Method for determining specific alpha-arabinofuranosidase activity in an animal feed product

Abstract

The invention relates to a method for determining α-arabinofuranosidase (abbreviated ABF) activity in a sample, referred to as the sample to be analyzed (p), of a product intended for animal feed and containing an additive that exhibits ABF activity, the ABF activity to be determined being specific to the additive, the method comprising the following steps: on the one hand, a sample, referred to as the control sample (c), of the product which is free of the additive, and, on the other hand, the sample to be analyzed (p), are provided; for each of the control sample (c) and the sample to be analyzed (p), respectively, an enzyme fraction exhibiting ABF activity is extracted under the same conditions, then the ABF activity of each of the extracts, respectively ABFc and ABFp, is measured; and ABFp - ABFc is calculated to obtain the specific ABF activity of the additive in the product; according to which method the ABF activity is measured by fluorescence by means of a substrate for the enzyme, 4-methylumbelliferyl-α-L-arabinofuranoside, which is hydrolyzable to fluorescent 4-methylumbelliferone.

Description

[0001] DESCRIPTION

[0002] TITLE: Method for determining specific α-arabinofuranosidase activity in an animal feed product

[0003] The present invention relates to the field of animal nutrition. More particularly, it concerns a method for evaluating the efficiency of an additive with enzymatic activity in an animal feed product.

[0004] Animal nutrition is a specialized field that aims to determine the nutritional needs of animals, particularly livestock, necessary for their growth and overall health, within the context of ever-increasing production of meat, dairy products, and other animal protein-based products. There are two main categories of livestock feed: forage and processed products. Forage is consumed by cattle in the form of hay or silage, while processed products are consumed by all livestock.Processed products include raw materials of plant origin generally consisting of feed cereals, such as maize, soybeans, sorghum, oats, barley, and plant by-products, and are generally supplemented with additives, in infinitesimal amounts, intended to improve the performance and health of animals and also to reduce the environmental impact of animal production, all or part of the additives being able to be integrated into said processed product or mixed with the previously processed product.

[0005] Among the most commonly used additives in animal production are nutritional additives, such as vitamins, amino acids and trace elements, and zootechnical additives including enzymes, and in particular those with α-arabinofuranosidase activity ("α-arabinofuranosidase" is hereinafter abbreviated "ABF" throughout the description of the invention) which contribute, alone or in association with other enzymes, to better overall digestibility of the product.

[0006] Given the complexity of the additive composition in animal feed, these additives are often custom-formulated as premixes to meet the specific needs of animals, taking into account their species and physiological stage. These highly concentrated premixes can be in solid or liquid form. They are intended to be mixed with plant-based raw materials by feed manufacturers or farmers preparing their own feed on the farm.

[0007] Animal feed products are available in various forms, such as cereal grains, flour, and pellets. Their preparation requires processing operations, such as grinding, pelleting, and mixing the raw materials with additives. This mixing phase can occur before, during, or after the raw materials are processed. Depending on whether they are solid or liquid, the additives are incorporated into the raw or processed material using mixers or by spraying. This operation is delicate because the additives are present in very small quantities relative to the large volumes of the raw material. The main challenge lies in ensuring their perfectly homogeneous distribution throughout the material. Since feed is distributed in rations, it is essential that each ration includes the additives that have been determined beforehand to meet the animals' needs.Measurements taken with manufacturers and especially with farmers managing the introduction of additives on their farms reveal that this is not always the case. The main reasons are a loss of these additives during feed manufacturing, for example due to heat treatment leading to additive degradation, and also an uneven distribution of additives within the feed.

[0008] Animal feed is most often consumed in pellet form due to its practicality compared to flour. This includes easier prehension by animals, space savings during storage and transport, no risk of segregation, good flow making handling easier, reduced dust, better preservation, greater digestibility, and the absence of pathogens, as these are destroyed during the manufacturing process. Pelletizing is carried out on cereals in flour form by steam injection, which involves temperatures reaching up to 90°C, followed by compression through a die. If additives are added before pelletizing, they undergo the same operating conditions.As previously mentioned, additives can include or consist of enzymes, and despite the availability of these enzymes in temperature-resistant variants, such as those obtained through mutagenesis or in a form protected, for example, by "core-shell" structures, higher temperatures cause the inhibition of at least some of them. Heating is not the only cause of enzyme inactivation; storage time can also affect enzyme stability. Consequently, the enzymatic activity of the feed does not reflect the quantities of enzymes initially introduced. If enzymes are not incorporated before or during pelleting, they can be added afterward, generally at the operator's facility, by spraying onto the pellets if they are liquid, or by mixing them with the pellets if they are solid.In this case too, an inhomogeneous distribution of additives in rations not conforming to the quantities of enzymes introduced was regularly observed.

