Method for producing fish cells

Abstract

The present invention relates to a method for producing fish cells by means of a cell medium, and to fish products obtainable therefrom and the use thereof.

Description

[0001] Methods for producing fish cells

[0002] Description

[0003] The present invention relates to a method for producing fish cells using a cell medium, and fish products obtainable therefrom and their use.

[0004] Meat is an important source of protein in the human diet. However, controversial animal welfare practices in the traditional meat industry, coupled with a growing global population and increasing demand for meat products, particularly fish (meat) products, make sustainable production alternatives essential. Cultured meat technology (especially so-called clean fish) offers the possibility of producing edible animal protein sources that are not associated with the environmental impacts of animal agriculture.

[0005] The production process of cultured meat can be described as follows:

[0006] All cellular substances contained in meat (muscle, connective tissue, fat) can be produced in respective bioreactors based on cell cultures, followed by a multi-stage process (McClements, D. J. (2023) Biotech Meat: Growing Meat from Cells. Meat Less - The Next Food Revolution, p. 149 ff. Springer-Verlag GmbH).

[0007] For the purposes of this invention, cell cultivation means the controlled in vitro proliferation of cells outside their original organism in sterile, closed systems (e.g., bioreactors) with a defined nutrient medium and under controlled environmental parameters. Isolated cells are maintained and multiplied in an artificial nutrient medium under defined conditions. The aim is to establish cell lines and / or to produce cell mass (biomass) for further applications.

[0008] Systems and conditions: Cell culture is usually carried out in closed, sterile bioreactors or culture vessels (e.g., cell culture flasks, dishes, spinners). The cells grow in a liquid nutrient medium containing all necessary amino acids, vitamins, growth factors, and dissolved gases (oxygen, carbon dioxide). Optimal conditions are continuously maintained in such systems—for example, through temperature and pH control, mechanical aeration and stirring, and the removal of metabolic waste. Strict asepsis is essential: the entire cultivation process takes place under sterile conditions to prevent microbial contamination.

[0009] According to the invention, cell cultivation can be explained in the following phases:

[0010] Phase 1, Collection Phase: Cells are harvested, usually via muscle biopsy, followed by cell selection and the creation of a donor cell bank using cell culture. In this cell culture, the selected stem cells can undergo the process of immortalization, allowing them to be used indefinitely for production.

[0011] Phase 2, Proliferation Phase: Cells from the donor cell bank are cultivated under controlled conditions using a nutrient solution containing nutrients and growth factors. Cultivation continues in the bioreactor under controlled and monitored conditions, particularly temperature, oxygen content, and pH, to promote cell proliferation. Phase 3, Maturation Phase: The cells are transferred to further bioreactors with the addition of specific additives such as nutrients and growth factors. Depending on the chosen additives, different tissues are produced: muscle cells, connective tissue cells, or fat cells. With the addition of scaffold materials, cell clusters resembling the natural cell clusters in real meat can form directly in the reactor.

[0012] After 4-6 weeks of maturation, the process is complete and the meat product can be produced from the resulting cells or cell clusters.

[0013] Furthermore, it is preferred in the prior art to use serum-free cell media, i.e. without fetal calf serum (FCS), particularly to avoid contamination risks and for better reproducibility (see e.g. EP 4 251 740 Al).

[0014] Distinguishing the cell culture method according to the invention from aquaculture in fish: Unlike aquaculture – the conventional fish farming method – cell culture does not involve raising live animals in water. Aquaculture, as defined by the Food and Agriculture Organization of the United Nations (FAO), encompasses the breeding of aquatic animals (fish, crustaceans, mollusks, etc.), where interventions such as stocking and feeding are carried out to increase yield. In contrast, in in-vitro cell culture, only isolated cells are cultivated – no complete, viable animal exists. The cells are supplied solely through the nutrient medium and the technology of the culture system.

[0015] Since cell culture does not use integral organisms, all physiological organ functions of a living animal are absent. The cultured cells possess no digestive, respiratory, or nervous systems; no animal respiration or movement takes place. Nutrients and oxygen are supplied diffusely via the medium or...

[0016] Gas phase regulation is applied and metabolic products are removed. Cell cultures, as defined in this invention, are completely artificial cell groups that grow in a fully controlled bioreactor or reaction vessel environment without external sensory control.

[0017] Consequently, the present invention and the cell cultivation according to the invention do not relate to aquaculture within the meaning of a disclaimer.

[0018] Fish flesh is preferred in this invention.

