Process for producing a feed enriched with at least one carotenoid or a feed component enriched with at least one carotenoid

Description

[0001] The present invention relates to a process for producing a feed or feed component enriched with astaxanthin and to such an enriched feed or feed component.

[0002] In the production of animal-based foods, the feed used for the farmed animals is an important factor. For example, in salmon farming in aquaculture, the feed should contain the carotenoid astaxanthin, which causes the typical reddish color of the fish meat and, as a natural antioxidant, has a variety of positive effects that contribute to the quality of the produced food. Under natural conditions, salmon usually absorb astaxanthin from small crustaceans. The small crustaceans, in turn, feed on microalgae, which naturally produce astaxanthin. This food chain does not exist in aquaculture. Therefore, in aquaculture, astaxanthin is added to the feed, which essentially covers the salmon's protein requirements through fishmeal. The astaxanthin used for this purpose is usually artificially synthesized from petroleum.However, this artificial astaxanthin is not permitted for use in organic aquaculture facilities. Furthermore, the nutritional composition of artificial astaxanthin is relatively unfavorable. Furthermore, artificially produced astaxanthin may contain toxic constituents and / or undesirable stereoisomers, which potentially pose a threat to food production.

[0003] An alternative to synthetically produced astaxanthin is natural astaxanthin, which can be extracted industrially, for example, from cultivated microalgae. Krill meal, which is primarily wild-caught, can also be used as an astaxanthin source, although this involves the consumption of limited natural resources. For the economical, large-scale production of astaxanthin from cultivated microalgae, optimized process control is required. The processing of the microalgae up to the extraction or purification of the astaxanthin is associated with high costs.Overall, the production of a feed for animal food production that is enriched with a valuable substance such as astaxanthin is very complex and associated with high costs, especially if the valuable substance is not a synthetic but a naturally produced one.

[0004] The article by Liland et al. (2017) "Modulation of nutrient composition of black soldier fly (Hermetia illucens) larvae by feeding seaweed-enriched media" (PLoS One 12(8): e0183188) focuses on black soldier fly larvae, which represent a promising source of proteins and lipids in animal feed. The authors describe the use of the brown seaweed. Ascophyllum nodosum in the substrate for the larvae.

[0005] The article by Dugas et al. (2013) "Carotenoid Supplementation Enhances Reproductive Success in Captive Strawberry Poison Frogs (Oophaga Pumilio)" (Zoo Biology 32(6): 655-658) discloses a carotenoid-enriched medium for fruit flies. Adult fruit flies are fed to strawberry poison frogs, and the effects on the reproductive success of the strawberry poison frogs are observed.

[0006] The article by Huangfu et al. (2013) "Antiaging Effects of Astaxanthin-Rich Alga Haematococcus pluvialis on Fruit Flies under Oxidative Stress" (J. Agric. Food Chem. 2013, 61: 7800-7804) describes antiaging effects in fruit flies under oxidative stress, where the fruit flies are bred with a substrate containing biomass from the astaxanthin-rich alga Haematococcus pluvialis enriched.

[0007] The invention therefore has the object of providing an improved process for producing a feed enriched with astaxanthin, which avoids the aforementioned disadvantages and allows the economical provision of a feed enriched with astaxanthin.

[0008] This object is achieved by a method for producing an astaxanthin-enriched feed or an astaxanthin-enriched feed component, as is the subject matter of claim 1. Preferred embodiments of this method are set out in the dependent claims. Furthermore, the object is achieved by an astaxanthin-enriched feed or an astaxanthin-enriched feed component, as is the subject matter of the independent claim.

[0009] In the proposed process, microorganisms are first cultivated to provide a microorganism biomass, whereby the microorganisms produce and / or accumulate at least one valuable substance, namely astaxanthin. The core of the process is that this microorganism biomass is fed to insect larvae. The insect larvae are larvae of the genus Hermeticism.The insect larvae, which store the valuable material, are then cultivated. These insect larvae are then processed into animal feed or a feed component. When reference is made to a valuable material in the following, this generally refers to astaxanthin. This process first economically produces the valuable material in the microorganisms. This valuable material is fed to the insect larvae together with the microorganisms. The insect larvae ingest the microorganisms, so that the breakdown of the microorganisms is usually carried out by the insect larvae themselves and does not have to be carried out through complex technical processes. Furthermore, it can be assumed that the valuable material accumulates within the insect larvae, since little or no metabolism of the valuable material is to be expected in the insect larvae.The insect larvae function, in a sense, as a natural bioreactor and simultaneously as a high-quality protein source during their subsequent processing into animal feed. The proposed combination of microorganism biotechnology and insect biotechnology makes it possible to produce a valuable feed that is particularly advantageous for the production of animal-based foods. The proposed process dispenses with synthetic production of the valuable material, thus avoiding the disadvantages of synthetically produced valuable materials described above. The process exploits the natural tendency of many microorganisms to extract valuable substances from food via the digestive system and to accumulate them, for example, as energy stores and / or reserve substances.At the same time, by using a food chain that is, to a certain extent, modeled on nature, it is possible to dispense with technically complex and cost-intensive processes for cell disruption and purification of the valuable material.

[0010] The feed or feed component is preferably a feed or feed component for animals. However, it is also possible, in principle, for the feed or feed component to be intended for human consumption, so that the process can also be used to produce a food or food component enriched with valuable substances for humans.

