Extractant free of organic solvents

Abstract

The invention relates to a sterile extractant free of an organic solvent for extracting mycotoxins, antibiotics or hormones present as contaminants in feed, food or food raw materials.

Description

[0001] Extractant free of organic solvents

[0002] The invention relates to an extractant free of organic solvents for extracting mycotoxins, antibiotics or hormones present as contaminants in feed, food or food raw materials.

[0003] BACKGROUND OF THE INVENTION

[0004] The safety of feed and food ingredients is critical to ensuring the health aspects of the food chain. The presence of various mycotoxins [e.g. aflatoxins, deoxynivalenol (hereinafter: DON)], antibiotics (e.g. gentamicin, beta-lactams, tetracyclines) and certain (e.g. steroid) hormones (e.g. estrogen, progesterone) can pose a serious health risk. The extraction techniques used so far do not always provide the sensitivity and selectivity required for the measurement of these substances, which makes the accurate detection and quantification of these substances difficult.

[0005] The presence of mycotoxins, antibiotics, hormones and other environmental contaminants in the food chain poses serious health and economic risks.

[0006] Health relevance of common environmental pollutants

[0007] Mycotoxins are common, naturally occurring, persistent organic pollutants affecting both animal and human populations. Mycotoxins are generally toxic compounds produced by various molds, mainly Aspergillus, Fusarium and Penicillium species. These toxins are commonly found in cereals, oilseeds, spices and other food ingredients, as well as in animal feed. The most commonly occurring mycotoxins include aflatoxins, DON, fumonisins, ochratoxins and zearalenone.

[0008] Mycotoxins can pose serious health risks to both humans and animals. For example, some aflatoxins are potent carcinogens that may cause liver cancer, while DON may cause gastrointestinal discomfort, vomiting, and diarrhea. In animals, mycotoxins often have immunosuppressive effects, can reduce feed efficiency, cause growth disorders, and even lead to death.

[0009] Mycotoxins can be particularly dangerous for children, the elderly, and people with weakened immune systems. Targeting them is therefore critical and in our common interest.

[0010] Antibiotics occurring as contaminants in feed and food can contribute to the development and spread of antibiotic resistance, which is a global public health problem, as it can promote the emergence and spread of (multi)resistant strains, and even make the treatment of infectious diseases increasingly difficult.

[0011] Hormones, derivatives, residues: hormones, such as estrogen and progesterone, occurring as contaminants in food, can disrupt the human hormonal balance, which can lead to hormonal imbalance, homeostatic disorders and, in the long term, serious diseases.

[0012] The economic relevance of the aforementioned environmental pollutants

[0013] The presence of these contaminants in food can have serious economic consequences, including product recalls, destruction, reduced agricultural production, and restrictions on international trade. This could have a particularly serious impact on the food and agricultural industries, especially in countries where these industries account for a significant share of exports.

[0014] It is important to note that mycotoxin contamination causes significant economic losses, as contaminated crops often have to be destroyed or sold at a lower price. In addition, producers and food processors have to comply with strict regulatory requirements, which increase costs. In animal husbandry, the presence of mycotoxins often reduces production efficiency and increases veterinary costs.

[0015] Risks and the need for appropriate testing

[0016] Regular and accurate measurement of mycotoxins, antibiotics and hormones present in food, raw materials, etc. is essential for food safety. Timely, simple, practical and cost- effective detection and removal of contaminants reduces public health risks and minimizes economic losses. The development and application of effective test methods is key to maintaining the safety of the food chain and contribute to the protection of consumer health and economic stability.

[0017] Accurate detection of mycotoxins and, for example, antibiotics is essential to ensure food and feed safety. Strict limits are set by international and local regulatory bodies (e.g. WHO, EU) and compliance with them is essential to protect public health and minimize economic losses. Modern measurement techniques such as LC-MS / MS, HPLC and ELISA enable the accurate detection of mycotoxins, contributing to maintaining the safety of the food chain.

[0018] Extraction and environmental impact

[0019] Environmentally friendly extraction agents without organic solvents are increasingly important in the field of food safety. These innovative solutions enable the efficient extraction of harmful chemicals such as mycotoxins and antibiotics from food matrices without causing environmental burden. The use of traditional, often target-specific, organic solvents often involves health risks and significant environmental burden, while "green", environmentally friendly chemical processes, such as alternative and / or more generally applicable extraction agents, reduce the generation of toxic by-products and waste management problems. They contribute to the development of sustainable food safety systems that protect both human health and the environment.

