KR100857246B1 - Preparation of functional fermented fish meal using dead fish and citrus peel - Google Patents

Abstract

A method for producing functional fermented fish meal by fermenting dried citrus meal as a moisture-conditioning agent and dead fish with fermenting bacteria is provided to prepare fish meal with effects of improving the immunity and reducing the death rate when fed to fish. Functional fermented fish meal is prepared by the steps of: putting 20kg citrus meal into a high temperature dryer, drying and grinding to 80meshes; adding the 5 to 10kg dried citrus meal as a moisture-conditioning agent(1) to 24kg dead fish(2), inoculating with fermenting bacteria in a weight ratio of 10,000:0.5 to 4, controlling the external steam temperature at 250 to 300deg.C, standing for 10 to 30min and fermenting when the internal temperature of the fermenter reaches 90±5deg.C; automatically stopping the fermentation when the moisture content of fish meal is 1.1 to 5.0%.

Description

Functional fermented fish meal using aquaculture harvest and citrus processed by-product and preparation method thereof {Preparation of functional fermented fish meal using dead fish and citrus peel}

1 is a flow chart of the production process of functional fermented fish meal using aquaculture farm harvest and citrus processed by-products

Figure 2 is a PCR confirmation result for the remaining fish disease infected bacteria for the safety evaluation of the prepared functional fermented fish meal,

Figure 3 is a measurement of free radical scavenging activity and hydrogen peroxide scavenging activity to determine the functionality of fermented fish meal derived from aquaculture farm and citrus gourd,

4 is a halibut-derived DNA protection effect of fermented fish meal derived from aquaculture farm and citrus gourd,

Figure 5 shows the growth rate of fish dietary fermented fish meal prepared by the addition of aquaculture plant by-products and citrus gourd,

Figure 6 shows the mortality and mortality of fish dietary fermented fish meal prepared by the addition of aquaculture plant by-product and citrus gourd,

7 is a phagocytosis test by leukocytes in the blood of the flounder bred with the functional fermented fish meal prepared by the addition of aquaculture plant by-product and citrus gourd,

Figure 8 shows the validation of lysozyme activity of the flounder bred with functional fermented fish meal prepared by the addition of aquaculture by-products and citrus gourd.

The present invention relates to a functional fermented fish meal using aquaculture farm harvest and citrus processed by-products, and to a method for producing the same, in more detail, by-products (waste fish) and citrus juice by-products occurring naturally after farming process Functional fermented fish meal is prepared by using dried product of phosphorus citrus gourd. In the conventional case, rice bran was mainly used as a moisture control agent in preparing fish meal, but when citrus gourd was processed for this purpose, it played a role as a moisture control agent. This citrus gourd contains flavonoids and vitamins, which are known to have excellent functions such as antioxidants. As a result, the citrus gourd was found to have improved immunity with reduced mortality of cultured fish. It was. Accordingly, the present invention relates to a functional fermented fish meal having a characteristic of reducing resource recycling and environmental pollution by efficient treatment and utilization of aquaculture farms and citrus processing by-products, and a method of manufacturing the same.

    Organic waste generated in Korea can be roughly classified into domestic waste and sewage sludge, agricultural waste, forest waste, industrial waste, and fish waste. It is reported that these organic wastes account for more than half of the total waste generated, and because of its high perishability, it is estimated that various organic wastes, such as odors and sewage leaks, are highly likely. Therefore, the treatment technology for stabilization, reduction and recycling of organic waste has been highlighted. In addition, organic wastes can be used directly or indirectly as a source of energy for living organisms, or have many alternative sources of fossil fuel (methane gasification) and recycling (feeding and composting), so that by-products can be stabilized or reduced by appropriate treatment technology. By recycling, the consumption of existing resources can be reduced to secure economic feasibility and contribute to environmental conservation.

