In-vitro Antimicrobial Activity of Essential Oils and Spices Powder of some Medicinal Plants Against Bacillus Species Isolated from Raw and Processed Meat

Figures & data

Table 1 Occurrence of Bacillus spp. in Samples Collected During the Three Periods

Food Sample	First Season				Second Season				Third Season
	Number of Samples Tested	Number of Presumptive Colonies	Occurrence of Bacillus spp. in Samples	% of Bacillus spp. in Samples	Number of Samples Tested	Number of Presumptive Colonies	Occurrence of Bacillus spp. in Samples	% of Bacillus spp. in Samples	Number of Samples Tested	Number of Presumptive Colonies	Occurrence of Bacillus spp. in Samples	%of Bacillus spp. in Samples
Fresh veal meat	2	9	-ve	0.0	2	3	-ve	0.0	2	12	-ve	0.0
Fresh chicken meat	2	14	1 isolate	50	2	4	1 isolate	50	2	6	-ve	0.0
Fresh mutton meat	1	11	-ve	0.0	1	7	-ve	0.0	1	10	-ve	0.0
Beef luncheon	4	13	1 isolate	25	4	15	1 isolate	25	4	15	1 isolate	25
Chicken luncheon	4	15	-ve	0.0	4	9	-ve	0.0	4	14	-ve	0.0
Ground beef	4	7	2 isolates	50	4	21	2 isolates	50	4	26	1 isolate	25
Basterma	2	16	1 isolate	50	2	13	-ve	0.0	2	7	-ve	0.0
Beef burger	1	22	1 isolate	100	1	23	1 isolate	100	1	5	1 isolate	100
Total	20	107	6 isolates	30	20	95	5 isolates	25%	20	95	3 isolates	15

Table 2 Biochemical Reaction Results of Selected Strains

Bacillus oceanisedimins		Brevibacillus invocatus
Test	Result	Test	Result
Isolation source	Minced beef	Isolation source	Minced beef
Motility	+ve	Motility	+ve
Morphology	Spore forming rods	Morphology	Spore-forming rods
Gram-stain	+ve	Gram-stain	+ve
Temperature for growth	37	Growth temperatures range	15−35
Temperature optimum range	25–45	pH range	6–8.5
pH for growth	7	Catalase	+ve
pH optimum range	6–9	Indole	Weight
NaCl for growth (%)	0–12	Nitrate reduction	-ve
Indole	-ve	Casein hydrolysis	-ve
Methyl red	+ve	Gelatin hydrolysis	-ve
Voges-Proskauer	-ve	Urea hydrolysis	-ve
Catalase	+ve	Starch hydrolysis	-ve
Glycerol	+ve	Assimilation of:
Ribose	+ve	D-Alanine	-ve
Galactose	+ve	L-Alanine	+ve
Glucose	+ve	L-Aspartate	-ve
Fructose	-ve	D-Fructose	-ve
Mannitol	-ve	Fumarate	-ve
Maltose	-ve	D-Gluconate	-ve
Lactose	-ve	Glutamate	+ve
Saccharose	-ve	DL-Glycerate	-ve
Strach	-ve	Lactulose	-ve
Gelatin	+ve	Maltose	-ve
		Sucrose	-ve

Abbreviations: +ve, positive; -ve, negative.

Table 3 Sequence Analysis of the 16S rDNA Gene by BLASTn Tool

Isolates	Name Of Closely Associated Strain	Identity %	Gene Bank Accession Number of Closely Associated Strain
AKM5	Bacillus oceanisediminis strain C22	96.18%	MT457440.1
AKM6	Brevibacillus invocatus strain B32	99.23%	MH587029.1

Figure 1 Phylogenetic dendrogram based on 16S rRNA gene sequences showing the site of strain AKM5 & AKM6 between members of diverse genus species. The evolutionary history was inferred using the UPGMA method.⁵⁹ The optimal tree with the sum of branch length = 0.13321378 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method⁶⁰ and are in the units of the number of base substitutions per site. The analysis involved 21 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 258 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.⁶¹

Table 4 Changes in Bacillus spp. Log₁₀ Count (CFU/g) of Minced Samples Treated with Essential Oils During Storage at 4±1°C Up to 15 Days

