TWI381842B - Orally igy embedded preparation and process thereof - Google Patents

Description

Egg yolk immunoglobulin (IgY) oral preparation and preparation method thereof

The present invention relates to the prevention and treatment of aquatic diseases.

The immunoglobulin in poultry eggs contains three kinds of IgA, IgM and IgY. Its source is transferred from the blood of the female poultry during the spawning process. IgA and IgM will be present in the protein, while IgY will migrate to the yolk. Part, it is called egg yolk immunoglobulin. Compared with other kinds of immunoglobulins, IgY has many advantages as an antibody source. For example, because of the large distance between mammals and poultry, it is difficult to cross-react with mammalian antibodies in immunoassay, and it is easier to produce relative antigens. Highly specific antibody. In addition, because the egg yolk contains only one antibody of IgY, it is simpler to separate the antibody. Currently, when a specific antibody source is to be obtained from an animal, the test mouse or rabbit is first immunized, and the serum is obtained by blood sampling to isolate the antibody. This invasive method of antibody production is prone to pain and stress in animals. However, IgY can be obtained by collecting the eggs every day after immunizing the birds, and then separating the antibodies from the egg yolk. Therefore, IgY is a better choice for specific antibody sources.

In the development of aquatic animal vaccines, the injection vaccines currently used on the market are more difficult to handle the injection of large quantities of small-sized aquatic animals such as larvae. If an oral vaccine can be used, feeding with the feed will greatly reduce the manpower and there will be no safety problems with adjuvant residues. The oral vaccine uses the oral delivery system of aquatic animals to directly mix the antibody or antigen into the feed and enter the animal body by oral administration. However, during the preparation process, the protein is easily denatured and may be hydrolyzed in water; even if it enters the body of the fish, it will be stomach acid. Digest most of it, and the protection effect is greatly reduced. Therefore, how to effectively deliver IgY antibodies into animals will be an urgent problem for the aquatic industry.

The present invention provides an oral IgY preparation for administration to a water-borne animal, wherein the egg powder containing the IgY antibody is embedded in an emulsion of a water-in-oil-in-water triple phase. The oral preparation has stable structure, convenient storage and transportation, is not easy to be hydrolyzed in water, and can protect IgY from gastric acid. After entering the aquatic animal, the IgY antibody is slowly released, thereby improving the prevention and protection effect.

Accordingly, one aspect of the present invention provides an oral preparation of IgY comprising a therapeutically or prophylactically effective amount of egg powder comprising an IgY antibody; an oil phase material of about 30-70% by weight, the oil phase material being present at room temperature Liquid, for embedding the egg powder; about 0.1-10% of the total weight ratio of the first emulsifier, which is added to the oil phase material; about 10-30% of the total weight ratio of the first aqueous phase material, Forming an internal aqueous phase which is coated with the oil phase material; about 20-40% of the total weight ratio of the second aqueous phase material, forming an external aqueous phase for coating the oil phase material; A second emulsifier is added to the outer aqueous phase in an amount of from about 0.1% to about 10% by weight.

Another aspect of the present invention provides a method of preparing an oral preparation of IgY as described above, the steps comprising: (1) providing an egg powder comprising an IgY antibody; (2) providing the egg powder with a total of about 30-70% The oil phase material is mixed with the liquid phase at a normal temperature, and then mixed with a first emulsifier of about 0.1-10% by weight to obtain an oil phase product; (3) the oil phase product is about 10-30% total. The weight ratio of the first aqueous phase material constitutes an internal aqueous phase, and after mixing in a weight ratio of 1:1 to 1:5, a water-in-oil emulsion product is obtained; (4) about 20-40 The second aqueous phase material of the total weight ratio constitutes an external aqueous phase, and is mixed with a second emulsifier of about 0.1-10% by weight to form a mixture; (5) the mixture and the step (3) The obtained water-in-oil emulsion product is mixed in a weight ratio of 1:1 to 1:5 to obtain an IgY oral preparation.

In another aspect of the invention there is provided an aquafeed comprising the oral formulation of IgY as described above.

Still another aspect of the present invention provides a method for preventing or treating a disease in an aquatic animal by orally administering an aquatic animal such as the above-described aquaculture feed.