[0020] For example, in a paper by Laszlo Kerekes (Elelmiszervizsgalati Kbzlemenyek 41, 1995 / 3, 215-224) a measurement system for the rapid determination of Fusarium F-2 and T-2 toxin contamination in wheat flours is described. He notes in the paper that methanol -water or acetonitrile-water solvent mixtures are usually used to extract Fusarium toxins from grain. In the latter case, the extraction is more efficient, preferably when 0.5% potassium chloride solution is added to the extraction mixture to avoid emulsion formation and 6% sulfuric acid is added to ensure a low pH. The method described in the paper uses the following extraction mixture as extraction solution: acetonitrile - 0.5% potassium chloride solution - 6% sulfuric acid (89: 10: 1).

[0021] Hungarian patent HU220703B1 describes a feed additive that can inactivate the mycotoxin content or at least a part thereof in feed. To check the effectiveness of the feed additive, the amount of residual mycotoxins in the gastric and intestinal juices of pigs was examined by high-pressure gas chromatography and thin-layer chromatography with different solvent mobile phases, for example acetonitrile in the case of zearalenone, a mixture of water: acetonitrile: acetic acid (525:450:25) in the case of ochratoxin, and a mixture of acetonitrile:water:acetic acid (47:52: 1) in the case of fumonisin.

[0022] European patent application EP4357752A1 describes a method for the extraction of mycotoxins from agricultural products. The method uses a mixture of formic acid (0.5%), acetonitrile (50-80%) and water as extraction solvent.

[0023] US Patent No. US7843566B2 discloses a device for the detection and quantification of food toxins, such as mycotoxins. The device disclosed in the document uses an 80:20 mixture of methanol and water as extraction mixture.

[0024] Chinese patent CN111707772B discloses a method for the simultaneous detection of various residual antibiotics in sewage sludge, which involves sequentially adding organic and inorganic extractants to the pretreated sludge. The organic extractant is a mixture of methanol, acetonitrile and ethyl acetate, and the inorganic extractant is a Na2EDTA- NaftPCU buffer solution with a pH of 3.0. US patent application US20210063375A1 describes a monitoring method for the detection of veterinary active agents and additives (e.g. sulfonamides, fluoroquinolones, hormones, penicillins, pesticides) in food of animal origin. According to the cited document, the method uses McIlvaine-lSfeEDTA buffer solution and acetonitrile to extract the substances to be detected. The Mcllvaine buffer is a mixture of citric acid and disodium hydrogen phosphate.

[0025] European patent application EP4285739A2 also discloses a method for the extraction of mycotoxin contaminants from food. According to the referenced document, an aqueous solution free of organic solvents is used for the extraction of mycotoxins, which comprises: about 1-15 g / 1 NaCl, about 5-20 g / 1 of Na2HPO4, about 0.1-2.0 g / 1 NaH2PO4 and about 10-150 g / 1 beta-cyclodextrin. The document does not disclose that the solution used for the extraction or any modification thereof may be suitable for the extraction of antibiotics or steroid hormones.

[0026] BRIEF DESCRIPTION OF THE FIGURE

[0027] Figure 1 : Testing of extraction from honey using a lateral flow system suitable for ampicillin measurement. Legend: AMP MEZ: honey spiked with 1 ng ampicillin; MULTI: honey spiked with antibiotics sulfadiazine (SD) and sulfamethizole (SM); MULTI MEZ: honey + Gentamycin, + Ampicillin; Control: antibiotic-free honey diluted with Saf extract. The test cassette also shows a negative result, so there are no false positives.

[0028] TECHNICAL PROBLEM TO BE SOLVED

[0029] The technical problem to be solved by the invention is to provide a sterile extraction buffer without organic solvents, which is universally suitable for the extraction of mycotoxins, antibiotics or hormones occurring as contaminants in feed, food or food raw materials for their qualitative and quantitative detection. The extraction buffer improves the detection sensitivity and accuracy of the extracted material and ensures the stability of the extracted materials.

[0030] It is important to note the environmentally friendly approach, because the solution protects both the environment and the health of laboratory workers. In addition, it also means a significant cost reduction, because the price of organic solvents and the packaging and / or shipping costs of these materials are much higher than that of the buffer described here, which can be prepared preferably at a concentration 10X higher than that used, thus reducing both packaging and shipping-licensing costs. An additional advantage may be that the use of the extractant described here may reduce the need for dedicated extractants for individual targets (it may sometimes function as a “common-universal” extractant).

