JPS6350992B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a solution composition comprising liquefied spirulina and a method for producing the same. More specifically, the present invention relates to a solution composition that is easily mixed and impregnated into animal feed and exhibits growth-promoting properties that further improve digestibility and absorption in the body, and a method for producing the same. Chlorella, a green alga, contains unknown physiologically active substances, and Spirulina, a blue-green alga, is estimated to contain nucleic acid-based growth factors that have a growth-promoting effect on animals. There is an urgent need to investigate. It is well known that Spirulina algae have a higher rate of digestion and absorption of nutrients and other active ingredients contained in the algae in animal bodies than Chlorella algae, which have strong cell walls. However, research has been conducted to extract and separate physiologically active substances from chlorella, and the use of this in a solution state has been developed. For example, it is known that chlorella extract, an unknown physiologically active substance contained in chlorella algae, has a growth-promoting effect on lactic acid bacteria, protozoa, plants, etc. Chlorella is extracted with hot water or an alkaline aqueous solution, and an enzyme is applied externally, such as trypsin.
Either act at PH7.2~8.4 or use cellulase at PH4~
Methods are known, such as digestion with Bacillus natto protease at pH 5 or pH 7, followed by extraction and separation. However, in the case of Spirulina, it is difficult to immediately apply the same extraction process as for Chlorella.
In other words, when Spirulina algae are subjected to hot water extraction to obtain the active ingredients, the active ingredients, such as vitamins, which are abundantly contained, are significantly reduced, and when the processing method uses external enzymes, the enzyme preparations are expensive. Therefore, the extract cannot be used as a feed additive for animals, such as fish. The purpose of the present invention is not to extract and separate the extract components in the algae, but to improve the digestion of Spirulina algae without reducing the nutrients and other active ingredients contained in the algae. An object of the present invention is to provide a solution composition comprising liquefied spirulina with improved absorbability and a method for producing the same. The present inventors have made extensive changes in their research on liquefying Spirulina at low temperatures in order to adapt it for use as feed for animals, especially larval fish for aquaculture, but also for growing fish larger than 20 to 30 mm. When conducting experiments under these conditions, it was found that the objective could be achieved by autolysis in a highly alkaline aqueous solution at around room temperature. In addition, Spirulina is not cultured aseptically, but in order to confirm that autolysis occurs, we cultured sterile seed Spirulina in an oversterilized culture solution that had been irradiated with ultraviolet light, and then placed it in a sterile box. Approximately 1 g of spirulina was recovered in terms of dry weight by aseptic separation, and 100 ml of a 0.5 molar sodium carbonate-sodium bicarbonate buffer solution (PH10) was collected.
0.5 ml of toluene was added and left at 30°C for 24 hours. When a portion of this dispersion was taken and observed under a microscope, it was found that the filaments were completely separated and only minute particles were observed. Furthermore, the ratio of the non-protein nitrogen content to the total nitrogen content of this dispersion was 65%, indicating that Spirulina was degraded by its own enzyme. At the same time, similar operations were performed on Spirulina cultured in a non-sterile manner, and similar results were obtained. The present invention is based on the knowledge obtained in the course of such research. The Spirulina algae used in the present invention can be learned from various known documents. For example, regarding the culture method, JP-A-56-64482 (title of invention: method and device for culturing microalgae) and JP-B No. 45-29430 (title of invention: method and culture tank for culturing algae) are described. According to the method, spirulina can be easily grown by inoculating spirulina into a culture solution containing dissolved nutrients and irradiating it with light while circulating the culture solution using a rotating body, blowing carbon dioxide-containing air, or driving force. and can be harvested. In addition, there are two known species of relatively large Spirulina that are currently cultivated using industrial culture methods: Spirulina platensis and Spirulina maxima. For details on the characteristics of
Publication No. 50-32996 (Title of invention: Fish breeding method)
You can see it in In addition, Spirulina Major (S.major), Spirulina Princess (S.
princeps), Spirulina laxissima (S.
laxissima), Spirulina butylsima (S.
subtillsima), Spirulina Caldaria (S.
Spirulina caldaria), S. curta and S. spirulissima are also known. Therefore, we will not discuss the cultivation method and morphological characteristics of Spirulina algae in detail here, but the component composition of Spirulina algae that has been confirmed to date is summarized in Table 1, and the main amino acid composition of protein components is summarized in Table 1. It is shown in Table 2. Note that the composition of the green alga Chlorella is also added to Tables 1 and 2 for comparison.

