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Antigenicity of Casein Enzymatic Hydrolysate
Antigenicity of Casein Enzymatic Hydrolysate M. TAKASE, Y. F U K U W A T A R I , K. KAWASE, I. K I Y O S A W A , K. OGASA, S. S U Z U K I I, and T. K U R O U M E 1
Central Research Laboratory Morinaga Milk Industry Co., Ltd. Tokyo, Japan
with clinical symptoms o f patients with milk allergy. Allergy to cow's milk protein has become more clearly defined in specific antibodies to the antigens in patients' sera (2). Developments of immunological techniques based on antigen-antibody reactions have contributed to detection of antigenicity (3). We prepared a soluble fraction from enzymatically hydrolyzed bovine casein to use as a source of amino acids as a substitute for cow's milk. This paper reports our efforts to use casein hydrolysates as substitutes for cow's milk in detecting antigenicity.
ABSTRACT
An enzymatic hydrolysate of bovine casein was studied for molecular size and antigenic properties. As determined by Sephadex G-25 gel filtration, polypeptide fractions having molecular weights greater than 1,000 were not detected in the soluble fraction of the casein hydrolysate. Antigenicity o f the fraction was not detected by heterologous passive cutaneous anaphylaxis in guinea pig-rabbit antiserum system mediated by immunoglobulin G types of antibody (guinea pig passive cutaneous anaphylaxis) and its inhibition test. Furthermore, it was not detected by rat long latency homologous mediated by immunoglobulin E antibody and its inhibition test. To prepare anticasein immunoglobulin E serum for rat long latency homologous passive cutaneous anaphylaxis, immunization of rats was by single subcutaneous injection of casein with aluminum hydroxide. Antiserum with highest passive cutaneous anaphylaxis titer (1:64) against bovine casein was obtained by a dose of 2,000/ag of the protein at day 12.
MATERIALS AND METHODS Casein
Casein was prepared from fresh skim milk (bovine) by four isoelectric precipitations as described by Nagasawa et al. (4). P roteolytic Enzymes
Proteolytic enzymes were pancreatin (Amano Pharmaceutical Co., Nagoya, Japan) and proteinases from Aspergillus oryzae (Amano Pharmaceutical Co., Nagoya, Japan) and Lactobacillus belveticus prepared by the method of Ogasa et al. (5).
INTRODUCTION
It is extremely important to destroy the antigenicity o f milk proteins for dietetic treatment of infants with milk allergy fed cow's milk formulas. Enzymatic digestion of milk protein is not only effective for destroying antigenicity, but it also is less liable to alter the nutritive value (1). The degree of antigenicity and the amount of hydrolysis of milk protein need to correlate
Received January 22, 1979. 1Department of Pediatrics, Gunma University, Gunma, Japan. 1979 J Dairy Sci 62:1570-1576
Experimental Animals
Sprague Dawley male rats, Hartley male guinea pigs, and male albino rabbits weighing about 250 g, 300 g, and 2.5 kg were used. Enzymatic Hydrolysis of Casein
A 10% casein solution adjusted to pH 7.0 with .5 N NaOH was pasteurized in a waterbath at 85 C for 15 rain. The solution was cooled to 50 C, and three enzymes were added at 1 part pancreatin, 4 parts proteinase of Aspergillus oryzae, and 2 parts proteinase of Lactobacillus helveticus to 150 parts casein. The solution was incubated at 50 C for 24 h
1570
ANTIGENICITY OF CASEIN HYDROLYSATE then heated at 85 C for 15 min to inactivate the proteolytic enzymes. The solution was cooled : to 5 C and centrifuged at 12,000 x g for 20 rain to remove the precipitate. About 91% of the total casein nitrogen remained in the supernatant. The supernatant containing the casein bydrolysate was freeze dried and stored until needed. The total nitrogen and formol nitrogen in the casein hydrolysate as determined by the Kjeldahl method and formol titration were 15.7g % and 6.3g %. Gel Filtration
Gel filtration was by 1.6 x 90-cm columns of Sephadex G-25 equilibrated with .5 N acetic acid. Blue dextran (Pharmacia, Uppsala, Sweden, tool wt 200,000), oxytocin (Sigma Chemical Co., mol wt 1,000), tyrosin (Sigma Chemical Co., mol wt 181) and tryptophan (Sigma Chemical Co., mol wt 204) were used as indicators of molecular size. Absorption of the eluted solution at 280 nm was determined in a Hitachi UV-VIS Spectrophotometer. The ninhydrin reaction was according to the method of Yemm et al. (6). Anticasein Rabbit Serum
Anticasein rabbit serum was prepared from the blood of a rabbit that had received four muscular injections of 10 mg of casein emulsified with Freund's complete adjuvant over 1 mo. Guinea Pig Passive Cutaneous Anaphylaxis (PCA)
Rabbit antiserum was diluted with saline so that a suitable amount of antibody was contained in .1 ml of the diluted solution. One-tenth milliliter of the diluted rabbit antiserum was injected intradermally into the shaved back of a normal guinea pig. After 4 h, the guinea pig was challenged by intraveneous injection of an antigen solution containing 5 mg of Evans-blue. The antigen solution was prepared by dissolving a specified amount of casein or casein hydrolysate with saline. The guinea pig was decapitated after 30 rain and the skin was reflected. The extent of extravasation of the dye was determined on the back of the skin. The PeA reactions were graded by the diameters of blueings as follows: less than 5 ram, - ; 5 to 10 ram, +; 10 to 15 ram, ++; 15 to 20 ram, +++; more than 20 mm, ++++. The PCA reactions
15 71
with blueings of more than 5 mm in a diameter were defined as positive. Rat Long Latency Homologous PCA
