In vitro evaluation of probiotic properties of yeasts and lactic acid bacteria isolated from ersho samples collected from different parts of Ethiopia

ABSTRACT

Ersho is an undefined starter culture used for Ethiopian injera production, and it is a good source of yeasts and lactic acid bacteria (LAB). However, few researchers have been done on the characterization of yeasts and LAB from ersho samples as a source of probiotics. The objective of the current work was to isolate and screen yeasts and LAB from ersho samples and in vitro characterization of their probiotic properties. An experimental research design was used. A total of 55 ersho samples were collected. Based on the ersho samples, 220 yeast colonies and 220 LAB colonies were isolated and purified. For the probiotic characterization, three yeasts (4A, 14D, 54A) and three LAB (1A, 13E, and 55A) were chosen. The isolates 4A, 14D, 54A, 1A, 13E, and 55A were identified as Saccharomyces cerevisiae KRSAN1, Saccharomyces cerevisiae JYC2577, Kazachstania humilis, Enterococcus lactis, Lactobacillus paracasei, and Lactobacillus species, respectively. All the isolates showed positive probiotic characteristics. They had very good acid resistance (percentage survival > 80%), high bile salt resistance (percentage survival > 140%), high cell surface hydrophobicity (>40%), high levels of auto-aggregation (>40%), variable co-aggregation with pathogens (ranged between 42% and 72%), variable antimicrobial activity (clear halo zone ranged from 4.0 to 31 mm), and variable antibiotic resistance responses. The results indicated that the ersho samples can be used as yeast and LAB sources for developing food and probiotic starter cultures.

KEYWORDS:

• Ersho
• food
• lactic acid bacteria
• probiotic
• starter culture
• yeast

Introduction

Probiotics are living microbes that benefit their hosts’ health. When we used them in appropriate quantities, they benefit consumer health.^[1] Many different species, including yeasts and bacteria, are used as probiotics. Lactobacillus species, Bifidobacterium species, Streptococcus species, Lactococcus lactis, and certain Enterococcus species are the most commonly used probiotic bacteria.^[2] Saccharomyces boulardii, Kluyveromyces lactis, Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Pichia kudriavzevii are examples of probiotic yeasts.^[3]

Dairy products, meat, vegetables, and other fermented foods can all be consumed with probiotics.^[4] Eating fermented foods with probiotics has many health advantages, including preventing and treating diarrheal diseases, preventing systemic infections, managing inflammatory bowel disease, immunomodulating, preventing and treating allergies, alleviating lactose intolerance, lowering cholesterol levels, being antipathogenic, antidiabetic, antiobesity, anti-inflammatory, anticancer, and antihypertensive.^[5]

Probiotic microorganisms have been chosen based on a variety of characteristics. To qualify as probiotics, microorganisms must meet a number of requirements, including being safe, viable, able to adhere to intestinal surfaces, colonization and survival in the gastrointestinal region, antimicrobial activity against pathogenic microbes, high tolerance to acid and bile, having an antibiotic resistance profile, and maintaining a balanced intestinal microbiota.^[6]

Yeasts and lactic acid bacteria (LAB) are the two most common microorganisms used as probiotic sources. Yeast species can be employed as probiotics. Due to their technological characteristics, such as their resistance to bile salt and low pH, ability to Growth at various temperatures and NaCl concentrations, and antagonistic action against food-borne pathogens,^[7] able to colonize and persist in the gastrointestinal region, and their immunomodulatory activities in simulated intestinal tract circumstances.^[8] Candidate probiotic bacteria known as LAB are frequently used in the food sector. Because LAB are generally recognized as safe (GRAS) by the WHO, they can be consumed without risk. They are crucial to the process of food fermentation because they prevent pathogenic and spoilage microorganisms from growing and enhance the taste, aroma, and texture of fermented foods.^[9]

It is commonly acknowledged that probiotics have been used to manage harmful microbes in humans, animals, and foods. However, the use of probiotics to treat animals or preserve food was not common in Ethiopia. The majority of fermented foods in Ethiopia are also produced by spontaneous fermentation, which left the diet lacking in sufficient number of probiotic microorganisms. Therefore, for the meal to provide any health benefits during its shelf life, there must be a sufficient number of active probiotic microorganisms. Thus, the goal of the current study was to characterize the probiotic properties of lactic acid bacteria and yeast isolates isolated from ersho samples in order to promote the use of probiotic starter cultures for the production of fermented foods and probiotic source.

Materials and methods

Study area

The study areas are Addis Ababa, Debre Berhan, Debre Markos, Bahir Dar, and Gondar, which all are located in Ethiopia.

Research design

An experimental research design was used to isolate, screen, and in vitro characterization of probiotic properties of yeast and lactic acid bacteria isolated from ersho samples.

Sample collection

A total of 55 ersho samples were collected from the study areas. Sterile 150 ml bottles were used to collect ersho samples. Sample numbers from 1–15, 16–25, 26–35, 36–45, and 46–55 were collected from Gondar, Bahir Dar, Debre Markos, Addis Ababa, and Debre Berhan cities, respectively. The collected ersho samples were transported to Gondar University, Ethiopia by ice box.

Isolation and purification of yeast and lactic acid bacteria

Yeast extract peptone dextrose agar plates (YPD) and de Man Rogosa and Sharpe (MRS) agar plates were used for the isolation and purification of yeasts and lactic acid bacteria from ersho samples, respectively.