[0019] The term "fish flesh" or "fish fillet" refers to the edible muscle tissue of fish without skin, bones and innards.

[0020] Depending on the natural fish species, fish flesh differs in taste, texture, and nutritional value. There are leaner varieties like cod or pike, as well as fattier ones like salmon and mackerel, which contain higher amounts of omega-3 fatty acids.

[0021] There is a need to make cultured fish meat comparable to native fish meat in shape, texture, color, taste and quality.

[0022] The production of in-vitro fish cells is described in the prior art. EP 2 500 412 Bl describes the production of fish cells, whereby the cultivation takes place at two different temperatures, and the cell culture medium contains an addition of at least one short-chain, polyunsaturated fatty acid from the group consisting of alpha-linolenic acid (ALA, C18:3n3), palmitoleic acid (C16:ln7),

[0023] Oleic acid (C18:ln9) and linoleic acid (C18:2n6) are selected, containing .

[0024] However, a disadvantage of the current state of the art is that fish cells cannot be produced cost-effectively in sufficient yield and quality.

[0025] The inventors have determined that the amino acid profile is essential for fish cell multiplication or proliferation, and leads to qualitatively improved fish cells without the need to use, for example, special fatty acids.

[0026] Surprisingly, hydrolysates of native plant proteins, preferably gluten and yeast, can provide advantageous amino acid profiles for a cell medium that promote fish cell proliferation, particularly in demanding fish cells such as salmon. A particular advantage is that the use of a serum (such as FCS) is not required.

[0027] Other native plant proteins are available, including those selected from the group of plants such as cereals, wheat, oats, soy, peas, lentils, beans, lupins, and hemp, particularly wheat gluten and gluten. However, gluten is especially preferred. Furthermore, mixtures or at least one plant protein may be present.

[0028] Plant proteins are obtained from plants (leaf, seed, root, flower, stem) by means of protein extraction, whereby the protein is dissolved from the other components (e.g. starch, fats, fiber) using methods such as water-soluble extraction at specific pH values ​​(alkaline or acidic), use of salt solutions (e.g. in lupin), enzymatic treatment to dissolve cell walls, or wet or dry separation of protein fractions.

[0029] Furthermore, due to the cell medium according to the invention, a particularly suitable and advantageous fatty acid spectrum for the cell culture is surprisingly achieved immediately, so that the cultured fish cells have a comparable lipid spectrum to, for example, shrimp (see figures).

[0030] Furthermore, the inventive method advantageously achieves a high ratio of polar to neutral lipids in the cultured fish cells. While in native fish muscle tissues polar lipids, especially phospholipids, typically constitute only 15-25 wt.% of the total lipids (Ackman, RG, Fish lipids: Composition, nutritive value, and metabolic roles, in WS Hoar, DJ Randall, & JR Brett (Eds.) , Fish Physiology, Vol. 2 (pp. 111-174) , Academic Press (1989) ), polar lipids, especially phospholipids, of more than 69 wt.% of the total lipids could be detected in the monolayer-cultured cells (lipid class analysis using latroscan TLC-FID).

[0031] The invention therefore relates to a method for producing fish cells, comprising the following steps: a) providing at least one proliferating fish cell, b) providing salts, vitamins and trace elements, c) providing a hydrolysate of at least one plant protein and yeast, d) combining c) with b) to obtain a cell medium and carrying out cell culture with a).

[0032] The inventive method allows the advantageous production, enrichment and multiplication of fish cells from at least one proliferating fish cell.

[0033] In a particularly preferred embodiment of the invention, the hydrolysate of c.) is provided by using at least one vegetable oil, preferably vegetable oils containing saturated and unsaturated fatty acids.

[0034] The hydrolysis to produce the hydrolysate of c) can be carried out chemically or enzymatically. Enzymatic hydrolysis using a protease is preferred.

[0035] Therefore, the invention relates to a hydrolysate of c.), which is an emulsion, and contains a vegetable oil, consequently to providing a hydrolysate of at least one vegetable protein and yeast, and at least one vegetable oil, wherein the hydrolysate is mixed with or contains a vegetable oil.

[0036] In another particularly preferred embodiment, the at least one vegetable oil is selected from the group consisting of wheat germ oil (174-176 mg / 100 g), sunflower oil (60 mg / 100 g), linseed oil (57 mg / 100 g), walnut oil (39 mg / 100 g), corn germ oil (31-34 mg / 100 g), safflower oil (29-45 mg / 100 g), sesame oil (28 mg / 100 g), peanut oil (25 mg / 100 g), rapeseed oil (25 mg / 100 g), almond oil (25 mg / 100 g), palm oil (25 mg / 100 g), rapeseed oil (20 mg / 100 g), soybean oil (17-25 mg / 100 g), and olive oil (12-40 mg / 100 g). Black salsify oil (6 mg / 100 g), linseed oil (5.8 mg / 100 g), avocado oil (13-15 mg / 100 g), macadamia oil (0.5-1.5 mg / 100 g) or

[0037] Algae oil (0.5-5 mg / 100 g) .

