Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 9: 5911 - 5922

DOI: https://doi.org/10.12418/CJAN2025.481

RESEARCH PAPER

Degradation Effects of Thermostable Acid Protease on Soybean Meal Antigen Protein and Its Effects on Growth Performance and Apparent Nutrient Metabolic Rates of Yellow Feathered Chickens

  • CHEN Dandie , 1 ,
  • WANG Min 1, 2 ,
  • LIU Ge 1 ,
  • HUANG Zhiyi 1, 2 ,
  • LI Yangyuan 1 ,
  • ZHANG Cheng 3 ,
  • DING Jianhua 4 ,
  • HONG Xiaohua 5 ,
  • GUO Yuguang , 1, 4, *
Expand
  • 1 Guangdong VTR Biotechnology Co., Ltd., Zhuhai 519060, China
  • 2 Zhuhai Hengqin Yitai Biotechnology Co., Ltd., Zhuhai 519060, China
  • 3 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
  • 4 Anhui Muxiang Poultry Industry Co., Ltd., Lu’an 237400, China
  • 5 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
* E-mail:

Received date: 2024-11-19

  Online published: 2025-09-12

Fold

Abstract

This experiment was conducted to investigate the temperature tolerance of thermostable acid protease (TAP), degradation characteristics of soybean meal antigen protein, and the effects of its supplementation in low protein corn-soybean meal type diet on growth performance and nutrient apparent metabolic rates of yellow feathered chickens. In vitro experiment was conducted to determine the temperature tolerance of TAP by water bath method and granulation method. The degradation effects of different addition amounts of TAP [0 (blank), 2, 4, 6 and 8 U/g] on antigen protein in soybean meal raw materials were analyzed. Meanwhile, the effects of TAP on the protein digestibility in corn-soybean meal type diet were determined through bionic digestion experiment. In vivo, 480 healthy one-day-old fast-growing yellow feathered male chickens were selected and randomly divided into 6 groups with 8 replicates in each group and 10 chickens in each replicate. Chickens in the normal protein group were fed a normal protein level corn-soybean meal type basal diet, those in the low protein group were fed a low protein level corn-soybean meal type diet, and those in experimental groups 1 to 4 were fed the diets supplemented with 2, 4, 6 and 8 U/g TAP on the basis of the low protein group diet, respectively. The experiment lasted for 56 days. The results showed as follows: 1) TAP still retained a relatively high activity (retention rate of 80.00%) under the granulation condition of 80 ℃ and had strong temperature tolerance. 2) Compared with the soybean meal raw materials and the blank group, the supplementation of different doses of TAP significantly increased the acid-soluble protein (ASP) content (P<0.05), and the supplementation of 4 to 8 U/g TAP significantly decreased the contents of glycinin and β-conglycinin (P<0.05). 3) In the bionic digestion experiment, compared with the blank group, the supplementation of 4 to 8 U/g TAP significantly increased the digestibilities of dry matter (DM) and crude protein (CP) (P<0.05). 4) From 1 to 21 days of age, compared with the normal protein group, the average daily feed intake (ADFI) and feed to gain ratio (F/G) in the experimental groups were significantly increased (P<0.05); compared with the low protein group, the ADFI in experimental groups 1 and 2 was significantly increased (P<0.05). From 22 to 42 days of age, compared with the normal protein group and the low protein group, the F/G in experimental group 2 was significantly decreased (P<0.05). From 43 to 56 days of age, compared with the normal protein group and the low protein group, there was a decreasing trend of F/G in experimental groups 2 to 4 (P=0.068). 5) Compared with the normal protein group, the apparent metabolic rates of DM, CP and gross energy (GE) in the experimental groups were significantly increased (P<0.05), and the apparent metabolic rate of phosphorus in experimental group 4 was significantly increased (P<0.05). Compared with the low protein group, the apparent metabolic rates of DM, CP and GE in experimental group 4 were significantly increased (P<0.05), and the apparent metabolic rate of calcium in experimental groups 2 to 4 was significantly increased (P<0.05). To sum up, TAP has strong temperature tolerance, which can increase the ASP content in soybean meal and reduce the antigen protein content. The supplementation of 4 to 8 U/g TAP in low protein diet of yellow feathered chickens can increase the nutrient apparent metabolic rates and improve their growth performance.

Key words： thermostable acid protease; antigen protein; low protein diet; yellow feathered chickens; growth performance; nutrient apparent metabolic rates

Cite this article

CHEN Dandie , WANG Min , LIU Ge , HUANG Zhiyi , LI Yangyuan , ZHANG Cheng , DING Jianhua , HONG Xiaohua , GUO Yuguang . Degradation Effects of Thermostable Acid Protease on Soybean Meal Antigen Protein and Its Effects on Growth Performance and Apparent Nutrient Metabolic Rates of Yellow Feathered Chickens[J]. Chinese Journal of Animal Nutrition, 2025 , 37(9) : 5911 -5922 . DOI: 10.12418/CJAN2025.481

