RU2721403C1 - Method of determining treatment effect in relation to nutritive value of raw materials for feed products - Google Patents

Abstract

FIELD: feed industry.

SUBSTANCE: invention relates to a method of determining treatment effect parameters with respect to quality of raw materials for a fodder product and/or feed products, according to which raw material processing conditions indicator for forage and/or fodder products is determined and specific assimilability of raw material amino acid for fodder product and/or fodder product in animal type is determined. Invention also relates to a method for optimizing fodder products with due allowance for certain indices of effect of treatment and thus produced and/or produced fodder products.

EFFECT: method for determination of treatment effect indices with respect to nutritive value of raw materials for fodder products is proposed.

13 cl, 30 dwg, 2 tbl, 2 ex

Description

The present invention relates to a method for determining the impact of processing on the nutritional value of raw materials for feed products and / or feed products, a method for optimizing feed products taking into account certain indicators of the impact of processing and the resulting and / or resulting feed products.

For many reasons, feed products can have negative effects on animals and related animal products, such as meat and milk, and in the worst case, people as their consumers. Examples of this are the wrong choice of feed products and the choice of diet and, consequently, the resulting nutrition and energy intake, contamination of feed products, biological load and / or load of toxins (mycotoxin) in spoiled feed and the so-called anti-nutritional factors in plant-based feed products .

Anti-nutritional factors are the result of secondary plant metabolism and are present only in certain plant species. They do not perform any important functions in the primary metabolism. Rather, their function is to protect against predators and pests, regulate and function as coloring agents and aromatic substances. The negative effects of anti-nutritional factors on animals when absorbed in food are a decrease in animal productivity, a change in the absorption of nutrients, metabolic disorders and their toxicity.

Anti-nutritional factors can be grouped into groups of substances of carbohydrates, proteins, phenols and derivatives of phenols, glucosides and glycosides, chelators and glucosinolates, as well as strumogenic factors, and individual compounds can be grouped into more than one class of substances.

Examples of anti-nutritional factors from the group of carbohydrate substances are:

non-starch polysaccharides that are present as part of the cell walls, such as pentosans, which are present in lupine, barley, corn and rye, beta-glucans, which are present in barley and rye, and pectins, which are present in sunflower varieties. Because of their ability to swell, these carbohydrates lead to the accumulation of water in animals, in particular in young birds, an increase in chyme viscosity, a decrease in specific caloric content in the feed product, a decrease in digestibility, and a decrease in terms of growth and productivity; and

- Non-digestible oligosaccharides, such as alpha-galactosides, such as raffinose, stachyose, verbacose and adjugose, which are present in the lupine, soybean and canola varieties and which are quickly converted by germs in the cecum / colon, leading to bloating and diarrhea .

Examples of anti-nutritional factors from the group of protein substances are:

- proteinase inhibitors found in beans, varieties of peas, lupins, soybeans, cyamopsis and rice (used for (young) chickens, piglets and carnivores), which inhibit the activity of trypsin and chymotrypsin and thus reduce the digestibility of protein; and

- lectins (hemagglutinins), which are found in Phaesolus species (beans, peas, soybeans and lupins (used for monogastric animals), and which bind to intestinal mucosa receptors, which leads to impaired resorption and in vitro agglutination of red blood cells.

WO 2011/109624 A1 discloses soybean species having a genetic allele for producing low levels of trypsin inhibitor in seeds. This document also discloses that soybean descendant plants of soybean varieties with an extremely low trypsin inhibitor content, where the offspring includes a combination of at least two types of soybean plants with the characteristics specified in WO 2011/109624 A1, so that said soybean descendant plant is not substantially differs in respect of these traits from soybean plants from WO 2011/109624 A1, as determined at a significance level of 5% when grown under the same environmental conditions.

Examples of anti-nutritional factors from the group of phenol substances and phenol derivatives are:

- tannins (derivatives of phenols), which are found in varieties of beans and peas (used for poultry, pigs and horses), which reduce feed intake, inhibit proteolytic enzymes and reduce protein digestibility, which leads to blockages in the stomach;

- alkylresorcinols that are in barley, often in triticale and very often in wheat (used for monogastric animals), which leads to reduced feed intake and growth; and

- gossypol, found in the seeds of cotton (used for all types of animals), which has a hemolytic effect because it binds to iron and leads to discoloration of the yolk, impaired protein metabolism and spermatogenesis, as well as damage to the liver and kidneys.

Examples of anti-nutritional factors from the group of glucoside and glycoside substances are:

- pyrimidine glucosides (vicin, convicin, etc.), which are found in varieties of beans and vetch (used for laying hens and sows), which lead to impaired fat metabolism, decreased parameters of laying and hatching, as well as impaired fertility and lactation;

- alpha-galactosides, which are in varieties of lupine, beans and peas;

- cyanogenic glucosides found in Phaesolus species (varieties of vetch seed, flax seed, tapioca and lupine species) (used for horses and all types of livestock), which lead to symptoms of intoxication due to the release of hydrocyanic acid (respiratory poison); and

- saponins found in varieties of beans, peas and lupins (used for: (young) chicken), which give a bitter taste, which leads to a decrease in feed intake, have a hemolytic effect and are also an antagonist of vitamin D.

Examples of anti-nutritional factors from the group of alkaloids are:

- alkaloids spartein, lupinin, hydroxylupanine, angustifolin, solanine, which are in varieties of lupine and, in particular, in varieties of tomatoes and potatoes (used for monogastric animals, pigs and cattle), ergot alkaloids, in particular, in rye, which give a bitter taste, which leads to a decrease in feed intake, but most should be noted, alkaloids are toxic, ergot alkaloids can lead to abortion, paralysis and spasms, in the area of cattle alkaloids, therefore, lead to a decrease in milk production; and

- sinapine found in rapeseed (used for hens, particularly hens, egg-laying brown-shelled) which intestinal bacteria was converted to t ri m etil and min (TMA), which is enriched in the liver, if the activity of TMA-oxidase in the liver not high enough and gives a fishy smell to the eggs.

Examples of anti-nutritional factors from the group of chelator substances are:

- phytic acid, which is found, for example, in corn, corn grains and waste extracts (used for monogastric animals, pigs and poultry), which reduces the availability of doubly charged ions, such as Ca ²⁺ , Zn ²⁺ and Fe ²⁺ , in the body by chelating these ions; and

- gossypol, found in the seeds of cotton (used for all types of animals), which has a hemolytic effect, since it binds to iron and leads to discoloration of the yolk, impaired protein metabolism and spermatogenesis, as well as damage to the liver and kidneys.

Examples of anti-nutritional factors from glucosinolate groups of substances are:

- glucobrassicin, gluconapine, glucobrassiconapine and progoytrin, all of them are in rapeseed (used for breeding animals, in particular pigs, poultry and calves, dairy cows), are enzymatically cleaved with the release of toxic compounds of isothiocyanate, thiocyanate and nitrile; other glucosinolates and their cleavage products lead to a decrease in feed intake, they impede the effectiveness of fertility and the production of thyroid hormones, contribute to the formation of goiter and cause the transfer of strumogenic factors into milk.

Strumogenic factors (found in soybean, flax seed and cabbage) also lead to an enlarged thyroid gland (= goiter).

The above non-limiting list of anti-nutritional factors and their negative effects in relation to animals shows that anti-nutritional factors are of great importance in feeding practice. Thus, in order to avoid the negative effects of anti-nutritional factors in relation to animals, anti-nutritional factors should be excluded from the raw materials used to obtain feed products. In the case when it is impossible to completely remove anti-nutritional factors from raw materials for feed products, the supply of anti-nutritional factors to animals must be limited in order to avoid harmful effects in relation to animals.

To remove anti-nutritional factors from raw materials for feed products or to reduce their presence in raw materials for feed products, the raw materials used in obtaining lump products are subjected to a treatment in which they are subjected to heat treatment, such as steaming or frying, which leads to removal , among others, inhibitors of proteinase and lectins, or treatment with alkali, which, for example, leads to the removal of synapine. Therefore, many raw materials for feed are heat treated. In addition, feed products are also heat treated to remove moisture. For example, the article “Feed extrusion process description” by Galen J. Rokey et al. (Revista Brasileira de Zootecnia, vol. 39, pp. 510-518, 2010) describes that extrusion steaming for the production of many products has a long history over the past three decades and provides a very useful and economical tool for processing animal diets. This treatment allows the best use of available cereal grains and vegetable and animal proteins in order to provide cost-effective and nutrient-rich diets with improved and unique nutritional characteristics. In addition, the articles additionally disclose that palatable, functional, and specially prepared feeds can be produced cost-effectively using raw material compositions, system configurations, and processing conditions.

However, such heat treatment can damage amino acids present in feed materials for feed. For example, compounds with an amino group, such as amino acids and proteins, undergo a Maillard reaction in the presence of reducing compounds, in particular reducing sugars. This is particularly true for lysine with an ε-amino group, which can react with a variety of ingredients in feed materials for feed. Compounds resulting from these reactions can be partially absorbed in the intestines of the animal, but they have no nutritional value. For example, the free ε-amino group of lysine molecules or lysine-containing proteins can react with the carbonyl group of reducing sugars, in particular hexoses, such as glucose, in a reversible condensation reaction, which initially gives Schiff base, which subsequently reacts in the process of irreversible Amadori rearrangement with the formation of ε -N-deoxyketosyllisine, sometimes referred to as Amadori's product or Maillard's early product. ε-N-Deoxyketosyl lysine can further react with the formation of brown pigments or melanoidins, which are nitrogen-containing organic compounds from yellow to brown or almost black. Schiff bases, at least formed from aliphatic aldehydes and reducing sugars, can be almost completely absorbed in the intestines of mammals. In comparison, the metabolism of the Amadori product of ε-N-deoxyketosyl is not significant. The conditions used in the processing of raw materials for feed products, in particular, high temperatures during steaming or frying, extreme pH values and high concentrations of reagents, contribute to the Maillard reaction. However, some of the reacted lysine derivatives are unstable acids and can be converted back to lysine during the acid hydrolysis step of a standard wet chemical amino acid analysis. This, however, does not occur in the digestive tract. Consequently, the amino acid concentrations in feed products determined using standard amino acid analysis will be misleading and lead to a reassessment of the real amino acid content and their availability in feed products damaged by heat treatment.

The Maillard reaction is considered the main cause of the destruction of amino acids and proteins containing amino acids in raw materials for feed products, in particular, lysine or lysine-containing proteins. However, in addition to the Maillard reaction, there are other reactions that lead to the destruction of amino acids and proteins containing amino acids. For example, strong heating of proteins in the absence of fats or (reducing) sugars leads to the reaction of lysine molecules with amino acid side chains of amino acids, such as asparagine and glutamine, with the formation of internal peptide bonds, the so-called isopeptides. In addition to reactions producing isopeptides, other reactions also occur, such as the formation of lysinoalanine, the reaction of lysine molecules with oxidized polyphenols, amino acid acylation, and amino acid racemization. In addition to modifying lysine molecules, processing feed materials for feeds also leads to protein denaturation and the formation of extensive cross-linking of proteins, intra- and intermolecular, as well as other amino acids other than lysine. The aforementioned reactions, including the Maillard reaction, can lead to a general loss of amino acids and a decrease in the digestibility of amino acids and proteins in feed materials for feed products, thereby reducing the consumption of amino acids, in particular lysine, and proteins.

Further processing of feed products may also lead to a decrease in the availability or solubility of proteins. For example, US 5733238 discloses a mixed source of organic and inorganic nitrogen with variable solubilities in the form of non-protein nitrogen, peptides, amino acids and intact protein obtained in the preferred embodiment of a feed additive according to US 5783218 from fermentable glutamic acid residues and / or fermentable corn residues, in which can be added carrier, additional amino acids and enzymes and which are superior to compositions of the prior art. This document further discloses that a conventional chemical analysis of feed additives based diets reflects the solubility value that would be obtained with raw materials, since a conventional chemical analysis of feed ingredients is not able to distinguish between changes in solubility rates. From a chemistry point of view, the modification of nitrogen solubility that occurs in a feed additive according to US Pat. No. 5,783,238 as a result of processing can be measured by measuring free chlorine. This analysis showed that only 33% of the non-protein nitrogen components in the mixture according to US 5733238 were readily soluble.

