Method for producing caviar or caviar-like products from mature living eggs of fish or crustaceans and products thereof

Abstract

The production of caviar from live sturgeon and the production of caviar-like products from live fish or live crustaceans is particularly sustainable and also efficient from an economic point of view. In the prior art, mature live eggs are treated with calcium cations, thus enzymatically forming a structurally hardened egg shell. In the method according to the invention, the egg shell of fish and crustaceans has three or more layers, in the potassium exposure step, mature live eggs are contacted in deionized water and a solution of potassium cations, wherein the concentration of potassium cations and the water temperature have no damaging effect on the live eggs. The eggs form a new stable layer (SS) in the egg shell by continuous electrical depolarization. The stable layer is elastic and provides the caviar or caviar-like product with new texture and properties for future use. The potassium cations used as a technical auxiliary agent are not present in the final product. A supplementary treatment with calcium cations can be used. The products manufactured with the invention have a particularly long shelf life, they can even be frozen without loss of quality.

Description

[0001] This invention relates to a method for producing caviar or caviar-like products from mature live eggs of fish or crustaceans, the mature live eggs being in a fertile but unfertilized state and having a natural potassium content in the egg plasma, by treating the mature live eggs in a salt solution that does not damage the eggs and then in at least one solution containing water and at least one cationic component dissolved therein, thereby stabilizing the eggshells of the mature live eggs to obtain caviar or caviar-like products.

[0002] Prepared unfertilized eggs, especially those of fish, are considered a delicacy and are increasingly being consumed. The term "roe" is used (in layman's terms) to describe eggs at any stage of maturity, from immature to mature, and the degree of development of an egg is not clearly defined. "Spawn" refers to the live, mature eggs for fertilization laid in water by female fish, lobsters, or other aquatic animals. Ovulated eggs are mature, fertile, live eggs that are released from the follicular cells of the ovary and into the body cavity. They then hatch or detach from there. According to the FAO Codex Alimentarius, caviar can only be produced from the eggs of female sturgeon of various species. In addition to wild sturgeon, sturgeon bred in freshwater aquaculture facilities are now also used for caviar production. With a few exceptions, sturgeon spawning occurs only in freshwater. The most famous sturgeon species (Acipenseridae) include the Siberian sturgeon (A. baerii), the Russian sturgeon (A. guldenstaedtii), the dauricus sturgeon (also known as the white sturgeon, or Beluga sturgeon), the transmontanus sturgeon (A. transmontanus), the ruthenus sturgeon, and their albino forms. However, hybrids of the female schrenckii sturgeon and the male dauricus sturgeon, and the closely related American "paddlefish" (Polydon spatula), are also named. Various types of caviar are known in the market, such as Sevruga, Osietra, and Beluga. White caviar (also known as "gold caviar") is obtained from albino sturgeon. Albino ruthenus sturgeon are sometimes used in aquaculture facilities to produce so-called "tsar caviar." However, this is not "real Tsar caviar," as it comes from the albino form of the original Dabry's sturgeon and is extremely rare.

[0003] Currently, caviar-like products are produced and sold from approximately 38 species of fish other than sturgeon, for example, see P Bronzi et al.: "Present and future sturgeon and caviar production and marketing: A global market overview" (Journal of Applied Ichthyology 2014, 30SI, 6, 1536-1546). These fish include tuna, roundhead fish, salmon, trout, herring, cod, carp, whitefish, and capelin, whose immature eggs are used to make caviar-like products (also known as "caviar substitutes" or "fake caviar"). The eggs of lobster, giant lobster, and other crustaceans can also be processed into caviar-like products. The methods and products claimed in this invention refer to these fish and crustaceans (and other suitable but not mentioned fish and crustaceans). Unless explicitly stated that it is caviar from sturgeon, the following generally refers to and includes caviar and caviar-like products from fish and crustaceans other than sturgeon, especially lobster and crawfish.

[0004] Caviar and caviar-like products are valuable foods. Caviar is rich in protein, containing a large amount of essential amino acids and fats. Caviar also contains vitamins D, E, and B. 12 It contains niacin; minerals such as sodium, potassium, magnesium, and calcium; and trace elements such as phosphorus, fluorine, iodine, and zinc. Furthermore, it has a high content of valuable cholesterol (HDL). Caviar and caviar-like products are used both as food and in the cosmetics industry or other industries that utilize this valuable substance. The size and firmness of the eggs depend largely on the type of fish or crustacean in question, their maturity, and the time of harvest.

[0005] Currently, some caviar products on the market are made from the mature eggs of sturgeon. However, today, caviar is still primarily derived from immature eggs and the ovaries of killed sturgeon. This is the conventional method of caviar extraction. Taking farmed sturgeon caviar as an example, it was initially thought that, like wild caviar of the past, the immature eggs would be strong enough to remove gonadal tissue residue without further invasive cleaning steps and be suitable for repackaging. However, experience from extracting caviar from farmed sturgeon over the past 20 years has shown that the immature eggs from these dead farmed sturgeon are too soft and require further processing using borax or other preservatives or pasteurization to create a product suitable for repackaging and preservation for 2 to 3 months or more.

[0006] The killing of females for caviar from wild catches, coupled with overfishing, pollution of waterways by industrial, agricultural, and domestic wastewater, and the construction of weirs and dams that obstruct migration routes to freshwater spawning grounds, has posed a significant threat to wild populations of approximately 27 different sturgeon species. Despite protection under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), poaching and the illegal black market trade persist in many areas. According to the World Sturgeon Conservation Society, costly sturgeon repopulation programs have been launched worldwide, but unfortunately, these programs have had limited success in China, Iran, and Russia. Only in the United States and Canada have measures to protect and restore populations begun to show results. In addition to allowing the capture of spawning animals, various farmed sturgeon species have been released into the wild with varying degrees of success as part of conservation programs to save endangered fish species. Crayfish and aristocratic crayfish populations are also severely threatened by environmental pollution and imported diseases such as crayfish plague. In aquaculture, the decisive progress and widespread stocking practices of crayfish farming have played a crucial role in protecting native populations. In aquaculture, females are kept alive to reproduce, and their eggs are obtained through evisceration. However, in aquaculture caviar production, female sturgeon are often simply slaughtered using traditional methods due to the lack of more gentle approaches. This completely ignores the fact that their reproductive capacity increases significantly with age. The "cesarean section method" is also partially practiced in Russia, which is by no means a gentle method, as it is associated with high mortality rates in sturgeon treated in this way. Existing technology

[0007] A method for producing pelleted caviar using spawned sturgeon roe is known from RU 2 232 523 C2. The harvested spawned roe is first treated with a hot 1.5% to 2% preservative aqueous solution in preparation for subsequent pasteurization at 65°C to 70°C. Besides the fact that each heating process significantly affects the flavor of the roe, using spawned roe with its very soft and viscous membrane cannot guarantee that they will withstand subsequent preservative treatment without breaking. However, because broken roe is difficult to remove, even a small fraction of broken roe can significantly reduce the quality of the caviar. Pasteurization denatures valuable proteins and imparts a powdery flavor to the caviar.

[0008] Regarding the extraction of ovulated eggs from sturgeon, the use of a catheter for this purpose is known, for example, from M. Szczepkowski et al.: "A simple method for collecting sturgeon eggs using a catheter" (Arch. Pole. Fich. (2011) 19: 123-128). As a result, the eggs can be easily removed by vacuum or suction. Removing the eggs from the sturgeon's abdominal cavity by simple massage is also well-known. This method, known as "peeling," is the gentlest harvesting method.

[0009] The closest prior art to this invention is disclosed in WO 2007 / 045233 A1. It describes a method for producing caviar or caviar-like products from the ovulated but unfertilized mature eggs of aquatic animals, preferably fish, by exogenously treating the mature eggs in a solution, wherein there is no endogenous morphological change in the cell membrane, allowing the egg cell (egg plasma with a surrounding serous membrane) to separate from the external environment and stabilize its structure. The solution used contains water and at least one cationic component (calcium cation Ca). ++ The cationic component dissolves in water at a predetermined concentration and induces structural stability upon contact with the egg. Calcium, a cell signaling molecule, induces calcium waves in the oocyte's oocytic plasma, leading to cortical responses and the release and activation of peroxidases. This enzyme ensures irreversible structural cross-linking of protein chains in the oocyte membrane by incorporating tyrosine molecules into both the interior and exterior of the radiation bands. The metabolic processes induced in live eggs result in the desired structural stability of the oocyte membrane. This stability cannot be achieved in immature eggs because the corresponding receptor and enzyme cascade reactions are not yet mature. For killed eggs, this process cannot be initiated at all, as metabolism ceases to occur. Due to the action of ovarian fluid, mature live eggs immediately form a sticky layer upon contact with water, allowing them to adhere to stones and plants in the spawning area. Therefore, before processing, the eggs are washed in a non-living (“physiological”) saline solution to remove ovarian fluid. Furthermore, mature live eggs have a natural potassium content in their oocytic plasma. For example, no harmful doses of potassium are added externally before harvesting (e.g., to induce ovulation).

