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Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum
Livestock Production Science 53 Ž1998. 183–190
Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum A. Marcin
a,)
, S. Kisidayova ˇ ´ b, V. Kmet´
b
a
b
Institute of Experimental Veterinary Medicine, HlinkoÕa 1 r A, 040 01 Kosice, ˇ SloÕak Republic ˇ ´ Institute of Animal Physiology-SloÕak Academy of Sciences, SoltesoÕej 3, 040 01 Kosice, ˇ SloÕak Republic Accepted 1 September 1997
Abstract The study of the wheat protein Žalbumin and globulin fraction. proteolysis was performed in the culture medium of the rumen protozoon Entodinium caudatum under anaerobic conditions in vitro. The rate of the wheat protein degradation was measured by the immunological sandwich technique dot-blot. The centrifugation of the protozoal culture media was performed at 500 g for 20 min. The resulting concentration of the protozoal cells was 20,000–24,000 per ml in the experimental culture medium. The samples were taken from the culture medium at 0, 2, 4, 6 and 24 h during incubation. After centrifugation at 500 g for 20 min, the supernatants were used for the determination of: Ž1. the total protein concentration, Ž2. the nonspecific proteolytic activity Žazoalbumin was used as a substrate. and Ž3. the wheat protein concentration in the culture medium by the immunological sandwich technique dot-blot. Subsequently, the results were used for the calculation of the degradation rate of the wheat protein. The rabbit polyclonal antiserum against the wheat protein was used as the first antibody in this method. The quantitation of this test was based on the spectrophotometric determination of the violet stain quantity on the nitrocellulose strip surfaces. The average rate of the wheat protein degradation in the culture medium E. caudatum was 0.014 mg mly1 hy1, whereas the average rate of the azoalbumin degradation was 0.003 mg mly1 hy1 in supernatant. Azoalbumin was more resistant to degradation in the supernatant of the culture medium in comparison with the wheat protein in the culture medium. The assay sensitivity Žs the limit of detection. of the simple immunological assay dot-blot was 104 ng mly1 Ži.e., 0.52 ng wheat protein applied to nitrocellulose foil. at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% Žcoefficient of variation. were 9.75% and 14%, respectively. The screening of the samples by dot-blot procedure proved to be an efficient way for the quantitation of the proteolytic activity in the rumen protozoa monoculture cultivated in vitro. q 1998 Elsevier Science B.V. Keywords: Rumen; Proteolysis; Protozoa; Entodinium; Dot-blot; Wheat protein
1. Introduction Some part of the dietary proteins entering the rumen are degraded by rumen microorganisms to peptide and amino acids. The first step of this degradation is done by proteases of bacteria. )
Corresponding author.
Ruminal protozoa may influence intestinal protein flow at a number of different levels. Protozoa can alter quantitatively ruminal degradation of dietary proteins and ruminal bacterial growth, consequently modifying the supply of both protein of feed and bacterial origin. However, the concentration of amino acids and ammonia are higher in faunated than in defaunated sheep ŽItabashi et al., 1990.. Rumen bac-
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A. Marcin et al.r LiÕestock Production Science 53 (1998) 183–190
teria seem to be primarily responsible for the degradation of plant protein. Rumen anaerobic fungi also attack plant proteins but they are reported to have only weak activity. Rumen ciliate protozoa can engulf rumen bacteria and insoluble proteins ŽOnodera and Kandatsu, 1970.. Entodiniomorphid protozoa are particularly efficient in taking up particulate matter suspended in ruminal fluid. Anatomically they possess to trap particles that are subsequently driven into a vestibulum and then into cytosomes ŽJouany, 1996.. On the other hand, Holotrichs protozoa have considerable capacity for taking up soluble compounds, they are less efficient than entodiniomorphs in the ingestion of insoluble plant particles ŽJouany, 1996.. Considering the differences in feeding strategies between protozoa and other ruminal microbes, it can be concluded that determining enzyme activities in autolyzed or sonicated cells is not appropriate for quantifying the contribution of ciliates to protein degradation in the in vivo rumen ŽJouany, 1996.. Thus, it may be that protozoal proteases have important roles in proteolysis within the rumen ecosystem. Results of biochemical, cultural and microscopic studies indicate that the contribution of protozoa depends on the complex interactions between protozoa, bacteria and dietary characteristics ŽJouany, 1989.. The objective was to study the wheat protein Žisolated from seeds. proteolysis in the monoculture of the rumen protozoon Entodinium caudatum during 24 h of incubation in vitro.
2. Materials and methods
by fresh medium. Protozoa were fed once a day using 0.75 mg wholemeal flour four times a week with a 300 m g dried grass and three times a week with 0.75 mg rice starch with 300 m g dried grass. The average concentration of the protozoal cells E. caudatum in the culture medium was 10,000–12,000 per ml. The protozoal number of the experimental culture medium was estimated by blowing 0.2 ml of the culture from 1 ml graduated pipette with a large hole at the tip into 2 ml of 0.02 mol ly1 iodine and counted microscopically all the protozoa in 0.1 ml of the mixture ŽColeman, 1978.. 2.2. Isolation of wheat protein from wheat flour The wheat protein was partly purified from commercially available wheat flour. The flour was defatted with acetone, dried and then homogenized in a mortar with Tris–NaCl buffer Ž0.02 mol ly1 Tris, 0.5 mol ly1 NaCl.. The isolation was repeated twice according to method of Spencer et al. Ž1988.. The albumin and globulin fractions were isolated from the wheat flour. The homogenate was centrifuged at 1000 g for 20 min and the supernatant fraction containing the extracted proteins was collected. The isolated proteins before the heat treatment consisted of the polypeptides with the molecular weights of 61, 36, 21 and 8 kD as it was confirmed by sodiumdodecylsulphate polyacrylamide gel electrophoresis ŽSDS-PAGE. ŽLaemmli, 1970.. Subsequently, the wheat protein was heat treated in the water bath Ž1008C for 5 min. and freeze dried. The heat treatment was performed to lower the solubility of the incubated wheat protein in the protozoa culture medium.