[0038] The brackets indicate the percentage of vitamin E in the respective oil.

[0039] In another formulation, in addition to vegetable oil, further excipients and additives may be included, such as antioxidants, pH stabilizers (especially buffers for pH values ​​of 5-7), etc., particularly those such as tocopherol (vitamin E), polyphenols, citric acid, etc.

[0040] A suitable composition for the provision of the hydrolysate contains 15-35 wt.%, in particular 23.8 wt.% vegetable protein, in particular wheat gluten, 15-35 wt.%, in particular 23.8 wt.% yeast extract, 35-65 wt.%, in particular 40-60 wt.%, in particular 47.6 wt.% vegetable oil, possibly further excipients and additives, such as optionally 1-5 wt.%, in particular 2.4 wt.% citric acid and optionally 1-5 wt.%, in particular 2.4 wt.% tocopherol (vitamin E).

[0041] In a preferred formulation, such a mixture is heated, since flour fractions, especially gluten, can contain bacterial and / or fungal spores that need to be inactivated to ensure sterility in the subsequent cell culture process. The antioxidant tocopherol can be added to the mixture to protect the lipids from oxidation at high temperatures.

[0042] This composition is preferably heated at 100–180 °C, particularly at 130–150 °C, for 10–180 minutes, especially 30–60 minutes. The resulting emulsion is then subjected to enzymatic or chemical hydrolysis. Enzymatic hydrolysis is carried out with a protease (peptidase, proteinase, or proteolytic enzyme) with the release of amino acids.

[0043] After completion of the hydrolysis, solid components can be removed by pelletizing in the centrifuge or filtration, and the oil-containing mixture can be emulsified using ultrasound.

[0044] Therefore, the invention relates to the production of the hydrolysate according to c . ), wherein at least one plant protein, in particular gluten and yeast, are mixed with a vegetable oil and heated, and subsequently chemically or enzymatically hydrolyzed.

[0045] The base medium or basal medium from a.) is then mixed with the hydrolysate (supra). The osmolarity of the resulting cell medium is adjusted to preferably 290-320 mOsmol / kg using sterile deionized water and a 30% NaCl solution.

[0046] The medium is advantageously stable during storage and shows no precipitation even after a longer period (6 weeks), which is often a problem in cell culture media production. It is also preferred to use calcium lactate in the cell medium according to the invention, since calcium lactate particularly supports the advantageous stabilization of the cell medium.

[0047] In particular, the hydrolysate according to the invention plays an essential role in the cell medium as an amino acid supplier. The amino acid profile of the wheat gluten / yeast extract hydrolysate preferred according to the invention was analyzed (Fig. 1). It turns out that yeast and gluten complete the amino acid profile to optimally provide the fish cells with the required amino acids.

[0048] Furthermore, the cell medium according to the invention can contain any carbon source, such as sugar, in particular glucose.

[0049] The proliferating fish cells intended for cell culture according to the invention can be of any type. Both known immortalized fish cell lines of the prior art and novel cell lines derived from fish tissue can be used. The starting tissue for a primary fish cell culture can be, for example, the total tissue of a fish larva or a specific organ of an adult target fish. Adult fish tissue can include, for example, tissue from a kidney, in particular the head kidney, the liver, the pancreas or the pyloric appendages, the intestine, the heart, the brain, the gonads, adipose tissue, the skin, or muscle tissue.

[0050] Suitable fish cell lines include, in particular, those such as:

[0051] Fish cell lines can be produced as shown in the examples.

[0052] In a preferred embodiment, the entire tissue of a fish larva is used. The term "fish larva" specifically includes fish eggs.

[0053] Proliferable fish cells used according to the invention are, but are not limited to, fish larvae or fish tissue from any fish species, but preferably those of the Osteichthyes, in particular species of the Teleostei. Examples include species of herring (Clupeoidei), salmonids (Salmonoidei), e.g., salmon, trout, especially brown trout, rainbow trout, char, huchen, whitefish, smelt, carp (Cyprinidae), eels (Anguillidae), perch (Percidae), cod (Gadidae), catfish (Siluroidae), flatfish (Pleuronectiformes), garfish (Beloniformes), sturgeon (Acipenseriformes), etc. However, sturgeon, herring, trout, salmon, eel, carp, mackerel, halibut or sardine are preferred.