One-tenth milliliter of suitably diluted antiserum was injected intradermally into the shaved back of a normal rat. After 48 h, the rat was challenged with an intraveneous injection of 1 ml of antigen solution containing 5 mg of Evans-blue. The PeA reaction was graded as described above. The PCA titer of the antiserum was determined by the PCA reaction as follows. Antiserum was serially diluted two fold and injected into the rat for passive sensitization. The PeA titer was expressed as the reciprocal of the maximum dilution of the serum that produced a positive PCA. In rat long latency homologous PeA (48 h), only IgE antibody in the antiserum reacts against the antigen. I nhibition Test
Inhibition tests were in guinea pig P e A and rat long latency homologous PCA. The test utilized the PCA reaction as an indication of the neutralization of sensitizing antibody by prior contact with an antigen. Anticasein serum was mixed with the material to be tested and incubated at 25 C for 1 h. The mixture was injected intradermally into an animal. The untreated antiserum was injected into the adjacent site on the same animal. The following procedures of the test were the same as the usual PeA test with a challenge with sufficient amounts of casein after 4 h in the guinea pig PeA and after 48 h in rat long latency homologous PeA. The neutralized antiserum failed to give a reaction or gave response smaller than that produced by untreated antiserum. RESULTS Gel Filtration
The elution pattern of the casein hydrolysate is in Figure 1. Two peaks appeared in the pattern measured spectrophotometrically at 280 nm. One peak was eluted with a volume equal to tyrosine, and the other one appeared in an effluent volume equal to tryptophane. In the elution pattern identified by the ninhidrin reaction, a major peak with a shoulder eluted between 100 and 150 ml. The shoulder emerged in an effluent volume equal to tyrosine. A Journal of Dairy Science Vol. 62, No. 10, 1979
1572
TAKASE ET AL. Inhibition Test on Guinea Pig PCA o : absorption at 280nrn • : ninhydrin absorption
1.0
D
A
c
B
0
.5
50
100
150
Etuted volume
200
250
-ml-
Figure 1. Gel filtration of casein hydrolysate on Sephadex G-25 column. The amount of casein hydrolysate applied was 50 mg. Arrows designate the position of markers (A:Blue dextran, B:oxytocin, C:tyrosine, D: tryptophan).
minor peak emerged in the same position as tryptophane. Little was eluted with a volume less than 100 ml which was an effluent volume required for oxytocin with a molecular weight of 1,000.
Antigenicity of the Casein Hydrolysete on Guinea Pig PCA
Sensitization of guinea pigs was with .1 ml of 100-fold diluted anticasein rabbit serum. One to 10 /ag of casein and 30 mg of casein hydrolysate were injected intraveneously into the guinea pigs. The results are in Table 1. All four guinea pigs challenged with 30 mg of the casein hydrolysate gave negative PeA. However, three of four guinea pigs challenged with 1/.tg of casein and all of four guinea pigs given more than 5/ag of casein gave positive PeA.
Anticasein rabbit serum diluted 1:50 was mixed with an equal volume of 1% casein hydrolysate solution and incubated at 25 C for 1 h. Controls also were prepared by mixing a 1:50 dilution of the antiserum with an equal volume of .01% or .001% casein solution. These mixtures were used for passive sensitization of guinea pigs. The P e A reaction was elicited by 5 /ag of casein, and the diameters of the blueings were compared. The results are in Figure 2. The antisera at S-1 and S-2 were neutralized thoroughly by casein solutions. The blueing of the antiserum treated with casein hydrolysate at S-3 was as large as that produced by untreated antiserum at R-3. This demonstrated that the casein hydrolysate had no capacity to neutralize the anticasein antibody in rabbit serum.
Preparation of the Rat Anticasein IgE Serum
Rats were injected subcutaneously with 2 ml of antigen solution containing various doses of casein (10 to 5,000 /ag) mixed with 50 mg of AI(OH)3 as an adjuvant. Blood samples from rats were obtained by heart puncture at intervals of 3 days from day 6 to day 24 after the injection, and the P e A titers were determined by rat long latency homologous PeA. The correlation between the doses of casein and the PeA titers is in Figure 3. The increase of PCA titer in rats was not by injections less than 500 /ag or more than 3,000 /.tg of casein. However, antisera with positive P e A titer were obtained from three of five rats injected with 1,000/ag of casein and all five rats injected with 2,000 /ag of casein. Antisera with the highest PeA titer (1:64) was obtained by injection with 2,000 /.tg of casein. Figure 4 shows changes in
TABLE 1. Antigenicity of casein hydrolysate on guinea pig PeA. Sample challenged to guinea pig
Casein hydrolysate Casein Casein Casein
Journal of Dairy Science Vol. 62, No. 10, 1979
Amount
30 mg 1 ug 5 ~zg 10 #g
Number of guinea pigs on intensity of skin reaction --
+
4 1
3
++
2
+++
++++
ANTIGENICITY OF CASEIN HYDROLYSATE
1573
128
R
S 1
s4 32
R
S2 o
R
~
S3 O0
Figure 2. Inhibitory effect o f casein hydrolysate on guinea pig PCA in casein-anticasein serum system. At R-l, R-2, and R-3, .1 ml of lO0-fold diluted anticasein rabbit serum were injected. Fiftyfold dilutions of the antiserum were mixed with equal volumes of .01% casein, .001% casein, or 1% casein hydrolysate and were injected at S-1, S-2, and S-3 by .1 ml (S-l: antiserum mixed with .01% casein, S-2:that with .001% casein, S-3:that with 1% casein).
