KR20080037457A - Compositions for addition to feed for fish comprising bacillus polyfermenticus, bacillus licheniformis and saccharomyces serevisiae - Google Patents

Abstract

A fish feed additive composition contains Bacillus polyfermenticus, Bacillus licheniformis and Saccharomyces cerevisiae is provided to increase the growth and nonspecific immunoreactivity of fish and feed efficiency when added to fish feed. A fish feed additive composition contains at least one of Bacillus polyfermenticus, Bacillus licheniformis and Saccharomyces cerevisiae. The Bacillus polyfermenticus, Bacillus licheniformis and Saccharomyces cerevisiae contain 1x10^8 to 10^10 CFU/kg, 1x10^8 to 10^10 CFU/kg and 1x10^7 to 10^9 CFU/kg respectively. The composition is added to feed for olive founder and Sebastes schlegeli.

Description

Compositions for addition to feed comprising fish Bacillus polyfermenticus, Bacillus polylichmenticus, Bacillus licheniformis and Saccharomyces serevisiae, including Bacillus polyfermentus, Bacillus rickeniformis and Saccharomyces cervisia

The present invention relates to a composition for fish feed comprising Bacillus polyfermentus, Bacillus rickeniformis and Saccharomyces cerbizi, more specifically Bacillus polyfermentus ( Bacillus polyfermenticus ), Bacillus licheniformis ( Bacillus licheniformis ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) relates to a composition for adding fish feed characterized in that it comprises at least one.

Bacillus polyfermentus bacteria, which are used as probiotics, are bacilli that form active spores and secrete various types of enzymes and have the property of inhibiting proliferation by lysing pathogenic bacteria such as typhoid bacteria, paratyphoid bacteria, and erythrocytes. In particular, it can be said that it is possible to prevent and treat cholera by ingesting marine fish which is a problem during summer as an excellent bacterium with cholera bacteria. In addition, since it forms active spores, it has a high resistance to gastric acid and thus has a high intestinal reach rate, which is an excellent strain showing an excellent effect in treating intestinal diseases. Bacillus rickenformis is also a spore-forming bacterium that inhibits growth against the diarrhea-causing Salmonella and Escherichia coli O-157, and it is used as an animal feed additive in Europe because it has the effect of decomposing prion protein, a causative agent of mad cow disease. . Saccharomyces serbidier strains have been ingested by humans for a long time, and they are a microbial group having so-called yeast-type cells among fungi that reproduce nutrients by the germination method and have a relatively long single cell generation and eukaryotic cell structure. have. Yeast has attracted great attention in the production of various fermented foods, as well as the production of nucleic acid-related substances in yeast cells obtained by mass culture and the use of proteins and vitamins in the cells.

Effect of Yeast Culture Addition in the Unit Animals Saccharomyces Since cerevisiae contains nucleic acid and such as nucleic acid salt and glutamic acid released during yeast cell growth in the medium, it improves palatability, and because it has a strong affinity for oxygen, it helps the growth of anaerobic bacteria by removing oxygen in the intestine. The feed efficiency is improved by the digestive enzymes produced by yeast, and the growth rate is improved by the improvement of fat digestion caused by the lipase activity of the yeast culture. In addition, the yeast cell wall has the ability to adsorb various substances, acting as a pH buffer, vitamin B group and amino acids produced by yeast improve the fertilization rate and hatching rate of laying hens.

