JPS6340771B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process for producing protein concentrates containing immunological factors of milk origin, in particular active immunoglobulins. It has already been proposed to introduce immunological factors of milk origin into nutritional products for newborns and infants by incorporating the immunological factors into the milk of newborns and infants; Oral injection of the substances is intended to allow the factors to pass into the infant's blood, but no indication is given of the method employed and the consequences of the systemic passive immunization. The milk of vaccinated cows is coagulated, the whey (lactoserum) is collected, and the lactoglobulins are selectively precipitated with ammonium sulfate, followed by dialysis against pure water, filtering, and drying. It has also been proposed to isolate immunolactoglobulins. In conjunction with those works, seroprotection studies in mice have shown that transdermal injection of these immune entities against certain enteropathogenic bacteria and viruses has shown that when animals are injected with these microorganisms by the same route, systemic passive It has been shown that protection occurs. The inventors have now found a method for producing protein concentrates containing immunoglobulins on a commercial scale without the immunoglobulins being denatured during the handling process. This product provides local passive immunity at the intestinal stage without reabsorption and without significant loss of activity within the gastrointestinal tract. The method according to the invention consists of the following steps: - collecting the milk of a hyperimmune milk-producing female; - separating the cream and impurities, coagulating the clarified and skimmed milk and separating the casein;
The product is evaporated and dried under conditions that sterilize the whey proteins, do not denature the immunoglobulins and maintain sterility. The accompanying drawings are a diagrammatic illustration of the various steps of the method. Milk-bearing females are members of the bovidae family, especially cows. The milk to be processed can be in various forms that are more or less enriched with antibodies. Colostrum (colostrnm),
transition milk (milk from 8 days after birth) or end-of-lactation milk
milk) (last two months of lactation). It is preferred to process the milk mixture to obtain high antibody levels for the longest possible period of time. Combination vaccination of cows, e.g. intracisternal instillation into the mammary gland, transdermal injection (subcutaneous, intravenous), injection into the posterior nodal system of the mammary gland,
Hyperimmunize by scarification, oral ingestion, or some combination of these modes. It is preferred to use a combination of these modalities in a carefully designed immunization regimen to obtain satisfactory antibody levels in each type of milk used. Thus, in the case of colostrum and transitional milk, vaccination is by the transdermal or intracisternal route 8-2 weeks before calving and by the oral route in the weeks following calving. In the case of late lactation, transdermal, topical and oral vaccinations are applied alternately for 2.5 months before the expected end of lactation. The vaccine used may be a mixture of the selected antigen and an adjuvant based on the homologous serum of the immunized cow, thus providing for detoxification of the vaccine and the formation of immune complexes. Because cows are used as source animals, the immunoglobulins (Ig's) contained in the protein concentrate
is mainly GIg′s (especially G ₁ Ig′s), except in breast milk where AIg′s is the predominant Ig′s. The Ig content in protein concentrates containing 70 to 80% by weight of protein is 25 to 35% by weight. Certain advantages are that, in the practical application of the method, Ig'S is not separated from the other proteins of the whey, so it is also present in the resulting concentrate. This means that selective precipitation and dialysis operations, for example with ammonium sulfate, are no longer necessary. In addition, certain bacteriostatic or antiviral factors present in whey, such as lactoferrin, or enzyme systems such as lactoperoxidase and ribonuclease, are also found in the protein concentrate. All kinds of antigens of viral or bacterial origin can be used, and the antibodies obtained will depend on the nature of the antigen injected into the cow. for example,
Incorporation of protein concentrates containing Ig's into any support suitable for oral administration provides protection against enteropathogenic bacteria and viruses that cause gastroenteritis and are associated with severe diarrhea and dehydration. be able to. This support can be supplemented with optional excipients or, preferably, with nutritional products such as milk for infants or malted milk, "humanised".
