燕窝低聚肽制备工艺及生物学评价
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摘要
采用生物酶解技术研究了燕窝低聚肽的制备工艺。研究了4种不同类型的酶对燕窝的降解行为,比较了燕窝酶解前后分子量范围、氨基酸组以及唾液酸含量变化,采用动物实验研究了燕窝低聚肽对小鼠免疫细胞活性的影响。结果表明,燕窝经60℃热变性处理后可通过胰蛋白酶和胃蛋白酶有效降解,酶解处理4. 0 h燕窝降解率分别达到96%和91%,碱性蛋白酶和中性蛋白酶可部分降解燕窝,4. 0 h后残留量分别为40%和36%。酶解后的燕窝低聚肽的分子量主要集中在1～3 ku,低聚肽中总蛋白含量高于燕窝,酶解后唾液酸含量为14. 3%。动物实验结果表明燕窝低聚肽增强刀豆蛋白A(Concanavalin A,Con A)诱导胸腺依赖性淋巴细胞(Thymus-dependent lymphocyte,T cell)增殖效果高于燕窝(P> 0. 05);燕窝与低聚肽在增强抑制自然杀伤细胞(Nature killer cell,NK cell)活性方面较为相近(P <0. 05)。
The enzymatic hydrolysis technique was used to study the preparation of bird's nest oligopeptides. The degradation behaviors of bird's nest were studied using four kinds of enzymes. The molecular weight range,amino acid composition and sialic acid content of the bird's nest were compared before and after enzyme digestion. Animal experiments were used to study the effects of bird' s nest oligopeptides on the activity of immune cells in mice. The results showed that the bird's nest could be effectively degraded by trypsin and pepsin after heat denaturation at 60℃ and the degradation rate was 96% and 91%,respectively after 4 hours of enzymatic treatment. However,alkaline protease and neutral protease could only partially degrade bird's nest. After 4 hours digestion,the residue rate of undissolved materials was 40% and 36%,respectively. The molecular weight of oligopeptides after enzymolysis was mainly concentrated in the range of 1 ku to 3 ku. The total protein content of oligopeptides was higher than that of bird's nests. The sialic acid content after enzymolysis was 14. 3%. The results of animal experiments showed that oligopeptides can significantly enhance the proliferation of concanavalin A( Con A)-induced T cell compared with bird's nest( P > 0. 05). There was no significant difference in the inhibition of NK cell activity between bird's nest and oligopeptides( P < 0. 05).
The enzymatic hydrolysis technique was used to study the preparation of bird's nest oligopeptides. The degradation behaviors of bird's nest were studied using four kinds of enzymes. The molecular weight range,amino acid composition and sialic acid content of the bird's nest were compared before and after enzyme digestion. Animal experiments were used to study the effects of bird' s nest oligopeptides on the activity of immune cells in mice. The results showed that the bird's nest could be effectively degraded by trypsin and pepsin after heat denaturation at 60℃ and the degradation rate was 96% and 91%,respectively after 4 hours of enzymatic treatment. However,alkaline protease and neutral protease could only partially degrade bird's nest. After 4 hours digestion,the residue rate of undissolved materials was 40% and 36%,respectively. The molecular weight of oligopeptides after enzymolysis was mainly concentrated in the range of 1 ku to 3 ku. The total protein content of oligopeptides was higher than that of bird's nests. The sialic acid content after enzymolysis was 14. 3%. The results of animal experiments showed that oligopeptides can significantly enhance the proliferation of concanavalin A( Con A)-induced T cell compared with bird's nest( P > 0. 05). There was no significant difference in the inhibition of NK cell activity between bird's nest and oligopeptides( P < 0. 05).
引文
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[8]刘琳,李修乐,高建萍,等.燕窝酶解产物中多肽的液相色谱质谱分析[J].中国中药杂志,2013,38(5):64-68.
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[11]王棘,战祥友,腾艳坤,等. DNFB柱前衍生化RP-HPLC法测定氨肽素的氨基酸含量[J].沈阳药科大学学报,2003,20(6):428-420.
[12]GB/T 22492-2008,大豆肽粉[S].中国国家标准化管理委员会,2008.
[13]王羚郦,李远彬,邱子博,等. 25种燕窝样品中唾液酸含量的测定与分析[J].中国实验方剂学杂志,2013,19(19):64-67.
[14]黄知几.燕窝对断乳后仔数智力水平及母鼠免疫功能的影响研究[D].福州:福建农林大学,2017.
