JP2020145984A - Abalone feed and method for producing abalone feed - Google Patents

Abstract

To provide new feed suitable for culture of abalone with excellent growth and survival rates of shellfish fry.SOLUTION: An abalone feed contains Coccomyxa microalgae.SELECTED DRAWING: None

Description

The present disclosure relates to abalone feeds and methods for producing abalone feeds.

There are two types of abalone farming: sea surface farming, in which a breeding basket is hung in the bay, and land farming, in which seawater is drawn into an inland concrete aquarium. Compared to sea surface aquaculture, land aquaculture is less susceptible to the effects of climate, weather, etc., and can control the breeding environment, so it has the advantage of being able to produce abalone in a stable manner.

As one of the land-based aquaculture, there is a closed-circulation type land-based aquaculture that circulates and filters seawater, and has been put into practical use. For example, Non-Patent Document 1 describes that closed-circulation aquaculture that reuses seawater has been put into practical use in abalone farming.

In aquaculture on land, juvenile abalone is placed in an inland aquarium or concrete aquarium, fed and cultivated until it reaches the shipping size. Feeds used for abalone farming include natural seaweed and commercially available compound feeds. Natural seaweeds are inexpensive, and most abalone farming feeds are natural seaweeds. However, natural seaweed has problems that the amount of resources has decreased due to shore burning in recent years, the composition of nutritional components is unstable because it is natural, and the growth of juveniles is slow. On the other hand, the compound feed has a high nutritional value and excellent growth of juvenile mussels, but on the other hand, it has a problem that it easily pollutes water and deteriorates water quality because it easily collapses (Non-Patent Document 2). In particular, abalone, which is an invertebrate, is much more sensitive to water quality than teleost fish, so how to maintain water quality is important. When the water quality deteriorates, the survival rate of juvenile mussels may decrease.

Yoichi Tsuji, "Abalone Seedling Production Development, Practical Use in Closed Circulation", Special Issue on Aquaculture Business "Understanding! Seedling Production and Breeding", 2014, p. 103-106 Yasuyuki Harada, Takayuki Kumagai, Keiichi Kozen, Kenji Yokoi, "Comparison of meat quality of abalone F1 hybrids cultivated with different diets", Journal of the Fisheries Society of Japan, 2012, Vol. 78, No. 5, p. 945-950

One aspect of the present disclosure provides a new feed suitable for abalone farming and a method for producing the same, which has excellent growth and survival rate of juvenile mussels.

One aspect of the present disclosure is a feed for abalone containing microalgae belonging to the genus Kokkomixa. The abalone feed according to one aspect of the present disclosure is excellent in the growth and survival rate of juvenile mussels.
One aspect of the present disclosure is a method for producing an abalone feed, which comprises culturing microalgae belonging to the genus Kokkomixa and producing a feed for abalone containing the cultured microalgae. According to the method for producing abalone feed according to one aspect of the present disclosure, it is possible to produce abalone feed having excellent growth and survival rate of juvenile clams.

It is a graph which shows the measurement result of TBARS. It is a graph which shows the measurement result of ω3 fatty acid (ω3 highly unsaturated fatty acid). FIG. 3A is a graph showing the measurement result of the umami component, FIG. 3B is a graph showing the measurement result of the sweetness component, and FIG. 3C is a graph showing the measurement result of the bitterness component. It is a radar chart which shows the measurement result of the content of each component.

The abalone feed according to one aspect of the present disclosure contains microalgae belonging to the genus Coccomyxa (hereinafter, also simply referred to as microalgae).
According to the study of the present inventor, the abalone feed according to one aspect of the present disclosure is excellent in the growth and survival rate of juvenile mussels. Further, according to the study of the present inventor, the abalone cultivated using the abalone feed according to one aspect of the present disclosure contains ω3 fatty acids such as α-linolenic acid, which are functional components in the meat. High content. Further, according to the study of the present inventor, the abalone cultivated using the abalone feed according to one aspect of the present disclosure has a large content of umami component and sweet component in the meat quality. Furthermore, according to the study of the present inventor, the immunity of abalone is high when the abalone feed of one aspect of the present disclosure is used.