[0054] The proliferating fish cells can be present as suspension cells, or growth can occur on (micro-)carriers (e.g., plant particles, etc.). Seeding can take place in a bioreactor (continuous perfusion culture may be necessary) with the cell medium according to the invention, or the proliferating fish cells can be brought into contact with the cell medium. In a further embodiment, the invention therefore relates to a method according to the invention, wherein at least one bioreactor is used.

[0055] Gluten is a valuable plant protein mixture found mainly in the grains wheat, rye, barley, and related grains such as spelt, and contains gliadins and glutenins. Wheat gluten is the preferred form.

[0056] “Yeast” or “yeast extract” is a valuable nutrient, rich in B vitamins, minerals and valuable proteins.

[0057] The bioreactors can be equipped with stirrers and other common auxiliary equipment. Suitable bioreactors for cultivation include hollow fiber bioreactors, stirred tank reactors (fermenters), fluidized bed reactors (aggregates or porous support materials), fixed-bed reactors with hollow fiber or flat-bed membranes (the process is tangential flow filtration), plug-flow reactors, and spinner bottle cultivation (see Horst Chmiel (ed.) Bioprocess Engineering (2011), Spektrum Akademischer Verlag, Heidelberg). The bioreactors can contain the medium, culture medium, or culture liquid according to the invention.

[0058] In another embodiment, the invention relates to cell-cultured fish cells that are cultured and obtainable or obtainable according to the inventive method, such that the proportion of polar lipids is at least 40, 50, 60, 65, 66, 67, 68, 69, 70 wt. % of the total lipids (100 wt. %) and / or the ratio of polar lipids (PL) to neutral lipids (NL) is > 2.5 : 1, in particular > 2.78:1 (see examples).

[0059] According to an example, the fish cells cultivated according to the invention possess an outstanding 69.4% polar lipids (PL) at only

[0060] 3.6% triglycerides (TG) and 21.4% cholesterol esters (CE), or a PL : NL ratio of 2.78 : 1.

[0061] Polar lipids are amphiphilic, especially those selected from the group of phospholipids, glycolipids and sphingolipids.

[0062] Phospholipid within the meaning of this invention is a molecule consisting of three main components: a glycerol molecule, two fatty acids and a phosphate group, which is usually linked to an additional molecule such as choline, serine, ethanolamine or inositol, and forms the essential or main proportion of the polar lipids in the cultured fish cells.

[0063] Neutral lipids within the meaning of this invention are a class of lipids (fats) that do not carry an electrical charge - i.e., unlike e.g. phospholipids, they are not polar or ionic, and consequently are hydrophobic, such as triglycerides (TG) and cholesterol esters (CE).

[0064] In cold water (e.g., salmonid larvae), membrane fluidity is crucial for the survival and growth of micro- and juvenile fish. A PL-rich diet (3-6% PL) significantly increases the survival rate and improves feed conversion. The cultured fish cells can therefore be used as a high-quality feed.

[0065] Phospholipid-bound EPA / DHA exhibits up to 1.5 times higher absorption than triglyceride-bound forms, as clinically demonstrated. The high PL profile supports both nutritional value and efficiency in feeding and nutrition (less loss through digestion). Furthermore, the invention relates to a method for obtaining fish products selected from the group consisting of fishmeal, fish proteins, fish oils and fatty acids, fish fat, and fish flesh, from fish cell cultures, comprising: i.) Cultivating and propagating proliferating

[0066] Fish cells in a cell culture medium according to the inventive method and obtaining fish cells, ii.) removal of the (obtained) fish cells, iii.) either freeze-drying and / or comminution and / or encoding of the removed fish cells, iv.) or providing the removed fish cells in a composite of fish cells for fish fat or fish meat or fish products thereof, in particular fish fillet, pods for fish burgers, battered fish, fish fingers, v.) or providing the removed fish cells, preferably in a composite of fish cells, for feed, in particular for juvenile fish.

[0067] “Removal” within the meaning of this invention means isolating or harvesting the cultured fish cells.

[0068] A composite of fish cells can be produced, for example, using presses or a 3D printer.

[0069] Fish products derived from such a network of fish cells are, in particular, processed products such as fish fillets, fish burger patties, battered fish, or fish fingers, possibly with the addition of further additives and processing aids. It should be noted that characteristics described in connection with an exemplary embodiment or an exemplary object can be combined with any other exemplary embodiment or with any other exemplary object.

