WO2023224507A1 - Detection of added water in a beverage or food product - Google Patents
Detection of added water in a beverage or food product Download PDFInfo
- Publication number
- WO2023224507A1 WO2023224507A1 PCT/RS2023/000007 RS2023000007W WO2023224507A1 WO 2023224507 A1 WO2023224507 A1 WO 2023224507A1 RS 2023000007 W RS2023000007 W RS 2023000007W WO 2023224507 A1 WO2023224507 A1 WO 2023224507A1
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- WO
- WIPO (PCT)
- Prior art keywords
- water
- ethanol
- product
- relative ratio
- tested
- Prior art date
Links
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/14—Beverages
- G01N33/146—Beverages containing alcohol
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the invention belongs to the field of chemical instrumental technique, i.e. the field of testing stable isotopes in analytes from food products, and for the purposes of determining the authenticity and detection of illegal practices, the origin of raw materials used in the production of food products, determining the geographical origin of products, as well as determining the authenticity of products in a narrower sense through determining the authenticity of original brands of food products.
- the invention is related to the process of preparing ethanol in water from the tested product, which is produced as a product of fermentation of a fermentable sugar reagent in isolated water from a food product (wine, fruit juices, fruit concentrates, fruit nectars and related products, fruit products and vegetables, milk and dairy products, meat, meat products and meat products, honey, etc.) under certain and specific analytical conditions, and further measuring the relative ratio of non-exchangeable stable isotopes of hydrogen in thus prepared and isolated ethanol using apparatus for determining the isotopic composition of non-exchangeable atoms of hydrogen and deuterium in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique.
- a food product wine, fruit juices, fruit concentrates, fruit nectars and related products, fruit products and vegetables, milk and dairy products, meat, meat products and meat products, honey, etc.
- isotopic methods proved to be the best analytical tool for determining the authenticity of food products by measuring the relative ratio of stable isotopes of light organic elements carbon, hydrogen, oxygen, nitrogen and sulfur (C, H, 0 , N and S).
- C, H, 0 , N and S stable isotopes of the mentioned elements
- determination of botanical origin and origin of raw materials used in the production process of certain food products wine, strong alcoholic beverages, fruit juices and related products, fruit products and vegetables, honey, milk and milk products, meat, meat raw materials and processed meat products, etc.
- determination and protection of the geographical origin of the aforementioned categories of food products 3. Determining the authenticity of original food products and production brands (original brands), and for the purposes of product certification and protection against plagiarism, forgeries and the gray zone of traffic on the market.
- Instrumental IRMS Isotope Ratio Mass Spectrometry
- peripheral units electrospray detectors
- gas-chromatographic peripherals for the preparation of sample analytes in continuous flow
- gas-chromatographic peripherals for the preparation of sample analytes in continuous flow
- gas-chromatographic peripherals for the preparation of sample analytes in continuous flow
- gas-chromatographic separation of components in continuous flow and their further combustion or pyrolysis
- liquid-chromatographic separation of components and their further conversion to elemental gases by oxidation-reduction or combustion, or pyrolysis and subsequent measurement of such gases on mass spectrometer detectors to determine the relative ratio of stable isotopes of light organic elements - IRMS.
- the detection of water was determined by measuring stable oxygen isotopes in product water using the equilibration technique, i.e. of a specific peripheral for sample equilibration, which is connected in continuous flow to the IRMS instrument.
- the current concept used for, for example, determining the authenticity of wine in Europe and in the world is based on the formation of extensive isotopic databases that, among other things, contain results for the values of the relative ratio of stable oxygen isotopes ( 6 18 O in wine water), and for the purposes of comparing the tested samples with the isotopic values of the samples that are stored in the reference databases.
- the work on these activities, the creation of isotopic bases is strictly and exclusively entrusted to national reference laboratories in the member states of the European Union and represent national secrets that are not shared with other EU member states precisely because there is no other way of determining the addition of water to wine.
- the values of S 18 O in wine water range from only 10 to 12 %o in relation to the international standard V-SMOW. This range is not sufficient to determine with certainty the addition of water in the wine production process, which represents an illegal practice, because with a small addition of water, these 6 18 O values move very little, and for these reasons it is necessary to compare the suspected of a sample with a known declaration, with a reference sample of the same wine from the database. Finally, if the 3 18 O value does not match, it does not necessarily mean that the wine was additionally diluted by the addition of water, but it could also be that another wine with a different 6 18 O value in the wine water was poured into the original bottle.
- This method is currently the only method used in practice, but it also means that there is currently no other method by which it is possible to quantitatively determine the addition of water in the final wine, fruit juices, to detect with certainty the quantitative addition of water to milk, or to detect the difference between fresh or reconstituted milk produced from milk powder with added water.
- isotopic databases are formed on an annual basis for all wines with indicated origin, which are the responsibility of the National Reference Laboratories of the EU member states.
- the central laboratory and database is located in Belgium in the town of Geel, where information is deposited for all European wines with designated geographical origin.
- the entire procedure is based on taking reference and representative samples of grapes from vineyards with a designated geographical origin, and then in the laboratory wine is made from those grape samples, which is analyzed for stable isotopes using IRMS and SNIF-NMR instrumental techniques, and then the results of those analyzes are sent to Geel (Belgium) where they are deposited in a central database. If a wine is suspected somewhere on the European market, further analysis and comparison with the reference sample for that wine provides information on its authenticity.
- milk can be classified into fresh milk and reconstituted milk produced from milk powder with added water. This procedure is carried out in industrial duplicators with mixers and dosers where all components (powdered milk, powdered whey, water) are added and mixed to a homogeneous consistency.
- the representation of one such court - duplicator is given in picture no. 3.
- the problem of milk authenticity has enormous consequences for the living standards of citizens from two aspects.
- the first aspect is related to the health safety of milk as a food product, and the second is related to problems in the relationship between cattle farmers (raw milk producers) and dairy farmers (milk processors).
- the problem related to determining the addition of water to fruit juices is related to the use of adequate raw materials in the production of fruit juices, and thus the correct declaration of such products.
- citrus fruits mainly citrus fruits (oranges, tangerines, grapefruit, passion fruit, lime, lemon, etc.) are grown in tropical or subtropical areas. Due to the long distance and time required to transport juices from these regions to Europe or North America, juices from these types of fruit are concentrated in order to preserve them during the journey and prevent them from spoiling or starting fermentation during transport.
- the raw material arrives at the destination where it will be used in the production of fruit juices, it is reconstituted with the repeated addition of water and the final product is reconstituted fruit juice made from fruit concentrate.
- fresh fruit juice is obtained by squeezing fresh fruit. According to the regulations on quality, it is forbidden to dilute fresh fruit juice with water, if such a product is declared as freshly squeezed fruit juice. Therefore, the price of such juices is higher compared to reconstituted fruit juices.
- Fraudsters and unfair competition very often declare reconstituted fruit juices as fresh fruit juices and thus commit economic fraud and mislead end consumers that they bought a higher quality product for their money for which they paid a higher price, and in return received a lower quality product, quality.
- aqueous solutions which may contain aromas, collagen and other binders
- the first closest state of the art for detecting the addition of water to food products is the official method for determining the relative ratio of stable oxygen isotopes in the water of the tested product sample.
- DI COMPENDIUM OF INTERNATIONAL METHODS OF ANALYSIS - O1V, Method for 18 O/ 16 O isotope ratio determination of water in wines and must, (Resolution OlV-Oeno 353/2009) - Method OIV-MA-AS2-12
- the document is an official method of the OIV that describes the principle, the area of application, as well as the work procedure and the method of determining the relative ratio of stable oxygen isotopes in wine water ( 5 18 O value in wine water).
- the second closest state of the art for conditionally determining the detection of water added to wine is the SNIF-NMR method (Site Natural Isotopic Fractionation - Nuclear Magnetic Resonance).
- D2 COMPENDIUM OF INTERNATIONAL ANALYSIS OF METHODS - OIV, Determination of the deuterium distribution in ethanol derived from fermentation of grape musts, concentrated grape musts, grape sugar ( rectified concentrated grape musts) and wines by application of nuclear magnetic resonance (SNIF-NMR/ RMN-FINS), (Oeno 426-2011) - Method OIV-MA-AS311-05.
- the document is an official method of the OIV that describes the principle, the area of application, as well as the procedure for working and determining the relative ratio of deuterium and hydrogen on the methyl and methylene group of ethanol ((D/H), and (D/H),, ).
- RS 52615 B describes the procedure for measuring non-exchangeable stable hydrogen isotopes in ethanol samples for the purposes of determining illegal practices of adding sugar and/or water to grape must before alcoholic fermentation.
- D3 Patent file, 52615 B, Apparatus for determining the isotopic composition of non-exchangeable stable hydrogen isotopes in ethanol samples and procedure for determining the authenticity and geographical origin of the product, Institute for Intellectual Property of the Republic of Serbia.
- Patent 52615 B deals with a technical solution and procedure for determining non-exchangeable stable hydrogen isotopes in ethanol samples from food products that already contain ethanol and/or fermentable sugars.
- the principle of the invention according to RS 52615 B is based on the isolation of already existing ethanol, which was created by stirring the sugars present in the examined food product, and then the dehydration of the thus separated ethanol and the creation of ethylene gas, further pyrolysis of ethylene to elementary hydrogen gas (H 2 ) and carbon , chromatographic separation, and measurement of the relative ratio of deuterium and hydrogen in the thus prepared hydrogen gas.
- This invention overcomes the existing barriers in the lack of technical analytical solutions known so far, and for easier detection of added water and determination of the origin of water in food products that contain water (wine, strong alcoholic beverages, fruit juices, fruit concentrates, coconut water, milk and dairy products, meat, meat raw materials, meat products and products, etc.).
- the principle of the invention is based on the fact that ethanol after alcoholic fermentation contains two types of information. Based on previously published scientific works on the topic of determining the origin of ethanol, as well as on the basis of the biochemical mechanism of ethanol formation during the glycolysis process, it was previously determined using Nuclear Magnetic Resonance that the deuterium atoms in the final ethanol, after the alcoholic fermentation is completed, will be found in the following positions:
- deuterium is found on the methyl group, then in the same ethanol molecule it will not be present on the methylene group and vice versa.
- the deuterium found on the hydroxyl group of ethanol is always exchangeable, which has the effect that ethanol as an organic compound is hygroscopic and binds water by forming hydrogen bridges.
- hydroxyl hydrogen and/or deuterium in the matrix in which ethanol is located By exchanging hydroxyl hydrogen and/or deuterium in the matrix in which ethanol is located, its overall ratio of deuterium and hydrogen atoms is constantly changing, i.e. it is always in dynamic balance with its environment.
