KR100994359B1 - A process for purifying aquaculture waste-seawater effluent and a process for producing polyunsaturated fatty acids, using a marine microorganism - Google Patents

Abstract

The present invention is Saizokytrium limasinum ( Schizochytrium) limacinum ) relates to a method for treating marine aquaculture sewage using a strain and a method for producing a high concentration of polyunsaturated fatty acids (PUFA) using the strain. Using the method of the present invention to treat marine aquaculture sewage biologically, not only can the high nitrogen, ammonia and phosphorus components in marine aquaculture sewage be effectively removed, but the high nitrogen, ammonia and phosphorus constituents in the sewage can be used as nutrients. Since the cultured Saizoquitium limasinum cells contain a high concentration of polyunsaturated fatty acids, the cells or polyunsaturated fatty acids produced therefrom can be usefully used as a feed composition for food or farmed fish.

Saizoquitium limasinum, polyunsaturated fatty acid, marine aquaculture sewage, nitrogen, ammonia, phosphorus

Description

A PROCESS FOR PURIFYING AQUACULTURE WASTE-SEAWATER EFFLUENT AND A PROCESS FOR PRODUCING POLYUNSATURATED FATTY ACIDS, USING A MARINE MICROORGANISM

The present invention relates to a method for treating marine cultured sewage using a Schizochytrium limacinum strain and a method for producing a high concentration of polyunsaturated fatty acids (PUFA) using the strain.

Wastewater produced in aquaculture is divided into sludge and sewage using membrane filters and sedimentation tanks. More than 80% of organic matter is present in the sludge. Most of the organic matter present in aquaculture wastewater is a waste produced by farmed fish or leftover feed leftovers, and if used continuously without being reprocessed, it will accumulate in the farm and cause major contamination of seawater and farm fish in the farm. The sludge is collected and reprocessed into feed or recycled as fertilizer.

On the other hand, as shown in Table 1, the concentrations of chemical oxygen demand (COD), total phosphorus, and ammonia nitrogen (NH ₃ -N) in aquaculture sewage produced in fish farms are determined by COD, total phosphorus, and ammonia nitrogen in general seawater. Very high than concentration In particular, ammonia concentrations in cultured sewage that are circulated in aquaculture can be fatal to farmed fish, so in order to control water quality in aquaculture, it is essential to improve aquatic fish health and prevent disease outbreaks. to be. Therefore, the quality of aquaculture sewage should be controlled at a certain level and can be a major source of marine pollution if discharged to nearby seas without any treatment.

Comparison of COD, Total Phosphorus, and Ammonia Nitrogen Concentrations in General Seawater and Aquaculture Sewage division COD Total phosphorus (TP) Ammonia Nitrogen (TN) General seawater 0.2-4.0 ppm 0.002-0.080 ppm 0.001 to 0.150 ppm Form sewage 1.0 to 4.3 ppm 0.050 to 0.380 ppm 0.030-0.240 ppm

General sewage can be purified by chemical and biological treatment methods, but aquaculture sewage containing sea water cannot be biologically or chemically treated due to the high concentration of salts and sulfur, and membrane filtration and All depends on the physical treatment methods, such as settling tanks. However, simply relying on physical reprocessing methods does not eliminate the major causes of environmental pollution such as high nitrogen and phosphorus present in the sewage.

On the other hand, existing polyunsaturated fatty acids have been mostly produced from fish extracts during processing of fish such as tuna and sardines. These polyunsaturated fatty acids have already been recognized for their value through the development of functional foods and drugs, and their safety has also been proven. However, the polyunsaturated fatty acids thus produced have problems such as the peculiar smell and aversion of fish. Recently, there have been many studies and movements to produce polyunsaturated fatty acids by culturing microorganisms.

