JP2012532629A - Marine-derived Bacillus barbaricus SCSIO02429 and method for preparing squid oligopeptide using the same - Google Patents

Abstract

The present invention relates to a marine-derived Bacillus barbaricus SCSIO 02429 CCTCC NO: M 2010213. The present invention also relates to a method for preparing a squid oligopeptide, characterized by adding Bacillus barbaricus SCSIO 02429 to an enzyme-producing fermentation-inducing medium and fermenting it to obtain a crude enzyme solution for hydrolysis. Crushed and added to the crude enzyme solution. Enzymatic digestion is performed to obtain a crude protein enzyme degradation product in which the glycosyl side chain is destroyed. The oil layer and the aqueous layer are separated, and the oil layer is removed and dried to obtain a squid oligopeptide. The yield of the squid oligopeptide of the present invention is 40 to 46%, and the yield of amino acids is 16 to 22%. This squid oligopeptide has the effect of lowering the seedling mortality rate of marine aquaculture animals and increasing the rate of weight gain, and it can replace conventional protein sources such as fish meal in the feed. Can be used as an additive.
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Description

  TECHNICAL FIELD The present invention relates to a Bacillus genus, and more specifically to a marine-derived Bacillus barbaricus SCSIO 02429 strain and a method for preparing a squid oligopeptide using this strain.

  With the rapid development of Japan's marine fishery, the annual production of squid has reached around 300,000 tons, making it one of Japan's important marine products. In the process of processing squid, about 15% of visceral waste is generated, and these wastes are rich in protein, but they are very easy to perish and deteriorate, and are difficult to store. In the usual treatment method, the waste from which squid oil has been extracted is buried, but recently, there is a report that a squid slurry is primarily processed to produce a squid slurry and used directly in fish feed. However, feeds that have not undergone such advanced processing are not highly bioavailable, pollute the water environment, and increase the chances of disease outbreaks in farmed animals. For this reason, it is important to promote the development and use of squid internal organs from the viewpoint of economic and environmental protection. Research on enzymatic degradation methods of squid viscera has also attracted widespread attention. Using bioenzyme technology, squid crude proteins are easily absorbed by aquatic animals and decomposed into protein sources of oligopeptides with a certain physiological function. This is one effective method and several studies have been conducted. For example, a study on the effects of factors such as enzyme type, dose, reaction temperature, reaction time, etc. on the crude protein conversion rate of squid viscera (Patent Literature 1) by Lake Changchun, Liu Chunxi et al. This includes the production of sea flavor components using decomposition, and the research on the removal method of heavy metals in the enzymatic decomposition solution of squid viscera by Zhangi et al.

  In the already reported method for enzymatic degradation of squid viscera, the types of enzymes used include trypsin, pepsin, neutral protease, alkaline protease, papain, bromelain and mixtures of these enzymes. However, there are still problems with the hydrolysis method of squid viscera at this stage, such that the hydrolysis rate of the crude protein is low and the residual crude protein of the enzymatic degradation product is difficult to be absorbed and used by marine aquaculture animals. Moreover, if the degree of hydrolysis of the crude protein is increased by measures such as increasing the reaction temperature, extending the reaction time, or increasing the amount of catalyst used, this will cause excessive degradation of the protein, which will greatly increase the yield of the target oligopeptide. Go down. For example, there is a literature report that the yield of amino acids by hydrolysis of squid viscera exceeds 70%, but the yield of oligopeptides is extremely low. If the yield of the target oligopeptide can be ensured while enhancing the hydrolysis efficiency of the crude protein, the utilization level of the squid viscera will be greatly improved.

Liu Chun, Lin Hong, Cao Tong-min, Shun Shun Wei, “Studies on Enzymatic Hydrolysis Technology of Squid Visceral Protein” (China), Food Industry Technology, 2004, Vol. 25, No. 9, p. 83-85

  An object of the present invention is to develop a new Bacillus genus from the ocean, and another object is to enzymatically decompose the squid viscera using this Bacillus genus and to increase the hydrolysis rate of the crude protein. It is to provide a method for preparing a squid oligopeptide with a high peptide yield.