2.1. Protozoal culture procedures The monocultured E. caudatum Žfamily Ophryoscolecidae. has been cultivated in vitro in the laboratory of the Institute of Animal Physiology Slovak Academy of Sciences ŽKosice, Slovak Reˇ public. for 5 yr using routine techniques described by Coleman Ž1964, 1978.. Briefly the cultures were maintained in the glass tubes Žvolume of cultivation medium 10 ml. at 398C under 100% carbon dioxide atmosphere. The protozoa were subcultured on caudatum-type medium ŽColeman, 1964, 1978.. Twice a week one half of the culture medium was replaced
2.3. Preparation of polyclonal antibody against wheat protein The isolated protein after the heat treatment in the water bath Ž1008C for 5 min. was freeze-dried and used for the immunization of two rabbits ŽNew Zealand white. according to usual immunization scheme ŽDunbar and Schwoebel, 1990.. The bleeding of the rabbits and processing of the serum was performed according to method of Dunbar and Schwoebel Ž1990.. The achieved titer of antibody in the wheat protein antiserum was 1:3000.
A. Marcin et al.r LiÕestock Production Science 53 (1998) 183–190
2.4. Incubation of wheat protein in the protozoal monoculture E. caudatum The anaerobic centrifugation of the protozoal culture medium E. caudatum was performed at the temperature 208C for 20 min at 500 g. Two thirds of the supernatant were discarded and the rest of the culture medium containing the protozoal cells Ž20,000–24,000 per ml. was incubated at the temperature 398C with the freeze-dried wheat protein in the cultivation vessel under CO 2 atmosphere for 24 h. The resulting concentration of the added wheat protein was 1.5 mg mly1 in the experimental culture medium. Two control culture media were used in the experiment, namely: Ž1. the culture medium with the protozoal cells Ž20,000–24,000 per ml. without wheat protein and Ž2. the protozoa-free supernatant with the wheat protein addition. The samples Ž1 ml. were taken from experimental and two control culture media at the intervals 0, 2, 4, 6 and 24 h during incubation. Samples were centrifugated at 500 g for 20 min. Subsequently, the supernatants were used for: Ža. the protein quantification by Bradford method ŽBradford, 1976., Žb. the determination of the nonspecific proteolytic activity Žazoalbumin was used as a substrate. ŽBroderick, 1987.. The mentioned azoprotein Ždiazotized bovine serum albumin-BSA. was degraded in the supernatant at the temperature 398C for 2 h, c. the determination of the wheat protein
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concentration by the modified immunological sandwich method dot-blot ŽTimmons and Dunbar, 1990.. Subsequently, the results obtained by the dot-blot analysis were used for the calculation of the degradation rate of the wheat protein in the culture medium of the rumen protozoa E. caudatum. The wheat protein degradation rate was calculated according to measured differences between the wheat protein concentrations in the culture medium at the beginning and at the end of the certain time interval Ž2 h and 18 h, respectively.. Consequently, the number of protozoal cells was determined in the experimental culture medium. The slight increase Žabout 32%. was observed at the end of incubations in comparison with the beginning. The samples, which were taken at the times above indicated, were applied to a dry nitrocellulose foil ŽFarby Laky n.p., Czech Republic, Praha. in triplicate each drop of 5 m l Žsee the legend of Fig. 1.. Two blanks containing Tris–NaCl buffer and BSA were also analyzed to determine the nonspecific binding to the solid phase. The plates were incubated at the room temperature Ž30 min. to allow binding of the antigen to the nitrocellulose surface. The protocol of immunoperoxidase staining is described in Table 1. Additionally, the nitrocellulose plates were cut in strips. Each strip contained one sample applied in triplicate, was inserted into 1 ml of acetone. The evaluation of the wheat protein concentration was performed according to the spectrophotometrical de-
Table 1 Dot-blot method ŽTimmons and Dunbar, 1990. for wheat protein quantitation in the culture media
Blocking of active spots Primary As Washing Secondary As Washing Substrate application Washing Washing a
Solution
Dilution
Time wminx
a
y y y y y y y y
15 20 3=5 20 3=5 d 5–7 3=5 10
b
TBS-Tween 20 SwaRrPx TBS-Tween 20 c 4-chloro-1-naphtol with H 2 O 2 TBS-Tween 20 bidistilled H 2 O
5% skim milk powder dissolved in TBS-Tween 20 buffer Ž10 mmol ly1 Tris–HCl, 0.9% NaCl, pH 7.2; Tween 20 0.02%.. Rabbit primary antiserum. c Substrate-dissolve 25 mg 4-chloro-1-naphtol in 5 ml ethanol, mix with 45 ml TBS buffer, filtrate and after 3 min add 0,1 ml H 2 O 2 Ž3%. to filtrate. d Staining will be induced in a few minutes. b
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termination of the violet stain intensity of the spots on the nitrocellulose foil at the wavelength 580 nm ŽVolker et al., 1989.. The measurement of the rate of the wheat protein proteolysis was based on the determination of the wheat protein concentration at 2 h, 4 h, 6 h and 24 h of incubation. Consequently, the differences between two levels of the wheat protein concentration at the beginning and at the end of the time interval of the incubation Ž2 h and 18 h respectively. were used for the calculation of the proteolysis rate. Data from 37 experiments were submitted to an analysis of variance. All Žin vitro. measurements and chemical analysis were carried out in triplicate. The differences between the measurements were assessed by t-test.
