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

Abstract

The invention relates to a method for determining α-arabinofuranosidase (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 having α-arabinofuranosidase activity, said α-arabinofuranosidase 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 enzymatic fraction having α-arabinofuranosidase activity is extracted under the same conditions, then the α-arabinofuranosidase 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, α-arabinofuranosidase activity is measured by fluorescence using an enzymatic substrate, 4-methylumbelliferyl-α-L-arabinofuranoside (4-MU-ABF), which is hydrolyzable into fluorescent 4-methylumbelliferone (4-MU).

Description

Title of the invention: Method for determining specific α-arabinofuranosidase activity in an animal feed product

[0001] 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.

[0002] Animal nutrition is a specialty that aims to determine the nutritional requirements of animals, particularly livestock, necessary for their growth and general health, within the context of ever-increasing production of meat, dairy products, and other animal protein-based products. There are two main categories of feed for livestock: forage and processed products. Forage is consumed in the form of hay or silage by cattle, and processed products 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 the said processed product or mixed with the previously processed product.

[0003] 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 which contribute, alone or in association with other enzymes, to better overall digestibility of the product.

[0004] Given the complexity of the additive fraction in feed, these additives are often custom-formulated as premixes to meet the specific needs of animals, particularly according to 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 feed on the farm.

[0005] Animal feed products are available in various forms, for example cereal grains, flour, pellets... Their preparation requires processing operations, such as grinding, pelleting, as well as mixing the raw materials with additives, this phase being able to intervene Before, during, or after the processing of raw materials, additives are incorporated into the raw or processed material, depending on whether they are solid or liquid. This process is carried out in mixers or by vaporization. This operation is delicate because the additives are present in very small quantities relative to the large volumes of the raw material; the main difficulty 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 determined beforehand to meet the animals' needs. However, 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 said additives during the manufacture of the food, for example due to heat treatment leading to the degradation of the additives, and also an uneven distribution of the additives in the food.

[0006] Feed is most often consumed in pellet form due to its practicality compared to flour, namely its easier prehension by animals, space savings during storage and transport, the absence of risk of segregation, its good flow making it easier to handle, reduced dust, better preservation, greater digestibility, and the absence of pathogens, as these are destroyed during manufacturing. Pelletizing is carried out on cereals in flour form by steam injection, thus involving temperatures that can reach 90°C, followed by compression through a die. If the additives, or at least some of them, are added before pelletizing, they undergo these 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-envelope 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 the rations, not conforming to the quantities of enzymes introduced, was regularly observed.

[0007] In this context, the Applicant wished to develop a tool to detect the level of α-arabinofuranosidase enzymes specifically provided by an additive in an animal feed product. The enzymes with activity α-Arabinofuranosidase promotes the digestibility of food products, especially when associated with other enzymes such as xynalases, but their levels are minute, on the order of a few tens of grams per ton of food and are difficult to detect.

[0008] The present invention solves this problem with a method for determining the quantity of enzymes with a-arabinofuranosidase activity in said food, said quantity resulting specifically from the input of additive initially introduced, and this for very low levels.

[0009] Thus, an object of the invention is a method for determining α-arabinofuranosidase (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 having α-arabinofuranosidase activity, said α-arabinofuranosidase activity to be determined being specific to said additive, said method comprising the following steps:

[0010] we have, on the one hand, a sample, called control sample (c), of said product free from said additive, and on the other hand, said sample to be analyzed (p);

[0011] For each of said control (c) and to be analyzed (p) samples, respectively, an enzymatic fraction having α-arabinofuranosidase activity is extracted under the same conditions, and then the α-arabinofuranosidase activity of each of the extracts, respectively ABFc and ABFp, is measured; and

[0012] ABFp - ABFc is calculated to obtain said specific ABF activity of said additive in said product;

[0013] according to which method, the α-arabinofuranosidase activity is measured by fluorescence using an enzymatic substrate, 4-methylumbelliferyl-αL-arabinofuranoside (4-MU-ABF), hydrolyzable into fluorescent 4-methylumbelliferone (4-MU).

