EA045115B1 - METHOD FOR PROCESSING Caviar FROM LIVE, MATURE, OVULATED FISH OR CRUSTACEAN EGGS AND RELATED PRODUCT - Google Patents

Description

The invention relates to a method for producing caviar or caviar product from live, mature eggs of fish or crustaceans, wherein the live, mature eggs are in a fertilizable, but unfertilized state, and have a natural potassium content in the cytoplasm, by processing the live, mature eggs in a solution of table salt that is harmless to them, and then at least in a solution containing water and at least one cationic component dissolved in it, acting as a stabilizing shell of the eggs of live, mature eggs, and to the caviar or caviar product.

Cooked, unfertilized eggs, particularly from fish, are prized as a delicacy and are consumed in increasing quantities. Fish eggs are called (in layman's terms) eggs in any degree of maturity, that is, from immature to mature, and the degree of development of the eggs is not clearly defined. Eggs are live, mature eggs that female fish, lobsters, or other aquatic animals lay in a body of water to fertilize. Ovulated eggs are mature, fertilizing, living eggs that have emerged from the follicular membranes in the ovaries and were transferred to the body cavity. From there they are then withdrawn or squeezed out. According to the Food Codex (Codex Alimentarius Commission) of the Food and Agriculture Organization of the United Nations (FAO), edible caviar can only be obtained from fish eggs of female fish of various sturgeon breeds. Meanwhile, along with wild sturgeon, sturgeon breeding in aquaculture hatcheries in fresh water is also used to obtain caviar. With a few exceptions, sturgeon spawn exclusively in fresh water. The best-known species of sturgeon (Acipenseridae) include, among others, the species A. baerii, A. guldenstaedtii, Huso huso (also called beluga), A. transmontanus, A. ruthenus, and their albinos. But hybrids between A. schrenckii (females) and A. dauricus (males), and with sturgeons, which are closely related to the American paddlefish (Polydon spatula), should also be mentioned. There are numerous varieties of caviar on the market, which, in particular, are called stellate sturgeon, sturgeon and beluga. White caviar (also golden caviar) is obtained from albino sturgeons. In some cases, so-called royal caviar is created from albinos of the A. ruthenus breed in aquaculture factories. But we are not talking about truly royal caviar, which originates from albino beluga sturgeon (Huso huso) and is very rare.

Currently, caviar products from about 38 other varieties of fish that are not sturgeon are obtained and sold on the market, for example, compare the publication of the authors P. Bronzi et al.: Present and future sturgeon and caviar production and marketing: A global market overview (Current and Future Status of Sturgeon and Caviar Production and Sales: General Market Overview) (Journal of Applied Ichthyology, 2014, SI Volume 30, Issue 6, pp. 1536-1546). These include caviar products from tuna, lumpfish, salmon, trout, herring, cod, carp, whitefish and capelin, from which immature caviar is extracted for caviar products (also called caviar substitute or fake caviar). The roe of lobster, large crayfish and other crustaceans can also be processed into caviar products. These specified (and also other suitable, but not named) fish and crustaceans are referred to by the method described in the invention and the products obtained by it. If a clear indication of sturgeon caviar is not given, then it should be kept in mind and always mean both caviar and caviar products of fish other than sturgeon, and crustaceans, in particular, lobster and broad-clawed crayfish.

Caviar and caviar products are valuable food products. Caviar is rich in protein with a high content of vital amino acids, and fat. Caviar contains vitamins D, E, B ₁₂ and nicotinic acid, minerals based on sodium, potassium, magnesium and calcium, as well as trace elements phosphorus, fluorine, iodine and zinc. In addition, it has a high content of good cholesterol (HDL). Caviar and caviar products can be used both as food and as a material in the production of cosmetics or in other industries that use similar valuable materials. The size and strength of the eggs largely depend both on the given breed of fish or crustacean, and on the maturity and thus on the time of its fishing.

There are currently some caviar products on the market made from mature ovulated sturgeon eggs. But at the present time, caviar is still mainly offered, in relation to which we are talking about immature fish eggs, which are obtained from the ovaries of killed sturgeons. We are talking about traditional methods of obtaining caviar. First of all, with regard to the caviar of aquaculture-grown sturgeon, we proceeded from the fact that here, as before with wild fish caviar, immature eggs without additional processing have sufficient strength under conditions of invasive processing in the washing stages to remove residual tissue of the gonads and for packaging. However, with the accumulation of industrial experience in obtaining sturgeon caviar from aquaculture over the past 20 years, it turned out that this immature fish roe of aquacultured killed sturgeon is too soft, and can only be processed using borax or other preservatives, or pasteurization, to obtain a product suitable for packaging and stored for more than 2-3 months.

The slaughter of female animals for caviar in the case of wild catches, together with severe overfishing, pollution of water bodies with industrial, agricultural and domestic wastewater, and the construction of gates and dams blocking migration routes to spawning grounds in fresh waters, leads to a huge threat to the population of almost 27 different breeds of sturgeon. In many

- 1,045,115 regions are still plagued by poaching and the illegal black market, despite sturgeon protections under the Washington Treaty for the Protection of Wildlife Species (CITES). Expensive re-introduction programs have been initiated around the world, but which, according to the World Sturgeon Conservation Society, have unfortunately proven to be of limited success, for example primarily in China, but also in Iran and Russia. Only conservation and repopulation efforts in the United States and Canada have shown initial success. Along with the permitted harvest of mature animals, sturgeon of various strains, also from aquaculture, have been released into open waters as part of recovery programs to save endangered stocks, with varying degrees of success. Stocks of crustaceans and European crayfish are also at great risk, among other things, due to environmental pollution and introduced diseases, such as crayfish plague. Decisive progress in crayfish aquaculture and extensive stocking of juveniles are important for the conservation of local stocks. In aquaculture, female animals are kept alive for breeding and the eggs are obtained by scraping. On the contrary, to obtain caviar in aquaculture, it is still traditional, as a rule, to simply kill female sturgeons, since gentle methods do not dominate. At the same time, they completely ignore the fact that they exhibit significantly improved reproductive function with age. Also, the methods of caesarean section partially practiced in Russia are by no means considered gentle methods, since they are accompanied by a high mortality rate of sturgeon treated in this way.

State of the art

From RU 2232523 C2 a method is known for producing granulated caviar from ovulated sturgeon caviar. In this case, the collected ovulated eggs are first treated in a hot aqueous solution of a preservative with a concentration of 1.5 to 2% in order to prepare them for subsequent pasteurization at temperatures from 65 to 70 ° C. In addition to the fact that each heating process significantly affects the taste of fish eggs, when using ovulated eggs, which have a very soft and sticky egg shell, it is not guaranteed that they will withstand subsequent treatments with preservatives without tearing. But even a small proportion of torn eggs significantly worsens the quality of the eggs, since torn eggs can only be removed with great difficulty. Pasteurization leads to denaturation of valuable proteins and gives caviar a mealy taste.

In connection with obtaining ovulated sturgeon caviar, for example, from the publication of the authors M. Szczepkowski et al.: A simple method for collecting sturgeon eggs using a catheter (Arch. Pol. Fich. (2011) , volume 19, pp. 123-128) there is a known application for this catheter. The eggs can thus be easily drained or sucked out under reduced pressure. It is also known to extract eggs from the abdominal cavity of sturgeon by simple massaging. This method is called squeezing, and is the most gentle method of collection.

The closest embodiment to the invention according to the prior art is disclosed in WO 2007/045233 A1. A method is described for obtaining caviar or caviar products from mature ovulated but unfertilized eggs of aquatic animals, preferably fish, by externally processing the mature eggs in solution, which results in an internal morphological change in the acellular egg membranes that separate the egg (the cytoplasm of the egg with the surrounding egg membrane) from the environment, with structural stabilization. In this case, the solution used contains water and at least one cationic component (calcium cations Ca~), which is dissolved in water at a predetermined concentration, and upon contact with the eggs causes structural stabilization. Calcium is involved in molecular signal transduction typical of cells, and in the egg cell a calcium wave is triggered in its cytoplasm, which again leads to a cortical reaction and to the release and activation of ovoperoxidase. This enzyme in the extracellular shell of the egg ensures irreversible structural cross-linking of protein chains as a result of the incorporation of tyrosine molecules into the internal radiate zone (Zona Radiata Externa) and the external radiate zone (Zona Radiata Externa). Thus, the induced process in a living, metabolic egg leads to structural stabilization of the egg shell. In immature eggs, such stabilization is not achieved, since the corresponding receptors and enzymatic cascades have not yet matured. In killed eggs the process cannot be initiated because metabolism no longer occurs. Living mature eggs, thanks to the ovarian fluid, upon contact with water, immediately form an adhesive layer so that in nature, at the spawning site, they are able to firmly adhere to stones and plants. Therefore, before processing, the eggs are washed with a (physiological) solution of table salt that does not damage living eggs to remove ovarian fluid. Due to this, living mature eggs have a natural potassium content in the cytoplasm. That is, for example, they were not given any harmful doses of potassium before collection (for example, to initiate ovulation).

