WO2004017751A1

WO2004017751A1 - Feed for fry young fishes and method of producing hydrolyzate of low-phytin vegetable protein to be used therein

Info

Publication number: WO2004017751A1
Application number: PCT/JP2003/009355
Authority: WO
Inventors: Toshihiro Nakamori; Hitoshi Furuta
Original assignee: Fuji Oil Company, Limited
Priority date: 2002-07-24
Filing date: 2003-07-23
Publication date: 2004-03-04
Also published as: KR101042124B1; JP4556868B2; AU2003248093A1; KR20110033314A; CN101066097A; CN1671294A; CN101066097B; CN100379354C; KR20050025138A; JPWO2004017751A1

Abstract

It is intended to provide a feed which is appropriate for enhancing the survival rate of young fry fishes with underdeveloped digestion and absorption functions or promoting the growth of them. It is also intended to obtain a vegetable protein hydrolyzate to be used in this feed. To obtain a low-phytin vegetable protein hydrolyzate, a process for producing the low-phytin vegetable protein hydrolyzate which comprises (a) the step of digesting a protein with the use of a protease and (b) the step of digesting phytic acid with the use of an enzyme digesting phytic acid has been completed. In the case where the vegetable protein is soybean protein, for example, it is preferable that the low-phytin soybean protein hydrolyzate has an average molecular weight of from 200 to 10,000. It is also preferable that the phytic acid content is 0.05% by weight or less (based on dry solid matters). A feed for young fry fishes containing this low-phytin soybean protein hydrolyzate makes it possible to provide a feed for young fry fishes which promotes the growth of young fry fishes and enhances the survival rate.

Description

Specification

Production method of feed for larvae and larvae and low phytin plant protein hydrolyzate used for the feed

The present invention provides a feed for larvae and larvae using a low-phytin plant protein hydrolyzate with reduced phytic acid. The present invention also relates to a method for producing a low-phytin vegetable protein hydrolyzate in which phytic acid is reduced. Background technology

Conventionally, soybean raw materials (soybean meal, soymilk, soybean protein, etc.) have been used as protein raw materials for fish farming feed.

It has become known that removing phytic acid contained in soybeans increases feed efficiency. .

Examples of the invention for removing or decomposing phytic acid include: (a) feed (JP-A-11-000164, JP-A-8-205785); and (b) soybean-derived feed such as defatted soybean and okara. Materials (Japanese Patent Application Laid-Open No. Hei 9-140334), (c) soy protein (Japanese Patent Application Laid-Open No. 2000-300185, Japanese Patent Application Laid-Open No. 4503002), and (d) soymilk (Japanese Patent Application Laid-open No. No. 166049, Japanese Patent Application Laid-Open No. 2000-245340 by the present applicant) and the like.

However, low phytin protein hydrolyzate has not been used in fish feed.

On the other hand, the present applicant has studied the use of a protein hydrolyzate obtained by hydrolyzing a soybean protein raw material using an enzyme as a feed for fish farming. For example, the invention described in JP-A-7-227223 and the invention described in JP-A-8-51937 have been made as baits for increasing the survival rate of larvae and fry. In order to further improve the survival rate of larvae and fry, we studied the improvement of soybean protein hydrolyzate. It is.

On the other hand, regarding the soybean protein hydrolyzate, the present applicant has disclosed a low-phytin plant protein hydrolyzate obtained by removing phytic acid using a resin for patients with renal disease (Japanese Patent Application Laid-Open No. Hei 8-09921).

On the other hand, it is also known to produce a low-phytin plant protein hydrolyzate by inoculating a soybean protein with a bacterium, fermenting the protein, and subjecting it to proteolysis and lyase treatment (JP-A-9-023822). .

In addition, there are inventions in which a protease is used in the same way as a protease because a crude enzyme is used when enzymatically treating a soybean protein raw material, or a combination of enzyme digestion and phytase treatment is used. It does not disclose or teach the use of degraded soy protein for fish farming (Japanese Patent Application Laid-Open Nos. 51-125300, 2002-51706, etc.).

As described above, the use of soybean protein hydrolyzate in fish feed has been studied by the present applicant, and low phytin soy protein hydrolyzate has also been studied. It is not known to be used for larvae or fry.

By the way, the method for removing phytic acid from soybeans is as follows: (1) A method for removing phytic acid during the aqueous extraction of soybean protein using a salt (JP-A-8-173052, JP-A-9-1212) 780), (2) a method of decomposing phytic acid with phytase or the like, and (3) a method of removing phytic acid by adsorbing it on a resin or the like (Japanese Patent Application Laid-Open No. 2001-163800). The method (2) is industrially advantageous because the process is less complicated than the methods (3) and (3).

Regarding (2) the method of decomposing phytic acid using fiuyase, etc., (a) many soybeans and defatted soybeans for use as feeds are known, and (b) Some isolated soy proteins are also known, but (c) little is known about plant protein hydrolyzate mixtures. For example, (a) feed (JP-A-11-000164, JP-A-8-205785), (b) soybean-derived feed material such as defatted soybean and okara (JP-A-9-140334) , Soybean protein (Japanese Patent Application Laid-Open Nos. 2000-300185 and 4503002 by the applicant of the present application) and soymilk (Japanese Patent Application Laid-Open No. 59-166049 and Japanese Patent Application Laid-Open No. 2000-245340 by the present applicant) It has been known.

