JPH03218318A - Production of treating agent for rotavirus infectious disease - Google Patents

Abstract

PURPOSE:To obtain native whey protein extremely effective for preventing and treating the title infectious disease of bovine newborns by blending colostrum of a great number of dairy cattle bred in plural zones, de-fatting, subjecting to rennet treatment, subjecting to ultrafiltration under a specific condition, concentrating and purifying. CONSTITUTION:Colostrum of more than 50 dairy cattle bred in plural zones is blended, de-fatted and subjected to rennet treatment to give colostrum whey, which is treated by an ultrafilter having 100,000 fractional molecular weight. Then the whey is concentrated until protein content exceeds 75% to purify a native whey protein rich in immunoglobulin, lactoferrin and lactoperoxidase and to give native whey protein useful for effectively preventing and treating rotavirus infectious diseases of bovine newborns. The protein is orally administered to bovine newborns within 36 hours from birth and a dose is at least 5g per time. Then, a dose of at least 5g/day is applied to the bovine newborns and the administration is continued for a week to effectively prevent rotavirus infectious diseases of bovine newborns.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention provides a method for producing unmodified whey protein (2) which is highly effective in preventing and treating rotavirus infection in newborn bovines, and a method produced by the method. The present invention relates to a method for preventing and treating rotavirus infection using unmodified whey protein.

``Conventional technology'' In the livestock industry, the production of high-quality beef by fattening Wagyu beef, especially Japanese black cattle, is one of the fields that holds hope for the future in Japan's livestock industry, which is facing a storm of liberalization. It is said. However, unlike humans, who receive immunoglobulins from their mothers via the placenta, newborn cows are born without any immunoglobulins. Therefore, it has been repeatedly argued that it is necessary to provide passive immunization by ingesting a sufficient amount of colostrum, which contains a large amount of immunoglobulin, immediately after birth.

On the other hand, as is clear from the fact that 10 to 15% of newborn cows die even if sufficient colostrum is fed, calves are surrounded by a wide variety of pathogens, and Situations may occur in which immunoglobulin alone is not sufficient to deal with these pathogens. Rotavirus is the first virus that infects newborn cows immediately after birth and is likely to cause diarrhea (3). This virus is a pathogenic virus that is common to humans, cows, and chickens, and it not only infects newborns and causes severe diarrhea, but also causes fatal mixed infections.

When seen under an electron microscope, rotavirus looks like a wheel, so it was named after the Latin word rota, meaning "wheel." Since it was discovered in the United States in 1969, its existence has been confirmed all over the world.

However, bovine rotavirus does not have a single antigenicity, and many types have been recognized, which makes prevention with vaccines extremely difficult.

Therefore, the conventional prophylactic method by vaccination is hardly ever adopted in practice, and to date there is no effective prophylactic treatment that can deal with all types of rotavirus.

The onset of diarrhea caused by rotavirus infection is characteristically concentrated in the winter, and epidemics are observed only in newborn babies. The feces of infected cows contain large amounts of virus (4), and the virus is excreted over a relatively long period of time.This, coupled with the fact that the virus is highly infectious, tends to spread the infection easily.

Once rotavirus invades, it is characteristic that it becomes endemic and outbreaks occur repeatedly. In cattle farms that have experienced infection, mother cows have a high rate of antibodies, and calves are given passive immunity through antibodies in their colostrum.

However, since antibodies in colostrum rapidly decrease and disappear, the incidence of disease increases from around 3 to 4 days after birth. This is said to be because it corresponds to the time when antibodies in milk disappear. In intestinal mucosal infections such as rotavirus infection, antibodies transferred from colostrum into the blood are ineffective in protecting against infection.

The onset of the disease can be as early as 12 hours after moving in, and the patient may suddenly pass yellow, watery stools, becoming dehydrated and weak, but usually recovers within 6 to 8 hours. The morbidity rate is high, but the mortality rate is low, θ~50%. However, mixed infections such as coronavirus, Escherichia coli, and pneumococcus are common and significantly worsen symptoms and prognosis. The lesion is localized to the small intestine, and significant changes occur in the trichome epithelial cells.