In this respect, Saizokitrium limasinum strain, a microalgae of marine origin, has a high ability to produce polyunsaturated fatty acids, which accounts for 17% of the total fatty acids (60% of the dry cells produced as fat) produced in seawater culture. Since the degree of polyunsaturated fatty acid is produced, it has recently attracted much attention as a research target, and has been developed in various fields for use as a food additive for food, human medicine, animal feed or aquaculture, including functionality.

The strain is capable of growing at high salt concentrations in seawater and shows different growth rates depending on the concentration of seawater. Further, the concentration and the various culture conditions of the sea water polyhydric generated according to (temperature, oxygen concentration, medium composition, etc.) different concentrations of the unsaturated fatty acid (as described in [Luying et al, ProcessBiochemistry, 42 (2):. 210 ~ 214, 2007]; and the literature [Shwu-Tzy Wu et al, ProcessBiochemistry, 40:. 3103 ~ 3108, 2005]). The strain uses glucose, glycerol and ammonium acetate as nutrients to accumulate polyunsaturated fatty acids (HA, DPA and palmitic acid) in its lipid body during heterotrophic growth (Yokochi T et al., ... Appl Mocrobiol Biotechnol, 49 : 72 ~ 76, 1998]; and the literature [Eiko Morita et al, MarineBiotechnol, 8: 319 ~ 327, 2006])...

DHA-rich lipids produced from S. cytotrium strains are being developed for use in food and animal feed, and various safety data and animal toxicity data (Ruben Abril et al., Regulatory) Toxicology and Pharmacology , 37: 73-82 , 2003), as well as safety as feed (Bruce G. Hammond et al., Regulatory Toxicology and Pharmacology , 33: 192-204, 2001], possibility of mutation (Bruce G. Hammond et al., Regulatory Toxicology and Pharmacology , 35: 255-265, 2002), and reproduction (Bruce G. Hammond et al., Regulatory Toxicology and Pharmacology , 33: 356–362, 2001] and many other useful data support safety.

In addition, when applied to aquaculture together with the sakyokitrium cells spray-dried algae than the seaweeds ( Mytilus) galloprovincialis ) showed a faster growth rate (Chris Langdon and Ebru Oenal, Aquaculture , 180: 283-294, 1999). Rotifer, Artemia nopli ( Artemia ) nutritionally fortified with Saizokitrium strains nauplii) the was was made using a fish feed got the results that enhance the growth of Atlantic halibut (flaunder), in hatchability of European sea bass have been reported to be effective noteworthy (as described in [Moti Harel et al, Aquaculture, 213.: 347-362, 2002).

In general, nutrients in aquaculture, such as sea bass, sea bream and tuna, flounder and flounder, sea bass, shrimp and crab, and scallops, mussels and abalones, are currently in artificial production. Unless developed, they rely on fatty acids or proteins from natural rotifers, artemia or copepods and the like. Moreover, the composition of fatty acids present in these foods absolutely affects the fertility and the growth rate of the fry. In particular, the polyunsaturated fatty acids such as arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be effective in promoting the development and growth of fry. Moti Harel et al., Aquaculture , 213: 347-362, 2002; Josianne Getal, Live foods in marine aquaculture, 2nd edition, Blackwell publishing, 96, 2003; and Jose R. Rainuzzo et al., Aquaculture , 155: 103-115, 1997].

However, naturally-used rotifers, copepods or artemias contain only small amounts of DHA or EPA (Josianne G et al., Live foods in marine aquaculture, 2nd edition, Blackwell publishing, 96, 2003); And Takashi Yamasaki et al., Journal of Bioscience and Bioengineering , 104 (3): 200 ~ 206, 2007]), and artificially incubating them with polyunsaturated fatty acids (HUFA or PUFA), proteins and amino acids, and enriched for a certain period of time (about 16 hours) after hatching from eggs. In order to increase the amount of useful ingredients temporarily and supply them to feed fry, many methods are actively researched around the world.

Therefore, the present inventors have been researching to develop a method for effectively removing high nitrogen, ammonia and phosphorus in aquaculture sewage. In addition, the present invention was completed by elucidating that polyunsaturated fatty acids can be obtained at high concentration by effectively culturing the strain using nitrogen, ammonia, and phosphorus in the sewage.