PCA t i t e r of t h e s e r u m f r o m day 6 to d a y 24 a f t e r t h e dose of 2 , 0 0 0 / . t g o f casein. T h e P C A titer o f t h e s e r u m increased to a m a x i m u m ( 1 : 6 4 ) at d a y 12, a n d t h e n d e c r e a s e d g r a d u a l l y to d a y 24. Antigenicity of Casein Hydrolysata on Rat Long Latency Homologous PCA
S e n s i t i z a t i o n o f rats were w i t h .1 ml o f t w o f o l d d i l u t e d a n t i s e r u m w i t h a PCA t i t e r o f 1:64. V a r i o u s doses of casein or 20 m g o f t h e casein h y d r o l y s a t e were i n j e c t e d i n t r a v e n e o u s l y into t h e sensitized rats. All t h r e e rats c h a l l e n g e d w i t h t h e casein h y d r o l y s a t e or 1 /ag o f casein gave negative PCA. H o w e v e r , all t h r e e rats challenged w i t h doses o f 5/~g or 1 0 / a g o f casein gave positive PCA. I nhibition Test on Rat Long Latency Homologous PCA
A n t i c a s e i n rat IgE s e r u m (PCA t i t e r 1 : 6 4 ) was m i x e d with a n e q u a l v o l u m e o f 1% casein h y d r o l y s a t e s o l u t i o n a n d i n c u b a t e d at 25 C for 1 h. T h e m i x t u r e was d i l u t e d serially t w o f o l d , a n d .1 ml was i n j e c t e d i n t r a d e r m a l l y i n t o a rat. U n t r e a t e d a n t i s e r a was d i l u t e d similarly w i t h saline a n d i n j e c t e d i n t o t h e s y m m e t r i c a l p o i n t s o f t h e rat. As a c o n t r o l , n e u t r a l i z a t i o n o f t h e
•
Dram
10
mnmm
aOOdb
102
oe
•
aO
5x102 103 Casein
mdba~
dbdSam
2x103 3xlO3 5x103 -)Jg -
Figure 3. PCA titers of anticasein IgE serum of rats with different doses of casein. Closed spots indicate rats injected by specified amount of casein. Experimental details are given in the text.
a n t i c a s e i n IgE s e r u m b y casein was t e s t e d in t h e same m a n n e r . T h e c o n c e n t r a t i o n o f casein solut i o n m i x e d w i t h a n t i c a s e i n IgE s e r u m was .001%. A P C A r e a c t i o n was elicited w i t h 1 m g o f casein. T h e results are in Figures 5 A a n d 5B. In Figure 5A, t h e a n t i s e r u m i n c u b a t e d w i t h t h e casein h y d r o l y s a t e f o r m e d b l u e i n g s as large as t h o s e o f t h e u n t r e a t e d a n t i s e r u m . In Figure 5B t h e a n t i s e r u m i n c u b a t e d w i t h .001% casein did n o t f o r m blueings. T h e a n t i s e r u m was n o t
64 32
:
16
<(.3 8 n
4 2
0
6
12 Days
18
24
Figure 4. PCA titer of anticasein rat lgE serum at different period after injection of 2,000 #g of casein. Journal of Dairy Science Vol. 62, No. 10, 1979
1574
TAKASE ET AL.
TABLE 2. Antigenicity of casein hydrolysate on rat long latency homologous PCA. Sample challenged to rat
Number of rats on intensity of skin reaction
Amount
--
Casein hydrolysate Casein Casein Casein
20 mg 1 ~g 5 ~g 10 #g
n e u t r a l i z e d t h o r o u g h l y b y t h e casein h y d r o l y sate.
DISCUSSION
T h e i m m u n o l o g i c a l activities o f p r o t e i n s a n d p e p t i d e s relate t o t h e i r m o l e c u l a r size. A n t i genicity of proteins and peptides with molecular w e i g h t s o f less t h a n 1 0 , 0 0 0 is w e a k (7). F o r
S
C
+
++
2
1 2
+++
++++
3 3 1
i n s t a n c e , h o g insulin w i t h a m o l e c u l a r w e i g h t o f 6 , 0 0 0 is usually i n j e c t e d i n t o h u m a n s w i t h o u t i m m u n o l o g i c a l r e s p o n s e (7). Similarly, vasopressin a n d o x y t o c i n , o c t a p e p t i d e s w i t h m o l e c u lar w e i g h t of a b o u t 1 , 0 0 0 , are n o t a n t i g e n i c (7). T h e casein h y d r o l y s a t e was n o t e l u t e d w i t h less t h a n 100 ml in a n e f f l u e n t v o l u m e of oxytocin. T h e r e f o r e , h y d r o l y s a t e did n o t contain peptides with molecular weights greater
R
C
Figure 5. Inhibitory effect of casein hydrolysate on rat long latency homologous PeA in casein-anticasein serum system. A:Anticasein lgE serum was mixed with equal volume of 1% casein hydrolysate. Twofold serial dilutions of the mixture (S) and antiserum control (C) were injected. Skin sites at the top row received an undiluted mixture and a 1:2 dilution of control antiserum. B:lnstead of 1% casein hydrolysate in the case of A, .O01% casein was used and the other respects of the test were same as A (R:the mixture of antiserum and .001% casein, C:antiserum control). Journal of Dairy Science Vol. 62, No. 10, 1979
ANTIGENICITY OF CASEIN HYDRO LYSATE than 1,000. However, the antigenic properties were not elucidated by molecular size alone. F r o m this point o f view, immunological analyses were done by PCA with anticasein rabbit IgG antibody and anticasein rat IgE antibody. Guinea pig PCA is a sensitive reaction elicited by the challenge of only 1 #g of casein. The casein hydrolysates did not produce any positive PCA by the challenge of 30 mg, which is 30,000 times greater than casein. However, it was indicated that immunoreactive fragments with one antigenic determinant, even if they existed, were not detected in the normal PCA (8). To detect such immunoreactive fragments, inhibition tests have been carried out (9, 10). Cebra separated an immunoreactive tetrapeptide from the hydrolysate of silk fibroin by an inhibition test of precipitin (9). Tokita et al. also detected