• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Lactose detection kit, on surfaces, water and food, comprising an extraction solution; a separation solution (17); a separation strip (19) of the lactose by chromatography, with a first line (20) for placing the sample, and a second end line (21) for the solution to rise; a first and a second display solutions; a sprayer and; a lactose pattern Lactose detection method, which comprises obtaining and homogenizing (1) the sample; introduction (7) of separation solution (17) into chamber; placement (8) of 5 μl of the filtrate in a separation strip (19); placement (9) of 5 μl of the lactose pattern; first drying (10); introduction (11) of separation strip (19) into chamber; second drying (12); Introduction (13) spray display solutions; spraying (14) of the separation strip (19); revealed (15); and interpretation (16) of the results. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2728137A1
    申请号:ES201830387
    申请日:2018-04-20
    公开日:2019-10-22
    发明作者:Saborit Florenci Cutrina
    申请人:Saborit Florenci Cutrina；
    IPC主号:
    专利说明:

    [0001]
    [0002] Lactose detection kit and detection procedure by applying it
    [0003]
    [0004] Technical Field of the Invention
    [0005]
    [0006] The present invention corresponds to the technical field of lactose detection tests, in particular to a detection kit on surfaces, water and food and the method of carrying it out.
    [0007]
    [0008] Background of the Invention
    [0009]
    [0010] Lactose is a sugar formed by the union of one molecule of glucose and another of galactose. It is present, in different proportions, in all mammalian milks (cow, goat, sheep) and, therefore, in many prepared foods, derivatives and additives.
    [0011]
    [0012] Lactose intolerance is a digestive disorder caused by deficiency in the small intestine of an enzyme called lactase, which is responsible for the splitting of lactose into the two simple sugars that compose it. When this conversion does not occur, lactose malabsorption is generated, causing various symptoms such as abdominal pain, bloating, diarrhea, constipation and / or vomiting.
    [0013]
    [0014] People diagnosed with lactose intolerance should follow a lactose-free or low-lactose diet (depending on the degree of intolerance), which is why and, based on the scientific opinion of the European Food Safety Authority (EFSA) ), on the maximum lactose contents in lactose intolerance and galactosemia, the Spanish Agency for Consumption, Food Safety and Nutrition (AECOSAN) has adopted guidelines for lactose content for the names "Lactose Free" (Products with a content lower lactose of 0.01%) and "Low lactose content" (Products with a lower lactose content of 1%).
    [0015]
    [0016] At present, therefore, there is a need to check the levels of lactose content, both for the food and for the surfaces on which it is treated and the cleaning waters of said surfaces.
    [0017] These checks should be as quick as possible, to hinder the minimum production process and, should not be very expensive, so that the price is not an impediment in the realization of these checks by companies.
    [0018]
    [0019] In the state of the art there are kits that allow the detection of milk proteins through relatively rapid methods such as ELISA. This large family of analyzes is related to showing the contamination of products that contain milk, but it has nothing to do with the detection of lactose, because in these kits it is sought to avoid an allergic reaction with respect to milk proteins, which may be present in products with or without lactose, while in the detection of lactose it is sought to avoid symptoms resulting from intolerance to this compound sugar.
    [0020]
    [0021] There are different alternatives in the market regarding the detection of lactose in surfaces, water and food, such as biosensors and the enzymatic method.
    [0022]
    [0023] Thus, the detection of lactose by biosensors is easy to use and achieves the specificity of detection of lactose with respect to other sugars that the sample may have. However, it presents as main drawbacks the price of equipment and consumables and the detection limit, which is above 100 ppm, which is the limit currently set by law.
    [0024]
    [0025] On the other hand, the enzymatic method is based on the enzymatic breakdown of lactose to analyze one of the two sugars that form it, galactose. It is an effective method for the exact quantification of the amount of lactose that a sample has, however it presents difficulty in handling, in the calibration adjustments, in the limitations of working with enzymes (it must be kept at freezing temperatures and refrigeration) and the need to have a spectrophotometer, which has a high price that not all companies can afford.
    [0026]
    [0027] The following state of the art documents RU2396560C1, CN106706798A, CN107315053A, CN10523216A, CN107462660A, CN106645132A, JP2008170428A, JPS5651663A and ES2361733 can be mentioned.
    [0028]
    [0029] Document RU2396560C1, defines a method of identification of carbohydrates, which involves the determination of the content of glucose, lactose, galactose, fructose and tagatose through thin layer chromatography on Sordfil plates.
    [0030] The plates are pre-saturated with a solution of sodium dihydrophosphate (NaH2PO4) 0.5 mol / dm3 and then dried. Next, a micro-syringe is used to collect 0.001 cm 3 of carbohydrate solution which is then deposited on the starting line on the chromatographic plate, dried in air and lowered to a solvent. The solvent used is a mixture of acetone, isopropyl alcohol and a solution of lactic acid. Chromatographic separation takes 80 to 90 minutes. When the solvent reaches the elution limit, the plate is dried again at room temperature until complete evaporation of the solvent.