WO 97/02489 A1 and NZ 312221 A disclose a method for determining the digestibility coefficient of reactive lysine for a food product. This method involves the steps of: a) introducing a marker into the analyzed food product, b) feeding the food product of a non-human subject for a predetermined period of time, c) obtaining a sample of the digested food product from the subject, d) determining the content of digestible reactive food lysine product using: i) introducing a derivatized lysine product into a food product; and ii) determining the content of digestible reactive lysine in a food product by measuring the equivalent content of derivatized lysine in a food product; e) determining the content of digestible reactive lysine in a digested food product by i) introducing a derivatizing lysine means for the epsilon-amino group of lysine in a digested food product and ii) determining the content of digestible reactive lysine in the digested food product by measuring the equivalent content of derivatized lysine in food the product and the digested food product, f) measuring the concentration of the marker in the food product and the digested food product, g) expressing the content of reactive lysine of both the food product and the digested food product per gram marker, and h) calculating the digestibility coefficient of reactive lysine.

Thermal exposure also has a significant effect on the amino acid content of other feed products that are obtained by high temperature treatment, such as the so-called DDGS (Refined Grain with Soluble Substances). Typically, DDGS is obtained in bioethanol plants based on starchy crops such as corn, wheat, barley and sorghum after distilling off ethanol and drying the remaining stillage, which is a by-product. Proteins, fibers and oils contained in bard are nutrients that determine its use as a feed product. However, only the dried by-product is suitable for storage and can also be fed to species other than ruminants. Typically, the dried by-product is granulated after drying, and the feed product thus obtained is commonly called DDGS. About a third of the crops used for bioethanol production result in DDGS. Each bushel of cereals used in bioethanol production (one bushel of cereals in the United States is 35.2391 liters) gives approximately 2.7 gallons of ethanol (1 gallon is 4.54609 liters), 18 pounds of DDGS (1 pound is 453.59237 g ) and 18 pounds of carbon dioxide. DDGS is characterized by a high content of cereal residues and residues of yeast proteins, minerals and vitamins and, therefore, a high residual energy value. Due to its high protein content of approximately 30% and its additional energy value, DDGS is a source of proteins and energy that are easily absorbed by beef cattle and dairy cows. In addition, DDGS can be used to feed poultry and pigs. The use of DDGS for feeding ruminants is especially common and well documented in the United States. In North America, approximately 80% of the volume of DDGS is used to feed cattle. However, the thermal effect during the distillation of ethanol formed during fermentation and drying of the remaining by-product leads to severe heat stress with respect to amino acids in the by-product, which can lead to a Maillard reaction of amino acids and proteins, the formation of isopeptides and lysinoalanines, the reaction of amino acids with oxidized polyphenols, acylation amino acids, racemization of amino acids, protein denaturation and the formation of a large number of protein cross-linking.

Typically, the amounts of amino acids in a feed material for a feed product are determined either using standard amino acid analysis methods or evaluated using near infrared spectroscopy. The standard methods for amino acid analysis are wet chemical methods in which the amino acids present in the feed materials of a feed product are either first boiled in hydrochloric acid to release the amino acids from the proteins with which they are mainly associated, followed by chromatographic separation of the hydrolyzate, or first oxidized, then subjected to hydrolysis and, finally, the hydrolyzate is subjected to chromatographic separation. The first alternative is applicable to all amino acids, with the exception of tryptophan, which is destroyed by hydrolysis, and methionine and cystine, which are partially decomposed by hydrolysis. However, sulfur-containing amino acids, methionine, cystine, and cysteine, can be quantified if they are oxidized at 0 ° C with permiric acid to methionine sulfone and cysteic acid before hydrolysis and if these derivatives are analyzed after hydrolysis. Both cystine and cysteine are determined as cysteic acid in the hydrolysates of an oxidized sample. During the hydrolysis of amino acids, asparagine and glutamine are completely converted to aspartic and glutamic acids and can be determined as such. Therefore, glutamine and asparagine are always determined together with naturally occurring glutamic acid and aspartic acid. Accordingly, certain values for glutamic acid and aspartic acid are summary parameters. The second alternative is applicable to all amino acids, with the exception of tyrosine, which, however, decomposes at the oxidation stage. Both alternatives allow you to accurately determine the amino acid content. However, the big disadvantage is that both alternatives are long and laborious. Therefore, these methods are not suitable for quick analyzes, in particular, not as routine methods. For comparison, near infrared spectroscopy is not suitable for accurate or even highly accurate determination of the amino acid content in a sample. Rather, this method only allows you to estimate or predict the amino acid content in the sample, but it is very light and very fast.

Evonik produces amino acids for feed and has over 50 years of experience in amino acid analysis. In 2012, using wet chemistry, the company investigated about 15,000 samples per year (see “Evonik's Amino NIR - NIR for the feed industry” by Richard Mills (http://nirperformance.com/2012/10/24/evoniks-amino -nir /). Although wet chemistry reference methods remain the gold standard for amino acid analysis, rapid tests using NIR are becoming increasingly important in providing customers with timely results to help create the best possible diets. NIR tools are networked with the Evonik laboratory in the center This network is constantly growing and currently includes about 870 NIR instruments located in feed mills and analytical laboratories around the world.

WO 01/15548 A1 and EP 1145645 A1 disclose a method for analyzing, selecting and improving raw materials for use in animal feed products in such a way as to eliminate systematic compositional failure, while guaranteeing the required level of nutrients in the supplemented product. In more detail, these documents disclose a method comprising the steps of analyzing the nutritional composition of batches of raw materials for use in an animal feed product, comprising measuring the amount of the last amino acids in a raw material using near infrared spectroscopy when comparing the nutritional composition with a predetermined nutritional composition, calculating the amount of additional nutrients needed to bring the batch composition into a predetermined nutritional composition, determining a threshold value for which there are clusters of raw materials that are both economically and nutritionally favorable, screening batches to reject those for which the required amount of additional nutrients exceed the threshold value, and approve those for which the required amount of additional nutrients is less than the threshold value, and supplement only approved batches of raw materials using the calculated amount of additional nutrients.

Numerous parameters are known for characterizing treatment influence indicators in relation to raw materials for a feed product, but experiments have shown that none of the parameters known in the literature is suitable for adequately characterizing food-related processing influence indicators in relation to raw materials for a food product. Among other things, this is due to the fact that individual parameters lead to different statements. For example, determination of urease activity is the most common test for assessing the quality of soybean processing. However, this test reveals only insufficient processing of raw materials for the feed product, but it is not suitable for detecting excessive processing of raw materials for the feed product. For comparison, the solubility of the sample proteins in alkali, in principle, makes it possible to distinguish excessively processed products from sufficiently processed products. However, this difference requires assumptions about the degree of thermal damage at specific values of the solubility of proteins in alkali. Therefore, assumptions already have a big impact on the categorization of the feed material for the feed product and / or feed product. In addition, separately, this method also leads to conflicting statements regarding the quality of the raw material for the feed product and / or feed product.

Therefore, it is not surprising that neither certain known parameters, nor a specific combination of parameters have been accepted in the feed industry as sufficient or even mandatory for characterizing food-related characteristics.

Thus, a need arose for a method that allows characterizing the parameters of the influence of processing in relation to the nutritional value of raw materials for a feed product on a global scale and regardless of the specific significance and, in particular, the strengths and weaknesses of individual methods.

According to the present invention, this problem is solved by obtaining a set of parameters that complement each other with respect to their significance and, therefore, are combinable. These parameters include, but are not limited to, trypsin inhibitor activity, urease activity, alkali protein solubility, protein dispersibility index and / or ratio of reactive lysine to total lysine. An additional parameter is at least one amino acid selected from the group consisting of methionine, cysteine, cystine, threonine, leucine, arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, aspartic acid and glutamic acid. These parameters are obtained by quantitative analysis of a series of samples of feed material for a feed product at different time points for processing a specific feed material for a feed product. For each of the defined parameters, a so-called processing condition indicator (PCI) is defined which describes all possible processing conditions for the feed material for the feed product, i.e. insufficient, sufficient or excessive processing. The thus obtained indicator of the processing conditions is then plotted on a graph to scale to facilitate the classification of the feed material for the feed product as under-processed, sufficiently processed or over-processed.

This procedure is not limited to any particular raw material for the feed product and, therefore, can also be used to determine the effects of processing on feed products, such as refined soluble solids (DDGS).

Thus, the aim of the present invention is a method for determining the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product, comprising the following stages:

a) exposing a sample of processed raw material for the feed product and / or feed product

a1) a quantitative analysis of at least one parameter selected from the group consisting of trypsin inhibitor activity, urease activity, alkali protein solubility, and protein dispersibility index;

a2) determining the ratio of the amount of reactive lysine to the total amount of lysine, which involves a quantitative analysis of the amount of reactive lysine and the total amount of lysine, followed by obtaining the ratio of the amount of reactive lysine to the total amount of lysine, and

a3) a quantitative analysis of the amount of at least one amino acid selected from the group consisting of methionine, cysteine, cystine, threonine, leucine, arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, asparaginic acids and glutamic acid;

b) plotting the parameters obtained in stages a1) to a3), depending on the processing times of the sample from stage a);

c) determining the region in the graph from step b), where the trypsin inhibitor activity value, expressed in mg of trypsin per g of sample, is more than 4, the increase in pH when determining the urease activity is more than 0.35, the protein solubility in alkali, expressed as the percentage of protein in the sample, soluble in an alkaline solution, is more than 85%, and / or the value of the dispersibility indicator of the protein, expressed as a percentage of the initial nitrogen content in the sample, is more than 40%, and assigning the region thus obtained to an insufficiently processed a sample;

d) determining the area in the graph from step b) where the ratio of the amount of reactive lysine to the total amount of lysine is less than 90%, the value of the protein dispersibility index, expressed as a percentage of the initial nitrogen content in the sample, is less than 15%, and / or the value of the solubility of the protein in alkali, expressed as the percentage of protein in the sample soluble in the alkaline solution, is less than 73%, and assigning the thus obtained region to an overly processed sample;

e) determining the area in the graph from step b) where the trypsin inhibitor activity, expressed in mg of trypsin per g of sample, is less than 4, the alkali protein solubility, expressed as the percentage of protein in the sample soluble in the alkaline solution, is from 73 to 85%, the value of the indicator of dispersibility of the protein, expressed as a percentage of the initial nitrogen content in the sample, is from 15 to 40%, and the ratio of the amount of reactive lysine to the total amount of lysine is at least 90%, and the classification of the resulting image of the area to a sufficiently processed sample;

 and / or

 subtracting the areas identified in stages c) and d) from graph b) and assigning the thus obtained area to a sufficiently processed sample;

f) obtaining a processing scale by standardizing the areas obtained in steps c) to e) to the same size, sorting them from over-processed to under-processed, or vice versa, and correlating the continuous scale and standardized and sorted areas;

g) substituting the values of the parameters obtained in stages a1) to a3) in a power series and calculating the average value based on the values obtained from each power series, the indicated average value being designated as an indicator of processing conditions (PCI),

 and

h) applying an indicator of the processing conditions obtained in step g) to the processing scale obtained in step f), determining whether the feed material for the feed product and / or the feed product is over-processed, sufficiently processed or under-processed.

In the context of the present invention, the term “amount of reactive lysine” is used to mean the amount of lysine that is actually available for the animal, in particular for digestion in the animal. For comparison, the term "total amount of lysine" is used in the context of the present invention in relation to the total amount of lysine that is actually available for the animal, in particular for digestion in the animal, and the amount of lysine that is not available for the animal, in particular not available for digestion in animal. The latter amount of lysine is usually due to lysine decomposition reactions, such as the Maillard reaction already mentioned.