Today, the fish farming industry is one of the fastest growing fisheries industries, and its economic importance is recognized worldwide due to the growing demand for seafood. In Korea, the three sides are the sea and almost all of the coastal area is used for aquaculture. In farmed fish, immunity is lowered due to dense breeding and deterioration of water quality, and therefore, they are often in an environment susceptible to infection by bacteria and parasites. Currently, various kinds of antibiotics are used in Korea alone or added to feed for disease treatment and prevention of infection, but more safety is required, and continuous use of antibiotics leads to an increase in resistant microorganisms. It can be transmitted, and the accumulation of antibiotics, along with the destruction of the intestinal flora of the farmed fish itself, can interfere with growth and adversely affect food consumption. In particular, the intestinal colony is not only important for metabolic processes but also essential for strengthening immunity and animal growth and maintaining health, and plays a beneficial role in the production of vitamins. Abnormalities in defense mechanisms facilitate the invasion of hospital microorganisms. Probiotics are known to have antimicrobial activity that inhibits the growth of pathogenic bacteria in various types of normal intestinal flora, and thus plays an important role in maintaining the balance of normal intestinal flora and suppressing the growth of pathogenic bacteria. However, the useful effects of these probiotics are mainly the experimental reports on humans and livestock, and the developed probiotics are also applied to humans and livestock. The optimal growth temperature for humans and livestock is 37 ℃, which is inappropriate for farmed fish growing at 15 to 25 ℃, especially for seawater farmed fish, which is able to withstand high salt concentrations. As the aquatic environment of fish is increasing rapidly, the water environment is getting worse and the frequency of fish disease is increasing.Therefore, there are reports showing the availability of probiotics that can substitute for antibiotics, and the effect of direct application to sea fish. Considering the reality of regulating the approval of new antibiotics in Korea, as a part of the development of probiotics that can suppress and replace the abuse of antibiotics in aquaculture fish, it is possible to form beneficial bacteria in fish farms, prevent growth and prevent disease and water quality. Development of probiotic for farmed fish for the purpose of pollution control is essential.

Recently, one of the methods for increasing the disease resistance of aquaculture fish and increasing its productivity, the vaccine method is involved in the specific immune response to a disease. However, fish disease vaccines, which have been successfully developed to date, have a limited scope and are too expensive to be used in terms of price. On the other hand, enhancing the nonspecific immune response of fish is very important in the sense that it can increase resistance to many kinds of fish diseases, and evaluation of immune enhancing substances to prevent diseases of farmed fish by enhancing the nonspecific immune response. Has been evaluated by many researchers in recent years. However, in order to increase fish productivity, research on growth accelerators and feed intake accelerators should be conducted together to bring optimal growth and survival rates of fish. These substances include polysaccharides, animal and plant extracts, probiotics, hormones, nucleic acid-related substances, organic acids, and sugars. These substances promote the growth of fish and improve feed efficiency or enhance the nonspecific immune response of fish to improve productivity. And it is evaluated as a substance for preventing the disease of fish. Therefore, it is necessary to investigate how these substances affect fish growth and nonspecific immune responses.

Therefore, in the present invention, as a result of intensive research efforts to overcome the problems of the prior art, the main form of feed additives in the single or mixed mixture of Bacillus polyfermentus, Bacillus rickeniformis, Saccharomyces cerbizier, etc. As a result of administering to the fish, halibut and jejubolak, it was confirmed that each of the above strains could have an excellent effect on reducing the mortality and increase, nonspecific immune response of the cultured fish by complementary action, and completed the present invention.

Accordingly, the present inventors have made intensive research to overcome the problems of the prior art, as a result, the composition for adding fish feed containing any one or more of Bacillus polyfermentus, Bacillus rickeniformis and Saccharomyces cerbizi It was confirmed that this fish could increase the growth, feed efficiency and nonspecific immune response, and completed the present invention.

Therefore, the main object of the present invention is to replace the feed additives added with antibiotics for the purpose of treating diseases used in aquaculture fish, and to provide a composition for adding fish feed that can increase the growth and non-specific immune response of fish There is.

According to one aspect of the invention, the invention is Bacillus polyfermenticus (BP), Bacillus rickenformis ( Bacillus licheniformis , BL) and Saccharomyces cerevisiae , SC) provides a composition for adding fish feed, characterized in that it comprises any one or more.