Variables	0	3	6	9	12	15	Mean±SD	P value
Control	3.333	3.349	3.372	3.379	3.405	3.439	3.380±0.038	–
Cumin 0.25%	3.101	2.978	2.904	2.654	0.00	0.00	1.940±1.509	0.0138
Cumin 0.50%	3.049	2.813	2.532	0.00	0.00	0.00	1.399±1.541	0.0010
Black seeds 0.25%	2.876	2.749	2.708	0.00	0.00	0.00	1.389±1.522	0.0011
Black seeds 0.50%	2.557	2.079	0.00	0.00	0.00	0.00	0.77±1.207	0.0001
Cloves 0.25%	2.939	2.881	2.741	2.579	0.00	0.00	1.857±1.444	0.0052
Cloves 0.50%	2.929	2.827	2.613	0.00	0.00	0.00	1.395±1.531	0.0013
Cinnamon 0.25%	3.194	2.898	2.807	2.699	0.00	0.00	1.933±1.506	0.0088
Cinnamon 0.50%	3.153	3.101	2.807	0.00	0.00	0.00	1.510±1.659	0.0037
Marjoram 0.25%	3.207	3.162	3.121	2.987	2.793	0.00	2.545±1.256	0.1412
Marjoram 0.50%	3.144	3.072	2.893	0.00	0.00	0.00	1.518±1.665	0.0047

Note: P: Significance between control group and each tested spices powder.

Abbreviation: S.D, std. deviation.

Figure 2 Changes in Bacillus spp. Log₁₀ counts (CFU/g) of treated minced samples with essential oils during storage at 6±1°C for up to 15 days.

Table 5 Changes in Bacillus spp. Log₁₀ Count (CFU/g) of Minced Samples Treated with Spices Powders During Storage at 4±1°C Up to 15 Days

Variables	0	3	6	9	12	15	Mean±SD	P value
Control	3.333	3.349	3.372	3.379	3.405	3.439	3.380±0.038	–
Cumin 0.50%	3.327	3.221	3.094	2.634	0.00	0.00	2.046±1.602	0.0229
Cumin 1.0%	3.309	3.128	2.717	0.00	0.00	0.00	1.526±1.682	0.0029
Black seeds 0.50%	3.188	3.049	2.833	2.506	0.00	0.00	1.929±1.512	0.0082
Black seeds 1.0%	3.089	2.448	0.00	0.00	0.00	0.00	0.923±1.444	<0.0001
Cloves 0.50%	3.272	3.128	2.935	2.613	0.00	0.00	1.991±1.558	0.0136
Cloves 1.0%	3.194	2.959	2.592	0.00	0.00	0.00	1.458±1.608	0.0014
Cinnamon 0.50%	3.215	2.935	2.741	2.569	2.128	0.00	2.265±1.168	0.0114
Cinnamon 1.0%	3.282	2.974	2.634	2.343	0.00	0.00	1.872±1.484	0.0044
Marjoram 0.50%	3.291	3.042	2.833	2.592	2.302	0.00	2.343±1.198	0.0184
Marjoram 1.0%	3.261	2.949	2.654	2.463	0.00	0.00	1.888±1.487	0.0049

Note: P: Significance between control group and each tested spices powder.

Abbreviation: S.D, standard deviation.

Figure 3 Changes in Bacillus spp. Log₁₀ counts (CFU/g) of treated minced samples with spices powder throughout storage at 6±1°C for up to 15 days.

Table 6 Antibacterial Activities of Some Essential Oils and Spices Powder Against Bacillus spp.

Essential Oil of the Plant	Inhibition Zone (mm)	Spices Powder of the Plant	Inhibition Zone (mm)
Cumin oil 50 mg/mL	16	Cumin 50 mg/mL	14
Cumin oil 100 mg/mL	18	Cumin 100 mg/mL	16
Black seeds oil 50 mg/mL	20	Black seeds 50 mg/mL	16
Black seeds oil 100 mg/mL	22	Black seeds 100 mg/mL	18
Cloves oil 50 mg/mL	16	Cloves 50 mg/mL	14
Cloves oil 100 mg/mL	20	Cloves 100 mg/mL	18
Cinnamon oil 50 mg/mL	14	Cinnamon 50 mg/mL	12
Cinnamon oil 100 mg/mL	14	Cinnamon 100 mg/mL	12
Marjoram oil 50 mg/mL	18	Marjoram 50 mg/mL	14
Marjoram oil 100 mg/mL	20	Marjoram 100 mg/mL	18

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