- the non-exchangeable deuterium and hydrogen atoms that are tightly bound to the carbon atoms on the methyl and methylene group can provide information under which conditions and from which raw materials that ethanol was created.
- alcoholic fermentation when due to the activity of the enzyme acetaldehyde-dehydrogenase in the presence of the oxido-reducing coenzyme NADH + H + , a pair of protons is transferred from the surrounding medium (water), where that pair of protons binds to the aldehyde group of acetaldehyde and thus creates an ethanol molecule as the final a product of alcoholic fermentation.
- the biochemical mechanism of alcoholic fermentation is shown in Figure 6.
- the principle is based on the preparation procedure and of making alcohol (ethanol) by taking water from the test sample, adding a fermentable sugar reagent and yeast and starting the alcoholic fermentation. It is necessary that the alcoholic fermentation of the sample be performed in anaerobic conditions without the presence of air, in order to secure the sample from potential losses of ethanol and eliminate the possibility of starting secondary aerobic fermentations, such as vinegar fermentation, etc.
- the basic and very important item is to always use the same fermentable sugar reagent, because in this way it is ensured that the contribution that would be made on the methyl (CH 3 -) group of ethanol formed in the water matrix, which represents the isolated water from the tested product, is blocked.
- the newly formed ethanol as a product of alcoholic fermentation if exo-water is added to the product, due to the change in the isotopic profile of the examined medium, that information is integrated on the methylene group of the newly formed ethanol, and then by isolating the ethanol produced under such specific conditions, its quantitative extraction from the overboiled medium and by determining the relative ratio of non-exchangeable stable isotopes of hydrogen using the apparatus for determining the isotopic composition of non- exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the IRMS instrumental technique, information is obtained on the origin of the water contained in the examined product.
- the second aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dn n value of ethanol from product water), and for the purpose of detecting added water in food products, if the product containing water, also also contains ethanol and/or fermentable sugars, in that case it is necessary to quantitatively separate water from ethanol and/or sugar in such a way that isotopic fractionation of water does not occur, which is the starting matrix for the preparation of ethanol for testing the origin of water in the tested product.
- the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( SDn,, value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product is wine containing alcohol (ethanol), it is necessary to perform a quantitative separation of alcohol first so that it does not affect and contaminate the preparatory matrix and thus affect the final test results, because the water must be sufficiently freed and without the presence of ethanol or fermentable sugars before the addition of the fermentable sugar reagent and yeast .
- the fermentable sugar reagent which is added to the aqueous matrix during alcoholic fermentation and is quantitatively converted into alcohol (ethanol) in order to obtain complete information from the examined matrix.
- the sixth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dn lt value of ethanol from product water), and for the purpose of detecting added water in food products, after the completed bioconversion of the fermentable sugar reagent in alcohol (ethanol), the quantitative separation of that alcohol (ethanol) from the aqueous matrix is carried out in the same way as previously described according to aspect four and aspect five of this invention.
- the seventh aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dn,i value of ethanol from product water), and for the purpose of detecting added water in food products, further determination of the relative ratio of non- exchangeable stable isotopes of hydrogen in isolated ethanol from the examined water matrix using the apparatus for determining the isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique.
- IRMS Isotope Ratio Mass Spectrometry
- the eighth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dn u value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product sample containing water contains fermentable sugars in addition to ethanol (for example semi-dry, semi-sweet or sweet wine, or any other product containing ethanol and fermentable sugars or only fermentable sugars) after the quantitative separation of ethanol, as described in aspects four and five of this section, it is necessary to quantitatively separate the water from the sugars present in the water matrix.
- fermentable sugars for example semi-dry, semi-sweet or sweet wine, or any other product containing ethanol and fermentable sugars or only fermentable sugars
- the ninth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( SDn value of ethanol from product water), and for the purpose of detecting added water in food products, quantitative separation of water from fermentable sugars is possible perform azeotropic distillation by forming an azeotropic mixture with toluene (or any other organic solvent with which water forms an azeotropic mixture) until complete quantitative separation of water from sugar.
- the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnn value of ethanol from product water), and in order to detect added water in food products, it is even possible to perform secondary alcoholic fermentation in order to convert the remaining fermentable sugar into ethanol, and after the completion of the alcoholic fermentation, quantitatively separate the alcohol (ethanol) again in the manner previously described in aspects four and five of this section, and then use the separated water further in the preparation as it is described in aspects six to eight of this section.
- the eleventh aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 5Dn u value of ethanol from product water), and for the purpose of detecting added water in food products if the examined product is milk or milk product, it is possible to add the fermentable sugar reagent directly to the tested product together with the yeast, without first separating the water from the other components of the milk, since milk does not contain fermentable sugars that turn into alcohol, as well as alcohol (ethanol).
- the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dn,i value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product is milk or milk product, for a better and easier performance of the ethanol in water preparation of the tested product sample, it is possible to coagulation milk proteins using rennet or another coagulating agent for easier separation of milk water from other milk components in milk.
- a suitable coagulating agent that does not contain hydrogen atoms in its composition that can end up in the water matrix and thus disrupt the isotopic profile of the water being prepared for testing.
- under fermentable sugar reagent can mean all monosaccharides or disaccharides that turn into alcohol (ethanol) during alcoholic fermentation, such as glucose, fructose, sucrose, etc., but it is also understood that using higher sugars (disaccharides, trisaccharides, as well as oligosaccharides) with the addition of hydrolase enzymes (amylase) or acids that would hydrolyze such sugars into monosaccharides which would then be fermentable and at the end of
- the fourteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( SDn,, value of ethanol from product water), and for the purpose of detecting added water in food products, for the preparation of ethanol in the water matrix of the tested of the product, it is also possible to do the conversion enzymatically by using the appropriate decarboxylase, dehydrogenase and coenzymes, thus simulating the steps of the biochemical conversion process and the creation of ethanol.
- pyruvic acid as a starting reagent, and instead of a fermentable sugar reagent and yeast, and by making a mixture with the enzymes pyruvatedecarboxylase and acetaldehyde-dehydrogenase and the oxido-reducing coenzyme nicotinamide adenine dinucleotide (NADH + H + ) with the aqueous matrix of the tested product, it is possible is to simulate the last two steps of alcoholic fermentation and produce ethanol that will contain information about the origin of water, as previously described and shown in Figure No. 6.
- the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dn value of ethanol from product water), and for the purpose of detecting added water in food products
- the procedure for the preparation of ethanol in water of the tested product applicable to all food products that contain water and in which it is possible to determine the origin of water, such as wine, fruit juices, fruit nectars, fruit concentrates, fruit pulps, fruit purees, refreshing soft drinks with and without fruit juice, food for babies and infants, strong alcoholic drinks, honey, coconut water, maple syrup, milk and milk products, meat and meat raw materials from beef, chicken, pork, lamb, veal, etc., as well as their offal (livers, kidneys, heart, stomach, etc.), meat products and meat products, fruit and vegetable products, spring and mineral waters, functional food products, vinegar (balsamic, wine, apple, alcoholic, diluted acetic acid
- the sixteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8 Dn value of ethanol from product water), and for the purpose of detecting added water in food products, for the purpose of better determining the authenticity of the examined product sample, it implies and bringing the obtained values of the relative ratio of non- exchangeable stable hydrogen isotopes in ethanol prepared in the water of the tested product sample ( 8Dn [( value of ethanol from food product water) into correlation with other values of isotopic analytical parameters, which are obtained by other already known isotopic techniques ( 8 13 C values of analytes in the tested sample, 8 1S O values of analytes in the tested sample, values of the relative ratio of non- exchangeable stable hydrogen isotopes in ethanol ( 8Dn, value of ethanol from the product) according to RS 52615 B, 8 15 N values in the tested sample and 8 32 S values in the examined sample).
- the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (6Dnn value of ethanol from product water), and for the purpose of detecting added water in food products is applicable for the following group of products, which includes: wine, fruit juices, fruit nectars, fruit concentrates, fruit pulps, fruit purees, refreshing soft drinks with and without fruit juice, food for babies and infants, strong alcoholic drinks, honey, coconut water, maple syrup, milk and milk products, meat and meat raw materials originating from beef, chicken, pork, lamb, veal, etc., as well as their offal (livers, kidneys, heart, stomach, etc.), meat products and meat products, fruit and vegetable products, and mineral waters, functional food products, vinegar (balsamic, wine, apple, alcoholic, diluted acetic acid, etc.), fish and fish products, as well as all related products and other food products containing water and any combinations thereof.
- the first step of the preparation procedure is based on the separation of water from the sample of the tested product from all other components that are present in the sample of the product, which can influence the results of the analysis by their presence. If alcohol (ethanol) and other easily volatile components are present in the tested product, they must be separated by distillation so that there is no separation and loss of water or its isotopic fractionation, and that ethanol does not remain in the water matrix.
- Separation of water from the tested product sample from ethanol and other easily volatile components that may be present in the tested product can be performed by liquid-liquid extraction, azeotropic distillation, as well as by any other physical and/or physico-chemical method or process or their combination.
- the separation of water from the mentioned components in the sample of the tested product is carried out by azeotropic distillation at atmospheric pressure or under reduced pressure (in a vacuum) with toluene or any other organic solvent that it does not dissolve in water, and at the same time forms an azeotropic mixture with water in the appropriate ratio and does not have exchangeable hydrogen atoms that could be exchanged with hydrogen atoms from the water of the product.
- This step of separating water from other components in the sample of the tested product can be done by liquid-liquid extraction, lyophilization with condensation and collection of the separated water, as well as by other physical or physico-chemical procedures and/or techniques and their combinations.
- the separation of water from other components present in the tested product can be done using coagulating and/or denaturing agents such as rennet or using acids, as well as microbiological activities naturally of microorganisms present in the tested product (for example raw milk) or by the addition of microorganisms to the sample of the tested milk or milk product in order to make the phase separation of the water-other component faster.
- product samples that are in a solid state such as meat or meat raw materials, fish and related and similar products, such product samples must first be crushed and homogenized as finely as possible, and then proceed with the extraction of water from the product sample in ways which are previously mentioned in this step of the preparation procedure.
- the second step of the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (6Dn,i value of ethanol from product water), and in order to detect added water in food products, is based on the addition of a fermentable sugar reagent and yeast to water from of the tested product and preparation of the mixture for alcoholic fermentation in order to stir the sugar reagent to alcohol (ethanol).
