CN108823190B - 一种降低烟叶特有亚硝胺的复合酶制剂及其制备方法和应用 - Google Patents
一种降低烟叶特有亚硝胺的复合酶制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种降低烟叶特有亚硝胺的复合酶制剂及其制备方法和应用,该复合酶制剂由中性蛋白酶、果胶酶、木聚糖酶、糖化酶、β‑葡聚糖酶、α‑淀粉酶、中温淀粉酶、纤维素酶、中性脂肪酶和烟草复合酶中的5种或5种以上,以及无菌蒸馏水组成。经过本发明的复合酶制剂的处理,晒黄烟调制烟叶中TSNAs含量降低了26.40%‑36.81%。本发明成本低,操作方便,易于大规模推广应用,提升烟叶的质量安全,在烟叶原料降害方面具有很大的应用价值。
Description
技术领域
本发明涉及一种复合酶制剂,尤其是一种降低烟叶特有亚硝胺的复合酶制剂,还涉及其制备方法和应用,属于烟叶加工领域。
背景技术
利用现代生物技术降解烟叶有害成分及其前体物质是卷烟减害的重要途径。近年来,随着人们对吸烟与健康关系的日益重视,烟草中的烟草特有亚硝胺(Tobacco-specificnitrosamines,简称TSNAs)因其潜在的致癌能力而备受关注,成为烟草公司降低烟草危害性的重要研究内容之一。TSNAs主要包括4种有害物质:N-亚硝基去甲基烟碱(简称NNN)、4-(N-甲基-亚硝基)-1-(3-吡啶基)-1-丁酮(简称NNK)、N-亚硝基假木贼碱(简称NAB)和N-亚硝基新烟草碱(简称NAT),大量研究证明TSNAs形成的主要前体物质有烟叶中的氮氧化物、硝酸盐和亚硝酸盐等。新鲜烟叶中几乎不含TSNAs,烟叶TSNAs是在烟叶采收后的调制和贮藏等过程中形成的,因此调制期是控制TSNAs形成的关键环节之一。
目前,国内外降低TSNAs含量的技术覆盖了品种选育、烟株田间管理、调制、醇化和储藏等从种子到烟叶加工的各个环节。在调制和醇化过程中,微生物菌剂是主要的控制烟叶TSNAs含量的生物技术。例如:祝明亮等将分离自白肋烟的6株反硝化细菌处理白肋烟烟叶,调制后烟叶TSNA量降低了27.56%-99.88%(微生物学报, 2004, 44(4):422-426);汪安云等将分离自白肋烟烟叶的根瘤土壤杆菌喷施于白肋烟烟叶经晾制后可显著降低烟叶TSNAs的含量,比同一时期对照烟叶中TSNA含量降低了81.3%(环境科学学报, 2006, 26(11): 1914-1920);专利03810579.9采用分离自白莱烟烟叶的可降解TSNAs的少动鞘氨醇单胞菌和/或荧光假单胞菌处理白莱烟烟叶从而降低烟叶TSNAs量;专利03135607.9将分离自烟草的内生细菌铜绿假单胞菌KenLXP30处理白肋烟烟叶显著降低烟叶TSNAs量;专利03135608.7将放射根瘤菌KenLXR34处理白肋烟烟叶显著降低烟叶TSNAs量;专利201110090798.X采用分离自烤烟烟叶的恶臭假单胞菌T2-2菌悬液处理烤烟烟叶,烟叶TSNAs中的NNN和NNK分别降低了28.33%和57.83%。然而,微生物菌剂在烟叶TSNAs含量控制中的应用存在以下几个方面的局限性:(1)微生物菌剂的发酵和制备工艺需要经过大量试验参数的反复优化,此外发酵和制备过程也耗时耗力;(2)微生物菌剂的制备成本较高;(3)微生物在烟叶上的定植能力和生长代谢活性,及环境条件等对其功能发挥影响较大,以上问题在不同程度上限制了微生物菌剂在TSNAs含量控制中的大规模推广应用。
酶具有靶标选择性强、催化效率高、作用条件温和等特点,利用酶处理改善烟叶及烟梗等从而提高其内在品质取得了较多研究成果。与烤烟的烘烤调制方式不同,晒黄烟主要采用在自然环境条件下的晾晒调制方式,调制时的自然环境条件有利于酶作用的发挥。利用酶处理调制期烟叶来降低烟叶调制过程中TSNAs含量的研究鲜见报道。
发明内容
为了克服现有技术问题的不足,本发明提供了一种降低烟叶特有亚硝胺的复合酶制剂,还提供其制备方法及应用,主要是通过喷施酶制剂的方式处理新鲜烟叶后进行烟叶的调制,从而降低调制烟叶中TSNAs的含量,提升烟叶的质量安全,为优质烟叶生产提供技术支持。本发明的技术方案如下:
一种降低烟叶特有亚硝胺的复合酶制剂,由中性蛋白酶、果胶酶、木聚糖酶、糖化酶、β-葡聚糖酶、α-淀粉酶、中温淀粉酶、纤维素酶、中性脂肪酶和烟草复合酶中的5种或5种以上,以及无菌蒸馏水组成,其中,各组分的质量百分比如下:
中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%、中性脂肪酶0.01%-0.02%、烟草复合酶0.1%-0.5%。
进一步地,复合酶制剂由如下质量百分比的组分组成:中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%,其余为无菌蒸馏水。
进一步地,作为另一种实施方式,复合酶制剂由如下质量百分比的组分组成:中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%,其余为无菌蒸馏水。
进一步地,作为另一种实施方式,复合酶制剂由如下质量百分比的组分组成:
烟草复合酶0.1%-0.5%、中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%、中性脂肪酶0.01%-0.02%,其余为无菌蒸馏水。
本发明涉及的上述的复合酶制剂的制备方法,包括如下步骤:
(1)按上述的组分准备原料;
(2)将步骤(1)的组分和无菌蒸馏水按照所述质量百分比进行搅拌混合,搅拌速度为50rpm-100rpm,混合温度为40℃±5℃,pH为5.5±0.3,至均匀获得复合酶制剂混悬液,即复合酶制剂。
本发明涉及的上述的复合酶制剂在晒黄烟调制中的应用,包括以下步骤:
(1)新鲜烟叶样品的制备:采集适熟的新鲜晒黄烟烟叶,划开主脉,编杆,杆长约120cm,每杆45-55片烟叶,置于晾晒棚内,杆距约20cm;
(2)复合酶制剂的烟叶喷施处理:将上述复合酶制剂均匀喷施至晾晒棚内的烟叶的两面,每杆烟叶喷施100-120mL复合酶制剂混悬液;
(3)烟叶调制:喷施处理的烟叶置于晾晒棚内,自然晾晒调制10-13d。
与现有技术相比,本发明具有以下突出的有益效果:
(1)本发明将各酶组分按照比例均匀混合后,以一定量均匀喷施处理新鲜晒黄烟烟叶后进行烟叶的调制,通过复合酶制剂中各酶组分的作用,烟叶中总氮、硝酸盐、降烟碱、麦斯明、假木贼烟碱、新烟碱等含量发生不同程度的降低,直接或间接地降低了TSNAs的前体物质,从而降低了调制过程中烟叶中TSNAs的含量。
(2)本发明所用酶为食品级生物酶,不使用其他化学添加剂,在烟叶上的使用不会带来安全隐患。
(3)本发明利用晒黄烟在自然环境中晾晒的调制方式,该调制方式适宜本申请复合酶制剂中酶作用的发挥,成本低,操作方便,易于大规模推广应用,能够有效降低晒黄烟调制烟叶中TSNAs含量,提升烟叶的质量安全,在烟叶原料降害方面具有很大的应用价值。
附图说明
图1是实施例1-3的复合酶制剂处理和对照组处理调制期晒黄烟烟叶中TSNAs含量的变化图。
具体实施方式
下面将结合本发明实施例,对本实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是对本发明一部分实例,而不是全部的实例。基于本发明中的实施例,本领域普通技术人员在没有付出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过购买获得的常规产品。
实施例1
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.03%、果胶酶0.02%、木聚糖酶0.01%、糖化酶0.02%、β-葡聚糖酶0.01%、无菌蒸馏水99.91%。
本实施例涉及的上述的复合酶制剂的制备方法,包括如下步骤:
(1)按上述的组分准备原料;
(2)将步骤(1)的组分和无菌蒸馏水按照所述质量百分比进行搅拌混合,搅拌速度为75rpm,混合温度为40℃,pH为5.5,至均匀获得复合酶制剂混悬液,即复合酶制剂。
本实施例涉及的上述的复合酶制剂在晒黄烟调制中应用,包括以下步骤:
(1)新鲜烟叶样品的制备:采集适熟的新鲜晒黄烟烟叶,划开主脉,编杆,杆长约120cm,每杆50片烟叶,置于晾晒棚内,杆距约20cm;
(2)复合酶制剂的烟叶喷施处理:将上述复合酶制剂混悬液均匀喷施至晾晒棚内的烟叶的两面,每杆烟叶喷施110mL复合酶制剂混悬液,对照组喷施等量的无菌蒸馏水;
(3)烟叶调制:喷施处理的烟叶置于晾晒棚内,自然晾晒调制13d。
