CN117288881A - 燕窝肽特征性肽段溯源数据库及其在燕窝肽鉴定中的应用 - Google Patents
燕窝肽特征性肽段溯源数据库及其在燕窝肽鉴定中的应用 Download PDFInfo
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Abstract
本发明提出了燕窝肽特征性肽段溯源数据库及其在燕窝肽鉴定中的应用,所述燕窝肽特征性肽段溯源数据库包括:燕窝肽19个特征性肽段的序列信息,所述燕窝肽19个特征性肽段的序列信息分别具有如SEQ ID NO:1‑19所示的氨基酸序列。利用本发明的燕窝肽特征性肽段溯源数据库可以实现快速、准确且高效地鉴定燕窝肽,具有广泛的推广应用价值。
Description
技术领域
本发明涉及生物领域。具体地,本发明涉及燕窝肽特征性肽段溯源数据库及其在燕窝肽鉴定中的应用。
背景技术
燕窝是一种中国传统美食,是一类具有滋补功效的功能性食品,具有多种药用价值,从中国到世界各地越来越备受关注。以食用燕窝为原料,经水解工艺生产的,主要成分的相对分子质量低于10000的产品称为燕窝肽,燕窝肽产品的主要成分为:蛋白质(以干基计)≥50g/100g、肽含量(以干基计)≥40g/100g、结合唾液酸≥50g/kg、相对分子质量<10000的燕窝肽比例≥80%。燕窝肽具有优良的促进细胞增殖、降血压、抗病毒等的功效。同时,燕窝肽由于具有较低的分子量被认为可以很容易被人体吸收,并具有透过表皮被吸收的能力。基于以上优势,燕窝肽在食品、药品、护肤品等领域被消费者、生产商、研究人员寄予厚望。
然而,还未有应用于燕窝肽鉴别的方法与技术,尤其是其特征性肽段还未明确。因此,研究燕窝肽特征性肽段及其在燕窝肽鉴定中的应用具有重要的实际意义。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题。为此,本发明提出了燕窝肽特征性肽段溯源数据库及其在鉴定燕窝肽中的用途和鉴定燕窝肽的方法,利用本发明的燕窝肽特征性肽段溯源数据库可以实现快速、准确且高效地鉴定燕窝肽,具有广泛的推广应用价值。
在本发明的一个方面,本发明提出了一种燕窝肽特征性肽段溯源数据库。根据本发明的实施例,所述燕窝肽特征性肽段溯源数据库包括:包括:燕窝肽19个特征性肽段的序列信息,所述燕窝肽19个特征性肽段分别具有如SEQ ID NO:1-19所示的氨基酸序列。
本发明的发明人采用不同批次、制备方法所得燕窝肽中识别出19个特征肽段,丰富了燕窝肽特征肽段的数目,建立了一种燕窝肽特征肽段的识别方法。将其肽段序列信息构建数据库,通过对待测肽段序列信息与数据库中的肽段序列信息比对分析,可以实现准确鉴定燕窝肽的目的。
SEQ ID NO: | 氨基酸序列 |
1 | AQYRPGLG |
2 | FDSP |
3 | FFL |
4 | FFR |
5 | KFL |
6 | LEF |
7 | LEVL |
8 | LFR |
9 | LKF |
10 | LRL |
11 | PLLE |
12 | QLLL |
13 | SPLL |
14 | VELE |
15 | VHF |
16 | WLK |
17 | WTF |
18 | YPM |
19 | YWL |
根据本发明的实施例,所述燕窝肽特征性肽段溯源数据库进一步包括:每种燕窝肽特征性肽段赋予的积分值,具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值与其余肽段序列赋予的积分值之比为5:1。在一些优选实施例中,具有SEQ IDNO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值为50分,具有SEQ ID NO:2-19所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值各为10分。发明人经过大量实验得到上述赋值方式,由此,可以基于与候选肽段匹配成功的燕窝肽特征性肽段所对应的积分值的总和,判断候选肽段是否为目标肽段。
在本发明的又一方面,本发明提出了前面所述燕窝肽特征性肽段溯源数据库在鉴定燕窝肽中的用途。
在本发明的又一方面,本发明提出了一种鉴定燕窝肽的方法。根据本发明的实施例,所述方法包括:将待测肽段样品进行提取处理,得到肽段提取液;将所述肽段提取液进行高效液相色谱和质谱检测,得到待测肽段序列信息;将所述待测肽段序列信息与前面所述燕窝肽特征性肽段溯源数据库进行比对分析,以便鉴定待测肽段样品是否为燕窝肽。由此,利用本发明的方法可以实现准确、高效且快速地鉴定燕窝肽。
根据本发明的实施例,所述提取处理包括:将待测肽段样品用水复溶,得到复溶液;将所述复溶液进行超滤和脱盐处理,得到肽段提取液。通过超滤处理可以更好地分离去除干扰物质,且提高分析的灵敏度和准确性。同时,可以更好地浓缩肽段,使其更容易被检测和分析。