3. Results The results obtained in dot-blot study of the wheat protein degradation in the culture medium of the protozoal monoculture E. caudatum are summarized in Fig. 1. The samples of the protozoal culture media were applied in triplicate to the nitrocellulose foil. The color intensity of the spots number 3 to 7 represents the actual wheat protein concentration in the samples at the time intervals 0, 2, 4, 6 and 24 h. The diluted wheat protein, the samples number 14 to 18, was used for the construction of the calibration curve. The time course of the wheat protein concentration in the culture media is illustrated in Fig. 2. It
Fig. 1. Immunological sandwich technique dot-blot was used in the study of the wheat protein degradation in the monoculture of rumen protozoa Entodinium caudatum Žkey - from left to right.: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 1
1
Tris–NaCl buffer Tris–NaCl buffer 2 sample with wheat protein Ž0 h incubation. 2 sample with wheat protein Ž2 h incubation. 2 sample with wheat protein Ž4 h incubation. 2 sample with wheat protein Ž6 h incubation. 2 sample with wheat protein Ž24 h incubation. 3 control sample without wheat protein Ž0 h incubation. 4 sample from supernatant with wheat protein Ž0 h incubation. 3 control sample without wheat protein Ž24 h incubation. 4 sample from supernatant with wheat protein Ž24 h incubation. 1 Tris–NaCl buffer 5 BSA Ž1 mg mly1 TBS. 6 wheat protein Ž2 mg mly1 . 6 wheat protein Ž1 mg mly1 . 6 wheat protein Ž0.5 mg mly1 . 6 wheat protein Ž0.25 mg mly1 . 6 wheat protein Ž0.125 mg mly1 . 5 BSA Ž1 mg mly1 TBS. 1 Tris–NaCl buffer 1
TrisrNaCl buffer Ž0.02 mol ly1 Tris, 0.5 mol ly1 NaCl.. 2 The samples were taken at the intervals 0, 2, 4, 6 and 24 h during incubation of the protozoal monoculture E. caudatum with protein addition. 3 The control samples were taken from the monoculture E. caudatum incubated without addition of wheat protein at 0 h and at 24 h. 4 The samples were taken from the supernatant after the centrifugation of the monoculture E. caudatum with the wheat protein addition at 0 h and 24 h of incubation. 5 Bovine serum albumin ŽBSA. 6 The freeze-dried wheat protein was used for the calibration.
A. Marcin et al.r LiÕestock Production Science 53 (1998) 183–190
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Fig. 2. Time course of the concentration decrease of wheat protein in the culture medium of the monoculture E. caudatum.
Fig. 3. Proteolytic activity of the monoculture E. caudatum detected as: Ža. degradation rate of the wheat protein in the culture medium, Žb. degradation rate of azoalbumin in the culture medium. A–E,G,I; C–E,G,I; E–G,I p - 0.01; G–I p - 0,05; B–D,F,H; D–H,F; F–H p - 0.001.
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was observed the decrease from 1.5 mg mly1 Ž"0.05 SD. at t s 0 h to 0.111 mg mly1 Ž"0.06 SD. at t s 24 h during 24 h of incubation. The proteolytic activity in the culture medium of the monoculture E. caudatum is summarized in Fig. 3. The highest value of the wheat protein degradation rate 0.324 mg mly1 hy1 was observed at t s 2 h Ž"0.015 SD, p - 0.01.. There was noticed the decrease to 0.169 mg mly1 hy1 Ž"0.01 SD, p - 0.01. after 4 h, 0.033 mg mly1 hy1 Ž"0.006 SD, p - 0.01. after 6 h, and 0.02 mg mly1 hy1 Ž"0.009 SD, p - 0.01. after 24 h of incubation. Whereas, the proteolysis of azoalbumin in supernatant of the culture medium achieved the highest value at t s 0 h Ž0.098 mg mly1 hy1 , "0.012 SD, p - 0.05.. There was observed the decrease to 0.053 mg mly1 hy1 Ž"0.009 SD, p - 0.05. at t s 2 h, 0.043 mg mly1 hy1 Ž"0.009 SD, p - 0.05. at t s 4 h, 0.026 mg mly1 hy1 Ž"0.006 SD, p - 0.01. at t s 6 h and 0.022 mg mly1 hy1 Ž"0.008 SD, p - 0.05. after t s 24 h of incubation. It was impossible to measure the wheat protein proteolysis at t s 0 h. The reason was the method of calculation which is described in the Section 2.4. The rate of the wheat protein degradation was higher in the protozoal culture medium in comparison with the azoalbumin proteolysis in the supernatant during 24 h of incubation. Azoalbumin was relatively resistant to degradation. From the above mentioned results it was concluded that the highest decrease of the wheat protein and azoalbumin concentration was observed during first 4 h of incubation. The assay sensitivity Žs the limit of detection. of the simple immunological assay dot-blot was 104 ng mly1 Ži.e., 0.52 ng wheat protein applied to nitrocellulose foil. at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% Žcoefficient of variation. were 9.75% and 14% respectively. The used rabbit antiwheat–antiserum showed no cross-reactivity or detectable binding with soya, pork, beef protein as well as egg albumin and casein. 4. Discussion Two substrates with the different solubility and degradability, the wheat protein and the azoalbumin
were used for the proteolytic activity evaluation in the culture media of the protozoal monoculture E. caudatum and the supernatant of the culture media respectively. The reason for the use of E. caudatum in the described experiments was the fact that the proteolytic activity of Entodiinae is greater than the proteolytic activity of Isotrichids ŽLockwood et al., 1988.. The wheat protein which consisted of albumin and globulin fractions was applied into the incubation medium in the form of insoluble particles. The lower solubility of this protein was caused by the heat treatment. According to the results of Ushida and Jouany Ž1985., the mixed A-type or B-type protozoal populations both stimulate degradation of low soluble, particulate dietary proteins in the rumen but are relatively inactive toward soluble proteins. The use of the immunological sandwich method dot-blot permitted under the described conditions the quantitative evaluation of the wheat protein degradation in the culture media of the protozoal monoculture E. caudatum. The second substrate was the diazotized bovine serum albumin Žazoalbumin. which solubility in the supernatant of the culture medium was higher. However, it is generally accepted that the use of diazotized proteins Žazoalbumin, azoglobulin and casein. as substrates for the proteolytic activity evaluation of the rumen microorganisms is considered for the suitable method ŽBrock et al., 1982.. There are some controversial information in the literature dealing with the problem if it is possible to measure the nonspecific proteolytic activity ŽBroderick, 1987. or specific proteolytic activity ŽBrock et al., 1982. with azoprotein. In the light of our results, the wheat protein proteolysis in the culture medium of the protozoal monoculture E. caudatum was significantly higher in comparison with the azoalbumin degradation in the supernatant. The wheat protein which solubility was significantly lower was applied into the incubation medium in the form of particles. The proteolysis of the wheat protein was mainly intracellular. On the contrary, Nagasawa et al. Ž1994. reported that usually released amino nitrogen, radioactivity or azosubstance from the various substrates, which were used to assay proteolytic activity, measure total pro-
