INTRODUCTION:

Carotenoids with a 40-carbon backbone that contains a large conjugated double-bond system, which play a vital role in photosynthesis and photoprotection^1,2. Most Carotenoids dissolve well in non-polar solvents^3,4 and are therefore liposoluble, a property that gives fruits, plants, birds and marine fauna some characteristic yellow, orange and red colours. This colouring capacity comes from a long series of conjugated double bonds that is also responsible for the antioxidant properties of these materials⁵.

Hydrocarbon Carotenoids are referred to as carotenes, while their oxygenated derivatives are called as xanthophylls. Within the latter group the oxygen atom can be present in the form of hydroxyl group (as in the case of lutein) or as oxy groups (as in the case of cantaxanthin) or as combination of both (as in astaxanthin)⁶.

Lutein, one of the main photosynthetic pigments, is a promising natural product for both the nutritional and pharmaceutical applications. The photosynthetic pigments present in photosynthetic microorganisms (such as microalgae) are known to play a key role in photosynthesis and photoprotection⁷. Lutein, together with zeaxanthin, imparts colour to the macula lutea, the spot in the human retina that allows the appreciation of fine details, and it is belived to have an important role as an antioxidant^8,9. This compound plays a critical role in the prevention of age-related macular degeneration (AMD). The consumption of fruits and vegetables rich in carotenoids, lutein and zeaxanthin, can be beneficial in delaying or protecting against AMD, through their antioxidative effects¹⁰. Lutein is an important isoprenoid polymer containing many conjugated double bonds, which can be readily isomerized, oxidized and degraded. Lutein is a lipophilic compound and is for the most part insoluble in water. The vicinity of the long chromophore of conjugated polyene chain gives the particular light-retaining properties. The polyene chain is vulnerable to oxidative debasement because is chemically unstable in acids and it depends on environmental conditions (by light/heat). Oxidative degradation, the main cause of extensive losses of carotenoids, depends on the availability of oxygen and is stimulated by light, enzymes, metals, and co-oxidation with lipid hydroperoxides. They have different susceptibilities to oxidation, being the more labile β-carotene, lutein, and violaxanthin¹¹.Formation of epoxides and apocarotenoids (carotenoids with shortened carbon skeleton) appears to be the initial step and total loss of color and biologic activities are the final consequences. Oxygen, is highly destructivein combination with light and heat, especially direct sunlight or ultraviolet light induces trans-cisphotoisomerization and photo destruction of carotenoids. Thus, work on carotenoids must be performed under subdued light¹². Since pure lutein is frequently used as a standard for identification and quantification, knowledge of its stability under various storage conditions is essential.

Many researchers have studied carotenoid stability in food or model systems. These molecules are known to be relatively stable in their natural environment, but become sensitive to light, temperature and chemical exposure during processing^13,14. Moreover, the stability of xanthophylls was found to be increasingly affected by the degree of their esterification and the nature of the fatty acids^15,16,17. In the present paper, the stability of lutein extracts was studied in response to physical and chemical parameters, such as light, temperature, antioxidant addition etc.

MATERIALS AND METHODS:

Culture condition:

Chlorella salina was obtained from CIBA, Chennai, Tamilnadu, India and cultivated using sterile f/2 medium under 4000 Lux illuminated with white fluorescent light for 12:12 h light and dark condition for 18 days.

Extraction of lutein from algal biomass:

Preparation of crude luteinwas carried out according to the literature¹⁸. In brief, 2ml of culture fluid was centrifuged at 3000 rpm for 10 min, and the supernatant was discarded. One ml of solution containing 2.5% ascorbic acid and 10 M KOH was added to the cell pellets, and the mixture is incubated at 60° C for 10m before cooled down to room temperature. 19 ml Methanol was subsequently added to the mixture for the extraction of lutein. The mixture was centrifuged at 3000 rpm for 15 min at 4° C, and the supernatant was collected and kept at-12° C for the subsequent determination of lutein stability.

