JP7738451B2 - Ergothioneine-containing feed and farmed fish fed with said feed - Google Patents

Description

The present invention relates to the technical fields of feed containing ergothioneine (hereinafter sometimes referred to as ERG) and the raising of farmed fish using such ERG-containing feed.

Food for farmed fish has traditionally consisted of fishmeal, which is high in protein and contains a balanced amount of amino acids required by fish, and fish oil, which contains essential fatty acids. However, the fish catches that are the source of fishmeal and fish oil have been declining worldwide, causing prices to soar. Against this backdrop, there is a need to develop sustainable alternatives to fishmeal. It is also expected that the use of such alternatives to fishmeal will add greater value to farmed fish. However, it is known that alternatives to fishmeal made from plant or animal ingredients can cause feeding disorders in farmed fish, particularly saltwater fish.

It has been reported that mushroom-derived ergothioneine inhibits the activity of polyphenol oxidase (PPO), which is involved in the blackening of crustaceans and the discoloration of fish blood vessels (Non-Patent Document 1: Food and Containers (2011) vol. 52, No. 7). Ergothioneine not only possesses potent antioxidant properties, but also has physiological effects such as elastase inhibition and tyrosinase inhibition, and is reported to be involved in the body's oxidation defense system. Applications of ergothioneine in the beauty and food fields, such as for skin whitening and wrinkle prevention, as well as in the medical field, are being attempted. Therefore, in addition to the goal of preventing fish discoloration, there is hope for the development of farmed fish feed formulated with ergothioneine, with the aim of developing farmed fish as functional foods containing ergothioneine.

Ergothioneine has been produced by extraction from basidiomycetes such as Pleurotus cornucopius, chemical synthesis, and fermentation using microorganisms. Extraction from basidiomycetes such as Pleurotus cornucopius takes time to obtain the raw materials, making it unsuitable for mass production. To obtain large quantities of ergothioneine, research has been conducted on fermentation using C1-compound-assimilating bacteria or yeast (Patent Document 1: WO 2016/104437), as well as fermentation using microorganisms overexpressing ergothioneine biosynthesis genes (Patent Document 2: WO 2017/150304).

However, ergothioneine obtained using these methods is extremely expensive, and adding it to feed for farmed fish is not cost-effective. Furthermore, when using genetically modified microorganisms, evaluation and management under the Feed Safety Act is required, which is an obstacle to feed development.

International Publication No. 2016/104437 International Publication No. 2017/150304

Food and Containers (2011) vol. 52, No.7, 432-438

When providing ERG-containing feed, we discovered that when using plant ingredients, if we try to include a high concentration of ERG, the amount of plant ingredient added becomes too large, which can lead to feeding disorders. Therefore, our goal is to provide plant-based ergothioneine-containing feed that can be used to raise farmed fish stably.

The present inventors have conducted extensive research in order to provide an ergothioneine-containing feed, and as a result, have developed a feed with a high ergothioneine content that does not cause feeding disorders in farmed fish by using a solid fermentation product of Aspergillus oryzae as a raw material, thereby arriving at the present invention.
Thus, the present invention relates to:
[1] A feed containing 0.001% or more ergothioneine and made from solid fermentation product of Aspergillus oryzae.
[2] The feed according to Item 2, wherein the koji mold solid fermentation product contains 0.35 g or more of ergothioneine per 1 kg of the koji mold solid fermentation product.
[3] The feed according to item 1 or 2, wherein the solid fermentation product of koji mold is blended in the feed so as to be 1 to 10% by mass.
[4] The feed according to any one of items 1 to 3, which is for raising farmed fish.
[5] The feed according to item 4, wherein the farmed fish are farmed tuna fish.
[6] A method for producing farmed fish, comprising raising farmed fish using the feed according to any one of items 1 to 5.
[7] The method according to item 6, comprising feeding the feed according to any one of items 1 to 5 within one month before landing.
[8] A farmed fish produced by the production method according to item 6 or 7.
[9] The farmed fish according to item 8, wherein the farmed fish is a farmed tuna fish.

By producing feed using solid-state fermentation product of koji mold with a high content of ergothioneine as a raw material, we have provided feed containing ergothioneine that does not cause eating disorders. Furthermore, farmed fish fed this feed had increased ergothioneine content and improved color and taste.

