Abstract The objective of this study was to determine bioactive compounds, nutrition, antioxidant activity, total phenolic content (TPC) and total flavonoid content (TFC) in shield aralia yoghurt. Shield aralia leaves (SAL) have traditionally been used as herbal plant. Fortification of shield aralia in yoghurt increased the health-benefits of yoghurt by the incorporated phytochemical constituent of herb such as polyphenol and flavonoid groups which can be used to treat various disease including atherosclerosis, obesity, diabetes mellitus, and cardiovascular disease. The Greek-style yoghurt with addition SAL resulted on new bioactive compounds belong to α-linoleic acid (ALA) including -hydroperoxy-octadecadienoic acid (9-HpODE), (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(s)-hydroperoxylinolenic acid (13(S)-HPOTrE), and (E)-6-hydroxyoctadec-4-eonoic acid or NP-011548. The proximate test results revealed that shield aralia yoghurt has high-protein content (15.46%), low-fat and carbohydrate content (0.16% and 0.88%, respectively). The antioxidant activity based on 2, 2-duphenylpicrylhydrazyl (DPPH) assay of shield aralia yoghurt was 68.66%, while TPC of SAL yogurt reached 62.61 mg GAE/g and the TFC was 201.05 mg QE/g. These results suggested that under fermentation by lactic-acid bacteria (LAB) increased the beneficial values of shield aralia leaves compared with shield aralia raw extract. Shield aralia yoghurt could be as promising supplementation for wellness care and also could be as exogenous antioxidant to increase body defense.

Keywords: Shield aralia, Greek-style yoghurt, Octadeconoic acid, Total phenolic content, Total flavonoid content

Citation: Pertami, S. B., Yunitasari, E., Budiono, B., Yulifah, R., Astuti, N.P., Herawati, T., Arifah, S.N., and Atho’illah, M.F. 2024. Bioactive compounds and antioxidant activity in greek-style yoghurt infused with shield aralia leaves. Natural and Life Sciences Communications. 23(3): e2024045.

INTRODUCTION

Yoghurt is diary product that fermented by lactic acid bacteria (LAB). Consumption of yoghurt has long been considered to body health especially for digestive tract (Adolfsson et al., 2004; Le Roy et al., 2022). Yoghurt referred to as the "food of the gods," has traversed centuries and cultures to become a staple of modern diets worldwide. Its unique blend of taste, texture, and rich of nutritional value such as protein, vitamin B12, calcium, zinc, vitamin D, and phosphorus (El-Abbadi et al., 2014; Fernandez and Marette, 2017). Yoghurt comes in various forms and varieties, each offering unique textures, flavors, and nutritional profiles. Yoghurt divided into various types based on the texture including regular yoghurt, Greek yoghurt (Fisberg and Machado, 2015), Icelandic skyr (Pereira et al., 2021), plant-based yoghurt (Gupta et al., 2022), and Bulgarian yoghurt (Velikova et al., 2018).

Greek-style yoghurt, renowned for its creamy texture and distinct tangy flavor, has garnered global acclaim for its nutritional richness and versatility. Greek yoghurt is produced by straining regular yoghurt to remove much of the whey, the liquid portion of the milk (Fisberg and Machado, 2015). The straining process concentrates the yoghurt's protein content (Nelios et al., 2023), making Greek yoghurt a popular choice for individuals seeking to increase protein intake. The straining process that creates Greek yoghurt also results in a reduction of lactose, the natural sugar found in milk. This reduction in lactose content can make Greek yoghurt easier to digest for individuals who are sensitive to lactose (Pei et al., 2017).