Treatments:

The lutein extract was subjected to various treatments including thermal treatment, antioxidant additives and light exposure. Colour changes of the treated samples were monitored from 0-3 weeks. Absorption spectra were monitored at 445 nm, using a UV-Vis spectrophotometer and the absorbance represents the lutein concentration in the extract. The absorbance is directly proportional to the lutein concentration, meaning that the higher the absorbance the higher the concentration.

Stability towards addition of antioxidant:

Ascorbic acid was added to the lutein extract with an increasing concentrations of 0.01% (W/V), 0.1% (W/V) and 0.5% (W/V). Each set of sample treatment was finally kept at 4°C, ambient temperature (25-28° C) and-20° C with and without light exposure. Control was prepared without addition of ascorbic acid. The percentage of colour was calculated for up to 3 weeks.

Stability towards temperature:

A preliminary study was conducted to determine the heat tolerance of lutein at different temperature 4°C,-20°C, ambient temperature (25-28°C) and 32°C respectively, with and without addition of antioxidant. Each set of sample treatment was finally kept in dark and with light exposure, and then percentage of colour loss was calculated.

Stability towards exposure to light:

The lutein samples without and with different concentration of antioxidant were exposed to continuous illumination at 4600 Lux provided with white fluorescent tubes at 25°C in an incubator. The percentage of colour was calculated for up to 3 weeks.

Stability towards Irradiation to UV:

The lutein samples without and with different concentration of antioxidant were exposed to ultraviolet light for up to 96 hrs. The percentage of colour was calculated for up to 3 weeks.

RESULTS AND DISCUSSION:

The parameters such as temperature, oxygen levels, and humidity had all been reported to affect the stability of carotenes in other foods during storage^19,20,21, very limited number of studies on the lutein stability were reported, so changes lutein during various storages were investigated.

The lutein stability was defined as the ratio of evolved lutein content in the microalga at a specific time period over the original lutein content at day 0, using a percentage scale. Freshly extracted and purified lutein was scanned through with a spectrophotometer between the wavelengths of 300-700nm. The absorption spectrum showed two major peaks corresponded to 444 and 471 nm (Fig: 1). Absorbance peak at 444 nm is characteristic absorption for lutein pigment. Least stability was observed when the extracts were exposed to light and at ambient temperature.

Effect of temperature:

Different storage temperature have different effects on stability. The absorption spectra of lutein after 3 weeks of storage at-20°C were slightly modified compared to those at 4°C. This was confirmed by the changes in absorbance spectra. Figure:1 shows the stability of lutein with added antioxidants at different storage temperature. At 4°C the extracts were stable up to 48h; at-20°C, the absorbance after 3 weeks were the same as at 0h. While at temperatures above these, the degradation of carotenoid increased gradually by increasing temperature. The highest degradation of lutein was observed at 32°C, followed by 25°C. This result showed that the higher the storage temperature, the faster was the degradation.

Fig: 1 Effect of storage condition on the stability of lutein with added antioxidants at different temperature

High temperature may cause the breakage of double bonds in the carotenoid molecule and caused pigment degradation^22,23. These results shows that thelutein was more stable at lower temperature and the results are in accordance with the previous results reported by Shi and Chen¹⁸.²⁴ Layug et al., indicated that the xanthophylls in Alfa alfa leaf extract was well protected at a low temperature (-18°C) for long term storage regardless of the antioxidant used. In addition the samples with ascorbic acid decayed much slower than that without. The addition of 0.01% of ascorbic acid showed greater stability followed by 0.1% and 0.5%, respectively. Therefore, the addition of a lesser amount of antioxidant after extraction could stabilize the pigment more effectively.