immunoreactive peptides in trypsin h y d r o l y s a t e s of cz-casein and /3-1actoglobulin (10). Immunoreactive fragments to anticasein antibodies might be present among various peptides in casein hydrolysates. In our studies, an inhibitory effect of the casein hydrolysate was not detectable by inhibition tests on guinea pig PCA. Prior to rat long latency homologous PCA with anticasein rat IgE antibody, the condition for the preparation of the antibodies was studied. Bloch et al. (11) prepared anti-egg albumin IgE serum and anti-hemocyanin IgE serum with a dose of 10/~g of antigen. In our work, anticasein rat IgE serum with the highest PCA titer (1:64) was obtained by a dose of 2,000 #g of casein. The IgE antibody was not produced by doses of 10 to 500/ag a n d 3,000 to 5,000 #g of casein. In rat long latency homologous PCA~ the reaction was positive with the challenge of 5 #g of casein, but unresponsive with 1 /~g. The reaction in this system was not as sensitive as guinea pig PCA. Recently the role of IgE antibodies in food allergies, including milk allergies, has been suggested as being extremely important. It is also necessary to know the antigenicity of casein hydrolysates against homologous IgE antibody systems. In the rat long latency homologous PCA and its inhibition test, casein hydrolysates did not have any reactivity with "IgE antibodies raised
1575
against whole casein. Spies et al. (12) found new antigens having determinants unrelated to/3-1actoglobulin after pepsin hydrolysis of the protein. Berrens et al. (13) reported increased skin reactivity of a browning reaction condensation product of /3-1actoglobulin and lactose. We have not studied the appearance of such a new antigen after enzymatic hydrolysis of casein. However, it has been demonstrated clinically that milk allergic infants with intractable diarrhea effectively tolerate an infant formula based on the casein hydrolysate which we prepared (14, 15). A flocculent precipitate was formed when casein was hydrolyzed with proteolytic enzymes. The precipitate contained about 9% of the starting casein nitrogen. The precipitate might consist of slightly soluble amino acids such as tyrosine along with insoluble peptides. Though characteristics of the insoluble peptides were not studied, the precipitate was removed by centrifugation for fear that it might carry immunoactive activities of casein or contain new antigens. The precipitate was not examined, but an infant formula based on the casein hydrolysate as a source of amino acids was given infants allergic to milk for a long time without producing new allergies. ACKNOWLEDGMENT
The authors are indebted to H. Inou, president of Morinaga Milk Industry Co., Ltd., for his interest and encouragement in the presentation of this manuscript. REFERENCES
1 Takahashi, F., and F. Tokita. 1970. Recent studies on antigenicities of milk proteins. Japan J. Dairy Sci. 24:A-1. 2 Hoffman, D. R., and Z. H. Haddad. 1974. Diagnosis of lgE-mediated reactions to food antigens by radioimmunoassay. J. Allergy Clin. Immunol. 54:165. 3 Hanson, L. A., and B. G. Johansson. 1970. Page 45 in Milk proteins, chemistry and molecular biology. Vol. 1. H. A. McKenzie, ed. Academic Press, New York and London. 4 Nagasawa, T., I. Kiyosawa, and K. Kuwahara. 1972. Amounts of lactoferrin in human colostrum and milk. J. Dairy Sci. 55:1651. 50gasa, K., 1. Kiyosawa, K. Kawase, Y. Fukuwatari, M. Takase, H. Yamamoto, and T. Tatematsu. 1977. Japanese Patent Gazette (Kokai), No. 79083. 6 Yemm, E. W., and E. C. Cocking. 1955. The determination of amino acids with ninhidrin. Analyst 80: 209. Journal of Dairy Science Vol. 62, No. 10, 1979
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T A K A S E ET AL.
7 Haurowitz, F. 1968. I m m u n o c h e m i s t r y and the Biosynthesis of antibodies. J o h n Wiley and Sons, Inc., New York. 8 Brocklehurst, W. E. 1973. Passive c u t a n e o u s anaphylaxis. Handbook of experimental i m m u nology. B. M. Weir, ed. Beckwell Scientific Publications Inc., Osney Mead, Oxford, 21-1. 9 Cebra, J. J. 1961. Studies on the combining sites of the protein antigen silk fibroin, III. Inhibition o f the silk fibroin-antifibroin system by peptides derived from the antigen. J. I m m u n o l . 86:205. 10 Tokita, F., and F. Takahashi. 1974. Isolation and characterization of an antigenetically active peptide from casein. Japan J. Dairy Sci. 23:A-31. 11 Bloch, K. J., J. L. Ohman, Jr., J. Waltin, and R. W. Cygan. 1973. Potentiated reagin response: Initiation
Journal of Dairy Science Vol. 62, No. 10, 1979
12
13
14 15
with m i n u t e doses of antigen and alum followed by infection with Nippostrongylus brasiliensis. J. I m m u n o l . 110:197. Spies, J. R., M. A. Stevan, W. J. Stein, and E. J. Coulson. 1970. The chemistry o f allergens. XX. New antigens generated by pepsin hydrolysis of bovine milk proteins. J. Allergy 45:208. Berrens, L., and E. Bleumink. 1966. Synthetic approaches to the biological activities o f #-lactoglobulin in h u m a n allergy to cow's milk. Nature, London, 212: 54-1. Asako, K., and Y. Yamashiro. 1977. A case of intractable diarrhea. Japan J. Ped. 30:653. Sekine, K., K. Maeda, T. Nakayama, and T. Nemoto. 1978. Successful t r e a t m e n t s o f two cases of intractable diarrhea. Ped. Jap. 19: 321.