在粮食作物和其他农作物中,我国玉米种植面积最大,且在畜牧业中的饲用量高达64%[1-2]。国内蛋白质饲料来源紧缺,大部分的豆粕依赖于国外进口,这使得成本控制、饲料资源的有效利用和动物生长性能的优化成为畜牧业的关注重点[3]。豆粕作为一种重要的植物蛋白质源,因其蛋白质含量高、氨基酸组成相对平衡而广泛应用于畜禽饲粮中[4-5]。然而,豆粕中含有大豆球蛋白和β-伴大豆球蛋白等抗原蛋白,其不仅难以被动物消化吸收,还可能引发肠道免疫反应,影响动物的生长性能和健康[6-8]。因此,如何有效降解豆粕中的抗原蛋白、提高其营养价值成为饲料科学领域的重要研究方向。
近年来,低蛋白质饲粮的应用备受关注。在家禽生产中,饲粮蛋白质含量超出适宜水平是不经济且有害于环境的[9],且多余的蛋白质被分解提供能量并以尿氮的形式排出[10]。有研究指出,幼龄动物肠道发育不完善、内源消化酶分泌不足,饲喂高蛋白饲粮易导致消化不良而引起腹泻,造成蛋白质资源浪费[11]。因此,为了降低生产成本以及减轻环境负担,可将动物饲粮配方中的蛋白质含量在不影响生长的情况下适当降低[12]。然而,低蛋白质饲粮中植物蛋白质源的抗营养因子(如豆粕抗原蛋白),可能进一步影响动物的消化吸收效率,引起过敏产生腹泻[13],从而降低动物对大豆蛋白质的消化率[14-16],抑制动物生长。因此,需要采取有效措施来降解这些抗营养因子,以提高饲料的利用率。研究表明,降低抗原蛋白含量可提高养分消化率[17]和动物生长性能[18]。马倩倩等[19]研究表明,在饲粮中添加外源性蛋白酶可以补充内源蛋白酶分泌的不足,提高蛋白质消化率,促进动物生长性能。朱青青等[20]研究表明,在生长猪饲粮中添加蛋白酶可用菜籽粕替代豆粕,并降低生产成本。
耐高温酸性蛋白酶(thermostable acid protease,TAP)作为一种能够在高温和酸性环境下保持活性的酶制剂,将其添加于饲粮中可提高养分消化率,降低饲料成本,并减少粪便中氮的排出[21]。TAP在高温制粒过程中仍能保持较活性,可为其在饲料行业中的应用提供重要优势。此外,TAP还可降解豆粕中的抗原蛋白,减少其对动物肠道的负面影响,从而在改善动物生长性能和提高养分消化率的同时降低饲料成本。黄羽肉鸡是我国重要的肉鸡品种之一,其生长性能和饲料转化效率直接影响养殖效益。然而,目前关于TAP耐温性和对豆粕抗原蛋白的降解效果,以及其应用于低蛋白质饲粮中对黄羽肉鸡生长性能和养分表观代谢率影响的研究尚不充分。因此,本试验旨在比较研究TAP的耐温性和对豆粕抗原蛋白的降解效果,并评估其应用于低蛋白质饲粮中对黄羽肉鸡生长性能和养分表观代谢率的影响,为TAP在黄羽肉鸡低蛋白质饲粮中的应用提供科学依据,以减少蛋白质资源的浪费。

1 材料与方法

1.1 体外试验

1.1.1 酸性蛋白酶(acid protease,AP)活性测定

参考GB/T 23527.1—2023,采用福林(Folin)法检测AP活性。在酸性(pH=3.0)条件下,将福林试剂还原,生成钼蓝和钨蓝,使用分光光度计于680 nm下测定溶液吸光度。酶活性与吸光度成正比,由此可计算产品的酶活性。

1.1.2 TAP耐温性测定

水浴法:将AP和TAP样品用pH=3.0的乳酸钠缓冲液提取并稀释,取5 mL酶液分别置于70、75和80 ℃的恒温水浴锅中准确处理3 min,取出迅速放入冰水中冷却3 min,按照1.1.1中方法测定处理和未处理的AP和TAP活性,并计算保留率。
制粒法:按照肉鸡饲粮的制粒流程,把AP和TAP分别按照2、4、6和8 U/g添加到饲粮中,经80 ℃制粒冷却后取样,按照1.1.1中方法测定AP和TAP活性,并计算保留率。

1.1.3 TAP对豆粕原料中抗原蛋白含量的影响

试验分为5组(每组6个重复),其中空白组不添加酶制剂,试验1组~试验4组分别添加2、4、6和8 U/g TAP。试验准确称取30 g豆粕原料于150 mL三角瓶中,按料液比3∶2与样品混合均匀,封上封口膜并置于60 ℃恒温水浴锅中水浴酶解12 h,结束后将样品转移至65 ℃烘箱中烘干,回潮24 h后制成风干样品,测定豆粕酶解前后的干物质(DM)、粗蛋白质(CP)、酸溶蛋白(acid-soluble protein,ASP)、大豆球蛋白和β-伴大豆球蛋白含量。

1.1.4 仿生消化添加TAP对玉米-豆粕型饲粮蛋白质消化率的影响

试验分为5组,每组5个重复,每个重复1根消化管。试验饲粮为玉米-豆粕型正常蛋白质水平基础饲粮,其中空白组不添加酶制剂,试验1组~试验4组分别添加2、4、6和8 U/g TAP。上机操作参考《单胃动物仿生消化系统操作手册(第4版)》[22]鸡程序,最后测定消化前饲粮与消化后残渣DM和CP含量并计算消化率。

1.2 体内试验

1.2.1 试验设计和饲养管理

选用健康1日龄快大型黄羽肉鸡公鸡480只,随机分为6组,每组8个重复,每个重复10只鸡。饲粮为玉米-豆粕型,其中正常蛋白质饲粮组为正对照组(下称正常蛋白质组),低蛋白质饲粮组为负对照组(下称低蛋白质组),试验1组~试验4组分别在低蛋白质组饲粮基础上添加2、4、6和8 U/g TAP。试验期56 d,分为1~21日龄、22~42日龄和43~56日龄3个阶段。TAP的产酶基因来自离蠕孢属(Bipolaris)菌,由毕赤酵母进行表达。
试验在广东溢多利生物科技股份有限公司动物试验基地的鸡舍进行,试验期间严格按管理手册与免疫程序进行管理和接种。肉鸡进行笼养,采取颗粒料饲喂,自由采食和饮水。所有试验程序均经广东溢多利生物技术有限公司动物护理和使用委员会批准(批准编号:2021307)。

1.2.2 试验饲粮

试验饲粮为玉米-豆粕型,参考《鸡饲养标准》(NY/T 33—2004)进行配制,其组成及营养水平见表1。
表1 饲粮组成及营养水平(风干基础)

