CN115010826B - 壳寡糖-羟基吡啶酮缀合物及其制备方法和应用 - Google Patents
壳寡糖-羟基吡啶酮缀合物及其制备方法和应用 Download PDFInfo
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Classifications
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- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
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- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
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- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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- A—HUMAN NECESSITIES
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Abstract
本发明公开了一种壳寡糖‑羟基吡啶酮缀合物,其结构通式为:通式中的R为或n的范围为2~20。本发明还同时公开了上述壳寡糖‑羟基吡啶酮缀合物的制备方法:以曲酸为原料制备含有羟基吡啶酮基的2‑氯甲基‑5‑羟基吡啶酮,2‑氯甲基‑5‑羟基吡啶酮和壳寡糖进行亲核取代反应,将羟基吡啶酮基偶联到壳寡糖上,从而生成壳寡糖‑羟基吡啶酮缀合物。壳寡糖‑羟基吡啶酮缀合物的用途是:作为抗氧化剂、酪氨酸酶抑制剂、抑菌剂。
Description
技术领域
本发明属于食品或化妆品等的保鲜技术领域,具体涉及一种新型的同时具有酪氨酸酶抑制活性、抗氧化和抗菌活性的壳寡糖-羟基吡啶酮缀合物及其制备方法和应用。
背景技术
南美白对虾味道鲜美,营养丰富,已经成为平衡膳食结构中重要的一部分,我国对于南美白对虾的需求量逐年增加。但是高蛋白、高水分的特点和多酚氧化酶的作用,导致虾容易滋生细菌发生腐败黑变,造成商业价值的降低。目前虾类主要使用的还是低温保鲜,冷藏保鲜时间短、效果差,冷冻保鲜影响其营养价值和风味。生物保鲜剂虽然安全无毒,但提取复杂,成本较高。所以通过人工合成的方法寻找一种高效、无毒的化学保鲜剂就成了目前的研究热点。
壳寡糖(COS)来源丰富,具有良好的生物相容性、降解性、无毒性,是天然的防腐剂,已经被用于食品保鲜领域。尽管壳寡糖有着独特的优势,但是由于抗菌、抗氧化等活性并不突出,还不足以代替传统的食品保鲜剂,因此,在食品行业的全面应用还需要加强其生物活性。壳寡糖上C-2位的氨基和C-6位的羟基反应活性较高,是理想的修饰位点,可以通过化学修饰的方法引入活性基团来提高壳寡糖的生物活性,获得一种高效,安全的新型食品保鲜剂。将活性基团引入壳寡糖分子中,拓展其应用范围极具研究意义。
目前现有的由壳寡糖改性得到的具有较强生物活性的缀合物的结构式举例如下:
壳寡糖缀合物1、2、3所用原料壳寡糖的n均为2~20,壳寡糖的重均分子量分别为1000、1500、1500Da,部分缀合物告知了具有抗氧化、抗菌活性。具体而言,缀合物1、3具有抗氧化活性,缀合物1、2、3具有抗菌活性;但是其性能仍然有待改进,且上述3者均未告知具有酪氨酸酶抑制活性。
发明内容