In the context of the present invention, a treatment that results in damage to the raw materials for the feed product and / or feed products and, in particular, to a decrease in amino acid amounts, is called over processing. In comparison, a treatment that does not provide complete or at least acceptable removal of anti-nutritional factors from the raw materials for the feed product and / or from the feed product is called insufficient processing. Finally, a treatment that results in complete or at least acceptable destruction of anti-nutritional factors without destroying the amino acids and / or proteins is called a sufficient treatment or a sufficient treatment.

A quantitative analysis of the activity of a trypsin inhibitor is based on the ability of inhibitors to complex with the trypsin enzyme and thus reduce its activity. Trypsin catalyzes the hydrolysis of synthetic substrates, N- and lf- b- benzoyl-D, L-arginine-p-nitroanilide (DL-BAPNA, IUPAC name, N- [5- (diaminomethylideneamino) -1- (4-nitroanilino) -1 -oxopentan-2-yl] benzylamide) and N- a lpha- b- benzoyl-L-arginine-p-nitroanilide (L-BAPNA, name according to IUPACN- [5- (diaminomethylideneamino) -1- (4-nitroanilino) -1 -oxopentan-2-yl] benzylamide). By catalyzed hydrolysis, a yellow product that does not contain p-nitroaniline is released, and therefore this leads to a change in the absorption rate. Trypsin activity is proportional to yellow staining. The concentration of p-nitroaniline can be determined using spectroscopy at a wavelength of 410 nm. According to the method of ISO 14902 (2001), L-BAPNA is usually used, and DL-BAPNA is usually used according to the method of AACC 22.40-01 (modification of the method originally invented by Hamerstrand in 1981).

According to ISO 14902, the sample is first finely ground with a 0.50 mm sieve. Heat should be avoided during grinding. The ground sample is mixed with an aqueous alkaline solution, for example, 1 g of the sample in 50 ml of sodium hydroxide solution (0.01 N), and the resulting solution, suspension, dispersion or emulsion is then stored for up to approximately 24 hours at a temperature not higher than 4 ° C. The mixture thus obtained has a pH from 9 to 10, in particular from 9.4 to 9.6. The resulting solution was diluted with water and allowed to settle. A sample of this solution, for example, 1 ml, is taken and diluted in accordance with its intended or previously approximate activity of a trypsin inhibitor, as a result of which 1 ml of a diluted solution will cause 40-60% inhibition of the enzymatic reaction. Trypsin stock solution, for example, 1 ml, is added to a mixture of L-BAPNA, water and a diluted sample extract solution, for example, 5 ml L-BAPNA, 2 ml (distilled) water and 1 ml of a respectively diluted sample extract solution. Then the samples are incubated for exactly 10 minutes at 37 ° C. The reaction is stopped by the addition of 1 ml of acetic acid (30%). A blank sample is prepared as described above, but trypsin is added after acetic acid. After centrifugation at 2.5 g, the absorbance is measured at a wavelength of 410 nm.

In AACC method 22-40.01, the sample is first finely ground using a 0.15 mm sieve. Heat should be avoided during grinding. The ground sample is mixed with an aqueous alkaline solution, for example, 1 g of the sample in 50 ml of sodium hydroxide solution (0.01 N) and slowly stirred for 3 hours at 20 ° C. The pH of the thus obtained solution, suspension, dispersion or emulsion should be from 8 to 11, preferably from 8.4 to 10. The resulting solution, suspension, dispersion or emulsion is diluted with water, shaken and allowed to settle. A sample of this solution, for example, 1 ml, is taken and diluted in accordance with its intended or previously approximate activity of a trypsin inhibitor, as a result of which 1 ml of a diluted solution will cause 40-60% inhibition of the enzymatic reaction. Trypsin stock solution, for example, 2 ml, is added to a mixture of D, L-BAPNA, water and a diluted sample extract solution, for example, 5 ml D, L-BAPNA, 1 ml of (distilled) water and 1 ml, respectively, of a diluted sample extract solution. Then the samples are incubated for exactly 10 minutes at 37 ° C. The reaction is stopped by the addition of 1 ml of acetic acid (30%). A blank sample is prepared as described above, but trypsin is added after acetic acid. After centrifugation at 2.5 g, the absorbance is measured at a wavelength of 410 nm.

Regardless of the method used, trypsin inhibitor activity is calculated as mg of trypsin inhibitor per g of trypsin according to the following formula:

,

,

i = percent inhibition (%);

Ar = standard absorption rate of the solution;

Abr = blank sample absorption standard;

As = absorption rate of the solution with the sample;

Abs = blank sample absorption rate with sample;

,

,

TIA = trypsin inhibitor activity (mg / g);

i = percent inhibition (%);

m0 = mass of the test sample (g);

m1 = trypsin mass (g);

f1 = dilution factor of sample extract; and

f2 = conversion factor based on trypsin purity.

One unit of trypsin is defined as the amount of enzyme that will increase the absorbance at 410 nm by 0.01 units after 10 minutes of reaction for every 1 ml of reaction volume. Trypsin inhibitor activity is defined as the number of inhibited trypsin units (TIU). TIU per ml is calculated by the formula

,

,

Where

A _blank = blank sample absorption rate

A _sample = absorption rate of the sample

V _Sample = volume of diluted sample solution in ml.

The TUI thus obtained is plotted against the volumes of the diluted sample solution, where an extrapolated inhibitor volume of up to 0 ml gives the final TUI [ml]. Finally, TUI per g of sample is calculated by the formula,

,

,

where d = dilution factor (final volume divided by the amount taken).

The results of this analytical method should not exceed 10% of the average for repeated samples.

Thus, a quantitative analysis of the activity of trypsin inhibitors preferably involves the following steps:

i) dissolving a sample of the feed product and / or feed material for the feed product in an alkaline solution;

ii) diluting an aliquot of the solution obtained in stage i) to obtain a mixture in which the concentration of the trypsin inhibitor is sufficient for approximately 40-60% inhibition of trypsin;

iii) adding a specific volume of trypsin solution to the mixture obtained in step ii);

i) adding BAPNA to the mixture obtained in step iii) to initiate the hydrolysis of BAPNA with trypsin;

ii) termination of the hydrolysis reaction;

iii) measuring the absorption index of the mixture obtained in stage v) at a wavelength of 410 nm and calculating the number of inhibited trypsin units (TUI) according to the formula,

,

,

Where

A _blank = blank sample absorption rate

A _sample = absorption rate of the sample

V _Sample = volume of diluted sample solution in ml;

and

plotting the TUI obtained in stage viii) on the volumes of the diluted sample solution, where an extrapolated inhibitor volume of up to 0 ml gives the final TUI [ml]; and / or

iv) TUI per g of sample according to the formula

,

,

where d = dilution factor (final volume divided by the amount taken).

The urease enzyme catalyzes the decomposition of urea to ammonia and carbon dioxide. Since urease is naturally found in soybean varieties, quantification of this enzyme is the most common test for assessing the quality of processed soybean varieties. Preferably, a quantitative analysis of urease is carried out in accordance with the method of ISO 5506 (1988) or AOCS Ba 9-58. Using the AOCS Ba 9-58 method, the residual urease activity is determined as an indirect indicator to assess whether protease inhibitors were destroyed during processing of the raw material for the feed product and / or feed product. The indicated residual urease activity is measured as an increase in pH in the test as a result of the release of an alkaline ammonia compound into the medium. The recommended level for the indicated increase in pH is an increase of 0.01-0.35 units (NOPA, 1997). A typical quantitative analysis of the urease activity of the raw material for a feed product and / or feed product is carried out as follows. First, a urea solution is prepared in a buffer containing NaH ₂ PO ₄ and KH ₂ PO ₄ , for example, 30 g of urea is added to 1 l of a buffer solution consisting of 4.45 g of Na ₂ HPO ₄ and 3.4 g of KH ₂ PO ₄ , and measure the pH value thus obtained. Then a sample of raw material for the feed product and / or feed product, for example, 0.2 g of soybean sample, is added to this solution. A test tube or beaker with the solution, suspension, dispersion or emulsion thus obtained is placed in a water bath, for example, at a temperature of 30 ± 5 ° C, preferably 30 ° C, for a period of 20 to 40 minutes, preferably 30 minutes. Finally, the pH of this solution, suspension, dispersion or emulsion is measured by comparison with the pH of the urea stock solution, and the difference is presented as an increase in pH.

Thus, a quantitative analysis of urease activity preferably involves the following steps:

i) obtaining a urea solution in a buffer containing Na ₂ HPO ₄ and KH ₂ PO ₄ ;

ii) measuring the pH of the solution from step i);

iii) adding a sample of raw material for the feed product and / or feed product to the urea-containing solution;

iv) maintaining the solution, suspension, dispersion or emulsion thus obtained at a constant temperature for a certain period of time, followed by measuring the pH of the solution, suspension, dispersion or emulsion, and

v) an expression of the difference between the pH values measured in steps ii) and iv) as an increase in pH.

The solubility of proteins in alkali, hereinafter also referred to as the solubility of proteins in an alkaline solution or the alkaline solubility of proteins, is an effective way to separate over-processed products from sufficiently processed products, for example, in accordance with DIN EN ISO 14244.

The solubility of proteins in alkali or alkaline solubility of proteins includes determining the percentage of protein that is dissolved in an alkaline solution. Before dissolving a sample with a known weight of the raw material for the feed product and / or feed product, the nitrogen content in the sample with a certain weight is determined using a standard method for determining nitrogen, such as the Kjeldahl or Dumas method. The nitrogen content thus determined is called the total nitrogen content. After that, a sample with the same weight and from the same source is suspended in an alkaline solution of a certain concentration, preferably in an alkaline hydroxide solution, in particular in a potassium hydroxide solution. An aliquot of the suspension thus obtained is collected and centrifuged. Again an aliquot of the suspension thus obtained was taken. The nitrogen content in this liquid fraction is determined using a standard nitrogen determination method, such as the Kjeldahl or Dumas method. The nitrogen content thus determined is compared with the total nitrogen content and expressed as a percentage of the initial nitrogen content of the sample.

Quantitative analysis of the alkaline solubility of proteins preferably includes the following steps:

i) determining the nitrogen content of the sample of raw material for the feed product and / or feed product, preferably using a method such as the Kjeldahl or Dumas method;

a) placing an aliquot of the sample from step i) in an alkaline solution, preferably in a solution of sodium hydroxide or potassium hydroxide, followed by stirring;

a) centrifuging the suspension, solution, dispersion or emulsion obtained in stage ii);

iv) determining the nitrogen content in an aliquot of the solution or supernatant of the suspension, solution, dispersion or emulsion obtained in step iii), preferably using a method such as the Kjeldahl or Dumas method; and

v) calculating the alkaline solubility of proteins in the form of the ratio of the nitrogen content determined in step iv) to the nitrogen content determined in step i).

Preferably, the alkaline solution used in step ii) is characterized by a pH of from 11 to 14, in particular from 12 to 13, for example, 12.5. The amount of alkali, such as sodium hydroxide or potassium hydroxide used to produce the alkali solution, depends on the volume of solution to be obtained.

A typical alkaline solution for determining the alkaline solubility of proteins is characterized by a pH value of, for example, 12.5, and is a potassium hydroxide solution with a concentration of 0.036 mol / L or 0.2% by weight. In step ii), 1.5 g of a soybean sample, for example, is placed in 75 ml of potassium hydroxide solution, followed by stirring at 8500 rpm. (rpm) for 20 minutes at 20 ° C. An aliquot of, for example, approximately 50 ml of the suspension, solution, dispersion or emulsion thus obtained is then taken and centrifuged immediately at 2500 g for 15 minutes. An aliquot of, for example, 10 ml of the supernatant of the suspension, solution, dispersion or emulsion thus obtained is taken, and the nitrogen content in the indicated aliquot is determined using standard nitrogen determination methods, such as the Kjeldahl or Dumas method. Finally, the results are expressed as a percentage of the nitrogen content of the sample.