- a fermentable sugar reagent in this step of the preparation procedure all monosaccharides or disaccharides, as well as their combinations, which turn into alcohol (ethanol) during alcoholic fermentation, such as glucose, fructose, sucrose, etc., can be used, but they can also higher sugars (disaccharides, trisaccharides and oligosaccharides), as well as their combinations, can be used, which with the addition of hydrolase enzymes (amylase) or acids would be converted and translated into fermentable sugars, and which would then at the end of the process of alcoholic fermentation or enzymatic bioconversions yielded alcohol (ethanol) and as such were suitable as a fermentable sugar reagent for the preparation of ethanol in water of the tested product.
- hydrolase enzymes as amylase
- fermentable sugar reagents can be of vegetable, animal or synthetic origin.
- Fermentable sugar reagent of plant origin means all fermentable sugars originating from grapes, fruits, industrial plants (sugar beet, sugar cane, corn, wheat, barley, rice, millet, sorghum, etc.), honey, other plants and other origins , as well as their combinations.
- the addition of pyruvic acid can be used as a starting reagent with the addition of pyruvate-decarboxylase, acetaldehyde-dehydrogenase and nicotinamide adenine dinucleotide oxidation-reducing coenzyme (NADH+H + ) and bioconversion of pyruvic acid via acetaldehyde to alcohol (ethanol).
- pyruvic acid as a starting reagent instead of a fermentable sugar reagent, pyruvic acid of the same isotopic profile of stable hydrogen isotopes is always used, so that its enzymatic bioconversion into ethanol in the product water always fixes the relative ratio of deuterium and hydrogen atoms, on the methyl (CH 3 -) group of the ethanol made in this way, i.e.
- the third step of the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (8Dn faced value of ethanol from product water), and with the aim of detecting added water in food products, is based on the separation of prepared alcohol (ethanol) from overboiled medium by distillation at atmospheric pressure or under vacuum.
- This step is not limited to the extraction of ethanol by distillation, but other physical and physicochemical procedures or techniques as well as their combination can be used.
- Quantitative separation of the prepared alcohol (ethanol) from the overheated medium can be performed using the distillation apparatus shown in Figure No. 13.
- the fourth step of preparing ethanol in product water and determining the relative ratio of non- exchangeable stable hydrogen isotopes in the ethanol thus prepared (6Dnu value of ethanol from product water), and with the aim of detecting added water in food products, is based on the measurement of the relative ratio of non-exchangeable stable hydrogen isotopes in the previously separated ethanol from the overboiled medium using the apparatus for determining the isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique, and then calculating the 6Dn u value of the prepared ethanol from the water of the tested product sample by comparing of the relative ratio of non-exchangeable stable hydrogen isotopes of ethanol from the water of the tested product sample with ethanol with the known relative ratio of non-exchangeable stable hydrogen isotopes obtained in the same way.
- IRMS Isotope Ratio Mass Spectrometry
- Figure no. 12 shows a graphic representation of the detection of water added to the final wine by measuring the relative ratio of non-exchangeable stable isotopes of hydrogen in ethanol prepared in wine water, i.e. wine from which wine ethanol has previously been separated ("alcohol free wine").
- Figure no. 14 shows a graphic representation of the results for the 8Dn u value of ethanol from milk water, with the aim of detecting the presence of reconstituted milk in fresh milk.
- Figures 17, 18, 19, 20 and 21 show graphical representations of the results obtained with this invention.
- Figure No. 15 shows the correlation of the results of SDn,, value of ethanol from product water in relation to 8 1S O value from wine water.
- Figure No. 16 shows a graphic representation of the correlation of the results of 8Dn K value of ethanol from wine water in relation to SDn, value of ethanol from wine, the values of which are obtained in accordance with the procedure for measuring the relative ratio of invariable of stable isotopes of hydrogen in ethanol samples and using the apparatus for determining the isotopic composition of non-exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the 1RMS instrumental technique.
- the procedure for preparing ethanol in product water and determining the relative ratio of non- exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dn N value of ethanol from product water), and for the purpose of detecting added water in food products, is used in chemical instrumental analysis for determining authenticity and determining the origin of water in samples of tested products, and can be used in correlation with the parameters of other isotopic species (C, O, H, N, S) and for the purposes of determining the geographical origin of products containing water.
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Abstract
For detecting added water in a beverage or food product, water is separated from a sample of said beverage or food product, sugar and yeast are added to said water, alcoholic fermentation is performed, the produced ethanol is extracted, and the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared and isolated ethanol is measured, based on IRMS (Isotope Ratio Mass Spectrometry).
Description
PROCEDURE FOR THE PREPARATION OF ETHANOL IN WATER OF THE TESTED PRODUCT AND DETERMINATION OF THE RELATIVE RATIO OF UNCHANGEABLE STABLE ISOTOPES OF HYDROGEN IN THUS PREPARED ETHANOL ( 6 Dn H VALUE OF ETHANOL FROM PRODUCT WATER), AND FOR THE PURPOSE OF DETECTION OF ADDED WATER IN FOOD PRODUCTS
Technical field to which the invention applies
The invention belongs to the field of chemical instrumental technique, i.e. the field of testing stable isotopes in analytes from food products, and for the purposes of determining the authenticity and detection of illegal practices, the origin of raw materials used in the production of food products, determining the geographical origin of products, as well as determining the authenticity of products in a narrower sense through determining the authenticity of original brands of food products. In this sense, the invention is related to the process of preparing ethanol in water from the tested product, which is produced as a product of fermentation of a fermentable sugar reagent in isolated water from a food product (wine, fruit juices, fruit concentrates, fruit nectars and related products, fruit products and vegetables, milk and dairy products, meat, meat products and meat products, honey, etc.) under certain and specific analytical conditions, and further measuring the relative ratio of non-exchangeable stable isotopes of hydrogen in thus prepared and isolated ethanol using apparatus for determining the isotopic composition of non-exchangeable atoms of hydrogen and deuterium in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique.
Technical problem
As explained in many scientific references in the previous period, isotopic methods proved to be the best analytical tool for determining the authenticity of food products by measuring the relative ratio of stable isotopes of light organic elements carbon, hydrogen, oxygen, nitrogen and sulfur (C, H, 0 , N and S). By determining the relative ratio of stable isotopes of the mentioned elements, it is possible to determine different levels of authenticity of food products through several levels: 1. determination of botanical origin and origin of raw materials used in the production process of certain food products (wine, strong alcoholic beverages, fruit juices and related products, fruit products and vegetables, honey, milk and milk products, meat, meat raw materials and processed meat products, etc.); 2. determination and protection of the geographical origin of the aforementioned categories of food products; 3. Determining the authenticity of original food products and production brands (original brands), and for the purposes of product certification and protection against plagiarism, forgeries and the gray zone of traffic on the market.
In the technical sense, instrumental analytical systems are already known, which are used to determine stable isotopes in products. Instrumental IRMS (Isotope Ratio Mass Spectrometry) technique with various peripheral units (elemental analyzer for sample combustion or pyrolysis, peripheral for equilibration of liquid and gas above the sample surface, gas-chromatographic peripherals, as well as liquid-chromatographic peripherals) for the preparation of sample analytes in continuous flow, which are based on several different principles of combustion, pyrolysis, sample equilibration, gas- chromatographic separation of components in continuous flow and their further combustion or pyrolysis, as well as liquid-chromatographic separation of components and their further conversion to elemental gases by oxidation-reduction or combustion, or pyrolysis, and subsequent measurement of
such gases on mass spectrometer detectors to determine the relative ratio of stable isotopes of light organic elements - IRMS.
The practices and procedures applied in the production of food products often leave rigorous consequences on the end product in terms of changing its chemical structure and, viewed from the perspective of isotopic chemistry, can provide information that can very often be deceptive or misleading and lead the analyst into the delusion that it is an illegal practice, when in fact it may be that the use of legal practices and procedures in the production process led to changes in the product itself. For these reasons, when developing analytical methods in the field of testing the authenticity of food products, it is very important to take into account all the variable factors in the production process that can affect the final analytes, and therefore the final results of the analysis.
In the previously granted patent under the number RS 52615 B, the main technical problems faced by analysts and which were the reasons for obtaining unrepeatable and non-reproducible results using conventional instrumental techniques based on measuring the relative ratios of stable isotopes in food analytes, and especially in determining the relative of stable hydrogen isotope ratios using conventional instrumental techniques based on an IRMS instrument.
The development of a new technique based on the apparatus for determining the isotopic composition of non-exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the IRMS instrument technique, under the commercial name EIM-IRMS (Ethanol Isotope Measurement - Isotope Ratio Mass Spectrometry), which was previously described in detail in the patent under the number RS 52615 B, the foundations were laid for the further development of this branch of instrumental analysis and the foundations for the development of new procedures for determining the authenticity of food, and this patent application represents precisely that development that expands the limits of the use of the aforementioned instrumental technique.
According to world experts in this field, the problem of detecting the addition of water to food products such as wine, but not only wine, but also fruit juices and related products, milk and milk products, as well as meat and meat products, in food products in general, represents the biggest challenge for the world's scientific community for decades. Due to the problems and the lack of adequate analytical technical solutions, this problem is still unsolvable, and as such it represents a great loss for all interested parties in the world food market, both in production and in trade.
The European Commission estimated that only because of the problem of falsification of wine and alcoholic beverages, the European budget suffers damage of 3.2 billion euros annually, which reflects negatively on the legal producers of wine and alcoholic beverages in Europe, because of the problem of falsification and the gray area circulation of uncontrolled and dubious products, legal producers cannot invest further in the development of their production, and therefore cannot hire new workers. Since the food industry is a generator of the economy that entails both primary agricultural production and the tertiary economic sector, which includes trade, catering, logistics, the packaging industry, marketing, etc., therefore, everyone in the chain is affected when it happens that the gray area of production and traffic starts to increase. In this sense, we should not forget the losses from the collection of fees, excise taxes and profit taxes, as well as contributions that flow into the state budget of every country in the world, including the Republic of Serbia.
1. Detection of added water in wine:
Until now, the detection of water was determined by measuring stable oxygen isotopes in product water using the equilibration technique, i.e. of a specific peripheral for sample equilibration, which is connected in continuous flow to the IRMS instrument.