It may be formed by milk that is specifically adapted to a particular group of neonates, such as milk for preterm infants or infants with a low birth weight. For example, if it is desired to use a concentrate containing prophylactic amounts of anti-E. coli Ig'S in milk as a support, 0.8 to 3 g
can be combined into 100g of dry matter,
An amount of concentrate up to 6 g is sufficient as a therapeutic amount. To carry out the process, it is necessary to carry out the degreasing and clarification operations very thoroughly in order to avoid subsequent blockage of the filter and ultrafilter. Defatting is preferably carried out in two stages, first at a low temperature to remove most of the fat and contaminants (e.g. blood often present in colostrum) and then at a high temperature to completely separate the fat and contaminants. It will be done. Coagulation can be carried out at the isoelectric point PH of the casein in the presence of rennet by addition of an acid such as citric acid. Coagulation is preferably carried out by addition of an acid, such as hydrochloric acid, up to a pH value of about 4.6 and by heating to a temperature of 40°C. Separation of casein involves clarification of the whey to allow separation of fines. On the other hand, it is important that operations requiring heat treatment (evaporation and drying) be carried out under conditions designed to prevent inactivation of Ig'S. The cream and curd were extracted with water to reduce the loss of Ig'S, some of which is removed with the cream during the skimming operation and other with the casein during the casein separation. ,
It is recommended to treat the wash water containing Ig'S to recover them. The method of the invention is illustrated in the following examples, in which the amounts and ratios indicated are as follows, unless otherwise indicated:
It depends on the weight. Example 1 Cows of various breeds are hyperimmunized using the vaccine whose preparation is described below, according to the following schedule, and milk collected during the last two months of lactation and during the first eight days after calving. did. Vaccine production The following gastrointestinal pathogenic E. coli serotypes were used: 018: K76 (B20) 0111: K58 (B4) 020: K17 (L) 0112: K68 (B11) 026: K60 (B6) 0119: K69 (B14) 044:K74(L) 0124:K72(B17) 055:K59(B5) 0125:K70(B15) 078:K80(-) 0126:K71(B16) 086:K61(B7) 0127:K63(B8 ) 0128:K68(B12) Various bacterial strains were obtained from hospitalized infant gastroenteritis patients. Serotyping was performed with polyvalent and monovalent antisera (Difco). Bacteria were cultured for 24 hours at +37° C. without shaking the bottles in a minimal amount of liquid medium containing 2% of amino acids derived from casein (defcocasamino acids) and 2% of glucose. To inactivate the microorganisms, the culture was separated into sedimented cells and supernatant layer by centrifugation. The cells were resuspended and incubated for 24 hours at +37°C in the presence of 0.5% formaldehyde, while the supernatant was inactivated as above in the presence of 0.05% formaldehyde. After recentrifugation and separation of the supernatant layer, the bacteria were resuspended in the original supernatant layer. This method allows the supernatant layer containing the bacterial exotoxin to be maintained as a vaccine. 1
- a vaccine containing 2×10 ⁹ bacteria/ml was obtained;
It was then diluted by mixing with a 2% solution of Al(OH) ₃ and homologous blood of the immunized cow. Immunization method A. Immunization regimen for obtaining colostrum and transitional milk containing anti-E. thusierichia coli Ig'S: Table B. Late-lactation milk (last 2 lactation milk) containing anti- E. coli Ig'S Immunization scheme for obtaining a 100-year-old cow.This immunization is carried out exclusively for the colostrum and transition milk period in cows that have already been immunized. [Table] [Table] Example 2 Milk collected during the first 8 days after calving from cows vaccinated according to Example 1 was processed as follows: - Defatting is carried out in two steps: Step A at low temperature.
and step B at high temperature. A: Put 1100kg of cooled milk into a centrifugal degreaser, and
It is directed to a 2500 volume buffer tank and then pumped into a high speed (approximately 11000 G) centrifugal clearer to remove blood and contaminants (dregs). B. Cooled clarified milk was stored in a 2500 capacity storage container, passed through a heater at 40 degrees C, and pumped into the centrifugal skim machine used earlier, thus producing 110 kg of cream.