【table】

【table】

[Table] In the present invention, Spirulina algae is one of Spirulina maxima and Spirulina platensis (both have similar compositions) having the composition shown in Tables 1 and 2 above, which are harvested by industrial cultivation. Or it is preferable to use both,
There is no problem in using other algal bodies of the genus Spirulina that are more similar in composition than in morphology. and,
Cake-like living algae collected from the culture tank, separated and washed with water are most suitable, but Spirulina algae that are washed with water and then crushed or dried without being crushed, preferably spray-dried at a hot air temperature of 90°C to 200°C. Spirulina algae that have been washed or freeze-dried after washing with water can also be used by mixing a part of the above-mentioned live algae. The present invention is made by autolyzing the above-mentioned Spirulina algae in a strongly alkaline aqueous solution.
The pH value of the aqueous solution is preferably 8.0 to 11.5.
More preferably, the pH is in the range of 9 to 10. If the PH value is less than 8 or more than 11.5, it is inappropriate because it is out of the active PH range of enzymes involved in the autolysis of Spirulina. Compounds or compound groups added to water to prepare such an alkaline aqueous solution include (a) Sodium carbonate - sodium bicarbonate (b) Potassium carbonate - potassium bicarbonate (c) Sodium phosphate - sodium hydrogen phosphate - Sodium dihydrogen phosphate - Sodium hydroxide (d) A combination of the above (c) sodium salt and potassium salt. (e) Glycine-sodium chloride-sodium hydroxide (f) One or more of glycine-potassium chloride-potassium hydroxide can be used. In addition, the concentration of the above compound or compound group in the aqueous solution is preferably 0.05 to 1M/PH
is adjusted with hydroxide such as sodium hydroxide. Regarding (a) to (e), one or more of the following compounds are added to such an alkaline aqueous solution for the purpose of promoting self-digestion of Spirulina algae and/or for antiseptic or antibacterial purposes.
~10Mol/, and (f) is preferably added at a concentration of 0.05 to 5%. (a) Inorganic salts such as sodium chloride and potassium chloride. (b) Sugars such as sucrose, fructose, lactose, glucose, galactose, sorbose, and maltose. (c) Sugar alcohols such as sorbitol and mannitol. (d) Organic acids and their salts such as gluconic acid, galactonic acid, lactic acid, acetic acid, propionic acid, citric acid, malic acid, fumaric acid. (e) Glutamic acid, aspartic acid, alanine,
Amino acids such as lysine and their salts. (f) Hydrophobic organic solvents such as toluene, ethyl acetate, xylene, n-hexane, ether, benzene. In the present invention, spirulina algae are added and dispersed in a dry weight of 10 to 100 g per 1 alkaline aqueous solution,
Autolysis is carried out for 0.5 to 48 hours with or without stirring while maintaining the temperature at 25 to 55°C, preferably 30 to 45°C. If the temperature of the solution during autolysis is lower than 25°C, the digestion progresses slowly, which is inappropriate; if it exceeds 55°C, enzyme deactivation occurs, which is inappropriate. After completion of autolysis, the desired product of the present invention can be obtained by adjusting the pH with mineral acid or organic acid depending on the intended use or as it is. The solution composition of the present invention will not deteriorate over a long period of time if stored in a light-shielding sealed container. In the liquefied Spirulina algae according to the present invention, the degree of autolysis of the algae can be determined, for example, by quantitatively analyzing the non-protein nitrogen content over time. In other words, although there is some variation depending on the culture conditions, the number of Spirulina algae is usually about 10 to 10.
Contains 11% nitrogen, taking this total nitrogen as 100.
