The isolation and characterization of indigenous microbes with probiotic potential from gatotan

Indigenous probiotics, which are more adaptive to ethnoecology such as human gut health are found in local food. Therefore, this study aimed to isolate, select, and characterize indigenous microbes as probiotic candidates namely gatotan , which is an Indonesian fermented food from cassava. The potential probiotic was dissolved in artificial gastric acid at pH 2.0 and incubated for 2 hrs, then the De Man Rogosa Sharpe and yeast in Malt Extract medium were used for selective isolation of lactic acid bacteria (LAB). The phenotype including morphological and physiological characteristics as well as the genotype such as PCR - fingerprinting was identified. The results showed that two of the three isolates have the potential to be developed as probiotics possibly growing in an acidic environment. The BGP (LAB) and YGK (yeast) isolates have the highest survival artificial gastric acid of pH 2.0. Furthermore, the BGP from cassava var. white ( Malang 2 ) had a milk - white colony, rod - shaped, undulate colony edges, gram - positive bacteria, non - motile, negative catalase, and heterofermentative. The YGK from cassava var. yellow ( Malang 4 ) also had white - colony characteristics, irregular shape, rough surface, opaque transparency, spheroidal cell morphology, and amylolytic index of 1.06±0.12 mm. Conclusively, the genotype characterization identified both isolates as Lactobacillus brevis and Pichia kudriavzevii, respectively.


Introduction
Over the years, probiotic foods have attracted many scientists because of their health benefits.The WHO has defined probiotics as a microorganism that provide health benefits to the host in appropriate amounts.They are mostly found in fermented local food one of which is the Indonesian gatotan.Previous studies identified microbes that have the potential as probiotic candidates (Emmawati et al., 2016).Astriani et al. (2018) isolated gatotan microbes as a starter to produce gatotan and those identified were Rhizopus oligosporus, Aspergillus niger, Bacillus licheniformis, Trichoderma sp., Lactobacillus manihotivorans, L. ferment and Bacillus brevis.Furthermore, the fermentation of gatotan occurs spontaneously, hence, it has the potential to support the growth of several microbes.The microorganisms present during fermentation affect the quality and safety of the final product.In the case of gatotan, it causes a change in black colour and soft texture.
Many superior probiotic LAB strains are found in local food sources such as spontaneously fermented products.The LAB exhibit functional and probiotic properties that are resistant to low pH, therefore they are classified as GRAS (Generally Recognized as Safe) bacteria by the US FDA or QPS (Qualified Presumption of Safety by EFSA) (Won et al., 2021).Another local food that has the potential as a candidate source of probiotics is "Mandai".Additionally, fermented products from dami or straw cimpedak fruit contain the probiotic microbe Lactobacillus plantarum which is resistant to bile salts and gastric acid (Emmawati et al., 2016).LAB probiotic isolates have long been developed from foods, and their functional properties stimulate the immune system (Emmawati et al., 2016).Lactobacilli and Bifidobacteria survive in the intestine and benefit health (Barakat et al., 2011).Probiotic microbes may provide enhanced growth, immune responses, nutrients, produce enzymes, and inhibit pathogens (Manhar et al., 2011).Meanwhile, the potential probiotic characteristics of FULL PAPER indigenous microbes from gatotan remain unknown.Therefore, this study attempted to isolate and identify the phenotype and genotype characteristics of probiotic candidates, which can survive in pH 2.0 of artificial gastric acid.

Gatotan preparation
Fresh cassava was peeled to separate the outermost cassava peel, which was then washed and dried in the sun.Dried cassava which is an ingredient for gatotan was mixed with sterile distilled water 1:3 (w/v) and spontaneously fermented anaerobically in a sterile jar for 5 days (28°C).During fermentation, the texture of the substrate changes to become soft, and the slurry was used as sample isolation of microbe from indigenous gatotan.The main ingredients used were cassava var.yellow (Malang 2) and cassava var.white (Malang 4).

Isolation of lactic acid bacteria and yeast
A total of 1 mL slurry was diluted using 0.85% physiological solution.Subsequently, the isolation of LAB was carried out aseptically in MRSA (Himedia, India) + CACO 3 and MEA (Himedia, India) yeast medium.Incubation was carried out aerobically for LAB and yeast at 37°C and 30°C, respectively for 48 hrs.The microbe colonies grown were selected and calculated, and then the LAB and yeast were purified using the quadrant method.Pure cultures were then streaked into slants of medium agar and stored at 4°C for further characterization.

