Microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food -

Microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food

The microencapsulation of oil soluble vitamins (A, D and E) using a one pot ultrasonic process and raw egg white proteins as a shell material. Green tea catechin/iron complex coating method was further developed to impart UV filtering property to the microcapsules in order to protect the encapsulated nutrients from photo degradation. The microcapsules showed antibacterial properties and long shelf-life. The encapsulated vitamins were protected from degradation upon heating, UV irradiation, simulated storage/transit and cooking processes. The in-vitro digestion study showed that functional vitamin D can be potentially released in the gastrointestinal tract improving vitamin D availability by more than 2-fold compared to the free vitamin. The low-cost egg white shell encapsulated vitamins can improve the nutritional value of staple food products to combat maternal and child malnutrition.

Material and methods

Egg whites were obtained from hen eggs purchased from a local market and separating the natural egg yolk. Oral vitamin D, ferrous sulfate tablet, vitamin A and E from brands Ostelin®, Fero-grad® and Blackmore®, respectively were purchased from a local pharmacy. Commercial Twinings® green tea bag and plain flour were purchased from a local market.

Formulation of vitamin filled microcapsules

A 20 kHz ultrasound generator with a standard titanium horn tip of 3 mm diameter was employed for synthesizing microcapsules. Vitamin D filled egg white shelled microcapsules were obtained by layering vitamin D (50 µL) on the surface of aqueous egg
white solution (1 mL) and then sonicating at 160 W for 1 min by placing the horn tip at the oil/aqueous (vitamin D/egg white solution) interface. The microcapsules were separated from the remaining protein solution by flotation and copious washing with Milli-Q water by repeating the process a few times. Vitamin A and E filled microcapsules were both synthesized using the same method.

Green tea (GT) infusions were prepared by submerging two tea bags in 200 mL hot (~80 ◦C) water for 5 min, cooled to room temperature (~25 ◦C), and filtered through a 0.22 µm membrane filter. Iron solution (10 mg/mL) was obtained by dissolving one ferrous sulfate tablet in 10 mL milli-Q water, filtered through 0.22 µm membrane filters to remove insoluble impurities. Subsequently, the GT infusions and the iron solution were mixed (ratio 4:1) under a vortex mixer for 10 s. The mixed solution was centrifuged at 855g for 1 min, and then the dark purple supernatant was collected. The collected green tea/iron (GT/FeII/III) complex solution (200 µL) was then added to the microcapsule’s suspension (1 mL) under a constant orbital shaker (40 rpm) for 2 min. The GT/FeII/III coated microcapsules were obtained by discarding the excess supernatant after centrifugation (95g, 1 min), and resuspended in 500 µL of Milli-Q water.

Antimicrobial activity of microcapsules

The EW microcapsules were analyzed by a turbidimetric Micrococcus luteus test of enzymatic activity. EW microcapsules (200 µL) were added to 3 mL of phosphate buffer containing 2 mg/ml Micrococcus lysodeikticus. The change in absorbance of the suspension at 450 nm was measured for 6 min.

Microstructural characterization is done by optical microscopy. The zeta potential of microcapsules/oil droplets before and after digestion was analyzed by Dynamic Light Scattering (DLS) with a Zetasizer Nano ZS. The pH value of microcapsules after exposure to different digestion phases was measured.

Microcapsules incorporated in bread making processes

Dough made with plain flour and water was used as an example food matrix. A suspension of vitamin D-loaded egg white microcapsules was added into the plain flour (ratio 3: 5) by following a kneading operation via 10-min hand or 2-min KitchenAid® machine. After this, a small piece of dough (microcapsules and flour mixture) was taken to be used as a thin slice for observing the microcapsules structures in the dough. For comparison, the same amount of Nile red labeled Vitamin D emulsion (free Vitamin D) was directly added into the dough, that subsequently went through kneading processes. The encapsulated/ free vitamin D embedded into the dough went through dry heating (15 min 220 ◦C) in an oven. This heating procedure simulates the biscuit/ bread baking process. After baking, the cooked dough was re-dispersed in methanol by using a blender (Breville®) for 5 min. After centrifugation (2375g, 5 min), the solid matrices were discarded, and the supernatant was filtered by 0.22 filter membrane for further HPLC analysis. Besides, to assess the actual temperature of microcapsules during the baking process, we then carried out the internal temperature measurement of the bread at different position by inserting a thermometer in the bread at different time.