By determining the protein dispersibility index (PDI), the solubility of proteins in water is measured after mixing the sample with water. This method also includes determining the nitrogen content in the sample with a known weight, which is usually performed in accordance with the same procedure as in the wet chemical analysis of proteins. The nitrogen content thus determined is also called the total nitrogen content. In addition, this method also involves obtaining a suspension of a sample of the same weight as that used to determine the nitrogen content, which is suspended in water, which is usually performed using a high-speed mixer. The suspension thus obtained is filtered, and the filtrate is subjected to centrifugation. The nitrogen content of the thus obtained supernatant is determined using a standard determination method, such as the Kjeldahl or Dumas method described above. The nitrogen content thus determined is also called the nitrogen content of the solution. The protein dispersibility index is ultimately calculated as the ratio of the nitrogen content of the solution to the total nitrogen content and expressed as a percentage of the original nitrogen content of the sample.

Quantitative analysis of the indicator of dispersibility of the protein preferably includes the following stages:

a) determining the nitrogen content of a sample of raw material for the feed product and / or feed product, preferably using a method such as the Kjeldahl or Dumas method;

ii) placing an aliquot of the sample from step i) in water;

iii) determining the nitrogen content of the dispersion obtained in step ii), preferably using a method such as the Kjeldahl or Dumas method; and

i) the calculation of the dispersibility index of the protein as the ratio of the nitrogen content determined in stage iii) to the nitrogen content determined in stage i).

Since the values of the protein dispersibility index increase with decreasing particle size, the results obtained when determining the protein dispersibility index depend on the particle size of the sample. Therefore, it is preferable to grind the sample to be determined for the dispersibility index of the protein, in particular with a sieve with a mesh size of 1 mm.

The procedure described above is in accordance with the American Petrochemical Society Official Method Ba 10-65 (A.O.C.S.), in which it is preferable to determine the protein dispersibility index. The nitrogen content of, for example, a soybean sample is determined using standard nitrogen determination methods, such as the Kjeldahl or Dumas method. An aliquot, for example, 20 g, of a soybean sample is placed in a mixer and (deionized) water, for example, 300 ml, is added at 25 ° C, followed by stirring, for example, at 8500 rpm. for 10 minutes. The suspension, solution, dispersion or emulsion thus obtained is filtered and the solution, dispersion or emulsion thus obtained is centrifuged, for example, at 1000 g for 10 minutes. Finally, the nitrogen content in the supernatant is determined using standard nitrogen determination methods, such as the Kjeldahl or Dumas method.

Many feed products are processed, which leads to possible damage to amino acids. This may make some amino acids inaccessible for their use in nutrition. This is especially true for lysine, which has an ε-amino group that can react with the carbonyl group of other compounds, for example, reducing sugars present in the diet, to produce compounds that can be partially absorbed from the intestine, but which have no nutritional value for the animal . The reaction of the ε-amino group of free and / or protein bound lysine with reducing sugars during heat treatment is known as the Maillard reaction. This reaction provides both early and late Maillard products. The early Maillard products are structurally altered lysine derivatives called Amadori compounds, and the later Maillard products are melanoidins. Melanoidins do not interfere with the normal analysis of lysine and do not affect the calculated digestibility values. They only lead to lower concentrations of absorbable lysine. Therefore, melanoidins are usually not identified by standard amino acid analysis. In comparison, Amadori compounds interfere with amino acid analysis and provide inaccurate lysine concentrations for the sample being analyzed. The lysine that binds in these compounds is called “blocked lysine” and is biologically inaccessible because it is resistant to any gastrointestinal enzymatic degradation.

The content of the reactive lysine in the sample can be determined using the Sanger reagent, i.e. 1- f torus 2,4- d and n Itro b enzola (FNDB). Therefore, lysine determined using this method is also called FDNB-lysine. The Sanger reagent converts lysine to yellow dinitrophenyl (DNP) lysine, which can be extracted and measured spectrophotometrically at a wavelength of 435 nm or by high performance liquid chromatography.

Alternatively, the content of reactive lysine in the sample can also be determined by guanidation using a mild O-methylisourea reagent. In this method, O-methylisourea only reacts with the ε-amino group of lysine, but does not interact with the α-amino group of lysine. Therefore, the guanidation reaction can be used to determine free lysine and peptide-bound lysine. Therefore, preference is given to guanidation reactions to determine reactive lysine. The lysine guanidation reaction provides for the formation of homoarginine, which is further derivatized with ninhydrin, and the resulting change in absorption can be measured at a wavelength of 570 nm. The derivatized sample is then hydrolyzed to obtain homoarginine again. The determination of reactive lysine can also be carried out using the guanidation reaction of intact protein bound lysine in an alkaline medium to obtain homoarginine. In this type of guanidation reaction is usually carried out under O - m and etil zo m -urea (OMIU).

Since this is a simpler method for use, preference is given to using the guanidation reaction to determine reactive lysine. The guanidation reaction involves incubating a sample of raw material for the feed product and / or feed product in O-methylisourea. Preferably, the ratio of O-methylisourea to lysine is greater than 1000. The sample thus obtained, obtained in step i), is dried and analyzed for homoarginine, preferably by high performance ion exchange liquid chromatography. Then the specified sample is derivatized using ninhydrin and the absorption coefficient of the derivatized sample is measured at a wavelength of 570 nm. After that, the specified sample is subjected to hydrolysis, followed by removal of the solvent to a dry state of the sample. The weight and molar amount of homoarginine in the sample are determined. Finally, the amount of reactive lysine is calculated based on the molar amount of homoarginine.

The guanidation reaction for the determination of reactive lysine thus preferably comprises the following steps:

a) incubating a sample of raw material for the feed product and / or feed product in O-methylisourea;

a) analyzing the sample obtained in step i) for homoarginine;

a) derivatization of the sample obtained in stage ii) with ninhydrin;

i) measuring the absorption coefficient of the sample obtained in stage iii) at a wavelength of 570 nm;

ii) subjecting the sample from step iv) to hydrolysis;

iii) determining the weight and molar amount of homoarginine in the hydrolyzed sample and

iv) determining the amount of reactive lysine based on the molar amount of homoarginine obtained in step vi).

However, not only lysine is susceptible to thermal damage when processing raw materials for the feed product and / or feed product, but also other amino acids. According to the method of the present invention, the amino acids quantitatively analyze the methionine, cysteine, cystine, threonine, leucine, arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, aspartic acid and glutamic acid in the raw material sample feed product and / or feed product. To a certain extent, amino acids are present not only in the form of separate compounds, but also in the form of oligopeptides, for example, dipeptides, tripeptides or higher peptides formed in an equilibrium reaction from two, three or even more amino acids. The amino group amino acid as nucleophilic is usually too weak to directly react with the carboxyl group of another amino acid, or it is present in protonated form (-NH ₃ ⁺ ). Therefore, the equilibrium of this reaction is usually on the left under standard conditions. Despite this, depending on the individual amino acids and the state of the sample solution, some determined amino acids may not be present as separate compounds, but to a certain extent in the form of oligopeptides, for example, dipeptides, tripeptides or higher peptides formed from two, three or even more amino acids . Therefore, the raw material sample for the feed product and / or feed product must be hydrolyzed, preferably acid or base hydrolyzed, using, for example, hydrochloric acid or barium hydroxide. To facilitate the separation of free amino acids and / or the identification and determination of amino acids, free amino acids are optionally derivatized with a chromogenic reagent. Suitable chromogenic reagents are known to those skilled in the art. Subsequently, free amino acids or derivatized free amino acids are subjected to chromatographic separation, in which different amino acids are separated from each other due to different retention times due to different functional groups of individual amino acids. Suitable chromatographic columns, for example, reverse phase columns, and a suitable eluent solvent for chromatographic separation of amino acids are known to those skilled in the art. The separated amino acids are ultimately determined in the eluates from the chromatography step by comparison with a calibrated standard obtained for analysis. Typically, amino acids that are eluted from a chromatographic column are detected using a suitable detector, for example, a conductivity detector, a mass specific detector or a fluorescent detector or UV / VIS detector, depending on when the amino acids were derivatized using a chromogenic reagent. The result is a chromatogram with peak areas and peak heights for individual amino acids. The determination of individual amino acids is performed by comparing peak areas and peak heights with a calibrated standard or a calibration curve for each amino acid. Because cystine (HO ₂ C (-H ₂ N) CH-CH ₂ -SS-CH ₂ -CH (NH ₂ ) -CO ₂ H) and cysteine (HS-CH ₂ -CH (NH ₂ ) -CO ₂ H) defined as cysteic acid (HO ₃ S-CH ₂ -CH (NH ₂ ) -CO ₂ H), quantitative analysis does not show any differences between the two amino acids. However, this does not seem to affect the accuracy of the quantitative analysis, since cysteine is usually very sensitive to oxidation and therefore is usually present in the form of cystine.

Quantitative analysis of at least one amino acid other than reactive lysine preferably comprises the following steps:

i) placing a sample of raw material for the feed product and / or feed product in an aqueous acidic solution;

ii) hydrolysis of the amino acids contained in said sample to release them;

iii) optionally derivatizing the free amino acids obtained in step ii) using a chromogenic reagent that enhances the separation and spectral properties of the amino acids;

iv) separation of free amino acids obtained in stage ii) and / or iii) using column chromatography and

v) determining the amount of separated amino acids in the eluates obtained in stage iv).

The above procedure is usually used to quantify the total amount of lysine that is required to determine the ratio of the amount of reactive lysine to the total amount of lysine, and to quantify at least one amino acid selected from the group consisting of methionine, cysteine, cystine, threonine, leucine , arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, aspartic acid and glutamic acid.

The most important point in the quantitative analysis of amino acids is the preparation of samples, which differs depending on the type of ingredients and amino acids of primary interest. Most amino acids can be hydrolyzed by hydrolysis in hydrochloric acid (6 mol / L) for a period of up to 24 hours. In the case of sulfur-containing amino acids, methionine, cysteine and cystine, hydrolysis is preceded by oxidation with formic acid. For quantitative analysis of tryptophan, hydrolysis is carried out using barium hydroxide (1.5 mol / l) for 20 hours.

Before quantitative analysis of amino acids, a sample of the raw material for the feed product and / or feed product is preferably finely ground. During the specified grinding of the feed material for the feed product and / or feed product, any heat evolution should be avoided in order to avoid further influence of heat with respect to the composition of the feed material for the feed product and / or feed product, in particular with respect to the parameter that is subjected to quantitative analysis from step a) of the method of the present invention.

The values obtained in relation to the parameters of the quantitative analysis performed in steps a1) to a3) in accordance with the present invention are plotted in step b) of the method in accordance with the present invention depending on the processing time of the samples that were subjected to quantitative analysis.

Next, in step c) of the method in accordance with the present invention, the region on the graph from step b) is determined, where

- the value of the activity of the trypsin inhibitor, expressed in mg of trypsin per g of sample, is more than 4,

- the increase in pH is more than 0.35,

- the value of the solubility of the protein in alkali, expressed as the percentage of protein in the sample, which is soluble in an alkaline solution, is more than 85%, and / or

- the value of the indicator of dispersibility of the protein, expressed as a percentage of the initial nitrogen content in the sample, is more than 40%,

and the areas on the graph from stage b), where at least one of these provisions is given, is referred to an insufficiently processed sample.

Then, in step d) of the method of the present invention, the region on the graph from step b) is determined, where

- the value of the ratio of the number of reactive lysine to the total amount of lysine is less than 90%,

- the value of the indicator of dispersibility of the protein, expressed as a percentage of the initial nitrogen content in the sample, is less than 15%, and / or

- the value of the solubility of the protein in alkali, expressed as the percentage of protein in the sample, which is soluble in an alkaline solution, is less than 73%,

and the areas on the graph from step b), where at least one of these provisions is given, refers to an over-processed sample.