The described sequence of reactions is called the second reaction in the literature. This is a slow metabolic reaction that, after the fusion of the first sperm with the egg, builds a permanent, physical-mechanical structure to protect the egg from aggregation on the egg with additional external sperm (polyspermy), but above all from environmentally harmful substances, microbes and mechanical damage to the resulting embryo . In the known method, this second reaction is triggered without fertilization by sperm. The achieved structural stabilization when consuming the product provides a bursting effect and explosive release of liquid cytoplasm. Preference for a specific popping effect is highly dependent on the use of the caviar and the nature of the consumers.

In addition, it is known from EP 2522226 B1 to preserve unripe unprocessed eggs of aquacultured fish in a composition of the flavonoid and antioxidant taxifolin (dihydroquercetin) and an organic salt, in particular also potassium citrate.

But the high concentrations of the composition used in this case lead to obvious changes in the ion content in the intracellular environment, which initiates programmed cell death (apoptosis). That is, caviar treated in this way immediately dies. The published documents SU 1662469 A1 and RU 2048111 C1 present methods for preserving sturgeon caviar, which use potassium compounds in such huge concentrations that apoptosis immediately develops in the eggs and they die. The same is also true for the document EP 2868207 B1 corresponding to the above-mentioned document RU 2232523 C2, and here additional denaturation due to heating also occurs.

The publication by G. E. Bledsoe et al.: Caviars and Fish Roe Products (Critical Reviews in Food Science and Nutrition, Vol. 43, No. 3, May 1, 2003, pp. 317-356) describes, within the fundamental use of nitrate in general as a preservative for the caviar of crabs, sturgeon and other fish, as well as the use of potassium nitrate. But this, as is usual in all preservation processes, is carried out at such high concentrations that a cell-destructive effect is exhibited, which sensitively disturbs the osmotic balance and immediately kills the treated eggs. In addition, only immature eggs at an early stage of development of gutted fish are preserved, which, firstly, need to be mechanically scraped out of the gonads, reconciling with damage, and, secondly, which do not yet have fully formed structures in the mature shell of the eggs, so this approach is not applicable for the invention.

From DE 2416685 A there is known a method for improved preservation of the red color when preserving salmon caviar or salmon by adding additives permitted by food legislation in the form of citrate. Upon completion of the process, it remains detectable in the final product and changes its composition. The high concentrations used in this case (from 5 to 10 wt.%) again kill live eggs immediately after being brought into contact. Since only immature caviar is used, it can be washed with water. In the case of mature caviar, the already mentioned sticky gel-like coating would form. Freezing unripe eggs, both before and after canning, always leads to damage to the frozen egg membrane and increased water loss, since the unformed and unstabilized egg shell cannot protect it. From JP S63 - 36763 A there is known a method for reducing the concentration of sodium chloride when preserving fish eggs in order to reduce the salty taste. In this case, sodium chloride is also replaced with various potassium compounds. True, this is again carried out at such high concentrations that the eggs, in relation to which we are talking about immature eggs, die, and no further metabolism can take place. From JP 2001 - 299 285 A there is known a method of processing caviar frozen in an unripe state to improve the texture. In this case, the eggs are washed at 5°C for up to 24 hours using potassium-containing chemicals. Such a long duration of treatment interrupts any metabolic process. Since the eggs were frozen without frost protection, the immature eggs no longer exhibit any metabolic activity and are also not capable of maturation. Therefore, they are also not applicable to the claimed invention. But the method presented by the invention does not deal with destructive discoloration or freezing caused by canning, but with the primary production of caviar and caviar products from unprocessed mature live eggs. Canning or freezing after processing live eggs are only optional additional steps for the invention. Discoloration is completely eliminated as it is not required.

From the publication of the authors Huang Hui et al.: Effect of Synthetic Preservatives on Volatile Flavor Compounds in Caviar of Sturgeon (Huso dauricusxA. schrenckii) ([J] FOOD SCIENCE, 2015, volume 36 (issue 12): pp. 97-103) there is a known desire to prevent loss of taste during storage of chilled caviar, for which synthetic preservation is carried out using preservatives such as potassium sorbate (E202, sorbic acid) and ascorbate (vitamin C). Potassium sorbate was used in different test groups at a constant level of 0.5 ppm. We used unripe eggs, which, as a result of preservative treatment, should receive an intense taste. Potassium sorbate acts as a mold and fermentation inhibitor, but can also reduce the flavor of the product.

Publication by L. Dufresne et al.: Kinetics of actin assembly attending fertilization or articial activiation of sea urchin eggs, Experimental Cell Research, Elsevier, Amsterdam, The Netherlands, volume 172, No. 1, September 1987, pp. 32-42) describes the ability of sea urchins to be artificially activated. Although the present invention does not deal with the processing of sea urchins, since they are not

- 3 045115 have the proper structure (egg shell with only two layers), this publication should be briefly discussed at this point. First, we used sea urchin caviar, which was obtained by injecting a solution of 0.5 M KCl with an extremely high concentration of potassium chloride into the abdominal cavity of the sea urchin. As a consequence, the egg has already been greatly influenced in terms of its state of electrical polarization, and the natural potassium content of the cytoplasm has clearly been altered. In addition, all sea urchin eggs were brought into contact with water before processing and formed a gel coating, which then had to be mechanically removed. As a result, the morphological and physiological properties of the acellular shell of the eggs are also fundamentally changed. Thus, sea urchin eggs, due not only to their fundamentally different structure, but also due to gross interference with metabolism by the action of potassium chloride during their extraction, are not applicable in the invention. In addition, calcium-free seawater is not deionized and contains per liter, among other things, over 10 g of sodium, 0.43 g of potassium and 1.3 g of magnesium, and 20 g of chlorine. Thus, washing eggs with calcium-free water also does not correspond to washing them with a solution of table salt that does not damage live, mature eggs.

The structure of the shell of fish eggs and crustacean eggs corresponds to the basic principle, and for the method will be described according to the terminology given in the publication of the authors Siddique et al.: A review of the structure of sturgeon egg membranes and of the associated terminology (Review of the structure of sturgeon egg membranes, and related terminology), (J. Appl. Ichthyol. (2014), pp. 1-10). To ensure consistency of concepts, it is worth briefly addressing this here.

In the case of mature eggs (oocytes, eggs) in animal ovaries, a follicle consisting of granulosa cells (also called follicular cells, follicular membranes, follicular epithelium) and theca cells (also called theca interna, theca externa) surrounds the egg to supply it with signaling signals. and nutrients. Between the granulosa cells and theca cells there is also a basement membrane (in science also called the perifollicular membrane, membrane, basement membrane). The egg plasma (oocytoplasm, Olemna, cytoplasm, egg interior) is surrounded by the egg membrane (oocytomembrane, PM plasma membrane, egg cell wall). During ovulation, the eggs are separated from the follicular cells and released into the abdominal cavity of the fish. The ovulated egg now retains its acellular egg shell (also called the extracellular matrix or extracellular membrane) formed during maturation itself, which is structurally formed, from the outside to the inside, from the alveolar layer AL (also called the gel coat, adhesive layer, gel coat in science, ( second outer) gelatinous membrane, layer 3, chorion (2)), outermost layer of the acellular egg membrane of the external zona radiata ZRE (in science also called the external vitelline zona (zona radiata = vitelline membrane), outer vitelline zona, outer vitelline membrane, layer 2, layer 2 of the chorion, Zona Pellucida Externa (Latin), Zona Radiata Externa (Latin), layer 1B of the shell, second shell), the outermost part of the vitelline shell, lying directly under the alveolar layer of the epitaxial EP layer (in science also called the epilayer 1, layer 4, outer layer of the first shell), which separates the ZRE from the ZRA, is not present in all types of eggs of the internal zona radiata ZRI (scientifically also called the inner vitelline shell, inner vitelline zone, inner vitelline membrane, layer 1 of the chorion, Zona Pellucida Interna (Latin), Zona Radiata Interna (Latin), layer 1A of the membrane, the inner layer of the first membrane), the innermost part of the vitelline membrane, closely associated with the ZRA, and the perivitelline space (in science also called the extraoocyte matrix, the gap with the microvilli processes cytoplasm), a thin space between the ZRI and the egg membrane, into which the cytoplasm protrudes numerous microvilli (MVs).