However, little is known about (C) a low-phytin plant protein hydrolyzate. For example, Japanese Patent Application Laid-Open No. H08-092123 by the present applicant discloses a method using a resin.

Japanese Patent Application Laid-Open No. 9-023822 discloses that isolated soybean protein is inoculated with a bacterium, fermented, and simultaneously subjected to enzymatic decomposition and phytase treatment to obtain a peptide product having a low phytic acid content. A method is disclosed.

However, there is no known method for reducing phytic acid below the detection limit as in the present invention. Disclosure of the invention

As the present inventors proceeded with their research, in order to use plant protein hydrolysates for larvae and fry with insufficient development of digestion and absorption functions, and for fish immediately after birth, peptide products must be used. It has been found that a plant protein hydrolyzate that is insufficient and has excellent digestibility and absorbability and that has extremely low phytic acid is required. Therefore, an object of the present invention was to obtain a low-phytin plant protein hydrolyzate having a very low phytic acid content, and more preferably a low-phytin plant protein hydrolyzate having a phytic acid content below the detection limit.

Further, the present invention aims at a feed for fry using such a low-phytin plant protein hydrolyzate.

The present inventors have conducted intensive studies and found that soy protein hydrolyzate used in feed By reducing the amount of phytic acid contained in the digest, the survival rate of the larvae and larvae was increased, and the present inventors obtained the knowledge of promoting the growth of the larvae and completed the present invention.

Based on the following findings, a low-phytin plant protein hydrolyzate was completed. That is, the present applicant has previously completed a low-phytic acid plant protein hydrolyzate (JP-A-8-092123) by resin treatment, but in order to reduce the phytic acid content below the detection limit, the resin treatment was carried out. Was complicated.

On the other hand, the present applicant produced a low phytic acid soybean protein by subjecting soybean protein to phytase treatment (Japanese Unexamined Patent Publication No. 2000-300185). It was difficult to obtain a soybean protein hydrolyzate with a very low phytic acid content below the detection limit.

Therefore, as a result of further intensive research, a soybean protein hydrolyzate with extremely low phytic acid (below the detection limit) was obtained by first enzymatically decomposing soybean protein to a specific molecular weight range and then subjecting it to lyase treatment. Was obtained. In addition, when enzymatically decomposing soybean protein, if soybean protein is extracted from the soybean material with water and then subjected to enzymatic digestion without drying, phytic acid can be detected even if it is first treated with lyase and then enzymatically degraded. We have obtained information that can be removed below the limit.

Surprisingly, it was found that the soybean protein hydrolyzate having a very low phytic acid content below the detection limit has a better flavor than the soybean protein hydrolyzate obtained by simply enzymatic degradation.

The present invention has been completed based on these findings.

That is, the present invention is a feed for larvae and juveniles, wherein the feed material contains a low-phytin plant protein hydrolyzate having a phytic acid content of 0.05% by weight or less (in dry solid content). .

As the low phytin plant protein hydrolyzate, a plant protein hydrolyzate having an average molecular weight of 200 to 10,000 is suitable. The present invention also provides a low phytin characterized by comprising (a) a step of degrading a protein using a protease and (b) a step of decomposing phytic acid by using an enzyme that degrades phytic acid. This is a method for producing a plant protein hydrolyzate. After decomposing the protein using a protease, it is preferable to decompose phytic acid using an enzyme that decomposes phytic acid.

After degrading the undried protein using an enzyme that degrades phytic acid, it is preferable to decompose the protein using a protease.

The enzyme that degrades phytic acid is preferably phytase.

The pH for phytase treatment is preferably 6-9.

The average molecular weight of the low phytin plant protein hydrolyzate is preferably from 200 to 100,000.

It is preferable that the content of phytic acid in the low-phytin vegetable protein hydrolyzate is such that phytic acid is not detected by vanadomolybdate spectrophotometry (detection limit: 5 mg / lOOg) per dry solid. BEST MODE FOR CARRYING OUT THE INVENTION

First, the feed for larvae and fry will be described.

The low phytin plant protein hydrolyzate used in the feed for larvae and larvae of the present invention has a phytic acid content of 0.05% by weight or less, preferably 0.01% by weight or less, more preferably 0.01% by weight or less in dry solids. 0 4% by weight or less (below the detection limit) is appropriate.

The smaller the amount of phytic acid contained in the plant protein hydrolyzate, the better the survival rate of larvae and juveniles, and thus it is preferable. It also has the effect of promoting the growth of fry, giving the dung stickiness and preventing turbidity due to water dung.

The average molecular weight of the low phytin plant protein hydrolyzate used in the larvae and feeds of the present invention is 200 to 100,000 (preferably 300 to 5,000). Things are appropriate. If the molecular weight is large, the effect of promoting the growth and survival rate of larvae and larvae is inferior, and if the molecular weight is reduced to amino acid, the osmotic pressure of the feed increases, and it becomes easy to dissolve, etc. Disappears.

A soybean protein hydrolyzate with a relatively large molecular weight may be used for fish farming feeds alone, but it is preferable that the average molecular weight be smaller as the fish farming fish is a larva, especially a larva that has just hatched from an egg. Oligopeptide mixtures are preferred.

The feed of the present invention suitably contains 40 to 70% by weight, preferably about 50 to 60% by weight of a protein component as a composition.