    In the 1990s, waste problems became socialized, and secondary pollution occurred during incineration and landfilling, increasing interest in food waste and sludge. Most of them are classified as organic wastes because they contain many organic substances that can be decomposed by microorganisms. Research on the use of such food waste as a fertilizer or feed has been conducted by many researchers for a long time, and specially manufactured food waste drying apparatus or fermentation apparatus has been devised for practical use. However, a major problem in these studies is the fact that food waste is not constant and the amount of salt added varies widely. This suggests that it is impossible to produce certain ingredients from food waste. Therefore, it is a reason why the use as a fertilizer increases rather than as a feed. Another problem that has been pointed out is that food waste has a number of different foreign matters that need to be efficiently sorted out.

    In 2001, toxic red tides resulted in a mass mortality of fish in the southern coastal area, and reported about one million dead fish (Hanrye-jeom newspaper, Aug. 28, 2001). In 2002, about 120,000 fish were killed at the Tongyeong aquaculture farm that suffered the first red tide in the year (Hanrye Newspaper, August 26, 2003). In early 2003, it was reported that as many as 600,000 mullet and 200,000 fry were killed in only four farms in Mandoli, Simwon-myeon, Gochang-gun, Gochang-gun, Jeollanam-do. In 2006, according to the Ministry of Oceans and Fisheries, the mortality rate of cultured fish in Jeju reached 34%. In light of this fact, it can be seen that the repetitive mass deaths of fish in Korea occur every year. As mentioned above, the killing of fish is caused not only by red tide and cold wave, but also by vibrio, edward, streptococci, and scotch insects in all farms, especially land tank farms. Approximately 20% of the farms are said to be dead. Most of the resulting debris is used as feed for livestock after being buried or boiled in the ground.

    Fishmeal is the highest source of protein for marine fish feed, and this fishmeal is a high quality protein source, but supplies are unstable and expensive. In addition, most farmed fish farms feed live feed such as frozen horse mackerel and canary, or use moist pellets (MP) to feed nutritional diseases, water pollution due to feed loss, and raw material costs. There are problems such as rising, causing many disadvantages.

   Meanwhile, fish processing residues (heads, tails, shells, intestines, etc.) obtained after the fish filleting process in fisheries processing plants are simply dried and used as fish meal components of fish farming feed. Although the production of fish meal for fish farming from fish processing residue is simple drying process, there is no technical difficulty, but there is a shortage of useful ingredient as fish meal due to lack of muscle protein of fish and the disadvantage of poor digestibility of fish meal due to simple drying. Can be. In addition, the simple drying process tends to give off bad smells due to evaporation of moisture, which tends to deteriorate the surrounding environment.

   Therefore, in the present invention, by using aquaculture farms harvested in aquaculture farms to produce fermented fish meal by drying at a high temperature fermentation conditions, dried products made by drying citrus gourd containing a large amount of flavonoids and vitamins as a moisture control agent during fermented fish meal production It is to develop functional fermented fish meal.

The present invention is a dry flounder farming waste produced in Jeju in large quantities every year (waste fish, 3,000 to 5,000 tons annually) and dried powder form of citrus fruit by-products citrus processing (hereinafter dried citrus gourd, 30,000 tons to 50,000 tons per year) To develop high-quality functional fermented fish meal with no odor, and is also a farm feed developed from by-products of farms, ensuring maximum efficiency and safety for pathogenic bacteria. It aims to spread functional fermented fish meal.

In order to achieve the above object, a functional fermented fish meal is prepared using dried by-products of citrus processing, a by-product (waste fish) and citrus juice process that occur naturally in aquaculture farms, and frozen farms collected from each aquaculture farm. Add 1 ~ 20 kg of dried citrus fruit, which is a moisture modifier with functionality based on 24 kg of harvest, and add the soil-derived bacteria as fermentation bacteria at a weight ratio of 10,000: 0.5 to 4 relative to the total mass. After adjusting the external steam temperature of the fermenter to 250-300 ° C. and waiting for 10-30 minutes, when the temperature inside the fermenter reaches 90 ± 5 ° C., the fermentation process for fish meal is started. The fermentation time is automatically terminated when the water content of the prepared fish meal is less than 5% after fermentation, and the total fermentation process time is measured. And the production rate is calculated by measuring the content of the functional fermented fishmeal derived from the aquaculture farm harvest, and to produce a functional fishmeal under the same conditions.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples, and various modifications can be made without departing from the scope and spirit of the present invention. Is possible.

