Effect of Biological and Biochemical Silage Additives on Final Nutritive , Hygienic and Fermentation Characteristics of Ensiled High Moisture Crimped Corn

The aim of this study was to determine the effect of different biological and biochemical additives on the final nutritive quality, fermentation process and concentration of mycotoxins of ensiled high moisture crimped corn. We created four variants for the experiment: control (UC), A1, and A2 (biological stimulators with the active principle of lactic acid bacteria) and variant B (combined additives with the active principle of lactic acid bacteria, benzoate sodium and active enzymatic complex of cellulases). After 6 months of storage in laboratory conditions, we determined in experimental silages the content of dry matter ranging from 608.9 to 613 g·kg-1. The significantly lower content of crude fibre was detected in silages with additives. In silages ensiled with additives we detected the highest content of nitrogen-free extract in variant B (834.3 g·kg-1 of DM, P < 0.05). A similar effect was determined also in the content of starch; significant differences were detected in variants A1 and B (P < 0.05) compared to the control variant. We detected a significantly (P < 0.05) higher content of total sugars in trial silages; the highest content was in variant A2 (6.1 g·kg-1 of DM). In the trial variants we determined significantly the lowest content of acetic acid in variant B (2.82 g·kg-1 of DM). In case of butyric acid, whose content in the control variant was 0.22 g·kg-1 of DM, we detected the lowest content in variant A1 conserved with homoand heterofermentative species of lactic acid bacteria. The lowest content of ammonia was determined in silages of variant B (0.074 g·kg-1 of DM). We found lower concentrations of DON and FUM (P > 0.05) after the application of biological and biochemical silage additives. In concentration of T-2 toxin we detected a significantly (P < 0.05) lower value in variant A1. In concentration of AFL we found significant differences between variants A1 and B, as well as in concentration of OT between untreated control variant (UC) and variants conserved by additives. Application of silage additives influenced the nutritive and hygienic quality of the conserved fodders. Feeds, maize, conservation, silage additives, mycotoxins Maize (Zea mays L.) is a major source of energy in feeding rations for ruminants in the Slovak Republic. Generally, it produces feed with different nutritional and hygienic indicators. The differences lie mainly in the energy content (Bíro 2001). Energy limits the nutritive value of feeds, except for fresh and green forages (Hoffman 1998). During the harvest period, the technology of high moisture corn is economically more efficient due to lower losses and processing costs (Volkov et al. 1999; Bíro and Juráček 2003). High moisture corn has a higher nutritive value and higher digestibility of organic matter compared to dry corn (Woodarce 2004). Starch supplied from corn and corn silage is an important source of dietary energy for lactating dairy cows and other ruminants. However, various sources of corn starch have highly variable ruminal and total tract digestibility (Ørskov 1986; Threuer 1986). Factors such as particle size (Remond et al. 2004), conservation method (Oba and Allen 2003) and type of corn endosperm (Correa et al. 2002; Šimko et al. 2008) can influence ruminal and total tract digestion of starch in lactating dairy cows ACTA VET. BRNO 2009, 78: 691-698; doi:10.2754/avb200978040691 Address for correspondence: Ing. Branislav Gálik, PhD. Department of Animal Nutrition Faculty of Agrobiology and Food Resources Slovak University of Agriculture in Nitra 949 76 Nitra, Slovak Republic Phone: +421 376 414 331 E-mail: Branislav.Galik@uniag.sk http://www.vfu.cz/acta-vet/actavet.htm (Blasel et al. 2006). Preservation system is based on quick anaerobic fermentation of plant carbohydrates, simultaneous rapid decrease of pH value and production of fermentation products and by-products (Merry et al. 1997). New approaches to development of ensiling additives lead to application of combined preparations based on several groups of lactic acid bacteria in order to reach high efficiency and large utilization range. Application of combined additives composite from homoand heterofermentative lactic acid bacteria stimulates silage fermentation and affects aerobic stability (Driehuis et al. 2001; Owen 2002). Many silage additives include microbial inoculants that extensively preserve feeds during ensiling (Bolsen et al. 1996). Biological additives containing different forms of lactic acid bacteria were used for forage preservation in many experiments of different authors (e.g. Chamberlain et al. 1987; Kung et al. 1999; Doležal and Zeman 2005). Inoculation of forages with selected lactic acid bacteria was recognized as a method to improve the fermentation process by stimulation and to ensure the aerobic stability of silage (Bolsen et al. 1996). Materials and Methods In the experiment, we conserved high moisture corn obtained from the University Experimental Farm in Kolíňany. Harvested corn grain (grain hybrid Latizana) was immediately mechanically processed and crushed by MURSKA 1000 HD grinder with the content of dry matter 613.3 g·kg-1. Four variants were analyzed in the experiment: control (UC) and experimental variants (A1, A2, B). Corn grain in experimental variants was ensiled with microbial (A1 and A2) and biochemical (B) additives, which we applied homogeneously to ensiled matter before ensiling into laboratory silos with the volume of 50 dm3. The additive used in variant A1 consisted of homoand heterofermentative species of lactic acid bacteria (Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri and Pediococcus pentosaceus: 2.5 × 1011 CFU·g-1). In variant A2 we applied the additive compound of 5 lactic acid bacteria species (Enterococcus faecium, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus buchneri and Pediococcus pentosaceus: 150 × 103 CFU·g-1). Inoculants used in variants A1 and A2 were in powder form. High moisture corn in variant B was ensiled by combined biochemical additive, where the biological part were lactic acid bacteria (Lactobacillus plantarum, Enterococcus faecium, Pediococcus pentosaceus and Lactococcus lactis: 166 × 109 CFU·g-1) and the chemical part were enzymatic complex of cellulases (Trichoderma viridae: activity 50 610 CU, 6478 IU) and preservative benzoate sodium. All variants were ensiled in 3 repetitions. After filling the matter into silos (density 1,100 kg·m-3), we sealed them and stored in the laboratory of feeds, conserved by the temperature of 18-20 °C. The nutritive characteristic of fresh high moisture corn is presented in Table 1. After termination of the fermentation process (6 months of storage) we opened the silos and in average laboratory samples we determined the indicators of nutritive value and fermentation process. For analysing organic and inorganic nutrients, we used standard methods according to the Regulation of Ministry of Agriculture of the Slovak Republic no. 2145/2004-100 about sampling feeds and about laboratory testing and evaluating of feeds. The content of fermentative carboxyl organic acids was determined on analyser EA 100 (Villa Labeco, SR) using the method of ionic electrophoresis.