[0031] THE FINDING FORMING THE BASIS OF THE INVENTION

[0032] It was found during our experiments that the addition of a non-ionic surfactant or detergent to the organic solvent-free extraction solution can facilitate the release of contaminants (so- called masked contaminants) enclosed in lipid droplets or bound to certain proteins by emulsification; and thus their detection and quantitative evaluation may be ensured, and the so-called matrix effect, which causes uncertainty and inaccuracy in terms of measurement, may be reduced.

[0033] BRIEF DESCRIPTION OF THE INVENTION

[0034] The invention provides an extractant that is free of organic solvents and comprises the following components: an alkali hydrogen phosphate, an alkali dihydrogen phosphate, an alkali chloride, a non-ionic surfactant and ultrapure or distilled water.

[0035] Preferably the extracting buffer consists essentially of or consists of the following components: an alkali hydrogen phosphate, an alkali dihydrogen phosphate, an alkali chloride, a non-ionic surfactant and ultrapure water.

[0036] Preferably the alkali hydrogen phosphate is Na2HPO4.

[0037] Preferably the alkali dihydrogen phosphate is KH2PO4.

[0038] Preferably the alkali chloride is NaCl.

[0039] Preferably the non-ionic surfactant is Triton X-100 (polyethylene glycol[4-(l, 1,3,3- tetramethylbuthyl)phenyl]ether or Tween 20 (polyoxyethylene (20) sorbitane monolaurate).

[0040] Preferably the amount of the alkali hydrogen phosphate is 0.020-0.070 mol / dm3, preferably 0.030-0.060 mol / dm3, preferably 0.035-0.045 mol / dm3, preferably 0.038-0.043 mol / dm3highly preferably 0.0405 mol / dm3.

[0041] Preferably the amount of the alkali dihydrogen phosphate is 0.005-0.01 mol / dm3, preferably 0.0060-0.0090 mol / dm3, preferably 0.0070-0.0077 mol / dm3, highly preferably 0,00735 mol / dm3.

[0042] Preferably the amount of the alkali chloride is 0.5-5 mol / dm3, preferably 1-4 mol / dm3, preferably 2-3 mol / dm3, preferably 2.4-2.7 mol / dm3, highly preferably 2.57 mol / dm3. Preferably the amount of the non-ionic surfactant is 0.1-0.5% (v / v), preferably 0.2-0.3 % (v / v), preferably 0.23-0.27 % (v / v), highly preferably 0.25% (v / v), based on the total volume of the extracting agent.

[0043] Preferably the pH of the extractant is 7-8, highly preferably 7.4.

[0044] Highly preferably the extractant consists essentially of or consists of the following:

[0045] - 0.0405 mol Na2HPO4,

[0046] - 0.00735 mol KH2PO4,

[0047] - 2.57 mol NaCl,

[0048] 0.25% (v / v) Triton X-100 (polyethylene glycol[4-(l, 1,3,3- tetramethylbuthyl)phenyl]ether), based on the total volume of the extractant,

[0049] - ultrapure water for 1000 ml total volume.

[0050] The invention relates to the use of the extractant according to the invention for the extraction of contaminants from animal and human food, such as feed and food raw materials.

[0051] Preferably, the contaminant is a mycotoxin (e.g. aflatoxins, DON), an antibiotic (preferably beta-lactams, e.g. ampicillin, preferably gentamicin, tetracyclines) or a hormone (preferably steroid hormones, preferably estrogens, e.g. estrogen, progesterone). Preferably, the contaminant is aflatoxin (Aflatoxin-Bl), DON, gentamicin, estrogen, estrol, estradiol, estriol or progesterone.

[0052] Preferably, the animal or human food is a plant-based food or a food of animal origin, preferably plant tissue or animal tissue or any mixture thereof. Preferably, the food is grain, sunflower, meat, animal tissue, honey, milk or dairy product.

[0053] The invention relates to the use of an extractant for the extraction of mycotoxins from a plant-based product, wherein the extractant consists of:

[0054] - 0.0405 mol Na2HPO4,

[0055] - 0.00735 mol KH2PO4,

[0056] - 2.57 mol NaCl,

[0057] 0.25% (v / v) Triton X-100 (polyethylene glycol[4-(l, 1,3,3- tetramethylbuthyl)phenyl]ether) based on the total volume of the extractant,

[0058] - ultrapure water for 1000 ml total volume.

[0059] Preferably, the mycotoxin is aflatoxin or deoxy nival enol.

