METHOD FOR THE PRODUCE OF CAVIAR OR A CAVIAR-SAME PRODUCT FROM LIVE, MATURE EGGS OF FISH OR CRUSTACEANS, AND SUCH PRODUCTS

Description

[0001] Process for producing caviar or a caviar-like product from live, mature, ovulated eggs of fish or crustaceans and such products.

[0002] The invention relates to a process for producing caviar or a caviar-like product from live, mature, ovulated eggs of fish or crustaceans which have three or more layers in the egg shell, the live, mature, ovulated eggs being in a fertilizable but unfertilized state and having a natural potassium content in the egg plasma, by treating the live, mature, ovulated eggs in a 0.6 percent to 1.0 percent saline solution and subsequently in at least one solution containing water and at least one cationic component dissolved therein which stabilizes the egg shell of the live, mature, ovulated eggs, and to caviar or a caviar-like product.

[0003] The processed, unfertilized eggs, especially those of fish, are valued as a delicacy and are increasingly consumed. "Roe" refers (in layman's terms) to eggs at any stage of maturity, from immature to mature, although the stage of development of the eggs is not clearly defined. "Spawn" refers to live, mature eggs laid by a female fish, lobster, or other aquatic animal into a body of water to be fertilized. Ovulated eggs are mature, fertilizable, live eggs released from the follicles in the ovaries and released into the body cavity. From there, they are then spawned or sloughed off. According to the FAO Codex Alimentarius, caviar may only be produced from the roe of female fish of various sturgeon species. In addition to wild sturgeon, sturgeon farmed in freshwater aquaculture facilities are now also used for caviar production.With a few exceptions, sturgeons spawn exclusively in freshwater. The most well-known sturgeon species include (. Acipenseridae ) include A. baerii, A. guldenstaedtii, Huso huso (also known as beluga sturgeon), A. transmontanus, A. ruthenus and its albino. Also worth mentioning are the hybrids between A. schrenckii (female) and A. dauricus (male) and the American "Paddlefish" ( Polydon spatula ). Various types of caviar are known on the market, including Sevruga, Osietra, and Beluga. White caviar (also called "golden caviar") is obtained from albino sturgeons. A. ruthenus albino, the so-called "Tsar's Caviar" is occasionally produced in aquaculture facilities. However, this is not the "real Tsar's Caviar" that comes from the albino of the Huso huso and is very rare.

[0004] Currently, caviar-like products are produced and marketed from approximately 38 other fish species that are not sturgeon species. See, for example, the publication by P. Bronzi et al.: "Present and future sturgeon and caviar production and marketing: A global market overview" (Journal of Applied Ichthyology 2014, 30 SI, 6, 1536-1546). These include tuna, lumpfish, salmon, trout, herring, cod, carp, whitefish, and capelin, whose immature roe is used to produce caviar-like products (also referred to as "caviar substitute" or "false caviar"). The roe of lobster, large crayfish, and other crustaceans can also be processed into caviar-like products. The process claimed by the invention and the manufacturable products relate to these (and other suitable, but not mentioned) fish and crustaceans.Unless sturgeon caviar is explicitly mentioned, the following shall generally refer to and include both caviar and caviar-like products from fish other than sturgeon and from crustaceans, in particular lobster and crayfish.

[0005] Caviar and caviar-like products are valuable foods. Caviar is rich in protein with a high content of essential amino acids and fat. Caviar contains vitamins D, E, B12, and niacin; the minerals sodium, potassium, magnesium, and calcium; and the trace elements phosphorus, fluorine, iodine, and zinc. It also has a high content of valuable cholesterol (HDL). Caviar and caviar-like products can be used both as food and as a substance in the cosmetics industry or in other industries that work with such valuable substances. The size and firmness of the eggs depend heavily on the specific species of fish or crustacean, as well as on their maturity and thus on the time of harvest.

[0006] There are currently some caviar products made from mature, ovulated sturgeon eggs on the market. However, the caviar offered today is still primarily immature roe, taken along with the ovaries from killed sturgeon. This is the conventional method of caviar production. Initially, it was assumed that, as was previously the case with wild caviar, the immature eggs from aquaculture sturgeon would be sufficiently strong without further treatment to withstand the invasive washing steps required to remove remnants of gonadal tissue and for repackaging.However, increasing experience in the production of caviar from sturgeon from aquaculture over the past 20 years has shown that this immature roe from killed aquaculture sturgeon is too soft and can only be further processed into a product suitable for repackaging and with a shelf life of more than 2 to 3 months by using borax or other preservatives or by pasteurisation.

[0007] The killing of wild-caught females for caviar extraction, combined with drastic overfishing, pollution of waters with industrial, agricultural, and domestic wastewater, and the construction of weirs and dams blocking migration routes to freshwater spawning grounds, has led to a massive threat to the wild populations of approximately 27 different sturgeon species. Despite the protection of sturgeons under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), poaching and illegal trade continue to be prevalent in many regions. Costly repopulation programs have been initiated worldwide, but according to reports from the World Sturgeon Conservation Society, these programs have unfortunately shown little success, particularly in China, but also in Iran and Russia. Only the conservation and repopulation measures in the USA and Canada are showing initial signs of success.In addition to the permitted catch of spawning stocks, various sturgeon species are also released from aquaculture into the wild as part of restoration programs with varying degrees of success in order to save populations threatened with extinction. Populations of crayfish and noble crayfish are also severely threatened by factors such as environmental pollution and introduced diseases such as crayfish plague. Significant advances in crayfish breeding in aquaculture and extensive stocking measures play an important role in the preservation of native stocks. In aquaculture, the females are left alive for breeding and the eggs are collected by stripping. In contrast, for caviar production in aquaculture the female sturgeons are generally simply killed because more gentle methods are not mastered. This completely ignores the fact that these sturgeons show significantly improved reproductive performance with increasing age.The "caesarean section method," which is sometimes practiced in Russia, is by no means a gentle method, as it is associated with a high mortality rate among the sturgeons treated in this way. Stand der Technik

[0008] A method for producing granular caviar from ovulated sturgeon roe is known from RU 2 232 523 C2. The harvested ovulated eggs are first treated in a hot, 1.5 percent to 2 percent aqueous solution of a preservative to prepare them for subsequent pasteurization at temperatures of 65°C to 70°C. Aside from the fact that each heating process significantly influences the flavor of the roe, there is no guarantee that ovulated eggs, which have a very soft and sticky egg shell, will survive subsequent treatments with preservatives without bursting. However, even a small proportion of burst eggs significantly impairs the quality of the caviar, as the burst eggs are difficult to remove. Pasteurization denatures the valuable proteins and gives the caviar a floury flavor.

[0009] In the context of collecting ovulated sturgeon eggs, for example, it is known from the publication by M. Szczepkowski et al.: "A simple method for collecting sturgeon eggs using a catheter" (Arch. Pol. Fich. (2011) 19:123-128) that a catheter can be used for this purpose. This allows the eggs to simply drain away or be suctioned out using negative pressure. It is also known to simply massage the eggs out of the sturgeon's abdominal cavity. This method is called "stripping" and is the most gentle harvesting method.

[0010] The closest prior art to the invention is disclosed in WO 2007 / 045233 A1. A process is described for producing caviar or caviar-like products from mature, ovulated but unfertilized eggs of aquatic animals, preferably fish, by exogenously treating the mature eggs in a solution, whereby an endogenous, morphological change in the acellular egg membrane, which separates the egg cell (egg plasma with surrounding egg membrane) from the environment, is induced by structural stabilization. The solution used contains water and at least one cationic component (calcium cations Ca ++< ), which is dissolved in the water at a predetermined concentration and induces structural stabilization upon contact with the eggs.Calcium is a cellular signal transduction molecule that triggers a calcium wave in the egg cell's ovum, which in turn leads to a cortical response and the release and activation of ovoperoxidase. This enzyme causes irreversible structural cross-linking of protein strands in the extracellular egg membrane by incorporating tyrosine molecules into the zona radiata interna and the zona radiata externa. The induced process in the living, metabolically active egg thus leads to the desired structural stabilization of the egg membrane. In immature eggs, such stabilization cannot be achieved because the corresponding receptors and enzyme cascades have not yet matured. In dead eggs, the process cannot be initiated at all because no metabolism is taking place.Live, mature eggs immediately develop a sticky layer upon contact with water due to the ovarian fluid, which allows them to adhere to rocks and vegetation in the spawning area in nature. Therefore, before treatment, the eggs are rinsed in a non-harmful ("physiological") saline solution to remove the ovarian fluid. Furthermore, the live, mature eggs have a natural potassium content in their egg plasma. Therefore, they were not exposed to harmful doses of potassium (e.g., to trigger ovulation) prior to harvest.

