Marine invertebrate feed, methods for manufacturing marine invertebrate feed, methods for raising marine invertebrates, methods for promoting shell length and methods for increasing fatty acid content.

Abstract

To provide a feed for marine invertebrates such as abalones and urchins, to provide a method for producing the feed for marine invertebrates, to provide a method for farming marine invertebrates, to provide a method for accelerating the length of shells, and to provide a method for increasing the content of fatty acids.SOLUTION: Provided are: a feed for marine invertebrates, characterized by containing microalgae belonging to the genus Euglena; a method for producing the feed for marine invertebrates, characterized by performing a solidification step of solidifying microalgae belonging to the genus Euglena; a method for farming marine invertebrates and a method for accelerating the length of shells, which comprise feeding microalgae belonging to the genus Euglena to marine invertebrates; and a method for increasing the content of fatty acids, which comprises feeding microalgae belonging to the genus Euglena to increase the content of fatty acids contained in edible parts of marine invertebrates.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a feed for marine invertebrates such as abalone and sea urchins, a method for producing a feed for marine invertebrates, a method for cultivating marine invertebrates, a method for promoting shell length, and a method for increasing fatty acid content. [Background technology]

[0002] When raising mammals, birds, fish, and other living creatures, especially those that are commercially valuable for food or ornamental purposes, it is extremely important to manage their health and physical condition. Among marine animals that are used for food, marine invertebrates such as abalone and sea urchins are extremely valuable aquatic resources.

[0003] Abalone habitats are shrinking due to a decrease in seaweed caused by rising seawater temperatures due to global warming. The limiting water temperature for adult Hokkaido abalone and black abalone during the high water temperature period (August) is 28°C, while for juveniles it is 25°C or 24°C, making them vulnerable to rising seawater temperatures. Furthermore, the seaweed necessary for abalone growth also has an optimum temperature range in the same water, so there are concerns that the habitat itself may disappear. Furthermore, it has been suggested that ocean acidification may make it more difficult for larvae to form shells.

[0004] Patent Document 1 describes a technique for feeding gel-form feed containing salmon growth hormone and alginic acid to juvenile shellfish such as abalone, with the aim of simply and efficiently promoting the growth of marine invertebrates, particularly shellfish such as abalone, and improving their productivity.

[0005] Sea urchins are declining in number due to a decrease in food and difficulty in forming shells. The limit temperature for the northern purple sea urchin during its high water temperature period (August) is 25°C, making it vulnerable to rising sea temperatures. Furthermore, the seaweed necessary for its growth also has an optimum temperature range in the same range, so there are concerns that the habitat itself may disappear. Furthermore, it has been suggested that ocean acidification may make it more difficult for larvae to form shells.

[0006] Patent Document 2 describes a technique for feeding sea urchins legumes in order to adjust the content of fatty acids, namely oleic acid, α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), contained in the edible parts of the sea urchin.

[0007] Meanwhile, Euglena (genus name: Euglena, Japanese name: Midorimushi) has been attracting attention as a promising biological resource for use as food, feed, fuel, etc. Patent Document 3 describes a feed additive for living organisms that contains a component derived from Euglena and is administered to mammals, birds, and fish. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-095104 [Patent Document 2] Japanese Patent Application Publication No. 2020-156418 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-027929 Summary of the Invention [Problem to be solved by the invention]

[0009] Furthermore, in recent years, a phenomenon known as "isoyake" (rocky coastal bleaching) has become more prevalent in coastal reef areas, where kelp, wakame seaweed, and other seaweed species have significantly decreased and stopped growing. This not only reduces the yield of economically valuable marine resources such as abalone and sea urchins, but also leads to a decline in the quality of the fish, including their meat content and flavor.

[0010] Therefore, there is a need for an effective feed for marine invertebrates, such as abalone and sea urchins, which are valuable marine resources. In the technology of Patent Document 3, a Euglena-derived component is administered to fish, but no study has been conducted on marine invertebrates.