[0009] In this context, the Applicant wished to develop a tool to detect the specific content of ABF-active enzymes provided by an additive in animal feed. ABF-active enzymes improve the digestibility of feed products, particularly when combined with other enzymes such as xynalases, but their levels are minute, on the order of a few tens of grams per ton of feed, and are difficult to detect.

[0010] The present invention solves this problem with a method for determining the quantity of ABF-active enzymes in said food, said quantity resulting specifically from the input of additive initially introduced, and this for very low levels.

[0011] Thus, an object of the invention is a method for determining an ABF activity in a sample, called the sample to be analyzed (p), of a product intended for animal feed and containing an additive having an ABF activity, said ABF activity to be determined being specific to said additive, said method comprising the following steps: on the one hand, a sample, called the control sample (c), of said product free from said additive, and on the other hand, said sample to be analyzed (p); for each of said control (c) and sample to be analyzed (p), respectively, an enzymatic fraction having an ABF activity is extracted under the same conditions, then the ABF activity of each of the extracts, respectively ABFc and ABFp, is measured; and ABFp - ABFc is calculated to obtain said ABF activity specific to said additive in said product;according to which method, ABF activity is measured by fluorescence using a substrate of the enzyme, 4-methylumbelliferyl-aL-arabinofuranoside (hereinafter abbreviated "4-MU-ABF"), hydrolyzable to fluorescent 4-methylumbelliferone (hereinafter abbreviated "(4-MU)").

[0012] ABF activity is expressed in ABF activity units, one ABF activity unit being defined as the amount of enzyme that releases one nanomole of 4-MU per minute per g or kg of sample from 4-MU-ABF, under given test conditions.

[0013] This method is reliable, sensitive and reproducible regardless of the presentation of the product intended for animal feed and regardless of the complexity of the additive fraction, including enzymatic additives.

[0014] Before describing the invention in more detail, some terms used in the text are defined below.

[0015] By product intended for animal feed, or animal feed product, we mean a supplemented feed that is ready to be given to the animal, in the form of a ration; we also mean a premix consisting of a mixture of additives ready to be incorporated into a feed, or consisting of a mixture of additives, for example a multi-enzyme complex, representing only a part of the fraction of additives.

[0016] Premixes of additives are not intended for direct animal feeding. They are highly concentrated mixtures that are blended with raw materials by feed manufacturers or farmers producing their own feed on the farm to obtain a finished product. The recovery rate of an additive is the ratio between the measured activity of that additive in the feed product and its theoretical activity in that product. For example, if the feed product is in pellet form, obtained by first mixing a raw material with additives and then granulating it, the recovery rate of one or more additives is the ratio between the measured activity of that additive in the pellets and its activity in the feed product before granulation, which is considered its theoretical activity.

[0017] The Applicant's work led her to select a fluorescence analysis method involving the fluorescent substrate 4-MU-ABF, which has a maximum excitation and emission wavelength of 320 nm and 385 nm, respectively. This substrate is hydrolyzed in the presence of an enzyme with ABF activity into fluorescent 4-MU, which has a maximum excitation and emission wavelength of 365 nm and 445 nm, respectively. This choice overcomes the problem of the technique's sensitivity given the small quantities of enzymes present in a sample; it also resolves the problem of specificity for ABF activity in a medium that generally contains a complex combination of enzymes.

[0018] It should be noted that other substrates are known to be suitable for determining ABF activity when the ABF enzyme is in pure form (i.e., not mixed at very low concentrations with other compounds, including other enzymes). However, as demonstrated below in the experimental section using the substrate 4-nitrophenyl αL-arabinofuranosidase (hereafter abbreviated as "pNP-ABF"), this information on such suitable substrates when the enzyme is in pure form cannot be applied to selecting substrates for determining ABF activity when the ABF enzyme is present at very low concentrations in a complex mixture of compounds (e.g., an animal feed product).

