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Full Research Article

Isolation and Identification of Saccharomyces Cerevisiae from Coconut Sap

G
Gunawan Wijonarko1
R
Rifda Naufalin1,*
I
Ike Sitoresmi Mulyo Purbowati1
K
Karseno1
1Department of Food Technology, Faculty of Agriculture, Jenderal Soedirman University, Grendeng, Purwokerto Utara, Banyumas, Jawa Tengah-53122, Indonesia.

ABSTRACT

Background: Coconut sap is a natural substrate with high sucrose content that supports the growth of various microorganisms, particularly yeasts. Saccharomyces cerevisiae is widely used in food fermentation and biotechnology; however, information regarding its presence and characteristics in coconut sap is still limited. This study aimed to determine the microbial profile of coconut sap and to isolate and identify Saccharomyces cerevisiae from coconut sap collected in Banyumas Regency, Indonesia.

Methods: Coconut sap samples were collected from traditional brown sugar producers and analyzed for total microbial, yeast and bacterial counts using plate count methods. Isolation of S. cerevisiae was conducted using the streak plate technique on potato dextrose agar supplemented with chloramphenicol and gentamicin. Morphological identification was performed using light microscopy based on cell shape and colony characteristics. Molecular identification was carried out by amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers. Phylogenetic analysis was constructed using the neighbor-joining method with MEGA 11 software.

Result: The native coconut sap showed higher total microbial and yeast counts compared to sulfite-treated and organic coconut sap. Five yeast isolates (GNS1, GNS3, GNS4, GNS9 and GNS14) exhibited morphological characteristics consistent with Saccharomyces species, including oval to round cell shapes. ITS rRNA gene sequencing confirmed that all isolates belonged to Saccharomyces cerevisiae. Phylogenetic analysis revealed that four isolates (GNS1, GNS3, GNS4 and GNS14) were closely related, while GNS9 represented a different strain within the same species.

INTRODUCTION

The coconut sap is a thick liquid taken from coconut tree flowers that has high sucrose content. Due to the high sucrose content, coconut sap contains an abundant quantity of microbes, especially Saccharomyces cerevisiae. S. cerevisiae has been studied intensively due to its significant role in human life. In food sectors, S. cerevisiae has been utilized for production of wine (Ganucci et al., 2018), beer (Kumari et al., 2019), bread (Olowonibi, 2017), fermented cereal and milk products (Motey et al., 2020), coconut toddy (Ahangangoda et al., 2019) and probiotic (Li et al., 2020). Meanwhile, the utilization of S. cerevisiae in the non-food sector has been widely studied, especially in bioethanol production. Previous studies reported that coconut inflorescence sap contains diverse fermentative microorganisms that contribute to spontaneous fermentation processes and microbial diversity in the sap (Mariyam et al., 2026).
       
S. cerevisiae are widely distributed in nature, found in organic and anorganic habitats which contain lots of carbohydrate. S. cerevisae can be found in organic habitat such as dates and molasses (Kechkar et al., 2019), wine (Ganucci et al., 2018), pineapple and oranges (Nasir et al., 2017), orange peels and spoilt tomato fruits (Milala et al., 2018), indigenous fermented foods (Menezes et al., 2020), kefir (Goktas et al., 2021) and nectarine (Noriko et al., 2019). S. cerevisiae can also be found in anorganic habitats such as in soil (Karki et al., 2017). In Indonesia, S. cerevisae can be found in Arenga pinnata sap (Periadnadi et al., 2018).
       
Based on several studies related to the use of S. cerevisiae in the food industry, there is no information about isolation of S. cerevisiae from coconut sap. In wine production, S. cerevisae is takenfrom grape (Senses-Ergul and Ozbas, 2016) and black raspberry extracts (Song et al., 2019). In beerindustry, S. cerevisiae has been successfully isolated from various habitats such as grapes, bread andstill apples (Rossi et al., 2018) cultures. The use of S. cerevisiae for probiotics has also been reportedby Motey et al. (2020) in cereals and fermented milk products and by Banik et al. (2019) in haria, churpi and khambir.
       