Central Research Laboratory Morinaga Milk Industry Co., Ltd. Tokyo, Japan
with clinical symptoms o f patients with milk allergy. Allergy to cow's milk protein has become more clearly defined in specific antibodies to the antigens in patients' sera (2). Developments of immunological techniques based on antigen-antibody reactions have contributed to detection of antigenicity (3). We prepared a soluble fraction from enzymatically hydrolyzed bovine casein to use as a source of amino acids as a substitute for cow's milk. This paper reports our efforts to use casein hydrolysates as substitutes for cow's milk in detecting antigenicity.
ABSTRACT
An enzymatic hydrolysate of bovine casein was studied for molecular size and antigenic properties. As determined by Sephadex G-25 gel filtration, polypeptide fractions having molecular weights greater than 1,000 were not detected in the soluble fraction of the casein hydrolysate. Antigenicity o f the fraction was not detected by heterologous passive cutaneous anaphylaxis in guinea pig-rabbit antiserum system mediated by immunoglobulin G types of antibody (guinea pig passive cutaneous anaphylaxis) and its inhibition test. Furthermore, it was not detected by rat long latency homologous mediated by immunoglobulin E antibody and its inhibition test. To prepare anticasein immunoglobulin E serum for rat long latency homologous passive cutaneous anaphylaxis, immunization of rats was by single subcutaneous injection of casein with aluminum hydroxide. Antiserum with highest passive cutaneous anaphylaxis titer (1:64) against bovine casein was obtained by a dose of 2,000/ag of the protein at day 12.
MATERIALS AND METHODS Casein
Casein was prepared from fresh skim milk (bovine) by four isoelectric precipitations as described by Nagasawa et al. (4). P roteolytic Enzymes
Proteolytic enzymes were pancreatin (Amano Pharmaceutical Co., Nagoya, Japan) and proteinases from Aspergillus oryzae (Amano Pharmaceutical Co., Nagoya, Japan) and Lactobacillus belveticus prepared by the method of Ogasa et al. (5).
INTRODUCTION
It is extremely important to destroy the antigenicity o f milk proteins for dietetic treatment of infants with milk allergy fed cow's milk formulas. Enzymatic digestion of milk protein is not only effective for destroying antigenicity, but it also is less liable to alter the nutritive value (1). The degree of antigenicity and the amount of hydrolysis of milk protein need to correlate
Received January 22, 1979. 1Department of Pediatrics, Gunma University, Gunma, Japan. 1979 J Dairy Sci 62:1570-1576
Experimental Animals
Sprague Dawley male rats, Hartley male guinea pigs, and male albino rabbits weighing about 250 g, 300 g, and 2.5 kg were used. Enzymatic Hydrolysis of Casein
A 10% casein solution adjusted to pH 7.0 with .5 N NaOH was pasteurized in a waterbath at 85 C for 15 rain. The solution was cooled to 50 C, and three enzymes were added at 1 part pancreatin, 4 parts proteinase of Aspergillus oryzae, and 2 parts proteinase of Lactobacillus helveticus to 150 parts casein. The solution was incubated at 50 C for 24 h
1570
ANTIGENICITY OF CASEIN HYDROLYSATE then heated at 85 C for 15 min to inactivate the proteolytic enzymes. The solution was cooled : to 5 C and centrifuged at 12,000 x g for 20 rain to remove the precipitate. About 91% of the total casein nitrogen remained in the supernatant. The supernatant containing the casein bydrolysate was freeze dried and stored until needed. The total nitrogen and formol nitrogen in the casein hydrolysate as determined by the Kjeldahl method and formol titration were 15.7g % and 6.3g %. Gel Filtration
Gel filtration was by 1.6 x 90-cm columns of Sephadex G-25 equilibrated with .5 N acetic acid. Blue dextran (Pharmacia, Uppsala, Sweden, tool wt 200,000), oxytocin (Sigma Chemical Co., mol wt 1,000), tyrosin (Sigma Chemical Co., mol wt 181) and tryptophan (Sigma Chemical Co., mol wt 204) were used as indicators of molecular size. Absorption of the eluted solution at 280 nm was determined in a Hitachi UV-VIS Spectrophotometer. The ninhydrin reaction was according to the method of Yemm et al. (6). Anticasein Rabbit Serum
Anticasein rabbit serum was prepared from the blood of a rabbit that had received four muscular injections of 10 mg of casein emulsified with Freund's complete adjuvant over 1 mo. Guinea Pig Passive Cutaneous Anaphylaxis (PCA)
Rabbit antiserum was diluted with saline so that a suitable amount of antibody was contained in .1 ml of the diluted solution. One-tenth milliliter of the diluted rabbit antiserum was injected intradermally into the shaved back of a normal guinea pig. After 4 h, the guinea pig was challenged by intraveneous injection of an antigen solution containing 5 mg of Evans-blue. The antigen solution was prepared by dissolving a specified amount of casein or casein hydrolysate with saline. The guinea pig was decapitated after 30 rain and the skin was reflected. The extent of extravasation of the dye was determined on the back of the skin. The PeA reactions were graded by the diameters of blueings as follows: less than 5 ram, - ; 5 to 10 ram, +; 10 to 15 ram, ++; 15 to 20 ram, +++; more than 20 mm, ++++. The PCA reactions
15 71
with blueings of more than 5 mm in a diameter were defined as positive. Rat Long Latency Homologous PCA