It is an object of the present invention to provide a method for effectively purifying marine aquaculture sewage.

Another object of the present invention is to provide a method for effectively producing polyunsaturated fatty acids.

Another object of the present invention is to provide a food or feed composition containing a high concentration of polyunsaturated fatty acids.

In order to achieve the above object, the present invention comprises the step of culturing the marine culture sewage septicus, glycerol and yeast extract in a culture medium containing lysochytrium limasinum strain, purifying marine culture sewage Provide a method.

More specifically, the present invention comprises the steps of: (1) culturing the S. pyritrium limasinum strain in a cell-recycling reactor in a medium comprising marine cultured sewage, glycerol and yeast extract, and (2) It provides a method for purifying marine culture sewage, comprising the step of precipitating the culture solution produced in the culture step to recover the cells to the incubator, and filtering the supernatant.

In order to achieve a further object, the present invention provides a method for producing polyunsaturated fatty acids by extracting the cyzokitrium limasinum cells or culture produced as a by-product of the method.

In order to achieve another further object, the present invention provides a composition for food or feed containing the cells, dried powder thereof or polyunsaturated fatty acid produced therefrom as an active ingredient.

Using the method of the present invention to reprocess marine aquaculture sewage by biological treatment using Saizoquitrium limasinum strain, it is circulated in aquaculture farms and is present in a high concentration in aquaculture seawater. Ammonia, which causes or stresses growth, and low levels of total nitrogen and total phosphorus, a major contributor to pollution and eutrophication, can effectively purify aquaculture sewage, as well as remove high nitrogen, ammonia and phosphorus Since the lysocytrium limasinum cells cultured as a nutrient source contain a high concentration of polyunsaturated fatty acids, the cells or polyunsaturated fatty acids produced therefrom can be usefully used as feed compositions for food or farmed fish.

The present invention is a microalgae of marine origin, Saizokitrium rimashinum, preferably Saizokitrium, for biological treatment in the process of reprocessing sewage containing a large amount of nitrogen, ammonia and phosphorus produced in seawater farms. It is characterized by culturing limasinum SR21 (ATCC MYA-1381) strain with cultured sewage.

In the present invention, in order to reprocess seawater culture sewage, the strain is incubated with marine culture sewage, and glycerol and yeast extract as a growth promoter in a cell recovery incubator, and then the culture solution is subjected to a density gradient sedimentation. The precipitated cells were precipitated in a precipitation tank, and the cells were recovered by the incubator, and the supernatant was passed through a membrane filter to obtain purified seawater (see FIGS. 1 and 2). At this time, the cell settling tank and the membrane filter retains the cells in the incubator and minimizes the loss of the cells through the outlet and at the same time discharges only the purified sewage out of the incubator to maintain the concentration of the cells in the incubator at all times to reprocess While maximizing the purification effect of sewage by the cells.

  In addition, the culturing may be performed by supplying glycerol and yeast extracts in a batch culture, fed-batch culture, semi-continuous culture or continuous culture. In particular, a semi-continuous or continuous culture method of feeding at a constant dilution rate is preferable. The dilution rate for supplying the glycerol and yeast extract is preferably 0.003 to 0.1 / hour, more preferably 0.003 to 0.03 / hour, depending on the cell concentration in the culture (Dilution rate (D) is also called dilution rate) , Defined as the feed rate (f, mL / hour) of fresh culture per volume (V, mL) of the incubator, ie D = f / V.

In this case, the glycerol may be preferably used at a concentration of 0.1 to 10% (w / v) with respect to the total medium, and the yeast extract is preferably at a concentration of 0.01 to 5% (w / v) with respect to the total medium. Can be used as

In addition, in the present invention, an additive selected from the group consisting of soy peptone, ammonium sulfate, ammonium citrate, and mixtures thereof may be further added to supply a nitrogen source in the culture.