[0070] When a term is referred to with an indefinite or definite article, such as "ein" in the singular, this also includes the term in the plural and vice versa, unless the context clearly indicates otherwise.

[0071] The expression "encompass" as used here does not only include the meaning of "contain", but can also mean "consist of" and "essentially consisting of".

[0072] Cell medium and cell culture medium are synonymous. The following examples and illustrations serve to further explain the invention, without, however, limiting the invention to these examples and illustrations.

[0073] Examples and illustrations:

[0074] Example 1:

[0075] Production of the cell medium according to the invention

[0076] To produce the cell (culture) medium (“GY”) according to the invention, a total of two steps are necessary to generate a complete medium. The first component is a mixture of inorganic salts, vitamins and trace elements, which are sterile filtered in the usual manner (“basic medium” - 33 components, see example below).

[0077] The second component consists of yeast extract and wheat gluten, which serve as an amino acid source, along with a vegetable oil, preferably rapeseed oil, which is also added. It is important to note that rapeseed oil serves both as a heat transfer medium in a preliminary sterilization process and as a fatty acid source for the cell culture. The next step is heating these components (composition), as flour fractions, especially gluten, can contain bacterial and / or fungal spores that need to be inactivated to ensure sterility in the subsequent cell culture process. These components are used in the following proportions (by mass): 23.8% wheat gluten, 23.8% yeast extract, 47.6% rapeseed oil, 2.4% citric acid, and 2.4% tocopherol (vitamin E). The antioxidant tocopherol is added to the mixture to protect the lipids from oxidation at high temperatures.Vitamin E is also added to meet the physiological needs of the fish cells. These components are heated at 130°C for 30 minutes. Sterilization and functionality of these samples were demonstrated using contact plates. The resulting emulsion is then used directly for the enzymatic hydrolysis step. Here, 10% (w%) of the hydrolysate is dissolved in a 50 mM acetic acid acetate buffer (pH 5.0), to which a protease from Aspergillus oryzae with at least 4000 units / L is added. Under sterile conditions, the mixture is then incubated at 50°C for 16 hours to release amino acids. The enzyme is then inactivated at 95°C for 10 minutes. Solid particles are removed by centrifugation, and the oil / water mixture is re-emulsified in an ultrasonic bath. The base medium is then combined with the hydrolysate, with an addition of between 1 and 10% (vol.-%) are used, the osmolarity of the complete medium (GY medium) is adjusted to 290-320 mOsmol / kg using sterile deionized water and 30% NaCl.

[0078] Example 2:

[0079] Salmon cells with a fatty acid profile similar to shrimp meat

[0080] Since no adaptation with the medium completely without serum had yet been performed at this time, at least a serum reduction to 2% FBS was achieved. The cells were cultured in the developed medium until an approximate wet mass of 150 mg per sample to be analyzed was reached. For comparison, with the exception of the rainbow trout (originating from the Thünen Institute's aquaculture facility), fish (fish flesh) and shrimp from the wild were acquired and subsequently also analyzed using the cell culture.

[0081] Using thin-layer chromatography (TLC) followed by flame ionization detection (FID), up to nine lipid classes expected to be present in fish lipids were simultaneously identified and quantified. Solutions of fish lipids are applied to silica-coated glass rods (chromarods), and the different lipid classes are separated by thin-layer chromatography. This requires three successive development steps, performed with different polar mobile phase mixtures. Following each development step, the chromarods are partially detected by FID to analyze the lipid classes relevant to that step. The amounts (in pg) of each lipid class can be determined from the integrals of the signals, resulting in a percentage distribution.As can be seen in the figures, the samples are very similar in their composition of neutral (Fig. 3) and polar (Fig. 4) lipids. This demonstrates that polar lipids, which play a crucial role in nutrition, are primarily found in the cultured cells. Example 3:

[0082] From the embodiment of a cell medium according to the invention (“GY”) Enzymatic hydrolysate 1-10 from wheat gluten and yeast extract and

[0083] vegetable oil

[0084] Example 4:

[0085] The fatty acid profiles of all cell samples are similar, as shown in Table 1. All are dominated by oleic acid (C18:ln9), which accounts for between 47 and 57% of the total fatty acids, followed by palmitic acid (C16:0) and stearic acid (C18:0). Such high oleic acid levels are only found in the fatty farmed fish (trout; Atlantic salmon) among the reference samples. Other fatty acids occur at a maximum of 5%. Polyunsaturated fatty acids were detected, albeit in significantly lower proportions than in the fish models. However, the fatty acid derivatives and proportions differ between the cells. While in king salmon (cultured with 2% FBS) mainly the C18-PUFA representatives could be detected, in the other cell samples proportions of up to 4% were the LC-PUFA ARA, EPA or DHA.However, their proportions are significantly lower than in fish samples with a comparable total lipid content (see Fig. 3). Comparable proportions of LC-PUFAs are only found in fatty fish, where, on the one hand, the ARA levels are significantly lower, and on the other hand, high absolute values ​​are reached for, for example, EPA and DHA. The corresponding differences are clearly visible in Figure 4.

[0086] Table 1: Fatty acid profiles of the comparison species and the samples from the cell culture. Mean values ​​of the respective species are shown (min. n=3).

[0087] All fatty acids that do not exceed the limit of quantification on average were not considered in the calculation of the percentage distribution and are not listed with values. Empty fields in the table correspond to results below the limit of quantification.

[0088] Example 5:

[0089] The lipid class distribution was determined using Latroscan TLC-FID (Flame Ionization Detection). For this purpose, cell pellets were lyophilized and the total lipids were extracted using the Bligh & Dyer method (chloroform : methanol : water, 2 : 2 : 1.8, v / v / v). Chromatographic separation was then performed in three development steps on silica gel rods: 1. Development 1: n-hexane : diethyl ether (68 : 2, v / v) - 30 min, detection until cholesterol ester peak.

[0090] 2. Development 2: n-Hexane : Diethyl ether : Formate (56 : 14 : 0.13, v / v / v) - 26 min, detection until cholesterol peak.

[0091] 3. Development 3: Chloroform : Methanol : Water (50 : 20 : 2, v / v / v) - 40 min, separation of polar lipids. Calibrated quantification was performed using external standards (nonadecane, cholesteryl palmitate, glyceryl tripalmitate, palmitic acid, cholesterol, 1,2-dipalm-PC, 1,2-

[0092] Dipalm-PE, sphingomyelin). Lipid classes were calculated as weight percent of the sum of all lipid classes (n > 3).

[0093] Table 2:

[0094] Mean values ​​from at least three independent measurements (n > 3) .

[0095] Legend:

[0096] • TG: Triglyceride

[0097] • CE: Cholesterol esters

[0098] • NL: Neutral lipids (= TG + CE)

[0099] • PL: Polar lipids (e.g. phospholipids)

[0100] • Aquaculture: Breeding under controlled conditions

[0101] • Wild-caught: Captured in natural habitats

[0102] • n > 3: Data from at least three independent cell cultures or fish samples.

[0103] Example 6:

[0104] Production of fish cell lines:

[0105] Under sterile conditions, a tissue sample is first taken from a healthy fish, for example, from muscle, fin, or skin tissue; alternatively, fertilized eggs can serve as starting material. The sample is immediately stored in an L-15-based transport medium containing antibiotics at 4–10 °C and processed further within 24 hours. In the cell culture workbench, the tissue is mechanically fragmented and then enzymatically incubated with a solution of trypsin and collagenase at room temperature for approximately 30–60 minutes to disperse cell aggregates. The resulting suspension is filtered through a 70 pm cell sieve to remove aggregates and obtain a homogeneous single-cell suspension. After centrifugation at approximately 300 x g for five minutes, the cell pellet is resuspended in fresh culture medium, such as Leibovitz's L-15 or DMEM / F-12.This medium contains 10-20% fetal bovine serum, 1% penicillin / streptomycin, and may optionally include growth factors such as bFGF, insulin, or IGF-1. The cell suspension is seeded into cell culture vessels and cultivated at a temperature of around 20°C without CO2 injection and at high humidity.

[0106] After 24–48 hours, fibroblasts and epithelial cells adhere to the substrate, while non-adherent cells are separated with the supernatant. The medium is changed every two to three days to replenish nutrients and remove cell debris. Once 70–80% confluency is reached in the culture, the cells are enzymatically dissolved with trypsin-EDTA and transferred to fresh medium at a ratio of 1:2 to 1:5. This pass-through process is repeated for at least 35 to 50 cycles to achieve spontaneous immortalization and selection of a stably proliferating cell population. Subsequently, the established cell line is characterized by morphological studies, determination of the doubling time, chromosome analysis, and analysis of cell-type-specific markers.For long-term storage, logarithmic growth cells are transferred to a cryopreservation medium consisting of 90% culture medium and 10% DMSO, aliquoted into cryovials, and cooled at a rate of -1 °C / min, initially to -80 °C, before being stored in liquid nitrogen (-196 °C). To resume the cultures, a cryovial is thawed, the medium is removed, and the cells are placed in fresh medium.