[0011] In a preferred embodiment of the method, the microorganism biomass is provided for feeding to the insect larvae without cell disruption of the microorganisms, in particular without technical cell disruption of the microorganisms. The cultivated microorganisms can therefore be fed directly to the insect larvae after harvesting. The insect larvae are often able to ingest and digest the microorganisms without prior cell disruption, so the actual cell exclusion occurs within the insect larvae.

[0012] Depending on the application, it may be particularly advantageous to further process the microorganism biomass before feeding it to the insect larvae, as the insect larvae may have limited ability to fully digest the microorganisms' cell walls. Therefore, it is particularly preferred that partial or complete technical cell disruption of the microorganisms be performed to provide the microorganism biomass before feeding the insect larvae. Technical cell disruption refers to process-related steps to effect cell disruption.Such technical cell disruption is possible using simple methods and can, if necessary, promote particularly efficient storage of the valuable material in the insect larvae in the further value chain. In this case, it is particularly preferable to perform only partial cell disruption or to simply weaken the cell wall structures. This is particularly advantageous in terms of energy consumption in terms of process technology. Furthermore, with partial cell disruption or with a merely weakened cell wall structure, the protective function of the cell structure for the valuable material is temporarily retained during the process.

[0013] Suitable technologies for the gentle weakening of cell wall structures or for partial cell disruption are known to those skilled in the art. Power ultrasound, for example, is suitable. Using precisely dosed ultrasound with preferably controlled adjustment of specific power input, amplitude, pulse sequence, residence time, pressure, and / or temperature, cell suspensions can be exposed and the cell wall structures weakened without completely disintegrating the cell. This has been demonstrated, for example, by the fact that cell aggregates (chains) of cyanobacteria of the species Arthrospira platensiswere separated without destroying the individual cells. Electroporation, for example, is also suitable. Cell walls can be reversibly or irreversibly permeabilized by various effects generated in an electric field (e.g., accelerated charge carriers). Furthermore, partial cell disruption or weakening of the cell wall can occur through temperature treatment. Cell walls can be partially damaged, particularly through freeze / thaw steps, as can brief heating. The use of freeze / thaw steps is particularly advantageous when intermediate storage and / or transport is planned for the provision of microorganism biomass for feeding to the insect larvae, whereby the biomass can be frozen for transport.Furthermore, it is possible to damage the cell walls through carefully dosed mechanical disruption or pressure swing processes to aid disruption in the insect larvae. Equipment for this includes ball mills, impact mills, or high-pressure homogenizers. Other options include chemical and biochemical methods, such as the use of enzymes or other, particularly decomposing, microorganisms.

[0014] Depending on the microorganism used for the proposed process and the respective cultivation conditions, it may happen that during the cultivation of the microorganisms, persistent stages, in the case of algae, for example, so-called cysts, form, which, as resistant persistence stages, have a particularly strong cell wall. For example, during the carotenogenesis of Haematococcus pluvialisvarious morphological and physiological changes in the cells, which, in addition to altered metabolic activity, also lead to the formation of a stable cell wall. The cells thus transition from a vegetative-flagellated cell stage to an immobile cyst stage. The cysts represent a resilient survival form, which, primarily through the stability and heterogeneity of the formed cell wall, are intended to optimally protect the organism from various abiotic environmental influences. Since the rigid cell wall structures make cell disruption and utilization of the microorganisms by the insect larvae more difficult, it can be particularly advantageous to convert the dormant stages of the microorganisms into reproducing, i.e., vegetative stages by changing the cultivation conditions.The cultivation conditions that influence this transition from the persistence stages to the reproductive stages include, for example, altered light conditions, altered salinity, and / or altered nutrient situation (see also Sun H. et al., Repeated cultivation: non-cell disruption extraction of astaxanthin for Haematococcus pluvialis. Sci. Rep. 6, 20578; doi: 10.1038 / srep20578 (2016)), which can be used in the proposed process depending on the specific circumstances. By influencing these conditions, the cell wall structure of the microorganisms can be weakened during cultivation, making the microorganism biomass more easily digestible by the insect larvae in the subsequent process step.

[0015] In a particularly preferred embodiment of the method, the microorganisms are conditioned before the microorganism biomass is provided. Through such conditioning, the production and / or enrichment of the valuable substance within the microorganisms can be stimulated or enhanced. For example, the production of certain valuable substances in the microorganisms can be triggered and / or enhanced by certain light conditions. Whether such conditioning is useful for the respective application depends on the respective microorganism and is apparent to the person skilled in the art. Through conditioning, the production of the valuable substance by the microorganisms can be significantly increased. The conditioning can take place within the framework of stress induction, whereby in particular the light conditions and / or the temperature conditions and / or the media composition (e.g. nutrient limitation) and / or salinity (e.g.increased salinity). Conditioning is preferably carried out after the actual cultivation or propagation phase of the microorganisms.

[0016] After culturing and, if necessary, conditioning the microorganisms and transferring them to vegetative propagation stages, the microorganisms are harvested and, if necessary, further processed to partially or completely disrupt the cells or to weaken the cell walls in order to facilitate cell disruption and / or processing within the insect larvae. For feeding the microorganism biomass to the insect larvae, the microorganism biomass can be mixed into a standard feed substrate for the insect larvae. This has the particular advantage that the microbiome typically present in the standard feed substrate can further disrupt the cells of the microorganisms, making the microorganism material even easier to process for the insect larvae.