In addition, in the present invention, the supernatant obtained as described above is reprecipitated in a secondary settling tank, and the obtained supernatant is filtered with a membrane filter to obtain clean seawater, and the obtained cells can be dried and used as aquaculture feed composition.

Using this method, ammonia is present in high concentrations in cultured seawater circulating in aquaculture, causing ammonia to cause disease or stress on growth, and total nitrogen and total phosphorus, which are major factors in pollution and eutrophication. Lowering to low concentrations (total phosphorus, total nitrogen and ammonia decreases by about 91%, 77.3% and 91% respectively; see FIG. 5) can effectively purify aquaculture sewage.

In addition, the present invention is characterized by using aquaculture wastewater as a source of protein, nitrogen, ammonia and phosphorus components for culturing the strain.

In one embodiment of the present invention was measured the dry cell mass of the cultured strain as described above, it was confirmed that the cell mass effectively increased (see Fig. 4).

In addition, since the strain cultured in the above process will contain a high concentration of polyunsaturated fatty acid as a by-product produced in the process of reprocessing the sewage, the present invention also provides a method for producing polyunsaturated fatty acid using the strain. .

The polyunsaturated fatty acid may be extracted from the cultured cells or culture medium in the reprocessing of cultured effluent, and the solvent used for the extraction may be chloroform, methanol, ethyl ether, methyl ether, ethyl acetate, hexane and a mixed solvent thereof. It may be selected from the group consisting of, preferably a mixed solvent of chloroform and methanol can be used.

In the extraction process, a polyunsaturated fatty acid can be extracted by conventional methods, ie, adding and homogenizing the extraction solvent, and then crushing the cells. After extraction, the cell lysate is removed by centrifugation, and the extraction solvent can be removed by distillation under reduced pressure. In addition, the production method of the present invention may include a conventional purification process.

In one embodiment of the present invention, the composition of the lipids and polyunsaturated fatty acids extracted as described above was analyzed using gas chromatograph (GC). As a result, the productivity of lipids was increased by about 60% when aquaculture was used. The use of seawater increased by about 53% (see Figure 6), and the proportion of polyunsaturated fatty acids above C ₂₂ in total lipids increased to 62.38% in aquaculture, but only 45.15% in clean seawater. (See Table 3). In continuous culture with cultured sewage, the productivity of lipids increased by 70-80% (50-55% in the past) per dry cell, and the proportion of C ₂₂ or more polyunsaturated fatty acids in total lipid production 20 to 30% (see Table 4).

Accordingly, the present invention also provides a food, feed composition or feed comprising as an active ingredient Saizokitrium limasinum cells containing a high concentration of polyunsaturated fatty acids, their dry powder or polyunsaturated fatty acids produced therefrom as an active ingredient. Provide additives.

The cells or dried cells may be used as feed for rotifers, artemia, copepods, larvae of shrimp, phytoplankton, and zooplankton to be bioencapsulated and used as feed for farmed fry.

In one embodiment of the present invention, after the bio-encapsulation of the cultured cells as described above to feed and shrimp Artemia, the increase in polyunsaturated fatty acid was analyzed using GC and compared with the fatty acid composition of Artemia used commercially . As a result, as the bioencapsulation progressed, the fatty acid composition of rotifers and Artemia increased gradually, and the content of polyunsaturated fatty acids, in particular C ₂₂ or more polyunsaturated fatty acids, increased compared to those of commercially used Artemia. 7).

The rotifers or artemias prepared as described above may be used as feed for farmed fish, and the dry powder of the cyzokitrium limasinum cell may be used to raise fry, fingering or larvae such as shellfish. have.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the invention only.

Example  1: cultivation of strains

Saizokytrium limasinum strain (ATCC MYA-1381) used in the present invention was incubated in SZ-1 medium having the composition of Table 2 for 24 hours, and then the cultured cells were recovered using sterilized clean seawater. Washed three times, concentrated 10 times again and divided by 200 ㎕ were stored in an ultra-low temperature freezer of -70 ℃, taken out if necessary was used after melting at 30 ℃.

