The 5th International Conference on Renewable Energy, Sustainable

Environmental, Agricultural and Artificial Intelligence Technologies

18th –20th, December 2023
Bangkok, Thailand

Effects of proportions and ingredients on the physicochemical properties and

anti-oxidant activity of Ardisia wine
Phanwasa Khamsuk, Noppawat Armattayawong, Benya Manochai, Jenjira Chumpookam*
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
*
Corresponding author E-mail: jchumpookam@gmail.com

Abstract: This study assessed the physicochemical characteristics and antioxidant capacities of six distinct
wine preparations obtained from Ardisia polycephala Wall. ex A.DC. (Ardisia berries). Each formula
consisted of 1,000 grams of Ardisia berries mixed with 3 liters of water. The only variations among the
formulas were the types of sugar and yeast used. Formula 1 contained 700 grams of brown sugar and 30
grams of SAF-INSTANT yeast. Formula 2 contained 700 grams of white sugar and 30 grams of SAF-
INSTANT yeast. Formula 3 contained 700 grams of brown sugar and 30 grams of Eagle Gold yeast.
Formula 4 contained 700 grams of white sugar and 30 grams of Eagle Gold yeast. Formula 5 contained 700
grams of brown sugar and 30 grams of Lalvin EC-1118 yeast. Formula 6 contained 700 grams of white
sugar and 30 grams of Lalvin EC-1118 yeast. The fermentation process lasted for 40 days for all
formulations. The investigation revealed no notable disparities in pH, total soluble solids, or alcohol content
among the six wine samples. Nevertheless, there was variation in the antioxidant activity, as Formulas 3,
4, and 6 displayed higher average antioxidant activities (ranging from 75.09% to 77.09%), which were
substantially different from the lower activities (ranging from 55.26% to 65.90%) observed in Formulas 1,
2, and 5. Results from sensory tests conducted with 120 inexperienced tasters showed that Formula 6 was
favored due to its color, flavor, sweetness, and overall satisfaction. The study suggests that Formula 6 is a
favorable foundation for developing Ardisia berry wine products that promote health.
Keywords: Ardisia polycephala, Wine, Sensory Evaluation, Antioxidant Activity

1. Introduction
Wine, a distinguished alcoholic beverage that sets itself apart from liquor and whiskey, is distinctive
in its production process, relying on fermentation instead of distillation [1]. This technique is crucial in
winemaking, particularly in places such as Thailand, which is fortunate to have a diverse range of ideal
fruits. Fruits suitable for winemaking exhibit a balanced combination of sweetness and acidity, brilliant
pigmentation, and alluring fragrances, occasionally complemented by a subtle astringent quality [2].
Common selections comprise grapes, pineapples, mangoes, okra, lukwa, cherries, and gooseberries. When
wine is drunk in a responsible manner, it can stimulate the appetite and assist in medical treatments. For
example, it can help to dilate blood vessels in individuals with low blood pressure and prevent the narrowing
of coronary arteries known as coronary artery stenosis [3]. This categorizes wine as a more popular drink,
especially among people prioritizing their health.
The ability of Saccharomyces cerevisiae, also known as baker's yeast, to convert carbohydrates into
ethanol and carbon dioxide [4, 5] in a short amount of time in both anaerobic and aerobic circumstances is
one of the most notable characteristics of this form of yeast [6]. Under the conditions of solely fermentative
development, acetaldehyde serves as the ultimate electron acceptor and is transformed into ethanol when
oxygen is not present or present in sufficient quantities [7]. S. cerevisiae continues to demonstrate alcoholic
fermentation even when the medium is depleted of sugar and glucose, even though respiration is possible
under aerobic circumstances with oxygen serving as the ultimate electron acceptor [8]. Therefore, the
fermentation process, which is essential in winemaking, is primarily influenced by yeast activity, with
Saccharomyces cerevisiae playing a particularly crucial role. This yeast species plays a crucial role in the
enchanting metamorphosis that occurs during the winemaking process. It efficiently transforms the inherent
sugars found in fruit juices into ethanol and carbon dioxide. This conversion is not merely a chemical
reaction; it is a process of culinary alchemy in which the basic sugars transform intricate alcohol molecules.
However, yeast's involvement in fermentation goes well beyond the mere creation of alcohol [9]. These