Although the problem of the instrumental technique itself for determining the relative ratio of stable oxygen isotopes 318O in the water of food products (wine, fruit juices, milk and dairy products, strong alcoholic beverages, etc.) will be discussed in the next section under "state of the art", in general, it should be noted that this analytical parameter has a very limited practical application due to the very small range of values in which it can move. For these reasons, the current concept used for, for example, determining the authenticity of wine in Europe and in the world, is based on the formation of extensive isotopic databases that, among other things, contain results for the values of the relative ratio of stable oxygen isotopes ( 618O in wine water), and for the purposes of comparing the tested samples with the isotopic values of the samples that are stored in the reference databases. The work on these activities, the creation of isotopic bases, is strictly and exclusively entrusted to national reference laboratories in the member states of the European Union and represent national secrets that are not shared with other EU member states precisely because there is no other way of determining the addition of water to wine.
A graphic example of such a database from earlier years is shown in Figure 1.
As can be seen on the ordinate from the attached graph, the values of S18O in wine water range from only 10 to 12 %o in relation to the international standard V-SMOW. This range is not sufficient to determine with certainty the addition of water in the wine production process, which represents an illegal practice, because with a small addition of water, these 618O values move very little, and for these reasons it is necessary to compare the suspected of a sample with a known declaration, with a reference sample of the same wine from the database. Finally, if the 318O value does not match, it does not necessarily mean that the wine was additionally diluted by the addition of water, but it could also be that another wine with a different 618O value in the wine water was poured into the original bottle.
This method is currently the only method used in practice, but it also means that there is currently no other method by which it is possible to quantitatively determine the addition of water in the final wine, fruit juices, to detect with certainty the quantitative addition of water to milk, or to detect the difference between fresh or reconstituted milk produced from milk powder with added water.
Furthermore, the International Organization for Wine and Vine, based in Dijon, France - OIV (International Organization for Wine and Wine) has been trying for many years to find a solution to detect added water in the final wine. Due to the increasing trend and the demands from the market that consumers are looking for products with lower, ie. reduced alcohol content, in the previous period the OIV adopted new resolutions concerning the newly accepted oenological practices for the reduction of alcohol in the final wine. This was a precedent that opened a pandora's box and created a big problem for the experts dealing with determining the authenticity of wines in the European Union.
Namely, as previously explained, in Europe, isotopic databases are formed on an annual basis for all wines with indicated origin, which are the responsibility of the National Reference Laboratories of the EU member states. The central laboratory and database is located in Belgium in the town of Geel, where information is deposited for all European wines with designated geographical origin.
The entire procedure is based on taking reference and representative samples of grapes from vineyards with a designated geographical origin, and then in the laboratory wine is made from those grape samples, which is analyzed for stable isotopes using IRMS and SNIF-NMR instrumental techniques, and then the results of those analyzes are sent to Geel (Belgium) where they are deposited in a central database. If a wine is suspected somewhere on the European market, further analysis and comparison with the reference sample for that wine provides information on its authenticity.
The problem arose at the moment when the OIV passed new resolutions that legalized the process of correcting the alcohol content in wine (OIV - Code of oenological practices 3.5.13. CORRECTION OF THE ALCOHOL CONTENT IN WINES (OENO 394B-2012)) , which state technical ways in which the reduction can be carried out. Among other things, the listed techniques include membrane techniques, including reverse osmosis, as well as vacuum distillation and other types of alcohol reduction in wine.
Namely, by using membrane techniques, which includes the use of reverse osmosis in the process of correcting the alcohol content in wine, one part of the water, the so-called "striping water", which is used as a counter current to extract alcohol from wine, due to the application of membranes and the process of diffusion in the opposite direction, ends up in wine which is actually processed for reduction, i.e. correction of the alcohol content, as shown in figure no. 2.
Since the enological practice of adding water is not allowed for wine "in stricto sensu", with the exception of technical water that is added with enological means and represents a negligible part in relation to the quantities of wine in production, the addition of water is legalized in some way by alcohol correction techniques, because during correction of alcohol results in the dilution of wine, and therefore the result of correction of alcohol is actually a combination of extracting alcohol, but also diluting the wine by diffusion of water in the opposite direction during reverse osmosis.
Due to the acceptance and legalization of such techniques in the process of wine production, another problem arose, because isotopic databases at the European level, which have been formed year after year for decades, and for the purposes of protecting wines with designated geographical origin, can no longer be used for comparison with samples from the market, because wine samples that are prepared in laboratories do not undergo the same treatment as grapes that go through the entire process and different oenological procedures in the wine production process. And that's where the problem arose.
Experts conducted comparative analyzes of such wines on isotopic analyzes and determined that due to such oenological practices, the original isotopic composition of the final wines changes, and therefore the entire concept of protection and determination of authenticity is called into question (Roberto Ferrarini et al., Variation of oxygen isotopic ratio during wine dealcoholization by membrane contactors: Experiments and modeling , Journal of Membrane Science, 498 (2016), 385-394, https://doi. ora/10.1016/i. memsci.2015.10.027 )
Also, the problem continued at the level of the European Commission, where the question of generally accepting the addition of water to the final wine as another item that will solve the problem in production, and the problem of climate change, which increasingly affects the production of grapes with a higher sugar content, was discussed. This precedent was not well received by legal wine producers in Europe, and especially by traditional producers, because the proposal included that water could be added to wines with protected geographical origin, and thus would open the back door to the possibility of an even greater presence of counterfeits on market, which would hit legal wine producers even more.
So, for example, it would be possible for a buyer to buy a wine in bulk, which according to the elaborations has been approved for the protection of geographical origin, and then to dilute it, increase the quantity, reduce the price a little and make a profit on the addition of water, with the fact that such a wine would could still bear the designation of geographical origin. Such a precedent would kill the wine industry in Europe, given that the current technical analytical capabilities are very modest and there is currently no possibility of determining the quantitative addition of water to wine. For these reasons, the decision on this matter is still pending the finding of a solution to the problem.
2. Detection of added water in milk:
The problems of determining the authenticity of milk are even greater than when we talk about the wine industry. Currently, there is no analytical method that can determine the origin of water in milk. According to the categories, milk can be classified into fresh milk and reconstituted milk produced from milk powder with added water. This procedure is carried out in industrial duplicators with mixers and dosers where all components (powdered milk, powdered whey, water) are added and mixed to a homogeneous consistency. The representation of one such court - duplicator is given in picture no. 3.
The problem of milk authenticity has enormous consequences for the living standards of citizens from two aspects. The first aspect is related to the health safety of milk as a food product, and the second is related to problems in the relationship between cattle farmers (raw milk producers) and dairy farmers (milk processors).
A case that happened in China back in 2008 and which caused great concern in September 2008 is well known in the world. According to the Ministry of Health of the People's Republic of China, in September 2008, there was a scandal when milk and baby formulas were adulterated with melamine. Several companies, including some dairy giants in China, were involved in this scandal, especially the baby milk produced by Sanlu, which sickened 4,294,000 babies, with almost 51,900 hospitalizations and six infant deaths by the end of November 2008.
On the other hand, in the Republic of Serbia over the past years, there has been a growing problem in the agreement between cattle farmers and dairy farmers regarding the purchase price of raw milk. Due to changes in economic parameters on the market due to the Corona virus pandemic and industry stagnation, the prices of raw materials and repro-materials have increased (fuel, artificial fertilizers, land lease, energy products, etc.), while the price of buying raw milk has remained at the same level from approx. 30 dinars for a liter of raw milk. Livestock farmers have been warning about the problems they are facing for several years, and many of them are shutting down their farms and sending their cattle to slaughterhouses, which is slowly killing the livestock of the Republic of Serbia. In the past 10 years, the livestock fund has been reduced by 80%, where the number of head 10 years ago was around one million, while now in 2022 it is around 200,000 head.
On the other hand, the import of raw materials and semi-finished products for the dairy industry in Serbia increased and at the end of 2021, the value of the import of milk powder, whey powder, concentrated milk, and milk fat amounted to about €185 million.
In these conditions, the market of dairy products in Serbia is debatable, because no one can determine the authenticity of the milk that is on the shelves in retail chains in Serbia. Declaring the product is a very important item, because any deception of end consumers is an economic fraud, where there is a
very high probability that reconstituted milk is mixed with fresh milk in a certain ratio and marketed as such under the name "fresh milk".
In that case, this problem directly affects livestock farmers, because then they only provide cover for dairies to have the input of raw materials into their factories.
By determining the authenticity of the milk, ie. determining the presence of added water in milk, i.e. diluting milk with water, would create conditions for the recovery of livestock production in Serbia and protect the national market from the dumping effect created by imported raw materials for the dairy industry. Although this is not a problem that affects only Serbian livestock farmers, there are similar examples in the rest of the world and other countries in the world.
3. Detection of added water in fruit juices:
The problem related to determining the addition of water to fruit juices is related to the use of adequate raw materials in the production of fruit juices, and thus the correct declaration of such products.
Freshly squeezed fruit juices (NFC - Non from concentrate) on the market fetch higher prices compared to fruit juices obtained by reconstituting fruit concentrates with the addition of water.
Various fruits that are not produced in Europe, mainly citrus fruits (oranges, tangerines, grapefruit, passion fruit, lime, lemon, etc.) are grown in tropical or subtropical areas. Due to the long distance and time required to transport juices from these regions to Europe or North America, juices from these types of fruit are concentrated in order to preserve them during the journey and prevent them from spoiling or starting fermentation during transport.
When the raw material arrives at the destination where it will be used in the production of fruit juices, it is reconstituted with the repeated addition of water and the final product is reconstituted fruit juice made from fruit concentrate.
On the other hand, fresh fruit juice is obtained by squeezing fresh fruit. According to the regulations on quality, it is forbidden to dilute fresh fruit juice with water, if such a product is declared as freshly squeezed fruit juice. Therefore, the price of such juices is higher compared to reconstituted fruit juices.
Fraudsters and unfair competition very often declare reconstituted fruit juices as fresh fruit juices and thus commit economic fraud and mislead end consumers that they bought a higher quality product for their money for which they paid a higher price, and in return received a lower quality product, quality.
The same story applies to related products that are declared "no added water", as well as to, for example, coconut water which, like fruit juices, can be made from coconut concentrate, or be fresh coconut water. This product is very popular in Latin American countries and is very susceptible to counterfeiting. Therefore, the determination of additional water is very important for all interested parties in the market, from legal producers to end consumers.
5. Detection of added water in meat, meat raw materials, processed products, meat products, fish and fish products:
Meat, as a product, is very prone to counterfeiting. Scams in meat trade can be different. There is a well- known case from 2013 in Europe where horse meat was mixed with beef and pork and as such ended up in declared beef burgers.
