**3. Results**

were transported into the upper acetonitrile layer, while the polar co-extract matrix (e.g. sugars or amino acids) remained in the aqueous phase [15]. The ultra-high performance liquid chromatograph Acquity UPLC System (Waters, USA), coupled with the tandem mass spectrometer LCT Premier XE (Waters, USA) with analyzer time-of-flight MS (TOFMS) was used for the identification and detection of analytes. Wider scale of *Fusarium* mycotoxins was determined deoxynivalenol (DON) and its conjugated forms, zearalenone (ZON) and its metabolites, enniatins, and so forth, but some of them only in trace amount. Only the mycotoxins, which were detected most frequently in our wheat grain samples—DON, deoxynivalenol-3-β-d-glucoside

Crude protein content (CP) in grain dry matter according to the Kjeldahl method (EN ISO 20483; ICC-Standard No. 105/2), wet gluten content (WG) in grain dry matter and gluten index (GI) using the apparatus Glutomatic Perten (ISO 5531), falling number (FN)—ISO 3093, sedimentation index—Zeleny test (ZS)—ISO 5529, volume weigh (VW)—ISO7971-2, and TKW (thousand kernels weight) were determined with the frame of the baking quality.

Protein and starch characteristics of the wheat flour (dough development, protein weakening, starch gelatinization, diastatics activity, and anti-stalling effect) were determined by the apparatus Mixolab (Chopin, Tripette et Renaud, Paris, France) according to the Mixolab protocol Chopin+ [16]. Evaluated flour was obtained by milling the cereal grain samples on a Bühler

A typical Mixolab curve, which is shown in **Figure 1**, is separated to the five stages repre-

(D3G), 3-acetyldeoxynivalenol (3-ADON), and ZON—are presented in this study.

**2.4. Standard technological quality parameters**

**2.5. Rheological characteristics**

332 Wheat Improvement, Management and Utilization

sented by five (C1–C5) points [16].

mill automat MLU 202.

**Figure 1.** Standard Mixolab curve.

#### **3.1. Content of mycotoxins and standard technological quality parameters**

An average content of evaluated *Fusarium* mycotoxins in grain is summarized in **Table 1**. Our results show significant differences in evaluated mycotoxins content between variants with natural contamination and artificial *Fusarium* spp. inoculation; on the other hand, differences between organic and conventional growing systems were statistically insignificant. Content of deoxynivalenol (DON) in grain was several times higher than content of other evaluated mycotoxins for both wheat species. Content of mycotoxins in *Triticum aestivum* grain was generally higher in comparison with grain of *Triticum spelta*.

Significant decrease of volume weight and thousand kernels weight but increase of protein and wet gluten content was observed in artificially inoculated variants for both wheat species—higher protein content in smaller grain is expectable. At the same time, general reductions of Zeleny sedimentation and gluten index in artificially inoculated variants were observed (**Table 2**). Similar situation—decreased falling number values were determined in artificially inoculated variants too.

Above mentioned findings were also confirmed by determined correlations between mycotoxins content and evaluated technological parameters (**Table 3**). Negative correlation coefficients were found between content of mycotoxins and most of the technological parameters for both wheat species. The most evident negative effect of mycotoxins content was seen on VW and TKW for the spelt wheat (correlation: −0.95\*\*; −0.97\*\*). On the other hand, positive correlation coefficients were found between content of mycotoxins, crude protein content, and wet gluten content in grain.


Values with different letter combinations are statistically significant at p ≤ 0.05; DON—deoxynivalenol; D3G deoxynivalenol-3-β-d-glucoside; ZON—zearalenone; 3-ADON—3-acetyldeoxynivalenol.

**Table 1.** Content of mycotoxins in the wheat grain of evaluated wheat species (average data of both cultivars).


Values with different letter combinations are statistically significant at p ≤ 0.05; CP—crude protein content in grain dry matter; WG—wet gluten content in grain dry matter; GI—gluten index; ZS—Zeleny sedimentation; FN—falling number; VW—volume weight; TKW—thousand kernels weight.

**Table 2.** Technological quality parameters of evaluated wheat species (average data of both cultivars).

#### **3.2. Mixolab**

#### *3.2.1. Triticum aestivum L.*

Resulted common wheat Mixolab parameters confirmed that *Fusarium* spp. infection markedly worsenes both protein and starch characteristics (**Table 4**). Average value of C2, which positively correlates with dough strength, was more than half lower after *Fusarium* spp.


Statistically significant for p ≤ 0.05(\*) and for p ≤ 0.01(\*\*).

**3.2. Mixolab**

*Triticum aestivum* L.

334 Wheat Improvement, Management and Utilization

Artificial inoculation

Natural contamination

Artificial inoculation

Natural contamination

*Triticum spelta* L.

*Triticum aestivum* L.

Natural contamination

Natural contamination

*Triticum spelta* L.