Screening the isolates

Growth rate, de Man Rogosa and Sharpe (MRS) broth acidification, flour extract broth acidification and hydrogen sulfide production were used for screening lactic acid bacteria isolates.^[10] Gas production,^[11] hydrogen sulfide production,^[12] growth rate,^[11] and dough raising parameters were used for screening yeast isolates.

Molecular identification of isolates

Isolate DNA extraction was done according to the protocol of Gen Elute genomic DNA purification kit. After extraction of DNA, they were subjected to polymerase-chain reaction amplification. The internal transcribed spacer regions (ITS1 and ITS4) of 5.8S of ribosomal DNA (5.8S rDNA) (for yeast) and 16S rDNA (for LAB) were amplified using a universal primer.^[13]

The reaction mixture for PCR amplification for each yeast isolate DNA was prepared using 20 μl volume containing 0.4 μl ITS-1 (5”-TCC GTA GGT GAA CCT GCG G-3‘), 0.4 μl ITS-4 (5’-TCC TCC GCT TAT TGA TAT GC-3”), 4.0 μl master mix (BioDyne5×FIREPolR master mix), 12.2 μl PCR water, and 3.0 μl DNA sample. The PCR conditions were an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 40 s, extension at 72°C for 1 min and final extension at 72°C for 5 min.

The reaction mixture for PCR amplification for each lactic acid bacterium isolate DNA was prepared using 20 μl volume containing 0.3 μl 27F - (AGAGTTTGATCMTGGCTCAG), 0.3 μl 1492 R - (TACCTTGTTACGACTT primer), 4.0 μl master mix (BioDyne5×FIREPolR master mix), 13.4 μl PCR water, and 2.0 μl DNA sample. The PCR conditions were an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 40 s, extension at 72°C for 2 min and final extension at 72°C for 7 min.

DNA extraction products and PCR products were separated on a 1% agarose gel containing 3 μl of ethidium bromide and visualized under UV light. After obtaining DNA sequencing, the sequencing errors were edited by using BioEdit Sequencer. After editing, the DNA sequences of yeast and LAB isolate samples were compared with GenBank National Center for Biotechnology Information (NCBI) database sequences by using basic alignment search tool (BLAST). Blasted sequences were aligned by using Clustal W. Phylogenetic tree of yeast, and LAB isolates were constructed by using molecular evolutionary genetic analysis 7 (MEGA 7), using a maximum likelihood algorithm character estimation method.^[13]

In vitro characterization of probiotic properties of isolates

Temperature tolerance

The effect of different temperature values on yeast and lactic acid bacteria isolate growth was measured by using spectrophotometer.^[14]

Acid and bile salt tolerance

For acid tolerance determination, 1 ml of a fresh MRS broth culture containing 1.5 × 10⁸ cfu/ml of lactic acid bacteria isolate suspension was added to 9 ml of MRS broth having pH 2.0, 2.5, 3.0, and incubated at 37°C for 3 h. After incubation, the optical density (OD) of LAB cultures was measured at 600 nm. Similarly, for bile salt tolerance determination, 1 ml of a fresh MRS broth LAB suspension was grown in 9 ml of fresh MRS broth having 0.3, 0.5, 1% bile salt and incubated at 37°C for 4 h. After incubation, the optical density of LAB culture was measured at 600 nm. Acid and bile salt tolerance of yeast isolates were measured. The acid and bile tolerance was estimated by determining the survival rate using the following equation: Survival Rate (%): [OD (After treatment)/OD (Before treatment)] × 100.^[15]

Cell surface hydrophobicity

The cell surface hydrophobicity of the LAB isolates were determined by following the procedure used by Sharma et al.^[16] and Prabhurajeshwar and Chandrakanth.^[17] The LAB isolates were inoculated into MRS broth and incubated at 37°C for 24 h followed by centrifugation at 5000 rpm for 15 min. After centrifugation, pellets were washed twice with PBS and adjusted for cell density to 0.65 OD by measuring their optical density (A) at 600 nm. After measuring OD, 5 ml of LAB suspension was mixed with 1 ml of solvent (chloroform, ethyl acetate, and xylene) and mixed by vortex for 5 min followed by incubation at 37°C for 1 h. After incubation, the solvent was discarded by using micropipette and then the optical density of the LAB suspension was measured. The percentage of cell surface hydrophobicity (CSH) of LAB was calculated as follows: CSH (%) = (1 -A_after/A_before) x100. Cell surface hydrophobicity of yeast isolates was also measured.^[3,18]

Auto-aggregation ability

Lactic acid bacteria (LAB) isolates were inoculated into MRS broth and incubated at 37°C for 24 h followed by centrifugation at 6000 rpm for 20 min, 4°C. LAB cells (pellets) were washed twice with phosphate buffer solution. Twenty milliliters of LAB suspension were added to the test tubes. The test tubes were mixed by vortex for 5 min and incubated at 37°C for 24 h. From each upper LAB suspension, their optical density was measured at 600 nm during 0, 5, and 24 h incubation. The auto-aggregation (AA) percentage of LAB isolates was calculated as follows: AA (%) = (1-(A_time/A_initial)) × 100, where: A time: absorbance at 5 h, 24 h and A initial: absorbance at 0 h incubation.^[3,18] The auto-aggregation ability of yeast isolates was also measured.^[19,20]

