CN115340988B - 一种提高葵花籽粕利用率的复合酶制剂及其应用 - Google Patents
一种提高葵花籽粕利用率的复合酶制剂及其应用 Download PDFInfo
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- A23K20/00—Accessory food factors for animal feeding-stuffs
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明涉及一种降解葵花籽粕中抗营养因子的复合酶制剂,属于酶制剂应用领域。所述复合酶制剂包括植酸酶、中性蛋白酶、木聚糖酶和纤维素酶,酶活比为1:25:30:80。本发明采用植酸酶、中性蛋白酶、木聚糖酶与纤维素酶进行复配,用于降解葵花籽粕中的抗营养因子如植酸、蛋白酶抑制因子、非淀粉多糖等以及降低粗纤维含量,提高葵花籽粕的消化利用率。
Description
技术领域
本发明涉及酶制剂及其应用,具体涉及一种提高葵花籽粕利用率的复合酶制剂。
背景技术
近年来,水产养殖业的一个重要研究领域就是寻求可替代鱼粉和豆粕的植物性蛋白源。葵花籽粕(sunflower seed meal),是以向日葵籽为原料,用预压浸提法或浸提抽取油后的副产物。其营养物质含量丰富,富含29%~43%的优质植物蛋白,且氨基酸组成相对平衡,蛋白组成为1%~4%醇溶蛋白、11%~17%谷蛋白、55%左右球蛋白和20%左右清蛋白。据报道,葵花籽粕中还含有大量的钙、磷和烟酸,大量的类脂、碳水化合物、还原糖、灰分等。
葵花籽粕蛋白质具有很好的蛋白特性。目前,已在水产饲料行业中得到应用。当日粮中鱼粉分别降低6.1%和12.2%,并分别添加10%和20%葵花籽粕(蛋白含量17%)时,对红点鲑(Salvelinus alpinus)的末重无显著影响,与对照组差异不显著。Smith等研究发现,当日粮中鱼粉分别降低3%和6%,且分别添加高蛋白葵花籽粕12.5%和25%(蛋白含量47%)时,替代3%鱼粉组体增重与对照组差异不显著,而替代6%鱼粉组与对照组相比,红点鲑体增重有所下降,且在绒毛高度和绒毛宽度方面表现出相同的趋势。同样,在肉鸡上的研究上也有类似的发现,当日粮中葵花籽粕添加量在14%以下时,肉鸡平均日采食量和平均日增重随着葵花籽粕添加量的增加而增加,料重比降低。当葵花籽粕添加达到28%时,肉鸡的生长性能降低。这可能是由于葵花籽粕中含有较高的粗纤维,当其在日粮中的添加水平过高时,高水平的粗纤维和非淀粉多糖增加肠道内容物黏度、延缓胃排空及降低肉鸡对干物质、粗蛋白质、粗脂肪的利用率等多种方式,降低饲料原料的营养价值[8]。另外,葵花籽粕因其含有绿原酸、植酸等抗营养因子、赖氨酸含量低等问题,使得其在饲料原料中的使用度较低,未能充分利用其经济价值。
目前,在生产中,常通过利用外源酶制剂的方法来催化降解植物性饲料原料中的抗营养因子,提高饲料原料的利用率。植酸酶、蛋白酶以及非淀粉多糖酶是目前市场中常用的几大酶类。植酸酶主要通过降解饲料原料中的植酸,释放出与之螯合的磷,进而增加P的吸收利用率;而蛋白酶的添加一方面可以将大分子蛋白水解成寡肽或氨基酸、另一方面能够刺激内源酶的分泌,从而提高植物蛋白的利用;非淀粉多糖酶一是能够破坏细胞壁的结构,从而使得细胞内容物被释放出来,提高营养成分利用率,二是催化降解植物性原料中的非淀粉多糖,缓解由非淀粉多糖所引起的食糜黏度增加对消化道吸收营养物质造成的阻碍。因此,可通过在饲料中添加多种酶制剂的方式来达到共同降解抗营养因子,促进营养物质的消化吸收,进而改善动物的生产性能。
目前,尚无一种专门酶解葵花籽粕中存在的抗营养因子,进而提高其利用率的复合酶制剂。
发明内容
本发明的目的是提供一种能够降解葵花籽粕中抗营养因子含量提高葵花籽粕的利用率的复合酶制剂。
为了实现上述目的,本发明提供了一种复合酶制剂,包括植酸酶、中性蛋白酶、木聚糖酶和纤维素酶,其中,所述植酸酶活性(U)与中性蛋白酶与木聚糖酶与纤维素酶之比为1:25:20~40:70~90。
根据本发明的复合酶制剂,其中,所述植酸酶活性(U)与中性蛋白酶与木聚糖酶与纤维素酶之比为1:25:30:70~90;
根据本发明的复合酶制剂,其中,所述植酸酶活性(U)与中性蛋白酶与木聚糖酶与纤维素酶之比为1:25:30:80。
本发明还提供该复合酶在草鱼饲料中的应用。
本发明的有益效果在于:采用植酸酶、中性蛋白酶、木聚糖酶与纤维素酶进行复配,降解葵花籽粕中的植酸、蛋白酶抑制因子、非淀粉多糖、纤维素等抗营养因子含量,极大的提高了葵花籽粕的消化利用率,减少了饲料原料的浪费,进一步提高饲料厂及养殖业的生产效益。
具体实施方式
1、实验试剂。
钒钼酸铵溶液:称取1.25g偏钒酸胺,加入200mL去离子水加热溶解,冷却后再加入250mL硝酸;另称取钼酸铵25g,加入400mL去离子水加热溶解,在冷却条件下,将两种溶液混合,用去离子水定容至1000mL,避光保存,若生成沉淀,则不能用。
