TWI721508B - Use of phycoerythrin for preparing composition for improving immunity and disease resistance of crustaceans - Google Patents

Abstract

The present invention provides a method for improving the immunity and disease resistance of crustaceans, which comprises administering a composition to the crustaceans; wherein the composition comprises a phycoerythrin; the method provided by the present invention can improve the crustaceans’ Immunity, more specifically, can improve the phagocytosis of crustaceans or the ability of protophenol oxidase system, and can improve the disease resistance of crustaceans, especially the ability of crustaceans to fight pathogens; the present invention also provides a phycoerythrin Used as an immune stimulant and its feed composition.

Description

A kind of phycoerythrin is used to prepare a group for improving the immunity and disease resistance of crustaceans Use of the compound

The present invention relates to the field of aquaculture, in particular to the field of aquaculture that improves the immunity and disease resistance of crustaceans.

Crustaceans are aquatic foods with a high unit price and a high market value. They are also key artificial breeding objects in countries all over the world. However, the immunity of the crustaceans itself is easily reduced due to the poor environment. When the breeding environment is not good, some pathogenic bacteria in the water often proliferate in large numbers and wait for opportunities to infect the crustaceans, causing the outbreak of infectious diseases. In 1992, a first generation of shrimp white spot disease broke out in Fujian, which severely damaged the entire Southeast Asian culture area. In 2016, the same situation occurred on the east coast of Australia. A large-scale outbreak of shrimp white spot disease caused significant economic losses. The Global aquaculture alliance (GAA) pointed out in a 2016 survey report that the biggest challenge currently facing the aquaculture industry is disease.

Nowadays, the strategies used to prevent or control the spread of diseases in various countries are still biased towards the use of antibiotics or chemicals. However, the environmental damage and food safety problems that antibiotics or chemicals may cause cannot be underestimated. Therefore, the aquaculture industry is in urgent need of an immunostimulant that can improve the disease resistance of crustaceans without harming the environment and causing food safety problems.

It can be seen from Table 1 that since 2004, some scholars have begun to propose immune stimulants that can increase the disease resistance of crustaceans. The time and survival rate in the table represent the time after challenge with Vibrio alginolyticus and the control group, which is significantly different from the survival rate at that time. It can be seen from Table 1 that most natural immune stimulants need at least 3-4 days to significantly improve the disease resistance of crustaceans. Therefore, providing a natural immune stimulant that can quickly improve the disease resistance of crustaceans is another problem to be solved in the aquaculture field.

Phycoerythrin (PE) is a type of phycobiliprotein, which is a water-soluble fluorescent protein that exists in algae such as red algae, green algae, cyanobacteria, and cryptophytes. Due to its various properties, phycoerythrin has been widely used in the development and research of various fields, including chemicals and biological agents, tumor treatment drugs, in vitro diagnostics, etc., especially for fluorescent research reagents. However, whether phycoerythrin can be used as an immune stimulus It is not known to improve the immunity and disease resistance of crustaceans.

The object of the present invention is to provide a method for improving the immunity and disease resistance of crustaceans, which comprises administering a composition to the crustaceans; wherein the composition comprises a phycoerythrin.

In order to achieve the purpose of the aforementioned invention, the immune ability is phagocytosis or protophenol oxidase system.

In order to achieve the purpose of the aforementioned invention, the disease resistance refers to the ability to resist pathogens; wherein the pathogens include viruses or bacteria.

In order to achieve the aforementioned purpose of the invention, the virus is white spot syndrome virus.

In order to achieve the purpose of the foregoing invention, the bacterium is Vibrio parahaemolyticus.

In order to achieve the aforementioned purpose of the invention, the crustaceans include shrimp, crab, crab, horseshoe crab, prawn or lobster.

In order to achieve the purpose of the foregoing invention, the effective dose of the phycoerythrin is 0.45 μg to 2.4 μg.

In order to achieve the aforementioned object of the invention, the administration method of the composition includes oral, immersion, spraying or injection.

The present invention also provides a use of phycoerythrin as an immune stimulant, which comprises administering a phycoerythrin to crustaceans; wherein the phycoerythrin can improve the immunity or disease resistance of the crustaceans.