Antimicrobial activity

The agar well-diffusion method was used to evaluate the antimicrobial activity of LAB and yeast isolates. The test organisms (Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Shigella dysenteriae) were obtained from the microbial laboratory, Institute of Biotechnology, Gondar University, Ethiopia. Four milliliters of yeast isolates and 4 ml of LAB isolates (0.5 McFarland) were inoculated into 45 ml YPD and 45 ml MRS broths, respectively. They were incubated at 37°C for 24 h. After incubation, they were centrifuged at 4000 rpm for 10 min at 4°C. The cell-free supernatants were mixed with 70% ammonium sulfate and incubated at 4°C for overnight. After incubation, they were centrifuged at 4000 rpm for 10 min. The pellets were mixed with 3 ml of distilled water, and the suspension was used as antagonistic study against test organisms. The pure cultures of test organisms were inoculated into nutrient broth and incubated at 37°C for 24 hr. After incubation, a volume of 100 µl of inoculum (0.5 McFarland) from each test organism was swabbed evenly over the surface of Muller Hinton agar plates with a sterile cotton swab. After spreading, a micropipette tip (9 ml in diameter) was used to prepare the agar well. Each well was filled with an isolated suspension. The plates were incubated at 37°C for 24 h. After incubation, the clear halo zone around the well was measured in mm.^[7,19]

Co-aggregation ability

Yeast isolates, LAB isolates, and test organisms were inoculated into YPD, MRS, and nutrient broth, respectively. They were incubated at 37°C for 24 h. Isolates and test organisms were adjusted to a density of 1.5 × 10⁸ cfu/ml. After cell density adjustment, 10 ml of yeast isolates, 10 ml of LAB isolates,10 ml of yeast-test organism mixture (1:1) and 10 ml LAB-test organism mixture (1:1) were incubated at 37°C for 24 h. Absorbance of yeast, LAB, yeast-test organism mixture, and LAB-test organism mixture were measured during 0, 4, and 24 h incubation. The percentage of co-aggregation (CA) was calculated as follows:

$\mathrm{C}\mathrm{A}\left(\mathrm{\%}\right)=\left(\left(\left({\mathrm{A}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{s}\mathrm{m}}+{\mathrm{A}}_{\mathrm{p}\mathrm{r}\mathrm{o}}\right)/2-{\mathrm{A}}_{\mathrm{m}\mathrm{i}\mathrm{x}}\right)/\left({\mathrm{A}}_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{s}\mathrm{m}}+{\mathrm{A}}_{\mathrm{p}\mathrm{r}\mathrm{o}}\right)/2\right)\mathrm{X}100$

Where: A_{test organism}, A_pro and A_mix represent absorbance of individual test organisms, pro, and their mixture after incubation for 5, 24 h, respectively.^[17]

Antibiotic resistance

The disc diffusion method was used for determining antibiotic resistance of yeast and LAB isolates against some antibiotic discs, including amoxicillin (45 μg), ampicillin (10 μg), chloramphenicol (30 μg), tetracycline (30 μg), gentamycine (10 μg), cotrimoxazole (25 μg), ciprofloxacin (5 μg), erythromycin (10 μg), vancomycine (30 μg), novobiocin (30 μg), and bacitracin. A volume of 100 µl cultures of isolates were swabbed evenly over the surface of Muller Hinton agar plates with a sterile cotton swab, and then antibiotic discs were placed. Plates inoculated with Lactic acid bacteria were incubated at 37°C for 24 h anaerobically. Plates inoculated with yeast isolates were incubated at 37°C for 24 h aerobically. After incubation, a clear halo zone around the discs was measured in mm and expressed as susceptible, S (≥21 mm); intermediate, I (16–20 mm) and resistance R (≤15 mm).^[21,22]

Hemolytic activity

Yeast and lactic acid bacterial isolates (24 h old cultures) were streaked on a blood agar base supplemented with 5% sheep blood and incubated at 37°C. After 48–72 h incubation, the hemolytic activity of yeast and LAB isolates was evaluated by observing partial hydrolysis of red blood cells and the production of a green zone (α-hemolysis), total hydrolysis of red blood cells producing a clear zone around the streaked isolate cultures (β-hemolysis) or no hydrolysis red blood cell, not form green and clear halo zone, growth normally (γ-hemolysis).^[23,24]

Gelatinase activity

A 200 µl of LAB cultures (24 h old cultures) were inoculated into 10 ml of gelatin broth (12% gelatin, 0.5% peptone, and 0.3% beef extract) and incubated at 37°C for 24 h. After the incubation, the cultures were placed in the refrigerator at 4°C for 1 h. After the cultures were refrigerated, if the cultures solidified, they were considered as negative response to gelatinase activity.^[25]

A 200 µl of yeast cultures (48 h old cultures) were inoculated into 10 ml of gelatin broth (g/L: tryptone 17.0, peptone 3.0, dextrose 2.5, NaCl 5.0, K₂HPO₄ 2.5, and 12% gelatin). The test tubes were incubated at 37°C for 3 days. After incubation, the cultures were incubated at 4°C for 1 h. After incubation, the cultures that form solidification were considered as negative response to gelatinase activity.^[26]

Biogenic amine production

Two hundred microliters of isolate suspension (LAB and yeast isolates separately) (0.5 McFarland at 600 nm) were inoculated into 20 ml of broth (peptone 5 g, yeast extract 3 g, glucose 1 g, bromocresol purple 0.02 g, amino acids 5 g (arginine, tyrosine, histidine, lysine), and distilled water 1000 ml). The test tubes were incubated at 37°C for 5 days. LAB and yeast isolates that do not form purple coloration, and they were considered as none-biogenic amine producers.^[27]

Beneficial role of isolates

Beneficial role of yeast and lactic acid bacteria isolates like proteolytic activity^[28] and exopolysaccharide production capacity was measured.^[29]

Data analysis

Lactic acid bacteria and yeast probiotic characteristics such as stress tolerance, adhesion ability, antimicrobial activity, and safety were presented in terms of mean and standard deviation. SPSS version 25 was used to calculate the mean and standard deviation. One way Anova (Duncan) is used to evaluate the presence or absence of statistically significant differences among lactic acid bacteria and yeast probiotic characteristics. The significance level was established as p ≤.05.

