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

Figures & data

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

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.

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.

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.

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.

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.

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		+	+	+	+	+	+

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).

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