S. cerevisiae can be isolated from its habitat using various methods. Methods that have been used to isolate S. cerevisiae are spread plate methods (Song et al., 2019), serial dilution (Kechkar et al., 2019), streak plate (Ahangangoda et al., 2019) and pour plate (Guimarães et al., 2006). All S. cerevisiae isolation methods used were proven to have satisfactory results. Several media thathave been used for the isolation of S. cerevisiae are YPG (Guimarães et al., 2006), MEM (Suranská et al., 2016), YMA (Kechkar et al., 2019), YPDA (Capece et al., 2016) and MEA (Pennacchia et al., 2008). Meanwhile, Olowonibi (2017) found that S. cerevisae could also be isolated using streak plate on PDA containing 0.05 mg/mL chloramphenicol and gentamicin.
       
Based on the background, this study was conducted with the aims to describe the microbial profile of coconut sap, then to isolate and to identify S. cerevisiae from coconut sap.

MATERIALS AND METHODS

The experimental work was conducted at The Laboratory of Food Microbiology, Faculty of Agriculture, Universitas Jenderal Soedirman, Purwokerto, Indonesia. Molecular analyses, including PCR amplification and sequencing, were performed using integrated molecular biology laboratory facilities. The research was carried out in 2023.
 
Sampling of coconut sap
 
Samples of coconut sap were collected from the traditional brown sugar farmer in Banyumas Regency, Indonesia. Coconut sap (500 mL) were collected using a simple random sampling technique. Samples of coconut sap were taken 30-60 mins after tapping using sterile plastic bottles and put into an ice box to prevent damage.
 
Total microbial and yeast count
 
A total of 50 mL of coconut sap sample was diluted using 450 mL of sterile 0.85% NaCl physiological solution. The total microbial count was performed by making series of dilutions in 9 mL of sterile 0.85% NaCl solution. One milliliter of each dilution series was taken to be grown on YMA medium. Incubation was performed at 37°C for 36 h. For total yeast count, 1 mL of each dilution series was grown on PDA medium containing 0.05 mg/mL chloramphenicol and gentamicin and was incubated at 37°C for 36 h (Feldsine et al., 2003).
 
Isolation of S. cerevisiae
 
Isolation of S. cerevisiae from coconut sap was performed using the streak plate method on PDA media containing 0.05 mg/mL chloramphenicol and gentamicin (Olowonibi, 2017). Each sample of coconut sap was previously shaken to make it homogeneous. Samples of coconut sap were taken using a loop needle and streaked 3 times on the media.
 
Morphological identification
 
Morphological characterization based on colony appearance and cell morphology remains an important preliminary approach for microbial identification (Bhutani et al., 2018). The morphological observations of the isolates were performed using light microscope at magnification of 400 ×. Using a needle, one of isolate was taken and then smeared on a glass object that had been previously dripped with polyphenol blue. Morphological identification of S. cerevisiae referred to “The Yeast: A taxonomic study” (Kreger-van, 1984).
 
Molecular identification
 
Genomic DNA was isolated from single colonies using Quick-DNA Fungal / Bacterial Miniprep Kit (Zymo Research, D6005, USA) according to the manufacturer’s protocol. Molecular identification of the yeast isolates was achieved with amplification of the internal transcribed spacer region with primers pairs ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) (White et al., 1989). PCR amplification was done with (2×) MyTaq HS Red Mix (Bioline, BIO-25048) and the expected PCR products’ size was 600-700 bp. The PCR products were checked on agarose gel electrophoresis and sent for sequencing. In order to construct a phylogenetic tree sequences with the highest similarity to the query, together with some sequences retrieved from the GenBank database were aligned and constructed from evolutionary distances by the neighbor-joining method using the software MEGA 11 (Tamura et al., 2021).
 