在烟叶调制过程中,每3天取样1次,按照五点取样法随机取5片烟叶,去除主脉,放入105℃的电热干燥烘箱内杀青15min后,将温度调至60℃下烘烤至干燥,然后使用植物粉碎机将样品粉碎后,按照YQ/T 29-2013《烟草及烟草制品 烟草特有N-亚硝胺的测定 高效液相色谱-串联质谱联用法》进行TSNAs检测。
检测结果显示,经过复合酶制剂处理,调制结束时晒黄烟烟叶中TSNAs含量与对照组相比降低了26.40%,如图1所示。
在本实施例中,针对晒黄烟新鲜烟叶在自然环境条件下进行晾晒的调制方式,晾晒棚内全天的温度在15-38℃之间,相对湿度在30%-95%之间,该温湿度差异主要由晒黄烟烟田基地的自然环境温湿度造成,此外烟叶的自然失水也对温湿度造成了一定的影响。该温湿度条件对酶的活性作用较为适宜。经过酶制剂对晒黄烟在调制过程中的处理,综合调节TSNAs形成的前体物,达到降低烟叶TSNAs含量的目的。
实施例2
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.02%、果胶酶0.01%、木聚糖酶0.005%、糖化酶0.01%、β-葡聚糖酶0.005%、α-淀粉酶0.01%、中温淀粉酶0.02%、纤维素酶0.02%、无菌蒸馏水99.90%。
本实施例涉及的上述的复合酶制剂的制备方法,包括如下步骤:
(1)按上述的组分准备原料;
(2)将步骤(1)的组分和无菌蒸馏水按照所述质量百分比进行搅拌混合,搅拌速度为50rpm,混合温度为35℃,pH为5.2,至均匀获得复合酶制剂混悬液,即复合酶制剂。
本实施例涉及的上述的复合酶制剂在晒黄烟调制中应用,包括以下步骤:
(1)新鲜烟叶样品的制备:采集适熟的新鲜晒黄烟烟叶,划开主脉,编杆,杆长约120cm,每杆45片烟叶,置于晾晒棚内,杆距约20cm;
(2)复合酶制剂的烟叶喷施处理:将上述复合酶制剂混悬液均匀喷施至晾晒棚内的烟叶的两面,每杆烟叶喷施100mL复合酶制剂混悬液,对照组喷施等量的无菌蒸馏水;
(3)烟叶调制:喷施处理的烟叶置于晾晒棚内,自然晾晒调制13d。
检测结果显示,经过复合酶制剂处理,调制结束时晒黄烟烟叶中TSNAs含量与对照组相比降低了36.81%,如图1所示。其余与实施例1相同。
实施例3
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:
烟草复合酶0.5%、中性蛋白酶0.01%、果胶酶0.005%、木聚糖酶0.0025%、糖化酶0.005%、β-葡聚糖酶0.0025%、α-淀粉酶0.005%、中温淀粉酶0.005%、纤维素酶0.005%、中性脂肪酶0.01%和无菌蒸馏水99.45%。
本实施例涉及的上述的复合酶制剂的制备方法,包括如下步骤:
(1)按上述的组分准备原料;
(2)将步骤(1)的组分和无菌蒸馏水按照所述质量百分比进行搅拌混合,搅拌速度为100rpm,混合温度为45℃,pH为5.8,至均匀获得复合酶制剂混悬液,即复合酶制剂。
本实施例涉及的上述的复合酶制剂在晒黄烟调制中应用,包括以下步骤:
(1)新鲜烟叶样品的制备:采集适熟的新鲜晒黄烟烟叶,划开主脉,编杆,杆长约120cm,每杆55片烟叶,置于晾晒棚内,杆距约20cm;
(2)复合酶制剂的烟叶喷施处理:将上述复合酶制剂混悬液均匀喷施至晾晒棚内的烟叶的两面,每杆烟叶喷施120mL复合酶制剂混悬液;
(3)烟叶调制:喷施处理的烟叶置于晾晒棚内,自然晾晒调制13d。
检测结果显示,经过复合酶制剂处理,调制结束时晒黄烟烟叶中TSNAs含量与对照组相比降低了29.98%,如图1所示。其余与实施例1相同。
实施例4
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.01%、果胶酶0.01%、木聚糖酶0.005%、糖化酶0.005%、β-葡聚糖酶0.005%和无菌蒸馏水99.965%。其余工艺与实施例1相同。
实施例5
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.02%、果胶酶0.02%、木聚糖酶0.01%、糖化酶0.02%、β-葡聚糖酶0.01%和无菌蒸馏水99.92%。其余工艺与实施例1相同。
实施例6