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microbes are the unacknowledged craftsmen of the winemaking industry, accountable for creating the
distinctive combination of flavors and scents that define every bottle of wine. Throughout fermentation,
yeast cells emit a complex array of chemicals - including esters, phenols, and higher alcohols - which
collectively enhance the intricate flavor characteristics and fragrance of the wine [1]. This procedure
imparts the wine with its own characteristics, ranging from the fruity aromas of a delicate white wine to the
strong, earthy flavors of a rich red wine. Yeast, during the process of fermentation, acts as both a biological
catalyst for alcohol synthesis and a creator of flavors [4], playing a crucial role in creating and improving
the sensory qualities of wine. The outcome is a beverage that transcends being merely an alcoholic drink,
but rather an intricate amalgamation of flavors and scents. Each glass serves as a tribute to the profound
impact of fermentation and the delicate craftsmanship of yeast.
The Ardisia polycephala Wall. ex A.DC. (Pilangasa), which belongs to the Myrsinaceae family, has
potential for winemaking in Thailand's tropical areas. This shrub, cultivated for its decorative, culinary, and
therapeutic properties, has fruits most enjoyable when fully matured, transitioning from deep crimson to
black. Despite their limited popularity due to their astringent flavor, these fruits offer substantial health
advantages, including treating numerous conditions ranging from diarrhea to dermatitis [10]. The Pilangasa
extract, obtained from a solution of 95% ethanol, has demonstrated antibacterial activities against pathogens
such as Salmonella spp. and Shigella spp., as well as efficacy in inhibiting the growth of lung and breast
cancer cells. The fully developed Pilangkasa fruit, rich in anthocyanins, possesses potent antioxidant
properties.
This study investigates the therapeutic properties of Pilangkasa, a plant found in the Bang Krachao
village in Samut Prakan province. The research also examines the possibility of enhancing its value by
processing. The study centers around the development of Pilangasa wine, exploring the ideal proportions
of ingredients, the influence of yeast in the fermentation process, and the analysis of the wine's
physicochemical attributes, such as acidity, total soluble solids, and alcohol level. The evaluation of
consumer approval is conducted utilizing the 5 5-point hedonic Scale methodology. The objective is to
disseminate this knowledge among local farmers, augmenting the worth of Pilangasa fruit by producing
Pilangasa wine, establishing a distinct identity, promoting community involvement, and earning revenue.

2. Material and Methods

2.1 Raw material preparation steps
The process of producing Pilangasa wine begins with the preparation of the Ardisia polycephala
Wall. ex A.DC. (Pilangasa) fruits. To experiment, it is necessary to use 1000 grams of completely mature
Pilangasa fruits for each of the six treatments. The fruits are first cleansed to eliminate soil, employing a
solution of pristine water combined with 15 grams of baking soda. Following a 15-minute soaking period,
the fruits are subsequently rinsed with fresh water. Subsequently, the process entails extracting the juice
from the Pilangasa fruits. The fruits are extracted, segregating the pulp from the juice. The extracted juice
serves as the foundation for fermenting the wine. The winemaking process is categorized into six
treatments, each characterized by different sugar types and yeast strains. These variants are intended to
investigate the influence of these variables on the ultimate wine product. The formulations of the treatments
are as follows:
Treatment 1 - Wine Formula 1:
• Pilangasa Fruit: 1000 grams
• Brown Sugar: 700 grams
• Yeast (SAF-INSTANT, Saccharomyces cerevisiae): 30 grams
• Clean Water: 3 liters
Treatment 2 - Wine Formula 2:
• Pilangasa Fruit: 1000 grams
• White Sugar: 700 grams
• Yeast (SAF-INSTANT, Saccharomyces cerevisiae): 30 grams
• Clean Water: 3 liters