FIG. 1 is a graph showing the change in ERG content with respect to the number of days of fermentation when fermentation was carried out using koji mold under fermentation conditions that resulted in a high ERG content in the koji mold solid-fermentation product. FIG. 2 is a graph showing the change in water content of the solid fermentation product of koji mold during fermentation. FIG. 3 is a graph showing the ERG content in the bodies of farmed fish raised on feed made from a solid fermentation product of koji mold with a high ERG content.

<ERG-containing feed>
A feed according to one embodiment of the present disclosure contains ergothioneine. More specifically, the feed contains 0.001% to 0.1% ergothioneine by dry weight. The feed is made from a solid fermentation product of koji mold with a high ERG content. The feed can be produced by mixing 1 to 50% of the solid fermentation product of koji mold with a commonly used feed such as moist pellets. The upper limit of the blending ratio of the solid fermentation product of ERG-rich koji mold can be 30%, 20%, or 10%, from the viewpoint of not inhibiting feeding. The lower limit of the blending ratio of the solid fermentation product of ERG-rich koji mold can be 1%, 5%, or 10%, from the viewpoint of increasing the ergothioneine content. Ingredients other than the solid fermentation product of koji mold with a high ERG content of the ERG-containing feed include ingredients commonly used in moist pellets, such as fish meal and fish oil. The ERG in the ERG-containing feed is typically derived from the solid fermentation product of koji mold with a high ERG content. More preferably, the ERG-containing feed of the present invention does not contain added ERG and/or mushroom-derived ERG. A sample made from a solid fermentation product of an ERG-rich koji mold as a raw material and containing 0.001% to 0.1% ergothioneine by dry weight is specifically referred to as an ERG-rich koji-containing feed.

ERG-containing feed is feed for fish. By feeding fish with a koji-containing feed with a high ERG content and cultivating them, it is possible to produce cultivated products with accumulated ergothioneine. The edible parts of farmed fish fed a koji-containing feed with a high ERG content are resistant to discoloration and deterioration in flavor, making them highly valuable products. Furthermore, farmed fish containing ergothioneine have excellent nutritional value because they provide ergothioneine, an antioxidant. Therefore, farmed fish containing ergothioneine that are cultivated on an ERG-containing feed can also be made into foods with functional claims. Such functional claims can display functions such as skin-beautifying or whitening effects based on ergothioneine's elastase inhibitory and tyrosinase inhibitory effects, preventive effects against lifestyle-related diseases based on the production of lipid peroxides, and preventive effects against dementia and Alzheimer's disease based on the removal of reactive oxygen species.

<Solid fermentation product of koji mold with high ERG content>
"ERG-rich koji mold solid fermentation" refers to a koji mold solid fermentation product produced by a method for increasing the ERG content in koji, as described in detail below. The ERG content of the ERG-rich koji mold solid fermentation product is at least 5 times, preferably at least 10 times, more preferably at least 20 times, even more preferably at least 50 times, and even more preferably at least 100 times higher than that of koji mold solid fermentation product fermented under normal fermentation conditions, for example, when the moisture content of the koji mold solid fermentation product is 25% or less. For example, the ERG-rich koji mold solid fermentation product contains at least 0.35 g ERG per kg of koji mold solid fermentation product, preferably at least 0.4 g ERG per kg of koji mold solid fermentation product, more preferably at least 0.5 g ERG per kg of koji mold solid fermentation product, even more preferably at least 0.8 g ERG per kg of koji mold solid fermentation product, and even more preferably at least 1.0 g ERG per kg of koji mold solid fermentation product. The ERG contained in the ERG-rich koji mold solid fermentation product of the present disclosure is produced by koji mold and is not added or concentrated. An ERG-rich koji mold solid-state fermentation product can be identified only by its production process, and it is impossible or impractical to identify the resulting ERG-rich koji mold solid-state fermentation product by its components or properties.

<Method for increasing ERG content>
The ERG-enhancing method according to one embodiment of the present disclosure refers to a method of solid-state fermentation of koji mold under fermentation conditions that increase the ERG content in the koji mold solid-fermentation product, as described in detail below. This ERG-enhancing method refers to a method for producing a koji mold solid-fermentation product having an increased ERG content compared to a koji mold solid-fermentation product produced using raw materials and under normal fermentation conditions.

