潜在的饲料添加剂在瘤胃液中的稳定性

1、毕 业 设 计（论 文）外 文 参 考 资 料 及 译 文潜在的饲料添加剂在瘤胃液中的稳定性学生姓名： 学号： 专业： 动物科学 所在学院： 动物科学与技术学院 指导教师： 职称： 副教授 stability and stabilization of potential feed additive enzymes in rumen fluiddiego p. morgavia, 1, c.james newbolda，david e. beeverb ，r.john wallaceabstractsome activities, including the -1,4-endoglucanase

2、 and xylanase from the extract derived from aspergillus niger, were stable for at least 6 h in rumen fluid. the same activities in the other extracts also retained substantial activity for several hours. -glucosidase and -xylosidase activities were much more labile. most being almost completely dest

3、royed after 1. and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most proteins in the extracts were digested extensively after up to 7 h of incubation. adding bovine serum albumin (0.5 g/l) to the incubation increased the half-life of trichoderma viride -glucosidase activi

4、ty from less than 0.5 h to 3 h. proteins extracted from plant materials, particularly the soybean 7s globulin fraction, also conferred protection from proteolytic breakdown，but none was as effective as bovine serum albumin. it was concluded that the stability of most fibrolytic enzymes in rumen flui

5、d, is not likely to be a limiting factor in the use of enzymes as feed additives for ruminants. but if the enzymes are not stable, means can be found for their stabilization.keywordsenzymes; ruminants; cellulase; xylanase; stability; proteolysis1. introductionindustrially produced enzymes are increa

6、singly being used as feed additives to improve the nutritional efficiency of farm animals. their application in ruminant diets is still under development. early results show promise, although many factors such as the precise nature of beneficial enzymes and the diet specificity of the response, rema

7、in to be resolved. a potential problem, which was identified previously by kopecny et al. is that protein in the diet, particularly soluble protein, is usually degraded rapidly in the rumen. thus, if dietary enzymes are to be effective as modifiers of rumen fermentation, the enzymes must resist prot

8、eolysis by rumen microorganisms for a time sufficiently long to affect digestion.the rate and extent of hydrolysis of individual proteins are affected by their chemical structure: most commercial enzyme preparations are the product of fungal fermentation, predominantly by trichoderma and aspergillus

9、 species, and they consist of a mixture of hydrolytic enzymes. as some of these are used as feed additives in nonruminants, they presumably resist degradation by gastric and pancreatic proteinases and may have structures that are also resistant to rumen microbial proteases.the objective of this stud

10、y was to determine the resistance of the enzyme activities in potential feed additives to rumen microbial proteolytic activity, and to investigate means of enhancing their resistance to proteolytic breakdown.2. materials and methods2.1. enzyme preparationsthe enzymes used were powdered commercial pr

11、eparations from fungal extracts that contained a variety of plant polysaccharide hydrolases. 2.2. incubations with rumen fluidrumen inocula were obtained from four rumen-fistulated sheep receiving a mixed diet consisting of hay, barley, molasses, fish meal, and minerals and vitamins fed in equal mea

12、ls of 500 g at 8 a.m. and 4 p.m. rumen fluid was removed before the morning feeding and strained through four layers of muslin. enzyme preparations, calculated to have similar carboxymethylcellulase (cmcase) activities, were added to hungate tubes and dissolved in 1 ml of 0.1 m anaerobic sodium phos

13、phate buffer, ph 6.5. then 9 ml of strained rumen fluid were added, the tubes were incubated at 39c under o2-free co2 and samples were removed periodically into microcentrifuge tubes on ice. protein concentration in the incubation mixture ranged from 0.15 to 0.30 g/l. particulate material was remove

14、d by centrifugation at 15 000 g for 15 min. the supernatant was stored at 80c until analyzed.2.3. stabilization of enzyme activitythe influence of bovine serum albumin (bsa, sigma, st. louis, mo, usa) or plant proteins on enzyme stability was examined by using the t. viride preparation. incubations

15、were performed as described above, with the experimental proteins being added to the enzyme solution before addition of rumen fluid. plant proteins used were maize zein (sigma) and those obtained from soybean and rice flours. soluble soybean proteins were extracted with 15 volumes (w/v) of 0.03 m tr

16、is-hcl buffer, ph 8, containing 10 mm2-mercaptoethanol for 1 h. the slurry was mixed thoroughly at 5-min intervals and centrifuged at 45 000 g for 20 min to obtain the soluble protein fraction in the supernatant.2.4. enzyme assaysendocellulase, xylanase, and amylase activities were assayed by using