Maize (Zea mays L.) is a major source of energy in feeding rations for ruminants in the Slovak Republic.Generally, it produces feed with different nutritional and hygienic indicators.The differences lie mainly in the energy content (Bíro 2001).Energy limits the nutritive value of feeds, except for fresh and green forages (Hoffman 1998).During the harvest period, the technology of high moisture corn is economically more efficient due to lower losses and processing costs (Volkov et al. 1999;Bíro and Juráček 2003).High moisture corn has a higher nutritive value and higher digestibility of organic matter compared to dry corn (Woodarce 2004).Starch supplied from corn and corn silage is an important source of dietary energy for lactating dairy cows and other ruminants.However, various sources of corn starch have highly variable ruminal and total tract digestibility (Ørskov 1986;Threuer 1986).Factors such as particle size (Remond et al. 2004), conservation method (Oba and Allen 2003) and type of corn endosperm (Correa et al. 2002;Šimko et al. 2008) can influence ruminal and total tract digestion of starch in lactating dairy cows (Blasel et al. 2006).Preservation system is based on quick anaerobic fermentation of plant carbohydrates, simultaneous rapid decrease of pH value and production of fermentation products and by-products (Merry et al. 1997).New approaches to development of ensiling additives lead to application of combined preparations based on several groups of lactic acid bacteria in order to reach high efficiency and large utilization range.Application of combined additives composite from homo-and heterofermentative lactic acid bacteria stimulates silage fermentation and affects aerobic stability (Driehuis et al. 2001;Owen 2002).Many silage additives include microbial inoculants that extensively preserve feeds during ensiling (Bolsen et al. 1996).Biological additives containing different forms of lactic acid bacteria were used for forage preservation in many experiments of different authors (e.g.Chamberlain et al. 1987;Kung et al. 1999;Doležal and Zeman 2005).Inoculation of forages with selected lactic acid bacteria was recognized as a method to improve the fermentation process by stimulation and to ensure the aerobic stability of silage (Bolsen et al. 1996).