The present invention provides an oral preparation of IgY comprising a therapeutically or prophylactically effective amount of egg powder comprising an IgY antibody; an oil phase material of about 30-70% by weight, the oil phase material being liquid at room temperature, used Embedding the egg powder; about 0.1-10% of the total weight ratio of the first emulsifier, which is added to the oil phase material; about 10-30% of the total weight ratio of the first aqueous phase material, forming an internal aqueous phase , which is coated with the oil phase material; about 20-40% of the total weight ratio of the second aqueous phase material constitutes an external aqueous phase, which is used to coat the oil phase material; and about 0.1-10% A total weight ratio of the second emulsifier which is added to the outer aqueous phase.

The IgY system used in the present invention is contained in an egg powder whose source is poultry such as chicken, duck or goose. A person skilled in the art can immunize poultry by immunizing poultry with a whole plant inactivated virus or a polypeptide fragment having an antigenic determining region according to their own knowledge or according to the literature according to the aquatic disease to be prevented or treated. The egg powder containing the IgY antibody is prepared from the eggs produced by the poultry. According to an embodiment of the present invention, the inactivated Taiwan Grouper iridovirus of Taiwan ( TGIV ) and the adjuvant are used to immunize the chicken several times, and the eggs produced by the chicken are collected and the egg yolk is partially frozen. After drying, the obtained egg powder containing IgY-resistant Taiwan grouper iridescent virus was obtained. The "effective amount" in the present invention means the content which can achieve the effect of preventing or treating diseases in aquatic animals. According to an embodiment of the invention, the egg powder comprising the IgY antibody is used in an amount of about 5-20% by weight of the oil phase material.

The term "oil phase substance" as used in the present invention refers to an ester or non-ester oily base which is commonly used in foods and medicines and which is liquid at normal temperature. Examples of the non-ester type oily base include mineral oil, and examples of the ester oily base include naturally occurring fatty acid esters such as liquid vegetable oils such as peanut oil, soybean oil, sunflower oil, corn oil, olive oil or rapeseed oil, and the like, and Liquid oil derived from animals such as lard, squid oil or fish oil. The above oils may be used singly or in combination for visual purposes. According to an embodiment of the invention, the oil phase material is fish oil.

The term "first emulsifier" as used in the present invention refers to a nonionic surfactant having an HLB value of less than 8 and an inversion point of between 10 and 40 °C. The "HLB value" refers to the hydrophile lipophile balance of a substance, and the lower the value, the worse the hydrophilicity. In the present invention, examples of the first emulsifier used for the preparation include hexitol anhydride stearate, hexitol anhydride oleate, sorbitan fatty acid ester, dioctyldodecyl-2lauroyl Glutamate, glyceryl monostearate or soybean phospholipid. Those skilled in the art can use the above-mentioned emulsifier alone or in combination according to different purposes. According to one embodiment of the invention, the first emulsifier is a sorbitan fatty acid ester (SPAMs).

The term "second emulsifier" as used in the present invention means a nonionic surfactant having an HLB value of more than 8 and an inversion point of between 10 and 40 ° C, that is, a hydrophilic emulsifier. . In the present invention, examples of the second emulsifier used for the preparation include hexitol anhydride laurate, hexitol anhydride palmitate, hexitol anhydride stearate, hexitol anhydride oleate, polyoxygen Ethylene sorbitan fatty acid ester, polyglyceryl monostearate-10 or polyglyceryl-10 stearate. Those skilled in the art can use the above-mentioned emulsifier alone or in combination according to different purposes. According to one embodiment of the invention, the second emulsifier is a polyoxyethylene sorbitan fatty acid ester (Tween).

The term "aqueous phase substance" as used in the present invention means an aqueous solution which may be water or a water-based solution. In the present invention, the aqueous phase material as the internal aqueous phase may further comprise a nutritional supplement, an amino acid, a protein powder, a vitamin, an immunostimulating agent, a trace mineral, a seaweed polysaccharide or a A water-soluble substance such as a vaccine or a combination of any two or more of the above. The purpose of the water-soluble substance is to enhance the effect of the IgY oral preparation, or for other purposes such as nutritional supplementation. Those skilled in the art can select the water-soluble substance to be used according to the needs.

