Microorganisms that produce eicosapentaenoic acid

Abstract

The present invention provides: a Shewanella sp. GI35 strain (National Institute of Technology and Evaluation, Patent Microorganisms Depositary, deposit number NITE BP-03244) or a mutant strain thereof; a feed containing said strain; a method for producing fish that produces eicosapentaenoic acid (EPA) in the body, the method being characterized by feeding fish with said feed; and the like.

Description

[[Technical Field]]

[0001] The present invention relates to a newly isolated strain GI35 of *Shewanella* sp., a bacterium that produces eicosapentaenoic acid (EPA) in significant quantities, or a mutant strain thereof, a feed comprising the same, and a method for producing fish that produce EPA in vivo, characterized by administering the feed, and the like. [[Background Art]]

[0002] EPA belongs to omega-3 polyunsaturated fatty acids (ω3-PUFA), and is an essential fatty acid for the developmental differentiation and growth of animals. It is a functional lipid that exhibits inhibitory effects on the onset of cardiovascular diseases such as cerebrovascular disorders and myocardial infarction by suppressing inflammatory responses and regulating immune functions (Non-Patent Documents 1 and 2). On the other hand, many vertebrates including humans do not have the group of fatty acid desaturases involved in the biosynthesis of ω3-PUFA, and cannot produce EPA by themselves (Non-Patent Document 3). For this reason, EPA must be ingested from the external environment through food or the like. Active intake of sardines, mackerel and other fish containing PUFA is recommended, and furthermore, many supplements and foods containing EPA are commercially available.

[0003] Regarding methods for preparing EPA, attempts have been made to develop methods involving purification from fish oil, and methods using labyrinthulids, which are marine microalgae, or high-EPA-producing bacteria isolated from low-temperature environments such as polar regions and the deep sea (Patent Document 1, Non-Patent Documents 4 and 5). On the other hand, EPA is a highly unsaturated fatty acid with 20 carbon atoms having five cis double bonds, and has the property of being easily oxidized by oxygen, light, temperature and the like. Since lipid peroxides generated by the decomposition of EPA exert harmful effects on living organisms, the separation, purification and mass production of EPA require enormous cost and time, leaving problems for their use as health foods and pharmaceuticals (Non-Patent Document 6).

[0004] Marine fish and diaphragmatic fish need to obtain EPA from external sources. External intake of EPA is also beneficial for freshwater fish. Therefore, in aquaculture, fishmeal is typically added to the feed of salmon, trout, and yellowtail at a rate of 40-50%. Fishmeal contains EPA and DHA. While the main ingredient of fishmeal is sardines, their catch has decreased significantly, leading to the development of feeds primarily made from soybeans and corn. However, plant-based feeds do not contain essential fatty acids such as EPA and DHA, leaving the securing of sufficient quantities of EPA and DHA needed for feed addition and the resolution of rising prices as urgent issues. Fishmeal is also added to the feed of juvenile fish to maintain healthy seedlings. When salmonid fry raised on EPA and DHA-fortified diets are released into rivers, it has been pointed out that the lack of sources of these highly unsaturated fatty acids in rivers can lead to increased mortality among juvenile fish due to EPA deficiency, resulting in a decrease in fish catches. Therefore, there is a need for new technologies to sustainably supply EPA, improve the health of juvenile fish, and establish stable mass production technologies (Non-Patent Document 7).

[0005] Fish feed containing EPA has also been studied. It has been reported that EPA can be enhanced by incorporating EPA-producing bacteria into rotifers and using the feed for juvenile fish (Patent Document 2). However, because this process involves incorporating EPA-producing bacteria into rotifers, the preparation of the feed is cumbersome. Moreover, there is no description or suggestion that EPA is continuously and stably produced in the bodies of juvenile fish given this feed. Furthermore, research on probiotics has shown that it is almost impossible for ingested live bacteria to survive in the intestinal tract and continuously exist there (Non-Patent Document 8). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Publication of a Patent Application of the People's Republic of China CN106434416A [Patent Document 2] Japanese Patent Application Publication H03-228652 [Non-patent literature]

[0007] [Non-Patent Document 1] Lancet 1, 1143-1145, 1971; J. Cardiol., 67, 22-27 2016. [Non-Patent Document 2] Biochem. Societ. Transac. 45, 1105-1115, 2017. [Non-Patent Document 3] Oikos, 125, 749-760, 2016. [Non-Patent Document 4] Appl. Environ. Microbiol. 82, 218-231, 2016. [Non-Patent Document 5] Methods Enzymol. 605, 3-32, 2018. [Non-Patent Document 6] Biotech. Advances 20, 491-515, 2003. [Non-Patent Document 7] Tomoharu Watanabe, "Enhancing the swimming ability of salmon and trout with feed supplemented with DHA-rich fish oil," Japanese, Hokkaido Research Organization, Research and Development Today, No. 877, 2019. [Non-Patent Document 8] Tomotari Mitsuoka, "The History and Evolution of Probiotics," Japanese Journal of Lactic Acid Bacteria 22, 26-37, 2011. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In light of the above circumstances, it is necessary to provide feed that enables sustained and stable EPA production within the animal body, as well as animals such as fish that sustainably and stably produce EPA within their bodies. [Means for solving the problem]

[0009] The inventors diligently conducted research to solve the above problems and succeeded in isolating EPA-producing bacteria of the genus Shewanella from the intestinal tract of Gymnogobius isaza, a goby fish species endemic to Lake Biwa, thus completing the present invention.