  We isolated Bacillus barbaricus SCSIO 02429 from deep sea sediments in the South Sea of China (South China Sea), and obtained a crude enzyme solution obtained by fermentation with Bacillus barbaricus SCSIO 02429. ) Side chains were broken and bromelain was added to catalyze the hydrolysis of protein peptide chains. The yield of the obtained squid oligopeptide was 40% to 46%, and the yield of amino acids was 16% to 22%, thereby realizing the object of the present invention.

  Bacillus barbaricus SCSIO 02429 is stored on August 31, 2010 at the China Center for Type Culture Collection (abbreviated as CCTCC), located in Wuchang, Wuchang, and storage number is CCTCC NO: M 2010213.

  The Bacillus barbaricus SCSIO 02429 was separated from the deep sea sedimentary environment (longitude: 119 ° 57.260 ′, latitude: 20 ° 59.877 ′, depth of 229 m) collected in 2009. It isolate | separated by the plate dilution method using the bacterial culture medium, and isolated by the streak method. Similarity analysis of the 16S rRNA nucleotide sequence revealed that there was a similarity of 99% (722/728 bp) with Bacillus barbaricus, which was differentiated from SCSIO 02429, one of the strains of Bacillus barbaricus species. This strain grows well at 28-37 ° C. in ISP2 medium (4.0 g yeast extract, 10.0 g wort, 4.0 g glucose, 20.0 g agar, pH 7.0 per liter distilled water) .

  In accordance with the standards, methods, and species classification characteristics of the “General Bacteriology Manual” and “Virges Bacteriology Manual”, tests such as bacterial morphology observation and physiological / biochemical tests were performed on the test strains. This strain also grows well in PYES medium (0.3% casein peptone, 0.3% yeast extract, 0.23% disodium succinate, pH 7.2) and is a brown, opaque, flat circular colony The maximum colony diameter is 5 mm. The colony has a complete boundary in the early stage of growth, but the boundary gradually disappears in the subsequent growth process. The cells are rod-shaped, the spores are oval, and the cells are gram positive. In PYES medium, all grow well at 28 to 37 ° C., and after 3 weeks of culture, obvious growth can be observed at 4 to 47 ° C. This bacterium can grow in a PYES medium containing 2% and 5% sodium chloride, and after culturing for 3 weeks, the growth can be observed in a PYES medium containing 12% NaCl. The strain can be observed to grow at pH 6.0, and can grow rapidly at pH 7.0, 8.0, and 9.5, indicating that this bacterium has alkali resistance. The results of the physiological and biochemical tests of the SCSIO 02429 strain are as shown in Table 1. “+” in the table indicates positive or usable, and “−” indicates negative or unavailable.

  SCSIO 02429 strain and the closest strain Bacillus barbaricus V2-BIII-A2 (references: Taubel M., Kampfer P, Buczolits S, Lubitz W, Busse H-J R. Bacillus barbolimus sp. wall painting. International Journal of Systemic and Evolutionary Microbiology, 2003, Vol. 53, p. 725-730. The characteristic number of Genus / EMBL / DDB42 in GenBank / EMBL / DDB42 is 21 in the Physiology 16S rDNA sequence. Mostly in Matching, but different D- ribose, citrate, utilization of sucrose, in the feature that the highest tolerable salt concentration of 5%. For this reason, SCSIO 02429 is distinguished from one of the strains of Bacillus barbaricus, and Bacillus barbaricus does not yet have a Chinese translation.

The preparation method of the squid oligopeptide of this invention is characterized by including the following procedures.
(1) Inoculating Bacillus barbaricus SCSIO 02429 into a bacterial species activation medium, culturing at 30-37 ° C. for 18-24 hours, and after activation, adding to a container containing an enzyme production induction fermentation medium, pH = 7 Fermentation is carried out for 12 to 24 hours under the conditions of 0.0 to 8.0 and 30 to 37 ° C., the protease activity of the fermentation solution reaches 2000 to 3000 units / mL, and the glycosaminoglycanase activity is 0.95 to 1.12. The step of obtaining a crude enzyme solution for glycosyl hydrolysis when the unit / mL is reached, wherein the enzyme production-inducing fermentation medium comprises squid visceral slurry, peptone, yeast extract, complex salt and water in a mass ratio of 20-30: It was prepared by mixing and heating at 5: 5: 10: 1000, and the composite salt was sodium chloride, potassium sulfate, magnesium chloride and salt Ammonium weight ratio 5-8: 2: 2: step is a mixture with 3,
(2) After crushing the squid viscera raw material into a slurry, it is mixed with the crude enzyme solution described in the procedure (1) at a volume ratio of 1: 1 to 2: 1 to perform enzymatic decomposition, and the pH at the start of the reaction is 6.5. -7.0, temperature is 40-50 ° C, time is 5-8 hours, obtaining a crude protein enzyme degradation product in which the glycosyl side chain is broken; and
(3) Bromelain is added to the crude protein enzyme degradation product obtained in step (2) at a ratio of adding 1000-1500 units (enzyme activity) of bromelain per gram of squid viscera, and the pH is adjusted to 6.5-7.0. And the reaction is carried out at 35 to 40 ° C. for 4 to 6 hours. The obtained enzyme degradation product is left standing, separated into a crude fat layer and an aqueous layer, the fat layer is removed and dried, and the squid oligopeptide is removed. Obtaining step.

