猪仿生消化中消化酶活性随消化时间变化的研究
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摘要
旨在探讨猪仿生消化过程中胃、小肠阶段消化酶的衰减以及补加消化酶后活性变化的规律,为仿生消化法评定猪饲料养分消化率提供参考。本研究中,试验一采用单因素完全随机设计,评价以玉米、大豆粕、小麦麸、玉米-大豆粕型饲粮为底物,在仿生消化中一次性注入模拟胃液和小肠液后,消化酶活性随消化时间的变化规律。胃阶段消化时间设0、1、 2、 3、4 h,测定消化液中胃蛋白酶的比活性及总活性。小肠消化时间设0、2、4、6、8、12、16 h,分析消化液中胰蛋白酶、糜蛋白酶和淀粉酶的活性。试验二根据试验一结果,在小肠消化4 h补加消化酶,评价补加酶后小肠消化4、6、8 h消化液中消化酶活性。每个时间点为一个处理,每个处理5个重复,每个重复1根消化管。结果表明:1)当模拟胃液与玉米、大豆粕、小麦麸或玉米-大豆粕型饲粮混合后,消化液中胃蛋白酶的比活性迅速降低(为模拟胃液活性的28.8%～59.1%),但在模拟消化1～4 h时,胃蛋白酶的比活性和总活性呈二次曲线增加(P<0.05),最终可达到模拟胃液比活性的78.0%～96.1%和总活性的87.2%～102.7%;在小肠消化阶段,模拟消化液中淀粉酶、胰蛋白酶、糜蛋白酶的活性均有不同程度的衰减(P<0.05),其中淀粉酶和糜蛋白酶活性的变化规律因消化底物不同而呈二次或其它非线性变化(P<0.05),而胰蛋白酶的活性均呈二次曲线下降(P<0.05)。胰蛋白酶的活性下降速度最快。2)补加消化酶后消化液中淀粉酶、胰蛋白酶、糜蛋白酶的活性均可恢复到初始值,但仍随消化时间而衰减(P<0.05),其中胰蛋白酶活性依然快速下降。综上所述,在仿生消化过程中,胃消化阶段无需补充胃蛋白酶;小肠消化阶段需要补充消化酶。
This experiment was conducted to investigate the variation of digestive enzyme activities in the simulated gastric and small intestinal digestion for pigs, which will provide a reference to develop the simulated digestion method for determining nutrients digestibility for pigs. In experiment 1, a single factor completely randomized design was used to investigate the variation of digestive enzyme activities depending on digestion time when the simulated gastric and small intestinal fluid were injected once during the simulated digestion for corn, soybean meal, wheat bran and corn-soybean meal. The digestion time of 0, 1, 2, 3 and 4 h was arranged for gastric digestion phase. The specific and total activities of pepsin in the digestive fluid were determined. The digestion time of 0, 2, 4, 6, 8, 12 and 16 h was arranged for the small intestine digestion phase. The specific and total activities of trypsin, chymotrypsin and amylase in the digestive fluid were determined. In experiment 2, the digestive enzymes were supplemented at 4 h of small intestine digestion phase according to the results of experiment 1. The digestive enzyme activities were determined at 4, 6 and 8 h of small intestine digestion phase after supplementing digestive enzymes. Each in vitro digestion time was considered as a treatment, and each treatment contained 5 replicates with 1 digestion tube per replicate. The results showed that: 1) When the simulated gastric fluid was mixed with corn, soybean meal, wheat bran or corn-soybean meal, the specific activity of pepsin in the digestive fluid was rapidly reduced(accounting for 28.8% to 59.1% of initial activity). However, from 1 to 4 h of the simulated digestion, the specific and total activities of pepsin were increased with the quadratic curve(P<0.05). The final values were 78.0% to 96.1% relative to initial specific activity and 87.2% to 102.7% relative to initial total activity of simulated gastric fluid, respectively. In the small intestine digestion phase, different extent of reduction of activities of amylase, trypsin and chymotrypsin in simulated digestive fluid was observed(P<0.05). The quadratic or non-linear curve variation of amylase and chymotrypsin activity in digestive fluid was observed with different substrates(P<0.05). The reduction of trypsin activity in digestive fluid with quadratic curve was observed(P<0.05). The greatest reduction of activity was observed in trypsin. 2) After the digestive enzymes were supplemented in simulated small intestinal digestion, the specific activities of amylase, trypsin and chymotrypsin in the digestive fluid could be recovered to the initial value, but they still decreased with the digestion time(P<0.05), among them, the trypsin activity still decreased rapidly. In conclusion, in the process of simulated digestion, there is no need to supplement pepsin in the gastric digestion stage; the digestive enzyme needs to be supplemented in the small intestine digestion stage.