Suitably, the feed of the present invention contains 1% to 30% by weight, preferably 3 to 25% by weight of a low phytin plant protein hydrolyzate. Usually, 3% by weight or more, preferably 10% to 80% by weight, of the protein component in the feed of the present invention is suitably replaced with the low-phytin plant protein hydrolyzate described above. When the replacement ratio increases, it becomes difficult to granulate the feed.

If the amount of low-phytin plant protein hydrolyzate in the feed of the present invention is low, the effect of increasing the survival rate of larvae and juveniles and the growth rate of juveniles is small. This is common to fish such as red sea bream and inoculated fish that are difficult to produce for aquaculture, and is different from other fish farming. The protein components other than the low-phytin vegetable protein hydrolyzate include krill, fish meal, processed egg, processed milk, gelatin, fish meal, seafood extract, yeast extract, and fish egg extract. Can be.

The feed of the present invention contains carbohydrates, fats, vitamins, minerals, n-3 highly unsaturated fatty acids, and phospholipids such as soybean lecithin, in addition to the aforementioned low-phytin vegetable protein hydrolysates and other proteins. Can be contained.

n-3 polyunsaturated fatty acids are essential for saltwater fish In addition, feed-based phospholipids such as soy lecithin can be included in the feed of the present invention since they are necessary components for larval and young fish culture.

The form of the feed of the present invention is preferably such that it has a particle size, buoyancy, sedimentation rate that is easy for fish to ingest, does not elute nutrients in water, and is digested and absorbed in the gastrointestinal tract. For larvae, it is preferable to prepare a fine particle feed by microencapsulation or the like.

As a method for encapsulating a low-phytin plant protein hydrolyzate having an average molecular weight of 200 to 100,000 by micro-mouth, for example, a method of spray-drying an aqueous solution of the hydrolyzate can be adopted. In addition, fats and oils can be added before spraying to adjust the dissolution, floating and dispersibility in seawater, if necessary. Alternatively, it can be prepared.

The feed of the present invention can be used in combination with a biological feed alone or in an appropriate amount at intervals of 30 minutes to 1 hour during the larval stage depending on the age of the larva.

Next, one of the methods for producing the low-phytin plant protein hydrolyzate used for the above larvae and fry feeds is described below.

(Plant protein raw material)

Known plant proteins can be used as the plant protein used in the present invention. Grains and oils are easily available, and soy protein is one of the preferred protein raw materials since soy protein is industrially produced in large quantities. It is.

Hereinafter, an example using a soybean protein containing a small amount of phytic acid will be described, but this method can be applied to other plant proteins.

The soybean protein raw material used for producing the low-phytin vegetable protein hydrolyzate of the present invention is a soybean protein such as soymilk (including skim soybean milk; the same applies hereinafter), concentrated soymilk, concentrated soybean protein, isolated soybean protein, defatted soybean, and the like. It is possible if it includes. Defatted soybeans are processed with little or no protein denaturation So-called low-denatured defatted soybeans that have been treated are preferred, and are not limited to varieties, production areas, and the like. In general, defatted soybeans which have been subjected to low-temperature extraction using n-hexane as an extraction solvent are suitable as raw materials, and particularly low denaturing with an NSI (nitrogen solubility coefficient) of 60 or more, preferably 80 or more. Defatted soy is preferred.

(Enzymatic degradation of soy protein)

Enzymatic degradation of soy protein can be obtained by hydrolyzing soy protein in an aqueous system (soy protein slurry or solution) using an enzyme.

For example, when low-denatured soybean protein is used, the concentration of the soybean protein solution to be subjected to the enzyme treatment is 1% by weight to 30% by weight, preferably 5 to 15% by weight, and more preferably 8 to 12% by weight. Appropriate. Even if the concentration is low, there is no problem in enzymatic decomposition, but productivity is unfavorably reduced.

The proteolytic enzyme (protease) used in the present invention may be exoprotease or endprotease alone or in combination, and may be of animal, plant or microbial origin. Specifically, serine proteases (trypsin, chymotoribsin derived from animals, subtilisin derived from microorganisms, lipoxypeptidase, etc.), thiol proteases (papine, ficin, bromelain, etc. derived from plants) And carboxyprotease (such as animal-derived pepsin) can be used. More specifically, “Protin FN” derived from Aspergillus oryzae (manufactured by Daiwa Kasei Co., Ltd.); “Alcalase” (manufactured by NOPO) and “Protin A” (manufactured by Daiwa Kasei Co., Ltd.) derived from Bacillus subtilis. Examples of endoproteinase-containing enzymes include “Protease S j” and “Protin AC-10” manufactured by Amano Pharmaceutical Co., Ltd., and “Protin AC-10” Biopase (Nagase Seikagaku Corporation) ) And Amino Pharmaceutical Co., Ltd., as a proteolytic enzyme containing exo and endoprotease. Ze "can be exemplified.

The hydrolysis conditions of the present invention vary somewhat depending on the type of protease used, but generally, hydrolysis of soybean protein is performed in the pH range, the temperature range, and the optimal reaction time of the protease. It is preferred to use a sufficient amount. When considering the use of a salt-restricted diet (for example, a tube feeding diet) together with the lipid metabolism improver, if the pH is 5 to 10, preferably 6 to 9, the salt by neutralization is used. It is preferable because the generation of can be reduced.