    [0031]
    [0032] In this document, two examples are used to demonstrate that the proposed method is better than another chromatography method, but the comparison is made under different conditions, since for the verification of the previous method they use a real food matrix, in particular a yogurt, to which apply a thin layer chromatography process with different plate impregnation reagents, solvents, developing solution ..., while in the verification of the method proposed in the document they use a pure laboratory mixture of different carbohydrates. In this way, both methods are not being carried out with equal conditions, since the behavior of a real matrix is not the same as that of a laboratory matrix, since there are multiple components in the matrix that can influence the final result.
    [0033]
    [0034] In addition, this document aims to identify the presence of carbohydrates in a sample, but at no time is the detection limit of the method determined. That is, it is not indicated what is the minimum value of the substances that can be detected with this method, so that if lactose is not detected, it may be that it does not exist in the sample or that it exists in an amount that does not It is detected by this method and yet it is above 100ppm in the initial sample. It is therefore a procedure that does not allow the detection of presence or absence of carbohydrates in a given concentration, so it is not valid for the purpose of this invention, which is to determine that the amount of lactose in a sample is lower or higher than the minimum value allowed to consider a food "lactose free" or "low lactose content".
    [0035]
    [0036] Likewise, a data extracted from the document, such as the amount of sample used of 1 microliters, indicates that it is working with concentrations of carbohydrates greater than 100ppm that currently mark the limit in the legislation.
    [0037] In addition, when trying to separate all carbohydrates, the length that the sample travels in the display strips is high, assuming times ranging from 80 to 90 minutes, excessive in practice, since they reduce productivity.
    [0038]
    [0039] In this method, in addition, since it is presented for ideal laboratory mixtures, it does not propose a pretreatment of the sample consisting of the extraction of lactose from the sample by dissolution, by precipitation of proteins and peptides. It is also a method that dilates excessively over time, due to drying at room temperature. This also detracts from standardization of the method, since each analyst can consider a dry plate after a different drying time.
    [0040]
    [0041] Another drawback is that it uses complicated handling reagents, because they are corrosive, carcinogenic or harmful. In addition, it adapts more to a laboratory method in which you have to adjust parameters, impregnate plates ... and not a quick kit that can be used by any company, in laboratories without large requirements of facilities and machinery, to know these parameters in a fast and simple way.
    [0042]
    [0043] Reference document CN106706798A defines a method for determining the lactose content that involves dissolving the sample to be measured in ultrasonic deionized water to obtain a mixed solution that is transferred to a volumetric flask and centrifuged. The dissolution and transfer process is repeated and the sample is replaced with a lactose standard to obtain a standard solution, using liquid chromatography to obtain the retention time and peak area of each substance.
    [0044]
    [0045] Liquid chromatography for the separation of carbohydrates is effective for a wide range of samples, however there are important limitations for use in the food industry. On the one hand the level of equipment / machinery necessary to carry it out (ultrasonic application, dedicated computer, chromatography column, centrifuge, etc ...), are a great inconvenience since more than 90% of the business food fabric are Small and medium enterprises and very few can afford the use of such techniques. On the other hand, the technical level of the personnel required to carry out this technique, once again, most companies cannot afford it. And finally, a large number of samples to be analyzed is required so that a laboratory could consider establishing such a technique.
    [0046] This patent, moreover, is aimed at detecting lactose in animal feed (mainly piglets), the sensitivity limits being completely different than in human feeding. The pretreatment that must be done to the sample, by ultrasound, centrifuge ... complicates the instruments.
    [0047]
    [0048] Reference document CN107315053A defines a method of analysis again by liquid chromatography to detect the appropriate amount of galactose, glucose, lactulose, sucrose and reference standard of lactose.
    [0049]
    [0050] It therefore presents the same technical, personnel, etc ... limitations as the previous document. Although in this case the document proposes an atomization system for the detection of substances at the end of liquid chromatography and is focused on the quality control of lactose and the investigation of related substances.
    [0051]
    [0052] Reference document CN105723216A, determines a method of detection and quantification of galacto-oligosaccharides in a sample that involves reacting a sample comprising galacto-oligosaccharides and dextrin with derivatization reagent, derivatizing dextrin and galacto-oligosaccharides in the sample and separating the Galactooligosaccharide component in the sample by high performance liquid chromatography using reverse phase 30C chromatography column.
    [0053]
    [0054] Again the method has the same technical, personnel, etc ... limitations and in this case even more because HPLC is used, which is an even more advanced technique than liquid chromatography, LC). This patent seeks the detection of two compounds (galactooligosaccharides) other than lactose, although one is a derivative thereof.
    [0055]
    [0056] The reference document CN107462660A, proposes an ion exchange chromatography that has the same limitations as in the previous cases. This document focuses on the detection of galacto-oligosaccharides in baby food and milk powder.
    [0057]
    [0058] Reference document CN106645132A uses an enzymatic method that breaks down lactose into its monosaccharides to measure galactose by chromogenic method (with a series of reagents the presence of color galactose is achieved). It is completely oriented to human samples and it is a type of methods that are usually totally qualitative, that is to say they value the presence / absence, but it is difficult to make a color change adjustment to assess the existence of a certain concentration.
    [0059] Reference document JP2008170428A makes an improvement in the technique of high performance liquid chromatography for the detection of traces of sugars and alcohols derived therefrom. The improvement consists in introducing a UV detector at the end of the chromatography column and benzoing the sugars / alcohols so that they absorb in UV (by themselves they do not). It has the same technical, personal, etc ... limitations as in the previous documents.