[0017] The insect larvae are larvae of the genus Hermeticism,especially larvae of Hermetia illucents (Black soldier fly). Larvae of Hermetia illucents are particularly suitable for biotechnological production on a larger scale, as they are robust and relatively undemanding. Larvae of The illuminating Hermetia, which can feed on almost all types of organic substances, are used as animal feed. Larvae of Hermetia illucents have high protein contents and high proportions of essential amino acids. Due to their special lifestyle, they are enormous feed converters. Larvae of Hermetia illucents can be mass-produced in a particularly advantageous manner with relatively little effort and feed. Furthermore, the suitability of larvae of this species for feeding fish has already been demonstrated. For example, studies have already been conducted on turbot ( Psetta maxima ) where at least 33% of the fishmeal used in the fish feed is replaced by protein from Hermetia illucentswere replaced (Kroeckel, S. et al., Aquaculture, Volumes 364-365, pages 345-362, 2012). Experiments by the inventors have shown that larvae of Hermetia illucents absorb cultivated microorganisms and enrich the valuable substances contained therein, so that larvae of Hermetia illucents are particularly suitable for the method according to the invention. Preferably, the insect larvae are cultivated in a conventional manner in an insect bioreactor, whereby it is particularly advantageous if the insect larvae are cultivated under controlled and / or regulated environmental conditions and under controlled and / or regulated feed supply conditions. Preferably, the larvae are used for further processing as feed before they reach the pupal stage.

[0018] According to the invention, the valuable substance is astaxanthin, a carotenoid. The term "astaxanthin" encompasses not only astaxanthin in its pure form, but also astaxanthin derivatives, such as, in particular, the ester forms of astaxanthin (e.g., mono- or diester forms of astaxanthin). In particular, the mono- and diester forms can occur naturally alongside the non-esterified pure form and are therefore also well suited for the purposes of the invention. It is also possible, for example, to enable the microorganisms to produce and / or enrich astaxanthin, e.g., in its non-esterified pure form, and / or astaxanthin derivatives, using recombinant methods, so that the proposed process can be used to produce feed or feed components enriched with the corresponding derivatives.In addition, it is possible that the astaxanthin may be derivatized during the process. In principle, other valuable substances can also be used to produce the feed or feed component in a manner analogous to the proposed process. Examples of other valuable substances are phycoerythrin, phycocyanin, other carotenoids and pigments, e.g. fucoxanthin, vitamins and amino acids, in particular essential or limiting amino acids. Depending on the desired valuable substance, corresponding microorganisms can then be used that naturally produce and / or enrich the respective valuable substance. The following list shows some selected valuable substances and corresponding microorganisms, in particular microalgae and cyanobacteria, which synthesize these valuable substances and which can be used in a manner analogous to the proposed process: . Phycoerythrin: Porphyridium purpureum, Porphyridium cruentum, Planktothrix rubescens Phycocyanin: Arthrospira platensis, Chlorella vulgaris, Aphanizomenon water flower Fucoxanthin: Phaeodactylum tricornutum, Chaetoceros gracilis, Odontella aurita Vitamin E, Vitamin C: Scenedesmus obiquus, Chlamydomonas reinhardtii, Chlorella vulgaris Amino acids MAA ( mycosporine-like amino acids ): Aphanizomenon water flower, Aphanizomenon spiralis, Chlorella sorokiniana

[0019] In other embodiments, genetically modified microorganisms can also be used, which are enabled, for example, by recombinant methods, to produce and / or enrich the valuable substance.

[0020] In a similar way to the proposed process, unsaturated fatty acids are particularly suitable as valuable materials, especially polyunsaturated fatty acids (PUFA - polyunsaturated fatty acids ) .For example, omega-3 fatty acids could be used as a valuable substance. Omega-3 fatty acids play a major role in human nutrition. Omega-3 fatty acids can, for example, be made available for human consumption through appropriately enriched eggs. The proposed process is therefore, in principle, suitable for the production of a feed or feed component for poultry, whereby this feed or component is enriched with omega-3 fatty acids as a valuable substance. The eggs laid by these poultry contain particularly high levels of omega-3 fatty acids and are therefore particularly suitable for human consumption. Microorganisms for this process can, for example, Chlamydomonas variabilis, Chlorella vulgaris or Nannochloropsis salina be used, whereby the provided microorganism biomass is used in the manner described above, for example, Hermetia larvaeThe larvae are then processed into a suitable feed or feed component for poultry.

[0021] The term "microorganisms" refers to microscopically small organisms that are invisible to the naked eye. These are essentially single-celled or few-celled organisms. It is advantageous for the process according to the invention if the microorganisms can be cultivated, for example, in bioreactors. The invention utilizes the property of many microorganisms to produce or accumulate certain substances that can be used by humans as valuable materials. In general, the term "valuable material" refers in particular to substances that play a special role in physiology, particularly in human nutrition and / or in the cultivation of livestock. Valuable materials can be understood, for example, as substances that are not synthesized by the organism itself or are synthesized only to a limited extent and that must therefore be ingested with food, e.g.certain vitamins, antioxidants, or unsaturated fatty acids (essential food components). In addition to their nutritional significance, these valuable substances can also have health or other functional significance for humans and / or the farm animals being cultivated. For example, in salmon farming in aquaculture, the main reason for adding astaxanthin is to achieve the typical salmon color. Salmon meat without this color would be practically unmarketable, even though the actual nutritional value is essentially identical. Furthermore, astaxanthin offers the additional benefit of being a highly effective antioxidant, although this usually plays only a minor role in marketability.