Separate it, collect it and discard 5Kg of waste. In a variant of this process, it is possible to use self-clarifying degreasers for low and high temperature degreasing operations, and clarifiers between these operations, thus reducing the degreasing time. - 4 square 750 with stirrer for coagulation operation with warm clarified skimmed milk (985Kg)
The volume is distributed to the coagulation tank. Add 1N hydrochloric acid solution at 37°C until the pH stabilizes at approximately 4.6, then reduce the temperature to 20°C with stirring.
Hold for a minute and then cool to about 15 degrees Celsius. -The next operation is the separation of casein. It consists of two consecutive centrifugations of slabbed milk that has been defatted and hardened into curds. The first one separates most of the coagulated casein by a self-cleaning clarifier which separates 150Kg of casein and the whey enters the buffer storage. A second centrifugation operation removes trace particles that could interfere with subsequent filtration and ultrafiltration, for example by the clarifier used in step A of the degreasing operation. 854.7Kg of clarified whey is obtained and pumped into a 2500 volume reservoir. - The whey is then successively transfiltrated and ultrafiltered. The filtration must be extremely sufficient to avoid the difficulties of ultrafiltration by separating the very fine particles of casein.
The straining is carried out in a Seitz Supra 100 strainer consisting of 20 x 20 cm plates. The liquid is 3000
It is stored in a container reservoir and then pumped into an ultrafilter. Ultrafiltration is carried out in two or three steps with retentate recycled to the reservoir. Two centrifugal pumps arranged in series operate at a pressure of about 7 bar on the inlet of the input compartment of the ultrafilter, and at the output end where the pressure is kept at about 4.5 bar. ). To prevent heating by the energy supplied to the pump, the holding material should be approximately 10−
Cool to 12 degrees C. The first step, i.e. ultrafiltration proper
The method separates the high molecular weight solutes and proteins retained on the membrane found in the retentate, while the lactose, vitamin mineral salts and water portion are removed in the permeate. Has 800 membranes
By using the DSS module (membrane surface = 7 m ² ), the retentate/permeate ratio is approximately 1:
3.1 and thus permeate 580Kg and retentate
274.7 Kg are obtained with an average permeate withdrawal of 14.17 Kg/m ² h. The second step is diafiltration, during which 825 Kg of water is continuously added to the retentate and the solution is circulated under the same conditions as in the first step. The diafiltration is stopped when the conductivity of the permeate reaches the required value, the ratio of retentate to added water being approximately 1:3. Thus, the permeate
825 Kg and 274.7 Kg retentate are separated with an average permeate withdrawal of 11.75 Kg/m ² h. The third step is
With desorption of 82.5 Kg of permeate, an optional concentration step is carried out by recycling the retentate to the membrane to a dry matter content of approximately 10%. Thus, 192.2Kg
A preconcentrated solution of . In a variant, this third step is omitted and instead proceeds directly to the sterilization step. The sterilization step is an important step in the present method as a whole, since heat treatment causes denaturation of Ig'S and cannot be applied to sterilization. The sterilization filter removes from the retentate any microorganisms that may still be present in it, most of which have already been removed during the separation of the cream, casein and dregs. Clarification by centrifugation and triple filtration of the retentate is necessary to prevent the sterilization filter from becoming closed. After clarification, the retentate was stored in a reservoir and passed through a filter line consisting of a prefiltration stage on three filters arranged in series (Seitz Spra 100, EK, EKS).