The strain was inoculated into an Erlenmeyer flask (300 mL) containing SZ-1 medium (50 mL), and then initially incubated for 24 hours at a rate of 150 rpm, and the initial culture medium was prepared in SZ- having the composition shown in Table 2 below. Inoculated in a stirred tank reactor (5 L) containing 2 medium (4 L) and the main culture (fermentation) for 6 days.

At this time, the culture was performed in a constant temperature chamber of 20 and 25 ℃, when the pH is lowered to 5.5 or less by the action of the bacteria was adjusted to pH 6.0 or more using 2 M caustic soda. In addition, the supply of oxygen was continuously supplied at the 1.0 v. V. M level.

SZ-3 medium having the composition of Table 2 was continuously fed to the strains cultured as described above at a dilution rate of 0.003 to 0.022 / hour, and at the same time excess medium was continuously removed through the outlet (continuous culture). The medium to be removed was separated into cells and culture medium through a settling tank and / or membrane filter, the cells were forced to recycle (recycling) using a pump back to the culture vessel.

At this time, the same culture was performed using clean seawater instead of marine aquaculture sewage as a comparative group.

Medium Composition of Saizokitrium Strains ingredient SZ-1 SZ-2 SZ-3 Sterilization Condition Yeast Extract (g) 1.0 10.0 1.0 30 to 60 minutes at 121 ° C and 1.5 bar Glycerol (g) 10.0 90.0 30.0 Marine Aquaculture Sewage (ℓ) 1.0 1.0 1.0 PH before sterilization 6.0 6.0 6.0 Usage Strain activation,
Initial culture medium Present culture medium Supply badge All marine aquaculture sewage used in the present invention was used by sterilizing the marine aquaculture effluent from Pukyong National University laboratory without filtration.

Example 1-1 : Optimization of culture conditions through flask culture.

  Each 10 ml of the culture medium incubated as in Example 1 was recovered for each hour, filtered through glass fiber filter paper (Whatmann, GF / C), and then dried in a drying oven at 105 ° C. for 12 hours. Dry cell mass was measured through the difference in weight between the filter paper containing the dried cells and the blank filter paper, and the results are shown in FIG. 3.

As shown in FIG. 3, the Cyzokitrium limasinum strain was grown faster at 25 ° C. than 20 ° C., and the cell mass was grown to 9.646 g / l in clean seawater and to 7.805 g / l in marine aquaculture sewage. In addition, the total dry cell mass did not change with the culture temperature.

In addition, the pH of cultured sewage decreased to 3.0 at the end of the culture, but the pH of the clean seawater did not change significantly around 7.0. Thus, this decrease in pH in aquaculture wastewater led to a sharp decrease in cell mass.

Example 1-2 Cell Growth Analysis in Continuous Culture

Dry cell mass was measured in the same manner as in Test Example 1 using the culture medium continuously cultured as in Example 1, and the results are shown in FIG.

As shown in FIG. 4, the total cell mass increased to 13.031 g / L as a result of the continuous culture.

Example 1-3 Analysis of Seawater Quality

In order to analyze the total nitrogen, total phosphorus and ammonia content in the seawater in ppm units, the continuous culture was filtered in the same manner as in Test Example 1, and then analyzed for the total nitrogen, total phosphorus and ammonia according to the standard method of water quality test. Was carried out and the results are shown in FIG. 4.

As shown in FIG. 5, total phosphorus, total nitrogen and ammonia decreased by about 91%, 77.3% and 91%, respectively.

Example 1-4 Analysis of Fatty Acid Composition

In the same manner as in Example 1 while culturing the strain in cultured sewage and clean seawater was analyzed the composition of fatty acids as follows.