[0011] The described reaction chain is referred to in the literature as the "second reaction." It is a slow metabolic reaction that, following the fusion of a first sperm with the egg, creates a permanent, physical-mechanical structure to protect the egg from further sperm aggregated outside the egg (polyspermy), but above all from environmental toxins, microbes, and mechanical damage to the developing embryo. In the known process, this second reaction is triggered without fertilization by a sperm having occurred. The achieved structural stabilization creates a "popping effect" and an explosive release of the liquid egg plasma when the product is consumed. The preference for a specific strength of the popping effect depends heavily on the use of the caviar and the consumer.

[0012] Furthermore, it is known from EP 2 522 226 B1 to preserve the immature roe of aquaculture-reared fish in a composition containing the flavonoid and antioxidant taxifolin (dihydroquercetin) and an organic salt, in particular potassium citrate. However, the high concentrations of the composition used lead to dramatic changes in the intracellular ionic environment, which induce programmed cell death (apoptosis). The roe treated in this way is thus killed immediately. Publications SU 1662469 A1 and RU 2 048 111 C1 also describe methods for preserving sturgeon eggs in which potassium compounds are used in such extremely high concentrations that apoptosis is immediately triggered in the eggs obtained by slaughter in an immature, non-ovulated state, causing them to die. The same applies to EP 2 868 207 B1 corresponding to the above-mentioned RU 2 232 523 C2 ,where additional denaturation occurs through heating.

[0013] The publication by GE Bledsoe et al.: "Caviars and Fish Roe Products" (Critical Reviews in Food Science and Nutrition, Vol. 43, No. 3, May 1, 2003, pages 317 to 356) discloses the use of potassium nitrate in the context of the primary use of nitrate as a preservative for crab, sturgeon, and other fish eggs. However, as is common with all preservation processes, this occurs in such high concentrations or in such inseparable combination with other preservatives that a cytotoxic effect occurs, severely disrupting the osmotic balance and immediately killing the treated eggs.Furthermore, only immature eggs at an early stage of development from slaughtered fish are preserved, which, on the one hand, have to be mechanically rubbed out of the gonads at the risk of damage and, on the other hand, do not yet have fully developed structures in the mature egg shell, so that they cannot be used in the invention.

[0014] The process described in DE 2 416 685 A aims to replace harmful nitrite-containing compounds to preserve the red color of salmonid roe strands. The nitrite- and nitrate-containing substances are replaced with compounds containing citrates and sulfites as anions. The fish product used is intact gonads obtained by slaughtering the fish, the so-called roe strands or roe sacs, which contain unovulated eggs. The intact gonads are first washed fresh or after thawing - if previously frozen - in water and a 3% saline solution. A 3% NaCl solution corresponds to more than three times the concentration of physiological saline and is therefore harmful to living cells.The color-stabilizing citrates, alone or in combination with sulfites as additives, are used as sodium or, optionally, potassium compounds in a saturated NaCl brine (36.5%), i.e., a concentration more than 40 times that of saline solution, to treat the roe strands. This leads directly to the death of the roe strand cells, thus inhibiting enzymatic autolysis (spoilage) and killing microorganisms through dehydration. Simultaneous color stabilization is achieved with sodium sulfite and sodium citrate at a maximum of 0.35% in the brine and, optionally, with 0.15% - 0.30% potassium citrate in the brine. These concentrations in the externally applied rinse correspond to an exposure of 15 mmol / l to 30 mmol / l K+< ions. An enrichment of citrate compounds between 300 and 700 ppm is achieved in the roe strands. These are additives that remain in the final product.The immature eggs are killed immediately.

[0015] JP S63 - 36 763 A discloses a process for reducing sodium chloride in the preservation of fish roe in order to reduce the salty taste. The sodium chloride is also substituted with various potassium compounds. However, this occurs in such high concentrations that the immature eggs are killed and can no longer perform any metabolic work. JP 2001 - 299 285 A discloses a process for treating immature frozen roe to improve its texture. The eggs are rinsed at 5°C for up to 24 hours using potassium-containing chemicals. Such a long treatment time interrupts any metabolic process. Since the roe was frozen without freeze protection, the immature eggs no longer have any metabolic activity and are also incapable of fertilization. They are therefore also not suitable for the claimed invention.However, the process claimed in the invention does not involve subsequent and fatal preservation, decolorization, or freezing, but rather the original production of caviar and caviar-like products from untreated mature live eggs. Preservation or freezing after treatment of the live eggs are merely optional additional steps in the invention. Decolorization is completely eliminated, as it is not required.

[0016] From the publication by Huang Hui et al.: "Effect of Synthetic Preservatives on Volatile Flavor Compounds in Caviar of Sturgeon" (Huso dauricus × ​​A. schrenckii) ([J] FOOD SCIENCE, 2015, 36(12): 97-103), it is known that the goal of preventing flavor loss during cool storage of caviar is to use synthetic preservation with the preservatives potassium sorbate (E202, sorbic acid) and ascorbate (vitamin C). A constant concentration of 0.5 parts per thousand of potassium sorbate was used in various test groups. Immature eggs were used, which are intended to acquire a more intense flavor through the preservative treatment. Potassium sorbate is considered to inhibit mold and fermentation, but can also impair the flavor of a product.

[0017] The publication by L. Dufresne et al.: "Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs" (Experimental Cell Research, Elsevier, Amsterdam, NL, Vol. 172, No. 1, September 1987, pages 32 to 42) deals with the artificial activation of sea urchin eggs. Although the present invention does not deal with the treatment of sea urchin eggs because they do not exhibit a suitable structure (only a two-layered egg shell), this publication will be briefly discussed here. Firstly, sea urchin eggs are used that were obtained by injecting a solution containing 0.5 M KCl with an extremely high potassium chloride concentration into the abdominal cavity of the sea urchin. This significantly influenced the egg's electrical polarization state and clearly altered its natural potassium content in the egg plasma.Furthermore, all sea urchin eggs come into contact with water prior to treatment and form a gel layer, which must then be mechanically removed. This fundamentally alters the morphological and physiological properties of the acellular egg shell. Therefore, sea urchin eggs are unusable in this invention not only because of their fundamentally different structure, but also because of the massive interference with metabolism caused by potassium chloride during their harvesting. Furthermore, calcium-free seawater is not deionized; per liter, it contains, among other things, more than 10 g of sodium, 0.43 g of potassium, 1.3 g of magnesium, and 20 g of chlorine. Thus, rinsing the eggs in calcium-free water is not the same as rinsing them in a saline solution that does not harm the living, mature eggs.

[0018] The structure of the egg membrane of fish and crustacean eggs follows a basic pattern and is described for the procedure using the terminology from the publication by Siddique et al.: "A review of the structure of sturgeon egg membranes and the associated terminology" (J. Appl. Ichthyol. (2014), 1-10). To establish terminological concordance, this will be briefly discussed here.

[0019] In maturing eggs (oocytes, egg cells) in the animal's ovaries, the follicle, consisting of granulosa cells (also called follicular cells, follicular membrane, follicular epithelium) and thecal cells (also called external and internal theca), surrounds the egg to supply it with signaling and nutrients. Between the granulosa cells and the thecal cells is the basal lamina (also known scientifically as the perifollicular membrane, membrane, basal lamina). The egg plasma (oocyte plasma, olemna, cytoplasm, egg interior) is surrounded by the egg membrane (oocyte membrane, plasma membrane PM, cellular egg membrane). During ovulation, the egg is released from the follicle cells and released into the fish's abdominal cavity. The ovulated egg retains only its acellular egg membrane (also known scientifically as the extracellular matrix or extracellular membrane), which it formed during egg maturation. The membrane is structurally composed, from the outside inward, of: Alveolar layer AL (scientifically also referred to as gel coat, adhesive layer, gel shell, (second external) gelatinous shell, layer 3, chorion (2)), outermost layer of the acellular egg shell Zona Radiata Externa ZRE (scientifically also referred to as external vitelline shell (zona radiata = vitelline shell), outer vitelline zone, external vitelline membrane, layer 2, chorion layer 2, zona pellucida externa lat., zona radiata externa lat., layer 1B of the shell, second shell), outer part of the vitelline shell, lies directly beneath the alveolar layer Epilayer EP (scientifically also referred to as epilayer 1, layer 4, outer layer of the first shell), separates the ZRE from the ZRA, not present in all egg types Zona Radiata Interna ZRI (scientifically also referred to as internal vitelline shell, inner vitelline zone, internal vitelline membrane, chorion layer 1, zona pellucida Interna lat., Zona Radiata Interna lat., layer 1A of the membrane, inner layer of the first membrane), inner part of the vitelline membrane, closely connected with the CRA and perivitelline cleft (in science also called extra-oocyte matrix, cleft with microvilli outpouchings of the egg plasma), narrow space between the CRI and the egg membrane, into which the egg plasma invaginates numerous microvilli (MV).