    [0060]
    [0061] Reference document JPS5651663A refers to a method for analyzing sugars, qualitatively and quantitatively by thin layer or paper chromatography, mainly in human samples (although it also names foods) and the application of ultraviolet rays after spraying the solution. It is a method that is not intended as a quick analysis kit and easy to use for people who do not expressly have high experience in thin layer chromatography.
    [0062]
    [0063] Finally, reference document ES2361733 is an industrial separation system of different components based on the serial use of different packed columns. It is not at any time to obtain a rapid lactose detection kit that can be used by food companies.
    [0064]
    [0065] There is therefore no process in the state of the art that can raise the detection of lactose in surfaces, water and food by a quick and simple test that can be presented as a rapid detection kit, suitable for application by any company in the sector, without requiring the specialization of operators.
    [0066]
    [0067] Description of the invention
    [0068]
    [0069] The lactose detection kit, on surfaces, water and food, on a sample obtained therefrom that is proposed herein comprises a solution for the extraction of lactose from the sample, where said extraction solution is contained in an extraction vessel and it has an amount of lactose between 0 and 50 mg / l.
    [0070]
    [0071] This extraction solution has 3 main functions that are to extract the lactose from the sample by dissolution in aqueous phase, precipitate and agglutinate the small proteins and peptides that the sample can contain to avoid interference in later parts, and adjust the sensitivity of the kit at a certain concentration depending on the Lactose content of the sample and the dilution applied thereto, through the addition of a small amount of lactose.
    [0072]
    [0073] This quantity of lactose that is contributed to the sample allows to increase the sensitivity of the method, being able to detect very small quantities that allow to know the existence of lactose from the levels determined by the legislation, which exceed what is allowed in foods that can be considered lactose free. Without this contribution, it would be impossible to detect lactose in the amounts of work used with this technique, since the sample is diluted and detection would not be allowed.
    [0074]
    [0075] The kit further comprises a separation solution and a hermetic separation chamber for placement thereof, where the separation solution is formed by a mixture containing at least one solvent suitable for the separation of sugars.
    [0076]
    [0077] It also comprises a strip of lactose separation from other soluble compounds in the sample, by chromatography.
    [0078]
    [0079] The separation solution helps to separate the lactose from other components of the sample and concentrate it in a very specific area of the strip, in the form of a band.
    [0080]
    [0081] The kit also has a first containment container with a first display solution and at least a second containment container with a second display solution. The first solution is formed by at least P-anisaldehyde and the second solution is formed by a mixture of sulfuric acid and absolute ethanol.
    [0082]
    [0083] A sprayer is also provided for mixing the first and second display solutions.
    [0084]
    [0085] Thus, the mixture of both visualization solutions has the function of developing agent of the separation strips, so that it allows to visualize different spots corresponding to the different compounds of the analyzed sample. Due to the instability of the mixture, they are presented as two separate solutions that are mixed just in the moment prior to use.
    [0086]
    [0087] Finally, the kit has a lactose pattern formed by a mixture of distilled water, lactose in a proportion greater than 15 ppm and a preservative.
    [0088] This pattern has a first main function consisting of internal quality control in each of the trials. Thus, the use of the lactose pattern makes it possible to ensure that the separation and visualization phases have been carried out properly, thus eliminating the obtaining of false negatives.
    [0089]
    [0090] Likewise, the pattern presents a second main function that resides in that it facilitates the visualization of the results, since the stain left by the pattern in the separation strip, allows to know exactly the height at which the lactose stain should appear. the sample in case it contains lactose. There are factors such as humidity, temperature, the sample itself ... which can vary slightly the final height of the spots, so that the lactose pattern allows to focus in each case what the value to be obtained should be in case of existence of lactose.
    [0091]
    [0092] This report also proposes a lactose detection procedure, on surfaces, water and food, by applying a detection kit as previously defined.
    [0093]
    [0094] Thus, the first phase is to obtain a sample and homogenize it.
    [0095]
    [0096] The second phase is formed by the introduction of the separation solution inside the separation chamber and hermetically sealed chamber for a time between 15 and 30min. It is important to place the separation solution inside the separation chamber for a time prior to its use, so that the solvent mixture of the liquid phase saturates the gas phase of the chamber.
    [0097]
    [0098] Next, the third phase consists of placing a quantity of 5 ^ l of the result of obtaining and homogenizing the sample, in a strip of separation, in the first indicative mark of the sample placement line.
    [0099]
    [0100] Likewise, as a fourth phase, an amount of 5 ^ l of the lactose pattern is placed, on the same separation strip, on the second mark indicative of the sample placement line.
    [0101]
    [0102] The fifth phase consists of a first drying of the separation strip at a temperature between 90 and 115 °, for a time between 30s and 10min.
    [0103] Next, the sixth phase of introducing the separation strip into the separation chamber and closing it again is carried out, for a time between 20 and 30 min, where the separation strip is arranged with a angle of inclination and oriented such that the first side is in a lower position with respect to the second side.