Hereinafter, an abalone feed according to one aspect of the present disclosure and a method for producing the same will be described in detail.
[Abalone food]
The abalone feed can be suitably used for abalone farming. The abalone in the present disclosure is a general term for shellfish belonging to the mollusc phylum gastropod. Abalones include abalone species such as black abalone, mega abalone, madaka abalone, and Ezo abalone, and genus Tokobushi such as Tokobushi, which are important resources in the fisheries industry.

As the microalgae belonging to the genus Kokkomixa, any strain belonging to the genus Kokkomixa can be used. Examples of the strain belonging to the genus Coccomyxa include Coccomyxa sp. The KJ strain (hereinafter referred to as KJ strain) is preferable. The KJ shares were deposited with the National Institute of Technology and Evaluation Patent Organisms Depositary (IPOD) on June 4, 2013 under the accession number FERM P-22254, and on June 2, 2015, the accession number FERM BP- It has been transferred to an international deposit under the Budapest Treaty as 22254. The KJ strain was previously classified into the genus Pseudococcomyxa, but is now classified into the genus Pseudococcomyxa.

The microalgae preferably contain 2% by mass or more and 5% by mass or less of α-linolenic acid in a dried state. That is, it is preferable that the dry powder obtained by drying the cultured microalgae contains α-linolenic acid of 2% by mass or more and 5% by mass or less. Abalones cultivated using abalone feeds containing such an amount of α-linolenic acid have been found to contain large amounts of α-linolenic acid, as shown in Examples below. There is. It is considered that this is because α-linolenic acid contained in the abalone feed was incorporated into the abalone. α-linolenic acid is one of the essential fatty acids and is said to have health effects such as lowering blood pressure. Therefore, abalone that is expected to have a high health effect can be produced by culturing using a diet for abalone using microalgae containing such an amount of α-linolenic acid. Microalgae containing such an amount of α-linolenic acid can be obtained by culturing in a state where nitrogen is not deficient. The method for culturing microalgae will be described in detail later.

The shape of the abalone feed is not particularly limited, but it is preferably solid. As the shape of the feed using the microalgae, for example, a concentrated solution obtained by concentrating the culture solution in which the microalgae are cultured can be considered. However, since the bottomed juveniles, especially the juveniles that have grown to a shell length of about 2 mm or more, have the property of feeding while peeling off the solid food with the radula, they are solid foods for abalone. Is preferable. Specific shapes of the abalone feed include pellets and jellies. Additives such as a binder may be added to the abalone feed, if necessary.

[Manufacturing method of abalone feed]
In the method for producing abalone feed, microalgae belonging to the genus Kokkomixa are cultivated to produce abalone feed containing the cultured microalgae. Specifically, the cultured microalgae are used to form the abalone feed into a desired shape. Hereinafter, a method for producing abalone feed will be described in detail.

<Culture of microalgae>
For culturing microalgae belonging to the genus Kokkomixa, a medium usually used for culturing microalgae can be used. Specifically, a known medium for freshwater microalgae or a medium for marine microalgae containing various nutrient salts, trace metal salts, vitamins and the like can be used. Examples of nutrient salts include nitrogen sources such as NaNO ₃ , KNO ₃ , NH ₄ Cl, and urea, and phosphorus sources such as K ₂ HPO ₄ , KH ₂ PO ₄ , and sodium glycerophosphate. Examples of trace metals include iron, magnesium, manganese, calcium, zinc and the like. Examples of vitamins include vitamin B1 and vitamin B12.

Specific examples of the medium include AF-6 medium. The composition of the AF-6 medium is as follows. The following numbers are the mass contained in 100 ml of medium.
NaNO ₃ : 14 mg
NH ₄ NO ₃ : 2.2 mg
_{MgSO 4 · 7H 2 O: 3} mg
KH ₂ PO ₄ : 1 mg
K ₂ HPO ₄ : 0.5 mg
_{CaCl 2 · 2H 2 O: 1} mg
CaCO ₃ : 1 mg
Fe-citrate: 0.2 mg
Citric acid: 0.2 mg
Biotin: 0.2 μg
Thiamine HCl: 1 μg
Vitamin B6: 0.1 μg
Vitamin B12: 0.1 μg
Trace metals: 0.5 mL
Distilled water: 99.5 mL
The medium is preferably adjusted to be acidic by adding an acid such as sulfuric acid as needed. When the medium is acidic, the growth of germs can be suppressed. The pH of the medium is preferably 4 or less, and more preferably 3 or more and 4 or less.