Example 1 Analysis of General Component Content

The analysis of the general components of the farm collection is water, crude protein, crude carbohydrate, crude lipid, ash, etc. Crude carbohydrates, crude fiber, crude fat, and vitamin C. According to the AOAC method (2002), the general ingredient analysis method was moisture drying at 105 ° C under atmospheric pressure, and crude protein was analyzed using Kjeltec ^TM 2300, Foss Co. Ltd., Denmark. By Sohxlet method (Sohxlet system 1046, TacatorAB, Sweden), crude ash was quantified after burning for 12 hours or more in a 550 ℃ incinerator. The crude fiber was analyzed by an automatic crude fiber analysis device, and vitamin C content was analyzed by Indophenol titration method. Carbohydrate is expressed as a difference value for the content of water, crude protein, crude fat and crude ash with a total content of 100%.

    The general components of the dead fish collected from each farm in Jeju Island and the citrus gourd and the high temperature dry citrus gourd collected from Jeju Island Development Corporation were analyzed and shown in Table 1.

The result was that the aquaculture by-products had a water content of about 73.3% and a crude protein of 18.2%, which is about 68% protein by dry weight. In the case of citrus gourd, the moisture content was 92.5%, mostly water, and crude carbohydrate containing crude fiber was 5.7%, which was 76% by dry weight. Vitamin C content of citrus gourd accounted for about 985 mg of 100 g of wet weight.

Example 2 Preparation of Functional Fermented Fish Meal Using Aquaculture Harvest and Citrus Foil

   The high-speed fermentation drying equipment used for the production of functional fermented fish meal using fish farm collection and citrus gourd is used to produce organic fertilizer from food waste. It was developed by Mitsubishi Electric Corporation in Nagasaki Prefecture, Japan. : BF-30) was used.

   On the other hand, the fermentation bacteria used to prepare a functional fermented fish meal using aquaculture harvest and citrus gourd in a high-speed fermentation dryer was used as aerobic high temperature bacteria belonging to the genus Bacillus, which was purchased from Seiwa, Japan. In addition, the characteristics of the bacteria used in the present invention is a heat-resistant soil-derived fermentation bacteria have the ability to decompose organic materials of plants and animals mainly at 80 ~ 90 ℃ the internal temperature of the high-speed fermentation drying apparatus was used in the present invention.

  In the preparation of dried citrus gourd 20 kg citrus gourd was injected into a high temperature dryer used for preparing fermented fish meal, followed by drying for 80 minutes at 120 ~ 130 ℃. After drying the citrus fruit pulverized to 80mesh, it was used as a water modifier (1) when preparing fish meal.

   On the basis of 24 kg of frozen farm collections (2) collected from each farm, 1-10 kg of dried citrus fruit (1), a moisture modifier (1) with added functionality, was used as a whole substrate. After adding at a weight ratio of 10,000: 0.5 to 4 relative to the base mass, the external steam temperature of the fermenter was adjusted to 250 to 300 ° C. for 10 to 30 minutes, and then the temperature inside the fermenter was set to 90 ± 5 ° C. The fermentation process for fish meal was started. The fermentation time is automatically terminated when the water content of the prepared fish meal is 1.1 ~ 5.0% after fermentation, and the total fermentation process time was measured. And the production rate was calculated by measuring the content of the functional fermented fish meal derived from aquaculture farm harvest, and produced functional fish meal under the same conditions. The general component analysis according to the citrus gourd content is shown in Table 2, and the general ingredient analysis according to the amount of fermentation bacteria is shown in Table 3.

The results showed that the protein content (31.1%) was the highest when 1 kg of citrus gourd per 24 kg of aquaculture farms was harvested, and the fermented bacteria showed the highest value of 32.2% at 10,000: 2 weight ratio compared to the whole substrate. Could. Therefore, the production of functional fermented fish meal for the flounder growth rate experiment was used to prepare a weight ratio of 24 kg of aquaculture farm harvest, 1 kg of citrus gourd as a moisture control agent and fermented strains 10,000: 2.