*3.2.1. Triticum aestivum L.*

VW—volume weight; TKW—thousand kernels weight.

Resulted common wheat Mixolab parameters confirmed that *Fusarium* spp. infection markedly worsenes both protein and starch characteristics (**Table 4**). Average value of C2, which positively correlates with dough strength, was more than half lower after *Fusarium* spp.

**Treatment Growing system DON (μg kg−1) D3G (μg kg−1) 3-ADON (μg kg−1) ZON (μg kg−1)**

Organic 19411.1b 2704.6b 472.3ab 2965.3b Conventional 26729.4b 4141.3b 1051.9b 3191.4b

Organic 304.7a 153.8a 7.5a 19.0a Conventional 257.5a 66.5a 7.5a 22.3a

Organic 12648.7b 2154.5b 149.8b 117.1<sup>b</sup> Conventional 14433.7b 2797.8b 169.8b 237.0b

Organic 25.6a 22.6a 7.5a 4.0a Conventional 260.1a 142.4<sup>a</sup> 7.5a 7.1<sup>a</sup> Values with different letter combinations are statistically significant at p ≤ 0.05; DON—deoxynivalenol; D3G—

**Table 1.** Content of mycotoxins in the wheat grain of evaluated wheat species (average data of both cultivars).

**Treatment Growing system CP (%) WG (%) GI ZS (ml) FN (s) VW (kg hl−1) TKW (g)**

Conventional 14.1<sup>b</sup> 32.5a 58.5ab 33.1a 259.3ab 47.2<sup>a</sup> 22.4<sup>a</sup>

Organic 12.0a 26.1a 88.3b 49.3b 283.8b 72.8<sup>b</sup> 47.3b Conventional 12.5ab 27.3a 83.8b 54.8b 279.8b 74.1<sup>b</sup> 49.5b

Conventional 20.6b 53.2a 21.0a 27.1<sup>a</sup> 321.0a 50.1a 20.9a

Organic 16.7a 49.1a 38.0a 39.1b 343.0a 73.0b 40.9b Conventional 18.8ab 52.8a 33.0a 34.7b 313.5a 74.0b 41.2<sup>b</sup>

Artificial inoculation Organic 13.7ab 30.0a 42.0a 34.0a 250.5a 50.4a 23.9a

Artificial inoculation Organic 19.6b 52.5a 24.5a 25.7a 307.0a 54.1a 22.1<sup>a</sup>

Values with different letter combinations are statistically significant at p ≤ 0.05; CP—crude protein content in grain dry matter; WG—wet gluten content in grain dry matter; GI—gluten index; ZS—Zeleny sedimentation; FN—falling number;

**Table 2.** Technological quality parameters of evaluated wheat species (average data of both cultivars).

deoxynivalenol-3-β-d-glucoside; ZON—zearalenone; 3-ADON—3-acetyldeoxynivalenol.

DON—deoxynivalenol; D3G—deoxynivalenol-3-β-d-glucoside; ZON—zearalenon; 3-ADON—3-acetyldeoxynivalenol; CP—crude protein content in grain dry matter; WG—wet gluten content in grain dry matter; GI—gluten index; ZS— Zeleny sedimentation; FN—falling number; VW—volume weight; TKW—thousand kernels weight.

**Table 3.** Correlation between mycotoxins content and technological parameters of evaluated wheat species.


Values with different letter combinations are statistically significant at p ≤ 0.05; C1—time required for maximum torque during mixing; C2—protein weakening; C3—starch gelatinization; C4—stability of gel; C5—starch retrogradation; C1C2 fall of protein strength; C3C4—diastatic activity; C5C4—anti-stalling effect; DS—time of dough stability before weakening.

**Table 4.** Mixolab characteristics of *Triticum aestivum* L. (average data of both cultivars).

inoculation for both growing systems. Therefore, higher rate of protein thermal weakening (C1C2) and visible shorter time of dough stability were found for inoculated variants.

It is evident from **Figure 2** that the inferior effect of infection on protein part of curve is especially obvious for variety Bohemia in 2011, where after dough development, there is rapid fall of the curve which implies low quality of gluten.

Dough heating and thus swelling of starch granules and increasing viscosity cause the increase of the curve—point C3. It was evident from our results that values of artificially inoculated variants were markedly lower than values of variants with natural *Fusarium* spp. contamination. Supposedly, this is due to damaged starch granules of inoculated variants. There was no statistical difference between organic and conventional growing systems. Although C5 parameters in the last section of the curve were slightly higher for naturally contamined variants than for inoculated variants, differences C5C4 representing retrogradation and consequently shelf life of end products were statistically comparable (**Table 4**).

Deteriorated rheological quality of inoculated variants was also confirmed by rated strong negative correlation coefficients between mycotoxins content and Mixolab parameters (**Table 5**).

