending on the variables evaluated during storage: chemical, such as compound formation 85 or degradation (chemical kinetics); physical such as a change in texture (sensory 86 deterioration); or microbial growth. Since the degradation mechanisms of vegetable oils and 87 natural pigments are mainly chemical (e.g., oxidation reactions), chemical kinetics models 88 are generally used to predict their shelf life under a certain storage condition. 89 90 Temperature is usually the most important storage variable affecting the shelf life of both 91 oils and pigments. Farhoosh and Hoseini-Yazdi (2013) evaluated 9 different oils at low and 92 high temperatures, finding that the oxidative stability of oil ranges from 6-26 months at 25°C 93 depending on the mathematical model used to analyze the data. The stability of isolated 94 carotenoids from natural sources has also been studied and modeled (Lombardelli et al., 95 2021; Mutsokoti et al., 2017). Moreover, the behavior of carotenoids applied as colorant in 96 5 processed foods during their storage has been studied in some matrices, such as fish sausage 97 (Chaijan & Panpipat, 2021) and linseed oil (Varas et al., 2020), finding specificity between 98 their stability and the matrix composition, besides the storage conditions (Sabuz et al., 2020; 99 Yuliati et al., 2019). However, the physicochemical and sensory stability of oil-based 100 colorants containing extracted carotenoids from bee pollen, considering the degradation 101 mechanisms of either the pigments and the solvent, has not been previously evaluated nor 102 modeled. 103 104 This study aimed at evaluating the stability of a carotenoid-rich extract from bee pollen 105 (CRE) as a function of storage temperature, monitoring the most relevant physicochemical 106 quality features, and using chemical kinetic models to predict the storage behavior of color. 107 This work contributes to the assessment of the marketability of a new type of natural colorant 108 for the food, pharmaceutical, and cosmetic industry. 109 110 2. Material and Methods 111 2.1 Carotenoid-rich extract (CRE) 112 Carotenoid-rich extract (CRE) was obtained by a microwave extraction process at 553 W for 113 2 min using an Advanced Microwave Extraction System ETHOS X (Milestone, Shelton, CT, 114 USA), equipped with a rotor of 12 Teflon hermetically closed vessels with magnetic stirring. 115 After extraction, the slurry was centrifuged at 10000 rpm for 10 min at ~20°C, and CRE was 116 frozen at -40°C until analyzed. 117 118 2.2 Evaluation of the physicochemical characteristics of the CRE as natural colorant 119 2.2.1 Total carotenoid content (TCC) 120 6 Preparation stock solution for calibration curve: 5.00±0.01 mg of β-carotene was weighed 121 and brought to 100.00±0.01 g with sunflower oil (Ordóñez-Santos, Pinzón-Zarate, and 122 González-Salcedo, 2015). The solution was stirred for one hour in the dark at room 123 temperature (17-23°C). It was then stored under refrigeration (4°C±1°C) for 15 days. After 124 this time, the shelf life of the stock solution was 5 days. 125 Calibration curve: six dilutions (0.5-6.5 µg/g oil) were prepared from the stock solution, and 126 the measurement was made spectrophotometrically by measuring the absorbance at 463 nm. 127 This value was determined after performing a sweep between 200-700 nm and finding the 128 wavelength of maximum absorbance of β-carotene in sunflower oil. The calibration curve 129 was performed in a spectrophotometer (Thermo Fisher Scientific, USA) using sunflower oil 130 as blank and reported in µg β-caroteneeq/g oil. 131 Determination of total carotenoids in extracts (TCC): extracts were measured directly or 132 diluted in sunflower oil, if necessary, at 463 nm in a spectrophotometer using sunflower oil 133 as blank. It was reported in µg β-caroteneeq/g oil, calculated by the following equation: 134 135 𝑇𝐶𝐶 (𝑚𝑔 𝛽−𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒𝑒𝑞 𝑘𝑔 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 ) = [µ𝑔 𝛽−𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒𝑒𝑞 𝑔 𝑜𝑖𝑙 ]𝑐𝑢𝑟𝑣𝑒 ∗𝐹𝐷 (1) 136 137 where FD is the dilution factor. 