The present invention also provides a feed composition comprising a phycoerythrin, wherein the phycoerythrin can improve the immunity or disease resistance of crustaceans.

The present invention provides a solution to the long-standing disease problem in the field. The phycoerythrin-containing composition provided by the present invention can increase the immunity of crustaceans, more specifically, can enhance the phagocytosis of crustaceans and non-specific immunity such as protophenol oxidase system. Applying the phycoerythrin-containing composition provided by the present invention to crustaceans can improve the disease resistance of crustaceans after being attacked by pathogens. In addition, the composition provided by the present invention uses phycoerythrin as an effective ingredient, which has the advantages of being natural, not causing food safety problems, and having low concerns about environmental damage.

Figure 1 shows the test flow chart of the effect of phycoerythrin on the ability of anti-pathogens.

Figure 2 shows the results of the phagocytosis test.

Figure 3 shows the test results of the original phenol oxidase system.

All technical and scientific terms mentioned in this manual, unless otherwise defined, are meanings that can be understood by the professionals in the field. The singular terms "one", "one", "the", "the said" "Unless otherwise stated, it can refer to more than one object. In addition, the terms "include" and "include" are both open-ended conjunctions.

The term "effective amount" in this specification refers to a sufficient amount of an effective active substance that can prevent or alleviate one or more disease symptoms or physiological conditions by the grantor; the result is to reduce and/or alleviate Signs, symptoms, or etiology, or intentional changes in other physiological systems.

The term "crustaceans" in this manual refers to arthropods belonging to the subphylum Crustacea (Crustacea), which includes but is not limited to shrimps, crabs, horseshoe crabs, horseshoe crabs, prawns or lobsters.

The term "pathogen" in this specification refers to a pathogenic substance that can cause crustacean diseases, which includes but is not limited to bacteria, fungi, viruses or parasites.

The composition used in the present invention, its dosage form includes but not limited to solution (Solution), emulsion (Emulsion), suspension (Suspension), powder (Powder), lozenge (Tablet), pill (Pill), tablet (Troche) ) Or capsule (Capsule) and other similar or suitable dosage forms of the present invention.

The composition of the present invention may further add an aquaculture additive, which includes but is not limited to vaccines, adjuvants, and immunostimulants; wherein the immunostimulants include but are not limited to chemical compounds, sugars Class or carbohydrate derivatives.

Suitable administration routes of the composition of the present invention include but are not limited to soaking, spraying, medicated bath, oral administration or injection.

The phycoerythrin (Phycoerythrin, PE) used in the embodiments of the present invention is commercially available, and those with ordinary knowledge in the field of the present invention can also extract the phycoerythrin referred to in the present invention from algae including red algae. In addition, unless otherwise specified, the materials used in the present invention are commercially available and readily available.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples.

Example 1: Test of the effect of phycoerythrin on immunity (1)

The immune mechanism of crustaceans is dominated by nonspecific immunity, which can be divided into humoral immunity and cellular immunity, and cellular immunity includes phagocytosis and other reactions. The present invention uses an in vitro test to test the effect of phycoerythrin on phagocytosis, as a basis for evaluating whether phycoerythrin can enhance the immunity of crustaceans.

Phagocytosis test: A phagocytosis reagent kit (#P35361, Life Technologies) was used to perform a phagocytosis test, which is an improved detection according to the method proposed by Neaga A. (2011). Take 100μL of quantified shrimp blood cell suspension (1x10 ⁶ cell/mL) or Shrimp salt solution buffer (SSS buffer) (replacing the blood cell suspension as the negative control group), add it to the 96-well plate with black bottom, and add 800 xg, 4 Centrifuge at ℃ for 20 minutes and remove the supernatant, then rinse once with SSS buffer, centrifuge again under the same conditions, and then add 100μL of phycoerythrin (0.013, 0.026, 0.052 and 0.116mg/mL) of different concentrations, respectively, control group Then add SSS buffer, incubate at room temperature for 60 minutes, centrifuge at 800xg, 4℃ for 20 minutes and remove the supernatant, then add 100μL of K-12 BioParticle ( Escherichia coli , fluorescent substance), incubate at room temperature After 20 minutes, use the ELISA Reader to detect the fluorescence value of 480nm (excitation light): 520nm (scattered light), and then substitute the following formula for calculation.