[0014] α-arabinofuranosidase activity is expressed in α-arabinofuranosidase activity units, one α-arabinofuranosidase activity unit being defined as the amount of enzyme that releases one nanomole of 4-methylumbelliferone (4-MU) per minute and per g or kg of sample from 4-methylumbelliferyl α-arabinofuranoside (4-MU-ABF), under given test conditions.

[0015] 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 fraction of additives, in particular enzymatic additives.

[0016] Before describing the invention in more detail, certain terms used in the text are defined below.

[0017] By animal feed product, or animal feed product, is meant a supplemented feed that is ready to be given to the animal in the form of a ration; it is also meant a premix consisting of a mixture of additives ready to be incorporated into a feed, or consisting of a mixture of additives, by for example a multi-enzyme complex, representing only a part of the fraction of additives.

[0018] Premixes of additives are not intended for direct animal feeding. They are highly concentrated mixtures that will be mixed with raw materials by feed manufacturers or farmers producing their own feed on the farm, in order to obtain a feed.

[0019] The recovery rate of an additive is defined as the ratio between the measured activity of said additive in the animal feed product and the theoretical activity of said additive in said product. For example, if the animal feed product is in the form of pellets obtained by first mixing a raw material with additives and then granulating, the recovery rate of one or more of the additives is the ratio between the measured activity of said additive in the pellets and the activity of said additive in said product before granulation, considered to be the theoretical activity.

[0020] The Applicant's work led her to select a fluorescence analysis method involving the fluorescent substrate 4-methylumbelliferyl-α-Arabinofuranoside (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 α-arabinofuranosidase activity to fluorescent 4-methylumbelliferone (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 amounts of enzymes present in a sample; it also resolves the problem of specificity for α-arabinofuranosidase activity in a medium that generally contains a complex combination of enzymes.

[0021] The α-arabinofuranosidase activity of a sample is measured using a standard curve prepared to correlate an optical absorbance value with the amount of 4-methylumbelliferone (4-MU) formed during the enzymatic hydrolysis of 4-methylumbelliferyl-α-arabinofuranoside (4-MU-ABF) catalyzed by an enzyme with α-arabinofuranosidase activity. For this purpose, at least two, preferably at least three, and even better at least four solutions of different concentrations of 4-MU are prepared and their fluorescent emission measured.

[0022] Preferred operating conditions have been determined to improve the sensitivity and specificity of the 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 α-arabinofuranosidase enzymes from the additive and a minimum of other enzymes with activity α-arabinofuranosidase from raw materials or other additives and said to be endogenous, which may interfere with the detection reaction.

[0023] 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 α-arabinofuranosidase activity, or without α-arabinofuranosidase activity, increases; as the pH approaches 2, the quantity of enzymes with α-arabinofuranosidase 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.

[0024] 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.

[0025] The extraction temperature is on the order of 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 on the order of 40°C.

[0026] 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.

[0027] Thus, the invention provides a method for evaluating the recovery rate of an additive present in a product intended for animal feed and which has α-arabinofuranosidase activity, said method comprising the following steps:

[0028] an a-arabinofuranosidase (ABF) activity is determined in a sample, referred to as the sample to be analyzed (p), of said product intended for animal feed, said a-arabinofuranosidase activity to be determined being specific to said additive, in accordance with the process of the invention as described above, including according to any of its particular variants;

[0029] a sample, called theoretical sample (t), of a product intended for animal feed is prepared from a sample, called control sample (c), of said product free from said additive to which said additive is added in the same content as that of said product containing said additive;

[0030] an enzyme fraction having α-arabinofuranosidase activity is extracted from this theoretical sample under the same conditions as the enzyme fractions of the control samples (c) and to be analyzed (p), then the α-arabinofuranosidase activity of said sample (ABFt) is measured under the same conditions as the measurement of ABFc and ABFp;

[0031] we calculate ABFt - ABFc;

[0032] if [ABFp - ABFc] is equal to [ABFt - ABFc], we conclude that the recovery rate of the additive is 100% in said product; and if [ABFp - ABFc] is less than [ABFt - ABFc], we conclude that the recovery rate of the additive is less than 100%.