In the present invention, the composition can be used as a feed additive for all fish commonly used, preferably, it is characterized in that it is added to the feed of the flounder, seaweed rockac. Probiotics used in humans and livestock have an optimal growth temperature of 37 ° C, which is inadequate for farmed fish growing at 15 to 25 ° C. Bacillus polyfermentus, Bacillus rikenimoformis of the present invention as a strain to form an active spore, can be active under the growth conditions of seawater cultured fish can exhibit the antibacterial effect of pathogenic bacteria. In addition, Saccharomyces cervage contains nucleic acid and has a nucleic acid salt and glutamic acid released during the growth of yeast cells, which improves palatability, and because of its high affinity for oxygen, anaerobic Helps grow bacteria The feed efficiency is improved by the digestive enzymes produced by yeast, and the growth rate is improved by the improvement of fat digestion caused by the lipase activity of the yeast culture. In addition, the yeast cell wall has the ability to adsorb various substances, acting as a pH buffer, vitamin B group and amino acids produced by yeast improve the fertilization rate and hatching rate of laying hens.

In the present invention, the Bacillus polyfermentus is 1 × 10 ⁸ ~ 10 ¹⁰ CFU / kg, Bacillus rickeniformis is 1 × 10 ⁸ ~ 10 ¹⁰ CFU / kg, Saccharomyces cervizi 1 × 10 It is preferred that the composition is characterized in that it comprises ⁷ to 10 ⁹ CFU / kg.

In the present invention, the composition is preferably a composition comprising all of Bacillus polyfermentus, Bacillus rickeniformis, Saccharomyces cervizier. Even when the strain is added alone in the composition can increase the growth and non-specific immune response of the fish, but when mixing the three strains may exhibit an increased effect due to the complementary action of each strain.

In the present invention, the composition is characterized in that it further comprises glucose as an excipient in addition to any one or more of Bacillus polyfermentus, Bacillus rickeniformis and Saccharomyces cerbizi. The excipient is mixed with the strains of the present invention to make a feed additive into a certain form, any excipients that are commonly used may be used, but preferably, glucose is used as an excipient.

In the present invention, the composition is preferably a composition characterized by improving fish growth and nonspecific immune response. Vaccination may be used to increase disease resistance and increase productivity of aquaculture fish, but it is involved in a specific immune response to a disease and the range of fish disease vaccines that have been successfully developed to date is limited. . While there are disadvantages that are too expensive to use in terms of price, enhancing the nonspecific immune response of fish is important in the sense that it can increase resistance to many kinds of fish diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these examples.

Example  1. Preparation of feed additives for fish

TABLE 1 Composition of Fish Feed Additives

1 kg of the feed additive of the composition table is mixed with 1 ton of mixed feed and administered to fish.

(Experimental example 1) Administration effect of the feed additive of Example 1 to the flounder flounder

This study was conducted to evaluate the effect of probiotics supplementation on the growth and immune response of flounder halibut diets. Experimental feed used white fish meal, corn gluten meal, and dehulled soybean meal as main protein sources. Wheat flour was used as a carbohydrate source and wheat flour was used as a lipid source. Squid liver oil containing a large amount of polyunsaturated fatty acid (n-3 HUFA) was used. In order to confirm the effect of adding probiotics, feed additives of Table 1 were added into the experimental feed. The crude protein content of the experimental feed was 50.0% and the available energy was 16.4 kJ / g. All experimental feeds were mixed with raw materials and extruded and molded into a pellet maker, dried by a forced draft dryer (15 ° C., 24 hours), and then particle sizes of 1,500 μm and 2,000 μm standard (sieve) to fit the size of the test fish. ) Evenly, then sealed and used while refrigerated at 4 ° C. After two weeks of preliminary breeding, the flounder, with an average weight of 18.0 ± 0.2 g (mean ± SD), was randomly placed in three 500 replicate tanks of 20 animals each for three experiments. 1.8 ± 0.5% (10 am, 4 pm) was supplied for 8 weeks. Flow rates ranged from 2 to 4 L / min, water temperature of 15 ± 2 ° C, and feed for 1.8 ± 0.5% of fish. The measurement items are as follows.