In the present invention, the aqueous phase material as the external aqueous phase may further comprise a water-soluble substance, for example, a structure for a stable oral preparation such as Sanxian gum, sodium alginate, k-carrageenan, sodium alginate, Soy lecithin, gum arabic or a combination of the other substances mentioned above.

The invention also provides a method for preparing an oral preparation of IgY as described above, the steps comprising: (1) providing an egg powder comprising an IgY antibody; (2) providing the egg powder at a normal temperature ratio of about 30-70% by weight After mixing the liquid phase material in a liquid state, mixing with the first emulsifier in an amount of about 0.1-10% by weight to obtain an oil phase product; (3) the oil phase product is in a total weight ratio of about 10-30% After the aqueous phase is mixed in a weight ratio ranging from 1:1 to 1:5, a water-in-oil emulsified product can be obtained; (4) about 20-40% of the total weight ratio is outside the aqueous phase and about 0.1-10% total After mixing with the second emulsifier, a mixture is formed; (5) the mixture is mixed with the water-in-oil emulsion obtained in the step (3) in a weight ratio ranging from 1:1 to 1:5 to obtain IgY orally. preparation.

The IgY oral preparation of the present invention has the advantages of structural stability and anti-acidicity, and is suitable for providing passive protection of aquatic animals against viruses by oral administration. The IgY oral preparation of the present invention can be mixed with aquatic feed, and then the feed is fed. Aquatic animals. The ratio of the IgY oral preparation to the aquaculture feed can be known by a person having ordinary skill in the art to which the present invention is based on experience and mixed feed types.

According to the above concept, the present invention also provides a method for preventing or treating diseases of aquatic animals, which is an orally administered aquatic animal such as the above-mentioned IgY oral preparation for aquaculture feed. Among them, the aquatic animal is a fish or a shrimp, and especially a small fish that is not easily injected is the most valuable.

The invention will be further illustrated by the following examples, but the actual invention is not limited by the examples shown.

Example 1: Preparation of anti-Taiwan grouper iridescent virus IgY

Chicken immunization and egg powder production

Use 3-4 month old hens to freely consume water and feed. The Taiwanese spotted iridescent virus (TGIV), a virus isolated from the squid grouper, was administered intramuscularly four times. The first immunization was performed by the addition of a complete adjuvant (sigma, USA), and the subsequent three immunizations were performed with incomplete adjuvant (sigma, USA), and each immunization was separated by two weeks. The eggs produced by the hens were collected before each injection and hydrated for one day at a ratio of 1:4 egg yolk to 0.1 times PBS solution, and the egg liquid was collected.

IgY price test

The TGIV antigen was diluted with 0.1 M NaHCO ₃ and added to a microplate of well 96, 50 μL of antigen (concentration of 100 ng / 50 μL) was added to each well, and placed in a workbench and dried at room temperature. After drying, 50 μL of a PBS solution containing 1% instant milk powder was added to each well and allowed to stand at 37 ° C for 2 hours. After the above liquid was dried, it was washed once with PBS/T mixed solution (PBS containing 0.05% Tween-20), and then washed twice with PBS solution. After drying naturally, the plate was stored at -20 ° C for use.

After the egg liquid was diluted twice in PBS solution containing 1% instant milk powder, 50 μL of the test solution was added to each well in a 96-well microplate, and after drying at 37 ° C for 2 hours, the liquid was dried. Wash 3 times with PBS/T buffer, wash 5 times with PBS solution and pat dry. Then, the goat-anti-chicken IgY antibody (Huahe Gene Company, Taiwan) linked to Horseradish Peroxidase was diluted at a ratio of 1:1000 with PBS solution containing 1% instant milk powder, and in each well. 50 μL was added and placed at 37 ° C for 1 hour. After the end of the reaction time, the above liquid was dried, washed 3 times with PBS/T buffer, washed 5 times with PBS solution and patted dry. Finally, 50 μL of O-phenylene diamine (OPD) (SIGMA, USA) was added to each well as a coloring substrate, and then reacted at room temperature for 30 minutes in the dark. After the coloration was completed, 50 μL of 3N HCl was added to each well to terminate the reaction, and the absorbance (OD) at 490 nm was detected. The reciprocal of the maximum dilution ratio of the sample is its price. The results showed that the IgY antibody was about 1:16000-64000.

