Effect of pasteurization time and temperature on bioactive compounds, antioxidant ability and color value of beverage produced from herbal extract (Passiflora foetida and embryo nelumbinis)

Herbal beverage is one of the natural products which are greatly preferred by customers. Passiflora foetida and Embryo Nelumbinis are two kinds of medicinal plants that possess many precious bioactive compounds that have the effect of supporting calmness, reducing stress, treating insomnia; and have been used in many folk remedies in Vietnam. The main objective of this study is to investigate the effects of heating temperature (75, 85 and 95°C) and pasteurization time (15, 30, 45 and 60 minutes) on the content of bioactive compounds (polyphenol, flavonoid, tannin, alkaloid and saponin), antioxidant ability (FRAP, DPPH) and color parameters (L*, a*, b* and ΔE) of product. The reseach was designed randomly with two factors and three replications. The content of bioactive compounds and antioxidant capacity of product were analyzed by standardization methods using spectrophotometer; and the color parameters were measured by the colorimeter. The result showed that the opitmal pasteurization temperature and time were 85°C and 30 minutes, at this heating process the product maintained high level of bioactive compounds and antioxidant ability. The content of polyphenol, flavonoid, tannin, saponin and alkaloid were 9.46 mgGAE/g, 4.44 mgQE/g, 4.85 mgTAE/g, 7.40 mgSE/g, 14.25 mgCE/g; the antioxidant capacity of product by DPPH and FRAP method were 64.50% and 28.25 μFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as LMFeSO4/g; the color parameters such as L*, a*, b* and ΔE were 31.58, -0.24, -0.47 and 61.25, respectively. The production process was successful in pilot equipment and the product can be used as one of the beverages for health strengthening.


Introduction
The busy lifestyle in developing countries force people have to work intensively.This causes negative effects on physical and mental health such as: exhaustion, stress, asthenia and insomnia.Suffering these phenomena in a long term will lead to serious diseases such as high blood pressure, depression, impaired cognitive ability, especially for middle-age and elderly people.Therefore, demands for natural products that could support for health are current trend of food research and food industry.1306 (2024) 012044 IOP Publishing doi:10.1088/1755-1315/1306/1/012044 2 Passiflora foetida distributed widely in high mountain, midlands and plains, is known as a traditional herbal plant in Vietnam.Its stem whole has a sweet and slightly bitter taste.It were used as refreshing drink, detoxifying and preventing diuretic and depression.The young shoots of the plant are often used as vegetable which could anti-inflammatory and reduce neurasthenia, insomnia, cough, edema, capsulitis [1,2].According to research by Yue et al. [3], the leaves of Passiflora foetida contain flavonoids such as: vitexin, isovitexin, saponarin, orientin, isoorientin, api-genin-8-C-diosgenindiglucoside, methylamino luteolin, lutein 7--D-glucoside.Subramani et al. [4] extracted whole Passiflora foetida stem with ethanol and petroleum, the extraction contained alkaloids, phenolics, tannins, flavonoids, glycosides, steroids and resins, these are natural functional components.Dried Passiflora foetida leaves contained ash, fiber, protein, fat and carbohydrate [5].The alkaloids extracted of Passiflora foetida have sedative effect on mice.The passerynum preparations (including Passiflora foetida, Erythrineme orientalis, lotus leaf, Cassia tora, Cuscuta chinensis lamk seed, mulberry leaf, Leucaena leucocephala seed and Eleutherine bulbosa) with a dose of 0.2 g/20 g, could reduce the nervine excited state of mice that have been given caffeine.Furthermore, the product has the effect of lowering blood pressure, increasing respiration of rabbits, and reducing heart rate.Passerynum has low toxicity and could be used at a dose of 75g/kg.In clinically tests, passerynum help patients to fall asleep easily, sleep well without effect on blood pressure [6].Lotus embryo is also an important traditional herb which is used in the treatment of high blood pressure, anxiety, insomnia, fever, and diaphoresis [2].Lotus embryo contains functional alkaloids (0.85-0.96%) such as asparagine, nelumbin, liensinin, izoliensinin, neferin, lotusin, methylcorypalin, nuxiferin, pronuxiferin [7].The lotus embryos have bitter taste, they could be used as refreshing drink and regulate heartbeat.Daily dose of lotus embryos are 2-4g as decoction and could be combined with other herbs [8].According to Do et al. [8], extraction of lotus embryo which are liensinin and neuciferin could regulate blood pressure, reduce insomnia, anti inflammatory, analgesic, and antitussive.Nuciferin, injected to mice at a dose of 1.32mg/kg for 3 days, did not affect on the mother's health embryonic development, with a dose of 60 mg/kg body weight on white mice, no negative effect on chromosomal mutations was found.The alkaloid extract of lotus heart also has sedative effects on rabbits [7].Early researches clearly showed health benefits of passiflora foetida and lotus embryo.In Vietnam these herbs have been used as traditional medicines and food to prevent the diseases, improve sleep and help sedation.However, these two ingredients are only used in raw as tea according to folk experience.So far, there have not been previous studies to process these two ingredients into processed food that could be used as functional beverage to reduce insomnia and improve antioxidant capacity.Therefore, it is necessary to research and develop a beverage product using passiflora foetida and lotus embryo as main material.This refreshing drink contain high ratio of biologically active substances that have health benefits, especially in the prevention of insomnia in the elderly.The drink could contribute to the diversification of beverage products on the food market.