[0022] The microorganisms are preferably algae, fungi, or bacteria, such as cyanobacteria. The microorganisms can be cultivated in a conventional manner, for example, in a photobioreactor. For example, conventional tubular photobioreactors or plate photobioreactors can be used. Overall, it is advantageous if the microorganisms are cultivated under controlled and / or regulated cultivation conditions in specially designed photobioreactors.

[0023] Microalgae are particularly advantageously suited for the proposed method. For example, green algae, in particular green algae of the genus Haematococcus, preferably green algae of the species Haematococcus pluvialis (Blood rain algae) can be used. Haematococcus pluvialisis traditionally one of the most important natural sources for the industrial production of astaxanthin. The astaxanthin is located intracellularly in the cells, with the astaxanthin within the cells in so-called Lipid bodies The algal cells usually have a very stable cell wall. In the industrial extraction of astaxanthin from Haematococcus pluvialisTherefore, cell disruption and the extraction of astaxanthin represent a crucial cost factor. This complex cell disruption and extraction of astaxanthin is largely avoided in the process according to the invention by feeding the cultivated algal cells themselves, possibly with a certain amount of processing, to the insect larvae, so that the algal cells within the insect larvae are broken down by their digestive system. For example, the algae can be fed directly to the insect larvae, i.e. fresh and without further processing and, for example, without freeze-drying, since the uptake and accumulation of astaxanthin in the larvae is generally already good. The uptake and storage of astaxanthin can be further improved if the algae are processed before feeding and, for example, partial cell disruption and / or a weakening of the cell walls is carried out.This can be achieved through simple technical measures, such as ultrasonic treatment or temperature treatment (as described above). The technical effort involved is considerably lower than for the industrial extraction of astaxanthin from algae.

[0024] In other preferred embodiments of the method, yeast fungi can be used as microorganisms, for example yeasts of the genus Phaffia, preferably yeasts of the species Phaffia rhodozyma. Also Phaffia rhodozyma is able to produce or enrich astaxanthin and is similar to Haematococcus pluvialis for producing an astaxanthin-enriched feed according to the process of the invention. Optionally, it may be provided to use genetically modified strains of Phaffia rhodozyma which show higher production rates of astaxanthin than the naturally occurring strains.

[0025] The feed or feed component producible using the process according to the invention can preferably be intended as a feed or feed component for fish and / or poultry. Depending on the intended purpose of the feed, appropriate adaptations to the feed can be made, for example, adjusting it to a specific protein content, adding certain additives, producing a specific dosage form, etc. The feed enriched with astaxanthin is particularly suitable as fish feed, especially for salmon farmed in aquaculture, since the astaxanthin produces the desired reddish color of the fish and also has beneficial nutritional properties for humans who consume the farmed fish.The feed produced according to the invention represents an economically and ecologically very advantageous alternative to astaxanthin-enriched fishmeal, which is currently used in large quantities for aquaculture. Fishmeal, as a conventional main feed ingredient for aquaculture, already threatens to overtax the natural fish resources of the world's oceans. With the process according to the invention, on the one hand, the protein component of the feed can be replaced to a considerable extent by cultivating the insect larvae. On the other hand, by feeding astaxanthin-producing microorganisms to the insect larvae, a suitable feed for aquaculture can be produced in a very efficient and sustainable manner in a resource-saving manner. Overall, natural fish stocks can be protected through the use of the process according to the invention.The process according to the invention eliminates the need for technically complex disruption of microorganisms, such as algae, extraction of the valuable substance, and its technical enrichment. If necessary, simplified cell disruption can be performed, for example, by freezing / thawing or other methods. Thus, technically complex methods for cell disruption and extraction can be dispensed with, since these conventionally technically complex steps are carried out by the insect larvae within a near-natural food chain. Using such a feed for salmon aquaculture can improve the nutritional properties of salmon meat by incorporating naturally produced astaxanthin and its beneficial constituents. This results in a novel and economically advantageous source of astaxanthin, even for organically operated aquaculture.

[0026] Preferably, the larval biomass is first dried during further processing of the insect larvae. The larval biomass can be dried to a specified residual moisture content. For example, a residual moisture content of up to 10% or up to 5% can be achieved. Preferably, the insect larvae are first killed before drying. The drying process can be carried out, for example, by treatment with hot air until the desired residual moisture content is reached. Drying the larval biomass has the advantage that, in principle, solids can be used in subsequent processing (rather than liquids or pastes).