3,5-二硝基水杨酸(DNS)试剂:称取3,5-二硝基水杨酸3.15g(化学纯),加水500mL,搅拌5s,水浴至45℃。然后逐步加入200g/L的氢氧化钠溶液100mL,同时不断搅拌,直到溶液清澈透明(注意:在加入氢氧化钠过程中,溶液温度不要超过48℃)。再逐步加入四水酒石酸钾钠91.0g、苯酚2.50g和无水亚硫酸钠2.50g。继续45℃水浴加热,同时补加水300mL,不断搅拌,直到加入的物质完全溶解。停止加热,冷却至室温后,用水定容至1000mL。用烧结玻璃过滤器过滤。取滤液,储存在棕色瓶中,避光保存。室温下存放7天后可以使用,有效期为6个月。
体外酶解缓冲液:分别称取磷酸氢二钠6.02g和磷酸二氢钠0.5g,加入去离子水900mL,搅拌均匀。用1mol/L盐酸溶液或者0.5mol/L氢氧化钠溶液调整至pH=7.5,定容至1000mL。
20%三氯乙酸;丙酮。
植酸酶、中性蛋白酶、木聚糖酶和纤维素酶均取自广东溢多利生物科技股份有限公司,植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成300U/mL;纤维素酶400000U/g,使用时配置成800U/mL。上述酶种的发酵菌种均为毕赤酵母,发酵培养基均为1%酵母粉、2%蛋白胨、2%葡萄糖。
2、实验方法
(1)称重一定质量的葵花籽粕,粉碎过40目筛,置于酶解反应容器中,加入不同组合的酶制剂、再加入缓冲液,使得料液比为1:10,其中葵花籽粕从市场获得。
(2)恒温恒速的摇床(40℃,150r/min)反应4h。
(3)水分和干物质采用水分采用105℃烘干法,粗脂肪采用索氏提取法。
(4)无机磷测定包括:取1mL上清液,加入100mL盐酸(盐酸:水=1:10),混匀,搅拌20min,离心取上清4mL,加入10mL钒钼酸胺显色液10mL,用去离子水稀释至50mL,混匀,4000r/min离心10min,400nm下测定吸光度。
(5)小肽含量测定包括:取上清液,加入15%三氯乙酸,混匀,静置,9500r/min离心10min,取上清,采用凯氏定氮仪测上清液中的氮含量。
(6)还原糖测定包括:取1mL上清液,加入2.5mL的DNS,混匀,沸水浴5min后取出流水冷却。加6.5mL去离子水,混匀后,540nm下测定吸光度。
(7)粗纤维的测定包括:样品测定前先采用丙酮进行脱脂处理。后称重1g脱脂完的样品放入滤袋,封口。之后按照仪器操作说明,进行粗纤维的测定。
实施例1
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成300U/mL;纤维素酶400000U/g,使用时配置成800U/mL。酶解反应时,酶液各取1mL。
实施例2
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成400U/mL;纤维素酶400000U/g,使用时配置成700U/mL。酶解反应时,酶液各取1mL。
实施例3
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成200U/mL;纤维素酶400000U/g,使用时配置成900U/mL。酶解反应时,酶液各取1mL。
实施例4
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成300U/mL;纤维素酶400000U/g,使用时配置成900U/mL。酶解反应时,酶液各取1mL。
对比例1
植酸酶2000U/g,使用时配置成10U/mL。酶解反应时,酶液取1mL。
对比例2
中性蛋白酶50000U/g,使用时配置成250U/mL。酶解反应时,酶液取1mL。
对比例3
木聚糖酶300000U/g,使用时配置成300U/mL;酶解反应时,酶液取1mL。
对比例4
纤维素酶400000U/g,使用时配置成800U/mL。酶解反应时,酶液各取1mL。
对比例5
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;酶解反应时,酶液各取1mL。
对比例6
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;木聚糖酶300000U/g,使用时配置成300U/mL。酶解反应时,酶液各取1mL。
对比例7
植酸酶2000U/g,使用时配置成10U/mL;中性蛋白酶50000U/g,使用时配置成250U/mL;纤维素酶400000U/g,使用时配置成800U/mL。酶解反应时,酶液各取1mL。
同时设置对照组,控制对照组反应条件与试验组一致。采用去离子水替代复合酶制剂的添加。
表1为本发明提供的复合酶制剂对葵花籽粕中水分、干物质、粗脂肪、无机磷、小肽、还原糖、粗纤维的影响