[0010] The described reaction chain is referred to in the literature as the "second reaction." This is a slow metabolic reaction that forms a permanent physical-mechanical structure after the first sperm fertilizes the egg, protecting the egg from external aggregation of other sperm (polyspermia), but more importantly, from environmental toxins, microorganisms, and mechanical damage to the emerging embryo. In known methods, this second reaction is initiated without sperm fertilization. The resulting structural stability provides a "creep effect" and an explosive release of liquid egg matter when the product is consumed. The preference for a specific intensity of the plop effect depends largely on the intended use of the caviar and the customer.

[0011] Furthermore, it is known from EP 2 522 226 B1 that immature fish eggs raised in aquaculture are preserved using a composition of flavonoids, the antioxidant paclitaxel (dihydroquercetin), and organic salts, particularly potassium citrate. However, the high concentration of the composition used causes significant changes in the intracellular ionic environment, thereby inducing programmed cell death (apoptosis). Therefore, fish eggs treated in this way are immediately killed. Publications SU 1662469 A1 and RU 2

[0012] 048 111C1 also shows a method for preserving sturgeon eggs, in which a very high concentration of potassium compound is used, causing immediate apoptosis and death of cells in the eggs. EP 2 868 207 B1, corresponding to RU 2 232 523 C2 above, is also applicable, where additional denaturation is performed by heating.

[0013] GEBledsoe 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), disclosed the use of potassium nitrate in its context as a general preservative for crab, sturgeon, and other fish eggs. However, as is customary in all preservation processes, at such high concentrations, cytotoxic effects occur, greatly disrupting osmotic balance and immediately killing the treated eggs. Furthermore, the slaughtered fish retain only immature eggs in the early stages of development, which, on the one hand, must be mechanically wiped from the gonads to withstand potential damage, and on the other hand, have not yet fully formed any mature egg membrane structures, so they cannot be used with the invention.

[0014] DE 2 416 685 A discloses a method for improving the preservation of salmon roe or salmon eggs by adding a food additive in the form of citrate, permitted by food law. After the method is completed, the latter remains detectable in the final product and alters its composition. The high concentration (5 to 10% by weight) used kills live eggs immediately upon contact. Only immature roe can be rinsed with water. As previously mentioned, mature roe will form a viscous gel layer. Since the undeveloped and unstable egg membrane cannot protect the eggs, freezing roe before and after preservation leads to freezing damage to the egg cells or membranes and causes moisture loss. JP S63-36763A discloses a method for reducing the sodium chloride content in the preservation of roe to reduce saltiness. Sodium chloride is also replaced by various potassium compounds. However, this again occurs at such high concentrations that the eggs, i.e., immature eggs, are no longer able to perform metabolic functions. JP 2001-299285A discloses a method for treating immature frozen eggs to improve texture. The eggs are rinsed at 5°C with a potassium-containing chemical for up to 24 hours. Such a long processing time would disrupt each metabolic process. Since the fish eggs are frozen without any antifreeze protection, the immature eggs no longer possess any metabolic activity and cannot reproduce. Therefore, they cannot be used in the claimed invention. However, the invention does not claim to involve subsequent preservation, decolorization, or freezing, but rather to the original production of caviar and caviar-like products from untreated, mature, live eggs. Preservation or freezing of the live eggs after treatment is merely an optional additional step in this invention. Decolorization is completely unnecessary because it is not required.

[0015] According to Huang Hui et al.: "Effect of Synthetic Preservatives on Volatile Flavor Compounds in Caviar of Sturgeon" (Huso dauricus × ​​A. schrenckii) ([J]FOODSCIENCE, 2015, 36(12): 97-103), flavor loss in caviar during refrigeration is preferably prevented by using the preservatives potassium sorbate (E202, sorbic acid) and ascorbate (vitamin C). A constant 0.5 / ml of potassium sorbate was used in each test group. Due to the preservative treatment, immature eggs were used to obtain a stronger flavor. Potassium sorbate is believed to inhibit mold growth and fermentation, but it can also impair the flavor of the product.

[0016] The artificial activation of sea urchin eggs is discussed in L. Dufresne et al.'s paper, "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). Although this invention does not relate to the treatment of sea urchin eggs because they do not exhibit a suitable structure (having only a two-layered egg membrane), this publication will be briefly discussed here. On one hand, sea urchin eggs obtained by injecting a very high concentration of 0.5M KCl solution into the peritoneal cavity of the sea urchin were used. As a result, the polarization state of the eggs was greatly affected, and the natural potassium content in the egg plasma was significantly altered. Furthermore, all sea urchin eggs were in contact with water and formed a gel layer before treatment, which had to be mechanically removed subsequently. This also fundamentally altered the morphology and physiological properties of the decellularized egg membrane. Sea urchin eggs cannot be used in this invention, not only because their structure is fundamentally different, but also because of the extensive metabolic intervention with potassium chloride during harvesting. Furthermore, calcium-free seawater is not deionized; it contains more than 10 grams of sodium, 0.43 grams of potassium, 1.3 grams of magnesium, and 20 grams of chlorine per liter, among other things. Therefore, rinsing eggs in calcium-free water is not the same as rinsing eggs in a salt solution that does not harm mature, live eggs.

[0017] The egg membrane structure of fish and crustaceans follows a basic pattern, and the method is described using the techniques described in Siddique et al.: "A review of the structure of sturgeon egg membranes and of the associated terminology", (J. Appl. Ichthyol. (2014), 1–10). The following is a brief explanation of how conceptual consistency is established.

[0018] In the mature egg (oocyte, ovum) of an animal ovary, the follicle is composed of granulosa cells (also called follicular cells, theca membrane, follicular epithelium) and sheath cells (also called inner and outer membranes), surrounding the egg to provide it with signaling substances and nutrients. Between the granulosa cells and sheath cells lies the basal layer (scientifically also called the follicular membrane, membrane, basal layer). The oocytoplasm (oocyte cytoplasm, sheath, cytoplasm, inner ovum) is surrounded by the egg membrane (oocyte membrane, plasma membrane PM, ovum membrane). During ovulation, the egg is removed from the follicular cells and released into the abdominal cavity of the fish. The ovulated egg retains only its non-cellular egg membrane (scientifically also called the extracellular matrix or extracellular membrane), which is formed during egg maturation and consists of the following components from the outside in:

[0019] ●Alveolar layer AL (scientifically also known as gel layer, adhesion layer, gel shell, (second outer) gel shell, third layer, chorion (2)), the outermost non-cellular membrane

[0020] ● The outer radial zone ZRE (scientifically also known as the outer vitelline sheath (radial zone = vitelline sheath), outer vitelline region, outer vitelline membrane, layer 2, chorionic layer 2, outer zona pellucida, outer radial zone, outer layer of the vitelline sheath, sheath layer 1B, second sheath layer) is the outer part of the vitelline sheath, located directly below the alveolar layer.

[0021] ●The epitaxial layer (EP, scientifically also known as epitaxial layer 1, layer 4, the outer layer of the 1st membrane) separates ZRE from ZRAZRI and is not present in all types of eggs.

[0022] ●The inner radiating zone ZRI (scientifically also known as the inner vitelline sheath, inner vitelline region, inner vitelline membrane, chorionic layer 1, inner zona pellucida, inner radiating zone, sheath layer 1A, inner layer of the first sheath) is located inside the vitelline membrane and is closely connected to the ZRE.

[0023] ● The perivitelline space (scientifically also known as the extracellular matrix of the oocyte, the space between the microvilli of the oocyte serosa), a narrow space between the ZRI and the serosa, into which many microvilli (MV) are inserted.

[0024] Viable ovulated oocytes can be electrostimulated via ion channels located on their plasma membrane. Furthermore, changes in the electrical properties of the plasma membrane are a prerequisite for oocyte activation and also affect the serous membrane. Pioneering studies in marine invertebrates have shown that the ion flow of potassium ions through the serous membrane leads to a transient change in the membrane potential (fertilization potential FP). This potential is generated by activating a transient voltage-dependent inward current entering the oocyte. Depolarization of the serous membrane potential (RP) is shown to be caused by the flow of ions through the serous membrane (ion current (fertilization current FC)). This current flows through the openings of nonspecific and highly conductive ion channels that can be activated by sperm or by artificial chemical or mechanical effects. To date, hypothetical models of the roles of different ion channels and associated ions have shown species-specific differences.

[0025] Compared to 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 plays a central role in the egg cell in nature. Potassium (K) + Potassium is the cation that determines the residual potential of the oocyte. The potassium gradient and ion permeability of the oocyte are regulated by transport proteins and ion channels. According to research by the Alfred Wegener Institute, the natural intracellular potassium concentration in mature unfertilized eggs of Siberian sturgeon is 50 mmol / L. On the other hand, extracellular calcium does not affect the resting potential / fertilization potential of the oocyte, nor does it participate in the first rapid electrical discharge (see below) itself. The ionic composition inside the oocyte differs from that in the surrounding environment. This separation between the intracellular and external media is essential for maintaining metabolic activity, thereby enabling cell survival. The different distribution of charge inside and outside the cell forms the gradient across the plasma membrane, which can be measured by the potential difference (resting potential).