Results

Isolation and purification of isolates

A total of 55 ersho samples were gathered from the study sites and used for the lactic acid bacteria and yeast isolation processes. Four colonies (labeled as A, B, C, and D), a total of 220 lactic acid bacteria colonies, and 220 yeast colonies were randomly selected and isolated from collected ersho samples. By repeatedly (three times) sub-culturing, the isolated lactic acid bacteria and yeast isolates were purified.

Screening the isolates

From a total of 220 lactic acid bacteria isolates, three isolates were chosen based on growth rate, hydrogen sulfide production, MRS (de Man, Rogosa, and Sharpe) broth acidification, and wheat flour extract broth acidification tests. Criteria like gas production, hydrogen sulfide formation, and growth rate were used for screening three yeast isolates from a total of 220 yeast isolates (Table 1).

Table 1. Parameters for screening lactic acid bacteria (LAB) and yeast isolates.

Isolates	Screening parameters	Total no. of isolates tested	Criteria for selection	Total number of isolates
LAB	Growth rate	220	Optical density ≥ 0.6	53
	Acidify MRS	53	Change of pH <4	37
	Acidify flour	37	Change of pH <4	11
	H2S production	11	None H2S producers	11
	Growth, acidification	11	High character	3
Yeasts	Gas production	220	Strong	4
			Medium	57
			Low	154
			None-producer	5
	H2S production	39	Green color	18
			Light brown	2
			Dark brown	14
			Black color	5
	Growth rate	20	Heavy	8
			Medium	12
			Low	0
	Dough raising	8	Highest dough raising	3

Molecular identification of isolates

Based on molecular analysis, the isolates 4A, 14D, 54A, 1A, 13E, and 55A were identified as Saccharomyces cerevisiae isolate KRSAN1, Saccharomyces cerevisiae strain JYC2577, Enterococcus lactis strain zy-75, Lactobacillus paracasei strain T1301, and Lactobacillus sp. CGMCC wqr2017–3, respectively (Table 2). Figure 1 depicts the evolutionary links among yeast and LAB isolates with other strains.

Figure 1. Phylogenetic tree analysis of yeast and lactic acid bacteria (LAB) isolates. a) Phylogenetic tree of yeasts isolates b) Phylogenetic tree of LAB isolates.

Table 2. Similarity percentage of yeast and LAB isolates with NCBI GenBank database sequence.

Isolates	GenBank accession number	Identity (%)	Closely related organisms	GenBank accession number
4A (MD1)	OQ834414	95	Saccharomyces cerevisiae isolate KRSAN1	OP681676
14D (MD2)	OQ834415	84	Saccharomyces cerevisiae strain JYC2577	MK044057
54A (MD3)	OQ834416	97	Kazachstania humilis clone Turkey strain 270_M45B3–3	OL454733
1A (MD1)	OQ872219	98	Enterococcus lactis strain zy-75	MH602296
13E (MD2)	OQ872220	95	Lacticaseibacillus paracasei strain T1301	MW243990
55A (MD3)	OQ872221	88	Lactobacillus sp. CGMCC wqr2017–3	KY766064

Key: NCBI: national center for biotechnology information.

In vitro characterization of probiotic properties of isolates

Temperature tolerance

Different yeast isolates were grown at various temperature ranges. They showed varying growth rates at various temperatures. They were grown at 25°C, 30°C, 37°C, and 40°C. Not every yeast isolate was cultivated at 45°C. Their fastest growth happened when the temperature reached at 30°C. There was not a significant difference in yeast isolate 4A growth between 25°C and 40°C and 30°C and 37°C at p >.05. At 30°C, yeast isolate 14D grew significantly more than it did at other temperatures at p ≤.05. At p ≤.05, there was a significant difference in the growth of yeast isolate 54A at various temperature treatments (Table 3).

Table 3. Effect of temperature on yeast and lactic acid bacteria growth.

Time	LAB	OD value at 25°C	OD value at 30°C	OD vale at 37°C	OD value at 45°C
24 h	1A	1.26 ± 0.01^a	1.34 ± 0.01^bc	1.37 ± 0.02^c	1.33 ± 0.03^b
	13E	1.29 ± 0.01^a	1.44 ± 0.01^b	1.51 ± 0.06^c	1.31 ± 0.03^b
	55A	1.21 ± 0.01^a	1.40 ± 0.02^b	1.45 ± 0.00^c	1.51 ± 0.01^d
48 h	1A	1.40 ± 0.00^a	1.52 ± 0.22^a	1.54 ± 0.01^a	1.52 ± 0.01^a
	13E	1.49 ± 0.05^b	1.51 ± 0.03^b	1.62 ± 0.11^b	1.43 ± 0.08^a
	55A	1.34 ± 0.00^a	1.48 ± 0.01^b	1.54 ± 0.05^c	1.75 ± 0.04^d
Time	Yeasts	OD at 25°C	OD at 30°C	OD at 37°C	OD at 40°C	OD at 45°C
24 h	4A	0.51 ± 0.00^a	0.69 ± 000^b	0.62 ± 0.01^b	0.49 ± 0.09^a	-
	14D	0.87 ± 0.02^b	0.96 ± 0.05^c	0.15 ± 0.04^a	0.12 ± 0.02^a	-
	54A	0.55 ± 0.04^a	0.71 ± 0.13^a	0.65 ± 0.14^a	0.60 ± 0.13^a	-
48 h	4A	0.73 ± 0.01^a	1.41 ± 0.12^c	0.87 ± 0.02^ab	0.10 ± 0.21^b	-
	14D	1.46 ± 0.05^b	1.64 ± 0.05^c	1.26 ± 0.26^b	0.50 ± 0.02^a	-
	54A	1.10 ± 0.01^a	1.49 ± 0.00^a	1.39 ± 0.23^a	1.15 ± 0.33^a	-