Living ovulated eggs are electrically excited through ion channels that are localized in their cell membrane. Changes in the electrical properties of the cell membrane, among other things, create the prerequisites for the activation of the egg and act in the egg shell. Pioneering work in marine invertebrates has demonstrated ionic currents of potassium cations across the egg membrane that cause a transient change in potential across the membrane (fertilization potential FP). This potential is generated by the activation of a temporary voltage-dependent internal current within the egg. It has been shown that depolarization of the membrane potential (RP) is caused by the flow of ions across the egg membrane (ion current (fertilization current FC)). This current flows through the openings of larger non-specific and highly conductive ion channels, which can be activated by sperm or artificial chemical or mechanical stimuli. Hypothetical models so far regarding the role of different ion channels and the corresponding ions show species-specific differences.

In natural eggs, potassium plays a central role, see E. Tosti et al: Electrical events during gamete maturation and fertilization in animals and humans (2004 Human Reproduction Update, vol. 10 , pp. 53-65). Potassium K+ is the cation that decisively determines the resting potential of the egg. At the same time, the potassium ⁺ -gradient and the permeability of the egg for regulative ions

- 4 045115 are regulated by transport proteins and ion channels. According to research by the Alfred Wegener Institute, in mature unfertilized eggs of the sturgeon species A. baerii, the natural intralet concentration of potassium cations is 50 mmol/l. In contrast, extracellular calcium has no effect on the resting potential/fertilization potential of the eggs, and therefore itself is not even involved in the first rapid electrical blockade (see below). The ionic composition inside the egg is different from the ionic composition of the external environment. This separation between the inside of the cell and the outside environment must be maintained for metabolic activity, and thus for the survival of the cell. The different distribution of electrical charges inside and outside the cell creates an electrical gradient across the cell membrane, which can be measured as a potential difference (resting potential).

The essence of the invention

Based on the prior art closest to the invention according to WO 2007/045233 A1, which in terms of the generic concept also serves for the production of caviar and caviar products, the object of the present invention should be seen in such an improvement of the method described there based on live, unfertilized, mature eggs of fish or crustaceans that a modification of the organoleptic perception regarding texture, taste, transportation, storage and deep freezing of mature eggs can be achieved. But at the same time, the above-described advantages with respect to caviar or caviar products obtained by this method or otherwise, which are also claimed by the invention, must remain unchanged. The solution to this problem follows from the independent claim of the invention. Preferred improvements to the invention are set out in the dependent claims and product-describing claims and are subsequently explained in more detail in connection with the invention.

The claimed method for producing caviar or caviar product based on live, mature eggs of fish or crustaceans according to the invention is characterized in that at the stage of exposure to potassium as a cationic component, potassium is dissolved in water to a concentration that does not damage live, mature eggs and does not change the natural potassium content in them, wherein the water before adding the potassium donor is deionized to form a cationic component, and has a temperature that is not damaging to live, mature eggs, and that the live, mature eggs are subjected to treatment in solution during the time of exposure to potassium until the desired elastic stabilization of the shell of the eggs is achieved.

The method claimed in the invention uses live, mature eggs, which can be obtained naturally, without harm to the fish or crustacean. Living, mature eggs are capable of fertilization, but are in an unfertilized state. Ovarian fluid has been removed by previous washing with a non-damaging solution of table salt to the living, mature eggs, so that an adhesive gel layer cannot form on the outer shell. In addition, eggs have a natural potassium content that has not been artificially altered. Concentrations of potassium cations that are extremely effective in relation to cellular physiology are used. Living eggs, which in the case of fish and crustaceans have more than two layers in the egg shell, that is, three or more layers, are electrically activated. The initial product is live, mature, fertilizing, but unfertilized eggs, in which the metabolism is fully functioning, so that even with insignificant concentrations of potassium cations, which do not cause harm and also do not leave any traces in the eggs, transport processes through the egg shell can operate and metabolic processes in the cytoplasm. The caviar or caviar products created or produced in this way exhibit a new texture with favorable stabilized elasticity through a new translucent extracellular layer (elastic stabilizing layer) in the egg shell, which softens somewhat at room temperature without reducing the stability of the caviar. The desired degree of elastic stabilization can be easily determined by personal testing (degree of elasticity of the eggs). The taste is pleasant, fresh and spicy, without a fishy aftertaste. Thanks to the purity of the eggs used, without the addition of preservatives, for example, borax, which is banned in many countries, they achieve a long shelf life (from 9 to 12 months) at standard temperatures between -2°C and -4°C. Eggs processed by the claimed method can also be frozen once without loss of quality, which results in enormous advantages in terms of storage and transportation, compare additionally below.

As a result of bringing live, mature eggs into contact with potassium cations (K ⁺ ) in a physiological, that is, cell-harmful concentration according to the invention, the eggs change within the framework of electrical effects, and the so-called first reaction is initiated, which in the shortest time (in the range from seconds to minutes) leads to electrically induced removal of stickiness upon contact with water, and during the subsequent course of processing causes the formation of a new, elastic stabilizing layer inside the egg shell. This new stabilizing layer gives the caviar shell elasticity, so that according to the invention, already at this point in time the caviar or caviar product is of the highest quality, so that it can be processed in optional technological stages, in particular canning and deep freezing (at -18° C), without loss of quality.

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Activation of eggs includes and goes through a whole series of cellular biological cascades. The second (slow) reaction (slow blockade) with the cortical reaction used in WO 2007/045233 A1 is accompanied there by a calcium-dependent enzymatic activation for strengthening and, finally, for a significant structural rearrangement of the egg shell as a result of irreversible tyrosine cross-linking of protein chains in Zona Radiata Interna and Zona Radiata Externa. In nature, this prepares the first cell division for the development of the embryo. In contrast, the first (fast) reaction used in the present invention (fast blockade, electrical blockade, fast electrical blockade) followed by depolarization/hyperpolarization and their continuity with varying durations depending on the animal species occurs at the beginning of all cell biological cascades. Both processes clearly differ depending on the substances used, namely, A) potassium cations for rapid electrical blocking with depolarization of the egg shell and the resulting activation of the egg and the formation of a separate, additional, new zone in the egg shell (formation of an elastic structured layer), and B ) calcium cations for slow mechanical blocking with enzymatically controlled morphological restructuring in the existing layers of the egg shell (formation of a structural stabilizing layer).

The first electrical event (act) is a rapid depolarization or even hyperpolarization within milliseconds, and in nature after fertilization should prevent the attachment of additional sperm. Rapid hyperpolarization is replaced by constant hyperpolarization within a subsequent time of up to 60 minutes (in some species of aquatic animals, such as lobsters, even up to 5 hours). The sperm remaining in the environment of the egg, although after a quick electrical blockade can still attach and fixate in the vitelline membrane of the egg shell (soft shell), cannot penetrate the cell membrane for the egg plasma for actual fertilization, as was shown in mollusks. With continued exposure to potassium cations according to the method claimed by the invention, in a living, fertilizing, but unfertilized and mature egg, the formation of a hitherto unknown, completely new, translucent (translucent, glassy, gel-like) zone (elastic stabilizing layer) is observed, which is GAG-positive (increasing appearance of glycosaminoglycans) and eosinophilic (stained with the red, acidic diagnostic dye eosin) to visualize cellular organelles, plasma proteins, connective tissue and their pre-stages) in which sperm would become stuck. The formation of this elastic stabilizing layer occurs during a constant hyperpolarization of 10 s, first in selected areas within the egg shell, and after completion it is located between the Zona Radiata Externa and the alveolar layer in living eggs with a structural configuration similar to that of fish and crustaceans (at least two layers in the egg shell). The reason for the formation of a new elastic stabilizing layer is seen in the continuous depolarization of the egg shell as a result of the supply of potassium cations in physiological concentrations in accordance with the invention.

According to European Union (EU) regulations regarding food additives for nutritional purposes, only the potassium compounds listed there are permitted, e.g. potassium bicarbonate (potassium bicarbonate, KHCO3) (CAS-No. 298-14-6), potassium carbonate (K2CO3) (CAS-No. 58408-7), Potassium Citrate (CaS-No. 6100-05-6), Potassium Hydroxide (KOH) (CaS-No. 1310-58-3), Potassium Chloride (KCl) (CaS-No. 7447-40-7), K, potassium iodide (KI) (CAS-No. 7681-11-0), potassium iodate (KIO3) (CAS-No. 7758-05-6). These compounds may also be added to foodstuffs according to the draft regulation of the European Parliament and of the Council of 10.11.2003 (finally KOM (2003) 671). Certain potassium compounds can also be added to food products for technological purposes, for example, potassium citrate (E 332), potassium lactate (E 326), potassium orthophosphate (E 340). By using the method according to the invention for treating live, mature fish or crustacean eggs with potassium cations in cell-acceptable concentrations (physiological concentrations, that is, not damaging the eggs) and without the formation of residues, caviar or caviar products can be created that satisfy all national and international requirements of authorities , trade organizations and consumers to quality. Studies of intracellular ion concentrations using optical emission spectroscopy (OES) in the cytoplasm of eggs at the Alfred Wegener Institute, in which potassium was used as a new substance to permanently depolarize the outer membrane of the eggs, showed no changes in the concentration in the egg plasma after treatment, even at different applied concentrations and length of processing time. The potassium cations used in the method according to the invention are therefore clearly considered to be technical auxiliary substances. The technical auxiliary substance is used in industrial processing and production of food products. Technical auxiliaries are food additives that are added to facilitate technological processes such as cutting and filtration. However, the final product must contain no technical auxiliary substances at all, or be present only in unavoidable (insignificant) residual quantities. Unlike modified food additives, which must also be declared on the packaging, technical excipients can no longer have any effect in the final product, which

- 6 045115 is a special advantage. Their use must be technically inevitable, not affecting the technological regime, not causing health concerns, and also harmless to smell and taste. Since the substances in processed foods are no longer present or effective, their use does not need to be labeled. This is also true for residues, reaction products or residual contents.