Table 1 Composition and nutrient levels of diets (air-dry basis) %

项目
Items		 1~21日龄
1 to 21 days of age		 22~42日龄
22 to 42 days of age		 43~56日龄
43 to 56 days of age
正常蛋白质组
Normal protein
group		 低蛋白质组
Low protein
group		 正常蛋白质组
Normal protein
group		 低蛋白质组
Low protein
group		 正常蛋白质组
Normal protein
group		 低蛋白质组
Low protein
group
原料Ingredients
玉米Corn 53.60 57.30 62.54 63.00 63.00 64.00
豆粕Soybean meal (42.5% CP) 36.00 33.20 31.32 27.80 27.78 25.10
次粉Wheat middling 4.00 3.50
米糠Rich bran 2.80 1.60 3.31
石粉Limestone 1.40 1.17 1.18 1.20 1.30 1.30
磷酸氢钙CaHPO4 1.60 1.70 1.50 1.50 1.32 1.31
大豆油Soybean oil 2.44 2.08 2.70 2.80 4.42 4.40
食盐NaCl 0.30 0.30 0.30 0.30 0.30 0.30
赖氨酸Lys (98.8%) 0.21 0.36 0.11 0.20
蛋氨酸Met (99%) 0.25 0.19 0.15 0.20 0.08 0.08
预混料Premix1) 0.20 0.20 0.20 0.20 0.20 0.20
合计Total 100.00 100.00 100.00 100.00 100.00 100.00
营养水平Nutrient levels2)
代谢能ME/(MJ/kg) 12.27 12.28 12.54 12.59 13.01 13.05
粗蛋白质CP 20.39 19.30 18.42 17.28 16.83 15.97
粗脂肪EE 4.80 4.34 5.12 5.39 7.20 7.15
钙Ca 0.92 0.85 0.86 0.86 0.84 0.70
总磷TP 0.60 0.62 0.60 0.61 0.62 0.64
赖氨酸Lys 1.24 1.33 1.03 1.05 0.85 0.80
蛋氨酸Met 0.57 0.50 0.45 0.49 0.37 0.36
蛋氨酸+半胱氨酸Met+Cys 0.93 0.84 0.79 0.81 0.68 0.66
色氨酸Trp 0.28 0.26 0.25 0.23 0.23 0.22
苏氨酸Thr 0.70 0.67 0.64 0.61 0.60 0.58

1)预混料为每千克饲粮提供 The premix provided the following per kg of diets:VA 8 000 IU,VD3 3 000 IU,VE 20 IU,VK3 2 mg,VB1 4 mg,VB2 3.6 mg,VB6 4 mg,VB12 0.02 mg,烟酸 nicotinic acid 40 mg,D-泛酸 D-pantothenic acid 12 mg,生物素 biotin 0.15 mg,叶酸 folic acid 1.0 mg,抗氧化剂 antioxidant 100 mg,Cu (as copper sulfate) 10 mg,Fe (as ferrous sulfate) 80 mg,Mn (as manganese sulfate) 80 mg,Zn (as zinc sulfate) 75 mg,I (as potassium iodide) 0.40 mg,Se (as sodium selenite) 0.30 mg。

2)粗蛋白质、粗脂肪、钙和总磷为实测值,其他营养水平根据《中国饲料成分及营养价值表(2012年第23版)》使用Brill配方软件计算。CP, EE, Ca and TP were measured values, while the other nutrient levels were calculated using Brill formula software based on Tables of Feed Composition and Nutritive Values in China (23rd edition, 2012).

1.3 测定指标及方法

1.3.1 ASP含量测定

ASP含量参考NY/T 3801—2020中方法适当修改后进行测定。准确称取3.6 g样品于50 mL离心管中,加入30 mL 15%的三氯乙酸(trichloroacetic acid,TCA)混合均匀静置20 min,随后于离心机2 810×g离心10 min,取10 mL上清液进行测定。
ASP含量计算公式如下:
ASP含量(%)=100×[(样品滴定盐酸体积-空白滴定盐酸体积)×盐酸浓度×0.014×6.25×(30/10)]/(样品质量×样品DM含量)。
式中:0.014为氮的毫摩尔质量;6.25为氮换算为蛋白质的平均系数;30为TCA体积;10为样品测定体积。

1.3.2 大豆球蛋白和β-伴大豆球蛋白含量测定

大豆球蛋白和β-伴大豆球蛋白含量采用酶联免疫吸附试验(ELISA)法测定,试剂盒购自天津龙科新域生物技术有限公司。

1.3.3 生长性能测定

分别于21、42和56日龄,空腹12 h后以重复为单位对试验鸡进行称重,并对余料进行统计,计算1~21日龄、22~42日龄、43~56日龄和1~56日龄的平均日增重(ADG)、平均日采食量(ADFI)和料重比(F/G);试验期间,记录试验鸡死淘情况,计算存活率。

1.3.4 养分表观代谢率测定

代谢试验采用外源指示剂法进行,提前2 d预饲含有指示剂二氧化钛(TiO2)的饲粮,52日龄09:00清粪,间隔24 h后于53~55日龄每天09:00各收1次排泄物,一共3次。将每天收集好的代谢排泄物以重复为单位混合均匀,剔除其中的皮屑、羽毛等,于65 ℃烘干、回潮24 h制成风干样品并粉碎过40目筛保存待测。
饲粮和排泄物样品中TiO2含量和养分含量的测定方法分别如下:TiO2含量参照邓雪娟等[23]的方法测定,DM含量参照GB/T 6435—2014中的方法测定,粗脂肪(EE)含量参照GB/T 6433—2006中的方法测定,CP含量参照GB/T 6432—2018中的方法测定,总能(GE)采用Parr 6400全自动氧弹热量计测定,钙含量参照NY/T 1944—2010中的方法测定,总磷含量参照GB/T 6437—2018中的方法测定。
养分表观代谢率计算公式如下:
某养分表观代谢率(%)=100-[100×(排泄物中该养分含量×饲粮中TiO2含量)/(饲粮中该养分含量×排泄物中TiO2含量)]。

1.4 数据统计分析

试验数据经Excel 2021初步整理后,采用SAS 9.2软件中的GLM模块进行单因素方差分析,并进行Duncan法多重比较。结果数据以平均值和均值标准误(SEM)表示,以P<0.05为差异显著,0.05<P<0.10为差异有显著趋势。

2 结果与分析

2.1 高温对TAP活性的影响

由表2可知,水浴法中AP和TAP活性保留率随着水浴温度的提高而降低,且TAP活性保留率高于AP;制粒法中TAP在80 ℃下依旧保留较高活性,耐温性较强。
表2 TAP高温条件下活性保留率

Table 2 Activity retention rate of TAP at high temperature %

项目
Items		 酶制剂类型
Enzyme preparation types		 保留率Retention rate
70 ℃ 3 min 75 ℃ 3 min 80 ℃ 3 min

