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Full Research Article

Study of Synbiotic Yogurt Raw Materials as Functional Food: Analysis of Goat Milk Quality, Lactic Acid Bacteria and Gowok (Syzygium polycephalum) Fruit Phytochemicals

S
Siti Rahmawati Zulaikhah1,2
T
Triana Setyawardani2,*
https://orcid.org/0009-0001-9488-9749
J
Juni Sumarmono2
1Department of Animal Husbandry, Faculty of Sains and Technology, Nahdlatul Ulama Purwokerto University. Sultan Agung Street No 42, Karangklesem, Purwokerto, Central Java, 53145, Indonesia.
2Faculty of Animal Science, Jenderal Soedirman University, Dr. Soeparno Street No. 60, Karangwangkal, Purwokerto, Central Java, 53122, Indonesia.

ABSTRACT

Background: The increase in cases of degenerative diseases has made people increasingly aware of the need to consume functional foods. Yogurt is an alternative source of functional food. Innovation in developing synbiotic yogurt as a quality functional food through the addition of gowok (Syzygium polycephalum), which is the local wisdom of Indonesia. Synbiotic yogurt will be made using goat milk which has several advantages compared to cow milk, including being non-allergenic because it has high digestibility.  The using of probiotic bacteria is also an effort to improve the functional properties of this yogurt, by adding Lactobacillus acidophilus bacteria. The using of gowok (Syzygium polycephalum) prebiotics by adding each part of the gowok (rind, flesh of fruit and whole seedless fruit) during the yogurt making process. This research is an initial study in preparing raw materials for making synbiotic yogurt. This study is meant to find out how good the raw materials for making synbiotic yogurt, which include: goat’s milk, lactic acid bacteria and gowok (Syzygium polycephalum).

Methods: This research began with testing the quality of goat milk using lactoscan, the quality of lactic acid bacteria using TPC (Total Plate Count) calculations, qualitative and quantitative testing of gowok (Syzygium polycephalum), which was previously extracted using the maceration method.

Result: The results of the study showed that the quality of goat milk to be used in making synbiotic yogurt had the criteria for good milk quality. The number of lactic acid bacteria colonies (Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus acidophilus) was 108CFU/ml, which met the minimum requirements for making yogurt, namely a minimum of 107CFU/ml. Gowok, also known as Syzygium polycephalum, has been shown to have active compounds like flavonoids, phenols, anthocyanins, terpenoids, tannins and alkaloids. These compounds can be used as a source of antioxidants and help increase the antioxidant activity in synbiotic yogurt so that it is hoped that it can produce yogurt with high functional properties.

KEYWORDS

INTRODUCTION

Functional foods have become a public concern recently, because they are believed to have health benefits.  Degenerative diseases such as coronary heart disease, diabetes, osteoporosis, cancer and others are increasingly being suffered by the public. This is a concern that needs to be anticipated, one way is by consuming functional foods that contain lots of antioxidants so that they are beneficial for health. Chandimali et al., (2024) stated that functional foods are foods or ingredients that have health functions beyond their basic nutritional value, for example bioactive components. One of the functional foods that is now widely known to the public is yogurt.
       
Yogurt is generally made from cow’s milk. This study used goat’s milk because of its many benefits, including a lack of lactose intolerance, improved digestibility, probiotic and prebiotic content that can maintain healthy gut microflora and protection against the negative effects of pathogenic bacterial infections and allergies (ALKaisy et al., 2023). Hammam et al., (2022) stated that goat milk and its products have health benefits including being anticarcinogenic, antidiabetic and antihypertensive.  Yogurt production requires the work of lactic acid bacteria (LAB), commonly used are Streptococcus thermophillus and Lactobacillus delbrueckii subsp. bulgaricus in order to increase its functional value, probiotics are used in the fermentation process, including Lactobacillus acidophillus. According to Hendarto  et al. (2019), with the presence of probiotics, yogurt can produce antioxidant activity derived from the work of these probiotics in the form of bioactive peptide compounds, vitamins and metabolites resulting from the fermentation process.
       
Efforts to improve the functional properties of yogurt by adding external ingredients containing natural antioxidants.  One of the ingredients added is gowok (Syzygium polycephalum), which ultimately makes the yogurt known as synbiotic yogurt.  Gowok (Syzygium polycephalum) is a local Indonesian potential that can be found once a year, because it is a seasonal fruit (Picture 1). Gowok contains flavonoids, alkaloids, tannins, terpenoids and saponins which have health benefits (Puspitasari and Sofandi, 2020).   This is reinforced by the statement Lubis et al., (2024) that gowok fruit flesh contains flavonoids, terpenoids, tannins and phenols, the IC50 value of ethanol extract of gowok fruit flesh is 58.082 ppm and the phenol content is 8.3686 mg GAE/g or 0.8386%. This research was conducted to explore gowok’s potential as an antioxidant and source of natural fiber while preserving local wisdom.

Picture 1: Gowok (Syzygium polycephalum).


       
This research aims to examine the raw materials for yogurt in the form of goat’s milk, lactic acid bacteria (LAB) and gowok (Syzygium polycephalum) which will be used in making yogurt. Knowledge of the quality of raw materials will be the capital in making the resulting yogurt.  Research on synbiotic yogurt by adding gowok (Syzygium polycephalum) with goat milk as the raw material and adding the probiotic bacteria Lactobacillus acidophillus is a novelty in itself, because there has been no previous research.

MATERIALS AND METHODS

The research materials consist of goat milk, lactic acid bacteria and gowok (Syzygium polycephalum).
 
Goat milk
 
The goat’s milk used in this study was Etawah crossbreed goat milk obtained from local breeders in the Lumbir area, Banyumas, Central Java, Indonesia. The quality testing of this goat milk uses a Lactoscan tool. The method used is to put a sample of approximately 50 ml into the Lactoscan device, then set it for quality testing and the test results can be read immediately. The quality of goat milk is based on Thai Agriculture Standard (TAS) No. 6006-2008 (Ratya et al., 2017).
 