To achieve high ERG content in a koji mold solid-fermentation product, it is important to control the initial moisture content of the mixture of solid medium and koji mold and the moisture content during fermentation for at least four days. More specifically, it is necessary to maintain the moisture content of the mixture of solid medium and koji mold at 25% or higher even after the fourth day of fermentation. If the moisture content of the mixture of solid medium and koji mold during fermentation is less than 25%, it is not possible to achieve a high ERG content in the koji mold solid-fermentation product. Therefore, in one embodiment of the present disclosure, a method for producing a koji mold solid-fermentation product with a high ERG content is characterized by maintaining the moisture content of the mixture at 25% or higher throughout the fermentation process during the process of mixing the solid medium and koji mold and fermenting. Furthermore, heat may be generated at the beginning of fermentation, causing a rapid loss of moisture. Therefore, considering the fermentation period and process, if the initial moisture content of the mixture of solid medium and koji mold at the start of cultivation is less than 45%, it is difficult to maintain the moisture content of the fermentation product at 25% or higher during fermentation (Figure 2). Therefore, a method for producing a solid fermentation product of koji mold with a high ERG content according to one embodiment of the present disclosure may include a step of adjusting the initial moisture content of the mixture of solid medium and koji mold to 45% or more.

The ERG-rich koji mold solid fermentation product is prepared by fermenting a conventional solid medium for culturing koji mold. Such a solid medium preferably contains 10% or more protein in the solid content. As raw materials for such a solid medium, beans, fish, meat, algae, and processed products thereof containing 20% or more protein can be used. Examples of processed products containing 20% or more protein include defatted soybeans, fish meal, meat meal, and spirulina powder. It is more preferable that the solid medium of the present invention contains raw materials that have been puffed. The solid medium can be prepared by steaming or simmering the above-mentioned raw materials in boiling water, and optionally crushing them.

Other fermentation conditions can be those typically used in this technical field. For example, the initial pH of the medium is adjusted to 5-10. The fermentation temperature is set to 20-40°C, and the fermentation time is 4 days or more, preferably 4-10 days, preferably 5-7 days, and more preferably 6-7 days. From the perspective of maintaining the moisture content of the koji mold solid-fermentation product during fermentation, it is preferable to culture it under high humidity conditions, and humidity can be adjusted to control the moisture content of the koji mold solid-fermentation product. Furthermore, since the fermentation is a long-term fermentation of 4 days or more and involves a high moisture content, it is desirable to carry out the fermentation in an environment that prevents the growth of unwanted bacteria. Containers that allow for aseptic cultivation, such as a Yamazaki-type koji-making apparatus or a drum koji-making apparatus, may also be used.

As an example, when using Aspergillus oryzae RIB326, a representative strain of koji mold, and producing an ERG-rich koji mold solid fermentation product using the production method of the present invention, it is possible to produce an ERG-rich koji mold solid fermentation product containing 0.35 g/kg or more of ERG, preferably 0.4 g/kg or more of ERG, more preferably 0.5 g/kg or more of ERG, even more preferably 0.8 g/kg or more of ERG, and even more preferably 1.0 g/kg or more of ERG (Figure 1).

<Koji mold>
As the koji mold, any fungus belonging to the genus Aspergillus can be used. Examples include Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus luchuensis, and Aspergillus tamarii. As the koji mold strain, for example, a publicly available strain such as Aspergillus oryzae RIB326, a strain contained in koji starter commercially available from koji starter makers, or a strain isolated from a food and beverage production environment such as a sake or soy sauce brewery can be used.

The koji mold may be a wild-type strain, or a mutant strain with high ERG production may be obtained by using a common mutagenesis method. Examples of mutagenesis methods include ultraviolet (UV) or X-ray irradiation, which physically damage DNA and introduce mutations, and treatment with alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), which chemically damage DNA and introduce mutations. From the perspective of food production, it is preferable to use a non-recombinant strain. The koji mold may be obtained by culturing koji mold in a medium, or by culturing koji mold and allowing spores to adhere and then drying. It may be dried together with the raw materials, or the spores alone may be collected.