[0020] Living ovulated eggs are electrically excitable through ion channels located in their plasma membrane. Changes in the electrical properties of the plasma membrane are, among other things, a prerequisite for egg activation and have an impact on the egg membrane. Pioneering work on marine invertebrates demonstrated ion currents of potassium cations through the egg membrane, causing a transient change in the potential across the membrane (fertilization potential FP). This potential is generated by the activation of a transient, voltage-dependent inward current into the egg interior. It has been shown that the depolarization of the membrane potential (RP) results from the flow of ions through the egg membrane (fertilization current FC). This current flows through the openings of large, nonspecific and highly conducting ion channels, which can be activated by sperm or artificial chemical or mechanical agents.The previously hypothetical models regarding the role of different ion channels and the relevant ions show species-specific differences.

[0021] Potassium plays a central role in egg cells in nature; see the publication by E. Tosti et al.: "Electrical events during gamete maturation and fertilization in animals and humans" (2004 Human Reproduction Update, Vol. 10, No. pp. 53-65). Potassium K +< is the cation that decisively determines the resting potential of the egg cell. The potassium +< gradient and the egg's permeability for ions are regulated by transport proteins and ion channels. According to studies at the Alfred Wegener Institute, mature unfertilized sturgeon eggs contain A. baerii Natural intracellular potassium cation concentrations are 50 mmol / l. Extracellular calcium, on the other hand, does not influence the resting potential / fertilization potential of the egg and is not itself involved in the first rapid electrical block (see below). The ionic composition inside the egg cell differs from the ionic composition in the external environment. This separation between the cell interior and the external medium 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 forms an electrical gradient across the egg membrane, which is measurable as a potential difference (resting potential). Aufgabenstellung

[0022] Based on the closest prior art to the invention according to WO 2007 / 045233 A1 ,which is also used for the production of caviar and caviar-like products, is the Aufgabe The aim of the present invention is to further develop the process described therein based on live, unfertilized, mature, ovulated eggs of fish or crustaceans in such a way that a modification of the sensory properties with regard to texture, taste, transport, storage, and deep-freezing of the mature, ovulated eggs can be achieved. However, the above-described advantages of the caviar or caviar-like products produced by the process or otherwise, which are also claimed in the invention, are to be retained. Lösung The main claim describes this problem. Advantageous developments of the invention are set forth in the subclaims and the product claims and are explained in more detail below in connection with the invention.

[0023] The claimed process for producing caviar or a caviar-like product based on live, mature, ovulated eggs of fish or crustaceans which have three or more layers in the egg shell is characterized according to the invention in that in a potassium exposure step potassium is dissolved in the water as the cationic component with a concentration of the potassium cations between 0.1 mmol / l and 3.0 mmol / l, wherein the water is deionized before the addition of a potassium donor for the formation of the cationic component and has a temperature in the range of the natural spawning temperature of the fish and crustaceans, and that the live, mature, ovulated eggs are treated in the solution for a potassium exposure time of between 5 min and 30 min, wherein finally the live, mature, ovulated eggs are dipped in a 0.6 percent to 1.0 percent sodium chloride solution.

[0024] The method claimed in the invention utilizes live, mature, ovulated eggs that can be obtained naturally without harming the fish or crustacean. The live, mature, ovulated eggs are capable of fertilization but unfertilized. The ovarian fluid has been removed by prior rinsing with a saline solution (0.6 percent to 1.0 percent) that is non-harmful to the live, mature, ovulated eggs, preventing a sticky gel layer from forming on the outer shell. Furthermore, the ovulated eggs have a natural, artificially unaltered potassium content. Only cell-physiologically effective concentrations of potassium cations are used. The live, ovulated eggs, which in fish and crustaceans have more than two layers in the egg shell, i.e., three or more layers, are electrically activated.The starting product is live, mature, ovulated, fertilizable, but unfertilized eggs whose metabolism is fully functional, so that even the smallest concentrations of potassium cations, which cause no harm and leave no traces in the egg, can trigger transport processes through the egg membrane and metabolic processes in the egg plasma. The caviar or caviar-like products produced or manufactured in this way exhibit a new texture with advantageous stabilizing elasticity due to a new hyaline, acellular layer (elastic stabilizing layer) in the egg membrane, which becomes slightly softer at room temperature without compromising the stability of the caviar. The desired degree of elastic stabilization can easily be determined by self-testing (degree of elasticity of the eggs). The taste is pleasantly fresh and spicy, without being "fishy."The purity of the eggs used results in a long shelf life (9 to 12 months) at standard temperatures between -2°C and -4°C, even without the addition of preservatives, such as borax, which is already banned in many countries. Furthermore, the ovulated eggs treated with the claimed process can be frozen for the first time without any loss of quality, resulting in enormous advantages regarding storage and transport (see below).

[0025] By bringing the living, mature, ovulated eggs into contact with the potassium cations (K +< ) in a physiological, i.e., cell-non-damaging concentration (between 0.1 mmol / l and 3.0 mmol / l), the ovulated eggs are altered by electrical events, eliciting the so-called "first reaction," which leads within a very short time (seconds to minutes) to the electrically induced elimination of stickiness upon contact with water and, during the further course of treatment, to the formation of a new, elastic stabilizing layer within the egg shell. This new stabilizing layer imparts elasticity to the egg shell, so that, according to the invention, a caviar or a caviar-like product of the highest quality is produced even at this stage. This product can also be subjected to optional processing steps, in particular preservation and deep-freezing (at -18°C), without any loss of quality.

[0026] Egg activation encompasses and proceeds through a whole series of cell biological cascades. The "second (slow) reaction" (slow block) used in WO 2007 / 045233 A1, with a cortical reaction, is followed by calcium-dependent enzyme activation for the reinforcement and ultimately the massive structural remodeling of the egg shell by irreversibly tyrosine-crosslinked protein strands in the zona radiata interna and the zona radiata externa. In nature, this prepares the first cell division for embryonic development. The "first (fast) reaction" used in the present invention (fast block, electrical block, fast electrical block), with a subsequent depolarization / hyperpolarization and its stabilization, which varies in duration depending on the species, is the beginning of all cell biological cascades.Both processes differ significantly due to the substances used, namely A) potassium cations for the rapid electrical block with depolarization of the egg shell and the resulting egg activation and formation of a single, additional, new zone in the egg shell (formation of an elastic structuring layer) and B) calcium cations for the slow mechanical block with an enzymatically controlled, morphological remodeling in the existing layers of the egg shell (formation of a structural stabilization layer).

[0027] The first electrical event is a rapid depolarization or even hyperpolarization within milliseconds, which, in nature, prevents further sperm from attaching to the egg after fertilization. This rapid hyperpolarization is followed by a sustained hyperpolarization within the following period of up to 60 minutes (in some aquatic species, such as lobsters, even up to 5 hours). Sperm remaining in the egg environment can still attach after the fast electrical block and become lodged in the vitelline coat of the egg shell (soft coat), but cannot penetrate the egg's plasma membrane for actual fertilization, as has been demonstrated in mollusks.If the potassium cation exposure according to the method claimed in the invention is continued, the formation of a completely new, hyaline (translucent, glassy, ​​gel-like) zone (elastic stabilization layer) is observed in the living, fertilizable, but unfertilized and mature, ovulated egg, which is hitherto unknown in the literature, which is GAG-positive (increased occurrence of glucosaminoglycans) and eosinophilic (stainable with the red, acidic diagnostic dye eosin) for the visualization of cell organelles, plasma proteins, connective tissue and its precursors), and in which sperm would become stuck.The formation of this elastic stabilization layer is first formed in partial areas across the egg shell during sustained hyperpolarization starting at 10 s. Once complete, it is located between the zona radiata externa and the alveolar layer in living eggs with a structural arrangement similar to that of fish and crustaceans (at least three layers in the egg shell). The cause of the formation of the new elastic stabilization layer is seen in the sustained depolarization of the egg shell through the inventive application of potassium cations at physiological concentrations (between 0.1 mmol / l and 3.0 mmol / l).

[0028] According to EU legislation of the Member States on food supplements, only the potassium compounds listed therein, such as potassium bicarbonate (potassium hydrogen carbonate, KHCO 3 ) (CAS No. 298-14-6), potassium carbonate (K 2 CO 3 ) (CAS No. 584-08-7), potassium citrate (CAS No. 6100-05-6 ), potassium hydroxide (KOH) (CAS No. 1310-58-3), potassium chloride (KCI) (CAS No. 7447-40-7), K, potassium iodide (KI) (CAS No. 7681-11-0), potassium iodate (KIO3) (CAS No. 7758-05-6), may be used for nutritional purposes. These compounds may also be added to foods according to the proposal for a regulation of the European Parliament and of the Council of November 10, 2003 (COM (2003) 671 final). Certain potassium compounds, such as potassium citrate (E 332), potassium lactate (E 326), and potassium orthophosphate (E 340), may also be added to foods for technological purposes.Using the method according to the invention for treating live, mature fish or crustacean eggs with potassium cations in cell-compatible concentrations (physiological concentrations, meaning they do not harm the egg) and without residue formation, caviar or caviar-like products can be produced that meet all national and international quality requirements imposed by authorities, distributors, and consumers. Investigations 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 sustain depolarization of the outer egg membrane, show no changes in concentration in the egg plasma after treatment, even with different concentrations and treatment durations. Thus, the potassium cations used in the method according to the invention are clearly considered a technical excipient.A technical aid is used in the industrial processing and production of food. Technical aids are food additives that are added to facilitate technical processes such as cutting and filtering. However, the technical aids must not be present in the final product at all or only in unavoidable (small) residues. In contrast to modifying food additives, which must also be declared on the packaging, technical aids must no longer have any effect in the final product, which is a particular advantage. Their use must be technically unavoidable, technologically ineffective, harmless to health, and harmless in terms of smell and taste. Since the substances are no longer present or effective in the treated food, their use does not need to be labeled. This also applies to residues, reaction products, or residual levels.