Lactic acid bacteria (LAB)
 
Lactic acid bacteria isolates Streptococcus thermophilus and Lactobacillus bulgaricus were obtained from the Inter-University Center (PAU) of Gadjah Mada University, with strains FNCC 0040 and FNCC 0041. The probiotic Lactobacillus acidophilus ATCC 4356 was obtained from Bogor Agricultural Institute. The number of lactic acid bacteria was calculated for each isolate. This isolate was grown in MRSB media, by preparing 10 ml of sterilized MRSB solution in a test tube, then 1 loop of the isolate was taken, then incubated at 40°C for 24-48 hours. BAL calculation uses TPC (Total Plate Count), which is previously diluted using sterilized distilled water and NaCl of 0.85gr/L. Each tube was filled with 9 ml of dilution solution, made for 101 to 107, as much as 1 ml of isolate from MRSB was put into the dilution solution to produce a dilution of 101, from 101 1ml was taken then put into the next tube to get a dilution of 102, from 102 1 ml was taken to get a dilution of 103 and so on. Next, prepare a growing medium from MRSA and place it in a petri dish. Then, 1 ml of each dilution, from 104 to 107, is taken and planted in the petri dish containing the MRSA. The petri dish is inverted and incubated at 40°C for 24 hours. Colonies are counted to determine the number of bacterial cells in CFU/ml.  Calculation of the number of lactic acid bacteria Steptococcus thermophillus, Lactobacillus bulgaricus and Lactobacillus acidophillus separately, as a starter (before inoculation) with each amount to 108 CFU/ml.
 
Gowok (Syzygium polycephalum)
 
Gowok (Syzygium polycephalum) was obtained from a fruit trader in Ajibarang, Banyumas, Central Java, Indonesia.  Gowok is prepared by separating the fruit: rind, flesh of fruit and a mixture of the rind and flesh of fruit, then dried at a temperature of 60°C using a Food Dehydrator until dry and broken. Next, grind it using a food grinder, so that you get flour from each part.
 
Qualitative test of gowok (Syzygium polycephalum)
 
Before testing, gowok flour was extracted using maceration referring to (Harborne, 1996). The samples were ground and 10 grams were taken and soaked in 96% ethanol at a height of 2.5 cm for 24 hours. The sample is filtered, then the ethanol is evaporated to obtain an extract.
 
Qualitative testing includes
 
a) Fenol hidrokuinon test (Harborne, 1996).
 
- Take 0.5 g of the extract sample and add 10 ml of homogenized 70% ethanol. 1 ml of the solution was taken and added with 2 drops of 5% FeCl3 solution. Observe the changes that occur, a positive reaction occurs if the color changes to green or green-blue.
 
b) Alkaloid test (Harborne, 1996).
 
-  100 µl of extract added with 50 µl of H2SO4 then vortexed. Add 100 µl of Dragendorf’s solution, then vortex and observe the color change after 30 minutes.
 
c) Tanin and Polifenol Test (Harborne, 1996).
 
- The extract is homogenized with water and boiled for several minutes, filtered. The filtrate was dropped with FeCl3. The blue to black color indicates the presence of tannin and/or polyphenol compounds. Uji Saponin (Harborne, 1996).
-  2 ml of extract plus 2 ml of hot distilled water, then shake  vigorously, observe whether there is foam or not.  If there is foam, add 2N HCl if the foam remains strong for 10 minutes, then it is positive for saponin.
 
d) Flavonoid test (Harborne, 1996)
 
- 100 µl of extract plus 50 µl of Zn or Mg plus 100 µl of 2N HCl solution, heated for 5-10 minutes. The filtrate was filtered and 50 µl of amyl alcohol was added. Observe the color change (yellow +, red ++, orange +++). Uji Terpenoid (Harborne, 1996).
- 1 gram of extract plus 1 ml of ethanol is boiled briefly, then filtered. Take 0.5 ml, add 0.2 ml diethylether and 0.3 ml sulfuric acid. If the color is reddish brown, it means positive.
 
Quantitative test of gowok (Syzygium polycephalum)
 
a) Flavonoid test (Harborne, 1996; Aminah et al., 2017).
 
- Create a standard curve. 2.5 ml quercetin plus 96% ethanol to a volume of 25 ml. Wuersetin levels 100 mg/l (100 ppm).  Make a standard solution of quercetin 20 ppm, 40 ppm, 40 ppm, 80 ppm. Take 500 µl each plus 2.5 ml distilled water.  After that, 1 ml was vortexed, plus 100 µl of 10 % AlCl3 and left for 6 minutes.  Add 100 µl of potassium acetate or 5 % NaNO2, incubate for 5 minutes in the dark room.

- The extract sample was weighed at 2.5 mg, dissolved in 2.5 ml of distilled water and 500 µl was taken, added to 2 ml of distilled water, then vortexed. Take 1 ml of the solution plus 100 µl of 10% AlCl3 and let it sit for 6 minutes. Add 100 µl of potassium acetate or 5% NaNO2, incubate for 5 minutes in a dark place. Read the absorption of the standard and the sample at a wavelength of 415 nm. Calculations by making a standard curve and the regression equation formula:
 
Y=ax+b
 
b) Total phenol test (Harborne, 1996; Sugiani et al., 2023).
 
- Weigh 50 mg of test sample. Added 250 µl of Folin Ciocalteu reagent.  After 1 minute, 750 µl of 20% Na carbonate was added, distilled water was added to a volume of 10 ml     using a root flask.  Incubated at 25°C for 2 hours.  Read the absorbance with a 760 nm wavelength spectrophotometer. A standard solution of 16 mg, 32, 64 and 128 mg gallic  acid was prepared. Create a correlation regression curve between gallic acid and the sample.
 
c) Antioxidant activity test (Harborne, 1996)
 
- Starting with making DPPH mother liquor.  4 mg DPPH powder was dissolved in methanol p.a. to a volume of 1 ml (DPPH concentration 0.1 µM). 200 µl of the DPPH solution   was taken plus methanol p.a. up to the 200 ml mark.

- Preparation of blank DPPH solution (control).  2 ml of 0.1 µM DPPH solution was put into a reaction tube, plus methano p.a. as much as 2 ml.  The mixture was shaken vigorously until it became uniform. It was then placed in a dark room for 30 minutes and measured using light at a wavelength of 515 nm. Preparation of test solutions from sample extracts. The extract weighed 5 mg was dissolved in 50 ml methanol to obtain an extract solution of 100 ppm. Then pipetted the extract solution as much as 0.5 ml, 1 ml, 1.5 ml and 2 ml, added methanol; p.a. up to a limit of 5 ml.  Finally obtained extract concentrations of 10, 20, 30, 40 ppm.