[0027] Depending on the application, it may be advantageous to separate the fat fraction from the protein fraction when processing insect larvae into feed or feed components, or to not separate the fat fraction from the protein fraction at all. Separating the fat fraction is particularly useful if the protein is to be obtained as a dry meal. Separating the fat and protein fractions generally also has the advantage that it allows for particularly precise mixing ratios and compensates for naturally occurring fluctuations in nutrient content. This makes it possible, for example, to produce a standardized and reproducible feed, for example in pelleted form.Depending on the material involved, omitting the fat fraction may have the advantage that the valuable material accumulates partially or essentially in the fat fraction of the insect larvae, and therefore, a certain proportion of the valuable material would be lost if the fat fraction were to be separated. In principle, it is also possible to use only the fat fraction for the feed, provided no protein source is desired in the feed or another protein source is to be added. Omitting the fat fraction may also be advantageous if damage to the valuable material is expected due to the required process-related parameters (e.g., temperature, pressure).

[0028] When processing insect larvae into feed, it is particularly preferred if the insect larvae enriched with the valuable substance represent only one component of the feed. The insect larvae are therefore first processed into a feed component, which is then processed into the final feed together with at least one other ingredient. In this way, the feed can be optimally adapted to various purposes, for example by using additional nutrients and / or additives for the production of the feed. Suitable additional components for a ready-to-use feed include, for example, wheat, wheat gluten, peas, horse beans, soy cake, soy concentrate, rapeseed meal, guar protein meal, sunflower cake, blood meal, brewer's yeast, krill meal, hydrolyzed fish protein, fish oil, rapeseed oil, vitamins and minerals such as monocalcium phosphate.Thus, in addition to the astaxanthin-enriched protein-rich component according to the invention, the ready-to-use feed may also contain, for example, wheat, fish oil, wheat gluten, krill meal, hydrolyzed fish protein, monocalcium phosphate, and brewer's yeast, making this feed particularly suitable for salmon cultures. A salmon trout feed may, for example, contain, in addition to the feed component according to the invention, blood meal, soybean cake, rapeseed meal, sunflower cake, wheat, fish oil, rapeseed oil, guar protein meal, peas, horse beans, soy concentrate, vitamins, and minerals. The nutrient contents of suitable feeds can, for example, be 40-60% total protein and 15-25% fat. In addition, carbohydrates, minerals, and vitamins, as well as indigestible components (burdens), are included in varying weight proportions.The astaxanthin content of the finished feed is preferably 50 - 100 mg / kg feed, preferably 70 - 80 mg / kg, in particular 75 mg / kg.

[0029] Pelleting is particularly preferred for the feed. This form of feed is suitable for a wide range of applications, for example, fish farming or poultry farming. In this form, the feed can be easily packaged, transported, and used for animal feeding. For pelleting, it is advantageous if the raw material produced from the insect larvae is dry and easy to store. It is advantageous to integrate the insect larvae into the feed with as little processing effort as possible to avoid losses of valuable substances that can occur, for example, during drying, defatting, and / or further purification.

[0030] Finally, the invention comprises an astaxanthin-enriched feed or an astaxanthin-enriched feed component that can be produced according to the described method. In preferred embodiments, this feed or feed component is characterized in that the protein content of the feed or the protein content of a component of the feed is provided on the basis of insect larvae, wherein the insect larvae are already enriched with astaxanthin due to their feeding with valuable substance-producing and / or enriching microorganisms according to the inventive concept. The feed component can in particular be an insect meal produced according to the invention. Furthermore, the feed component can be a fat fraction (insect fat) from the processing of the insects enriched with the valuable substance.Through appropriate processing of the insect larvae, the resulting feed (or a feed component) is characterized by being enriched with astaxanthin. Such feeds are particularly suitable as feed in animal breeding or animal husbandry. The astaxanthin content of the enriched feed is preferably approximately 50-100 mg / kg of feed, preferably 70-80 mg / kg, and especially 75 mg / kg.

[0031] In preferred embodiments, the feed can in principle be based on a conventional feed recipe, wherein in particular the protein content and / or the fat content of the recipe are completely or partially replaced by the insect meal and / or the fat fraction (insect fat) producible according to the invention. Depending on the specific valuable substance content of the insect component(s), the amounts of the insect components can be selected accordingly depending on the desired valuable substance content of the finished feed. For example, 5-50%, in particular 10-25%, preferably 19-20% of the dry mass of the feed can be formed by the insect meal. Furthermore, additionally or alternatively, 5-10%, in particular 7%, of the dry mass of the feed can be formed by the insect fat.

[0032] In principle, the feeds produced according to the invention are also suitable as food or as food components for human nutrition.

[0033] Further features and advantages of the invention will become apparent from the following description of exemplary embodiments in conjunction with the drawings. The individual features can be implemented individually or in combination with one another.

[0034] The figure illustrates schematically the method according to the invention. Description of implementation examples

[0035] The process according to the invention, which combines microorganism biotechnology and insect biotechnology and is intended for the production of an astaxanthin-enriched feed or an astaxanthin-enriched feed component, can be divided into three main phases: a) Induction of valuable substances - microorganisms that produce and / or accumulate astaxanthin are cultivated; b) Accumulation of valuable substances - the essentially unprocessed microorganism biomass is fed to insect larvae, with astaxanthin being stored within the insect larvae; c) Transfer of valuable substances - the valuable substance, together with the insect larvae as a protein source, is transferred into a formulation suitable for animal feed with essentially no loss.

[0036] The figureillustrates schematically the method according to the invention. In the first step 10, the microorganisms that produce and / or accumulate astaxanthin are cultivated to provide a microorganism biomass. This step can include conditioning, in which the synthesis or production of the valuable substance is stimulated by setting appropriate conditions. After harvesting and, if necessary, further processing of the microorganisms, in step 20 the microorganism biomass is passed on to the insect larvae, i.e. the larvae of the genus Hermetia, fed, and the insect larvae are cultured. In step 30, the insect larvae are processed into a feed or feed component.