#### *3.2.2. Triticum spelta L*

Final Mixolab characteristics of *T. spelta* L. (**Table 6**) imply slightly better resistance to *Fusarium* spp. infection compare to the reaction of *T. aestivum* variants. It is evident especially in case of Mixolab characteristics, representing protein part of curve (values of C2 and C1–C2).

Average value of C2, which represents the weakening of the protein, was lower in artificially inoculated variants for both wheat species, but in spelt, the difference between naturally contamined and artificially inoculated variants was not so high. At the same time, higher rate of protein thermal weakening (C1C2) and thus shorter time of dough stability were found in

**Figure 2.** An example of Mixolab curve—common wheat cultivar Bohemia.


inoculation for both growing systems. Therefore, higher rate of protein thermal weakening

It is evident from **Figure 2** that the inferior effect of infection on protein part of curve is especially obvious for variety Bohemia in 2011, where after dough development, there is rapid fall

Dough heating and thus swelling of starch granules and increasing viscosity cause the increase of the curve—point C3. It was evident from our results that values of artificially inoculated variants were markedly lower than values of variants with natural *Fusarium* spp. contamination. Supposedly, this is due to damaged starch granules of inoculated variants. There was no statistical difference between organic and conventional growing systems. Although C5 parameters in the last section of the curve were slightly higher for naturally contamined variants than for inoculated variants, differences C5C4 representing retrogradation and conse-

Deteriorated rheological quality of inoculated variants was also confirmed by rated strong negative correlation coefficients between mycotoxins content and Mixolab parameters

Final Mixolab characteristics of *T. spelta* L. (**Table 6**) imply slightly better resistance to *Fusarium* spp. infection compare to the reaction of *T. aestivum* variants. It is evident especially in case of

Average value of C2, which represents the weakening of the protein, was lower in artificially inoculated variants for both wheat species, but in spelt, the difference between naturally contamined and artificially inoculated variants was not so high. At the same time, higher rate of protein thermal weakening (C1C2) and thus shorter time of dough stability were found in

Mixolab characteristics, representing protein part of curve (values of C2 and C1–C2).

(C1C2) and visible shorter time of dough stability were found for inoculated variants.

quently shelf life of end products were statistically comparable (**Table 4**).

**Figure 2.** An example of Mixolab curve—common wheat cultivar Bohemia.

of the curve which implies low quality of gluten.

336 Wheat Improvement, Management and Utilization

(**Table 5**).

*3.2.2. Triticum spelta L*

Statistically significant for p ≤ 0.05(\*) and for p ≤ 0.01(\*\*); DON—deoxynivalenol; D3G—deoxynivalenol-3-β-d-glucoside; ZON—zearalenone; 3-ADON—3-acetyldeoxynivalenol; C1—time required for max. torque during mixing; C2—protein weakening; C3—starch gelatinization; C4—gel stability; C5—starch retrogradation; C1C2—fall of protein strength; C3C4—diastatic activity; C5C4—anti-stalling effect; DS—dough stability time.

**Table 5.** Correlation between Mixolab parameters and mycotoxins content for *Triticum aestivum* L. in both growing systems.

inoculated variants for both wheat species, but in spelt, the difference between naturally contamined and artificially inoculated variants was slightly lower compared to common wheat.

Correlation between Mixolab parameters and mycotoxins content for *T. spelta* shows **Table 7**. Although there was a deflection of inoculated variants particularly evident for parameters C2 and C1–C2, affirmed by significant negative correlation between mycotoxins content and C2


Values with different letter combinations are statistically significant at p ≤ 0.05; C1—time required for max. torque during mixing; C2—protein weakening; C3—starch gelatinization; C4—stability of gel; C5—starch retrogradation; C1C2—fall of protein strength; C3C4—diastatic activity; C5C4—anti-stalling effect; DS—dough stability time.

**Table 6.** Mixolab characteristics of *Triticum spelta* L. (average data of both cultivars).


Statistically significant for p ≤ 0.05(\*) and for p ≤ 0.01(\*\*); DON—deoxynivalenol; D3G—deoxynivalenol-3-β-d-glucoside; ZON—zearalenone; 3-ADON—3-acetyldeoxynivalenol; C1—time for maximum torque during mixing; C2—protein weakening; C3—starch gelatinization; C4—gel stability; C5—starch retrogradation; C1C2—fall of protein strength; C3C4—diastatic activity; C5C4—anti-stalling effect; DS—dough stability before weakening.

**Table 7.** Correlation between Mixolab parameters and mycotoxins content for *Triticum spelta* L. in both growing systems.

and C1–C2 values, generally *Fusarium* effect was less pronounced in comparison with common wheat.

It is evident from **Figure 3** that despite the shifts of individual curves for variety Ceralio, majority of resulting Mixolab parameters for various type of treatment were statistically insignificant. Just characteristics C2 and dough stability for the control from organic treatment were preferable to the conventional variant.

**Figure 3.** An example of Mixolab curve—spelt wheat cultivar Ceralio.