It contains about 2.8% non-protein nitrogen, and since the non-protein nitrogen content increases as autolysis progresses, the degree of digestion can be easily determined. For example, it has been confirmed that the ratio of non-protein nitrogen content to the total nitrogen content of liquefied Spirulina algae, which has slowed down to the extent of digestion after about 42 hours had passed since the start of autolysis, was 42.2%. This indicates that 40.5% of protein nitrogen was self-digested by enzymatic decomposition. Such a highly decomposed solution of proteins cannot be obtained from algae that have lost enzymatic activity by cold water or hot water extraction, or by strong alkaline extraction (pH around 11.5). The total nitrogen content in the present invention is determined by calculating the total content of the solution as normal Kjeldahl nitrogen, and the non-protein nitrogen is determined by calculating the nitrogen soluble in 10% trichloroacetic acid by the Kjeldahl method. Note that when nitrogen compounds such as glycine and urea are added to the Spirulina dispersion medium, both the total nitrogen content and the soluble nitrogen content will be greatly increased. Calculate each nitrogen content and find the ratio. The liquefied Spirulina algae body according to the present invention has a proportion of non-protein nitrogen in the total nitrogen of at least 20%,
It is important that it is preferably 30% or more, and 20
If the value is less than 20%, large fragments of Spirulina algae are present in the liquefied product, which is not preferable for use in, for example, larvae. When the proportion of non-protein nitrogen in the total nitrogen exceeds 30%, no large fragments (about 10 μm or more) are found in the liquefied product. Furthermore, in the present invention, the degree of autolysis of algae is
It can also be determined by optically measuring the substances that are eluted and liberated into the liquid during digestion. That is, there is a substance that exhibits a maximum absorption pattern around 260 nm, which the present inventors estimate is a nucleic acid-based substance, and this can be determined by measuring the degree of increase in absorbance at 260 nm. For example, perchloric acid is added to a solution in which a certain amount of Spirulina algae is autolysed in a certain amount of digestive fluid by the method described above so that the final concentration is 0.4N, and the resulting coagulation precipitate of soluble protein and algae are The body residue was separated and removed using a centrifuge (10000G, 10 minutes), and the resulting solution was measured with a spectrophotometer to measure the absorbance at 260 nm, and the relationship between decomposition and absorbance was determined from a calibration curve that had been determined in advance. The degree of decomposition can be grasped. The solution composition according to the present invention can be used for animals, especially coho salmon, red sea bream, carp, Japanese loach, flounder, and rainbow trout.
It is suitable for use as feed for aquaculture larvae such as amago, blowfish, horse mackerel, yellowtail, amberjack, telapia, ayu, eel, etc., but it can also be used for various aquaculture fish that are in the growth stage of 20 to 30 mm or more. can. Furthermore, the solution composition according to the present invention can be used not only as a feed material for the above-mentioned fish, but also for other animals such as cows, pigs, horses, sheep, mink, etc.
Appropriately used as feed for livestock such as goats, experimental animals (guinea pigs, rats), and stray animals such as chickens and small birds, as well as protozoan rotifers such as green beetles and gourd bugs, and crustaceans such as watermelons and Kuruma shrimp. can be used. In particular, it is suitable to use it by adding it to solid feed or liquid feed for the larval stage of these animals. The composition of the present invention can be added to feed by methods such as spraying, impregnating, dipping, and mixing, but the important point is that as long as it is evenly dispersed and added to feed and efficiently administered to and ingested by individual animals and farmed fish. Any method of addition can be used. In particular, a mixed solution of a feed spreader and a solution composition whose main component is alginic acid, such as Gel Binder (manufactured by Kimitsu Chemical Co., Ltd.), Mailitschi (manufactured by Tanabe Seiyaku Co., Ltd.), and Stanguard (manufactured by Taito Pfizer Co., Ltd.). It is preferable to use it by spraying or mixing it with the feed. According to the present invention as described above, a solution composition in which most of the active ingredients contained in Spirulina algae are dissolved in the solution can be obtained, so that the digestibility for animals is significantly improved compared to when the algae are intact. Moreover, since it is in a solution state, it has the advantage of being easier to handle when added to basic feed. The present invention will be explained in more detail by showing Examples and usage examples of solution compositions below. Example 1 A cake made of living Spirulina maxima algae collected from the culture tank and dehydrated (30 g in dry weight)
