Isolation and identification of gastric acid - tolerant yeast from tapai

Lactobacilli and Bifidobacteria are the most extensively employed bacterial strains in marketable probiotic supplements . However, another probiotic was recently developed from yeast screening based on tolerance against gastric acid. This research aimed to isolate yeasts from traditional Indonesian fermented food (tapai). Screening of probiotic yeasts was based on their survival in gastric acid of pH 2.0. Yeast strains were cultured in malt extract agar


Introduction
From 2004 to 2015, Indonesia was the sixth-largest producer of cassava (Manihot esculenta) (Yuliati et al., 2019). Up to 32.8 million tons of cassava were produced in 2018, which equates to an average productivity of 21.85 tons/ha. The agricultural sector in Jember regency has a wide range of potential commodities and considers the availability of a large harvest area of cropping cassava to offer resources for generating healthier foods. Fermented foods have increased nutritional bioavailability in the industrialized world by regulating microbiota and altering certain target activities for host health. For example, tapai, a popular fermented cassava meal in Jember, imparts a range of sweet-sour flavours, soft textures, and yeast as a starter culture (Nuraida and Owens, 2014). Yeast in tapai consists of diverse species, including Trichosporon sp., Clamydomucor sp., Candida sp., and Saccharomyces sp. (Tamang et al., 2016).
Currently, microbes such as yeast and bacteria have been investigated to contain probiotic properties on human health (Czerucka et al., 2007;de Melo Pereira et al., 2018;Xu et al., 2018). In 2002, the Food and Agriculture Organization (FAO) and World Health Organization (WHO) defined probiotics as live microorganisms that possess health outcomes and effects when administered in balanced amounts (Halder and Mandal, 2015).
Lactic acid bacteria (LAB) are the most common and well-studied probiotics. However, certain yeast species have recently been promoted as an effective probiotic in many functional clinical studies (Salminen et al., 2010;Syal and Vohra, 2013;Xu et al., 2018). Some popular commercial yeasts include Saccharomyces boulardii (Edwards et al., 2007) and Saccharomyces cerevisiae, extensively identified due to their significant contribution to the performance of probiotics (Czerucka et al., 2007;Didari et al., 2014). Widyatmoko et al. (2018) isolated and identified the indigenous yeast from Jember's favourite tapai products. The screening results from using amylolytic yeast to improve the cassava starch fermentation process. Indigenous yeasts used as probiotics are expected to resist gastric acidity and bile salts, anti-microbial activity against pathogens, presence of gastrointestinal enzymes, and body temperature at 37°C (Gil-Rodríguez et al., 2015;Johansen et al., 2019). However, the presence of indigenous tapai yeast (ITY) as a potential probiotic candidate remains unknown. This study isolates and identifies indigenous yeast's phenotype and genotype characteristics from tapai. It evaluates the potential of probiotic sources with tolerance to artificial gastric acid of pH 2 to develop the probiotic from yeast rather than lactic acid bacteria.

Screening the yeast from favorite tapai products
Four types of favourite tapai products were collected from Jember and Bondowoso regions in Indonesia. Tapai Sumber Madu and Sari Madu were obtained from Jember, while Handayani and Tapai Manis BWS were obtained from the Bondowoso district of East Java, Indonesia. The artificial gastric acid and 5 M buffered hydrochloric acid (pH 2) were used with the following contents: NaCl 8 g; KCL 0.2 g; Na 2 HPO 4 .2H 2 O 8.25 g; NaHPO 4 14.35 g; CaCl 2 .2H 2 O 0.1 g; MgCl 2 .6H 2 O 0.18 g. A total of 10 g of tapai sample was homogenized in 90 mL of sterile distilled water after three days of incubation. Approximately 1 mL of the solution was diluted in a 9 mL physiological solution of 0.85% NaCl (w/v). A total of 1 mL was transferred to a malt extract agar (MEA) medium using the pour plate method. The samples were incubated for 48 hrs at 30°C until growth was achieved (Ebabhi et al., 2013). After the incubation, 5 mL of gastric acid was dropped in the plate and incubated for 24 and 48 hrs at 30°C. Then, 2 mL of solution from the plate was pipetted, inoculated separately in MEA medium, and incubated at 30°C for 48 hrs.

Determination of macroscopic and microscopic morphological of yeast
Macroscopic characters were recorded based on colony colour, convexity, shape, surface, and elevation (Meyer et al., 1984;Widiastutik and Alami, 2014). The appearance of the colony was achieved using streaking methods of the colony on MEA medium after 48 hrs incubation at 30°C. The microscopic character was conducted using crystal violet dye, staining yeast cells fixed on a glass object. Yeast cells were seen using a microscope with a magnification of 400×.