Statistic analysis
 
Data were analyzed through independent t-test and one-way ANOVA using SPSS version 20 software.

RESULTS AND DISCUSSION

Microbial profile of coconut sap
 
The native coconut sap had a higher total microbes than sulfite coconut sap and organic coconut sap (Table 1). Total microbes in sulfite coconut sap and organic coconut sap were 6.24 and 6.57 log CFU/mL, respectively. Such finding indicated that sulfite and organic solvents were equally effective in killing microbes. Sulfite had a low antimicrobial effect by 0.02% but was able to inhibit microbial growth in tempeh. Fatisa (2013) reported that minimum inhibitory concentration (MIC) of pulasan skin (Nephelium mutabile) against Staphylococcus aureus and Escherichia coli were 0.76 mg/mL and 0.816, respectively.

Table 1: Microbial profile of fresh coconut sap.


       
The total yeast in the three types of sap revealed that the native sap (5.54 log CFU/mL) had a higher total yeast compared to the total yeast in the sulfite sap (4.39 log CFU/mL) and organic sap (4.63 log CFU/mL). Such finding indicated that both sulfite and organic laru were effective in reducing the amount of yeast in coconut sap. Mangosteen rind and jackfruit wood contained in organic solvents have antimicrobial properties that can inhibit microbial growth (Bouarab-Chibane et al., 2019). On the other hand, Irwin et al. (2017) found that the test bacteria experienced significant growth at sulfite concentrations between 250-500 ppm. The total bacteria in the sulfite sap (1.85 log CFU/mL) were lower than the total bacteria in the native sap (2.02 log CFU/mL) but there was no difference with the organic sap (1.94 log CFU/mL). Such finding indicated that sulfite was slightly more effective than organic laru in reducing the number of bacteria in coconut sap. The effectiveness of sulfite compounds were 1.4 times higher than H2O2 compounds and natural preservatives (Irwin et al., 2017).
 
Isolation
 
The results of S. cerevisiae isolation from 15 samples of coconut sap using streak plate method are presented in Fig 1. The appearance of the white colonies showed the same characteristic as the standard colonies. Therefore, samples with the same appearance were further analyzed morphologically using a light microscope. The appearance of the sample isolates was similar to the standard isolate under a light microscope at 400× magnification. It can be observed that the cells were oval, round or rod in shape (data not shown).

Fig 1: Isolation of S. cerevisiae.


 
Morphological characteristics
 
Morphological characteristics were observed after isolation of S. cerevisiae from 15 samples of coconut sap from afternoon tapping. It can be observed under a microscope that the cells were oval, round or rod in shape. However, oval cells were mostly found. There were 9 isolates which were oval in shape. Cell size varied in length between 12.5-17.8 µm and in width between 0.8-4.6 µm. The dimension of GNS9 cell was 15.2 µm × 4.5 µm (data not shown). The color of the cells was between white and cream and the cell surface was dull and clear. Kreger-van (1984) reported that the genus Saccharomyces is characterized by round, elliptical, or cylindrical cell shapes, white colonies, dull surfaces and raised elevations. Genus Saccharomyces reproduces through multilateral budding, with the larger size of the stem cells compared to the tillers.
 
Molecular characteristics
 
Based on conoly appearance and microscopic observations, five isolates coded as GNS1, GNS3, GNS4, GNS9 and GNS14 suspected to belong to the genus Saccharomyces. To confirm the preliminary identification results, molecular characterization was carried out through amplification and sequencing of the ITS region. PCR amplification using ITS1 and ITS4 primers was successfully performed for all isolates, producing DNA bands of approximately 600-700 bp, as presented in Fig 2. The obtained sequences were analyzed using BLAST against sequences available in the NCBI GenBank database. The comparison results showed that all isolates had high similarity with Saccharomyces cerevisiae sequences, confirming their species identity (Table 2). Furthermore, phylogenetic analysis based on ITS sequences was constructed using the neighbor-joining method to evaluate the genetic relationship among the isolates. The phylogenetic tree in Fig 3 demonstrated that isolates GNS1, GNS3, GNS4 and GNS14 were grouped within the same cluster, indicating close genetic relatedness (Fig 3). Meanwhile, isolate GNS9 formed a different branch, suggesting that this isolate may represent a different strain of S. cerevisiae. Molecular techniques provide more precise identification compared to conventional morphological observations alone (Waseem et al., 2020). In this study, sequencing analysis confirmed that all isolates belonged to Saccharomyces cerevisiae.These results confirmed that coconut sap collected from banyumas regency contains genetically diverse S. cerevisiae strains with potential applications in fermentation and biotechnology.