17、medium viscosity carboxymethyl cellulose (cmc), oat spelt xylan, and soluble wheat starch as substrates, respectively, and all were obtained from sigma. assays were carried out by adding 50 l of sample to a tube containing 100 l of 0.1 m sodium citrate/phosphate buffer, ph 5, and 50 l of 2% cmc, 2%

18、oat spelt xylan, or 1% wheat starch, respectively. the mixture was incubated at 39c for 1 h for cmcase, 30 min for xylanase or 4 h for amylase. the reaction was stopped by adding somogyi reagent, and reducing sugars generated were measured by the nelsonsomogyi method. incubations were also carried o

19、ut with sample in the absence of added substrate and with enzyme solutions alone plus buffer in place of rumen fluid. each sample was incubated in duplicate and results are expressed as means of samples from four sheep.glycosidase activities were measured in 96-well plates. each well contained 10 1

20、of sample, 80 l of 0.1 m sodium citrate/phosphate buffer, ph 5, and 10 l of 50 m 4-methylumbelliferyl-d-glucopyranoside(sigma) for -glucosidase and -xylosidase, respectively. substrates were prepared by diluting 20 l of 25 mm stock dimethylformamide solution in 10 ml of water. the plates were incuba

21、ted at 39c for 5 min for -glucosidase or 60 min for -xylosidase, then the reaction was stopped by adding 100 l of 1 m glycine-naoh buffer, ph 10.6. release of 4-methylumbelliferone was measured fluorometrically at 365-nm excitation and 450-nm emission. each assay was carried out in triplicate. resul

22、ts are expressed as means samples from four sheep.the proteolytic activity of strained rumen fluid was measured by using digesta taken immediately before feeding, and also using digesta taken 2 h after feeding. proteolytic activity was measured by the rate of breakdown of casein, which had been labe

23、led with 14c by reductive methylation。 the results are means of duplicate incubations with each sheep.2.5. electrophoretic analysis of enzymic propertieselectrophoresis was performed with a multiphor ii electrophoresis system and excelgel sds 818% precast gradient polyacrylamide gels (pharmacia, st.

24、 albans, herts al1 3aw, uk). samples were treated with sds under reduced (in the presence of dl-dithiothreitol) or nonreduced conditions as described by pharmacia. proteins were stained with coomassie blue or silver staining and glycoproteins were detected with a glycoprotein detection kit (sigma) b

25、ased on the periodic acid-schiff reagent method. samples were obtained after incubation of the enzyme preparations with rumen fluid. rumen fluid from three animals obtained as described above was pooled and incubated with the enzyme preparations under co2 for 7 h in a shaking water bath at 39c. afte

26、r incubation, particulate material was removed by centrifugation and the supernatant was dialyzed for 24 h against distilled water. sample aliquots were stored at 80c until electrophoresis. for 0 h incubations, chilled rumen fluid was added to the enzymes and kept on ice for 1 to 2 h to allow enzyme

27、 solubilization.3. results3.1. addition of enzymes to rumen fluidthe enzyme mixtures were added to rumen fluid in quantities sufficient to give a significant increase in the total extracellular cmcase activity of rumen fluid. as a consequence, xylanase, amylase, -glucosidase, and -xylosidase activit

28、ies also increased. all of these enzyme activities were measured against a background of similar activities already present in rumen fluid. however, because the assays were carried out by using supernatants after centrifugation, from which microorganisms had been removed, the interference in measuri

29、ng the added enzymes was not great. furthermore, all enzyme activities present in extracellular rumen fluid remained relatively constant throughout the incubations 。3.2. proteinase activity of rumen fluidthe proteinase activity of strained rumen fluid was measured by using 14c-labeled casein. proteo

30、lytic activity in the four sheep was 2.10, sd 0.54 and 2.19, sd 0.57 mg casein hydrolyzed/h/ml strained rumen fluid immediately before and 2 h after feeding, respectively.3.3. stability of enzyme activity in rumen fluid-1,4-endoglucanase activity in the a. niger preparation, measured by the release

31、of reducing sugars from cmc, was particularly stable when added to rumen fluid in vitro, with no apparent decrease in activity after 6 h of incubation in rumen fluid. the other enzyme preparations had a half-life of 2 h for t. viride and about 4 h for i. lacteus and preparation m. activity against x

32、ylan was more stable than the cmcase activities, except for i. lacteus, which lost 60% of its xylanase after 2 h of incubation. a. niger xylanase was not affected by the incubation, whereas t. viride and preparation m retained nearly 75% of the original activity at the end of the incubation period.