[0029] In the invention, the ovulated eggs are treated in a solution bath (an aqueous solution, a solution with water). The ovulated eggs are placed there and left in the solution bath until - depending on the type of egg used - the desired degree of stabilization (elastic and, if applicable, structural) has been achieved. The eggs are then simply removed from the bath. To reliably prevent undesirable further stabilization after removal by cations in the still adhering solution, the invention involves dipping (briefly immersing) the living, mature, ovulated eggs in a non-damaging saline solution (physiological saline solution, 0.6 percent to 1.0 percent) to remove the introduced cations from the mature, ovulated eggs.This flushes away the cations and immediately interrupts the stabilization processes they have caused. The (desired) degree of stabilization of the egg shell achieved up to that point is reliably maintained as the final state.

[0030] It is particularly preferred and advantageous if at least one potassium salt, preferably the salt of citric acid (potassium citrate E332) and / or the salt of hydrochloric acid (potassium chloride E508) and / or the salt of sorbic acid (potassium sorbate E202), is dissolved in water as a potassium donor for the formation of the cationic component. A potassium donor is understood to be a compound containing potassium which, upon dissolution in water, yields the potassium cations, the concentration of which is determined by the concentration of the respective potassium compound and its structural formula in the water. The potassium salts mentioned are all even approved as food additives with E numbers, although in this invention they are used only as technical auxiliaries that no longer occur in the final product and are not subject to declaration.According to a subsequent process modification, a particularly advantageous and preferred concentration of potassium cations in one solution with previously deionized water 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. All ranges specified within the scope of this invention (including other parameters) should always include all marginal and intermediate values ​​(integer and non-integer). In order to produce the stated potassium cation concentrations in water, the water must be deionized. However, since water molecules are constantly decomposing in water, it is understood that only a degree of deionization that can be achieved by technical means can be achieved (electrical conductivity in water between 1 µS and 15 µS at 25°C as a measure of the achievable deionization).

[0031] Furthermore, the potassium exposure time in the potassium exposure step is preferably and advantageously 10 min, 12 min, 15 min, 20 min, or 25 min. However, other potassium exposure times within the specified range are also readily selectable. The formation of the new elastic stabilizing layer that seals the egg membrane begins just a few seconds (up to 10 s) after the start of treatment. However, since the ovulated eggs do not all react simultaneously with sustained depolarization and formation of the stabilizing layer in partial areas of the membrane surface, an extended treatment time of up to 10 min is recommended to achieve sustained depolarization in all treated eggs. With increasing treatment time, it migrates outwards and is ultimately localized between the zona radiata externa and the alveolar layer in the entire egg membrane surrounding the egg.As a result, the invention elastically stabilizes the live, mature, ovulated eggs in such a way that they can be salted, repackaged, and frozen without any problems.

[0032] As a further optional modification, the method claimed in the invention additionally provides for a calcium exposure step, which can be carried out after or prior to the potassium exposure step. The changes to the egg shell brought about in each sequence occur independently of one another in their described characteristics. Preferably and advantageously, in the calcium exposure step, calcium is dissolved in another solution with water as a cationic component in a concentration (between 0.1 mmol / l and 3 mmol / l) that is non-harmful to the living, mature eggs (i.e., physiological), wherein the water is deionized prior to the addition of a calcium donor for the formation of the cationic component. The living, mature, ovulated eggs are treated in the calcium exposure step for a time (between 9 min and 30 min) until the desired structural stabilization of the egg shell is achieved.The desired degree of structural stabilization can easily be determined by self-testing (the degree of popping of the eggs). The temperature is within the natural spawning temperature range of fish and crustaceans, between 1°C and 29°C. Finally, the live, mature, ovulated eggs are dipped in a 0.6 percent to 1.0 percent saline solution.

[0033] In the calcium exposure step, it is advantageous and preferable to use at least one calcium salt, preferably calcium citrate, calcium chloride, and / or calcium sorbate, as a calcium donor (calcium supplier; for definition, see potassium donor). Calcium salts are approved in the European Union as food additives under the numbers E333 and E509 without a maximum limit and E203 with a maximum limit. To better distinguish the use of the two ion types, the spellings "potassium" and "calcium" (not calcium) were chosen.

[0034] Calcium is already physiologically present in the egg cell and is an essential component of cellular metabolism. From the above-mentioned WO 2007 / 045233 A1, it is known to use calcium chloride to achieve structural strengthening of the egg shell through irreversible cross-linking of proteins through the incorporation of tyrosine molecules. In addition to the improved and adjustable elasticity of the egg shell fundamentally achieved with the invention, the calcium exposure step can further mechanically strengthen the egg shell. Thus, an optimal, stabilizing combination for certain types of caviar and caviar substitutes can be achieved.This is particularly advantageous for very large, unstable ovulated eggs (greater than 3.2 mm in diameter, for example, beluga or white sturgeon eggs) or for those that are particularly soft in the mature, ovulated state (maximum force less than or equal to 0.3 N until bursting in the hardness test, for example, sterlet sturgeon eggs). The application of both treatment steps results in a very high-quality caviar or caviar-like product for problematic (size, softness) ovulated eggs.

[0035] Advantageously and preferably, the concentration of calcium cations in the other 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. The calcium exposure time is preferably 10 min, 12.5 min, 15 min, 16 min, 20 min, or 25 min. When selecting the treatment duration, it must be taken into account that the strength of the egg shell increases steadily with increasing calcium exposure time until a limit is reached. In nature, fertilized fish eggs reach a strongly hardened egg shell after approximately 60 minutes, which is no longer suitable for consumption. In lobsters, this can take up to 24 hours.

[0036] A key process parameter in the process claimed by the invention is the temperature of the solutions in which the mature eggs are treated. This temperature should be physiological, meaning that it does not interfere with the natural processes in the living eggs. In the claimed process, the temperature of the solutions is always within the range of the natural spawning temperature of fish or crustaceans. This ensures that the electrical activation of the egg shell induced in the potassium exposure step, with a depolarization starting at the resting potential, occurs reliably. At unnatural spawning temperatures, for example, in fish or crustaceans from polar regions above 15°C, no electrical egg activation occurs, and the living, mature, ovulated eggs cannot be electrically or enzymatically stabilized. They become atretic.The same applies to ovulated eggs of fish or crustaceans from temperate and tropical zones. Generally, at solution temperatures above 35°C, the ovulated eggs experience significant loss of quality due to degeneration.

[0037] To adapt the temperatures of the solutions to their natural habitats, the present invention roughly divides the habitats of fish and crustaceans during the periods of natural reproduction, whose live, mature, ovulated eggs can be used, into three climatic zones: polar zones (at the poles), temperate zones (between the polar zones and the tropical zone), and tropical zone (around the equator). In the present invention, it is preferred and advantageous to set the temperature of one solution (potassium exposure) and / or the other solution (calcium exposure) in a polar temperature range between 1°C and 15°C, preferably between 5°C and 12°C, particularly preferably 10°C, a temperate temperature range between 10 and 20°C, preferably 15°C, particularly preferably 12°C, or a tropical temperature range between 20°C and 29°C, preferably 27°C, particularly preferably 21°C.Temperatures that lead to changes - degeneration, cell death - in the eggs, as is the case, for example, during pasteurization by heating to temperatures above 40°C, are avoided at every stage of the process in the invention.

[0038] Since the cation concentrations used in the process according to the invention trigger animal-specific physiological reactions of an electrical (potassium exposure) and metabolic (calcium exposure) nature and thus influence the processing into a stable, edible end product, deionized water must always be assumed in the solution in order to achieve an exact concentration of electrically (potassium) or metabolically (calcium) active cations (positively charged). It is therefore technically achievable and thus preferred and advantageous if the deionized water at 25°C has an electrical conductivity between 1 µS / cm and 15 µS / cm, preferably 10 µS / cm or below, and particularly preferably 1 µS / cm. Depending on the regional source, drinking water and well water consist of a greatly varying composition of various ions, which can under certain circumstances even have antagonistic effects on cellular metabolism.At a temperature of 25°C, for example, the electrical conductivity of ultrapure water is 0.055 µS / cm, deionized water 1 µS / cm, rainwater 50 µS / cm, or drinking water 500 µS / cm. To obtain reproducible results with the claimed method, it is important to know the electrical conductivity of deionized water.