[0060] Preferably, the plant-based product is a grain- or sunflower-based product. The invention relates to the use of an extractant for extracting an antibiotic from a product of animal origin, wherein the extractant consists of:

[0061] - 0.0405 mol Na2HPO4,

[0062] - 0.00735 mol KH2PO4,

[0063] - 2.57 mol NaCl,

[0064] - 0.25% (v / v) Triton X-100

[0065] (polyethylene glycol[4-(l,l,3,3-tetramethylbuthyl)phenyl]ether) based on the total volume of the extractant,

[0066] - ultrapure water for 1000 ml total volume.

[0067] Preferably, the animal product is meat, animal offal, dairy product or honey.

[0068] Preferably, the antibiotic is gentamicin or a beta-lactam antibiotic, preferably ampicillin.

[0069] The invention relates to the use of an extractant for extracting estrogens from a product of animal origin, wherein the extractant consists of:

[0070] - 0.0405 mol Na2HPO4,

[0071] - 0.00735 mol KH2PO4,

[0072] - 2.57 mol NaCl,

[0073] - 0.25% (v / v) Triton X-100

[0074] (polyethylene glycol[4-(l,l,3,3-tetramethylbuthyl)phenyl]ether) based on the total volume of the extractant,

[0075] - ultrapure water for 1000 ml total volume.

[0076] Preferably, the animal product is meat, animal offal or dairy product.

[0077] DETAILED DESCRIPTION OF THE INVENTION

[0078] The purpose of the invention is to develop a special, environmentally friendly extraction buffer (“Saf extract”) that is capable of, even in general use, the efficient extraction of mycotoxins, antibiotics and hormones from various food matrices, such as feed, food and food raw materials. The buffer is also intended to improve the detection sensitivity and accuracy in laboratory analyses and to ensure the structural stability of the extracted substances.

[0079] The matrix is the material in which the compound(s) to be analyzed (e.g. aflatoxins) are located. In the case of maize (Zea mays), for example, the matrix is extremely complex, containing many different components, including: • starch: the most abundant component of maize, which influences the interaction with the solvent and the extraction of the contaminant(s) to be detected, e.g. aflatoxins,

[0080] • lipids: oily components can also affect the extraction efficiency and cause interference during detection,

[0081] • proteins: some proteins are able to bind the contaminant(s) to be detected, e.g. aflatoxin(s), thus making its extraction more difficult,

[0082] • pigments: natural pigments in maize (e.g. carotenoids) can absorb and interfere with the radiation on the detector (in the case of optical, spectroscopic-based detection), which can cause a disturbing background,

[0083] • other mycotoxins: other mycotoxins may also occur in maize, which may cause interference (e.g. possible antibody cross-linking, etc.) during aflatoxin analysis,

[0084] • water: the moisture content of maize affects the extraction efficiency (inducing different “dilution”) and the stability of the contaminants to be detected, e.g. aflatoxins.

[0085] Matrix elements can affect the extraction of the contaminant(s) to be detected, e.g. aflatoxins, in several ways:

[0086] • may reduce extraction efficiency: matrix components may bind the contaminant to be detected, e.g. aflatoxin, making it more difficult to dissolve in the extraction solvent,

[0087] • may increase interference: matrix components may absorb, modify the radiation on the detector or otherwise interfere with the detection of the contaminant (e.g. by additional emission),

[0088] • may affect the stability of the contaminant(s) to be detected, e.g. aflatoxins: some matrix components may accelerate their degradation. Semi-sterility (use of sterile extraction buffer) is also an important aspect, as it helps to minimize the amount of microorganisms in the extract, which may be involved e.g. in the degradation of targets.

[0089] The extractant described herein was designed taking into account the effect of the matrix.

[0090] The detergent present in the extraction agent is able to reveal non-polar toxin and steroid targets from lipid droplets, and can improve the recovery of mycotoxins in the sample in protein-bound form, e.g. mycotoxin molecules bound to transport proteins, such as serum albumin or other globulins, from animal tissues or biological fluids.

[0091] The invention relates to an extractant which is free of organic solvents and which comprises or consists of the following components: alkali hydrogen phosphate, alkali dihydrogen phosphate, alkali chloride, nonionic surfactant and ultrapure water. The organic solvent-free extraction buffer according to the invention comprises or consists of the following components:

[0092] - alkali hydrogen phosphate in an amount of 0.038-0.043 mol / dm3,

[0093] - alkali dihydrogen phosphate in an amount of 0.0070-0.0077 mol / dm3,

[0094] - alkali chloride in an amount of 2.4-2.7 mol / dm3,

[0095] - nonionic surfactant in an amount of 0.23-0.27 % (v / v) based on the total volume of the extraction buffer, wherein the solvent is ultrapure water.