was dispersed in an aqueous solution (PH10) containing 20% by weight of common salt and sodium carbonate-sodium bicarbonate so that the sodium concentration was nomolar. This dispersion solution was kept at a controlled temperature of 30°C for 48 hours to allow the algae to self-digest. When we observed changes in the dispersion solution over time using a microscope, we found that the spiral shape was fragmented after about 18 hours, and after about 26 hours, digestion had been completed to the extent that 4 to 5 fragments of one unit cell remained in one field of view. Digestion progressed to the extent that these fragments disappeared after 40 hours. Also,
After 48 hours, the ratio of non-protein nitrogen content to total nitrogen content was 45%. Furthermore, perchloric acid was added to the solution after the digestion treatment to a final concentration of 0.4N to coagulate the soluble proteins, and then centrifuged (10,000G) for 10 minutes to separate the algae residue and the coagulated precipitate. After removal, the absorbance was measured at wavelengths from 220 nm to 350 nm, and a curve as shown in FIG. 1 was obtained, having an absorption maximum at 260 nm. When the absorbance at 260 nm (cell thickness: 1 cm) was measured as the autolysis time of the algae increased according to the method described above, a curve as shown in FIG. 2 was obtained. {In this case, when 1 g of spirulina is dispersed in 100 ml in terms of dry matter and autolyzed, the OD260nm (1 cm cell) is 12.5. Comparative example a Solution in which the pH of the dispersion solution was controlled to 7 (a) and PH of
Example 1 except that a solution (b) controlled at 12 was prepared.
The operation was repeated. Digestion progressed slowly in both solution (a) and solution (b), and the degree of digestion progress after 48 hours was equivalent to that after 10 hours in Example 1, indicating that the production method is practical. was not recognized. Example 2 200 g of potassium chloride and 18 g of sodium hydrogen phosphate (Na ₂ HPO ₄ .12H ₂ O) were dissolved in water, further diluted with water to a pH of 1, and 10N sodium hydroxide solution was added to adjust the pH to 10. did. Add and disperse a cake made of living Spirulina platensis algae (60 g in dry weight) to this solution, and reduce the temperature to 35
The mixture was maintained at a controlled temperature for 24 hours to allow autolysis. Additionally, as the digestion progressed, the pH was measured from time to time, and 5N sodium hydroxide was added dropwise to maintain the pH at 10. The ratio of non-protein nitrogen content to the total nitrogen content of this solution composition was 41%. Example 3 Sodium hydrogen phosphate ( _Na2HPO4・_12H2O ) ₁₈
After dissolving 825 g of sucrose in 400 ml of water, 825 g of sucrose was added and dissolved by heating, and the pH was adjusted to 10 by adding 10N sodium hydroxide solution. Add this solution to a cake consisting of living algae of Spirulina maxima (25% on dry weight basis).
g) and a cake made of living algae of Spirulina platensis (25 g in terms of dry weight) were added and dispersed and mixed, and the temperature was controlled at 35° C. and maintained for 48 hours to allow self-digestion. The ratio of non-protein nitrogen content to the total nitrogen content of this solution composition was 38%. Comparative Example b The operation of Example 3 was repeated. However, the temperature of the dispersion solution is 13℃ (condition-1) and the same temperature is 57℃.
(Condition-2), and only the autolysis conditions of Spirulina algae were changed. In the case of condition-1, the ratio of non-protein nitrogen content to the total nitrogen content in the solution composition after 48 hours was approximately 10%, and the progress of digestion was extremely slow. Further, in the case of condition-2, although the nitrogen content ratio reached approximately 15% after 6 hours, no increase was observed after that, indicating that the enzyme necessary for autolysis had been deactivated. Examples 4 to 8 Dispersed solution composition, Spirulina algae and temperature/
The liquefied spirulina solution composition of the present invention was produced by changing the combination of autolysis conditions such as retention time as shown in Table 3.