表1复合酶制剂酶解对葵花籽粕中营养物质含量的影响(%)
水分 | 干物质 | 粗脂肪 | 无机磷 | 小肽 | 还原糖 | |
对照组 | 8.52 | 91.48 | 1.98 | 0.135 | 2.18 | 0.83 |
实施例1 | 3.28 | 96.72 | 1.99 | 0.285 | 21.34 | 2.86 |
实施例2 | 3.32 | 96.68 | 1.99 | 0.279 | 21.28 | 2.77 |
实施例3 | 3.38 | 96.62 | 1.99 | 0.28 | 21.31 | 2.8 |
实施例4 | 3.34 | 96.66 | 1.98 | 0.281 | 21.33 | 2.82 |
对比例1 | 6.98 | 93.02 | 1.98 | 0.211 | 5.67 | 1.77 |
对比例2 | 7.01 | 92.99 | 1.98 | 0.148 | 19.79 | 1.21 |
对比例3 | 7.66 | 92.34 | 1.98 | 0.151 | 4.88 | 1.66 |
对比例4 | 7.54 | 92.46 | 1.98 | 0.153 | 4.21 | 1.54 |
对比例5 | 5.45 | 94.55 | 1.99 | 0.229 | 20.18 | 2.01 |
对比例6 | 5.21 | 94.79 | 1.99 | 0.235 | 20.65 | 2.23 |
对比例7 | 5.14 | 94.86 | 1.99 | 0.233 | 20.77 | 2.19 |
结果显示,复合酶制剂的加入,能够降解葵花籽粕中非淀粉多糖、植酸和粗纤维、提高小肽含量,且实施例1-4的效果优于对比例1~7。
实施例1-4以及对比例1-7对草鱼消化能力的影响
以葵花籽粕为底物,对上述酶制剂进行草鱼肠道仿生消化评估,分别测定所述底物的干物质消化率、蛋白消化率和能量消化率。仿生消化为在体外模拟体内消化环境进行物质消化的试验,也称为立体消化技术。本发明实施例以葵花籽粕为底物,进行仿生消化评估。具体的仿生消化试验采用如下试验步骤进行。
(1)配制仿生消化试剂
模拟肠道液:分别称重胰蛋白酶、糜蛋白酶和淀粉酶溶于去离子水中,保证总体系为25mL。临用前配置。
肠道前段缓冲液:称取101.38g无水磷酸氢二钠、44.89g无水磷酸二氢钠、11.55g氯化钠和1.45g氯化钾,青霉素160万U,溶于去离子水中,并在25℃下调节pH至6.95,定容到2000mL。
肠道后段缓冲液:称取100.06g无水磷酸氢二钠、47.76g无水磷酸二氢钠、11.98g氯化钠和0.82g氯化钾,青霉素160万U,溶于去离子水中,并在25℃下调节pH至6.92,定容到2000mL。
仪器:单胃动物仿生消化系(SDS-2)。
(2)二步法酶解
采用肠道前段-肠道后段二步法酶解。
肠道阶段模拟消化的参数为:温度25℃,缓冲液流速120mL/min,肠道前段消化时间9h,肠道后段消化时间7h,清洗液1500mL/次,每次清洗40min,共清洗6次。
(3)试验操作
准备称重2g(精确到0.0002)样品,置于装有透析袋的模拟消化器中,再往透析袋中加入20mL模拟肠液,将模拟肠液置于单胃动物仿生消化仪(SDS-2)单胃动物仿生消化系统中。仿生消化结束后,用注射器吸取约5mL去离子水将透析袋中的未消化残渣无损失的转移到已知绝干重的90mm培养皿中,在65℃烘干至无水痕,约12h,再在105℃烘干至恒重,留样,待测。
(4)检测指标及方法
检测原料及残渣蛋白质、能量含量,蛋白质采用凯氏定氮仪测定,能量采用HWR-15C自动快速热量计测定仪测定,计算降解率,所有消化率均以干物质为基础。
其计算方式如下:
干物质消化率=(称样量-消化完残渣干重)/称样量*100。
蛋白质消化率=(饲料中蛋白质含量-消化完残渣中蛋白质含量)/饲料中蛋白质含量*100。
能量消化率=(饲料中能量含量-消化完残渣中能量含量)/饲料中能量含量*100。
表2复合酶制剂对葵花籽粕干物质、蛋白质和代谢能消化率的影响
结果显示,复合酶制剂的添加能够提高葵花籽粕在草鱼肠道中干物质、蛋白质和能量的利用率,且实施例的提高效果优于对比例1~7。
以上实施例仅用于解释本申请的技术方案,不限定本申请的保护范围。
Claims (2)
1.一种降解葵花籽粕中抗营养因子的复合酶制剂,其特征在于,所述复合酶制剂由植酸酶、中性蛋白酶、木聚糖酶和纤维素酶组成;所述植酸酶、中性蛋白酶、木聚糖酶、纤维素酶的酶活比为1:25:30:80,其中,所述植酸酶、中性蛋白酶、木聚糖酶、纤维素酶的酶活比依据酶活单位U计算。
2.权利要求1所述解葵花籽粕中抗营养因子的复合酶制剂在草鱼饲料制备中的应用。
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