[0039] Since the method claimed in the invention treats living cells in the form of activatable mature, ovulated egg cells, it is important, among other things, that the solutions are adapted to the metabolism of the cells so that the metabolic processes induced by the method can also take place. Therefore, it is advantageous and preferred if one and / or the other solution has a (physiological, non-detrimental to the living organism) pH between 6.8 and 8.0, preferably between 7.0 and 7.9, particularly preferably 7.2, 7.4, or 7.5. In particular, the pH adjusted in the solution is relevant for the slow metabolic reaction in the calcium exposure step. Since enzymatic processes in the cell are strongly regulated by the pH, the intracellular pH in the potassium exposure step (electrical process) was also investigated.However, the pH value in the egg plasma of the eggs treated with the various potassium-containing substances in different concentrations and for different durations remains essentially unchanged in the pH optimum between 7 and 8 and shows the expected individual differences in individual fish and crustaceans.

[0040] In the invention, the various exposure steps serve to endogenously stabilize (elastically and optionally structurally) the egg shell of the live, mature, ovulated eggs. This means that the caviar or caviar-like product is ready for further processing, such as salting and packaging. During ovulation, the live, mature eggs are expelled from the follicle cells, leaving no traces of blood vessel tissue or follicle cell residue adhering to them, which could harbor bacteria or fungi. Therefore, harvested ovulated eggs are extremely pure and thus ideally suited for a long shelf life. This is reliably ensured if, following the final exposure step, a mild salting with a sodium chloride concentration of 2.0% to 3.8%, preferably 3.5%, based on the quantity of caviar or caviar-like product, is carried out for preservation and flavor intensification.The sodium chloride must be free of potassium and calcium donors, such as those found in anti-caking agents, as this prevents uncontrolled changes in the egg shell of the treated ovulated eggs due to salting. For caviar made from sturgeon eggs, dry salting is carried out with simple table salt (sodium chloride NaCl), while wet salting is often used when processing the roe of other fish species into caviar-like products such as salmon and trout caviar. The salting process used in the invention in the mentioned range is a very light salting, also referred to as "malossol," and is a clear indication of high quality. Pasteurization or heating to a temperature of 60°C and above is completely unnecessary for the caviar or caviar-like product claimed in the invention, as this is unnecessary and would only impair the quality of the product and its sensory properties.Malossol salting gives caviar or caviar-like products produced using the claimed process a minimum shelf life of at least 9 to 12 months when stored at -2°C. Due to the light salting, they do not freeze.

[0041] A further improvement in the quality of the produced caviar or caviar-like product is achieved with the invention if, according to a further modification, the caviar or caviar-like product is preferably and advantageously stored in hermetically sealed glass containers for several months, preferably one to three months, following preservation and flavor intensification. Through storage, the caviar "ripens" and gains flavor intensity depending on the degree of ripeness. However, this ripening is to be viewed as a further development of flavor (as with cheese, for example) and has nothing to do with the "maturity" of the mature eggs used in the invention in the sense of biological development. Here, maturity refers to the possibility of fertilization and thus to the stage of development of the living eggs.During the flavor maturation process, the caviar is stored in glass containers, which provide sufficient space for maturation because it is not compressed (as is the case with packaging in metal, snap-lid containers), thus retaining its intensely flavorful oils. Caviar packaged in this glass according to the invention is not to be confused with pasteurized caviar, which is also frequently packaged in glass. Furthermore, storage in environmentally friendly glass containers avoids the often-criticized metallic taste of caviar packaged conventionally in metal containers.

[0042] According to a further process modification, it is preferred and advantageous if, following preservation and flavor intensification or storage and flavor maturation, the caviar or caviar-like product is frozen at a temperature range between -20°C and -15°C, preferably at -18°C. Ideally, the caviar matures to the desired degree of maturity for human consumption and is frozen either fresh, 14 days after production, or after a maximum of 3-4 months of maturation. The caviar is frozen either in 500g glass jars prior to repackaging or, after repackaging for the end customer, in 30g, 50g, 125g, 250g, or 500g (possibly up to 1000g) vacuum-sealed jars. Caviar obtained by conventional slaughter cannot be frozen.Pasteurized or heated caviar can be frozen, but suffers from severe deterioration in quality due to the heat treatment. The ability to freeze caviar or caviar-like products according to the invention enables optimal caviar marketing that meets today's convenience food demands. Until now, marketing has encountered limitations due to the specific, precisely maintained transport and storage temperatures of -2°C to -4°C, which most suppliers do not maintain. Therefore, conventionally produced caviar is treated with questionable preservatives such as borax or pasteurized to ensure its shelf life for at least 12 months or longer. The caviar produced with the present invention, however, can simply be frozen and thus stored and kept fresh for a longer period.Tests have shown that caviar thawed slowly in the refrigerator at temperatures between +4°C and +7°C does not show any loss of flavor or change in texture.

[0043] The live, mature, ovulated eggs used in the invention are capable of fertilization but unfertilized. Furthermore, they are unwetted by water and have a natural potassium content in their egg plasma. Such live eggs can be released from the gonads into the fish's abdominal cavity and harvested from there through the genital opening. This can be achieved, for example, through natural spawning, by stripping (external massage of the abdominal cavity), or using a catheter through which the eggs are drained or suctioned out of the abdominal cavity. Eggs released from the gonads into the abdominal cavity are referred to as ovulated eggs (maturity level 5) that are still surrounded by a mucous ovulation fluid. To prevent the formation of a sticky layer on the eggs upon contact with water, the ovulation fluid is rinsed off with physiological saline solution (0.6 percent to 1.0 percent) before treatment begins.Ovulated eggs can be harvested from the live animal, which is particularly sustainable. A good overview of the different stages of maturity in cod can be found in the publication by IG Katsiadaki et al.: "Assessment of quality of cod roes and a relationships between quality and maturity stage" (J. Sci Food Agric 79:1249-1259 (1999)), particularly in Table 1. A numerical definition of the stage of maturity is possible using the so-called "polarization index." This 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 the vegetative poles (semi-major axis). Therefore, according to a next embodiment of the invention, it is preferred and advantageous if live, mature, ovulated eggs of fish or crustaceans are treated with a polarization index PI of 0.05 ≤ PI ≤ 0.15, preferably 0.05 ≤ PI ≤ 0.12.Eggs with this PI are particularly suitable for harvesting for treatment according to the invention. Further information on the polarization index (PI) of eggs can be found, for example, in the Guidelines for Sturgeon Breeding (Publication . FAO Ankara 2011 Fisheries and Aquaculture Technical Paper 570 "Sturgeon Hatchery Practices and Management for Release - Guidelines").

[0044] With the method claimed in the invention, the live, mature eggs of fish and crustaceans (scientific name Crustacea ) whose eggs have the basic structure required for the invention (more than two layers in the egg shell) and which are suitable for consumption in the form of caviar or caviar-like products. Preferably and advantageously, live, mature, ovulated eggs from wild-caught or aquaculture-bred fish or crustaceans can be treated, which were obtained in the ovulated state by stripping or other targeted harvesting, such as catheterization. In this case, it is also possible, for example, to harvest animals that are intended for restocking in the wild, for example in a restocking project. The proceeds from the sale of caviar and caviar-like products can then be used for restocking measures.It is particularly preferred and advantageous if the claimed method is used to treat living, mature, ovulated eggs from recent and primitive living bony fish, preferably from living sturgeon. (Genuine) caviar of the highest quality can then be produced using the method. However, caviar-like products from lobster or other crustaceans, e.g., noble crayfish, can also be produced in the highest quality using the claimed method according to the invention. Furthermore, very large (over 3.2 mm in diameter) or soft, unstable, ovulated eggs (texture in the hardness test below 0.3 N, above which the eggs burst) can preferably and advantageously be treated, since the method claimed in the present invention can optionally also comprise two exposure steps with both an elastic (electrically stimulated) and a structural (enzymatically stimulated) stabilization of the egg shell.

[0045] Finally, the invention also claims various products made from live, mature, ovulated eggs of fish or crustaceans, which have three or more layers in the egg shell, which can be produced using the claimed process but also using other processes. The products are characteristically characterized by the additional elastic stabilizing layer in the egg shell in the form of an eosinophilic, hyaline layer with embedded glycosaminoglycans. However, the live egg is unfertilized, which is why the elastic stabilizing layer does not occur naturally. In the invention, the elastic stabilizing layer lies between the zona radiata interna and the alveolar layer, preferably between the zona radiata externa and the alveolar layer. It can therefore only occur in live eggs with an egg shell structure of more than two layers.Sea urchins, for example, have exactly two layers in their egg shell. The new stabilizing layer is transparent, gel-like, and elastic, and can be histologically stained with eosin red and alcian blue. When produced using the method claimed in the invention, its appearance is influenced by the potassium cation concentration used in the potassium exposure step, and its position is influenced by the potassium exposure time.