  The drying described in the procedure (3) may be spray drying. The peptone and yeast extract used in the present invention are commercially available.

  The present invention destroys the glycosaminoglycan side chain connected to the peptide chain in the visceral protein by enzymatic degradation of the crude enzyme produced by Bacillus barbaricus SCSIO 02429, and protects the protein peptide chain from hydrolysis by the glycosyl group. In mild conditions that prevent excessive hydrolysis of the reaction, the yield of squid oligopeptide reaches 40-46% and the yield of amino acid reaches 16-22%. This squid oligopeptide has the effect of lowering the seedling mortality rate of marine aquaculture animals and increasing the weight gain rate, and can be replaced by conventional protein sources such as fish meal in feed. Moreover, since it has a certain physiological activity and can significantly increase the weight gain rate and survival rate of aquatic animals, it can be used as a functional protein source, an additive and the like for a marine aquaculture feed.

  The following examples are further illustrations of the present invention and are not a limitation on the present invention.

  In the examples, Bacillus barbaricus SCSIO 02429 is stored in the Chinese Typical Culture Storage Center, and the storage number is CCTCC NO: M 2010213. The squid used was purchased from the Guangzhou Huangsha seafood market, identified as squid (Ommastrefeth bartrami) by differentiation, the internal organs were collected, stored at -18 ° C, and thawed at 4 ° C 24 hours prior to use. The bromelain used was produced by Nanning Panbo Biotechnology Co., Ltd., with 800,000 units (enzyme activity) / g. Peptone was produced by Oxide, and yeast extract was purchased from Ryukyuten Biotechnology Institute.

  To 1 L of water, 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride and 15 g of agar were added to adjust the pH to 7.0 to obtain a bacterial species activation medium.

  Take 100 g of squid visceral slurry, 25 g of peptone, 25 g of yeast extract and 50 g of complex salt, add 5000 mL of deionized water, heat to dissolve, adjust pH to 7.0, transfer to fermenter, steam sterilize The enzyme production fermentation medium was obtained. The complex salt is composed of 20.9 g of sodium chloride, 8.3 g of potassium sulfate, 8.3 g of magnesium chloride and 12.5 g of ammonium chloride.

  The bacterial species of Bacillus barbaricus SCSIO 02429 was inoculated into the bacterial species activation medium and cultured at 30 ° C. for 24 hours. After activation, the bacterial species is added to a fermentor containing 5 L of enzyme-producing fermentation medium, and fermented for 24 hours under the conditions of pH = 7.0, 30 ° C., stirring rate 100 r / min, and ventilation rate 0.2, and protease activity When 2000 units / mL and glycosaminoglycanase activity reached 0.95 units / mL, the fermentation was completed, and 4.7 L of a crude enzyme solution for hydrolysis was obtained.

  Method for measuring protease activity: The protease activity was measured by the method of the professional standard of the People's Republic of China “Protease activity measurement method SB / T 10317-1999”.

  Definition of unit of glycosaminoglycanase (enzyme activity): the amount of enzyme per minute required to form 1 μmol of glucosamine at 60 ° C., pH 5.2, 30 minutes of reaction. Method for measuring glycosaminoglycanase activity: Weigh out a certain amount of glycosaminoglycan, dissolve in 0.2 mol / L acetic acid solution, and adjust pH to 5.2 with 0.2 mol / L sodium acetate. And formulated into a 0.5% glycosaminoglycan solution. Inhale 1.5 mL of glycosaminoglycan solution, incubate at 60 ° C for 2 minutes, add 0.5 mL of enzyme solution, shake, react for 30 min, add 3 mL of potassium ferricyanide reagent to stop the reaction. The amount of reducing sugar was measured by the Imoto method, and the degradation rate of glycosaminoglycan was calculated.