Finally, in step e) of the method of the present invention, a region on the graph from step b) is determined, where

- the value of the activity of the trypsin inhibitor, expressed in mg of trypsin per g of sample, is less than 4,

- the value of the solubility of the protein in alkali, expressed as the percentage of protein in the sample, which is soluble in an alkaline solution, is from 73% to 85%,

- the value of the indicator of dispersibility of the protein, expressed as a percentage of the nitrogen content in the sample, is from 15 to 40%, and

- the ratio of the reactive amount of lysine to the total amount of lysine is at least 90%,

and the areas on the graph from stage b), where at least one of these provisions is given, refers to a sufficiently processed sample.

In addition or alternatively, in step e) of the method according to the present invention, the regions obtained in steps c) and d) are subtracted from the graph from step b), and the regions thus obtained are assigned to a sufficiently processed sample.

In the rare case when the implementation of both alternatives in step e) provides different areas, the average size is determined for these areas.

In order to facilitate the classification of the raw material for the feed product and / or feed product subjected to the method according to the present invention into over-processed, insufficiently or sufficiently processed, it is additionally necessary to obtain a processing scale on which the indicator of the processing conditions from step f) is finally applied. The size of the regions determined in steps c) to e) of the method of the present invention may vary in size, in particular with respect to their height (expansion of regions in the y direction or along the ordinate) and / or their length (expansion of regions in the x direction or along the abscissa). Therefore, in step f) of the method according to the present invention, the regions defined in steps c) to e) are standardized to an equal size, and the standardized regions are subsequently sorted from over-processed to under-processed or vice versa. In addition, a continuous scale refers to standardized and sorted areas.

According to the present invention, the parameter values obtained in stages a1) to a3) of the method according to the present invention are introduced in step g) of the method of the present invention in a power series, and the values thus obtained are used to determine the average value, which is the so-called indicator of conditions processing (PCI).

A typical power series corresponds to the formula

,

,

Where

 i = maximum number of analyzed parameters;

 n = specific parameter;

x _n = value of a specific parameter and

a _n = weighting factor for the parameter.

In the context of the present invention, the weighting coefficient is preferably an integer. Preferably, the weighting factor is an integer from 1 to 10.

Given the formation of an average value based on values from power series, the so-called processing condition indicator (PCI) is obtained using the formula,

,

,

Where

i = maximum number of analyzed parameters;

 n = specific parameter;

x _n = value of a specific parameter and

a _n = weighting factor for the parameter.

Finally, the indicator of the processing conditions obtained in step g) is then finally applied in step h) of the method according to the present invention to the processing scale obtained in step f) to determine whether the feed material for the feed product and / or the feed product is excessively processed, sufficiently processed, or insufficiently processed.

Preferably, a series of samples of the processed raw material for the feed product and / or feed product at different points in time of processing is exposed to the method in accordance with the present invention in order to ensure a comprehensive sampling. Preferably, said series of samples includes at least 100 samples, in particular 200, 300, 400, 500 or more samples. In the case of a series of samples, the type of raw material for the feed product and / or feed product is preferably of the same type. In addition, it is preferable to expose more than one series of samples, preferably of the same type, of the feed material for the feed product and / or feed product to the method according to the present invention. This also has the advantage that a series of samples from different regions of the world can be subjected to the method according to the present invention. This allows you to get a comprehensive set of data, which also allows you to determine the effect of indicators of the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product from different regions of the world. Thus, the method according to the present invention also takes into account various climatic conditions in different regions of the world, which together with the processing also have an effect on the nutritional value of the raw material for the feed product and / or feed product.

Animal growth requires a food source of amino acids. However, the amino acids present in the feed are not fully digestible. Rather, the digestibility of amino acids varies depending on the raw materials for the feed product or feed product and, in addition, it also varies among amino acids. For example, the content of anti-nutritional factors or fibers in the matrix of raw material for a feed product can provide a decrease in the digestibility of amino acids in some species of animals. Amino acids are absorbed in the small intestine. Digestible amino acids are absorbed through the walls of the small intestine. Undigested materials pass through the colon and are excreted in feces, at least theoretically. However, the colon microflora can metabolize some undigested amino acids for its own growth and development. As a result, the absorption of amino acids in some animal species cannot be determined by simply subtracting the amino acid content in feces from the amino acid content in the diet fed to the animal. In order to avoid distortion of the results under the influence of microbes of the intestine, the digestibility of amino acids in monogastric animals is most correctly measured at the end of the small intestine. This part of the intestine is also called the ileum. Therefore, in the area of animal nutrition, the corresponding digestibility of amino acids is also called digestibility in the ileum or digestibility coefficient in the ileum. By the method of analysis in the ileum, the difference between the amount of each of the amino acids in the diet and in the contents of the ileum, divided by the amount of each amino acid in the diet, is measured. However, the contents collected at the end of the small intestine contain large amounts of endogenous proteins, and, depending on the relative contribution from the loss of endogenous amino acids, the apparent coefficients of assimilation of amino acids in the ileum are affected to varying degrees. The expression that appears in the context of the assimilation of amino acids in the ileum, therefore, reflects the fact that the coefficients are not regulated by the loss of endogenous nitrogen and amino acids. The so-called true ileum digestibility coefficients of amino acids or the ileum digestibility rate (AID) are calculated using the formula:

,

,

Where

AA _absorbed = the amount of a single amino acid received by the animal as part of the diet, and

AA _output = amount of a single amino acid in the contents of the ileum.

Losses of endogenous protein and amino acids can be divided into basic (minimal) and additional specific loss. Basic loss is non-specific and is associated with dry matter consumption, while specific loss is associated with intrinsic factors in feed products, for example, fibers and anti-nutritional factors such as trypsin inhibitors, lectins and tannins. Types of endogenous secretion come from various sources, including saliva, pancreatic secrets, exfoliated epithelial cells and mucin. The amount of basic loss of endogenous protein and amino acids in the contents of the ileum can be determined using various methods. These methods include protein-free feeding diets, feeding diets containing protein sources that are believed to be fully (100%) absorbed by complete absorption of amino acids, and a regression technique.

Inaccuracies in apparent ileal digestibility are overcome when apparent ileal digestibility ratios are standardized by adjusting them for baseline loss of endogenous amino acids. The standardized or standard ileum digestion coefficients obtained in this way are independent of the level of amino acids in the diet. A key issue for determining the standardized digestibility of amino acids in the ileum is the quantification of the baseline loss of endogenous amino acids in the contents collected at the end of the small intestine. The standard ileal amino acid digestibility coefficients or the standard ileal digestibility coefficients (SID) are calculated using the formula:

Where

AA _absorbed = amount of a single amino acid received by the animal as part of the diet,

AA _output = amount of a single amino acid in the contents of the ileum, and

AA _{base end} = amount of base loss of endogenous amino acid.

To date, to evaluate the nutritional value of the specified raw material for a feed product and / or feed product for a certain animal species, in practice, only one single standard coefficient of digestibility of a specific amino acid is used in the feed material for a feed product and / or in a feed product for a certain animal species, independently from the origin of the feed material for the feed product and / or feed product. Accordingly, standard digestion coefficients in modern practice do not take into account any regional impacts in relation to nutritional value, nor do they take into account any impacts of differences in the processing of raw materials for the feed product and / or feed product.

However, without taking into account additional exposure parameters in relation to nutritional value, the current practice of using a single standard digestibility coefficient does not allow a reliable and meaningful assessment of the nutritional value of the raw material for the feed product and / or feed product.

For comparison, the method according to the present invention takes into account this effect, since it provides a correlation of the standard coefficient of assimilation of amino acids in the ileum of the feed material for the feed product and / or feed product for animals with an indicator of the processing conditions obtained in the case of the same feed material for the feed product and / or a feed product that already reflects the processing influence parameters with respect to the nutritional value of the feed material for the feed product and / or feed product.

In one embodiment, the method according to the present invention further comprises the following steps:

i) determining a standardized ileum digestibility coefficient (SID) of an amino acid in a feed material for a feed product and / or in a feed product for a certain animal species using:

i1) a quantitative analysis of the amount of said amino acid (AA _absorbed ) in the same sample as in step a);

i2) introducing the specified sample to a certain animal species and determining the endogenous loss of the specified amino acid (AA _{basic, deduced.} ) and the outflow of the amino acid from the ileum (AA _{sublingual, outflow} ) and

i3) substituting the parameter values obtained in stages i1) and i2) into the general formula (II):

,

,

and

b) plotting a standardized digestion coefficient in the ileum obtained in stage i), depending on the indicator of the processing conditions obtained in step g), and / or expressing the specified standard coefficient of digestibility in the ileum in the equation, depending on the indicator of the processing conditions, obtained in stage g).

Examples of calibration equations that give the ileum digestibility coefficient (SID _AA ) versus PCI are given below. These equations give the ileum digestibility coefficient for a particular amino acid in full-fat soybeans in poultry or pigs:

- standardized digestion coefficient of methionine in the ileum (SID _Met ) from full-fat soybeans in poultry:

- standardized _digestion coefficient of cystine in the ileum (SID _cys ) from full-fat soybeans in poultry:

- standardized digestion coefficient of methionine and cystine in the ileum (SID _{Met + Cystine) of} full-fat soy in poultry:

- standardized digestion coefficient of lysine in the ileum (SID _Lys ) from full-fat soybeans in poultry:

- standardized digestibility coefficient of threonine in the ileum (SID _Thr ) from full-fat soybeans in poultry:

- standardized digestibility coefficient of tryptophan in the ileum (SID _Trp ) from full-fat soybeans in poultry:

- standardized assimilation coefficient of arginine in the ileum (SID _Arg ) from full-fat soybeans in poultry:

- standardized digestion coefficient of isoleucine in the ileum (SID _Ile ) from full-fat soybeans in poultry:

R² = 0,9657,

R ² = 0.9657,

- standardized digestion coefficient of leucine in the ileum (SID _Leu ) from full-fat soybeans in poultry:

- standardized digestibility coefficient of valine in the ileum (SID _Val ) from full-fat soybeans in poultry:

- standardized digestion coefficient of histidine in the ileum (SID _His ) from full-fat soybeans in poultry:

- standardized digestion coefficient of phenylalanine in the ileum (SID _Phe ) from full-fat soybeans in poultry:

- standardized digestion coefficient of methionine in the ileum (SID _Met) from full-fat soybeans in pigs:

- standardized _digestion coefficient of cystine in the ileum (SID _cys ) from full-fat soybeans in pigs:

- standardized digestion coefficient of methionine and cystine in the ileum (SID _{Met + Cystine)} from full-fat soybeans in pigs:

- standardized digestion coefficient of lysine in the ileum (SID _Lys ) from full-fat soybeans in pigs:

- standardized digestibility coefficient of threonine in the ileum (SID _Thr ) from full-fat soybeans in pigs:

- standardized digestion coefficient of tryptophan in the ileum (SID _Trp ) from full-fat soybeans in pigs:

- standardized assimilation coefficient of arginine in the ileum (SID _Arg ) from full-fat soybeans in pigs:

- standardized digestion coefficient of isoleucine in the ileum (SID _Ile ) from full-fat soybeans in pigs:

- standardized digestion coefficient of leucine in the ileum (SID _Leu ) from full-fat soybeans in pigs:

- standardized digestibility coefficient of valine in the ileum (SID _Val ) from full-fat soybeans in pigs:

- standardized digestion coefficient of histidine in the ileum (SID _His ) from full-fat soybeans in pigs:

- standardized digestion coefficient of phenylalanine in the ileum (SID _Phe ) from full-fat soybeans in pigs:

- standardized digestion coefficient of glycine in the ileum (SID _Gly ) from full-fat soybeans in pigs:

- standardized digestion coefficient of serine in the ileum (SID _Ser ) from full-fat soybeans in pigs:

- standardized digestion coefficient of proline in the ileum (SID _Pro ) from full-fat soybeans in pigs:

- standardized digestibility coefficient of alanine in the ileum (SID _Ala ) from full-fat soybeans in pigs:

- standardized digestibility coefficient of aspartic acid in the ileum (SID _Asp ) from full-fat soybeans in pigs:

and

- standardized digestion coefficient of glutamic acid in the ileum (SID _Glu ) from full-fat soybeans in pigs:

For each calibration, the corresponding determination coefficient is given, denoted as R ² . In statistics, the coefficient of determination is a number that indicates how accurately the data fit the statistical model — sometimes just a line or curve. A value of R ² equal to 1 indicates that the regression line corresponds well to the data, and a value of R ² equal to 0 means that these lines do not correspond to the data at all. In all cases, the standardized digestibility of amino acids in the ileum is characterized by R ² , which is very close to 1. Accordingly, the statistical model is very well consistent with the data.