    [0104]
    [0105] Said separation strip must then be subjected to a seventh phase formed by a second drying at a temperature between 90 and 115 °, for a time between 30s and 10min;
    [0106]
    [0107] The eighth phase is the introduction of the total of the second visualization solution contained in the second containment container, into a sprayer and, subsequently, the introduction of 0.1 ml of the total of the first visualization solution contained in the first containment container, over the previous one in the same sprayer and, stirred the mixture.
    [0108]
    [0109] Once the mixture of both first and second display solutions is prepared, spraying the separation strip with it is carried out uniformly.
    [0110]
    [0111] Finally, a development phase of the separation strip is carried out for a time between 5 and 15 min, at a temperature between 100 and 120 ° and a final phase of interpretation of the results.
    [0112]
    [0113] With the lactose detection kit and the detection procedure by application thereof proposed here, a significant improvement in the state of the art is obtained.
    [0114]
    [0115] This is thus a way of detecting lactose in food as well as in water and surfaces, fast and easy to use, which allows the presentation of it in a kit that can be used by people who do not need a specialization to achieve it.
    [0116]
    [0117] With this kit it is possible to control the efficiency of cleaning on work surfaces and CIP systems, that is, on-site cleaning systems, without disassembling equipment or pipes, in addition to validating the production chain by analysis of semi-finished products and products. finished and release finished product.
    [0118] This kit is based on the principles of thin layer chromatography for the separation and detection of lactose in surfaces, water and food. In addition to extracting lactose from the analysis matrix, it separates it from other soluble components and visualizes the presence or absence of lactose in the sample.
    [0119]
    [0120] Thanks to the application of an extraction solution, it is possible to adjust the sensitivity of the kit to a certain concentration of lactose, by adding a small amount of it. Thus, since currently the minimum value allowed to be able to consider a "lactose-free" sample is 100ppm according to the legislation, the kit adjusts to these values, but if the minimum value varies, the kit would be prepared with a appropriate sensitivity for the new value.
    [0121]
    [0122] In this way the proposed kit and its application procedure achieve not only the detection of the existence or not of lactose, but also and as a significant advantage, it achieves the identification of the amount of lactose with a clear cut-off point legal, which is currently 100ppm, to be able to name a product "lactose free".
    [0123]
    [0124] In the application of the kit for the detection of lactose, a controlled drying is carried out and at high temperatures, by means of a heater, oven or similar, so that on the one hand a faster process realization is achieved and on the other a standardization of drying at the same performance conditions, which subsequently affects the results obtained.
    [0125]
    [0126] Regarding the speed of the separation process, this kit separates the possible amount of lactose in the sample and does not act on the rest of the substances, so the separation process is faster and has a duration of about 25-30 minutes, compared to 80-90 minutes of other processes.
    [0127]
    [0128] In addition, the kit presents all the elements that are required for the detection of lactose, none of them being a harmful material that should be handled with extreme caution, so it is a simple kit that can be used by personnel who do not necessarily have to have a special qualification for it.
    [0129]
    [0130] The elements it contains are not very expensive, so this kit is accessible to all companies that require a detection of lactose presence and They can apply it themselves, without resorting to external companies, so you get a reduced cost and faster.
    [0131]
    [0132] In addition, the process is carried out following guidelines on the elements provided in the kit, without impregnating and / or cutting plates, preparing reagents, adjusting parameters such as dilution ..., so it is easier and more apt to apply in the company itself, without the need to send the samples to a laboratory.
    [0133]
    [0134] Therefore, it is a very effective kit, suitable for application in disparate conditions of temperature and humidity, depending on where it is used, since the application of the lactose pattern provides the measure in each specific case according to the environmental and concrete conditions of the measurement. This is a relevant advantage over the standard thin layer chromatography method, since having to always maintain the same environmental conditions is an important limitation.
    [0135]
    [0136] It is also able to detect the presence or absence of lactose with the necessary sensitivity to determine if the existing amount is in a proportion that exceeds a legislative limit.
    [0137]
    [0138] Brief description of the drawings
    [0139]
    [0140] In order to help a better understanding of the features of the invention, according to a preferred example of practical implementation thereof, an integral part of said description is provided, a series of drawings where, for illustrative and non-limiting purposes, represented the following:
    [0141]
    [0142] Figure 1 shows a plan view of a lactose separation strip, for a preferred embodiment of the invention.
    [0143]
    [0144] Figures 2.1 to 2.4.- Shows a plan view of lactose separation strips in which the results obtained respectively, have been positive, negative, and two possible cases of invalid, for a preferred embodiment of the invention. .
    [0145]
    [0146] Figure 3.1 and 3.2.- Shows elevation and profile views of the separation chamber in the phase of introducing the separation strip into a lactose separation strip, for a preferred embodiment of the invention. .
    [0147] Figure 4.- Shows a block diagram of the lactose detection process, in surfaces, water and food, by means of a kit as defined, for a preferred embodiment of the invention.
    [0148]
    [0149] Detailed description of a preferred embodiment of the invention
    [0150]
    [0151] In view of the figures provided, it can be seen how in a preferred embodiment of the invention, the lactose detection kit, on surfaces, water and food, on a sample obtained therefrom, presents several elements which are detailed below.