[0096] The extraction buffer according to the invention has a pH of approximately 7.3 -7.5. In one embodiment of the extractant of the invention, the nonionic surfactant is Triton X-100 (polyethylene glycol [4-(l,l,3,3-tetramethylbutyl)phenyl] ether, Merck Millipore] or Tween 20 (polyoxyethylene (20) sorbitan monolaurate, Sigma-Aldrich).

[0097] In one embodiment of the invention, the extraction agent (“Saf extract”) contains the following components:

[0098] - 0.0405 mol Na2HPO4,

[0099] - 0.00735 mol KH2PO4,

[0100] - 2.57 mol NaCl,

[0101] - 0.25% (v / v) Triton X-100 based on the total volume of the extraction agent,

[0102] - ultrapure water to a final volume of 1000 mL [e.g. MilliQ distilled water (dH2O)].

[0103] The pH of the extractant is 7.4; it is sterile filtered.

[0104] The extraction buffer (“SafeXtract”) enables the efficient extraction of targeted contaminants, namely mycotoxins, antibiotics or hormones, from various matrices, reducing the amount of interfering substances (e.g. oil or fat phase or certain proteins) and thus improving the accuracy and reliability of detection.

[0105] The extraction buffer of the invention can be prepared by a process comprising the following steps: a) weigh out the alkali hydrogen phosphate and alkali dihydrogen phosphate and mix them; b) add the weighed alkali chloride to the resulting mixture; c) the weighed nonionic surfactant is mixed with a first portion of the previously determined amount of ultrapure water; d) the solid mixture obtained in step b) is dissolved in the solution obtained in step c); e) adding a second portion of ultrapure water to adjust the predetermined pH using a pH meter; f) filtering the resulting solution to ensure microbiological purity; g) diluting the solution obtained in point f) if necessary, for example 10-fold.

[0106] The buffer thus produced ("SafeXtract") is suitable for the efficient extraction of various mycotoxins (e.g. aflatoxins, DON), antibiotics (e.g. gentamicin, beta-lactams, tetracyclines) and hormones (e.g. estrogen, progesterone) from feed and food ingredients. The advantageous effects of the extraction buffer according to the invention are: a) efficient extraction: the composition of the extraction buffer ensures a high level of extraction of the targeted contaminants; b) stability and sensitivity: the use of the extraction buffer minimizes matrix effects, thereby improving the accuracy and sensitivity of analytical tests; c) broad applicability: the extraction buffer can be used in various food matrices, including feed and other food ingredients; d) it is of less burden to the environment and cost-effective.

[0107] EXAMPLES

[0108] Example 1 : Preparation of the extraction buffer (SafeXtract)Na2HPO4 (0.0405 mol, 5.75 g) and KH2PO4 (0.00735 mol, 1 g) were measured and mixed together. NaCl (2.57 mol, 150 g) was added to the mixture. 2.5 ml (0.25 % (v / v) calculated for a final volume of 1000 ml) Triton X-100 was dissolved in 900 ml MilliQ distilled water, and the solid mixture obtained previously was dissolved in the resulting solution. After dissolution, the resulting solution was diluted to 1000 ml with MilliQ distilled water. If necessary, pH = 7.4 was adjusted using a pH meter by adding a small amount of acid or base. The resulting solution was then sterile filtered in a Class II Type Biosafety Cabinet as follows. The resulting solution was passed through a 0.22 pm PES filter system (SteriCup, SteriTop) under vacuum control into a sterile bottle, and the bottle was then closed with a sterile cap. Optionally, the sterilized solution can be distributed into additional, smaller sterile containers in a booth, for example, using a serological pipettor and sterile tips.

[0109] In the studies presented in the following examples, the extraction buffer prepared in this example was used as the extraction buffer.

[0110] Example 2: Mycotoxin measurement from com matrix Extraction (recovery) was performed on corn (maize; Zea mays; seed) samples using the extraction buffer (SafeXtract) and a mixture of acetonitrile (ACN) and distilled water as a reference for comparison. A predetermined amount of aflatoxin Bl was added to the sample and its recovery was measured.