Table 2: Top 10 hit blast result toward NCBI database.



Fig 2: PCR product of five isolates of S. cerevisiae.



Fig 3: The tree was inferred from ITS sequence and constructed using neighbor-joining method.

CONCLUSION

The results of the isolation of S. cerevisiae in coconut sap from Banyumas Regency show that coconut sap contains S. cerevisiae. Based on the results of morphological and genetic identification, 5 isolates of S. cerevisiae were obtained which were coded as GNS1, GNS3, GNS4, GNS9 and GNS14 isolates.Based on the microbial profile of coconut sap, it can be said that coconut sap is a very potential source of yeast. This can be seen from the total yeast in the three types of coconut sap used. In native sap total yeast was 5.54 log CHU/ml, sulfite sap was 4.39 log CFU/ml and organic sap was 4.63 log CFU/ml.

ACKNOWLEDGEMENT

Financial support for this research was provided by a grant from the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia. The author would like to thank Prof. Dr. Rifda Naufalin, Karseno Ph.D and Dr. Ike Sitoresmi Mulyo Purbowati for the guidance in conducting the research. The authors also thank to Riyan Anggriawan Ph.D and Ali Maksum, S.P.,M.P.  for their advice in preparing the manuscript.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Isolation and Identification of Saccharomyces Cerevisiae from Coconut Sap

    G
    Gunawan Wijonarko1
    R
    Rifda Naufalin1,*
    I
    Ike Sitoresmi Mulyo Purbowati1
    K
    Karseno1
    1Department of Food Technology, Faculty of Agriculture, Jenderal Soedirman University, Grendeng, Purwokerto Utara, Banyumas, Jawa Tengah-53122, Indonesia.

    ABSTRACT

    Background: Coconut sap is a natural substrate with high sucrose content that supports the growth of various microorganisms, particularly yeasts. Saccharomyces cerevisiae is widely used in food fermentation and biotechnology; however, information regarding its presence and characteristics in coconut sap is still limited. This study aimed to determine the microbial profile of coconut sap and to isolate and identify Saccharomyces cerevisiae from coconut sap collected in Banyumas Regency, Indonesia.

    Methods: Coconut sap samples were collected from traditional brown sugar producers and analyzed for total microbial, yeast and bacterial counts using plate count methods. Isolation of S. cerevisiae was conducted using the streak plate technique on potato dextrose agar supplemented with chloramphenicol and gentamicin. Morphological identification was performed using light microscopy based on cell shape and colony characteristics. Molecular identification was carried out by amplification and sequencing of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers. Phylogenetic analysis was constructed using the neighbor-joining method with MEGA 11 software.

    Result: The native coconut sap showed higher total microbial and yeast counts compared to sulfite-treated and organic coconut sap. Five yeast isolates (GNS1, GNS3, GNS4, GNS9 and GNS14) exhibited morphological characteristics consistent with Saccharomyces species, including oval to round cell shapes. ITS rRNA gene sequencing confirmed that all isolates belonged to Saccharomyces cerevisiae. Phylogenetic analysis revealed that four isolates (GNS1, GNS3, GNS4 and GNS14) were closely related, while GNS9 represented a different strain within the same species.