Subsequent sterilization filtration is carried out on Seitz Spra 20 and Millipore HA (0.45μ) filters arranged in series (previously sterilized with superheated water). Sterilized retentate is stored in a 500 volume sterile reservoir. All of the following operations are performed under sterile conditions. The subsequent evaporation step can be carried out, for example, under sterilized, compressed and strained nitrogen. The evaporation step is carried out in a thin-layer evaporator (with a falling film with a surface of 1 m ² ) at a heating temperature of 75° C. to a dry matter content of at least 20%, the temperature of the product being approximately 30° C. To avoid infection, the transfer is made by peristaltic pumps from the retentate reservoir to the concentrate reservoir connected to the loop containing the evaporator, and the concentration operation takes place in a closed circuit. This operation
Does not affect Ig′S activity. The concentrate (96 g) is then dried by any suitable means, such as freezing followed by lyophilization. Freezing can be carried out at -40°C on the plate. Ig'S is -30 degrees C
It can be stored for about 45 days without any loss of activity.
The product at -30°C is lyophilized at 0.5 torr on a plate in a chamber with a condenser temperature of -50°C and warmed to a temperature of 30 to 35°C.
The method used for drying does not affect the activity of Ig'S. 19.1 Kg of dry product containing 25-35% Ig'S is thus obtained and packaged under sterile conditions. Example 3 By employing the method described in Example 2 for processing milk collected during the first 8 days after calving and during the last 4 weeks of lactation, a protein concentrate with the following average composition was obtained: Contains: Protein 75±5% Immunoglobulins 30±5% α-lactoglobulins 15±5% β-lactoglobulins 35±5% Serum albumins 5±2% Other trace proteins 5±2% Residual water 4 ±0.5% Lactose 10±2% Inorganic salts 5±2% Non-protein nitrogenous substances 5±2% Example 4 The method involves extracting cream and casein with water to recover some lost Ig'S. As in Example 2, except the water is recycled to the production line. - Cold defatting (step A) is carried out in a parallel water extraction line to extract the protein fraction containing Ig'S entrained in the cream. The cream (115 Kg) resulting from the first centrifugation operation was stored in a 1000 volume reservoir equipped with an agitator and 40
Mix with 150 kg of lukewarm water at degree C, stir the whole thing for 30 minutes and centrifuge in a degreaser. cream
115Kg and 150Kg of solution are thus separated,
The solution is defatted and recombined with clarified milk. 1130 kg of liquid are thus obtained for thermal degreasing (step B) and coagulation. - Separation of casein from 1145 kg of defatted, rennet-added and acidified milk, 991 kg of whey
and gives 154Kg of casein. with water from curdled milk
Parallel lines are used to extract the protein fraction containing Ig′S. Upon removal from the self-cleaning clarifier, the curds are stored in a reservoir with a stirrer and stirred with 300 kg of lukewarm water at 40 degrees C for 30 minutes and transferred to a colloid mill. The suspension of crushed curd is then separated in a self-cleaning clarifier. washed casein
154Kg and 300Kg of washing liquid are recovered and the washing liquid is recombined with whey for subsequent operations. 1291Kg of whey was filtered and ultrafiltrated and the permeate (,
and) obtain 2010Kg and preconcentrate 216Kg.
Evaporation gives 108 Kg of concentrate, which is then dried to give 21.6 Kg of dry product. This represents an increase in Ig″S of about 14% compared to the method described in Example 2. Example 5 1 Kg of the powder prepared according to Example 2 or 4, or 2 Kg of the powder prepared according to Example 3, was added to 100 Kg of milk powder.
Ig-concentrate prepared according to Example 2 or 4 by homogeneously mixing with
250mg/Kg/day or Ig prepared according to Example 3
- 140 cal/Kg/day equivalent to 500 mg/Kg/day of concentrate
Obtaining a product containing a prophylactic amount of Ig″S for a daily isocaloric feed. Example 6 - Milk Ig″S has the antibody specificity normally found in human Ig″S, and this specificity Various in vitro and in vivo tests were carried out to demonstrate that the specificity of milk Ig'S is maintained during the manufacturing process and that the specific milk Ig'S exhibits protective activity by interfering with the pathogenic mechanisms of enteropathogenic microorganisms. Example 2, hereinafter referred to as “Ig-concentrate”
or protein concentrate according to 4. Passive hemagglutination Red blood cells from sheep were sensitized with a urea extract of a specific E. coli serotype. This extract was prepared using the Brodhage method [Brodhage H.,
(1961), Journal of the Hygiene (J.