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 Treatment 3 - Wine Formula 3:
• Pilangasa Fruit: 1000 grams
• Brown Sugar: 700 grams
• Yeast (Eagle Gold, Saccharomyces cerevisiae): 30 grams
• Clean Water: 3 liters
Treatment 4 - Wine Formula 4:
• Pilangasa Fruit: 1000 grams
• White Sugar: 700 grams
• Yeast (Eagle Gold, Saccharomyces cerevisiae): 30 grams
• Clean Water: 3 liters
Treatment 5 - Wine Recipe 5:
• Pilangasa Fruit: 1000 grams
• Brown Sugar: 700 grams
• Yeast (Lalvin EC-1118, Saccharomyces cerevisiae bayanus): 30 grams
• Clean Water: 3 liters
Treatment 6 - Wine Recipe 6:
• Pilangasa Fruit: 1000 grams
• White Sugar: 700 grams
• Yeast (Lalvin EC-1118, Saccharomyces cerevisiae bayanus): 30 grams
• Clean Water: 3 liters
Subsequently, every mixture undergoes the fermentation process. The objective of these treatments
is to analyze the fluctuations in flavor, alcohol level, and other physicochemical characteristics of the wine
produced, in order to ascertain the optimal blend of components for Pilangasa wine. Bring the squeezed
pilangasa juice from each treatment. (formula) above. Add a 6-liter fermentation jar by filtering Pilangkasa
water with a thin white cloth to prevent contamination of pulp and seeds. Add 3 liters of pure water . Add
different types of sugar according to the treatment, stir the sugar to dissolve, then take the finished yeast
but treatment and dissolve it in 100 milliliters of pure water. 3-minute timer to accelerate yeast activity.
When the time is up, pour the yeast into a jar and marinate each wine. Cover the jar with 1 layer of thin
white cloth and leave to marinate for 40 days. Store wine jars in a secure area. It has no light and stores at
room temperature. At the end of the due date, take the plentiful wine, pegged with a thin white cloth and a
siphon to separate the residue and sediment. Filtered wine is sterilized by the pasteurization method (70°C)
for 20 minutes to reduce microbial content to a safe level for consumers and inhibit yeast activity. Wait for
the wine to drop to room temperature. Place the filling into a narrow-mouth bottle and close the lid tightly.

2.2 Physico-chemical properties analysis

pH is measured by a pH meter (model PH20, manufactured by Apera Instrument, China). Analysis
of Total Soluble Solids (TSS) was measured in Brix degrees (◦Brix) with a sweetness meter (Hand
Refractometer, manufactured by Atago, Japan). The alcohol content in wine is measured using an alcohol
refractometer (produced by Yieryi, China) by dropping the wine checked into the measuring sheet and then
looking at the refractive value. Measured in % (percent) of alcohol units (%v /v). PPH radical scavenging
activity Analysis of antioxidant activity according to the method of Ingkasupart et al. [11]. Prepare a
solution by weighing 0.029 g of 1,2-diphenyl-1-picrylhydrazyl (DPPH) mixed with 500 ml of ethanol. In
a bottle, adjust the volume under dark conditions. Place on a Magnetic Stirrer for 30 minutes until the
solution is completely drained—store at 4°C. Then take 22 μl of pilangasa wine per sample. Repeat 10
times for each treatment. Vacuum the prepared DPPH solution into a sample of 200 μl per sample into a
microplate. Then wrap the foil to replace it in a dark state and place it on a shaker for 30 minutes. Measure
absorption with a microplate reader at a wavelength of 517 nm. To calculate the antioxidant activity
(%DPPH scavenging activity) as follows:

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 ABlank - ASample
AA(%) = x 100
ABlank

When AA(%) = Antioxidant Activity

ABlank = absorption value of solution DPPH without samples
A Sample = The absorption value of the pilangasa wine sample.

2.3 Comprehensive methodology for Pilangasa wine evaluation and analysis

The approach employed for the Pilangasa Wine study is intricately organized and incorporates
various crucial elements. The initial element, Pilangasa Wine Tasting, utilizes the 5 Point Hedonic Scale to
assign scores ranging from 1 point (Minimum Acceptability) to 5 points (Excellent). This evaluation
method assesses various characteristics including aroma, flavor, sweetness, alcohol concentration, and
quality. The Sensory Quality Assessment entails a randomized sample of 120 participants and evaluates the
wine's color, clarity, aroma, and flavor. This section of the study is structured as a fully randomized
experiment with three repetitions, and the average data are compared using Duncan's Multiple Range Test
(DMRT) at a 95% confidence level. In addition, the Analysis of Experimental Results is conducted using
SAS version 9.2, and comparisons are made with DMRT at a 95% confidence level to assure statistical
precision. The project is carried out at two primary sites: the Laboratory for Fruit Tree Production
Physiology, Experimental Plot 2, and the Laboratory for Medicinal Plants, both located under the
Department of Horticulture, Faculty of Agriculture at Kasetsart University. The trial time for this extensive
study extends from April 12, 2022, to January 11, 2023, enabling a meticulous examination and evaluation
of the sensory characteristics and overall desirability of Pilangasa wine.

