RO130200A0 - Process for preparing fodder from bio-ethanol preparation by-products - Google Patents

Abstract

The invention relates to a process for preparing fodder for monogastric and/or ruminant animals. According to the invention, the process comprises the vinasse normalization up to 15% dry substance, the yeast cells plasmolysis and the humification of vinasse melanoidin compounds by treatment with laccase and intermittent homogenization under pressure, followed by drying the distiller's wash and the fermentation yeast to result in a distiller's dry wash with soluble products, mixing 95 parts of distiller's dry wash with 5 parts of vinasse, densifying the mixture by pressing, to finally result in a granulated fodder.

Description

The present invention relates to a process for obtaining feeds for monogastric and / or ruminants, by utilizing by-products from the manufacture of (bio) ethanol, respectively borhot and vinasa.

Processes for obtaining monogastric and / or ruminant feed from by-products from (bio) ethanol manufacture are known. Borhot results in the use of grain cereals as a raw material, and the vinegar is formed when using molasses, sugar beet or cane. In the case of the bramble, the usual process of conversion into fodder is to dry it together with the fermentation yeast. Dry distillers with soluble products (DDGS) are formed in the case of the usual dry milling process, and high-protein distillers dried grains (HPDDG), when the milling process is used wet or the variant of processing of the dry mill for the recovery of a part of fibers and of the germs of cereals. In the case of molasses, the usual process is concentration by evaporation.

The main technical problem in the use as by-products of by-products from the manufacture of bio-ethanol is that of concentrating in these by-products some potentially toxic contaminants or compounds formed during industrialization - see for example. Granby et al., 2012, Potential contamination issues arising from the use of biofuel and food industry by-products in animal feed, in Animal feed contamination: effects on livestock and food safety, ed. Fink-Gremmels

J., Pag: 514-539, Woodhead Publishing, Cambridge, UK. Mycotoxins formed in cereals during vegetation can be concentrated up to three times in the borot (Wu and Munkvold, 2008, Journal of Agricultural and Food Chemistry, 56 (11), 3900-3911). In melanoma, melanohydrylic compounds, formed as a result of repeated heat treatments, reduce the bioavailability of some nutrients and have a high mutagenic potential (Stemme et al. 2005, Journal of Animal Physiology and Animal Nutrition, 89 (3-6), 179-183), and the concentration by evaporation amplifies the formation of such compounds.

US Patent No. 8,496,984 B2 describes the use of micronized lignocellulosic material, present in the bio-ethanol fabrication booth, as in other by-products, such as. sugar cane stalks, to adsorb mycotoxins (\ 2 Ο 1 4 0 0 2 6 9 - '·-20 · 20U present in fodder. Microriization of lignocellulosic material is performed by enzymatic and / or mineral acid treatment, followed by grinding. wet in a disk mill and spray drying.

By micronization, the specific surface of the lignocellulosic material increases and an adsorbent layer is formed which has the high capacity to fix a wide range of mycotoxins, especially those difficult to bind, represented by ochratoxin, deoxynivalenol and T2 toxin.

In the embodiments of the invention, the binding of aflatoxins to the adsorbent by micronization of lignocellulosic material is not described. It is considered that these fungal mycotoxins from the Aspergillus Section Flavi group are adsorbed by the cell walls of the fermentation yeasts, present in the fermenter where the fermentation yeast is found, and thus will no longer cause negative effects. Dramatic situations of milk contamination with aflatoxins, which follow after the drought years, and which are a direct consequence of the use in the feeding of lactating cows of dry pork distillery with soluble maize products, whose significant contamination with aflatoxins is favored by drought (Piva et al., 2006, in Mycotoxin Factbook: Food & Feed Topics, eds Barug et al, 3rd World Mycotoxin Forum, Noordwijk, Netherlands, pp. 139-153), shows that this binding of yeast cell walls is not effective in the case high levels of aflatoxins. Digestion of micronized lignocellulosic material in rumen, under the action of cellulosolytic symbiont microorganisms, can cause the release of adsorbed mycotoxins, so the proposed adsorbent is not very suitable for ruminants.