Specifically, the sample culture was dried overnight at 105 ℃, extracted lipids from the dry cells with a chloroform: methanol (2: 1) mixture, and then evaporated the solvent again under vacuum overnight, the total amount of lipid (%, w / w) was measured (see FIG. 6).

The lipids extracted as above were methylated in methanol containing HCl (10%, v / v) at 80 ° C. for 1 hour. Fatty acid methyl esters (FAME) were extracted with n-hexane for 1 hour, the n-hexane layer containing FAME was evaporated under vacuum, and then FAME was dissolved in dichloromethane and injected into GC. (2 μl). GC consisted of a FID detector (250 ° C.). The injection section (separated type: 100%) was set at 250 ° C., and the column was capillary column, and analyzed at a temperature of 75 to 250 ° C. (stayed at 75 ° C. for 5 minutes, and then at 140 ° C. at a rate of 14 ° C./min. Up to 250 [deg.] C. at a rate of 15 [deg.] C./min.

The results analyzed as described above are shown in Table 3, Table 4, and FIG.

Changes in Fatty Acid Composition of Saizoquitrium in Cultured Sewage and Clean Seawater by Culture Period Incubation time
(Work) Fatty acid composition (%) in aquaculture Fatty acid composition in clean sea water (%) C ₁₄ C ₁₆ C ₁₈ C ₂₂ C ₁₄ C ₁₆ C ₁₈ C ₂₂ 4 4.62 57.80 10.35 25.78 4.07 54.25 7.25 31.23 7 2.71 27.83 3.25 60.00 3.29 45.86 3.16 44.44 10 1.93 27.48 4.26 62.38 2.57 39.94 6.29 45.15

※ The ratio of the corresponding fatty acid content to total fatty acid content is expressed as a percentage.

Changes in Fatty Acid Composition of Saizoquitrium by Continuous Culture Incubation time
(time) Fatty acid composition (%) Myristic oleic acid
(C ₁₄ ) Palmitic acid
(C ₁₆ ) Palmit
Oleic acid
(C ₁₆ ) Oleic acid
(C ₁₈ ) Linoleic acid
(C ₁₈ ) C ₂₂ DPA DHA 353 3.03 0.93 42.87 2.22 7.26 9.46 2.16 27.39 377 3.48 1.23 47.73 2.2 6.8 4.34 4.18 25.96 401 3.3 1.4 46.69 1.6 7.31 5.16 4.17 25.65 429 3.27 1.35 46.57 1.55 6.61 5.21 4.18 26.46 455 3.3 1.41 48.79 1.19 5.23 3.94 4.91 28.77 473 3.37 1.42 50.21 1.43 6.7 3.97 4.49 26.72 520 3.83 1.68 56.42 1.34 5.48 3.41 3.87 22.09 Average 3.37 1.35 48.47 1.65 6.48 5.07 3.99 26.1

 ※ The ratio of the corresponding fatty acid content to total fatty acid content is expressed as a percentage.

As shown in Figure 6, the use of aquaculture sewage increased the productivity of the lipid up to about 60%, while using clean seawater increased to about 53%.

In addition, as shown in Table 3, the proportion of polyunsaturated fatty acids of C ₂₂ or more in the total produced lipids increased by 62.38% in aquaculture sewage, but only by 45.15% in clean seawater.

In addition, as shown in Table 4, in continuous culture using aquaculture wastewater, the productivity of lipids increased by 70-80% per dry cell, and the proportion of polyunsaturated fatty acids of C ₂₂ or more in the total lipid production was 20-30%. .

Example  2: Bioencapsulation of Cell Cultured in Cultured Sewage Using Roots

<2-1> Mass culture of rotifers

The culture system was run on a weekly cycle including initial inoculation, production, and washing.

Initial inoculation of rotifers ( Brachionous.plicatilis or B. rotundiformis ) for cultivation is carried out at a volume of 200 L initial culture so that the volume of the culture is 1/5 of the total culture vessel (for example, 1 ton culture vessel). Start), the initial inoculation was inoculated to 200 ~ 300 / ㎖, and divided into four times a day to feed the baker's yeast periodically.