[0046] To remove the ovarian fluid, the live, mature, ovulated eggs are treated with a saline solution (0.6 percent to 1.0 percent) that is harmless to the eggs. This is a physiological saline solution. Furthermore, it is advantageous and preferred if the saline solution is a 0.9 percent saline solution. To prepare a 0.9 percent saline solution, 9 g of sodium chloride (NaCl) are dissolved in 1 liter of water. This concentration corresponds to the natural occurrence in the human organism; therefore, it is referred to as a "physiological" saline solution.

[0047] Furthermore, caviar or a caviar-like product made from unfertilized, mature, ovulated eggs of fish or crustaceans with three or more layers in the egg shell is claimed, which is characterized by the additional irreversible cross-linking of protein strands formed in the egg shell by incorporated tyrosine molecules. This additional irreversible cross-linking is localized in the zona radiata interna and the zona radiata externa of the living, ovulated eggs of fish or crustaceans. The irreversible cross-linking leads to additional structural stabilization of the egg shell. Together with the existing elastic stabilization, it can also be used to treat particularly large or soft ovulated eggs.The caviar or caviar-like product can be produced using the claimed process, with the degree of structural stabilization in the egg shell then depending on the calcium exposure time and the calcium cation concentration in the calcium exposure step. Other processes for producing caviar or a caviar-like product with the same degree of irreversible protein cross-linking in the egg shell are also applicable. Further details on the processes and products claimed by the invention can be found in the following specific description of the embodiments. Ausführungsbeispiele

[0048] The process for producing caviar or a caviar-like product from live, mature, ovulated eggs of fish or crustaceans and such products according to the invention and their advantageous modifications for a further understanding of the invention are explained in more detail below using exemplary embodiments and figures. Fig 1A, B, CREM images comparing obtained live eggs in the immature and mature states (state of the art), Fig 2a first table of egg shell thickness measurements for live, mature eggs of Siberian sturgeon, Fig. 3a second table showing egg shell thickness measurements when treating live, mature beluga sturgeon eggs, Fig. 4A, B, C, DREM images of the treatment series for the formation of the stabilizing layer of differently treated live, mature sturgeon eggs by potassium cation treatment compared to the double treatment with potassium and calcium cations and calcium cations alone, Fig. 5A, B, C, DTEM images of the structure of the egg shell layers with the formation of the new stabilizing layer (SS) between the zona radiata externa (ZRE) and the alveolar layer (AL), Fig. 6A, B, C, DTEM images of the cortical granules of untreated and differently treated (potassium cations alone, double treatment with potassium and calcium cations, calcium cations alone) mature sturgeon eggs, Fig 7A, B, C, DLight micrographs of untreated mature Siberian sturgeon eggs and of live, mature Siberian sturgeon eggs treated with potassium ions, Fig. 8A, B, C, DLight micrographs of live, mature eggs treated with calcium cations and of live, mature eggs treated with potassium cations and calcium cations from Siberian sturgeon and Fig. 9A, BREM and light microscopy images of the structure of the egg shell of beluga sturgeon after treatment of live, mature eggs with potassium and calcium cations.

[0049] Studies of the relationship between the weight and age of sturgeons and the size of the caviar grain or the amount of caviar harvested have shown that with increasing weight and age of the sturgeons, the diameter of the egg increases and with it the quality of the caviar. Furthermore, with increasing weight and age of the sturgeons, the amount of caviar harvested increases and with it the economic success. In the natural environment, sturgeons do not reach sexual maturity for the first time until they are 12 to 26 years old, depending on the species. Most sturgeons spend the growth phase up to their first reproduction in the sea or in estuaries and then migrate into rivers to find their spawning grounds on rocky bottoms in freshwater. But even in aquaculture, sturgeons need around 5 to 16 years, depending on the sturgeon species, until they reach sexual maturity and thus the first caviar harvest.The repeated harvesting of caviar in aquaculture from live females over many years requires animal-friendly husbandry with optimal feeding and low stocking densities. Due to their late sexual maturity and long lifespan, it is always economically and ecologically viable. Caviar production in the economically viable tonnage range is easily achievable with a coordinated workflow from harvesting to processing the stripped eggs into caviar. With appropriate upscaling measures, a daily production of 80 kg or more can be achieved, depending on the age of the fish and the associated caviar yield.

[0050] The claimed process uses live, mature eggs that have previously been cleaned with physiological saline solution. These are ovulated eggs that, due to their stage of maturity (the stage of ovulation readiness and fertilization capacity), are previously pushed out of the gonad by fine muscle fibers of the follicular cells, a process known as ovulation. The ovulated eggs are released into the oviducts and abdominal cavity of the fish without any cell debris or other residues. From there, they can be removed by massaging the abdomen without affecting the fish's life. The completely clean surface of the eggs offers no niches or folds for bacterial and fungal infestation, resulting in a long shelf life for the caviar or caviar-like product. The use of preservation methods, such as borax, which is harmful to human health, is not required. Fig. 1A , B, C are state-of-the-art scanning electron microscopy (SEM) images of a living egg and the ovulation process. Fig. 1A shows an immature egg cell with follicular cells, as they occur in conventional caviar from killed sturgeon. Fig. 1B shows in-situ ovulation and release of the mature egg cell from the surrounding follicle cell. Fig. 1C then shows a live, mature sturgeon egg that is obviously completely smooth and clean.

[0051] Using an embodiment example with live, mature eggs after ovulation, a possible process sequence of the claimed method with some optional additional steps is explained in more detail below: Stripping live eggs at stage V of maturation from the live female sturgeon after the lysis of the germinal vesicles, immediate transport of the stripped live, ovulated eggs together with the ovarian fluid to a caviar laboratory (standing times are largely avoided; unavoidable standing times occur on ice and under exclusion of oxygen by covering the ovarian fluid with an air-impermeable plastic film), immediate thorough rinsing of the live, ovulated eggs in 0.9 percent physiological saline solution until the ovarian fluid is completely removed, carrying out the potassium exposure step: preparing a 0.1 to 2 millimolar potassium cation solution from potassium citrate in deionized water with a conductivity of 10 µS / cm (at 25°C) and a temperature in the polar temperature range of 10°C, introducing the live, mature,ovulated eggs into the solution for a potassium exposure time of 10 min and removing the treated eggs from the solution and briefly dipping the treated eggs into a 0.9 percent physiological saline solution.

[0052] Other methods of obtaining mature, ovulated eggs are also possible. Even when using live, mature, ovulated eggs from a previously killed animal, blood and fat must be rinsed off, which is achieved by pretreatment with a preferably physiological saline solution. Dipping allows the elasticity and diameter of the stabilizing layer to be additionally controlled (in addition to the choice of exposure time). Due to the electrical influence of the introduced potassium cations, the described treatment causes a hyaline, elastic stabilizing layer to form between the zona radiata externa and the alveolar layer in the egg shell of the live ovulated eggs. For ovulated eggs of normal size and softness, treatment with the potassium exposure step is sufficient. However, if particularly large, soft, or sensitive ovulated eggs of some species of sturgeons, for example Huso huso, Acipenser transmontanus or Acipenser ruthenus, used, a calcium exposure step can be added (or preceded): additionally performing the calcium exposure step: preparing another 0.5 to 2 millimolar calcium cation solution from calcium chloride in deionized water with a conductivity of 10 µS / cm (at 25°C) and a temperature in the polar temperature range of 10°C, placing the live, mature, ovulated eggs in the other solution for a calcium exposure time of 12 minutes and removing the treated ovulated eggs from the other solution and briefly dipping the treated ovulated eggs in a 0.9 percent physiological saline solution.

[0053] In addition to the elastic stabilizing layer from the potassium exposure step, a structural protein crosslinking of the egg shell forms, which in fish and crustaceans is located in the already existing radiata interna and radiata externa zones of the egg shell. This additional structural protein crosslinking of the egg shell through tyrosine residues lends plastic strength, especially to large eggs and eggs that were soft or fragile prior to treatment—in addition to the elasticity from the potassium exposure step. Here, too, dipping provides additional control. The resulting product is (real) caviar from live, mature, ovulated eggs, which can then be further processed as follows: Mixing the caviar with dry table salt NaCl (3.5g / 100g caviar, 3.5%), which contains neither potassium nor calcium donors (K +< - and Ca ++< -free flow agents), which corresponds to a malossol salting for preservation, filling the lightly salted caviar into glass containers, preferably 500g maturation jars, and hermetically vacuum sealing the containers with screw lids and labeling, storing the glass containers at -2°C for 2 to 4 months for further maturation of the caviar and optionally freezing the fresh caviar or the caviar matured according to customer requirements in the glass containers at -18°C.

[0054] The live, mature, ovulated eggs treated in the potassium exposure step form a completely new zone due to the treatment: the stabilizing layer, which is elastic and hyaline (gel-like). The stabilizing layer is easily stained for detection. It is located between the alveolar layer AL and the zona radiata externa ZRE and has not been described in the literature to date. For the structural organization of the egg of fish and crustaceans, please refer to the introduction to the description with the corresponding glossary after Siddique pointed out.