    INTRODUCTION

    The coconut sap is a thick liquid taken from coconut tree flowers that has high sucrose content. Due to the high sucrose content, coconut sap contains an abundant quantity of microbes, especially Saccharomyces cerevisiae. S. cerevisiae has been studied intensively due to its significant role in human life. In food sectors, S. cerevisiae has been utilized for production of wine (Ganucci et al., 2018), beer (Kumari et al., 2019), bread (Olowonibi, 2017), fermented cereal and milk products (Motey et al., 2020), coconut toddy (Ahangangoda et al., 2019) and probiotic (Li et al., 2020). Meanwhile, the utilization of S. cerevisiae in the non-food sector has been widely studied, especially in bioethanol production. Previous studies reported that coconut inflorescence sap contains diverse fermentative microorganisms that contribute to spontaneous fermentation processes and microbial diversity in the sap (Mariyam et al., 2026).
           
    S.     cerevisiae are widely distributed in nature, found in organic and anorganic habitats which contain lots of carbohydrate. S. cerevisae can be found in organic habitat such as dates and molasses (Kechkar et al., 2019), wine (Ganucci et al., 2018), pineapple and oranges (Nasir et al., 2017), orange peels and spoilt tomato fruits (Milala et al., 2018), indigenous fermented foods (Menezes et al., 2020), kefir (Goktas et al., 2021) and nectarine (Noriko et al., 2019). S. cerevisiae can also be found in anorganic habitats such as in soil (Karki et al., 2017). In Indonesia, S. cerevisae can be found in Arenga pinnata sap (Periadnadi et al., 2018).
           
    Based on several studies related to the use of S. cerevisiae in the food industry, there is no information about isolation of S. cerevisiae from coconut sap. In wine production, S. cerevisae is takenfrom grape (Senses-Ergul and Ozbas, 2016) and black raspberry extracts (Song et al., 2019). In beerindustry, S. cerevisiae has been successfully isolated from various habitats such as grapes, bread andstill apples (Rossi et al., 2018) cultures. The use of S. cerevisiae for probiotics has also been reportedby Motey et al. (2020) in cereals and fermented milk products and by Banik et al. (2019) in haria, churpi and khambir.
           
    S. cerevisiae     can be isolated from its habitat using various methods. Methods that have been used to isolate S. cerevisiae are spread plate methods (Song et al., 2019), serial dilution (Kechkar et al., 2019), streak plate (Ahangangoda et al., 2019) and pour plate (Guimarães et al., 2006). All S. cerevisiae isolation methods used were proven to have satisfactory results. Several media thathave been used for the isolation of S. cerevisiae are YPG (Guimarães et al., 2006), MEM (Suranská et al., 2016), YMA (Kechkar et al., 2019), YPDA (Capece et al., 2016) and MEA (Pennacchia et al., 2008). Meanwhile, Olowonibi (2017) found that S. cerevisae could also be isolated using streak plate on PDA containing 0.05 mg/mL chloramphenicol and gentamicin.
           
    Based on the background, this study was conducted with the aims to describe the microbial profile of coconut sap, then to isolate and to identify S. cerevisiae from coconut sap.

    MATERIALS AND METHODS

    The experimental work was conducted at The Laboratory of Food Microbiology, Faculty of Agriculture, Universitas Jenderal Soedirman, Purwokerto, Indonesia. Molecular analyses, including PCR amplification and sequencing, were performed using integrated molecular biology laboratory facilities. The research was carried out in 2023.
     
    Sampling of coconut sap
     
    Samples of coconut sap were collected from the traditional brown sugar farmer in Banyumas Regency, Indonesia. Coconut sap (500 mL) were collected using a simple random sampling technique. Samples of coconut sap were taken 30-60 mins after tapping using sterile plastic bottles and put into an ice box to prevent damage.
     
