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Optimization of Extraction Time and Temperature for Java Tea (Orthosiphon aristatus BI. Miq) based Functional Drink Production

Johanes Michael1, Chutamat Niwat2*, Christofora Hanny Wijaya1

1 Food science and Technology program, Bogor Agricultural University, Bogor 16136, Indonesia
2 Food technology program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand, School of Agro-Industry Mae Fah Luang University, Chiang Rai, Thailand

CitationCitation COPIED

© 2019 Michael J, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Copyright: © 2019 Hanny Wijaya C. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Abstract

Java tea based functional drink, consisting of java tea, sappan wood, ginger, temulawak, lime and kaffir lime, has been claimed rich in bioactive compounds and antioxidant activities. The objective of this research was to study the effect of different extraction time and temperature of mixed simplicia on bioactive compounds and antioxidant activity in order to get the optimum process. Simplicia was made by drying each herb separately until it reached certain moisture content. Thesimplisia were mixed according to formula, extracted and then used as the ingredients for the production of the functional drink. Eleven extraction conditions were obtained from Design-Expert®7.00 software using two factors, time (15-60 min) and temp (60-95°C). The analyzed responses were total phenolic content and antioxidant activity, which affected significantly by time and temperature of extraction (p<0.05). The highest TPC (211.58 ± 4.87 mg GAE/L) was achieved by extracting simplicia with 78°C for 15 min, while highest DPPH (745.32 ± 17.44 µmoles Trolox/L) was achieved by extracting simplicia with 90°C for 21 min 36 sec (p<0.05). Different time and temperature of simplicia extraction did not significantly affected the pH and total soluble solid (p>0.05). Three treatments with the highest total phenolic content and antioxidant activity, (Run 4, 10 and 11) were analyzed using microbial (Total Plate Count, yeast and mold, and Total Coli form) and sensory evaluation. Microbial analysis showed that three highest treatment of functional drink were according to the regulation. Sensory analysis (using a 9-point hedonic scales method with 30 untrained panelists) showed no significant difference between three highest desirability treatment (p>0.05). Thus, 90°C for 21 min 36 sec, with desirability of 0.979, was considered to be the optimal condition for mixed simplicia extraction.

Keywords

Bioactive compounds; Extraction; Functional drink; Java tea

Practical Application

Infusion is one of the most commonly used extraction method to get phytochemicals from herbal plants. This research developed optimum extraction time and temperature for mixed simplicia to make java tea based functional drink. The optimum condition was obtained from the highest bioactive compound and antioxidant activity contained in the functional drink.

Introduction

Traditional herbal consumption has been established as part of health adaptation system, including prevention of diseases, reduction of symptoms and rehabilitation from disease [1]. Thousands of phytochemicals from plants have been claimed as safe and broadly effective alternatives with less adverse effect [2]. Anti-inflammatory, antioxidant, analgesic and wound healing are some biological activity associated with phytochemicals from herbal plants. Natural bioactive compounds have been found to take part in prevention and inhibition of cancer development [3].

The tip of java tea (Orthosiphon aristatus BI. Miq) leaves has been utilized to make herbal tea because it contains phenolic compounds including flavonoids and phenyl, propanoids as the major constituents (Yuliana et al., 2009). Sappanwood (Caesalpinia sappan L) extract has been used as anti-diarrhea, anti-inflammatory and potential for reducing blood glucose level in white rats [4]. According to [5] ginger (Zingiber officinale Roscoe.) contains active phenolic compounds such as gingerol, paradol, and shogaol that have antioxidant and anticancer activity. In addition, temulawak (Curcuma xanthorrhiza Roxb) has been traditionally consumed to treat liver disease, diabetes, cancer, stomach and heart disorders [6].