水浴法
Water bath method		 酸性蛋白酶AP 40.00 <10.00 0.00
耐高温酸性蛋白酶TAP 85.00 55.00 35.00

制粒法
Granulation method		 酸性蛋白酶AP 30.00
耐高温酸性蛋白酶TAP 80.00

2.2 TAP对豆粕ASP和抗原蛋白含量的影响

由表3可知,与豆粕原料和空白组相比,试验组ASP含量均显著提高(P<0.05);同时,试验3组和试验4组ASP含量显著高于试验1组和试验2组(P<0.05),且试验3组与试验4组之间无显著差异(P>0.05)。与豆粕原料和空白组相比,试验2组、试验3组和试验4组大豆球蛋白和β-伴大豆球蛋白含量显著降低(P<0.05);同时,试验4组大豆球蛋白和β-伴大豆球蛋白含量显著低于其他试验组(P<0.05)。由此可知,在本试验条件下,豆粕中ASP含量随着TAP添加量的提高而提高,进而提高ASP占CP的百分比;同时,TAP能够降低豆粕原料中抗原蛋白含量,且添加8 U/g TAP较适宜。
表3 TAP对豆粕ASP和抗原蛋白含量的影响

Table 3 Effects of TAP on contents of ASP and antigen protein in soybean meal

项目
Items		 豆粕原料
Soybean
meal raw
materials		 空白组
Blank
group		 试验1组
Experimental
group 1		 试验2组
Experimental
group 2		 试验3组
Experimental
group 3		 试验4组
Experimental
group 4		 均值
标准误
SEM		 P值
P-value
粗蛋白质CP/% DM 50.78 50.95 50.85 50.85 50.70 50.80 0.234 0.984
酸溶蛋白ASP/% DM 1.77e 2.05d 2.49c 2.70b 2.86a 2.91a 0.020 <0.001
酸溶蛋白/粗蛋白质
ASP/CP/%		 3.53e 3.99d 4.90c 5.31b 5.65a 5.68a 0.026 <0.001
大豆球蛋白
Glycinin/(mg/g DM)		 283.23a 279.71a 255.93b 254.63b 247.46c 215.08d 2.418 <0.001
β-伴大豆球蛋白
β-conglycinin/
(mg/g DM)		 119.69a 104.83b 101.14bc 99.05cd 96.54d 91.23e 1.280 <0.001

同行数据肩标相同字母或无字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下表同。

In the same row, values with the same letter or no letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as below.

2.3 仿生消化添加TAP对玉米-豆粕型饲粮蛋白质消化率的影响

由表4可知,在仿生消化系统鸡程序中,DM和CP消化率随着TAP添加量的提高而提高,其中试验4组(添加8 U/g TAP)DM和CP消化率分别比空白组提高了1.73%和5.52%(P<0.05)。
表4 仿生消化添加TAP对玉米-豆粕型饲粮蛋白质消化率的影响

Table 4 Effects of TAP supplementation on protein digestibility in corn-soybean meal type diet by bionic digestion %

项目
Items		 空白组
Blank
group		 试验1组
Experimental
group 1		 试验2组
Experimental
group 2		 试验3组
Experimental
group 3		 试验4组
Experimental
group 4		 均值
标准误
SEM		 P值
P-value
干物质消化率DM digestibility 78.98c 79.22c 79.85b 79.92b 80.35a 0.116 <0.001
粗蛋白质消化率CP digestibility 73.85c 74.02c 76.31b 76.56b 77.93a 0.186 <0.001

2.4 低蛋白质饲粮中添加TAP对黄羽肉鸡生长性能的影响

由表5可知,1~21日龄,与正常蛋白质组相比,试验组ADFI和F/G显著提高(P<0.05);与低蛋白质组相比,试验1组和试验2组ADFI显著提高(P<0.05),试验组F/G无显著差异(P>0.05);试验组之间ADFI和F/G无显著差异(P>0.05)。22~42日龄,与正常蛋白质组和低蛋白质组相比,试验2组F/G显著降低(P<0.05),其他试验组F/G无显著差异(P>0.05)。43~56日龄,与正常蛋白质组和低蛋白质组相比,试验2组~试验4组F/G有降低趋势(P=0.068)。1~56日龄,各组间生长性能指标无显著差异(P>0.05)。由此可知,黄羽肉鸡1~21日龄低蛋白质饲粮中可不添加TAP,22~56日龄低蛋白质饲粮中添加4 U/g TAP可降低F/G,提高其生长性能。
表5 低蛋白质饲粮中添加TAP对黄羽肉鸡生长性能的影响

Table 5 Effects of TAP supplementation in low protein diet on growth performance of yellow feathered chickens

项目
Items		 正常蛋
白质组
Normal
protein
group		 低蛋白
质组
Low protein
group		 试验1组
Experimental
group 1		 试验2组
Experimental
group 2		 试验3组
Experimental
group 3		 试验4组
Experimental
group 4		 均值
标准误
SEM		 P值
P-value
1~21日龄1 to 21 days of age
1日龄体重
BW at 1 day of age/g		 33.64 33.70 33.66 33.80 33.65 33.66 0.082 0.753
21日龄体重
BW at 21 days of age/g		 457.50 439.63 438.25 442.00 446.69 443.63 5.696 0.210
平均日增重
ADG/(g/d)		 20.18 19.33 19.27 19.44 19.67 19.52 0.272 0.209
平均日采食量
ADFI/(g/d)		 32.96c 33.24bc 33.99a 34.01a 33.69ab 33.84ab 0.241 0.016
料重比F/G 1.63b 1.72a 1.77a 1.75a 1.71a 1.74a 0.025 0.009
22~42日龄22 to 42 days of age
42日龄体重
BW at 42 days of age/g		 1 360.00 1 319.13 1 317.25 1 348.50 1 332.38 1 320.38 15.476 0.278
平均日增重
ADG/(g/d)		 42.97 41.88 41.86 43.17 42.18 41.75 0.692 0.562
平均日采食量
ADFI/(g/d)		 92.36 89.20 89.31 88.74 91.24 90.98 1.025 0.108
料重比F/G 2.15a 2.14a 2.13a 2.06b 2.16a 2.18a 0.026 0.034
43~56日龄43 to 56 days of age
56日龄体重
BW at 56 days of age/g		 2 018.51 1 973.65 1 962.44 2 029.00 2 028.25 2 018.19 28.350 0.395
平均日增重
ADG/(g/d)		 47.04 46.75 46.08 48.61 49.71 49.84 1.215 0.147
平均日采食量
ADFI/(g/d)		 126.20 122.26 126.04 125.05 128.19 127.51 1.855 0.293
料重比F/G 2.70 2.62 2.74 2.58 2.59 2.56 0.049 0.068
1~56日龄1 to 56 days of age
平均日增重
ADG/(g/d)		 35.45 34.64 34.44 35.63 35.62 35.44 0.506 0.395
平均日采食量
ADFI/(g/d)		 78.54 76.48 77.75 77.29 78.90 78.69 0.791 0.239
料重比F/G 2.22 2.21 2.26 2.17 2.22 2.22 0.019 0.100
存活率
Survival rate/%		 98.75 98.75 98.75 100.00 100.00 100.00 0.884 0.700