[0010] In other words, the present invention provides the following: (1) Shewanella sp. GI35 strain (NITE BP-03244, Patent Microorganism Depositary Center, National Institute of Technology and Evaluation) or a mutant strain thereof. (2) Feed containing Shewanella sp. GI35 strain or a variant thereof. (3) A method for producing fish in which Shewanella sp. GI35 strain or a mutant strain is present in the intestinal tract, characterized by administering Shewanella sp. GI35 strain or a mutant strain to fish. (4) A method for producing fish that produce eicosapentaenoic acid (EPA) in their bodies, characterized by administering Shewanella sp. GI35 strain or a mutant strain to fish. (5) Fish in which the Shewanella sp. GI35 strain or its variants are present in the intestinal tract (excluding the Isaza fish in which the GShewanella sp. GI35 strain is present in the intestinal tract). (6) Fish that have Shewanella sp. GI35 strain or its variant in their intestinal tract and produce EPA in their bodies (excluding Isaza fish that have Shewanella sp. GI35 strain in their intestinal tract). (7) A method for producing fish with accelerated growth, characterized by administering Shewanella sp. GI35 strain or a mutant strain thereof to fish. (8) A method for producing fish with an altered intestinal flora, characterized by administering Shewanella sp. GI35 strain or a mutant strain to fish. (9) A method for producing EPA, characterized by culturing Shewanella sp. GI35 strain or a mutant strain thereof. (10) A method for producing EPA, which comprises culturing a host cell into which a gene group involved in EPA production in Shewanella sp. strain GI35 or a variant of said gene group has been introduced. (11) A cell into which a gene group involved in EPA production in Shewanella sp. strain GI35 or a variant of said gene group has been introduced. (12) A food or drink comprising Shewanella sp. strain GI35 or a mutant strain thereof. (13) A food or drink obtained by processing a fish according to any one of (5) to (8). Advantageous Effects of the Invention

[0011] The Shewanella GI35 strain of the present invention produces a large amount of EPA and survives well in the intestinal tract. When a feed containing the present bacterium is administered to an animal, EPA is continuously and stably supplied to the host from the strain that survives in the intestinal tract and becomes persistently present therein. That is, the host becomes capable of producing EPA in the body. Thus, an animal rich in EPA is provided. By eating such an animal, a person can ingest EPA, which is an omega-3 polyunsaturated fatty acid, which maintains and improves health, and is expected to prevent cardiovascular diseases, lifestyle-related diseases, and the like. In addition, administration of a feed containing the present bacterium can promote growth of animals and / or modify the intestinal flora. Brief Description of the Drawings

[0012] [Figure 1] Figure 1 shows the results of examining the effect of temperature on the growth of Shewanella GI35 strain isolated from the intestinal tract of salamander trout. [Figure 2] Figure 2 shows the results of gas chromatography examination of EPA production by the Shewanella GI35 strain at low temperature (4°C) and high temperature (18°C) (upper chart and lower chart, respectively). [Figure 3] Figure 3 shows a scheme of an expression vector into which the pfa operon of Shewanella GI35 strain has been introduced. [Figure 4]The lower chart in Figure 4 shows the results of gas chromatography analysis of EPA production in E. coli transformed with an expression vector containing the pfa operon of Shewanella strain GI35 (lower chart). The upper chart in Figure 4 shows the results of gas chromatography analysis of EPA production in E. coli introduced with a pBlueScript II KS(+) plasmid without the incorporation of foreign genes, used as a control. [Figure 5] Figure 5 shows the results of examining the polyunsaturated fatty acid content in the lipids of rainbow trout fed a fish feed containing the Shewanella GI35 strain. PC stands for phosphatidylcholine. EPA written as a superscript to the right of the molecular species name means that the molecular species contains EPA. DHA written as a superscript to the right of the molecular species name means that the molecular species contains DHA. Fold-change indicates how many times more GI35-fed is compared to the control. PUFA-containing PC (≧n) is the percentage of PC molecules with n or more double bonds in the fatty acid chain to the total PC molecules. The p-value is the value obtained from the t-test. [Figure 6] Figure 6 is a graph showing the effect of feed containing Shewanella GI35 strain on promoting the growth of rainbow trout fry (after 3 months of rearing). [Figure 7] Figure 7 is a graph showing the effect of feed containing Shewanella GI35 strain on promoting the growth of rainbow trout fry (after 6 months of rearing). [Figure 8] Figure 8 is a chart illustrating the procedure for metagenomic analysis. [Figure 9] Figure 9 is a graph showing the results of analyzing the diversity of the microbial community using principal coordinate analysis (results of β diversity analysis: Weighted UniFracs distance). [Figure 10] Figure 10 is a graph comparing the functional profiles of the gut microbiota using predictive metagenomic analysis by PICRUSt. The three bars in each group indicate the abundance of bacteria possessing amylase (left panel) or nitrite reductase (right panel) in the gut microbiota of three individuals randomly selected from each group. [Modes for carrying out the invention]

[0013] As described above, the inventors have successfully isolated a new species of Shewanella bacterium that produces a significant amount of EPA from the intestinal tract of the goby fish *Isaza*, a species endemic to Lake Biwa. The inventors named this bacterium Shewanella sp. GI35 strain. This strain was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan, in accordance with the Budapest Convention on the International Recognition of the Deposit of Microorganisms for Patent Procedure. It was assigned receipt number NITE ABP-03244 on July 8, 2020, and accession number NITE BP-03244 on August 25, 2020. In this specification, Shewanella sp. GI35 strain may be referred to as "GI35 strain".