  Take a 5 kg slice of squid viscera and grind it at 2000 r / min for 10 minutes using a homogenizer to obtain about 4.7 L of a paste, which is transferred to a fermentor containing the enzyme-producing fermentation product, The pH of the solution was adjusted to 6.5 with 2.0 mol / L acetic acid, the temperature was adjusted to 40 ° C., and the reaction was performed at a stirring rate of 100 r / min for 8 hours to obtain a crude protein enzyme degradation product in which the glycosyl side chain was broken. It was.

  Bromelain (6.25 g) was added to the crude protein enzyme degradation product in which the glycosyl side chain was broken, and the enzyme dose was adjusted to about 1000 units (enzyme activity) per gram of squid viscera material. After stirring until uniform, the reaction temperature was set to 40 ° C., the stirring speed was 50 revolutions / minute, the pH was 7.0, and the reaction time was 6 hours. After the reaction is completed, stirring is stopped, the product is allowed to stand to separate into an oil layer and an aqueous layer, the crude oil in the upper layer is removed, and sampling is performed to measure the yield of squid oligopeptide and amino acid yield. Were 40% and 16%, respectively. The enzyme degradation product was immediately spray-dried to obtain 853 g of squid oligopeptide powder.

The yield of oligopeptide and amino acid yield were measured by gel chromatography. Here, oligopeptide yield (%) = N ₂ / N ₁ × 100, amino acid yield% = N ₃ / N1 × 100, where N ₁ is the total amount of amino acid nitrogen contained in the enzymatic degradation solution, N ₂ Is the amount of amino acid nitrogen (g) contained in the effluent of peptides having a molecular mass of 300 to 1000 Da, and N ₃ is the amount of amino acid nitrogen (g) contained in the effluent of molecular weight 100 to 250 Da. N1 was measured by Kjeldahl nitrogen determination. Enzymatic degradation products are degreased, centrifuged to remove insoluble matter, weighed, dissolved in 0.2 mol / L sodium phosphate buffer, loaded onto Sephadex LH 20 gel column, and 0.2 mol / L sodium phosphate buffer. Elution was performed, and the effluent of each holding volume was collected in order, and then the molecular mass distribution of each effluent was measured by gel permeation HPLC. The combined spillage in 300~1000Da molecular mass range is an oligopeptide fragment, a measure of the amino acid nitrogen content in it in Kjeldahl nitrogen determination method is N _2. A combination of effluents within the molecular mass range of 100 to 250 Da is a free amino acid fragment, and N ₃ is the amount of amino acid nitrogen contained in the Kjeldahl nitrogen determination method. The molecular mass distribution of each effluent was measured by gel permeation HPLC. At this time, the calculation formula is V _e = −b′lgM _w + c ′, where V _e is the retention volume, M _w is the molecular mass, and b ′ and c ′ are constants. The constants b ′ and c ′ were equilibrated with a chromatographic column (PL aquagel-OH 30 8um, SEC, UK) at 1 mL / min using 0.2 mol / L sodium phosphate buffer, and constant at an absorbance of 214 nm. The sample was injected using a blue dextran solution, V ₀ (dead volume) was measured, the sample was injected using a glycine solution, V _t (total volume of the gel column bed) was measured, and the sample was prepared using a standard protein mixture. It is obtained by injecting, recording the elution volume V _e of various standard proteins at a flow rate of 1.0 mL / min, and creating a calibration curve of logarithm of molecular weight-V _e . Each effluent obtained by direct chromatographic separation on a Sephadex LH-20 column was used as a sample, the sample was injected, the retention volume _Ve was measured, and the molecular mass distribution range was calculated according to the formula.

  To 1 L of water, 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride and 15 g of agar were added, and the pH was adjusted to 7.0 to obtain a bacterial species activation medium.