A. Marcin et al.r LiÕestock Production Science 53 (1998) 183–190
teolytic activity of rumen protozoa including endopeptidase and exopeptidase activities. Nagasawa et al. Ž1994. observed that mixed rumen ciliate protozoa Žmainly Entodiniinae. from goats have two kinds of protease; one has a pH optimum at 3.0, the other is active at neutral or alkaline pH. In addition, their findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein. The degradation of the used substrates accelerated with time as a result of increasing proteolysis and disruption in protein structures as it was previously demonstrated by Broderick and Craig Ž1989.. In spite of the fact that the solubility of azoalbumin was higher than wheat protein the degradation rate was lower. However, the digestion of the natural proteins is not in direct correlation with its solubility in the case of rumen degradation caused by protozoa and bacteria ŽMuszynski and Michalowski, 1987.. Degradation rates of proteins were shown to be in portion to the disulphide content of each protein, the greater the number of disulphide bonds, the higher the resistance to degradation ŽBroderick et al., 1991.. BSA has a complex tertiary structure with 6% cysteine disulphide bonds and possesses a greater resistance to degradation. In spite of the fact that the solubility of the heat treated protein was lower it was possible to quantify the concentration of the peptides from the wheat protein in the culture media using the dot-blot method. It must be pointed out that the curve of the time coarse of wheat protein concentration in the culture media has not the lag phase. It is typical for in situ and in vivo substrate degradation ŽTerramoccia et al., 1992.. There was no observed total disappearance of the wheat protein concentration in the culture media after 24 h of incubation. In addition, there was observed some small proteolytic activity in these media which was measured by the degradation of both used substrates at the end of incubation. Jouany et al. Ž1992. concluded that the protozoal effect on protein degradation varies according to the type of protozoa present and the nature of the protein. Another approach for the proteolysis determination are the methods which use synthetic substrates. The use of these substrates does not simulate sufficiently the degradation in vitro as the use of the
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natural substrates. In this case a lot of proteolytic enzymes are not used.
5. Conclusion A simple immunological technique dot-blot with the spectrophotometric quantitation was used for the study of the wheat protein Žisolated from seeds. degradation in the monoculture of the rumen protozoon E. caudatum under anaerobic conditions in vitro. The coarse of the concentration decrease of the wheat protein was gradual in the protozoal culture medium during the whole time of incubation. The highest decrease of the wheat protein concentration Žfrom 1.5 mg mly1 to 0.514 mg mly1 . was observed during first 4 h of incubation. The total release of the wheat protein was observed in the medium after 24 h of incubation. In addition to the above mentioned, the rate of the wheat protein degradation was higher in the protozoal culture medium in comparison with the azoalbumin proteolysis in the supernatant during 24 h of incubation. The average rate of the wheat protein proteolysis was higher Ž0.1365 mg mly1 hy1 . in the protozoal culture medium in comparison with the azoalbumin proteolysis Ž0.0484 mg mly1 hy1 . in the supernatant during 24 h of incubation. Azoalbumin was relatively more resistant to degradation than the wheat protein. The screening of the samples by dot-blot procedure with the spectrophotometric quantization proved to be an efficient way for the quantization of the proteolytic activity of the rumen protozoa cultivated in vitro as well as for the determination of the protein degradability under in vitro Žin the protozoal culture medium. or in vivo Žin the rumen. conditions.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Brock, F.M., Forsberg, C.W., Buchanan Smith, J.G., 1982. Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors. Appl. Environ. Microbiol. 44, 561–569.
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Broderick, G.A., 1987. Determination of protein degradation rates using a rumen in vitro system containing inhibitors of microbial nitrogen metabolism. Brit. J. Nutr. 58, 463–475. Broderick, G.A., Craig, W.M., 1989. Metabolism of peptides and amino acids during in vitro protein degradation by mixed rumen organisms. J. Dairy Sci. 72, 2540–2548. Broderick, G.A., Wallace, R.J., Orskov, E.R., 1991. Control of rate and extent of protein degradation. In: Tsuda, T., Sasaki, Y., Kawashima, R. ŽEds.., Physiological Aspects of Digestion and Metabolism in Ruminants, Proceedings of the Seventh International Symposium on Ruminant Physiology. Academic Press, Tokyo, pp. 541–582. Coleman, G.S., 1964. The metabolism of Escherichia coli and other bacteria by Entodinium caudatum. J. Gen. Microbiol. 37, 209–223. Coleman, G.S., 1978. Rumen entodiniomorphid protozoa. In: Taylor, A.E.R., Baker, J.R. ŽEds.., Methods in Cultivating Parasites in vitro. Academic Press, London, pp. 39–54. Dunbar, B.S., Schwoebel, E., 1990. Preparation of polyclonal antibodies. Method. Enzymol. 182, 663–670. Itabashi, H., Kobayashi, T., Takenaka, T., Matsumoto, M., 1990. The microbial metabolism and its regulation. In: Hoshino, S., Onodera, R., Minato, H., Itabashi, H. ŽEds.., The Rumen Ecosystem. Japan Scientific Societies Press, Tokyo, pp. 169– 176. Jouany, J.P., 1989. Effects of diet on populations of rumen protozoa in relation to fibre digestion. In: Nolan, J.V., Leng, R.A., Demeyer, D.I. ŽEds.., The Roles of Protozoa and Fungi in Ruminant Digestion. Armidale, Penambul Books, pp. 59–74. Jouany, J.P., 1996. Effect of rumen protozoa on nitrogen utilization by ruminants. J. Nutr. 126, 1335–1346. Jouany, J.P., Ivan, M., Papon, Y., Lassalas, B., 1992. Effects of Isotricha, Eudiplodinium, Epidinium, Entodinium and a mixed population of rumen protozoa om the in vitro degradation of fish meal, soybean meal and casein. Can. J. Anim. Sci. 72, 871–880.