<Method of ERG analysis>
ERG can be extracted from the ERG-rich koji mold solid-fermentation product or ERG-containing feed using methods well known in the art. The extraction solvent is not particularly limited as long as it dissolves ERG, and examples include organic solvents such as methanol, ethanol, isopropanol, and acetone; aqueous organic solvents obtained by mixing these organic solvents with water; water, warm water, and hot water. After adding the solvent, ERG can be extracted by optionally performing a crushing treatment. The extraction solvent temperature can be set within the range of room temperature to 100°C.

One embodiment of the ERG extraction method involves adding a solid fermentation product of koji mold with a high ERG content to water to prepare a suspension, then heating the resulting suspension at 98-100°C for 15 minutes, for example, followed by centrifugation to recover the supernatant, which is then filtered to remove insoluble matter. Alternatively, the heat-treated suspension may be filtered without being centrifuged.

In addition, instead of the above-mentioned heating treatment, bacterial cells may be subjected to disruption treatments such as disrupting the bacterial cells using disruption means such as an ultrasonic disrupter, French press, Dynomill, or mortar; dissolving the bacterial cell walls using a cell wall-lytic enzyme such as Yatalase; or dissolving the bacterial cells using a surfactant such as SDS or Triton X-100. These methods can be used alone or in combination.

The resulting extract can be purified by subjecting it to purification processes such as centrifugation, filter filtration, ultrafiltration, gel filtration, separation based on differences in solubility, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography, reverse phase chromatography, etc.), crystallization, activated carbon treatment, and membrane treatment.

Qualitative or quantitative analysis of ERG can be performed by any method, including, but not limited to, HPLC. HPLC separation conditions can be selected appropriately by those skilled in the art; for example, analysis can be performed under the conditions described in the Examples below.

<Method of farmed fish production>
A method for producing cultured fish according to an embodiment of the present disclosure includes raising cultured fish using an ERG-containing feed. The method for producing cultured fish can also be simply referred to as a rearing method. Cultured fish raised and produced according to the method for producing cultured fish are referred to as cultured fish according to an embodiment of the present disclosure. For example, the ERG-containing feed is continuously fed at least 10 times. Feeding can be performed as a satiation feeding once a day during normal times and once every two days during winter. The ERG-containing feed may be fed at any timing, but from the viewpoint of improving taste, it is preferable to provide a feeding period with the ERG-containing feed before landing. More specifically, the feeding period is within six months, for example, within three months, within one month, within two weeks, within 12 days, or within 10 days. From the viewpoint of exerting a sufficient effect of improving color and/or taste, the feeding period can be several days or more, for example, 10 days or more. The water temperature during the rearing period is selected appropriately depending on the fish species, but as an example, it is set to 10°C to 32°C for cultivating tuna.

Any tuna species may be farmed, but as an example, tuna weighing less than 40 kg and less than four years old are desirable. Farmed tuna weighing less than 40 kg and less than four years old generally have a stronger sour taste than farmed tuna that have grown for four years or more, making them unsuitable for consumption. On the other hand, by providing a feeding period with a high-ergothioneine diet before landing, the ergothioneine content in the fish can be increased (Figure 3) and the sourness can be reduced (Example 3). The longer the feeding period with a high-ERG koji diet before landing, the more the ergothioneine content can be increased and the more the sourness can be reduced in farmed tuna, particularly farmed fish less than four years old. The increase in ergothioneine content varies depending on the ERG content of the high-ERG koji diet or the feeding period. As an example, farmed fish fed a feed containing koji with a high ERG content have an increase in ERG content of 10% or more, preferably 20% or more, more preferably 30% or more, and even more preferably 50% or more, compared to a control fed a feed not containing koji with a high ERG content.

In addition, shortening the farming period before landing can reduce the risk of devastating damage to farmed tuna fish caused by red tides and other factors. With this rearing method, as long as the fish are fed with a high-ERG diet for the entire rearing period described above before landing, there may be a period during which other feed is fed. Examples of other feed include live feed (horse mackerel, mackerel, etc.), moist pellets, etc.