It is particularly preferred and advantageous when at least one potassium salt, preferably a citric acid salt (potassium citrate E332), and/or a hydrochloric acid salt (potassium chloride E508), and/or sorbic acid salt (potassium sorbate E202). By potassium donor is meant a compound that, after being dissolved in water, supplies potassium cations, their concentration being regulated by the concentration of the potassium compound in question in water and in accordance with its structural formula. Even all of the potassium salts mentioned are acceptable as food additives with E-numbers, although according to the invention they are used only as technical auxiliary substances that are no longer contained in the final product and are not subject to notification. A particularly favorable and preferred concentration of potassium cations in solution using pre-deionized water, according to the following modification of the method, is between 0.1 and 3.0 mmol/l, preferably 0.1 mmol/l, 0.5 mmol/l, 0 .65 mmol/L, 1.6 mmol/L, or 2.0 mmol/L, especially preferably 0.1 mmol/L and 1.5 mmol/L. In this case, in all ranges of values (also of other parameters) given within the framework of this invention, all edge and intermediate values (integer and non-integer) are always taken into account. In order for the indicated concentrations of potassium cations in water to be obtained, it must be deionized. But since dissociation of water molecules also constantly occurs in water, it is clear that a feasible degree of deionization can only be achieved by technical means (electrical conductivity in water between 1 μS and 15 μS at 25°C as a measure of achievable deionization).

Moreover, the duration of potassium exposure in the potassium exposure step is preferably and advantageously between 5 and 30 minutes, preferably for 10 minutes, 12 minutes, 15 minutes, 20 minutes or 25 minutes. But other durations of potassium exposure can also be chosen. When processing mature lobster (crustacean) eggs, the duration of exposure can be up to 50 minutes or more. The formation of a new elastic stabilizing layer, which compacts the egg shell, begins within a few seconds (up to 10 s) after the start of processing. But since not all eggs simultaneously react with constant depolarization and with the formation of a stabilizing layer in partial areas of the surface of the egg shell, longer treatment times of up to 10 minutes are recommended to achieve constant depolarization in all treated eggs. As the duration of treatment increases, it moves outward and is finally localized between the Zona Radiata Externa and the alveolar layer in the entire egg shell around the egg. In this way, live, mature eggs uniformly achieve elastic stabilization so that they can be salted, packaged and deep-frozen without any problems.

As a further optional modification, the method claimed by the invention may additionally include a calcium exposure step, which can be carried out after the potassium exposure step, or can be carried out as a precursor to it. The resulting changes in each case on the egg shell in both sequences occur independently of each other with respect to their described characteristics. In the calcium exposure step, calcium is preferably and advantageously dissolved in water to form another solution of calcium as the cationic component at a concentration that is not damaging to live, mature eggs (i.e., physiological) and the water is deionized before adding the calcium donor to form the cationic component. Live, mature eggs at the calcium stage are treated for as long as possible until the desired structural stabilization of the egg shell is achieved. The desired degree of structural stabilization can be determined without problems by in-house testing (degree of calf bursting effect). In the calcium action step, at least one calcium salt, preferably calcium citrate, calcium chloride and/or calcium sorbate, is preferably and advantageously used as a calcium donor (calcium supplier, definition see as for potassium donor). Calcium salts as food additives are authorized in the European Union under the numbers E333 and E509, without maximum quantity limitation, and E203 with maximum quantity limitation. To better distinguish the addition of both types of ions, the orthographic forms potassium and calcium (not Kalzium) were chosen.

Calcium is physiologically already present in the egg and is an essential part of cellular metabolism. From the document WO 2007/045233 A1 already cited above, it is known to use calcium chloride, and thereby achieve structural strengthening of the egg shell as a result of irreversible cross-linking of proteins by the incorporation of tyrosine molecules. In addition to the improved and adjustable elasticity of the egg shell, which is fundamentally achieved in the invention, it can also be structurally strengthened mechanically by carrying out a calcium treatment step. In this way, an optimal stabilizing combination for certain types of caviar and caviar substitutes can be achieved. In particular, this is advantageous in the case of very large unstable eggs (more than 3.2 mm in diameter, for example, beluga sturgeon eggs, or white sturgeon), or those that are particularly soft when ripe (at maximum strength, less than or equal to 0.3 N before breaking in a strength test, for example, sterlet caviar as sturgeon). Moreover, as a result of using both stages of processing, very high-quality caviar or caviar products are obtained without caviar that creates problems (in terms of size, softness).

Advantageously and preferably, the concentration of calcium cations in the other solution is between 0.1 mmol/L and 3.0 mmol/L, preferably 0.1 mmol/L, 0.5 mmol/L, 0.8 mmol/L, 1. 0 mmol/L, 1.5 mmol/L, 1.6 mmol/L or 2.0 mmol/L. The duration of calcium treatment is preferably between 9 and 30 minutes, preferably for 10 minutes, 12.5 minutes, 15 minutes, 16 minutes, 20 minutes or 25 minutes. When choosing the duration of treatment, it should be taken into account that the strength of the egg shell constantly increases with increasing duration of exposure to calcium up to the limiting value. In nature, in fertilized fish eggs, a highly hardened egg shell is reached after about 60 minutes, so that it becomes unsuitable for nutrition. For a lobster this can last up to 24 hours.

An essential technological parameter for the method claimed by the invention is the temperature of the solutions in which mature eggs are processed. It must be physiological, that is, it does not interfere with the natural processes in living eggs. In the claimed method, the temperature of the solutions is always within the range of the natural temperature of fish or crustacean eggs. This ensures that the electrical activation of the egg shell, induced during the potassium treatment stage, reliably occurs with depolarization starting from the resting potential. At unnatural egg temperatures, for example in fish or crustaceans from polar regions, above 15°C, on the contrary, electrical activation does not occur, and live mature eggs cannot be stabilized electrically or enzymatically. They are atretic. The same is true for fish eggs or crustaceans from temperate and tropical climates. In principle, it is true that at solution temperatures above 35°C, eggs undergo severe quality losses due to degeneration.

To harmonize the temperatures of the solutions with the conditions of the natural environment, in the present invention, the habitat areas of fish and crustaceans during periods of natural reproduction, during which live, mature eggs can be used, are roughly divided into three climatic zones: polar zones (at the poles), temperate zones ( between the polar zones and the tropical zone), the tropical zone (near the equator). In the present invention, it is preferably and advantageously provided that the temperature of one solution (when treated with potassium) and/or the other solution (when treated with calcium) in the temperature range of the polar region is taken to be between 1°C and 15°C, preferably between 5°C and 12°C, especially preferably 10°C, in the temperature range of the temperate region is taken to be between 10 and 20°C, preferably 15°C, especially preferably 12°C, or in the temperature range of the tropical region between 20°C and 29° C, preferably 27°C, especially preferably 21°C. Temperatures that lead to changes - degeneration, death of cells - eggs, for example, such as those that occur during pasteurization by heating at temperatures above 40 ° C, are not allowed in the invention at any time during the execution of the method.

Since the concentrations of cations used in the method according to the invention excite breed-specific physiological reactions of an electrical (effect of potassium cations) and metabolic (effect of calcium cations) nature, and thereby influence the processing with the formation of a stable final product suitable for nutrition, it is always necessary to proceed from deionized water in solution to achieve precise concentrations of electrically (potassium cations) and metabolically (calcium cations) active (positively charged) cations. It is therefore technically achievable and thereby preferred or advantageous for deionized water at 25° C. to have an electrical conductivity of between 1 μS/cm and 15 μS/cm, preferably 10 μS/cm or lower, particularly preferably 1 μS/cm. Drinking and artesian water, depending on regional sources, has a highly variable content of various ions, which, depending on the circumstances, can even have opposite effects on cellular metabolism. At a temperature of 25°C, the electrical conductivity of, for example, the purest water is 0.055 µS/cm, deionized water 1 µS/cm, rain water 50 µS/cm or drinking water 500 µS/cm. To be able to obtain reproducible results when implementing the claimed method, it is important to know the electrical conductivity of deionized water.