  Take 150 g of squid slurry, 25 g of peptone, 25 g of yeast extract, 50 g of complex salt, add 5000 mL of deionized water, dissolve by heating, adjust pH to 8.0, transfer to fermentor, steam sterilize, An enzyme-producing fermentation medium was obtained. The complex salt in this consists of 26.6 g of sodium chloride, 6.7 g of potassium sulfate, 6.7 g of magnesium chloride and 10.0 g of ammonium chloride.

  Bacillus barbaricus SCSIO 02429 was inoculated into the activation medium and cultured at 37 ° C. for 18 hours. Once activated, the bacterial species is added to a fermentor containing 5 L of enzyme-producing fermentation medium and fermented for 12 hours under the conditions of pH = 8.0, 37 ° C., stirring rate 100 r / min, ventilation rate 0.2, and protease activity Was 3000 units / mL and the glycosaminoglycanase activity was 1.12 units / mL, the fermentation was completed, and 4.7 L of a crude enzyme solution for hydrolysis was obtained. Protease activity and glycosaminoglycanase activity were measured by the method of Example 1.

  Take a 10 kg slice of squid viscera and grind it at 2000 r / min for 10 minutes using a homogenizer to obtain a paste of about 9.4 L, transfer to a fermentor containing the enzyme-produced fermentation product, 2 The pH of the solution was adjusted to 7.0 with 0.0 mol / L acetic acid, the temperature was adjusted to 50 ° C., and the mixture was reacted at a stirring speed of 100 r / min for 5 hours to obtain a crude protein enzyme degradation product in which the glycosyl side chain was broken. .

  Bromelain (18.15 g) is added to the crude protein enzyme degradation product in which the glycosyl side chain is broken, and the enzyme dose is about 1500 units (enzyme activity) per g of squid viscera raw material. After stirring until uniform, the reaction temperature was set to 35 ° C., the stirring speed was 50 r / min, the pH was 6.5, and the reaction time was 4 hours. When the reaction is over, the stirring is stopped, the product is allowed to stand to separate into an oil layer and an aqueous layer, the crude oil in the upper layer is removed, and then the yield of the squid oligopeptide and the amino acid yield are measured by sampling. The results were 46% and 22%, respectively. The enzyme degradation product was immediately spray-dried to obtain 1630 g of squid oligopeptide powder. The oligopeptide yield and amino acid yield were calculated by the method of Example 1.

  Hybrid tilapia (Oreochromis niloticus x O. aureus) seedlings are provided by Guangdong Mandala non-fish seedling farm and are the same group of hybrid tilapia larvae on the day of hatching. Prior to the experiment, the larvae were first released into a 50 liter plastic tank and acclimatized for 2 days, and were not fed during the acclimatization period.

  Hybrid tilapia seedlings were divided into one control group and four experimental groups. Of these, the feed composition used for the control group is 46% fish meal, 15% malt root, 19% tapioca starch, 3% yeast, 1% soy lecithin, 0.5% choline, 0.5% calcium hydrogen phosphate, and 0% mixed vitamin .4%, formulated mineral salt 0.6%, cellulose 8%, soybean oil 1%, sodium alginate 1%, gelatin 4%, feed composition used in Experiment Group 1 was 41% fish meal, squid obtained in Example 1 Oligopeptide 5%, malt root 15%, tapioca starch 19%, yeast 3%, soybean lecithin 1%, choline 0.5%, calcium hydrogen phosphate 0.5%, formulated vitamin 0.4%, formulated mineral salt 0 .6%, cellulose 8%, soybean oil 1%, sodium alginate 1%, gelatin 4%, feed composition used in experimental group 2 is fish meal 41%, squid oligopeptide 5% obtained in Example 2, malt root 15 %, Pioca starch 19%, yeast 3%, soy lecithin 1%, choline 0.5%, calcium hydrogen phosphate 0.5%, formulated vitamin 0.4%, formulated mineral salt 0.6%, cellulose 8%, soybean oil 1%, sodium alginate 1%, gelatin 4%, feed composition used in Experimental Group 3 is fish meal 36%, squid oligopeptide 10% obtained in Example 1, malt root 15%, tapioca starch 19%, yeast 3% 1% soybean lecithin, 0.5% choline, 0.5% calcium hydrogen phosphate, 0.4% formulated vitamin, 0.6% formulated mineral salt, 8% cellulose, 1% soybean oil, 1% sodium alginate, Gelatin 4%, feed composition used in experimental group 4 is fish meal 36%, squid oligopeptide 10% obtained in Example 2, malt root 15%, tapioca starch 19%, yeast 3%, soybean lecithin 1%, coli 0.5%, 0.5% calcium hydrogen phosphate, formulated vitamins 0.4%, 0.6% Formulation mineral salts, cellulose 8%, 1% soybean oil, sodium alginate 1% and 4% gelatin. The corresponding feed composition was mixed in a high moisture condition to form a slurry, dried at 50 ° C. negative pressure (0.097 mPa), crushed, sieved to a particle size of 60-80 μm, and the resulting feed was 20 ° C. Stored in the refrigerator.