Example 3 Risk Assessment for Bacteria of Functional Fermented Fish Meal Prepared Using Aquaculture Harvest and Citrus Foil

     The safety of fish disease bacteria from aquaculture harvested fermented fish meal was analyzed for quality safety. That is, the isolated fish disease bacteria were identified from the by-products collected from each farm, and fermented fish meal was prepared under optimal fermentation conditions. DNA was isolated from the fermented fish meal prepared with bacterial culture and identified by bacterial PCR.

In other words, this study was performed on halibut samples suspected of being infected with streptococcosis, Edward's disease, Vibrio disease, and S. aureus, reported as major bacterial diseases of flounder. Streptococcus iniae , Streptococcus parauberis , the causative agent of streptococcosis , Edwardsiella tarda , the causative agent of Edward's disease, Flexibacter maritimus , reported as the causative agent of glide bacterium, was isolated. First, the separation of Streptococcus niaea , Streptococcus parauberis, and Edward ciellatada After aseptic dissection of the flounder, the kidney and liver tissues were plated on a plate of 2.5% NaCl-BHIA (Difco, USA), 2.5% NaCl-SS agar (Difco, USA), blood agar medium, 30 ± 0.5 ° C, 24 After incubation for ˜48 hours. The isolates were isolated from colonies grown on each plate and subjected to biochemical properties such as gram staining, oxidase test, and catalase test using MacFaddin (2000). After briefly identifying Edward Siella spp. , Streptococcus ina , Streptococcus parauberis, and Edward ciellata were identified by PCR. Isolation and identification of Flexibacter maximus was performed by microscopic examination of the surface ulcers of fish to identify the glial bacteria, and then cultured in Hsu-Shotts medium (Bullock, 1986), followed by separation of pale yellow colonies and biochemical properties. It was identified through PCR. Identification of the isolate strain using PCR used the primer set and conditions shown in FIG. At this time, MFX6100 (TOYOBO, Japan) was used for DNA extraction for PCR.

   After the isolation and identification of the causative bacteria from each aquaculture by-products, the number of bacteria of infected lung fish was confirmed, and the killing of fish disease bacteria of fermented fish meal produced through fermentation under the optimum fermentation conditions. The killing of fish disease bacteria was confirmed by diluting the fermented fish meal powder sample using sterile physiological saline and filtration using a membrane to extract total DNA and PCR using the primer set shown in FIG. 3. In order to check the contamination level of the general bacteria on the prepared fish meal, the number of living cells was measured. The viable cell count was measured by plate smearing. In other words, the prepared fish meal was incubated at 30 ± 0.5 ° C. and 24 ± 2 hours by injecting plate count agar (Difco, USA) and marine agar 2216 medium (Difco, USA) into each 1 ml of the sample diluted in sterile saline. The viable cell count was then measured.

Four isolates of S. iniae, S. parauberis, E. tarda, and F. maritimus were harvested from the farms and artificially left for 1 hour after adding about 5 × 10 ⁷ cfu / g. The fermented fish meal was prepared in a fermentation drying apparatus, and the infection of the fish bottle was examined by the colony counter method and is shown in FIG.

In this result, when a certain amount of sample was taken from the fermented fish meal derived from aquaculture by-products (1%) and then diluted 10- and 1000-fold, it was found that no bacteria were grown.

Table 4 shows the number of fish disease infectious bacteria detected in the functional fermented fish meal extract prepared by the addition of aquaculture plant by-product and citrus gourd. When the extract was incubated in agar plate medium without dilution, slight bacterial growth was observed.

This showed that some colonies were growing when the extract extract was filtered as shown in Table 4.

Figure 2 shows the results of PCR confirmation for the residual fish disease infected bacteria for the safety evaluation of the prepared functional fermented fish meal, it will be described in more detail,

Line 1 of (A) is a DNA size marker, lines 2 and 3 are extracts of fermented fishmeal prepared from aquaculture farms infected with Streptococcus pauberis, and lines 4 and 5 are streptococcus inies infected. An extract of fermented fishmeal prepared from aquaculture farm harvest, line 6 is a marker indicating Streptococcus parauberis, and line 7 is a marker indicating Streptococcus inae.