US Patent No. 8,507,019 B2 refers to the use as a mycotoxin-specific adsorbent of leonardite, an oxidized lignite containing 90% humic substances, with at least 45% humic acid, and metal ions, oxides and clay minerals. The adsorbent is effective in limiting the estrogenic syndrome produced by zearalenone in sows and reduces the adsorption of fumonisin and aflatoxins in chickens.

However, the use of fossil coal as a source of humic acids generates the risk of introducing potentially toxic elements (heavy metals) into the food chain, so it is necessary to use sources of humic substances with a low risk of contamination.

Another major problem in the use of borhot as feed, especially that resulting from the dry milling process, is the one determined by the small size of the particles. These small dimensions create problems with handling, transportation and storage and raise the cost of transport. US Patent 7,695 c 1 4 0 0 2 6 9 -0 ΐ -04- 20U

747 B2 presents an agglomeration process that involves the extrusion of the borot and the liquid residue from distillation, followed by microwave drying of the extrudate. Extrusion, however, tends to lead to the expansion of some compounds from the composition of borhot and soluble residue / fermentation yeasts, which make extrusion densification not very pronounced. Advanced densification procedures are required, whereby the volumetric mass of the feed produced on the basis of by-products from the manufacture of cereal bio-ethanol is significantly increased.

The technical problem solved by the invention is to carry out a process for obtaining feed from by-products from (bio) ethanol manufacture, through which a higher densification and a significant inactivation of the contaminants present in the respective byproducts, mycotoxins from cereal and melanoidinic compounds from vinegar.

It is another object of this invention to realize the adsorption of mycotoxins from borhot and / or melanoidin compounds from vinegar into structures that are not metabolized in the digestive system of domestic animals.

The process according to the invention consists of the following steps:

x Normalization of vinegar to 15% dry matter, of which at least 10% of the total dry matter, respectively 1,5% of the total vinegar, is represented by yeast, by evaporation under vacuum;

x Plasmolysis of yeast cells and humification of melanoidinic compounds from vinegar, by laccase treatment, 0.1 parts enzymatically prepared with an activity of 120 LAMU / g per 1000 parts vinous, for 18 hours at 55 ° C, and intermittent homogenization under pressure, every 6 hours, in a piston homogenizer, 5 cycles at 150 MPa;

X Drying of the distillation hopper and fermentation yeast on the roller dryer to form the dry distillation hopper with soluble products, DDGS, with a residual humidity of max. 12%;

x Mixing of the dry distillery pod with plasmolized and humidified vinegar, in a proportion of 95 parts dry distillery to 5 parts vinegar;

x Densification of dry borer mixture - plasmolized and humidified vinegar, by pressing in a pellet press with horizontal molds, to form pellets with a length of approx. 15 mm and the diameter of 5 ... 8 mm.

The proposed process has the following advantages:

^ ° ν, .η ⁶⁹ ·· n ν Reduces the content of soluble melanoidin compounds from vinegar, which have potential deleterious effects, by laccase-catalyzed oxidation / humification of soluble melanoidins;

J Forms humic structures by condensing amino acids / peptides from yeast plasmolysis with humic acids resulting from oxidation of vinyl melanoidins, which have high absorption capacity of mycotoxins in absorption complexes, which are not digested by domestic, ruminant or monogastric animals;

v It favors the process of densification by pelleting, due to the presence in the plasmolized and humidified vinegar of components, respectively of yeast cell wall fragments and of polypeptides, which react with lignocellulosic material, stabilizing the structure of the pellets;

J Significantly increases the final feed, reducing transport, handling and storage costs.

The following is an embodiment illustrating the invention without limitation.

Example. 75 liters of vinegar resulting from the (bio) ethanol manufacture of sugar beet molasses is concentrated by evaporation using a Simax® 251 / h Evaporator (Kavalier, Sazava, Czech Republic). The dry matter was checked, using a Rudolph J257 digital refractometer (Rudolph Research Analytical, Hackettstown, NJ, USA) and the yeast biomass content, by centrifugation at 10,000 xg, for 15 min., Followed by weighing the centrifuge tubes, after constant mass drying at 105 ° C. Vinasa is normalized to 15% dry matter, of which at least 10% of the total dry matter, respectively 1,5% of the total vinegar, is represented by yeast. The result is 52 kg of concentrated syrup, which is transferred into a 100 liter Simax® glass vessel (Kavalier, Sazava, Czech Republic), equipped with a thermostat and mechanical shake. It is treated with a laccase preparation, 0.1 g enzyme preparation with an activity of 120 LAMU / g per 1000 g vinegar.