Effect of antioxidant addition:

In this present study, the time course of lutein stability with different concentration of antioxidant and temperatures are represented. Ascorbic acid was added to the lutein pigment at various concentrations such as 0.01%, 0.1% and 0.5%. Comparing the initial and final lutein stabilities, the decay of the lutein was faster at higher temperature than at 4°C and-20°C, regardless of the antioxidant added. Addition of ascorbic acid to the concentration of 0.01% managed to preserve the colour in all the dark storage conditions, more preferably at lower temperatures. Ascorbic acid added to 0.5% on the other hand did not offer the promising observation except for-20°C dark storage. These results were in accordance with the reported literature Chang et al.²⁵

Exposure to Light:

Photostability of the extracted lutein was investigated at 4°C under continuous illumination of 4000 Lux for 3 weeks. With the light illumination, the lutein stability was examined under different conditions (ie.) with added antioxidant (0.01%, 0.1% and 0.5%) and the same without light exposure. Samples stored under light showed faster decay than that stored at dark. Samples without the addition of ascorbic acid, the degradation of lutein was even faster, which is about 70 % within 7 days and nearly complete degradation was observed with 2 weeks. Those that with the addition of antioxidant retarded lutein decay; i.e, only 30-40% decay was observed. These observations confirmed that lutein is susceptible to oxidation upon exposure to light²⁶ and the addition of antioxidant could retard lutein degradation.

Light and temperature played an important role in keeping the stability of lutein Structure. When exposed to light lutein showed a greater tendency to lose its chromophore. Figure: 2 shows the contents of lutein during storage in the dark. The contents of lutein were found to decrease following the increase of storage temperature, implying that degradation may still proceed even in the absence of light. Figure: 3 shows the concentrations of lutein during storage under light. After storage for 1 week at 4, 25 and 32°C under light, the amounts of lutein decreased by 0.39, 0.26 and 0.29 µg/g, respectively. These results indicates that a more pronounced degradation of lutein occurred during light storage than during dark storage.

Fig: 2 Effect of storage condition on the stability of lutein at different temperature without light (dark)

Fig: 3 Effect of storage condition on the stability of lutein at different temperature with light

Effect of UV-irradiation:

The structure of the lutein, changed during a continuous prolonged irradiation with UV-C light (300 nm) as evidenced by the changes in their absorption spectra. With an absorption maximum in the Vis region, carotenoids are obviously not efficient UV-absorbers but still are able to perform a protective function against UV-light in plants^27,28. The other very important function of carotenoids, of global character, is their anti-oxidant function (this is one of the reasons for the wide use of carotenoids in the food industry^29,30. For such a purpose, carotenoids may act in a preventive manner, i.e., they inhibit the formation of reactive oxygen species (ROS) by reacting directly with oxygen, or, if radicals are already present, they act as chain-breaking anti-oxidants³¹. Even in this simplest solution system, carotenoids undergo degradation, i.e., bleaching, when exposed to prolonged UV-irradiation. A 40% decay within a period of 10 days was observed with continuous irradiation with UV-A. A significant decrease in the concentration of lutein was observed from day 3 in UV-A treated sample. At the end of the experiment the bleaching rate is high with the UV-A treated samples. The lutein (%) retention of the treated samples were around 70% with UV-C and 60% with UV-B respectively.

The effect of light, (UV and visible light), towards lutein stability is due to the excitation of electron of the chromophores to a more energetic state, led to higher reactivity or lowered activation energy of the molecule³². The basic linear and symmetrical skeleton of the carotenoids can be cyclized at one or both ends. The shift is responsible for the yellow, orange or red colour of these compounds. The presence of oxygen in the structure might be responsible for the degradation.

CONCLUSION:

In this study, the amount of the degradation of natural lutein produced by Chlorella salina in different environmental temperature, different irradiation periods and exposure to UV for 3 weeks was investigated. In conclusion, temperature, light and UV irradiance have great impact to the colour degradation of lutein pigment. In the present study, light has been the major factor of colour deterioration. Exposure of light caused colour loss up to 50% after one week of storage in room temperature. In fact, high temperature (32°C) led to almost 80% of colour loss in the initial stage of storage. Storage with ascorbic acid exhibited a greatest stability when stored at lower temperature (4°C) even after 3 weeks of storage. Finally we observed the best condition in preserving the lutein pigment was at 4°C with 0.01% of antioxidant.

CONFLICT OF INTEREST:

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Received on 27.03.2018 Modified on 16.05.2018

Research J. Pharm. and Tech 2018; 11(8): 3308-3312.

DOI: 10.5958/0974-360X.2018.00608.X