Phagocytosis activity (%) = (experimental group/control group) x 100%

The results of the phagocytosis test are shown in Figure 2. The phagocytosis effect of the experimental group treated with phycoerythrin is better than that of the untreated control group, and the higher the concentration of phycoerythrin treated, the better the phagocytosis effect. This result shows that phycoerythrin has the effect of enhancing the ability of phagocytosis.

Example 2: Test of the effect of phycoerythrin on immunity (2)

The prophenoloxidase system (proPO system) is an enzyme immune system similar to the complement system in crustaceans, and it is also an important indicator factor for evaluating the immunity of crustaceans. The present invention further uses an in vitro test to test the effect of phycoerythrin on the protophenol oxidase system as another basis for evaluating whether phycoerythrin can enhance the immunity of crustaceans.

Protophenol oxidizing enzyme system test: According to the Hernández-López (1996) method for improved detection, the shrimp blood cells were suspended in the shrimp blood cell culture medium (modified complete Hank's balance salt solution, MCHBSS), and 500 μL of the quantified shrimp blood cell suspension (1x106 cell) /mL), add 500μL of different concentrations of phycoerythrin (0.013, 0.026, 0.052 and 0.116mg/mL), incubate at room temperature for 30 minutes, centrifuge at 800 xg, 4℃ for 10 minutes, and then remove the shrimp blood cell culture medium And phycoerythrin, add 1mL cacodylate buffer (cacodylate buffer, CA buffer) to suspend the shrimp blood cells, then ultrasonically shake for 5 seconds, a total of 4 times, and then centrifuge at 800xg, 4℃ 10 minutes, take 50μL of supernatant to 96-well plate, add 50μL of 0.1% trypsin (trypsin) was used for stimulation, and the control group was added with 50μL of dimethylarsine buffer as a control. After reaction for 10 minutes, 50μL of L-Dopa (L-Dopa) was added to each well as a receptor. After reacting for 15 minutes, detect the absorbance at 492nm with an enzyme immunoassay reader (ELISA Reader), and calculate the activity using the following formula.

Protophenol oxidase activity = (absorbance value of experimental group)-(absorbance value of control group)

The results of the protophenol oxidase system test are shown in Figure 3. The protophenol oxidase system functions better than the untreated control group in the experimental group with phycoerythrin, and the higher the concentration of phycoerythrin treated, the protophenol oxidizes The better the function of the enzyme system. This result shows that phycoerythrin has the effect of enhancing the function of protophenol oxidase system.

Example 3: Test of the influence of phycoerythrin on disease resistance (1)

Phycoerythrin is added to feed orally or injected into shrimps, and then the effect of phycoerythrin on the ability of crustaceans to resist pathogens is evaluated through a bacterial challenge test. The specific implementation is based on the method proposed by Siririrustananun, N. (2011), after improvement. The experiment uses Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) as the bacteria for the challenge. As shown in Figure 1, the experimental group was injected with 20μL of phycoerythrin (0.026, 0.052 and 0.116mg/mL) at different concentrations the day before the challenge. Inject 20μL of Vibrio parahaemolyticus every other day, and the injection volume of each shrimp is 5×10 ⁶ CFU; the control group is the shrimp that has not been treated with phycoerythrin and directly injected with Vibrio parahaemolyticus; in addition, only 20μL SSS is injected The buffered shrimp served as the non-challenged control group; there were five groups in total, with 11 shrimps in each group. The shrimp survival rate was observed at 12, 24, 36, 48, 60 and 72 hours after the challenge.

The results are shown in Table 2. The survival rate of shrimps without phycoerythrin treatment is only 27.27% after being challenged by Vibrio parahaemolyticus for 1 day, while the survival rate of shrimps can be increased to 50% by phycoerythrin treatment. % Above, and the higher the phycoerythrin concentration, the higher the survival rate of the shrimp. The results confirmed that phycoerythrin can effectively improve shrimp's ability to fight bacteria.