本发明要解决的技术问题是提供一种壳寡糖-羟基吡啶酮缀合物及其制备方法,该壳寡糖衍生物具有良好的酪氨酸酶抑制活性、抗氧化活性和抗菌活性。
为了解决上述技术问题,本发明提供一种壳寡糖-羟基吡啶酮缀合物(同时具备抗氧化、抑制酪氨酸酶和抑菌性能的壳寡糖-羟基吡啶酮缀合物),其结构通式为:
通式中的R为n的范围为2~20。
本发明还同时提供了上述壳寡糖-羟基吡啶酮缀合物的制备方法,包括:以曲酸为原料制备含有羟基吡啶酮基的2-氯甲基-5-羟基吡啶酮;2-氯甲基-5-羟基吡啶酮和壳寡糖进行亲核取代反应,将羟基吡啶酮基偶联到壳寡糖上,从而生成壳寡糖-羟基吡啶酮缀合物(带有羟基吡啶酮结构的壳寡糖-羟基吡啶酮缀合物)。
说明:以曲酸为原料通过苄基保护、氨水缩合、氯代反应后脱去苄基得到含有羟基吡啶酮基的2-氯甲基-5-羟基吡啶酮(即,化合物5),此为现有的已知技术。
作为本发明的壳寡糖-羟基吡啶酮缀合物的制备方法的改进:
将2-氯甲基-5-羟基吡啶酮和壳寡糖溶于溶剂中,加入氢氧化钠做缚酸剂,于室温搅拌反应12±1h,2-氯甲基-5-羟基吡啶酮:壳寡糖=1:2或2:1的摩尔比;2-氯甲基-5-羟基吡啶酮:氢氧化钠=1:2的摩尔比;
然后在反应所得物中加入乙醇(无水乙醇)使产物沉淀;所得的沉淀用去离子水透析,冷冻干燥,得到壳寡糖-羟基吡啶酮缀合物。
作为本发明的壳寡糖-羟基吡啶酮缀合物的制备方法的进一步改进:作为原料的壳寡糖的重均分子量为800~1000Da,脱乙酰度85%~90%。
作为本发明的壳寡糖-羟基吡啶酮缀合物的制备方法的进一步改进:
溶剂为二甲基亚砜(DMSO)水溶液,所述DMSO水溶液中,DMSO:水=1:1的体积比。
作为本发明的壳寡糖-羟基吡啶酮缀合物的制备方法的进一步改进:每1g的壳寡糖配用20±5mL的DMSO水溶液。
本发明还同时提供了上述壳寡糖-羟基吡啶酮缀合物的用途,至少为以下任一用途:
抗氧化剂、酪氨酸酶抑制剂、抑菌剂。
作为本发明的壳寡糖-羟基吡啶酮缀合物的用途的改进:用于食品的保鲜或化妆品的配制。
本发明的含羟基吡啶酮结构的壳寡糖缀合物的制备路线为:
(1)、化合物2和3的合成
以曲酸(1)为原料并按文献报道方法合成(Design and synthesis ofhydroxypyridinone-L-phenylalanine conjugates as potential tyrosinaseinhibitors.Journal of Agricultural and Food Chemistry 2013,61(27),6597-6603),依次得到化合物2和3。
化合物2:5-苄氧基-2-羟甲基-吡喃-4-酮,产率为83.8%。
化合物3:5-苄氧基-2-羟甲基-吡啶-4-酮,产率为73.5%。
(2)、化合物4的合成
称取20g化合物3(5-苄氧基-2-羟甲基-吡啶-4-酮)溶于100mL氯化亚砜(SOCl2)中,于室温反应2h,反应结束后抽滤得到大量淡黄色沉淀,再用大量丙酮洗涤,得到化合物4。
化合物4:5-苄氧基-2-氯甲基-吡喃-4-酮,产率为79.0%。
(3)、化合物5的合成
称取10mmol(2.5g)化合物4于三颈烧瓶中,加入100mL CH2Cl2搅拌成分散均匀的悬浮液,充入氮气。在冰浴条件下,使用恒压滴液漏斗缓慢滴加100mL含有1M BCl3的CH2Cl2溶液。反应结束后加入20mL甲醇使沉淀完全溶解,然后旋蒸除去溶剂,再用甲醇乙醚(甲醇:乙醚体积比=1:5)重结晶2~3次,得到化合物5。
化合物5:2-氯甲基-5-羟基吡啶酮,产率为94.0%。
(4)化合物6的合成