The quantitative analysis from stages a1) to a3) of the method according to the present invention is rather laborious and costly. Near-infrared (NIR) measurements of the respective feed material for the feed product and / or feed product would be a more effective alternative in terms of time and cost for determining the effect of processing on the nutritional value of the feed material for the feed product and / or feed product. However, near infrared spectroscopy does not provide the results with the desired accuracy; rather, it often leads to conflicting results. Accordingly, neither quantitative analysis nor near infrared spectroscopy per se is suitable for efficiently determining, in terms of time and cost, the effect of processing on the nutritional value of the raw material for the feed product and / or feed product.

In accordance with the present invention, this problem is solved by the fact that the absorption values in the near infrared region obtained for a sample of raw material for the feed product and / or feed product correlate with the corresponding values of the quantitative analysis of the above. The thus obtained correlation of the values of the quantitative analysis with the absorption values of the NIR measurements is preferably depicted or displayed in the form of a calibration graph, which facilitates the comparison of the absorption values of the NIR measurements of another sample with the corresponding exact values for the parameters based on the quantitative analysis.

Another objective of the present invention, therefore, is a method of evaluating the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product, comprising the following steps:

A) exposing the sample to the same raw material for the feed product and / or feed product as in step a) of the method for determining the processing effect with respect to the nutritional value of the feed material for the feed product and / or feed product, near infrared spectroscopy (NIR );

B) comparing the values of the absorption intensity at the corresponding wavelengths or wave numbers in the NIR spectrum obtained in stage A) with the corresponding parameters and their values determined in stages a1) to a3), and

C) plotting the comparison of stage B) in the form of a calibration graph and / or expressing the parameters determined in stages a1) - a3) in the calibration equation depending on the values of absorption intensity at the corresponding wavelengths or wave numbers compared in stage B).

Depending on the spectrometer used, the near-infrared (NIR) spectra of stage A) can be recorded at wavelengths from 400 to 2500 nm using any suitable infrared spectroscopes operating either on a monochromator principle or on a Fourier transform basis. Preferably, NIR spectra are recorded from 1000 to 2500 nm. The wavelengths are easily converted to the corresponding wave numbers, and therefore, NIR spectra, of course, can also be recorded with the corresponding wave numbers. Since the organic compounds to be determined according to the method in accordance with the present invention, i.e. proteins and amino acids, are rich in O-H bonds, C-H bonds and N-H bonds, they are suitable for detection by near infrared spectroscopy. However, a biological sample, such as a feed product, contains many different organic compounds and, therefore, is a complex matrix. Despite this, each biological substance has a unique spectrum in the near infrared region, which can be compared with an individual fingerprint. Therefore, we can assume that two biological substances with the same spectrum have the same physical and chemical composition and, therefore, are identical. On the other hand, if two biological substances have different spectra, it can be assumed that they are different in terms of either their physical or chemical characteristics or in terms of both. Owing to their individual and highly specific absorption bands, the signals of organic compounds and their intensity values in the NIR spectra can be easily assigned and correlated with a specific organic compound and its concentration in a sample with a known weight. Thus, NIR spectroscopy makes it possible to reliably predict or evaluate, for example, the amount of amino acids and proteins in a sample. Since the same sample of a specific raw material for a feed product and / or feed product is subjected to quantitative analysis in stage a) and NIR spectroscopy in stage A), it is also possible to determine and correlate the absorption values and their intensity values in the NIR spectrum with the parameters such as trypsin inhibitor activity, urease activity, alkali protein solubility, and protein dispersibility index, as well as their meanings and changes. After the values of the absorption intensity at the corresponding wavelengths or wave numbers were successfully agreed upon, i.e., assigned and correlated with the parameters of interest and their values, NIR spectroscopy makes it possible to reliably predict or evaluate the effect of processing in relation to the nutritional value of the raw material for a feed product and / or feed product. For this purpose, a large number of NIR spectra are recorded, for example, 100, 200, 300, 400, 500 or more, raw material for the feed product and / or feed product, and the absorption intensities at the corresponding wavelengths or wave numbers are compared with the corresponding parameters and their meanings. When the sample from the sample of step A) is not translucent, the reflection coefficient of the emitted light from the sample is measured, and the difference between the emitted light and the reflected light is set as absorption. The absorption intensities thus obtained are used in the following steps, for example, in step B) above and in steps D) and G) below.

In one embodiment, the method of evaluating an effect of processing in relation to the nutritional value of a feed material for a feed product and / or feed product further comprises the following steps:

D) comparing the values of the absorption intensity at the corresponding wavelengths or wave numbers in the NIR spectrum of the sample obtained in stage B) with an indicator of the processing conditions obtained for the same sample in stage g) of the method for determining the treatment effect in relation to the nutritional value of the raw material for feed product and / or feed product; and

E) constructing a comparison graph according to stage D) in the form of a calibration graph and / or expressing an indicator of the processing conditions in the calibration equation depending on the values of absorption intensity at the corresponding wavelengths or wave numbers compared in stage D).

After completing NIR calibrations, NIR spectroscopy can be used as a standard method to evaluate the effect of processing on the nutritional value of the feed material for the feed product and / or feed product.

In an additional embodiment, the method of evaluating the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product further comprises the following steps:

F) exposing the sample to the same raw material for the feed product and / or feed product as in step a) of the method for determining an effect of processing regarding the nutritional value of the feed material for the feed product and / or feed product, NIR spectroscopy;

G) deriving the values of at least one of the parameters from stages a1) to a3) corresponding to the absorption values in the NIR spectrum obtained in stage F), based on the calibration graph according to stage C), and / or substituting the values of the absorption intensity at the corresponding lengths waves or wavenumbers in the NIR spectrum obtained in stage F) into the calibration equation according to stage C) to obtain values for the parameters from stages a1) to a3);

H) substituting the values for the parameters obtained in stage G) into a power series and calculating the average value based on the values obtained from each power series, the indicated average value being designated as an indicator of processing conditions (PCI), and / or

I) removing PCI from the calibration graph according to stage E) and / or substituting the values of the absorption intensity at the corresponding wavelengths or wave numbers in the calibration equation according to stage E) to obtain an indicator of processing conditions; and

J) the application of an indicator of the processing conditions obtained in stages H) and / or I) on the processing scale of the method for determining the indicator of the effect of processing in relation to the nutritional value of the feed material for the feed product and / or feed product, determining whether the feed material is for feed product and / or feed product over-processed, adequately processed or insufficiently processed.

Preferably, in step G), the same parameters are obtained as in steps a1) to a3).

Based on the calibrations already obtained for the indicator of the processing conditions and the specific digestibility coefficient, the method according to the present invention also makes it possible to determine the specific digestibility coefficient by NIR spectroscopy.

In another embodiment, the method of evaluating the effects of processing in relation to the nutritional value of the feed material for the feed product and / or feed product, thus further comprising the step of

K) substituting the indicator of processing conditions obtained in stage H) into the calibration equation according to stage j) and / or deriving the functional value for the indicator of processing conditions obtained in stage I) to obtain the specific amino acid digestibility coefficient (D _AA ) in the raw material for the feed product and / or feed product from step F).

Based on the data set and corresponding calibrations obtained in the methods of the present invention, the method of the present invention also makes it possible to evaluate the effect of processing on the nutritional value of the raw material for the feed product and / or feed product of unknown origin. Alternatively, the sample in step F) of the method for evaluating the impact of processing in relation to the nutritional value of the feed material and / or feed product has the same origin as in step a) of the method for determining the influence of processing in relation to the nutritional value of the feed material for feed product and / or feed product.

In one embodiment, the sample used in step F) of the method for evaluating the effects of processing on the nutritional value of the feed material and / or feed product has an unknown origin or the same origin as in step a) of the method for determining the effects of processing in relation to the nutritional value of the raw material for the feed product and / or feed product.

In the case of the feed material for the feed product and / or the feed product, which is considered over-processed, the method also allows you to determine the difference between the required value and the actual amino acid content in the feed material for the feed product and / or in the feed product by comparing the maximum digestibility coefficient in the ileum the intestine of an amino acid from a raw material for a feed product and / or from a feed product in a certain animal species using a specific amino acid digestibility coefficient obtained using the method of the present invention for a particular sample.

The method of evaluating the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product of the present invention, therefore, further provides a step

L) determining the differential amount between the desired value and the actual value of the amount of amino acid in the feed material for the feed product and / or feed product based on the difference between the maximum digestibility coefficient in the ileum of the specified amino acid according to the method of determining the impact of processing in relation to the nutritional value of the feed material for feed product and / or feed product and specific digestibility of the specified amino acid obtained in stage K).

Using the specific digestibility coefficient (D _AA ) obtained using the method according to the present invention for a particular amino acid in a particular sample of raw material for a feed product and / or feed product, it is also possible to determine the digestible amount of amino acid in said sample. Said digestible amount of amino acid can simply be obtained by multiplying the amount of amino acid of a sample of raw material for the feed product and / or feed product obtained in step G) by an indicator of the processing conditions obtained in step K)

In a further embodiment, the method of evaluating the effects of processing in relation to the nutritional value of the raw material for the feed product and / or feed product of the present invention, thus further comprising the step of

A) determining the digestible amount of the amino acid in the sample of raw material for the feed product and / or feed product by multiplying the amount of the specified amino acid in the sample of raw material for the feed product and / or feed product obtained in stage G) by the specific digestibility coefficient obtained in stage K).

The feed material for the feed product and / or feed product that is used according to the methods of the present invention is preferably soybeans, soybeans, preferably full-fat soybeans and / or soya products, preferably soybean meal and soybean meal / press cake from the expeller. This is due to the fact that soybeans, soybeans and soya products are the most suitable raw materials for feed products and / or feed products.

In one embodiment of the methods of the present invention, the feed material for the feed product and / or the feed product is soy, soybeans, or soya product.

The determination of the standard coefficient of digestibility in the ileum and the specific coefficient of digestibility obtained using the methods of the present invention is not subject to any restrictions with respect to some species of animals. Rather, these methods can be used to determine and / or evaluate a standardized digestion coefficient in the ileum and a specific digestibility coefficient for any possible animal species. Despite this, the preferred animal species in the context of the present invention are monogastric animals, i.e., animals having a single chamber stomach, including omnivores, such as pigs, poultry, for example, turkeys and chicken, carnivores, such as cats and herbivores such as horses, deer and rabbits, and ruminants such as cows, goats and sheep.

In one embodiment of the methods of the present invention, the animal species refers to omnivores, carnivores, herbivores and / or ruminants.

The method according to the present invention can be carried out using a computer. This allows the method according to the present invention to be implemented as a standard method. In this case, the calibration equations obtained in steps C) and j) of the methods of the present invention are stored on a computer, so that only steps F) to J) are performed by a computer, and optionally step K) of the method according to the present invention. Preferably, a near infrared spectrometer is also controlled by a computer in the case of step F) of the method according to the present invention. Additionally or alternatively, the computer by which steps F) -J) and optionally step K) of the method according to the present invention are performed, and the computer on which the calibration equations are stored are not identical. In this case, the first computer by which steps F) -J) and optionally step K) of the present invention are performed, and the second computer on which the calibration equation is stored form a network. Additionally or alternatively, the data set and the calibration curve are stored in a cloud storage, which is accessible on the first computer, in which case the first computer and cloud storage form a kind of network.

Therefore, another objective of the present invention is a computerized method for determining the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product, where stages F) - J) are carried out using a computer, and the calibration equations according to stage C) and / or according to step E) is stored on a computer or in cloud storage.