在质谱分析中,样品中的盐类会引起一系列问题,例如干扰质谱信号、降低质谱信号强度、产生杂质峰等,这些干扰会影响对肽段的鉴定和定量分析结果。通过脱盐处理,可以有效去除样品中的盐类,消除干扰因素,提高质谱信号的质量和强度。这样可以获得更准确的特征性肽段质谱图,有助于对蛋白质的鉴定和定量分析。
根据本发明的实施例,所述超滤采用的滤膜孔径为1~20kDa,收集流出液。由此,可以更好地分离去除干扰物质,且提高分析的灵敏度和准确性。同时,可以更好地浓缩肽段,使其更容易被检测和分析。
根据本发明的实施例,所述脱盐处理采用的透析袋截留分子量为500~1500Da。由此,可以更佳有效地去除样品中的盐类,消除干扰因素,提高质谱信号的质量和强度,有助于对肽段的鉴定和定量分析。
根据本发明的实施例,所述液相色谱采用的流动相包括流动相A和流动相B,所述流动相A为0.05~0.2体积%的甲酸水溶液,所述流动相B为0.05~0.2体积%的甲酸和80~88体积%乙腈的水溶液。由此,在此条件下可以实现有效分离上述19种燕窝肽特征肽段,从而有助于后续质谱检测。
根据本发明的实施例,所述液相色谱采用的流动相洗脱条件如下。由此,在此条件下可以实现有效分离上述19种燕窝肽特征肽段,从而有助于后续质谱检测。
时间 | 流动相A | 流动相B |
0-50min | 96%-50% | 4%~50% |
50min-54min | 50%-0% | 50%~100% |
54min-60min | 0% | 100% |
根据本发明的实施例,所述质谱选自二级串联质谱,数据采集模式使用每次全扫描后采集10个碎片图谱;一级质谱扫描范围设置为300 1800m/z,扫描分辨率设置为70,000@m/z 200;二级质谱扫描范围固定起点为110m/z,二级扫描分辨率设置为17,500@m/z200。由此,可以准确鉴定出19种燕窝肽特征肽段序列信息。
根据本发明的实施例,所述比对分析包括:将所述待测肽段序列信息与所述燕窝肽特征性肽段溯源数据库中的肽段序列信息进行比对,将所有比对成功的肽段序列对应的积分值相加,得到总积分值;当所述总积分值大于等于N分时,则所述待测肽段样品为燕窝肽;当所述总积分值小于N分时,则所述待测肽段样品不为燕窝肽;N=100×具有SEQ IDNO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值/50;或者N=100×所述燕窝肽特征性肽段溯源数据库中除具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段以外的其余任一燕窝肽特征性肽段赋予的积分值/10。在一些优选实施例中,N选自100。
根据本发明的实施例,所述燕窝肽的分子量不超过10000。
本发明还可以具有如下技术效果:
1.准确性高:本发明采用质谱技术对燕窝肽进行分析,识别出燕窝肽特征性肽段,提高了燕窝肽鉴定的准确性。
2.快速高效:基于燕窝肽特征性肽段溯源数据库,本发明可以实现对燕窝肽的快速鉴定,大大减少了检测时间。
3.操作简便:本发明采用常用的生物分析技术,如质谱技术,操作简便,易于实现。
4.可推广应用:本发明适用于燕窝市场的监管部门、燕窝生产企业和科研机构进行燕窝肽鉴定,具有广泛的推广应用价值。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,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,均为可以通过市购获得的常规产品。
实施例1
1、将由厦门市燕之屋丝浓食品有限公司提供的燕窝分别利用碱性蛋白酶和风味蛋白酶在不同酶解时间(0.5h、1h、2h、3h、4h、5h和6h)条件下进行酶解,得到燕窝肽。
碱性蛋白酶酶解条件:温度:50~55℃、pH值>8.5、酶添加量:4000U/g~8000U/g。
风味蛋白酶酶解条件:温度:53℃、pH值5.5~7.5、酶添加量:4000U/g~6000U/g。
分别将不同酶解方式得到的燕窝肽进行后续步骤。
2、取约50μL肽溶液样品加入200μL UA buffer(8M Urea,150mMTris-HCL pH8.0)混匀,转入10kD超滤离心管,离心14000g 15min,采用的透析袋截留分子量为1000Da进行脱盐处理后,加入200μL UA buffer离心14000g 15min,弃滤液。如入100μL IAA(50Mm IAA inUA),600rpm振荡1min,避光室温30min,离心14000g 10min,加入100μL UA buffer,离心14000g 10min重复2次。加入100μLTris-HCl缓冲液,离心14000g 10min重复2次。加入40μLTrypsin buffer缓冲液(2μg Trypsin in 40μLTris-HCl缓冲液),600rpm振荡1min,37℃16-18h,换新收集管,离心14000g 10min,取滤液得到待测肽段。