- Measurement of the absorbance of the extract solution. Take 2 ml of the 10-40 ppm extract test solution and add 2 ml of 0.1 µM DPPH solution (so that a total volume of 4 ml  is obtained), vortex to make it homogeneous and store in the dark for 30 minutes. Next, the absorbance was measured at 515 nm.

- Determination of inhibition. Free radical scavenging activity is expressed as percent inhibition with the formula:
 
  
 
To determine the IC50 value, the sample concentration and percent inhibition are plotted on the X and Y axes respectively in the linear regression equation. This equation is used to calculate the IC50 of each sample expressed by a Y value of 50 and the X value obtained as IC50.  The IC50 criteria are:

IC50: <50 ppm Very strong antioxidant.
IC50: 50-100 ppm strong antioxidant.
IC50: 100-150 ppm moderate antioxidant.
IC50: 150-200 ppm weak antioxidant.
IC50: >200 ppm shows very weak antioxidant activity.

RESULTS AND DISCUSSION

Goat milk
 
Data was taken by testing the quality of crossbreed etawa goat milk using Lactoscan which was repeated 3 times. The results from testing the quality of crossbreed Etawa goat milk are shown in Table 1.

Table 1: Goat milk quality.


              
Indonesia already has a quality standard for cow’s milk, but there is no quality standard for goat’s milk. Therefore, the Thai Agriculture Standard (TAS) No. 6006-2008 can be used as standard for goat’s milk, as conducted by Ratya et al., (2017). Based on this TAS, the quality of goat’s milk used in this study has met the requirements, including protein content >3.7%, SNF >8.25%, fat content almost 4%, pH 6.5-6.8.
       
Based on the results of the crossbreed etawa goat milk quality test, it shows that the milk is in good condition and corresponds to the composition of goat’s milk which is usually used in yoghurt processing. This is supported by the statement Monteiro et al., (2019), goat’s milk used as raw material for yoghurt has a fat content of between 4.25-3.8%, protein 2.9-3.52%, lactose 4.08-4.27%, non-fat dry extract (SNF) 8.68-8.90% and pH 6.3 to 6.7.  It was also explained that yoghurt from goat’s milk has a weakness, namely that its structure is weaker than sheep’s or cow’s milk, because it contains fewer types of casein and many non-protein nitrogen ingredients are higher. The 3.93% lactose content in goat’s milk in the study was a substrate for LAB fermentation, which would convert lactose into lactic acid, decreasing the pH. Low pH causes protein agglutination, resulting in a thick and soft texture.  Fat will contribute to the texture and aroma of fermented yogurt.  Supported by the statement of Jankowska et al., (2026) the basic parameters of goat milk (pH, fat, protein) directly affect the fermentation process and texture of yogurt. Goat’s milk has a lower αs1-casein content than cow’s milk, but relatively higher β-casein. This causes goat milk protein to form less strong protein gels during lactic acid bacteria coagulation, making syneresis easier to occur. Fat contributes to the gel structure of yogurt because the fat globules support the protein network formed during coagulation.  Goat’s milk generally has smaller fat globules, making it easier to emulsify and interact in a gel system during fermentation. However, the resulting gel structure tends to be weaker and less viscous than cow’s milk yogurt due to the different fat and protein profiles, making syneresis easier to occur. During fermentation, because lactose changes into lactic acid, the pH will decrease.  This decreasing in pH results in protein denaturation, making the proteins susceptible to interacting with each other, thus forming a gel network. If the pH drops too quickly, the gel network will become brittle, making it susceptible to a soft and watery texture. Patel et al., (2017) also stated that goat’s milk yogurt has a slightly thinner consistency and texture due to its higher mineral content.
 
Lactic acid bacteria
 
The lactic acid bacteria commonly used in making yoghurt are Lactobacillus bulgaricus and Streptococcus thermophilus, but to further improve their functional properties, probiotic bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum and Bifidobacterium are added. Adriani et al., (2024) stated that yogurt with Bifidobacterium sp. and Lactobacillus acidophilus starters had higher lipase activity (0.45 units/ml) compared to Lactobacillus bulgaricus and Streptococcus thermophilus (0.18 units/ml). This proves that Bifidobacterium sp. and Lactobacillus acidophilus can increase the functional value of yogurt because they have health benefits. In this study, Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus acidophilus were used. Based on the results of microbiological tests by counting the number of bacterial colonies before use in making yoghurt, the number of colonies found in each BAL sample was 108 CFU per milliliter. This number is in accordance with the LAB number requirements for yoghurt processing, which are stipulated by Badan Standarisasi Nasional, (2009) on yogurt, namely the minimum requirement for yoghurt starter bacteria is 107 CFU/ml. According to Birollo et al., (2000) the minimum number of lactic acid bacteria required for yoghurt during shelf life is 5 x 108 CFU/ml (France and Spain), 106 CFU/ml (Switzerland and Italy), 107 CFU/ml (Japan and Argentina).  In this study, cell counts were obtained for Lactobacillus bulgaricus, Streptococcus thermophillus and Lactobacillus acidophillus, which means they meet the requirements for making yoghurt.
       
The number of LAB starter colonies each starting at 108 CFU/ml is in line with the research of Pratama  et al.  (2020) which used three types of starters (Lactobacillus fermentum, Streptococcus thermophillus and Pediococcus acidilactici) with each colony number of 108 CFU/ml. A study by Emmawati  et al. (2021) found that the first LAB count was 106 CFU/ml, but after yogurt fermentation, the LAB count increased to 108 CFU/ml. This indicates an increasing in the LAB count after yogurt fermentation.
 
Qualitative test results of gowok
 
The results of the qualitative gowok test can be seen in Table 2.

Table 2: Gowok (Syzygium polycephallum) qualitative test results.