[0111] 1) Reference measurement: extractant: 84% ACN and 16% dELO; extraction: 5 g corn sample + 25 ml extractant, shaking for 15 minutes, extract filtered with a Whatman filter. The extract was diluted 10X with IX PBS (100 pl extract + 900 pl PBS).

[0112] 2) Measurement with the extraction buffer (SafeXtract): extraction: 5 g com sample + 25 ml IX SafeXtract, shaking for 15 minutes, filtering the extract with a Whatman filter.3.) Measurement of mycotoxins from the extracts (points 1 and 2) using the Toxi -Watch Total Aflatoxin ELISA Kit, according to the manufacturer's protocol. The results obtained are shown in Tables 1 and 2 below.

[0113] Table 1 : Results of measurements using the reference extractant (acetonitrile-water mixture) from maize

[0114] SD: standard deviation; SEM: standard error of the mean, ppb: parts per billion

[0115] Table 2: Results of the measurements using the extracting agent (SafeXtract) from maize

[0116] SD: standard deviation; SEM: standard error of the mean, ppb: parts per billion Conclusion: There was no significant difference between the two extractants; two-sample t-test, equal variance p=0.112.

[0117] Example 3 : Mycotoxin measurement from sunflower matrix Extraction (recovery) was performed on sunflower (Helianthus annuus; seed) samples using the extraction buffer (SafeXtract) and a reference extractant (acetonitrile (ACN) and distilled water) for comparison. A predetermined amount of aflatoxin Bl was added to the sample and its recovery was measured.

[0118] 1) Reference measurement: extractant: 84% ACN and 16% dELO, extraction: 5 g sunflower sample + 25 ml extractant, shaking for 15 minutes, extract filtration with a Whatman filter. The extract was diluted 10X with IX PBS (100 p extract + 900 pl PBS).

[0119] 2) Measurement with the extraction buffer of the invention (SafeXtract): extraction: 5 g sunflower sample + 25 ml IX SafeXtract, shaking for 15 minutes, filtering the extract with a Whatman filter.3.) Measurement of mycotoxins from the extracts (points 1 and 2) using the Toxi-Watch Total Aflatoxin ELISA Kit, according to the manufacturer's protocol.

[0120] Table 3: Results of measurements using the reference extractant (acetonitrile-water mixture) from sunflower

[0121] SD: standard deviation; SEM: standard error of the mean, ppb: parts per billion

[0122] Table 4: Results of the measurements using the extracting agent (SafeXtract) from sunflower

[0123] SD: standard deviation; SEM: standard error of the mean, ppb: parts per billion Conclusion: in the case of sunflower with high oil content, the extraction buffer improved the recovery, two-sample t-test p=0.004. Example 4: Testing the extraction buffer (SafeXtract) according to the invention on a water-soluble mycotoxin, DON

[0124] This example describes the detection of known levels of DON contaminants in a mixture of wheat (Triticum) and maize (Zea mays).

[0125] During extraction buffer testing, the mycotoxin was detected using the Toxi-Watch DON ELISA kit.

[0126] Extraction: 5 g ground grain (wheat and maize; seed) + 25 ml SafeXtract buffer 15 min shaking and filtration with a Whatman filter.

[0127] Mycotoxin measurement from extracts using Toxi-Watch DON ELISA Kit, according to the manufacturer's protocol.

[0128] The measurement results are summarized in Table 5.

[0129] Table 5

[0130] : Samples with known DON content

[0131] Example 5: Extraction from muscle, liver and fat tissue from fallow deer Biopsy samples were taken from the animals according to standards and the samples were stored properly until processing. A predetermined amount of aflatoxin Bl was added to the sample and its recovery was measured.

[0132] Tissue homogenized by a standard blender / thurax device was extracted with 2 ml of SafeXtract buffer on 1 g of sample, followed by macrobead digestion (MP Biomedicals, Fast prep 24., 20,000 rpm 2X20 seconds), followed by 15 min of shaking and centrifugation (7000 g for 15 min at + 4 °C). ELISA measurements were performed on the extracts. The results are shown in Table 6 below. Table 6

[0133] Example 6: Extraction of gentamicin and other antibiotics from various animal food samples (meat, milk, honey) and detection using immunoanalytical methods.

[0134] Example 6.1 : Honey measurements

[0135] A predetermined amount of gentamicin sulfate was added to the sample and its recovery was measured.

[0136] 1 g of a sample of Hungarian acacia honey, confirmed to be antibiotic-negative by HPLC measurement method, purchased on the market, was shaken with 2 ml of the extraction buffer according to the invention for 20 minutes, while checking for complete dissolution. Following the extraction, ELISA measurements were performed.