    Total microbial and yeast count
     
    A total of 50 mL of coconut sap sample was diluted using 450 mL of sterile 0.85% NaCl physiological solution. The total microbial count was performed by making series of dilutions in 9 mL of sterile 0.85% NaCl solution. One milliliter of each dilution series was taken to be grown on YMA medium. Incubation was performed at 37°C for 36 h. For total yeast count, 1 mL of each dilution series was grown on PDA medium containing 0.05 mg/mL chloramphenicol and gentamicin and was incubated at 37°C for 36 h (Feldsine et al., 2003).
     
    Isolation of S. cerevisiae
     
    Isolation of S. cerevisiae from coconut sap was performed using the streak plate method on PDA media containing 0.05 mg/mL chloramphenicol and gentamicin (Olowonibi, 2017). Each sample of coconut sap was previously shaken to make it homogeneous. Samples of coconut sap were taken using a loop needle and streaked 3 times on the media.
     
    Morphological identification
     
    Morphological characterization based on colony appearance and cell morphology remains an important preliminary approach for microbial identification (Bhutani et al., 2018). The morphological observations of the isolates were performed using light microscope at magnification of 400 ×. Using a needle, one of isolate was taken and then smeared on a glass object that had been previously dripped with polyphenol blue. Morphological identification of S. cerevisiae referred to “The Yeast: A taxonomic study” (Kreger-van, 1984).
     
    Molecular identification
     
    Genomic DNA was isolated from single colonies using Quick-DNA Fungal / Bacterial Miniprep Kit (Zymo Research, D6005, USA) according to the manufacturer’s protocol. Molecular identification of the yeast isolates was achieved with amplification of the internal transcribed spacer region with primers pairs ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) (White et al., 1989). PCR amplification was done with (2×) MyTaq HS Red Mix (Bioline, BIO-25048) and the expected PCR products’ size was 600-700 bp. The PCR products were checked on agarose gel electrophoresis and sent for sequencing. In order to construct a phylogenetic tree sequences with the highest similarity to the query, together with some sequences retrieved from the GenBank database were aligned and constructed from evolutionary distances by the neighbor-joining method using the software MEGA 11 (Tamura et al., 2021).
     
    Statistic analysis
     
    Data were analyzed through independent t-test and one-way ANOVA using SPSS version 20 software.

    RESULTS AND DISCUSSION

    Microbial profile of coconut sap
     
    The native coconut sap had a higher total microbes than sulfite coconut sap and organic coconut sap (Table 1). Total microbes in sulfite coconut sap and organic coconut sap were 6.24 and 6.57 log CFU/mL, respectively. Such finding indicated that sulfite and organic solvents were equally effective in killing microbes. Sulfite had a low antimicrobial effect by 0.02% but was able to inhibit microbial growth in tempeh. Fatisa (2013) reported that minimum inhibitory concentration (MIC) of pulasan skin (Nephelium mutabile) against Staphylococcus aureus and Escherichia coli were 0.76 mg/mL and 0.816, respectively.

    Table 1: Microbial profile of fresh coconut sap.


           
    The total yeast in the three types of sap revealed that the native sap (5.54 log CFU/mL) had a higher total yeast compared to the total yeast in the sulfite sap (4.39 log CFU/mL) and organic sap (4.63 log CFU/mL). Such finding indicated that both sulfite and organic laru were effective in reducing the amount of yeast in coconut sap. Mangosteen rind and jackfruit wood contained in organic solvents have antimicrobial properties that can inhibit microbial growth (Bouarab-Chibane et al., 2019). On the other hand, Irwin et al. (2017) found that the test bacteria experienced significant growth at sulfite concentrations between 250-500 ppm. The total bacteria in the sulfite sap (1.85 log CFU/mL) were lower than the total bacteria in the native sap (2.02 log CFU/mL) but there was no difference with the organic sap (1.94 log CFU/mL). Such finding indicated that sulfite was slightly more effective than organic laru in reducing the number of bacteria in coconut sap. The effectiveness of sulfite compounds were 1.4 times higher than H2O2 compounds and natural preservatives (Irwin et al., 2017).
     