Since the method claimed in the invention treats living cells in the form of activated mature eggs, then, among other things, it is also important that the solutions are coordinated with the metabolism of the cells, thereby the metabolic processes induced during the execution of the method can also occur. It is therefore favorable and preferred when one and/or the other solution has a (physiological, not harmful to the living organism) pH value between 6.8 and 8.0, preferably between 7.0 and 7.9, especially preferably 7.2, or 7.4, or 7.5. In particular, established in

- 8 045115 solution pH value is important for the slow metabolic reaction during the calcium treatment stage. Since enzymatic processes in the cell are largely regulated by pH, the intracellular pH value was also studied at the stage of potassium treatment (electrical process). But the pH value in the cytoplasm of eggs treated with various potassium-containing substances at different concentrations and for different durations remains essentially unchanged with an optimal pH value between 7 and 8, and exhibits the expected individual differences in individual fish and crustaceans.

According to the invention, the various stages of exposure serve to endogenously stabilize (elastic and optionally structural) the egg shell of living, mature eggs. Thus, the caviar or caviar product is already ready for further processing, such as salting and packaging. During ovulation, live, mature, ovulated eggs are squeezed out of the follicular cells so that there is no longer any tissue debris from blood vessels or follicular cell debris that bacteria or fungi can settle on. Therefore, the collected eggs have high purity, and thus the best prerequisites for long-term preservation. This is reliably ensured when, in the last stage of processing for preservation and intensification of taste, moderate salting with sodium chloride is carried out in an amount, relative to the amount of caviar or caviar product, from 2.0 to 3.8%, preferably 3.5%. In this case, sodium chloride should not contain potassium and calcium donors, for example, such as those contained in anti-caking agents, since this prevents uncontrolled changes in the shell of the eggs of the processed eggs during salting. In the case of sturgeon caviar, dry salting is carried out with simple table salt (sodium chloride NaCl), while wet salting is often performed when processing the eggs of other fish species to obtain caviar products, such as salmon and trout caviar. The salting carried out according to the invention in the specified range is a completely light salting, which is also called lightly salted and is an unambiguous sign of high quality. Pasteurization or heating at a temperature of 60°C and above for the caviar or caviar product claimed by the invention is completely excluded, since this is unnecessary and would only worsen the quality of the product and its organoleptic properties. Lightly salted processing during salting of caviar or caviar products obtained by the stated method ensures a minimum shelf life when stored at -2°C of at least 9 to 12 months. At the same time, thanks to only a light salting, they do not freeze.

An additional improvement in the quality of the resulting caviar or caviar product according to the invention is achieved when, according to an additional modification, immediately after preservation and intensification of taste, the caviar or caviar product is preferably and favorably stored in sealed airtight glass vessels for several months, preferably from one to three months . As a result of storage, caviar matures and, depending on the degree of ripeness, acquires an intense taste. But this ripening should be considered in the sense of an additional improvement in taste (for example, as in the case of cheese), and has nothing to do with the degree of maturity of the mature caviar used in the invention in the sense of biological development. Here, maturity refers to suitability for fertilization, and thus to the developmental state of living eggs. When ripened in terms of taste, storage is carried out in glass vessels, which leave the caviar enough space to ripen, since it is not compressed (as when packaged in metal cans with a lid placed on top), and thereby retains its flavor-intensifying oils. The caviar thus packaged in glass according to the invention should not be confused with pasteurized caviar, which is also often packaged in glass. Additionally, when stored in environmentally friendly glass containers, the often-criticized metallic taste of caviar traditionally packaged in metal cans is prevented.

According to the following modification of the method, it is preferable and favorable when, after canning and flavor intensification or storage and ripening in terms of taste, the caviar or caviar product is frozen at a temperature in the range between -20°C and -15°C, preferably at -18°C . Ideally, caviar is matured in flavor for human consumption to the consumer's desired degree of maturity and is frozen either fresh after 14 days of receipt or after a maximum of 3-4 months of ripening. The caviar is frozen either in 500g glass containers before being placed in an outer container, or after being placed in an outer container for end consumers in glass containers of 30g, 50g, 125g, 250g or 500g (if necessary, up to 1000g), which can be evacuated. Caviar obtained by traditional slaughter cannot be frozen. Although pasteurized or heated caviar can be frozen, it exhibits extreme deterioration in quality due to heat treatment. Thanks to the possibility of freezing caviar or caviar product according to the invention, optimal preparation for sale of caviar can be ensured, which meets modern requirements for ready-to-eat products. Preparation for sale, due to the specific, precisely maintained temperatures during transport and storage from -2°C to -4°C, still faces restrictions, since most providers do not comply with them. Therefore, traditionally the resulting caviar is treated with questionable preservatives, such as borax, or pasteurized to allow it to be preserved for at least a period of 12 months or longer. Meanwhile, the caviar obtained according to the present invention can be simply frozen, and thereby be stored and remain fresh for a longer period of time. Tests have shown that caviar slowly thawed at a temperature of +4°C to +7°C in the refrigerator does not show any loss of taste or changes in texture.

The processing of eggs according to the invention is carried out in a bath with a solution (aqueous solution, solution in water). Eggs are added and kept in a bath with a solution for such a time until, depending on the type of caviar used, the desired degree of stabilization (elastic and, as appropriate, structural) is achieved. Then the eggs are simply removed from the bath. In order to reliably avoid undesirable further stabilization by cations in the still adhering solution after extraction, according to an additional modification of the method, it is preferable and advantageous that, after achieving the desired elastic (and, optionally, structural) stabilization, immersion (short-term dipping) is carried out to remove the corresponding introduced cations from the mature eggs. live, mature eggs in a solution of table salt that does not damage them (physiological solution of table salt). As a result, the cations are washed away and the stabilization processes caused by them are immediately stopped. The previously achieved (desired) degree of stabilization of the egg shell is reliably obtained as the final state.

The live, mature eggs used according to the invention are capable of fertilization, but are not fertilized. In addition, they are not wetted with water and have a natural potassium content in the cytoplasm. Such live eggs can be hatched either from the gonads in the abdominal cavity of the fish, and from there collected through the genital opening. For example, this can be done by natural spawning, squeezing (massage of the abdominal cavity from the outside) or the use of a catheter through which the eggs flow out or are sucked out of the abdominal cavity. In relation to eggs extracted from the gonads in the abdominal cavity, they speak of ovulated eggs (5th degree of maturity), which are still surrounded by the mucous ovulation fluid. To prevent the formation of an adhesive layer on the eggs upon contact with water, the ovulation fluid is washed off with a physiological solution of table salt before starting treatment. Ovulated eggs can be obtained from a living animal, which is particularly time-consuming. But according to the invention, live, mature eggs of the 3rd or 4th degree of maturity can also be used, which are still extracted from the gonads of a killed animal and then sorted. A good overview of the different degrees of maturity in cod is given by I.G. Katsiadaki et al.: Assessment of quality of cod roes an relationship between quality and maturity stage (J. Sci Food Agric, vol. 79: pp. 1249-1259 (1999)) , in particular, there in the table. 1. Numerical determination of the degree of maturity can be done using the so-called polarization coefficient. It is calculated from the ratio of the distance between the cell nucleus and the cell membrane to the diameter of the egg between the animal and vegetal poles (semimajor axis). Therefore, according to a further embodiment of the invention, it is preferred and advantageous when live, mature fish or crustacean eggs are treated with a polarization coefficient PI of 0.05<PI<0.15, preferably 0.05<PI<0.12. Eggs with this PI are particularly suitable for collection for processing according to the invention. More detailed information on the PI polarization coefficient of eggs can be found, for example, in the guidelines for sturgeon farming (FAO Ankara publication 2011, Fisheries and Aquaculture Technical Paper 570 Sturgeon Hatchery Practices and Management for Release -Guidelines management of sturgeon breeding for release - instructions)).

The method claimed in the invention can process live, mature eggs of fish and crustaceans (scientific name Crustacea), the eggs of which have the basic structure necessary for the invention (more than two layers in the egg shell), and are suitable for consumption in the form of caviar or caviar products. Preferably and advantageously, live, mature fish and crustacean eggs from wild-caught or aquaculture culture that have been obtained in the ovulated state by extrusion or other targeted collection methods, such as using a catheter, can be processed. In this case, for example, the collection can also be carried out from such animals that are intended for re-release into the wild, for example, as part of a restocking project. Then the proceeds from the sale of caviar and caviar products can again be used for measures to stock juvenile fish. It is particularly preferable and advantageous when the claimed method involves processing live, mature eggs from modern and ancient bony fish, preferably from live sturgeon. Then (genuine) caviar of the highest quality can be obtained using this method. But the claimed method according to the invention can also produce high-quality caviar products from lobster or other crustaceans, for example, crayfish. In addition, very large (with a diameter greater than 3.2 mm) or soft, unstable eggs (with a texture in the hardness test below 0.3 N, with tearing at a higher value) can be processed preferably and favorably, as claimed in the present invention the non- 10 045115 method may also necessarily include two stages of influence for both elastic (electrically stimulated) and structural (enzymatically stimulated) stabilization of the egg shell.