[0107] Culture medium is reconstituted. In this way, robust cell lines are created that can be used in adherence or suspension culture for applications such as the production of cultured fish tissue, drug and toxicity tests, or immunological investigations.

[0108] 1. Sample collection

[0109] Under strictly sterile conditions, a tissue sample is taken from a healthy, possibly anesthetized, fish. The starting material can be, for example, muscle, fin, or skin tissue, or fertilized eggs. The sample is immediately stored in a transport medium (e.g., Leibovitz's L-15 with antibiotics) at 4–10 °C and processed further within 24 hours.

[0110] 2. Tissue preparation

[0111] In a cell culture workbench, the sample is mechanically fragmented using sterile scalpels or scissors. The tissue suspension is then enzymatically treated (e.g., with 0.05% trypsin and 0.1% collagenase for 30–60 minutes at room temperature) to break down cell aggregates. The suspension is then passed through a 70 pm cell sieve to remove aggregates and obtain a homogeneous single-cell suspension.

[0112] 3. Cell isolation and sowing

[0113] The single-cell suspension is centrifuged (approx. 300 x g, 5 min), the supernatant discarded, and the cell pellet resuspended in fresh medium. Depending on the cell line, Leibovitz's L-15 or DMEM / F-12 is used, supplemented with 10–20% FBS, 1% penicillin / streptomycin, and optional growth factors (e.g., bFGF 10 ng / ml, insulin 5 pg / ml, IGF-1 10 ng / ml). The cells are seeded into culture vessels (e.g., T25 flasks) and cultured at ~20 °C (incubated without CO2, relative humidity ~95%).

[0114] 4. Primary culture

[0115] After 24-48 hours, fibroblasts and epithelial cells adhere to the substrate, while non-adherent cells are separated in the supernatant. The medium is changed every 2-3 days. Under continuous observation, dead cells and cell debris are removed by gently pouring off the supernatant.

[0116] 5. Passengering and Immortalization

[0117] Once the culture reaches 70-80% confluency, the cells are enzymatically dissolved (e.g., 0.05% trypsin-EDTA, 3-5 min.), inactivated (by adding serum-containing medium), and passaged further in a ratio of 1:2 to 1:5. Cultivation is performed for at least 35-50 passages to select for spontaneous immortalization. Only when consistently stable proliferation is observed beyond this number of passages is a cell line considered established.

[0118] 6. Characterization

[0119] The established cell line is validated with regard to morphology (microscopy), growth kinetics (doubling time), karyotype (chromosome analysis), and phenotypic markers (e.g., immunofluorescence for cell-type-specific proteins). Differentiation potentials (myogenesis, adipogenesis) are investigated as needed.

[0120] 7. Cryopreservation

[0121] For long-term storage, cells are detached at logarithmic growth (approx. 70% confluence), resuspended in cryobuffer (90% medium + 10% DMSO), and aliquoted into cryovials. The vitreous tubes are cooled incrementally (-1 °C / min) to -80 °C and then stored in liquid nitrogen (-196 °C).

[0122] 8. Long-term cultivation and application

[0123] The frozen cell stocks serve as master and working banks. For further use, cryotubes are thawed, seeded into a medium, and adapted under identical culture conditions. The cell lines can be cultured in adherence or suspension, genetically modified, differentiated, or used in bioreactors (e.g., hollow fiber or microparticle systems) for the production of cultured fish tissue, drug screening, or toxicity testing.

[0124] Figure 1: Determined amino acid profile of the enzymatic wheat gluten / yeast extract hydrolysate. The levels of the 20 proteinogenic amino acids in the hydrolysates were determined using standards via ion chromatography. The levels of three replicates (PI to P3, n=3) are shown.

[0125] Figure 2: Confluence of CHSE-214 cells after seeding in DMEM / F12 and the medium according to the invention. The cells were previously cultured in DMEM / F12, trypsinized, and centrifuged; the pellets were then resuspended and seeded into the respective medium. Zeiss Labscope software with the AI ​​Gell Confluency module was used for confluence analysis, and the measurements were manually confirmed.

[0126] Figure 3: Percentage of neutral lipids in the total lipid content. Shown are the mean values ​​of all comparison fish, as well as shrimp and the cell culture.