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680– 685. Lockwood, B.C., Coombs, G.H., Williams, A.G., 1988. Proteinase activity in rumen ciliate protozoa. J. Gen. Microbiol. 134, 2605–2614. Muszynski, P., Michalowski, T., 1987. The effect of protein solubility on the growth of rumen ciliates in vitro. In: Bod’a, K. ŽEd.., Physiology of Ruminant Nutrition, Proceedings of the IV International Symposium on Physiology of Ruminant Nutrition. Slovak Academy of Sciences-Institute of Animal Physiology, Kosice, Slovak Republic, pp. 345–357. ˇ Nagasawa, T., Nagase, T., Onodera, R., 1994. High molecular weight protease in rumen ciliate protozoa. Biosci. Biotech. Biochem. 58, 2020–2023. Onodera, R., Kandatsu, M., 1970. Amino acids and protein metabolism of rumen ciliate protozoa: IV. Metabolism of casein. Jpn. Zootech. Sci. 41, 307–313. Spencer, D., Higgins, T.J.V., Freer, M., Dove, H., Coombe, J.B., 1988. Monitoring the fate of dietary proteins in rumen fluid using gel electrophoresis. Brit. J. Nutr. 60, 241–247. Terramoccia, S., Puppo, S., Rizzi, L., Martillotti, F., 1992. Comparison between in sacco and in vitro protein rumen degradability. Ann. Zootech., 41, s. 20. Timmons, T.M., Dunbar, B.S., 1990. Protein blotting and immunodetection. Method. Enzymol. 182, 679–688. Ushida, K.J., Jouany, J.P., 1985. Effect of protozoa on rumen protein degradation in sheep. Reprod. Nutr. Dev. ´ 25, 1075– 1081. Volker, T.A., Herman, E.M., Chrispeels, M.J., 1989. In vitro mutated phytohemaglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability. Plant Cell 1, 95–104.
Wheat protein proteolysis in the monoculture of rumen protozoon Entodinium caudatum A. Marcin
a,)
, S. Kisidayova ˇ ´ b, V. Kmet´
b
a
b
Institute of Experimental Veterinary Medicine, HlinkoÕa 1 r A, 040 01 Kosice, ˇ SloÕak Republic ˇ ´ Institute of Animal Physiology-SloÕak Academy of Sciences, SoltesoÕej 3, 040 01 Kosice, ˇ SloÕak Republic Accepted 1 September 1997
Abstract The study of the wheat protein Žalbumin and globulin fraction. proteolysis was performed in the culture medium of the rumen protozoon Entodinium caudatum under anaerobic conditions in vitro. The rate of the wheat protein degradation was measured by the immunological sandwich technique dot-blot. The centrifugation of the protozoal culture media was performed at 500 g for 20 min. The resulting concentration of the protozoal cells was 20,000–24,000 per ml in the experimental culture medium. The samples were taken from the culture medium at 0, 2, 4, 6 and 24 h during incubation. After centrifugation at 500 g for 20 min, the supernatants were used for the determination of: Ž1. the total protein concentration, Ž2. the nonspecific proteolytic activity Žazoalbumin was used as a substrate. and Ž3. the wheat protein concentration in the culture medium by the immunological sandwich technique dot-blot. Subsequently, the results were used for the calculation of the degradation rate of the wheat protein. The rabbit polyclonal antiserum against the wheat protein was used as the first antibody in this method. The quantitation of this test was based on the spectrophotometric determination of the violet stain quantity on the nitrocellulose strip surfaces. The average rate of the wheat protein degradation in the culture medium E. caudatum was 0.014 mg mly1 hy1, whereas the average rate of the azoalbumin degradation was 0.003 mg mly1 hy1 in supernatant. Azoalbumin was more resistant to degradation in the supernatant of the culture medium in comparison with the wheat protein in the culture medium. The assay sensitivity Žs the limit of detection. of the simple immunological assay dot-blot was 104 ng mly1 Ži.e., 0.52 ng wheat protein applied to nitrocellulose foil. at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% Žcoefficient of variation. were 9.75% and 14%, respectively. The screening of the samples by dot-blot procedure proved to be an efficient way for the quantitation of the proteolytic activity in the rumen protozoa monoculture cultivated in vitro. q 1998 Elsevier Science B.V. Keywords: Rumen; Proteolysis; Protozoa; Entodinium; Dot-blot; Wheat protein
1. Introduction Some part of the dietary proteins entering the rumen are degraded by rumen microorganisms to peptide and amino acids. The first step of this degradation is done by proteases of bacteria. )
Corresponding author.
Ruminal protozoa may influence intestinal protein flow at a number of different levels. Protozoa can alter quantitatively ruminal degradation of dietary proteins and ruminal bacterial growth, consequently modifying the supply of both protein of feed and bacterial origin. However, the concentration of amino acids and ammonia are higher in faunated than in defaunated sheep ŽItabashi et al., 1990.. Rumen bac-
0301-6226r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 1 - 6 2 2 6 Ž 9 7 . 0 0 1 3 9 - 5
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teria seem to be primarily responsible for the degradation of plant protein. Rumen anaerobic fungi also attack plant proteins but they are reported to have only weak activity. Rumen ciliate protozoa can engulf rumen bacteria and insoluble proteins ŽOnodera and Kandatsu, 1970.. Entodiniomorphid protozoa are particularly efficient in taking up particulate matter suspended in ruminal fluid. Anatomically they possess to trap particles that are subsequently driven into a vestibulum and then into cytosomes ŽJouany, 1996.. On the other hand, Holotrichs protozoa have considerable capacity for taking up soluble compounds, they are less efficient than entodiniomorphs in the ingestion of insoluble plant particles ŽJouany, 1996.. Considering the differences in feeding strategies between protozoa and other ruminal microbes, it can be concluded that determining enzyme activities in autolyzed or sonicated cells is not appropriate for quantifying the contribution of ciliates to protein degradation in the in vivo rumen ŽJouany, 1996.. Thus, it may be that protozoal proteases have important roles in proteolysis within the rumen ecosystem. Results of biochemical, cultural and microscopic studies indicate that the contribution of protozoa depends on the complex interactions between protozoa, bacteria and dietary characteristics ŽJouany, 1989.. The objective was to study the wheat protein Žisolated from seeds. proteolysis in the monoculture of the rumen protozoon Entodinium caudatum during 24 h of incubation in vitro.