Flavored Greek yoghurt is a variation of traditional Greek yoghurt that has been infused with natural or artificial flavors to create a wide range of taste experiences and enhancing yoghurt properties. These flavors can be achieved using natural fruit extracts, spices, sweeteners, or other ingredients (Rashwan et al., 2023). Natural flavors from natural ingredients have antioxidant defense over reactive oxygen species (ROS) that can prevent various diseases including cardiovascular disease, atherosclerosis, cancer, diabetes mellitus, and other metabolic disorder (Arifah et al., 2020; Meziane et al., 2023; Ziółkiewicz al., 2023). Shield aralia (Polyscias scutellaria) is a tropical shrub with 2-6 meters in height and commonly grown as a native plant in Southwest Pacific island. Shield aralia leaves has bitter taste, aromatic odor and been traditionally used in various cultures for its potential health benefits including anti-inflammatory, antioxidant, immune support, and could be used to increase breast milk production. Pertami et al., (2021) reported that bioactive compounds in aqueous shield aralia leaves such as kaempferol, and afzelin involved to enhance the breast milk production by helping the serotonin reuptake thus synergistically increase the prolactin production. Flavonoids and tannins constitute the main groups of plant polyphenols that play numerous molecular and biochemical roles in plants, such as signalling, free radical scavenging, mediating auxin transport, and plant defence (Kiani et al., 2021). Flavonoids have long been considered to have health-related functions in the human body, including antioxidant, antimicrobial, and anticancer properties (Soleimani et al., 2022).  Polyphenol content in natural resource also plays important part in human nutrition due to the positive effect of polyphenol for health (Petkova et al., 2023). Quercetin, rutin also has been evaluated the administration of aqueous shield aralia leaves extract on lactating rats resulting on increasing the prolactin and oxytocin levels Budiono et al., (2023).

Fortification of shield aralia leaves in Greek-style yoghurt is not been evaluated yet. This research aimed to explored and analyze the new flavored Greek-style yoghurt by shield aralia leaves. The purpose of this study is to formulate shield aralia leaves into functional food product which easily to accept by society especially for breastfeeding mother. Evaluation on bioactive compounds, nutritional, antioxidant activity is necessary to health benefit of shield aralia yoghurt.

MATERIALS AND METHODS

Materials

Fresh shield aralia (P.scutellaria) leaves (SAL) were obtained from Materia Medica Batu (MMB), Indonesia. Shield aralia leaves was harvested at July, 2023. Greek-style yoghurt starter used Biokul Greek Yoghurt (PT. Jaya Utama Santikah, Jakarta, Indonesia), fresh milk, 2,2-diphenylpicrylhydrazyl (DPPH) (D9132, Sigma Aldrich, USA), folin-ciocalteu reagent (1.09001.0500, Merck, USA), distilled water, sodium carbonate (NaCO₃), aluminium chloride (AlCl₃), sodium nitrite (NaNO₂) and sodium hydroxide (NaOH).

Yoghurt production

Briefly, 3% of fresh SAL were mixed with fresh milk (w/v) using blender. The mixture was blended until the mixture mixed well. SAL milk was added with 6% of sucrose then pasteurized at temperature 85°C for 30 minutes (Sah et al., 2014). The pasteurized SAL milk was cooled until the temperature decreased at 40°C. 2% of Biokul Greek-yogurt (PT. Jaya Utama Santikah, Indonesia) starter (w/v) was added into pasteurized SAL milk, then incubated at 37°C for 24 hours. Biokul Greek-yogurt starter contain Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum. Yoghurt was harvested then kept in refrigerated (4°C) for further analysis. Original yoghurt was made without adding SAL in fresh milk.

Viable cell count of lactic acid bacteria

0.5 mL of each original yoghurt and SAL yoghurt were diluted in 4.5 mL of sterile 0.9% NaCl (Dan et al., 2019). The diluted sample were then homogenized using vortex and performed for serial dilution. Viable bacterial counts of S. thermophillusm L. bulgaricus, L. acidophilus, and B.longum were determined by culturing the diluted sample in MRS agar then incubated under anaerobic incubation for 48 hours at 37°C. The viability of bacteria in each yoghurt was replicated for 3 times at each dilution (Huang et al., 2022).

Screening of bioactive compound by LC-HRMS

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was used to determine bioactive compounds in shield aralia yoghurt. LC-HRMS analysis was performed using UHPLC Vanquish Tandem Q Exactive Plus Orbitrap HRMS Thermoscientific with column accucore C18, 100x2.1 mm, 1.5 µm (Thermoscientific). Vanquish method was used for the LC-HRMS analysis. Briefly, dissolved shield aralia yoghurt with methanol (1:1), centrifuged at 13,000 rpm for 10 min, then filtered using 0.2 µm of polytetrafluoroethylene (PTFE). The solvent for LC-HRMS analysis included solvent A contain water, 0.1% formic acid (A) and acetonitrile, and solvent B contain with formic acid B. The analytical flow rate was 0.2 mL/min with a column temperature of 30°C. Mass range of LC-HRMS used 100-1,500 m/z. The mzCloud MS/MS library was used to generate data of LC-HRMS (Budiono et al., 2023; Goy et al., 2022).