[0055] The table in the Fig. 2 are based on measurements of the diameter (in µm) of the extracellular egg shell of mature, ovulated eggs of the Siberian sturgeon using cryosections of constant layer thickness (10 µm) using computer-controlled image analysis (Zeiss) under the influence of different treatments for stabilizing the egg shell. The table shows the formation of a new stabilization layer. SS and the diameter of existing layers (ZRI, ZRE, AL) of the egg shell under treatment with different concentrations in mmol / l of potassium cations K +< alone (from potassium citrate) and in combination with calcium cations Ca ++< (from calcium chloride) during the various exposure steps according to the invention. Live, mature, ovulated eggs from Siberian sturgeon were Acipenser baerii In addition to the previous and Siddique explained acronyms still means Mw Mean, Std Standard deviation. The values ​​for the new elastic stabilization layer are stored SS . Furthermore, reference is made to the prior art according to the above-mentioned WO 2007 / 045233 A1.

[0056] Quality controls after treatment showed that only at concentrations of 1 mmol / l and 1.5 mmol / l potassium cations does an egg shell thickness of at least 12 µm occur, resulting in an intermediate product that has lost its stickiness and is stable enough for further caviar processing. Furthermore, it was shown that a treatment duration of preferably 10 minutes is advisable to ensure that all live eggs in the solution undergo a metabolic reaction. A treatment quantity of 2.5 kg of caviar (in approximately 25 l of solution) was achieved in one treatment unit. Sensory testing of the caviar after the inventive treatment with potassium cations showed that the elastic texture of Siberian sturgeon caviar did not differ with concentration variations between 1 mmol / l and 1.5 mmol / l.In contrast, the ovulated eggs treated in one solution with lower potassium cation concentrations vary in texture, and only a few eggs are stable, while the untreated eggs are very soft and burst. According to the sensory tests conducted, a treatment with two exposure steps (potassium and calcium cations) results in a firm, pearly product, also referred to as a "superplop" in Siberian sturgeon eggs.

[0057] The table in Fig. 3 shows the presence and diameters (in µm) of layers of the extracellular egg shell with different treatments according to the invention of the live large eggs of the beluga sturgeon Huso huso The formation of the new eosinophil stabilization layer was also observed when treating this caviar with potassium cations (from potassium citrate). SS in the extracellular egg shell, which is also located between the ZRE and the AL is located. For the large beluga sturgeon eggs, the sensory tests conducted showed that a double treatment with potassium and calcium cations leads to optimal results regarding the texture of the fragile live, mature, ovulated eggs.

[0058] The Fig. 4A B, C, and D show SEM images of the changes in the structure of the extracellular egg shell of live, mature, ovulated eggs, exemplified by Siberian sturgeon eggs, under various treatments. Two magnifications are shown: 6000x on the left and 12000x on the right (inset). The treatments were always performed on the live egg, which was snap-frozen in hexane at -80°C to preserve the native state for histological cryosections. Fig. 4AIn untreated mature, ovulated eggs, the zones of the egg shell do not show a clear separation from each other (state of the art). Fig. 4BUnder the influence of 0.5 mmol / l potassium, the new stabilization layer is already formed SS between the Zona Radiata Externa ZRE and the alveolar layer AL, while the Zona Radiata Interna ZRI and Zona Radiata Externa ZRE show an unchanged loose protein structure as in untreated eggs. Fig. 4CThe consecutive double treatment of the ovulated eggs with potassium and calcium cations shows in the SEM both characteristic morphological features, namely the stabilization layer SS by the potassium treatment AND the twisting and cross-linking of the protein strands in the Zona Radiata Interna ZRI and the Zona Radiata Externa ZRE, which is characteristic of calcium treatment, compare Fig. 4D . Fig, 4D Calcium treatment alone leads to strong twisting and cross-linking of the loose protein strands in the Zona Radiata Interna ZRI and the Zona Radiata Externa ZRE (state of the art), compare with Fig. 4A und Fig. 4B without calcium treatment.

[0059] The Fig. 5A , B, C, D show transmission electron micrographs (TEM, 3000x magnification) of the multilayered structure of the egg shell of mature sturgeon eggs treated with 1.0 mmol / l potassium cations. Fig. 5A shows the Zona Radiata Interna ZRI with loose, cloudy fibrils that pass through the epilayer EP from the Zona Radiata Externa ZRE separated (phenomenon is only ultrastructurally identifiable). The Zona Radiata Externa ZRE is characterized by a filamentous network of elongated fibrils. Fig. 5B shows the ultrastructural formation of the new stabilization layer SS with a fine-granular structure directly between the Zona Radiata Externa ZRE and - according to Fig. 5C - the alveolar layer AL, which towards the periphery of the egg shell - according to Fig. 5D - is penetrated by small ducts (small channels, canals). Ultrastructural analyses confirm that treatment with potassium cations (1.0 mmol / l) according to the invention leads to the formation of a previously unknown new stabilizing layer SS with amorphous structure and positioning in fish and crustaceans between the Zona Radiata Externa ZRE and the alveolar layer AL leads.

[0060] In the Fig. 6A , B, C, D are TEM images (3000x magnification) of the cortical granules CG in the peripheral egg plasma within the plasma membrane of the mature ovulated eggs, where the Fig. 6A an untreated egg (state of the art), the Fig. 6B an egg treated with potassium cations, the Fig. 6C an egg treated with potassium and calcium cations and the Fig. 6D shows an egg treated only with calcium cations (state of the art).

[0061] Cortical granules are secretory organelles (structurally distinct regions) found in eggs and are closely associated with the fertilization event. Cortical granules contain enzymes, such as peroxidase, and structural elements for tyrosine cross-linking of the zona radiata interna. ZRI and the Zona Radiata Externa ZRE. As analyzed using TEM under the influence of various treatments, the cortical response and the release of its contents are triggered exclusively by treatment with calcium cations. An identical process also occurs during natural fertilization through the sperm-induced calcium wave at the egg plasma membrane. In the untreated egg ( Fig. 6A ) are the cortical granules in the peripheral egg plasma CG with their enzyme content are clearly recognizable as large, round vesicles that also contain structural elements. The cortical granules are also CG in the potassium treatment alone according to the invention ( Fig. 6B ) are still present unchanged. However, a strong vesicular transport at the egg membrane from the egg plasma into the acellular egg shell can be observed. In the Fig. 6A und Fig. 6B is with D the yolk. In a double treatment with potassium and calcium cations ( Fig. 6C ) there is a release (arrows) of the contents of the cortical granules CG and also to form the new stabilization layer SS Treatment with calcium cations alone ( Fig. 6D ) in turn only leads to the cortical reaction, whereby the contents are released into the acellular egg shell and the enzymatic cross-linking of the Zona Radiata Interna ZRI and Zona Radiata Externa ZRE initiated by tyrosine residues. Empty vacuoles remain in the egg plasma V.

[0062] For diagnostic screening of cryosections for the potassium effect in the invention, the prior art in the Fig. 7A und Fig. 7B light micrographs (400x magnification) of untreated live eggs of the Siberian sturgeon Acipenser baerii The left photo according to Fig. 7A shows a HE stain (hematoxylin-eosin stain), the right photo according to Fig. 7B shows a staining with Alcian blue. This test stains glucosaminoglycans GAG, hyaluronic acid, and fibrin. It can be seen that Alcian blue is absent in the various layers of the egg membrane, but is prominent in the ooplasm. OP is present. The individual layers are labeled as described above and their thicknesses are indicated by double arrows.

[0063] In the Fig. 7C und Fig. 7D are, however, photographs of cryosections of live, mature eggs in the ovulated state of the Siberian sturgeon treated with the method claimed in the invention in the potassium exposure step Acipenser baerii The eggs were treated with a concentration of 1.5 mmol / l potassium cations (from potassium citrate). The appearance of the new stabilizing layer is striking. SS between the Zona Radiata Externa ZRE and the alveolar layer AL in the extracellular egg shell. In the left photo according to Fig. 7C After staining with eosin, it is evident that this new stabilizing layer SS is particularly eosinophilic. In the right photo according to Fig. 7D After staining with Alcian blue, it is evident that this new stabilizing layer SS is particularly rich in GAG. This explains the particularly advantageous elasticity of the new stabilizing layer SS derive.