Line 1 of (B) is a DNA size marker, lines 2, 3, 4 and 5 are extracts of fermented fishmeal prepared by Edward Sielatada infected farm harvest, and line 6 shows Edward ciellatada. Indicated marker.

Line 1 of (C) is a DNA size marker, lines 2, 3, 4, and 5 are extracts of fermented fishmeal prepared from aquaculture farms infected with Plexibacter maximus, and line 6 is a flexibacter maximus Indicated marker.

As a result, DNA was extracted from the cultured chalet cultured by fermented fish meal extract derived from aquaculture by-products to confirm whether the bacteria were artificially infected by PCR method is shown in Figure 2. As a result of PCR, it was confirmed that the bacteria found when the stock solution was cultured were not artificially added four kinds of fish disease bacteria, but other bacteria incorporated in the manufacturing process. Although some bacteria were found from the stock solution of fermented fish meal derived from aquaculture by-products, it was confirmed that this was not a fish-borne bacterium. It was confirmed that it was a safe product, and it would be possible to produce a complete product just by inhibiting the influx of bacteria in the process.

Experimental Example 4 Measurement of DPPH Radical Scavenging Activity for Maximization of Functionality of Fermented Fish Meal Prepared Using Aquaculture Harvest and Citrus Foil

Figure 3 shows the measurement of free radical scavenging activity and hydrogen peroxide scavenging activity in order to determine the functionality of fermented fish meal derived from aquaculture farm and citrus gourd, produced by high temperature fermentation of aquaculture harvest and citrus gourd Free oxygen radical scavenging activity of functional fermented fish meal was measured by Electron donating ability (EDA) by modifying the method of Blois (1958). 0.1 ml of each seaweed extract was added to 2.9 ml of 4.0 × 10 ^-4 M DPPH (1,1-diphenyl-2-picrylhydrazyl) solution, stirred for 5 seconds, and reacted for 30 minutes, and then the absorbance was measured at 516 nm. The electron donating ability was shown as a reduction ratio.

    The results are shown in Figure 3, the fermented fish meal added citrus gourd showed a DPPH free radical radical scavenging activity than the rice bran added fermented fish meal. In addition, methanol and water extracts of fermented fish meal containing citrus gourd were measured and DPPH free radical activity was measured. The methanol extract showed high radical scavenging activity, and both extracts had a concentration of 2 mg / ml or higher. The scavenging rate was over 50%. When the concentration of the extract was 4 mg / ml, the scavenging rate of water and methanol extract was about 80% and 90%, respectively. On the other hand, DPPH free radical radical scavenging activity was less than 8% at all concentrations in fermented fish meal processed under the same conditions without adding citrus fruit.

Example 5 Determination of Hydrogen Peroxide Scavenging Activity for Functional Maximization of Fermented Fish Meal Prepared Using Aquaculture Harvest and Citrus Foil

       Hydrogen peroxide scavenging activity of functional fermented fish meal prepared by aquaculture fermentation of dried farms and citrus peels was measured according to the method of M et al. (1985). 100 μL of 0.1 M phosphate buffer (pH 5.0) and samples were mixed in a 96 hole microwell plate. Then, 20 μL of hydrogen peroxide (hydrogen peroxide) is added and reacted at 37 ° C. for 5 minutes. Finally, 30 μL of 1.25 mM EBITS (ABTS) and peroxidase (peroxidase, 1 unit / mL) were added and reacted at 37 ° C. for 10 minutes and the activity was measured at 405 nm.

Hydrogen peroxide scavenging activity, like DPPH free radical scavenging activity, was higher than that of water soluble extract, and the scavenging rate increased with increasing concentration of sample. In particular, when the concentration of the sample was 4 mg / ml showed a high scavenging activity of about 80% or more ( Figure 3 ).

