Optimization and extraction of pomegranate peel extract, its nanophytosomal encapsulation and investigation of anti-fungal, physico-chemical and sensory properties of yogurt enriched with it

Abstract

Abstract Aim: The purpose of the present study was to optimize the extraction of pomegranate peel extract (PPE), its encapsulation in the form of phytosomal nano-system with the aim of enriching foods, and investigating its effect on the physico-chemical and sensory properties of yogurt and its antimicrobial effect in enriched yogurt Background: It seems that PPE, due to its beneficial properties, can be used as a natural additive in order to increase the level of food safety and improve the health of consumers. However, due to some defects of PPE such as low bioavailability and high sensitivity to environmental conditons, the application of the encapsulation method seems to be necessary. The thin layer hidration-sonication method is a useful encapsulation technique with the ability to increase bioactive compounds' bioavailability and stability, to disguise their unfavorable taste and prevent their reaction with some food components such as iron and calcium. Also, its enrichment in a food product that is widely consumed and suitable for all age groups as a food supplement with added value and with a higher level of safety is logical and justifiable. Material and methods: In the first phase, with the use of Design Expert software, the effect of sounication time and temperature as well as stirring speed on extraction efficiency, antioxidant activity, and total phenolic content of hydroethanolic PPE (ethanol/water (60/40 v/v) solvent) was investigated. Then, the optimal treatment in terms of the highest extraction efficiency, antioxidant activity, and total phenolic content was selected for preparing a phytosomal delivery system consisting of different ratios of gamma-oryzanol and lecithin by thin hydration-sonication method. Structural characteristics and stability of nanophytosomes were evaluated in terms of particle size and PPE leakage during one month of storage at 4°C. The formulation with the highest stability, the lowest polydispesity index, and the smallest average particle size was selected as the optimal formulation. The antioxidant activity and the antioxidant stability during one month, the release rate in human gastero-intestinal model (in the presence of digestive enzymes), and the cytotoxicity of the optimal formulation were evaluated on normal and cancer cell lines. Free and nanoencapsulated PPE (optimal formulation) was added to 100 g of stirred yogurt in the amount of 200 mg PPE, and its antifungal, physico-chemical, and organoleptic properties were evaluated during 4 weeks. It is worth mentioning that in order to uniformly distribute PPE in the stirred yogurt, nanoencapsulated PPE was added to the yogurt in the form of a suspension and not a powder. Results: The optimized conditions to obtain maximum extraction efficiency (38.14%), antioxidant activity (92.15%), and total phenol content (283.18 mg GAE/g PPE DW), were determined as sonication time = 70 min, sonication temperature = 61.8°C, and the stirring speed = 1000 rpm. The results suggested that the extraction of PPE by a blend of ultrasonication as a modern method and dynamic maceration as a conventional method could improve its extractability. In other words, these two methods together have intensifying effects in increasing extraction efficiency, which could further be utilized in food and agricultural industries. So that, the extraction yield, DPPH inhibitory activity and total phenol content variables from 28%, 78%, and 167 mg GAE/g PPE DW just by using the dynamic maceration method, increased up to 38.14%, 92.15% and 283.18 mg GAE/g PPE DW, respectively by using combined ultrasonication/dynamic maceration method. Also, the extraction time was reduced from up to five days in traditional methods to one day in the combined method. The optimal formulation of nanophytosomes was obtained in a weight-to-weight ratio of 8:2:2 lecithin: gamma-oryzanol: PPE. The optimized formulation was stable during one-month of storing at 4 oC and changes in the size of particles and PPE retention rate were insignificant (p > 0.05). The optimal formulation showed a favorable antioxidant stability during one month of storage at 4 °C, and it seemed that the gamma-oryzanol in addition to increasing the stability of the delivery system, strengthened the antioxidant activity of the PPE. The release of PPE from the nanophytosomes in the simulated human gasterointestinal model was gradual, and after two hours in the simulated gastric fluid, only 22.66 ± 2.51% of the initially loaded PPE was released. The cytotoxicity of nanoencapsulated PPE on cancer cell lines (MCF-7) was significantly higher than that of free PPE (IC50 = 103 µg/mL). The nanoencapsulated PPE added to yogurt prevented the growth and proliferation of Aspergillus parasiticus inoculated into yogurt during 4 weeks of storage at 4 °C, and it had not adverse effects on the sensory and physico-chemical properties of stirred yogurt samples during 4 weeks of storage compared to the control sample (without extract). Conclusion: The designed method for extracting phenolic compounds from pomegranate peel, in addition to its simplicity, and low investment cost, showed reduced extraction time and significantly increased PPE extraction efficiency compared to traditional methods. Compared to other encapsulation methods, thin layer hydration-sonication demonstrated higher encapsulation efficiency, structural and antioxidant stability, and controlled release behaviour. It seems that yogurt enriched with nano-encapsulated PPE has the enough capacity to be produced on an industrial scale in terms of the availability of raw materials that are mainly obtained from agricultural and food industries wastes in order to enhance the safety level of the food product and improve the health of consumers.

Key words: Gamma-oryzanol, Lecithin, Nanophytosome, Pomegranate peel extract, Srirred yogurt