2.5 低蛋白质饲粮中添加TAP对黄羽肉鸡养分表观代谢率的影响

由表6可知,与正常蛋白质组相比,试验组DM、CP和GE表观代谢率显著提高(P<0.05),试验4组磷表观代谢率显著提高(P<0.05);与低蛋白质组相比,试验4组DM、CP和GE表观代谢率显著提高(P<0.05);试验2组~试验4组钙表观代谢率显著提高(P<0.05),且与正常蛋白质组相比无显著差异(P>0.05);与正常蛋白质组和低蛋白质组相比,除试验3组EE表观代谢率显著降低(P<0.05)外,其他试验组EE表观代谢率无显著差异(P>0.05)。此外,试验4组DM、CP和GE表观代谢率显著高于其他试验组(P<0.05),磷表观代谢率显著高于试验1组和试验3组(P<0.05)。
表6 低蛋白质饲粮中添加TAP对黄羽肉鸡养分表观代谢率的影响

Table 6 Effects of TAP supplementation in low protein diet on nutrient apparent metabolic rates of yellow feathered chickens %

项目
Items		 正常蛋白质组
Normal protein
group		 低蛋
白质组
Low protein
group		 试验1组
Experimental
group 1		 试验2组
Experimental
group 2		 试验3组
Experimental
group 3		 试验4组
Experimental
group 4		 均值
标准误
SEM		 P值
P-value
干物质DM 65.68d 71.95b 70.01c 71.34bc 71.15bc 74.76a 0.446 <0.001
粗蛋白质CP 54.64c 60.04b 63.88b 60.63b 64.86b 69.44a 1.993 <0.001
粗脂肪EE 86.00a 87.92a 86.69a 88.46a 81.98b 87.18a 1.082 0.007
钙Ca 41.16ab 32.93c 32.45c 41.20ab 45.47a 39.29b 1.267 <0.001
磷P 62.05b 65.04ab 62.91b 63.85ab 62.35b 66.34a 0.908 0.033
总能GE 73.03d 77.77b 75.76c 77.46bc 76.43bc 79.55a 0.550 <0.001

3 讨论

3.1 AP酶学性质的比较

不同的蛋白酶来源于微生物发酵[24],酶在生产过程中的产量及其酶学性质均受菌株种类间的差异而影响,不同菌株发酵的AP酶学性质存在略微区别[25]。王鑫等[26]研究表明,黑曲霉酸性蛋白酶的最佳反应条件为50 ℃、pH=3。岳晓平[27]研究表明,米曲霉酸性蛋白酶最适反应条件为50 ℃、pH=4.5。袁江兰等[28]研究表明,安卡红曲霉酸性蛋白酶最适反应条件为50 ℃、pH=3.0。本试验所用TAP最适反应条件为75 ℃、pH=2.5,在高温制粒条件下可保留80%活性。此结果与李西波等[29]得出的TAP在80 ℃下仍能保留74.20%活性的研究结果相似,这表明其在高温环境下的稳定性显著优于普通蛋白酶。良好的耐高温特性使得TAP在饲料加工过程中具有广泛的应用前景,尤其是在高温制粒过程中,能够有效保持酶的活性,从而提高饲粮的营养价值。

3.2 TAP对豆粕ASP和抗原蛋白含量的影响

ASP分子质量较小,可间接反映抗原蛋白被降解的程度,其含量高则表明抗原蛋白减少[30]。酶解可有效降解豆粕中的抗原蛋白,以减少其对肠道发育不完全的幼龄动物的危害[5,31]。李旺等[32]研究表明,豆粕经过酶制剂酶解后可提高豆粕中ASP含量,降低抗原蛋白含量。龚阿琼等[33]研究表明,豆粕中添加AP可降解53.60%的β-伴大豆球蛋白。本试验中,豆粕中添加8 U/g TAP后ASP含量提高了64.41%,同时,大豆球蛋白和β-伴大豆球蛋白含量分别降低了24.06%和23.78%,这与Medeiros等[30]和李旺等[32]的研究报道一致,表明AP能够有效降解豆粕中的抗原蛋白,提高豆粕中ASP含量,改善豆粕的消化特性,从而提高其营养价值。

3.3 低蛋白质饲粮中添加TAP对黄羽肉鸡生长性能的影响

动物试验法评定饲用酶制剂有效性是最客观且真实的方法[34]。在动物生产中,对动物生长性能相关的各项指标进行统计与分析,能够更加直观地体现出饲养效果,其中生长性能相关指标包括ADFI、ADG和F/G等[35]。张巍等[36]研究表明,降低饲粮蛋白质水平会严重影响肉鸡生长性能。而Mohammadigheisar等[37]研究表明,低蛋白质饲粮中添加外源蛋白酶可提高养分消化率,从而改善肉鸡的生长性能。在本试验条件下,低蛋白质饲粮中添加TAP仅提高了仔鸡阶段的ADFI,而刘松柏等[38]研究表明,低蛋白质饲粮中添加125 g/t蛋白酶不仅降低了矮脚黄羽肉仔鸡F/G,还提高了ADG,2个试验结果不一致可能是鸡的品种、酶制剂活性、菌种来源或当地饲养天气不一致导致;本试验在低蛋白质饲粮中添加TAP有降低43~56日龄F/G的趋势,提高ADG和56日龄体重,提高1~56日龄黄羽肉鸡生长性能,这与李东东等[39]、Rehman等[40]和Freitas等[41]在低蛋白质饲粮中添加蛋白酶改善肉鸡生长性能,且主要是提高ADFI和ADG的结论相似;在22~56日龄阶段,在低蛋白质饲粮中添加4 U/g TAP降低F/G,表明添加TAP能够提高饲料利用率。综上所述,在1~21日龄黄羽肉鸡饲粮中可不添加TAP并饲喂正常蛋白质水平的饲粮,而在22~56日龄低蛋白质饲粮中添加4~6 U/g TAP可提高黄羽肉鸡生长性能。