[0014] The GI35 strain produces a remarkable amount of EPA (several times more than the EPA-highly producing Shewanella livingstonesis Ac10 strain – compared to literature values), survives well in the intestinal tract, and becomes continuously present. Moreover, the GI35 strain grows well and produces a large amount of EPA even at relatively high temperatures (room temperature, e.g., about 18°C) compared to other Shewanella species. The GI35 strain is a novel bacterial strain characterized by these special properties.

[0015] Accordingly, in one embodiment, the present invention provides the GI35 strain or a mutant strain thereof. The mutant strain of the GI35 strain is a mutant strain derived from the GI35 strain. The mutant strain of the GI35 strain may be a naturally occurring mutant or an artificial mutant. Methods for producing artificial mutant strains are known and include, but are not limited to, genetic recombination, genome editing, treatment with drugs such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonic acid (EMS), and ultraviolet irradiation. Examples of mutant strains of the GI35 strain include, but are not limited to, strains with higher EPA production capacity than the GI35 strain, strains that proliferate well at higher temperatures, and strains with excellent colonization ability in the intestinal tract. The mutant strain of the GI35 strain may have a whole genome sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more, to the whole genome sequence of the GI35 strain. Sequence homology between genomes can be examined using known programs such as FASTA and BLAST. However, the mutant of the GI35 strain has EPA production capacity equivalent to that of the GI35 strain. Here, equivalent EPA production capacity means 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 100% or more, and most preferably 120% or more.

[0016] In a further embodiment, the present invention provides a feed containing the GI35 strain or a mutant strain thereof. The animals to which the feed of the present invention is administered may be of any kind and are not particularly limited. Examples of animals to which the feed of the present invention is administered may include fish, poultry such as chickens, quail, turkeys, and ducks, livestock such as cattle, pigs, goats, sheep, horses, and donkeys, and pets such as dogs, cats, rabbits, and hamsters. By administering the feed of the present invention to animals, the GI35 strain or a mutant strain thereof survives and continuously exists in the intestinal tract of the animals, and EPA is continuously produced in the animal's body, which is thought to be beneficial for promoting the animal's health. Preferably, the feed of the present invention is administered to fish. Fish is a collective term for the classes Haliotis, Cephalaspida, Chondrichthyes, and Osteichthyes. In this specification, fish and fish are synonymous. The feed of the present invention can be administered to any kind of fish. The feed of the present invention can be administered to freshwater fish, saltwater fish, and anaphylactic fish. Marine fish and anadromous fish (such as salmon and trout) either lack one of the enzymes necessary for EPA biosynthesis or have weak activity of those enzymes and are unable to produce EPA themselves. Therefore, administering the feed of the present invention to marine fish and anadromous fish is effective. The feed of the present invention can be administered to juvenile, immature, and adult fish. Preferably, the feed of the present invention is administered to juvenile and immature fish. Typically, the feed of the present invention is administered to farmed fish. Examples of farmed fish include, but are not limited to, salmon, trout, yellowtail, sea bream, amberjack, bluefin tuna, pufferfish, flounder, striped jack, horse mackerel, kingfish, striped beakfish, filefish, sea bass, black rockfish, carp, rainbow trout, cherry salmon, eel, and sweetfish.

[0017] The form of the feed containing the GI35 strain or its mutant is not particularly limited, but may be similar to that of known animal feeds. Examples of the form of the feed of the present invention include, but are not limited to, moist pellets, dry pellets, powder, crumble, and paste feed. The feed of the present invention can be produced by adding, mixing, etc., the GI35 strain or its mutant to the raw materials of animal feed, during the manufacturing process of animal feed, or to animal feed products. Methods for adding, mixing, etc., the GI35 strain or its mutant are well known. The required amount of bacterial cells can be obtained by culturing the GI35 strain or its mutant. The cultivation of the GI35 strain or its mutant will be described later. The GI35 strain or its mutant obtained by culturing can be separated from the culture medium by methods such as centrifugation. The obtained GI35 strain or its mutant can also be dried by methods such as freeze-drying. Freeze-dried products of the GI35 strain or its mutant or culture solutions of the GI35 strain or its mutant may be mixed in the manufacturing process of animal feed. Alternatively, the prepared animal feed may be impregnated with a culture solution of the GI35 strain or its variants, or sprinkled with freeze-dried products of the GI35 strain or its variants. The feed of the present invention is manufactured so that all or part of the GI35 strain or its variants in the feed can reach the animal's intestines as live bacteria.

[0018] The amount of GI35 strain or its variants in the feed can be appropriately adjusted depending on the type and size of the animal, the components of the feed, etc. The dosage of the feed containing GI35 strain or its variants can also be appropriately adjusted depending on the type and size of the animal. For example, the dosage of the feed containing GI35 strain or its variants may be the same as that of regular feed.

[0019] The feed of the present invention may be used in combination with other feeds.

[0020] Fishmeal contains EPA and DHA, and is incorporated into aquaculture feed. However, due to a significant decline in the catch of sardines, the raw material for fishmeal, it has become difficult to incorporate fishmeal into aquaculture feed, forcing a reduction in its amount. As a result, the development of fishmeal substitute feeds using soybeans and corn as the main ingredients is underway. However, plant-based ingredients do not contain essential fatty acids such as EPA and DHA. Therefore, by manufacturing and using the feed of the present invention by mixing plant-based ingredients with the GI35 strain or its mutant strain, the above problems can be solved. Furthermore, when salmonid fish fry raised on EPA / DHA-fortified diets are released into rivers, the deficiency of EPA leads to an increase in the mortality rate of juvenile fish, resulting in a decrease in catch. Under these circumstances, if the feed of the present invention is administered to salmonid fish fry, EPA can be continuously and stably produced within the fry even when released into rivers, reducing the mortality rate and preventing a decline in catch.