Hydrogen peroxide scavenging activity The scavenging activity of fermented fish meal which was processed under the same conditions without addition of citrus gourd showed little scavenging activity. These results indicate that when fermented fish meal is prepared using only citrus meal instead of rice bran, which is a moisture modifier, the content of functional substances contained in citrus gourd may be relatively high, especially vitamin C (Vitamin C), which is known to have high antioxidant activity. It may be due to the effect of flavonoid compounds.

Example 6 Measurement of DNA Damage Inhibitory Activity for Maximization of Functionality of Fermented Fish Meal Prepared from Aquaculture Harvest and Citrus Foil

Blood samples were drawn from healthy fish. 5 mL of blood was mixed with 5 mL of phosphate buffered saline (PBS) and 5 mL of histopac was added. After centrifugation at 40 g for 30 minutes, lymphocytes were isolated and washed with phosphate buffered saline (PBS). Finally, the cells were suspended in medium (90% fetal calf serum, 10% dimethyl sulfoxide) at a concentration of 6 × 10 ⁶ cells / mL.

The prepared fishmeal was diluted to 0, 1, 10, 25, 50 μg / mL using phosphate buffered saline (PBS), and lymphocytes at 2 × 10 ⁴ cells / mL with 1 mL of sample were added. Incubated at 25 ° C. for 60 minutes. After preculture, cells were centrifuged at 380 g for 5 minutes. The harvested cells were resuspended at 0 ° C. for 5 minutes using 50 μM hydrogen peroxide-treated phosphate buffered saline (PBS), and the control group was treated with phosphate buffered saline (PBS) not treated with hydrogen peroxide. Was suspended using. Cells were then centrifuged and washed with 1 mL phosphate buffered saline (PBS).

Alkaline comet assay was performed by supplementing the method such as Sign (Singh). Cells were mixed with 75 μL of 0.5% low melting agarose (LMA) and then dispersed on slides precoated with normal melting agarose (NMA) with 1% melting point. Once the gel had solidified, add 75 μL of 0.5% LMA onto the slide again, cover the cover glass, and remove the lysis buffer (2.5 M NaCl, 100 mM IDT, 10 mM Tris, and 1% sodium laurylma). Soak in sodium laurylasarcosine (1% Triton X-100 and 10% dimethylsulfoxide) and soak for 1 hour at 4 ° C. After drying, the slides were arranged in an electrophoretic tank containing 300 mM sodium hydroxide and 10 mM sodium (Na ₂ EDTA, pH 13.0) and unpacked for 40 minutes. DNA electrophoresis was performed for 20 minutes at a voltage of 25 V / 300 mA. Slides were rinsed three times with 5 minutes in neutralizing buffer (0.4 M Tris, pH 7.5), washed for 5 minutes with ethanol and stained with 50 μL of ethidium bromide at 20 μg / mL. . Measurements were performed with an image analyzer (kinetic Imaging Komet 5.0, UK) and a fluorescence microscope (LEICA DMLB, Germany). The degree of DNA damage in lymphocytes was measured in the tail at the distance or tail length of DNA fragments moved from the nucleus. The tail instantaneous value multiplied by the percentage of DNA contained was measured, and two slides were made for each subject to measure the degree of DNA damage in a total of 100 lymphocytes, 50 each.

Figure 4 shows the DNA protection effect of the flounder derived from fermented fish meal derived from aquaculture farms and citrus gourd, the bar graph shows the length of% tail, the sun graph shows the effect of inhibiting cell damage. When the concentrations of 10 μg / ml and 50 μg / ml were treated in the lymphocytes of the flounder with the fermented fish meal extract derived from the aquaculture by-products, the protective effect was about 70% and 80%, respectively. At the concentration of μg / ml, DNA protection was about 90%, whereas the lymphocytes of fish treated with the extract of fermented fish meal without the addition of dry citrus gourd added the rice bran to 80% of DNA damage regardless of the concentration of the extract. More than% happened.

Based on the above results, the use of citrus gourds from fermented fish meal from aquaculture by-products is expected to enable the production of functional fish meals using citrus gourds, which produce about 30,000 to 50,000 tons annually in Jeju. If it is used as a water conditioner instead of rice bran, it uses only farm by-products and dried citrus gourds, which not only simplifies the manufacturing process but also lowers the manufacturing cost. It is thought that the production of functional fermented fish meal will be sufficiently possible.