       
The results of qualitative tests show that gowok has many phenolic compounds (phenols, alkaloids, tannins, saponins, flavonoids and terpenoids), this proves that gowok is a source of natural antioxidants derived from plants. This research is the same as the research results of Puspitasari and Sofandi (2020) which stated that gowok fruit contains flavonoid, alkaloid, tannin and terpenoid compounds. This is also in line with research by Lubis et al., (2024), which states that gowok fruit contains tannins and phenols. This study is more detailed because it differentiates the phenolic compound content in the Flesh of fruit, Rind and whole seedless fruit of gowok. In this study, the highest tannin and phenol content was found in the rind, while the saponin content was negative in the flesh of fruit, the same results as (Nurmalasari et al., 2016).  According to Wahid (2020), tannins are phenolic compounds found in many plants that can dissolve in water and organic solvents, have the ability to precipitate proteins and have the potential for treatment because they act as antioxidants, antibacterials and antifungals.
       
This research found that saponin compounds were not found in the flesh of the gowok fruit, but were present in the skin, the mixture was present due to the influence of the skin. The presence of saponins gives a bitter taste, can be used as an antifungal, anti-inflammatory, so it can be used to treat thrush, vaginal discharge, boils, diarrhea and dysentery (Nurkhasanah and Dhurhania, 2023). The presence of terpenoids, which are secondary metabolites from plants in gowok, means that gowok also has the potential to be a source of antioxidants, anti-inflammatory, anti-allergic, anti-cancer, so it also has the potential to be applied in the world of food, cosmetics, medicine and pharmacy (Câmaraet_al2024).  Based on these qualitative results, gowok has great potential to be utilized because it contains many bioactive compounds as a source of antioxidant activity. 

Quantitative test results of gowok
 
The results of the quantitative gowok test can be seen in Table 3.

Table 3: Gowok (Syzygium polycephallum) quantitative test results.


       
Based on the results of this quantitative test, it was found that the flesh of the gowok fruit had a strong activity of 76.42 ppm, the same results were obtained in the research of Nurmalasari et al., (2016) which stated that the antioxidant activity of gowok fruit was classified as strong at 60.187 (strong: 50-100 ppm). The results of the IC50 antioxidant activity in rind of gowok (Syzygium polycephalum) 12.95 ppm, which is classified as very strong, are in line with the research of Irnawati et al., (2017) who obtained the IC50 antioxidant activity in rind of ruhruhi fruit (Syzygium polycephalum Merr.), which is also classified as very strong at 7.92 mg/L. The high antioxidant activity in the rind is in line with the high total phenol content in that part. The highest total phenol content was in the gowok rind at 69.02 mg GAE/g or 6.902%, followed by the fruit flesh at 51.74 mg GAE/g or 5.174%. This result was greater than the research of Lubis et al., (2024) which obtained a total phenol content in the gowok fruit flesh of 8.3686 mg GAE/g or 0.8386%.  These results demonstrate that gowok fruit has the potential as a source of antioxidants capable of combating free radicals, due to its three mechanisms: 1) reducing the formation of Reactive Oxygen Species (ROS, 2) eliminating ROS and 3) regulating and protecting with antioxidants (Widiasriani et al., 2024). The results of the quantitative test of the highest total phenol content in this rind are in line with the high anthocyanin content in the rind of 48.59 mg/L. Anthocyanin according to Irnawati et al., (2017) is one of the polyphenols included in the flavonoid group, found in abundance in fruit, providing red, blue and purple pigments in fruit and flowers, so it has the potential as a natural dye and source of antioxidants. The anthocyanin content in the rind is higher than in the flesh of fruit, because the color pigment is more in the rind and the flesh of the gowok fruit is white. 
       
The results of quantitative analysis of bioactive compounds show that gowok (Syzygium polycephalum) has a high total phenol content with the dominant fraction being flavonoids, which as a source of natural antioxidants.  These characteristics make gowok have great potential to be used in the development of functional foods. The use of goat milk as a raw material for synbiotic yogurt offers advantages in terms of protein structure and digestibility, although it has several limitations related to sensory properties and product stability. The addition of gowok fruit peel as a by-product rich in phenolic compounds into the goat milk-based synbiotic yogurt formulation is a strategic approach in diversifying functional food products, while increasing added value and sustainable utilization of local resources.

CONCLUSION

This research resulted in the raw material of Etawah crossbred goat milk having good quality because it meets the standards for making yogurt. The lactic acid bacteria used are Streptococcus thermophilus, Lactobacillus bulgaricus and probiotic Lactobacillus acidophilus. The number of cells has met the minimum criteria for the number of lactic acid bacteria used in making yogurt, which is 108 CFU/ml.  Gowok (Syzygium polycephalum) is a fruit native to Indonesia, which contains active compounds (phenols, alkaloids, tannins, polyphenols, saponins, terpenoids, flavonoids and anthocyanins), making it a potential source of antioxidants. The rind of gowok fruit (Syzygium polycephallum) and whole seedless fruit have very strong antioxidant activity because they have IC50 = <50 ppm.

ACKNOWLEDGEMENT

The current study received support from the Directorate of Research and Community Service (DPPM) for the 2025 budget year.
 
Disclaimers
 
The authors confirm that this manuscript titled ”Study of Synbiotic Yogurt Raw Materials as Functional Food: Analysis of Goat Milk Quality, Lactic Acid Bacteria and Gowok (Syzygium polycephalum) Fruit Phytochemicals” is an original contribution and has not been published before,  nor is it currently being reviewed for publication elsewhere. Each author has  played a  vital role in the conception, design,  implementation and interpretation of the research study and consents to the  manuscript’s  content. The authors also mention that there is no conflict of interest related to the research, authorship, or publication of this article. The information provided in this research is  precise and gathered through legitimate and ethical investigative methods. All sources of information and references utilized in this manuscript have been  properly acknowledged. By submitting this article, the authors take complete responsibility for the authenticity and integrity of the research work provided.
 
Informed consent
 
This study did not include any people or animals.

CONFLICT OF INTEREST

The authors say there are no conflicts of interest when it comes to publishing this article. No money or support affected how the study was planned, the data was gathered, the results were analyzed, the decision to publish was made, or the manuscript was written.