Growth survey: weight gain, feed efficiency, protein efficiency ratio, specific growth rate, condition factor, hepatosomatic index, survival rate Survival rate

-Nutritional Analysis: Analysis of general ingredients (protein, fat, ash, water) of feed and fish

Serological studies: hemoglobin, hematocrit, total protein, glucose, GOT, GPT

-Nonspecific immune response and disease resistance investigation: phagocytic respiratory burst test isolated from the head of fish, serum lysozyme activity test, challenge test (survival rate using pathogenic bacteria)

The addition of probiotics showed a significant increase in the probiotics administration group in gain, feed efficiency, daily growth rate and protein conversion efficiency [Table 2]. In particular, the BP + SC administration group was the most effective.

TABLE 2 Weight Gain, Feed Efficiency, Daily Growth Rate, and Protein Conversion Efficiency of 8-week Feed Additives for Olive Flounder

TABLE 3 Analysis of Fish Components of Olive Flounder According to Feeding for 8 Weeks

There were no significant differences in fish body composition according to probiotic supplementation among all experimental groups [Table 3].

TABLE 4 Hematological and Serological Analyzes Following Feeding for 8 Weeks

It is reported that the blood components of fish can be used in terms of nutrition, health status and stress analysis, and also change depending on the lack of essential nutrients in the feed or the habitat environment and growth of the species (Garrido et al., 1990). Siddiqui, 1977). The blood and serum components of the flounder fry fed the probiotic-added feed of this experiment are shown in [Table 4]. Hemoglobin, Hematocrit, Serum Total protein, Serum glucose, and Serum GPT did not show any significant difference in all experiments. However, in Serum GPT, the experimental group fed BP + SC was lower than the BL and BP fed groups, and there was no significant difference between Cont, BP and BL experimental groups.

TABLE 5 Nonspecific Immune Responses of Olive Flounder Following 8 Weeks of Feeding

Phospocyte activity measurement is often used as an essential measure to best reflect how a nonspecific immune response has been enhanced in fish. In addition, serum lysozyme activity and complement activity are used, and pathogenic bacteria are actually inoculated with fish to evaluate resistance. To date, methods used to measure phagocytic activity include changes in phagocytosis, changes in chemotaxis and changes in respiration.

NBT activity of BP + SC fed group was significantly higher than that of the control group (P <0.05), but there was no significant difference between the experimental groups fed BP, BL and BP + SC. However, there was no significant difference in serum lysozyme activity in all experiments [Table 5]. This resulted in an increase in nonspecific immune response due to the mixed addition of Bacillus polyfermentus and Saccharomyces cerbiz in halibut diets. This result is consistent with the report that the supply of Saccharomyces cerbizi to Shrimp (Penaeus vannamei) increased the nonspecific immune response and increased resistance to Vibrio (Scholz U et al., 1999). In the following attack experiment, the mortality of the experimental group fed with BP + SC was the lowest.

TABLE 6 Edwardsiella for halibut Cumulative mortality after tarda attack

The results of the attack experiment (accumulated mortality due to an artificial infection) are shown in [Table 6]. Our company Edwardsiella One day after inoculation of tarda , it started 7 days later. E. tardar was positively isolated from all the dead individuals, and the initial mortality rate was lower in all feeds fed probiotics than the control. From day 5 to the end of the attack experiment, the experimental group fed BP + SC was significantly lower than the experimental groups fed BP and BL, and the experimental groups fed BP, BL and BP + SC were Significantly lower than Cont).

Through the results of this experiment, the addition of Bacillus polyperimentus, Bacillus rickenformis and Bacillus polyperimentus and Saccharomyces cervix enhances the non-specific immune response of the flounder, resulting in rapid expansion of aquaculture and In addition, it is expected to improve the resistance to many fish disease incidence factors caused by high density farming.

(Experimental example 2) Administration effect of the feed additive of Example 1 with respect to juvenile pulp

Experiments were carried out on the zodiac rock rock in the same manner as in Experimental Example 1.