2. Materials and methods
by fresh medium. Protozoa were fed once a day using 0.75 mg wholemeal flour four times a week with a 300 m g dried grass and three times a week with 0.75 mg rice starch with 300 m g dried grass. The average concentration of the protozoal cells E. caudatum in the culture medium was 10,000–12,000 per ml. The protozoal number of the experimental culture medium was estimated by blowing 0.2 ml of the culture from 1 ml graduated pipette with a large hole at the tip into 2 ml of 0.02 mol ly1 iodine and counted microscopically all the protozoa in 0.1 ml of the mixture ŽColeman, 1978.. 2.2. Isolation of wheat protein from wheat flour The wheat protein was partly purified from commercially available wheat flour. The flour was defatted with acetone, dried and then homogenized in a mortar with Tris–NaCl buffer Ž0.02 mol ly1 Tris, 0.5 mol ly1 NaCl.. The isolation was repeated twice according to method of Spencer et al. Ž1988.. The albumin and globulin fractions were isolated from the wheat flour. The homogenate was centrifuged at 1000 g for 20 min and the supernatant fraction containing the extracted proteins was collected. The isolated proteins before the heat treatment consisted of the polypeptides with the molecular weights of 61, 36, 21 and 8 kD as it was confirmed by sodiumdodecylsulphate polyacrylamide gel electrophoresis ŽSDS-PAGE. ŽLaemmli, 1970.. Subsequently, the wheat protein was heat treated in the water bath Ž1008C for 5 min. and freeze dried. The heat treatment was performed to lower the solubility of the incubated wheat protein in the protozoa culture medium.
2.1. Protozoal culture procedures The monocultured E. caudatum Žfamily Ophryoscolecidae. has been cultivated in vitro in the laboratory of the Institute of Animal Physiology Slovak Academy of Sciences ŽKosice, Slovak Reˇ public. for 5 yr using routine techniques described by Coleman Ž1964, 1978.. Briefly the cultures were maintained in the glass tubes Žvolume of cultivation medium 10 ml. at 398C under 100% carbon dioxide atmosphere. The protozoa were subcultured on caudatum-type medium ŽColeman, 1964, 1978.. Twice a week one half of the culture medium was replaced
2.3. Preparation of polyclonal antibody against wheat protein The isolated protein after the heat treatment in the water bath Ž1008C for 5 min. was freeze-dried and used for the immunization of two rabbits ŽNew Zealand white. according to usual immunization scheme ŽDunbar and Schwoebel, 1990.. The bleeding of the rabbits and processing of the serum was performed according to method of Dunbar and Schwoebel Ž1990.. The achieved titer of antibody in the wheat protein antiserum was 1:3000.
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2.4. Incubation of wheat protein in the protozoal monoculture E. caudatum The anaerobic centrifugation of the protozoal culture medium E. caudatum was performed at the temperature 208C for 20 min at 500 g. Two thirds of the supernatant were discarded and the rest of the culture medium containing the protozoal cells Ž20,000–24,000 per ml. was incubated at the temperature 398C with the freeze-dried wheat protein in the cultivation vessel under CO 2 atmosphere for 24 h. The resulting concentration of the added wheat protein was 1.5 mg mly1 in the experimental culture medium. Two control culture media were used in the experiment, namely: Ž1. the culture medium with the protozoal cells Ž20,000–24,000 per ml. without wheat protein and Ž2. the protozoa-free supernatant with the wheat protein addition. The samples Ž1 ml. were taken from experimental and two control culture media at the intervals 0, 2, 4, 6 and 24 h during incubation. Samples were centrifugated at 500 g for 20 min. Subsequently, the supernatants were used for: Ža. the protein quantification by Bradford method ŽBradford, 1976., Žb. the determination of the nonspecific proteolytic activity Žazoalbumin was used as a substrate. ŽBroderick, 1987.. The mentioned azoprotein Ždiazotized bovine serum albumin-BSA. was degraded in the supernatant at the temperature 398C for 2 h, c. the determination of the wheat protein
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concentration by the modified immunological sandwich method dot-blot ŽTimmons and Dunbar, 1990.. Subsequently, the results obtained by the dot-blot analysis were used for the calculation of the degradation rate of the wheat protein in the culture medium of the rumen protozoa E. caudatum. The wheat protein degradation rate was calculated according to measured differences between the wheat protein concentrations in the culture medium at the beginning and at the end of the certain time interval Ž2 h and 18 h, respectively.. Consequently, the number of protozoal cells was determined in the experimental culture medium. The slight increase Žabout 32%. was observed at the end of incubations in comparison with the beginning. The samples, which were taken at the times above indicated, were applied to a dry nitrocellulose foil ŽFarby Laky n.p., Czech Republic, Praha. in triplicate each drop of 5 m l Žsee the legend of Fig. 1.. Two blanks containing Tris–NaCl buffer and BSA were also analyzed to determine the nonspecific binding to the solid phase. The plates were incubated at the room temperature Ž30 min. to allow binding of the antigen to the nitrocellulose surface. The protocol of immunoperoxidase staining is described in Table 1. Additionally, the nitrocellulose plates were cut in strips. Each strip contained one sample applied in triplicate, was inserted into 1 ml of acetone. The evaluation of the wheat protein concentration was performed according to the spectrophotometrical de-
Table 1 Dot-blot method ŽTimmons and Dunbar, 1990. for wheat protein quantitation in the culture media
Blocking of active spots Primary As Washing Secondary As Washing Substrate application Washing Washing a
Solution
Dilution
Time wminx
a
y y y y y y y y
15 20 3=5 20 3=5 d 5–7 3=5 10
b
TBS-Tween 20 SwaRrPx TBS-Tween 20 c 4-chloro-1-naphtol with H 2 O 2 TBS-Tween 20 bidistilled H 2 O
5% skim milk powder dissolved in TBS-Tween 20 buffer Ž10 mmol ly1 Tris–HCl, 0.9% NaCl, pH 7.2; Tween 20 0.02%.. Rabbit primary antiserum. c Substrate-dissolve 25 mg 4-chloro-1-naphtol in 5 ml ethanol, mix with 45 ml TBS buffer, filtrate and after 3 min add 0,1 ml H 2 O 2 Ž3%. to filtrate. d Staining will be induced in a few minutes. b
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termination of the violet stain intensity of the spots on the nitrocellulose foil at the wavelength 580 nm ŽVolker et al., 1989.. The measurement of the rate of the wheat protein proteolysis was based on the determination of the wheat protein concentration at 2 h, 4 h, 6 h and 24 h of incubation. Consequently, the differences between two levels of the wheat protein concentration at the beginning and at the end of the time interval of the incubation Ž2 h and 18 h respectively. were used for the calculation of the proteolysis rate. Data from 37 experiments were submitted to an analysis of variance. All Žin vitro. measurements and chemical analysis were carried out in triplicate. The differences between the measurements were assessed by t-test.