Preparation of sample and experimental design
Dried Passiflora foetida plants (cutted into 3-5 cm) were purchased from a herbal shop at Long Xuyen market, An Giang.The ratio (w/w) of Embryo Nelumbinis/Passiflora foetida was 1/12, and raw material/water ratio (w/v) was 1/80.They were extracted at 85 o C for 45 minutes.The extract was filtered through a cloth filter the supernatant was collected, and was added with 8% of rock sugar.After that, the solution was filled in 250 mL glass bottle, then sealed and waited for the pasteurization process.The experiment was designed randomly with two factors and three replications.The pasteurization temperature was 75, 85 and 95 o C and pasteurization times were 15, 30, 45 and 60 minutes.Parameters of color (L * , a * , b * and E), content of bioactive compounds (polyphenols, flavonoids, tannins, alkaloids and saponins), and antioxidant capacity (FRAP and DPPH) of the product were analyzed.

Analytical methods
The content of bioactive compounds: The polyphenol content was determined by Folin-Ciocalteu reagent method and the results were expressed as milligrams of gallic acid equivalents per gram dry matter (mgGAE/g DM) through a standard curve (y = 0.0692x + 0.0993, R 2 = 0.9993) [9].The aluminum chloride colorimetric method was used for flavonoids determination and the amount of flavonoid was calculated as quercetin equivalent per gram (mgQE/g DM) based on standard equation y = 0.0054x + 0.0026, R 2 = 0.9995 [10].Tannin content was determined by Folin-Denis method and the results were showed as milligrams of tannic acid equivalents per gram (mgTAE/g DM) based on standard curve (y = 0.0089x + 0.0748, R 2 = 0.9988) [11].The content of saponin was determined by equation y = 200*OD/0.389(in which y: standard saponin concentrate, OD: absorbance of standard saponin solution), results were calculated as milligrams of saponin equivalents per gram (mgSE/g DM) [12].The content of alkaloid was calculated following standard curve (y=0.0035x+ 0.7552, R 2 =0.9983) and results were expressed as milligram of colchicine equivalents per gram (mgCE/g DM) [13].
The antioxidant activity: Determination of DPPH free radical scavenging capacity according to the method described by Aluko et al. [14], and DPPH (%) = [(Abs control -Abs sample)/Abs control] x 100, where Abs control is the absorbance of DPPH solution mixed in ethanol, Abs sample is the absorbance of extracts + DPPH or standard substance.Determination of ferrous reducing ability by FRAP method [15] based standard equation of FeSO4.7H2O(y = 0.5177x + 0.0855, R 2 = 0.9981), results were expressed as micromol ferric sulfate per gram (µM FeSO4/g DM).
The color parameters of product: The Hunter L * (lightness/darkness), a * (redness/greenness) and b * (yellowness/blueness) values of samples were measured using a Minolta colorimeter (Model No. CR-400, Konica Minolta AC-A350, Japan).The colorimeter was calibrated for internal light (D65), using a while calibration plate before carrying out color measurements.Triplicate measurements were used for each determination.Total color difference (E) was calculated by Equation 1. (1) Where Lo, ao and bo represented the reading of blank sample, and L * , a * and b * represented the instantaneous individual reading of product [16].

Experimental data analysis
Using the software Microsoft Excel and Statgraphic Centurion XVI for calculate, drawing graphs and analysis of variance ANOVA, test the significant difference of the treatments through LSD (Least Significant Difference).