The degree of hydrolysis is suitably an average molecular weight of from 200 to 100,000, preferably from 300 to 500,000. The degree of hydrolysis can be adjusted depending on the purpose and use. For example, in the case of feed or feed, there is no problem even with a relatively large molecular weight, but when used as a feed for fish farming, when used as a feed for larvae and larvae, a low molecular weight that is easy to digest is preferable, and the average molecular weight is It is suitably from 200 to 500, more preferably about 200 to 2000. (Phytic acid degradation by phytic acid degrading enzyme)

Examples of the enzyme for decomposing phytic acid used in the present invention include enzymes derived from plants such as wheat and potato or enzymes derived from animal organs such as intestinal tract, bacteria, yeast, mold, and enzymes derived from microorganisms such as actinomycetes. Enzymes such as phytase-phosphatase having phytic acid decomposition activity can be used.

As the enzyme for decomposing phytic acid, phytophosphatase is suitable, but phytase is more preferable. Fischerichia can be used from various strains having a phytase-producing ability, such as Aspergillus, Rhizopus, Saccharomyces, Mucor, and Geotricum. Preferably it is derived from the genus Aspergillus, more preferably the genus Aspergillus: a phytase derived from Aspergillus ficuum and a phytase derived from Aspergillus niger. Ze and Aspergillus Terreus The phytase from (Aspergillus terreus) can be selected from just the group. In order to decompose phytic acid in soybean into inositol, it is necessary to cleave the ester group, and the enzyme that does this is phytase.

It is also possible to use a fungal acid phosphatase as the acid phosphatase. That is, it is selected from the group consisting of acid phosphatase derived from Aspergillus ficuum, acid phosphatase derived from Aspergillus niger, and acid phosphatase derived from Aspergillus terreus. I can do it.

The degradation of phytic acid by enzymatic treatment can be carried out under very mild conditions, and therefore has very little effect on proteins. For example, the enzyme reaction of the present invention may be performed at 30 to 60 ° C. for 0.1 to 30 hours.

In the present invention, the pH during the phytic acid decomposition reaction is particularly important, and the pH is preferably 6 to 9, preferably 6.2 to 8.5, more preferably 6.2 to 7 pH. Soy protein treated at a pH of less than 6.0 is not preferred because its solubility is reduced and the flavor becomes worse. Further, if the pH exceeds 9.0, the flavor deteriorates, which is not preferable. By decomposing phytic acid within the above pH range, it is possible to produce a soybean protein with better reduced phytic acid.

Therefore, the enzyme suitably used in the present invention is preferably an enzyme capable of decomposing phytic acid and phytate in a neutral to alkaline pH range of pH 6 or more, but the origin is not particularly limited and the above-mentioned enzymes are not limited. You can use enzymes.

The enzyme can be used irrespective of the form of powder or liquid. The enzyme is used in an amount of about 0.01 to 10% by weight, preferably about 0.05 to 2% by weight, more preferably about 0.1 to 1% by weight based on the weight of the crude protein in the soybean protein. The enzyme titer is 0.1 to 100 U / g crude soy protein, preferably 0.5 to 20 U / g crude soy protein, more preferably Preferably, phytase of about l to 10 U / g crude soybean protein is added. The enzyme activity was determined by reacting a reaction mixture consisting of 0.4 ml of 0.2 M Tris-HCl buffer containing 4 mM sodium phytate (pH 6.5), 0.5 ml of distilled water, and 0.1 ml of enzyme solution at 37 ° C for 30 minutes. After the reaction, add 10% TCA 1. Oml to stop the reaction. The inorganic phosphoric acid content in this reaction solution was determined by the Fiske-Subbarow method. The amount of enzyme that releases 1 μηι 1 of inorganic phosphoric acid per minute under the above conditions was defined as 1 unit (U).

In the present invention, as long as the method includes a step of decomposing a protein using a protease and a step of decomposing phytic acid using an enzyme that decomposes phytic acid, the order may be combined in any manner. Through these steps, phytin can be reduced, that is, the phytic acid content in the plant protein hydrolyzate can be reduced to 0.5% or less and 0.2% or less per dry solid content. Furthermore, in order to reduce the phytic acid content to a detection limit of 5 mg / 100 g or less, it is necessary to subject the protein to a phytase treatment after enzymatic degradation of the protein. It is more preferable in the production of On the other hand, even if the protein is subjected to phytase treatment and then subjected to enzymatic decomposition, it is difficult to reduce the phytic acid content to a detection limit of 5 mgZ10Og or less. However, if undenatured soybean protein is used as described above, phytase is allowed to act on the soybean protein solution (not powder-dried) to decompose phytic acid as described above, and then, as described above, Even when decomposed, a target low-phytin plant protein hydrolyzate having a phytic acid content below the detection limit can be obtained.

The average molecular weight of the low phytin plant protein hydrolyzate obtained as described above is preferably from 200 to 100,000, and more preferably from 300 to 500,000. The phytic acid content was determined by the vanadomolybdate spectrophotometry (detection limit: 5 mgZl 0 g) in the dry solid content of the low phytin vegetable protein hydrolyzate. 5% or less, preferably one in which phytic acid is not detected.

(Example)

Hereinafter, embodiments of the present invention will be described with reference to examples.

First, the bait is explained.

[Production Example 1] (Production of low phytic acid soybean protein hydrolyzate)

Water is added 7 times to 10 parts by weight of defatted soybeans, and extracted while stirring at 50 ° C and pH 7 for 30 minutes.Okara and soymilk are separated by a centrifugal separator, and the soymilk is adjusted to pH with sulfuric acid. After adjusting to 4.5, centrifuged and separated into isoelectric protein and whey protein.After adding 4 times water to isoelectric protein and adjusting to pH 6.0 with NaOH. Then, 50 parts by weight of an 8% concentration soybean protein solution was prepared.