[0127] (CHSE-214 with the medium according to the invention). From left to right, the fat content of the comparison fish samples decreases.

[0128] Average fat content / sample size: Atlantic salmon: Lip% = 12.4%, n = 4; Rainbow trout (Thünen): Lip% = 11.7%, n = 3; Herring: Lip% = 9.1%, n = 5; Rainbow trout: Lip% = 8.44%, n = 3; Chunk salmon: Lip% = 1.45%, n = 4; Alaska pollock: Lip% = 0.74%, n = 4; Prawn: Lip% = 1.0%, n = 4; Cell culture (2% / 10% FBS): Lip% = 0.6%, n = 2.

[0129] Figure 4: Percentage of polar lipids in total lipid. Shown are the mean values ​​of all comparison fish, as well as shrimp and the cell culture. From left to right, the fat content of the comparison fish samples decreases.

[0130] Cell culture refers to the CHSE-214 cell line that was cultivated with the cell medium according to the invention.

Claims

Patent claims 1. A method for producing fish cells, characterized in that the following steps are carried out: a) providing at least one proliferating fish cell, b) providing salts, vitamins and trace elements, c) providing a hydrolysate of at least one plant protein and yeast, d) mixing c) with b) to obtain a cell medium and carrying out cell culture with a).

2. Method according to claim 1, characterized in that the at least one plant protein is selected from the group of plants cereals, wheat, oats, soy, peas, lentils, beans, lupins, hemp, in particular the plant proteins wheat gluten, gluten.

3. Method according to claim 1 or claim 2, characterized in that the hydrolysate from c.) contains at least one vegetable oil, in particular selected from the group consisting of wheat germ oil, sunflower oil, linseed oil, walnut oil, corn germ oil, safflower oil, sesame oil, peanut oil, rapeseed oil, almond oil, palm oil, rapeseed oil, soybean oil, olive oil, black salsify oil, linseed oil, avocado oil, macadamia oil or algae oil.

4. Method according to one of the preceding claims, wherein, to produce the hydrolysate according to c.), at least one plant protein and yeast are mixed with at least one plant oil and heated, and subsequently hydrolyzed chemically or enzymatically.

5. Method according to claim 4, wherein for the production of the hydrolysate according to c.) , wherein the at least one plant protein is gluten or wheat gluten and yeast is a yeast extract.

6. The method according to claim 4, wherein the heating takes place at 100 to 180 degrees Celsius.

7. Method according to one of the preceding claims, wherein for the production of the hydrolysate according to c.), wherein 15-35 wt. % at least one vegetable protein, in particular 15-35 wt. % wheat gluten and 15-35 wt. % yeast extract are combined with 35-65 wt. %, in particular 40-60 wt. % vegetable oil and optionally further auxiliary and additive substances.

8. The method according to claim 7, wherein the further auxiliary and additive substances contain 1-5 wt.% citric acid or 1-5 wt.% tocopherol.

9. Method according to any of the preceding claims, wherein the at least one proliferating fish cell is selected from the group consisting of sturgeon, herring, trout, salmon, eel, carp, mackerel, halibut or sardine, and fish cell line.

10. Method according to one of the preceding claims, wherein the cell culture takes place in at least one bioreactor.

11. Methods for obtaining fish products, selected from the group consisting of fishmeal, fish proteins, fish oils and fatty acids, fish fat, fish flesh, from fish cell cultures, comprising: i.) Cultivating and propagating proliferating Fish cells in a cell culture medium according to the method of any one of claims 1 to 8 and obtaining fish cells, ii.) removal of the fish cells, iii.) either freeze-drying and / or comminution and / or encoding of the removed fish cells, iv.) or providing the removed fish cells in a composite of fish cells for fish fat or fish meat or fish products thereof, in particular fish fillet, pods for fish burgers, battered fish, fish fingers, v.) or providing the removed fish cells, preferably in a composite of fish cells, for feed, in particular for juvenile fish.

12. Cell medium comprising salts, vitamins and trace elements and a hydrolysate of at least one plant protein, in particular gluten and yeast, and a vegetable oil, in particular calcium lactate.

13. Cell medium according to claim 12 according to the embodiment according to example 3.

14. Use of a cell medium according to claim 12 or claim 13 for the cultivation of fish cells.

15. Cell-cultured fish cells obtainable by a method according to any one of claims 1 to 9, wherein the proportion of polar lipids is at least 40, 50, 60, 65, 66, 67, 68, 69, 70 wt. % of total lipids (100 wt. %) or the ratio of polar lipids to neutral lipids is > 2.5:1.

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