    [0152]
    [0153] Thus, it comprises a lactose extraction solution from the sample, wherein said extraction solution is contained in an extraction vessel and has an amount of lactose between 0 and 50 mg / l. Depending on the dilution used and the type of sample, it is necessary to provide a greater or lesser amount of lactose, because if it has 100ppm, when the amount is diluted it decreases and is more difficult to detect, since it can be below the minimum necessary for subsequent visualization on the separation strip (19), whereby the contribution of lactose from the extraction solution allows detection to be possible.
    [0154]
    [0155] In this preferred embodiment of the invention, a dilution of 1/5 is made, so if the product had an amount less than or equal to 100ppm of lactose, the diluted sample would have 20ppm. Thus, the extraction solution will present in this case 20mg / l of lactose, so that the concentration in the sample rises to 40ppm in case there is really a presence of lactose, and this value is already detectable by this detection method .
    [0156]
    [0157] In this preferred embodiment of the invention, it is considered that the sample to be analyzed comes from a food. Thus, the extraction solution also comprises lactose, distilled water, a preservative formed by benzoic acid and a deproteinization reagent consisting of zinc acetate, sodium tungstate, sulfuric acid, trichloroacetic acid, perchloric acid, ether or acetone.
    [0158]
    [0159] In this preferred embodiment of the invention, the deproteinization reagent used is zinc acetate.
    [0160] The detection kit also comprises a separation solution (17) and an airtight separation chamber (18) for placement thereof. This separation solution (17) is formed by a mixture containing at least one solvent suitable for the separation of sugars.
    [0161]
    [0162] In this preferred embodiment of the invention, the separation solution (17) is formed by a solvent which in particular is acetone and further comprises distilled water.
    [0163]
    [0164] In other embodiments, the solvent may be formed by ethyl acetate, isopropanol, N-Butanol, phosphate buffer, methanol, chloroform, ammonium hydroxide, acetic acid and / or N-propanol.
    [0165]
    [0166] As shown in Figure 1, this kit also has a separation strip (19) of the lactose with respect to other soluble compounds in the sample, by chromatography. This separation strip (19) has a rectangular shape with a first and second sides (19.1, 19.2) of shorter lengths.
    [0167]
    [0168] Parallel to both the first and second sides (19.1, 19.2) presents a first line (20) of placing the sample close to the first side (19.1) and a second line (21) to end the rise of the separation solution (17), close to the second side (19.2).
    [0169]
    [0170] In this preferred embodiment of the invention, the separation strip (19) of the lactose is composed of a silica gel chromatopholium 60, but in other embodiments it may be composed of cellulose, kieselguhr, or a combination of silica gel and kieselguhr.
    [0171]
    [0172] Likewise, in this preferred embodiment of the invention, the separation strip (19) of the lactose has an impregnation of sodium acetate, but in other embodiments in which the composition of the separation strip is different, It may have an impregnation in sodium borate, boric acid or sodium bisulfite.
    [0173]
    [0174] As can be seen in Figure 1, the separation strip (19) of the lactose has on the first line (20) of the sample, a first and a second mark (22, 23) indicative of the place of placement of the sample. sample and lactose pattern, respectively, where both first and second indicative marks (22, 23) are separated from each other.
    [0175]
    [0176] The detection kit comprises a first containment container with a first display solution and at least a second containment container with a second display solution.
    [0177]
    [0178] The first solution is formed at least by P-anisaldehyde, being in this embodiment formed only by P-anisaldehyde and, the second solution is formed by a mixture of sulfuric acid and absolute ethanol.
    [0179]
    [0180] Thus, in this preferred embodiment of the invention, the first containment container of the first display solution comprises an amount between 2 and 3 ml thereof, in this case being 2.5 ml and, the second container of Containment of the second solution comprises a mixture of 1.8ml of absolute ethanol and 0.1ml of sulfuric acid.
    [0181]
    [0182] In this preferred embodiment of the invention, the detection kit comprises a first containment container and 25 second containment containers, so that it is valid to perform 25 detection tests. In other embodiments, the kit may contain different quantities, valid for a different number of detection tests.
    [0183]
    [0184] The kit further comprises a sprayer for mixing the first and second display solutions and a lactose pattern formed by a mixture of distilled water, lactose in a proportion greater than 15 ppm and a preservative. In this preferred embodiment of the invention a lactose standard with a concentration of 100ppm thereof is considered.
    [0185]
    [0186] In this preferred embodiment of the invention, the lactose standard preservative is formed by benzoic acid.
    [0187]
    [0188] This report also proposes a lactose detection procedure, in surfaces, water and food, by applying a detection kit as previously defined.
    [0189] This procedure presents a first phase consisting of obtaining and homogenizing (1) a sample.
    [0190]
    [0191] In this preferred embodiment of the invention, in which it has been considered that the detection of lactose is carried out on a food, said obtaining and homogenization phase (1) of the sample comprises a first stage of removal or crushing (2) of food.
    [0192]
    [0193] Likewise, said first phase comprises a second stage of introduction (3) of a portion of the sample in an extraction vessel containing an extraction solution to then stir the container until homogenization of the assembly is obtained and, a third stage of filtration (4) of the previous homogenized assembly, using a filter paper. For this, the portion of the sample to be used is between 0.5 and 1g, since it is a solid food (AS). In this embodiment we take 0.5g.