Key: Numbers are expressed as means ± standard deviation in triplicates. The numbers with different superscript lower case letters in the same rows are significantly different at p ≤.05. OD: optical density.

Isolates of lactic acid bacteria (LAB) were subjected to varying temperatures (25°C, 30°C, 37°C, and 45°C). LAB isolates 1A and 13E performed best at a temperature of 37°C, while LAB isolate 55A performed best at a temperature of 45°C. At p ≤.05, the growth of LAB isolate 55A varied significantly with respect to temperature. The growth of LAB isolate 13E was significantly different at p ≤.05 at both 25°C and 45°C. At 25°C, LAB isolate 1A’s growth was significantly lower than it was at other temperatures at p ≤.05 (Table 3).

Acid and bile tolerance

All of the isolates were cultivated at various pH levels. They were shown various survival rates. The growth of isolates was boosted when the pH value was raised from 2 to 3. Yeast isolates grew more rapidly than lactic acid bacteria did. Isolates 14D and 1A had the highest and lowest percentages of survival, respectively. At p >.05, there was not a significant difference in the growth of 4A and 54A isolates at all pH levels. However, at p ≤.05, there was a significant difference in the growth of the bacterial isolates at the various pH levels (Table 4).

Table 4. Percentage survival (%) of isolates in different pH and bile salt values for 3 h.

Yeast and LAB isolates		OD at pH 2	OD at pH 2.5	OD at pH 3
Yeast isolates	4A	133 ± 0.01^a	152 ± 0.04^a	154 ± 0.00^a
	14D	195 ± 0.00^a	211 ± 0.02^ab	226 ± 0.03^b
	54A	164 ± 0.00^a	179 ± 0.00^a	191 ± 0.01^a
LAB isolates	1A	64 ± 0.00^a	79 ± 0.00^b	89 ± 0.00^c
	13E	68 ± 0.00^a	82 ± 0.00^b	95 ± 0.00^c
	55A	80 ± 0.00^a	91 ± 0.00^b	98 ± 0.00^c
Yeast and LAB isolates		OD at 0.3% bile salt	OD at 0.5% bile salt	OD at 1% bile salt
Yeast isolates	4A	152 ± 0.01^b	131 ± 0.000^a	104 ± 0.00^a
	14D	157 ± 0.00^b	143 ± 0.00^b	115 ± 0.00^a
	54A	167 ± 0.00^c	145 ± 0.00^b	138 ± 0.02^a
LAB isolates	1A	185 ± 0.00^b	146 ± 0.00^ab	135 ± 0.01^a
	13E	156 ± 0.01^c	142 ± 0.00^b	111 ± 0.00^a
	55A	186 ± 0.00^a	172 ± 0.00^a	160 ± 0.00^a

Key: Numbers are expressed as means ± standard deviation in triplicates. The numbers with different superscript letters in the same rows are significantly different at p ≤.05, OD: optical density.

Isolates were grown at different bile salt concentrations. But their percentage of survival decreased when the bile salt concentration was raised from 0.3% to 1%. At 0.3%, 0.5%, and 1% bile salt concentration, lactic acid bacterium isolate 55A had survival rates of 186, 172, and 160, respectively. At p ≤.05, there was a statistically significant difference between the growth of 13E and 54A in the various bile salt concentrations (Table 4).

Hydrophobicity and auto-aggregation ability

When the isolates were attached to various solvents, such as xylene, ethyl acetate, and chloroform, they displayed variable degrees of adhesion. Ethyl acetate showed the highest isolated attachment compared to other solvents. The adherence percentages of 4A, 14D, 54A, 1A, 13E, and 55A were 77, 74, 82, 65, 42, and 45, respectively (Figure 2a). All isolates of yeast and lactic acid bacteria showed the ability to aggregate. The ability of yeast isolates to aggregate was greater than the ability of bacteria to aggregate. The incubation time was raised from 5 h to 24 h, the isolates’ capacity for aggregation was improved (Figure 2b).

Figure 2. Cell surface adhesion and aggregation: a) Percentage of cell surface hydrophobicity of yeast and LAB isolates b) Percentage auto-aggregation of yeast and LAB isolates.

Co-aggregation with pathogens

The capacity of the isolates for co-aggregation differed. The ability of isolates to co-aggregate was enhanced when the incubation time was increased from 4 h to 24 h. After 24 h of incubation, isolates 1A, 55A, 13E, 54A, 14D, and 4A showed the best co-aggregation ability with Salmonella typhimurium ATCC 13,311 in increasing order (Figure 3a). Similar to this, at 24 h, the highest co-aggregation with Shigella dysenteriae was observed in isolates 4A (Figure 3b); the highest co-aggregation with Escherichia coli ATCC 25,922 in isolate 54A (Figure 3c) and the highest co-aggregation ability with Staphylococcus aureus ATCC 25,923 was observed in isolate 4A (Figure 3d).