Original formula of java tea based functional drink was found by Wijaya et al. The drink combined herbal extracts which are java tea, ginger, sappanwood, temulawak, lime and kaffir lime. Beside rich in antioxidants, java tea based functional drink also exhibits anti hyperglycemic activity. Several researches were conducted to improve the consumer acceptance by using combination of lime and kaffir lime for the drink and to study the anti hyperglycemic activity the functional drink [7], which result in lower blood glucose level of diabetic white rat after the administration of functional drink.

Heat treatment is required to extract phytochemicals from plants. Temperature of extraction can affect the rate of process sufficiency and availability of bioactive compounds [8]. Therefore, time and temperature optimization of extraction process is essential to produce java tea based functional drink with the highest bioactive compounds extracted from the ingredients with minimal deterioration. Consequently, the aim of this work was to investigate the effect of different extraction time and temperature on the bioactive compounds and antioxidant activity of java tea based functional drink in order to get the optimum process.

Materials and Methods

Chemicals and reagents
Folin-Ciocalteu’s phenol reagents were purchased from Loba ChemieTM, India. Sodium carbonates (Na2 CO3 ) were purchased from QRëC®, New Zealand. Trolox (6-Hydroxyl-2,5,7,8-tetramethylchromane-2-carboxylic acid), Gallic acid and DPPH (1,1-diphenyl2-picrylhydrazyl), were purchased from Sigma-Aldrich®, China. Methanol was purchased from Ajax Finechem®, Seven Hills, Australia. All other chemicals and solvents in this study were of analytical grade.

Collection of raw material
 Java tea (Orthosiphon aristatus BI. Miq), sappanwood (Caesalpinia sappan Linn) and temulawak (Curcuma xantorrhiza) were received from Tropical Biopharmaca Research Center (Trop BRC) Bogor, Indonesia. Ginger (Zingiber officinale Roscoe.), kaffir lime (Citrus hystrix DC), and lime (Citrus aurantifolia Swingle) were purchased from local market in Chiang Rai, Thailand. Food additives used to make the functional drink were sucrose, asesulfame potassium and sucralose, which are received from PT Chemco Prima Mandiri, Indonesia; sodium benzoate, xanthan gum received from PT SOHO Industry Pharmasi, Indonesia, salt, Lemon flavor and flavor enhancer (IMP:GMP) received from PT Ajinomoto, Indonesia.

Preparation of dried java tea, ginger, sappanwood, and temulawak 
Java tea, ginger, sappanwood and temulawak were dried individually into simplicia by tray dryer at 50°C, until it reached moisture content below 5% for java tea, below 8% for temulawak and ginger, and below 10% for sappanwood (Indonesian Ministry of Health, 2000). The dried simplicia were separately grinded and sieved until the particle size was 80 mesh. Dried simplicia was stored in sealed plastic container, in room temperature until extraction.

Extraction of mixed simplicia with different condition 
Each simplicia was weighed and extracted based on condition given in Table 1. Optimum condition of factors, which are time and temperature of extraction were achieved using Response Surface Methodology (RSM) within Design-Expert®7.00 program. Rotatable Central Composite Design (CCD) was used and analysis was done in triplicates with three replicates at the center point, which consists of two factors and two responses as follows:

Factors

  • 15 min ≤ time ≤ 60 min
  • 60°C ≤ temperature ≤ 95°C Responses (Y)
  • Y1 = DPPH : µmoles Trolox/mL
  • Y2 = Total Phenolic Content (TPC): µg Gallic Acid Equivalent/mL Model as function time and temperature was as follows: 
    Y=f(X1,X2)+ε,
    Where ε defined as the error observed in the response (Y). Response surface was defined by f(x1 ,x2 ).

After the extraction, filtration was done using a cheese cloth. Extraction of citrus was done using citrus squeezer and by removing the pulp and seed. All the ingredients were mixed together and pasteurized for 3 minutes at 80°C. The functional drink was then stored in a aluminum foil wrapped glass bottle using hot filling method and refrigerated in dark room for further analysis.