Finally, the invention also claims various products from live, mature eggs of fish or crustaceans, which can be obtained by the claimed method, but also by other methods. The products typically differ in that an elastic stabilizing layer is additionally formed in the egg shell in the form of an eosinophilic, translucent layer with embedded glycosaminoglycans. But at the same time, a living egg is unfertilized, so an elastic stabilizing layer does not arise in nature. According to the invention, the elastic stabilizing layer is located between the Zona Radiata Interna and the alveolar layer, preferably between the Zona Radiata Externa and the alveolar layer. That is, it can only occur in living eggs with more than a two-layer egg shell structure. For example, sea urchins have only exactly two layers in their egg shell. The new stabilizing layer is transparent, gel-like and elastic, and can be stained with eosin red and alcian blue when determined histologically. When produced by the method claimed in the invention, its manifestation is influenced by the concentration of potassium cations used at the stage of exposure to potassium, and its position is affected by the duration of exposure to potassium.

To remove ovarian fluid, live mature eggs are treated with a solution of table salt that does not damage the eggs before processing. In this case, we are preferably and favorably talking about a physiological solution of table salt. Moreover, it is advantageous and preferred when the sodium chloride solution is formed as a sodium chloride solution with a concentration of 0.6% to 1.0%, especially preferably as a 0.9% sodium chloride solution. To obtain a 0.9% solution of table salt, 9 g of sodium chloride (NaCl) is dissolved in 1 liter of water used. This concentration corresponds to the natural level in the human body, which is why a solution of table salt is called physiological.

In addition, caviar or a caviar product is claimed from unfertilized, mature eggs from aquatic animals, which differ in that in the egg shell, irreversible cross-linking of protein chains is additionally carried out by incorporating tyrosine molecules. Moreover, such additional irreversible cross-linking is localized in the Zona Radiata Interna and Zona Radiata Externa of living fish or crustacean eggs. Irreversible cross-linking leads to additional structural stabilization of the egg shell. In addition to the existing elastic stabilization, particularly large or soft eggs can be processed in this way. Caviar or caviar product can be obtained by the claimed method, and the degree of structural stabilization in the shell of the caviar then depends on the duration of exposure to calcium and the concentration of calcium cations at the stage of exposure to calcium. Other methods for producing caviar or caviar product with the same manifestation of irreversible cross-linking of proteins in the shell of the caviar are also applicable. Additional embodiments of the method claimed in the invention and products can be borrowed from the following special descriptive part of the implementation examples.

Examples of implementation of the invention

The method of obtaining caviar or caviar product from live, mature eggs of aquatic animals, and similar products according to the invention, and their preferred modifications, is further explained in more detail in order to further understand the invention by means of exemplary embodiments and figures. As shown here,

Fig. 1A, B, C represent scanning electron microscope (REM) photographs for comparison of obtained live eggs in immature and mature states (according to the state of the art),

Fig. 2 presents the first table of measurements of the thickness of the egg shell when processing live, mature Siberian sturgeon eggs,

Fig. 3 presents a second table of measurements of the thickness of the egg shell when processing live, mature beluga eggs of the sturgeon family,

Fig. 4A, B, C, D represent REM photographs of treatment cycles for the formation of a stabilizing layer of differently treated live, mature sturgeon eggs by treatment with potassium cations, in comparison with double treatment with potassium and calcium cations, also with calcium cations only,

Fig. 5A, B, C, D represent transmission electron microscope (TEM) photographs of the formation of egg shell layers with the formation of a new stabilizing layer (SS) between the Zona Radiata Externa (ZRE) and the alveolar layer (AL),

Fig. 6A, B, C, D represent TEM photographs of cortical granules and differently (potassium cations only, double treatment with potassium and calcium cations, calcium cations only) treated mature sturgeon eggs,

Fig. 7A, B, C, D represent optical microscope photographs of untreated, mature Siberian sturgeon eggs and potassium-treated live, mature Siberian sturgeon eggs,

Fig. 8A, B, C, D represent optical microscope photographs of live, mature eggs treated with calcium cations, and live, mature eggs of Siberian sturgeon treated with potassium cations and calcium cations, and

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Fig. 9A, B represent photographs obtained using REM and optical microscopes of the structure of the egg shell of beluga sturgeon after treatment of live, mature eggs with potassium and calcium cations.

From studies of the relationship between the weight and, accordingly, the age of sturgeon and the size of the eggs and, accordingly, the amount of collected caviar, it is known that with increasing weight and, accordingly, the age of the sturgeon, the diameter of the eggs increases, and thereby the quality of the caviar. In addition, as the weight and, accordingly, age of the sturgeon increases, the amount of caviar collected increases, and thus economic success. Sturgeons in natural conditions first reach sexual maturity, depending on the species, only at 12-26 years. Many sturgeon spend the growth phase until the first reproductive age at sea or in estuaries, and then migrate into rivers to find their spawning areas on rocky freshwater substrates. But in aquaculture also, sturgeon require from about 5 to 16 years, depending on the species of sturgeon, until the first sexual maturity, and thus until the first collection of caviar. Repeated collection of eggs in aquaculture from live females over many years involves careful handling of fish with optimal feeding and low numbers per unit area, and due to late puberty and long life expectancy, is always economically and environmentally feasible. The production of caviar in an economically interesting range in tons is easily achievable with a coordinated technological regime for collecting and processing the isolated eggs to form caviar. With suitable scaling measures, daily production of up to 80 kg or more can be achieved, depending on the age of the fish and thus the associated quantity of eggs.

In the claimed method, live, mature eggs are used, which were previously cleaned with a solution of table salt. In this case, we are talking about ovulated eggs, which were previously squeezed out of the gonads by thin muscle fibers of the follicular cells, in accordance with their stage of maturity (the stage of readiness for ovulation and the ability to fertilize), in a process called ovulation. Ovulated eggs are released into the oviducts and abdominal cavity free of cell debris and other debris. From there they can then be squeezed out by massaging the belly, without harming the life of the fish. A completely cleaned surface of the eggs does not leave any niches or folds for bacteria or fungi to attack, which results in high preservation of the caviar or caviar product. The use of preservation methods, for example, using borax, which is harmful to human health, is not required. In fig. 1A, B, C show scanning electron microscopy (REM) photographs of live eggs and the ovulation process according to the state of the art. Fig. Figure 1A shows an immature egg with a follicular cell, such as is found in traditional caviar from killed sturgeon. Fig. 1B shows in-situ ovulation and release of a mature egg from the surrounding follicular cell. Then FIG. 1C shows a live, mature sturgeon egg that appears completely smooth and clean.

Next, using one example of execution with live, mature eggs after ovulation, the possible sequence of stages of the claimed method with some optional additional stages is explained in more detail:

extraction of live eggs from living female sturgeon at stage V of maturity after lysis of terminal vesicles, immediate transfer of the extracted live eggs along with ovarian fluid to the laboratory for egg processing (with every possible avoidance of downtime, with inevitable breaks in handling eggs, ensuring the exclusion of oxygen access by covering the ovarian fluid airtight polymer film), direct thorough washing of live eggs with a 0.9% physiological solution of table salt, up to the complete removal of ovarian fluid, carrying out the stage of exposure to potassium:

obtaining a 0.1 to 2 millimolar solution of potassium cations from potassium citrate in deionized water with a specific conductivity of 10 µS/cm (at 25°C) at a polar temperature of 10°C, placing live, mature eggs in the solution for 10 minutes of exposure to potassium, and removing the treated eggs from the solution, and briefly immersing the treated eggs in a 0.9 percent physiological solution of table salt.

Other methods for obtaining mature eggs are also possible. When using live, mature eggs from a previously killed animal, the blood and fat must also be washed off, or the eggs must even be squeezed out of the gonads, which is achieved by pre-treatment, preferably with a physiological solution of table salt. Through immersion, the elasticity and diameter of the stabilizing layer can be further controlled (that is, along with the choice of duration of exposure). As a result of the described treatment in living eggs, due to the electrical influence of the introduced potassium cations, a translucent, elastic stabilizing layer is formed between the Zona Radiata Externa and the alveolar layer in the egg shell. For eggs of normal size and softness, treatment with potassium at the stage of exposure is sufficient. But if particularly large, soft or sensitive eggs of some sturgeon breeds are used, for example, Huso huso, Acipenser transmontanus or Acipenser ruthenus, a calcium exposure stage can also be added (or carried out previously):

Carrying out an additional stage of exposure to calcium:

obtaining another 0.5- to 2-millimolar solution of calcium cations from calcium chloride in deionized water with a specific conductivity of 10 μS/cm (at 25 ° C) at a polar temperature of 10 ° C, placing live, mature eggs in the solution for 12 minutes of exposure to calcium, and removing the treated eggs from another solution, and briefly immersing the treated eggs in a 0.9 percent saline solution.