Figure 3. Co-aggregation a) Co-aggregation of isolates with Salmonella typhimurium b) Co-aggregation of isolates with Shigella dysenteriae, c) Co-aggregation of isolates with E. coli, and d) Co-aggregation of isolates with S. aureus.

Safety and beneficial role of isolates

All isolated yeast and lactic acid bacteria responded negatively to gelatinase activity, hemolytic activity, and biogenic amine synthesis. Because there was no liquefied broth formation, no distinct halo zone development around streaked culture, and no purplish color formation, respectively. Proteolytic activity was observed in all isolated yeast and lactic acid bacteria due to the formation of a clear halo zone in skim milk agar plates. They were diacetyl producers, and all isolates showed a red color. All yeast and LAB isolates were exopolysaccharide producers (Table 5).

Table 5. Safety and beneficial role of LAB and yeast isolates.

		LAB response			Yeasts response
Activity of LAB and yeast isolates		1A	13E	55A	4A	14D	54A
Hemolytic (sheep blood)		-	-	-	-	-	-
Gelatinase (gelatin)		-	-	-	-	-	-
Biogenic amine production	Histidine	-	-	-	-	-	-
	Tyrosine	-	-	-	-	-	-
	Lysine	-	-	-	-	-	-
	Arginine	-	-	-	-	-	-
Exopolysaccharide production (mg/100ml)		24	41	32	147	96	138
Diacetyl production		+	+	+	+	+	+
Proteolytic activity		+	+	+	+	+	+

Antibiotic resistance

All yeast isolates (4A, 14D, and 54A) showed antibiotic resistance. They did not form a clear halo zone around the antibiotic disc. But lactic acid bacteria (LAB) isolates had different responses to different antibiotic treatments. LAB isolate 55A was susceptible to chloramphenicol and cotrimoxazole antibiotic discs. Similarly, LAB isolate 1A was susceptible to cotrimoxazole and ciprofloxacin, while LAB isolate 13E was susceptible to cotrimoxazole antibiotic disc. All LAB isolates were resistant to amoxicillin, ampicillin, erythromycin, vancomycin, bacitracin, and novobiocin antibiotic discs (Table 6).

Table 6. Antibiotic susceptibility of yeast and lactic acid bacteria isolates (mm on average).

Antibiotics	1A	13E	55A	4A	14D	54A
Amoxicillin	R	R	R	R	R	R
Ampicillin	R	R	R	R	R	R
Chloramphenicol	R	I	S	R	R	R
Tetracycline	R	I	I	R	R	R
Gentamycine	I	I	I	R	R	R
Cotrimoxazole	S	R	S	R	R	R
Ciprofloxacin	S	S	I	R	R	R
Erythromycine	R	R	R	R	R	R
Vancomycin	R	R	R	R	R	R
Novobiocin	R	R	R	R	R	R
Bacetracin	R	R	R	R	R	R

Key: Susceptible S (≥21 mm); intermediate, I (16–20 mm) and resistance R (≤15 ml).

Antimicrobial activity

The level of each isolate’s antimicrobial activity against pathogens varied. The yeast isolate 4A was more effective than other isolates at killing off Salmonella typhimurium and Escherichia coli. It was more effective than the control to kill these pathogens. The lactic acid bacterium isolate 1A was more effective than other isolates at inhibiting the growth of pathogens (Shigella dysenteriae and Staphylococcus aureus). Both yeast isolates 4A and 14D were more effective than other isolates at controlling the growth of Candida albicans (Table 7).

Table 7. Inhibition zones of isolates against food-borne pathogens (in cm).

Pathogens	Yeast isolates			LAB isolates
	4A	14D	54A	1A	13E	55A	Control
Salmonella typhimurium	2.7 ± 0.10^d	2.5 ± 0.10^cd	2.4 ± 0.10^bc	2.2 ± 0.26^b	0.5 ± 0.12^a	0.4 ± 0.15^a	2.6 ± 0.12^cd
Shigella dysentriae	2.9 ± 0.15^de	2.6 ± 0.15^bc	2.5 ± 0.10^b	3.1 ± 0.10^e	0.7 ± 0.17^a	0.5 ± 0.10^a	2.8 ± 0.12^cd
E. coli	2.6 ± 0.10^d	2.1 ± 0.15^c	2.0 ± 0.26^c	2.1 ± 0.15^c	0.7 ± 0.10^b	0.4 ± 0.17^a	2.5 ± 0.10^d
S. aureus	2.5 ± 0.20^d	2.1 ± 0.15^c	2.1 ± 0.17^c	2.6 ± 0.21^d	0.9 ± 0.15^b	0.3 ± 0.15^a	2.5 ± 0.15^d
Candida albicans	3.1 ± 0.06^c	3.1 ± 0.15^b	2.8 ± 0.12^b	0.8 ± 0.10^a	0.9 ± 0.15^a	0.9 ± 0.06^a	4.2 ± 0.10^d

Discussion

In the current study, all of the isolates negatively responded to hemolytic activity, gelatinase activity, and biogenic amine production. In gelatin medium broth, there was a solidified broth observation that indicates that the isolates did not produce gelatinase enzyme. Similarly, there was no purple color formation in amino acid medium broth, which indicates the isolates did not produce biogenic amines.