TABLE 7 Weight gain, feed efficiency, daily growth rate and protein conversion efficiency of feed additives for 12 weeks

The results of the 12-week breeding experiment were shown in [Table 7]. The experimental group fed BP + SC in weight gain, feed efficiency, daily growth rate, and protein conversion efficiency was higher than the control group (P <0.05), but BP, BL and BP + SC were supplied. There was no significant difference in one experimental section. These results indicate that Streptococcus spp. Or the addition of Bacillus spp. Increased the growth of rainbow trout, common snook, tilapia and carp. (Vazquez-Juarez et al., 1993; Mohanty et al., 1996; Bogut et al., 1998; Kennedy et al., 1998). It was also consistent with reports that probiotic addition to turbot and tilapia feed increased feed efficiency (Naik et al., 1999; De Schrijver & Ollevier, 2000). Bougon et al. (1988), Rychen & Nunes. (1994) and Gil. (1998) reported that the addition of probiotics in feed increased digestibility in animals, resulting in increased protein conversion efficiency, feed efficiency and growth. Maurilio Lara-Flores et al. (2003) explained. There was no significant difference in survival among all experimental sections.

TABLE 8 Analysis of Fish Components of Olive Flounder According to Feeding for 12 Weeks

The general component analysis values of the whole body are shown in [Table 8]. In the whole body fat content, the control group was higher than the other groups (P <0.05), and there was no significant difference in the whole body protein, ash and water contents.

TABLE 9 Hematological and Serological Analysis of Zobololak Rocks Following 12 Week Feeding

Blood components were examined to evaluate the effect of probiotic added to the physiological status of experimental fish in the rockfish lacquer feed [Table 9]. Hemoglobin, hematocrit, GOT, GPT and TP did not show any significant differences in all experimental sections. However, the content of glucose (TG) in plasma was significantly lower in the experimental group fed with BP + SC than in the control group (P <0.05), but the experimental group fed with BP, BL and BP + SC was significantly lower. No difference was seen.

TABLE 10. Nonspecific Immune Responses of Zobololak Rocks Following 12 Week Feeding

The addition of probiotics in Zibobolak feed showed a significant effect on NBT and serum lyszyme activity, which is a measure of nonspecific immune response [Table 10]. In respiratory burst activity (NBT assay), the experimental group fed BP + SC, BL and BP was significantly higher than the control group (P <0.05), and the experimental section fed BP, BL and BP + SC. There was no significant difference in. Serum lysozyme activity was higher in the control group fed with BP, BL and BP + SC than the control group (P <0.05), and there was no significant difference between the experimental groups fed BP, BL and BP + SC. Did. These results were found in rainbow trout, turbot, and European eel in Bacillus spp., Enterococcus spp., Yersinia spp. And reports that increased nonspecific immune response when fed Streptococcus spp. (Anderson et al., 1979; Griffin, 1983; Olesen NJ, 1991; Ring, E et al., 1996; Austin, B., 1998 O'Sullivan, DJ, 1999; Chang & Liu., 2002). This suggests that probiotics affect the activation of antioxidant-related enzymes in fish bodies, directly and indirectly, thereby neutralizing oxygen radicals, which also affects the production of reactive oxygen intermediates (ROIs) that occur during phagocytic respiration. Is thought to be crazy. Fish lysozyme is an antimicrobial-like, mucolytic enzyme derived from lymphocytes, which is found in serum, mucus, and certain tissues. It is mainly produced in neutrophils and monocytes, and only a small amount is detected in macrophage (Hansen, 1974). Therefore, lysozyme is part of the endogenous defense mechanisms for parasitic, bacterial and viral infections in fish (Ingram, 1980). The nonspecific immune response enhancement effect of these probiotics is believed to be due to the low mortality in the challenge experiment.

TABLE 11 Cumulative mortality after Edwardsiella tarda attack on the rockfish

The attack test results are shown in [Table 11]. The mortality started 1 day after the inoculation of Edwardsiella tarda and ended 11 days. The initial mortality rate was lower in all experimental groups treated with probiotics than the control group. From the 8th day to the end of the challenge experiment, the experimental group fed BP + SC was significantly lower than the control group (P <0.05), and the experimental group fed BP, BL, and BP + SC was significantly lower. There was no difference.