Examples of the method for culturing microalgae belonging to the genus Kokkomixa include a method of supplying CO ₂ (carbon dioxide) and stirring under aeration conditions. At that time, for example, light irradiation with a light-dark cycle or continuous light irradiation can be performed for culturing. Examples of the light-dark cycle include a light-dark cycle in which a 12-hour light irradiation with a fluorescent lamp and a 12-hour dark condition are repeated.

Microalgae belonging to the genus Kokkomixa are preferably cultured in a state where they are not deficient in nitrogen. Specifically, the mass of microalgae cultivated in the medium per unit volume, for example, the mass in a dry state, is predetermined with respect to the total mass of nitrogen of the nitrogen source contained in the medium per unit volume. It is preferable to culture under conditions that do not exceed the value of.

This predetermined value is appropriately set as follows. Microalgae produce oil containing triglyceride as a main component when cultured in a state of being deficient in nitrogen as a nutrient. This oil is expected to be used as an algae oil in various fields such as biofuels and cosmetics. On the other hand, when cultured in a state where nitrogen as a nutrient is not deficient, microalgae do not produce such oil. The above-mentioned predetermined value is a condition in which when the value is exceeded, the microalgae produce oil, resulting in a nitrogen-deficient state. When cultured in a nitrogen-free state, microalgae are characterized by producing a large amount of α-linolenic acid instead of producing oil.

As the culture in a state where nitrogen is not deficient, for example, it is preferable to culture while maintaining the condition that N / DW is 2% by mass or more. Here, N is the total mass of nitrogen of the nitrogen source added to the medium per unit volume, and DW is the mass of microalgae contained in the medium per unit volume in a dry state. N can be determined from the nitrogen source added to the medium and its mass. DW can be determined, for example, from the absorbance of the medium. Specifically, the absorbance of the medium in light having a wavelength of 720 nm is measured. The relationship between the absorbance of the medium and the mass of the fine algae powder obtained by removing water from the medium per unit volume and drying it is determined in advance as a calibration curve. The DW can be determined from the measured absorbance based on this calibration curve. When cultured while maintaining a state in which N / DW is 2% by mass or more and nitrogen is not deficient, microalgae containing a large amount of α-linolenic acid can be obtained as described above.

As a method of culturing in a state where nitrogen is not deficient, for example, before the mass of microalgae cultivated in the medium reaches the above-mentioned predetermined value with respect to the total mass of nitrogen of the nitrogen source first added to the medium. In addition, there is a method of removing microalgae from the medium.

<Molding of abalone feed>
The cultured microalgae are used to shape the abalone feed into the desired shape.
First, the cultured microalgae are concentrated by removing at least a part of the water contained in the medium. Concentration can be carried out by a method such as centrifugation or membrane separation. Next, the concentrated microalgae are dried to obtain a dry powder. Then, the obtained dry powder of microalgae is used to shape the abalone feed into a desired shape. For example, pelletized abalone feed can be produced by granulating using a pellet mill or an extruder. In addition, a jelly-like abalone feed can be produced by mixing dry powder of fine algae with agar or the like and hardening the powder.

[1. Manufacture of abalone feed]
The KJ strain was cultured according to the following procedure.
(1) AF-6 medium was prepared. The pH of the medium was adjusted to 3.5.
(2) The medium was sterilized at 121 ° C. for 20 minutes, and the KJ strain was planted.
(3) 2% CO ₂ was aerated.
(4) The cells were cultured at room temperature while being irradiated with light. The light intensity was about 100 μmol / m ² · s, and the room temperature was 25 ° C. In addition, as described above, the cells were cultured while maintaining a state in which nitrogen was not deficient.