称取不同摩尔比的2-氯甲基-5-羟基吡啶酮和壳寡糖溶于DMSO水溶液中,加入适量氢氧化钠做缚酸剂,于室温下过夜反应,用过量无水乙醇沉淀、反复洗涤,用去离子水透析后冻干得到化合物6,即壳寡糖-羟基吡啶酮缀合物。壳寡糖-羟基吡啶酮缀合物I和II分别简写为COS-HPO1和COS-HPO2。
化合物6:COS-HPO1和COS-HPO2得率为分别为52.7%和74.7%。
本发明具有以下技术优势:
1、本发明获得了一种新的壳寡糖-羟基吡啶酮缀合物,其同时具备抗氧化、抑制酪氨酸酶和抑菌性能。其抗氧化活性明显强于背景技术所述的缀合物1和3,抗菌谱更广且抗菌活性明显强于缀合物1和2。此外,本发明中所引入的羟基吡啶酮结构是酪氨酸酶抑制剂的重要支架,本发明所述的壳寡糖-羟基吡啶酮缀合物还具有显著的酪氨酸酶抑制活性。
2、此类新的壳寡糖-羟基吡啶酮缀合物(COS-HPO2)无细胞毒性且有良好的水溶性,在食品、医药、化妆品等领域有广泛的应用价值。
可作为食品保鲜剂应用于食品工业,作为抗菌剂应用于医药领域,还可作为美白剂应用于化妆品领域。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为作为原料的壳寡糖(A)、中间产物的2-氯甲基-5-羟基吡啶(B)、作为产物的壳寡糖缀合物COS-HPO1(C)和作为产物的COS-HPO2(D)的1H-NMR谱(核磁共振氢谱图);
图2为壳寡糖-羟基吡啶酮缀合物对单酚酶的抑制作用;
图3为壳寡糖-羟基吡啶酮缀合物对二酚酶活力的影响;
图4为壳寡糖-羟基吡啶酮缀合物对细胞存活率的影响;即,壳寡糖和壳寡糖-羟基吡啶酮缀合物COS-HPO2对RAW 264.7(A)和MRC-5(B)细胞的细胞毒性;
图5为南美白对虾在4℃储藏过程中细菌总数的变化;
图6为南美白对虾在4℃储藏过程中挥发性盐基氮TVB-N的变化;
图7为南美白对虾在4℃储藏过程中颜色的变化。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此。
本发明中:DMSO水溶液中,DMSO和水的体积比为1:1。
羟基吡啶酮结构的壳寡糖缀合物为:
由重均分子量800~1000Da,脱乙酰度85%~90%的壳寡糖制备而成,R为羟基吡啶酮基,n为2~20,壳寡糖的氨基和羟基上同时引入了羟基吡啶酮基。
实施例1、一种壳寡糖-羟基吡啶酮缀合物的制备方法:
称取2g(12.4mmol)壳寡糖(重均分子量1000Da,脱乙酰度85%)和0.99g(6.2mmol)2-氯甲基-5-羟基吡啶酮于圆底烧瓶中。依次加入20mL DMSO和20mL水搅拌至溶解,然后加入0.49g氢氧化钠(12.4mmol)做缚酸剂。室温搅拌反应12h,反应结束后用过量无水乙醇(约200mL)沉淀,所得沉淀加水(约20mL)复溶,滴加1mol/L的稀盐酸调pH至弱酸性(即,Ph约为5.5)。再次醇沉(加入约100mL无水乙醇沉淀)、抽滤,重复三次。将得到的固体溶于少量(约20mL)去离子水后用纤维素透析袋(截留分子量500)于蒸馏水中透析48h,真空冷冻干燥24h(真空度1Pa、温度-60℃)得到壳寡糖-羟基吡啶酮缀合物Ⅰ,用核磁共振氢谱鉴定结构并通过积分分析其取代度。终产物的得率为52.7%,取代度为0.43。
该含羟基吡啶酮结构的壳寡糖缀合物Ⅰ为:
n为2~20(n的范围与原料壳寡糖一致)。
含羟基吡啶酮结构的壳寡糖缀合物得率的计算公式为:
含羟基吡啶酮结构的壳寡糖缀合物取代度计算公式为:
实施例2、