[0037] In a particularly preferred embodiment, microalgae, in particular algae of the species Haematococcus pluvialiscapable of producing astaxanthin. The insect larvae used are the larvae of the species Hermetia illucens The enriched feed produced using this process is particularly suitable as fish feed or as a supplement to fish feed. It is particularly suitable for use in salmon farming in aquaculture.

[0038] Astaxanthin is currently the most important high-value product that can be obtained from microalgae. Nevertheless, the global market share of commercially used, algae-based astaxanthin is less than 1%. The majority of the market is held by synthetic astaxanthin synthesized from petrochemical sources. However, synthetic astaxanthin contains an unnatural ratio of the compound's stereoisomers, which is unfavorable for nutritional purposes and is particularly prohibited in organically certified foods. Despite growing consumer interest in natural food production chains and the disadvantages of synthetic astaxanthin, the current production price of algal astaxanthin is difficult to compete with its synthetic counterpart. In a conventional algae cultivation process for the production of astaxanthin, harvesting, cell disruption, and product extraction represent enormous costs.A crucial cost factor here is the mechanical cell disruption and extraction of astaxanthin, which is usually achieved by supercritical CO2 extraction. Extraction is traditionally carried out on dried biomaterial, so a drying step, such as spray drying, must take place beforehand. These are all very energy-intensive processes. The method according to the invention, in contrast, has the crucial advantage that these energy-intensive process steps can be partially or completely dispensed with. In the method according to the invention, the insect larvae, i.e. the . Hermetia larvae,the astaxanthin-producing microalgae directly. During the digestion of Hermetia larvae, a natural cell disruption and storage of astaxanthin essentially takes place. This natural cell disruption can be supported by suitable processing of the algae. Before the algal biomass is fed to the insect larvae, partial or, if necessary, complete cell disruption or a weakening of the cell wall structures occurs through simple technical measures. The result is a beneficial transfer of valuable substances from microalgae into the feed or feed component. This process can be used to produce a feed or foodstuff enriched with high-quality, naturally produced astaxanthin, and which entails significantly lower production costs than comparable conventional processes. Storage of astaxanthin in Hermetia larvae

[0039] For initial experiments, a commercially available astaxanthin preparation derived from algal biomass with an astaxanthin content of approximately 5% (AstaReal ®< A1010) was used. This material was added to a basic feed for rearing squid at concentrations of 0.5% (Group A) and 0.25% (Group B) Hermetia larvae For this purpose, several test series were conducted, each with 500 animals. Shortly before entering the pupal stage, the animals were washed and killed by deep freezing. Subsequently, an analysis of astaxanthin in the larval tissue was performed.

[0040] For the determination of the astaxanthin concentration in the Hermetia larvaeThese were mechanically crushed in their native state using a mortar and pestle. The astaxanthin was extracted from the material using acetone. Subsequently, a photometric determination and calculation of the astaxanthin concentration was performed using known absorption coefficients. The following table summarizes the analysis results: experimental group Algal biomass in basic feed Astaxanthin in basic feed Astaxanthin in larval material Enrichment factor A 0.56% (5.6 mg / g feed) 0.028% (0.28 mg / g feed) 4.96% (49.6 mg / g larva) 177 B 0.25% (2.8 mg / g feed) 0.014% (0.14 mg / g feed) 2.71% (27.1 mg / g larva) 194

[0041] These results demonstrate a significant accumulation of the valuable substance astaxanthin in the larvae, as well as a correlation with the feeding rate. Microscopic observations of prepared larvae show significant pigment deposition in tissue compartments.

[0042] Overall, these results demonstrate that the valuable substance astaxanthin accumulates or is stored in the larva and that astaxanthin is essentially not metabolized. Dependence of astaxanthin storage on the algae dosage form

[0043] For further experiments, different dosage forms of an algal biomass ( Haematococcus pluvialis ) for feeding Hermetia larvae (fresh and freeze-dried). A total of ~ 56 mg of astaxanthin in the form of fresh or freeze-dried algal biomass (2.8 g biomass dry matter) was mixed with 410 g of larval basic feed and used to feed 230 Hermetia larvaeused. The larvae were cultured on the medium for 12 to 15 days before harvesting. At the time of harvest, some of the larvae had already reached the pupal stage. After harvesting, the larvae and pupae were killed by cold and used for further analysis. The different developmental stages were examined separately for astaxanthin concentration. For this purpose, the larvae and pupae were first freeze-dried for 48 hours and then processed into a homogeneous sample mixture using a ball mill. Defined amounts were weighed from the homogenized samples, and mechanical cell disruption was then carried out. After cell disruption, the astaxanthin was extracted from the samples using acetone. The astaxanthin concentration was determined using a photometric measurement at 420 nm.