[0019] Thus, quite surprisingly, it turned out that the 4-MU-ABF substrate was perfectly suited to determine the ABF activity of a sample of animal feed containing an additive with ABF activity.

[0020] The ABF activity of a sample is measured using a standard curve designed to correlate an optical absorbance value with the amount of 4-MU formed during the enzymatic hydrolysis of 4-MU-ABF catalyzed by an enzyme with ABF activity. For this purpose, at least two, preferably at least three, and even better at least four solutions of different 4-MU concentrations are prepared and their fluorescent emission measured.

[0021] Preferred operating conditions have been determined to improve the sensitivity and specificity of detection. These are indicated below and can be considered individually or in combination. While they relate to both sample preparation and the enzymatic detection reaction, those concerning sample preparation are more critical, as they must allow for the extraction from the product sample of both a maximum of ABF-active enzymes from the additive and a minimum of other ABF-active enzymes from raw materials or other additives, known as endogenous enzymes, which could interfere with the detection reaction.

[0022] The pH of an extraction solution for obtaining the sample is advantageously 2–6, and even better, below 4. At 4 or above, the risk of extracting a large number of enzymes with endogenous ABF activity, or with no ABF activity at all, increases; approaching pH 2, the quantity of enzymes with ABF activity decreases, and therefore the fluorescent signal obtained. A preferred pH for the extraction solution is in the range of 3 to 3.5, or even 3.3.

[0023] The buffer of the extraction solution can also influence the performance of the process. A preferred buffer is chosen from among the following: citrate, acetate, citrate / phosphate, glycine / HCl, succinate, aconitate, phthalate.

[0024] The extraction temperature is around ambient temperature, advantageously between 10°C and 30°C, preferably between 10°C and 20°C. The temperature of the enzymatic reaction is preferably from 30°C to 60°C; more advantageously, it is from 35°C to 45°C, and even better, it is around 40°C.

[0025] The process of the invention is of particular interest in evaluating the recovery rate of an additive present in a product intended for animal feed.

[0026] Thus, the invention provides a method for evaluating the recovery rate of an additive that is present in a product intended for animal feed and that has an ABF activity, said method comprising the following steps: an ABF activity is determined in a sample, called the sample to be analyzed (p), of said product intended for animal feed, said ABF activity to be determined being specific to said additive, in accordance with the method of the invention as described above, including according to any of its particular variants; a sample, called the theoretical sample (t), of a product intended for animal feed is prepared from a sample, called the control sample (c), of said product free from said additive to which said additive is added in the same quantity as that of said product containing said additive;An enzymatic fraction with ABF activity is extracted from this theoretical sample under the same conditions as the enzymatic fractions of the control (c) and sample to be analyzed (p), then the ABF activity of said sample (ABFt) is measured under the same conditions as the measurement of ABFc and ABFp; ABFt - ABFc is calculated; if [ABFp - ABFc] is equal to [ABFt - ABFc], it is concluded that the recovery rate of the additive is 100% in said product; and if [ABFp - ABFc] is less than [ABFt - ABFc], it is concluded that the recovery rate of the additive is less than 100%.

[0027] A recovery rate of less than 100% of the additive may result from uneven distribution of the additive within the product. In this case, it is necessary to consult with the manufacturer to adjust the equipment. It may also be due to a degraded enzyme; in this case, a heat-stable or protected enzyme should be considered to make it resistant to the product's processing.

[0028] In the process for determining ABF activity in a sample described above, as well as in the process for evaluating the recovery rate of an additive present in a product intended for animal feed and having ABF activity also described above, the enzyme fractions having α-arabinofuranosidase activity can be extracted from the samples under at least one, preferably all of the following conditions:

[0029] - in a buffer chosen from among the following buffers: citrate, acetate, citrate / phosphate, glycine / HCl, succinate, aconitate, phthalate,

[0030] - at a pH of 2 to 6, preferably below 4,

[0031] - at a temperature of 10°C to 30°C.

[0032] Preferably, the pH is 3.3.

[0033] Preferably, ABF activity is measured at a temperature of 30°C-60°C, more advantageously from 35°C to 45°C, and even better at around 40°C.