[0021] As shown in the examples, the GI35 strain survives well in the intestinal tract of fish and becomes continuously present there. Therefore, by administering the GI35 strain or its mutant strain to fish, it is possible to obtain fish in which the GI35 strain or its mutant strain exists in the intestinal tract. The method of administering the GI35 strain or its mutant strain to fish can be any method and is not particularly limited, but generally, the GI35 strain or its mutant strain is administered by mixing it with feed. Fish in which the GI35 strain or its mutant strain exists in the intestinal tract can continuously and stably produce EPA in their bodies.

[0022] Therefore, in a further embodiment, the present invention provides a method for producing fish in which the GI35 strain or its mutant strain is present in the intestinal tract, characterized by administering the GI35 strain or its mutant strain to fish.

[0023] In a further embodiment, the present invention provides a method for producing fish that produce EPA in their bodies, characterized by administering the GI35 strain or a mutant strain to the fish.

[0024] Administration in these embodiments of the invention may be carried out by administering the above-mentioned feed.

[0025] In a further embodiment, the present invention provides fish (excluding Isaza fish) in which the GI35 strain or its mutant strain is present in the intestinal tract, and fish (excluding Isaza fish) in which the GI35 strain or its mutant strain is present in the intestinal tract and which produce EPA in their bodies. These fish can continuously and stably produce EPA in their bodies, and their meat also has a high EPA content.

[0026] Marine fish and anadromous fish cannot produce EPA on their own. Freshwater fish can only produce small amounts of EPA on their own. In contrast, fish fed the feed of the present invention, whether marine, anadromous, or freshwater, will be able to continuously and stably produce EPA in their bodies. In other words, by feeding the feed of the present invention, it is possible to continuously and stably obtain EPA-rich fish. It is expected that consuming EPA-rich fish will maintain and improve health and contribute to the prevention of cardiovascular diseases and lifestyle-related diseases.

[0027] In a further embodiment, the present invention provides a method for producing fish with accelerated growth, characterized by administering the GI35 strain or its mutant strain to fish. By administering the GI35 strain or its mutant strain to fish, the growth of the fish can be accelerated. For example, the GI35 strain or its mutant strain may be administered throughout the juvenile stage, or it may be administered transiently during the juvenile stage. Fish obtained by this method may be rich in EPA.

[0028] In a further embodiment, the present invention provides a method for producing fish with a modified gut microbiota, characterized by administering the GI35 strain or a mutant strain to fish. By administering the GI35 strain or a mutant strain to fish, the gut microbiota after growth can be modified. The administration of the GI35 strain or a mutant strain is as described above. By modifying the gut microbiota using this method, the activity of various enzymes present in the gut microbiota can be enhanced or suppressed. For example, the activity of enzymes related to promoting the digestion and absorption of food may be enhanced, or the activity of enzymes that contribute to improving meat quality may be enhanced. Fish obtained by this method may be rich in EPA.

[0029] In a further embodiment, the present invention provides a method for producing EPA, characterized by culturing the GI35 strain or a mutant strain thereof.

[0030] Any culture method is acceptable for culturing strain GI35 or its mutants, as long as it allows the strain to grow and produce EPA. Strain GI35 or its mutants may be cultured in a manner similar to that used for known Shewanella bacteria. For example, strain GI35 or its mutants may be cultured in a medium containing glucose, peptone, yeast extract, sodium chloride, and other inorganic salts. The medium may be either a liquid or solid medium. In the case of liquid culture, methods such as shaking culture, stirring culture, or static culture may be used. Flasks, jars, tanks, etc., may be used as culture vessels. Strain GI35 can grow at approximately 4°C to 37°C, with a suitable growth temperature of approximately 18°C ​​to 30°C. On the other hand, a preferred culture temperature that is compatible with sufficiently high EPA production is approximately 4°C to 20°C. Those skilled in the art can select and determine suitable culture conditions for strain GI35 or its mutants. The amount of EPA in the medium can be measured, for example, using gas chromatography. The produced EPA can be recovered from the culture medium or bacterial cells by known methods.

[0031] The preservation method for strain GI35 or its variants may be the same as that for known Shewanella bacteria. Examples of preservation methods include, but are not limited to, slant storage and freeze-drying.

[0032] In yet another aspect, the present invention provides a method for producing EPA, characterized by culturing host cells into which a group of genes involved in EPA production of the GI35 strain or a mutant of said gene group has been introduced.

[0033] The inventors have successfully cloned the entire genome of the pfa operon, a group of genes involved in EPA production in the GI35 strain. By incorporating this operon into an expression vector, introducing the vector into a host cell (e.g., E. coli), and culturing the host cell, EPA can be produced. Each component of the pfa operon may also be incorporated into separate expression vectors. Various expression vectors and host cells that can be used in this method are known and can be selected as appropriate.