Determination of growth temperature of tapai yeast isolates
Two loopful of yeast isolates were transferred to 1 mL of sterile physiological solution. Additionally, a 10 µL liquid sample was diluted in a 1 mL malt extract broth (MEB) medium and homogenized using a vortex. The growth temperature of each ITY isolate was incubated at 10°C, 28°C , 37°C , 40°C , and 45°C for 48 hrs. The results were assessed based on the turbidity of the MEB medium in the cell culture tube.

Analysis of survival yeast on artificial gastric acid
The pre-selected yeasts from the previous analysis were further characterized for their resistance to gastric acid of pH 2 using the method of Rajkowska and Kunicka-Styczynska (2010) with some modifications. Samples were collected at 0 resemble, as the initial population and after 2 hrs of incubation in 5 M buffered hydrochloric acid (pH 2) as the final population of ITY. Two loopful of ITY were dissolved in 5 mL sterile MEB broth and incubated at 30°C overnight. A 1 mL solution was transferred to a 9 mL physiological solution of 0.85% NaCl (w/v) to determine initial yeast survival. The solution (1 mL) was diluted in 9 mL buffered hydrochloric acid (pH 2) and incubated for 2 hrs at room temperature to measure final yeast survival. For initial and final yeast survival, seven serial dilutions of yeast isolates were collected in 9 mL saline water (0.85% NaCl). Then, 1 mL of the dilutions were pour-plated into MEA at 30 o C for 48 hrs. The percentage survival of yeast strains was calculated using the equation given by the following formula:
The DNA strands NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL-4 (5'-GGTCCGTGTTTCAAGACGG-3') of the D1/D2 28S rRNA gene were read using the PCR primers from PT. Genetika Science, Indonesia. The PCR reaction is under the following condition: 3 mins of initial denaturation at 95°C (1 cycle The genes were amplified using primers, and DNA fragments produced were sequenced. DNA sequencing was performed from purified PCR products, and Bidirectional sequencing was used. The results were then compared to the sequences included in the GenBank database using BLASTN (Basic Local Alignment Search Tool) at www.ncbi.nlm.nih.gov/BLAST (Altschul et al., 1990). The NCBI Blast Tree Method was used to generate the phylogenetic tree using Neighbor-Joining (Unrooted Tree).

Microscopic and macroscopic morphological of tapai yeast isolates
The five yeast isolates were cultured using an MEA medium incubated at 30 o C for 48 hrs. The morphological characteristic of ITY is represented in Table 1. SUL and SUP isolates were generated from the tapai Sumber Madu brand, while SM isolates were from the tapai Sari Madu brand. TM isolate was isolated from Tapai Manis BWS and H isolate from tapai Handayani brand. The microscopic and macroscopic appearance is represented in Figure 1 and Figure 2.
The macroscopic morphology of yeast has been reported with white colour, cream-grained texture, opaque colony surface, and convex elevation (Widiastutik and Alami 2014). According to Moon et al. (2014), yeasts also identified the morphology characteristics with white colour, circular shape, entire colony edge, convex elevation, growing at 37°C, with an elongated cell having pseudo mycelium. A similar result in morphological characteristics of yeast has been observed in the previous study, where Candida tropicalis has a white-cream colour, round colony shape, convex elevation, flat edge, smooth surface, and oval cell shape (Suryaningsih et al., 2018).

Growth temperature of tapai yeast isolates
The yeast isolates were cultured in MRSB (Man Ragosa Sharpe Broth) medium for 24 and 48 hrs incubation at various temperatures. When selecting a yeast strain starter for industrial fermentation, stress resistance to pH and temperature is required. Suriasih et al. (2012) proved that higher yeast counts increased with higher incubation temperature (28±2°C). However, potential probiotic yeast should resist viability at the body temperature (37°C) (Gil-Rodríguez et al., 2015;Johansen et al., 2019). In this study, all strains showed specific growth in the presence of temperature stress at 37-40°C. It provides information regarding resistance to the fermentation temperature of tapai. During the fermentation process, heat production was caused by the exothermic reaction to increase the optimal temperature of tapai by 35-40°C (Kanino, 2019).