[0034] In the process for determining ABF activity in a sample described above, as well as in the process for evaluating the recovery rate of an additive that is present in a product intended for animal feed and that has an ABF activity also described above, the ABF activity can be measured using a 4-MU standard curve that is expressed in nmol / L.

[0035] The product may be a supplemented feed or a premix of additives. The product may be a supplemented feed or a premix of additives for monogastric animals.

[0036] The advantages provided by the invention are further detailed in Example 1 according to the invention which follows in support of Figure 1, as well as in Comparative Example 2 in support of Figures 2 and 3.

[0037] Example 1: Implementation of the method for determining ABF activity according to the invention in a sample of an animal feed product

[0038] 1) Preparation of an enzymatic sample

[0039] The same protocol was followed whether the sample was the one to be analyzed (p) or the control sample (c), the following steps were carried out: approximately 10.0 g ± 0.5 g of a ground sample of said product were weighed and introduced into a 125 mL Erlenmeyer flask; using a HandyStep® type pipette, 50 mL of a citrate buffer solution, pH 3.3 (0.425 mol / L citric acid and sodium citrate solution, with 0.05% (w / v) bovine serum albumin, BSA) was added; the mixture was stirred using a magnetic stir bar at 500 rpm for 30 minutes at room temperature; approximately 5 mL of the solution was transferred into a single-use plastic tube and centrifuged for 10 minutes at 7000 rpm at 15°C. The resulting supernatant was introduced into a syringe and then filtered using a 0.45 µm syringe filter; if necessary, it can be diluted in the citrate buffer, pH 3.3, above.

[0040] The final solution was named the "analysis solution".

[0041] 2) Preparation of substrate solution (at 100 umol / L 4-MU-ABF)

[0042] First, a stock solution of 4-MU-ABF (at approximately 675 pmol / L) was prepared by introducing 15 mg of 4-MU-ABF into a 125 mL bottle, then, using a HandyStep® type pipette, 72 mL of a 0.1 mol / L sodium acetate buffer solution pH 4.5 was added.

[0043] For the analysis, a fraction of this stock solution was taken and diluted in the citrate buffer solution, pH 3.3 detailed above in point 1), namely 0.425 mol / L, with 0.05% (w / v) of BSA, in order to reach a concentration of 100 pmol / L.

[0044] 3) Preparation of the 4-MU (sodium salt) standard curve

[0045] In a 60 mL bottle, approximately 15 mg ± 0.01 mg of 4-MU sodium salt was weighed out, and then 50 mL of water was added using a HandyStep® type pipette. The mixture was homogenized to obtain a "SOL1" solution.

[0046] In a 60 mL bottle, 0.27 mL of SOL1 was introduced, then, using a HandyStep® type pipette, the volume was made up to 50 mL with the citrate buffer solution, pH 3.3 (detailed above in point 1), namely 0.425 mol / L, with 0.05% (w / v) of BSA. The mixture was homogenized to obtain a "SOL2" solution.

[0047] In a 15 mL bottle, 1 mL of SOL2 was introduced and then, using a HandyStep® type pipette, 7 mL of the citrate buffer solution, pH 3.3 detailed above in point 1), namely 0.425 mol / L, with 0.05% (w / v) of BSA was added to obtain a 4-MU 1 pmol / L solution.

[0048] A range of dilutions was prepared from the 4-MU 1 pmol / L solution according to Table 1 below. Table 1

[0049] Each of the solutions in Table 1 above was poured into a cuvette for a fluorometer and the fluorescence was measured (excitation at 365 nm and emission at 445 nm; 400 V).

[0050] The resulting calibration curve is shown in Figure 1.