33、amylase was not a predominant activity in any the preparations. the highest amylase activities were detected in preparation m and t. viride, but they were not stable. the a. nigerenzyme was again the most stable 。3.4. relation between enzyme stability and chemical structurethe electrophoretic mobili

34、ty of proteins present in the enzyme extracts was determined in sds-page, with and without reduction, and before and after incubation with rumen fluid. multiple protein bands were present in all of the extracts, particularly i. lacteus, and most of these bands disappeared after incubation in rumen f

35、luid. however, certain bands appeared to survive after 7 h of incubation. the bands that survived this incubation were different in the presence and absence of dithiothreitol, indicating that they were cross-linked by disulfide bonds. in general, however, the banding patterns in the presence and abs

36、ence of dithiothreitol were completely different, and it was not possible to correlate the banding patterns. an attempt was made to associate this characteristic with the presence of cystine in the protein molecule, by analysis of bands excised from the gels, but no conclusive results were found. ge

37、ls were stained for glycosylated proteins: glycosidation was a common feature in all the enzyme preparations (results not shown). however, it appeared that there was no direct relation between resistance to proteolysis and the presence of sugar moieties in proteins.4. discussionthe effectiveness of

38、dietary enzymes in nonruminants is assumed to stem partly from their marked resistance to proteolytic degradation if enzymes are to be effective in diets fed to ruminants, it is reasonable to assume that similar resistance will have to be among their properties. indications from an earlier study wer

39、e that cellulases from t. viride were not stable when incubated with rumen digesta. the rumen microbial population is generally perceived, because of the significance of rumen proteolysis in depriving the host animal of much of the protein it consumes, as being highly proteolytic. in fact, in microb

40、ial terms the activity is not particularly high; it is the high microbial biomass and long residence time of protein in the digesta, which causes the high extent of breakdown of susceptible proteins. not all proteins are susceptible to rapid breakdown, depending on their solubility and structure. th

41、e susceptibility to ruminal breakdown of the proteins and enzyme activities present in several commercial enzyme preparations was therefore measured in order to assess whether fibrolytic enzymes were likely to be sensitive to ruminal proteolysis, and if this was likely to be an obstacle to their pot

42、ential application as feed additives for ruminants.the experiments were done by adding enzyme mixtures to rumen fluid taken from sheep receiving a grass hay/concentrate diet. the rumen fluid was strained through muslin to remove large particulate material and therefore to aid pipetting. the rumen fl

43、uid was also taken before the morning feeding in order to minimize problems associated with soluble sugars being present in the rumen fluid after feeding, which would affect the subsequent measurement of glycanase activities. it was established that the proteolytic activity of rumen fluid immediatel

44、y before and 2 h after feeding was similar, suggesting that variation in proteinase according to the time of sampling would not have a large bearing on the results; however, the straining process would remove many of the solids-associated microorganisms, and the proteolytic activity of the strained

45、rumen fluid would inevitably be lower than whole digesta. the extent of this underestimate was not determined; however, another advantage of taking prefeeding samples would be that the solids-associated microbial population would presumably be minimal at this time.after incubation of the enzyme prep

46、arations with the strained rumen fluid, small particulate material, principally microorganisms, was removed by centrifugation. the background cell-free activity due to rumen fluid was usually much lower than the activity of enzymes added, and it was in any case fairly stable. it should be noted that

47、 enzyme activity determinations were performed at ph 5, closer to the optimal ph of most fungal enzymes than the usual ph of rumen fluid, which is 66.5.it is concluded that the stability of fibrolytic enzymes in rumen fluid is unlikely to be a limiting factor in their application as feed additives f

48、or ruminants. particular enzyme activities may, however, be more labile than others. if such an activity is a key component of the mode of action of feed-additive enzymes for ruminants, it would appear likely that enzyme activity might be protected by simple means. the emphasis should now be placed

49、on identifying which, if any, fibrolytic activity is limiting in the rumen and on devising means of amplifying that activity. applying enzymes as feed supplements promises to be the simplest technology for achieving such an amplification in the immediate future.潜在的饲料添加剂在瘤胃液中的稳定性摘要来源于黑曲霉的提取物如-1,4-葡聚糖

50、酶和木聚糖酶在瘤胃液中的稳定性长达至少六个小时，来自其他提取物的相同酶的活性也能维持数小时。-葡萄糖苷酶和-木糖苷酶的活性更加不稳定，大部分是一小时之后完全被破坏。十二烷基硫酸钠聚丙烯酰胺凝胶电泳表明,长达7小时之后，提取物中的大部分蛋白被充分消化. 潜伏期中添加牛血清白蛋白（0.5克/升）可增加木霉-葡萄糖苷酶的活性半衰期小于0.5小时至3小时.从植物中提取的蛋白质尤其是大豆7s球蛋白部分,也避免了蛋白的水解作用，但没有血清蛋白那么有效。得出的结论是瘤胃液中大多数纤维素酶的稳定性,作为反刍动物的饲料添加剂纤维素酶似乎不是酶使用的限制性因素。如果酶使用不稳定，可以找到方法使它们变得更稳定。.关