138 139 2.2.2 Carotenoid profile 140 The extraction and saponification of carotenoids were carried out following the method by 141 Tiwari et al. (2019) with modifications. Four hundred (400) mg of CRE were placed in a 142 Falcon tube with 15 mL ethanol and 4 mL methanolic KOH solution (35% w/v). The mixture 143 7 was saponified during 30 min at 70°C under dim light. Once the saponification was finished, 144 5 mL of NaCl solution (5% w/v) were added. Immediately, 15 mL of the extraction solution 145 (hexane:ethyl acetate 85:15 v/v) were added, mixed and centrifuged at 4000 rpm for 5 min 146 at 4°C. The organic phase was collected, and the extraction procedure was repeated twice 147 over the residue. The organic-colored fractions were pooled and subsequently evaporated to 148 dryness at a temperature <30°C. The extract was dissolved in 1 mL hexane; 1 mL NaCl 149 solution (5% w/v) was added, mixed, and centrifuged at 4000 rpm for 5 min at 4°C. This 150 procedure was repeated twice. The organic phases were recovered and concentrated until 151 dryness at a temperature <30°C. The residue was re-dissolved in 200 µL of ethyl acetate, 152 centrifuged and analyzed by Rapid Resolution Liquid Chromatography (RRLC) in an Agilent 153 1260 system with diode array detection operating in the UV-vis range (Agilent, USA), 154 equipped with a C30 YMC pre-column (10×4 mm, 3 µm) and a YMC C30 (150x4.6 mm, 3 155 µm) column (Dinslaken, Germany). The mobile phase was methanol, methyl-tert-butyl ether 156 and miliQ water (Stinco et al., 2019). The selected wavelengths were 285 nm for α- 157 tocopherol and phytoene, and 450 nm for the rest of carotenoids. The identification of the 158 carotenoid compounds was carried out by comparison of their chromatographic and UV-vis 159 spectroscopic characteristics with their corresponding standards and data reported by other 160 authors (Aman et al., 2005; Song et al., 2016; Zhang et al., 2016). External calibration was 161 used for quantification and results were reported in mg carotenoid/kg oil. 162 163 2.2.3 Tristimulus colorimetry 164 Samples were brought to room temperature (17°C-23°C), and the measurement was 165 performed directly in a liquid cell in a CM-5 colorimeter (Konica Minolta, Japan). Results 166 were expressed in the CIELAB system (Rodríguez-Pulido et al., 2021) for CIE standard 167 8 illuminant D65 and CIE 1964 Standard Observer (10° angle of vision). The equipment 168 records Cartesian (L, a*, b*) and polar coordinates (C*, hab). 169 170 2.2.4 Lipid oxidation indexes 171 Peroxide value (PV): this variable was assessed according to AOAC 965.33 (Official 172 Methods of Analysis, 2012). Briefly, 5±0.5 g of sample was weighed, and 30 mL of 173 chloroform: acetic acid mixture (2:3) was added. It was stirred gently, 0.5 mL of a saturated 174 KI solution was added, stirred again, and 30 mL of distilled water was added. Finally, 0.5 175 mL of starch (1%) was added, stirred again, and titrated with Na2S2O3 (0.1 or 0.01 N) until 176 the blue color disappeared. The peroxides value was calculated as follows: 177 178 𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑒 𝑣𝑎𝑙𝑢𝑒 (𝑃𝑉,𝑚𝑒𝑞 𝑂2 𝑘𝑔 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 ) = 𝑆∗𝑁∗1000 𝑚 (2) 179 180 Where S is the volume of Na2S2O3, mL; N is Na2S2O3 concentration, N and m is the sample 181 weight, g. 182 183 p-anisidine value (AV) was performed according to ISO 6885-2016 (2016). First, 0.4±0.05 g 184 of the extract was weighed and brought to 25 mL with iso-octane. Three dilutions for analysis 185 were then prepared: 186 (i) Dilution with reaction (A1): 5 mL of the solution was transferred to a test tube, and 1 mL 187 of p-anisidine reagent (Alfa Aesar, USA) (0.125 g/50 mL acetic acid) was added, shaken, 188 and kept in the dark for 10 min. 189 15 bottle was selected as packaging to protect CRE from oxygen and light to avoid degradation 334 of the carotenoids and polyunsaturated fatty acids (PUFAs) of the oil; this container has been 335 used in similar products. Furthermore, since glass is impermeable to gases, humidity, light, 336 and oxygen, the coloring extract will be isolated from the environment. Therefore, only the 337 nature of the product (own degradation reactions) and the storage temperature should be 338 considered for the stability study and determination of shelf life. 339 340 Figure 1 shows the behavior of the different parameters over time. Since the enriched oil will 341 be used mainly as a natural colorant, the hue coordinate (hab) stability was analyzed. 