[0034] An example of the gene group involved in EPA production in the GI35 strain that can be used in the EPA production method of the present invention is the one having the nucleotide sequence shown in SEQ ID NO: 1 (pfa operon). The gene group involved in EPA production in the GI35 strain includes five genes: pfaA, pfaB, pfaC, pfaD, and pfaE. The nucleotide sequence of pfaA is shown as nucleotides 2413 to 10503 in SEQ ID NO: 1. The nucleotide sequence of pfaB is shown as nucleotides 10500 to 12794 in SEQ ID NO: 1. The nucleotide sequence of pfaC is shown as nucleotides 12791 to 18724 in SEQ ID NO: 1. The nucleotide sequence of pfaD is shown as nucleotides 18835 to 20481 in SEQ ID NO: 1. The complementary strand sequence of the nucleotide sequence of pfaE is shown as nucleotides 30 to 899 in SEQ ID NO: 1. A variant of the GI35 strain gene group involved in EPA production may include genes corresponding to pfaA, pfaB, pfaC, pfaD, and pfaE of the GI35 strain. The nucleotide sequences of the genes corresponding to pfaA, pfaB, pfaC, pfaD, and pfaE may have 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more homology to the nucleotide sequences of pfaA, pfaB, pfaC, pfaD, and pfaE of the PI35 strain, respectively (except in cases where all five genes have 100% homology). Furthermore, a variant of the GI35 strain gene group involved in EPA production may have 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more homology to the nucleotide sequence shown by Sequence ID: 1. Sequence homology between genes can be examined using known programs such as FASTA or BLAST. Furthermore, mutants of the gene group involved in EPA production in the GI35 strain may have a nucleotide sequence corresponding to the nucleotide sequence shown in Sequence ID No. 1 of the mutant strain of the GI35 strain.However, mutants of the GI35 strain's EPA-producing genes produce 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 100% or more, and most preferably 120% or more of EPA compared to using the GI35 strain's EPA-producing genes. Mutants of the GI35 strain's EPA-producing genes can be produced by known methods such as site-directed mutagenesis, genome editing, and chemical methods.

[0035] The introduction of genes involved in EPA production into host cells is usually carried out by introducing an expression vector containing these genes into the cells. The types of expression vectors, methods for incorporating the gene groups into the expression vectors, and methods for introduction are well known and can be appropriately selected depending on the type of host cell and the size and base sequence of the introduced genes. pfaA, pfaB, pfaC, pfaD, and pfaE may all be incorporated into a single expression vector and introduced into host cells, or they may be incorporated into multiple expression vectors and these vectors introduced into cells.

[0036] In yet another aspect, the present invention provides cells into which a group of genes involved in EPA production of the GI35 strain or a mutant of said gene group has been introduced. Such cells can be cultured to produce EPA. The cells may be microbial cells, animal cells, or plant cells, and are not particularly limited, but typical examples include bacterial cells such as Escherichia coli cells and Bacillus subtilis cells.

[0037] In yet another aspect, the present invention provides food and beverages containing the GI35 strain or its variants. Food and beverages include foods, beverages, and health foods such as supplements and so-called Foods for Specified Health Uses (FOSHU). By ingesting the food and beverages of the present invention, the GI35 strain or its variants survive in the intestinal tract and continue to be present in the intestinal tract, leading to the continuous production of EPA in the body. This is expected to maintain and improve health and contribute to the prevention of cardiovascular diseases and lifestyle-related diseases. Specifically, effects such as a reduction in triglycerides and inhibition of platelet aggregation can be expected. Since the GI35 strain is a bacterium that inhabits the intestinal tract of the edible fish Isaza, feed and food and beverages containing it are highly safe.

[0038] The food and beverages of the present invention may be produced by adding or mixing the GI35 strain or its variants to the raw materials of the food and beverages, during the manufacturing process of the food and beverages, or in the food and beverage products. A freeze-dried product of the GI35 strain or its variants or a culture solution of the GI35 strain or its variants may be mixed during the manufacturing process of the food and beverages. Alternatively, the culture solution of the GI35 strain or its variants may be impregnated into the finished food and beverages, or the freeze-dried product of the GI35 strain or its variants may be sprinkled on them. The food and beverages of the present invention are produced in such a way that all or part of the GI35 strain or its variants in the food and beverages can reach the intestines of animals as live bacteria. Supplements and health foods may be produced in the same manner as or similar to the production of known pharmaceuticals.

[0039] The food or beverage of the present invention may take any shape; for example, it may take the same shape as existing food or beverages, or it may take the form of a drink, paste, cream, tablet, powder, granules, capsule, etc. Furthermore, the food or beverage of the present invention may be used as a food additive.

[0040] As described above, the food and beverages of the present invention are highly safe, so there are no particular restrictions on the amount of food and beverages consumed.

[0041] In yet another aspect, the present invention provides processed food and beverages made from fish (excluding Isaza fish) in which the GI35 strain or its variant is present in the intestinal tract, or from fish (excluding Isaza fish) in which the GI35 strain or its variant is present in the intestinal tract and which produce EPA in their bodies. These fish are rich in EPA. Therefore, processed food and beverages made from these fish are also rich in EPA, and it is expected that consuming them will increase EPA intake, maintain and improve health, and lead to the prevention of cardiovascular diseases and lifestyle-related diseases. Specifically, effects such as a reduction in triglycerides and inhibition of platelet aggregation can be expected.