This soybean protein solution was heated to 50 ° C., and 0.04 parts by weight of phytase-degrading enzyme “Sumiteam PHY” manufactured by Shin Nippon Chemical Co., Ltd. was added and reacted for 60 minutes. The reaction solution was sterilized at 150 ° C for 7 seconds, cooled to 50 t, adjusted to pH 7.0 with Na〇H, and proteases ”from Amano Pharmaceutical Co., Ltd. 0.16 Parts by weight were added and reacted for 5 hours. The pH was adjusted to 6.5, sterilized at 150 ° C for 7 seconds, and immediately powder-dried with a spray dryer.

The content of phytic acid (mesoinositoxyhexalic acid) in this powder was not detected when measured by vanadomolybdic acid absorption spectrophotometry (detection limit: 5 mg Z100 g). The average molecular weight measured by electrophoresis was about 500.

[Production Example 2] (Production of soybean protein hydrolyzate without phytic acid removal) Separate soybean protein (“Fujipro-R” manufactured by Fuji Oil Co., Ltd.) Protin FN (Daiwa Kasei Co., Ltd.) Manufactured by Aspergillus) One part was used to decompose the enzyme at 50 ° C for 5 hours, heated at 70 ° C for 30 minutes to deactivate the enzyme, cooled, and centrifuged. The supernatant was spray-dried to produce a soybean protein hydrolyzate. This product had a TCA (trichloromouth acetic acid) solubility (15% TCA soluble nitrogen Z total nitrogen value multiplied by 100) of 100 and an average molecular weight of 100%. 6 7 6

[Example 1 and Comparative Example 1]

• Example 1

38 Five-day-old larvae and larvae were transferred from the preliminary breeding aquarium to a 300-liter, 100-L experimental aquarium. The temperature of the seawater was maintained at 17 ° C during the experiment.

A low-phytin soybean protein hydrolyzate produced in the same manner as in Production Example 1 was used as an experimental feed in the aquaculture feed of Table 1 in an amount of 0, 2.5, 5.0, 10.0, and 20.0 parts by weight of casein. And added as shown in Table 1 to obtain a fine particle feed by a conventional method. Table 1 shows the composition of the experimental feed. The unit is parts by weight.

Group 5 is the control.

The experiment was started at the age of 39 days, and the animals were fed eight times an hour from 9:00 am until 16 o'clock, and at 17 o'clock, fed Artemia as a biological feed at nine times a day. The feed amount was 0.31 g-0.50 g / time for Z fish, and 55 to 80 animals / time for Artemia, as the age of day passed.

On the 14th and 52th days after the start of the experiment, about 50 larvae and juveniles were measured for their length, number of surviving tails, and pigmentation rate.

• Comparative Example 1

As an experimental feed, a soybean protein hydrolyzate without phytic acid removal produced in the same manner as in Production Example 2 was used, and the same method as in Example 1 was used. Farmed inflammation

(table 1 )

Table 2 shows the results of Example 1 and Comparative Example 1.

(Table 2) 52 days old results

From the results of Example 1, it was found that the growth of the 14 group to which the low-phytate soybean protein hydrolyzate was added was promoted as compared to the 5 group of the control, and a significant difference was observed particularly in the 13 group. . The survival rate was better in the 14 group than in the 5 group. The pigment abnormality rate did not differ between the groups, and no abnormality was observed in the group to which the soybean protein hydrolyzate was given.

In addition, the survival rate of the soybean protein hydrolyzate (oligopeptide mixture) having a lower phytic acid content was significantly higher than that of Comparative Example 1 below. (Production Example 3)

Separated soybean protein (Fuji Oil Co., Ltd. “New Fuji Pro-I”) 100 parts by weight was dissolved in 900 parts of water, and proteolytic enzyme (Dainkasei Co., Ltd. “Protin”) was added to 2 parts. After incubating at 50 ° C for 5 hours, insoluble materials were removed by centrifugation (5000 rpm × 30 minutes), and the mixture was heated at 80 ° C for 30 minutes to inactivate and sterilize the enzyme. Lyophilization yielded an enzyme digest (SH).

An acrylic weakly basic anion exchange resin (“KA890” manufactured by Sumitomo Chemical Co., Ltd.) is packed into a column with a diameter of 1.4 cm to a height of 15 cm (resin volume 23 cm2), and 50 ml of a 5% sodium hydroxide solution and ion exchange. Washing was carried out by passing 500 ml of water. On the other hand, the above-mentioned enzyme hydrolyzate (SH) was dissolved in ion-exchanged water, the pH was adjusted to 4.5 with 10% hydrochloric acid, and adjusted with ion-exchanged water so that the final protein concentration was 10%. .

The above-prepared solution was passed at 51 ml / hr from the top of the column which had been filled with KA890 and washed, and the treatment solution eluted from the bottom of the column was collected. Pass the enzymatic digestion solution through until the volume reaches 1219 ml (121.9 g protein, 5.3 g per ml resin), that is, phytic acid is completely converted to resin. The eluate that had been adsorbed and removed was collected and freeze-dried to obtain a low phosphorus content enzyme digest (SHR). Phytic acid content was measured by the method of Mohammed et al. (Cereal Chem. 63. 475.1986). As a result, phytic acid was not detected (detection limit: 0.005% by weight).