This experiment was conducted to investigate the variation of digestive enzyme activities in the simulated gastric and small intestinal digestion for pigs, which will provide a reference to develop the simulated digestion method for determining nutrients digestibility for pigs. In experiment 1, a single factor completely randomized design was used to investigate the variation of digestive enzyme activities depending on digestion time when the simulated gastric and small intestinal fluid were injected once during the simulated digestion for corn, soybean meal, wheat bran and corn-soybean meal. The digestion time of 0, 1, 2, 3 and 4 h was arranged for gastric digestion phase. The specific and total activities of pepsin in the digestive fluid were determined. The digestion time of 0, 2, 4, 6, 8, 12 and 16 h was arranged for the small intestine digestion phase. The specific and total activities of trypsin, chymotrypsin and amylase in the digestive fluid were determined. In experiment 2, the digestive enzymes were supplemented at 4 h of small intestine digestion phase according to the results of experiment 1. The digestive enzyme activities were determined at 4, 6 and 8 h of small intestine digestion phase after supplementing digestive enzymes. Each in vitro digestion time was considered as a treatment, and each treatment contained 5 replicates with 1 digestion tube per replicate. The results showed that: 1) When the simulated gastric fluid was mixed with corn, soybean meal, wheat bran or corn-soybean meal, the specific activity of pepsin in the digestive fluid was rapidly reduced(accounting for 28.8% to 59.1% of initial activity). However, from 1 to 4 h of the simulated digestion, the specific and total activities of pepsin were increased with the quadratic curve(P<0.05). The final values were 78.0% to 96.1% relative to initial specific activity and 87.2% to 102.7% relative to initial total activity of simulated gastric fluid, respectively. In the small intestine digestion phase, different extent of reduction of activities of amylase, trypsin and chymotrypsin in simulated digestive fluid was observed(P<0.05). The quadratic or non-linear curve variation of amylase and chymotrypsin activity in digestive fluid was observed with different substrates(P<0.05). The reduction of trypsin activity in digestive fluid with quadratic curve was observed(P<0.05). The greatest reduction of activity was observed in trypsin. 2) After the digestive enzymes were supplemented in simulated small intestinal digestion, the specific activities of amylase, trypsin and chymotrypsin in the digestive fluid could be recovered to the initial value, but they still decreased with the digestion time(P<0.05), among them, the trypsin activity still decreased rapidly. In conclusion, in the process of simulated digestion, there is no need to supplement pepsin in the gastric digestion stage; the digestive enzyme needs to be supplemented in the small intestine digestion stage.
引文
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[2] YEGANI M,KORVER D R.Effects of corn source and exogenous enzymes on growth performance and nutrient digestibility in broiler chickens[J].Poult Sci,2013,92(5):1208-1220.
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[12] WILFART A,JAGUELIN-PEYRAUD Y,SIMMINS H,et al.A step-wise in vitro method to estimate kinetics of hydrolysis of feeds[J].Livest Sci,2007,109(1-3):179-181.
[13] WILFART A,JAGUELIN-PEYRAUD Y,SIMMINS H,et al.Kinetics of enzymatic digestion of feeds as estimated by a stepwise in vitro method[J].Anim Feed Sci Technol,2008,141(1-2):171-183.
[14] SOPADE P A,GIDLEY M J.A rapid in-vitro digestibility assay based on glucometry for investigating kinetics of starch digestion[J].Starch-St?rke,2009,61(5):245-255.
[15] PéREZ-VENDRELL A M,TORRALLARDONA D.In vitro digestibility kinetics of diets containing different cereal sources[J].Livest Sci,2010,134(1-3):47-49.
[16] 王钰明.猪模拟小肠液的制备及仿生消化法测定饲料可消化养分含量的研究[D].北京:中国农业科学院,2015.WANG Y M.Study on the preparation of mimic intestinal fluid and simulated digestion method for determination of digestible nutrient content of feeds for pigs[D].Beijing:Chinese Academy of Agricultural Sciences,2015.(in Chinese)
[17] ANSON M L.The estimation of pepsin,trypsin,papain,and cathepsin with hemoglobin[J].J Gen Physiol,1938,22(1):79-89.
[18] DAHLQVIST A.A method for the determination of amylase in intestinal content[J].Scand J Clin Lab Invest,1962,14(2):145-151.
[19] WIRNT R.Trypsin:measurements with Nα-p-toluene-sulphonyl-L-arginine methyl ester as substrate[M]//BERGMEYER H U.Methods of Enzymatic Analysis.New York:Verlag Chemie,1974:1021-1024.
[20] WIRNT R.Chymotrypsin measurements with N-benzoyl-L-tyrosin methyl ester as substrate[M]//BERGMEYER H U.Methods of Enzymatic Analysis.Weinheinm:Verlag Chemie,1974:1009-1012.