[0042] The processed foods and beverages made from the above-mentioned fish also include foods, beverages, and health foods such as supplements and so-called Foods for Specified Health Uses (FOSHU). The processed foods and beverages made from the above-mentioned fish may take any form. The processed foods and beverages may be made by cooking all or part of the fish according to normal cooking methods (e.g., boiling, grilling, steaming, making sashimi, etc.), or by mixing all or part of the fish with other ingredients. Alternatively, the processed foods and beverages may be extracts of all or part of the fish (e.g., in the form of capsules containing the extract), or by drying all or part of the fish and making it into powder, granules, tablets, flakes, etc. There are no particular restrictions on the amount of processed foods and beverages made from the above-mentioned fish that can be consumed.

[0043] Unless otherwise specified, terms used herein shall be understood in the sense commonly understood in the fields of biology, microbiology, biochemistry, fisheries science, etc.

[0044] The present invention will be described in more detail and specifically below with reference to examples, but these examples are not intended to limit the scope of the present invention. [Examples]

[0045] (1) Isolation and identification of GI35, a strain that produces a large amount of EPA, from the intestinal tract of the Japanese sand eel. The inventors discovered that the Isaza fish, a goby species endemic to Lake Biwa, exceptionally accumulates large amounts of EPA among freshwater fish. On the other hand, the inventors revealed that the metabolic activity of EPA-producing enzymes in the Isaza fish is low. Therefore, as part of the EPA intake pathway of the Isaza fish, the inventors analyzed the intestinal microbiota and found that marine bacteria of the genus Shewanella, which produce high levels of EPA, are present in the intestinal tract of the Isaza fish. On the other hand, since these bacteria were not detected in individuals of the Honmoroko (Gnathopogon caerulescens), a species endemic to Lake Biwa that migrates in the mid-water column, it was considered that these bacteria are intestinal bacteria unique to the Isaza fish.

[0046] Furthermore, we attempted to identify EPA-producing bacteria from the intestinal tract of the Isaza fish and successfully isolated and cultured bacteria of the genus Shewanella. Based on the EPA-producing ability of the isolated strains, we identified useful strains and conducted a detailed analysis of strain GI35, which is a highly EPA-producing bacterium.

[0047] First, the whole genome sequence of strain GI35 was determined to identify the species. PCR was performed using prokaryotic rDNA universal primers (8EF and 1492R), and approximately 1.5 kb, almost the entire length of the 16S rDNA, was obtained. Molecular phylogenetic analysis of this sequence showed 91.1% homology with the sequence of Shewanella putrefaciens. This result indicated that strain GI35 is a closely related species to Shewanella putrefaciens. Next, the whole genome sequence of strain GI35, approximately 5.56 Mb, was determined using whole-genome shotgun sequencing with the NextSeq system (Illumina). Species identification using the ANI method (Average Nucleotide Identity) showed an ANI value of 86% (less than 95%), indicating that it is a different species, and it was revealed that strain GI35 of the genus Shewanella is a new species. As explained above, the GI35 strain was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, and was assigned receipt number NITE ABP-03244 on July 8, 2020, and accession number NITE BP-03244 on August 25, 2020.

[0048] (2) Temperature-dependent growth and EPA production mode of the GI35 strain The temperature-dependent growth of the GI35 strain was investigated. The growth of the GI35 strain in liquid culture at different temperatures was tracked by the increment in turbidity. The results are shown in Figure 1. It was found that the GI35 strain can grow at temperatures from 4°C to 37°C, with 18°C ​​to 30°C being the optimal temperature for growth.

[0049] The EPA production mechanism of the GI35 strain was investigated. The bacteria were cultured in LB medium (10g tryptone / 5g yeast extract / 10g NaCl / 1L) at 4°C for approximately 24 hours and at 18°C ​​for approximately 12 hours. The results showed that the GI35 strain produced large amounts of EPA under both low-temperature (4°C) and high-temperature (18°C) culture conditions (Figure 2). The EPA content in the phospholipid constituent fatty acids produced was as follows. For comparison, literature values ​​for Shewanella livingstonesis Ac10 strain, a highly EPA-producing bacterium isolated from Antarctic seawater (Kawamoto et al. (2009) Journal of Bacteriology 191, 632-640) are also shown. GI35 strain: 4.2% (18℃); 12.3% (4℃) Ac10 strain: 0.7% (18℃); 5.1% (4℃) These results demonstrate that the GI35 strain possesses extremely high EPA production capacity. Furthermore, it was concluded that the optimal culture temperature for achieving sufficiently high EPA production is approximately 4°C to 20°C.

[0050] (3) Whole genome cloning of the Pfa operon, a group of genes involved in EPA production from the GI35 strain, and EPA production in E. coli into which the Pfa operon has been introduced. We successfully cloned the entire genome of the Pfa operon, a group of genes involved in EPA production, from the GI35 strain. The region containing the full-length Pfa operon (SEQ ID NO: 1), predicted from the whole genome sequence of the GI35 strain, was amplified by PCR and incorporated into pBlueScript II KS+ at the Not I and Kpn I sites to obtain an expression vector (Figure 3). When transformants were created by introducing this expression vector into E. coli (E. coli) that lacked EPA-producing ability, these E. coli cells were found to produce EPA (Figure 4, bottom). As a control, E. coli cells introduced into a pBlueScript II KS(+) plasmid without the foreign gene did not produce EPA (Figure 4, top).

