بررسی ویژگی‌های فیزیکوشیمیایی و بیولوژیکی نوشیدنی انار پروبیوتیک فراسودمند غنی‌شده با ژل آلوئه ورا

Introduction: The increasing awareness of consumers regarding health and nutrition has led to a growing demand for functional foods and beverages. Herbal and fruit-based drinks play a significant role in meeting nutritional needs and promoting health due to their bioactive compounds and antioxidant properties (Rajendran et al., 2025). Pomegranate juice (Punica granatum L.) rich in polyphenols, flavonoids, and anthocyanins has strong antioxidant properties and reported to be effective in reducing cardiovascular diseases, diabetes, and certain cancers (Keshvarzad et al., 2023). Additionally, Aloe vera gel has extensive applications in the food and pharmaceutical industries due to bioactive carbohydrates, vitamins, minerals, antioxidants, and amino acids. Aloe vera gel used as a natural preservative and an alternative to sulfur oxide to increase the shelf life of food products (Chelu et al., 2023). Moreover, adding probiotic bacteria such as Lactobacillus plantarum (L. plantarum) to herbal beverages enhances digestive health and strengthens the immune system. Studies have shown that compounds present in beverages influence the viability of these bacteria under different storage conditions (Nouri & Amin Moghaddasi, 2019). The previous researches have been conducted on adding Aloe vera gel to anola and ginger (Sasi Kumar et al., 2013), orange-carrot nectars (Hamid et al., 2014), and papaya (Ramachandran & Nagarajan, 2014) to enrich the beverages. In the past studies, health-promoting behaviors, chemical compositions, and antioxidant activity (Esposto et al., 2021), anti-inflammatory and anticancer capabilities (Habib et al., 2023), and probiotic viability of lactic acid bacteria (Rajendran et al., 2025) have been studied in pomegranate beverages. However, studies depicted that no research has been performed so far on the production of probiotic pomegranate beverage enriched with Aloe vera gel, thus highlighting the necessity and importance of conducting the present investigation.
Materials and methods: Fresh pomegranates (Saveh variety) purchased from a local market in Tehran, Iran. Fresh Aloe vera leaves sourced from a local farm. L. plantarum pTCC1058 probiotic strain obtained from Tak Gene Zist Company. Other materials included sugar, pectin, microbiological culture media (De Man, Rogosa and Sharpe (MRS) agar), and standard laboratory reagents. Aloe vera gel preparation: Aloe vera gel extraction followed the method described by Graili et al. (2021). After washing the leaves, the epidermal layer removed, and the inner gel extracted. The gel was soaked in distilled water for 5 minutes to reduce bitterness, then dried and cut into 4×4 mm pieces. Fresh pomegranate juice extracted and adjusted to 13° Brix. The 5.8 % sugar and 0.3 % pectin added to juice and homogenized (Bakhshizadeh et al., 2023). L. plantarum cultured in MRS agar and, after 48 h of incubation, added to the juice at a 1% concentration. The juice was prepared with four different Aloe vera gel concentrations (0, 10, 30, and 50 %). Pasteurization performed at 85 °C for 30 seconds, and samples were stored at 4 °C. The conducted following tests: pH and acidity measured using a pH meter and titration method (Iranian National Standard, 2008), respectively. The potentiometric method was used to measure acidity (Graili et al., 2021) and soluble solids were determined with a refractometer (Atago, Co., Tokyo, Japan) at ambient temperature (Iranian National Standard No. 2685). The ash of samples were calculated using the Iranian National Standard No. 2685 and a furnace (Fan Azma Gostar, Iran) at a temperature of 500 °C. Ascorbic acid (Vitamin C) quantified using the 2, 6-Dichlorophenol Indophenol method (Mazumdar & Majumdar, 2003). Total phenols and anthocyanins were measured using spectrophotometry (Rababah et al., 2023). Antioxidant activity assessed by the DPPH radical scavenging method (Habib et al., 2023). MRS agar containing sorbitol was used for enumeration and L. plantarum was cultured using a double-layer method and then maintained at 37 °C under anaerobic conditions for 48 h (Nouri & Amin Moghaddasi, 2019). Overall acceptance, taste and mouthfeel were assessed using a hedonic method with a 1 to 5 rating scale and 15-trained panelists were asked to evaluate the beverage samples (Hekmat et al., 2015). Fourier transform infrared spectroscopy (FTIR) spectroscopy used to identify functional groups (Chelu et al., 2023). Statistical analysis for the data was performed using SPSS software and one-way variance analysis and the presence or absence of Duncan's significant difference was examined at the 95 % level.