(Example 2 and Comparative Example 2)

Using the low-phytin soybean protein hydrolyzate obtained in Production Example 3 and the soybean protein hydrolyzate from which Phytic acid has not been removed obtained in Production Example 2, the fertilized egg obtained from the parent shrimp is hatched. Pre-bred up to Zoea 1 stage A breeding experiment was performed using

As shown in Table 3, 100 zoos of Zoeal were housed in a 1 liter beaker and bred at room temperature using a particulate feed. The composition of the test feed is as shown in Table 4.

As a test feed, 10.0 parts by weight of each soybean protein hydrolyzate produced in the same manner as in Production Example 3 and Production Example 2 was added to the prawn cultivated feed shown in Table 4 by replacing casein (control) with 10.0 parts by weight. This was used as a fine particle feed.

m

The effect of addition was determined based on the growth index of the larva (Growth index: reached growth stage) and the survival rate. The results are as shown in Table 5. G

(Table 3) Rearing conditions of prawns

Condition Description

Larva (at the start of breeding) Stage of Zoea 1

Rearing time Until the post-larvae is reached Rearing density 100 fish / tank (ll)

Water temperature

Rearing water pH 8.4 ± 0.2

100% / day

Feeding frequency 2 times / day

Feed particle size

Zoeal stage 53μΠ1

Mys s stage 125μΙ11

Pos t-larvae 250μηι

Feeding rate (mg feed / Larva / day)

Zoeal stage 0.16

Mys is stage 0.20

Pos t-larvae 0.24 (Table 4) Composition of test feed

1 (2 0 ω 3) (2 2 3) = 3

(Table 5) Results of breeding experiments on prawns

Growth index = (lnl + 2n2 + 3n3 + 4n4

+ 5n5 + 6n6 + 7n7) / N (where N = nl + n2

+ · · ·-+ N7. )

nl, n2, n3, n4, n5, n6 and n7 are Zoea 1, Zoea2,

Zoea 3, My sis 1, My sis 2, My sis 3 and Post larvae

1 Indicates the number of larvae. As a result, the body length (measured on the 11th day) of the bostora was different from that in the case where the low-fitin soybean protein hydrolyzate-added group in Production Example 3 was added or that in the case where the soybean protein hydrolyzate-added group in Production Example 2 was added. There was no difference between them, but the survival rate and growth index The low phytin soybean protein hydrolyzate-added group tended to be good.

(Example 3)

From 70 to 80 days after birth, three larvae were transferred to a 20-L experimental aquarium with 20 fish per larva per group, and breeding experiments were performed. The breeding water temperature was maintained at 18: during the experiment.

The experimental feed was prepared based on a commercially available flounder feed (EP feed manufactured by Higashimaru Co., Ltd., S-6 for seeds and seedlings). That is, a commercially available feed is heated, and the low-phytin soybean protein hydrolyzate produced in Production Example 1 and the unfiltered soybean protein hydrolyzate produced in Production Example 2 are attached thereto. After coating, it was dried and used as experimental feed. Table 6 shows the composition of commercial feed and the composition of experimental feed supplemented with soybean protein hydrolyzate.

(Table 6)

(Composition of commercial feed)

7 5.5% fish meal, oak milk

1 2.7% flour, starch, corn flour

1 1.8% feed yeast, refined fish oil, soy lecithin, yeast extract, calcium dihydrogen phosphate, trace elements, vitamins

(Composition of soybean protein hydrolyzate supplemented feed)

6 7.5% fish meal, krill meal

10% soy protein hydrolyzate

1 1.7% flour, starch, corn flour

1 0.8% feed yeast, refined fish oil, soy lecithin, yeast extract, calcium dihydrogen phosphate, trace elements, vitamins

(Composition of low phytin soybean protein hydrolyzate supplemented feed)

6 7.5% fish meal, krill meal

10% low phytin soy protein hydrolyzate

1 1.7% flour, starch, corn flour

10.8% feed yeast, refined fish oil, soy lecithin, yeast extract, calcium dihydrogen phosphate, trace elements, vitamins 80.3 mm in average length, 4.4 g fish weight, body weight / Total length = 5 5 The experiment was started using 5 mg / mm fry, and the animals were fed four times a day every 6 hours from 9 am. The body length and fish weight of the 60 larvae and larvae 10 days and 20 days after the start of the experiment were measured, and the weight / total length was calculated from the measurement results. (Table 7) Day 0 of measurement (60 fish tested)

Remarks: Changes in parentheses are for comparison

(Table 8) Day 10 of measurement start (60 test fish, 60 survivors)

Remarks: Changes in parentheses are for comparison (Table 9) Day 20 of measurement start (60 test fish, 60 survivors)

Remarks: In parentheses, the increase / decrease from the control group. As a result, the average body weight Z-body length (mg Zmni) at day 10 was lower in the control group and in the group with low phytin soybean protein hydrolyzate than in the group with soybean protein hydrolyzate. The growth was high and the effect of promoting growth was recognized. In addition, on day 20 the largest individual in weight / length (mg / mm) was also observed. The minimum value was higher than that of the control group and the addition of soybean protein hydrolyzate, but the solids also varied widely due to the concentration of food in large fish. It became.

(Breeding progress)

The water temperature, feeding amount, and feeding status during the rearing period are described below.