称取2g(12.4mmol)壳寡糖(重均分子量1000Da,脱乙酰度85%)和3.96g(24.8mmol)2-氯甲基-5-羟基吡啶酮于圆底烧瓶中。依次加入20mL DMSO和20mL水搅拌至溶解,然后加入49.6mmol氢氧化钠做缚酸剂。室温搅拌反应12h,反应结束后用过量无水乙醇沉淀,加水复溶,滴加适量1mol/L的氢氧化钠溶液调pH至弱碱性(即,pH约为8.5)。再次醇沉、抽滤,重复三次。将得到的固体溶于少量去离子水后用纤维素透析袋(截留分子量500)于蒸馏水透析48h,真空冷冻干燥24h得到壳寡糖-羟基吡啶酮缀合物Ⅱ,用核磁共振氢谱鉴定结构并通过积分分析其取代度。终产物的得率为74.7%,取代度为1.2。
该含羟基吡啶酮结构的壳寡糖缀合物Ⅱ为:
n为2~20。
图1中A-D分别为壳寡糖、2-氯甲基-5-羟基吡啶酮和本发明所得产物的壳寡糖-羟基吡啶酮缀合物COS-HPO1和COS-HPO2的核磁共振氢谱图。
图1-B为2-氯甲基-5-羟基吡啶酮的氢谱图,化学位移为8.19ppm和7.44ppm处的峰为吡啶酮环上H6和H3质子峰,4.94ppm处则对应-CH2(H7)的质子峰。
与壳寡糖相比,两个缀合物的氢谱图中均出现了新的质子峰。图1-C中化学位移7.95ppm、7.79ppm和7.04ppm、6.86ppm处的峰分别对应为羟基吡啶酮骨架上H6和H3的质子峰,4.36ppm、4.41ppm归属为-CH2(H7)的化学位移。图1-D中化学位移7.44ppm、7.38ppm和6.47ppm、6.31ppm的峰分别对应为羟基吡啶酮骨架上H6和H3的质子峰,4.51ppm、4.46ppm归属为-CH2(H7)的化学位移。这几个峰的存在,证明2-氯甲基-5-羟基吡啶酮与壳寡糖发生了反应,目标产物的形成。
实验1、抗氧化活性的测定
通过测定壳寡糖-羟基吡啶酮缀合物对DPPH自由基、ABTS自由基和羟基自由基的清除率来评价其抗氧化活性。
1、DPPH自由基清除率的测定
参考文献方法(Antioxidant and hypolipidemic activities of pectinisolated from citrus canning processing water.LWT,2022,15,113203)进行测定。
2、ABTS自由基清除率的测定
参考文献方法(Antioxidant activity of water-soluble chitosanderivatives.Bioorganic&Medicinal Chemistry Letters,2001,11(13),1699-1701)进行测定。
3、羟基自由基清除率的测定
参考文献方法(Purification,characterization and antioxidant activityof polysaccharides from Porphyra haitanensis.International Journal ofBiological Macromolecules,2020,165,2116-2125)进行测定。
含羟基吡啶酮结构的两个壳寡糖缀合物对三种自由基都表现出良好的清除能力,它们对DPPH、ABTS和羟基自由基的清除活性远强于壳寡糖,其中COS-HPO2强于COS-HPO1(表1)。
表1、壳寡糖-羟基吡啶酮缀合物的自由基清除活性(IC50(mg/mL))
将背景技术中所述的壳寡糖缀合物1~壳寡糖缀合物3按照上述方法进行检测,就DPPH自由基的半数清除浓度(IC50)而言:现有的壳寡糖缀合物1~壳寡糖缀合物3中效果最佳的壳寡糖缀合物1的IC50仅约为0.32mg/mL,其自由基清除能力均弱于本发明实施例1、实施例2所得产物。
实验2、酪氨酸酶抑制抑制活性的测定
1、缀合物对蘑菇酪氨酸酶单酚酶抑制活性的测定