[0051] (4) EPA production in rainbow trout juveniles administered with GI35 strain We investigated whether sustained EPA supply from within the body is possible by feeding fish with the GI35 strain, allowing it to survive and multiply in the intestinal tract, and maintaining its continuous presence there. Specifically, we divided rainbow trout fry into two groups: one group that ingested the GI35 strain along with a standard feed containing fish meal (Nisshin Marubeni Trout Feed Super A) for one week (GI35-fed), and a control group that did not ingest the strain. After administration of the GI35 strain, the fish were fed a standard feed without the bacteria for two weeks. Furthermore, the feed was switched to a defatted feed from which lipid components such as highly unsaturated fatty acids were removed, and after two weeks of rearing, the highly unsaturated fatty acid content in the lipids (phosphatidylcholine) of the individual fish was compared between the GI35 strain-ing group and the control group. As a result, a significant increase in EPA and DHA content was observed in the GI35 strain-ing group (Figure 5). Rainbow trout do not possess the Δ5 fatty acid desaturase enzyme that biosynthesizes EPA, but they do possess the Δ6 fatty acid desaturase enzyme that biosynthesizes DHA from EPA. Therefore, it was considered that the EPA supplied by the GI35 strain was converted to DHA (see Aquaculture 315, 131-143, 2011; Scientific Reports 7, 3889, 2017 for information on the conversion of EPA to DHA). On the other hand, in individuals fed a normal diet for two weeks after ingesting the GI35 strain, no significant difference in polyunsaturated fatty acid content was observed between the GI35-fed group and the control group (data not shown). This result is thought to be because the normal diet contains a large amount of polyunsaturated fatty acids, so the supply of EPA from the GI35 strain did not produce a significant difference. These findings reveal that the GI35 strain, ingested for one week, survives in the intestinal tract, continues to exist, and sustainably supplies EPA within the individual even after four weeks. [Examples]

[0052] (1) Promoting the growth of rainbow trout fry using feed containing GI35 strain (3 months rearing). (i) Experimental method (a) Feeding conditions Standard feed group: The trout were raised for 3 months on rainbow trout feed (Nisshin Marubeni Trout Feed Super A) (standard feed: contains approximately 40% fish meal). Regular feed → GI35 group: Raise on regular feed for one month, then add GI35 strain (~3x10 9 The animals were raised for two months on a standard diet (GI35 supplemented diet) containing cells / g. GI35 → Standard Feed Group: The fish were fed a GI35 supplemented diet for one month, and then fed a standard diet for two months. (b) Weight measurement We grouped five individuals together and measured the weight of all individuals in the tank. The vertical axis in Figure 6 represents the average weight per group. The number of measurements for each group is: Standard feeding groups: 20 groups Regular feed → GI35 group: 11 groups GI35 → Normal feed group: 15 groups That was the case.

[0053] (ii) Results The results are shown in Figure 6. Both the normal feed → GI35 group and the GI35 → normal feed group showed significantly greater growth (weight gain) compared to the normal feed group (p<0.01). No significant difference was observed in weight gain between the normal feed → GI35 group and the GI35 → normal feed group, indicating that early administration of GI35 significantly promotes growth. No juvenile fish mortality was observed in either the normal feed → GI35 group or the GI35 → normal feed group, indicating no toxicity from long-term intake of the GI35 strain. From these results, we can conclude the following: No long-term toxicity has been observed from ingesting the GI35 strain. • Ingestion of the GI35 strain significantly promotes growth. • It was revealed that transient intake of the GI35 strain maintained growth promotion even two months later.

[0054] (2) Promoting the growth of rainbow trout fry using feed containing GI35 strain (6 months rearing) (i) Experimental method (a) Feeding conditions Standard feed group: Raised for 6 months on rainbow trout feed (Nisshin Marubeni Trout Feed Super A and Trout Fry Super) (Standard feed: contains approximately 40% fish meal). Regular feed → GI35 group: Raise on regular feed for one month, then add GI35 strain (~3x10 9 They were raised for 5 months on a regular diet (GI35 supplemented diet) containing cells / g. GI35 → Regular Feed Group: Raised on GI35 supplemented feed for 1 month, then on regular feed for 5 months. (b) Weight measurement Twenty individuals were randomly selected from each tank and their weight was measured. The vertical axis in Figure 7 shows the average weight per animal. The number of measurements for each group is: Regular feeding group: 20 fish Regular feed → GI35 group: 20 fish GI35 → Regular feed group: 20 fish That was the case.

[0055] (ii) Results The results are shown in Figure 7. Significant growth promotion (p<0.01) was observed in the GI35 → normal diet group (initial intake group). While significant growth promotion (p<0.05) was observed in the group that received regular feed followed by the GI35 group (long-term intake group), the effect was less pronounced compared to the initial intake group. No juvenile fish mortality was observed in either the normal feed → GI35 group or the GI35 → normal feed group, and no toxicity was observed from further long-term intake of the GI35 strain. From these results, we can conclude the following: No toxicity was observed even after long-term administration of the GI35 strain over a period of five months. In fact, administration of the GI35 strain was continued for another six months, and no deaths were observed until the end of the rearing period. 2) The growth-promoting effect of ingesting the GI35 strain is remarkable. 3) The growth-promoting effect of the GI35 strain does not require continuous intake; the effect is sustained by a single intake. [Examples]

[0056] Metagenomic analysis of the gut microbiota of rainbow trout juveniles administered with GI35 strain. Metagenomic analysis of the gut microbiota was performed on juvenile fish from the normal feed group and juvenile fish from the GI35 → normal feed group, obtained by the method shown in (2) of Example 2, after 6 months of rearing, to identify changes in the gut microbiota due to GI35 intake and the resulting changes in metabolic activity that were predicted to occur.