Thus, in addition to the elastic stabilizing layer from the stage of exposure to potassium, structural cross-linking of the egg shell proteins occurs, which in fish and crustaceans is localized in the already existing Radiata Interna and Radiata Externa zones of the egg shell. This additional structural cross-linking of the egg shell proteins with tyrosine residues primarily imparts plastic strength to large and soft eggs before processing or sensitive eggs - along with elasticity after the potassium treatment stage. Here, too, the optional immersion again serves for additional adjustability. The resulting product is (genuine) caviar from live, mature eggs, which can then be processed as follows:

mixing caviar with dry table salt NaCl (3.5 g/100 g of caviar, 3.5%), which does not contain either potassium donors or calcium donors (not containing K+ and Ca ⁺⁺ anti-caking agents) (3.5 g/100 g of caviar, 3.5%), which corresponds to lightly salted salting for canning, filling lightly salted caviar into glass containers, preferably 500g glass ripening jars, and airtight vacuum sealing the jars with screw caps and labeling, storing the glass jars at -2°C for a period of 2 to 4 months to further ripen the caviar, and, optionally, freezing fresh caviar or ripened caviar at the consumer's request in glass jars at -18°C.

Live, mature eggs treated at the stage of exposure to potassium as a result of treatment form a completely new zone: a stabilizing layer, which is formed elastic and translucent (gel-like). To confirm this, the stabilizing layer can simply be painted. It is localized between the alveolar layer AL and the Zona Radiata Externa ZRE, and has not yet been described in the literature. With regard to the structural structure of the eggs of fish and crustaceans, a link to the introductory part of the description should be provided in the corresponding glossary of the author Siddique.

Based on the table in Fig. 2 measures the diameter (in µm) of the extracellular egg shell on mature Siberian sturgeon eggs by cryosectioning into layers of constant thickness (10 µm), using computer-controlled image analysis (Zeiss), under the influence of various processing conditions to stabilize the egg shell. The table shows the formation of a new stabilizing layer SS and the diameter of the existing layers (ZRI, ZRE, AL) of the egg shell when treated with additives with various concentrations in mmol/l of potassium cations K ⁺ only (from potassium citrate), and in combination with calcium cations Ca ^{+ +} (from calcium chloride), during the various stages of action according to the invention. Live, mature eggs of the Siberian sturgeon Acipenser baerii were subjected to processing. Along with the abbreviations already previously indicated and explained in the work by the author Siddique, the abbreviation Mw also means the average value, Std standard deviation. Values are given for the new elastic stabilizing layer SS. In addition, reference is made to the prior art according to the above-mentioned document WO 2007/045233 A1.

Quality tests after treatments have shown that only at concentrations of potassium cations above 1 mmol/l and 1.5 mmol/l does the thickness of the egg shell reach at least 12 μm, and an intermediate layer appears, that stickiness is lost, and the egg is stable enough for subsequent processing. In addition, it has been found that a treatment duration of preferably 10 minutes is advisable, and thus all live eggs present in the solution also react metabolically. It was possible to achieve in one processing unit the amount of processed caviar at the level of 2.5 kg (in approximately 25 liters of solution). Organoleptic testing of caviar after treatment with potassium cations according to the invention showed that the elastic texture of Siberian sturgeon caviar did not change with concentration variations between 1 mmol/l and 1.5 mmol/l. In contrast, eggs treated in a solution with lower concentrations of potassium cations vary in texture and only a few eggs are stable, while untreated eggs are very soft and ruptured. According to the organoleptic tests carried out, the treatment in two stages of action (potassium and calcium cations) leads to a strong, pearly product, also called

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Superplop (with excellent popping) in the case of Siberian sturgeon eggs.

Table in Fig. 3 shows the presence and diameter (in microns) of the layers of the extracellular membrane of the eggs on mature eggs under various processing conditions according to the invention of live large eggs of the beluga breed Huso huso sturgeon. When this egg was treated with potassium cations (from potassium citrate), the formation of a new eosinophilic stabilizing layer SS was observed in the extracellular membrane of the egg, which was also localized between ZRE and AL. Organoleptic tests of large beluga eggs showed that double treatment with potassium and calcium cations leads to optimal results in terms of the texture of fragile, live, mature eggs.

Fig. 4A, B, C, D show REM photographs of changes in the structure of the extracellular membranes of eggs taken as an example of living, mature Siberian sturgeon eggs under various processing conditions. Views are shown at two magnification levels: on the left with 6000x, and on the right (in fragments) with 12000x. In this case, treatments were always carried out on live eggs, which were flash-frozen in hexane at -80°C to obtain histological cryosection to preserve the native state.

Fig. 4A In untreated mature eggs, the shell zones of the eggs do not show clear separation from each other (prior art).

Fig. 4B Under the influence of 0.5 mmol/l potassium, a new stabilizing layer SS already appears between the Zona Radiata Externa ZRE and the alveolar layer AL, while the Zona Radiata Interna ZRI and Zona Radiata Externa ZRE exhibit an unchanged loose protein connection, as in untreated eggs.

Fig. 4C Successive double treatment of eggs with potassium and calcium cations shows in REM both characteristic morphological features, namely the stabilizing SS layer as a result of potassium treatment and the twisting and cross-linking of protein chains in Zona Radiata Interna ZRI and Zona Radiata Externa ZRE, which is characteristic of calcium treatment , compare fig. 4D.

Fig. 4D Treatment with calcium alone results in strong coiling and cross-linking of loose protein chains in Zona Radiata Interna ZRI and Zona Radiata Externa ZRE (prior art), compared to FIG. 4A and FIG. 4B without calcium treatment. Fig. 5A, B, C, D show transmission electron microscope photographs (TEM, 3000x magnification) of the multilayer structure of the egg shell of mature sturgeon eggs when treated with potassium cations at a concentration of 1.0 mmol/L. Fig. 5A shows Zona Radiata Interna ZRI with unbound, cloudy fibrils, which is separated from Zona Radiata Externa ZRE by an EP epitaxial layer (the phenomenon can only be identified at the ultrastructural level). Zona Radiata Externa ZRE can be characterized by a fibrous network structure of long, elongated fibrils. Fig. 5B shows the ultrastructural formation of a new stabilizing layer SS with a fine-grained structure directly between the Zona Radiata Externa ZRE and - according to FIG. 5C - alveolar layer AL, which penetrates to the periphery of the egg shell - according to FIG. 5D - small ductules (small ducts, tubules). Ultrastructural analyzes confirmed that treatment with potassium cations (1.0 mmol/l) according to the invention leads to the formation of a hitherto unknown new stabilizing layer SS with an amorphous structure and positioning in fish and crustaceans between the Zona Radiata Externa ZRE and the alveolar layer AL.

In fig. 6A, B, C, D show TEM photographs (3000x magnification) of cortical CG granules in the peripheral cytoplasm within the plasma membrane of mature eggs, with FIG. 6A shows unprocessed eggs (prior art), FIG. 6B shows an egg treated with potassium cations, FIG. 6C shows eggs treated with potassium and calcium cations, and FIG. 6D shows an egg treated with calcium cations only (prior art).

Cortical granules are secretory organelles (structurally confined areas) that are found in eggs and are closely associated with the fertilization event. Cortical granules contain enzymes such as peroxidase and the tyrosine cross-linking building blocks Zona Radiata Interna ZRI and Zona Radiata Externa ZRE. As the effect of different treatment conditions was analyzed using TEM, a cortical reaction is initiated and its contents are released solely due to treatment with calcium cations. An identical process also occurs during natural fertilization with a sperm-induced calcium wave on the cell membrane of the egg. In the untreated egg (Fig. 6A), in the peripheral cytoplasm, cortical CG granules are clearly distinguishable by their enzymatic equipment as large, round vesicles (vesicles), which also contain structural elements. Similarly, cortical CG granules when treated with potassium alone according to the invention (Fig. 6B) remain unchanged as before. True, one can observe strong vesicular transport on the egg membrane from the cytoplasm to the extracellular membrane of the egg. In fig. 6A and FIG. 6B the symbol D represents the yolk. When dually treated with potassium and calcium cations (Fig. 6C), this results in both the release (arrows) of the contents of cortical CG granules and the formation of a new stabilizing SS layer. Treatment with calcium cations alone (Fig. 6D) again results in a cortical reaction, with the contents being released into the extracellular egg shell and enzymatic cross-linking of Zona Radiata Interna ZRI and Zona Radiata Externa ZRE by tyrosine residues being initiated. Empty vacuoles remain in the cytoplasm V.