[21] LOW A G.Nutritional regulation of gastric secretion,digestion and emptying[J].Nutr Res Rev,1990,3(1):229-252.
[22] LAWRENCE T L J.The effect of certain dietary factors on in vivo pH changes and pepsin activity in the stomach of the growing pig[J].Br Vet J,1972,128(8):402-411.
[23] LAWLOR P G,LYNCHP B,CAFFREY P J,et al.Measurements of the acid-binding capacity of ingredients used in pig diets[J].Ir Vet J,2005,58(8):447-452.
[24] MAKKINK C A,NEGULESCU G P,QIN G X,et al.Effect of dietary protein source on feed intake,growth,pancreatic enzyme activities and jejunal morphology in newly-weaned piglets[J].Br J Nutr,1994,72(3):353-368.
[25] ZEBROWSKA T,LOW A G,ZEBROWSKA H.Studies on gastric digestion of protein and carbohydrate,gastric secretion and exocrine pancreatic secretion in the growing pig[J].Br J Nutr,1983,49(3):401-410.
[26] PARTRIDGE I G,LOW A G,SAMBROOK I E,et al.The influence of diet on the exocrine pancreatic secretion of growing pigs[J].Br J Nutr,1982,48(1):137-145.
[27] BOTERMANS J A M,PIERZYNOWSKI S G.Relations between body weight,feed intake,daily weight gain,and exocrine pancreatic secretion in chronically catheterized growing pigs[J].J Anim Sci,1999,77(2):450-456.
[28] THAELA M J,PIERZYNOWSKI S G,JENSEN M S,et al.The pattern of the circadian rhythm of pancreatic secretion in fed pigs[J].J Anim Sci,1995,73(11):3402-3408.
[29] DAL BORGO G,SALMAN A J,PUBOLS M H,et al.Exocrine function of the chick pancreas as affected by dietary soybean meal and carbohydrate[J].Proc Soc Exp Biol Med,1968,129(3):877-881.
[30] JOHNSON L R.Gastrointestinal Physiology[M].Philadelphia:Mosby Elsevier,2007.
[31] WENK C.The role of dietary fibre in the digestive physiology of the pig[J].Anim Feed Sci Technol,2001,90(1-2):21-33.
[32] LOW A G.The activity of pepsin,chymotrypsin and trypsin during 24 h periods in the small intestine of growing pigs[J].Br J Nutr,1982,48(1):147-159.
[33] 茹炳根,杜锦珠,曾耀辉,等.猪胰蛋白酶自溶后的活性产物[J].北京大学学报,1979(4):32-47.RU B G,DU J Z,ZENG Y H,et al.Active products of porcine trypsin after autolysis[J].Acta Scicentiarum Naturalum Universitis Pekinesis,1979(4):32-47.(in Chinese)
[2] YEGANI M,KORVER D R.Effects of corn source and exogenous enzymes on growth performance and nutrient digestibility in broiler chickens[J].Poult Sci,2013,92(5):1208-1220.
[3] ZHAO F,REN L Q,MI B M,et al.Developing a computer-controlled simulated digestion system to predict the concentration of metabolizable energy of feedstuffs for rooster[J].J Anim Sci,2014,92(4):1537-1547.
[4] ZHAO F,ZHANG L,MI B M,et al.Using a computer-controlled simulated digestion system to predict the energetic value of corn for ducks[J].Poult Sci,2014,93(6):1410-1420.
[5] BOISEN S,FERNáNDEZ J A.Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses[J].Anim Feed Sci Technol,1997,68(3-4):277-286.
[6] PAN L,MA H,PIAO X S,et al.A computer-controlled simulated digestion system is a promising in vitro digestibility technique to predict digestible energy of corn grain for growing pigs[J].Anim Feed Sci Technol,2018,235:43-49.
[7] CLUNIES M,LEESON S.In vitro estimation of dry matter and crude protein digestibility[J].Poult Sci,1984,63(1):89-96.
[8] JOHNSTON J,COON C N.The use of varying levels of pepsin for pepsin digestion studies with animal proteins[J].Poult Sci,1979,58(5):1271-1273.
[9] HUANG R L,TAN Z L,XING T X,et al.An in vitro method for the estimation of ileal crude protein and amino acids digestibility using the dialysis tubing for pig feedstuffs[J].Anim Feed Sci Technol,2000,88(1-2):79-89.
[10] PIERZYNOWSKI S G,WESTR?M B R,KARLSSON B W,et al.Pancreatic cannulation of young pigs for long-term study of exocrine pancreatic function[J].Can J Anim Sci,1988,68(3):953-959.
[11] THAELA M J,JENSEN M S,CORNéLISSEN G,et al.Circadian and ultradian variation in pancreatic secretion of meal-fed pigs after weaning[J].J Anim Sci,1998,76(4):1131-1139.