Other methods of economic fraud in meat trade are related to the addition of aqueous solutions (which may contain aromas, collagen and other binders), which are used to treat meat in order to gain weight in cases where raw meat is sold per kilogram in butchers. Meat treated in this way gains volume (pork, chicken) and looks fresh and attractive to the end customer and consumer, but when thermally processed, the end product is not even half the size of the one bought in the shop (butchers). By the time the end consumer realizes that he has been deceived, it is already too late. Economic frauds on the weight of meat are the most widespread in the world and currently there is no analytical way to determine the addition of water to meat raw materials (chicken, pork, veal, etc.) as well as to meat products (bacon, dry neck, prosciutto, etc. ).
With a new procedure for determining the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol prepared in product water, which is created by stirring the added fermentable sugar reagent in the isolated water of the food product, and further determination of the relative ratio of stable hydrogen isotopes using the apparatus for determining the isotopic composition of non-exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the IRMS instrumental technique, it is possible to determine the origin of water in food products such as wine, fruit juices, fresh fruit juices, milk and dairy products, meat and meat raw materials, meat products and products, strong alcoholic beverages, etc. .
State of the art
The first closest state of the art for detecting the addition of water to food products is the official method for determining the relative ratio of stable oxygen isotopes in the water of the tested product sample.
As previously stated in the "Technical problem" section, currently in the world for the detection of added water in wine, fruit juices, and other liquid matrices, determination of the relative ratio of stable oxygen isotopes in the water of the investigated product is used. The technique is based on the equilibration of the sample with carbon dioxide, which is in a mixture with the inert gas Helium in the "head-space" space in the vial for 24 hours, and then the gas, after equilibration with water from the sample, is transferred in a stream of helium in a continuous flow through non-fionic filters, a gas chromatographic column containing silica gel, all with the aim of extracting moisture from the gas, and by further introducing carbon dioxide into the IRMS, it is measured on the detectors of the instrument and obtains the relative ratio of 18O/16O in the examined carbon dioxide gas , and which corresponds to the relative ratio of stable oxygen isotopes 18O/16O in the water of the tested sample. The scheme of the equilibration technique for testing stable oxygen isotopes and determining the 818O value in the water of the tested product sample is given in figure no. 4.
The reference document related to the technique described above is:
DI = COMPENDIUM OF INTERNATIONAL METHODS OF ANALYSIS - O1V, Method for 18O/16O isotope ratio determination of water in wines and must, (Resolution OlV-Oeno 353/2009) - Method OIV-MA-AS2-12
The document is an official method of the OIV that describes the principle, the area of application, as well as the work procedure and the method of determining the relative ratio of stable oxygen isotopes in wine water ( 518O value in wine water).
The second closest state of the art for conditionally determining the detection of water added to wine is the SNIF-NMR method (Site Natural Isotopic Fractionation - Nuclear Magnetic Resonance).
Using nuclear magnetic resonance, intermolecular scanning of the methylene group of ethanol, it is possible to determine what the conditions were during alcoholic fermentation, and therefore the origin of water during alcoholic fermentation. In this way, the analytical parameter (D/H)u is obtained, which represents the relative ratio of deuterium and hydrogen on the methylene group of ethanol. The problem associated with this parameter is that the values of this parameter move within very narrow limits, and the measurement unit itself is in ppm. Over the years, the reference central laboratory in Geel (Belgium) stopped using this parameter as a reliable indicator of added water, due to the narrow range, as well as the large standard deviation obtained when scanning this ethanol site.
The reference document regarding the SNIF-NMR method is:
D2 = COMPENDIUM OF INTERNATIONAL ANALYSIS OF METHODS - OIV, Determination of the deuterium distribution in ethanol derived from fermentation of grape musts, concentrated grape musts, grape sugar ( rectified concentrated grape musts) and wines by application of nuclear magnetic resonance (SNIF-NMR/ RMN-FINS), (Oeno 426-2011) - Method OIV-MA-AS311-05.
The document is an official method of the OIV that describes the principle, the area of application, as well as the procedure for working and determining the relative ratio of deuterium and hydrogen on the methyl and methylene group of ethanol ((D/H), and (D/H),, ).
Furthermore, the third closest state of the art is the patent numbered RS 52615 B, which describes the procedure for measuring non-exchangeable stable hydrogen isotopes in ethanol samples for the purposes of determining illegal practices of adding sugar and/or water to grape must before alcoholic fermentation.
D3 = Patent file, 52615 B, Apparatus for determining the isotopic composition of non-exchangeable stable hydrogen isotopes in ethanol samples and procedure for determining the authenticity and geographical origin of the product, Institute for Intellectual Property of the Republic of Serbia.
Patent 52615 B deals with a technical solution and procedure for determining non-exchangeable stable hydrogen isotopes in ethanol samples from food products that already contain ethanol and/or fermentable sugars.
The principle of the invention according to RS 52615 B is based on the isolation of already existing ethanol, which was created by stirring the sugars present in the examined food product, and then the dehydration of the thus separated ethanol and the creation of ethylene gas, further pyrolysis of ethylene to elementary hydrogen gas (H2) and carbon , chromatographic separation, and measurement of the relative ratio of deuterium and hydrogen in the thus prepared hydrogen gas.
Presentation of the essence of the invention
This invention overcomes the existing barriers in the lack of technical analytical solutions known so far, and for easier detection of added water and determination of the origin of water in food products that contain water (wine, strong alcoholic beverages, fruit juices, fruit concentrates, coconut water, milk and dairy products, meat, meat raw materials, meat products and products, etc.).
Scientific foundations on which the essence of the invention is based:
The principle of the invention is based on the fact that ethanol after alcoholic fermentation contains two types of information. Based on previously published scientific works on the topic of determining the origin of ethanol, as well as on the basis of the biochemical mechanism of ethanol formation during the glycolysis process, it was previously determined using Nuclear Magnetic Resonance that the deuterium atoms in the final ethanol, after the alcoholic fermentation is completed, will be found in the following positions:
If deuterium is found on the methyl group, then in the same ethanol molecule it will not be present on the methylene group and vice versa. The deuterium found on the hydroxyl group of ethanol is always exchangeable, which has the effect that ethanol as an organic compound is hygroscopic and binds water by forming hydrogen bridges. By exchanging hydroxyl hydrogen and/or deuterium in the matrix in which ethanol is located, its overall ratio of deuterium and hydrogen atoms is constantly changing, i.e. it is always in dynamic balance with its environment. On the other hand, the non-exchangeable deuterium and hydrogen atoms that are tightly bound to the carbon atoms on the methyl and methylene group can provide information under which conditions and from which raw materials that ethanol was created.
According to the attached diagram in Figure 5, deuterium atoms located in certain positions in sugar, after alcoholic fermentation, end up on methyl and methylene groups.
According to the previous model proposed by prof. dr. Gerard Martin's thesis was that hydrogen and deuterium atoms, which are attached to the 1, 6, 6' positions of glucose, will end up on the methyl group of ethanol after alcoholic fermentation.
However, 80% of the hydrogen and deuterium atoms from the hydroxyl groups of grape sugar are in constant dynamic equilibrium with the surrounding water in the grape must (grape juice) during alcoholic fermentation, which means that these atoms will be redistributed on the methylene group in the ethanol molecule.
From these facts it follows that the relative ratio of deuterium and hydrogen on the methyl group of ethanol will give information about the botanical origin of the sugar from which that ethanol was made (grapes, fruits, sugar beets, sugar cane, corn, wheat, rice, wheat, potatoes, etc. ), while the relative ratio of deuterium and hydrogen on the methylene group will provide information about the type of water in which ethanol was formed. This fact is mostly a consequence of the last step in the glycolysis process, i.e. alcoholic fermentation, when due to the activity of the enzyme acetaldehyde-dehydrogenase in the presence of the oxido-reducing coenzyme NADH + H+ , a pair of protons is transferred from the surrounding medium (water), where that pair of protons binds to the aldehyde group of acetaldehyde and thus creates an ethanol molecule as the final a product of alcoholic fermentation. The biochemical mechanism of alcoholic fermentation is shown in Figure 6.
This also means that if the aqueous matrix is altered and water is added to it, it will disturb the biochemical dynamic balance and a new balance will be established. Thus, the ratio of exchangeable deuterium and hydrogen atoms in the matrix will be changed, which will result in a change in the
relative ratio of deuterium and hydrogen on the methylene group (CH2 - j in the newly formed ethanol molecule.
All these scientific facts represent the starting point for the new invention of detecting the addition of water and determining the origin of water in food products containing water such as wine, fruit juices, strong alcoholic beverages, milk and milk products, meat and meat raw materials, meat products and meat products , etc.
For example, if water is added to the final wine in which the alcoholic fermentation has already been completed and all the sugar has boiled off into ethanol, then such addition of water will no longer have an effect on the irreplaceable stable isotopes of hydrogen in the already formed ethanol, but it will in any case change the isotopic profile wine water of such diluted wine.
This addition will cause a change in the relative ratio of stable oxygen isotopes ( 318O value in wine water), but this change, due to the narrow range in which these values move, will not be able to give us quantitative information about the amount of added water. Figures 7, 8, 9, 10, and 11 show graphical representations of the change in 318O values in wine water with the addition of exo-water to the final wine.
According to the first aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dnu value of ethanol from product water), and for the purpose of detecting added water in food products, the principle is based on the preparation procedure and of making alcohol (ethanol) by taking water from the test sample, adding a fermentable sugar reagent and yeast and starting the alcoholic fermentation. It is necessary that the alcoholic fermentation of the sample be performed in anaerobic conditions without the presence of air, in order to secure the sample from potential losses of ethanol and eliminate the possibility of starting secondary aerobic fermentations, such as vinegar fermentation, etc. The basic and very important item is to always use the same fermentable sugar reagent, because in this way it is ensured that the contribution that would be made on the methyl (CH3 -) group of ethanol formed in the water matrix, which represents the isolated water from the tested product, is blocked. In this way, the newly formed ethanol as a product of alcoholic fermentation, if exo-water is added to the product, due to the change in the isotopic profile of the examined medium, that information is integrated on the methylene group of the newly formed ethanol, and then by isolating the ethanol produced under such specific conditions, its quantitative extraction from the overboiled medium and by determining the relative ratio of non-exchangeable stable isotopes of hydrogen using the apparatus for determining the isotopic composition of non- exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the IRMS instrumental technique, information is obtained on the origin of the water contained in the examined product.
According to the second aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnn value of ethanol from product water), and for the purpose of detecting added water in food products, if the product containing water, also also contains ethanol and/or fermentable sugars, in that case it is necessary to quantitatively separate water from ethanol and/or sugar in such a way that isotopic fractionation of water does not occur, which is the starting matrix for the preparation of ethanol for testing the origin of water in the tested product.