In an embodiment, the computerized method according to the present invention further provides that the additional steps K) and / or any of steps L) and M) of the method according to the present invention are also carried out using a computer.

The advantage of this is that using a computerized method, it is not only determined whether the raw material for the feed product and / or the feed product under study is insufficiently processed, sufficiently processed or over-processed, but also determine what quantities of a particular amino acid are necessary to ensure an optimal diet for specific animal species in the case of insufficiently processed raw material for the feed product and / or feed product. It also allows you to control with your computer the installation for receiving or mixing feed.

Thus, another objective of the present invention is also a method of obtaining a feed product, comprising stages F) -L) of the method according to the present invention, where the method further comprises at least one of the stages:

N) additional processing of the raw material for the feed product and / or feed product, if the raw material for the feed product and / or feed product is defined as not sufficiently processed, and / or

O) adding a differential amount of the amino acid obtained in accordance with stage L) to the feed material for the feed product and / or feed product, if the feed material for the feed product and / or feed product is defined as over-processed.

This method facilitates the provision of feed products that do not contain a critical amount of anti-nutritional factors, preferably less than 4 mg of trypsin inhibitors per gram of feed product, and, on the other hand, contain the required amount of amino acids for the species of animals to which they are provided. The required amount of amino acids for animal species is regulated using stage N).

Figures

Figures 1-12 show the coefficients of assimilation of amino acids in the ileum (SID _AA ) from full-fat soybeans for poultry depending on the indicator of processing conditions (the term in brackets is a mathematical equation for the corresponding calibration equation). The rhombs in these figures (designated as statistical series 1) correspond to the individual PCI values of the corresponding processed full-fat soybeans, and the straight line (indicated as polynomial) represents a graph of the function of the individual SIDs for the corresponding amino acid.

FIG. 1. The coefficient of assimilation of methionine in the ileum in full-fat soybeans for poultry:

(SID _Met = - 0.3581 × PCI ² + 8.679 × PCI + 33.624).

FIG. 2. The coefficient of assimilation of cystine in the ileum in full-fat soybeans for poultry:

(SID _Cystine = - 0.442 × PCI ² + 11.983 × PCI + 13.905).

FIG. 3. The coefficient of assimilation of methionine and cystine in the ileum in full-fat soybeans for poultry:

(SID _{Met + Cystine} = - 0.3861 × PCI ² + 9.8435 × PCI + 13.53).

FIG. 4. The coefficient of assimilation of lysine in the ileum in full-fat soybeans for poultry:

(SID _Lys = - 0.4187 × PCI ² + 11.462 × PCI + 5.6474).

FIG. 5. The coefficient of assimilation of threonine in the ileum in full-fat soybeans for poultry:

(SID _Thr = - 0.368 × PCI ² + 9.2054 × PCI + 12.772).

FIG. 6. The coefficient of digestibility of tryptophan in the ileum in full-fat soybeans for poultry:

(SID _Trp = - 0.4046 × PCI ² + 9.7674 × PCI + 23.052).

FIG. 7. The coefficient of assimilation of arginine in the ileum in full-fat soybeans for poultry:

(SID _Arg = - 0.3033 × PCI ² + 7.3008 × PCI + 41.512).

FIG. 8. The coefficient of assimilation of isoleucine in the ileum in full-fat soybeans for poultry:

(SID _Ile = - 0.3974 × PCI ² + 9.211 × PCI + 29.802).

FIG. 9. The coefficient of assimilation of leucine in the ileum in full-fat soybeans for poultry:

(D _Leu = - 0.3639 × PCI ² + 8.3187 × PCI + 35.843).

FIG. 10. The coefficient of assimilation of valine in the ileum in full-fat soybeans for poultry:

(SID _Val = - 0.388 × PCI ² + 9.0608 × PCI + 29.464).

FIG. 11. The coefficient of digestibility of histidine in the ileum in full-fat soybeans for poultry:

(SID _His = - 0.3554 × PCI ² + 9.1547 × PCI + 25.938).

FIG. 12. The coefficient of assimilation of phenylalanine in the ileum in full-fat soybeans for poultry:

(SID _Phe = - 0.3523 × PCI ² + 8.0374 × PCI + 37.432).

Figures 13-30 show the coefficients of digestibility of amino acids in the ileum (SID _AA ) from full-fat soybeans for pigs depending on the indicator of processing conditions (the term in brackets is a mathematical equation for the corresponding calibration equation). The rhombs in these figures (designated as statistical series 1) correspond to the individual PCI values of the corresponding processed full-fat soybeans, and the straight line (indicated as polynomial) represents a graph of the function of the individual SIDs for the corresponding amino acid.

FIG. 13. The standard coefficient of assimilation of methionine in the ileum (SID _Met ) from full-fat soybeans in pigs:

(SID _Met = - 0.3286 × PCI ² + 7.3561 × PCI + 43.444).

FIG. 14. The standard coefficient of assimilation of cystine in the ileum (SID _Cys ) from full-fat soybeans in pigs:

(SID _Cys = - 0.4982 × PCI ² + 13.115 × PCI - 11.392).

FIG. 15. The standard coefficient of assimilation of lysine in the ileum (SID _{Met + Cystine} ) from full-fat soybeans in pigs:

(SID _{Met + Cystine} = - 0.4237 × PCI2 + 10.534 × PCI + 14.77).

FIG. 16. The standard coefficient of assimilation of lysine in the ileum (SID _Lys ) from full-fat soybeans in pigs:

(SID _Lys = - 0.4397 × PCI ² + 11.359 × PCI + 11.75).

FIG. 17. The standard coefficient of digestibility of threonine in the ileum (SID _Lys ) from full-fat soybeans in pigs:

(SID _Thr = - 0.291 × PCI ² + 6.2769 × PCI + 44.594).

FIG. 18. Standard assimilation coefficient of tryptophan in the ileum (SID _Trp ) from full-fat soybeans in pigs:

(SID _Trp = - 0.3167 × PCI ² + 6.6559 × PCI + 45.534).

FIG. 19. The standard coefficient of assimilation of arginine in the ileum (SID _Arg ) from full-fat soybeans in pigs:

(SID _Arg = - 0.261 × PCI ² + 5.3573 × PCI + 63.685).

FIG. 20. The standard coefficient of assimilation of isoleucine in the ileum (SID _Ile ) from full-fat soybeans in pigs:

(SID _Ile = - 0.3204 × PCI ² + 6.7739 × PCI + 48.135).

FIG. 21. The standard coefficient of assimilation of leucine in the ileum (SID _Leu ) from full-fat soybeans in pigs:

(SID _Leu = - 0.2901 × PCI ² + 5.7556 × PCI + 55.925).

FIG. 22. The standard coefficient of assimilation of valine in the ileum (SID _Val ) from full-fat soybeans in pigs:

(SID _Val = - 0.2801 × PCI ² + 5.8136 × PCI + 52.234).

FIG. 23. The standard coefficient of digestibility of histidine in the ileum (SID _His ) from full-fat soybeans in pigs:

(SID _His = - 0.2915 × PCI ² + 6.548 × PCI + 48.067).

FIG. 24. The standard coefficient of digestion of histidine in the ileum (SID _Phe ) from full-fat soybeans in pigs:

(SID _Phe = - 0.2676 × PCI ² + 4.9292 × PCI + 62.59).

FIG. 25. The standard coefficient of digestibility of glycine in the ileum (SID _Gly ) from full-fat soybeans in pigs:

(SID _Gly = - 0.3377 × PCI ² + 7.7741 × PCI + 35.285).

FIG. 26. The standard coefficient of digestibility of serine in the ileum (SID _Ser ) from full-fat soybeans in pigs:

(SID _Ser = - 0.3257 × PCI ² + 6.9689 × PCI + 44.913).

FIG. 27. The standard coefficient of assimilation of proline in the ileum (SID _Pro ) from full-fat soybeans in pigs:

(SID _Pro = - 0.4428 × PCI ² + 10.473 × PCI + 36.719).

FIG. 28. The standard coefficient of digestibility of alanine in the ileum (SID _Ala ) from full-fat soybeans in pigs:

(SID _Ala = - 0.3002 × PCI ² + 6.6179 × PCI + 44.817).

FIG. 29. The standard coefficient of assimilation of aspartic acid in the ileum (SID _Asp ) from full-fat soybeans in pigs:

(SID _Asp = - 0.4159 × PCI ² + 10.756 × PCI + 9.9347).

FIG. 30. The standard coefficient of assimilation of glutamic acid in the ileum (SID _Glu ) from full-fat soybeans in pigs:

(SID _Glu = - 0.3041 × PCI ² + 6.9635 × PCI + 44.434).

Examples

1. Determination of the effects of processing on the nutritional value of full-fat soybeans and the standardized coefficient of digestibility of amino acids in the ileum of poultry

Full-fat soybeans (FFSB), obtained from one batch, were used to determine the effect of various heat treatments on the food composition and standardized digestion of amino acids in the ileum (SID) in poultry. Untreated FFSB (K0) was subjected to short-term treatment using wet heating at 80 ° C for 1 minute (K1) or long-term treatment at 100 ° C for 6 minutes (K2) or at 100 ° C for 16 minutes (K3) s followed by further expansion at 115 ° C for 15 seconds (K1 / K2 / K3-115) or at 125 ° C for 15 seconds using an HL OEE 15.2 extruder from Amandus Kahl GmbH & Co. KG, Hamburg, Germany. K3 sub-samples were further autoclaved at 110 ° C for 15 minutes (Z1), 30 minutes (Z2), 45 minutes (Z3), 60 minutes (Z4), 120 minutes (Z5), 180 minutes (Z6), 240 minutes (Z7), 300 minutes (Z8) or 360 minutes (Z9). Exit from the expander treated with FFSB was transferred at a temperature of about 90 ° C for 20 seconds in a dryer, where the FFSB was dried for 5 minutes with a temperature gradient of 85 ° C to 40 ° C. After the drying step, the FFSB was allowed to cool to a temperature of 20 ° C. for 5 minutes.

The total number of amino acids and the amount of reactive lysine in variously processed FFSB and an indicator of the processing conditions (PCI) of amino acids in poultry were determined using the method according to the present invention.

Variously processed FFSBs, specific amounts of individual amino acids, and an indicator of processing conditions (PCI) of amino acids in poultry are summarized in Table 1.

The standardized assimilation in the ileum (SID) for each amino acid in poultry depending on PCI is shown in figures 1-12.

PCI FFSB was compared with the SID curve for each amino acid in figures 1-12. This comparison shows that PCI FFSB, designated as Z1 or Z2, are always SID, which corresponds to the maximum on a separate curve or at least close to it. Thus, FFSBs designated as Z1 or Z2 are considered sufficiently processed. In comparison, FFSBs designated as K0, K1-115 / 125, K2-115 / 125 and K3-115 / 125 have always been characterized by the SID, which is located to the right of the maximum on a separate curve and, therefore, are considered insufficiently processed. In addition, the SIDs of FFSB, designated as Z3-Z9, were always located to the left of the maximum on a separate curve and, therefore, they are considered overly processed.

A study of the standardized digestion coefficients of amino acids in the ileum, summarized in Table 1, proves that the classification of FFSB, designated as Z1 and Z2, as sufficiently processed, FFSB, designated as K0, K1-115 / 125, K2-115 / 125 and K3- 115/125, as insufficiently processed and FFSB, designated as Z3-Z9, as over-processed, is correct, because FFSB, designated as Z1 and Z2, had the highest digestibility rates. For comparison, all other FFSBs had lower digestibility factors. This proves that the use of PCI is a useful tool for describing the effect of processing conditions on the quality of the feed material for the feed product and / or feed product.

Table 1. Brief description of the various FFSB-treated, standardized ileum digestibility factors for individual amino acids and processing condition indicator (PCI) in poultry.