The laccase preparation used is Denilite® II S (Novozyme A / S, Bagvaerd, Denmark), which has an activity of 120 LAMU / g. A LAMU unit is defined as the amount of enzyme that converts 1 pmol of syringaldazine per minute at pH 7.5 and 30 ° C, with tetramethoxy-azo-bis (methylene-quinone) formation, which is quantified by absorption at 530 nm.

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Hold for 18 hours at 55 ° C. Every 6 hours, respectively at 6 and 12 hours and at the end of the enzymatic oxidation, a pressure homogenization is carried out. A piston homogenizer, GEA Niro Soavi Arriete NS2006 (GEA Niro Soavi, Parma, Italy), equipped with a "knife edge" type valve, 5 cycles at 150 MPa is used.

In parallel, it is dried on a roller dryer with GMF Gouda D5 / 5 (Andritz Separation Gouda, Waddinxveen, The Netherlands), which has an active surface of 0.75 sqm, a diameter of 500 mm and a length of 500 mm, an amount of 4000 kg of distillery hopper which also contains fermentation yeast, to obtain about 1000 kg of dry distillery hopper with soluble products, DDGS, with a humidity of max. 12%.

Mix the dry distillery pod with plasmolized and humidified vinegar, in proportion of 95 parts dry distillery to 5 parts vinegar, respectively 950 kg dry borot, DDGS, with 50 kg plasmolized and humidified vinegar. The resulting mixture of dried borot - plasmolized and humidified vinegar, is densified using a pellet press (mill) with horizontal dies, model Kahl 14-175 (Amandus Kahl, Reinbek / Hamburg, Germany), at a specific power of 1 kW for 0.015 ... 0.02 m ² , maintaining the temperature of the pelletized mixture at about 65 ° C, to form pellets with a length of approx. 15 mm and the diameter of 5..8 mm, resulting in the granulated feed F.

An experiment was performed to test the desorption of contaminants, potentials present in the feeds resulting from the co-products from the manufacture of bio-ethanol, from the structures formed in the granulated feed F. The experiments were carried out by incubation in test solutions that simulate digestion in animals. monogastric and in ruminants, using samples of granulated feed F.

In order to simulate the passage through the digestive tract of the monogastric animals, respectively oral cavity, stomach and duodenum, a pH gradient incubation of 7.4, 2.0 and 8.4 was performed, with the addition of salivary amylase, and pepsin, respectively. and trypsin. 3 g of F feed, ground to 20 mesh (0.85 mm) granulation, using a Retsch mill (Grindomix, Retsch, Haan, Germany), was incubated for 15 min, with shaking and at 37 ° C. , with 30 ml of 0.9% saline, pH 7.4, and 200 units of salivary arrylase (type IX-A, Sigma Aldrich, St. Louis, Mo, USA). One unit of amylase used is that amount of enzyme that releases 1 mg of maltose from starch in three minutes at pH 6.9 and 20 ° C. A 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined. The pH of the solution was reduced to 3.0 by the addition of 1 M HCl. 25 ml of saline and 1000 5 were added.

Ο 1 4 U Ο 2 6 9 - Ο 7 -04- 2014 pepsin units (Sigma Aldrich, 1 enzyme unit produces a variation of absorbance at 280 nm of 0.001 per min at pH 2.0 and 37 ° C, measured as soluble products in TCA, using hemoglobin as substrate). It was incubated for 3 hours, with stirring and at 37 ° C, after which a 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined. The pH was changed to 8.4 with 1 M NaOH, after which 1000 units of trypsin were added (Sigma Aldrich, one unit determines in one minute a variation of extinction at 253 nm of 0.001, when the substrate used is Ν-α-benzoyl-L-arginine ethyl ester, BAEE). It was incubated for 3 hours, with stirring and at 37 ° C, after which a 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined.