Hyg.), 1961, 148 , 94]. Bacteria in Roux bottles, nutrient agar (Defco)
Top, cultured at 37 degrees C for 30 hours. The culture was mixed with 10 ml of phosphate buffered salt solution (8.8 g NaCl) to pH 7.2.
+ _{1.86gNa2HPO4 ・ 2H2O} + _{0.43gKH2PO4 /
1000mlH2O} ). Urea (Merck) 10g
Following the addition of , the suspension was gently stirred at 37° C. for 90 hours. After centrifugation, the supernatant layer was thoroughly dialyzed against a salt solution buffered with phosphate to PH 7.2, diluted to a final volume of 50 ml with a salt solution buffered with phosphate to PH 7.2, and aliquoted in small portions. -20 degrees C
It was frozen. Sheep red blood cells were collected using the method of Avrameas et al. [Avrameas S., Taudou B., Chuilon S. Immunochemistry, 1969]
6, 67] and the method of Otto et al. [Otto H., Takamiya H., Vogt A., J.Immunol.Methods,
1973, 3 , 137]. Sheep red blood cells were pretreated with glutaraldehyde (final concentration, 5% sheep red blood cells and 0.5% glutaraldehyde), washed, and suspended in salt solution buffered with phosphate to PH 7.2 for 20 min.
% suspension was formed and then mixed with the same volume of urea extract. After incubating for 16 hours at 20 degrees C with gentle agitation, the sensitized sheep red blood cells were washed several times and suspended at 1% level for direct use. Sheep red blood cells in the form of a 10% suspension can be stored for several weeks at 4 degrees Celsius. Before incubation, each specimen to be tested must be absorbed into sheep red blood cells pretreated with glutaraldehyde (0.1 ml of precipitated sheep red blood cells per ml of Ig-concentrate, 37° C., 60 minutes). Titration was carried out under a microtiter system using a polystyrene plate with a V-shaped depression [Sever JL, J. Immunol., 1962, 88 ;
320, and Conrath TB, Handbook of Microtiter Procedures,
1972). A series of 50μ dilutions in normal rabbit serum diluted 1/200 was prepared and 25μ of 1% sensitized sheep red blood cells was added to each dilution. Other series were prepared with non-sensitized sheep red blood cells as a non-specific agglutination control. Results were read after 2 hours and hemocyte aggregation titers were obtained at the final dilution leading to a clear positive reaction. The results obtained for the five E. coli serotypes are shown in the following table: E. coli serotype Agglutination titer O 55 1/256 O 111 1/64 O 119 1/128 O 127 1/128 O 128 1 /64 Control - Bacteriostatic activity in vitro The bacteriostatic activity on the growth of various E. coli serotypes was tested by adding Ig-concentrates to the culture medium. A culture compatible microtiter system was used to allow a minimum amount of specimen to be used in the test and a maximum number of specimens to be tested at the same time. Each culture had a final volume of 0.25 ml in either minimal or enriched medium containing 1% glucose. For each value of incubation time tested, two samples were taken to reduce the risk of error due to volume changes. The results were evaluated by double counting of 10μ sections of nutrient agar plates. The bacterial inoculum consists of a dilution of a 16-hour culture in minimal medium containing 2×10 ³ bacteria/ml. Incubation was carried out at a temperature of 37° C. in a humid atmosphere. Ig-concentrates at final concentrations of 1 μg/ml, 10 μg/ml, 100 μg/ml and 1 mg/ml were added to the medium prior to inoculation with 2×10 ³ E. coli/ml. At a concentration of 10 μg/ml of Ig-concentrate, clear inhibition of E. coli O111: _B4 was observed during the first 4 hours of incubation. Compared to the control formed by the medium itself, greater growth was observed in normal whey 1 obtained from non-immunized cows.