Probiotic screening of the artificial simulated gastric acid pH 2
The LAB and yeast isolates can tolerate simulated gastrointestinal (GI) tract conditions (Zhai et al., 2015).Furthermore, the screening of probiotic candidates used two types of gatotan from cassava namely Malang 4 and 2. Gastric acid juice with the following contents (g/L): 8 NaCl; 0.2 KCL; 8.25 Na 2 HPO 4 .2H 2 O; 14.35 Na 2 HPO 4 ; 0.1 CaCl 2 .2H 2 O; 0.18 MgCl 2 .6H 2 O is used as hydrochloric acid buffer.A volume of 1 mL slurry was added with 5 mL artificial gastric acid at pH 2 and suspended in a medium to measure the survival microbe.After 2 hours of incubation, the culture with the treatment of 1 mL was added to 9 mL of artificial simulated gastric acid at pH 2. The cultures were then incubated at 37 and 30°C (48 hrs).The percentage survival of strains was calculated as follows (Luang-In et al., 2021):

Characterization of gatotan lactic acid bacteria of tolerance gastric acid
Gatotan LAB isolates of gastric acid tolerance were characterized based on their phenotype (morphological and physiological) and genotype (PCR-fingerprinting).The results from probiotic screening obtained one LAB (BGP) strain from two gatot variants, which was isolated from cassava var.white.Furthermore, the morphological characteristics of LAB indigenous gatotan include macroscopic and microscopic.The macroscopic observations were made with a magnification of 400× and 1,000× and it included colony edges, shape, and colour, as well as their cell form.A gram-positive and gram-negative bacterium is indicated by purple and red colour, respectively.LAB's physiological properties include catalase test, motility, and type of fermentation.The catalase aims to determine the ability of bacteria to degrade H 2 O 2 ; when this test shows the presence of oxygen bubbles, it is a catalase-positive (aerobic) bacterial isolate and catalase-negative (anaerobic) did not show bubbles.The formation of oxygen bubbles indicates the production of catalase enzyme by the test bacterium.Additionally, motility aims to determine the growth of bacteria, which is indicated by spreading growth and non-motile for positive and negative, respectively.The fermentation type test is used to determine the group of bacteria.Heterofermentative bacteria produce oxygen bubbles in the Durham tube and homofermentative will not.

Characterization of gatotan yeast of tolerance gastric acid
The candidates for probiotic yeast were obtained from two gatotan variants and the characterization used YGK samples with the highest survival rate.The YGK strain was isolated from cassava var.yellow and the macroscopic observations were carried out by observing FULL PAPER yeast colonies growing directly on the surface of the media, including their surface, colour, transparency, and cell morphology (Nurhayati et al., 2018).Furthermore, the microscopic observations were carried out at 400× magnification with a microscope by staining with crystal violet solution and the cells fixed on a glass object.Yeast was cultured on extract agar medium with starch (yeast 0.3%; malt 0.3%; starch 2%; and agar 1.5%) and incubated for 2 days at 30°C.Afterwards, Lugol's iodine solution (1 g I 2 , 2 g KI in 300 mL of distilled water) was added to the culture (Nurhartadi and Rahayu, 2011).The clear zone that appears around the colonies indicates amylolytic activity from the yeast and was calculated as follows (N1: clear zone diameter that appears around the colonies; N2: colonies diameter): The molecular analysis of yeast was performed using the 28S ribosomal rRNA, the genomic DNA was extracted using Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, D6005) and PCR amplification was carried out with (2×) MyTaq HS Red Mix (BIO-25048).The PCR master mix consists of dd H 2 O 9.5 mL, myTaq HS Red Mix (2×) 12.5 mL, 20 µM NL-1 primer, 20 µM NL-4 primer, and 1 mL DNA template.PCR amplification of yeast using primer NL1: 5'-GCATATCAATAAGCGGAGGAA AG3-' and primer NL4: 5'-GGTCCGTGTTTCAAGACGAG-3' from PT. Genetika Science, Indonesia.Also, the PCR conditions of yeast included initial denaturation at 95°C for 3 minutes (1 cycle) and 10s (35 cycles), annealing at 52°C for 30s (35 cycles), extension at 72°C for 45s (35 cycles), and held at 4°C (1 cycle).

