Isolation of a novel deep-sea Bacillus circulus strain and uniform design for optimization of its anti-aflatoxigenic bioactive metabolites production

Figures & data

Table 1. Factors and their levels in the uniform design.

Factors	Level of Factors
X1 inoculation size (%)	1	2	4	8
X2 cultivation temperature (°C)	20	28	36
X3 cultivation time (d)	2	6	10
X4 pH of medium	6	7	8	9	10
X5 content of seawater (%)	0	20	40	60	80	100
X6 yeast extract (g/L)	1	2	3	4	5	6	7	8
X7 maltose (g/L)	2	4	8	10	12	14	16	20
X8 glucose (g/L)	1	2	4	8	12

Figure 1. The phylogenetic tree of the genus Bacillus showing the position of the novel deep-sea strain FA13.

Table 2. Anti-fungal and anti-aflatoxigenic activities of the 10 times diluted supernatants obtained from different experiment runs designed by uniform design.

experiment runs	Mycelia growth inhibition ratio(%)	Aflatoxin inhibition ratio(%)
N1	18.30 ± 11.70	94.10 ± 0.20
N2	6.80 ± 4.60	60.50 ± 3.30
N3	0.00 ± 0.00	60.50 ± 14.60
N4	10.10 ± 8.90	87.80 ± 4.30
N5	0.00 ± 0.00	77.60 ± 9.50
N6	7.20 ± 11.10	76.80 ± 5.10
N7	8.30 ± 8.20	27.90 ± 14.70
N8	14.60 ± 13.10	61.70 ± 6.30
N9	11.40 ± 11.90	91.30 ± 4.70
N10	16.20 ± 9.50	30.40 ± 9.60
N11	17.30 ± 11.60	71.50 ± 16.90
N12	0.00 ± 0.00	81.10 ± 1.80
N13	0.00 ± 0.00	31.90 ± 5.10
N14	6.60 ± 7.20	87.90 ± 4.20
N15	0.00 ± 0.00	26.40 ± 12.10
N16	3.90 ± 3.70	57.00 ± 15.00
control	0.00 ± 0.00	70.90 ± 18.60

Table 3. Results of the multifactor and reciprocal regression analysis with the highest mycelia growth inhibition ratio.

Factors	Partial correlation index	T test value	P-value
r(y, X3) =	1.0150	18.7096	0.0032
r(y, X4) =	1.0158	30.7664	0.0013
r(y, X6) =	1.0165	49.3600	0.0005
r(y, X1*X3) =	−1.0319	94.3020	0.0001
r(y, X1*X5) =	1.0169	150.3063	0.0001
r(y, X2*X3) =	−1.0440	178.8622	0.0000
r(y, X2*X5) =	−1.0405	50.7267	0.0005
r(y, X3*X5) =	1.0739	146.6788	0.0000
r(y, X3*X6) =	1.0176	55.0063	0.0004
r(y, X4*X6) =	−1.0309	32.9933	0.0011
r(y, X5*X6) =	−1.0410	367.0966	0.0000
r(y, X6*X7) =	−1.0608	71.0856	0.0002
r(y, X6*X8) =	1.0740	295.1917	0.0000

Table 4. Results of multifactor and square regression analysis with the highest aflatoxin inhibition ratio.

Factors	Partial correlation index	T test value	P-value
r(y,X1) =	1.0120	180.2885	0.0042
r(y,X2) =	1.0119	107.6849	0.0069
r(y,X3) =	1.0920	124.9897	0.0060
r(y,X6) =	1.0640	159.6755	0.0043
r(y,X7) =	1.0809	90.3913	0.0078
r(y,X8) =	1.0949	86.4505	0.0085
r(y,X1*X1) =	−1.0730	197.1295	0.0038
r(y,X2*X2) =	−1.0639	93.8400	0.0077
r(y,X3*X3) =	−1.0949	98.5193	0.0074
r(y,X4*X4) =	1.0730	240.0177	0.0031
r(y,X5*X5) =	−1.0120	236.3563	0.0030
r(y,X6*X6) =	−1.0730	118.4645	0.0063
r(y,X7*X7) =	−1.0049	100.7431	0.0072
r(y,X8*X8) =	−1.0120	121.5531	0.0063

Table 5. Comparison of predicted values and measured values of different optimized fermentation protocols.

	mycelia growth inhibition ratio%		aflatoxin inhibition ratio%
Protocol	predicted value	measured value	predicted value	measured value
1	73.231	100	-	100
2	-	0	137.1	12.0 ± 17.6
3	-	26.7 ± 9.7	151.57	72.1 ± 9.8

Table 6. Inhibition ratios of mycelia growth and aflatoxin of the 20 times diluted supernatants obtained from three batches of the protocol 1.

	Mycelia growth inhibition ratio(%)	aflatoxin inhibition ratio(%)
Batch 1
protocol 1	100.00 ± 0.00	100.00 ± 0.00
Control(un-optimized)	23.1 ± 7.9	71.9 ± 1.9
Batch 2
protocol 1	100.00 ± 0.00	100.00 ± 0.00
Control(un-optimized)	13.89 ± 3.5	62.5 ± 2.7
Batch 3
Protocol 1	100.00 ± 0.00	100.00 ± 0.00
Control(un-optimized)	0.00 ± 0.00	50.4 ± 19.5

Figure 2. tip culture assays of the 20 times diluted supernatants obtained from three batches of the optimized protocol 1.

Table 7. Comparison of various factors between the optimized protocol and the original protocol.

	Cultivation temperature (°C)	Cultivation time (day)	Inoculation size %	Seawater content%	pH	glucose(g/L)	maltose(g/L)	Yeast extract(g/L)	Medium cost (local price) (￥/L)
protocol 1	21.24	10.1799	1.06	0	6.192	13.806	2.082	9.369	2.819778
Original protocol	28.0	6	1.0	100	7.2–7.4	4	10	4	4.632

Figure 3. Effect of different heat treatment of fermented cell-free supernatants on the inhibition of mycelia growth and red pigment production (CK stands for GY medium).