Probiotic Characterization of <i>Enterococcus</i> spp. Isolated from Raw Goat Milk

The aim of this research was to determine the probiotic potential and safety of lactic acid bacteria (LAB) isolated from raw goat milk. Gram positive and catalase negative bacteria were isolated from raw goat milk (n = 61) and identified as LAB. LAB isolates were screened for antimicrobial, probiotic and technological characteristics. LAB isolates showed antimicrobial activity against foodborne pathogens (Staphylococcus aureus, Escherichia coli and Salmonella Typhimurium) and high survival rate at pH 2 (93.54-100.38% after 4h), in the presence of 0.3% bile salts (100.85-108.96% after 4h) and simulated gastric fluid (74.16-80.13% after 3h). Three LAB isolates (1, 3 and 13) with high antimicrobial activity against all foodborne pathogens and probiotics characteristics were subjected to 16S rRNA sequencing and identified as Enterococcus faecium strains. Enterococcus spp. exhibited milk coagulation potential, amylolytic activity, susceptibility to antibiotics and no evidence of hemolysis. Enterococcus spp. isolated from goat milk showed probiotic and technological characteristics and can be used as a starter culture after further safety evaluation.

Keywords:
Lactic acid bacteria; Probiotics; Antimicrobial activity; Raw goat milk; Enterococcus spp

Isolate No.	Isolates Code	Growth Condition
1	S4	Anaerobic
2	S9	Aerobic
3	S11	Aerobic
4	S17A	Aerobic
5	S17B	Anaerobic
6	S19A	Aerobic
7	S19B	Anaerobic
8	S21A	Aerobic
9	S21B	Anaerobic
10	S22	Anaerobic
11	S25	Anaerobic
12	S43	Anaerobic
13	S49	Anaerobic
14	S51	Anaerobic
15	S52	Anaerobic
16	S56A	Aerobic
17	S56B	Anaerobic
18	S57	Aerobic
19	S58A	Aerobic
20	S58B	Anaerobic
21	S59	Aerobic
22	S60A	Aerobic
23	S60B	Anaerobic
24	S61A	Aerobic
25	S61B	Anaerobic

Isolates	Spot on lawn assay (mm)			Well diffusion assay (mm)
Isolates	E. coli	S. aureus	S. Typhimurium	E. coli	S. aureus	S. Typhimurium
1	27.33 ± 0.58^bA	24 ± 2.00^fghA	25.33 ± 3.51^cdeA	18 ±3.00^aA	20.33 ±1.15^aA	25 ±4.58^aA
3	33.33 ± 1.53^aB	38.33 ± 1.53^abA	35.33 ± 0.58^abAB	19.67 ±3.06^aA	20.33 ±4.17^aA	19.33 ±2.08^abA
4	35.67 ± 0.58^aA	37.67 ± 2.5^abcA	35.00 ± 1.00^abA	-	17.33 ±2.08^aA	17 ±2.00^bA
5	22.67 ± 0.58^bcdeB	40.00 ± 0.00^aA	25.33 ± 3.51^cdeB	-	-	-
6	24.67 ± 0.58^bcdB	26.67 ± 0.15^efgA	-	-	-	-
8	23.00 ± 3.61^bcdeA	23.33 ± 3.31^fghA	20.00 ± 2.00^eA	-	-	-
12	25.33 ± 0.58^bcdC	30.33 ± 0.58^cdefB	35.53 ± 0.58^abA	-	-	-
13	23.67 ± 0.58^bcdeB	30.67 ± 0.50^bcdefA	29.33 ± 2.52^bcA	21.00 ±3.61^aA	18.33 ±0.58^aA	16.67 ±1.53^bA
14	21.33 ± 5.86^cdeA	20.33 ± 5.58^ghA	29 ± 2.65^bcdA	-	-	-
15	26.33 ± 0.58^bcA	28.67 ± 0.53^defA	-	-	-	-
16	23.67 ± 1.53^bcdeB	25.00 ± 1.36^fghB	39.67 ± 0.58^aA	-	-	-
18	24.67 ± 1.53^bcdA	25.67 ± 1.5^efgA	28.33 ± 1.53^bcdA	-	18.00 ±1.00^aA	15.67 ±1.53^bA
19	18.00 ± 1.00^eB	17.67 ± 1.53^hB	21.67 ± 1.15^deA	-	-	-
21	33.67 ± 2.08^aA	33.00 ± 2.0^abcdeA	29.67 ± 1.53^bcA	-	-	-
22	20.00 ± 1.00^deA	26.00 ± 1.93^efgA	28.33 ± 2.89^bcdA	-	-	-
23	22.33 ± 1.53^bcdeB	35.33 ± 1.5^abcdA	28.33 ± 5.51^bcdAB	17 ±1.00^aA	-	15 ±2.65^bA