[0051] 4) Sample analysis procedure

[0052] The same protocol was followed whether the sample was the one to be analyzed (p) or the control sample (c) as obtained in step 1), and for each, the following steps were carried out: two tubes, labeled Tomin and T20min, were prepared; 100 pL of the analytical solution prepared in step 1) was introduced into tube T20min; the Tomin and T20min tubes were placed in a water bath at 40°C ± 0.2°C and incubated for 2 minutes; 700 pL of the 100 pmol / L 4-MU-ABF substrate solution prepared in step 2) was added to tube T20min without mixing, and then to tube Tomin at 20-second intervals. The timer was started as soon as the substrate solution was added to T20min; After exactly 20 minutes, in order to stop the enzymatic reaction, 2.4 mL of a 1 mol / L sodium carbonate solution was added to the T20min tube and then to the Tomin tube, respecting the same time interval.The tubes were shaken and placed on a rack outside the thermostatic bath; 100 µL of the analytical solution prepared in step 1) was added to the Tomin tube and shaken; after transferring 250 µL of each solution into fluorometer cuvettes, the fluorescence of each solution was measured (excitation at 365 nm and emission at 445 nm; 400 V). The spectrophotometer was zeroed with demineralized water. 5) Determination of ABFp and ABFc activities.

[0053] The ABFp and ABFc activities were calculated according to the following steps: a) from the calibration solutions, the slope (a) of the calibration line shown in Figure 1 was calculated; b) from the fluorescent intensities of the analytical solutions for each sample, the difference in intensity between T20min and Tomin (Al) was calculated; c) the enzymatic activity is proportional to the fluorescent intensity; it is expressed in U per kg of food sample and was calculated according to the equation below:

[0054] AZ dilution factor 1

[0055] Activity (U / kg) = — X - - - x - a Test portion 20min in which (a) is defined at point a), Al is defined at point b), the test portion is the mass of the weighed sample.

[0056] In this example,

[0057] - for the sample to be analyzed (p) of an animal feed product containing an additive with ABF activity: test portion = 10.13 g, dilution factor = 5000 mL, slope (a) of the calibration curve shown in Figure 1 = 25.85, fluorescent intensity T20min = 3709 and fluorescent intensity T20min = 512, i.e.

[0058] Al = 3197,

[0059] 3197 5000 1

[0060] Activity (vU / 7 k yq) = — — - x - x — = 3052 U / kq 25.85 10.13 20 ' y

[0061] ABFp was therefore 3052 U / kg.

[0062] - for the control sample (c) of said animal feed product but which was free of said additive having ABF activity: test portion = 10.16 g, dilution factor = 5000 mL, slope (a) of the calibration curve shown in Figure 1 = 25.85, fluorescent intensity T20min = 2208 and fluorescent intensity T20min = 486, i.e.

[0063] Al = 1722,

[0064] 1722 5000 1

[0065] Activity (vU / 7 k yq) = — — - x - x — = 1639 U / kq 25.85 10.16 20 ' y

[0066] ABFc was therefore 1639 U / kg. ABFp-ABFc = 3052- 1639 = 1413 U / kg, that is to say the specific ABF activity of said additive in said product.

[0067] 6) Determination of the recovery rate of the additive present in the animal feed product

[0068] A sample, called the theoretical sample (t) of the animal feed product was prepared in the following manner: in a sample, called the control sample (c), of a product which was free of said ABF activity additive, said additive was added at a level identical to that of said additive in the product to be analyzed which contains said ABF activity additive.

[0069] An enzymatic fraction with ABF activity was extracted from this theoretical sample (t) under the same conditions as the enzymatic fractions of the control samples (c) and to be analyzed (p).

[0070] The ABF activity of said sample (ABFt) was measured under the same conditions as the measurement of ABFc and ABFp.

[0071] ABFt was 3075 U / kg.

[0072] ABFt - ABFc = 1436 U / kg.

[0073] [ABFp - ABFc] being equal to 1413 U / kg, we conclude that the recovery rate of the additive is 98% in said product.

[0074] Example 2: Comparative example: Determination of ABF activity in a sample of an animal feed product using pNP-ABF as a substrate

[0075] In this comparative example, the pNP-ABF substrate was used to determine the ABF activity of a sample of an animal feed product.

[0076] Indeed, it is known to use this substrate to determine ABF activity when the enzyme is in its pure form. More specifically, the action of ABF, under specific temperature and pH conditions, on the pNP-ABF substrate releases para-nitrophenol, which is measurable by fluorescence at 405 nm in a basic medium.