- 14 045115

For diagnostic screening of cryosections to determine the effect of potassium according to the invention, FIG. 7A and FIG. 7B shows optical microscope photographs (400x magnification) of state-of-the-art unprocessed live eggs of the Siberian sturgeon Acipenser baerii. Left photo according to Fig. 7A shows HE staining (hematoxylineosin staining), the right photograph according to FIG. 7B shows Alcian blue staining. This test stains the glycosaminoglycans GAG, hyaluron, and fibrin. It can be discerned that Alcian blue is absent in the various layers of the egg shell, but clearly appears in the ooplasm of the OR. The individual layers are designated according to the above embodiments and their thicknesses are indicated by double-headed arrows.

On the contrary, in FIG. 7C and FIG. 7D shows photographs of cryosections processed by the method claimed in the invention at the stage of exposure to potassium of live, mature eggs in the ovulated state of the Siberian sturgeon Acipenser baerii. The eggs were treated with potassium cations at a concentration of 1.5 mmol/l (from potassium citrate). The appearance of a new stabilizing layer SS between the Zona Radiata Externa ZRE and the alveolar layer AL in the extracellular membrane of the egg is clearly visible. In the left photo according to Fig. 7C after eosin staining shows that this new SS stabilizing layer is particularly eosinophilic. In the right photo according to Fig. 7D after Alcian blue staining clearly shows that this new SS stabilizing layer is particularly enriched in GAG. This results in a particularly favorable elasticity of the new stabilizing layer SS.

For diagnostic screening of cryosections to determine the effect of calcium alone and the effect of double treatment with potassium and calcium cations, FIG. 8A and FIG. 8B shows photographs of cryosections of mature eggs of the Siberian sturgeon Acipenser baerii processed in accordance with the prior art in accordance with the document WO 2007/045233 A1 with 400x magnification. Left photo according to Fig. 8A shows HE staining (hematoxylin-eosin staining), the right photograph according to FIG. 8B shows Alcian blue staining. Ovulated Siberian sturgeon eggs were processed. In the left photo according to Fig. 8A with HE staining, one can distinguish protein chains cross-linked with tyrosine molecules in Zona Radiata Intema ZRI and Zona Radiata Extema ZRE. In addition, the division between both zones is clearly visible. As a result of cross-linking, structural stabilization of the egg shell is obtained. In the right photo according to Fig. 8B with Alcian blue staining, it can be recognized that Zona Radiata Interna ZRI and Zona Radiata Externa ZRE are only weakly colored, from which we can conclude that the GAG content is low, and less elasticity can be judged, while in the ooplasm of OR, strong staining indicates very large amount of GAG.

In fig. 8C and FIG. 8D shows photographs of cryosections of live, ovulated eggs processed by the claimed method, which were treated at an additional stage of exposure to calcium using the method according to document WO 2007/045233 A1. Live, ovulated eggs of Siberian sturgeon Acipenser baerii were treated at the potassium stage with 1.5 mmol/L potassium cations (from potassium citrate), and at the calcium stage with 1.6 mmol/L calcium cations (from calcium chloride) . In addition to what is shown in the photographs according to FIGS. 8A and 8B, the strengthening stitching of the egg shell can now be distinguished, also shown in the photographs according to FIGS. 8C and 8D the new translucent SS stabilizing layer with its elastic stabilizing effect on the egg shell. Processed live, mature Siberian sturgeon eggs are thus both elastic (due to GAGs) and structurally (as a result of protein cross-linking) stabilized, and form excellent caviar.

In fig. 9A shows a characteristic REM photograph (at 12,000x magnification) of the egg shell of live, mature, ovulated eggs of the beluga sturgeon species Huso huso after double processing according to the invention. Fig. 9B shows an optical microscope photograph (at 400x magnification) of the egg shell of live, mature, ovulated beluga Huso huso eggs after double treatment according to the invention. Both photos show the new SS stabilizing layer and the crosslinking of Zona Radiata Interna ZRI and Zona Radiata Externa ZRE. In addition, it is clearly visible that the eggs of the species Huso huso have an exceptionally pronounced alveolar layer AL with large vacuoles V. Screening using cryosections with H&E staining according to the invention confirms the formation of a new elastic stabilizing layer SS during the stage of exposure to potassium cations and additional cross-linking of proteins in egg shell in two stages of exposure to potassium and calcium cations.

List of reference items

AL - alveolar layer

Ca++ - calcium cations

CG - cortical granules

CYT - cytoplasm (OP)

D - yolk

EP - epitaxial layer

K+ - potassium cations

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Claims (12)

Mw - average value OR - egg cytoplasm (egg plasma) pAL - alveolar layer (to the periphery of the egg shell) PO - plasma membrane of the oocyte (egg) SS - stabilizing layer Std - standard deviation V - vacuole ZRI - internal radiant zone (Zona Radiata Interna) ZRE - external radiant zone (Zona Radiata Externa) CLAIM 1. A method for processing caviar from live, mature, ovulated eggs of fish or crustaceans, which have three or more layers in the egg shell, and the live, mature, ovulated eggs are in a fertilizable, but unfertilized state, and have a natural potassium content in cytoplasm, in a 0.6-1.0% solution of table salt, and then at least in a solution containing water and at least one cationic component dissolved in it, acting as a stabilizing shell of the eggs of living, mature, ovulated eggs, characterized by that carry out a potassium exposure step, in which potassium as a cationic component is dissolved in water with a concentration of potassium cations from 0.1 to 3.0 mmol/l, and the water before adding the potassium donor is deionized to form the cationic component and has a range the natural temperature of fish or crustacean eggs is a temperature of from 1 to 29 ° C, and that live, mature, ovulated eggs are subjected to treatment in a solution for exposure to potassium for a time of 5 to 30 minutes, and in conclusion, the live, mature, ovulated eggs are immersed in 0.6-1.0% solution of table salt. 2. The method according to claim 1, characterized in that at least one potassium salt is used as a potassium donor, preferably a citric acid salt and/or a hydrochloric acid salt and/or a sorbic acid salt. 3. Method according to claim 1 or 2, characterized in that the concentration of potassium cations in the solution is 0.1 mmol/l, 0.5 mmol/l, 0.65 mmol/l, 1.6 mmol/l or 2, 0 mmol/l, preferably 1.0 mmol/l or 1.5 mmol/l. 4. The method according to one of the preceding paragraphs, characterized in that the duration of potassium exposure at the potassium exposure stage is 10 minutes, 12 minutes, 15 minutes, 20 minutes or 25 minutes. 5. The method according to one of the previous paragraphs, characterized in that after or before the stage of exposure to potassium, an additional stage of exposure to calcium is carried out, in which calcium is dissolved as a cationic component in water to form another solution with a concentration of calcium cations from 0.1 to 3.0 mmol/l, wherein the water before adding the calcium donor is deionized to form a cationic component and has a temperature ranging from 1 to 29°C within the natural temperature range of fish or crustacean eggs, and that live, mature, ovulated eggs are subjected to treatment in solution for a period of time exposure to calcium from 9 to 30 minutes, and in conclusion, live, mature, ovulated eggs are immersed in a 0.6-1.0% solution of table salt. 6. The method according to claim 5, characterized in that at least one calcium salt is used as a calcium donor, preferably calcium citrate, calcium chloride and/or calcium sorbate. 7. Method according to claim 5 or 6, characterized in that the concentration of calcium cations in another solution is 0.5 mmol/l, 0.8 mmol/l, 1.0 mmol/l, 1.5 mmol/l, 1 .6 mmol/l or 2.0 mmol/l. 8. The method according to one of claims 5-7, characterized in that the duration of exposure to calcium at the stage of exposure to calcium is 10 minutes, 12.5 minutes, 15 minutes, 16 minutes, 20 minutes or 25 minutes. 9. Method according to one of the preceding claims, characterized in that the temperature of one solution when exposed to potassium and/or the other solution when exposed to calcium in the temperature range of the polar region is between 1 and 15°C, preferably between 5 and 12°C, especially preferably 10°C, in the temperate region temperature range between 10 and 20°C, preferably 15°C, especially preferably 12°C, or in the tropical temperature range between 20 and 29°C, preferably 27°C, especially preferably 21° WITH. 10. Method according to one of the preceding claims, characterized in that the deionized water at 25°C has an electrical conductivity of between 1 and 15 μS/cm, preferably 10 μS/cm or lower, particularly preferably 1 μS/cm. 11. Method according to one of the preceding claims, characterized in that one and/or the other solution has a pH value between 6.8 and 8.0, preferably between 7.0 and 7.9, especially preferably 7.2 or 7, 4, or 7.5. 12. The method according to one of the preceding paragraphs, characterized in that then, at the last stage of influence for preserving and intensifying the taste, skillfully -