    [0194]
    [0195] In another preferred embodiment of the invention, in which the product to be analyzed is a liquid (AL) or semi-liquid food, the second and third stages are the same as in the case of solid foods (AS), and the sample portion to use is 2ml.
    [0196]
    [0197] In another embodiment, in which the product to be analyzed is a surface (S), the sampling and homogenization phase (1) of the sample comprises a first stage of taking a swab (5.1), moistened (5.2) of the same with extraction and rubbing solution (5.3) of an area of the surface to be tested equivalent to 5cm2 and, a second and third stages equal to the previous cases, the sample portion being used to use one end of the swab.
    [0198]
    [0199] In another preferred embodiment, in which the product to be analyzed is cleaning water (A), the phase of obtaining and homogenizing (1) of the sample comprises a first stage of taking a direct amount (6) thereof and a second and third stages equal to the previous cases, the sample portion being 2ml.
    [0200]
    [0201] The second phase consists of the introduction (7) of the separation solution (17) into the separation chamber (18) and airtight closure thereof for a time between 15 and 30min. In this preferred embodiment of the invention, a time of 20min is maintained.
    [0202] Then, a third phase of placement (8) of an amount of 5 ^ 1 of the result of obtaining and homogenizing the sample is carried out, in a separation strip (19), in the first mark (22) indicative of the place of placing the sample, followed by a fourth phase of placement (9) of an amount of 5 ^ l of the lactose pattern, on the same separation strip (19), on the second mark (23) indicative of the place of placement of the lactose pattern.
    [0203]
    [0204] The fifth phase consists of a first drying (10) of the separation strip (19) at a temperature between 90 and 115 °, for a time between 30s and 10min. In this preferred embodiment of the invention, drying is carried out by means of a heater at a temperature of 100 ° C, for 45s.
    [0205]
    [0206] And, once the separation strip (19) is dried with the samples, the sixth phase of introduction (11) of the same is carried out inside the separation chamber (18) and closed of the chamber again, during a Time between 20 and 30 min. The separation strip (19) must be positioned as shown in Figures 3.1 and 3.2, with an angle of inclination and oriented such that the first side (19.1) is in a lower position with respect to the second side (19.2). In this preferred embodiment of the invention, the separation strip is kept inside the separation chamber for a time of 26min.
    [0207]
    [0208] In this phase, the rise of the separation solution (17) by the separation strip (19) takes place, until the second line (21) of the end of the rise of the separation solution (17).
    [0209]
    [0210] Next, the seventh phase consists of a second drying (12) at a temperature between 90 and 115 °, for a time between 30s and 10min. In this preferred embodiment of the invention, drying is carried out by means of a heater at a temperature of 100 ° C, for 6min.
    [0211]
    [0212] With the separation strip (19) already dry, an eighth phase of introduction (13) of the total of the second display solution contained in the second containment container, in a sprayer and then introduction of 0.1 ml of the total is carried out of the first visualization solution contained in the first containment container, over the previous one in the same sprayer and, stirred of the mixture.
    [0213] In this preferred embodiment of the invention, the amount of the second display solution contained in the second containment vessel has been indicated to be a mixture of 1.8 ml of absolute ethanol and 0.1 ml of sulfuric acid. This amount contributed is that necessary for the performance of a test. Also, to this second solution, once it is introduced into the sprayer, 0.1 ml of P-anisaldehyde is added, which is taken with a pipette of the total of the first visualization solution contained in the first container.
    [0214]
    [0215] With this mixture of visualization solutions, the ninth spray phase (14) is carried out uniformly of the separation strip (19) with the mixture of the first and the second visualization solutions.
    [0216]
    [0217] The separation strip (19) is sprayed until it is soaked and an eleventh development phase (15) of the separation strip is carried out for a time between 5 and 15 min, at a temperature between 100 and 120 °. In this preferred embodiment of the invention, the development takes place for 10min, at a temperature of 110 ° C.
    [0218]
    [0219] Since the sample and lactose pattern is always deposited on the separation strip (19), the lactose spot (24) that appears on the separation strip (19) as a result of the lactose pattern will show the height at that a similar band (25) should appear for the sample, in the case that lactose is present in a proportion equal to or greater than the limit of the legislation, in this case 100ppm.
    [0220]
    [0221] The last phase is the interpretation (16) of the results.
    [0222]
    [0223] Thus, it may happen that the result obtained from a sample is the one shown in Figure 2.1. In this case, as can be seen, in said Figure 2.1, in the vertical of the sample a band (25) appears at the same height as the lactose spot (24) corresponding to the lactose pattern, whereby the result obtained is positive and it can be affirmed that the sample contains a concentration equal to or greater than 100ppm of lactose.
    [0224]
    [0225] The rest of the spots (26) that appear on the vertical corresponding to the sample, represent the different carbohydrates of the analyzed sample.
    [0226]
    [0227] It may also happen that the result obtained is that shown in Figure 2.2, in which case, it is observed that in the vertical of the sample no band appears at the same height as the lactose spot (24) corresponding to the lactose pattern, so it can be stated that there is no lactose concentration in the sample that reaches 100ppm.