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    Full Research Article

    Study of Synbiotic Yogurt Raw Materials as Functional Food: Analysis of Goat Milk Quality, Lactic Acid Bacteria and Gowok (Syzygium polycephalum) Fruit Phytochemicals

    S
    Siti Rahmawati Zulaikhah1,2
    T
    Triana Setyawardani2,*
    https://orcid.org/0009-0001-9488-9749
    J
    Juni Sumarmono2
    1Department of Animal Husbandry, Faculty of Sains and Technology, Nahdlatul Ulama Purwokerto University. Sultan Agung Street No 42, Karangklesem, Purwokerto, Central Java, 53145, Indonesia.
    2Faculty of Animal Science, Jenderal Soedirman University, Dr. Soeparno Street No. 60, Karangwangkal, Purwokerto, Central Java, 53122, Indonesia.

    ABSTRACT

    Background: The increase in cases of degenerative diseases has made people increasingly aware of the need to consume functional foods. Yogurt is an alternative source of functional food. Innovation in developing synbiotic yogurt as a quality functional food through the addition of gowok (Syzygium polycephalum), which is the local wisdom of Indonesia. Synbiotic yogurt will be made using goat milk which has several advantages compared to cow milk, including being non-allergenic because it has high digestibility.  The using of probiotic bacteria is also an effort to improve the functional properties of this yogurt, by adding Lactobacillus acidophilus bacteria. The using of gowok (Syzygium polycephalum) prebiotics by adding each part of the gowok (rind, flesh of fruit and whole seedless fruit) during the yogurt making process. This research is an initial study in preparing raw materials for making synbiotic yogurt. This study is meant to find out how good the raw materials for making synbiotic yogurt, which include: goat’s milk, lactic acid bacteria and gowok (Syzygium polycephalum).

    Methods: This research began with testing the quality of goat milk using lactoscan, the quality of lactic acid bacteria using TPC (Total Plate Count) calculations, qualitative and quantitative testing of gowok (Syzygium polycephalum), which was previously extracted using the maceration method.

    Result: The results of the study showed that the quality of goat milk to be used in making synbiotic yogurt had the criteria for good milk quality. The number of lactic acid bacteria colonies (Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus acidophilus) was 108CFU/ml, which met the minimum requirements for making yogurt, namely a minimum of 107CFU/ml. Gowok, also known as Syzygium polycephalum, has been shown to have active compounds like flavonoids, phenols, anthocyanins, terpenoids, tannins and alkaloids. These compounds can be used as a source of antioxidants and help increase the antioxidant activity in synbiotic yogurt so that it is hoped that it can produce yogurt with high functional properties.

    KEYWORDS

    INTRODUCTION

    Functional foods have become a public concern recently, because they are believed to have health benefits.  Degenerative diseases such as coronary heart disease, diabetes, osteoporosis, cancer and others are increasingly being suffered by the public. This is a concern that needs to be anticipated, one way is by consuming functional foods that contain lots of antioxidants so that they are beneficial for health. Chandimali et al., (2024) stated that functional foods are foods or ingredients that have health functions beyond their basic nutritional value, for example bioactive components. One of the functional foods that is now widely known to the public is yogurt.
           
    Yogurt is generally made from cow’s milk. This study used goat’s milk because of its many benefits, including a lack of lactose intolerance, improved digestibility, probiotic and prebiotic content that can maintain healthy gut microflora and protection against the negative effects of pathogenic bacterial infections and allergies (ALKaisy et al., 2023). Hammam et al., (2022) stated that goat milk and its products have health benefits including being anticarcinogenic, antidiabetic and antihypertensive.  Yogurt production requires the work of lactic acid bacteria (LAB), commonly used are Streptococcus thermophillus and Lactobacillus delbrueckii subsp. bulgaricus in order to increase its functional value, probiotics are used in the fermentation process, including Lactobacillus acidophillus. According to Hendarto  et al. (2019), with the presence of probiotics, yogurt can produce antioxidant activity derived from the work of these probiotics in the form of bioactive peptide compounds, vitamins and metabolites resulting from the fermentation process.
           
    Efforts to improve the functional properties of yogurt by adding external ingredients containing natural antioxidants.  One of the ingredients added is gowok (Syzygium polycephalum), which ultimately makes the yogurt known as synbiotic yogurt.  Gowok (Syzygium polycephalum) is a local Indonesian potential that can be found once a year, because it is a seasonal fruit (Picture 1). Gowok contains flavonoids, alkaloids, tannins, terpenoids and saponins which have health benefits (Puspitasari and Sofandi, 2020).   This is reinforced by the statement Lubis et al., (2024) that gowok fruit flesh contains flavonoids, terpenoids, tannins and phenols, the IC50 value of ethanol extract of gowok fruit flesh is 58.082 ppm and the phenol content is 8.3686 mg GAE/g or 0.8386%. This research was conducted to explore gowok’s potential as an antioxidant and source of natural fiber while preserving local wisdom.

    Picture 1: Gowok (Syzygium polycephalum).


           
    This research aims to examine the raw materials for yogurt in the form of goat’s milk, lactic acid bacteria (LAB) and gowok (Syzygium polycephalum) which will be used in making yogurt. Knowledge of the quality of raw materials will be the capital in making the resulting yogurt.  Research on synbiotic yogurt by adding gowok (Syzygium polycephalum) with goat milk as the raw material and adding the probiotic bacteria Lactobacillus acidophillus is a novelty in itself, because there has been no previous research.

    MATERIALS AND METHODS

    The research materials consist of goat milk, lactic acid bacteria and gowok (Syzygium polycephalum).
     
    Goat milk
     
    The goat’s milk used in this study was Etawah crossbreed goat milk obtained from local breeders in the Lumbir area, Banyumas, Central Java, Indonesia. The quality testing of this goat milk uses a Lactoscan tool. The method used is to put a sample of approximately 50 ml into the Lactoscan device, then set it for quality testing and the test results can be read immediately. The quality of goat milk is based on Thai Agriculture Standard (TAS) No. 6006-2008 (Ratya et al., 2017).
     