[0011] The present invention has been made in view of the above problems, and an object of the present invention is to provide a feed for marine invertebrates such as abalone and sea urchins, a method for producing a feed for marine invertebrates, a method for cultivating marine invertebrates, a method for promoting shell length, and a method for increasing fatty acid content. [Means for solving the problem]

[0012] As a result of extensive research, the present inventors have found that Euglena serves as a good food source for marine invertebrates such as abalone and sea urchins, and that Euglena has the effect of promoting shell length and increasing fatty acid content in marine invertebrates.

[0013] Therefore, the above-mentioned problems are solved by a feed for marine invertebrates, which contains microalgae belonging to the genus Euglena. In this case, the marine invertebrate is preferably an abalone or a sea urchin. In this case, the microalgae are preferably defatted Euglena algae. In this case, the marine invertebrate feed is preferably used to promote shell growth of the marine invertebrates. In this case, the feed for marine invertebrates is preferably used to increase the content of fatty acids contained in the edible parts of the marine invertebrates. In this case, the fatty acid is preferably oleic acid or linoleic acid.

[0014] Furthermore, the above-mentioned problems can be solved by the method for producing feed for marine invertebrates of the present invention by carrying out a solidification step of solidifying microalgae belonging to the genus Euglena.

[0015] Furthermore, the above-mentioned problems are solved by a method for cultivating marine invertebrates according to the present invention, which is characterized in that microalgae belonging to the genus Euglena are fed to marine invertebrates.

[0016] Furthermore, according to the method for promoting shell length of the present invention, the above-mentioned problems can be solved by feeding marine invertebrates microalgae belonging to the genus Euglena.

[0017] Furthermore, according to the method for increasing fatty acid content of the present invention, the above-mentioned problems can be solved by feeding microalgae belonging to the genus Euglena to marine invertebrates, thereby increasing the fatty acid content in the edible parts of the marine invertebrates. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide a feed for marine invertebrates such as abalone and sea urchins, a method for producing a feed for marine invertebrates, a method for cultivating marine invertebrates, a method for promoting shell length, and a method for increasing fatty acid content. [Brief explanation of the drawings]

[0019] [Figure 1] (Left) Black abalone feeding on Euglena compound feed (solidified feed), (Right) Solidified feed being fed by black abalone. [Figure 2] 1 is a table showing the composition of Euglena formulated feed. [Figure 3] This is the survival curve for 6 months of rearing. [Figure 4] Conditions of the breeding water over the six months of breeding: (top left) water temperature, (top right) pH, (bottom left) dissolved oxygen concentration, (bottom right) ammonia nitrogen concentration. [Figure 5] Changes in shell length and individual weight over 6 months of rearing: (left) shell length, (right) individual weight. [Figure 6] 1 is a table showing the nutritional composition of each feed. [Figure 7] 1 is a table showing the free amino acid composition of each feed. [Figure 8] 1 is a table showing the fatty acid composition of each feed. [Figure 9] 1 is a table showing the nutritional composition of individual abalone fed each feed over a 6-month period. [Figure 10] 1 is a table showing the free amino acid composition of individual abalone fed each feed over a 6-month period. [Figure 11] 1 is a table showing the fatty acid composition of individual abalone fed each feed over a 6-month period. [Figure 12A]FIG. 1 shows the start of abalone feeding-attractant microalgae screening. [Figure 12B] FIG. 1 shows the results of screening for abalone feeding-attracting microalgae. [Figure 13A] FIG. 1 shows the start of abalone feeding-attractant microalgae screening. [Figure 13B] FIG. 1 shows the results of screening for abalone feeding-attracting microalgae. [Figure 14] FIG. 1 shows the results of an abalone feeding attraction test for Euglena. [Figure 15] FIG. 1 is a diagram showing an outline of a screening test for sea urchin feeding-attracting microalgae. [Figure 16] FIG. 1 shows the results of screening for sea urchin feeding-attracting microalgae. DETAILED DESCRIPTION OF THE INVENTION

[0020] Hereinafter, embodiments of the present invention will be described with reference to Figures 1 to 16. The embodiments relate to a feed for marine invertebrates such as abalone and sea urchins, a method for producing a feed for marine invertebrates, a method for cultivating marine invertebrates, a method for promoting shell length, and a method for increasing fatty acid content.