Example 7 Breeding of Olive Flounder Using Functional Fermented Fish Meal Prepared from Farmed Fish and Citrus Foil

     Flounder growth experiment using the present invention was carried out in a farm located in Seogwipo, Jeju Island, the flounder used before the start of the experiment was adjusted to feed the commercial feed for about 3 weeks to adjust to the experimental environment. The average weight of experimental fish was 230 ± 15 g and 140 ± 10 g before starting the experiment. The same experiment was carried out on two different fish weights and was repeated twice. The breeding tank used a concrete square tank of 10 (W) × 10 (L) × 1 (H) (m) and housed about 2,000 birds in each tank. Breeding quantity was returned 18 times a day, and aeration for sufficient oxygen supply. During the experiment, the water temperature, dissolved oxygen (DO), pH, and salinity were measured every day. The dissolved oxygen was measured using a dissolved oxygen meter and the pH was measured using a pH meter. During the experiment, the breeding water temperature was 18 ~ 20 ℃, the minimum water temperature was 18 ℃ and the maximum water temperature was 20 ℃. Breeding water was used seawater containing 35 ± 5% of groundwater, dissolved oxygen was 7.0 ± 0.5 mg / l, pH was 7.5 ± 0.5. Forage was used for 24 kg of aquaculture farms, 1 kg of dried citrus gourd as a moisture conditioner, and fermented strains were prepared at a weight ratio of 10,000: 2, with 7% added to the amount of wet feed. . Twice a day (7 AM and 5 PM) were fed full stomach (supplied until swelling), experimental breeding was carried out for 12 weeks, growth was measured at 4, 8 and 12 weeks, respectively.

Figure 5 shows the growth rate of the fish dietary fermented fish meal prepared by the addition of aquaculture by-products and citrus gourd, the left figure is about 140 g of the initial body weight of fish before the start of breeding, the right was about 230 g, Both the control and the fish meal added with the functional fermented fish meal using citrus gourd showed good growth, while the control group showed a slightly higher growth rate than the fish meal added with the fermented fish meal. , 140 g of the experimental group, fermented fish meal intake fed the fish meal supplements showed a slightly higher growth rate, but was not significant (p> 0.05, Figure 5).

Figure 6 shows the mortality and mortality of fish fed the functional fermented fish meal prepared by the addition of aquaculture plant by-product and citrus gourd, the above figure is about 140 g of the initial fish weight of the fish before the start of breeding, the right is about 230 g.

In mortality, the control group showed the highest mortality at 4 weeks in both groups with different fish weights, and the highest mortality at 8 weeks in the fish meal diets containing fermented fish meal. However, it was confirmed that the fish flour added diet fermented fish meal was lower than the control (nonfermented fish meal added) in both groups. This result is thought that fermented fish meal lowered the mortality by improving the physiological function of the fish and is shown in FIG.

Example 8 Immune Enhancement Effect of Fishes Fed Functional Fermented Fish Meal

In the present invention, not only the growth rate of the fish but also the immune enhancing effects of the fish were examined. To investigate the phagocytosis caused by radicals, respiratory hyperactivity was analyzed according to Secombes (1990). Whole blood and 0.2% NBT reagent were mixed in a 1: 1 ratio in a micro tube, and then reacted at room temperature for 30 minutes, and then 1 ml dimetyl formamide was added. 200 μl of the reaction was dispensed into the microplate, and the plate was finally measured by microreader (Tecan ^™ ) at 560 nm to analyze the respiratory activity of the experimental fish.

Figure 7 shows the phagocytosis verification by leukocytes in the blood of the flounder bred with functional fermented fish meal prepared by the addition of aquaculture plant by-product and citrus gourd, NBT activity of the experimental diet fed the fermented fish meal added compared to the control It was possible to signify what was shown more than twice as high (Fig. 7). Therefore, the results of this experiment suggest that the fish fed FFM-added fish may have a higher phagocytosis effect than the intake of raw foods, and thus have higher potential resistance to foreign substances.