酪氨酸酶单酚酶活力测定参考文献(Solid-phase synthesis of kojic acid-tripeptides and their tyrosinase inhibitory activity,storage stability,andtoxicity.Bioorganic&Medicinal Chemistry Letters,2004,14(11),2843-2846)的方法并作修改:将磷酸缓冲液(pH 6.86)和L-酪氨酸溶液(2mM)放置在30℃的水浴锅中保温。用移液枪分别吸取180μL磷酸缓冲液、100μL L-酪氨酸于96孔板中,加入10μL不同浓度(终浓度为0.1、0.2、0.4、0.8、1.6mg/mL)的抑制剂(分别为本发明所得的COS-HPO1和COS-HPO2,以及作为对照的COS和α-熊果苷(α-Arbutin)),最后快速加入10μL蘑菇酪氨酸酶溶液(1000μ/mL),水平混匀后迅速放入30℃的恒温培养箱保温10min,于475nm处记录吸光度值。α-熊果苷和COS作阳性对照;每个浓度设置3个平行取平均值。不同组的反应体系如表2。
表2、反应液体系
缀合物对酪氨酸酶单酚酶活性的抑制率按如下公式进行计算,所得结果如图2所示。
抑制率(%)=[1-(OD3-OD4)/(OD1-OD2)]×100%
其中OD1、OD2、OD3和OD4分别为第一到第四组溶液的吸光度值。
由图2可以看出,两个缀合物对酪氨酸酶单酚酶活性的抑制作用随浓度的增加而增大,COS-HPO1和COS-HPO2的单酚酶抑制活性远强于壳寡糖,半抑制浓度(IC50)分别为0.67和0.28mg/mL。
COS-HPO1和COS-HPO2的单酚酶抑制活性明显强于传统的酪氨酸酶抑制剂α-Arbutin(IC50值为0.95mg/mL)。
2、缀合物对蘑菇酪氨酸酶二酚酶抑制活性的测定
酪氨酸酶二酚酶活力测定方法参考文献(A Novel Inhibitor Against MushroomTyrosinase with a Double Action Mode and Its Application in Controlling theBrowning of Potato.Food and Bioprocess Technology,2017,(3),1-10)并作修改:将磷酸缓冲液(pH 6.86)和L-多巴溶液(0.5mM)和放置在30℃的水浴锅中保温。用移液枪分别吸取180μL磷酸缓冲液、100μLL-多巴于96孔板中,加入10μL不同浓度(终浓度为0.1、0.2、0.4、0.8、1.6mg/mL)的抑制剂,最后快速加入10μL蘑菇酪氨酸酶溶液(1000μ/mL),水平混匀后迅速在30℃恒温环境中用酶标仪于475nm处监测反应体系的吸光度值,30s的间隔测定10min,以吸光度值对时间作图,所得曲线的直线部分的斜率即为酶活力,再以酶的相对剩余活力对抑制剂浓度作图,所得结果如图3所示。
二酚酶的相对活力随抑制剂浓度增大而不同幅度的降低,说明缀合物对二酚酶的抑制效果与浓度呈正比。两个缀合物COS-HPO1和COS-HPO2的半抑制浓度(IC50)分别为0.73mg/mL和0.30mg/mL,COS-HPO2对二酚酶活力的抑制能力强于COS-HPO1。
3、缀合物对酪氨酸酶二酚酶抑制类型和抑制常数的测定
测定方法与上述测定方法基本相同,固定酪氨酸酶的浓度,改变加入的L-多巴的量,测定不同浓度缀合物对二酚酶酶活力的影响。采用Lineweaver-Burk双倒数作图法,以反应速率的倒数1/v为横坐标,底物浓度的倒数1/[S]为纵坐标作图,直线于Y轴的截距为1/Vmax,于X轴的截距为1/Km的绝对值,通过得到的拟合直线来判断抑制类型,再分别以斜率和截距对抑制剂浓度进行二次作图,计算出酶促反应的各种动力学参数:米氏常数Km、最大反应速度Vm和抑制常数KI、KIS。