[0137] The measurement results are summarized in Table 7 below. Table 7

[0138] Example 6.2: Milk measurements

[0139] A known amount of gentamicin sulfate was added to the sample and its recovery was measured.

[0140] 1) Extraction: 2 ml of raw, market-purchased, Hungarian, producer bovine milk sample + 2 ml of SafeXtract buffer, then shaking for 15 minutes at 150 rpm.

[0141] 2) Defatting: Centrifugation of 1 ml of “milk extract” for 15 minutes at + 4°C at 10,000 rpm.

[0142] 3) 2X dilution of the fraction below the fat layer: 100 pl of defatted extract + 100 pl of 0.01M PBS buffer.

[0143] 4) Following extraction, ELISA measurements were performed.

[0144] The measurement results are shown in Table 8 below.

[0145] Table 8

[0146] Example 6.3: Additional testing for honey measurements - beta-lactam antibiotic test results with lateral flow tests

[0147] 1) Extraction: 1 g honey sample + 1 ml SafeXtract buffer, then vortex until completely dissolved.

[0148] 2) Testing of honey extract with a factory -made, immunoassay-based lateral flow system for the specific detection of beta-lactam antibiotics.

[0149] 1 ng of ampicillin (AMP) was added to HPLC-negative acacia honey.

[0150] The test setup is shown in Figure 1. Ampicillin (AMP) (beta-lactam antibiotic) sample: HPLC negative (antibiotic-free) acacia honey was spiked with 1 ng of AMP. Multi spike (MULTI) sample: 1 ng of AMP and 25 mg of each of the sulfonamide antibiotics sulfadiazine (SD) and sulfamethizole (SM) were spiked. The purpose of MULTI is to verify that other non-beta-lactam antibiotics do not interfere with the specific anti-AMP antibody extract on the test cassette.

[0151] The extraction buffer of the invention also detected the concentration of 1 ng / ml from the mixed sample containing SM, SD and Ampicillin.

[0152] Example 7: Extraction of estrogen and progesterone hormones from milk and dairy products and quantitative determination by ELISA

[0153] Example 7.1 : Testing cow's milk

[0154] We performed our study on raw, market-purchased, Hungarian, producer bovine milk samples. We added a known amount of progesterone to the sample and measured its recovery.

[0155] 1) Extraction: 2 ml milk sample + 2 ml SafeXtract buffer, then shake for 15 minutes at 150 rpm.

[0156] 2) Defatting: Centrifugation of 1 ml of “milk extract” for 15 minutes at 4°C at 10,000 rpm.

[0157] 3) 2X dilution of the fraction below the fat layer: 100 pl defatted extract + 100 pl 0.01M PBS buffer.

[0158] 4) After extraction, measurements were performed.

[0159] The measurement results are shown in Table 9 below.

[0160] Table 9 ppb: parts per billion

[0161] Example 7.2: Testing 3.5% fat yogurt

[0162] We performed our test on raw, market-purchased, Hungarian, producer yogurt samples of known fat content, produced from bovine milk.

[0163] 1) Extraction: 2 ml yogurt sample + 2 ml SafeXtract buffer, then shaking for 15 minutes at 150 rpm.

[0164] 2) Centrifuging the mixture for 15 minutes at 4°C at 10,000 rpm. 3) Further diluting the whey generated during sample preparation: 100 pl whey + 300 pl 0.01 M PBS buffer.

[0165] 4) After extraction, measurements were performed.

[0166] The measurement results are shown in Table 10 (E2 = estradiol).

[0167] Table 10

[0168] 12: estradiol, ppt: parts per trillion

[0169] INDUSTRIAL APPLICABILITY

[0170] The extraction buffer of the invention ("SafeXtract") can be generally used in various food safety tests, for the detection and measurement of mycotoxins, antibiotics and certain hormones in feed and food ingredients [e.g. meat, milk, dairy products (e.g. cheese and yoghurt), honey]. Its fields of application include laboratory immuno-analytical methods, such as lateral flow (dip stick) test, ELISA, and tests based on bacterial inhibition.

Claims

Claims1. Extracting agent, which is free of organic solvents and comprises an alkali hydrogen phosphate, an alkali dihydrogen phosphate, an alkali chloride, a non-ionic surfactant and water.

2. The extracting agent according to claim 1, consisting essentially of or consisting of an alkali hydrogen phosphate, an alkali dihydrogen phosphate, an alkali chloride, a non-ionic surfactant and water.