[12] WILFART A,JAGUELIN-PEYRAUD Y,SIMMINS H,et al.A step-wise in vitro method to estimate kinetics of hydrolysis of feeds[J].Livest Sci,2007,109(1-3):179-181.
[13] WILFART A,JAGUELIN-PEYRAUD Y,SIMMINS H,et al.Kinetics of enzymatic digestion of feeds as estimated by a stepwise in vitro method[J].Anim Feed Sci Technol,2008,141(1-2):171-183.
[14] SOPADE P A,GIDLEY M J.A rapid in-vitro digestibility assay based on glucometry for investigating kinetics of starch digestion[J].Starch-St?rke,2009,61(5):245-255.
[15] PéREZ-VENDRELL A M,TORRALLARDONA D.In vitro digestibility kinetics of diets containing different cereal sources[J].Livest Sci,2010,134(1-3):47-49.
[16] 王钰明.猪模拟小肠液的制备及仿生消化法测定饲料可消化养分含量的研究[D].北京:中国农业科学院,2015.WANG Y M.Study on the preparation of mimic intestinal fluid and simulated digestion method for determination of digestible nutrient content of feeds for pigs[D].Beijing:Chinese Academy of Agricultural Sciences,2015.(in Chinese)
[17] ANSON M L.The estimation of pepsin,trypsin,papain,and cathepsin with hemoglobin[J].J Gen Physiol,1938,22(1):79-89.
[18] DAHLQVIST A.A method for the determination of amylase in intestinal content[J].Scand J Clin Lab Invest,1962,14(2):145-151.
[19] WIRNT R.Trypsin:measurements with Nα-p-toluene-sulphonyl-L-arginine methyl ester as substrate[M]//BERGMEYER H U.Methods of Enzymatic Analysis.New York:Verlag Chemie,1974:1021-1024.
[20] WIRNT R.Chymotrypsin measurements with N-benzoyl-L-tyrosin methyl ester as substrate[M]//BERGMEYER H U.Methods of Enzymatic Analysis.Weinheinm:Verlag Chemie,1974:1009-1012.
[21] LOW A G.Nutritional regulation of gastric secretion,digestion and emptying[J].Nutr Res Rev,1990,3(1):229-252.
[22] LAWRENCE T L J.The effect of certain dietary factors on in vivo pH changes and pepsin activity in the stomach of the growing pig[J].Br Vet J,1972,128(8):402-411.
[23] LAWLOR P G,LYNCHP B,CAFFREY P J,et al.Measurements of the acid-binding capacity of ingredients used in pig diets[J].Ir Vet J,2005,58(8):447-452.
[24] MAKKINK C A,NEGULESCU G P,QIN G X,et al.Effect of dietary protein source on feed intake,growth,pancreatic enzyme activities and jejunal morphology in newly-weaned piglets[J].Br J Nutr,1994,72(3):353-368.
[25] ZEBROWSKA T,LOW A G,ZEBROWSKA H.Studies on gastric digestion of protein and carbohydrate,gastric secretion and exocrine pancreatic secretion in the growing pig[J].Br J Nutr,1983,49(3):401-410.
[26] PARTRIDGE I G,LOW A G,SAMBROOK I E,et al.The influence of diet on the exocrine pancreatic secretion of growing pigs[J].Br J Nutr,1982,48(1):137-145.
[27] BOTERMANS J A M,PIERZYNOWSKI S G.Relations between body weight,feed intake,daily weight gain,and exocrine pancreatic secretion in chronically catheterized growing pigs[J].J Anim Sci,1999,77(2):450-456.
[28] THAELA M J,PIERZYNOWSKI S G,JENSEN M S,et al.The pattern of the circadian rhythm of pancreatic secretion in fed pigs[J].J Anim Sci,1995,73(11):3402-3408.
[29] DAL BORGO G,SALMAN A J,PUBOLS M H,et al.Exocrine function of the chick pancreas as affected by dietary soybean meal and carbohydrate[J].Proc Soc Exp Biol Med,1968,129(3):877-881.
[30] JOHNSON L R.Gastrointestinal Physiology[M].Philadelphia:Mosby Elsevier,2007.
[31] WENK C.The role of dietary fibre in the digestive physiology of the pig[J].Anim Feed Sci Technol,2001,90(1-2):21-33.
[32] LOW A G.The activity of pepsin,chymotrypsin and trypsin during 24 h periods in the small intestine of growing pigs[J].Br J Nutr,1982,48(1):147-159.
[33] 茹炳根,杜锦珠,曾耀辉,等.猪胰蛋白酶自溶后的活性产物[J].北京大学学报,1979(4):32-47.RU B G,DU J Z,ZENG Y H,et al.Active products of porcine trypsin after autolysis[J].Acta Scicentiarum Naturalum Universitis Pekinesis,1979(4):32-47.(in Chinese)