[Table] Yes.
Usage Example 1 Hydrochloric acid was added to the solution composition of the present invention produced in Example 1 to neutralize it to almost neutrality, and the entire solution was used in the next breeding experiment. The target fish were young coho salmon, and approximately 25,000 fish per section were raised in a 90 m ² tank at a water injection rate of 2.5 tons per minute. Pellets for rainbow trout (trade name, manufactured by Showa Sangyo Co., Ltd.) were used as the basic feed, and while the pellets were stirred in a mixer, an aqueous feed spreader solution and an alkaline solution with the composition used in Example 1 were added to the control feed. The neutralizing solution was prepared by uniformly spraying a mixed solution of a feed spreader aqueous solution and the solution composition of the present invention on the feed in the test group using a sprayer, and then adding feed oil to the feed in both groups and allowing it to be absorbed. I fed it. The blending ratio of each material component used is as shown in Table 4 below.

【table】

[Table] The feeding rate was set at 1.3% and the feeding amount was increased temporarily. For feeding, the same amount of pellets was impregnated with feed oil (trade name: Riken Feed Oil Ω, manufactured by Riken Pittamin Co., Ltd.) and administered to both the control and test plots. The test results were as shown in Table 5. Control group-2 in Table 5 is the same except that instead of 100 parts by weight of the liquefied Spirulina solution composition in the test group feed in Table 4, 3 parts by weight of Spirulina maxima live algae was added in terms of dry weight. These are the results of preparing a feed with the same composition and subjecting it to a feeding test.

[Table] In addition, the autolysis solution prepared under the condition-2 of Comparative Example-(b) above (non-protein nitrogen content/total nitrogen content =
0.15) was also applied to coho salmon breeding in the same way as in this usage example, but only results comparable to control group-2 were obtained. Number of rearing days (t) 36 days Total feeding weight (F) (pellets + feed oil) Calculation formula f=F/{(Wo+Wt) /2・(No+Nt)/2・t} (%) I=Wt−Wo/Wo+W ₂ /2・1/t(%) E=I/f(%) R=F/(Wt-Wo)・Nt+No/2 Increase in total fish weight in test area compared to control area 1
28.1% and 13% higher than Control Group 2, respectively, which indicates that the solution composition of the present invention contributes to growth promotion. Use Example 2 After neutralizing the solution composition produced in Example 2 with hydrochloric acid, a breeding test was conducted using target fish as young red sea bream. The feed was shrimp paste and other ingredients were added and kneaded. The blending ratio of each material was as shown in Table 6 below.

[Table] (Unit: parts by weight)
Rearing was carried out for 35 days, with 1,500 juveniles released 30 days after hatching into small sea-surface net pens (2 x 2 x 2 m). The results are shown in Table 7 below, showing that feeding was particularly active in the early stages in the test plots.

[Table] Usage Example 3 The liquefied spirulina solution composition obtained in Example 3 was neutralized with hydrochloric acid and subjected to the following rearing test on juvenile red carp. The basic feed is crumble for carp (product name:
A for Yamato fry (manufactured by Nisshin Feed Co., Ltd.) and other ingredients were atomized using a munger and uniformly added to the crumble being stirred in a mixer. The blending ratio of each material is as shown in Table 8 below, and water, a feed spreader, and a liquefied spirulina solution composition were mixed and used as a solution that was dispersed and dissolved.

[Table] (Unit: parts by weight)
A rearing test was carried out by placing 1000 fry in a 5 x 8 m aquarium for 30 days, and the results shown in Table 9 were obtained. The feeding rate was initially set at 6 to 7% and adjusted as necessary, and the same amount was fed to both groups, resulting in a total feeding amount of 3420 g as crumble. In addition, the amount of autolysed fluid supplied in the test group feed was equivalent to approximately 0.02% spirulina in the crumble on a dry weight basis.

【table】 [Brief explanation of drawings]

FIG. 1 is an ultraviolet absorption curve diagram of the components dissolved in the solution composition of the present invention, and FIG. 2 shows the maximum absorption wavelength region (260n
It is a curve diagram showing the change in absorbance in m).

Claims (1)

[Claims] 1. The Spirulina algae are self-digested by controlling the temperature at 25-55°C while maintaining the pH of a dispersion solution in which Spirulina live algae are dispersed in an alkaline aqueous solution in the range of 8.0-11.5. , a liquefied spirulina solution composition having a non-protein nitrogen content of at least 20% relative to the total nitrogen content of the dispersion solution. 2. The liquefied spirulina solution composition according to claim 1, wherein the autolyzed solution of spirulina algae contains a substance that exhibits maximum absorption at 260 nm in optical properties. 3. The liquefied Spirulina solution composition according to claim 1 or 2, wherein the Spirulina algae is one or both of Spirulina maxima and Spirulina platensis. 4 While maintaining the pH of the dispersion solution of Spirulina living algae dispersed in an alkaline aqueous solution in the range of 8.0 to 11.5, the temperature is controlled at 25 to 55°C to autolyze the Spirulina algae, and all of the dispersion solution is A method for producing a liquefied spirulina solution composition that increases the non-protein nitrogen content to nitrogen content by at least 20%. 5. The method for producing a liquefied Spirulina solution composition according to claim 4, wherein the Spirulina algae is one or both of Spirulina maxima and Spirulina platensis.