随着缀合物COS-HPO1和COS-HPO2浓度的增大,米氏常数Km值增大,酶促反应的最大速率Vmax值减小,说明两个缀合物对二酚酶均为混合型抑制。两个缀合物对酪氨酸酶二酚酶抑制类型和抑制常数见表3。
表3、缀合物的酪氨酸酶二酚酶抑制类型及抑制常数
实验3、壳寡糖缀合物抗菌活性测定
通过测定抑菌圈直径大小、最小抑菌浓度(MIC)最小杀菌浓度(MBC)实验研究壳寡糖及其缀合物对腐败希瓦氏菌(Shewanella putrefaciens)、铜绿假单胞菌(Staphylococcus aureus)、大肠杆菌(Listeria monocytogenes)、金黄色葡萄球菌(Escherichia coli)和单增李斯特菌(Pseudomonas aeruginosa)的抑菌活性。
1、抑菌圈试验(牛津杯法)
将灭菌后的牛津杯摆放在平板培养皿上,待琼脂培养基(每管20mL)冷却至50℃左右时,加入1mL的菌液(105CFU/mL),轻轻摇匀后倒入培养皿中,待凝固后用无菌镊子将牛津杯慢慢取出。平板上每个孔中分别加入100μL(40mg/mL)的COS、COS-HPO1、COS-HPO2和ε-PL水溶液,无菌水作空白对照。然后放置在恒温培养箱中37℃(腐败希瓦氏菌30℃)培养24h后,观察并测量抑菌圈的直径,每组平行3次,取平均值。实验结果如表4所示。
表4、壳寡糖-羟基缀合物的抑菌圈实验结果
2、最小抑菌浓度(MIC)和最小杀菌浓度(MBC)测定
在无菌试管(13×100mm)中将样品溶液(0.96mL,66.67mg/mL)与1.0mL灭菌的大豆肉汤混合,然后进行两倍梯度稀释。将每种细菌悬浮液(40μL,105CFU/mL)添加到每个稀释溶液(1.96mL)中,以达到0.0625、0.125、0.25、0.5、1、2、4、8、16、32mg/mL的最终样品浓度。不加样品的试管作阳性对照,不加菌液和样品的作阴性对照,培养24h后观察未发生浑浊试管所代表的样品溶液终浓度即为最小抑菌浓度MIC,每组试验平行三次。
选取最小抑菌实验中未产生浑浊的试管(抑制剂的终浓度≥MBC),吸取100μL培养液至固体培养基上,用涂布棒涂布均匀后在37℃(腐败希瓦氏菌30℃)下培养24h,没有菌落生长的平板对应的最小样品浓度即为最小杀菌浓度MBC。
最小抑菌浓度(MIC)和最小杀菌浓度(MBC)实验结果见表5和表6。
表5、壳寡糖-羟基吡啶酮缀合物的MIC(mg/mL)
表6、壳寡糖-羟基吡啶酮缀合物的MBC(mg/mL)
由表5和表6可知,COS-HPO1和COS-HPO2对五种菌的抑制作用明显强于原料壳寡糖,其中COS-HPO2强于COS-HPO1。
实验4、细胞毒性实验(CCK-8法)
以实施例2所得壳寡糖缀合物COS-HPO2为例,通过CCK-8法评估了其细胞毒性。将RAW264.7和MRC-5细胞溶液(100μL,约2×104个细胞/mL)接种到96孔板中。37℃培养24h后,弃上清。每孔分别加入新鲜培养基(90μL;RAW264.7是DMEM培养基;MRC-5是MEM培养基)和样品(10μL;20、50、100、200μg/mL)溶液,培养24h,加入CCK-8试剂(10μL)并孵育4h,于450nm处记录每个孔的吸光度值。每个浓度设置5个复孔。其中Asample为样品组吸光度,Ablank是空白组吸光度(含有培养基和CCK-8溶液),Acontrol为对照组吸光度(含细胞和CCK-8溶液)。细胞存活率用如下公式进行计算,实验结果如图4所示。