IgY neutralization test

The egg liquid was first filtered through a 0.45 μm filter, and then serially diluted from 1:20 to 1:10240 in PBS solution, and added with 200 TCID ₅₀ /mL of virus solution at 25 ° C for one hour, and additionally with PBS solution. The same concentration of virus solution was mixed as a control group. Then, the two kinds of the mixture were separately added to the 96 well microplate of the grouper cell line, and after two hours of adsorption, the liquid was removed, and the L ₁₅ cell culture containing 2% fetal bovine serum (Hyclone, USA) was added. liquid. If the antibody is unable to effectively neutralize the virus, there will be symptoms such as rounding of the cells, marginalization of the nuclear nucleus, and finally rupture of the cells. The cytopathic condition is observed and recorded under a microscope every day. As a result, it was found that at the dilution ratios of 1:80, 1:160, 1:320, and 1:640, the cells did not produce lesions, and it was shown that IgY had the best neutralizing effect at the above dilution ratio.

Example 2: Preparation of IgY oral preparation

The first stage first prepares a water/oil emulsion. First, 30 mL of commercial fish oil was prepared, which was purchased from Taiwan Yihua Industrial Co., Ltd., and the product code was FO, which was refined by various fish. Different concentrations of egg powder (5%, 10%, 15%, respectively) were added to the fish oil, and 0.9 g of a lipophilic emulsifier (Span, purchased from Nippon Pure Chemical Industries, Ltd.) was added, and the mixture was uniformly mixed. The homogenizer (Silverson, England) was homogenized at a speed of 8,000 rpm while adding an internal phase aqueous solution (10 mL). A water/oil emulsion was formed after 2 minutes of homogenization.

In the second stage, 40 mL of the water/oil emulsion formed in the first stage was added to a 20 mL aqueous solution containing 1 g of a hydrophilic emulsifier (Tween, purchased from Nippon Pure Chemical Industries, Ltd.), and homogenized at 5000 rpm for 2 minutes. After homogenization, a water-in-oil-in-water type IgY oral preparation can be obtained.

Example 3: Preparation characteristics of IgY oral preparation

Formation rate

After proper dilution of the IgY oral preparation, 5 photos were taken by the photographic system, and each photo had at least 50 drops. The percentage of the triple emulsion droplets in the total emulsion droplets was calculated. The results are shown in Table 1. The formation rate of the group containing 5% and 10% egg powder was 85% or more, and the group containing 15% egg powder also had a formation rate of 78%.

Emulsion stability

10 mL of each phase of the multiphase emulsion was taken up in a 15 mL centrifuge tube and placed at 4 ° C, and the changes were periodically observed and recorded. The results are shown in Table 2. The higher the egg powder content, the less stable the stability, but the low dose group can reach more than 45 days.

Example 4: Evaluation of the in vivo effect of IgY oral preparation against Taiwan grouper iridescent virus (TGIV)

The experimental spotted grouper was purchased from 868 Aquatic Animal Biotechnology Co., Ltd., and its grouper was from the Gaoheping area. It was kept at a salinity of 30~33% _o and a temperature of 26~30 °C. The commercial squid feed was fed three times a day, and the experiment was carried out one week after the animal was raised. Grouper was randomly selected before the start of the experiment, and the spleen and kidney samples were tested for TGIV polymerase chain reaction. The reaction conditions of each cycle were: first at 94 ° C for 2 minutes, then 94 ° C for 40 seconds, bonding temperature 52 ° C for 30 seconds and 72 ° C for 1 minute, a total of 35 cycles of amplification reaction, and finally at 72 ° C The unfinished reaction is completed in 10 minutes. The experiment was confirmed after no TGIV infection. The primer sequence for polymerase chain reaction detection is as follows: forward primer: 5"CCCTT CCTTG TGTCG TGTCT 3" (sequence number: 1); reverse primer: 5"GCC ACC GTA ATC AGT TCG AT 3" (sequence number: 2).