<Farmed fish>
In one embodiment of the present disclosure, the farmed fish may be any fish species that can be farmed. As an example, it is preferable to use the feed as feed for tunas, yellowtails, sea bream, pufferfish, salmon, trout, and flounder. Examples of farmed tunas include the Thunnus family and the Bonito family. Examples of the Thunnus family include the Thunnus genus, the Bonito genus, the Scomber genus, and the Bonito genus, and examples of the Bonito family include the Dogtooth Thunnus genus and the Bonito genus. Examples of tunas include albacore, bluefin tuna, southern bluefin tuna, Atlantic bluefin tuna, Atlantic bluefin tuna, yellowfin tuna, bigeye tuna, and longfin tuna of the genus Thunnus, skipjack tuna of the genus Skipjack tuna, flathead tuna and frigate tuna of the genus Bonito, and bigeye tuna of the genus Sparrut, and skipjack tuna of the genus Bonito, or albacore, bluefin tuna, southern bluefin tuna, Atlantic bluefin tuna, yellowfin tuna, bigeye tuna, longfin tuna, skipjack tuna, and skipjack tuna. Preferred examples include albacore, bluefin tuna, Atlantic bluefin tuna, Atlantic bluefin tuna, yellowfin tuna, bigeye tuna, longfin tuna, skipjack tuna, and skipjack tuna.

<Method of manufacturing edible part>
A method for producing edible parts according to one embodiment of the present disclosure includes preparing farmed tuna fish obtained by the breeding method of the embodiment described above, harvesting edible parts from the prepared farmed tuna fish, and placing the edible parts in a container, and may include other steps as necessary.

Examples of processed forms include the so-called headless form, in which the head and tail have been removed from farmed tuna fish, and the so-called loin form, in which the headless form is divided into left and right halves and dorsal and ventral sides and cut into quarters.

Farmed tuna fish and their edible parts can also be used as animal feed for livestock such as cattle, pigs, and poultry, other farmed fish, and pet food.

All references cited in this disclosure are incorporated herein by reference in their entirety. In this disclosure, % means % by weight unless otherwise specified.

The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications to the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.

Example 1: Production of ERG-rich koji mold solid-fermentation product using defatted soybeans as a medium. 640 g of puffed defatted soybeans (protein content: approximately 48%) were placed in a plastic bag, and 480 ml of boiling water was added and steamed (protein content of the solid medium after steaming with hot water: approximately 27%). After cooling to room temperature, 2.4 g of Aspergillus oryzae RIB326 seed culture (wheat bran culture) was added and mixed well, and the mixture was placed evenly on a lid. Fermentation was carried out at 95% humidity and 32°C. When the temperature of the koji mold solid-fermentation product reached 40°C, the room temperature was changed to 25°C, humidity control was stopped, and fermentation was continued for 7 days to obtain an ERG-rich koji mold solid-fermentation product. The koji mold solid-fermentation product was weighed on days 1, 3, 4, and 7 after the start of fermentation, and the moisture content was calculated (Figure 2). In addition, portions of the solid fermented koji mold product were collected on the 1st, 2nd, 3rd, and 6th days after the start of fermentation.

Example 2: ERG analysis method (1) ERG extraction 5 g of ERG-rich koji mold solid fermentation product was weighed out, 75% ethanol was added, and the ERG-rich fermentation product was crushed and left at room temperature for 1 day to extract ERG from the ERG-rich koji mold solid fermentation product. The ERG content was measured under the following analytical conditions, and the ERG content per kg of ERG-rich koji mold solid fermentation product was found to be 1.1 g. Similarly, the ERG content was measured for the fermentation product on days 1, 2, 3, and 6 after the start of fermentation, and the results are shown in a graph ( FIG. 1 ).
As a reference example, ERG was similarly extracted from Miyako Koji Square (manufactured by Isesosha Co., Ltd.), Rice Koji H (manufactured by Kose Foods Co., Ltd.), and Rice Koji S (manufactured by Kose Foods Co., Ltd.), and the ERG content was measured under the following analytical conditions. The ergothioneine content of each was as follows:

(2) LCMS analysis conditions analyzer: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mm D x 150 mm
Solvent A: Acetonitrile Solvent B: 5 mM ammonium acetate/H ₂ O
Flow rate: 0.5 ml/min 80% solvent A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
MS Scan Mode

(3) Ergothioneine analysis conditions by LC-MS/MS Analytical equipment: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mm D x 150 mm
Solvent A: 0.1% formic acid/acetonitrile Solvent B: 0.1% formic acid/H ₂ O
Flow rate: 0.5ml/min 80%A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
Collision energy: 11V
Trace: m/z 230.1>186.1
Collision energy: 20V
Trace: m/z 230.1>127.0

Example 3: Raising farmed tuna fish with ERG-containing feed (1) Production of ERG-containing feed An ERG-containing feed was produced by mixing an average of 2.69% (2.43-3.05%) of the ERG-rich koji mold solid-fermentation product produced in Example 1 with moist pellet feed (sold by:). The ERG content of the ERG-containing feed was 0.011% by dry weight.