342 Likewise, the carotenoid content and the peroxide value, since these parameters are 343 responsible for the color and the quality in vegetable oils, respectively. 344 345 The color of the extract, determined by the hue value, remained stable throughout the 90 days 346 of storage (Figure 1a), both for the extracts and the control samples (sunflower oil). These 347 results are similar to those reported in the stability study conducted on tomatoes and carrots 348 for 6 months (Mutsokoti et al., 2017). 349 350 In contrast, TCC decreased over time (Figure 1b), with more significant losses as temperature 351 increased. At 30°C, the loss of carotenoids was 10.7%, while for 45 and 60°C, they were 16.8 352 and 27.5%, respectively. Losses are mainly due to conjugated double bonds degradation, 353 which is higher at higher temperatures (Ordóñez-Santos et al., 2020). The results obtained in 354 this work were contrasted with those of Mutsokoti et al. (2017) who did not find significant 355 changes in carotenoids during 6 months of storage (T: 20, 30 and 40°C). The stability of 356 carotenoids was caused by the low oil addition (5%) to the mixture of tomato and carrot; this 357 16 avoided carotenoid degradation to prevent oil deterioration. Temperature-dependent losses 358 are common to other fat-soluble bioactive compounds, including certain polyphenols, which 359 can decrease 10% and 15% in olive oil stored at 6°C and 26°C, respectively, during 125 days 360 (Sanmartin et al., 2018). Importantly, the fact that the color remained stable during the 361 observed period despite the total carotenoid losses indicates that these losses are not sensory- 362 relevant. 363 364 The peroxide vale (PV) increased over time (Figure 1c), which agrees with the food 365 deterioration processes. This is an expected trend for oils stored above room temperature, 366 with and without the addition of bioactive compounds (Farhoosh & Hoseini-Yazdi, 2013; 367 Varas et al., 2020). However, there was not a clear dependence of the PV with the storage 368 temperature, suggesting complex oxidation mechanisms, probably involving secondary 369 oxidation reactions. It is essential to notice that PV never exceeded 15 meqO2/kg CRE, the 370 maximum limit according to the Codex Alimentarius standard for oil quality (Chutia & 371 Mahanta, 2020; Varas et al., 2020). 372 373 3.3 CRE shelf life determination 374 3.3.1 Reaction kinetics 375 As mentioned above, the shelf life determination requires knowledge of the degradation 376 reaction kinetics of the evaluated variables, in this case hab, TCC, and PV. From the equations 377 of the zero-, first- and second-order models, the correlation coefficients presented in Table 2 378 were obtained using linearized regression plots of the Eq. (5). 379 380 17 The data in bold (Table 2) represent the highest values obtained from the coefficients for 381 each of the temperatures evaluated. The kinetic orders that presented two or more 382 temperatures with coefficients in bold were selected as global reaction kinetics. Most of the 383 evaluated variables had a kinetic characterized by a zero-reaction order, except the hue for 384 the control and TCC for CRE, which appeared to behaver according second-order kinetics. 385 In the case of TCC, reaction order 2 was selected because the reaction constant at 30°C 386 presented values that indicate this model does not fit the experimental data. 387 388 The results of the present study differ from those reported by Varas et al. (2020) and Borguini 389 et al. (2021), who enriched linseed, soybean, and olive oils with carotenoids, respectively. 390 They found that the oxidation mechanism for PV is described by first-order kinetics, whereas 391 for CRE, PV has zero-order kinetics. Furthermore, the degradation of carotenoids in soybean 392 oil presents zero-order kinetics (Borguini et al., 2021) and 1.5-order in canola and palm oils 393 (Mba et al., 2017). Those results contradict the present work where the TCC degradation 394 presented second-order kinetics. 395 396 As expected, the reaction constants k increased with temperature. Additionally, PV reaction 397 constants were the highest, indicating that they will degrade faster than carotenoids or color. 398 In fact, PUFAs in sunflower oil are susceptible to oxidation with increasing temperatures 399 generating the hydroperoxides, which decompose themselves; this generates more free 400 radicals that will accelerate the propagation reactions (Farhoosh & Hoseini-Yazdi, 2013). 