[0026] Technical issues

[0027] Based on the closest prior art to the present invention according to WO 2007 / 045233 A1, which is also used in the production of caviar and caviar-like products, the problem of the present invention is to further develop the methods described therein, based on live, unfertilized, mature eggs of fish or crustaceans, in such a way that the sensory characteristics of the mature eggs in terms of texture, flavor, transport, storage, and deep freezing can be improved. However, the caviar or caviar-like products that can be produced by this or other methods, as claimed by the present invention, will also retain the aforementioned advantages. The solution to this task is obtained from the main claims. Advantageous further embodiments of the invention are shown in the dependent and product claims, and are described in more detail below in conjunction with the invention.

[0028] The claimed method for producing caviar or caviar-like products based on mature live eggs of fish or crustaceans according to the present invention is characterized in that, in the potassium exposure step, potassium is dissolved in water as a cationic component of the water, the concentration of which does not harm the mature live eggs and does not change their natural potassium content; the water is deionized before adding potassium donors to form cationic components; and the water is at a temperature that is harmless to the mature live eggs. The mature eggs are then treated in solution for potassium exposure time until the desired egg membrane elastic stability is achieved.

[0029] In the method claimed in this invention, mature live eggs are used, which can be obtained naturally without harming the fish or crustaceans. The mature live eggs are fertile but unfertilized. Ovarian fluid is removed beforehand by rinsing with a saline solution that does not harm the mature live eggs, thus preventing the formation of a sticky gel layer on the shell. Furthermore, these eggs have a natural, unaltered potassium content. Only physiologically effective concentrations of potassium ions are used. The live eggs have more than two, i.e., three or more, layers in the egg membrane of fish and crustaceans, and are electrically activated. The starting product is a fresh, mature, fertile but unfertilized egg with complete functional metabolism, so even the lowest concentration of potassium ions will not cause damage or leave any trace in the egg, triggering transport processes through the serous membrane and metabolic processes within the serous plasma. Caviar or caviar-like products produced in this way have a novel texture, possessing advantageous stable elasticity through a new transparent, acellular layer (elastic stabilizing layer) in the egg membrane, which softens at room temperature without limiting the stability of the caviar. The required elastic stability can be easily determined through self-testing (egg elasticity). The taste is fresh and spicy, without any "fishy" smell. Even without the addition of preservatives such as borax, which is banned in many countries, the purity of the eggs used allows for a long shelf life (9 to 12 months) at standard temperatures of -2°C to -4°C. Eggs treated in this way can also be frozen without losing quality, offering significant advantages in storage and transportation, as detailed below.

[0030] According to the present invention, mature live eggs are subjected to a physiological, i.e., non-destructive concentration of potassium cations (K). + Upon contact with water, the egg undergoes changes within the framework of an electrical event, triggering what is known as the "first reaction." This reaction removes the stickiness of contact with water within a very short time (seconds to minutes) through electrical induction, and in further processing, forms a new, elastic, stabilizing layer within the egg membrane. This new stabilizing layer imparts elasticity to the egg membrane, thus enabling the production of the highest quality caviar or caviar-like products at this stage, which can be further processed, particularly with preservation and deep freezing (at -18°C), without any loss of quality.

[0031] Egg activation involves and proceeds through a series of cellular biological cascades. Following the “second (slow) reaction” (slow blocking) with cortical activity, as used in WO 2007 / 045233 A1, is calcium-dependent enzyme activation via irreversible tyrosine-linked protein chains in the inner and outer radiation zones, for the expansion and eventual large-scale structure of the egg membrane. In nature, this prepares the embryo for the first cell division. Conversely, the “first (fast) reaction” (fast blocking, resistive interruption, fast resistive interruption) with subsequent depolarization / hyperpolarization, and its stabilization for varying durations depending on the animal species, as used in some embodiments of the invention, occurs at the beginning of all cellular biological cascades. These two processes differ significantly due to the substances used: A) potassium ions, for fast resistive interruption of egg membrane depolarization, thereby triggering egg activation and the formation of a single, new region within the egg membrane (forming an elastic stabilizing layer); and B) calcium ions, for slow mechanical blockage and enzyme-controlled morphological transformation within the existing egg membrane layer (forming a structural stabilizing layer).

[0032] The first electrical event involves rapid depolarization, even hyperpolarization, within milliseconds, designed to prevent further sperm adhesion to the egg after natural fertilization. Over the next 60 minutes (or even up to 5 hours for some aquatic animals, such as lobsters), the rapid hyperpolarization dissociates from the stable hyperpolarization. Although sperm remaining near the egg can still adhere to and remain within the vitelline membrane (soft membrane) of the egg after the rapid electrical interruption, it cannot penetrate the plasma membrane for actual fertilization, as can be observed in mollusks. If potassium exposure is continued according to certain embodiments of the invention, a novel, transparent (clear, glassy, ​​gel-like) region (elastic stabilizing layer) is observed in live, fertile, but unfertilized, and mature eggs. This region, previously unreported, is GAG-positive (increased glycosaminoglycan content) and eosinophilic (stainable with eosin, a red acidic diagnostic dye), and can be used for visualization of organelles, plasma proteins, connective tissue, and their precursors, within which sperm become trapped. The formation of this elastic stabilizing layer first occurs in a portion of the cell membrane during hyperpolarization lasting 10 seconds or longer, and after completion, it lies between the outer radial zone and the alveolar layer in the egg. Live eggs have a structural design similar to that of fish and crustaceans (with more than two layers in the egg membrane). The reason for the formation of this new elastic stabilizing layer is the continuous depolarization of the serous membrane supplied with physiologically concentrated potassium ions according to the invention.

[0033] According to EU member state legislation on food supplements, only potassium compounds listed therein, such as potassium bicarbonate (KHCO3) (CAS No. 298-14-6), potassium carbonate (K2CO3) (CAS No. 584-08-7), potassium citrate (CAS No. 6100-05-6), potassium hydroxide (KOH) (CAS No. 1310-58-3), potassium chloride (KCl) (CAS No. 7447-40-7), potassium iodide (KI) (CAS No. 7681-11-0), and potassium iodate (KIO3) (CAS No. 7758-05-6), are permitted for nutritional purposes. Similarly, the European Parliament and Council Regulation Proposal of 10 November 2003 (COM(2003)671final) allows these compounds to be added to food. Certain potassium compounds, such as potassium citrate (E 332), potassium lactate (E 326), and potassium orthophosphate (E340), may also be added to food for technical purposes. According to the method of the present invention, mature live eggs of fish or crustaceans are treated with potassium ions at a cell-compatible concentration (physiological concentration, i.e., harmless to the egg) without leaving residues, allowing the production of caviar or caviar-like products to meet all national and international quality requirements of competent authorities, distributors, and customers. At the Alfred Wegener Institute, intracellular ion concentrations in the cytoplasm of egg cells were studied using optical emission spectroscopy (OES), in which potassium was used as a novel substance to continuously depolarize the plasma membrane, with no change in concentration in the egg plasma after treatment, even with varying concentrations and times of treatment. Therefore, potassium ions used in certain embodiments of the method according to the present invention clearly function as processing aids. Processing aids are used in the industrial processing and production of food. Processing aids are food additives added for technical processes such as cutting and filtering. However, in the final product, processing aids must be completely absent or present only in unavoidable (small) residues. Unlike food additives that must be declared on packaging, processing aids are particularly advantageous if they no longer have any effect on the final product. Their use must be technically unavoidable, technically ineffective, harmless to health, and odorless and tasteless. Since these substances are no longer present or inactive in processed foods, it is not necessary to declare their use. This also applies to residues, reaction products, or residual content.

[0034] Preferred and advantageously, at least one potassium salt, preferably a salt of citric acid (potassium citrate E332) and / or a salt of hydrochloric acid (potassium chloride E508) and / or a salt of sorbic acid (potassium sorbate E202), dissolved in water as a potassium donor for forming the cationic component. A potassium donor is a potassium-containing compound that, upon dissolution in water, provides potassium ions, the concentration of which depends on the concentration of the specific potassium compound in the water and its structural formula. All of the mentioned potassium salts are even approved as food additives with an E value, although in some embodiments of the invention they are used only as processing aids and are not present in the final product, and no declaration is required. An advantageous and preferred concentration of potassium ions in a solution containing pre-deionized water is from 0.1 mmol / L to 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, particularly 1.0 mmol / L or 1.5 mmol / L according to embodiments. All boundary and intermediate values ​​(integers and non-integers) should also always be included within the scope of this invention (as well as other parameters). To achieve the potassium ion concentration mentioned above in water, it must be deionized. However, since water molecules are constantly breaking down in water, it is understandable that only a certain degree of deionization can be achieved through technical means (the conductivity of water at 25°C is between 1 μS and 15 μS, serving as a measurement standard for achieving deionization).

