Impact of Erwinia gerundensis as a Biocontrol Agent on the Sanitary and Technological Quality of Barley Malt

Figures & data

Table 1. Culture media composition and suppliers.

Culture medium	Composition	Supplier
Oxytetracycline-Glucose-Yeast Extract Agar (OGYE Agar)	Yeast extract: 5 g/L, Glucose: 20 g/L, Oxytetracycline: 0.1 g/L	Biokar Diagnostics (France)
Potato Dextrose Agar (PDA)	Potato Starch (from infusion): 4 g/L, Dextrose: 20 g/L, Agar: 15 g/L	Difco Labs. (France)
Spezieller Nährstoffärmer Agar (SNA)	K2HPO4: 1 g/L, KNO3: 1 g/L, MgSO4.7H2O: 0.5 g/L, KCl: 0.5 g/L, Glucose: 0.2 g/L, Saccharose: 0.2 g/L, Agar: 20 g/L	–
Yeast Malt Broth (YM)	Glucose: 10 g/L, Malt extract: 3 g/L, Peptic digest of animal tissue: 5 g/L, Yeast extract: 3 g/L	Sigma Aldrich (India)
Yeast Malt Agar (YMA)	Agar: 20 g/L, Glucose: 10 g/L, Malt extract: 3 g/L, Peptic digest of animal tissue: 5 g/L, Yeast extract: 3 g/L	Sigma Aldrich (India)

Figure 1. Enniatin (ENN) concentration in control (F. tricinctum alone) and co-culture (F. tricinctum + E. gerundensis) experiments in liquid medium. Panel A: Co-culture between F. tricinctum 2399 and E. gerundensis 6E4; Panel B: Co-culture between F. tricinctum 2502 and Erwinia gerundensis 6E4. Histograms (mean ± standard deviation) with different letters (a, b, c) are significantly different (p < 0.05). *Total ENN refers to the sum of enniatins A, A1, B, and B1.

Table 2. Biomass and enniatin concentrations during co-culture on barley kernels. For total ENN, values (mean ± standard deviation) with different letters (a, b, c, d) are significantly different (p < 0.05).

	E. gerundensis			F. tricinctum 2399		F. tricinctum 2502
	Control	vs. TRI 2399	vs. TRI 2502	Control	vs. EG	Control	vs. EG
Initial biomass (CFU/kg)	4.0 x 10^10	4.0 x 10^10	1.0 x 10^10	1.0 x 10^7	1.0 x 10^7	1.0 x 10^7	1.0 x 10^7
Final biomass (CFU/kg)	1.3 x 10^13	1.4 x 10^13	1.2 x 10^13	1.8 x 10^10	3.2 x 10^9	5.5 x 10^9	6.0 x 10^9
Total ENN (µg/kg)	NA	NA	NA	625 ± 89^c	199 ± 55^d	3507 ± 74^a	2167 ± 704^b

TRI: Fusarium tricinctum; EG: Erwinia gerundensis; ENN: enniatin; NA: Not Applicable.

Figure 2. Relative abundance of E. gerundensis (EG) throughout the malting process. Expressed in number of reads of EG sequences per a total of 23136 reads. M1: no EG treatment; M2: barley treated with 4 x 10¹⁰ CFU EG/kg; M3: Barley treated with 4 x 10⁷ CFU EG/kg; M4: barley treated with 4 x 10⁴ CFU/kg; Seed: unmalted barley.

Figure 3. Effect of E. gerundensis on the total fungal load of malt. Error bars represent the standard error.

Figure 4. Effect of E. gerundensis (EG) on mycotoxin content of malt. A: ENN; B: DON; C: DON3G; D: T2 + HT2 toxins. Error bars represent the uncertainty of the LC-MS/MS method (15%).

Table 3. Technological quality of malted barley with different doses of E. gerundensis (EG) applied before steeping.

Malt quality parameters	Reference range*	Untreated malt	Malt with 4 x 10^4 CFU EG/kg barley	Malt with 4 x 10^7 CFU EG/kg barley	Malt with 4 x 10^10 CFU EG/kg barley
Diastatic Power (°WK)	> 250	290	300	290	250
Extract fine grind (% DM)	> 80	83.7	83.3	83.2	83.7
Friability (% flour)	> 80	90	87	88	90
Total β-glucan (mg/L)	< 200	219	233	210	173
Wort viscosity (mPa.s)	1.5 − 1.7	1.54	1.52	1.54	1.55
Free amino nitrogen (mg/L)	> 140	191	179	179	196
Total protein (% DM)	10 − 11.5	10.4	10.2	10.3	10.2
Soluble protein (% DM)	3.5 − 4.5	4.8	4.8	4.8	4.8
Kolbach index (%)	> 35	46	47	47	47
Filtration time (min per 350 g of wort)	NA	33.8	30.2	31.9	30.4
Moisture content (%)	< 4.5	3.6	3.6	3.6	3.6
Wort pH	5.7 − 6	5.8	5.8	5.8	5.8

*Reference range was provided by the French Institute of Beverages, Brewing and Malting (IFBM), member of the Analysis Committee of the European Brewery Convention; NA: Not Applicable.

Table 4. Effect of E. gerundensis on the mycotoxin content of malt. Values (mean ± standard deviation) with the same letters are not significantly different (p > 0.05).

Conditions	ENN (µg/kg)	T2 + HT2 (µg/kg)
Unmalted barley	491 ± 256^a	84 ± 57^a
Untreated malt	259 ± 265^a	33 ± 34^a
Malt treated with E. gerundensis	160 ± 66^a	26 ± 14^a
P-value	0.236	0.361

Table 5. Effect of E. gerundensis on the technological quality parameters of malt. Values (mean ± standard deviation) with different letters are significantly different (p < 0.05).

Malt quality parameters	Reference range*	Untreated malt	Malt treated with E. gerundensis	P-value
Diastatic Power (°WK)	> 250	273 ± 6^a	280 ± 0^a	0.116
Extract fine grind (% DM)	> 80	82 ± 0.3^a	82 ± 0.2^a	0.877
Friability (% flour)	> 80	92 ± 0.6^a	92 ± 0.6^a	1.000
Total β-glucan (mg/L)	< 200	132 ± 14^a	127 ± 3^a	0.585
Wort viscosity (mPa.s)	1.5 − 1.7	1.51 ± 0.0^a	1.49 ± 0.0^b	0.047
Free amino nitrogen (mg/L)	> 140	192 ± 1.7^a	190 ± 0.6^a	0.189
Total protein (% DM)	10 − 11.5	10.4 ± 0.0^a	10.4 ± 0.2^a	0.725
Soluble protein (% DM)	3.5 − 4.5	5.0 ± 0.2^a	5.1 ± 0.0^a	0.374
Kolbach index (%)	> 35	48 ± 2^a	49 ± 1^a	0.275
Filtration time (min per 350 g of wort)	NA	26.3 ± 2.9^a	21.6 ± 4.6^a	0.205
Moisture content (%)	< 4.5	3.8 ± 0.0^a	3.9 ± 0.1^a	0.116
Wort pH	5.7 − 6	5.8 ± 0.0^a	5.6 ± 0.0^b	< 0.001

*Reference range was provided by the French Institute of Beverages, Brewing and Malting (IFBM), member of the Analysis Committee of the European Brewery Convention; NA: Not Applicable.