One-tenth milliliter of suitably diluted antiserum was injected intradermally into the shaved back of a normal rat. After 48 h, the rat was challenged with an intraveneous injection of 1 ml of antigen solution containing 5 mg of Evans-blue. The PeA reaction was graded as described above. The PCA titer of the antiserum was determined by the PCA reaction as follows. Antiserum was serially diluted two fold and injected into the rat for passive sensitization. The PeA titer was expressed as the reciprocal of the maximum dilution of the serum that produced a positive PCA. In rat long latency homologous PeA (48 h), only IgE antibody in the antiserum reacts against the antigen. I nhibition Test
Inhibition tests were in guinea pig P e A and rat long latency homologous PCA. The test utilized the PCA reaction as an indication of the neutralization of sensitizing antibody by prior contact with an antigen. Anticasein serum was mixed with the material to be tested and incubated at 25 C for 1 h. The mixture was injected intradermally into an animal. The untreated antiserum was injected into the adjacent site on the same animal. The following procedures of the test were the same as the usual PeA test with a challenge with sufficient amounts of casein after 4 h in the guinea pig PeA and after 48 h in rat long latency homologous PeA. The neutralized antiserum failed to give a reaction or gave response smaller than that produced by untreated antiserum. RESULTS Gel Filtration
The elution pattern of the casein hydrolysate is in Figure 1. Two peaks appeared in the pattern measured spectrophotometrically at 280 nm. One peak was eluted with a volume equal to tyrosine, and the other one appeared in an effluent volume equal to tryptophane. In the elution pattern identified by the ninhidrin reaction, a major peak with a shoulder eluted between 100 and 150 ml. The shoulder emerged in an effluent volume equal to tyrosine. A Journal of Dairy Science Vol. 62, No. 10, 1979
1572
TAKASE ET AL. Inhibition Test on Guinea Pig PCA o : absorption at 280nrn • : ninhydrin absorption
1.0
D
A
c
B
0
.5
50
100
150
Etuted volume
200
250
-ml-
Figure 1. Gel filtration of casein hydrolysate on Sephadex G-25 column. The amount of casein hydrolysate applied was 50 mg. Arrows designate the position of markers (A:Blue dextran, B:oxytocin, C:tyrosine, D: tryptophan).
minor peak emerged in the same position as tryptophane. Little was eluted with a volume less than 100 ml which was an effluent volume required for oxytocin with a molecular weight of 1,000.
Antigenicity of the Casein Hydrolysete on Guinea Pig PCA
Sensitization of guinea pigs was with .1 ml of 100-fold diluted anticasein rabbit serum. One to 10 /ag of casein and 30 mg of casein hydrolysate were injected intraveneously into the guinea pigs. The results are in Table 1. All four guinea pigs challenged with 30 mg of the casein hydrolysate gave negative PeA. However, three of four guinea pigs challenged with 1/.tg of casein and all of four guinea pigs given more than 5/ag of casein gave positive PeA.
Anticasein rabbit serum diluted 1:50 was mixed with an equal volume of 1% casein hydrolysate solution and incubated at 25 C for 1 h. Controls also were prepared by mixing a 1:50 dilution of the antiserum with an equal volume of .01% or .001% casein solution. These mixtures were used for passive sensitization of guinea pigs. The P e A reaction was elicited by 5 /ag of casein, and the diameters of the blueings were compared. The results are in Figure 2. The antisera at S-1 and S-2 were neutralized thoroughly by casein solutions. The blueing of the antiserum treated with casein hydrolysate at S-3 was as large as that produced by untreated antiserum at R-3. This demonstrated that the casein hydrolysate had no capacity to neutralize the anticasein antibody in rabbit serum.
Preparation of the Rat Anticasein IgE Serum
Rats were injected subcutaneously with 2 ml of antigen solution containing various doses of casein (10 to 5,000 /ag) mixed with 50 mg of AI(OH)3 as an adjuvant. Blood samples from rats were obtained by heart puncture at intervals of 3 days from day 6 to day 24 after the injection, and the P e A titers were determined by rat long latency homologous PeA. The correlation between the doses of casein and the PeA titers is in Figure 3. The increase of PCA titer in rats was not by injections less than 500 /ag or more than 3,000 /.tg of casein. However, antisera with positive P e A titer were obtained from three of five rats injected with 1,000/ag of casein and all five rats injected with 2,000 /ag of casein. Antisera with the highest PeA titer (1:64) was obtained by injection with 2,000 /.tg of casein. Figure 4 shows changes in
TABLE 1. Antigenicity of casein hydrolysate on guinea pig PeA. Sample challenged to guinea pig
Casein hydrolysate Casein Casein Casein
Journal of Dairy Science Vol. 62, No. 10, 1979
Amount
30 mg 1 ug 5 ~zg 10 #g
Number of guinea pigs on intensity of skin reaction --
+
4 1
3
++
2
+++
++++
ANTIGENICITY OF CASEIN HYDROLYSATE
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128
R
S 1
s4 32
R
S2 o
R
~
S3 O0
Figure 2. Inhibitory effect o f casein hydrolysate on guinea pig PCA in casein-anticasein serum system. At R-l, R-2, and R-3, .1 ml of lO0-fold diluted anticasein rabbit serum were injected. Fiftyfold dilutions of the antiserum were mixed with equal volumes of .01% casein, .001% casein, or 1% casein hydrolysate and were injected at S-1, S-2, and S-3 by .1 ml (S-l: antiserum mixed with .01% casein, S-2:that with .001% casein, S-3:that with 1% casein).