All yeast and lactic acid bacteria isolates did not produce hemolytic activity because both a clear halo zone and a green-hued zone were not observed around the streaked cultures on the sheep blood agar plates. This result was consistent with the finding of Pereira et al.^[30] who reported that all yeast isolates negatively responded to hemolytic and gelatinase activity. Supaporn et al.^[31] selected 8 isolates out of 14 lactic acid bacteria through the hemolytic activity of the isolates.

All isolates were grown at body temperature; they had acid and bile salt resistance; antimicrobial activity; cell surface hydrophobicity; auto-aggregation; and co-aggregation ability. One of the parameters for choosing a probiotic source is that it should grow at body temperature. In the current study, both yeast and lactic acid bacteria (LAB) isolates were grown at body temperature. The growth of LAB isolates 1A and 13E were significantly higher at body temperature than other temperature values at p ≤.05. There was no significant difference of yeast isolates 4A and 54A growth at their maximum growth temperature (30°C) and body temperature (37°C) at p ≤.05 (Table 3). These results were in accordance with the findings of Aswani et al.^[14] who observed that isolates were grown at different temperatures, including body temperature.

Low pH affects the growth of microbes. By producing hydrogen ions in the cytoplasm of microbes, stomach acidity damages microbial DNA, enzymes, and proteins and breaks down microbial DNA. Probiotic microbes should be low pH resistant in order to perform their function in GIT. Probiotics had different survival percentages. Probiotics’ percentage survival rates are divided into four groups based on how well they grow at pH 2.5: survival rates below 10% indicate susceptibility; survival rates between 10% and 60% indicate moderate resistance; survival rates between 61% and 80% indicate good resistance; and survival rates above 80% indicate very good resistance.^[32] In the present study, all isolates of yeast and lactic acid bacteria were categorized as having very good resistance to pH 2.5. The percentage of isolates that survived at pH 2.5 ranged from 82% to 211%. Almost, there was no significance difference among the growth of yeast isolates at all pH values tested at p ≤.05. However, there was a significant difference among the growth of LAB isolates at all pH values tested at p ≤.05 (Table 4). These results were consistent with that of Sadeghi et al.^[15]

All yeast isolates in the current study had a higher acid survival percentage than all lactic acid bacteria did (Table 4). According to Mehdi et al.^[33] certain lactic acid bacteria isolates had a lower acid survival percentage than yeast isolates. Isolated yeast and lactic acid bacteria displayed varying survival rates at various pH levels. Probiotic microbes survive at low pH, and they adapt to it through a variety of mechanisms, including cell membrane alteration, biofilm formation, alkali production, H⁺-ATPase activation, amino acid decarboxylase, metabolic regulation, and macromolecule repair.^[34] Cell wall remodeling, activation of cell wall integrity, and activation of general stress response pathways are the main mechanisms by which the probiotic yeasts are able to survive in stomach acidity.

Bile salts have antimicrobial substances that can damage DNA, degrade bacterial membranes, denatured proteins, chelate calcium and iron, and cause harm to microorganisms. Therefore, a microbe must be resistant to bile salts in order to be considered a probiotic. Probiotics have different percentages of survival at 0.3% bile salt concentration. Based on how well they grow in bile salt concentrations of 0.3%, probiotics are defined as having a survival rate of 0%, 1–19%, 20–59%, 60–99%, 100–139%, or greater than or equal to 140%.^[35] Based on this classification, the survival rate for all yeast and lactic acid bacteria isolates was >140%. The isolates’ survival rates ranged from 152% to 186% at 0.3% bile salt concentration. Almost, the growth of all isolates at 0.3% bile concentration was significantly higher than other bile salt concentrations (0.5%, 1%) at p ≤.05 (Table 4). This result was consistent with the finding of Menezes et al.^[36] who reported that isolates with >140% were chosen to be used as probiotic sources.

In the current investigation, the isolates’ survival rates varied. The percentage survival of isolates decreased when the bile concentration was increased from 0.3% to 1%, and vice versa (Table 4). This result was in line with the finding of Ruiz et al.^[37] who observed that with increasing bile concentration, the percentage survival of isolates decreased. The bile tolerance of LAB isolates was almost as high as the bile tolerance of yeast isolates (Table 4). This result contradicted the finding of Mehdi et al.^[33] who observed that the percentage survival of LAB isolates at different bile concentrations was lower than the percentage survival of yeast isolates.

The responses of yeast and LAB isolates to bile salt concentrations were diverse. The reasons might be the natural diversity of yeast and LAB isolates. Probiotic bacteria use a variety of mechanisms for bile salt tolerance, including the production of stress response proteins, protective bio-polymers, bile hydrolyzing enzymes, alterations in the composition of cell membranes, and the biosynthesis of nitrogenous bases and amino acids.^[38] In order to live in bile salt stress environments, probiotic yeasts employ a variety of strategies, including the production of organic molecules such as glycerol and trehalose.^[39]

In the current investigation, isolates’ hydrophobicity percentages of chloroform, ethyl acetate, and xylene ranged from 33% to 51%, 42% to 82%, and 16% to 41%, respectively (Figure 2a). The isolates with ethyl acetate had a percentage of hydrophobicity that fulfilled the minimum standard requirement for using probiotic sources. A probiotic microbe must have a minimum hydrophobicity value of 40%.^[27] According to Lara-Hidalgo et al.,^[40,41] the probiotic microbes with >40% hydrophobicity are hydrophobic.