In the control group, the water in the aquarium became turbid due to diarrhea in the feces after feeding in the control group, whereas the feces in the group in which the soybean protein hydrolyzate and low phytin soybean protein hydrolyzate were added had a shape. The water in the aquarium remained clean, with soft stool maintained. In addition, the low phytin soybean protein hydrolyzate tended to have more dung and more dung. (Table 10)

Next, examples of the low-phytin soybean protein hydrolyzate will be described.

(Example 4)

Water is added seven times to 10 kg of defatted soybeans, extracted after stirring at 50 ° C and pH 7 for 30 minutes, and then separated from okara and soymilk by a centrifugal separator. After adjusting to 4.5, centrifuged and separated into isoelectric precipitate protein and whey protein.After adding 4 times water to the isoelectric precipitate protein, adjusting the pH to 7.0 with NaOH, 50 liters of an 8% protein solution was prepared.

To this protein solution, 160 g of each of the proteolytic enzymes “proteases” and “protease MJ (manufactured by Amano Pharmaceutical Co., Ltd.) were added, reacted at 50 ° C. for 5 hours, and hydrolyzed (1). Then, the pH was adjusted to 6.0, and 40 g of phytate-degrading enzyme “Sumitim P HY” (manufactured by Shin Nippon Chemical Co., Ltd.) was added. After the addition, the mixture was reacted at 50 ° C for 60 minutes, adjusted to pH 6.5 with NaOH, sterilized at 150 ° C for 7 seconds, and immediately dried with a spray dryer.

The content of phytic acid (mesoinositoxyhexalic acid) in this powder was not detected (detection limit: 5 mg / 100 g) by vanadomolybdic acid absorption spectrophotometry, and phytic acid was reduced very well. did it.

(Example 5)

Water is added 7-fold to 10 kg of defatted soybeans, extracted with stirring at 50 and pH 7 for 30 minutes, and then separated into okara and soymilk by a centrifugal separator. After adjusting to 5 and centrifuging to separate the isoelectric precipitate protein and whey protein, add 4 fold water to the isoelectric precipitate protein, adjust to pH 6.0 with Na〇H, and adjust to 8% A concentration of 50 liters of protein solution was adjusted.

This protein solution was heated to 50 ° C., and lipase-degrading enzyme “Sumitim PHY” was added with 40 g from Shin Nippon Chemical Co., Ltd., and allowed to react for 60 minutes. The pH was adjusted to 7.0 with H, and 160 g of “Proteases” (Amano Pharmaceutical Co., Ltd.) was added and reacted for 5 hours. The pH was adjusted to 6.5, sterilized at 150 ° C for 7 seconds, and immediately dried with a spray drier.

The content of phytic acid (mesoinosittohexalic acid) in this powder was not detected by vanadomolybdic acid absorption spectrophotometry (detection limit: 5 mg / 100 g), reducing phytic acid extremely well did it.

(Example 6)

Water is added 7-fold to 10 kg of defatted soybeans, extracted at 50 :, pH 7 with stirring for 30 minutes, separated into okara and soymilk by a centrifuge, and the soymilk is PH4 After adjusting the pH to 5, centrifuged and separated into isoelectric precipitate protein and whey protein.After adding 4 fold water to the isoelectric precipitate protein, adjusting the pH to 6.0 with NaOH, 8 50 liters of a 50% protein solution was adjusted and immediately powder-dried with a spray dryer. This protein solution was adjusted to an 8% solution, heated to 50 ° C, added with 40 g of Sumitomo P HY, a protease degrading enzyme, manufactured by Shin Nippon Chemical Co., Ltd., and reacted for 60 minutes. After the reaction, the pH was adjusted to 7.0 with NaOH, and 160 g of "Proteases" (manufactured by Amano Pharmaceutical Co., Ltd.) was added, followed by reaction for 5 hours. The pH was adjusted to 6.5, sterilized at 150 ° C for 7 seconds, and immediately dried with a spray drier.

When the content of phytic acid (mesoinosittohexalic acid) in this powder was measured by vanadomolybdic acid spectrophotometry, phytic acid was reduced to 0.5% (detection limit: 5 mg / 100%). 0 g).

(Example 7)

Add 7 times water to 10 kg of defatted soybeans, and stir at 50 and pH 7 for 30 minutes. After extracting the okara and soymilk using a centrifuge, the soymilk was adjusted to pH 4.5 with sulfuric acid, centrifuged, separated into isoelectric precipitation protein and whey protein, and then isoelectrically precipitated. After four-fold water addition to the protein, the pH was adjusted to 6.0 with NaOH, and 50 liters of an 8% protein solution was prepared.

This protein solution was heated to 50 ° C, and the phytase-degrading enzyme “Sumitim PHY”, 40 g, manufactured by Shin Nippon Chemical Industry Co., Ltd. was added and reacted for 60 minutes. Was dried with a spray dryer.

The solution was adjusted to 8%, adjusted to pH 7.0 with NaOH, and added with 160 g of Proteaze M "Amano Pharmaceutical Co., Ltd., and allowed to react for 5 hours. After sterilization for 7 seconds, the powder was immediately dried with a spray dryer.

When the content of phytic acid (mesoinosittohexalic acid) in this powder was measured by vanadomolybdic acid spectrophotometry, phytic acid was reduced to 0.2% (detection limit: 5 mg / min. g).