Materials and Methods
In the experiment, we conserved high moisture corn obtained from the University Experimental Farm in Kolíňany.Harvested corn grain (grain hybrid Latizana) was immediately mechanically processed and crushed by MURSKA 1000 HD grinder with the content of dry matter 613.3 g•kg -1 .
Four variants were analyzed in the experiment: control (UC) and experimental variants (A1, A2, B).Corn grain in experimental variants was ensiled with microbial (A1 and A2) and biochemical (B) additives, which we applied homogeneously to ensiled matter before ensiling into laboratory silos with the volume of 50 dm 3 .The additive used in variant A1 consisted of homo-and heterofermentative species of lactic acid bacteria (Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri and Pediococcus pentosaceus: 2.5 × 10 11 CFU•g -1 ).In variant A2 we applied the additive compound of 5 lactic acid bacteria species (Enterococcus faecium, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus buchneri and Pediococcus pentosaceus: 150 × 10 3 CFU•g -1 ).Inoculants used in variants A1 and A2 were in powder form.High moisture corn in variant B was ensiled by combined biochemical additive, where the biological part were lactic acid bacteria (Lactobacillus plantarum, Enterococcus faecium, Pediococcus pentosaceus and Lactococcus lactis: 166 × 10 9 CFU•g -1 ) and the chemical part were enzymatic complex of cellulases (Trichoderma viridae: activity 50 610 CU, 6478 IU) and preservative benzoate sodium.All variants were ensiled in 3 repetitions.After filling the matter into silos (density 1,100 kg•m -3 ), we sealed them and stored in the laboratory of feeds, conserved by the temperature of 18-20 °C.The nutritive characteristic of fresh high moisture corn is presented in Table 1.After termination of the fermentation process (6 months of storage) we opened the silos and in average laboratory samples we determined the indicators of nutritive value and fermentation process.For analysing organic and inorganic nutrients, we used standard methods according to the Regulation of Ministry of Agriculture of the Slovak Republic no.2145/2004-100 about sampling feeds and about laboratory testing and evaluating of feeds.The content of fermentative carboxyl organic acids was determined on analyser EA 100 (Villa Labeco, SR) using the method of ionic electrophoresis.Contents of alcohols and ammonia were detected by Conway microdiffusion method, titration acidity by alkalimetric titration and active acidity by the electrometric method.Energy (NEL and NEG) and protein (PDI) values were calculated by regression scheme (Petrikovič and Sommer 2002).After opening the laboratory silos, we sensorically observed the occurrence of fungi.Concentration of mycotoxins (FUM: fumonisins, AFL: aflatoxins, ZON: zearalenone, DON: deoxynivalenol, T-2 toxin and OT: ochratoxin) was detected using the immunoenzymatic method in screening quantitative test of ELISA Reader (NEOGEN, U.S.A.).Before spectrophotometric measuring of concentration, the samples were processed by extraction in distilled water (DON), 70% methanol (FUM, ZON, AFL), respectively in 50% methanol (OT and T-2 toxin).
Significance of determined differences was tested by single-factor analysis of variance (ANOVA).The evidence of differences of the mean values was assessed by t-test.