Bioactive compound analysis

Analysis of Total Phenolic Content (TPC)
Total Phenolic was determined by colorimetric assay using Folin Ciocalteu’s phenol reagent. Analysis began with mixing 1.0 ml sample with 5.0 mL of 10% Folin-Ciocalteu’s reagent. The mixture was vortexed, then 4 mL of 7.5% w/v Na2 CO3 solution was added, and vortexed again. The absorbance was determined using spectrophotometer UV-Vis at 765 nm after 60 min incubation in room temperature. Gallic acid, with concentration ranging from 0 to 1000 mg/L, was used to get the standard concentration of phenolic content in samples. The total phenolic content of samples was calculated and reported as gallic acid equivalents (mg GAE/L sample).

Antioxidant analysis by DPPH assay
Measurement of antioxidant activity was based on DPPH scavenging activity according to the modification method from Anesini et al. and Molyneux. Analysis began with mixing 50 µL of sample with 1950 µL of a 60 µM DPPH (1,1-diphenyl-2-picrylhydrazyl C18H12N5 O6 M=394.33) solutions in methanol. The absorbance was measured at 517 nm by using spectrophotometer UV after incubation for 30 min at dark place. Trolox was diluted in methanol to make standard solution (1000 µmoles/L). The antioxidant activity of samples was calculated and reported as Trolox equivalents (µmoles Trolox/L sample). 

Physical and chemical analysis

Color measurement
The color of all samples was measured by a colorimeter using the CIELAB system—namely, L* (lightness), +a (redness), -a (greenness), +b (yellowness), and -b (blueness). The colorimetric measurements performed three times for the java tea based functional drink.

Turbidity measurement
Turbidity analysis was performed with Turbidity meter HI 93703. The measurement unit was expressed as Formazine Turbidity Unit (FTU). Each sample was measured three times and the average value calculated.

pH measurement
According to AOAC method (1995), pH meter must be calibrated using buffer solution pH 4.0 and pH 7.0 prior to usage. Sample approximately 30–50 mL was placed in a beaker glass and then measured using pH meter.

Total Soluble Solid measurement
Total soluble solid was measured using hand refractometer. Sample was dropped on the prism of refractometer which has been stabilized before. The scale displayed by the refractometer was reported. Total soluble solid was reported as °Brix.

Microbial analysis

Total Plate Count measurement
Total plate count analysis was according to USFDA. From each dilution level of sample, 1 mL solution was pipetted aseptically into sterilized petri dish and then sterilized Plate Count Agar (PCA) was also added into the petri dish. Petri dish then was moved in circular motion. After the media was hardened, petri dish was incubated in 37°C for 48 hours. The total microbe was reported as Colony Forming Unit (CFU).

Yeast and Mold measurement
Yeast and mold analysis was according to USFDA (1998). From each dilution level of sample, 1 mL solution was pipetted aseptically into sterilized petri dish and then sterilized Potato Dextrose Agar (PDA) was also added into the petri dish. After pouring the media, petri dish was moved in circular motion. After the media was hardened, petri dish was incubated in 30°C for 48 hrs. The result was reported as Colony Forming Unit (CFU/g).

Total Coliform measurement
Total coliform by Most Probable Number (MPN) method was determined according to Bacteriological Analytical Manual. From each dilution of sample, 1 mL solution was pipetted aseptically into sterilized test tube containing 9 mL sterilized Lauryl Tryptose Broth (LTB) and Durham tube. The tubes then incubated at 37°C for 24 hrs. The positive tubes, determined by the formation of gas and turbidity, then transferred as much as one loop to 10 mL sterilized Brilliant Green Lactose Bile (BGLB) broth. The tubes then incubated at 37°C for 48 hrs. Coliform are reported as MPN.

Sensory evaluation 
From eleven treatments of functional drink, three samples with highest responses in total phenolic compound and antioxidant activity were chosen. Those samples were tested using 9 points hedonic scale of appearance, aroma, color, flavor, after taste, and overall liking. Sample size of 20 mL was served in a small clear glass cup and analyzed using 30 untrained panelists.