[0035] Furthermore, the potassium exposure time in the potassium exposure step is preferably and advantageously between 5 and 30 minutes, more preferably 10, 12, 15, 20, or 25 minutes. Other potassium exposure times within this range are also readily selectable. When processing mature lobster eggs (crustaceans), exposure times as long as 50 minutes or more may even occur. A new elastic stabilizing layer begins to form within seconds (maximum 10 seconds) after the start of treatment, which improves the egg membrane. However, since the eggs cannot simultaneously form a stabilizing layer on a portion of the egg membrane surface via continuous depolarization reactions and exocytosis of glycoproteins, it is recommended to extend the treatment time to 10 minutes to achieve continuous depolarization in all treated eggs. Ultimately, the new elastic layer is located between the outer radial zone and the alveolar layer throughout the egg membrane surrounding the egg. As a result, mature live eggs are elastically stabilized by this invention in such a manner that they can be salted, repackaged, and deep-frozen without problems.

[0036] As a further optional improvement, certain embodiments of the method according to the invention also provide a calcium exposure step, which can be performed after or before the potassium exposure step. The respective changes in the egg membrane occur independently of each other in the two processes, in accordance with their described characteristics. In the calcium exposure step, calcium is preferably and advantageously dissolved as a cationic component in another solution containing water at a concentration that does not damage mature live eggs (i.e., physiological concentration), wherein the water is deionized before the addition of the calcium donor. Mature live eggs are treated in the calcium exposure step until the desired egg membrane structural stability is achieved. The desired degree of structural stability can be easily determined by self-testing (the degree of egg plop effect). In the calcium exposure step, at least one calcium salt, preferably calcium citrate, calcium chloride, and / or calcium sorbate, is advantageously and preferably used as a calcium donor (calcium donor, see potassium donor for definition). Calcium salts are authorized as food additives in the European Union under numbers E333 and E509, without quantity restrictions, and number E203, without quantity restrictions. In German, the spellings “Kalium” and “Calcium” (instead of “Kalzium”) are chosen to better distinguish the two ion types.

[0037] Calcium is physiologically present in egg cells and is an essential component of cellular metabolism. As known from WO2007 / 045233A1, calcium chloride is used to structurally strengthen the egg membrane by irreversibly cross-linking proteins through the incorporation of tyrosine molecules. In addition to achieving essentially improved and tunable elasticity of the egg membrane through this invention, it can also be structurally mechanically solidified through a calcium exposure step. Therefore, an optimal, stable combination can be achieved for certain caviar types and caviar substitutes. This is particularly advantageous for very large, unstable eggs (diameter greater than 3.2 mm, e.g., eggs from beluga whales or sturgeon) or eggs that are particularly soft when mature (maximum force less than or equal to 0.3 N until they burst in a hardness test, e.g., eggs from small sturgeon). The application of these two treatment steps can provide high-quality caviar or caviar-like products for eggs with problems (size, softness).

[0038] The concentration of calcium ions in the other solution is advantageously 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 calcium exposure time is preferably between 9 and 30 minutes, more preferably 10, 12.5, 15, 16, 20, or 25 minutes. The selection of the treatment time should take into account the fact that the strength of the egg membrane increases with increasing calcium exposure time, until a limit is reached. In nature, fish fertilized eggs develop a hardened egg membrane after approximately 60 minutes, rendering them unsuitable for consumption. Lobster eggs may require up to 24 hours.

[0039] In some embodiments of the method according to the invention, an important process parameter is the temperature of the solution used to treat mature eggs. This is said to be physiological, meaning it does not hinder natural processes in live eggs. In some embodiments, the solution temperature is always within the natural spawning temperature range of fish or crustaceans. This ensures that the electroactivation of the egg membrane induced in the potassium exposure step proceeds reliably, and that depolarization begins at the quiescent potential. At non-natural spawning temperatures, such as in polar regions above 15°C for fish or crustaceans, electroactivation of the eggs does not occur, and mature live eggs cannot be stabilized electrically or enzymatically. They become atretic. The same applies to eggs from fish or crustaceans from mixed and tropical regions. A fundamental rule is that at temperatures above 35°C, solution denaturation leads to a severe decline in egg quality.

[0040] To adapt the solution temperature to their natural habitat, the present invention broadly divides the living areas of fish and crustaceans whose eggs can be used during natural reproduction into three climatic zones: polar (at the poles), temperate (between the poles and the tropics), and tropical (around the equator). The present invention preferably and advantageously utilizes the fact that the temperature of one solution (potassium exposure) and / or another solution (calcium exposure) is selected from a polar temperature range between 1°C and 15°C, preferably between 5°C and 12°C, particularly preferably 10°C; a moderate temperature range between 10°C 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. The present invention preferably avoids temperature-induced changes in the eggs—denaturation, cell death, for example, in cases of pasteurization by heating to temperatures above 40°C. The present invention preferably avoids this at any point in the process.

[0041] Because the cation concentration used in certain embodiments of the method according to the invention triggers animal-specific physiological responses of electrochemical (potassium exposure) and metabolic (calcium exposure), and thus affects the processing to produce a stable edible end product, it is assumed that deionized water is present in the solution to achieve a precise concentration of electrochemical (potassium) or metabolic (calcium) active cations (positively charged). Therefore, it is technically feasible and therefore preferred and advantageous if the conductivity of deionized water at 25°C is between 1 μS / cm and 15 μS / cm, preferably 10 μS / cm or less, particularly preferably 1 μS / cm. Drinking water and well water from regional sources vary greatly in their composition of different ions, and in some cases may even have antagonistic effects on cellular metabolism. For example, at a temperature of 25°C, the conductivity of pure water is 0.055 μS / cm, deionized water is 1 μS / cm, rainwater is 50 μS / cm, or drinking water is 500 μS / cm. Knowing the conductivity of deionized water is important to obtain reproducible results.

[0042] Since living cells in the form of activated mature oocytes are treated with certain embodiments of the method of the present invention, it is particularly important that the solution is adapted to the metabolism of the cells, thereby inducing metabolic processes to occur in the method. Therefore, it is advantageous and preferred that one and / or another solution has a pH (physiological and biologically harmless) between 6.8 and 8.0, preferably between 7.0 and 7.9, and particularly preferably between 7.2, 7.4, or 7.5. In particular, the pH regulated in the solution is associated with the slow metabolic reactions in the calcium exposure step. Since enzymatic processes in cells are highly regulated by pH, the intracellular pH in the potassium exposure step (electrochemical process) was also examined. However, the pH in the oocyte plasma of eggs treated with various potassium-based substances of different concentrations and durations remained substantially constant between the optimal pH of 7 and 8, and showed the expected individual variability in the case of individual fish and crustaceans.

[0043] In embodiments of the invention, different exposure steps are used to endogenously stabilize the egg membrane of mature, live eggs (structurally elastic and optionally). Thus, the caviar or caviar-like product is ready for further processing, such as curing and packaging. During ovulation, fresh, mature, ovulated eggs are expelled from the follicular cells, so that no vascular or follicular cell tissue residues adhere to bacteria or fungi. Therefore, the harvested ovulated eggs have good purity, thus providing optimal conditions for long shelf life. This can be reliably ensured if, after the final exposure step for preservation and flavoring, mild salinization with sodium chloride at 2.0% to 3.8%, preferably 3.5%, relative to the content of the caviar or caviar-like product, is performed. The sodium chloride should preferably be free of potassium and calcium donors, such as those contained in the additives, as this prevents uncontrolled changes in the egg membrane due to salt. Sturgeon roe caviar is cured with simple common salt (sodium chloride, NaCl) using dry salting. When processing the roe of other fish into caviar-like products (e.g., salmon and trout caviar), wet salting is typically used. The salting process, which can be performed within a designated area using the method of the present invention, is a very light salting process, also known as "malossol," a clear indicator of high quality. Pasteurization or heating to 60°C or higher is preferably completely reduced for the caviar or caviar-like products claimed by the present invention, as this is unnecessary and would only harm the quality and sensory characteristics of the product. Due to malossol salting, caviar or caviar-like products produced using the above method have a minimum shelf life of at least 9 to 12 months if stored at -2°C. During processing, it does not freeze due to the light salt content.