3. Results and Discussion

Fermentation is an age-old natural process harnessed by humans for millennia, primarily for the
production of alcoholic beverages, bread, and various by-products. Biochemically, fermentation involves
an organism transforming a carbohydrate like starch or sugar into alcohol or acid. For instance, yeast
undergoes fermentation to convert sugar into alcohol, extracting energy in the process [12]. This
biochemical phenomenon was spontaneously utilized long before its scientific understanding was
established. Traditionally, the fermentation of wines, beers, and ciders involves strains of S. cerevisiae, the
most common and commercially used yeast. This yeast is renowned for its fermentative capabilities and
technological properties, ensuring consistent and high-quality products. Beyond alcoholic beverages,
fermentation is pivotal in producing various industrial products such as yogurt, cheese, bread, and coffee.
Additionally, yeasts play a crucial role in environmental applications like wastewater treatment and biofuel
production. At the biochemical level, fermentation occurs when yeasts (and some bacteria) break down
pyruvate, a product of glucose metabolism, into ethanol and carbon dioxide [13].
A eukaryotic microbe, yeasts live in water, soil, air, and on plants and fruits. They are essential to
ripe fruit breakdown and fermentation. Yeast thrives on fermentable carbohydrates, amino acids, vitamins,
minerals, and oxygen, unlike lactic acid bacteria [14, 15]. The most frequent yeast shapes are spherical,
ellipsoidal, or oval. Identification usually begins with a microscopic assessment, then microbiological and
biochemical investigations [16]. Classification tests sugar fermentation, amino acid assimilation, ethanol,
organic acid, and SO2 generation and tolerance. Most yeasts reproduce asexually by budding, creating
genetically identical cells [17]. Some Saccharomyces species reproduce by cell fission. Yeasts can sporulate
to withstand food scarcity, causing genetic diversity [18-19]. Asexual reproduction is desirable in industrial
fermentation for stability. Yeast may ferment sugars in ripe fruit and processed grains and withstand low
pH [20]. Technology divides yeasts into Saccharomyces and non-saccharomyces. Saccharomyces,
especially the well-studied S. cerevisiae, is employed in wine and beer production due to its effective

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fermentative capacity, quick growth, and flexibility, including tolerance to SO2 levels that kill most other
yeasts. However, not all S. cerevisiae strains have these benefits.

Table 1. Physiochemical properties of 6 different formulas of Ardisia wine.

Scavenging
TSS Alcohol activity of
Formula pH
(◦ Brix) (%) DPPH radical
1 4.6 9.67 9.67 (%)
55.26 c1/
2 4.6 9.33 9.67 65.90 b
3 4.7 9.67 9.67 75.09 a
4 4.6 9.67 10.00 76.90 a
5 4.6 9.67 10.00 51.11 d
6 4.6 9.67 10.00 77.09 a
F-test ns ns ns *
CV (%) 0.17 6.49 4.15 4.83
1/
: Means in the same column followed by different letters are significant by different (P≤0.05) by
DMRT. *: Means statistically significant difference P ≤ 0.05. ns: Means not statistically different.

Quality test results of various pilangasa wines aged for 40 days showed that the pH of the total soluble
solids There was no statistical difference between the TSS and alcohol content of the six wine formulations.
The average value is in the range of 4.6-4.7. The average is in the range of 9.33-3.67 degrees Brix and the
alcohol content is in the range of 9.67-10.00%. Formula 3, Formula 4 and Formula 6 wines have no
difference in average antioxidant activity of 75.09-77.09%, which is a statistically significant difference
with Formula 1, Formula 2, and Formula 5 wines with an average of 51.11-65.90% (Table 1). Sensory
test results of various pilangasa wine recipes aged for 40 days from the sensory test results of all 6 formulas
of pilangasa wine at random of 120 testers, giving 5 levels of satisfaction rating. It was found that in terms
of color, there was a significant statistical difference in satisfaction scores from all 6 wine formulas, namely
that there was no difference between Formula 4 and Formula 6 wines. It has an average color score of 4.13-
4.25, which is statistically significantly different from other formulas.
Formula 4 had the highest average clarity score of 3.95 points, followed by Formula 1 with a clarity
score of 3.75 points, while other formulas had an average score of 3.01-3.51 points (Table 2). Satisfaction
rating of 6 wines with different ingredient rates In terms of taste, the satisfaction score from all 6 wines
was statistically significantly different, with Formula 6 having the highest average taste score of 3.82
points, followed by Formula 4 with a taste score of 3.48 points, while others had an average score of 2.98-
3.38 points (Table 2). Other formulas have an average score of 2.81-3.32 (Table 2) for alcohol content.
Satisfaction scores from all six wine recipes differed statistically significantly. There was no statistical
difference. It has an average score of 3.39-3.57 points, but statistically significantly different from the
formula. 2 Achieved the lowest average alcohol content satisfaction score of 3.07 points (Table 2) In terms
of overall quality, the satisfaction score from all 6 wines differed statistically significantly: Formula 6 had
the highest average score of 3.83 points, followed by Formula 4 with 3.67 points, while other formulas had
an average overall quality score of 3.08-3.46 points (Table 2).
According to the study of all 6 wine formulas, the pH is in the range of 4.6-4.7, which is slightly
higher than that of conventional wines (Figure 1). A typical wine has a pH between 2.9-4.2, low acidity
results in a darker, more stable wine color than high acidity. Sugar is a food source for yeast to grow and
convert into alcohol. Typical tropical fruits, when diluted with water, tend to have little sugar left. This