[0057] (1) Experimental method Library preparation and sequencing were performed according to the following steps 1-6. 1. Three individuals were randomly selected from each group, and DNA was extracted and purified from their intestinal contents using the QIAamp DNA Microbiome Kit. 2. Quantitative measurement of DNA solution: The concentration of the DNA solution was measured using Synergy LX (BioTek) and Quanti Fluor dsDNA System (Promega). 3. Library preparation: A 16s rDNA library was prepared using the 2-steptailed PCR method. 4. Library quantification: The concentrations of the prepared libraries were measured using Synergy H1 (BioTek) and the QuantiFluor dsDNA System. 5. Library Quality Verification: The quality of the prepared libraries was verified using the Fragment Analyzer dsDNA 915 Reagent Kit (Advanced Analytical Technologies). 6. Sequencing Analysis: Sequencing was performed using the MiSeq system and MiSeq Reagent Kit v3 (Illumina) under conditions of 2x300bp.

[0058] The metagenomic analysis was performed using the procedure shown in Figure 8.

[0059] (2) Experimental results (i) Analysis of microbiota diversity using principal coordinate analysis: β diversity analysis As shown in Figure 9, the bacterial flora differed significantly between the normal feed group and the GI35 → normal feed group. This result indicates that administering the GI35 strain to juvenile fish can alter the gut microbiota after growth. Furthermore, this effect was found to be achievable with transient administration of the GI35 strain.

[0060] (ii) Comparison of bacterial flora functional profiles using predictive metagenomic analysis with PICRUSt The results are shown in Figure 10. Compared to the standard feed group, an increase in bacterial species exhibiting alpha-amylase activity and nitrite reductase activity was observed in the GI35 → standard feed group. Increased alpha-amylase activity in the intestinal tract is presumed to greatly contribute to individual growth by aiding in the digestion and absorption of starch contained in feed. Increased nitrite reductase activity in the intestinal tract promotes the removal of nitrite and is considered important for maintaining homeostasis in individuals. Furthermore, nitric oxide (NO), produced by the action of nitrite reductase, is a strong signaling molecule that has the function of activating various physiological functions, including thickening of the gastrointestinal mucosa and promotion of blood circulation. Therefore, it is assumed that increased nitrite reductase activity in the intestinal tract promotes the digestion and absorption of food. In summary, it was found that administering the GI35 strain to fish can increase the gut microbiota that promotes the digestion and absorption of feed. Furthermore, this effect was found to be achieved through transient administration of the GI35 strain. [Industrial applicability]

[0061] By administering the GI35 strain or its mutants, or a feed containing the same, to animals, animals rich in EPA can be sustainably and stably supplied. Furthermore, by administering the GI35 strain or its mutants, or a feed containing the same, the growth of animals can be promoted and / or the gut microbiota can be modified. Therefore, the present invention is extremely useful in livestock farming, fisheries, particularly aquaculture, and the food industry. [Accession Number]

[0062] The GI35 strain was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture. It was assigned receipt number NITE ABP-03244 on July 8, 2020, and accession number NITE BP-03244 on August 25, 2020. [Sequence Listing Free Text]

[0063] Sequence ID 1 shows the full-length nucleotide sequence of the Pfa operon predicted from the whole genome sequence of the GI35 strain.

[0064] This application is a priority claim application based on Japanese Patent Application No. 2020-123809 filed on July 20, 2020, and incorporates the entire contents of said Japanese Patent Application by reference.

Claims

1. Shewanella genus strain GI35 (Shewanella sp. GI35 strain) (Accession number NITE BP-03244, Patent Microorganism Depositary Center, National Institute of Technology and Evaluation) or a mutant strain of Shewanella, wherein the whole genome sequence of the mutant strain has 95% or more homology to the whole genome sequence of the GI35 strain, and the mutant strain contains genes corresponding to pfaA, pfaB, pfaC, pfaD and pfaE of the GI35 strain.

2. Feed containing the strain described in claim 1.

3. A method for producing fish in which bacteria present in the intestinal tract have been modified, characterized by administering the strain described in claim 1 to the fish.

4. A method for producing fish with an increased amount of eicosapentaenoic acid (EPA) in their bodies, characterized by administering the strain described in claim 1 to the fish.

5. Fish in which the strain described in claim 1 is present in the intestinal tract (excluding Isaza in which the Shewanella sp. GI35 strain is present in the intestinal tract).

6. Fish that have the strain described in claim 1 in their intestinal tract and produce EPA in their bodies (excluding Isaza fish that have the Shewanella sp. GI35 strain in their intestinal tract).

7. A method for producing fish with accelerated growth, characterized by administering the strain described in claim 1 to fish.

8. A method for producing fish with a modified intestinal flora, characterized by administering the strain described in claim 1 to fish.

9. A method for producing EPA, characterized by administering the strain described in claim 1 to fish.

10. A method for producing EPA, comprising culturing host cells into which a gene group involved in EPA production of the strain described in claim 1 or a mutant of said gene group has been introduced, wherein the mutant includes genes corresponding to pfaA, pfaB, pfaC, pfaD, and pfaE of the GI35 strain.

11. Cells into which a group of genes involved in EPA production of the strain described in claim 1 or a mutant of said gene group has been introduced, wherein the mutant includes genes corresponding to pfaA, pfaB, pfaC, pfaD and pfaE of the GI35 strain.

12. Food or beverage containing the Shewanella sp. GI35 strain or a mutant strain as described in claim 1.

13. A food or beverage made from fish obtained by the method described in claim 5 or 6, or by the manufacturing method described in claim 7 or 8, and containing the strain or components derived from the strain.

Images