Statistical analysis 
Data was expressed as means ± standard deviation. Data was analyzed using analysis of variance (ANOVA) and then Duncan multiple range tests using IBM SPSS Statistics 22.0 with significance level of 95%


Table 1: Treatment design by Response Surface Methods (Design Expert® 7.00)

Results and Discussions

Bioactive compound and antioxidant activity of simplicia
The total phenolic and DPPH content of each simplicia extract can be seen in Table 2. From the ingredients, java tea shared the highest in total phenolic (949.53 ± 7.97 mg GAE/100 g) and antioxidant activity (7459.78 ± 76.00 µmoles Trolox/100 g), while temulawak showed the lowest content of total phenolic (55.47 ± 0.52 mg GAE/100 g) and antioxidant activity (1998.46 ± 160.66 µmoles Trolox/100 g). The major bioactive compound of each herbs are ursolic acid and oleanolic acid from java tea, brazilein from sappanwood, gingerol from ginger, and curcumin from temulawak [4,9-11].

Bioactive compound and antioxidant activity of functional drink
The total phenolic and antioxidant activity of functional drink are shown in Table 3. The response surface for total phenolic using 2FI Polynomial model is significant (p<0.05) with the equation given:
TPC=-21.81+2.98*Te+5.56*Ti-0.08*Te *Ti
With R2 = 0.7465 (Te =temperature, Ti =time).

From the equation, extraction time showed higher effect to the total phenolic of the drink rather than temperature of extraction. Different extraction conditions caused different phenolic profiles because phenolic compounds have a different degree of labiality [12].

The response surface for antioxidant activity using 2FI Polynomial model is also significant (p<0.05) with the equation given: AA = 75.82+ 8.42*Te +10.48*Ti -0.16*Te *Ti and R2 = 0.8029 (AA=antioxidant activity)

Extraction time also showed higher effect to antioxidant activity rather than the temperature of extraction. These findings differed from the research conducted by Vergara-Salinas JR [12]. They reported that extraction temperature of Thymus vulgaris was the only factor sharing statistically significant effect. The difference between this finding and Vergara-Salinas research may occur because each research used different range of time and temperature. The extraction of T. vulgaris by Vergara-Salinas has wider range of temperature (50- 200°C), while the extraction of mixed simplicia has wider range of time (15-95 min). Several researches stated that in the extraction process, the amount of phenolic compound and antioxidant activity increased to some point and then started to decrease as the further extent of time and temperature [13-15].

Optimization of time and temperature of extraction
The optimization used response surface to generate the most desirable conditions based on several criteria. The criteria are antioxidant activity maximize (priority +++++) and total phenolic maximize (priority +++++). The Design-Expert®7.00 program gave the optimum solution based on highest desirability value of antioxidant activity and total phenolic (data not shown). Treatment 10 (90°C for 21 min 36 sec) was suggested to have the highest desirability value (0.979). Together with treatment 4 and 11, treatment 10 went through sensory evaluation and microbial analysis.

Physical and chemical properties of functional drink
Physical and chemical properties of eleven treatments are shown in Table 4. Turbidity measurement showed that there was a significant difference at p<0.05. Turbidity reflects the cloudiness of a fluid, due to insoluble solid particles invisible to the eyes. These insoluble particles in the java tea functional drink mostly were from mixed simplicia extract and citrus extract, which cannot be separated using a cheese cloth. Xanthan gum used in the drink helped the suspension of those insoluble components [16]. A slight different in turbidity measurement between treatments might happen because the filtration process for mixed simplicia extract using cheesecloth was not consistent in terms of the mesh size.