细胞存活率(%)=(Asample-Ablank)/(Acontrol-Ablank)×100%
根据图4,两种细胞在20-200μg/mL浓度范围内的样品环境中存活率均在95%以上,说明COS和COS-HPO2在细胞水平上是安全的。
实验5、南美白对虾的保鲜实验
以实施例2所得壳寡糖缀合物COS-HPO2为例,并以ε-聚赖氨酸作为阳性对照进行虾保鲜实验。南美白对虾平均体长为10±0.5cm,平均体重为9.5±0.5g,随机分为5组分别在壳寡糖溶液(0.5%)、缀合物COS-HPO2溶液(0.5%)、ε-聚赖氨酸溶液(0.1%)、缀合物COS-HPO2(0.5%)+ε-聚赖氨酸(0.1%)溶液和超纯水(空白对照)中浸泡10min后捞出沥干用保鲜膜包裹分装无菌保鲜袋中,于4℃冰箱储藏。在储藏期内(0、2、4、6、8、10、12天)测定不同处理组虾样品的新鲜度指标。
1、细菌总数的测定
细菌总数的测定方法参考GB 4789.2-2016。具体方法为:取5g虾肉和45mL无菌生理盐水于无菌均质袋中,用均质器将虾肉拍打成匀浆并用无菌生理盐水稀释为10-2、10-3、10-4、10-5、10-6。分别吸取100μL在营养琼脂平板上涂布,每个稀释度平行3次,于37℃恒温培养箱中培养48±2h。选取菌落数在30CFU~300CFU范围内的平板进行计数。
不同样品处理后南美白对虾在储藏期间总细菌总数变化如图5所示。参照国标GB2733-2015,水产品菌落总数不超过5lg(CFU/g)都被认为是新鲜的,超过6lg(CFU/g)便不可再食用。储藏初期虾的菌落总数为3.70±0.05lg(CFU/g),第6天时空白组达到了6.77lg(CFU/g),不可再食用。而COS、ε-PL、COS-HPO2和COS-HPO2+ε-PL处理组分别在第8(6.6)、8(6.15)、10(5.94)和第12(5.97)天达到腐败最大限度。总体来说,COS-HPO2处理组菌落总数显著小于(P<0.05)COS处理组,而又显著高于COS-HPO2+ε-PL处理组。COS-HPO2+ε-PL处理组表现出最强的抑菌作用。
2、挥发性盐基氮(TVB-N)的测定
参考GB 5009.228-2016的方法测定不同储藏时间下虾肉的挥发性盐基氮含量。
南美白对虾在4℃保藏过程中TVB-N的变化趋势如图6所示,空白组在前面4天内增长缓慢,主要是由于储存初期微生物数量较少,腐败程度较低。之后微生物开始大量繁殖,TVB-N含量快速增加,在6天时就达到了36mg/100g,超过国标GB 2733-2015的限定值30mg/100g。而经过COS、ε-PL、COS-HPO2与COS-HPO2+ε-PL处理后四组虾TVB-N含量在第6天分别为25.14、24.77、17.50和15.25mg/100g。COS和ε-PL处理组均分别在第8天达到35.70和33.24mg/100g,达到腐败限度。COS-HPO2处理组在第10天达到29.50mg/100g,而COS-HPO2+ε-PL处理组在第12天才达到28.13mg/100g。上述结果表明COS-HPO2+ε-PL组可以有效的减少南美白对虾TVB-N的产生。
3、颜色变化的测定
用白板校准后的色差仪测定南美白对虾头胸甲处颜色的变化。基于CIE Lab表色系统来表示样品的色泽,指标分别为L*,a*,b*,并根据以下公式计算ΔE值。
其中L0、a0、b0为虾样品的初始值
如图7-A所示,各组的L*都随着时间的延长而下降,表明虾发生不同程度的黑变,其中空白组下降趋势最为明显。从初始41.6到储藏末期的26.5。COS-HPO2处理组和COS-HPO2+ε-PL的L*值显著高于其他组(P<0.05),说明COS-HPO2可以有效的抑制虾体内的多酚氧化酶的活性,延缓虾的黑变。