2. Determination of indicators of the effect of processing on the nutritional value of full-fat soybeans and the standard coefficient of digestibility of amino acids in the ileum in pigs

Full-fat soybeans (FFSB) obtained from one batch were used to determine the effect of various heat treatment procedures on the food composition and standardized digestion of amino acids in the ileum (SID) in pigs. Untreated FFSB (K0) was subjected to a short treatment using wet heating at 80 ° C for 1 minute followed by further expansion at 125 ° C for approximately 15 seconds (K4), a long treatment at 100 ° C for 6 minutes, followed by further expanding at 125 ° C for approximately 15 seconds (K5) or continuous processing at 100 ° C for 16 minutes followed by further expansion at 125 ° C for approximately 15 seconds (K6) using an OEE 15.2 extruder from Amandus Kahl GmbH & Co. KG, Hamburg, Germany. K6 sub-samples were further autoclaved at 110 ° C for 15 minutes (Z10), 30 minutes (Z11), 45 minutes (Z12) and 60 minutes (Z13). Exit from the expander treated with FFSB was transferred at a temperature of about 90 ° C for 20 seconds in a dryer, where the FFSB was dried for 5 minutes with a temperature gradient of 85 ° C to 40 ° C. After the drying step, the FFSB was allowed to cool to a temperature of 20 ° C. for 5 minutes. Another portion of the untreated FFSB (K0) was heat treated at 110 ° C in an autoclave for 15 minutes (Z14) or for 30 minutes (Z15) or heat treated at 150 ° C in an autoclave for 3 minutes (Z16), 6 minutes (Z17), 9 minutes (Z18) or 12 minutes (Z19).

The total number of amino acids and the amount of reactive lysine in variously processed FFSB and an indicator of the processing conditions (PCI) of amino acids in pigs were determined using the method according to the present invention.

Variously processed FFSBs, a specific reactive amount of individual amino acids, and an indicator of processing conditions (PCI) of amino acids in pigs are summarized in Table 2.

The standardized ileum digestibility coefficient (SID) for each amino acid in pigs depending on PCI is shown in figures 13-30.

PCI FFSB was compared with the SID curve for each amino acid in figures 13-30. This comparison shows that PCI FFSBs, designated as Z11 and Z12, always had a SID that corresponds to a maximum or at least close to a maximum on a separate curve. Thus, FFSBs designated as Z11-12 are considered sufficiently processed. In comparison, the FFSBs designated as K0 and K4-K6 were always characterized by SID, which is located to the right of the maximum on a separate curve and, thus, they are considered insufficiently processed. In addition, the SIDs of FFSB, designated as Z13-19, were always located to the left of the maximum on a separate curve and, thus, they are considered overly processed.

A study of the standardized digestion coefficients of amino acids in the ileum, summarized in Table 2, proves that the classification of FFSB, designated as Z11 and Z12, as sufficiently processed, FFSB, designated as K0 and K4-K6, as insufficiently processed, and FFSB, designated as Z13- Z19, as over-processed, is correct because the FFSBs designated as Z11 and Z12 had the highest amino acid digestibility ratios. For comparison, all other FFSBs had lower amino acid digestibility ratios. This proves that the use of PCI is a useful tool for describing the effect of processing conditions on the quality of the feed material for the feed product and / or feed product.

Table 2. A brief description of the variously processed FFSBs, the defined standardized digestion coefficients in the ileum of individual amino acids, and the indicator of processing conditions (PCI) in pigs.

Claims (53)

1. The method of determining the impact of processing in relation to the nutritional value of the raw material for the feed product and / or feed product, comprising the following stages: a) exposing a sample of processed raw material for the feed product and / or feed product a1) a quantitative analysis of at least one parameter selected from the group consisting of trypsin inhibitor activity, urease activity, alkali protein solubility, and protein dispersibility index; a2) determining the ratio of the amount of reactive lysine to the total amount of lysine, which involves a quantitative analysis of the amount of reactive lysine and the total amount of lysine, followed by obtaining the ratio of the number of reactive lysine to the total amount of lysine, and a3) a quantitative analysis of the amount of at least one amino acid selected from the group consisting of methionine, cysteine, cystine, threonine, leucine, arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, asparaginic acids and glutamic acid; b) plotting the parameters obtained in stages a1) to a3), depending on the processing times of the sample from stage a); c) determining the area in the graph from stage b), where the trypsin inhibitor activity value, expressed in mg of trypsin per g of sample, is more than 4, the increase in pH when determining the urease activity is more than 0.35, the protein solubility in alkali, expressed as the percentage of protein soluble in the alkaline solution in the sample is more than 85%, and / or the value of the protein dispersibility index, expressed as a percentage of the initial nitrogen content in the sample, is more than 40%, and assigning the region thus obtained to is insufficient processed sample; d) determining the area in the graph from step b), where the ratio of the amount of reactive lysine to the total amount of lysine is less than 90%, the value of the protein dispersibility index, expressed as a percentage of the initial nitrogen content in the sample, is less than 15%, and / or the value of the solubility of the protein in alkali, expressed as the percentage of protein soluble in the alkaline solution in the sample, is less than 73%, and assigning the region thus obtained to an overly processed sample; e) determining the region in the graph from step b) where the trypsin inhibitor activity, expressed in mg of trypsin per g of sample, is less than 4, the alkali protein solubility, expressed as the percentage of protein soluble in the alkaline solution in the sample, is from 73 to 85%, the value of the indicator of dispersibility of the protein, expressed as a percentage of the initial nitrogen content in the sample, is from 15 to 40%, and / or the ratio of the number of reactive lysine to the total amount of lysine is at least 90% , and assigning the thus obtained region to a sufficiently processed sample;  and / or  subtracting the areas identified in stages c) and d) from graph b) and assigning the thus obtained area to a sufficiently processed sample; f) obtaining a processing scale by standardizing the areas obtained in steps c) to e) to the same size, sorting them from over-processed to under-processed, or vice versa, and correlating the continuous scale and standardized and sorted areas; g) substituting the parameter values obtained in steps a1) to a3) in a power series and calculating the average value based on the values obtained from each power series, the indicated average value being designated as an indicator of processing conditions (PCI);  and h) applying an indicator of the processing conditions obtained in step g) to the processing scale obtained in step f), determining whether the feed material for the feed product and / or the feed product is over-processed, sufficiently processed or under-processed. 2. The method according to p. 1, further comprising the following steps: i) determining a standardized ileum digestibility coefficient (SID) of an amino acid in a feed material for a feed product and / or in a feed product for a certain animal species using: i1) a quantitative analysis of the amount of said amino acid (AA _absorbed ) in the same sample as in step a) according to claim 1; i2) introducing the specified sample to a certain animal species and determining the endogenous loss of the specified amino acid (AA _{basic, deduced.} ) and the outflow of the amino acid from the ileum (AA _{sublingual, outflow} ) and i3) substituting the values of the parameters obtained in stages i1) and i2) into the general formula (II),

, and j) plotting a standardized digestion coefficient in the ileum obtained in stage i), depending on the indicator of the processing conditions obtained in step g) according to paragraph 1, and / or expressing the specified standard digestion coefficient in the ileum in the calibration equation, depending from the indicator of the processing conditions obtained in stage g) according to paragraph 1. 3. A method of evaluating the impact of processing in relation to the nutritional value of the raw material for a feed product and / or feed product, which is carried out using a computer, comprising the following steps: A) exposing the sample to the same raw material for the feed product and / or feed product as in step a) according to claim 1, near infrared spectroscopy (NIR); B) comparing the values of the absorption intensity at the corresponding wavelengths or wave numbers in the NIR spectrum obtained in stage A) with the corresponding parameters and their values determined at the stages of sample exposure a1) a quantitative analysis of at least one parameter selected from the group consisting of trypsin inhibitor activity, urease activity, alkali protein solubility, and protein dispersibility index; a2) determining the ratio of the amount of reactive lysine to the total amount of lysine, which involves a quantitative analysis of the amount of reactive lysine and the total amount of lysine, followed by obtaining the ratio of the number of reactive lysine to the total amount of lysine, and a3) a quantitative analysis of the amount of at least one amino acid selected from the group consisting of methionine, cysteine, cystine, threonine, leucine, arginine, isoleucine, valine, histidine, phenylalanine, tyrosine, tryptophan, glycine, serine, proline, alanine, asparaginic acids and glutamic acid; and C) plotting the comparison graph according to stage B) in the form of a calibration graph and / or expressing the parameters determined in stages a1) to a3) in the calibration equation depending on the values of absorption intensity at the corresponding wavelengths or wave numbers compared in stage B) . 4. The method according to p. 3, further comprising the following steps: D) comparing the values of the absorption intensity at the corresponding wavelengths or wave numbers in the NIR spectrum of the sample obtained in step B) according to claim 3, with an indicator of the processing conditions obtained for the same sample in step g) according to claim 1, and E) constructing a comparison graph according to stage D) in the form of a calibration graph and / or expressing an indicator of the processing conditions in the calibration equation depending on the values of absorption intensity at the corresponding wavelengths or wave numbers compared in stage D). 5. The method according to p. 3 or 4, further comprising the following steps: F) exposing a sample of raw material for the feed product and / or feed product of unknown origin or of the same origin as in step a) according to claim 1, NIR spectroscopy; G) deriving the values of at least one of the parameters from stages a1) to a3) corresponding to the absorption values in the NIR spectrum obtained in stage F), based on the calibration graph according to stage C), and / or substituting the values of the absorption intensity at the corresponding lengths waves or wavenumbers in the NIR spectrum obtained in stage F) into the calibration equation according to stage C) to obtain values for the parameters from stages a1) to a3); H) substituting the values for the parameters obtained in stage G) into a power series and calculating the average value based on the values obtained from each power series, the indicated average value being designated as an indicator of processing conditions (PCI), and / or I) removing PCI from the calibration graph according to stage E) and / or substituting the values of the absorption intensity at the corresponding wavelengths or wave numbers in the calibration equation according to stage E) to obtain an indicator of processing conditions; and J) applying an indicator of the processing conditions obtained in stage H) and / or I) on the processing scale specified in paragraph 1, determining whether the raw material for the feed product and / or the feed product is over-processed, sufficiently processed or insufficiently processed. 6. The method according to p. 5, where in stage G) receive the same parameters as in stages a1) to a3). 7. The method according to p. 5 or 6, further comprising a stage K) substituting the indicator of the processing conditions obtained in stage H) into the calibration equation according to stage j) and / or deriving the functional value for the indicator of the processing conditions obtained in stage I) to obtain the specific digestibility coefficient (D _AA ) of the amino acid in the raw material for the feed product and / or feed product from step F). 8. The method according to p. 7, further comprising a stage L) determining the differential amount between the desired value and the actual value of the amount of amino acid in the feed material for the feed product and / or feed product based on the difference between the maximum digestibility coefficient in the ileum of the specified amino acid according to claim 2 and the specific digestibility coefficient of the specified amino acid obtained in stage K). 9. The method according to any one of paragraphs. 1-8, providing for the stages M) determining the digestible amount of amino acid in the sample of raw material for the feed product and / or feed product by multiplying the amount of said amino acid in the sample of raw material for the feed product and / or feed product obtained in stage G) by the specific digestibility coefficient obtained in stage K). 10. The method according to any one of paragraphs. 1-9, where the raw material for the feed product and / or the feed product is soy, soybeans or soya product. 11. The method according to any one of paragraphs. 1-10, where the species of animals refers to omnivores, carnivores, herbivores and / or ruminants. 12. The method according to any one of paragraphs. 3-11, where the calibration graphs and / or calibration equations from step C) according to clause 3 and / or from step E) according to clause 4 are stored on a computer or in a cloud storage. 13. A method of obtaining a feed product, comprising stages F) - L) according to any one of paragraphs. 5-12, where the method further comprises at least one of the stages N) additional processing of the raw material for the feed product and / or feed product, if the raw material for the feed product and / or feed product is defined as not sufficiently processed, and / or O) adding a differential amount of the amino acid obtained in step L) to the feed material for the feed product and / or the feed product, if the feed material for the feed product and / or the feed product is determined to be over-processed.

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