At the beginning of the cultivation, the freshwater introduced daily increased the volume of the culture solution in the culture tank to increase the growth rate (1/5 volume per day). Eventually, on the fifth day, the volume of the culture medium increased to 1 ton, and the density of the rotifers in the culture medium was 300-500 / ml.

On the last day, one ton of culture was concentrated by sieving, whereupon the sieve and the concentration vessel were pre-chemically sterilized in chlorite solution for 24 hours and washed for 5-15 minutes in sterilized distilled water before use. This culture enriched rotifer was again used as seeding seed for high density mass culture.

The main cultivation of rotifers for mass cultivation was carried out in batch culture before feeding of larvae or larvae in a 1 ton culture tank every three days.

The initial inoculation rotifer concentration was 5,000 to 10,000 / ml, and was incubated in a 1,000-l culture tank every three days. The concentrated chilled chlorella and a small amount of vitamin 12 were fed four times a day at regular intervals. Supplied. After 3 days the rots in the culture increased to 20,000-30,000 / ml.

<2-2> bioencapsulation

Saizoquitium limasinum cells cultured in marine sewage as in Example 1 were aseptically stored at 20 to 23 ℃ before bioencapsulation, the temperature of the tank for bioencapsulation to 20 to 25 ℃, salinity of sea water 20 To 25 ppt.

In a 100-l culture tank containing the rotifer 500 × 10 ³ / l incubated as described in <2-1>, the Cyzoquitrium limasinum cells at a concentration of 0.3 g / l for a total of 16 hours every 4 hours. Bioencapsulation was performed while feeding and maintaining the concentration of dissolved oxygen at 30% saturation concentration.

Example  3: Artemia  Bioencapsulation of Cell Cultured in Cultured Sewage

Supplying cyzokitrium limasinum cells to a culture tank containing 200 × 10 ³ / L of Brine shrimp artemia nauplii of the instar I stage, and the temperature of the tank for bioencapsulation is 25 to Bioencapsulation was carried out in the same manner as in <2-2> of Example 2, except that the temperature was maintained at 28 ° C.

Example 2,3-1 : Analysis of fatty acid composition of bioencapsulated rotifers and Artemia

In Examples 2 and 3, the rotifers and artemias undergoing bioencapsulation were collected every hour, collected using a nylon mesh of 50 μm and 150 μm, respectively, and then fatty acids were extracted in the same manner as in Test Example 4. Analysis was performed by GC, and the results are shown in Tables 5 and 6. Table 5 shows the analysis results of the fatty acid composition of Yunchung and Table 6 shows the analysis results of the fatty acid composition of Artemia.

In addition, the present invention compares the fatty acid composition of the bio-encapsulated Artemia and commercial Artemia, and the results are shown in Table 7.

Fatty Acid Composition of Cyclone Encapsulated Cyclochitrium by Hours Incubation time
(time) Fatty acid composition (%) C ₁₆ C ₁₈ C ₂₀ C ₂₂ 0 35.8 20.6 28.8 0 8 24.5 9.0 17.2 26.5 16 22.3 9.9 18.0 29.6 24 22.5 3.5 16.0 27.9

※ The ratio of the corresponding fatty acid content to total fatty acid content is expressed as a percentage.

Fatty Acid Composition of Artemiaia Bio-encapsulated Saizotririum Incubation time
(time) Fatty acid composition (%) C ₁₆ C ₁₈ C ₂₀ C ₂₂ 0 16.5 75.7 5.5 0.0 8 16.9 70.9 7.0 6.1 16 13.3 60.6 10.9 14.3 24 15.5 55.9 8.3 18.3

 ※ The ratio of the corresponding fatty acid content to total fatty acid content is expressed as a percentage.