[0044] According to further variations, storing caviar or caviar-like products in sealed glass containers for several months, preferably one to three months, can further improve the quality of the produced caviar or caviar-like products in certain embodiments of the invention, preferably and advantageously after preservation and flavor enhancement. Caviar "matures" through storage, and depending on the degree of maturity, the flavor intensity increases. However, this maturity is in the sense of further development of flavor (e.g., cheese), and is not related to the "maturity" of the mature eggs used in the invention in a biological developmental sense. Here, maturity refers to the possibility of fertilization, and therefore the developmental state of the live eggs. When caviar matures relative to its flavor, it is stored in glass containers, which provides sufficient space for the maturation of the caviar because it is not compressed (as in metal snap-top jars), thus retaining its rich, oily flavor. Therefore, caviar packaged in glass according to certain embodiments of the invention should not be confused with pasteurized caviar, which is also frequently packaged in glass. Furthermore, storing it in environmentally friendly glass containers avoids the metallic taste often criticized in caviar typically packaged in metal containers.

[0045] According to another variation of this method, it is preferred and advantageous to freeze caviar or caviar-like products in a temperature range of -20°C to -15°C, preferably at -18°C, and then preserve and enhance the flavor or store and mature the flavor. Ideally, the caviar should be flavorful enough for human consumption and reach the maturity desired by the respective customers, and should be freshly frozen 14 days after production or up to 3-4 months after maturation. The caviar is frozen in 500g glass containers before or after repackaging, so that the end customer can place it in 30g, 50g, 125g, 250g, or 500g (possibly up to 1000g) glass containers, which are then vacuum-sealed. Caviar obtained in conventional slaughter cannot be frozen. Although pasteurized or heated caviar can be frozen, the heat treatment results in extremely high quality loss. The possibility of freezing caviar or caviar-like products according to the invention enables optimal caviar sales that meet the current demands of convenience foods. Because specific transport and storage temperatures are required to be between -2°C and -4°C, marketing has reached its limits and these temperatures must be strictly adhered to, as most suppliers cannot maintain them. Therefore, conventionally obtained caviar is treated with harmful preservatives such as borax or pasteurized to preserve it for at least 12 months or longer. However, caviar produced using embodiments of the present invention can be simply frozen, thus preserving it and keeping it fresh for an even longer period. Experiments have shown that caviar thawing slowly in a refrigerator at +4°C to +7°C does not lose its flavor or texture.

[0046] In some embodiments of the invention, the eggs are treated in a solution bath (aqueous solution, aqueous solution). The eggs are added to and remain in the solution bath until the desired stability (elastic, optionally structural) is achieved, depending on the type of eggs used. The eggs are then removed from the bath. To reliably prevent unwanted further stabilization after removal of cations still adhering to the solution, in some embodiments of the invention, according to a further variation of the method, it is preferred and advantageous to immerse (briefly soak) the mature, live eggs in a saline solution (physiological saline solution) that does not damage them, if the cations introduced from the mature eggs are removed after achieving the desired elastic (and optionally structural) stability. This washes away the cations and immediately disrupts the stabilization process they induce. The (desired) stability of the egg membrane obtained to date is reliably preserved as its final state.

[0047] In some embodiments of the invention, the mature live eggs used are fertile but not fertile. They are generally not wetted by water and have a natural potassium content in the oocyst. Such live eggs can be released from the gonads into the abdominal cavity of the fish and harvested from there through the genital opening. This can be accomplished, for example, by natural spawning, peeling (massaging the abdominal cavity from the outside), or using a duct to expel or aspirate the eggs from the abdominal cavity. Eggs released from the gonads into the abdominal cavity are called ovulated eggs (maturity level 5) and are still surrounded by a mucous ovulation fluid. To avoid the formation of an adhesive layer after the eggs come into contact with water, the ovulation fluid is rinsed with a saline solution before processing begins. Ovulated eggs can be obtained from live animals, which is particularly sustainable. However, mature live eggs at maturity level 3 or 4 can also be used in the invention, which are taken from the gonads of dead animals and then separated. IG Katsiadaki et al., "Assessment of quality of cod roes and relationship between quality and maturity stage" (J. Sci Food Agric 79:1249-1259 (1999)), particularly Table 1, provides a good overview of different maturity levels in cod. A numerical definition of maturity is possible with the aid of the so-called "polarization index." This is calculated based on the ratio of the distance between the cell nucleus and the plasma membrane to the egg diameter (major axis) between the animal and nutrient poles. Therefore, according to a next embodiment of the invention, it is preferred and advantageous to treat mature live eggs of fish or crustaceans 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 the treatment according to the invention. More information on the polarization index (PI) of eggs can be found, for example, in the Sturgeon Breeding Guide (published FAO Ankara 2011 Fisheries and Aquaculture Technical Paper 570 "Sturgeon Hatchery Practises and Management for Release - Guidelines").

[0048] The method claimed in this invention can be used to process mature live eggs of fish and crustaceans (scientific name Crustacea) that have the basic structure required by the invention (more than two layers in the egg membrane) and are suitable for consumption in the form of caviar or caviar-like products. Preferred and advantageously, mature live eggs of fish or crustaceans caught in the wild or aquaculture are processed, harvested in an ovum-opening state by spawning or detachment, for example, by cannulation. Thus, for example, animals intended for refeeding from the wild, such as those from refeeding projects, can also be harvested. Proceeds from the sale of caviar and caviar-like products can then be returned to storage facilities. In some embodiments of the invention, it is particularly preferred and advantageous to process mature live eggs of the nearest and natural live-boned fish, preferably live sturgeon. Embodiments of the invention can then be used to produce the highest quality (pure) caviar. According to embodiments of the method of the invention, other caviar-like products from lobsters or other crustaceans, such as crawfish, can also be produced in the highest quality. Furthermore, since the implementation of the method of the present invention may optionally include two exposure steps to stabilize the egg membrane by elasticity (electric stimulation) and structure (enzymatic stabilization), it is preferably and advantageous to process very large (diameter greater than 3.2 mm) or soft, unstable eggs (hardness in the hardness test is less than 0.3 N, above which the egg will rupture).

[0049] Finally, embodiments of the invention also include a product different from mature live eggs of fish or crustaceans, which can be produced by the method claimed in this invention, or by other methods. This product is characterized by the additional formation of an elastic stabilizing layer in the form of an eosinophilic transparent layer within the egg membrane, incorporating glycosaminoglycans. However, in this case, the live egg is unfertilized, which is why the elastic stabilizing layer does not occur naturally. In some embodiments of the invention, the elastic stabilizing layer is located between the inner radial zone and the alveolar layer, preferably between the outer radial zone and the alveolar layer. Therefore, it can only occur in live eggs with more than two layers of egg membrane structure. For example, sea urchins only show two layers within the egg membrane. The new stabilizing layer is transparent, gel-like, and elastic, and can be histologically stained red with eosin and blue with Alsin. During the production process using some embodiments of the invention, its characteristics are affected by the concentration of potassium cations used in the potassium exposure step, and its location is affected by the duration of potassium exposure.

[0050] To remove ovarian fluid, mature, viable eggs are treated with a saline solution that does not damage the eggs before further treatment. Preferably and advantageously, this is a physiological saline solution. Furthermore, it is advantageous and preferred that the saline solution is prepared as a 0.6% to 1.0% solution, particularly preferably a 0.9% solution. For example, to prepare a 0.9% saline solution, 9g of sodium chloride (NaCl) is dissolved for every 1 liter of water used. This concentration corresponds to the naturally occurring saline solution in human organisms and is therefore referred to as a "physiological" saline solution.

[0051] Some embodiments of the present invention also relate to caviar or caviar-like products from unfertilized mature eggs of aquatic animals, characterized by the additional irreversible cross-linking formed in the egg membrane by incorporated tyrosine molecules. This additional irreversible cross-linking is located between the inner and outer radial bands of the live egg of a fish or crustacean. The irreversible cross-linking results in additional structural stability of the egg membrane. Combined with existing elastic stabilizing layers, it can also be used to process particularly large or soft eggs. Caviar or caviar-like products can be prepared according to embodiments of the present invention, wherein the structural stability in the egg membrane then depends on the calcium exposure time and calcium cation concentration in the calcium exposure step. Other methods for preparing caviar or caviar-like products with the same irreversible protein cross-linking properties in the egg membrane are also applicable. Other embodiments of the methods and products of the present invention can be found in the following specific descriptive sections relating to exemplary embodiments, but the scope of the invention is in no way limited to such exemplary embodiments. Example

[0052] The following description, through examples and accompanying drawings, further explains the method for producing caviar or caviar-like products from mature live eggs of aquatic animals according to the present invention, as well as the products and advantageous variations thereof, to further illustrate the invention. This demonstrates...

[0053] Figure 1A Images A, B, and C are used to compare SEM images (pre-existing technology) of live eggs in immature and mature states.

[0054] Figure 2 An initial table of measurements of egg membrane thickness during the processing of mature live eggs from Siberian sturgeon.

[0055] Figure 3 The second table shows the measurements of egg membrane thickness during the processing of mature live beluga sturgeon eggs.

[0056] Figure 4A SEM images of sturgeon eggs undergoing different treatments (B, C, D) to compare the formation of a stable layer in live, mature eggs after dual treatment with potassium and calcium ions, treatment with calcium ions alone, and treatment with potassium ions.