Total soluble solid using Brix scale measures the percentage of sugar and other dissolved solids in solution [17]. The sugar and other dissolved solids in the functional drink mostly were from the sucrose and citrus used. For pH, citrus contributed to lowering pH of functional drink since the lime juice itself has pH of 2.00-2.35. Since the formula used was the same between conditions, there was no significant difference. Color measurement of the functional drink showed a significant difference at p<0.05, between treatment 5 with the lowest extraction temperature used and treatment 11 with highest extraction temperature. The pigment components contained in the drink was brazilein from sappan wood, together with chlorophyll from java tea and curcumin from temulawak.

The natural color of brazilin was yellow but oxidation reaction and light effect changed it to brazilein which has red color and stable in high temperature [18]. According to [19], curcumin showed maximum brightness and yellowness when it was being treated with heat above 80°C. According to [20], heat treatment can cause degradation of chlorophyll, changing the color from green to dull olive green. The degradation of pigments caused the lightness of the drink to increase.

Microbial analysis
The result of microbial analysis for three selected treatments (4, 10, 11) were shown in Table 5. The analysis of total plate count, yeast and mold, and coliform of functional drink complied with SNI 01-3719-1995, with total plate count less than 1.0 x 104 CFU/g, yeast and mold less than 1.0 x 102 CFU/g and coliform less than 2.0 x 101 CFU/g. Pasteurization process is only effective in eliminating pathogens and spoilage bacteria, therefore sometimes a product can still contain other microbes such as yeast and mold [21]. The use of preservative, in this case, sodium benzoate and also the presence of natural acidulant created a hurdle effect that can inhibit the growth of those microbes.

Sensory evaluation

The result of sensory evaluation for three selected treatments (4, 10, 11) were shown in Table 6. A total of 30 panelists are categorized based on nationality, consisted of Thai (63.4%), Indonesian (13.3%), Malaysian (13.3%), Vietnamese (6.7%), and Bruneian (3.3%), and age: 26.7% (17-20 years old); 60.0% (21- 25 years old); 13.3% (>25 years old). The result showed that there was no significant difference in appearance, aroma, color, flavor, aftertaste, and overall liking of the product (p<0.05). Thus, treatment using 90°C for 21 min 36 sec, min was considered the best based on antioxidant activity and total phenolic content.

Verification of optimization process
After getting the selected treatment, it needs to be verified in order to analyze the precision of optimization process. Table 7 shown the verification result of optimization result. The response of DPPH showed the value was within the range of Confidence Interval (CI) and Prediction Interval (PI), while the response of total phenolic was only within the range of PI and slightly out of CI range. CI is a range that computed from the mean observed with a significance level of 95% while PI is a range that shows where the next response can be expected with a significance level of 5%. Based on the verification result, extraction of mixed simplicia with 90°C for 21 min 36 sec was verified and can be used. 


Values are expressed as means ± SD (n=3).
Table 2: Total phenolic and antioxidant activity of simplicia extract


Values are expressed as means ± SD (n=3).
Different letters in the same column indicate significant difference at p<0.05.
Table 3: Total phenolic content and antioxidant activity of eleven treatments of functional drink


Different letters in the same column indicate significant difference at p<0.05.
Values are expressed as means ± SD (n=3); ns=not significant; T=treatment
Table 4: Chemical and physical analysis of eleven treatments of functional drink


ND=Not Detected
Table 5: Microbial analysis of selected functional drink


Values are expressed as means ± SD (n=3); ns=not significant;
Different letters in the same column indicate significant difference at p<0.05 
Table 6: Sensory evaluation of selected functional drink


Values are expressed as means ± SD (n=3)
Table 7: Prediction and verification result of treatment 10 of functional drink 

Conclusion

Different conditions of extraction significantly affected the bioactive compound, antioxidant activity and the color of java tea based functional drink, but not the sensory attributes, other analyzed physical and chemical properties. The optimum condition for mixed simplicia extraction was 90°C for 21 min 36 sec. The findings of this study are expected to get a higher level of antioxidant activity and bioactive compound for functional drink production.

Acknowledgments

The authors would like to express a sincere gratitude to Mae Fah Luang University and Bogor Agricultural University for the funding and support throughout this research.

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