    Lactic acid bacteria (LAB)
     
    Lactic acid bacteria isolates Streptococcus thermophilus and Lactobacillus bulgaricus were obtained from the Inter-University Center (PAU) of Gadjah Mada University, with strains FNCC 0040 and FNCC 0041. The probiotic Lactobacillus acidophilus ATCC 4356 was obtained from Bogor Agricultural Institute. The number of lactic acid bacteria was calculated for each isolate. This isolate was grown in MRSB media, by preparing 10 ml of sterilized MRSB solution in a test tube, then 1 loop of the isolate was taken, then incubated at 40°C for 24-48 hours. BAL calculation uses TPC (Total Plate Count), which is previously diluted using sterilized distilled water and NaCl of 0.85gr/L. Each tube was filled with 9 ml of dilution solution, made for 101 to 107, as much as 1 ml of isolate from MRSB was put into the dilution solution to produce a dilution of 101, from 101 1ml was taken then put into the next tube to get a dilution of 102, from 102 1 ml was taken to get a dilution of 103 and so on. Next, prepare a growing medium from MRSA and place it in a petri dish. Then, 1 ml of each dilution, from 104 to 107, is taken and planted in the petri dish containing the MRSA. The petri dish is inverted and incubated at 40°C for 24 hours. Colonies are counted to determine the number of bacterial cells in CFU/ml.  Calculation of the number of lactic acid bacteria Steptococcus thermophillus, Lactobacillus bulgaricus and Lactobacillus acidophillus separately, as a starter (before inoculation) with each amount to 108 CFU/ml.
     
    Gowok (Syzygium polycephalum)
     
    Gowok (Syzygium polycephalum) was obtained from a fruit trader in Ajibarang, Banyumas, Central Java, Indonesia.  Gowok is prepared by separating the fruit: rind, flesh of fruit and a mixture of the rind and flesh of fruit, then dried at a temperature of 60°C using a Food Dehydrator until dry and broken. Next, grind it using a food grinder, so that you get flour from each part.
     
    Qualitative test of gowok (Syzygium polycephalum)
     
    Before testing, gowok flour was extracted using maceration referring to (Harborne, 1996). The samples were ground and 10 grams were taken and soaked in 96% ethanol at a height of 2.5 cm for 24 hours. The sample is filtered, then the ethanol is evaporated to obtain an extract.
     
    Qualitative testing includes
     
    a) Fenol hidrokuinon test (Harborne, 1996).
     
    - Take 0.5 g of the extract sample and add 10 ml of homogenized 70% ethanol. 1 ml of the solution was taken and added with 2 drops of 5% FeCl3 solution. Observe the changes that occur, a positive reaction occurs if the color changes to green or green-blue.
     
    b) Alkaloid test (Harborne, 1996).
     
    -  100 µl of extract added with 50 µl of H2SO4 then vortexed. Add 100 µl of Dragendorf’s solution, then vortex and observe the color change after 30 minutes.
     
    c) Tanin and Polifenol Test (Harborne, 1996).
     
    - The extract is homogenized with water and boiled for several minutes, filtered. The filtrate was dropped with FeCl3. The blue to black color indicates the presence of tannin and/or polyphenol compounds. Uji Saponin (Harborne, 1996).
    -  2 ml of extract plus 2 ml of hot distilled water, then shake  vigorously, observe whether there is foam or not.  If there is foam, add 2N HCl if the foam remains strong for 10 minutes, then it is positive for saponin.
     
    d) Flavonoid test (Harborne, 1996)
     
    - 100 µl of extract plus 50 µl of Zn or Mg plus 100 µl of 2N HCl solution, heated for 5-10 minutes. The filtrate was filtered and 50 µl of amyl alcohol was added. Observe the color change (yellow +, red ++, orange +++). Uji Terpenoid (Harborne, 1996).
    - 1 gram of extract plus 1 ml of ethanol is boiled briefly, then filtered. Take 0.5 ml, add 0.2 ml diethylether and 0.3 ml sulfuric acid. If the color is reddish brown, it means positive.
     
    Quantitative test of gowok (Syzygium polycephalum)
     
    a) Flavonoid test (Harborne, 1996; Aminah et al., 2017).
     
    - Create a standard curve. 2.5 ml quercetin plus 96% ethanol to a volume of 25 ml. Wuersetin levels 100 mg/l (100 ppm).  Make a standard solution of quercetin 20 ppm, 40 ppm, 40 ppm, 80 ppm. Take 500 µl each plus 2.5 ml distilled water.  After that, 1 ml was vortexed, plus 100 µl of 10 % AlCl3 and left for 6 minutes.  Add 100 µl of potassium acetate or 5 % NaNO2, incubate for 5 minutes in the dark room.

    - The extract sample was weighed at 2.5 mg, dissolved in 2.5 ml of distilled water and 500 µl was taken, added to 2 ml of distilled water, then vortexed. Take 1 ml of the solution plus 100 µl of 10% AlCl3 and let it sit for 6 minutes. Add 100 µl of potassium acetate or 5% NaNO2, incubate for 5 minutes in a dark place. Read the absorption of the standard and the sample at a wavelength of 415 nm. Calculations by making a standard curve and the regression equation formula:
     
    Y=ax+b
     
    b) Total phenol test (Harborne, 1996; Sugiani et al., 2023).
     
    - Weigh 50 mg of test sample. Added 250 µl of Folin Ciocalteu reagent.  After 1 minute, 750 µl of 20% Na carbonate was added, distilled water was added to a volume of 10 ml     using a root flask.  Incubated at 25°C for 2 hours.  Read the absorbance with a 760 nm wavelength spectrophotometer. A standard solution of 16 mg, 32, 64 and 128 mg gallic  acid was prepared. Create a correlation regression curve between gallic acid and the sample.
     
    c) Antioxidant activity test (Harborne, 1996)
     
    - Starting with making DPPH mother liquor.  4 mg DPPH powder was dissolved in methanol p.a. to a volume of 1 ml (DPPH concentration 0.1 µM). 200 µl of the DPPH solution   was taken plus methanol p.a. up to the 200 ml mark.

    - Preparation of blank DPPH solution (control).  2 ml of 0.1 µM DPPH solution was put into a reaction tube, plus methano p.a. as much as 2 ml.  The mixture was shaken vigorously until it became uniform. It was then placed in a dark room for 30 minutes and measured using light at a wavelength of 515 nm. Preparation of test solutions from sample extracts. The extract weighed 5 mg was dissolved in 50 ml methanol to obtain an extract solution of 100 ppm. Then pipetted the extract solution as much as 0.5 ml, 1 ml, 1.5 ml and 2 ml, added methanol; p.a. up to a limit of 5 ml.  Finally obtained extract concentrations of 10, 20, 30, 40 ppm.