[0077] Experiments were therefore carried out with an animal feed containing 50 g of an additive with ABF activity per tonne of said feed. This additive comprised, as a mass percentage relative to the mass of said additive, 8% enzymes, of which 0.55% was ABF. This mass concentration of ABF in an animal feed product is quite normal. 1) Preparation of 5 analytical solutions:

[0078] Three analytical solutions, hereinafter referred to as "test feed 1", "test feed 2" and "test feed 3", were prepared by adding 10 g of said feed (which therefore contained 500 pg of additive, including 2.75 pg of ABF) to 100 mL of a sodium acetate buffer solution at a concentration of 0.1 mol / L and pH 4.5. The concentration of ABF was thus only 27.5 pg of ABF per liter of buffer solution for each of these tests feed 1 to 3.

[0079] Furthermore, a 4 ème The analytical solution was prepared by adding a specific quantity of the additive, which has ABF activity, to 100 mL of the aforementioned buffer solution, such that the ABF concentration was 27.5 pg per liter of the buffer solution. This 4 ème the solution hereafter referred to as the "additive test" was therefore devoid of the other constituents of the food and comprised the additive at a concentration identical to that of the solutions of food tests 1 to 3.

[0080] Finally, a 5 ème The analytical solution was prepared by adding 10 g of a feed containing all the components of the feed solutions from tests 1 to 3, with the sole exception of the additive, to 100 mL of the aforementioned buffer solution. This 5 ème The solution without the additive is hereinafter referred to as the "control test".

[0081] 2) Preparation of pNP-ABF substrate solution at 4 mmol / L

[0082] To obtain 10 mL of substrate, 10.85 mg of pNP-ABF was weighed into a 15 mL bottle and 10 mL of the buffer solution described in point 1) above was added.

[0083] 3) Dosage procedure for the 5 tests

[0084] For each of the 5 detailed tests above, the following procedure was followed for each of the 3 enzymatic reaction times of 2 hours, 20 hours, and 23 hours, i.e., incubation times: 2 tubes, labeled Tomin and Tincubation, were prepared; 40 pL of the analytical solution prepared in step 1) was introduced into tube Tincubation; the Tomin and Tincubation tubes were placed in a water bath at 40°C ± 0.2°C; 360 pL of the substrate solution prepared in step 2) were added to tube Tincubation without mixing, and then to tube Tomin at 10-second intervals. The timer was started as soon as the substrate solution was added to Tincubation; After exactly the incubation time (i.e., 2 hours or 20 hours or 23 hours), in order to stop the enzymatic reaction, 1.2 mL of a 1 mol / L sodium carbonate solution was added to the Tincubation tube and then to the Tomin tube, respecting the same time interval.The tubes were shaken and placed on a rack outside the thermostatic bath; 40 µL of the analytical solution prepared in step 1) was added to the Tomin tube and shaken; after transferring 250 µL of each solution into fluorometer cuvettes, the fluorescence of each solution was measured at 405 nm. The spectrophotometer was zeroed with demineralized water.

[0085] Table 2 below details for each test the absorbance values ​​measured at 405 nm at incubation times of 0, 2 hours, 20 hours and 23 hours.

[0086] Table 2

[0087] Figure 2 represents a graph of absorbance as a function of incubation time (or in other words, the duration of the enzymatic reaction) for each of the 5 trials.

[0088] Based on the graph in Figure 2 and the detailed results in Table 2 above, it can be seen that for the additive test, the absorbance is very low and considered to be zero, as it is below the detection limit of 0.114 (i.e., equal to 3 x Absorbance). H20 (= 3 x 0.038 = 0.114). Under these conditions, ABF was not able to produce enough para-nitrophenol to be detectable by fluorescence.

[0089] Furthermore, a change in absorbance was observed for feed trials 1 to 3 as a function of incubation time. However, this change was identical to that of the control trial, which, as a reminder, contained no additive. Therefore, the observed change in absorbance is not related to the activity of the ABF present in the additive.

[0090] Thus, during this experiment, ABF activity could not be determined. This highlights that the pNP-ABF substrate is not a suitable substrate for determining ABF activity when the ABF enzyme is present at very low concentrations in animal feed.

[0091] In addition, absorbance spectra between 300 nm and 550 nm were recorded for the 5 tests. Figure 3 shows the absorbance spectra between 300 nm and 550 nm for food tests 1 to 3, the control test, and the additive test.