Phylogenetic analysis
The amplification of the gene was carried out using primer and sequencing of the obtained DNA fragments.Subsequently, DNA sequencing was performed from purified PCR products using Bi-directional sequencing.Isolates characterization aims to determine the genus and strain (Suryani et al., 2021).The assembly of PCR-16S rRNA and 28S rRNA was then carried out on May 28, 2021, and analyzed using BLASTN (Basic Local Alignment Search Tool Nucleotides) (http:// www.ncbi.nlm.nlh.gov/), which was also used to present the DNA sequencing results in the FASTA.Sequence similarity was estimated by homology and the phylogenetic tree was constructed at National Center for Biotechnology Information (NCBI) database.

Gatotan microbial tolerance to artificial gastric acid
The probiotic must have resistance to gastric acid pH using a bile salt solution test (1%) and low pH (2) (Jafari -Nasab et al., 2021).The results of the gatotan microbial resistance test against artificial gastric acid can be seen in Table 1.Furthermore, the surviving gatotan microbes were characterized as shown in Table 2.
The results showed differences in the type of growing microbes.Gatotan var.white indicates the growth of LAB and yeast, while gatotan var.yellow only shows yeast.This is also due to the composition of the cassava content of the white variety which is different from the yellow variety.Ayetigbo et al. (2018) reported that yellow cassava has a yellower colour and is sweeter than white.
According to Astriani et al. (2018), the types of microbes identified in gatotan are molds including R. oligosporus, A. niger, and Trichoderma sp. as well as LAB namely L. manihotivorans, L. ferment, B. licheniformis, and B. brevis.Figure 1 shows the difference in the appearance of the whole cassava, peeled cassava, dried gatotan and submerged gatotan.The slurry gatotan has pH of 5.3 in both variants.Three isolates were obtained from slurry gatotan (yellow and white var. of cassava).Furthermore, the high survival rate of microbe, which includes BGP (90.29%) and YGK (88.51%) indicates that they can live at low pH.The lowest was 85.88% (YGP).The results showed that probiotic bacteria and yeast only grow on var.white indicating that cassava varieties affect their growth.The microbial population decreased in time intervals (0-2 hrs) proving that all isolates had the same acidity tolerance, and an increasing acidic condition affects their viability.It showed that the indigenous gatotan microbial has potential as probiotic candidate.

Characteristics of gatotan microbial resistance to artificial gastric acid
Gatotan LAB isolation results of tolerance gastric acid were obtained from the gatotan var.white.Meanwhile, LAB is known as a commercial species and an important group of bacteria in the food industry such as dairy products (Elzeini et al., 2021).Various LABs are considered safe in food or probiotics and improve nutrition for human health (Aplevicz et al., 2014).Those found in the cassava var.white can survive at pH 2, hence, they have the potential as probiotics.(Aplevicz et al., 2014).
The results of LAB morphology are consistent with the reports of Aplevicz et al. (2014).The morphology of LAB was able to produce lactic acid as the end product, which was gram-positive, negative catalase, non-spore forming rods, and non-motile.Negative catalase indicates that they are anaerobic, hence cannot grow in the presence of O₂ or acidic substances due to the formation of H₂O₂ in the atmosphere, which is toxic to bacteria.In spontaneously fermented amaranth samples, 15 gram-positive and catalase-negative, eight coccishaped, and seven rod-shaped isolates were identified.They included L. plantarum, Lactococcus garvieae, and Weissella cibaria (Iruene et al., 2021).LAB isolated from gatotan is homofermentative bacteria and can only produce lactic acid as the main product of fermentation (Anjum et al., 2014).