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.01%、果胶酶0.005%、木聚糖酶0.005%、糖化酶0.005%、β-葡聚糖酶0.005%、α-淀粉酶0.005%、中温淀粉酶0.005%、纤维素酶0.01%和无菌蒸馏水99.95%。其余工艺与实施例2相同。
实施例7
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:中性蛋白酶0.03%、果胶酶0.02%、木聚糖酶0.01%、糖化酶0.02%、β-葡聚糖酶0.01%、α-淀粉酶0.01%、中温淀粉酶0.02%、纤维素酶0.02%和无菌蒸馏水99.86%。其余工艺与实施例2相同。
实施例8
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:
烟草复合酶0.1%、中性蛋白酶0.01%、果胶酶0.01%、木聚糖酶0.002%、糖化酶0.005%、β-葡聚糖酶0.003%、α-淀粉酶0.005%、中温淀粉酶0.005%、纤维素酶0.005%、中性脂肪酶0.01%和无菌蒸馏水99.845%。其余工艺与实施例3相同。
实施例9
本实施例的降低烟叶特有亚硝胺的复合酶制剂,由如下质量百分比的组分组成:
烟草复合酶0.5%、中性蛋白酶0.03%、果胶酶0.02%、木聚糖酶0.01%、糖化酶0.02%、β-葡聚糖酶0.01%、α-淀粉酶0.01%、中温淀粉酶0.02%、纤维素酶0.02%、中性脂肪酶0.02%和无菌蒸馏水99.34%。其余工艺与实施例3相同。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种降低烟叶特有亚硝胺的复合酶制剂,其特征在于:由中性蛋白酶、果胶酶、木聚糖酶、糖化酶、β-葡聚糖酶、α-淀粉酶、中温淀粉酶、纤维素酶和中性脂肪酶中的5种或5种以上,以及无菌蒸馏水组成,其中,各组分的质量百分比如下:
中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%、中性脂肪酶0.01%-0.02%;
上述复合酶制剂用于均匀喷施至晾晒棚内的烟叶的两面。
2.权利要求1所述的一种降低烟叶特有亚硝胺的复合酶制剂,其特征在于:其组成成分及质量百分比含量为:
中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%,其余为无菌蒸馏水。
3.权利要求1所述的一种降低烟叶特有亚硝胺的复合酶制剂,其特征在于:其组成成分及质量百分比含量为:
中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%,其余为无菌蒸馏水。
4.权利要求1所述的一种降低烟叶特有亚硝胺的复合酶制剂,其特征在于:其组成成分及质量百分比含量为:
烟草复合酶0.1%-0.5%、中性蛋白酶0.01%-0.03%、果胶酶0.005%-0.02%、木聚糖酶0.0025%-0.01%、糖化酶0.005%-0.02%、β-葡聚糖酶0.0025%-0.01%、α-淀粉酶0.005%-0.01%、中温淀粉酶0.005%-0.02%、纤维素酶0.005%-0.02%、中性脂肪酶0.01%-0.02%,其余为无菌蒸馏水。
5.权利要求1-4之一所述的复合酶制剂的制备方法,其特征在于:包括如下步骤:
(1)、按权利要求1-4之一的组分准备原料;
(2)、将步骤(1)的组分和无菌蒸馏水按照所述质量百分比进行搅拌混合,搅拌速度为50rpm-100rpm,混合温度为40℃±5℃,pH为5.5±0.3,至均匀获得复合酶制剂混悬液,即复合酶制剂。
6.权利要求1-4之一所述的复合酶制剂或者权利要求5的制备方法得到的复合酶制剂在晒黄烟调制中应用,其特征在于:包括以下步骤:
(1)、新鲜烟叶样品的制备:采集适熟的新鲜晒黄烟烟叶,划开主脉,编杆,杆长120cm,每杆45-55片烟叶,置于晾晒棚内,杆距20cm;
(2)、复合酶制剂的烟叶喷施处理:将上述复合酶制剂均匀喷施至晾晒棚内的烟叶的两面,每杆烟叶喷施100-120mL;
(3)、烟叶调制:喷施处理后的烟叶置于晾晒棚内,自然晾晒调制10-13d。
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