[0021] <Marine invertebrates> In this embodiment, the term "marine invertebrates" refers to animals that live in the ocean, other than animals with vertebrates such as jawless worms, fish, amphibians, reptiles, birds, and mammals. Examples of marine invertebrates include marine invertebrates belonging to the phylum Mollusca, Echinodermata, Arthropoda, Cnidaria, and Annelida. Among marine invertebrates, marine invertebrates that can ingest microalgae belonging to the genus Euglena, particularly marine invertebrates that can ingest solid food, are preferred.

[0022] Examples of marine invertebrate species include abalone, turban shells, turban shells, barnacles, oysters, scallops, and other crustaceans such as shrimp and crabs, and sea urchins, with abalone being particularly preferred. However, the species is not limited to these, and other invertebrates that live in freshwater, such as pond snails and Japanese river snails, can also be included.

[0023] (Abalone) "Abalone" is a general term for gastropods belonging to the class Gastropoda of the phylum Mollusca, and includes, for example, gastropods of the genus Abalone, such as the black abalone, the mega-i abalone, the Madaka abalone, and the Ezo abalone, and gastropods of the genus Abalone, such as the tokobushi abalone.

[0024] (Sea urchins) "Sea urchins" is a general term for echinoderms belonging to the class Urchinida in the phylum Echinodermata, including, for example, the purple sea urchin, the northern purple sea urchin, the green sea urchin, the northern sea urchin, the red sea urchin, etc. The edible parts of sea urchins are the gonads consisting of the ovaries or testes.

[0025] <Euglena> In this embodiment, "Euglena" includes microorganisms taxonomically classified in the genus Euglena, their varieties, mutants, and closely related species in the family Euglenaceae. Here, the genus Euglena refers to a group of organisms belonging to the Excavata, phylum Euglenozoa, class Euglenophyceae, order Euglenales, and family Euglenaceae among eukaryotes.

[0026] Specific examples of species belonging to the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis, etc. As Euglena, Euglena gracilis, particularly Euglena gracilis Z strain, can be used. However, other Euglena species such as Euglena gracilis Z strain mutant SM-ZK strain (chloroplast-deficient strain) and E. gracilis var. bacillaris, genetic mutants such as chloroplast mutants of these species, and Astasia longa can also be used.

[0027] The genus Euglena is widely distributed in freshwater such as ponds and marshes, and may be isolated from these and used. Alternatively, any Euglena species that has already been isolated may be used. The genus Euglena encompasses all mutant strains thereof. These mutant strains also include those obtained by genetic methods such as recombination, transduction, and transformation.

[0028] (Euglena algae) In this embodiment, Euglena algae bodies can be used as Euglena. Euglena live cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are. Euglena live cells can be used as they are after harvesting from the culture tank, but it is preferable to wash them with water or physiological saline. Euglena algae bodies may also be used in the form of a dispersion in which they are dispersed in a liquid such as water. In this embodiment, dried Euglena algae bodies (Euglena powder) obtained by freeze-drying or spray-drying live Euglena cells are preferably used as Euglena algae.

[0029] Furthermore, mechanically treated Euglena algae cells obtained by subjecting living Euglena cells to mechanical treatment such as ultrasonic irradiation or homogenization may be used as the Euglena algae. Furthermore, the mechanically treated Euglena algae may be dried and used as a dried product. Furthermore, defatted Euglena powder may be obtained by degreasing Euglena algae using, for example, a nonpolar solvent.

[0030] <Food for marine invertebrates> The feed for marine invertebrates according to this embodiment contains microalgae belonging to the genus Euglena. The feed for marine invertebrates may contain alginic acid powder for solidification and additives commonly used as feed components, such as antibiotics, vitamins, and minerals.

[0031] The marine invertebrate feed according to this embodiment can be used to promote shell length in marine invertebrates. Here, shell length refers to the length from the apex of the shell, which is the starting point for shell growth at the top of a snail shell, to the tip of the water canal at the bottom of the shell, and is the length of the longest part of the shell of an abalone, and is synonymous with shell height.