    Isolation
     
    The results of S. cerevisiae isolation from 15 samples of coconut sap using streak plate method are presented in Fig 1. The appearance of the white colonies showed the same characteristic as the standard colonies. Therefore, samples with the same appearance were further analyzed morphologically using a light microscope. The appearance of the sample isolates was similar to the standard isolate under a light microscope at 400× magnification. It can be observed that the cells were oval, round or rod in shape (data not shown).

    Fig 1: Isolation of S. cerevisiae.


     
    Morphological characteristics
     
    Morphological characteristics were observed after isolation of S. cerevisiae from 15 samples of coconut sap from afternoon tapping. It can be observed under a microscope that the cells were oval, round or rod in shape. However, oval cells were mostly found. There were 9 isolates which were oval in shape. Cell size varied in length between 12.5-17.8 µm and in width between 0.8-4.6 µm. The dimension of GNS9 cell was 15.2 µm × 4.5 µm (data not shown). The color of the cells was between white and cream and the cell surface was dull and clear. Kreger-van (1984) reported that the genus Saccharomyces is characterized by round, elliptical, or cylindrical cell shapes, white colonies, dull surfaces and raised elevations. Genus Saccharomyces reproduces through multilateral budding, with the larger size of the stem cells compared to the tillers.
     
    Molecular characteristics
     
    Based on conoly appearance and microscopic observations, five isolates coded as GNS1, GNS3, GNS4, GNS9 and GNS14 suspected to belong to the genus Saccharomyces. To confirm the preliminary identification results, molecular characterization was carried out through amplification and sequencing of the ITS region. PCR amplification using ITS1 and ITS4 primers was successfully performed for all isolates, producing DNA bands of approximately 600-700 bp, as presented in Fig 2. The obtained sequences were analyzed using BLAST against sequences available in the NCBI GenBank database. The comparison results showed that all isolates had high similarity with Saccharomyces cerevisiae sequences, confirming their species identity (Table 2). Furthermore, phylogenetic analysis based on ITS sequences was constructed using the neighbor-joining method to evaluate the genetic relationship among the isolates. The phylogenetic tree in Fig 3 demonstrated that isolates GNS1, GNS3, GNS4 and GNS14 were grouped within the same cluster, indicating close genetic relatedness (Fig 3). Meanwhile, isolate GNS9 formed a different branch, suggesting that this isolate may represent a different strain of S. cerevisiae. Molecular techniques provide more precise identification compared to conventional morphological observations alone (Waseem et al., 2020). In this study, sequencing analysis confirmed that all isolates belonged to Saccharomyces cerevisiae.These results confirmed that coconut sap collected from banyumas regency contains genetically diverse S. cerevisiae strains with potential applications in fermentation and biotechnology.

    Table 2: Top 10 hit blast result toward NCBI database.



    Fig 2: PCR product of five isolates of S. cerevisiae.



    Fig 3: The tree was inferred from ITS sequence and constructed using neighbor-joining method.

    CONCLUSION

    The results of the isolation of S. cerevisiae in coconut sap from Banyumas Regency show that coconut sap contains S. cerevisiae. Based on the results of morphological and genetic identification, 5 isolates of S. cerevisiae were obtained which were coded as GNS1, GNS3, GNS4, GNS9 and GNS14 isolates.Based on the microbial profile of coconut sap, it can be said that coconut sap is a very potential source of yeast. This can be seen from the total yeast in the three types of coconut sap used. In native sap total yeast was 5.54 log CHU/ml, sulfite sap was 4.39 log CFU/ml and organic sap was 4.63 log CFU/ml.

    ACKNOWLEDGEMENT

    Financial support for this research was provided by a grant from the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia. The author would like to thank Prof. Dr. Rifda Naufalin, Karseno Ph.D and Dr. Ike Sitoresmi Mulyo Purbowati for the guidance in conducting the research. The authors also thank to Riyan Anggriawan Ph.D and Ali Maksum, S.P.,M.P.  for their advice in preparing the manuscript.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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