Results
The nutritive characteristic of high moisture corn is presented in Table 1.The content of nutrients in silages from high moisture corn is given in Table 2.After 6 months of storage we detected the content of dry matter in high moisture corn silages ranging from 596.3 (variant A1) to 607.4 g•kg -1 (variant A2).In the crude protein content, which is typically deficient in corn, we did not detect significant differences influenced by additives.The highest content of crude protein was in silages of variant A1 (95.4 g•kg -1 of DM).For corn grain low content of crude fibre is also typical; this is in negative correlation with  digestibility of organic matter.In our experiment, we determined a positive decrease of crude fibre content in silages with applied additives; the differences found were significant (P < 0.05).The lowest content of crude fibre (23.5 g•kg -1 of DM) was detected in silages from high moisture corn conserved by combined biochemical inoculant in variant B. In the content of ash (13.5-14.1 g•kg -1 of DM) we did not detect significant differences influenced by additives.Nutritionally, corn grain is valued also for its high content of easily digestible carbohydrates as nitrogen-free extract (NFE).Additives positively influenced the content of NFE in silages.Significantly the highest content of NFE (P < 0.05) was in silages conserved by combined biochemical additive (variant B).Likewise, in the content of starch as source of energy in corn grain, we determined a positive influence of additives.Compared to the variant UC, in which we ensiled high moisture corn without additives, we detected a significantly higher content of starch in silages of variant A1 and B (P < 0.05).The content of total sugars analysed according to Luff-Schoorl ranged from 1.0 to 6.1 g•kg -1 of DM.Detected differences between variant UC and variants with applied additives were significant (P < 0.05).We did not detect significant differences in the energetic and protein value of conserved silages from high moisture corn.
Detected values of fermentation process indicators are presented in Table 3.In silages with additives we determined a lower content of lactic acid compared to the variant without additives, mainly in variant A1 (10.55 g•kg -1 of DM).The content of acetic acid was low in all variants and it did not exceed 5.0 g•kg -1 of DM.In variant A1 we found a lower content of dry matter with a lower content of total sugars, and higher value of pH compared to variant A2.These factors affected lower production of lactic acid and a higher content of acetic acid in variant A1.The content of undesirable butyric acid was low in silages, non-significantly the highest concentration was in silages of variant A2 (0.38 g•kg -1 of DM), in this variant we applied a biological additive for stimulation of the fermentation process.Active acidity (pH) of water extracts ranged from 3.70 (variant B) to 3.85 (variant A1).The highest pH together with the lowest titration acidity (TA) was found in silages in which we detected the highest content of acetic acid (variant A1).Positive non-significant influence of applied additives was detected in the content of ammonia (NH 3 ), which compared to the control variant (0.416 g•kg -1 of DM) ranged in trial variants from 0.074 (variant A2) to 0.101 g•kg -1 of DM (variant A1).Concentration of mycotoxins in high moisture corn before ensiling is presented in Table 4.In fresh high moisture corn we did not detect the concentration of deoxynivalenol at the detection level of mg•kg -1 .The most prevalent were Fusarium toxins: FUM, followed by ZON and T-2.The samples of high moisture corn before ensiling were the least contaminated by toxin producers of the genera Penicillium and Aspergillus.Concentration of mycotoxins in silages of high moisture corn is given in Table 5.The lowest concentration of zearalenone (29.83 µg•kg -1 ) was determined in silages of the variant conserved by the combined biochemical additive (variant B).In concentration of deoxynivalenol, we detected lower values in all variants conserved by different silage additives (0.067 mg•kg -1 ).Average concentration of deoxynivalenol in untreated control variant was 0.133 mg•kg -1 .The same effect of silage additives was found in concentration of fumonisins.Significantly the lowest concentration of T-2 toxin was in variant A1 which we conserved by homo-and heterofermentative species of lactic acid bacteria.The positive effect of the biochemical additive (variant B) was detected also in the concentration of aflatoxins (2.13 µg•kg -1 ).Significant differences in the concentration of aflatoxins were found between variants A1 and B. The concentrations of ochratoxin were lower in silages of variants conserved by biological and biochemical additives (the lowest in variant A2, 0.533 µg•kg -1 ).Compared to untreated control variant, the differences were significant (P < 0.05).

Discussion
In a similar experiment, Doležal and Zeman (2005) determined the average content of dry matter of 603.4 g•kg -1 which partially corresponds with our results.In the content of crude protein, we confirmed the results of Zebrowska et al. (1997) who determined the average content of crude protein in corn of 100 g•kg -1 of DM.In the content of crude protein we did not detect significant effects of additives.Similar results were also reported by Wardynski et al. (1993).The content of crude fibre ranged in silages from 23.5 to

Table 2 .
Nutrient contents of high moisture corn silage * DM -dry matter, CP -crude protein, F -fat, CF -crude fiber, A -ash, NFE -nitrogen free extract, OM -organic matter, S -starch, TS -total sugars, NEL -net energy of lactation , NEG -net energy of gain, PDIE, PDIN -protein digestible in intestine Values with identical superscripts within one column are significant at P < 0.05x x x x

Table 3 .
Results of fermentation process of high moisture corn silages * DM: dry matter, LA: lactic acid, AA: acetic acid, BA: butyric acid, PA: propionic acid, FA: formic acid, TA: titration acidity, pH: active acidity, NH 3 : ammonia, Alc: alcohols Values with identical superscripts within one column are significant at P < 0.05 x x x x