According to the third aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( SDn,, value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product is wine containing alcohol (ethanol), it is necessary to perform a quantitative separation of alcohol first so that it does not affect and contaminate the preparatory matrix and thus affect the final test results, because the water must be sufficiently freed and without the presence of ethanol or fermentable sugars before the addition of the fermentable sugar reagent and yeast .
According to the fourth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnn value of ethanol from product water), and for the purpose of detecting added water in food products, it is understood that other methods of quantitative extraction of ethanol from a product containing alcohol (ethanol), such as vacuum distillation, liquidliquid extraction, azeotropic distillation as well as other techniques, as long as it is achieved that alcohol (ethanol) is quantitatively extracted from the examined matrix and while the extraction technique itself does not create isotopic fractionation of the water matrix that needs to be further used for the preparation of ethanol to determine the origin of the water in the examined product sample.
According to the fifth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnn value of ethanol from product water), and for the purpose of detecting added water in food products, it is imperative that the fermentable sugar reagent which is added to the aqueous matrix during alcoholic fermentation and is quantitatively converted into alcohol (ethanol) in order to obtain complete information from the examined matrix.
According to the sixth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnlt value of ethanol from product water), and for the purpose of detecting added water in food products, after the completed bioconversion of the fermentable sugar reagent in alcohol (ethanol), the quantitative separation of that alcohol (ethanol) from the aqueous matrix is carried out in the same way as previously described according to aspect four and aspect five of this invention.
According to the seventh aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dn,i value of ethanol from product water), and for the purpose of detecting added water in food products, further determination of the relative ratio of non- exchangeable stable isotopes of hydrogen in isolated ethanol from the examined water matrix using the apparatus for determining the isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique.
According to the eighth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnu value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product sample containing water contains
fermentable sugars in addition to ethanol (for example semi-dry, semi-sweet or sweet wine, or any other product containing ethanol and fermentable sugars or only fermentable sugars) after the quantitative separation of ethanol, as described in aspects four and five of this section, it is necessary to quantitatively separate the water from the sugars present in the water matrix.
According to the ninth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( SDn value of ethanol from product water), and for the purpose of detecting added water in food products, quantitative separation of water from fermentable sugars is possible perform azeotropic distillation by forming an azeotropic mixture with toluene (or any other organic solvent with which water forms an azeotropic mixture) until complete quantitative separation of water from sugar. It goes without saying that other ways of quantitatively separating water from fermentable sugars using other physical methods and techniques are possible, as long as it is achieved that water and fermentable sugars are quantitatively separated and the extraction technique itself does not create isotopic fractionation of the water matrix that needs to be further prepared for analysis. It will be respected that any other method of separating water from impurities (ethanol and/or fermentable sugars), which may be present in the water of the product and thus contaminate the water matrix for the preparation of ethanol to determine the origin of the water in the tested product, does not change the essence, the principle and the ultimate goal of this invention.
According to the tenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8Dnn value of ethanol from product water), and in order to detect added water in food products, it is even possible to perform secondary alcoholic fermentation in order to convert the remaining fermentable sugar into ethanol, and after the completion of the alcoholic fermentation, quantitatively separate the alcohol (ethanol) again in the manner previously described in aspects four and five of this section, and then use the separated water further in the preparation as it is described in aspects six to eight of this section.
According to the eleventh aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 5Dnu value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product is milk or milk product, it is possible to add the fermentable sugar reagent directly to the tested product together with the yeast, without first separating the water from the other components of the milk, since milk does not contain fermentable sugars that turn into alcohol, as well as alcohol (ethanol).
According to the twelfth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dn,i value of ethanol from product water), and for the purpose of detecting added water in food products, if the examined product is milk or milk product, for a better and easier performance of the ethanol in water preparation of the tested product sample, it is possible to coagulation milk proteins using rennet or another coagulating agent for easier separation of milk water from other milk components in milk. Also on that occasion, it is very important to use a suitable coagulating agent that does not contain hydrogen atoms in its composition that can end up in the water matrix and thus disrupt the isotopic profile of the water being prepared for testing.
According to the thirteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 5DnN value of ethanol from product water), and for the purpose of detecting added water in food products, it is important to emphasize that under fermentable sugar reagent can mean all monosaccharides or disaccharides that turn into alcohol (ethanol) during alcoholic fermentation, such as glucose, fructose, sucrose, etc., but it is also understood that using higher sugars (disaccharides, trisaccharides, as well as oligosaccharides) with the addition of hydrolase enzymes (amylase) or acids that would hydrolyze such sugars into monosaccharides which would then be fermentable and at the end of the alcoholic fermentation process give alcohol (ethanol) can be considered suitable for the preparation of ethanol in product water, and in accordance with this aspect of the invention .
According to the fourteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( SDn,, value of ethanol from product water), and for the purpose of detecting added water in food products, for the preparation of ethanol in the water matrix of the tested of the product, it is also possible to do the conversion enzymatically by using the appropriate decarboxylase, dehydrogenase and coenzymes, thus simulating the steps of the biochemical conversion process and the creation of ethanol. For example, by adding pyruvic acid as a starting reagent, and instead of a fermentable sugar reagent and yeast, and by making a mixture with the enzymes pyruvatedecarboxylase and acetaldehyde-dehydrogenase and the oxido-reducing coenzyme nicotinamide adenine dinucleotide (NADH + H+ ) with the aqueous matrix of the tested product, it is possible is to simulate the last two steps of alcoholic fermentation and produce ethanol that will contain information about the origin of water, as previously described and shown in Figure No. 6. According to this aspect of the invention, it will be respected that modifications are possible and that if instead of pyruvic acid as a starting reagent for the preparation of ethanol in the aqueous matrix of the tested sample, any intermediate metabolite in the chain of the biochemical mechanism of the formation of ethanol in the glycolysis process is taken, and then appropriate mixtures of enzymes and coenzymes are added in order to complete the bioconversion process to ethanol as the end product of glycolysis, it will be considered part of the same solution described here.
According to the fifteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8Dn value of ethanol from product water), and for the purpose of detecting added water in food products, it will be considered that the procedure for the preparation of ethanol in water of the tested product applicable to all food products that contain water and in which it is possible to determine the origin of water, such as wine, fruit juices, fruit nectars, fruit concentrates, fruit pulps, fruit purees, refreshing soft drinks with and without fruit juice, food for babies and infants, strong alcoholic drinks, honey, coconut water, maple syrup, milk and milk products, meat and meat raw materials from beef, chicken, pork, lamb, veal, etc., as well as their offal (livers, kidneys, heart, stomach, etc.), meat products and meat products, fruit and vegetable products, spring and mineral waters, functional food products, vinegar (balsamic, wine, apple, alcoholic, diluted acetic acid, etc.), fish and fish products. Meat and fish also include frozen processed or semi-processed raw materials. Also in terms of this aspect of the invention, the list of categories of food products is not limited only to the mentioned
categories of food products, but includes all related products as well as other food products that contain water.
According to the sixteenth aspect of the invention of the procedure for the preparation of ethanol in product water for the purposes of determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8 Dn value of ethanol from product water), and for the purpose of detecting added water in food products, for the purpose of better determining the authenticity of the examined product sample, it implies and bringing the obtained values of the relative ratio of non- exchangeable stable hydrogen isotopes in ethanol prepared in the water of the tested product sample ( 8Dn[( value of ethanol from food product water) into correlation with other values of isotopic analytical parameters, which are obtained by other already known isotopic techniques ( 813C values of analytes in the tested sample, 81SO values of analytes in the tested sample, values of the relative ratio of non- exchangeable stable hydrogen isotopes in ethanol ( 8Dn, value of ethanol from the product) according to RS 52615 B, 815N values in the tested sample and 832S values in the examined sample). According to this aspect of the invention, by correlating the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol prepared in the water of the tested product sample ( 8Dn value of ethanol from the water of the food product) with the values of the other mentioned isotopic species, new information is obtained about the overall authenticity of the product.
Detailed description of the invention
In accordance with the basic idea of the invention of the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (6Dnu value of ethanol from product water), and with the aim of detecting added water in food products, it is based on the bioconversion of the initial fermentable sugar reagents in the presence of yeast to the end product of alcoholic fermentation - alcohol (ethanol) in product water, further separating the produced ethanol from the overboiled medium, and then subsequently determining the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol from product water ( 5Dnn values in ethanol from water product) using the apparatus for determining the isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples.
It is important to note that the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (6Dnn value of ethanol from product water), and for the purpose of detecting added water in food products, is applicable for the following group of products, which includes: wine, fruit juices, fruit nectars, fruit concentrates, fruit pulps, fruit purees, refreshing soft drinks with and without fruit juice, food for babies and infants, strong alcoholic drinks, honey, coconut water, maple syrup, milk and milk products, meat and meat raw materials originating from beef, chicken, pork, lamb, veal, etc., as well as their offal (livers, kidneys, heart, stomach, etc.), meat products and meat products, fruit and vegetable products, and mineral waters, functional food products, vinegar (balsamic, wine, apple, alcoholic, diluted acetic acid, etc.), fish and fish products, as well as all related products and other food products containing water and any combinations thereof.
The first step of the preparation procedure is based on the separation of water from the sample of the tested product from all other components that are present in the sample of the product, which can influence the results of the analysis by their presence. If alcohol (ethanol) and other easily volatile components are present in the tested product, they must be separated by distillation so that there is no
separation and loss of water or its isotopic fractionation, and that ethanol does not remain in the water matrix.
In the case of wine or any other product containing alcohol (ethanol), its quantitative extraction can be performed using the distillation apparatus shown in Figure No. 13.
Separation of water from the tested product sample from ethanol and other easily volatile components that may be present in the tested product can be performed by liquid-liquid extraction, azeotropic distillation, as well as by any other physical and/or physico-chemical method or process or their combination. If the sample of the tested product contains a certain amount of fermentable sugars, proteins, fats, acids, the separation of water from the mentioned components in the sample of the tested product is carried out by azeotropic distillation at atmospheric pressure or under reduced pressure (in a vacuum) with toluene or any other organic solvent that it does not dissolve in water, and at the same time forms an azeotropic mixture with water in the appropriate ratio and does not have exchangeable hydrogen atoms that could be exchanged with hydrogen atoms from the water of the product. This step of separating water from other components in the sample of the tested product can be done by liquid-liquid extraction, lyophilization with condensation and collection of the separated water, as well as by other physical or physico-chemical procedures and/or techniques and their combinations. When it comes to liquid products containing proteins and fats (such as milk and dairy products), the separation of water from other components present in the tested product can be done using coagulating and/or denaturing agents such as rennet or using acids, as well as microbiological activities naturally of microorganisms present in the tested product (for example raw milk) or by the addition of microorganisms to the sample of the tested milk or milk product in order to make the phase separation of the water-other component faster. When it comes to product samples that are in a solid state, such as meat or meat raw materials, fish and related and similar products, such product samples must first be crushed and homogenized as finely as possible, and then proceed with the extraction of water from the product sample in ways which are previously mentioned in this step of the preparation procedure.