Determination of vitamin recovery

Vitamin D microcapsules and emulsion were suspended in water and then heated at 40 ◦C for 20 h in water bath to determine the stability of vitamin D. The samples were irradiated by UV light (22 V, 700 mA) for 8 h to study the protection of egg whites from vitamin D degradation. To mimic the cooking condition, microcapsules and emulsion were embedded into flour dough for baking at 220 ◦C for 15 min. After the treatment, samples were collected for nutrients recovery determination. Microcapsule samples were broken down using high intensity ultrasound (120 W) for 30s. Then methanol (ratio to sample 2:1) was used to dissolve Vitamin D, followed by centrifugation and filtration to remove the egg white shell (0.22 µm filter). HPLC analyses of vitamin A and E recovery was performed using acetone as an eluent at 25 ◦C with UV detector set at 325 nm.

Results 

Formulation of vitamin filled egg white microcapsules

The average size of vitamin D filled microcapsules was 5.2 ± 1.6 µm. In the current study, to avoid external toxic chemical reducing agents, egg white (EW) suspension was treated by heating (5 min, 90 ◦C) to thermally denature the proteins. For comparison, untreated egg white was also emulsified by ultrasound to evaluate the difference in morphology and stability of the obtained microcapsules. These ultrasonically denatured proteins containing sufficient free thiol groups can be adsorbed and crosslinked at the vitamin D droplet interface, resulting in a continuous and stable shell stabilized by hydrophobic interactions and disulfide bond linkages. The surface tension measurements (EW- vitamin D) also showed that the native EW solution possesses better surfactant properties (20.10 ± 0.28 mN/m) for vitamin D than pre-heated EW (26.85 ± 1.06 mN/m). This observation indicates that native EW proteins better adsorbed at the oildroplets interface to form a homogenous and more stable shells. Overall, this result suggests that the availability of free thiol group in solution is crucial to form a robust shell during microcapsules fabrication process.

Antimicrobial activity and shelf-life

Lysozyme from egg white has been reported to act as an antibacterial protein, which is known to degrade the peptidoglycan layer in the cell walls of Gram-positive bacteria by hydrolyzing the bond between Nacetyl muramic acid and N-acetyl glucosamine.

A decrease in the turbidity as a function of time was observed when the mixture of bacteria and microcapsules was analyzed, indicating the lysis of Micrococcus lysodeikticus. The reduction in the turbidity demonstrated the EW microcapsules still possess antimicrobial activity due to the presence of lysozyme. Therefore, these results confirm that the degradation of bacteria was induced by antibacterial function of EW microcapsules. The size of fresh, 3 months and 6 months stored microcapsules were 5 ± 2 μm, 6 ± 2 μm, and 6 ± 2 μm, respectively. The high colloidal stability combined with the antibacterial property of EW microcapsules potentially allows EW microcapsules to have long-shelf lives during transit and storage processes. Apart from Vitamin D filled microcapsules, other lipophilic (oil-soluble) micronutrients such vitamin A and vitamin E have also been successfully encapsulated into egg white shells using the same ultrasonic method. The size of vitamin A and vitamin E filled microcapsules is 5.3 ± 1.3 µm and 5.3 ± 1.2 µm, respectively.

Stability and functionality testing of microcapsules

Many nutrients are sensitive to temperature, moisture and ultraviolet light, which can cause degradation and thus limited absorption after ingestion. Although the high colloidal stability allows EW microcapsules to maintain their structure well up to 6 months, the role of microcapsules in improving encapsulated micronutrients stability against detrimental effects is vital; hence required a comprehensive investigation. To improve the ability of UV absorption, developed an additional coating method using green tea/iron complex on top of the egg whites’ shells.

Conclusions

The ultrasound-assisted formation of oil-soluble vitamin (A, D and E) filled microcapsules from raw hen egg whites has been successfully achieved. It is noted that the high availability of free thiol groups in protein solution is crucial to form stable microcapsules with robust shells. The thermal stability and nutrients functionality testing demonstrated that the microcapsules could be embedded into food products while maintaining the nutrients functionality. The long-shelf microcapsules can protect micronutrients payloads during exposure to high temperature, moisture, UV light irradiation, mechanical stress and enable slow release of nutrients after ingestion. Such highly stable micronutrient filled microcapsules are promising to be applied in food industry to enhance nutritional value of staple foods.

Reference

Zhu, H., Mettu, S., Cavalieri, F. and Ashokkumar, M., 2021. Ultrasonic microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food. Food chemistry, 353, p.129432.