3.4 低蛋白质饲粮中添加TAP对黄羽肉鸡养分表观代谢率的影响

饲粮中的营养成分不能被动物充分的吸收利用[42],所以在研究中通常通过对饲粮和动物代谢物中养分进行测定,并计算养分代谢率来评估动物对饲粮养分的消化和吸收性能[43]。体外仿生消化法可对饲用酶制剂有效性进行快速的评定,并具有一定的参考价值[34]。在体外仿生消化试验中,在正常蛋白质饲粮中添加8 U/g TAP,DM和CP消化率分别比空白组提高了1.73%和5.52%。McCafferty等[44]研究表明,在玉米-豆粕型饲粮中添加外源蛋白酶可提高肉鸡DM和GE表观代谢率。同时,外源酶制剂对饲粮中大分子蛋白和蛋白类抗营养物质具有分解作用,进而提高CP消化率[45-46],促进动物生长性能的提高。Lei等[47]研究表明,在育肥猪低蛋白质饲粮中添加蛋白酶可提高CP表观全肠道消化率,提高其生长性能。Angel等[48]研究表明,在肉鸡饲粮中添加蛋白酶可提高生长前期DM、CP和GE代谢率。在动物试验中,本试验在低蛋白质饲粮中添加8 U/g TAP提高了黄羽肉鸡DM、CP、钙、磷和GE表观代谢率。综上可知,在低蛋白质饲粮中添加TAP可促进机体对养分的消化吸收,从而提高黄羽肉鸡生长性能。

4 结论

① TAP经高温制粒后,其酶活性保留率比水浴法高,可解决饲料高温制粒酶失活的问题;
② TAP可提高豆粕中ASP含量,降低抗原蛋白含量;
③ 基于仿生消化,TAP可提高玉米-豆粕型饲粮DM和CP消化率;
④ 在低蛋白质玉米-豆粕型饲粮中添加4~8 U/g TAP可提高饲粮养分表观代谢率,进而提高黄羽肉鸡生长性能。

References

Publishing order | Descend order by publishing year | Descend order by cited within
[1]
陈甜. 玉米价格分析与预报[J]. 山西农经, 2022(16):8-12,34.

CHEN T. Analysis and forecast of corn price[J]. Shanxi Agricultural Economy, 2022(16):8-12,34. (in Chinese)

[2]
张万祥. 鸡蛋、玉米、豆粕期货价格联动与套利分析——基于产业链视角[D]. 硕士学位论文. 兰州: 兰州大学, 2021.

ZHANG W X. The analysis of price linkage and arbitrage of egg,corn and soy bean meal futures-based on the industry chain perspective[D]. Master’s Thesis. Lanzhou: Lanzhou University, 2021. (in Chinese)

[3]
鲁兆宁, 岳增华. 低豆粕、低蛋白水平日粮对尼古拉火鸡生长性能的影响研究[J]. 现代畜牧兽医, 2024(6):42-46.

LU Z N, YUE Z H. Effects of low soybean meal and low protein level diet on growth performance of Nicholas turkey[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2024(6):42-46. (in Chinese)

[4]
STEIN H H, BERGER L L, DRACKLEY J K, et al. Nutritional properties and feeding values of soybeans and their coproducts[M]// JOHNSONL A, WHITEP J, GALLOWAYR. processing,andutilization. New York: Academic Press and AOCS Press,2008:613-660.

[5]
MUKHERJEE R, CHAKRABORTY R, DUTTA A. Role of fermentation in improving nutritional quality of soybean meal-a review[J]. Asian-Australasian Journal of Animal Sciences, 2016, 29(11):1523-1529.

[6]
SOTAK-PEPER K M, GONZALEZ-VEGA J C, STEIN H H, et al. Concentrations of digestible,metabolizable,and net energy in soybean meal produced in different areas of the United States and fed to pigs[J]. Journal of Animal Science, 2015, 93(12):5694-5701.

[7]
KLUESS J W, KONINKX J, BIKKER P, et al. The effect of PHA-lectins and diet composition on performance and intestinal development in weaning piglets[J]. Livestock Science, 2007, 108(1/2/3):288-291.

[8]
ZHAO Y, QIN G X, SUN Z W, et al. Effects of glycinin and β-conglycinin on enterocyte apoptosis,proliferation and migration of piglets[J]. Food and Agricultural Immunology, 2010, 21(3):209-218.

[9]
SONG X R, ANAS M A, KURNIAWATI A, et al. Effects of reduced-protein diets with protease supplementation on growth,carcass yield,intestinal morphology,organ development,nutrient digestibility,and blood biochemical of broiler chickens[J]. Translational Animal Science, 2023, 7(1):txad098.

[10]
QIU K, CHEN J, ZHANG G M, et al. Effects of dietary crude protein and protease levels on performance,immunity capacity,and AA digestibility of broilers[J]. Agriculture, 2023, 13(3):703.

[11]
李洪滨, 刘延杰, 陈宁, 等. 不同蛋白质水平饲粮中添加酸性蛋白酶对肉鸡生长性能和养分表观消化率的影响[J]. 动物营养学报, 2023, 35(5):2943-2955.

DOI

LI H B, LIU Y J, CHEN N, et al. Effects of acid protease supplementation in diets with different protein levels on growth performance and nutrient apparent digestibility of broilers[J]. Chinese Journal of Animal Nutrition, 2023, 35(5):2943-2955. (in Chinese)

DOI

[12]
KOBAYASHI H, NAKASHIMA K, ISHIDA A, et al. Effects of low protein diet and low protein diet supplemented with synthetic essential amino acids on meat quality of broiler chickens[J]. Animal Science Journal, 2013, 84(6):489-495.