[0057] Figure 5AImages B, C, and D contain TEM images of the noncellular oocyte membrane layer structure in which a new stabilizing layer (SS) forms between the inner zone of radiation (ZRE) and the alveolar layer (AL).

[0058] Figure 6A TEM images of cortical granules in mature sturgeon eggs (B, C, D, untreated, and treated with different methods: potassium ion treatment only, dual treatment with potassium and calcium cations, and calcium cation treatment only).

[0059] Figure 7A Optical microscopic images of untreated mature live eggs from Siberian sturgeon (A, B, C, D) and mature eggs from Siberian sturgeon treated with potassium ions.

[0060] Figure 8A Optical microscopic images of mature live eggs from Siberian sturgeon, including those treated with calcium ions (B), potassium ions and calcium cations (C), and D.

[0061] Figure 9A SEM and optical micrographs of the egg membrane structure of beluga sturgeon after treatment with potassium and calcium cations.

[0062] Studies on the relationship between sturgeon weight and / or age and caviar seed size or the amount of caviar harvested show that egg diameter and caviar quality increase with sturgeon weight and / or age. Furthermore, as sturgeon weight and age increase, caviar yield also increases, thus improving economic efficiency. Depending on the species, sturgeon in their natural environment do not reach sexual maturity until they are 12 to 26 years old. Most sturgeon spend their growth and development period until they first reproduce in the sea or estuary, then migrate to rivers to find their spawning grounds on rocky freshwater surfaces. Moreover, in aquaculture, sturgeon require approximately 5 to 16 years, depending on the species, to reach first sexual maturity and thus their first caviar harvest. The premise for repeated harvesting of caviar from live females in aquaculture over many years is that the animals are fed optimally with low stocking densities, and that it is always economically and ecologically sound due to their late sexual maturity and long lifespan. From the harvesting and processing of molted eggs to the coordinated workflow of caviar production, it is easy to produce economically viable tonnes of caviar. Depending on the age of the fish and the associated quantity of caviar, appropriate scale-up measures can enable daily production of 80 kg or more.

[0063] Some embodiments of the invention use mature, live eggs that have been pre-washed with a saline solution. These are ovulating eggs, which, due to their maturation stages (ovulation preparation and fertilization stages), have previously been expelled from the gonads by the fine muscle fibers of the follicular cells—a process known as ovulation. The ovulating eggs are released into the oviducts and abdominal cavity of the fish without any cellular or other residue. They can then be removed by massaging the abdomen without affecting the fish's lifespan. The surface of the eggs is completely clean, allowing no niches or folds for bacterial or fungal contamination, thus extending the shelf life of caviar or caviar-like products. Protective methods harmful to human health, such as borax, are unnecessary. Figure 1A Images B and C show prior art scanning electron microscope (SEM) images of live eggs and the ovulation process. Figure 1A Immature oocytes with follicular cells are shown, such as those found in the regular caviar of killed sturgeon. Figure 1B The in situ release of mature oocytes from surrounding follicular cells is shown. Then, Figure 1C shows a live, mature sturgeon egg, which is clearly completely smooth and clean.

[0064] In an exemplary embodiment of the mature, viable ovum after ovulation, the possible method workflow according to embodiments of the present invention is explained in more detail below with some optional additional steps:

[0065] ●After the blastocyst dissolves, during the maturation stage, V separates the live female fish from the live eggs.

[0066] ● The live eggs and ovarian fluid to be removed are immediately transported to the caviar laboratory (largely avoiding waiting time, which is unavoidable when the ovarian fluid is covered with an airtight plastic film and placed on ice to remove oxygen).

[0067] ● Immediately rinse the live eggs thoroughly in 0.9% saline solution until the ovarian fluid is completely removed.

[0068] ● Perform potassium exposure procedures:

[0069] • Prepare potassium cation solutions of 0.1 to 2 mmol using potassium citrate in deionized water with a conductivity of 10 μS / cm (at 25 °C) and a polarity temperature range of 10 °C.

[0070] • Introduce mature, live eggs into the solution, with a potassium exposure time of 10 minutes.

[0071] • Remove the treated eggs from the solution; and

[0072] ● Briefly immerse the eggs that have undergone the above treatment in a 0.9% saline solution.

[0073] Other methods of extracting mature eggs are also possible. Even when using mature live eggs from previously killed animals, blood and fat must be washed away, or even the eggs must be wiped from the gonads, which can be achieved by pretreatment with a preferred physiological saline solution. Immersion allows for additional control over the elasticity and diameter of the stabilizing layer (i.e., in addition to selecting the duration of exposure). Due to the catalytic effect of the introduced potassium ions, the described treatment influences the formation of a transparent, elastic stabilizing layer between the outer radial zone of the egg membrane and the alveolar layer. In eggs of normal condition and softness, treatment with a potassium exposure step is sufficient. However, if using particularly large, soft, or sensitive eggs from certain sturgeons such as the Kaluga sturgeon, the shin sturgeon, or the small sturgeon, a calcium exposure step may also be incorporated (or placed beforehand):

[0074] ● Additional calcium exposure steps:

[0075] • Another calcium cation solution of 0.5 to 2 mmol was prepared using calcium chloride in deionized water with a conductivity of 10 μS / cm (at 25 °C) and a polarity temperature range of 10 °C.

[0076] • Introduce mature, live eggs into the solution, with a potassium exposure time of 10 minutes.

[0077] • Remove the treated eggs from the solution; and

[0078] ● Briefly immerse the eggs that have undergone the above treatment in a 0.9% saline solution.

[0079] In addition to the elastic stabilizing layer from the potassium exposure step, this also forms structural protein cross-links in the egg membrane of fish and crustaceans, which are already present in the inner and outer radial bands of the egg membrane. This additional structural protein cross-linking of the egg membrane via tyrosine residues also particularly imparts plasticity to large, soft, or sensitive eggs—in addition to the elasticity from the potassium exposure step. An optional impregnation function can also be used here to achieve additional controllability. The resulting product is (genuine) caviar from mature live eggs, which can then be further processed as follows:

[0080] ● Combine caviar with dried (K-free) + and Ca ++ A mixture of sodium chloride (NaCl) (3.5g / 100g caviar, 3.5%) and a drip aid is used, which is equivalent to the amount of marossol salt used for preservation.

[0081] ● Fill a glass container (preferably 500g aged glass) with lightly salted caviar, and vacuum seal the container with a screw cap and pull strap.

[0082] ● Store the caviar in glass containers at -2°C for 2 to 4 months to allow it to further mature and optionally…

[0083] ●Fresh or mature caviar is frozen in glass containers at -18°C, according to customer requirements.

[0084] Due to the treatment, mature live eggs treated with the potassium exposure step form a novel region: the stabilizing layer SS, which is elastic and transparent (gel-like). The stabilizing layer SS can be easily stained for detection. It is located between the alveolar layer AL and the outer radial zone ZRE, and has not been described in the literature to date. For a descriptive introduction to the structural design of fish and crustacean eggs, please refer to the corresponding Siddique glossary.

[0085] Figure 2 The table shows measurements (in μm) of the extracellular egg membrane on mature Siberian sturgeon eggs using computer-controlled image analysis (Zeiss), based on frozen sections with a constant layer thickness (10 μm) under the influence of different treatment methods to stabilize the egg membrane. The table displays the formation of the new stabilizing layer SS and the diameters of the existing layers (ZRI, ZRE, AL) within the egg membrane, which are respectively treated with potassium cations (K) at mmol / L in various exposure steps according to the present invention. + (from potassium citrate) and with calcium cation Ca ++ Treatment with additives of varying concentrations (from calcium chloride) was performed on mature live eggs from the Siberian sturgeon *Acipenser baerii*. Except for the previously explained acronyms and in the case of *Siddique*, Mv represents the mean and Std represents the standard deviation. The values ​​given are for the new elastic stabilizing layer SS. Furthermore, reference is made to the prior art according to WO 2007 / 045233 A1 above.

[0086] Post-treatment quality control showed that only at potassium ion concentrations of 1 mmol / L and 1.5 mmol / L could the egg membrane thickness reach at least 12 μm, and an intermediate caviar product was formed that had lost its viscosity and was sufficiently stable for further caviar processing. Furthermore, it was shown that a preferred treatment time of 10 minutes was reasonable to allow all live eggs in the solution to undergo metabolic reactions. A processing yield of 2.5 kg of caviar (approximately 25 liters of solution) could be achieved in the treatment unit. Sensory testing of the potassium-treated caviar according to the invention showed that the elastic texture of Siberian sturgeon caviar did not show any difference between the 1 mmol / L and 1.5 mmol / L concentrations. On the other hand, the texture of eggs treated with a solution of lower potassium ion concentration was different; only a few eggs were stable, while untreated eggs were very soft and brittle. According to the sensory tests conducted, treatment with two exposure steps (potassium and calcium ions) produced a solid, pearly product, also known as "super-puff" in the case of Siberian sturgeon eggs.