    Lysozyme, a factor involved in the humoral immune response, is an enzyme that kills bacteria by separating beta-1,4 bonds between acetylmuramic acid and acetylglucosamine on the bacterial wall, and increases antibody and complement activity against gram-positive bacteria. Increase phagocytosis of phagocytes.

In the experimental method, M. lysodeikticus solution was prepared by adding Micrococcus lysodeikticus to 0.02 M sodium citrate buffer (pH 5.5) according to Jaya and Sahoo. 15 μl of serum was dispensed into 96-well plates, and immediately 150 μl of M. lysodeikticus solution was measured and absorbance was measured at 450 nm. After the reaction at 0.5 ° C. for 0.5 and 1 hour, the absorbance values were obtained at the same wavelength. Find the difference between the absorbance values before and after. The active unit of lysozyme was defined as the amount of enzyme that showed a decrease in absorbance of 0.001 per minute.

Figure 8 shows the validation of lysozyme activity of the flounder bred with functional fermented fish meal prepared by the addition of aquaculture plant by-products and citrus gourd.

As a result of this experiment, it is thought that the flounder fed with fermented fish meal increased the immune system more than the flounder fed with only raw feed, thus increasing the ability to cope with potential diseases ( FIG. 8 ). .

[Reference Example] Statistical Analysis

     Data was analyzed using a statistical program SPSS (version 10). The figures are expressed as mean ± standard error (SE). One-way analysis of variance (ANOVA) was performed to test the significance of each group.

     As described above, although the present invention has been described with reference to the above embodiments, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.

Referring to the effects of the above-described configuration and operation in detail as follows. As described above, as a result of actually applying the functional fermented fish meal produced using the farm harvest and citrus gourd according to the present invention in the farm, as well as the growth rate of the fish when the present invention is added than when using only raw feed Immunity increased and mortality decreased. Therefore, we can conclude a tentative conclusion that healthy fish can be farmed, and can also be used as a feed manufacturer by manufacturing and supplying protein feed sources in Korea, which mostly depend on imports.

Claims (4)

In the production method of functional fermented fish meal using aquaculture farm harvest and citrus processed by-products, As a water conditioner (1) during fish meal preparation, 20 kg of citrus fruit paste was poured into a high temperature dryer and dried at 120 to 130 ° C. for 80 minutes. The dried citrus fruit was pulverized with 80 mesh, and the dried citrus fruit was used as a moisture modifier (1), and on the basis of 24 kg of the aquaculture farm (2), the citrus fruit was dried as a moisture modifier (1). 5 to 10 kg was added to the aquaculture farm harvest (2), and the mixed mixture was used as a whole substrate. The fermentation bacteria were added at a weight ratio of 10,000: 0.5 to 4 relative to the total mass, and then the external steam temperature of the fermenter was 250. After adjusting to ~ 300 ℃, after waiting for 10-30 minutes, when the temperature inside the fermenter is 90 ± 5 ℃ to start the fermentation process for fish meal production, The fermentation time of the fishmeal prepared through the fermentation process is functional fermentation using aquaculture plant harvest and citrus processed by-products, characterized in that the fermentation is finished after the fermentation is completed, when the water content of the fishmeal is 1.1 ~ 5.0% Method of preparing fish meal. According to the production method of claim 1, the fermentation bacteria are added to the mixture of the dried citrus fruit and the aquaculture plant harvester (2) with a water conditioner (1), fermented by a fermenter and produced as a fish meal, characterized in that Functional fermented fish meal using citrus processed by-products. 3. The functional fermented fish meal according to claim 2, wherein the methanol extract of the functional fermented fish meal using the aquaculture plant harvest and citrus processed by-product has an effect on radical scavenging activity and hydrogen peroxide scavenging activity. [Claim 3] The cultured fermented fish meal and citrus processed by-products of claim 2, wherein the functional fermented fish meal using the fish farm and the citrus processed by-products have the effect of combating diseases by improving the physiological function of the fish and increasing the immune system. Functional fermented fish meal using.

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