DOI PMID

[13]
侯金丽. 豆粕抗营养因子的检测技术研究[J]. 中国饲料, 2024(8):56-59.

HOU J L. Research on the detection technology of anti nutritional factors in soybean meal feed[J]. China Feed, 2024(8):56-59. (in Chinese)

[14]
CHENG Y H, HSIAO F S H, WEN C M, et al. Mixed fermentation of soybean meal by protease and probiotics and its effects on the growth performance and immune response in broilers[J]. Journal of Applied Animal Research, 2019, 47(1):339-348.

[15]
周天骄, 谯仕彦, 马曦, 等. 大豆饲料产品中主要抗营养因子含量的检测与分析[J]. 动物营养学报, 2015, 27(1):221-229.

DOI

ZHOU T J, QIAO S Y, MA X, et al. Detection and analysis of main antinutritional factors content in soybean products[J]. Chinese Journal of Animal Nutrition, 2015, 27(1):221-229. (in Chinese)

[16]
ERDAW M M, WU S B, IJI P A, et al. Growth and physiological responses of broiler chickens to diets containing raw,full-fat soybean and supplemented with a high-impact microbial protease[J]. Asian Austral Journal Animal, 2017, 30(9):1303-1313.

[17]
SOUMEH E A, MOHEBODINI H, TOGHYANI M, et al. Synergistic effects of fermented soybean meal and mannan-oligosaccharide on growth performance,digestive functions,and hepatic gene expression in broiler chickens[J]. Poultry Science, 2019, 98(12):6797-6807.

[18]
ZHU J J, GAO M X, ZHANG R L, et al. Effects of soybean meal fermented by L. plantarum,B. subtilis and S. cerevisieae on growth,immune function and intestinal morphology in weaned piglets[J]. Microbial Cell Factories, 2017, 16(1):191.

[19]
马倩倩, 王兆明, 陈雪颖, 等. 蛋白酶在动物生产中的应用研究进展[J]. 黑龙江畜牧兽医, 2020(23):48-51.

MA Q Q, WANG Z M, CHEN X Y, et al. Progress in the application of protease in animal production[J]. Heilongjiang Animal Science and Veterinary Medicine, 2020(23):48-51. (in Chinese)

[20]
朱青青, 余冰, 何军, 等. 豆粕和菜籽粕饲粮中添加新型蛋白酶对生长猪生长性能、养分消化率和血清生化指标的影响[J]. 动物营养学报, 2023, 35(6):3563-3573.

DOI

ZHU Q Q, YU B, HE J, et al. Effects of novel protease supplementation in soybean meal and rapeseed meal diets on growth performance,nutrient digestibility and serum biochemical indices of growing pigs[J]. Chinese Journal of Animal Nutrition, 2023, 35(6):3563-3573. (in Chinese)

[21]
KIM Y J, KIM T H, SONG M H, et al. Effects of different levels of crude protein and protease on nitrogen utilization,nutrient digestibility,and growth performance in growing pigs[J]. Journal of Animal Science and Technology, 2020, 62(5):659-667.

[22]
赵峰, 王钰明, 张虎, 等. 单胃动物仿生消化系统操作手册[M]. 4版. 北京: 中国农业科学院, 2021.

ZHAO F, WANG Y M, ZHANG H, et al. Manual instruction of monogastric animal bionic digestion system[M]. 4th ed. Beijing: Chinese Academy of Agricultural Sciences, 2021. (in Chinese)

[23]
邓雪娟, 刘国华, 蔡辉益, 等. 分光光度计法测定家禽饲料和食糜中二氧化钛[J]. 饲料工业, 2008, 29(2):57-58.

DENG X J, LIU G H, CAI H Y, et al. Spectrophotometric determination of titanium dioxide in poultry feed and chyme[J]. Feed Industry, 2008, 29(2):57-58. (in Chinese)

[24]
李昊. 酸性蛋白酶酶学性质及其在蓝湿革软化过程中的应用研究[D]. 硕士学位论文. 济南: 齐鲁工业大学, 2020.

LI H. The properties of acid protease and its application in the bating process of wet-blue[D]. Master’s Thesis. Ji’nan: Qilu University of Technology, 2020. (in Chinese)

[25]
HSU W H, PAN T M. Monascus purpureus-fermented products and oral cancer:a review[J]. Applied Microbiology and Biotechnology, 2012, 93(5):1831-1842.

[26]
王鑫, 金鹏, 宋鹏, 等. 黑曲霉酸性蛋白酶EXPA的克隆表达与酶学性质解析[J]. 食品与发酵工业, 2019, 45(3):40-46.

DOI

WANG X, JIN P, SONG P, et al. Cloning,expression and biochemical characterization of a novel acid protease EXPA from Aspergillus niger[J]. Food and Fermentation Industries, 2019, 45(3):40-46. (in Chinese)

DOI

[27]
岳晓平. 酸性蛋白酶在毕赤酵母中的表达及其酶学性质[D]. 硕士学位论文. 天津: 天津科技大学, 2019.

YUE X P. Expression and characterization of acid protease in Pichia pastoris[D]. Master’s Thesis.Tianjin: Tianjin University of Science and Technology, 2019. (in Chinese)

[28]
袁江兰, 何首春, 康旭, 等. 安卡红曲霉酸性蛋白酶分离纯化及部分酶学性质分析[J]. 食品科学, 2018, 39(10):118-124.

DOI

YUAN J L, HE S C, KANG X, et al. Purification and Some enzymatic properties of acid protease from Monascus anka[J]. Food Science, 2018, 39(10):118-124. (in Chinese)

[29]
李西波, 张旭, 杨柳, 等. 高产酸性蛋白酶菌株的选育及酶学性质研究[J]. 中国调味品, 2018, 43(3):28-33,40.

LI X B, ZHANG X, YANG L, et al. Study on breeding of strain for producing high-yield acid proteinase and enzymatic properties[J]. China Condiment, 2018, 43(3):28-33,40.

[30]
MEDEIROS S, XIE J J, DYCE P W, et al. Isolation of bacteria from fermented food and grass carp intestine and their efficiencies in improving nutrient value of soybean meal in solid state fermentation[J]. Journal of Animal Science and Biotechnology, 2018,9:29.