如图7-B所示,五组虾的a*值都呈现上升趋势为先慢后快。对照组上升速度显著高于其他组(P<0.05),在第6天的时候a*值已经达到了1.8,COS、ε-PL、COS-HPO2和COS-HPO2+ε-PL处理组分别为1.41、1.19、0.69和0.61。在12天时,COS-HPO2处理组和COS-HPO2+ε-PL处理组分别为2.52和2.19,显著低于COS处理组的3.8(P<0.05)。
由图7-C可以看出,五组虾b*的随着储藏时间的延长逐步增大,表明虾发生了黄变,COS-HPO2处理组和COS-HPO2+ε-PL处理组在整个储藏期间的b*都显著小于其他3组(P<0.05),在第12天时分别为5.31和4.39,显著小于COS处理组的8.13(P<0.05)。
图7-D为各处理组ΔE值的变化趋势,ΔE值可以反映虾整体的颜色变化,与前面3个指标趋势相同,COS-HPO2+ε-PL处理明显抑制了ΔE的增长,在储存末期(12天)空白组、COS、ε-PL、COS-HPO2和COS-HPO2+ε-PL处理组的ΔE值分别为18.4、12.97、12.61和6.53,而COS-HPO2+ε-PL处理组仅为4.52。综合以上结果,证明缀合物COS-HPO2和ε-PL协同使用可以最有效的减缓南美白对虾在储藏期间黑变、红变、黄变和总体色差的变化,保持虾的感官品质,延长货架期。
综上,本发明中,壳寡糖经过改性以后抗氧化活性、酪氨酸酶抑制活性和抑菌活性明显提高,在制备的缀合物COS-HPO2的基础上与ε-聚赖氨酸复配使用,可以进一步提升保鲜效果,将南美白对虾的货架期延长至12天。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (7)
1.壳寡糖-羟基吡啶酮缀合物,其特征在于结构通式为:;通式中的R为/>或/>;
n的范围为2~20;
壳寡糖-羟基吡啶酮缀合物的制备方法,包括:以曲酸为原料制备含有羟基吡啶酮基的2-氯甲基-5-羟基吡啶酮,2-氯甲基-5-羟基吡啶酮和壳寡糖进行亲核取代反应,将羟基吡啶酮基偶联到壳寡糖上,从而生成壳寡糖-羟基吡啶酮缀合物。
2.如权利要求1所述的壳寡糖-羟基吡啶酮缀合物的制备方法,其特征在于:
将2-氯甲基-5-羟基吡啶酮和壳寡糖溶于溶剂中,加入氢氧化钠做缚酸剂,于室温搅拌反应12±1 h,2-氯甲基-5-羟基吡啶酮:壳寡糖=1:2或2:1的摩尔比;2-氯甲基-5-羟基吡啶酮:氢氧化钠=1:2的摩尔比;
然后在反应所得物中加入乙醇使产物沉淀;所得的沉淀用去离子水透析,冷冻干燥,得到壳寡糖-羟基吡啶酮缀合物。
3.根据权利要求2所述的壳寡糖-羟基吡啶酮缀合物的制备方法,其特征在于:作为原料的壳寡糖的重均分子量为800~1000 Da,脱乙酰度85%~90%。
4.根据权利要求2或3所述的壳寡糖-羟基吡啶酮缀合物的制备方法,其特征在于:
溶剂为二甲基亚砜水溶液,所述二甲基亚砜水溶液中, 二甲基亚砜:水=1:1的体积比。
5.根据权利要求4所述的壳寡糖-羟基吡啶酮缀合物的制备方法,其特征在于:每1 g的壳寡糖配用20±5 mL的二甲基亚砜水溶液。
6.如权利要求1所述的壳寡糖-羟基吡啶酮缀合物的用途,其特征是至少为以下任一用途:
制备抗氧化剂或酪氨酸酶抑制剂或抑菌剂。
7.根据权利要求6所述的壳寡糖-羟基吡啶酮缀合物的用途,其特征是:用于食品的保鲜或化妆品的配制。
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