Example 4 Test of the influence of phycoerythrin on disease resistance (2)

Phycoerythrin is added to feed orally or injected into shrimps, and then through a virus challenge test to evaluate the effect of phycoerythrin on the ability of crustaceans to resist pathogens. The specific implementation is based on the method proposed by Siririrustananun, N. (2011), after improvement. The test uses White Spot Syndrome Virus (WSSV) as the virus for the challenge. As shown in Figure 1, the experimental group was injected with 20μL of phycoerythrin (0.026, 0.052 and 0.116) at different concentrations on the day before the challenge. mg/mL), another 20μL of white spot virus was injected every other day, the injection volume of each shrimp was 9.19×10 ⁴ copies number; the control group was the shrimp that had not received the phycoerythrin treatment but directly injected the white spot virus; Shrimp that received only 20μL of SSS buffer was used as the non-challenged control group; there were five groups in total, with 11 shrimps in each group. The shrimp survival rate was observed at 12, 24, 36, 48, 60, 72, 84 and 96 hours after the challenge.

The results are shown in Table 3. The shrimps that were not treated with phycoerythrin all died 3 days after being challenged with White Spot Syndrome Virus (WSSV), while the survival rate of shrimps treated with phycoerythrin was as low as Survival time has improved. The results confirmed that phycoerythrin can effectively improve the ability of shrimp to fight viruses.

According to the results of Example 3 and Example 4, it can be calculated that the effective dose of phycoerythrin can effectively enhance the ability of crustaceans to resist pathogens is 0.45μg~2.4μg; among them, the preferred effective dose range is 0.52μg~2.32μg ; Among them, the better effective dose range is 1.04μg~2.32μg; among them, the best effective dose is 2.32μg.

In summary, the present invention provides a method for using phycoerythrin as an immune stimulant to improve the immunity and disease resistance of crustaceans. It can be seen from the examples of the present invention that the use of phycoerythrin as an immune stimulant can significantly improve the disease resistance of crustaceans in only 1.5 days, and the survival rate of the experimental group (63.64%) of the phycoerythrin treatment is higher than that of the control group. (27.27%) is much higher. Compared with traditional immune stimulants (Table 1), using phycoerythrin as an immune stimulant has the advantage of rapidly improving the disease resistance of crustaceans, and it is also less likely to cause food safety problems and harm the environment.

From the content disclosed in the preferred embodiments of this specification, those with ordinary knowledge in the field of the present invention can clearly understand that the foregoing embodiments are only examples; those with ordinary knowledge in the technical field of the present invention can implement it through many changes and substitutions. It does not differ from the technical features of the present invention. According to the embodiments of the specification, the present invention can have many variations without hindering its implementation. The claims provided in this specification define the scope of the present invention, which covers the aforementioned methods and structures and their equivalent inventions.

The above-mentioned multiple functions are in fact fully in line with the statutory patent requirements for novelty and advancement. You file an application in accordance with the law, and I implore your office to approve this invention patent application to encourage invention.

Claims (7)

A use of phycoerythrin is used to prepare a composition for improving the immunity and disease resistance of crustaceans, wherein the effective dose of the phycoerythrin is 0.45 μg to 2.4 μg. The use as described in item 1 of the scope of patent application, wherein the immune ability is phagocytosis or protophenol oxidase system. The use as described in item 1 of the scope of patent application, wherein the disease resistance refers to the ability to resist pathogens; wherein the pathogens include viruses or bacteria. The use as described in item 3 of the scope of patent application, wherein the virus is white spot syndrome virus. The use described in item 3 of the scope of patent application, wherein the bacterium is Vibrio parahaemolyticus. The use described in item 1 of the scope of the patent application, wherein the crustaceans include shrimp, crab, horseshoe crab, horseshoe crab, prawns, or lobster. The use according to any one of items 1 to 6 in the scope of the patent application, wherein the administration method of the composition includes oral, soaking, spraying or injection.

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