Effect of pasteurization temperature and time on bioactive compounds of product
Natural antioxidants are abundant in edible plants, especially in spices and herbs [17].Most herbal plants have strong antioxidant capacity [18] and free radical scavenging capacity [19].Pasteurization is an important process in beverage processing, affecting not only the microbial safety but also the quality of the product, especially the content of bioactive compounds.In order to limit reducing product quality, it is necessary to find the optimal combination of the two factors "temperature -time".Research results presented in Table 1 showed that when the pasteurization temperature increased from 75 o C to 95 o C, the content of bioactive compounds as tannins, polyphenols, flavonoids, alkaloids and saponins tended to increase and reached to the highest value in 85 o C.These values were 4.82 mgTAE/g, 9.36 mgGAE/g; 4.42 mgQE/g; 14.03 mgCE/g and 7.44 mgSE/g, respectively; then they were decreased slightly at 95 o C and there was no statistically significant difference.At the pasteurization temperature of 75 o C, the content of bioactive compounds was low level, that might be due to the fact that microorganisms have not been completely destroyed, so they can decompose or oxidize biological compounds.When the pasteurization time increased from 15 to 60 minutes, the content of bioactive compounds tended to increase and reached to the highest value at 30 minutes.The content of tannins, polyphenols, flavonoids, alkaloids and saponins were 4.84 mgTAE/g; 9.41 mgGAE/g; 4.41 mgQE/g; 14.27 mgCE/g and 7.38 mgSE/g, respectively.When the pasteurization time was extended to 45 and 60 minutes, the content of bioactive compounds decreased.This thing proved that prolonged time would degrade bioactive compounds.According to Laslo et al. [20] showed a significant increase in phenolic content when the pasteurization time was extended from 0 to 25 minutes at 80 o C. A research by Saini et al. [21] showed that there was an increase in total phenolic and flavonoid content when the treatment temperature increseased from 50 to 70 o C, and there was a gradually decreasing when the temperature was over 80 o C; or there was an increase in the content of these compounds when the heat treatment time was prolonged from 15 to 45 minutes, and decreased when the treatment time was extended to 60 minutes.Similarly, the study of Laslo et al. [22] showed the phenolic content was decreased when pasteurization conditon performed at 80 o C for 25 min, but the content of flavonoids tended to increase.This was thought to have the degradation of phenolic compounds during heat treatment [23].

Effect of pasteurization temperature and time on antioxidant activities of product
There was many methods to measure total antioxidant capacity directly from plant extracts and biological fluids [24].In the study, the FRAP and DPPH methods were selected to evaluate the antioxidant activity of herbal beverage.Because FRAP was a sensitive method for measuring the total antioxidant capacity of fresh biological fluids and pharmacologically active plant products [25].Besides, DPPH was a stable free radical and very popular for studying natural antioxidants [26].The results in Figure 1 and Figure 2 showed when the pasteurization temperature increased from 75 to 95 o C, the ferrous reducing ability of the product tended to increase and reached to the highest value at 85 o C, which was 28.19 M FeSO4/g; while the ability of free radical scavenging DPPH reached to the highest value that was 62.73% at 95 o C, but there was no statistically significant difference (P0.05) between the pasteurization temperature of 85 o C and 95 o C. Besides, when the pasteurization time was extended from 15 to 30 minutes, the ferric reducing ability by FRAP method and DPPH free radicals also tended to increase and reached the highest value that was 28.50 M FeSO4/g and 64.68% at 30 minutes, respectively; then there was decreased as it was expanded to 60 minutes.According to Laslo et al. [20] showed there was a significant increasing in antioxidant activity when the pasteurization time was extended from 0 to 25 minutes at 80 o C. Saini et al. [21] showed that there was an increase in DPPH free radical scavenging activity when the treatment temperature was increased from 50 to 70 o C and decreasing at 80 o C.There was an increase in DPPH free radical scavenging when the heat treatment time was prolonged from 15 to 45 minutes, and there was decreased when pasteurization time was expanded to 60 minutes.A study by Laslo et al. [22] showed that the pasteurization at 80 o C for 15 to 25 min did not significantly affect DPPH free radical scavenging activity, ferric reducing activity by FRAP decreased but total antioxidant capacity by TEAC increased.The studies of Bhatacherjee et al. [27], Oliveira et al. [28] and Oliveira et al. [29] showed that the phenolic content and antioxidant activity were not significantly changed when pasteurization temperature was higher than 80 o C for 10 minutes.Bhattacherjee et al. [27] reported when studying the effect of different pasteurization temperatures on the quality of aonla juice came to the conclusion that the temperature of 80°C was optimal to obtain juice rich in bioactive compounds.On the other hand, the study also developed regression equations to predict the content of bioactive compounds (polyphenols, flavonoids, tannins, alkaloids and saponins) and antioxidant capacity (DPPH and FRAP) of the product with the form quadratic equation Z = a + bX + cY + b'X 2 + c'Y 2 + dXY (Table 2).The equations found the correlation coefficients of R 2 and R 2 adj were higher than 0.8694, according to Guan & Yao [30], regression models with correlation coefficient of R 2  0.8 had good compatibility between predicted model and experiment.Hence, these equations could be used to predict the change in bioactive compounds content and antioxidant capacity of the product according to the surveyed pasteurization temperature and time.This change is clearly shown in Figure 3 and Figure 4.
IOP  The statistical results showed significant effects of pasteurization temperatures and heating times (P≤0.01) on bioactive compounds (polyphenol, flavonoid, tannin, alkaloid and saponin) as well as antioxidant activities (DPPH and FRAP) of the product.The variance analysis of regression model had high R square values up to 0.9161, R 2 adj was 0.8694 and P value less than 0.01.The relationship between the independent variables, bioactive compounds and antioxidant activities can be found in the Table 2.
The response and contour graphs in the Figure 3 and 4 showed changes of bioactive compounds and antioxidant activities of the product under effect of pasteurization temperatures and heating times.They would have different increase or decrease, and optimal area was expressed orange areas in charts.