The in vitro experimental model used to simulate the passage through the digestive tract of ruminants involved the use of 125 ml Wheaton fermentation vials, in which 3 g of ground F feed and 30 ml of brine were introduced. The three grams were incubated for 30 min, with shaking and at 37 ° C, with 25 ml of 0.9% saline and 300 IU of salivary α-amylase (type IX-A, Sigma Aldrich). A 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined. The remaining medium was bubbled for 10 minutes with 99.8% carbon dioxide, followed by 30 ml of filtered ruminal fluid from non-lactating rumen cannulated in rumen. It was re-bubbled for 10 minutes with 99.8% carbon dioxide, and then incubated for 3 hours at 39 ° C in anaerobiosis. A 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined. The pH was changed to 8.4 with 1 M NaOH, after which 1000 units of trypsin (Sigma Aldrich) were added. It was incubated for 3 hours, with stirring and at 37 ° C, after which a 5 ml aliquot was taken in which the content of mycotoxins and soluble melanoidin compounds was determined.

Similar experiments were carried out using the dry distillery borot, taken from the drying stage, mixed with vinegar with 15% dry matter, non-plasmolized and non-humidified, in proportion of 95 parts dry 15 parts vinyl. The resulting mixture was air-dried at 40 ° C, brought to the same 20 mesh granulation by milling by Retsch milling and used as reference in in vitro desorption experiments by incubation in environments that simulate passage through the digestive tract of animals. farm, monogastric and ruminant, respectively.

To determine aflatoxin from the aliquots taken, the filtrate was extracted three times with 25 ml of chloroform. The cloformic fractions were combined, evaporated 6 ^ 2014 U02 & S '

1 -04- 2014

to dryness, the residue is taken up in methanol and brought to 1 ml in a volumetric flask. Samples from this extract were analyzed on an Agilent 6224 high pressure chromatographic fluid (Agilent Technologies, Santa Clara, CA, USA) using a ternary isocratic mixture of water, methanol and acetonitrile, 50/40/10 , on a ZORBAC Eclipse XBD-C18 column, 4.6 mm x 150 mm x 3.5 pm, with fluorescence detection (AOAC Official Method 994.08, 2000, 17th edition, volume II, AOAC International, Gaithersburg, Maryland, USA ). As a standard, a mixture of aflatoxins provided by Sigma Aldrich - Supelco was used (Sigma Aldrich, the mixture contained aflatoxin Bi 1 pg / ml; aflatoxin B ₂ 0.3 pg / ml; aflatoxin Θί 1 pg / ml; aflatoxin G ₂ 0.3 pg / ml) , and the quantification was done by reporting the surface of the corresponding peaks, from the analyzed samples with those of the standard.

The determination of trichothecenes, respectively 3-acetyl deoxynivalenol (ADON) and deoxinivalenol (DON), from the aliquots taken was performed after repeated extraction three times in ethyl acetate, evaporation to dry, resumption in acetonitrile and determination by HPLC with fluorescence detector ( Matthew et al., 2002, J. Chromatogr., A, 955: 245).

The determination of soluble melanoidin compounds from the aliquots were determined by high pressure chromatography with differential refractometric detector (Shen etal., 2007, Food Chem., 102, 281-287).

Samples of initially representative F granulated feed grains were also sampled, which were ground to 20 mesh granulation (0.85 rpm) using a Retsch mill. The samples determined the total aflatoxins content, after extraction with water-methylene chloride, 1:10 v / v, and trichothecene respectively, after extraction in acetonitrile - water solution (84:16, v / v), pre-purification with multi-functional cleaning column (Mycosep ™, Romer Labs Inc., Union, MT, USA), and HPLC determination with fluorescence detector, and differential refractometric detector, respectively.

To determine the total melanoidinic compounds, humic acids (soluble melanoidinic compounds in alkaline solutions) and fulvic acids (soluble compounds), samples were taken from the granulated feed F, which were extracted with 0.1 M sodium pyrophosphate and 0% sodium hydroxide. , 1 M. The supernatant was acidified to pH 2 with HCl and kept overnight at 24 hours at room temperature. To separate humic acids from fulvic acids, the solution was centrifuged and the humic acid precipitate was dissolved with 2 M sodium hydroxide (Yeomans and Bremner, 1988, Comm. Soil Sci. Plant Anal., 19: 1467-1476) . The carbon content of humic acids and fulvic acids was determined by the method of Sims and Adonis, 1971, Soil Sci., 112: 137-141.