(2) Rearing Method The ERG-containing feed was fed to 3-year-old tuna once a day for 12 days, a total of 12 times.
In the control group, moist pellet feed containing no ERG-rich koji was fed to three-year-old tuna fish for the same period and frequency.

(3) Fish measurements Three fish were taken from the test area and two from the control area by fishing.
Immediately after being caught, the tuna was bled, nerve-killed, and the internal organs and gills were removed on board the boat, and it was then stored in ice water until it was processed into loins at the processing plant.
The tuna taken were treated as described above, and in the test area, individual 1 had a fish weight of 31.9 kg and a fork length of 119 cm, individual 2 had a fish weight of 30.6 kg and a fork length of 118 cm, and individual 3 had a fish weight of 34.7 kg and a fork length of 126 cm; in the control area, individual 1 had a fish weight of 29.4 kg and a fork length of 116 cm, and individual 2 had a fish weight of 34.6 kg and a fork length of 122 cm.
The fish bodies stored in ice water were processed into loins and transported and stored on ice until sensory evaluation.

(4) Sensory Evaluation Sensory evaluation was conducted by analytical and preference-based evaluation using 24 evaluators who were selected and trained in-house. Each item in the sensory evaluation was scored using absolute evaluation. Evaluators who were trained to evaluate each item in the sensory evaluation according to a set standard scored sourness on a 5-point scale from 0 to 1, 2, 3, and 4, with increasing sourness, and hardness on a 7-point scale from soft to hard, with -3, -2, -1, 0, +1, +2, and +3.

As a result of the sensory evaluation, the test group showed a significant decrease in sourness (P<0.05) compared to the control group. Also, the hardness of the test group showed a significant tendency (P=0.08), but it was decreased compared to the control group (Table 2).

In the sensory evaluation (preference-type evaluation), the taste was evaluated comprehensively and the evaluation was converted into a score as an "overall evaluation" (Table 3).

(5) Measurement of ERG content in tuna fish The fish from the test and control groups were weighed, and after adding 75% ethanol, the fish were crushed and left at room temperature for one day to extract ERG. The ERG content was measured under the following analytical conditions, and the results of the ERG content per gram of fish are shown in a graph (Figure 3).

(5-1) LCMS analysis conditions analyzer: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mm D x 150 mm
Solvent A: Acetonitrile Solvent B: 5 mM ammonium acetate/H ₂ O
Flow rate: 0.5 ml/min 80% solvent A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
MS Scan Mode

(5-2) Conditions for ergothioneine analysis by LC-MS/MS Analytical equipment: UPLC CQ micro; Waters
UPLC
Column: 2.5 HILIC 3.0 mm D x 150 mm
Solvent A: 0.1% formic acid/acetonitrile Solvent B: 0.1% formic acid/H ₂ O
Flow rate: 0.5ml/min 80%A
Inject: 2μL
Mass spectrometer ESI: ES+
Cone V: 21V
Capillary V: 4.5kV
Source temperature: 120℃
Desolvation temperature: 400℃
Collision energy: 11V
Trace: m/z 230.1>186.1
Collision energy: 20V
Trace: m/z 230.1>127.0

Claims (8)

A feed containing 0.001% or more of ergothioneine and made from a solid fermentation product of koji mold, wherein the solid fermentation product of koji mold contains 0.35 g or more of ergothioneine per kg of the solid fermentation product of koji mold . The feed according to claim 1, in which the solid fermentation product of koji mold is blended into the feed at a concentration of 1 to 10% by mass. The feed according to claim 1 or 2 , which is for raising farmed fish. The feed according to claim 3, wherein the farmed fish is farmed tuna fish. A method for producing farmed fish, comprising raising farmed fish using the feed according to any one of claims 1 to 4 . The production method according to claim 5, comprising a step of feeding the feed according to any one of claims 1 to 4 within one month before landing. A farmed fish produced by the production method according to claim 5 or 6 . The farmed fish according to claim 7 , wherein the farmed fish is a farmed tuna fish.