After completion of the culture, a part of the water contained in the medium was removed by centrifugation and concentrated, and the obtained concentrate was dried.
Then, using the obtained dry powder, a pellet-shaped abalone feed and a jelly-shaped abalone feed were produced. The pellet-shaped abalone feed was produced by molding into pellets having a diameter of 2 mm and a length of 6 mm using a pellet mill die roller. The content ratio of microalgae in the obtained pelletized abalone feed was 100% by mass. The jelly-like abalone feed was produced by adding 2 g of agar powder and 5 g of dry powder of microalgae to 70 g of water, mixing them, and hardening them. The content ratio of microalgae in the obtained jelly-like abalone feed was 6.5% by mass.

[2. Feeding test]
Divided into the following four sections, 100 juvenile abalone (mega abalone) with a shell length of about 32 mm and a total mass of 3.7 g or more and 3.8 g or less are stored in a 120 L aquarium with a hiding place, and raw seawater. It was bred by a free-flowing method using. The salinity of the raw seawater was 32 psu, and the water temperature was 15 ° C. or higher and 20 ° C. or lower. The rotation rate of raw seawater was 15 to 20 rotations / day. In each ward, 20 g was fed once every two days. The breeding was carried out for 6 months, and the total feeding amount was 1800 g. In addition, each time before feeding, a siphon was used to remove dirt such as residual food that had sunk on the bottom of the aquarium.

(I) Seaweed plot As a control plot, dried arame and Ecklonia cava returned to seawater were used as feed.
(Ii) Pellet group As an example, the produced pellet-shaped abalone feed was used as the feed.

(Iii) Jelly plot As an example, the produced jelly-like abalone feed was used as the feed.
(Iv) Mixed feed group As a comparative example, a general commercially available mixed feed for abalone was used as the feed.

The breeding results are shown in Table 1 below.

As shown in Table 1, in the pellet group and the jelly group, abalone having a larger shell length and weight than the seaweed group in the control group was obtained, and the abalone growth was excellent. On the other hand, in the pellet group and the jelly group, the growth of abalone was smaller than that in the compound feed group, but the survival rate was excellent. A high survival rate is preferable because the number of shipments can be increased. It is probable that the low survival rate in the mixed feed plot was due to the fact that the mixed feed collapsed in water and the finely divided feed clogged the gills of the abalone. The total production of abalone was the lowest in the seaweed plot of the control plot, and was about the same in the pellet plot, jelly plot and compound feed plot.

[3. Measurement of abalone TBARS]
TBARS was measured for the abalone edible meat obtained in each plot by the following procedure. A Food TBARS Assay kit manufactured by Oxford Biomedical Research was used for the measurement of TBARS. TBARS is an abbreviation for 2-thiobarbituric acid-reactive substance, and is a general term for substances whose concentration increases in response to oxidative stress. The lower the measured value of the obtained TBARS, the stronger the resistance to oxidative stress and the higher the immunity.

(1) A 5 g sample was accurately weighed from the abalone edible meat, 5 ml of distilled water was added, and homogenized with a homogenizer.
(2) The inside of the container and the homogenizer were washed with 5 ml of distilled water, and the washing solution was added to the sample solution to prepare a total of 10 ml of the sample solution.

(3) 1 ml of the sample solution was separated, 1 ml of the indicator was added, and the mixture was stirred.
(4) The sample solution to which the indicator was added was allowed to stand at room temperature for 60 minutes to react.
(5) The sample solution after the reaction was centrifuged for 5 minutes under the conditions of a centrifugal force of 15,000 × g and a temperature of 22 ° C. or higher and 25 ° C. or lower, and a supernatant was collected.

(6) The absorbance of light having a wavelength of 532 nm was measured for the supernatant.
(7) The measured value of TBARS was calculated by comparing with the calibration curve prepared with the standard reagent.
The measurement results are shown in FIG. As shown in FIG. 1, in the pellet group and the jelly group, abalone having a lower TBARS measurement value and higher immunity than the seaweed group and the compound feed group in the control group was produced.

[4. Measurement of abalone ω3 fatty acids]
According to the following procedure, the content ratio of ω3 fatty acids in the fatty acid composition contained in the edible meat of abalone obtained in each section was measured.

(1) A sample of abalone edible meat was minced with a mixer.
(2) A predetermined amount of the minced sample was weighed into a 200 ml Erlenmeyer flask, 100 ml of a solution of chloroform: methanol = 2: 1 was added, and the mixture was extracted at 65 ° C. for 1 hour with stirring.