[0033] A recovery rate of less than 100% of the additive may result from a non-homogeneous distribution of the additive in the product; in this case, it is necessary to reconfigure the equipment with the manufacturer. It may also result from a degraded enzyme; in this case, a heat-stable or protected enzyme should be considered to make it resistant to the product treatment(s).

[0034] The invention is further detailed in the following example in support of [Fig. 1]

[0035] Example: Protocol for determining α-arabinofuranosidase activity in a sample of animal feed 1) Preparation of an enzymatic sample

[0036] The same protocol is followed whether the sample is the one to be analyzed (p) or the control sample (c), the following steps are carried out:

[0037] Approximately 10.0 g ± 0.5 g of a ground sample of said product is weighed and introduced into an Erlenmeyer or a 125 mL furnace flask;

[0038] Using a HandyStep® type pipette, 50 mL of citrate buffer, pH 3.3 (0.425 M citric acid and sodium citrate solution, with 0.05% (w / v) bovine serum albumin, BSA) is added;

[0039] it is stirred using a magnetic stir bar at 500 rpm for 30 minutes at room temperature;

[0040] Approximately 5 mL of the solution is transferred into a single-use plastic tube and centrifuged for 10 minutes at 7000 rpm at 15°C.

[0041] the resulting supernatant is introduced into a syringe and then filtered using a 0.45 µm syringe filter;

[0042] If necessary, it is diluted in the citrate buffer, pH 3.3, above. The final solution is called the analytical solution.

[0043] 2) Preparation of the substrate solution (at 100 pM of 4-MU-ABF)

[0044] First, a stock solution of 4-MU-ABF (at approximately 675 pM) is prepared by introducing 15 mg of 4-MU-ABF into a 125 mL bottle, then, using a HandyStep® type pipette, 72 mL of 0.1 M sodium acetate buffer pH 4.5 is added.

[0045] For the analysis, a fraction of this stock solution is taken and diluted in the citrate buffer, pH 3.3 above (0.425 M, with 0.05% (w / v) of BSA) to reach a concentration of 100 pM.

[0046] 3) Preparation of the 4-MU (sodium salt) standard curve

[0047] In a 60 mL bottle, approximately 15 mg ± 0.01 mg of 4-MU sodium salt is weighed out, and then, using a HandyStep® type pipette, 50 mL of water is added. The mixture is homogenized to obtain a SOL1 solution.

[0048] In a 60 mL bottle, 0.27 mL of SOL1 is introduced, then, using a HandyStep® type pipette, the volume is brought up to 50 mL with citrate buffer, pH 3.3, as described above (0.425 M, with 0.05% (w / v) BSA). The mixture is homogenized to obtain a SOL2 solution.

[0049] In a 15 mL bottle, 1 mL of SOL2 is introduced, then, using a HandyStep® type pipette, 7 mL of citrate buffer, pH 3.3 (0.425 M, with 0.05% (w / v) BSA), is added to obtain a 4-MU 1 pM solution.

[0050] From the 4-MU 1 pM solution, a range of dilutions is prepared according to the table below.

[0051] [Tables 1] [4-MU] (nM) 4-MU 1 pM (pL) Buffer (pL) Na2CO3 1 M (pL) 0.0 0 800 2400 12.5 40 760 2400 62.5 200 600 2400 125.0 400 400 2400 250.0 800 0 2400

[0052] Each of the solutions in the table above is poured into a cuvette for a fluorometer and the fluorescence is measured (excitation at 365 nm and emission at 445 nm; 400 V).