observed in the presence of mg/ml. Clearance of E. coli in mice Eight mice (male Swiss white mice weighing 20 to 24 g) were used for each test, two served as controls, and six were used in duplicate for three tests. Ta. All mice were injected subcutaneously with 0.1 ml of heparin (500 UI/ml). A 16-hour culture of E. coli O111:B4 was diluted in a 1:10 ratio with sterile salt solution and then in a 1:10 ratio for the control and containing three selected concentrations of Ig-concentrates. The sample was diluted to a ratio of 1:100 by the addition of fetal calf serum (Defco). 0.2 ml of the resulting solution was injected intraperitoneally into the tail of each mouse (2 mice as control and 2×
3 for testing) received approximately 2×10 ⁶ bacteria, and the original bacterial suspension contained 10 ⁹ bacteria/ml.
Immediately after the injection (to time) and 20, 40 and 60 minutes later, 50μ of blood was collected from the ophthalmic plexus into a calibrated heparin-lined capillary tube.
capillary tubes) and the specimen immediately transferred to 5 ml of sterile saline solution (hemodilution).
10 ⁻² ), which was then diluted to 10 ⁻³ and 10 ⁻⁴ in salt solution. Counts were performed on agar plates (Defco) with 1 ml of each dilution, and the phagocytic index K was determined using Biozzi G., Stiffel G., Halpern BM, Le Minorl. .)Mauton
D.), J. Immunol., 1961, 87 , 296): K = logC ₁ −logC ₂ /T ₂ −T ₁ C ₁ and C ₂ correspond to the counts at T ₁ and T ₂ hours do. Thus, in the presence of fetal calf serum, a normal reduction in bacterial numbers was observed due to detachment in the retinal endothelial system, whereas addition of 10 μg of Ig-concentrate to the injection solution removed blood from the bloodstream within 20 minutes. resulted in 99.6% disappearance of bacteria. The results are shown in the following table: [Table] In order to determine the specificity of the opsonization process, tests were carried out by exhaustive absorption of Ig-concentrates with O111:B4 serotype of E. coli,
And this absorption was shown to destroy virtually all of the activity in increasing phagocytosis even in an amount of 1 mg of Ig-concentrate. The following table shows the phagocytosis index K obtained after 20 minutes: [Table] Protection test in mice 10 Logarithm of a culture of E. coli O111:B4 in nutrient medium (Defco) containing ⁹ bacteria/ml Dilute (log ₁₀ ) and prepare 5% mucin (granular mucin to enhance bacterial pathogenicity, 1701-W).
Mold, Wilson Laboratories
Laboratories), Chicago, Illinois)
10 ^-4 , 10 ^-5 , 10 ^-6 and 10 ^-7 dilutions were obtained. 3 ml of each dilution followed by 0.6 ml of the solution to be tested;
Namely, they were mixed with saline solution, Ig-concentrate and normal whey protein (non-immune cow), respectively. Five mice were used for each dilution, resulting in 0.6 ml of the mixture.
was injected intraperitoneally into each mouse. The results of the evaluation of the number of dead mice after 48 hours among the 5 treated mice are shown in the following table. [Table] μ
*Calculated as total protein containing 35 to 45% milk Ig'S.
100 μg of Ig-concentrate produced complete protection for all bacterial concentrations tested, whereas 100 μg of whey protein isolated from the milk of non-immunized cows produced no protection. Example 7 A first series of clinical studies showed that milk Ig'S resists proteolytic breakdown into inactive fragments in the intestinal tract. 11 infants (9 males) who were hospitalized for various reasons but had never had gastroenteritis, ranging from 2 weeks to 1 year old.