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makes the wine tasteless and the yeast will stop reacting quickly and result in a low alcohol content, so
additional sugar must be added. White sugar is most suitable for making wine because it will not change
the color and aroma of the wine. White sugar wine formulated with white sugar tends to get higher sensory
test scores than pilangasa wines. Brown sugar, and in the research of studying the source of sucrose on the
fermentation kinetics of smooth orange wine, described the effect of white sugar in the fermentation of
smooth orange wine as an ideal sugar for yeast cell growth, S. cerevisiae and alcohol production due to its
microbial growth rate. The duration of microbial multiplication of the product and the maximum rate of
product occurrence.

Table 2. Sensory evaluation of 6 different formulas of Ardisia wine

Overall
Sweet
Formula Color Clarity Odor Flavor Alcohol satis
ness
faction
1 3.7 3.3 3.1 3.39 3.1
2 3.8 3.1 3.07 3.0
3 2.9 3.1 3.2 3.2 3.44
4 4.1 3.4 3.4 3.57
5 2.7 3.2 3.3 3.39 3.3
6 4.2 3.4 3.49 3.8
F- * * ns * * * *
C 4.1 7.0 5.8 6.8 4.9 3.71 7.3
1/
: Means in the same column followed by different letters are significant by different
(P≤0.05) by DMRT.*: Means statistically significant difference P ≤ 0.05.ns: Means not statistically
different.

Formula1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6

Figure 1. Comparison of the color and clarity of Ardisia wine.

Yeast S. cerevisiae is a yeast that has been reported to be fermented. It can withstand conditions with
high alcohol content and high sugar concentration and low pH, so it is commonly used as the main yeast
that is popular for fermentation (Liquor Organization, Excise Department, 2017). Grossmann et al., [14]
and Sommer et al. [15] reported that wines obtained from grape juice fermentation use yeast mixtures of

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Saccharomyces and non-Saccharomyces. In terms of satisfaction, each person will prefer different flavors
depending on how each person feels. Therefore, the quality and satisfaction of consumers towards Pilangasa
wine is different. Each recipe is different (Figure 1). Formula 6 wines have quality and sensory test scores
with color, taste, and taste scores. Therefore, the composition rate of Formula 6 wine has the most suitable
properties to develop into Pilangasa wine beverage products in order to create uniqueness and generate
income for the community.

4. Conclusion
The comprehensive quality assessment of Pilangkasa wine revealed no significant statistical
discrepancies in terms of pH levels, total dissolved solids, and alcohol concentration across all
tested wine formulations. Furthermore, the DPPH assay results showed that Formulas 3, 4, and 6
had similar antioxidant capacity, with an average antioxidant activity ranging from 75.09% to
77.09%. These values were significantly higher compared to the results obtained for Formulas 1,
2, and 5. A group of 120 evaluators conducted sensory assessments to grade the sensory
characteristics of Formula 6, including its color, taste, scent, and general palatability. The results
indicate that Formula 6 has unique characteristics that make it well-suited for future development
as a Pilangkasa wine beverage product, perhaps providing improved health advantages.

Acknowledgements

The authors thank the Department of Horticulture, Faculty of Agriculture, Kasetsart University,
Bangkok, Thailand for providing the facilities.

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