Characteristics of gatotan yeast of tolerance gastric acid
Yeast is a safe eukaryotic microorganism widely used in fermented foods (Wulan et al., 2021).Previous studies have identified probiotic yeasts in fermented foods such as kefir, cocoa, cheese, and other products.According to Hsiung et al. (2021), yeast can grow at 37°C , survive at the pH of gastric acid and bile salts, and aggregate with various pathogenic microbes.In this study, YGK was isolated from gatotan var.yellow which can survive at pH 2, hence it has the potential as a probiotic candidate.Generally, several species of yeast are probiotics, an example is the Saccharomyces boulardii, which is used to treat gastrointestinal (GI) (Zanello et al., 2009).The reasons for the exploration of probiotic yeast include (1) even though yeast cell size is roughly 10 times greater than bacterial, it can only contribute about <0.1% of microbiota in the GI tract, suggesting that it can represent stearate inhibition which is significant for bacteria; (2) differences in the composition of the cell wall of yeast and bacteria, the response is called immunobiotic (Huffnagle and Noverr, 2013).Subsequently, the morphological characteristics of yeast include colonies, surface, colour, transparency, and cell morphology.In a study by (Moon et al., 2014), yeast was isolated and identified in kimchi that had irregular or circular colonies; rough or smooth surface; ivory, white, and pink colours; translucent or opaque transparency; oval or spheroidal cell morphology.They include Rhodotorula mucilaginosa, Kazachstania bulderi, K. exigua, K. servazzii and Pichia kudriavzevii.
The fermentation of gatotan involves various types of microorganisms such as molds, LAB, and yeasts.Furthermore, the raw material for gatotan is cassava which contains starch causing changes in components during fermentation.Microorganisms such as fungi, yeast, and bacteria found in cassava can decompose starch through the production of an enzyme called amylase (Damayanti et al., 2021).Cassava starch is a polysaccharide that includes amylose and amylopectin polymers and it is a branched polymer of glucose molecules having 1.6 glycoside bonds.The results showed that the amylolytic index of YGK was 1.06±0.12mm with a microbial and clear zone diameter of 5.61 mm and 11.50 mm, respectively.This result is consistent with the reports of Nurhartadi and Rahayu (2011) that tape contains two amylolytic yeast isolates namely Saccharomycopsis fibuligera and Pichia burtonii.The YGK isolate was identified as P. kudriavzevii, which had 100% similarity and the phylogenetic tree construction of P. kudriavzevii contained four groups (Figure 3).The first and second consist of P. kudriavzevii SAG (MN595038.1)and CK12 (MN712334.1),respectively.The third group includes P. kudriavzevii CK9 (MN710524.1),Leg2 (MH472647.1),CK10 (MN710525.1),HCM-NM79 (MK101219.1),CK8 (MN710523.1),and strain CR-Y103 (KX118628.1),and the last include P. kudriavzevii SL1-7 (HM123747.1),and 3.4-2 (FJ455113.1).Nucleotide BLAST homology of yeast was determined using assembly sequences 578 bp.This result is consistent with the report of Kurtzman (2011) that P. kudriavzevii is distributed in spontaneous fermentation and is proven in this study using gatotan.According to Rodriguez et al. (2021), P. kudriavzevii can be a probiotic candidate from a food environment.

Conclusion
Lactobacillus brevis and P. kudriavzevii were microbes isolated from gatotan that survived in artificial simulated gastric acid, with a survival rate of 88.51% and 90.29%, respectively.The characteristics of L. brevis from the isolation of gatotan var.white include milkwhite colony, rod-shaped, undulate edges, gram-positive bacteria, non-motile, heterofermentative, and negative catalase.Similarly, P. kudriavzevii characteristics from the isolation of gatotan var.yellow include white-colony and irregular colony, rough surface, opaque transparency, and spheroidal cell morphology with an amylolytic index of 1.06±0.12mm.Subsequently, the indigenous microbe from gatotan obtained LAB and yeast with promising probiotic properties for further evaluation, hence in vitro testing is recommended.

Table 1 showed
the survival of According to Mulaw et al. (2019), fermented food contains probiotic microorganisms that can degrade carbohydrates, it was

Table 1 .
The microbial survival on the artificial gastric acid at pH 2.0

Table 2 .
The characteristics of BGP and YGK isolates.as a sample to obtain LAB isolates that have the potential as probiotics.The main LAB species in fermented foods is the genus Lactobacillus (8 log CFU/ g) https://doi.org/10.26656/fr.2017.7(S1).9© 2023 The Authors.Published by Rynnye Lyan Resources FULL PAPER used