[0032] The marine invertebrate feed according to this embodiment can be used to increase the fatty acid content in the edible parts of marine invertebrates. The fatty acids contained in the edible parts of marine invertebrates are not particularly limited, but examples thereof include n-3 unsaturated fatty acids such as oleic acid, linoleic acid, linolenic acid, EPA, and DHA.

[0033] <Method for producing feed for marine invertebrates> The method for producing food for marine invertebrates according to this embodiment is characterized by a solidification step of solidifying microalgae belonging to the genus Euglena. The solidification step is carried out by solidifying the microalgae belonging to the genus Euglena using alginic acid powder or the like.

[0034] <Marine invertebrate cultivation method, shell length promotion method, fatty acid content improvement method> The method for cultivating marine invertebrates according to this embodiment is characterized in that it comprises feeding microalgae belonging to the genus Euglena to marine invertebrates. Conventional methods can be employed for cultivating marine invertebrates, except for feeding microalgae belonging to the genus Euglena. Feeding marine invertebrates with microalgae belonging to the genus Euglena can improve the survival rate of the marine invertebrates, promote the growth of the shell length, which is the size of individual marine invertebrates, and increase the fatty acid content in the edible parts of the marine invertebrates.

[0035] That is, the method for cultivating marine invertebrates according to this embodiment is characterized by feeding marine invertebrates microalgae belonging to the genus Euglena. Furthermore, the method for increasing fatty acid content according to this embodiment is characterized by increasing the fatty acid content in the edible portions of marine invertebrates by feeding them microalgae belonging to the genus Euglena. The fatty acids whose content is to be increased are not particularly limited, but examples include oleic acid and linoleic acid. To achieve this goal, it is sufficient to feed microalgae belonging to the genus Euglena until the content of oleic acid or linoleic acid in the edible portions of marine invertebrates exceeds the content of n-3 unsaturated fatty acids such as oleic acid, linoleic acid, linolenic acid, EPA, and DHA typically contained in the edible portions of marine invertebrates. [Example]

[0036] The present invention will be described in detail below based on specific examples, but the present invention is not limited to these.

[0037] (Euglena powder) Euglena gracilis powder (Euglena algae, manufactured by Euglena Co., Ltd.) is dead Euglena algae (dead Euglena cells) that have been dried and mechanically treated to remove live Euglena cells.

[0038] (Defatted Euglena powder) The defatted Euglena powder was prepared by culturing Euglena algae through anaerobic fermentation to increase the wax ester content, and then extracting the wax ester from the Euglena powder using an organic solvent such as hexane.

[0039] <Test 1: Abalone farming test using Euglena powder-containing feed> In Experiment 1, we developed alginate-solidified feed for black abalone farming, containing Euglena powder or defatted Euglena powder (Figure 1). A six-month feeding test using this solidified feed was conducted in a closed circulating tank.

[0040] (Test feed administration experiment in a closed circulation breeding tank) i) Test feed composition for abalone farming In a feeding attraction test using abalone juveniles, we found that they ingested Euglena significantly (with activity equivalent to that of Chlorella), so we added 0.5% (w / w) of the defatted and untreated Euglena powder shown in Figure 2 to Macfield's abalone feed for a breeding test. The feed was solidified using alginate powder from Ina Food Industry Co., Ltd.

[0041] ii) Rearing conditions ii)-1: Survival rate At the end of the six-month feeding experiment, the mortality rates were as follows: solid formula feed (MF) group: 38 / 100 (62%); Euglena (EU) group: 18 / 100 (82%); and defatted Euglena (DE) group: 54 / 100 (46%) (Figure 3). These results suggest that feeding Euglena (EU) diet increases survival rate, but that this effect is drastically reduced when the diet is degreased. In other words, it is inferred that the lipid components contribute to the improvement of survival rate.