[0044] The measurement results showed that the larval stage at harvest has a significant influence on the astaxanthin concentration within the larvae. The analyses showed that the astaxanthin concentration in the pupae was comparatively lower than in the larvae. The concentrations varied between 1 and 200 µg of astaxanthin per gram of dry biomass of the larvae. Furthermore, conclusions could be drawn from the experiments regarding the optimal dosage form of the algal biomass. The test results showed that the astaxanthin concentration in the larvae fed with fresh algal biomass was 2–2.5 times higher than in those fed with freeze-dried algal biomass. Production of an astaxanthin-enriched feed based on Hermetia- Larvae

[0045] The cultivation of green algae Haematococcus pluvialisis carried out in a conventional manner. For example, cultivation can be carried out in a two-stage process. First, the algal biomass is cultivated under optimal growth conditions in a suitable photobioreactor. Light and nutrient conditions correspond to the algae's needs. Cultivation is preferably carried out on a mineral salt medium and nitrate as the N source at a temperature of 20 - 30 °C. Lighting is preferably provided by PAR light (PAR - Photosynthetically Active Radiation) and photon flux densities of 50 - 350 µmol / (m 2< s). Fumigation is carried out with CO 2 - enriched air in the range of 0.1 - 2% CO 2 . During this phase, the algal cells are motile, growing and multiplying by cell division. The cultivation phase generally takes place over a period of 5 - 15 days. Once the maximum biomass concentration has been reached, the algal cells are exposed to limiting and / or inhibiting stress conditions (conditioning). These preferably include light stress, salinity and nutrient limitation. The algae then induce astaxanthin biosynthesis. Astaxanthin formation is preferably triggered by irradiation with strong blue light and photon flux densities of 300 - 1500 µmol / (m 2< s) with the addition of salt and a limitation of the nitrogen source. The resulting carotenogenesis lasts between 2 - 5 days under these conditions.The cells form persistent forms (cysts), which can be easily harvested by separation in a gravitational field (sedimentation, centrifugation). Preferably, the obtained fresh biomass can be used directly for feeding to the . Hermetia larvae used, e.g., as a 1-2% aqueous suspension. Alternatively, the algal biomass can be made storable by freezing, freeze-drying, or drying.

[0046] The cultivation of Hermetia larvae takes place in a standard breeding container, with newly hatched young larvae of Hermetia illucensare fed with a standard larval feed substrate, such as pig fattening feed, supplemented with food sources from suitable biomass, such as industrial waste streams (plant residues, food waste, fermentation residues). For the breeding process, the feed composition is mixed with drinking water to a water content of approximately 70% and transferred to the breeding tank. The newly hatched L1 larvae (newly hatched juvenile larvae) are then introduced into a bioreactor. The larvae are cultivated at a temperature range between 28°C and 35°C with continuous addition and removal of metabolic gases. Depending on the development and growth of the larvae, the algal biomass, which contains the valuable substance astaxanthin, is added after one to seven days. After a further twelve days, the final larval stage is reached.The larvae are separated from the remaining substrate by sieving and wind sifting and then processed further.

[0047] The materials enriched with the valuable Hermetia larvae are converted into a dry, powdered, and storable formulation that can be incorporated into a feed as a feed component. This feed component is characterized, on the one hand, by its high astaxanthin content. On the other hand, the insect larvae provide a high protein content to this feed component. Depending on the application, in addition to this feed component, which is produced according to the process according to the invention, further ingredients can be added to the ready-to-use feed. Their compositions can be adapted to the respective application in a conventional manner, for example, in terms of further value and nutrient content, etc.

[0048] A possible alternative to Haematococcus pluvialisprovides the yeast Phaffia rhodozyma which is also suitable for the production of astaxanthin.

[0049] For further processing of the insect larvae, a preferred embodiment of the process first involves drying the killed insect larvae, particularly with hot air, so that most of the water is removed from the larvae. The dried insect mass can then be further processed in an oil press, where oil is separated under increased pressure and temperature. This produces two main products, each of which can be used entirely as high-quality feed additives. These products are, on the one hand, insect fat and, on the other, a presscake made from the hydrophilic components of the insect biomass. Both products contain astaxanthin, which was transferred to the insects via the algae. Free astaxanthin is extracted with the insect fat. Protein-bound forms are found in the presscake. The presscake consists of approximately 60% crude protein and is about 90% digestible.It is completely dry, which has a beneficial effect on stability, storage capacity, and further processing. To optimize mixing processes, the press cake can be ground into insect meal. Both products can be used as feed components. Depending on requirements, they can be added to the feed being produced in varying weight proportions along with other ingredients. This allows the feed to be produced in a standardized and reproducible manner.

[0050] In detail, for the further processing of the harvested Hermetia larvaeThe larvae are first killed and then dried to a maximum residual moisture content of 5% (24 hours in warm air at 80°C). To separate the fat fraction from the protein fraction, the dried larvae are heated to 60°C. The larvae are pressed in an extruder process, with temperatures between 80°C and 100°C being set in the screw flight of the press. To prepare the protein fraction, the protein-rich press cake is ground into a flour using a hammer mill or impact mill (insect meal). To prepare the fat fraction, the remaining components are separated into liquid and solid components by sieving at room temperature, with optional refining to the desired degree of separation (insect fat). The press cake contains approximately 53% crude protein, approximately 18% carbohydrates, approximately 15.7% crude ash, and approximately 9.7% fat.Although astaxanthin is fat-soluble and therefore present to a significant extent in the fat fraction, a certain proportion is also present in the press cake, since the press cake also contains a certain amount of fat and since astaxanthin can also be bound to proteins or carbohydrates.