(5) On the other hand, it is known that a large amount of immunoglobulin is secreted in the milk of chewing mammals, especially in the colostrum. As mentioned above, rotavirus prefers to infect young animals with immature immune systems, so even if milk contains antibodies that recognize and bind to rotavirus antigens, No wonder. In fact, on average, cow's milk, the most common milk, has been found to contain high titers of antibodies against bovine rotavirus.

Therefore, the anti-rotavirus antibodies contained in colostrum,
It is considered that it can be applied to the prevention and treatment of rotavirus infection in newborn cows. The main problems encountered in doing so are the preparation of neutralizing antibodies that are compatible with all rotavirus types from ■■ to ■, ■timing of antibody administration, ■antibody dosage, and ■antibody formulation method. There are four points. As a result of intensive research, the present inventors solved these problems, established an effective method for preventing and treating rotavirus infection in newborn cows, and completed the present invention. This will be explained in more detail below.

(6) ``Means to solve the problem'' Rotaviruses are classified into serotypes 1 to ■, but there are regional differences in their prevalence. Therefore, even if an immunoglobulin has a high neutralizing antibody titer against type ■ rotavirus, it may not necessarily be able to effectively protect against type ■ rotavirus infection. It is also known that there are large individual differences in the antibody status of dairy cows. Therefore, in order to orally administer immunoglobulin to neonatal cattle to provide passive immunity against rotavirus, it is desirable to have immunoglobulins that have highly neutralizing antibodies against all serotypes of rotavirus. Such immunoglobulins can be obtained by mixing colostrum collected from as many cows as possible from various regions and extracting the immunoglobulins.

In addition, although it has been argued that it is necessary to provide newborn cows with sufficient colostrum, it was unclear how much should be given. Furthermore, since mother cows do not necessarily have neutralizing antibodies against all rotaviruses, it is not known how much or when to feed whey protein, which is rich in immunoglobulins (7) extracted from colostrum. It wasn't. The present inventors investigated this point and found that newborn cows are given sufficient colostrum immediately after birth, and then fed whey protein from colostrum containing 50 to 65% immunoglobulin until 24 days after birth. It has been found that if 5 g is given orally once a day within hours, the onset of diarrhea caused by rotavirus can be effectively suppressed and further complications can be prevented.

Formulation is also essential when administering immunoglobulins orally to newborn cows. And, the formulation is not just a dosage form that newborn cows prefer to take orally.
It is also necessary that the dosage form is such that the physiologically active proteins in the colostrum, including immunoglobulin, can reach the action site in the gastrointestinal tract and exert their effects. For example, the digestive tract of mammals is covered with a mucus layer, and if a physiologically active protein is simply dissolved in water and given, it does not necessarily reach the intestinal mucosa, which is the site of action. The present inventors have discovered the following two dosage forms that meet this purpose.

The first dosage forms consisted of immunoglobulin-rich colostrum milk (8), which was powdered and mixed with various electrolyte solutions, such as Ringer's or Tyrode's powders, and soaked in water before use. It is a method of dissolving.

This is because immunoglobulin is very difficult to dissolve in water, but relatively well soluble in electrolyte solutions. Furthermore, for newborn cows that have lost water and electrolytes due to rotavirus-induced diarrhea, supplementing these components at the same time as immunoglobulin is an effective therapeutic means.

Furthermore, in order to further enhance the efficacy of the immunoglobulin preparation, the following method may be employed.

After dissolving the above-mentioned physiologically active protein in 10 to 50 times the amount of Tyrode's solution enriched with glucose to 5 to 8%, oil or fat equivalent to the same amount to 3 times the amount of immunoglobulin is added, and lecithin or milk is used as an emulsifier. Add fat globule coating and emulsify. The effect of this immunoglobulin emulsion on preventing rotavirus infection in newborn cows is two to three times higher than when immunoglobulin is simply given as dissolved in an electrolyte solution. What is also noteworthy is that despite being a large molecule, a significant amount of (9) immunoglobulin is absorbed into the body across the absorption barrier of the intestinal tract.

Therefore, the technique of emulsifying immunoglobulin with oil and ingesting it orally is worthy of attention as a method for absorbing immunoglobulin from the intestinal tract.