3. Results The results obtained in dot-blot study of the wheat protein degradation in the culture medium of the protozoal monoculture E. caudatum are summarized in Fig. 1. The samples of the protozoal culture media were applied in triplicate to the nitrocellulose foil. The color intensity of the spots number 3 to 7 represents the actual wheat protein concentration in the samples at the time intervals 0, 2, 4, 6 and 24 h. The diluted wheat protein, the samples number 14 to 18, was used for the construction of the calibration curve. The time course of the wheat protein concentration in the culture media is illustrated in Fig. 2. It
Fig. 1. Immunological sandwich technique dot-blot was used in the study of the wheat protein degradation in the monoculture of rumen protozoa Entodinium caudatum Žkey - from left to right.: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 1
1
Tris–NaCl buffer Tris–NaCl buffer 2 sample with wheat protein Ž0 h incubation. 2 sample with wheat protein Ž2 h incubation. 2 sample with wheat protein Ž4 h incubation. 2 sample with wheat protein Ž6 h incubation. 2 sample with wheat protein Ž24 h incubation. 3 control sample without wheat protein Ž0 h incubation. 4 sample from supernatant with wheat protein Ž0 h incubation. 3 control sample without wheat protein Ž24 h incubation. 4 sample from supernatant with wheat protein Ž24 h incubation. 1 Tris–NaCl buffer 5 BSA Ž1 mg mly1 TBS. 6 wheat protein Ž2 mg mly1 . 6 wheat protein Ž1 mg mly1 . 6 wheat protein Ž0.5 mg mly1 . 6 wheat protein Ž0.25 mg mly1 . 6 wheat protein Ž0.125 mg mly1 . 5 BSA Ž1 mg mly1 TBS. 1 Tris–NaCl buffer 1
TrisrNaCl buffer Ž0.02 mol ly1 Tris, 0.5 mol ly1 NaCl.. 2 The samples were taken at the intervals 0, 2, 4, 6 and 24 h during incubation of the protozoal monoculture E. caudatum with protein addition. 3 The control samples were taken from the monoculture E. caudatum incubated without addition of wheat protein at 0 h and at 24 h. 4 The samples were taken from the supernatant after the centrifugation of the monoculture E. caudatum with the wheat protein addition at 0 h and 24 h of incubation. 5 Bovine serum albumin ŽBSA. 6 The freeze-dried wheat protein was used for the calibration.
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Fig. 2. Time course of the concentration decrease of wheat protein in the culture medium of the monoculture E. caudatum.
Fig. 3. Proteolytic activity of the monoculture E. caudatum detected as: Ža. degradation rate of the wheat protein in the culture medium, Žb. degradation rate of azoalbumin in the culture medium. A–E,G,I; C–E,G,I; E–G,I p - 0.01; G–I p - 0,05; B–D,F,H; D–H,F; F–H p - 0.001.