[0064] For diagnostic screening on cryosections for calcium effect alone and double treatment effect by potassium and calcium cations, Fig. 8A und Fig. 8B from the prior art photos of cryosections of mature eggs of the Siberian sturgeon treated with the method according to WO 2007 / 045233 A1 Acipenser baerii shown at 400x magnification. The left photo according to Fig. 8A shows a HE stain (hematoxylin-eosin stain), the right photo according to Fig. 8B shows staining with Alcian blue. Ovulated eggs of the Siberian sturgeon were treated. In the left photo, according to Fig. 8A With HE staining, the protein strands cross-linked by tyrosine molecules in the Zona Radiata Interna are ZRI and Zona Radiata Externa ZRE can be seen. Furthermore, a clear separation between the two zones is visible. The cross-linking results in a structural stabilization of the egg shell. In the right photo according to Fig. 8B With the Alcian blue stain it can be seen that the Zona Radiata Interna ZRI and the Zona Radiata Externa ZRE are only very weakly colored, which indicates little GAG ​​and lower elasticity, whereas in the ooplasm OP a strong color indicates a lot of GAG.

[0065] In the Fig. 8C und Fig. 8D Photographs of cryosections of live, ovulated eggs processed by the claimed method are shown, which were treated in the additional calcium exposure step with the method according to WO 2007 / 045233 A1. The mature ovulated eggs of the Siberian sturgeon Acipenser baerii were treated with 1.5 mmol / l potassium cations (from potassium citrate) in the potassium exposure step and with 1.6 mmol / l calcium cations (from calcium chloride) in the calcium exposure step. In addition to the exposure shown in the photos according to Fig. 8A und Fig. 8B The strengthening cross-linking of the egg shell shown in the photos according to Fig. 8C und Fig. 8D demonstrated new hyaline stabilization layer SS with their elastic stabilizing function of the egg shell. The treated live, mature, ovulated eggs of the Siberian sturgeon are thus stabilized both elastically (by GAGs) and structurally (by protein cross-linking), forming perfect caviar.

[0066] In the Fig. 9A is a characteristic SEM image (12000x magnification) of the egg shell of live, mature ovulated eggs of the beluga sturgeon Huso huso after the double treatment according to the invention. The Fig. 9B shows a light microscopic image (400x magnification) of the egg shell of live, mature, ovulated eggs of the beluga sturgeon Huso huso after the double treatment according to the invention. Both images show the new stabilization layer SS and the networking of the Zona Radiata Interna ZRI and Zona Radiata Externa ZRE. Furthermore, it is noticeable that the eggs of the Huso huso an extremely pronounced alveolar layer AL with large vacuoles V Screening of cryosections with H&E staining confirms the formation of the new elastic stabilization layer in the invention SS with one potassium cation exposure step and the additional protein cross-linking in the egg shell with two exposure steps with potassium and calcium cations. Bezugszeichenliste

[0067] AL Alveolar layer Ca ++< Calcium cations CG Cortical granules CYT Cytoplasm (OP) D yolk EP Epilayer K +< Potassium cations Mw mean OP Oocyte plasma (egg plasma) pAL Alveolar layer (towards the periphery of the egg shell) MONTH Plasma membrane of the oocyte (egg cell) SS Stabilization layer Std Standard deviation V vacuole ZRIInternal Radiated Zone ZRA External Radiated Area

Claims

1. A method for producing caviar or a caviar-like product from live, mature, ovulated eggs from fish or crustaceans which have three or more layers in the egg envelope, wherein the live, mature, ovulated eggs are in a fertile but unfertilised state and have a natural potassium content in the egg plasma, by treatment of the live, mature, ovulated eggs in a 0.6 percent to 1.0 percent saline solution and subsequently in at least one solution containing water and at least one cationic component dissolved therein which acts to stabilise the egg envelope of the live, mature, ovulated eggs, characterized in that in a potassium exposure step, potassium is dissolved in water as the cationic component in a concentration of potassium cations of between 0.1 mmol / l and 3.0 mmol / l, wherein prior to the addition of a potassium donor for the formation of the cationic component, the water is deionised and is at a temperature in the natural spawning temperature range for the fish and crustaceans of between 1°C and 29°C, and in that the live, mature, ovulated eggs are treated in the solution for a potassium exposure time of between 5 min and 30 min, wherein finally, dipping of the live, mature, ovulated eggs is carried out in a 0.6 percent to 1.0 percent saline solution.

2. The method as claimed in claim 1, characterized in that the potassium donor employed is at least one potassium salt, preferably the salt of citric acid and / or the salt of hydrochloric acid and / or the salt of sorbic acid.

3. The method as claimed in claim 1 or claim 2, characterized in that the concentration of the potassium cations in the one 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 as claimed in one of the preceding claims, characterized in that the potassium exposure time in the potassium exposure step is between 10 min, 12 min, 15 min, 20 min or 25 min.

5. The method as claimed in one of the preceding claims, characterized in that after or before the potassium exposure step, in a calcium exposure step in another solution, calcium is dissolved in water as the cationic component in a concentration of the calcium cations of between 0.1 mmol / l and 3 mmol / l, wherein prior to the addition of a calcium donor for the formation of the cationic component, the water is deionised and is at a temperature of between 1°C and 29°C which is in the natural spawning temperature range for fish and crustaceans, and in that the live, mature, ovulated eggs are treated in the solution for a calcium exposure time of between 9 min and 30 min, wherein finally, dipping of the live, mature, ovulated eggs is carried out in a 0.6 percent to 1.0 percent saline solution.

6. The method as claimed in claim 5, characterized in that the calcium donor employed is at least one calcium salt, preferably calcium citrate, calcium chloride and / or calcium sorbate.

7. The method as claimed in claim 5 or claim 6, characterized in that the concentration of the calcium cations in the other 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 as claimed in one of claims 5 to 7, characterized in that the calcium exposure time in the calcium exposure step is 10 min, 12.5 min, 15 min, 16 min, 20 min or 25 min.

9. The method as claimed in one of the preceding claim, characterized in that the temperature of the one solution in the potassium exposure step and / or of the other solution in the calcium exposure step in a polar temperature range is between 1°C and 15°C, preferably between 5°C and 12°C, particularly preferably 10°C, in a moderate temperature range is between 10°C and 20°C, preferably 15°C, particularly preferably 12°C, or in a tropical temperature range is between 20°C and 29°C, preferably 27°C, particularly preferably 21°C.

10. The method as claimed in one of the preceding claims, characterized in that the deionised water has an electrical conductivity at 25°C of between 1 µS / cm and 15 µS / cm, preferably 10 µS / cm or below, particularly preferably 1 µS / cm.

11. The method as claimed in one of the preceding claims, characterized in that the one and / or the other solution has a pH of between 6.8 and 8.0, preferably between 7.0 and 7.9, particularly preferably 7.2 or 7.4 or 7.5.

12. The method as claimed in one of the preceding claims, characterized in that subsequent to the last exposure step carried out for the purposes of preservation and flavour intensification, mild salting is carried out with 2.0% to 3.8%, preferably 3.5% sodium chloride with respect to the quantity of caviar or caviar substitute product, wherein the sodium chloride is free from potassium and calcium donors.

13. The method as claimed in claim 12, characterized in that subsequent to the preservation and flavour intensification, storage of the caviar or caviar-like product is carried out in hermetically sealed glass containers over several months, preferably one to three months, at a temperature between -2°C and -4°C.

14. The method as claimed in claim 12 or claim 13, characterized in that subsequent to the preservation and flavour intensification or storage, the caviar or the caviar-like product is frozen at a temperature in the range between -20°C and -15°C, preferably at -18°C.

15. The method as claimed in one of the preceding claims, characterized in that live, mature, ovulated eggs from fish or crustaceans with a polarization index "PI" of 0.05 ≤ PI ≤ 0.15, preferably 0.05 ≤ PI ≤ 0.12, are treated.

16. The method as claimed in one of the preceding claims, characterized in that live, mature, ovulated eggs from fish or crustaceans caught in the wild or from aquaculture farming and which are harvested in an ovulated state by spawning or stripping are treated.

17. The method as claimed in claim 16, characterized in that live, mature, ovulated eggs from recent and primitive bony fish, preferably from sturgeon species, or live, mature eggs from crustaceans, preferably from lobsters, are harvested.

18. The method as claimed in one of the preceding claims 6 to 17, characterized in that very large live, mature, ovulated eggs with a grain size of greater than or equal to 3.2 mm in diameter or soft, unstable, live, mature, ovulated eggs with a load before bursting of less than or equal to 0.3 N are treated.

19. The method as claimed in one of the preceding claims, characterized in that the saline solution is a 0.9 percent saline solution.

20. A caviar or caviar-like product from live, mature, ovulated eggs from fish or crustaceans which have three or more layers in the egg envelope, wherein the live, mature, ovulated eggs are in a fertile but unfertilised state and have a natural potassium content in the egg plasma, characterized in that an elastic stabilizing layer (SL) is formed in the egg envelope between the zona radiata interna (ZRI) and the alveolar layer (AL), preferably between the zona radiata externa (ZRE) and the alveolar layer (AL), and has eosinophilic and hyalinic properties and is incorporated into the glucosaminoglycans.

21. The caviar or caviar-like product as claimed in claim 20, characterized in that in addition, an irreversible cross-linking of protein strands by integrated tyrosine molecules develops in the egg envelope in the zona radiata interna (ZRI) and in the zona radiata externa (ZRE).