"Example" Hereinafter, an example of the invention achieved by the present inventors using immunoglobulin-rich whey protein extracted from colostrum will be described.
Examples will be described from the viewpoints of setting the dosage and timing of administration for prevention and treatment of rotavirus infection, (1) method for formulating whey protein rich in immunoglobulins, and (2) method for enhancing activity.

[Example 1] Frozen colostrum collected from Southern Kyushu, Nagano Prefecture, and Tokachi Plain, Hokkaido, each weighing 150 kg, was mixed and dissolved by heating at 40"C, and acetic acid was added to adjust the pH to 4. Then, centrifugation was performed. 0.01% of rennet and 0.02% of CaC1 2 were added to the skimmed colostrum from which the cream had been separated and the cream was removed, and the casein was completely turned into curd while stirring for 1 hour.

This liquid was further centrifuged to separate 130 kg of the aggregated casein curd portion (10) and 300 kg of the whey portion. After adding NaOH to the whey to correct the pH to 6-7, it was filtered through a filter press to obtain a filtrate. This filtrate was passed through an ultrafiltration membrane with a molecular weight cut off of 100,000, and 3-fold concentration was repeated five times to obtain 80 kg of a concentrated solution. This concentrate was sterile filtered and freeze-dried to obtain about 8 kg of powdered colostrum whey protein. The protein content of this powder is 95%
The immunoglobulin content was 65%, and the lactoferrin content was 5.7%.

(11) Expressed as a dilution factor that reduces the amount of rotavirus virus infecting cells by 60%.

As shown in Table 1, colostrum immunoglobulins collected from dairy cows in northern and southern Japan have unique neutralizing antibody titers against rotavirus, and those from southern Kyushu have high neutralizing antibody titers against types I and ■. In Japan, the neutralizing antibody titers against these types are low, but the neutralizing antibody titers against the ■type and ■types are high. The antibody produced in Nagano Prefecture, located in the center of Honshu, showed a neutralizing antibody titer between the two.

Therefore, by mixing the colostrum collected from each of these regions, it is possible to obtain whey protein powder that exhibits strong antiviral activity on average for each serotype, as shown in Table 1.

Using whey protein extracted by mixing the colostrum collected from the Miike area mentioned above, we investigated the infection prevention effect by orally administering it to newborn Japanese black breeds in various places during the winter when rotavirus infection is prevalent. . In addition, the first dose of immunoglobulin is 5g within 24 hours of birth, containing 300IIlj! containing 6% glucose. The drug was dissolved in an electrolyte replenisher and administered orally, and then administered orally once a day for 3 to 5 consecutive days (12). The control group received electrolyte replenisher 3 containing 6% glucose.
00m j! gave only. The calves were kept and observed for one month, and the presence or absence of diarrhea and death due to infectious diseases were recorded.

Table 2 Results of Colostrum Whey Protein in Japanese Black Breed Newborns As is clear from Table 2, when colostrum whey protein was given, diarrhea and death due to infectious diseases were highly and significantly suppressed.

[Example 2] To 1 part of immunoglobulin-rich whey protein separated from colostrum, concentrated, and powdered by the method of Example 1, 6 parts of Tyrode's solution powder enriched with glucose is added and mixed. The composition of the glucose-enriched Tyrode's solution powder (13) is as follows. Glucose 5.045 g, sodium chloride 0.800 g, potassium chloride 0.02 g, magnesium chloride 0.01 g, calcium chloride 0.02
g, sodium monophosphate 0.005 g, sodium bicarbonate 0.100 g. When mixing is complete, add 3 of the total amount.
Lightly moisten the powder by spraying distilled water equivalent to ~4% evenly, mix thoroughly, and mix with 2 va (
16-18mesh) to granulate it in an extrusion granulator, and then in a fluidized fluid dryer at a temperature of 70°C.
It was dried at ℃ for 25 minutes. The moisture content of the granules thus prepared was 5.5-7.5%.

[Example 3] 37.5 g of the colostrum immunoglobulin-rich whey protein preparation prepared by the method of Example 2 was dissolved in 500 mj+ water and orally administered to Holstein neonates within 24 hours of birth, and thereafter 37.5g once a day for 5 consecutive days with 5g of water
It was dissolved in 00w 12 and administered orally. The control group received only electrolyte replenishment without whey protein.