[0087] Figure 3 The table shows the presence and diameter (in μm) of the extracellular egg membrane layer in different treatments according to the invention of live large eggs from beluga sturgeon (Kaluga dauricus). During treatment of the caviar with potassium cations (from potassium citrate), the formation of a new eosinophilic stabilizing layer SS in the extracellular egg membrane was also observed, which is also located between ZRE and AL. Sensory tests performed on large eggs from beluga sturgeon indicated that dual treatment with potassium and calcium ions optimizes the texture of the delicate, mature, live eggs.

[0088] Figure 4A Images A, B, C, and D show SEM images of changes in the extracellular membrane structure of mature live eggs, such as those from Siberian sturgeon under various treatments. Two magnifications are shown: left 6000x and right (cutout) 12000x. Live eggs were always treated by flash-freezing them in hexane at -80°C to produce cryosections of tissue that retain their natural state.

[0089] Figure 4A In untreated mature eggs, the regions of the egg membrane are not clearly separated from each other (pre-existing technology).

[0090] Figure 4B Under the influence of 0.5 mmol / L potassium, a new stable layer SS has formed between the outer radiation zone ZRE and the alveolar layer AL, while the inner radiation zone ZRI and the outer radiation zone ZRE show unchanged loose protein structures as in untreated eggs.

[0091] Figure 4CTwo consecutive treatments of the egg with potassium and calcium cations revealed characteristic morphological features in SEM: the formation of a stable SS layer and the twisting and cross-linking of protein chains in the inner and outer radial bands ZRI and ZRE, respectively, which were characteristic of calcium treatment. Figure 4D .

[0092] Figure 4D Calcium treatment alone leads to twisting and cross-linking of loose protein chains in the inner radiation zone ZRI and outer radiation zone ZRE (existing technology), compared to no calcium treatment. Figure 4A and Figure 4B .

[0093] Figure 5A Images A, B, C, and D show transmission electron microscopy (TEM, 3000x magnification) images of the multilayer structure of the egg membrane of mature sturgeon eggs during treatment with 1.0 mmol / L potassium ions. Figure 5A The inner radial band ZRI (the phenomenon can only be identified as a superstructure) with loose, turbid fibers is shown, separated from the outer radial band ZRE by the epitaxial layer EP. The outer radial band ZRE is characterized by a filamentous network of elongated protofibrils. Figure 5B The formation of a new stable layer SS with a fine-grained structure is shown, which is directly located in the outer radiation zone ZRE and - according to Figure 5C -Between the alveolar layers (AL), it permeates to the periphery of the oocyte membrane-According to Figure 5D -Through small tubes (small conduits, channels). Superstructural analysis confirmed that treatment with potassium ions (1.0 mmol / L) according to certain embodiments of the invention resulted in the formation of a previously unknown, stable layer SS with an amorphous structure, located between the outer radial zone ZRE and the alveolar layer AL in fish and crustaceans.

[0094] Figure 6A Images B, C, and D show TEM images (3000x magnification) of cortical granules CG in the peripheral oocyte plasma within the plasma membrane of a mature egg. Figure 6A The image shows an untreated egg (prior art). Figure 6B Eggs treated with potassium cations are shown. Figure 6C Eggs treated with potassium and calcium cations are shown. Figure 6D Eggs treated with calcium cations only are shown (prior art).

[0095] Cortical granules are secretory organelles (structurally definable regions) found in the oocyte and are closely related to fertilization. Cortical granules contain enzymes such as peroxidases and structural elements for tyrosine cross-linking of the inner radial band ZRI and outer radial band ZRE. As analyzed by TEM under various treatments, cortical reactions and the release of their contents are triggered by calcium ion treatment. The same process occurs during sperm-induced calcium waves in the oocyte plasma membrane during natural fertilization. In untreated oocytes (… Figure 6A In this study, the enzyme-containing cortical granules (CG) are clearly identifiable as large, round vesicles (bubbles) in the peripheral ootheca, which also contain structural components. Similarly, during potassium treatment alone according to the invention, the cortical granules (CG) remain unchanged. Figure 6B However, strong vesicular transport from the egg plasma to the non-cellular egg membrane can be observed on the serous membrane. Figure 6A and Figure 6B In this study, the egg yolk was labeled with D. In the dual treatment with potassium and calcium ions ( Figure 6C The discharge of contents from cortical granules (CG) (arrow) and the formation of a new stable layer (SS) both occurred. Treatment with calcium ions alone ( Figure 6D This only leads to a cortical reaction, in which the contents are expelled into the non-cellular egg membrane, and tyrosine residues trigger enzymatic cross-linking of the inner radial band ZRI and the outer radial band ZRE. Empty vacuole V remains in the egg plasma.

[0096] In order to perform diagnostic screening of potassium effects in frozen sections in this invention, Figure 7A and Figure 7B An optical microscope image (400x magnification) of untreated live eggs of the Siberian sturgeon Acipenser baerii is shown in the prior art. Figure 7A The left image shows HE staining (hematoxylin-eosin staining). Figure 7B The right figure shows alicin blue staining. This assay stains glycosaminoglycans (GAG), hyaluronic acid, and fibrin. It can be seen that alicin blue is absent in all layers of the oocyte membrane, but is clearly present in the oocyte ploid. Each layer is characterized according to the above-described embodiments, and its thickness is marked with double arrows.

[0097] on the other hand, Figure 7C and Figure 7D Cryosection images of live, mature Siberian sturgeon (Acipenser baerii) eggs in ovulation state, treated using the potassium exposure method claimed in this invention. The eggs were treated with 1.5 mmol / L potassium ions (from potassium citrate). A new stabilizing layer (SS) is clearly visible between the outer radial zone (ZRE) of the oocyte membrane and the alveolar layer (AL). Figure 7C The left image shows that the new stable layer SS is particularly eosinophilic after eosin staining. According to... Figure 7DThe right figure shows that the new stabilizing layer SS, after being stained with alexandrite blue, is particularly rich in GAG. This results in the particularly advantageous elasticity of the new stabilizing layer SS.

[0098] To determine the role of calcium alone and the combined effects of potassium and calcium ion treatment, diagnostic screening was performed on frozen sections. Figure 8A and Figure 8B Photographs of frozen sections of mature Siberian sturgeon Acipenser baerii eggs, treated according to the method in WO 2007 / 045233 A1, are shown, magnified 400 times. According to... Figure 8A The left image shows HE staining (hematoxylin-eosin staining), according to Figure 8B The right image shows Alsin blue staining. The eggs of a Siberian sturgeon were treated. According to... Figure 8A The HE staining in the left image shows protein chains cross-linked with tyrosine molecules in the inner radial zone (ZRI) and outer radial zone (ZRE). A clear separation between the two regions is visible. Cross-linking contributes to the structural stability of the non-cellular egg membrane. Based on... Figure 8B In the right image, stained with Alsin blue, it can be seen that the inner radial band ZRI and outer radial band ZRE are stained only very weakly, which results in very few GAGs and low elasticity, while in the oocyte OP, strong staining indicates a great many GAGs.

[0099] exist Figure 8C and Figure 8D The image shows a photograph of a frozen section of live ovulated eggs treated using the method claimed for protection. These eggs had been treated in an additional calcium exposure step according to the method described in WO 2007 / 045233 A1. Mature ovulated eggs of the Siberian sturgeon *Acipenser baerii* were treated with 1.5 mmol / L potassium ions (from potassium citrate) in the potassium exposure step and 1.6 mmol / L calcium ions (from calcium chloride) in the calcium exposure step. In addition to curing... Figure 8A and Figure 8B In addition to the cross-linking of the egg membrane shown in the photograph, it is now also possible to... Figure 8C and Figure 8D The photograph shows a novel transparent membrane stabilizing layer (SS) with stabilizing function in the egg membrane. Therefore, the mature, live eggs of treated Siberian sturgeon are stabilized both elastically (via GAG) and structurally (via protein cross-linking), forming perfect caviar.

[0100] Figure 9A The image shows a characteristic SEM image (12000x magnification) of the egg membrane of a live, mature, ovulating egg from the beluga sturgeon (Kaluga dauricus) after dual processing according to the present invention. Figure 9BOptical microscopic images (400x magnification) of the egg membrane of a live, mature, ovulating egg from the beluga sturgeon (Kaluga dauricus) after dual treatment according to the invention are shown. Both images show the new stabilizing layer SS and the cross-linking of the inner radial band ZRI and outer radial band ZRE. Furthermore, it is noteworthy that the Kaluga dauricus egg possesses a very distinctive alveolar layer AL with large vacuolars V. Screening of frozen sections by H&E staining confirmed the formation of the new elastic stabilizing layer SS through a potassium ion exposure step, and the additional protein cross-linking formed in the egg membrane through exposure to potassium and calcium ions.