肽段用液相色谱流动相A相溶解后采用纳升流速HPLC液相系统Easy nLC进行分离,流动相A液为0.1%甲酸水溶液,流动相B液为0.1%甲酸乙腈水溶液(乙腈为84%);色谱柱以95%的流动相A平衡,相关液相梯度设置如下:0---50min,4%~50%流动相B;50---54min,50%~100%流动相B;54---60min,流向相B维持在100%,流速为250nL/min;肽段经由毛细管高效液相色谱分离后用Q-Exactive质谱仪(ThermoFinnigan)进行质谱分析;分析时长:60min,检测方式:正离子,母离子描范围:300-1800m/z,一级质谱分辨率:70,000@m/z200,AGC target;3e6,一级Maximum IT:10ms,Number of scan ranges:1,Dynamicexclusion:40.0s。多肽和多肽碎片的质量电荷比按照下列方法采集:毎次全扫描(fullscan)后采集10个碎片图谱(MS2 scan),MS2 Acitivation Type:HCD,Isolation window:2m/z,二级质谱分辨率:17,500@m/z 200,Microscans:1,二级Maximum IT:60ms,Normalized collision energy:30eV,Underfill ratio:0.1%。
二级质谱数据使用软件Mascot 2.2进行数据库检索。检索参数设置如下表:
表1 Mascot查库参数
Item | Value |
Enzyme | Trypsin |
Max Missed Cleavages | 2 |
Fixed modifications | Carbamidomethyl(C) |
Variable modifications | Oxidation(M) |
Peptide Mass Tolerance | ±20ppm |
Fragment Mass Tolerance | 0.1Da |
Database | UniProt_Vibrio_cholerae_114251_20201104 |
Database pattern | Reverse |
Mascot Score | >20 |
利用Mascot数据库,在3次重复上中得到了不同批次、不同酶解方式所得燕窝肽的共有特征性肽段19个,具体序列如下表所示。
表2肽段序列信息
利用该序列信息构建了一个燕窝肽特征性肽段溯源数据库,此数据库包含了上述19个特征性肽段及其积分值,该数据库可用于测试样品肽是否为燕窝肽。其中,NO:1(AQYRPGLG)肽段氨基酸序列积50分,其余NO:2~NO:19氨基酸序列各积10分,当积分≥100分时即判定测试样品肽为燕窝肽。如积分≤100分,则认为该测试样品肽不是燕窝肽。
利用该数据库,在3次重复实验中共鉴定出肽段数和得分情况如下表所示。可以看出,利用本发明的燕窝肽特征性肽段和鉴定方法可以准确地鉴定出燕窝肽。
表3分别利用碱性蛋白酶和风味蛋白酶在不同酶解时间获得的肽段和得分
实施例2
将由厦门市燕之屋丝浓食品有限公司利用不同酶制剂(碱性蛋白酶(4000U/g~8000U/g)、风味蛋白酶(4000U/g~6000U/g)、胰酶(2000U/g~4000U/g)、胃蛋白酶(2000U/g~4000U/g)、碱性蛋白酶加胰酶(4000U/g~6000U/g)、风味蛋白酶加胰酶(4000U/g~6000U/g))进行酶解处理所得酶解产物燕窝肽进行鉴定,具体步骤参考实施例1。
利用燕窝肽特征性肽段溯源数据库,在3次重复实验中共鉴定出肽段数和得分情况如下表所示。可以看出,利用本发明的燕窝肽特征性肽段和鉴定方法可以准确地鉴定出燕窝肽。
表4分别利用不同酶酶解获得的肽段和得分
样品 | 酶制剂 | 肽段个数 | 得分 |
1 | 碱性蛋白酶 | 1946 | 180 |
2 | 风味蛋白酶 | 1545 | 170 |
3 | 胰酶 | 1246 | 160 |
4 | 胃蛋白酶 | 1159 | 160 |
5 | 碱性蛋白酶加胰酶 | 1742 | 170 |
6 | 风味蛋白酶加胰酶 | 1432 | 150 |
对比例1
选取市售大豆蛋白肽、胶原蛋白肽、玉米蛋白肽、松茸蛋白肽、鳕鱼蛋白肽和猪胶原蛋白肽等20种其他肽类物质,按照实施例1的步骤2进行鉴定。