PCA t i t e r of t h e s e r u m f r o m day 6 to d a y 24 a f t e r t h e dose of 2 , 0 0 0 / . t g o f casein. T h e P C A titer o f t h e s e r u m increased to a m a x i m u m ( 1 : 6 4 ) at d a y 12, a n d t h e n d e c r e a s e d g r a d u a l l y to d a y 24. Antigenicity of Casein Hydrolysata on Rat Long Latency Homologous PCA
S e n s i t i z a t i o n o f rats were w i t h .1 ml o f t w o f o l d d i l u t e d a n t i s e r u m w i t h a PCA t i t e r o f 1:64. V a r i o u s doses of casein or 20 m g o f t h e casein h y d r o l y s a t e were i n j e c t e d i n t r a v e n e o u s l y into t h e sensitized rats. All t h r e e rats c h a l l e n g e d w i t h t h e casein h y d r o l y s a t e or 1 /ag o f casein gave negative PCA. H o w e v e r , all t h r e e rats challenged w i t h doses o f 5/~g or 1 0 / a g o f casein gave positive PCA. I nhibition Test on Rat Long Latency Homologous PCA
A n t i c a s e i n rat IgE s e r u m (PCA t i t e r 1 : 6 4 ) was m i x e d with a n e q u a l v o l u m e o f 1% casein h y d r o l y s a t e s o l u t i o n a n d i n c u b a t e d at 25 C for 1 h. T h e m i x t u r e was d i l u t e d serially t w o f o l d , a n d .1 ml was i n j e c t e d i n t r a d e r m a l l y i n t o a rat. U n t r e a t e d a n t i s e r a was d i l u t e d similarly w i t h saline a n d i n j e c t e d i n t o t h e s y m m e t r i c a l p o i n t s o f t h e rat. As a c o n t r o l , n e u t r a l i z a t i o n o f t h e
•
Dram
10
mnmm
aOOdb
102
oe
•
aO
5x102 103 Casein
mdba~
dbdSam
2x103 3xlO3 5x103 -)Jg -
Figure 3. PCA titers of anticasein IgE serum of rats with different doses of casein. Closed spots indicate rats injected by specified amount of casein. Experimental details are given in the text.
a n t i c a s e i n IgE s e r u m b y casein was t e s t e d in t h e same m a n n e r . T h e c o n c e n t r a t i o n o f casein solut i o n m i x e d w i t h a n t i c a s e i n IgE s e r u m was .001%. A P C A r e a c t i o n was elicited w i t h 1 m g o f casein. T h e results are in Figures 5 A a n d 5B. In Figure 5A, t h e a n t i s e r u m i n c u b a t e d w i t h t h e casein h y d r o l y s a t e f o r m e d b l u e i n g s as large as t h o s e o f t h e u n t r e a t e d a n t i s e r u m . In Figure 5B t h e a n t i s e r u m i n c u b a t e d w i t h .001% casein did n o t f o r m blueings. T h e a n t i s e r u m was n o t
64 32
:
16
<(.3 8 n
4 2
0
6
12 Days
18
24
Figure 4. PCA titer of anticasein rat lgE serum at different period after injection of 2,000 #g of casein. Journal of Dairy Science Vol. 62, No. 10, 1979
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TAKASE ET AL.
TABLE 2. Antigenicity of casein hydrolysate on rat long latency homologous PCA. Sample challenged to rat
Number of rats on intensity of skin reaction
Amount
--
Casein hydrolysate Casein Casein Casein
20 mg 1 ~g 5 ~g 10 #g
n e u t r a l i z e d t h o r o u g h l y b y t h e casein h y d r o l y sate.
DISCUSSION
T h e i m m u n o l o g i c a l activities o f p r o t e i n s a n d p e p t i d e s relate t o t h e i r m o l e c u l a r size. A n t i genicity of proteins and peptides with molecular w e i g h t s o f less t h a n 1 0 , 0 0 0 is w e a k (7). F o r
S
C
+
++
2
1 2
+++
++++
3 3 1
i n s t a n c e , h o g insulin w i t h a m o l e c u l a r w e i g h t o f 6 , 0 0 0 is usually i n j e c t e d i n t o h u m a n s w i t h o u t i m m u n o l o g i c a l r e s p o n s e (7). Similarly, vasopressin a n d o x y t o c i n , o c t a p e p t i d e s w i t h m o l e c u lar w e i g h t of a b o u t 1 , 0 0 0 , are n o t a n t i g e n i c (7). T h e casein h y d r o l y s a t e was n o t e l u t e d w i t h less t h a n 100 ml in a n e f f l u e n t v o l u m e of oxytocin. T h e r e f o r e , h y d r o l y s a t e did n o t contain peptides with molecular weights greater
R
C
Figure 5. Inhibitory effect of casein hydrolysate on rat long latency homologous PeA in casein-anticasein serum system. A:Anticasein lgE serum was mixed with equal volume of 1% casein hydrolysate. Twofold serial dilutions of the mixture (S) and antiserum control (C) were injected. Skin sites at the top row received an undiluted mixture and a 1:2 dilution of control antiserum. B:lnstead of 1% casein hydrolysate in the case of A, .O01% casein was used and the other respects of the test were same as A (R:the mixture of antiserum and .001% casein, C:antiserum control). Journal of Dairy Science Vol. 62, No. 10, 1979
ANTIGENICITY OF CASEIN HYDRO LYSATE than 1,000. However, the antigenic properties were not elucidated by molecular size alone. F r o m this point o f view, immunological analyses were done by PCA with anticasein rabbit IgG antibody and anticasein rat IgE antibody. Guinea pig PCA is a sensitive reaction elicited by the challenge of only 1 #g of casein. The casein hydrolysates did not produce any positive PCA by the challenge of 30 mg, which is 30,000 times greater than casein. However, it was indicated that immunoreactive fragments with one antigenic determinant, even if they existed, were not detected in the normal PCA (8). To detect such immunoreactive fragments, inhibition tests have been carried out (9, 10). Cebra separated an immunoreactive tetrapeptide from the hydrolysate of silk fibroin by an inhibition test of precipitin (9). Tokita et al. also detected immunoreactive peptides in trypsin h y d r o l y s a t e s of cz-casein and /3-1actoglobulin (10). Immunoreactive fragments to anticasein antibodies might be present among various peptides in casein hydrolysates. In our studies, an inhibitory effect of the casein hydrolysate was not detectable by inhibition tests on guinea pig PCA. Prior to rat long latency homologous PCA with anticasein rat IgE antibody, the condition for the preparation of the antibodies was studied. Bloch et al. (11) prepared anti-egg albumin IgE serum and anti-hemocyanin IgE serum with a dose of 10/~g of antigen. In our work, anticasein rat IgE serum with the highest PCA titer (1:64) was obtained by a dose of 2,000 #g of casein. The IgE antibody was not produced by doses of 10 to 500/ag a n d 3,000 to 5,000 #g of casein. In rat long latency homologous PCA~ the reaction was positive with the challenge of 5 #g of casein, but unresponsive with 1 /~g. The reaction in this system was not as sensitive as guinea pig PCA. Recently the role of IgE antibodies in food allergies, including milk allergies, has been suggested as being extremely important. It is also necessary to know the antigenicity of casein hydrolysates against homologous IgE antibody systems. In the rat long latency homologous PCA and its inhibition test, casein hydrolysates did not have any reactivity with "IgE antibodies raised
1575
against whole casein. Spies et al. (12) found new antigens having determinants unrelated to/3-1actoglobulin after pepsin hydrolysis of the protein. Berrens et al. (13) reported increased skin reactivity of a browning reaction condensation product of /3-1actoglobulin and lactose. We have not studied the appearance of such a new antigen after enzymatic hydrolysis of casein. However, it has been demonstrated clinically that milk allergic infants with intractable diarrhea effectively tolerate an infant formula based on the casein hydrolysate which we prepared (14, 15). A flocculent precipitate was formed when casein was hydrolyzed with proteolytic enzymes. The precipitate contained about 9% of the starting casein nitrogen. The precipitate might consist of slightly soluble amino acids such as tyrosine along with insoluble peptides. Though characteristics of the insoluble peptides were not studied, the precipitate was removed by centrifugation for fear that it might carry immunoactive activities of casein or contain new antigens. The precipitate was not examined, but an infant formula based on the casein hydrolysate as a source of amino acids was given infants allergic to milk for a long time without producing new allergies. ACKNOWLEDGMENT
The authors are indebted to H. Inou, president of Morinaga Milk Industry Co., Ltd., for his interest and encouragement in the presentation of this manuscript. REFERENCES
1 Takahashi, F., and F. Tokita. 1970. Recent studies on antigenicities of milk proteins. Japan J. Dairy Sci. 24:A-1. 2 Hoffman, D. R., and Z. H. Haddad. 1974. Diagnosis of lgE-mediated reactions to food antigens by radioimmunoassay. J. Allergy Clin. Immunol. 54:165. 3 Hanson, L. A., and B. G. Johansson. 1970. Page 45 in Milk proteins, chemistry and molecular biology. Vol. 1. H. A. McKenzie, ed. Academic Press, New York and London. 4 Nagasawa, T., I. Kiyosawa, and K. Kuwahara. 1972. Amounts of lactoferrin in human colostrum and milk. J. Dairy Sci. 55:1651. 50gasa, K., 1. Kiyosawa, K. Kawase, Y. Fukuwatari, M. Takase, H. Yamamoto, and T. Tatematsu. 1977. Japanese Patent Gazette (Kokai), No. 79083. 6 Yemm, E. W., and E. C. Cocking. 1955. The determination of amino acids with ninhidrin. Analyst 80: 209. Journal of Dairy Science Vol. 62, No. 10, 1979
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7 Haurowitz, F. 1968. I m m u n o c h e m i s t r y and the Biosynthesis of antibodies. J o h n Wiley and Sons, Inc., New York. 8 Brocklehurst, W. E. 1973. Passive c u t a n e o u s anaphylaxis. Handbook of experimental i m m u nology. B. M. Weir, ed. Beckwell Scientific Publications Inc., Osney Mead, Oxford, 21-1. 9 Cebra, J. J. 1961. Studies on the combining sites of the protein antigen silk fibroin, III. Inhibition o f the silk fibroin-antifibroin system by peptides derived from the antigen. J. I m m u n o l . 86:205. 10 Tokita, F., and F. Takahashi. 1974. Isolation and characterization of an antigenetically active peptide from casein. Japan J. Dairy Sci. 23:A-31. 11 Bloch, K. J., J. L. Ohman, Jr., J. Waltin, and R. W. Cygan. 1973. Potentiated reagin response: Initiation
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12
13
14 15
with m i n u t e doses of antigen and alum followed by infection with Nippostrongylus brasiliensis. J. I m m u n o l . 110:197. Spies, J. R., M. A. Stevan, W. J. Stein, and E. J. Coulson. 1970. The chemistry o f allergens. XX. New antigens generated by pepsin hydrolysis of bovine milk proteins. J. Allergy 45:208. Berrens, L., and E. Bleumink. 1966. Synthetic approaches to the biological activities o f #-lactoglobulin in h u m a n allergy to cow's milk. Nature, London, 212: 54-1. Asako, K., and Y. Yamashiro. 1977. A case of intractable diarrhea. Japan J. Ped. 30:653. Sekine, K., K. Maeda, T. Nakayama, and T. Nemoto. 1978. Successful t r e a t m e n t s o f two cases of intractable diarrhea. Ped. Jap. 19: 321.