    - Measurement of the absorbance of the extract solution. Take 2 ml of the 10-40 ppm extract test solution and add 2 ml of 0.1 µM DPPH solution (so that a total volume of 4 ml  is obtained), vortex to make it homogeneous and store in the dark for 30 minutes. Next, the absorbance was measured at 515 nm.

    - Determination of inhibition. Free radical scavenging activity is expressed as percent inhibition with the formula:
     
      
     
    To determine the IC50 value, the sample concentration and percent inhibition are plotted on the X and Y axes respectively in the linear regression equation. This equation is used to calculate the IC50 of each sample expressed by a Y value of 50 and the X value obtained as IC50.  The IC50 criteria are:

    IC50: <50 ppm Very strong antioxidant.
    IC50: 50-100 ppm strong antioxidant.
    IC50: 100-150 ppm moderate antioxidant.
    IC50: 150-200 ppm weak antioxidant.
    IC50: >200 ppm shows very weak antioxidant activity.

    RESULTS AND DISCUSSION

    Goat milk
     
    Data was taken by testing the quality of crossbreed etawa goat milk using Lactoscan which was repeated 3 times. The results from testing the quality of crossbreed Etawa goat milk are shown in Table 1.

    Table 1: Goat milk quality.


                  
    Indonesia already has a quality standard for cow’s milk, but there is no quality standard for goat’s milk. Therefore, the Thai Agriculture Standard (TAS) No. 6006-2008 can be used as standard for goat’s milk, as conducted by Ratya et al., (2017). Based on this TAS, the quality of goat’s milk used in this study has met the requirements, including protein content >3.7%, SNF >8.25%, fat content almost 4%, pH 6.5-6.8.
           
    Based on the results of the crossbreed etawa goat milk quality test, it shows that the milk is in good condition and corresponds to the composition of goat’s milk which is usually used in yoghurt processing. This is supported by the statement Monteiro et al., (2019), goat’s milk used as raw material for yoghurt has a fat content of between 4.25-3.8%, protein 2.9-3.52%, lactose 4.08-4.27%, non-fat dry extract (SNF) 8.68-8.90% and pH 6.3 to 6.7.  It was also explained that yoghurt from goat’s milk has a weakness, namely that its structure is weaker than sheep’s or cow’s milk, because it contains fewer types of casein and many non-protein nitrogen ingredients are higher. The 3.93% lactose content in goat’s milk in the study was a substrate for LAB fermentation, which would convert lactose into lactic acid, decreasing the pH. Low pH causes protein agglutination, resulting in a thick and soft texture.  Fat will contribute to the texture and aroma of fermented yogurt.  Supported by the statement of Jankowska et al., (2026) the basic parameters of goat milk (pH, fat, protein) directly affect the fermentation process and texture of yogurt. Goat’s milk has a lower αs1-casein content than cow’s milk, but relatively higher β-casein. This causes goat milk protein to form less strong protein gels during lactic acid bacteria coagulation, making syneresis easier to occur. Fat contributes to the gel structure of yogurt because the fat globules support the protein network formed during coagulation.  Goat’s milk generally has smaller fat globules, making it easier to emulsify and interact in a gel system during fermentation. However, the resulting gel structure tends to be weaker and less viscous than cow’s milk yogurt due to the different fat and protein profiles, making syneresis easier to occur. During fermentation, because lactose changes into lactic acid, the pH will decrease.  This decreasing in pH results in protein denaturation, making the proteins susceptible to interacting with each other, thus forming a gel network. If the pH drops too quickly, the gel network will become brittle, making it susceptible to a soft and watery texture. Patel et al., (2017) also stated that goat’s milk yogurt has a slightly thinner consistency and texture due to its higher mineral content.
     
    Lactic acid bacteria
     
    The lactic acid bacteria commonly used in making yoghurt are Lactobacillus bulgaricus and Streptococcus thermophilus, but to further improve their functional properties, probiotic bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum and Bifidobacterium are added. Adriani et al., (2024) stated that yogurt with Bifidobacterium sp. and Lactobacillus acidophilus starters had higher lipase activity (0.45 units/ml) compared to Lactobacillus bulgaricus and Streptococcus thermophilus (0.18 units/ml). This proves that Bifidobacterium sp. and Lactobacillus acidophilus can increase the functional value of yogurt because they have health benefits. In this study, Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus acidophilus were used. Based on the results of microbiological tests by counting the number of bacterial colonies before use in making yoghurt, the number of colonies found in each BAL sample was 108 CFU per milliliter. This number is in accordance with the LAB number requirements for yoghurt processing, which are stipulated by Badan Standarisasi Nasional, (2009) on yogurt, namely the minimum requirement for yoghurt starter bacteria is 107 CFU/ml. According to Birollo et al., (2000) the minimum number of lactic acid bacteria required for yoghurt during shelf life is 5 x 108 CFU/ml (France and Spain), 106 CFU/ml (Switzerland and Italy), 107 CFU/ml (Japan and Argentina).  In this study, cell counts were obtained for Lactobacillus bulgaricus, Streptococcus thermophillus and Lactobacillus acidophillus, which means they meet the requirements for making yoghurt.
           
    The number of LAB starter colonies each starting at 108 CFU/ml is in line with the research of Pratama  et al.  (2020) which used three types of starters (Lactobacillus fermentum, Streptococcus thermophillus and Pediococcus acidilactici) with each colony number of 108 CFU/ml. A study by Emmawati  et al. (2021) found that the first LAB count was 106 CFU/ml, but after yogurt fermentation, the LAB count increased to 108 CFU/ml. This indicates an increasing in the LAB count after yogurt fermentation.
     
    Qualitative test results of gowok
     
    The results of the qualitative gowok test can be seen in Table 2.

    Table 2: Gowok (Syzygium polycephallum) qualitative test results.