Ig produced according to Example 2 or 4
- fed with milk containing concentrate in isocaloric doses equivalent to 2 g/Kg body weight equally distributed over 24 hours. The first and last diets combined with Ig-concentrate were labeled with 0.2 g of Carmine red. At least one stool was collected prior to administration of the I-concentrate, and subsequent stools were collected systematically over a 72 hour period, and the stools were stored at -20 degrees Celsius.
Extracts of feces were prepared by adding 2 parts of feces to 1 part of a salt solution buffered with phosphate to PH 7.2 and then dispersing the feces with a glass rod at 0 degrees Celsius. The suspension was then stirred (400 rpm) for 20 minutes at room temperature using a mini-stirrer. The suspension was then heated to 0 degrees C.
and centrifuged (3500G) for 15 minutes at that temperature. The supernatant layer was then carefully removed. The presence of active Ig'S in fecal extracts was confirmed by double immunodiffusion (Ochterlony test) and immunoelectrophoresis. The antiserum used in this test was prepared by repeated subcutaneous and intravenous injections of 1 mg of milk antibody or 0.1 mg of G ₁ Ig into rabbits. For the Ochterlony test, punch 4 mm indentations spaced apart at 9.5 mm intervals [LKB-Producteur
A glass plate (94 x 84 mm) formed by
1 in a salt solution buffered to PH7.2 with phosphate.
% agar gel and a bovine colostrum whey anti-protein antiserum and a cow's milk monovalent specific anti-G ₁ Ig antiserum were used. For immunoelectrophoresis, a glass plate (100 x 85
mm), 0.05M barbiturate (sodium barbiturate per 1 water, buffered to PH 8.3)
10.3g + citric acid 0.5g + oxalic acid 0.5g)
% agar gel and electrophoretic separation was carried out for 90 minutes at room temperature and 4 volts/cm.
After antiserum diffusion (24 hours), plates were washed with salt solution, dried, and azocarmine B (azocarmine B).
Color was developed using B). Milky Ig'S was observed to be present in feces despite considerable variation in the time of their appearance and duration of Ig-secretion. Furthermore, there was good correspondence between the appearance and disappearance of carmine red label and Ig'S. To confirm the activity of these milk Ig'S, an in vivo seroprotection test was performed in mice (as described in Example 6) using 6 fecal extracts for each strain, and the results are shown in the following table. : [Table] There is a clear correlation between the positive immunoelectrophoresis results and the protective ability of the substances contained in the fecal extract. In particular, the concentration of milk Ig'S in the extract (infant MD) and (infant SG) is estimated to correspond to at least 1 mg of Ig concentrate. A second series of clinical trials was conducted to determine the therapeutic and prophylactic properties of the Ig-concentrates prepared according to Examples 2 or 4. Therapeutic properties This study was conducted in infants up to the age of 5 months with benine or acute gastroenteritis caused by E. coli. In each series of studies, a total of 152 patients received Ig-concentrate 2g/Kg/day for 5 days or 1 day via their diets.
g/Kg/day for 10 days. Patients were not given any medications, such as sulfonamides or antibiotics, either orally or transdermally. Fecal cultures were prepared the day after each dose, the last being 36 to 48 hours after the final dose of Ig-concentrate. 43 patients were treated similarly but instead of Ig-concentrate.
Treated with whey protein from non-immune cows. Negative fecal cultures were obtained in 90 infants (57.7%) during the treatment period. In the control series,
Only 14 infants (27.7%) showed spontaneous disappearance of E. coli in fecal cultures.