[0101] List of Marker Symbols

[0102] AL alveolar layer

[0103] Ca ++ Calcium cations

[0104] CG Cortical Granules

[0105] D egg yolk

[0106] EP surface

[0107] K + potassium ions

[0108] Mv average

[0109] OP oocyte cytoplasm (oocyte cytoplasm)

[0110] Plasma membrane of PO oocyte (egg cell)

[0111] SS stable layer

[0112] Std standard deviation

[0113] V-bubble

[0114] ZRI Inner Radiation Zone

[0115] ZRE outer radiation zone

Claims

1. A method for producing caviar or caviar-like products from fresh, mature, ovulated eggs of fish or crustaceans, characterized in that, in, The fresh, mature, ovulated oocytes are in a fertile but unfertilized state and have a natural potassium content in the oocyte plasma. The method involves treating the fresh, mature, ovulated oocytes in a 0.6% to 1.0% saline solution without damaging the oocytes, followed by treating the mature oocytes in a potassium exposure step in a solution containing water and at least one cationic component dissolved therein, thereby stabilizing the oocyte membrane metabolism of the fresh, mature, ovulated oocytes. In the potassium exposure step, potassium is dissolved in water as a cationic component at a concentration that does not harm the live, mature, ovulating eggs or alter their natural potassium content. The potassium ion concentration in the solution is between 0.1 mmol / L and 3.0 mmol / L. The water is deionized and kept at a temperature harmless to the live, mature, ovulating eggs before the addition of the potassium donor to form the cationic component. This temperature is within the natural spawning temperature range of fish or crustaceans, from 1°C to 29°C. The live, mature, ovulating eggs are treated in the solution for potassium exposure for 5 to 30 minutes until the desired elastic stability of the egg membrane is achieved. After achieving the desired elastic stability, the mature, live eggs are immersed in a 0.6% to 1.0% salt solution that does not damage them. A new elastic stabilizing layer (SS) is formed between the inner radial zone (ZRI) and alveolar layer (AL) of the egg membrane. This SS has acidophilic and transparent properties and is incorporated with glycosaminoglycans.

2. The method according to claim 1, characterized in that, At least one potassium salt is used as the potassium donor, wherein the at least one potassium salt is a salt of citric acid and / or a salt of hydrochloric acid and / or a salt of sorbic acid.

3. The method according to claim 1 or 2, characterized in that, The concentration of potassium ions in a solution is 0.1 mmol / L, 0.5 mmol / L, 0.65 mmol / L, 1.6 mmol / L or 2.0 mmol / L.

4. The method according to claim 1 or 2, characterized in that, The concentration of potassium ions in a solution is 1.0 mmol / L or 1.5 mmol / L.

5. The method according to claim 1, characterized in that, The potassium exposure time in the potassium exposure step is 10 minutes, 12 minutes, 15 minutes, 20 minutes, or 25 minutes.

6. The method according to claim 1, characterized in that, In the calcium exposure step, either before or after the potassium exposure step, calcium is dissolved as a cationic component in another aqueous solution at a concentration that does not damage mature live eggs. The water is deionized before the calcium donor is added and its temperature does not harm the mature live eggs. The mature live eggs are treated in the solution for calcium exposure time until the desired egg membrane structure is stabilized.

7. The method according to claim 6, characterized in that, At least one calcium salt is used as a calcium donor, and the at least one calcium salt is calcium citrate, calcium chloride and / or calcium sorbate.

8. The method according to claim 6 or 7, characterized in that, The concentration of calcium ions in the other solution ranged from 0.1 mmol / L to 3.0 mmol / L.

9. The method according to claim 6 or 7, characterized in that, The concentration of calcium ions in the other solution is 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.

10. The method according to claim 6 or 7, characterized in that, In the calcium exposure step, the calcium exposure time is 9 to 30 minutes.

11. The method according to claim 6 or 7, characterized in that, The calcium exposure time is 10 minutes, 12.5 minutes, 15 minutes, 16 minutes, 20 minutes, or 25 minutes.

12. The method according to claim 6 or 7, characterized in that, The temperatures of one solution in the potassium exposure step and / or another solution in the calcium exposure step are adapted to the habitats of fish and crustaceans capable of using their eggs during natural reproduction, said habitats including polar, temperate, and tropical regions. When the living area is the polar region, the temperature is within the polar temperature range of 1°C to 15°C; or When the living area is in the temperate zone, the temperature is within the moderate temperature range of 10°C to 20°C; or When the living area is in the tropics, the temperature is in the tropical temperature range of 20°C to 29°C.

13. The method according to claim 12, characterized in that, When the living area is the polar region, the temperature of one solution in the potassium exposure step and / or another solution in the calcium exposure step is within the polar temperature range of 5°C to 12°C.

14. The method according to claim 13, characterized in that, When the living area is the polar region, the temperature of one solution in the potassium exposure step and / or the other solution in the calcium exposure step is within a polar temperature range of 10°C.

15. The method according to claim 12, characterized in that, When the living area is in the temperate zone, the temperature of one solution in the potassium exposure step and / or the other solution in the calcium exposure step is in the medium temperature range of 15°C or 12°C.

16. The method according to claim 12, characterized in that, When the living area is tropical, the temperature of one solution in the potassium exposure step and / or the other solution in the calcium exposure step is within the tropical temperature range of 27°C or 21°C.

17. The method according to claim 1 or 2, characterized in that, The conductivity of deionized water at 25°C is between 1 μS / cm and 15 μS / cm.

18. The method according to claim 17, characterized in that, The conductivity of the deionized water at 25°C is 10 μS / cm or 1 μS / cm.

19. The method according to claim 1 or 2, characterized in that, The pH of one and / or the other solution is between 6.8 and 8.

0.

20. The method according to claim 19, characterized in that, The pH of one and / or another solution is between 7.0 and 7.

9.

21. The method according to claim 20, characterized in that, The pH of one and / or another solution is 7.2, 7.4, or 7.

5.

22. The method according to claim 1, characterized in that, Following the final exposure step for preservation and flavor enhancement, the product is gently salted with sodium chloride at 2.0% to 3.8% of the caviar or caviar-like product content, wherein the sodium chloride is free of potassium and calcium donors.

23. The method according to claim 22, characterized in that, Mild salting is performed with 3.5% sodium chloride relative to the content of caviar or caviar-like products, wherein the sodium chloride is free of potassium and calcium donors.

24. The method according to claim 22, characterized in that, After preservation and flavor enhancement, store caviar or caviar-like products in sealed glass containers at a temperature between -2°C and -4°C for one to three months.

25. The method according to any one of claims 22 to 24, characterized in that, After preservation, flavor enhancement, or storage, caviar or caviar-like products are frozen at a temperature range of -20°C to -15°C.

26. The method according to claim 25, characterized in that, The caviar or caviar-like product is frozen at -18°C.

27. The method according to claim 6, characterized in that, After achieving the required elasticity and structural stability, mature live eggs are immersed in a salt solution that will not damage them.

28. The method according to claim 1 or 2, characterized in that, Treat mature live eggs of fish or crustaceans with a polarization index PI of 0.05 ≤ PI ≤ 0.

15.

29. The method according to claim 28, characterized in that, The polarization index PI is 0.05 ≤ PI ≤ 0.

12.

30. The method according to claim 1 or 2, characterized in that, Processing mature live eggs of fish or crustaceans caught in the wild or in aquaculture, the eggs being harvested in an ovum-laying state by spawning or peeling.

31. The method according to claim 30, characterized in that, Process the mature live eggs of recent and natural live bony fish, or process the mature live eggs of crustaceans.

32. The method according to claim 31, characterized in that, The most recent and natural live eggs of the live-bone fish were captured from sturgeon.

33. The method according to claim 31, characterized in that, The mature, live eggs of the crustaceans were captured from lobsters.

34. The method according to claim 7, characterized in that, Process very large mature live eggs with a diameter greater than or equal to 3.2 mm, or soft, unstable mature live eggs with a load capacity of less than or equal to 0.3 N before rupture.

35. The method according to claim 1, characterized in that, The salt solution is a 0.9% salt solution.

36. A caviar or caviar-like product of mature live eggs of a fish or crustacean, produced from fresh, mature, ovulated eggs of a fish or crustacean by the method described in any one of claims 1-35, wherein the mature live eggs are in a fertile but unfertilized state and have a natural potassium content in the roe plasma. Its features are, An elastic stabilizing layer (SS) is formed between the inner radial zone (ZRI) and alveolar layer (AL) of the egg membrane. This SS has acidophilic and transparent properties and is incorporated with glycosaminoglycans.

37. The caviar or caviar-like product according to claim 36, characterized in that, The elastic stabilizing layer is formed between the outer radial zone (ZRE) of the oocyte membrane and the alveolar layer (AL).

38. The caviar or caviar-like product according to claim 36 or 37, characterized in that, The incorporated tyrosine molecules create additional irreversible protein chain crosslinks in the inner and outer radiation zones (ZRI and ZRE) of the egg membrane.

Images