           
    The results of qualitative tests show that gowok has many phenolic compounds (phenols, alkaloids, tannins, saponins, flavonoids and terpenoids), this proves that gowok is a source of natural antioxidants derived from plants. This research is the same as the research results of Puspitasari and Sofandi (2020) which stated that gowok fruit contains flavonoid, alkaloid, tannin and terpenoid compounds. This is also in line with research by Lubis et al., (2024), which states that gowok fruit contains tannins and phenols. This study is more detailed because it differentiates the phenolic compound content in the Flesh of fruit, Rind and whole seedless fruit of gowok. In this study, the highest tannin and phenol content was found in the rind, while the saponin content was negative in the flesh of fruit, the same results as (Nurmalasari et al., 2016).  According to Wahid (2020), tannins are phenolic compounds found in many plants that can dissolve in water and organic solvents, have the ability to precipitate proteins and have the potential for treatment because they act as antioxidants, antibacterials and antifungals.
           
    This research found that saponin compounds were not found in the flesh of the gowok fruit, but were present in the skin, the mixture was present due to the influence of the skin. The presence of saponins gives a bitter taste, can be used as an antifungal, anti-inflammatory, so it can be used to treat thrush, vaginal discharge, boils, diarrhea and dysentery (Nurkhasanah and Dhurhania, 2023). The presence of terpenoids, which are secondary metabolites from plants in gowok, means that gowok also has the potential to be a source of antioxidants, anti-inflammatory, anti-allergic, anti-cancer, so it also has the potential to be applied in the world of food, cosmetics, medicine and pharmacy (Câmaraet_al2024).  Based on these qualitative results, gowok has great potential to be utilized because it contains many bioactive compounds as a source of antioxidant activity. 

    Quantitative test results of gowok
     
    The results of the quantitative gowok test can be seen in Table 3.

    Table 3: Gowok (Syzygium polycephallum) quantitative test results.


           
    Based on the results of this quantitative test, it was found that the flesh of the gowok fruit had a strong activity of 76.42 ppm, the same results were obtained in the research of Nurmalasari et al., (2016) which stated that the antioxidant activity of gowok fruit was classified as strong at 60.187 (strong: 50-100 ppm). The results of the IC50 antioxidant activity in rind of gowok (Syzygium polycephalum) 12.95 ppm, which is classified as very strong, are in line with the research of Irnawati et al., (2017) who obtained the IC50 antioxidant activity in rind of ruhruhi fruit (Syzygium polycephalum Merr.), which is also classified as very strong at 7.92 mg/L. The high antioxidant activity in the rind is in line with the high total phenol content in that part. The highest total phenol content was in the gowok rind at 69.02 mg GAE/g or 6.902%, followed by the fruit flesh at 51.74 mg GAE/g or 5.174%. This result was greater than the research of Lubis et al., (2024) which obtained a total phenol content in the gowok fruit flesh of 8.3686 mg GAE/g or 0.8386%.  These results demonstrate that gowok fruit has the potential as a source of antioxidants capable of combating free radicals, due to its three mechanisms: 1) reducing the formation of Reactive Oxygen Species (ROS, 2) eliminating ROS and 3) regulating and protecting with antioxidants (Widiasriani et al., 2024). The results of the quantitative test of the highest total phenol content in this rind are in line with the high anthocyanin content in the rind of 48.59 mg/L. Anthocyanin according to Irnawati et al., (2017) is one of the polyphenols included in the flavonoid group, found in abundance in fruit, providing red, blue and purple pigments in fruit and flowers, so it has the potential as a natural dye and source of antioxidants. The anthocyanin content in the rind is higher than in the flesh of fruit, because the color pigment is more in the rind and the flesh of the gowok fruit is white. 
           
    The results of quantitative analysis of bioactive compounds show that gowok (Syzygium polycephalum) has a high total phenol content with the dominant fraction being flavonoids, which as a source of natural antioxidants.  These characteristics make gowok have great potential to be used in the development of functional foods. The use of goat milk as a raw material for synbiotic yogurt offers advantages in terms of protein structure and digestibility, although it has several limitations related to sensory properties and product stability. The addition of gowok fruit peel as a by-product rich in phenolic compounds into the goat milk-based synbiotic yogurt formulation is a strategic approach in diversifying functional food products, while increasing added value and sustainable utilization of local resources.

    CONCLUSION

    This research resulted in the raw material of Etawah crossbred goat milk having good quality because it meets the standards for making yogurt. The lactic acid bacteria used are Streptococcus thermophilus, Lactobacillus bulgaricus and probiotic Lactobacillus acidophilus. The number of cells has met the minimum criteria for the number of lactic acid bacteria used in making yogurt, which is 108 CFU/ml.  Gowok (Syzygium polycephalum) is a fruit native to Indonesia, which contains active compounds (phenols, alkaloids, tannins, polyphenols, saponins, terpenoids, flavonoids and anthocyanins), making it a potential source of antioxidants. The rind of gowok fruit (Syzygium polycephallum) and whole seedless fruit have very strong antioxidant activity because they have IC50 = <50 ppm.

    ACKNOWLEDGEMENT

    The current study received support from the Directorate of Research and Community Service (DPPM) for the 2025 budget year.
     
    Disclaimers
     
    The authors confirm that this manuscript titled ”Study of Synbiotic Yogurt Raw Materials as Functional Food: Analysis of Goat Milk Quality, Lactic Acid Bacteria and Gowok (Syzygium polycephalum) Fruit Phytochemicals” is an original contribution and has not been published before,  nor is it currently being reviewed for publication elsewhere. Each author has  played a  vital role in the conception, design,  implementation and interpretation of the research study and consents to the  manuscript’s  content. The authors also mention that there is no conflict of interest related to the research, authorship, or publication of this article. The information provided in this research is  precise and gathered through legitimate and ethical investigative methods. All sources of information and references utilized in this manuscript have been  properly acknowledged. By submitting this article, the authors take complete responsibility for the authenticity and integrity of the research work provided.
     
    Informed consent
     
    This study did not include any people or animals.

    CONFLICT OF INTEREST

    The authors say there are no conflicts of interest when it comes to publishing this article. No money or support affected how the study was planned, the data was gathered, the results were analyzed, the decision to publish was made, or the manuscript was written.

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