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The same types of determinations, mycotoxins and melanoidinic compounds, were also performed on samples taken from a dry borer mixture of distillery with non-humidified and non-plasmolized vinyl. The results are presented in tables 1 and 2 below, the values determined being normalized to the quantity of sample taken. The feed resulting from the described process significantly reduces the desorption of contaminants in environments that simulate digestion in monogastric and ruminant, respectively.

Tab. 1. Desorption of contaminants from granulated feed F made according to the example in simulation experiments of digestion in farm animals.

contaminant F1 granulated feed content Monogastric simulation Ruminant simulation Oral cavity Stomach Duodenum Oral cavity ruddy Duodenum Total aflatoxin, pg / kg 3.82 (100%) 0 0 0 0 10.99% 12.57% Aflatoxin B) 1.28 (100%) 0 0 0 0 11.72% 14.84% Total t ricotecene, pg / kg 415.9 (100%) 5.53% 8.80% 12.62% 6.18% 9.28% 12.94% ADON, pg / kg 109.2 (100%) 4.40% 7.51% 11.72% 4.85% 9.52% 11.81% DON, pg / kg 306.7 (100%) 5.93% 9.26% 14.57% 6.65% 10.60% 12.68% Total melanoidine 525.9 (100%) 1.52% 2.00% 2.28% 1.37% 2.28% 2.72% Humic acids 423.3 (100%) 0.66% 0.73% 0.85% 0.57% 0.90% 0.97% Fulvic acids 102.6 (100%) 5.07% 7.21% 8.19% 4.68% 7.99% 9.94%

Tab. 2. The desorption of the contaminants from the dry distillery mix mixed with non-humidified vinegar in digestion simulation experiments on farm animals.

contaminant Fodder content Monogastric simulation Ruminant simulation Oral cavity Stomach Duodenum Oral cavity ruddy Duodenum Total aflatoxin, pg / kg 3.87 (100%) 0.00% 16.54% 21.19% 7.24% 36.69% 52.20% Aflatoxin B) 1.24 (100%) 0.00% 19.35% 33.06% 0.00% 30.65% 54.03% Total trichothecene, pg / kg 415.9 (100%) 26.71% 53.21% 82.23% 25.44% 44.60% 80.81% ADON, pg / kg 109.2 (100%) 31.78% 84.80% 89.10% 32.42% 39.56% 87.27% DON, pg / kg 306.7 (100%) 24.91% 41.96% 79.78% 22.95% 46.40% 78.51% Total melanoidine 533.2 (100%) 20.26% 43.21% 58.48% 18.68% 51.22% 60.67% Humic acids 112.6 (100%) 2.49% 2.75% 3.20% 3.02% 4.26% 4.53% Fulvic acids 420.6 (100% 25.01% 54.04% 73.28% 22.87% 63.79% 75.70%

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Claims (1)

Claim 1. Process according to the invention characterized in that it consists of the following steps: normalization of vinegar to 15% dry matter, of which at least 10% of the total dry matter, respectively 1,5% of the total vinegar, is represented by yeast, by evaporation under vid; yeast cell plasmolysis and humification of melanoidinic compounds from vinegar, by laccase treatment, 0.1 parts enzymatically prepared with activity of 120 LAMU / g per 1000 parts vinous, for 18 hours at 55 ° C, and intermittent homogenization below pressure, every 6 hours, in a homogenizer with piston, 5 cycles at 150 Mpa; drying on the roller dryer of the distillation hopper and fermentation yeast, to form the dry distillery hopper with soluble products, DDGS, with a residual humidity of max. 12%; mixing of the dry distillery pod with plasmolized and humidified vinegar, in a proportion of 95 parts dry distillery to 5 parts vinegar; densification of the dry borot mixture - plasmolized and humidified vinegar, by pressing in a pellet press with horizontal molds, to form pellets with a length of approx. 15 mm and the diameter of 5 ... 8 mm.