[0092] Based on the shape of the absorbance curves in Figure 3, it can be seen that compounds absorb at a wavelength of 405 nm in tests 1 to 3 and the control test. These compounds do not correspond to the para-nitrophenol produced by the enzymatic reaction. Rather, they are phenol-type compounds, sugars present in foods (particularly animal feed) that also absorb at a wavelength of 405 nm. This result also highlights that the pNP-ABF substrate is not a suitable substrate for determining ABF activity when the ABF enzyme is present at very low concentrations in animal feed.

[0093] In conclusion, this comparative example has demonstrated that the detection of the ABF enzyme present in an animal feed product (namely a very complex mixture of different compounds), and at a very low concentration (2.75 pg per 10 g of feed) could not be achieved by fluorescence using the pNP-ABF substrate.

[0094] This comparative example demonstrates that substrates known to be used to determine ABF activity in its pure form (i.e., in isolation and not in association with other compounds, including other enzymes) may not be suitable for detecting this ABF enzyme at very low concentrations in complex foods such as animal feed. In other words, the knowledge about suitable substrates for determining ABF activity in its pure form cannot be applied to identifying a suitable substrate for determining ABF activity when the ABF enzyme is present in very small quantities in a complex mixture of compounds such as an animal feed product.In view of this, it was therefore not at all obvious that the 4-MU-ABF substrate would be a perfectly suitable substrate for determining the ABF activity of a sample of animal feed product in which ABF is present at very low concentration.

Claims

DEMANDS 1. A method for determining α-arabinofuranosidase activity (hereinafter abbreviated as "ABF") in a sample, referred to as the sample to be analyzed (p), of a product intended for animal feed and containing an additive having ABF activity, said ABF activity to be determined being specific to said additive, said method comprising the following steps: a sample, referred to as the control sample (c), of said product free from said additive is available on the one hand, and said sample to be analyzed (p) is available on the other hand; for each of said control (c) and sample to be analyzed (p), respectively, an enzyme fraction having ABF activity is extracted under the same conditions, and then the ABF activity of each of the extracts, respectively ABFc and ABFp, is measured; and ABFp - ABFc is calculated to obtain said ABF activity specific to said additive in said product;according to which method, ABF activity is measured by fluorescence using a substrate of the enzyme, 4-methylumbelliferyl-aL-arabinofuranoside), hydrolyzable to fluorescent 4-methylumbelliferone (hereafter abbreviated "4-MU").

2. A method for evaluating the recovery rate of an additive which is present in a product intended for animal feed and which has an ABF activity, said method comprising the following steps: determining an ABF activity in a sample, referred to as the sample to be analyzed (p), of said product intended for animal feed, said ABF activity to be determined being specific to said additive, according to the method of claim 1; preparing a sample, referred to as the theoretical sample (t), of a product intended for animal feed, from a sample, referred to as the control sample (c), of said product free from said additive to which said additive is added in the same quantity as that of said product containing said additive;An enzymatic fraction with ABF activity is extracted from this theoretical sample under the same conditions as the enzymatic fractions of the control (c) and sample to be analyzed (p), then the ABF activity of said sample (ABFt) is measured under the same conditions as the measurement of ABFc and ABFp; ABFt - ABFc is calculated; if [ABFp - ABFc] is equal to [ABFt - ABFc], it is concluded that the recovery rate of the additive is 100% in said product; and if [ABFp - ABFc] is less than [ABFt - ABFc], it is concluded that the recovery rate of the additive is less than 100%.

3. A process according to claim 1 or 2, characterized in that the enzymatic fractions having ABF activity are extracted from the samples under at least one, preferably all of the following conditions: in a buffer selected from citrate, acetate, citrate / phosphate, glycine / HCl, succinate, aconitate, phthalate buffers, at a pH of 2 to 6, preferably less than 4, at a temperature of 10°C to 30°C.

4. Process according to claim 3, characterized in that the pH is 3.

3.

5. A method according to any one of claims 1 to 4, characterized in that the ABF activity is measured at a temperature of 30°C-60°C, more advantageously, from 35°C to 45°C, and even better in the order of 40°C.

6. A method according to any one of claims 1 to 5, characterized in that the ABF activity is measured using a 4-MU standard range which is expressed in nmol / L.

7. A process according to any one of claims 1 to 6, characterized in that the product is a supplemented food or a premixture of additives.

8. A process according to claim 7, characterized in that the product is a supplemented feed or a premix of additives for monogastric animals.

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