[0042] ii)-2: Rearing water parameters: Rearing water temperature, pH, dissolved oxygen, ammonia nitrogen The rearing water temperature was 17.4°C, pH 8.2-8.3, dissolved oxygen 7.1-7.8 mg / L, and ammonia nitrogen 0.05 mg / L or less, providing a stable environment throughout the feeding test (Figure 4). The measurement methods for each water quality item are described below.

[0043] Water temperature: Digital water thermometer (Tetra) pH: Portable water quality meter pH (DKK Toa Corporation DM-32p) Dissolved oxygen: Portable water quality meter pH (DKK Toa Corporation DM-32p) Ammonia nitrogen: Portable medium-concentration ammonia nitrogen meter (Hanna Instruments Japan Co., Ltd. HI96715) Total hardness, calcium, nitrate and nitrite concentrations: Tetra Test 5-in-1 (water quality test paper)

[0044] ii)-3: Growth changes of individuals in each group Figure 5 shows the changes in shell length and weight over a six-month period. No significant differences were confirmed between the three groups for either shell length or weight. There was a tendency for the shell length of the defatted individuals to gradually increase, but this result was probably due to an increase in individual deaths, which resulted in a lower individual density in the rearing space compared to the other two groups.

[0045] (Nutrient composition of the feed used in the aquaculture test and differences in the nutritional composition of individuals reared for 6 months) Test feed analysis The nutritional composition of the feed used in the aquaculture experiment is shown in Figure 6, the free amino acid composition in Figure 7, and the fatty acid composition in Figure 8. The slightly higher calorie count in the Euglena (EU) and defatted Euglena (DE) diet groups is presumably due to the addition of 2% to the solid formulated feed (MF). The protein composition was slightly higher in the Euglena (EU) and defatted Euglena (DE) diets, and the Euglena (EU) diet contained twice the amount of lipids (Figure 6). The defatted Euglena (DE) diet contained 1.1 to 1.2 times the carbohydrates of the solid formulated feed (MF) and Euglena (EU) diets (Figure 6). Almost no free amino acids were detected in the feed (Figure 7). Regarding free fatty acids, high levels of C14:0 myristic acid were detected in the Euglena (EU) diet (Figure 8).

[0046] (Analysis of individuals reared for 6 months) Nutritional analysis of individuals raised on the three types of diets showed that in the equal energy feeding test, the protein content was slightly higher in the defatted Euglena (DE) diet group (Figure 9). Conversely, the carbohydrate content was half that of the solid formula feed (MF) and Euglena (EU) diet groups (Figure 9). Although abalone contains a high amount of arginine, Konosu et al. reported that arginine does not contribute significantly to the umami flavor of abalone (Food Industry Journal 20, 432-439 (1973)). Furthermore, there were no differences in the amounts of glycine, alanine, and glutamic acid, which contribute to the sweetness of abalone, between the diets (Figure 10). Regarding the fatty acid composition (Figure 11), since the fat content was very low, it is unlikely that this is a factor that causes a significant difference in taste. However, the high levels of oleic acid and linoleic acid in the defatted Euglena (DE) diet group may contribute, albeit slightly, to the stronger perception of umami via the oil receptors (CD36, GPR120) present on the human tongue.

[0047] (Summary of Exam 1) It was suggested that Euglena food contributes to improving the survival rate of abalone. In addition, defatted Euglena was shown to promote shell length, making it a good feed material when used in combination with Euglena.

[0048] <Test 2: Changes in the intestinal flora after feeding alginate-solidified chlorella and euglena diets> In Experiment 2, the changes in the intestinal flora of black abalone and purple sea urchin individuals due to Euglena feeding were examined using next-generation sequencers. Changes in the intestinal flora due to Euglena feeding were examined using black abalone (from Owase, Mie Prefecture) and northern purple sea urchin (from Hirono Town, Iwate Prefecture).

[0049] To allow the animals to acclimate to their environment, the black abalone were fed a commercial solid feed (Macfield) and the northern sea urchins were fed seaweed (Eisenia bicolor). After two weeks of acclimation, the animals were switched to solid feed containing Euglena powder or Chlorella powder solidified with alginic acid to a concentration of 5% (w / v). Since the influence of alginic acid was also suspected, a group fed an alginic acid diet was also set up, and changes in the intestinal bacterial flora were examined in four samples of each species, including the group fed before the test. The environmental bacterial flora was also measured at the start and end of each rearing test period.