Isolates	Acid tolerance				Bile tolerance (0.3% bile)	SGF with pepsin at pH 2		Heat resistance
	pH 2.0		pH 4.0		Bile tolerance (0.3% bile)	SGF with pepsin at pH 2		Heat resistance
	2h	4h	2h	4h	4h	1h	3h
1	103.31 ± 2.24^a	96.85 ± 3.11^a	95.95 ± 1.26^b	80.79 ± 2.47^c	102.62 ± 3.23^a	82.40 ± 2.18^c	76.66 ± 3.55^a	95.26 ± 0.28^a
3	95.22 ± 4.92^a	93.54 ± 4.85^a	96.47 ± 2.15^ab	91.48 ± 4.97^b	100.85 ± 3.82^a	93.56 ± 1.98^ab	78.97 ± 4.23^a	95.56 ± 3.65^a
4	79.97 ± 2.96^b	97.25 ± 2.72^a	104.55 ± 4.55^a	100.26 ± 3.34^ab	103.20 ± 3.55^a	87.33 ± 2.79^bc	74.16 ± 3.06^a	96.30 ± 3.15^a
13	82.27 ± 3.48^b	100.38 ± 1.23^a	88.86 ± 2.62^b	108.40 ± 4.53^a	104.19 ± 0.69^a	91.99 ± 4.04^ab	78.94 ± 3.71^a	97.72 ± 0.43^a
18	96.34 ± 2.68^a	97.43 ± 4.76^a	90.30 ± 4.59^b	94.33 ± 4.61^b	108.96 ± 4.55^a	98.33 ± 2.93^a	74.23 ± 3.36^a	96.54 ± 2.79^a
23	^-	^-	97.21 ± 2.36^ab	97.67 ± 2.37^ab	103.80 ± 4.21^a	97.83 ± 3.66^a	80.13 ± 3.45^a	96.05 ± 1.00^a

Isolates	Autoaggregation %		Coaggregation %
Isolates	3 h	24 h	2 h	24 h			Clear zones around colonies
1	8.5 ± 0.17^c	28.45 ± 0.30^f	7.49 ± 0.35^d		47.16 ± 0.85^c	9.21 ± 1.73^a		+
3	10.29 ± 0.12^a	55.36 ± 0.34^a	9.44 ± 0.29^b		35.16 ± 0.89^e	8.88 ± 2.03^a		+
4	9.31 ± 0.15^b	51.45 ± 0.28^b	8.39 ± 0.27^c		59.27 ± 0.35^a	6.41 ± 0.66^a		-
13	10.50 ± 0.39^a	30.49 ± 0.31^e	8.19 ± 0.15^cd		44.33 ± 1.07^d	9.83 ± 0.64^a		+
18	6.50 ± 0.32^d	47.22 ± 0.17^c	10.40 ± 0.33^a		50.87 ± 0.96^b	9.16 ± 2.07^a		-
23	9.64 ± 0.14^b	41.36 ± 0.46^d	9.84 ± 0.16^ab		58.15 ± 0.97^a	6.18 ± 2.29^a		+

Isolates	Zone of inhibition (mm)
	Ampicillin		Vancomycin	Tetracycline	Ciprofloxacin	Streptomycin	Septran	Gentamicin	Chloramphenicol	Amikacin
1	21.67 ± 2.08^bC	23.67 ± 1.53^aC		35.67 ± 3.06^aA	12 ± 2.00^bD	9.67 ± 1.15^aD	-	13 ± 2.00^aD	29.33 ± 0.58^aB	18.67 ± 0.58^aC
3	26.67 ± 2.08^abA	21.67 ± 0.58^aB		28.67 ± 1.15^bA	9.33 ± 1.15^bC	-	-	10.67 ± 1.53^aC	19.33 ± 1.15^bB	-
13	28.33 ± 2.08^aB	21.67 ± 0.58^aC		33.33 ± 2.08^abA	17.67 ± 1.53^aD	-	-	-	27 ± 1.73^aB	-

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