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was observed the decrease from 1.5 mg mly1 Ž"0.05 SD. at t s 0 h to 0.111 mg mly1 Ž"0.06 SD. at t s 24 h during 24 h of incubation. The proteolytic activity in the culture medium of the monoculture E. caudatum is summarized in Fig. 3. The highest value of the wheat protein degradation rate 0.324 mg mly1 hy1 was observed at t s 2 h Ž"0.015 SD, p - 0.01.. There was noticed the decrease to 0.169 mg mly1 hy1 Ž"0.01 SD, p - 0.01. after 4 h, 0.033 mg mly1 hy1 Ž"0.006 SD, p - 0.01. after 6 h, and 0.02 mg mly1 hy1 Ž"0.009 SD, p - 0.01. after 24 h of incubation. Whereas, the proteolysis of azoalbumin in supernatant of the culture medium achieved the highest value at t s 0 h Ž0.098 mg mly1 hy1 , "0.012 SD, p - 0.05.. There was observed the decrease to 0.053 mg mly1 hy1 Ž"0.009 SD, p - 0.05. at t s 2 h, 0.043 mg mly1 hy1 Ž"0.009 SD, p - 0.05. at t s 4 h, 0.026 mg mly1 hy1 Ž"0.006 SD, p - 0.01. at t s 6 h and 0.022 mg mly1 hy1 Ž"0.008 SD, p - 0.05. after t s 24 h of incubation. It was impossible to measure the wheat protein proteolysis at t s 0 h. The reason was the method of calculation which is described in the Section 2.4. The rate of the wheat protein degradation was higher in the protozoal culture medium in comparison with the azoalbumin proteolysis in the supernatant during 24 h of incubation. Azoalbumin was relatively resistant to degradation. From the above mentioned results it was concluded that the highest decrease of the wheat protein and azoalbumin concentration was observed during first 4 h of incubation. The assay sensitivity Žs the limit of detection. of the simple immunological assay dot-blot was 104 ng mly1 Ži.e., 0.52 ng wheat protein applied to nitrocellulose foil. at 88% recovery. The accuracy and reproducibility characterized as intra-assay and inter-assay CV% Žcoefficient of variation. were 9.75% and 14% respectively. The used rabbit antiwheat–antiserum showed no cross-reactivity or detectable binding with soya, pork, beef protein as well as egg albumin and casein. 4. Discussion Two substrates with the different solubility and degradability, the wheat protein and the azoalbumin
were used for the proteolytic activity evaluation in the culture media of the protozoal monoculture E. caudatum and the supernatant of the culture media respectively. The reason for the use of E. caudatum in the described experiments was the fact that the proteolytic activity of Entodiinae is greater than the proteolytic activity of Isotrichids ŽLockwood et al., 1988.. The wheat protein which consisted of albumin and globulin fractions was applied into the incubation medium in the form of insoluble particles. The lower solubility of this protein was caused by the heat treatment. According to the results of Ushida and Jouany Ž1985., the mixed A-type or B-type protozoal populations both stimulate degradation of low soluble, particulate dietary proteins in the rumen but are relatively inactive toward soluble proteins. The use of the immunological sandwich method dot-blot permitted under the described conditions the quantitative evaluation of the wheat protein degradation in the culture media of the protozoal monoculture E. caudatum. The second substrate was the diazotized bovine serum albumin Žazoalbumin. which solubility in the supernatant of the culture medium was higher. However, it is generally accepted that the use of diazotized proteins Žazoalbumin, azoglobulin and casein. as substrates for the proteolytic activity evaluation of the rumen microorganisms is considered for the suitable method ŽBrock et al., 1982.. There are some controversial information in the literature dealing with the problem if it is possible to measure the nonspecific proteolytic activity ŽBroderick, 1987. or specific proteolytic activity ŽBrock et al., 1982. with azoprotein. In the light of our results, the wheat protein proteolysis in the culture medium of the protozoal monoculture E. caudatum was significantly higher in comparison with the azoalbumin degradation in the supernatant. The wheat protein which solubility was significantly lower was applied into the incubation medium in the form of particles. The proteolysis of the wheat protein was mainly intracellular. On the contrary, Nagasawa et al. Ž1994. reported that usually released amino nitrogen, radioactivity or azosubstance from the various substrates, which were used to assay proteolytic activity, measure total pro-
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teolytic activity of rumen protozoa including endopeptidase and exopeptidase activities. Nagasawa et al. Ž1994. observed that mixed rumen ciliate protozoa Žmainly Entodiniinae. from goats have two kinds of protease; one has a pH optimum at 3.0, the other is active at neutral or alkaline pH. In addition, their findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein. The degradation of the used substrates accelerated with time as a result of increasing proteolysis and disruption in protein structures as it was previously demonstrated by Broderick and Craig Ž1989.. In spite of the fact that the solubility of azoalbumin was higher than wheat protein the degradation rate was lower. However, the digestion of the natural proteins is not in direct correlation with its solubility in the case of rumen degradation caused by protozoa and bacteria ŽMuszynski and Michalowski, 1987.. Degradation rates of proteins were shown to be in portion to the disulphide content of each protein, the greater the number of disulphide bonds, the higher the resistance to degradation ŽBroderick et al., 1991.. BSA has a complex tertiary structure with 6% cysteine disulphide bonds and possesses a greater resistance to degradation. In spite of the fact that the solubility of the heat treated protein was lower it was possible to quantify the concentration of the peptides from the wheat protein in the culture media using the dot-blot method. It must be pointed out that the curve of the time coarse of wheat protein concentration in the culture media has not the lag phase. It is typical for in situ and in vivo substrate degradation ŽTerramoccia et al., 1992.. There was no observed total disappearance of the wheat protein concentration in the culture media after 24 h of incubation. In addition, there was observed some small proteolytic activity in these media which was measured by the degradation of both used substrates at the end of incubation. Jouany et al. Ž1992. concluded that the protozoal effect on protein degradation varies according to the type of protozoa present and the nature of the protein. Another approach for the proteolysis determination are the methods which use synthetic substrates. The use of these substrates does not simulate sufficiently the degradation in vitro as the use of the
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natural substrates. In this case a lot of proteolytic enzymes are not used.
5. Conclusion A simple immunological technique dot-blot with the spectrophotometric quantitation was used for the study of the wheat protein Žisolated from seeds. degradation in the monoculture of the rumen protozoon E. caudatum under anaerobic conditions in vitro. The coarse of the concentration decrease of the wheat protein was gradual in the protozoal culture medium during the whole time of incubation. The highest decrease of the wheat protein concentration Žfrom 1.5 mg mly1 to 0.514 mg mly1 . was observed during first 4 h of incubation. The total release of the wheat protein was observed in the medium after 24 h of incubation. In addition to the above mentioned, the rate of the wheat protein degradation was higher in the protozoal culture medium in comparison with the azoalbumin proteolysis in the supernatant during 24 h of incubation. The average rate of the wheat protein proteolysis was higher Ž0.1365 mg mly1 hy1 . in the protozoal culture medium in comparison with the azoalbumin proteolysis Ž0.0484 mg mly1 hy1 . in the supernatant during 24 h of incubation. Azoalbumin was relatively more resistant to degradation than the wheat protein. The screening of the samples by dot-blot procedure with the spectrophotometric quantization proved to be an efficient way for the quantization of the proteolytic activity of the rumen protozoa cultivated in vitro as well as for the determination of the protein degradability under in vitro Žin the protozoal culture medium. or in vivo Žin the rumen. conditions.
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