[0053] The resulting calibration curve is shown in [Fig. 1]. 4) Sample analysis procedure

[0054] The same protocol is followed whether the sample is the one to be analyzed (p) or the control sample (c) as obtained in point 1) and, for each of them, the following steps are carried out and for each analytical solution, the following steps are carried out:

[0055] 2 tubes named TOmin and T2omin are prepared;

[0056] 100 pL of the analysis solution prepared at point 1) is introduced into the T20mm tube;

[0057] The TOmin and T20min tubes are placed in a water bath at 40°C ± 0.2°C and left incubate for 2 minutes;

[0058] 700 pL of the 100 pM 4-MU-ABF substrate solution prepared in step 2) is added to the T20min tube without mixing, then to the TOmin tube at a time interval (e.g., 20 s). The timer is started as soon as the substrate solution is added to the T20min tube;

[0059] After exactly 20 minutes, 2.4 mL of 1 M sodium carbonate solution is added to tube T20min and then to tube TOmin, respecting the same time interval. The tubes are shaken and placed on a rack outside the thermostatic bath;

[0060] 100 pL of the analysis solution prepared at point 1) is added to the TOmin tube and shaken;

[0061] after pouring each solution into cuvettes for fluorimeter, the fluorescence of each solution is measured (excitation at 365 nm and emission at 445 nm; 400V).

[0062] 5) Determination of α-arabinofuranosidase activities ABFp and ABFc

[0063] The α-arabinofuranosidase activities ABFp and ABFc are calculated according to the following steps:

[0064] a) from the calibration solutions, the slope (a) of the calibration line shown on the is calculated;

[0065] b) from the fluorescent intensities of the analysis solutions of each sample, the difference in intensity between T20min and TOmin (AI) is calculated;

[0066] c) The enzymatic activity is proportional to the fluorescent intensity; it is expressed in U per kg of food sample and is calculated according to the equation below:

[0067] [Math.l] Activity (Ulkg) = % x x _L i"; Zuimn test socket

[0068] in which (a) is defined in point a), AI is defined in point b), the test portion is the weighed sample mass.

[0069] In the present example,

[0070] test portion = 10.0028 g

[0071] dilution factor = 800 mL

[0072] slope (a) = 26.94 nmol / L

[0073] fluorescent intensity T20min = 5596 and fluorescent intensity TOmin = 1939, i.e. AI = 3657,

[0074] [Math.2] Activity (^Mg)=^XTS8X^ = 543 IU / kg

[0075] The ABFP ratio of 543 U / kg to ABFt calculated for a theoretical sample (t) is then calculated: if the ratio is less than 100%, then actions can be taken to improve the recovery rate of the tested additive.

Claims

Demands

1. A method for determining α-arabinofuranosidase (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 having α-arabinofuranosidase activity, said α-arabinofuranosidase 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 enzymatic fraction having α-arabinofuranosidase activity is extracted under the same conditions, and then the α-arabinofuranosidase activity of each of the extracts, respectively ABFc and ABFp, is measured; and ABFp - ABFc is calculated to obtain said specific ABF activity of said additive in said product;According to which method, α-arabinofuranosidase activity is measured by fluorescence using an enzymatic substrate, 4-methylumbelliferyl-αL-arabinofuranoside (4-MU-ABF), which is hydrolyzable into fluorescent 4-methylumbelliferone (4-MU).

2. A method for evaluating the recovery rate of an additive present in an animal feed product and having α-arabinofuranosidase activity, said method comprising the following steps: determining α-arabinofuranosidase (ABF) activity in a sample, referred to as the sample to be analyzed (p), of said animal feed product, said α-arabinofuranosidase 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 an animal feed product from a sample, referred to as the control sample (c), of said product free of said additive, to which said additive is added in the same proportion as that of said product containing said additive; and extracting from this theoretical sample, an enzyme fraction having α-arabinofuranosidase activity under the same conditions that the enzymatic fractions of the control (c) and to be analyzed (p) samples are measured, then the α-arabinofuranosidase 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 α-arabinofuranosidase activity are extracted from the samples under at least one, preferably all of the following conditions: in a buffer selected from the following buffers: citrate, acetate, citrate / phosphate, glycine / HCl, succinate, aconitate, phthalate, at a pH of 2 to 6, preferably less than 4, at a temperature of 10°C to 30°C.

4. The method according to claim 3, characterized in that the pH is a

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

6. A method according to any one of claims 1 to 5, characterized in that the α-arabinofuranosidase activity is measured using a 4-methylumbelliferone standard curve and 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.