Effect of pasteurization temperature and time on color parameters of product
Color parameters of L*, a*, b* and E could be applied to express the color degradation and provide useful data for quality control in plant products [31].The color change of the processed samples related to the initial conditions.The color parameters of the products after pasteurization were also analyzed, the results are presented in Table 3.  Results showed that when the pasteurization temperature was increased from 75 to 95 o C, the L * value and total color difference E of the product have not changed statistically significant (P0.05), the values of a * and b * tended to increase, that is, the product color was more reddish brown and there was statistically significant difference (P≤0.01).When the pasteurization time was extended from 15 to 60 minutes, the L * value tended to increase, while the total color difference E tended to decrease and there was statistically significant difference (P≤0.05), and the a * and b * values had a little change but there was not statistically significant difference (P0.05).At pasteurization temperature and time were 85 o C and 30 minutes, the color parameters of product had suitable values that the making to product was good color.According to Kong et al. [32], the pasteurization changed color parameters of pomegranate juice, it increased a * value, decreased b * and L * values while E values did not change significantly.In addition, a study of Buniowska et al [33] showed that there were an increasing of L * and b * values, and decreasing of a * value after heating fruit beverages.These showed that changes of color parameters (L * , a * , b * and E) depend on chemical composition of beverage as well as temperature and time of pasteurization process.

Conclusions
The study found that the optimal pasteurization temperature and time for Passiflora foetida and Embryo Nelumbinis drink was 85 o C during 30 minutes.At these optimal pasteurization conditions, the herbal beverage maintained high levels of bioactive compounds and antioxidant activity.The content of polyphenol, flavonoid, tannin, saponin and alkaloid were 9.46 mgGAE/g, 4.44 mgQE/g, 4.85 mgTAE/g, 7.40 mgSE/g, 14.25 mgCE/g; and the antioxidant ability of product by FRAP and DPPH methods were 64.50% and 28.25 MFeSO4/g, respectively.The color parameters such as L * , a * , b * and E also obtained suitable values that thet were 31.58,-0.24, -0.47 and 61.25, respectively.The production process was successful in pilot equipment and the product can be used as one of the beverages for health strengthening.In future, the product could be a potential functional beverage for customer.

Figure 1 .
Figure 1.Charts show ability of ferrous scavenging by FRAP according to temperature (a) and time (b) of pasteurization

Figure 2 .
Figure 2. Charts show ability of free radical scavenging DPPH according to temperature (a) and time (b) of pasteurization

Table 1 .
Effect of pasteurization temperature and time on the content of bioactive compounds Notes: Mean (n=3); Means with different letters in the same column express statistically significant differences with ( ns ) P  0.05, (*) P 0.05 and (**) P 0.01.

Table 2 .
Regression equations predicted change of bioactive compounds content and antioxidant activity of product

Table 3 .
Effect of pasteurization temperature and time on color parameters of product Notes: Mean (n=3); Means with different letters in the same column express statistically significant differences with ( ns ) P  0.05, (*) P 0.05 and (**) P 0.01.