(3) The obtained extract was allowed to cool and then filtered, and the filtrate liquid was distilled off with an evaporator.
(4) 50 ml of petroleum ether was added to the residue obtained by distillation, an appropriate amount of sodium sulfate was added, and the mixture was stirred well and allowed to stand.

(5) 1 ml of the supernatant was separated, placed in a screw cap test tube, and dried by an evaporator to prepare a measurement sample.
(6) For the measurement sample, the content ratio of ω3 fatty acid was measured using GCMS (Gas Chromatograph Mass Spectrometer). As a pretreatment for the measurement, the measurement feed was methylated using a fatty acid methylation kit manufactured by Nacalai Tesque. For cleanup before GCMS measurement, a methylated fatty acid purification kit manufactured by Nacalai Tesque was used.

The measurement results are shown in FIG. In FIG. 2, the unit on the vertical axis is α-linolenic acid, EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) ω3 fatty acids contained in the obtained measurement sample, and the total mass ratio of these. .. As shown in FIG. 2, abalone having a remarkably high α-linolenic acid content and a high ω3 fatty acid content was produced in the pellet group and the jelly group.

[5. Evaluation of abalone taste]
The content of free amino acids contained in the edible meat of abalone was measured by the following procedure.
(1) To a predetermined amount of abalone edible meat sample, 10 ml of 0.1 M hydrochloric acid was added, and homogenized with a homogenizer.
(2) The sample solution was centrifuged, allowed to stand, and then the supernatant was separated.
(3) Degreasing was performed by adding hexane to the supernatant, stirring well, and then removing hexane. This process was repeated 3 times.

(4) 600 μL of acetonitrile was added to 200 μL of the degreased solution, and centrifugation was performed. The supernatant was collected and used as a measurement sample. In addition, this step was performed in order to remove a protein.
(5) HPLC (High Performance Liquid Chromatography) analysis of free amino acids was performed using the Derivatization Reagent Kit "AccQ Tag Ultra Derivatization Kit" manufactured by Waters.

The measurement results are shown in FIG. FIG. 3A shows the total content of glutamic acid, aspartic acid, arginine, and leucine, which correspond to the umami component in abalone, among the measured free amino acids. FIG. 3B shows the total content of arginine, glycine, taurine, alanine, serine, and glutamine, which correspond to the sweetening component in abalone, among the measured free amino acids. FIG. 3C shows the total content of lysine, tyrosine, methionine, valine, isoleucine, and phenylalanine, which correspond to the bitterness component in abalone, among the measured free amino acids.

As shown in FIG. 3, in the pellet group and the jelly group, the umami component and the sweet component were more than those in the seaweed group and the compound feed group in the control group, and the bitterness component was less than in the compound feed group.

[6. Comprehensive evaluation]
FIG. 4 shows the content of the functional component, the umami component, the sweet component, and the bitter component in each of the pellet group, the jelly group, and the compound feed group based on the seaweed group of the control group. The radar chart to be represented is shown. The functional components include the content of ω3 fatty acids obtained by measuring the ω fatty acids of abalone in item 4 above, and GABA (γ-aminobutyric acid) and GABA (γ-aminobutyric acid) obtained by measuring free amino acids in item 5 above. It is the total content with the content of taurine. In addition, ω3 fatty acid and GABA are functional components expected to have health effects such as lowering blood pressure, and taurine is a functional component expected to have health effects such as recovery from fatigue.

As shown in FIG. 4, the pellet group and the jelly group contained more umami components, sweet components and functional components than the seaweed group and the mixed feed group of the control group.

Claims (5)

A diet for abalone containing microalgae belonging to the genus Kokkomixa. The abalone feed according to claim 1, wherein the microalgae contain 2% by mass or more and 5% by mass or less of α-linolenic acid in a dried state. The abalone feed according to claim 1 or 2, which is in a solid state. Cultivate microalgae belonging to the genus Kokkomixa
A method for producing an abalone feed, which comprises producing the abalone feed containing the cultured microalgae. The method for producing abalone feed according to claim 4, wherein the microalgae are cultured in a state where the medium is not deficient in nitrogen.

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