51、键字酶活性; 反刍动物; 纤维素酶，木聚糖酶的稳定性; 蛋白水解1. 介绍酶工业化生产越来越多地被用作饲料添加剂，以改善农场动物营养效率。它们在反刍动物中的应用仍处于开发阶段。早期的研究结果表明虽然许多因素如有益的酶的精确性质和和相应的饮食特性仍然需要解决。一个潜在的问题，这是以前由kopecny等提出的。这个问题是关于饮食中的蛋白质问题，尤其是可溶性蛋白会在瘤胃中迅速降解。因此，如果应用的酶和瘤胃发酵的修饰物一样有效，这些酶必须抵制蛋白酶水解，而蛋白酶水解是由于瘤胃微生物污染引起，长此以往会影响消化。个别蛋白质水解的速度和程度受其化学结构影响。大多数的商用酶制剂是真菌发酵产品，这些真菌产品主

52、要是木酶和黑种，它们构成了水解酶混合物。其中一些作为饲料添加剂在瘤胃中使用。他们大概抵抗胃和胰蛋白酶的降解，并可能有抗瘤胃微生物蛋白酶的结构。本研究的目的是确定潜在的饲料添加剂的酶活动抵抗瘤胃微生物蛋白活性，并调查它们抵抗水解破坏这一能力提高的方法。2. 材料与方法2.1 酶制剂商业制剂，是含有多种植物多糖水解酶的真菌提取物粉末使用的酶。待添加的隐藏文字内容22.2 瘤胃液培养瘤胃接种体是从绵羊的四个瘤胃瘘管中获得的，这些绵羊所接受的混合饲料是由干草，大麦，糖蜜，鱼粉还有多种矿物质，维它命组成等于500克的膳食喂养上午8时和下午4点，瘤胃液在早晨喂奶前被清除掉并通过四层纱布过滤。酶制剂，计算有

53、类似carboxymethylcellulase（纤维素酶）的活动，被添加到hungate管和溶解于1毫升0.1米厌氧钠磷酸盐缓冲液，ph值6.5。过滤的瘤胃液为9毫升，管培养在39c下充满二氧化碳，保持厌氧状态。样品排到离心管中，这些离心管放在冰上。蛋白质在发酵物中的浓度介于0.15至0.30克/升。除掉颗粒物质，在15 000g下离心15分钟。上清液保存在-80c，直到分析。2.3 酶活性的稳定性使用绿色木霉制剂对牛血清白蛋白（bsa，sigma公司，圣路易斯，密苏里州，美国）或植物蛋白质酶的稳定性的影响进行了检查。实验的蛋白质再被添加到瘤胃液之前被添加到酶液中。使用的植物蛋白即玉米醇溶蛋

54、白（sigma公司）是从大豆和大米面粉获得的。0.03l的tris-hcl缓冲液，ph8含2- 巯基乙醇为1小时10ml可提取可溶性大豆蛋白。泥浆混匀，每隔5分钟和45 000g下离心20分钟获得上清可溶性蛋白含量。2.4酶活性的测定通过使用介质粘度羧甲基纤维素（cmc）对纤维素酶，木聚糖酶，淀粉酶活性进行检测。将燕麦木聚糖和可溶性小麦淀粉各自作为底物，这些都来自sigma公司。在50l的样品管中加入含有100l0.1 m的柠檬酸钠/磷酸盐缓冲液，ph值5，和50l2的cmc或1，2，燕麦木聚糖，小麦淀粉，分别进行了检测。混合物在39c，纤维素酶，木聚糖酶或4小时的淀粉酶30分钟为1培养。反应

55、停止加somogyi试剂，和还原糖生成的nelson-somogyi法11测定。，在没有增加基板和酶液加缓冲瘤胃的基础上样品发酵液开始进行。每个样品重复培养，结果以来自四只羊样品作为方法进行表示。糖苷酶的活性测定在96孔板。每口井含有10180l0.1 m的柠檬酸钠/磷酸盐缓冲液，ph值5，分别含10l50m4-methylumbelliferyl-d-葡萄糖苷-葡萄糖苷酶和-木糖苷酶（sigma公司）的样本。基板制备20l25毫米股票二甲基甲酰胺溶液的稀释在10毫升的水。该板块在39c孵育-葡萄糖苷酶达60分钟，-木糖苷酶达5分钟，然后停止反应，加入100l1m甘氨酸-naoh缓冲，ph值10.6。荧光分光光度法在365nm激发，450nm发射的4 -甲基伞形酮释放。每个检测进行了一式三份。结果表示以从四个羊的样品为方法。过滤的瘤胃液中蛋白活性测定通过在饲喂前立即采