Comparison of Fatty Acid Compositions of Artemia with Bio-encapsulated Cyzokitrium and Commercial Artemia division Fatty acid composition (%) C ₁₆ C ₁₈ C ₂₀ C ₂₂ Example 3 15.5 55.9 8.3 18.3 Cyst 16.6 51.8 3.2 0.1 Nopley 17.3 to 26.8 54.6 to 84.9 3.5 to 8.9 Not detected

※ The ratio of the corresponding fatty acid content to total fatty acid content is expressed as a percentage.

As shown in Tables 5 and 6, it was confirmed that the fatty acid of bioencapsulated yunchung and Artemia according to the present invention gradually increased as the bioencapsulation progressed.

In addition, as shown in Table 7, the bioencapsulated Artemia according to the present invention was found to have a higher polyunsaturated fatty acid content, especially C ₂₂ or more polyunsaturated fatty acid content compared to the fatty acids of commercially available Artemia.

1 shows a process for reprocessing marine aquaculture sewage according to the method of the present invention.

Figure 2 shows in detail the cell recovery incubator.

Figure 3 compares the dry cell weight of the cells cultured using clean seawater and cultured filthy water at 20 and 25 ℃ in the present culture of the present invention.

Figure 4 shows the concentration change of the cells according to the dilution rate in the continuous culture of the present invention.

Figure 5 shows the change in the content of total phosphorus, ammonia and total nitrogen of the seawater retreated according to the present invention.

Figure 6 compares the amount of lipid per dry cell weight of the cultured cells in the reprocessing process of the present invention compared to the case using clean seawater.

Claims (10)

(1) culturing a Schizochytrium limacinum strain in a cell containing marine culture sewage, glycerol and yeast extract in a cell-recycling reactor; And (2) sedimentation of the culture broth produced in step (1) to recover the sizokytrium limasinum cells back to the incubator, and the supernatant is filtered, marine culture sewage using the sizoquiritri limasinum strain How to cleanse. The method of claim 1, A method for purifying marine aquaculture sewage using a Cyzokitrium limasinum strain, which supplies glycerol and yeast extracts batchwise, fed-batch, semi-continuous or continuous. The method according to claim 1 or 2, A method for purifying marine aquaculture sewage using a Cyzokitrium limasinum strain, wherein glycerol is used at a concentration of 0.1 to 10% (w / v) relative to the total medium. The method according to claim 1 or 2, A method for purifying marine aquaculture sewage using a cyzokitrium limasinum strain, wherein the yeast extract is used at a concentration of 0.01 to 5% (w / v) relative to the total medium. The method of claim 2, A method for purifying marine aquaculture sewage using a Cyzokitrium limasinum strain, wherein the glycerol and yeast extracts are fed semicontinuously or continuously at a dilution rate of 0.003 to 0.1 / hour. The method according to claim 1 or 2, A method for purifying marine aquaculture sewage using a cyzokitrium limasinum strain, which further adds an additive selected from the group consisting of soypeptone, ammonium sulfate, ammonium citrate and a mixture of two or more thereof to the medium. The method according to claim 1 or 2, A method of purifying marine aquaculture sewage using a cyzokitrium limasinum strain, further comprising reprecipitating the supernatant in a secondary settling tank. (1) cultivating the Cytochytrium limasinum strain in a cell recovery incubator in a medium comprising marine aquaculture sewage, glycerol and yeast extracts; (2) A method of producing polyunsaturated fatty acids by extracting the cyzoquitrium lycinum bacteria obtained by precipitating the culture solution produced in step (1). (1) cultivating the Cytochytrium limasinum strain in a cell recovery incubator in a medium comprising marine aquaculture sewage, glycerol and yeast extracts; (2) A food or feed composition comprising polysaccharide unsaturated fatty acid extracted from the cyzokitrium rimasinum cell, obtained by precipitating the culture solution produced in step (1). The method of claim 9, A food or feed composition, characterized in that the bio-encapsulated cyzotritri limasinum cells or dried powder thereof into larvae of crustaceans including rotifers and artemia.

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