    [0228]
    [0229] Likewise, two other cases represented in Figures 2.3 and 2.4 may occur. In both Figures it is observed that there is an absence of spots in the vertical of the lactose pattern, which in Figure 2.4 also adds to an absence of spots also in the vertical of the sample. In both cases the result is not valid, as it means that the test has not been developed properly and therefore this result must be invalidated.
    [0230]
    [0231] The described embodiment constitutes only an example of the present invention, therefore, the specific details, terms and phrases used herein are not to be considered as limiting, but are only to be understood as a basis for the claims and as a representative basis that provides an understandable description as well as sufficient information to the person skilled in the art to apply the present invention.
    权利要求:
    Claims (1)
    [0001]
    1- Lactose detection kit, on surfaces, water and food, on a sample obtained from them, characterized in that it comprises
    - a solution for the extraction of lactose from the sample, wherein said extraction solution is contained in an extraction vessel and has an amount of lactose between 0 and 50 mg / l;
    - a separation solution (17) and a hermetic separation chamber (18) for placing it, where the separation solution (17) is formed by a mixture containing at least one solvent suitable for the separation of sugars; - a separation strip (19) of the lactose with respect to other soluble compounds in the sample, by chromatography;
    - a first containment container with a first visualization solution and at least a second containment container with a second visualization solution, where the first solution is formed by at least P-anisaldehyde and the second solution is formed by a mixture of acid sulfuric and absolute ethanol;
    - a sprayer for mixing the first and second display solutions, and;
    - a lactose pattern formed by a mixture of distilled water, lactose in a proportion of greater than 15ppm and a preservative.
    2- Lactose detection kit according to claim 1, characterized in that the extraction solution further comprises distilled water, a preservative formed by benzoic acid and a deproteinization reagent formed by zinc acetate, sodium tungstate, sulfuric acid, acid trichloroacetic acid, perchloric acid, ether or acetone.
    3- Lactose detection kit according to any of the preceding claims, characterized in that the at least one solvent of the separation solution (17) is formed by ethyl acetate, isopropanol, N-Butanol, acetone, phosphate buffer, methanol, chloroform, ammonium hydroxide, acetic acid and / or N-propanol.
    4- Lactose detection kit according to claim 3, characterized in that the separation solution (17) comprises distilled water.
    5- Lactose detection kit, according to any of the preceding claims, characterized in that the separating strip (19) of the lactose has a rectangular shape with opposite first and second sides (19.1, 19.2) of shorter length, a first line (20) of placing the sample close to the first side (19.1) and parallel to it, and a second line (21) to end the rise of the separation solution (17) near the second side (19.2) and parallel to the same.
    6- Lactose detection kit, according to any of the preceding claims, characterized in that the separation strip (19) of the lactose is composed of a chromatofolium of silica gel 60, cellulose, kieselguhr, or a combination of silica gel and kieselguhr.
    7- Lactose detection kit according to claim 6, characterized in that the separation strip (19) of the lactose has an impregnation of sodium acetate, sodium borate, boric acid or sodium bisulfite.
    8- Lactose detection kit according to any of the preceding claims, characterized in that the separation strip (19) of the lactose has on the first line (20) of the sample, a first and a second mark (22) , 23) indicative of the place of placement of the sample and of the lactose pattern respectively, where said first and second indicative marks (22, 23) are separated from each other.
    9- Lactose detection kit according to any of the preceding claims, characterized in that the first containment container of the first display solution comprises an amount between 2 and 3 ml thereof and the second containment container of the second solution It comprises a mixture of 1.8ml of absolute ethanol and 0.1ml of sulfuric acid.
    10- Lactose detection kit according to any of the preceding claims, characterized in that the lactose standard preservative is formed by benzoic acid.
    11- Lactose detection procedure, in surfaces, water and food, by applying a detection kit as defined in claims 1 to 10, characterized in that it comprises the phases
    - obtaining and homogenization (1) of a sample;
    - introduction (7) of the separation solution (17) into the separation chamber (18) and airtight closure thereof for a time between 15 and 30min;
    - placing (8) an amount of 5 ^ l of the result of obtaining and homogenizing, in a separation strip (19), in the first mark (22) indicative of the first line (21);
    - placing (9) an amount of 5 ^ l of the lactose pattern, on the same separation strip (19), on the second mark (23) indicative of the second line (21);
    - first drying (10) of the separation strip (19) at a temperature between 90 and 115 °, for a time between 30s and 10min;
    - introduction (11) of the separation strip (19) into the separation chamber (18) and closed again, for a time between 20 and 30 min, where the separation strip (19) it is arranged with an angle of inclination and oriented such that the first side (19.1) is in a lower position with respect to the second side (19.2);
    - second drying (12) at a temperature between 90 and 115 °, for a time between 30s and 10min;
    - introduction (13) of the total of the second visualization solution contained in the second containment container in a sprayer and then introduction of 0.1 ml of the first visualization solution over the previous one into the same sprayer and agitation of the mixture;
    - spraying (14) uniformly of the separation strip (19) with the mixture of the first and the second display solutions;
    - developing (15) of the separation strip (19) for a time between 5 and 15 min, at a temperature between 100 and 120 °;
    - interpretation (16) of the results.