  An air stone was placed in the juvenile fish breeding tank, air was supplied for 24 hours, 50% of water was changed every day, waste at the bottom of the tank was sucked (sucked out), and the temperature was controlled at 27 ° C. with a thermostat. Each day, the diet containing 10% of the larvae's body weight was fed three times, and the experiment cycle was 21 days, which was repeated three times.

Mortality and weight gain were calculated by the following methods.
The weight of the hybrid tilapia (accurately to 0.001 g) and the number of juveniles are measured at the start and end of the experiment, respectively.
Mortality = (number of larvae at start of experiment-number of larvae at end of experiment) / number of seedlings at start of experiment x 100%
Absolute weight gain rate = (weight of young fish at the end of experiment (g) −weight of young fish at the start of experiment (g)) / number of days of experiment

  As a result, the mortality rate of the young fish in the control group was 53% ± 8%, the absolute weight gain rate was 0.0053 ± 0.0012 g / day, the mortality rate of the young fish in the experimental group 1 was 42% ± 10%, and the absolute weight gain rate. 0.0062 ± 0.0005 g / day, experimental group 2 larval mortality 45% ± 10%, absolute weight gain 0.0068 ± 0.0011 g / day, experimental group 3 larval mortality 38% ± 2%, absolute weight gain rate 0.0075 ± 0.0015 g / day, experimental group 4 larval mortality rate was 39% ± 6%, absolute weight gain rate 0.0069 ± 0.0012 g / day.

Claims (3)

  Strain of Bacillus barbaricus SCSIO 02429, Chinese typical culture storage center management number M 2010213. A method for preparing squid oligopeptides, comprising:
(1) The Bacillus barbaricus SCSIO 02429 bacterial species according to claim 1 was transferred to a bacterial species activation medium, cultured at 30 to 37 ° C. for 18 to 24 hours, and after activation, an enzyme production induction fermentation medium was added. In addition to the container, fermentation is carried out for 12 to 24 hours under the conditions of pH = 7.0 to 8.0 and 30 to 37 ° C., the protease activity of the fermentation solution reaches 2000 to 3000 units / mL, and the glycosaminoglycanase activity is A step of obtaining a crude enzyme solution for glycosyl group hydrolysis when reaching 0.95 to 1.12 units / mL, wherein the enzyme production induction fermentation medium comprises squid visceral slurry, peptone, yeast extract, complex salt and It is made by mixing and heating water at a mass ratio of 20-30: 5: 5: 10: 1000 and dissolving, and the complex salt is made of sodium chloride, potassium sulfate, chloride Magnesium and ammonium chloride weight ratio 5-8: 2: 2: step is a mixture with 3,
(2) After crushing the squid viscera raw material into a slurry, it is mixed with the crude enzyme solution described in the procedure (1) at a volume ratio of 1: 1 to 2: 1 to perform enzymatic decomposition, and the pH at the start of the reaction is 6.5. -7.0, obtaining a crude protein enzyme degradation product having a glycosyl side chain broken at a temperature of 40-50 ° C for 5-8 hours, and
(3) Bromelain is added to the crude protein enzyme degradation product obtained in step (2) at a ratio of 1000-1500 units (enzyme activity) of bromelain per gram of squid viscera, and the pH is adjusted to 6.5-7.0. Adjusting, reacting at 35 to 40 ° C. for 4 to 6 hours, allowing the obtained enzyme degradation product to stand to separate into a crude fat layer and an aqueous layer, removing the fat layer and drying to obtain a squid oligopeptide Including methods.   The method for preparing a squid oligopeptide according to claim 2, wherein the drying described in the procedure (3) is spray drying.