Yeast survival on the artificial gastric acid
The best-demonstrated survival percentage of indigenous tapai yeast as probiotic agents under stressful Note: SUL, SUP, and SM were isolated from tapai favorite products of Jember; TM and H were isolated from tapai favorite products of Bondowoso  FULL PAPER pH 2 were SUL (82.71%) and SM isolates (79.74%). At the end of 2 hrs incubation at artificial gastric acid, these isolates had a growth capacity of 6.20±0.35 CFU/mL and 5.75±0.45 CFU/mL. In artificial gastric acid, the probiotic survival percentage for the other three presented only a slight variation of 52.68% (SUP), 54.81% (TM), and 54.40% (H). However, the growth rate of the yeasts equal 3.98±0.03 CFU/mL for SUP, 3.81±0.31 CFU/mL for TM, and 3.99±0.07 CFU/mL for H. All isolates have the same acidity tolerance, with increasing acidity affecting probiotic viability. This pattern is evident at all time intervals (0 and 2 hrs), as tests showed a drop in viable yeast counts as the acidity level of the sample increased.
Wickerhamomyces anomalus LV-6, the yeast strain isolated from the fermented excreta of broilers, has been investigated with a survival percentage of 98.30% and viability of yeast of 7.50±0.11 CFU/mL under gastric acid of pH 2 for 3 hrs incubation (García-Hernández et al., 2012). Pichia kudriavzevii OM11 had the highest survival of 93.46% at pH 2 after an exposure period of 3 hrs at 37°C. The crucial characteristic of a good probiotic source is tolerating high acid levels (pH) in the stomach ranging from 2-5. Each yeast isolate is significant in low pH tolerance because the probiotic source can survive at 37°C, colonize the gastrointestinal system, and the presence of bile salt (Karasu-Yalcin et al., 2019). Sahadeva et al. (2011) demonstrated that microorganisms from cultured milk drinks in the Malaysian marketplace survive at pH 3.0 and make them good probiotic sources. Brands A, B, and C had a growth capacity of 6.94 CFU/mL, 6.60 CFU/mL, and 9.40 CFU/ mL at 37°C for 3 hrs incubations.
The probiotics should be present in sufficient quantities to benefit the intestinal epithelium until they are adhered to and colonized by the intestinal epithelium for a fermented food designated as probiotics. Probiotics' positive effect has generated tremendous interest due to the proteins, vitamins, minerals, and different immunestimulating chemicals (proteases, β-glucans, and mannan oligosaccharides) found in yeast (Gil-Rodríguez et al., 2015;Azhar et al., 2019;Wulan et al., 2021). During sugar fermentation, microbiota as probiotics reduce the environment's pH and inhibit the growth of undesired microorganisms. They also contribute to food preservation by generating secondary metabolites such as lactic acid, fatty acid, and bacteriocin (Evivie et al., 2017). According to Fernández et al. (2003), probiotic sources should be able to withstand a pH of at least 3.0 and greater than 1.5 while fasting with high amounts of acid. Yeast ability of SUL and SM isolate has a good tolerance of the artificial gastric acid of pH 2, closely related to their strain specification (Lin et al., 2006).

Genotypic characteristics of tapai yeast
The divergent D1/D2 domain of the 28S rRNA of SUL and SM for the highest survival as the probiotic source was amplified. The sequences were compared to the nucleotide database using the BLAST program from the National Centre for Biotechnology Information (NCBI). The % identity from BLAST shows that SUL was classified as cluster Kodamaea ohmeri (99-100%), while SM shows (99-100%) identity with Pichia kudriavzevii (Figures 3 and 4). The PCR fragments of SUL and SM prove a distinctive band of approximately 500-600 bp.
Based on pioneering studies, P. kudriavzevii was found in fruits, soil, and miscellaneous fermented food and beverages, capable of producing ethanol and growing at temperatures of 45°C (Yuangsaard et al., 2013;Mbuk et al., 2016). Pichia sp. isolated from traditional Indian fermented foods (idli and jalebi batter), has beneficial properties for present viable probiotic agents and can be widely employed as food and feed supplements (Syal and Vohra, 2013). Meanwhile, K. ohmeri is a yeast isolated from fermented glutinous rice and can produce ethanol (Sumerta and Kanti, 2017). Experiments by Azhar et al. (2019) have shown that Kodamaea sp is used as a probiotic source and consumed as a fermented beverage by the Malaysian people. In a previous study, K. ohmeri and P. kudriavzevii also showed good tolerance to temperature stress after 24 hrs of incubation at 30°C and 37°C (Amoikon et al., 2018). The yeast strains were associated with various biotechnological applications, including probiotic    (Fleet, 2007;García-Hernández et al., 2012).

Conclusion
The approaches for identifying yeast as a probiotic source were explored using genotype and phenotype features extracted from tapai as fermented cassava. Survival percentage under artificial gastric acid, the SUL isolate identified as K. ohmeri, exhibited the highest survival percentage of 82.71%, with a growth capacity of 6.20 CFU/mL. The SM isolate was identified as P. kudriavzevii with a survival percentage of 79.74%and growth capacity of 5.75 CFU/mL after 2 hrs incubation in gastric acid of pH 2. The yeast strains P. kudriavzevii and K. ohmeri isolated from the fermented food of tapai have shown promising properties to be further evaluated as probiotic candidates through in vitro methods.

Conflict of interest
The authors declare no conflict of interest.