Proximate test

The proximate test included moisture, ash, crude protein, crude fat, and carbohydrate based on Official Methods of Analysis (AOAC). The proximate was performed using the Kjeldahl method with conversion factor of 6.5 (N x 6.25). The crude fat was determined using Soxhlet method, while the moisture contents were analyzed using oven drying. The temperature of oven drying (Memmert GmBH+Co.KG, Germany) was 105°C until the constant weight was provided. The ash content was determined by muffle furnace at 600°C approximately for 4 hours. The total carbohydrate was achieved by following equation (Mohammad et al., 2021):

% carbohydrate = 100% - (% moisture + % fat/lipid + % protein + % ash)

Extraction of yoghurt

The yoghurt extraction was following procedure by Muniandy et al., (2016) with slight modification. Briefly, 10 g of shield aralia yoghurt were homogenized with 2.5 mL sterile distilled water and adjusted to pH 4.0 by adding 0.1 M of HCl. Yoghurt were incubated in waterbath at temperature 45°C for 10 min, then centrifugated at 3,500 rpm 4°C for 10 min. The supernatant was collected then adjusted to pH 7.0 using 0.1 M NaOH. Centrifugated at 3,500 rpm 4°C for 10 min, the clear supernatant was collected into sterile tube and kept in freezer (-20°C) for antioxidant activity, total phenol, and total flavonoid assay.

Antioxidant activity

The antioxidant activity of shield aralia yoghurt was measured by DPPH reagent (Budiono et al., 2023). Briefly, 40 µL of shield aralia yoghurt extract were added with 200 µL of 50 µM DPPH solution (dissolved in methanol). The mixture was shaken and incubated in darkness room for 30 min. Methanol was used as blank, and 50 µM DPPH solution was used as control. The absorbance was measured at 517 nm of wavelength (Thilavech et al., 2023; Warinthip et al., 2023). The percentage inhibition activity was calculated according to the following equation:

% inhibition = (A_control  -  A_sample)/A_control

Total phenolic content (TPC) assay

Total phenolic content (TPC) was measured using Folin-Ciocalteu method. 40 µL of yoghurt extract and 100 µL of Folin-Ciocalteu reagent (diluted 10-fold) were mixed and incubated for 5 min (Pewlong et al., 2021). Added with 75 µL of sodium carbonate (75 g/L) then incubated for 2 hours in darkness at room temperature (Sirisa-ard et al., 2023). Gallic acid (GA) was used as standard dan construct linear regression line. Distilled water was used as blank. The TPC analysis was measured at 740 nm wavelength and the TPC value was estimated as mg gallic acid equivalent (mg GAE)/g of dry extract (Ablat et al., 2014).  

Total flavonoid contents (TFC) assay

Total flavonoid contents (TFC) in shield aralia yoghurt was measured according to Doungsaard et al., (2023) with slight modification. 50 µL of shield aralia yoghurt extract were mixed with 70 µL distilled water and 15 µL of 5% NaNO₂, incubated for 5 min at room temperature. 15 µL of 10% AlCl₃ solution was added into the mixture, then incubated for 6 min at room temperature. 100µL of 1 M NaOH solution were added into the mixture, and measured the absorbance at 510 nm wavelength. Distilled water was used as blank and quercetin was used to determine standard curve (200-1,000µg/mL). The data were expresses as mg quercetin equivalent (mg QE)/g of dry extract.

RESULTS

Viable counts of lactic acid bacteria

The total viable bacteria count in original yoghurt was 1.8 x 10⁷ CFU/mL while SAL yoghurt had total viable bacteria 2.4 x 10⁶ CFU/mL (Table 1). This result shows that fortification of SAL only decreased total viable bacteria less than 10 CFU/mL. Fortification of SAL in yoghurt could be suggested are safe for maintaining viability of lactic acid bacteria in yoghurt.

Qualitative determination of bioactive compounds in shield aralia yoghurt by LCHRMS

LC-HRMS analysis (Figure 1) revealed that shield aralia yoghurt contains a compound belonging to polyunsaturated fatty acid (PUFA) linoleic acid, which only appears while the shield aralia is under fermentation by LAB. The PUFA in shield aralia yoghurt namely 9-hydroperoxy-octadecadienoic acid (9-HpODE), (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(s)-hydroperoxylinolenic acid (13(S)-HPOTrE), and (E)-6-hydroxyoctadec-4-eonoic acid or NP-011548.