The feed preparation was divided into six groups, namely: A-embedded group: mixed with 10% egg powder and multi-phase emulsion and commercial glutinous rice powder (Tairong Industry Co., Ltd., Taiwan) Sexual feed; B is not embedded in the high-dose group: the powdered meal is mixed into a wet feed, and the added egg powder dose accounts for 30% of the total mixture weight; C is not embedded in the low-dose group: the egg powder is powdered The mixture was formulated into a wet feed, and the dosage of the added egg powder was about 5% of the total mixture weight; and the positive control groups of the above blank powder containing no antibody were AV, BV and CV, respectively. The IgY antibody content of the unembedded high dose group was about 6 times that of the embedded group.

The method of virus challenge test was to take 10 fish in each group, and the group was divided into intraperitoneal injection group and soaking attack group. The doses of the virus used in the injection group were 10 ^3.3 and 10 ^2.3 TCID ₅₀ /0.05 mL per fish, while the virus doses used in the infusion group were 10 ⁵ and 10 ⁴ TCID ₅₀ /1500 mL, soaked for two hours. The death was observed and recorded daily after the attack, and the cumulative mortality was calculated.

Preventive effect experiment

Grouped groupers were grouped as Table 3. Each group of 40 fry, with an average length of 5.5 cm and a weight of 3.9 g, was placed in a 2-foot tank with two-fold flow-through seawater.

Each group of feeds was fed three meals a day, and each group of self-made wet feeds with a total weight of more than 5% in each group was fed on the same day. On the first day or the fifth day after feeding for three days, 10 rats in the experimental group and the positive control group were removed on the 1st, 5th, 10th, and 20th day, and the TGIV intraperitoneal injection challenge test was performed. The dose was 10 ^3.3 TCID _50. /0.05mL/fish, the negative control group was treated with intraperitoneal injection of L15 medium instead of virus, and the death was observed daily and the relative survival rate was calculated. The results are shown in Figures 1 and 2.

Figure 1 shows the virus attack on the first day after feeding the three groups of feeds, and recorded daily deaths to calculate the relative survival results. As shown in Figure 1, after 14 days of virus challenge, there was a 50% survival rate in addition to the IgY oral preparation group (A) and the unembedded high dose group (B), and the survival rates of the other groups were 0. %. In addition, Figure 1 also shows that the IgY oral formulation of the present invention is comparable to the unencapsulated high dose group.

Figure 2 shows the virus attack on the fifth day after feeding the three groups of feeds, and recorded daily deaths, and calculated the relative survival results. As shown in Fig. 2, on the fourteenth day of the virus challenge, the survival rate of the IgY oral preparation group (A) of the present invention was almost 100%, and the unembedded high dose group (B) had a survival rate of about 80%, and Except for the D group fed the general feed, the survival rate of the other groups was only 10-20%.

It can be seen from the above data that the oral preparation of IgY of the present invention has a remarkable effect on resistance to viral attack in a living test, and can greatly improve the survival rate of aquatic animals, and the survival rate is equal to or higher than that of the unembedded high dose. .

Changes and modifications may be made in accordance with the embodiments without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited by the scope of the embodiments disclosed herein,

The foregoing description and the embodiments may achieve better illustrative effects by the additional drawings. To enhance the description of the invention, the drawings of the appropriate embodiments are set forth herein. It is to be noted that the present invention is not limited to the descriptions set forth herein.

Figure 1 shows the results of a virus attack on the first day after feeding the grouper with three days of feed, recording the relative survival rate after daily death.

Figure 2 shows the results of a virus attack on the fifth day after feeding the grouper with three days of feed, recording the relative survival rate after daily death.

Claims (21)