Example  2. Preparation of Fish Feed Additives

TABLE 12. Composition of Fish Feed Additives

In Example 1, the efficacy of the BP + SC additive was excellent, and the efficacy of the SC alone group and the mixed administration group between the bacteria was verified. 1 kg of the feed additive of the composition table is mixed with 1 ton of mixed feed and administered to fish.

(Experimental Example 3) Administration Effect of the Feed Additive of the Present Invention for Juvenile Flounder

It carried out by the same method as Experimental example 1.

TABLE 13 Weight Gain, Feed Efficiency, Daily Growth Rate, and Protein Conversion Efficiency of 8-week Feed Additives for Olive Flounder

The increase of feed rate, feed efficiency, daily growth rate and protein conversion efficiency were higher in all of the experimental groups fed with probiotics than the control, and the experimental group fed with BP + BL + SC showed the best effect [ Table 13].

TABLE 14 Analysis of Fish Components of Olive Flounder According to Feeding for 8 Weeks

There were no significant differences in fish body composition according to probiotic supplementation among all experimental groups [Table 14].

TABLE 15. Hematological and Serological Analyzes Following Feeding for 8 Weeks

The blood and serum components of the flounder fry fed the probiotic-added feed of this experiment are shown in [Table 15]. Hemoglobin, Hematocrit, Serum Total protein, Serum glucose, and Serum GPT did not show any significant difference in all experiments.

TABLE 16. Nonspecific Immune Responses of Olive Flounder Following 8 Weeks of Feeding

The NBT activity of the experimental group fed with probiotics was significantly higher than that of the control, but there was no significant difference between the experimental groups fed with BP + SC and BL + SC. The spheres showed the highest activity. However, the experimental group fed with BP + BL + SC showed the highest activity in serum lysozyme activity, but there was no significant difference in the other experimental groups. This resulted in an increase in nonspecific immune response due to the mixed addition of Bacillus polyfermentus, Bacillus rickenformis and Saccharomyces cerbiz in halibut diets.

TABLE 17. Cumulative mortality after Edwardsiella tarda attack on flounder

The results of the attack experiment (accumulated mortality due to human infection) are shown in [Table 17]. The mortality started 1 day after inoculation of Edwardsiella tarda and ended 7 days. E. tardar was positively isolated from all the dead individuals, and the initial mortality rate was lower in all feeds fed probiotics than the control. The mortality rate was lower in the experimental group that was administered in combination than when SC was administered alone, and BP + BL + SC mixed showed the best results.

Through the results of this experiment, the addition or combination of Bacillus polyfermentus, Bacillus rickenformus and Bacillus polyfermentus and Saccharomyces cerbizi increases the growth and feed efficiency of flounder, and it is also non-specific. Increasing the immune response to improve disease resistance to E. tarda showed that it can be used as a feed additive.

As described above, according to the present invention, the composition of the present invention can increase the growth of fish, feed efficiency and nonspecific immune response when added to the feed of fish of the fry. In addition, it is possible to suppress the abuse of antibiotics that have been used for disease treatment and prevention of infections of fish, and to promote growth by forming beneficial bacteria in the fish farm, and water pollution with high feed efficiency. Effects such as suppression can be expected.

[references]

Claims (5)

Bacillus polyfermentus polyfermenticus ), Bacillus licheniformis ( Bacillus licheniformis ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) A composition for adding fish feed, characterized in that it comprises at least one. According to claim 1, wherein the composition is a composition characterized in that it is added to the feed of halibut, skinball rock. The method according to claim 1, wherein the Bacillus polyfermentus is 1 × 10 ⁸ ~ 10 ¹⁰ CFU / kg, Bacillus rickenformis is 1 × 10 ⁸ ~ 10 ¹⁰ CFU / kg, Saccharomyces cervizi 1 × 10 ⁷ to 10 ⁹ CFU / kg composition. The composition of claim 1, wherein the composition comprises all of Bacillus polyfermentus, Bacillus rickeniformis, and Saccharomyces cerevisiae. The composition of claim 1, wherein the composition enhances the growth of fish and a nonspecific immune response.