[31]
WU J J, CAO C M, REN D D, et al. Effects of soybean antigen proteins on intestinal permeability,5-hydroxytryptamine 1evels and secretory IgA distribution in the intestine of weaned piglets[J]. Italian Journal of Animal Science, 2016, 15(1):174-180.

[32]
李旺, 何万领, 丁轲, 等. 不同处理方法对豆粕中抗原蛋白和酸溶蛋白的影响[J]. 西北农林科技大学学报(自然科学版), 2020, 48(1):25-32.

LI W, HE W L, DING K, et al. Effect of different treatment methods on antigen protein and acid soluble protein in soybean meal[J]. Journal of Northwest A&F University(Natural Science Edition), 2020, 48(1):25-32. (in Chinese)

[33]
龚阿琼, 高震, 陈敬帮, 等. 不同酶制剂对豆粕中抗原蛋白的影响[J]. 中国饲料, 2019(23):120-123.

GONG A Q, GAO Z, CHEN J B, et al. The influence of different enzyme preparation of antigen protein in soybean meal[J]. China Feed, 2019(23):120-123. (in Chinese)

[34]
高研, 张远龙, 刘金爱, 等. 饲用酶制剂应用有效性评价方法研究[J]. 中国饲料添加剂, 2018(4):1-5.

GAO Y, ZHANG Y L, LIU J A, et al. Research on evaluation methods for the effectiveness of feed enzyme applications[J]. China Feed Additive, 2018(4):1-5. (in Chinese)

[35]
陈丹蝶, 冯定远, 王敏, 等. 复合葡萄糖氧化酶与过氧化氢酶对肉鸡生长性能、养分消化率、血清指标及肠道形态的影响[J]. 动物营养学报, 2023, 35(12):7748-7758.

DOI

CHEN D D, FENG D Y, WANG M, et al. Effects of combination of glucose oxidase and catalase on growth performance,nutrient digestibility,serum indices and intestinal morphology of broilers[J]. Chinese Journal of Animal Nutrition, 2023, 35(12):7748-7758. (in Chinese)

[36]
张巍, 郭万正, 严念东, 等. 低蛋白日粮对肉鸡生长性能及氮排泄的影响[J]. 饲料研究, 2012(6):61-62.

ZHANG W, GUO W Z, YAN N D, et al. Effects of low protein diet on growth performance and nitrogen excretion of broilers[J]. Feed Research, 2012(6):61-62. (in Chinese)

[37]
MOHAMMADIGHEISAR M, KIM I H. Addition of a protease to low crude protein density diets of broiler chickens[J]. Journal of Applied Animal Research, 2018, 46(1):1377-1381.

[38]
刘松柏, 谭会泽, 温志芬, 等. 低蛋白质日粮添加蛋白酶对黄羽肉鸡生长性能的影响[J]. 粮食与饲料工业, 2020(3):44-47.

LIU S B, TAN H Z, WEN Z F, et al. Effects of protease on growth performance of yellow feathered broilers fed low protein diet[J]. Cereal & Feed Industry, 2020(3):44-47. (in Chinese)

[39]
李东东, 李宗锐, 丁雪梅, 等. 不同粗蛋白质水平饲粮添加外源蛋白酶对肉鸡生产性能、血清生化指标和抗氧化功能的影响[J]. 动物营养学报, 2015, 27(9):2820-2831.

DOI

LI D D, LI Z R, DING X M, et al. Effects of different crude protein levels diet supplemented with exogenous protease on performance,serum biochemical indices and antioxidant function of broilers[J]. Chinese Journal of Animal Nutrition, 2015, 27(9):2820-2831. (in Chinese)

[40]
REHMAN Z U, KAMRAN J, EL-HACK M E A, et al. Influence of low-protein and low-amino acid diets with different sources of protease on performance,carcasses and nitrogen retention of broiler chickens[J]. Animal Production Science, 2018, 58(9):1625-1631.

[41]
FREITAS D M, VIEIRA S L, ANGEL C R, et al. Performance and nutrient utilization of broilers fed diets supplemented with a novel mono-component protease[J]. Journal of Applied Poultry Research, 2011, 20(3):322-334.

[42]
王本会, 张凤娟, 袁村玉. 益生菌制剂对仔猪养分消化率的影响[J]. 中国猪业, 2022, 17(3):54-56.

WANG B H, ZHANG F J, YUAN C Y. Effects of probiotics on nutrient digestibility of piglets[J]. China Swine Industry, 2022, 17(3):54-56. (in Chinese)

[43]
李秀丽. 菌酶混合型饲料添加剂对肉牛生长性能、营养物质表观消化率及经济效益的影响[J]. 饲料研究, 2021, 44(23):12-15.

LI X L. Effect of bacterial enzyme mixed feed additives on growth performance,apparent digestibility of nutrients and economic benefit of beef cattle[J]. Feed Research, 2021, 44(23):12-15. (in Chinese)

[44]
MCCAFFERTY K W, TOGHYANI M, MORGAN N K, et al. Effects of protease supplementation and diet type on jejunal and ileal digestibility and total tract metabolisability of nitrogen,starch,and energy in broilers[J]. British Poultry Science, 2022, 63(3):386-394.

[45]
BARLETTA A. Introduction:current market and expected developments[M]// BEDFORDM R, PARTRIDGEG G. Enzymesin farm animal nutrition. 2nd ed.Wallingford:CABI,2010:1-11.

[46]
SLOMINSKI B A. Recent advances in research on enzymes for poultry diets[J]. Poultry Science, 2011, 90(9):2013-2023.

DOI PMID

[47]
LEI X J, CHEONG J Y, PARK J H, et al. Supplementation of protease,alone and in combination with fructooligosaccharide to low protein diet for finishing pigs[J]. Animal Science Journal, 2017, 88(12):1987-1993.

[48]
ANGEL C R, SAYLOR W, VIEIRA S L, et al. Effects of a mono-component protease on performance and protein utilization 7 to 22 day old broiler chickens[J]. Poultry Science, 2011, 90(10):2281-2286.

Options
Outlines

/

〈 〉

京ICP备17050989号-2
Copyright © Chinese Journal of Animal Nutrition, All Rights Reserved.
Tel: 010－62817823　E-mail: cjan@vip.163.com
Powered by Beijing Magtech Co., Ltd.