An oral preparation of IgY comprising a therapeutically or prophylactically effective amount of egg powder comprising an IgY antibody; an oil phase material of about 30-70% by weight, the oil phase material being liquid at room temperature for embedding the The egg powder, wherein the amount of the egg powder of the IgY antibody is about 5% to 10% by weight in the oil phase material; about 0.1-10% of the total weight ratio of the first emulsifier, which is added to the oil phase material; About 10-30% of the total weight ratio of the first aqueous phase material constitutes an internal aqueous phase which is coated with the oil phase material; about 20-40% of the total weight ratio of the second aqueous phase material constitutes an outer An aqueous phase for coating the oil phase material; and a second emulsifier in an amount of from about 0.1% to about 10% by weight, which is added to the outer aqueous phase. The IgY oral preparation of claim 1, wherein the first emulsifier is a nonionic surfactant having an HLB value of less than 8. The oral preparation of IgY of claim 2, wherein the first emulsifier is hexitol anhydride stearate, hexitol anhydride oleate, sorbitan fatty acid ester, dioctyldodecyl - 2 month lauric acid glutamic acid, glyceryl monostearate, soybean phospholipid or a combination of any two or more of the above. An oral preparation of IgY according to claim 3, wherein the first emulsifier is a sorbitan fatty acid ester. The IgY oral preparation of claim 1, wherein the second emulsifier is a nonionic surfactant having an HLB value of greater than 8. An oral preparation of IgY according to claim 5, wherein the second emulsifier is hexitol anhydride laurate, hexitol anhydride palmitate, hexitol anhydride stearate, hexitol anhydride oleic acid An ester, a polyoxyethylene sorbitan fatty acid ester, a polyglyceryl monostearate-10, a polyglyceryl-10 stearate or a combination of any two or more of the foregoing. An oral preparation of IgY according to claim 6 wherein the second emulsifier is a polyoxyethylene sorbitan fatty acid ester. An IgY oral preparation according to claim 1, wherein the internal aqueous phase further comprises a water-soluble substance. The IgY oral preparation according to claim 8 , wherein the water-soluble substance is a nutritional supplement, an amino acid, a protein powder, a vitamin, an immunostimulating agent, a trace mineral, a seaweed polysaccharide, A vaccine or a combination of any two or more of the above. A method for preparing an IgY oral preparation according to claim 1, wherein the step comprises: (1) providing an egg powder comprising an IgY antibody; and (2) providing the egg powder to a total weight ratio of about 30-70% The liquid phase material in a liquid state is mixed at a normal temperature, so that the amount of the egg powder of the IgY antibody is about 5% to 10% by weight in the oil phase substance, and then mixed with the first emulsifier in an amount of about 0.1-10% by weight. Obtaining an oil phase product; (3) obtaining an oil after mixing the oil phase product with a total weight ratio of about 10-30% by weight of the first aqueous phase material in a weight ratio ranging from 1:1 to 1:5 a water-in-water emulsion product; (4) mixing about 20-40% of the total weight ratio of the second aqueous phase material with about 0.1-10% of the total weight ratio of the second emulsifier to form a mixture; (5) the mixture The water-in-oil emulsion product obtained in the step (3) is mixed in a weight ratio ranging from 1:1 to 1:5 to obtain an IgY oral preparation. The method of claim 10, wherein the oil phase material which is liquid at normal temperature is vegetable oil, animal oil or mineral oil. The method of claim 10, wherein the first emulsifier is a nonionic surfactant having an HLB value of less than 8. The method of claim 12, wherein the first emulsifier is hexitol anhydride stearate, hexitol anhydride oleate, sorbitan fatty acid ester, dioctyldodecyl-2 Lauryl glutamic acid, glyceryl monostearate, soybean phospholipid or a combination of any two or more of the above. . The method of claim 13, wherein the first emulsifier is a sorbitan fatty acid ester. The method of claim 10, wherein the second emulsifier is a nonionic surfactant having an HLB value greater than 8. The method of claim 15, wherein the second emulsifier is hexitol anhydride laurate, hexitol anhydride palmitate, hexitol anhydride stearate, hexitol anhydride oleate, Polyoxyethylene sorbitan fatty acid ester, polyglyceryl monostearate-10, polyglyceryl-10 stearate or any two of the above or The combination of the substances. The method of claim 16, wherein the second emulsifier is a polyoxyethylene sorbitan fatty acid ester. The method of claim 10, wherein the internal aqueous phase further comprises a water soluble material. The method of claim 18, wherein the water-soluble substance is a nutritional supplement, an amino acid, a protein powder, a vitamin, an immunostimulating agent, a trace mineral, a seaweed polysaccharide, a vaccine. Or a combination of any two or more of the above. An aquaculture feed comprising an oral preparation of IgY as in claim 1 of the patent application. An use of an oral preparation of IgY as claimed in claim 1 for the manufacture of aquaculture feed for the prevention or treatment of diseases in aquatic animals.