One must take into account the fact that the natural protein of whey from non-immune cows contains small amounts of anti-E. coli Ig. It was also observed that treatment was more often unsuccessful when the highest doses were administered for a shorter period of time. Fecal consistency was tested in each case. The study showed disappearance of diarrhea and significant clinical improvement in many cases, even in cases where feces remained positive. If the stool culture is negative,
Fecal consistency improved more rapidly when the Ig-concentrate was administered in a milk formula with low lactose content. Preventive Properties Preterm infants are an extremely susceptible group as far as gastroenteritis caused by E. coli is concerned. It was this group that was selected for the clinical prevention trial. Each study was conducted for 6 months in two rooms with eight incubators in each room. Tests and controls in each room were distributed so that all patients were exposed to the same conditions, with four incubators used for testing and four for control purposes. Each patient remained for an average of 41 days, and infants, regardless of their group (test or control), were transferred and replaced after recovery. Seventy cases were followed up, 36 cases served as controls, and 34 cases served as the test group. The test group received Ig-concentrate in each meal distributed over 24 hours in an amount of 0.25 g/Kg/day. Fecal cultures were prepared and tested for each 5 days from the beginning of the study. Of the total number of 666 fecal cultures, 339 belonged to the control group and 327 to the test group. Of the latter, 33 (10.1%) showed the presence of E. coli, while 96 (28.3%) of the 339 control cultures were positive. Serotype Essierich coli often associated with acute forms of gastroenteritis
When O119:B14 was tested per se, the frequency of positive fecal cultures was 5.5% in the test group compared to 23.3% in the control group. It can be concluded that incorporation of a protein concentrate containing specific anti-E. coli milk Ig'S into the milk of preterm infants significantly reduces the extent of infection by E. coli in this particularly susceptible group of neonates.

[Brief explanation of drawings]

The drawing is a diagram for explaining the method for producing a protein concentrate of the present invention.

Claims (1)

[Scope of Claims] 1. A method for producing a protein concentrate of milk origin containing native and active IgG immunoglobulin effective against enteropathogenic E.Coli in infants, comprising: Cows were vaccinated with a series of consecutive doses of antigen by parenteral and topical routes from 8 weeks to 2 weeks and by oral administration for about the last 2 weeks before calving, and for end secretion about 2 weeks before the estimated end of lactation. At 2.5 months, a series of serial challenge doses, alternating parenteral, topical and oral, is started and milk is collected from the first 8 days after calving, optionally from the last 2 months of lactation, (b) first at low temperatures; , followed by a high temperature two-step skimming operation to separate the cream and contaminants, (c) coagulate the clarified and defatted milk, (d) separate the casein, filter and ultrafiltrate, and The above method, characterized in that the whey protein is sterilized by filtration, the preconcentration and sterilized whey is evaporated under sterile conditions, and the product is dried under conditions that do not denature immunoglobulins and maintain sterility. Production method. 2 Step (d) is: Separate the casein, filter and ultrafiltrate in three steps, namely ultrafiltration proper, diafiltration and preconcentration steps, sterilize the whey protein by filtration and close under sterile conditions. Claim 1, wherein the circuit evaporates the preconcentrated, sterilized whey and then freezes the evaporated whey, followed by drying by lyophilization and then packaging the product under aseptic conditions.
The method described in section. 3. The method of claim 1, wherein the vaccine is mixed with homologous serum of vaccinated cattle prior to topical administration. 4 Non-denatured active IgG effective against gastroenteritis in infants
A process for producing a milk-origin protein concentrate containing immunoglobulins, comprising: (a) administering the antigen by parenteral and topical routes from about 8 weeks to 2 weeks before delivery and by oral administration for about the last 2 weeks before delivery; and, for end secretion, a series of serial challenge doses, alternating parenteral, topical and oral, approximately 2.5 months before the estimated end of lactation, and for the first 8 months after calving. milk is collected from day one, optionally from the last two months of lactation, (b) separated from cream and contaminants by a two-step skimming operation, first at low temperature and then at high temperature; (c) clarified; (d) the casein is separated, filtered, ultrafiltered, and the whey protein is sterilized by filtration; (e) the immunoglobulins lost during the skimming operation and casein separation are cream and casein; (f) evaporate the products under conditions that do not denature the immunoglobulins and preserve sterility. , the above-mentioned manufacturing method, which is characterized by drying.