3. The extracting agent according to claim 1, comprising 0.020-0.070 mol / dm3alkali hydrogen phosphate, 0.005-0.01 mol / dm3alkali dihydrogen phosphate, 0.5-5 mol / dm3alkali chloride, 0.1-0.5% (v / v) non-ionic surfactant, water.

4. The extracting agent according to claim 3, consisting essentially of or consisting of 0.020-0.070 mol / dm3alkali hydrogen phosphate, 0.005-0.01 mol / dm3alkali dihydrogen phosphate, 0.5-5 mol / dm3alkali chloride, 0.1-0.5% (v / v) non-ionic surfactant, water.

5. The extracting agent according to any one of the preceding claims, wherein the non- ionic surfactant is Triton X-100 (polyethylene glycol[4-(l, 1,3,3- tetramethylbuthyl)phenyl]ether) or Tween 20 (polyoxyethylene (20) sorbitane monolaurate).

6. The extracting agent according to any one of the preceding claims, wherein the alkali hydrogen phosphate is Na2HPO4.

7. The extracting agent according to any one of the preceding claims, wherein the alkali dihydrogen phosphate is KH2PO4.

8. The extracting agent according to any one of the preceding claims, wherein the alkali chloride is NaCl.

9. The extracting agent according to any one of the preceding claims, wherein the nonionic surfactant is Tween 20.

10. The extracting agent according to any one of the preceding claims, wherein the alkali hydrogen phosphate is 0.038-0.043 mol / dm3ISfeHPCh.

11. The extracting agent according to any one of the preceding claims, wherein the alkali dihydrogen phosphate is 0.0070-0.0077 mol / dm3KH2PO4.

12. The extracting agent according to any one of the preceding claims, wherein the alkali chloride is 2.4-2.7 mol / dm3NaCl.

13. The extracting agent according to any one of the preceding claims, wherein the amount of the nonionic surfactant is 0.23-0.27% (v / v) based on the total volume of the extracting agent.

14. The extracting agent according to any one of the preceding claims, wherein the extracting agent consist essentially of or consists of- 0.0405 mol / dm3Na2HPO4,- 0.00735 mol / dm3KH2PO4,- 2.57 mol / dm3NaCl,- 0.25% (v / v) Triton X-100 based on the total volume of the extracting agent,- ultrapure or distilled water.

15. Use of the extracting agent according to any one of the preceding claims for extracting contaminants from animal feed and human food.

16. The use according to claim 15, wherein the contaminant is a mycotoxin, an antibiotic and / or a hormone.

17. The use according to claim 16, wherein the contaminant is a mycotoxin, an antibiotic and a hormone.

18. Use of an extracting agent for the extraction of mycotoxins from a plant-based product, wherein the extracting agent consists essentially of or consists of:- 0.0405 mol / dm3Na2HPO4,- 0.00735 mol / dm3KH2PO4,- 2.57 mol / dm3NaCl,- 0.25% (v / v) Triton X-100 based on the total volume of the extracting agent,- ultrapure or distilled water.

19. The use according to claim 18, wherein the mycotoxin is aflatoxin or deoxy nival enol and the plant-based product is a cereal- or sunflower seed-based product.

20. Use of an extracting agent for extracting an antibiotic from a product of animal origin, wherein the extracting agent consists essentially of or consists of- 0.0405 mol / dm3Na2HPO4,- 0.00735 mol / dm3KH2PO4,- 2.57 mol / dm3NaCl,- 0.25% (v / v) Triton X-100 based on the total volume of the extracting agent,- ultrapure or distilled water.

21. The use according to claim 20, wherein the animal product is meat, animal offal, dairy product or honey and the antibiotic is gentamicin.

22. Use of an extracting agent for the extraction of estrogens from a product of animal origin, wherein the extracting agent consists essentially of or consists of:- 0.0405 mol / dm3Na2HPO4,- 0.00735 mol / dm3KH2PO4,- 2.57 mol / dm3NaCl,- 0.25% (v / v) Triton X-100 based on the total volume of the extracting agent,- ultrapure or distilled water.

23. The use according to claim 22, wherein the animal product is preferably meat, animal offal or a dairy product.

24. The extracting agent or use according to any one of the preceding claims, wherein the extracting agent is sterile.

25. The extracting agent or use according to any one of the preceding claims, wherein the water is ultrapure water.

26. The extracting agent or use according to any one of the preceding claims, wherein the pH of the extracting agents is about 7.3-7.5.

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