Figure 1. LC-HRMS result from shield aralia yoghurt (A). We found 4 compounds belong to octadeconoic acid which appeared after fermentation by LAB namely  9-HpODE (B), (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid (C), 13(S)-HPOTrE (D), and NP-011548 (E).

Shield aralia yoghurt nutrition

Based on proximate test analysis (Table 1), original yoghurt has 76.5% moisture content, 1.5% ash, 6% fat, 15% crude protein, and 1% carbohydrate. Furthermore, SAL yoghurt, the moisture content reached up to 82.67% while ash content was only 0.83%. The shield aralia yoghurt has a low-fat content of only 0.16% and low carbohydrate content of 0.88%. Conversely, the crude protein content is quite high, at 15.46%. The proximate test results suggested that consumption of shield aralia yoghurt is good for health because it contains high protein, low fat, and low carbohydrate.

Table 1. Proximate test result of shield aralia yoghurt.

Antioxidant activity, phenolic and flavonoid content

Table 2 shows that the antioxidant activity of original and SAL yoghurt is 43.47 and 68.66%, respectively. Meanwhile the TPC and TFC of original yoghurt are 53.72 mg GAE/g and 132.74 mg QE/g. SAL yoghurt has 62.71 mg GAE/g of TPC and 201.06 mg QE/g of TFC. Addition of SAL in yoghurt increases antioxidant activity and the bioactive compound including phenol and flavonoid contents.

Table 2. Antioxidant, TPC, and TFC measurement in shield aralia yoghurt.

DISCUSSION

Shield aralia yoghurt could be the novel product of fortification in yoghurt product. The LC-HRMS results showed that some bioactive compounds belong to α-linoleic acid (LA) derivates appear after fermentation. Octadecenoic acids are hydroxy fatty acid (FA) that derivates from LA that fermented by LAB. Lactobacillus acidophilus has ability to convert LA into 13(S)-hydroxy-9-octadecenoic acid and 10,13-dihydroxyoctadecanoic acid (Kishimoto et al., 2003). Based on the previous study showed that aqueous shield aralia extract has flavonoid compounds including rutin, quercetin-3β-D-glucoside, quercetin, quercitrin, kaempferol, hyperoside, afzelin, quercetin-3-O-rhamnoside-7-O-glucoside, and kaempferol-3-O-β-glucopyranosyl-7-O-α-rhamnopyranoside (Budiono et al., 2023). Aqueous shield aralia extract also contain bioactive compounds belonging to terpenes including D-(+)-camphor, ursolic acid, oleanolic acid, (-)-caryophyllene oxide, and illicic acid. After fermentation by LAB, the PUFA compounds including 9-HpODE, (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(S)-HPOTrE, and NP-011548 are believed to increase the benefit of yoghurt. Shi et al. (2023) reported that three octadecenoic acid compounds that isolated from avocado peel extract, namely (9)-HpODE, (15Z)-9,12,13-Truhydroxy-15-octadecenoic acid and NP-011548 have anti-bacterial activity against Staphylococcus aureus. A study by Kumar et al. (2016) revealed that treatment using 13(S)-HPOTrE increased the survival rate of mouse induced with endotoxin-mediated septic shock and polymicrobial sepsis. 13(S)-HPOTrE has anti-inflammatory effect by inactivating NLRP3 inflammasome complex through PPAR-γ pathway. The 9-HpODE as a product of linoleic acid peroxidation induced intracellular glutathione oxidation and mediated by glutathione peroxidase 4 (GPx4) in radical scavenging activity. 9-HpODE, (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(S)-HPOTrE, and NP-011548 are known for their ability to maintain health, including their role in increasing endogenous glutathione peroxidase (GPx) enzyme which involved radical scavenging activity (Corteselli et al., 2019). Based on these study reports, it could be suggested that 9-HpODE, (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(S)-HPOTrE, and NP-011548 found in shield aralia yoghurt could gain the health benefit of yoghurt value.

 The viable LAB demonstrated a slight decrease in SAL than original yoghurt (Table 2). The decrease of LAB viability might be attributed from the presence of linoleic acid and its derivatives, which act as antimicrobial components and sensitive to gram-positive bacteria (Atwaa et al., 2022). The proximate test (Table 2) informed that shield aralia yoghurt has low fat and high protein content. Greek-style yoghurt is a yoghurt style with thicker and creamier texture due to high protein content. Based on Codex standard for fermented milk, high-protein yoghurt should consist with minimum of 2.7% protein and less than 15% fat (Jørgensen et al., 2019). High-protein yoghurt has beneficial for health including for the elderly or sports nutrition. Consumption of yoghurt for athletes could increase muscle protein synthesis (Tipton et al., 2007) and plasma amino acids (Hall et al., 2003). Moreover, high-protein content also has a correlation with weight wellness trends. This combination of nutrition is good, especially to prevent obesity and diabetes mellitus.