Yeast isolate 14D and lactic acid bacterium isolate 1A had highest adhesion ability with chloroform solvent. This adhesion ability indicates that the isolates have positive charge and could attach with mucus of gastro intestinal tract. This is due to mucus has a net negative charge.^[42] The solvent with the lowest percentage of hydrophobicity among the others was xylene (Figure 2a). This result was consistent with the finding of Alkalbani et al.^[39] According to Pereira et al.,^[30] xylene was the solvent where isolates had the least adhesion ability. The degree to which yeast and lactic acid bacteria isolates adhered to the solvent varied. The reason might be variation in hydrophilic, hydrophobic, and cell components of the cell wall of isolates. Probiotics can bind to the host GIT in a number of different ways, including lipoteichoic acid, pili, exopolysaccharides, surface proteins, and fimbriae.^[40]

Based on the percentage of auto-aggregation (AA%) at 5 h, probiotics were classified as low (AA% < 30%), intermediate (AA% between 30% and 60%), and high (AA% > 60%).^[36] In this investigation, the isolates’ percentages of auto-aggregation ranged from 51% to 85% at 5 h (Figure 2b). Two isolates (13E and 55A) had intermediate auto-aggregation ability, whereas four isolates (4A, 14D, 54A, and 1A) had high auto-aggregation ability. It has been suggested that the auto-aggregation properties should be greater than 40% for excellent probiotic isolates.^[43] Based on this, all isolated yeasts and LAB had excellent auto-aggregation abilities. Increasing the incubation time resulted in yeast and LAB isolates being more capable of self-aggregation (Figure 2b). This result was consistent with the finding of Alkalbani et al.,^[39] who reported that yeasts’ capacity for auto-aggregation increased as incubation time rose.

Auto-aggregation ability of yeast isolates was higher than isolates of LAB auto-aggregation (Figure 2b). This result was consistent with the finding of Hsiung et al.^[44] who noted that yeast isolates had a larger percentage of auto-aggregation than LAB isolates. Because their cells are bigger and heavier, yeast strains have a superior aggregation capacity than bacteria that precipitate more quickly. Probiotics have the ability to self-assemble due to the presence of exopolysaccharides, surface proteins, and S-layer proteins. Variations in cell wall composition, the presence and type of cell appendages, and projecting macromolecules from the cell wall all affect the auto-aggregation ability of isolates.^[45]

In the present study, isolates showed different co-aggregation percentages (Figure 3). These results were in agreement with the findings of Bhushan et al.^[46] who reported that isolates had a wide range of co-aggregation values against pathogens. Bruna et al. ^[48] who reported that yeast and lactic acid bacteria isolates could aggregate with different pathogens. Co-aggregation ability is one mechanism of probiotic for controlling the colonization of pathogens to the intestine.

The main mediators for the co-aggregation of probiotic microorganisms with pathogens are cell surface proteins, S-layer components, fimbriae, and lipopolysaccharides.^[47] The primary components of the yeast cell wall, mannose, glucans, and chitin, may all be connected to yeast co-aggregation with pathogens.^[39] Co-aggregating bacteria and microbial pathogens adhering to one another within cells and the development of biofilms of auto-aggregating bacteria on the intestinal mucosa.^[48]

All of the yeast isolates used in the investigation were resistant to antibiotics. However, LAB isolates had different responses to antibiotic resistance (Table 6). This result was consistent with the finding of Pereira et al.^[30] who discovered that all yeast isolates were resistant to antibiotics. Mohammed & Con^[49] made the observation that all LAB isolates were ampicillin susceptible. The coding genes for antibiotic resistance were found in chromosomes and plasmids. Antibiotic resistance genes found in plasmids are passed to other microbes through horizontal gene transfer. However, chromosomal antibiotic resistance genes cannot be passed on to other organisms.

All of the isolates in the current study showed inhibitory activity against food-borne pathogens. There was no significant difference between isolate 4A and control inhibition zone for Salmonella typhimurium, and E. coli treatment at p > .05. The inhibition clear halo zones of the various isolates varied (Table 7). This result was in accordance with the findings of Bruna et al.^[18] who reported that yeast and lactic acid bacteria isolates showed different degree of antimicrobial activity. Variations in the incubation period for the generation of antimicrobial metabolites,^[50] strain diversity,^[51] and target variation^[52] are possible causes.

Conclusion

Potential probiotic yeast and lactic acid bacteria strains were isolated and purified from ersho samples. They were fulfilled the criteria for selecting a candidate probiotic starter culture in vitro characterization such as growth in body temperature, low acid tolerance, bile salt tolerance, lack of hemolytic and gelatinase activity, and with no biogenic amine production. Furthermore, they showed advantageous probiotic characteristics such as high cell surface hydrophobicity, high auto-aggregation capability, high co-aggregation ability with food-borne pathogens, and high antimicrobial activity. The present study goal was also achieved by screening yeast stains (Saccharomyces cerevisiae KRSAN1, Saccharomyces cerevisiae JYC2577, Kazachstania humilis) and lactic acid bacteria strains (Enterococcus lactis, Lactobacillus paracasei, and Lactobacillus species) from ersho samples that are used as suitable for probiotic starter culture development. However, further in vivo study will be needed to use them for practical application at industrial level.

Acknowledgments

We would like to acknowledge the Department of Industrial and Environmental Biotechnology, Institute of Biotechnology for providing laboratory space, laboratory chemicals, and laboratory media for doing this research.

Disclosure statement

The authors declare no conflict of interest.