    12- Lactose detection method according to claim 11, characterized in that in the case where the product to be analyzed is formed by liquid or solid foods (AL, AS), the phase of obtaining and homogenizing (1) the sample It comprises a first stage of stirring or crushing (2) of the food.
    13. Lactose detection method according to claim 11, characterized in that in the case of surfaces (S), the phase of obtaining and homogenizing (1) of the sample comprises a first step of taking a swab (5.1), moistened (5.2) thereof with extraction solution and rubbing (5.3) of an area of the surface to be tested equivalent to 5cm2.
    14. Method for detecting lactose, according to claim 11, characterized in that in the case of water (A), the phase of obtaining and homogenizing (1) of the sample comprises a first stage of taking (6) of a direct amount from the same.
    15. Method for detecting lactose, according to any of claims 12 to 14, characterized in that the phase of obtaining and homogenizing (1) of the sample comprises a second stage of introduction and dilution (3) of a portion of the sample in an extraction vessel containing an extraction solution, and stirring the vessel until homogenization of the assembly and a third filtration stage (4) of the above homogenized assembly is obtained, by means of a filter paper, where the portion of the sample is formed by one end of the swab in the case of surfaces (S), between 0.5 and 1g in the case of solid foods (AS) or per 2ml in the case of water (A) or liquid (AL) or semi-liquid foods respectively.
    类似技术:
    公开号 | 公开日 | 专利标题
    BR112018015408A2|2018-12-18|methods and apparatus for detecting compounds in liquids
    Fu et al.2019|A PET/paper chip platform for high resolution sulphur dioxide detection in foods
    Pavlíček et al.2014|Very fast electrophoretic determination of creatinine and uric acid in human urine using a combination of two capillaries with different internal diameters
    CN102680689A|2012-09-19|Kit for detecting creatine kinase isoenzyme and preparation and use methods thereof
    Lin et al.2006|Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography
    KR910010187A|1991-06-29|Appendicitis detection method by measuring σ-hydroxy hypuric acid
    CN111896730A|2020-11-06|Dry-type immunofluorescence quantitative method Heparin Binding Protein | detection kit
    Phonchai et al.2020|A portable sol-gel urea colorimetric method for the determination of urea in feedstuffs
    CN103048471A|2013-04-17|Method for quantitatively detecting protein acetylation level
    ES2728137A1|2019-10-22|Lactose detection kit and detection procedure by applying it |
    Berry et al.1968|Detection of metabolic disorders: Chromatographic procedures and interpretation of results
    CN104502606B|2017-05-03|Application of 1-pyrenyl-carbohydrazide in specific detection of glycoprotein
    CN206177961U|2017-05-17|Chloramphenicol colloidal gold method immunity chromatography test paper card
    CN103983733A|2014-08-13|Method for detecting biogenic amine in yellow wine by using chromatographic sheet
    BR112014021862B1|2020-03-31|PROCESS AND KIT FOR DETERMINING AN ANALYTICAL TEST ELEMENT AND ITS USE
    CN109884306B|2022-02-01|Small molecule detection test strip, kit and detection method thereof
    de Souza Silva et al.2007|Sequential injection spectrophotometric determination of metoclopramide in pharmaceutical preparations
    CN108241059B|2021-04-06|Colloidal gold rapid test card and kit for detecting fumonisins, and method for detecting fumonisins
    FI57665C|1980-09-10|KUVETTENHET
    CN104569398B|2016-06-22|The enzyme linked immunological kit of detection ethoxy quinoline and application thereof
    CN103869036A|2014-06-18|Quantitative detection method for sennoside A and sennoside B in medicament
    US20180321202A1|2018-11-08|Methods and devices for detecting methanol poisoning using formate oxidase
    CN204101565U|2015-01-14|A kind of colloidal gold test paper card simultaneously detecting lincomycin and nitroimidazoles medicine
    CN202794176U|2013-03-13|Amikacin ELISA | detection kit
    El‐Sherif et al.2005|Stability‐indicating methods for the determination of lansoprazole
    同族专利:
    公开号 | 公开日
    ES2728137B2|2020-03-03|
    WO2019202193A1|2019-10-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP0323605A2|1987-12-21|1989-07-12|Abbott Laboratories|Chromatographic binding assay devices and methods|
    WO2002089946A1|2001-05-09|2002-11-14|Danisco Sweeteners Oy|Separation process by simulated moving bed chromatography|
    CN107315053A|2017-05-31|2017-11-03|南京威尔药业股份有限公司|About the EFI fog detector liquid phase chromatography analytical method of material in a kind of lactose|
    法律状态:
    2019-10-22| BA2A| Patent application published|Ref document number: 2728137 Country of ref document: ES Kind code of ref document: A1 Effective date: 20191022 |
    2020-03-03| FG2A| Definitive protection|Ref document number: 2728137 Country of ref document: ES Kind code of ref document: B2 Effective date: 20200303 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201830387A|ES2728137B2|2018-04-20|2018-04-20|Lactose detection kit and detection procedure by applying it|ES201830387A| ES2728137B2|2018-04-20|2018-04-20|Lactose detection kit and detection procedure by applying it|
    PCT/ES2019/070264| WO2019202193A1|2018-04-20|2019-04-16|Kit for detecting lactose and detection method using same|
    [返回顶部]