(Example 8)

Water is added seven times to 10 kg of defatted soybeans, extracted after stirring at 50 ° C and pH 7 for 30 minutes, and then separated from okara and soymilk by a centrifugal separator. After adjusting to 4.5, centrifuged and separated into isoelectric precipitate protein and whey protein.After adding 4 times water to the isoelectric precipitate protein, adjusting the pH to 6.0 with NaOH, 50 liters of an 8% protein solution was prepared.

Heat this protein solution to 50 ° C, add 40 g of Sumiteam PHY, a enzyme degrading enzyme from Shin Nippon Chemical Co., Ltd., react for 60 minutes, and spray immediately. The powder was dried with a dryer.

This solution was adjusted to 8%, adjusted to pH 7.0 with NaOH, and added with 160 g of Protease (Amano Pharmaceutical Co., Ltd.) and reacted for 5 hours (pH 6. 2) Then, again, phytase-degrading enzyme "Sumiteam P HY" After adding 40 g manufactured by Nippon Kagaku Kogyo Co., Ltd. and reacting for 60 minutes, the mixture was sterilized at 150 ° C. for 7 seconds and immediately dried by a spray drier.

The content of phytic acid (mesoinosittohexalic acid) in this powder was measured by vanadomolybdic acid spectrophotometry, and it was found that phytic acid was reduced to 0.2% (detection limit: 5 mg / 1 0 0 g).

(Example 9)

Once powder-dried isolated soy protein (Fuji Oil Co., Ltd., “New Fuji R”) 30 kg was converted to a 10% aqueous solution with a pH of 7.0, and the proteolytic enzyme “Puchi Tease” Amano Pharmaceutical 1.2 kg of Protease M and 0.3 kg of Amano Pharmaceutical Co., Ltd. were hydrolyzed at 50 ° C for 5 hours (15%, 8% solubility). Thereafter, the pH was adjusted to 6.0, and 0.6 kg of the fuzease-degrading enzyme “Sumiteam P HY” manufactured by Shin Nippon Chemical Co., Ltd. was added, followed by hydrolysis at 45 ° C for 2 hours. .

The decomposition solution was adjusted to a high-speed centrifugal separator capable of continuous processing at a feed speed of 100 liters, and the sedimentation component generated was separated and removed. The obtained centrifugal supernatant (solid content: 70%) was adjusted to pH 6.5, sterilized at 150 ° C for 7 seconds, and immediately dried by a spray dryer.

The content of phytic acid (mesoinosittohexalic acid) in this powder was not detected by vanadomolybdic acid absorption spectrophotometry (detection limit: 5 mg / 100 g). Could be reduced.

Industrial applicability

By feeding the feed of the present invention, the growth of larvae and larvae can be promoted, and the survival rate can be greatly increased.

In particular, it may be possible to increase the survival rate of larvae and fry that are difficult to fish. In this way, fish cultivation, which has been difficult to fish conventionally, has become possible. It also promotes the growth of fry that has grown to some extent, makes the dung sticky, prevents water turbidity, and improves the growth environment. In addition, a low-phytin plant protein hydrolyzate suitable for such a quantity has become possible.

That is, according to the present invention, a low-phytin vegetable protein hydrolyzate having an extremely low phytic acid content and being below the detection limit by vanadomolybdic acid absorption spectrophotometry has become possible. In addition, the low-phytin vegetable protein hydrolyzate according to the method of the present invention has an excellent flavor (it is presumed to be due to the decomposition of phytic acid) as compared with the low-phytin soybean protein hydrolyzate obtained by the resin adsorption method.

In addition, the low-phytin plant protein hydrolyzate of the present invention has a small molecular weight and extremely low phytic acid, so that it can be digested and absorbed when used in fry with insufficient development of digestion and absorption functions or animal feeds immediately after birth. It is highly effective and promotes the absorption of trace metals such as calcium.

Claims

The scope of the claims

A feed for larvae and larvae, comprising a plant protein hydrolyzate having a phytic acid content of 0.05% by weight or less (in dry solid content) as a feed material.

2. The feed for larvae and larvae according to claim 1, wherein the plant protein hydrolyzate is a plant protein hydrolyzate having an average molecular weight of 200 to 100,000.

(3) A fine-particle diet encapsulated in a micro-mouth so that the form of the feed is easy to ingest for larvae and larvae, and has a particle size, buoyancy, sedimentation rate, and does not elute nutrients in water and can be digested and absorbed in the digestive tract. A feed for larvae and larvae according to any one of claims 1 and 2.

4. A low-phytin protein protein hydrolyzate characterized by comprising (a) a step of degrading a protein using a protease and (b) a step of degrading phytic acid using an enzyme that degrades phytic acid. Production method of decomposition products.

5. The method according to claim 4, wherein the soybean protein is decomposed using a protease, and then phytic acid is decomposed using an enzyme that decomposes phytic acid.

6. The method according to claim 4, wherein the undried soybean protein is decomposed using an enzyme that decomposes phytic acid, and then the protein is decomposed using a protease.

7. The method according to any one of claims 4 to 6, wherein the enzyme that degrades phytic acid is phytase.

8. The process according to any one of claims 4 to 7, wherein the pH to be filtered is 6 to 9.

9. The method according to any one of claims 4 to 8, wherein the low phytin plant protein hydrolyzate has an average molecular weight of 200 to 100,000.

10. The phytic acid content in the low-phytin plant protein hydrolyzate is not detected by vanadomolybdic acid spectrophotometry (detection limit 5 mg Z 100 g) per dry solid content. Any manufacturing method.