The second step of the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (6Dn,i value of ethanol from product water), and in order to detect added water in food products, is based on the addition of a fermentable sugar reagent and yeast to water from of the tested product and preparation of the mixture for alcoholic fermentation in order to stir the sugar reagent to alcohol (ethanol). As a fermentable sugar reagent in this step of the preparation procedure, all monosaccharides or disaccharides, as well as their combinations, which turn into alcohol (ethanol) during alcoholic fermentation, such as glucose, fructose, sucrose, etc., can be used, but they can also higher sugars (disaccharides, trisaccharides and oligosaccharides), as well as their combinations, can be used, which with the addition of hydrolase enzymes (amylase) or acids would be converted and translated into fermentable sugars, and which would then at the end of the process of alcoholic fermentation or enzymatic bioconversions yielded alcohol (ethanol) and as such were suitable as a fermentable sugar reagent for the preparation of ethanol in water of the tested product. It is important to emphasize that fermentable sugar reagents can be of vegetable, animal or synthetic origin. Fermentable sugar reagent of plant origin means all fermentable sugars originating from grapes, fruits, industrial plants (sugar beet, sugar cane, corn, wheat, barley, rice, millet, sorghum, etc.), honey, other plants and other origins , as well as their combinations.
Furthermore, it is important to emphasize that during the process of preparing ethanol in product water and determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus
prepared (6Dn „ value of ethanol from product water), and in order to detect added water in food products, it is important that as fermentable the sugar reagent always uses the same reagent with a known and constant isotopic profile of stable hydrogen isotopes, so that by turning it into ethanol in the water of the product, the relative ratio of deuterium and hydrogen atoms on the methyl (CH3 -) group of the ethanol made in this way is always fixed , i.e. thus preventing a change in the relative ratio of deuterium and hydrogen on the methyl group (CH3 -) of ethanol, which means that in that case any change in the relative ratio of total non-exchangeable stable isotopes of hydrogen in the prepared ethanol ( SDn,, values of the prepared ethanol) from of water in the tested product be a consequence and depend only on the change in the relative ratio of deuterium and hydrogen atoms on the methylene group (CH2 -) of ethanol, which provides information about the origin of the water in the tested product and thus detect the addition of additional water in the tested product.
Also, in this step of the process of preparing ethanol in product water and determining the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol thus prepared (6Dn„ value of ethanol from product water), and in order to detect added water in food products, it is possible instead of using a fermentable sugar reagent and yeast, use other appropriate reagents, which can also yield alcohol (ethanol) through bioconversion. In this sense, the addition of pyruvic acid can be used as a starting reagent with the addition of pyruvate-decarboxylase, acetaldehyde-dehydrogenase and nicotinamide adenine dinucleotide oxidation-reducing coenzyme (NADH+H+ ) and bioconversion of pyruvic acid via acetaldehyde to alcohol (ethanol). In this case too, it is important that by using pyruvic acid as a starting reagent instead of a fermentable sugar reagent, pyruvic acid of the same isotopic profile of stable hydrogen isotopes is always used, so that its enzymatic bioconversion into ethanol in the product water always fixes the relative ratio of deuterium and hydrogen atoms, on the methyl (CH3 -) group of the ethanol made in this way, i.e. to prevent a change in the relative ratio of deuterium and hydrogen on the methyl group (CH3 -) of ethanol, which means that in that case any change in the relative ratio of total non-exchangeable of stable hydrogen isotopes in the prepared ethanol ( SDn,, values of the prepared ethanol) from the water of the tested product be a consequence and depend only on the change in the relative ratio of deuterium and hydrogen atoms on the methylene group (CH2 - > of ethanol, which provides information about the origin of the water in the tested product and thus detect the addition of added water in the tested product.
Furthermore, it is understood and will be accepted that it is possible, according to the same biochemical principle, instead of a fermentable sugar reagent as a starting reagent, to use the addition of any intermediate metabolite from the process of the biochemical mechanism of the formation of ethanol from sugar as a starting reagent and the addition of appropriate enzymes and coenzymes, and in order to bioconversion led to the final product of alcoholic fermentation (ethanol). And so on, that by using any intermediate metabolite as a starting reagent from the process of the biochemical mechanism of the formation of ethanol from sugar with the addition of all appropriate enzymes and coenzymes, and instead of fermentable sugar reagent and yeast, it always uses an intermediate metabolite of the same isotopic profile of stable hydrogen isotopes so that its enzymatic bioconversion into ethanol in the water of the product always fixes the relative ratio of deuterium and hydrogen atoms on the methyl (CH3 . ) group of the ethanol thus made, i.e. that the change in the relative ratio of deuterium and hydrogen on the methyl group is prevented in this way (CH3 -) of ethanol, which means that in that case any change in the relative ratio of the total non-exchangeable stable isotopes of hydrogen in the prepared ethanol ( SDn,, values of the prepared ethanol) from the water of the tested product will be a consequence and depend only on the change in the relative ratio of atoms of deuterium and hydrogen on the methylene group (CH2 -) of ethanol, which provides information on the origin of water in the tested product and thus determine the addition of additional water in the tested product.
The third step of the procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol (8Dn „ value of ethanol from product water), and with the aim of detecting added water in food products, is based on the separation of prepared alcohol (ethanol) from overboiled medium by distillation at atmospheric pressure or under vacuum. This step is not limited to the extraction of ethanol by distillation, but other physical and physicochemical procedures or techniques as well as their combination can be used.
Quantitative separation of the prepared alcohol (ethanol) from the overheated medium can be performed using the distillation apparatus shown in Figure No. 13.
The fourth step of preparing ethanol in product water and determining the relative ratio of non- exchangeable stable hydrogen isotopes in the ethanol thus prepared (6Dnu value of ethanol from product water), and with the aim of detecting added water in food products, is based on the measurement of the relative ratio of non-exchangeable stable hydrogen isotopes in the previously separated ethanol from the overboiled medium using the apparatus for determining the isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples, which is based on the IRMS (Isotope Ratio Mass Spectrometry) instrumental technique, and then calculating the 6Dnu value of the prepared ethanol from the water of the tested product sample by comparing of the relative ratio of non-exchangeable stable hydrogen isotopes of ethanol from the water of the tested product sample with ethanol with the known relative ratio of non-exchangeable stable hydrogen isotopes obtained in the same way.
Figure no. 12 shows a graphic representation of the detection of water added to the final wine by measuring the relative ratio of non-exchangeable stable isotopes of hydrogen in ethanol prepared in wine water, i.e. wine from which wine ethanol has previously been separated ("alcohol free wine").
Figure no. 14 shows a graphic representation of the results for the 8Dnu value of ethanol from milk water, with the aim of detecting the presence of reconstituted milk in fresh milk.
Figures 17, 18, 19, 20 and 21 show graphical representations of the results obtained with this invention.
After determining the SDn,, value of the ethanol prepared from the water of the tested product sample, and in order to better determine the authenticity of the tested product sample, it is also implied to bring the obtained values of the relative ratio of non-exchangeable stable hydrogen isotopes in the ethanol prepared in the water of the tested product sample ( 6DnN value of ethanol from water food product) in correlation with other values of isotopic analytical parameters, which are obtained by other already known isotopic techniques ( 813C values of analytes in the tested sample, 818O values of analytes in the tested sample, values of the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol ( SDn, value of ethanol from the product) according to the procedure of measuring the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol samples, 81SN values in the tested sample and 832S values in the tested sample). According to this aspect of the invention, by correlating the relative ratio of non-exchangeable stable hydrogen isotopes in ethanol prepared in the water of the tested product sample ( aDn,, value of ethanol from the water of the food product) with the values of the other mentioned isotopic species, new information is obtained about the overall authenticity of the product.
Figure No. 15 shows the correlation of the results of SDn,, value of ethanol from product water in relation to 81SO value from wine water.
Furthermore, also in accordance with this aspect of the invention, Figure No. 16 shows a graphic representation of the correlation of the results of 8DnK value of ethanol from wine water in relation to SDn, value of ethanol from wine, the values of which are obtained in accordance with the procedure for measuring the relative ratio of invariable of stable isotopes of hydrogen in ethanol samples and using
the apparatus for determining the isotopic composition of non-exchangeable stable isotopes of hydrogen and deuterium in ethanol samples based on the 1RMS instrumental technique.
Method of application of the invention
The procedure for preparing ethanol in product water and determining the relative ratio of non- exchangeable stable hydrogen isotopes in the ethanol thus prepared ( 8DnN value of ethanol from product water), and for the purpose of detecting added water in food products, is used in chemical instrumental analysis for determining authenticity and determining the origin of water in samples of tested products, and can be used in correlation with the parameters of other isotopic species (C, O, H, N, S) and for the purposes of determining the geographical origin of products containing water.
Claims
Claim 1.
The procedure for the preparation of ethanol in product water and the determination of the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol ( 8 Dn ii value of ethanol from product water), and for the purpose of detecting added water in food products, comprsing by including:
- Separation of the water from the tested sample of the product from all substances that could affect the contamination of the matrix for the preparation of new ethanol in the water of the tested product,
- addition of fermentable sugar reagent,
- adding yeast,
- starting the alcoholic fermentation of the mixture prepared in this way, which lasts until the complete bioconversion of the fermentable sugar reagent into alcohol (ethanol),
- extracting the produced ethanol from the overheated medium,
- measurement of the relative ratio of non-exchangeable stable isotopes of hydrogen in isolated alcohol (ethanol).
Claim 2.
The method according to claim 1, comprising that it further consists in calculating the 8DnN value of the prepared ethanol from the water of the tested product sample by comparing the relative ratio of non-exchangeable stable hydrogen isotopes of ethanol from the water of the tested product sample to ethanol with the known relative ratio of non-exchangeable stable hydrogen isotopes obtained on the same the way.
Claim 3.
The procedure according to claim 1, comprising that the separation of water from other substances present in the tested product sample, which could affect the contamination of the matrix for the preparation of new ethanol in the product water, includes the separation of potentially present ethanol and all other easily volatile and/or fermentable substances sugar, protein, fat, acid, etc.
Claim 4.