Fat content is distinguished into full fat with 8.8-20% of fat, low-fat with <2% of fat, and non-fat with <0.05% of fat (Jørgensen et al., 2019). Based on the proximate test can conclude that shield aralia yoghurt is categorized as a low-fat product. Consumption of low-fat dairy products believed could be used to maintain the metabolic process in normal state. Sun et al. (2007) reported an increasing incidence of coronary vascular disease (CVD) after consuming high fat dairy product. Shield aralia yoghurt as a dairy product with low-fat content is beneficial, especially in some diseases related to obesity and CVD (Soltani and Vafa, 2017).

The DPPH scavenging activity of SAL yoghurt has percentage inhibition value at 68.66%, while the TPC was 62.61 mg GAE/g and the TFC was 201.06 mg QE/g.  Meanwhile, the antioxidant activity of original yoghurt only reached 43.47% with TPC value was 54.72 mg GAE/g, and the TFC value was 132.74 mg QE/g of dry extract. The previous study using shield aralia water extract, absolute ethanol extract, and 70% ethanol extract showed the TPC were 16.35, 24.49, and 15.37 mg GAE/g dry extract, respectively (Budiono et al., 2023). The TPC in shield aralia yoghurt significantly differs from shield aralia extracted using 3 solvents. Furthermore, the TFC values for SAL yoghurt was 201.07 mg QE/g of dry extract. This results also higher than TFC value in shield aralia water, absolute ethanol, and 70% ethanol extract which only have TFC value at 59.89, 63.66. and 58.42, consecutively (Budiono et al., 2023). Fortification in yoghurt using other natural ingredients has been done in various yoghurt, including fortification with vegetables, fruits, herbs, and spices. Antioxidant and phytochemical content between original yoghurt and SAL yoghurt (Table 2) showed that SAL yoghurt is better than original yoghurt. The higher levels of TPC and TFC in the SAL yoghurt also supported with SAL itself which also contain with phenolic and flavonoid compound (Budiono et al., 2023). In conclusion, fortification of SAL in the yoghurt can increase antioxidant activity, TPC, and TFC in yoghurt. Another study about fortified yoghurt has been reported by El-Said et al. (2014) where the antioxidant activity in yoghurt added with pomegranate peel water extract was 89-90% and the TPC reached up to 16.34 mg GAE/g while the TFC value was up to 6.86 mg rutin/g of dry extract. Kim et al., (2019) also reported that in fortification of lotus leaves yoghurt had TPC value 24.0 mg GAE/g and DPPH activity was 48.81%. Based on comparative study results suggested that shield aralia yoghurt is more beneficial for health than only in form of shield aralia extract with fermentation by LAB increased the TPC and TFC value of shield aralia leaves.

CONCLUSION

In summary, there are four specific compounds that belong to linoleic acid derivatives, including 9-hydroperoxy-octadecadienoic acid (9-HpODE), (15Z)-9,12,13-Trihydroxy-15-octadeconoic acid, 13(s)- hydroperoxylinolenic acid (13(S)-HPOTrE), and (E)-6-hydroxyoctadec-4-eonoic acid. SAL yoghurt has higher moisture (82.67%) and low fat (0.16%) than original yoghurt. The antioxidant activity, TPC, and TFC of SAL demonstrated higher than original yoghurt with values of 68.66%, 62.71 mg GAE/g, and 201.06 mg QE/g, respectively. Therefore, the fortification of shield aralia leaves increased the health benefit value of yoghurt. Further experiments are needed to discover the molecular mechanism of SAL yoghurt for maintaining body weight and also could develop as a functional food to prevent metabolic syndrome disease.

AUTHOR CONTRIBUTIONS

Sumirah Budi Pertami, Esti Yunitasari, Budiono Budiono, Tutik Herawati designed experiments, analysis and interpretation, supervised all the experiments, and writing the manuscript Rita Yulifah and Nurul Puji Astuti performed data statistical analysis and interpretation. Siti Nur Arifah and Mochammad Fitri Atho’illah data collection and processing, writing the manuscript, and critical review. All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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