[0050] Next-generation sequence analysis was outsourced to Seibu Giken Co., Ltd. using MiSeq under 2 x 300 bp conditions. To identify the bacteria, specific primers were designed for the V3-V4 region of nine V regions (V1 to V9) in the 16S rRNA, which has a gene length of approximately 1.5 kbp and whose sequence differs depending on the type of bacteria, and PCR was performed, followed by rapid and large-scale DNA sequence reading using a next-generation sequencer.

[0051] As a result, in both the black abalone and the sea urchin, Euglenozoa and Chlorophyta were dominant species when fed Euglena or Chlorella. Although it is not clear whether the growth of each species was stimulated by feeding Euglena powder, the profile of the intestinal microbiota changed.

[0052] <Test 3: Screening of feeding-attractant microalgae> In Test 3, various microalgae were examined for their ability to attract black abalone and sea urchins. Five grams of each microalgae powder was collected in a 50 mL centrifuge tube, and 40 mL (8 volumes) of each was subjected to the Bligh & Dyer method to extract the lipid-soluble components. The extracted lipid-soluble components were concentrated under reduced pressure using a rotary evaporator (water bath 40°C). Then, 100 μL of the concentrate was applied to an Avicel plate (TLC cellulose Merrk-Millipore) (36 mm diameter circle) to a concentration of 100 mg / mL. After application, the Avicel plate was scanned.

[0053] Black abalone specimens with shell lengths of 22-26 mm and sea urchin specimens with diameters of 30-40 mm were selected and fasted for 24 hours the day before the feeding attraction test. Avicel plates with a 30 mm diameter circle of sample applied were submerged in the rearing tanks, and 10 black abalone specimens and 8 sea urchin specimens were released into each tank and allowed to feed for 24 hours. After 24 hours, the Avicel plates were collected, dried, and photographed using a scanner. The cellulose surface scraped off by the sea urchins on the Avicel plates was digitized using Image J software. The ratio of the scraped area to the applied area before contact was calculated using the following formula to rank the feeding attraction of the microalgae.

[0054] Feeding attraction activity (%) = ((scraped area) / (application area before contact)) x 100 The ranking is -: feeding attractant activity 0-5%, +: feeding attractant activity 5-20%, ++: feeding attractant activity 30-40%, +++: feeding attractant activity 50-70%, ++++: feeding attractant activity 80-100%.

[0055] The results are shown in Figures 12A to 16. It was found that Euglena effectively attracted both black abalone and sea urchin.

Claims

1. A feed containing microalgae belonging to the genus Euglena, which is used to increase the content of oleic acid or linoleic acid in the edible parts of marine invertebrates.

2. 2. The feed for marine invertebrates according to claim 1, wherein the marine invertebrates are abalones or sea urchins.

3. 3. The feed for marine invertebrates according to claim 2, wherein the microalgae are defatted Euglena algae.

4. 4. The marine invertebrate feed according to claim 3, which is used to promote shell growth of the marine invertebrates.

5. The feed described in claim 1, wherein the marine invertebrate is a sea urchin.

6. A method for producing the feed according to claim 1 or 2, comprising: The above method, which includes a solidification step of solidifying microalgae belonging to the genus Euglena.

7. A method for cultivating marine invertebrates, comprising feeding the marine invertebrates microalgae belonging to the genus Euglena, thereby increasing the content of oleic acid or linoleic acid in the edible parts of the marine invertebrates.

8. A method for promoting shell length, comprising feeding a marine invertebrate microalgae belonging to the genus Euglena to the marine invertebrate, thereby increasing the fatty acid content in the edible portion of the marine invertebrate.

9. A method for increasing the content of oleic acid or linoleic acid in the edible parts of marine invertebrates by feeding them microalgae belonging to the genus Euglena.

Images