The method according to claim 3, comprising that the separation of water from ethanol and other volatile substances includes distillation of the sample with the separation of ethanol and all other accompanying volatile substances.
Claim 5.
The method according to claim 3, comprising that the separation of water from ethanol and other easily volatile substances and/or fermentable sugars includes separation by other physical and physico-chemical techniques, such as liquid-liquid extraction, azeotropic distillation, lyophilization with condensation and reception of the separated water, as well as other techniques and procedures or their combinations.
Claim 6.
The method according to claim 5, comprising that the separation of water from the potentially present fermentable sugars in the sample of the tested product, and after the previously extracted ethanol from the sample of the product includes the stirring of the present fermentable sugars into ethanol in the presence of yeast, and then the separation of that ethanol by distillation or any other by physical or physico-chemical process or technique or their combinations.
Claim 7.
The method according to claim 1, comprising that if the tested product contains fermentable sugars, proteins, fats, acids (such as acetic acid, etc.) and other components, the separation of water from other components from the tested product sample includes azeotropic distillation with toluene or any with any other organic solvent that forms an azeotropic mixture with water in a certain ratio, without dissolving in water and not possessing exchangeable hydrogen atoms, at atmospheric pressure or under vacuum, as well as lyophilization with condensation and reception of separated water, as well as any other physical procedure and/or separation technique or a combination thereof.
Claim 8.
The method according to claim 1, comprising that as a fermentable sugar reagent, the same reagent with a known and constant isotopic profile of stable hydrogen isotopes is always used, so that when it is stirred by the yeast into ethanol in the water of the product, the relative ratio of deuterium and hydrogen atoms is always fixed in this way to the methyl (CH3 -) group of ethanol made in this way, i.e. to prevent a change in the relative ratio of deuterium and hydrogen on the methyl group (CH3 -) of ethanol, which means that in that case any change in the relative ratio of the total unchangeable stable of hydrogen isotopes in the prepared ethanol ( 5DnN values of the prepared ethanol) from the water of the tested product be a consequence and depend only on the change in the relative ratio of deuterium and hydrogen atoms on the methylene group (CH2 - ) of ethanol, which provides information about the origin of the water in the tested product and on thus detect the addition of added water in the tested product.
Claim 9.
The method according to claim 1, comprising that the fermentation sugar reagent includes all monosaccharides or disaccharides, as well as their combinations, which turn into alcohol (ethanol) during alcoholic fermentation, such as glucose, fructose, sucrose, etc., but also includes and the use of higher sugars (disaccharides, trisaccharides and oligosaccharides), as well as their combinations, with the addition of hydrolase enzymes (amylase) or acids that would hydrolyze such sugars into monosaccharides, which would then be fermentable at the end of the alcoholic fermentation process or enzymatic bioconversions yielded alcohol (ethanol) and as such were suitable as a fermentable sugar reagent for the preparation of ethanol in water of the tested product.
Claim 10.
The method according to claim 9, comprising that the fermentation sugar reagent includes all carbohydrates (sugars) of plant, animal or synthetic origin.
Claim 11.
The method according to claim 10, comprising that the fermentation sugar reagent of plant origin includes sugars from grapes, fruits, industrial plants (sugar beet, sugar cane, corn, wheat, barley, rice, millet, sorghum, etc.), honey and other origins , as well as their combinations.
Claim 12.
The method according to claim 1, comprising that during the preparation of ethanol in water of the tested product, instead of the addition of fermentable sugar reagent and yeast, the addition of pyruvic acid is used as a starting reagent with the addition of pyruvate-decarboxylase, acetaldehyde-dehydrogenase and the oxidoreducing coenzyme of nicotinamide adenine dinucleotide (NADH+H + ) and perform bioconversion of pyruvic acid via acetaldehyde to alcohol (ethanol).
Claim 13.
The method according to claim 1, comprising that during the preparation of ethanol in water of the tested product, instead of the addition of fermentable sugar reagent and yeast, the addition of any intermediate metabolite from the process of the biochemical mechanism of the formation of ethanol from sugar is used as a starting reagent and the addition of appropriate enzymes and coenzymes, and how bioconversion would lead to the final product of alcoholic fermentation (ethanol).
Claim 14.
The method according to claim 12, comprising by the use of pyruvic acid as a starting reagent and the addition of the enzyme pyruvate decarboxylase, acetaldehyde dehydrogenase and the oxidoreducting coenzyme nicotinamide adenine dinucleotide (NADH+H+) , and instead of fermentable sugar reagent and yeast, pyruvic acid is always used of the same isotopic profile of stable hydrogen isotopes, so that its enzymatic bioconversion into ethanol in the water of the product always fixes the relative ratio of deuterium and hydrogen atoms on the methyl (CH3 -) group of the ethanol made in this way, i.e. the change in the relative ratio is prevented of deuterium and hydrogen on the methyl group (CH3 -) of ethanol, which means that in that case any change in the relative ratio of total non-exchangeable stable isotopes of hydrogen in the prepared ethanol ( SDn,, values of the prepared ethanol) from the water of the tested product will be a consequence and depend only from the change in the relative ratio of deuterium and hydrogen atoms on the methylene group (CH2 -) of ethanol, which gives information about the origin of water in the tested product and thus detect the addition of additional water in the tested product.
Claim 15.
The method according to claim 13, comprising that by using any intermediate metabolite as a starting reagent from the process of the biochemical mechanism of the formation of ethanol from sugar with the addition of all appropriate enzymes and coenzymes, and instead of the fermentable sugar reagent and yeast, an intermediate metabolite of the same isotopic profile is always used of stable isotopes of hydrogen so that its enzymatic bioconversion into ethanol in the product water always fixes the relative ratio of deuterium and hydrogen atoms on the methyl (CH3 -) group of the ethanol thus produced, i.e. preventing a change in the relative ratio of deuterium and hydrogen on the methyl group (CH3 -) of ethanol, which means that in that case any change in the relative ratio of the total non-exchangeable stable isotopes of hydrogen in the prepared ethanol (5Dnn values of the prepared ethanol) from the water of the tested product will be a consequence and depend only on the change of the relative ratio of deuterium and hydrogen atoms on the methylene group (CH2 -) of ethanol, which provides information on the origin of water in the tested product and thus determine the addition of added water in the tested product.
Claim 16.
The method according to claim 1, comprising that the product containing water is selected from the following group of products, which includes: wine, fruit juices, fruit nectars, fruit concentrates, fruit pulps, fruit purees, refreshing soft drinks with and without fruit juice, baby food and infants, strong alcoholic beverages, honey, coconut water, maple syrup, milk and milk products, meat and meat raw materials originating from beef, chicken, pork, lamb, veal, etc., as well as their offal (livers, kidneys, heart, stomach, etc.), meat products and meat products, fruit and vegetable products, spring and mineral waters, functional food products, vinegar (balsamic, wine, apple, alcoholic, diluted acetic acid, etc.), fish and fish products, as well as all related products and other food products containing water and any combination thereof.
Claim 17.
The method according to claim 1, comprising that if the tested product is milk or milk products, the separation of water from other components present in the tested milk sample includes coagulation and/or denaturation of milk proteins using rennet or some other coagulating agent for easier separation of milk water from other components in milk.
Claim 18.
The method according to claim 17, comprising that the separation of water from other components in the tested milk sample also includes the separation of phases due to the activity of microorganisms that may be natural in the tested product (raw milk), or by the addition of microorganisms to the tested milk sample.
Claim 19.
The method according to claim 17, comprising that the separation of water from other components contained in the tested milk sample includes the possibility of using other physical and physico-chemical techniques and procedures such as azeotropic distillation with toluene or
any other organic solvent that with water in a certain relationship forms an azeotropic mixture, without dissolving in water and not possessing exchangeable hydrogen atoms, at atmospheric pressure or under vacuum, lyophilization with condensation and reception of separated water, as well as other techniques and procedures or their combinations.
Claim 20.
The procedure according to claim 1, comprising by the separation of water from the tested product sample from other components of the tested product, if the tested product is in a solid state, such as meat and meat raw materials and offal, fish, as well as similar and related products, as well as all other products that are in a solid state and contain water, are carried out by azeotropic distillation with toluene or any other organic solvent that forms an azeotropic mixture with water in a certain ratio, without dissolving in water and not possessing exchangeable hydrogen atoms, at atmospheric pressure or under vacuum, as well as through lyophilization with condensation and collection of separated water, as well as any other physical process and/or separation technique or their combination in a previously finely divided and homogenized sample.
Claim 21.
The method according to claim 1 comprising that the process of preparing ethanol in product water and determining the relative ratio of non-exchangeable stable hydrogen isotopes in the thus prepared ethanol from product water ( 5 Dn „ value of ethanol from product water), and for the purpose of detecting added water in food products further includes measurement at least one isotopic composition of the product, i.e. relative ratios of isotopes, from the group consisting of:
- Oxygen in the water molecules of the product,
- Carbon in the molecules of the various tested product analytes,
- Oxygen in the alcohol molecules of the product, i
- Non-exchangeable hydrogen in the alcohol molecules of the product, i
Comparison of the measured isotopic composition of the product with the corresponding known isotopic composition of the product of known origin.
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| RS20220459A RS20220459A1 (en) | 2022-05-16 | 2022-05-16 | PROCEDURE OF ETHANOL PREPARATION IN THE WATE OF THE TEST PRODUCT AND DETERMINATION OF THE RELATIVE RATIO OF NON-EXCHANGABLE STABLE ISOTOPES OF HYDROGEN IN SO PREPARED ETHANOL (δDnII VALUE OF ETHANOL FROM THE PRODUCT WATER) AND FOR THE PURPOSE OF DETECTION OF ADDED WATER TO FOOD PRODUCTS |
| RSP-2022/0459 | 2022-05-16 |
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| US20110136097A1 (en) * | 2008-05-15 | 2011-06-09 | Ivan Smajlovic | Method for determining origin of alcohol or sugar containing products |
| RS52615B (en) | 2008-05-15 | 2013-04-30 | Ivan SMAJLOVIĆ | Apparatus for determination of isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples and procedure for determination authenticity and geographical origin of products |
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2022
- 2022-05-16 RS RS20220459A patent/RS20220459A1/en unknown
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| US20110136097A1 (en) * | 2008-05-15 | 2011-06-09 | Ivan Smajlovic | Method for determining origin of alcohol or sugar containing products |
| RS52615B (en) | 2008-05-15 | 2013-04-30 | Ivan SMAJLOVIĆ | Apparatus for determination of isotopic composition of non-exchangeable hydrogen and deuterium atoms in ethanol samples and procedure for determination authenticity and geographical origin of products |
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