The calves were observed for 4 weeks after administration for the presence or absence of diarrhea and for death due to infection.

(14) Table 3 Colostrum whey protein preparation for Holstein newborns From Table 3, it is clear that whey protein extracted from colostrum has a preventive effect on diarrhea.

[Example 4] In order to improve the antiviral activity of whey protein rich in immunoglobulins extracted from colostrum produced in Nagano Prefecture, it was formulated as follows. First, preparation 2 of colostrum whey protein prepared in Example 2
2.5 g was dissolved in 30 On+ 1 water, and 6 g of soybean oil and 0.3 g of milk fat globule coating were added and emulsified by ultrasonication. This emulsion is 300 liters 17! was orally administered to Holstein newborns within 24 hours of birth, and then from the next day onwards once a day for 30 hours for 5 consecutive days. The emulsion of (15) was orally administered. The calves were then observed for 4 weeks to check for rotavirus-induced diarrhea. In addition, only the replacement fluid was orally administered to the calves in the control group. At this time, the analytical values of the colostrum whey protein used are as follows.

(16) As shown in Table 5, even when whey protein was emulsified and orally administered, diarrhea caused by rotavirus infection was highly and significantly suppressed. In this test, although the dose of whey protein was reduced from 5 g per day in Example 3 to 3 g, the diarrhea preventive effect was equivalent to or slightly superior to that in Example 3.

[Example 5] An attempt was made to powderize the colostrum whey protein prepared in Example 4. Add 3 liters of lactose to 5 liters of the emulsion prepared by the method shown in Example 4.
00 g, whey protein manufactured by Bioisolate (Wh
ey protein isolate. WPI) 1
00 g was added, dissolved, and freeze-dried to obtain 800 g of hygroscopic powder. 16g of this powder and 100Ilj of water! In addition, by stirring well, an emulsion containing 1 g of colostrum whey protein can be easily regenerated.

"Effects of the Invention" According to the present invention, it is possible to produce an intact whey protein containing all immunoglobulins having highly neutralizing antibodies against each serotype of rotavirus. Furthermore, rotavirus infection can be effectively prevented or treated by orally administering the unmodified whey protein to newborn cows (17). Furthermore, by preparing the undenatured whey protein as a powder and mixing it with a powder of an electrolyte solution to form a formulation, the physiologically active protein can be delivered to the intestinal mucosa, which is the site of action.

Furthermore, if a neutral fat is added to the unmodified whey protein and the protein is emulsified using an emulsifier, an even higher effect can be obtained than when it is dissolved in an electrolyte solution.

(18)

Claims (5)

[Claims] (1) Colostrum collected from more than 50 dairy cows raised in multiple regions is mixed, and colostrum whey prepared through defatting and rennet treatment is passed through an ultrafiltration membrane with a molecular weight cutoff of 100,000. For the prevention and treatment of rotavirus infection in newborns, which is characterized by processing and further concentrating until the protein content exceeds 75% to purify undenatured whey protein rich in immunoglobulins, lactoferrin, and lactoperoxidase. Method for producing undenatured whey protein. (2) 5 g of undenatured whey protein produced according to the invention described in claim (1) is orally administered at least once within 36 hours of birth of the calf, and thereafter, at least 5 g is continuously administered every day for one week. A method for preventing rotavirus infection in newborn cattle, characterized by: (3) The undenatured whey protein produced by the invention according to claim (1) is powdered and mixed with powder of an electrolyte replenisher such as Ringer's or Tyrode, dissolved at the time of use, and orally administered. Prevention and treatment of diarrhea in calves. (4) A prophylactic and therapeutic agent for calf diarrhea, which comprises adding undenatured whey protein from colostrum separated and prepared according to the invention according to claim (1) to calf milk replacer. (5) One part or more of a neutral whey protein such as butter oil or soybean oil per one part of undenatured whey protein rich in immunoglobulin, lactoferrin, and lactoperoxidase purified from bovine colostrum according to the invention described in claim (1). Add fat and emulsify with emulsifiers such as lecithin and milk fat globule coating,
A method for preventing and treating rotavirus infection in newborn cows, which comprises orally administering at least 3 g of immunoglobulin per calf per day on consecutive days.