[0051] Both the protein meal and the fat fraction can be used for further processing as a feed component. For further processing, this material can be mixed with conventional feed components to achieve the desired concentration of valuable substances and nutrients and then processed into a pelleted feed, for example, by extrusion. Suitable additional components for a ready-to-use feed include wheat, wheat gluten, peas, horse beans, soybean cake, soybean concentrate, rapeseed meal, guar protein meal, sunflower cake, blood meal, brewer's yeast, krill meal, hydrolyzed fish protein, fish oil, rapeseed oil, vitamins, and minerals such as monocalcium phosphate.Such substances for a feed can, for example, be used as a basic recipe, with individual components of this basic recipe being completely or partially replaced by the feed components according to the invention, which are based on the insect larvae enriched with a carotenoid (in particular insect meal and insect fat, see above). In particular, fish meal and fish oil and / or vegetable oil of a conventional basic recipe can be partially or completely replaced by the insect meal according to the invention and / or the fat fraction according to the invention, so that the resulting carotenoid concentration in the finished feed is, for example, 50-100 mg / kg of feed, preferably 70-80 mg / kg. In general, for example, up to about 70% of the protein and fat content in the feed can be replaced by insect protein and insect fat according to the invention.In 1 kg of dry matter of the feed, for example, 5 - 50%, e.g. approximately 20%, of insect meal according to the invention and, for example, 5 - 10%, e.g. approximately 7%, of insect fat according to the invention can be added.

[0052] For 1 kg of feed, for example, a total of 262 g (26.2%) of the protein meal according to the invention (approximately 19% of the dry matter) and the fat fraction according to the invention (approximately 7% of the dry matter), obtained using the method described above, can be used. An exemplary composition of a feed according to the invention suitable as fish feed is listed below: Feed components Feed % dry matter Fish meal (Clupea sp.) 3,92 Carotenoid-enriched insect meal 19,16 Blood meal 5,00 gelatin 5,00 Pea protein isolate 14,00 Wheat gluten 10,00 Biolysin 0,21 Wheat starch 13,49 cornstarch 9,50 Rapeseed oil 1,30 Fish oil 3,00 Carotenoid-enriched insect fat 7,00 Vitamin mix 0,50 Mineral mix 0,40 Monocalcium phosphate 0,50 Fibers (bentonite / cellulose / diamol) 7,02 sum 100,00

[0053] The astaxanthin-enriched feed can be fed particularly to farmed fish, such as salmon. Similarly, it is possible to produce a feed enriched with omega-3 fatty acids, which is particularly suitable for feeding poultry, especially chickens and turkeys.

[0054] Alternatively, the method according to the invention can be used to produce a near-natural feed, wherein the larvae and / or prepupae are fed directly. For this purpose, the harvested larvae and / or prepupae can be killed, for example, by freezing at -18°C. Alternatively, killing by oxygen deprivation in a nitrogen or carbon dioxide atmosphere, for example, is possible. The essentially unprocessed insect larvae are suitable as fresh feed, for example, for the cultivation of farmed fish and, correspondingly, also for poultry or other farm animals.

Claims

1. A method for preparing an astaxanthin-enriched feed or an astaxanthin-enriched feed component, comprising the following method steps: - Cultivation of microorganisms that produce and / or accumulate astaxanthin to provide a microorganism biomass, - feeding the microorganism biomass to insect larvae of the genus Hermetia and cultivating the insect larvae, and - processing the insect larvae into a feed or a feed component.

2. The method according to claim 1, characterized in that the provision of the microorganism biomass for the feeding to the insect larvae is carried out without a technical cell disruption of the microorganisms.

3. The method according to claim 1, characterized in that the provision of the microorganism biomass for the feeding to the insect larvae is carried out with a partial or complete technical cell disruption of the microorganisms.

4. The method according to any one of the preceding claims, characterized in that during the cultivation of the microorganisms, permanent stages of the microorganisms are converted into vegetative stages by changing the cultivation conditions.

5. The method according to any one of the preceding claims, characterized in that prior to providing the microorganism biomass, the microorganisms are conditioned by a stress induction to stimulate or enhance the production and / or accumulation of astaxanthin.

6. The method according to any one of the preceding claims, characterized in that the insect larvae are larvae of Hermetia illucens.

7. The method according to any one of the preceding claims, characterized in that the microorganisms are algae or fungi.

8. The method according to claim 7, characterized in that the algae are green algae, in particular green algae of the genus Haematococcus, preferably green algae of the species Haematococcus pluvialis.

9. The method according to claim 7, characterized in that the fungi are yeasts of the genus Phaffia, preferably yeasts of the species Phaffia rhodozyma.

10. The method according to any one of the preceding claims, characterized in that during processing of the insect larvae a drying ot the insect larvae is carried out.

11. The method according to any one of the preceding claims, characterized in that the fat fraction is not separated from the protein fraction during processing of the insect larvae.

12. The method according to any one of claims 1 to 10, characterized in that the fat fraction is separated from the protein fraction during processing of the insect larvae.

13. The method according to any one of the preceding claims, characterized in that the insect larvae are processed into the feed component, which is processed together with at least one further ingredient into a feed.

14. An astaxanthin-enriched feed or an astaxanthin-enriched feed component obtainable by a method according to any one of claims 1 to 13.