利用燕窝肽特征性肽段溯源数据库,在3次重复实验中共鉴定出肽段数和得分情况如下表所示。可以看出,利用本发明的燕窝肽鉴定方法可以准确鉴定出上述非燕窝肽物质。
表5其他物种的肽段鉴定结果
样品 | 酶制剂 | 肽段个数 | 得分 |
1 | 大豆蛋白肽 | 1763 | 80 |
2 | 胶原蛋白肽 | 1623 | 70 |
3 | 玉米蛋白肽 | 1235 | 70 |
4 | 松茸蛋白肽 | 1123 | 90 |
5 | 鳕鱼蛋白肽 | 1245 | 70 |
6 | 猪胶原蛋白肽 | 1456 | 80 |
7 | 鱼胶原蛋白肽 | 1526 | 60 |
8 | 海参肽 | 1214 | 70 |
9 | 牡蛎肽 | 1123 | 60 |
10 | 花胶肽 | 1348 | 80 |
11 | 扇贝肽 | 1453 | 80 |
12 | 蛤蜊肽 | 1432 | 70 |
13 | 牛骨肽 | 1689 | 80 |
14 | 阿胶肽 | 1589 | 90 |
15 | 羊肚菌肽肽 | 1783 | 80 |
16 | 苦瓜肽 | 1348 | 60 |
17 | 芝麻肽 | 1521 | 50 |
18 | 核桃肽 | 1457 | 60 |
19 | 大米肽 | 1567 | 50 |
20 | 枯草菌肽 | 1553 | 70 |
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (10)
1.一种燕窝肽特征性肽段溯源数据库,其特征在于,包括:燕窝肽19个特征性肽段的序列信息,所述燕窝肽19个特征性肽段分别具有如SEQ ID NO:1-19所示的氨基酸序列。
2.根据权利要求1所述的燕窝肽特征性肽段溯源数据库,其特征在于,进一步包括:每种燕窝肽特征性肽段赋予的积分值;
具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值与其余肽段序列赋予的积分值之比为5:1;
任选地,具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值为50分,具有SEQ ID NO:2-19所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值各为10分。
3.权利要求1或2所述燕窝肽特征性肽段溯源数据库在鉴定燕窝肽中的用途。
4.一种鉴定燕窝肽的方法,其特征在于,包括:
将待测肽段样品进行提取处理,得到肽段提取液;
将所述肽段提取液进行高效液相色谱和质谱检测,得到待测肽段序列信息;
将所述待测肽段序列信息与权利要求1或2所述燕窝肽特征性肽段溯源数据库进行比对分析,以便鉴定待测肽段样品是否为燕窝肽。
5.根据权利要求4所述的方法,其特征在于,所述提取处理包括:
将待测肽段样品用水复溶,得到复溶液;
将所述复溶液进行超滤和脱盐处理,得到肽段提取液。
6.根据权利要求5所述的方法,其特征在于,所述超滤采用的滤膜孔径为1~20kDa,收集流出液;
所述脱盐处理采用的透析袋截留分子量为500~1500Da。
7.根据权利要求4所述的方法,其特征在于,所述高效液相色谱采用的流动相包括流动相A和流动相B,所述流动相A为0.05~0.2体积%的甲酸水溶液,所述流动相B为0.05~0.2体积%的甲酸和80~88体积%乙腈的水溶液;
任选地,所述液相色谱采用的流动相洗脱条件如下:
8.根据权利要求4所述的方法,其特征在于,所述质谱选自二级串联质谱,数据采集模式使用每次全扫描后采集10个碎片图谱;一级质谱扫描范围设置为3001800m/z,扫描分辨率设置为70,000@m/z 200;二级质谱扫描范围固定起点为110m/z,二级扫描分辨率设置为17,500@m/z 200。
9.根据权利要求4所述的方法,其特征在于,所述比对分析包括:
将所述待测肽段序列信息与所述燕窝肽特征性肽段溯源数据库中的肽段序列信息进行比对,将所有比对成功的肽段序列对应的积分值相加,得到总积分值;
当所述总积分值大于等于N分时,则所述待测肽段样品为燕窝肽;
当所述总积分值小于N分时,则所述待测肽段样品不为燕窝肽;
N=100×A/50,A为具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段赋予的积分值;或者
N=100×B/10,B为所述燕窝肽特征性肽段溯源数据库中,除具有SEQ ID NO:1所示的氨基酸序列的燕窝肽特征性肽段以外的其余任一燕窝肽特征性肽段赋予的积分值;
优选地,N选自100。
10.根据权利要求3所述用途或权利要求4~9任一项所述的方法,其特征在于,所述燕窝肽的分子量不超过10000。
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