401 Similar to the studies of Rahimi and Mikani (2019) and Varas et al. (2020) made with the 402 addition of lycopene extract in sunflower and linseed oil, respectively, bee pollen carotenoids 403 decreased the reaction rate of peroxides, as the constants are lower in CRE than in pure oil, 404 18 evidencing the positive effect of including carotenoids in vegetable oils in terms of oxidative 405 quality. This effect could be related to the antioxidant mechanism of carotenoids. Carotenoids 406 are antioxidant substances because they are able to reduce oxidative stress via capture 407 radicals by electron transfer. In this mechanism, carotenoids give electrons more easily than 408 the radicals, and only could be consider as an antioxidant if the radical accept the electron 409 (Meléndez-Martínez, 2018). Due to the acceptance of electrons by peroxides, carotenoids in 410 CRE could be considered as good antioxidant substances. 411 412 3.3.2 Effect of temperature on the reaction rate and shelf life determination 413 The effect of temperature on the degradation rate of the parameters was evaluated as the 414 activation energy (Ea), using the values of k and and the Arrhenius equation (Eq. 6) after 415 linearization (Eq. 7). The results are presented in Figure 2 and Table 3. Ea indicates the 416 minimum energy required to carry out a chemical reaction. The lower value of Ea for hab in 417 CRE and for PV in the oil indicate that these parameters are less sensitive to temperature, i.e. 418 a larger change in temperature is needed for there to be a change in the reaction rate (Varas 419 et al., 2020). The Ea of TCC was lower than that reported by Mba et al. (2017) (56-71 420 kJ/mol), while the Ea of PV in CRE was higher than that reported by Varas et al. (2020) (24.8 421 kJ/mol). 422 423 𝑙𝑛𝑘 = K𝑜−𝐸𝑎𝑅 ⁄∗1 𝑇 ⁄ (7) 424 425 Using the Arrhenius equation, the activation energy, the Ko constant, and a room temperature 426 of 20°C, the reaction constant k was calculated. Once the color degradation (measured as 427 hue) follows a zero-order kinetic, the equation of zero order was used, considering a critical 428 19 value for hab of 70 to avoid excessive degradation in hue; this could be observed by the color 429 change from orange to yellow. The CRE shelf life was 37 months at storage conditions: i.e., 430 room temperature packed in amber glass jars. These results demonstrated the efficiency of 431 bee pollen carotenoids in combination with oil lipids in color preservation. 432 433 4. Conclusion 434 An oil-based natural carotenoid-rich colorant (CRE) was obtained by microwave-assisted 435 extraction of bee pollen using sunflower oil. The CRE is rich in several carotenoids, mainly 436 xanthophylls, and vitamin E, and it can be used as colorant in orange to red hues. The 437 behavior of the CRE under accelerated conditions (30°C-60°C during 3 moths) demonstrated 438 (i) its color persistence (constant hue values throughout storage), (ii) the occurrence of lipid 439 oxidation following complex mechanisms, involving primary and secondary oxidative 440 reactions, but maintaining an adequate oxidative quality (peroxide value < 10 meqO2/kg oil), 441 and (iii) a carotenoid loss of maximum 27%. Considering a loss of color up to a maximum 442 hue value of 70 in the CIE color space, the CRE has a shelf life of 37 months stored in amber 443 glass bottles at 20°C, representing a competitive advantage in the market concerning natural 444 colorants in general. 445 446 ACKNOWLEDGMENTS 447 448 This work was supported by the [Fondo Francisco José de Caldas - FJDC, derivado del 449 Convenio 785 de 2017 (MINCIENCIAS - Departamento de Cundinamarca)]; under Grant 450 [802-2018]. Proyect "Technological strategies for the assessment of bee pollen produced in 451 20 Cundinamarca as a source of fat-soluble bioactive components of nutritional and antioxidant 452 interest for inclusion in food matrices". 453 454 Declarations of interest: none 455 456 REFERENCES 457 458 AENOR. (2016). Aceites y grasas de origen animal y vegetal. Determinación del índice de 459 anisidina. ISO 6885:2016 (p. 14). AENOR. 460 Aman, R., Biehl, J., Carle, R., Conrad, J., Beifuss, U., & Schieber, A. (2005). 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