Results and discussion: Physicochemical: pH increased from 4.11 in the control sample to 5.03 in the 50 % Aloe vera gel sample. Acidity decreased from 0.40 to 0.30. Brix and ash increased from 11.25 to 18.01 % and 9.41 to 10.60 %. The ranges of pH and acidity for pomegranate beverage due to acidic nature detected between 2.87 to 4.14 and 0.35 to 3.36, which were consistent with previous results (Akbarpour et al., 2009; Rababah et al., 2023). The lowest and highest Brix levels observed 11.25 and 18.01 % in D0 and also D50 treatments, respectively. This factor improved by adding more Aloe vera gel in orange beverage (Graili et al., 2021) and orange-carrot nectars (Hamid et al., 2014), which were in line with present results. The minimum and maximum ash contents distinguished 9.41 and 10.60 % in D0 and D50 treatments, respectively. The results outlined that addition of more Aloe vera gel caused an enhancement in ash level. Aloe vera gel contains 18 % minerals such as calcium, magnesium, phosphorus and potassium (Mousavi et al., 2011). Biological characteristics: Vitamin C improved from 5.13 to 16.06 mg per 100 mL. Total phenols and anthocyanins decreased. Antioxidant activity showed no significant difference. The amount of vitamin C increased by enhancing Aloe vera gel in mango nectar (Elbandy et al., 2014) and orange beverage (Graili et al., 2021). The highest and lowest levels of total phenol contents found 2.32 in D0 and 1.25 for D50 and also, a significant difference was observed among all samples (p < 0.05). In a study, 243.89 and 72.41 mg of gallic acid per 100 mL were observed in 100 % pomegranate and lemon juice, respectively (González-Molina et al., 2009). In accordance with present results, a decrease in total phenol content was observed by adding Aloe vera gel to quinoa beverage (Bhargawa et al., 2014). The most and least anthocyanin compounds were detected 12.40 for D0 and 6.07 mg/L in D50 and also a significant difference was observed among all samples (p < 0.05). The amount of anthocyanin decreased with increasing milk in pomegranate beverage (Qasem Tabar et al., 2013). According to present results, the anthocyanin range was 11.02 to 3.66 mg/g for pomegranate juice (Rababah et al., 2023). The maximum and minimum antioxidant contents were observed in D0 (50.13 %) and D50 (45.27 %). The anthocyanins, polyphenols, and vitamin C and their possible synergism indicated high antioxidant levels in pomegranate juice (Anahita et al., 2015; Chelu et al., 2023). Probiotic viability: Stable for the first 7 days, then gradually decreased. The 30 % Aloe vera gel sample showed the best bacterial viability. There was a positive and direct correlation between the more bacterial population and extract concentration, which was attributed to the polyphenol compounds and their radical scavenging role (Rababah et al., 2023). In the present study, reducing pomegranate juice led to a decrease in phenolic compounds and the survival of probiotic bacteria. On the other hand, the extensive of shelf life had an effect on decreasing survival, which was in line with previous researches (Mousavi et al., 2011; Hekmat et al., 2015; Nouri & Amin Moghaddasi, 2019). Sensory evaluation: The 30 % Aloe vera gel sample received the highest scores for taste, texture, and overall acceptance. The 50 % Aloe vera gel sample received the lowest scores due to increased bitterness. The results indicated that excessive increase in Aloe vera gel (more than 30 %) caused higher bitterness and beverage concentration and led to a reduction in desirability from the consumers point of view, which was in accordance with previous researches (Sasi Kumar et al., 2013; Graili et al., 2021). FTIR Spectroscopy Analysis: The presence of antioxidant and polyphenol functional groups confirmed. Peaks around 3200 to 3600 (cm-1) indicated the presence of hydroxyl groups, polyphenols in pomegranate juice (Chaud, 2020) and hydroxyl stretching groups in carbohydrate monomers of Aloe vera (such as uronic acid, mannose or galacturonic acid or phenolic compounds in anthraquinones (Chelu et al., 2023), which were more intense in the D50 sample.
Conclusion: This study demonstrated that adding Aloe vera gel enhanced pH, Brix, and vitamin C levels while reducing probiotic viability and phenolic compounds at higher concentrations. Based on physicochemical, biological, and sensory evaluations, 30 % Aloe vera gel sample was the best formulation for producing industrial functional probiotic beverages.