[15]YAN C,ZHI H,XIAO M L,et al. In vitro biocompatibility and osteoblast differentiation of an injectable chitosan/nano-hydroxyapatite/collagen scaffold[J]. J Nanomater,2012,2012:1-6.
[16]马天宇,苗惠,张舒峰,等. MTS-PMS法测定脾细胞增殖及NK细胞活性的实验条件优化[J].毒理学杂志,2016,30(1):83-85.
[17]张诚,陈幸华,张曦,等. WST-8检测培养小鼠脾NK细胞增殖及杀伤活性的研究[J].西南国防医药,2008,18(3):313-315.
[18]林洁茹,周华,赖小平.燕窝研究概述[J].中药材,2006,29(1):85
[19]HIROKAZU Y,NAOKO Y,SHIN Y Y,et al. The expression of sialylated high-antennary N-glycans in edible bird's nest[J]. Carbohyd Res,2008,343:1373-1377.
[20]崔慧娥,李昌模,张燕,等.燕窝糖蛋白纯化及多克隆抗体的制备[J].食品工业科技,2009,30(7):307-309.
[2]SHI J Y,HU X T,ZOU X B,et al. A rapid and nondestructive method to determine the distribution map of protein,carbohydrate and sialic acid on edible bird's nest by hyper-spectral imaging and chemometrics[J]. Food Chem,2017,229:235-241.
[3]MA F C,LIU D C,Sketch of the edible bird's nest and its important bioactivities[J]. Food Res Int,2012,48:559-567
[4]WARASRI S,NARUMOL M,NIRUNDORN M. Nutritional composition of the farmed edible bird's nest(Collocalia fuciphaga)in Thailand[J]. J Food Comp Anal,2013,31:41-45.
[5]SHIM E K S,CHANDRA G F,LEE S Y. Thermal analysis methods for the rapid identification and authentication of swiftlet(Aerodramus fuciphagus)edible bird's nest-A mucin glycoprotein[J]. Food Res Int,2017,95:9-18.
[6]NURFATIN M H,ETTY S I K,ALIAH N,et al. Effect of enzymatic hydrolysis on angiotensin converting enzyme(ACE)inhibitory activity in swiftlet saliva[J]. Int Food Res J,2016,23(1):141-146.
[7]KONG H K,WONG K M,LO S C L. Identification of peptides released from hot water insoluble fraction of edible bird's nest under simulated gastro-intestinal conditions[J]. Food Rese Int,2016,85:19-25.
[8]刘琳,李修乐,高建萍,等.燕窝酶解产物中多肽的液相色谱质谱分析[J].中国中药杂志,2013,38(5):64-68.
[9]CHUA K H,LEE T H,NAGANDRAN K,et al. Edible bird's nest extract as a chondro-protective agent for human chondrocytes isolated from osteoarthritic knee:in vitro study[J]. BMC Complementary Alternative Med,2013,13:19-27.
[10]GUO C T,TAKAHASHI T,BUKAWA W,et al. Edible bird's nest extract inhibits influenza virus infection[J]. Antiviral Res,2006,70:140-146.
[11]王棘,战祥友,腾艳坤,等. DNFB柱前衍生化RP-HPLC法测定氨肽素的氨基酸含量[J].沈阳药科大学学报,2003,20(6):428-420.
[12]GB/T 22492-2008,大豆肽粉[S].中国国家标准化管理委员会,2008.
[13]王羚郦,李远彬,邱子博,等. 25种燕窝样品中唾液酸含量的测定与分析[J].中国实验方剂学杂志,2013,19(19):64-67.
[14]黄知几.燕窝对断乳后仔数智力水平及母鼠免疫功能的影响研究[D].福州:福建农林大学,2017.
[15]YAN C,ZHI H,XIAO M L,et al. In vitro biocompatibility and osteoblast differentiation of an injectable chitosan/nano-hydroxyapatite/collagen scaffold[J]. J Nanomater,2012,2012:1-6.
[16]马天宇,苗惠,张舒峰,等. MTS-PMS法测定脾细胞增殖及NK细胞活性的实验条件优化[J].毒理学杂志,2016,30(1):83-85.
[17]张诚,陈幸华,张曦,等. WST-8检测培养小鼠脾NK细胞增殖及杀伤活性的研究[J].西南国防医药,2008,18(3):313-315.
[18]林洁茹,周华,赖小平.燕窝研究概述[J].中药材,2006,29(1):85
[19]HIROKAZU Y,NAOKO Y,SHIN Y Y,et al. The expression of sialylated high-antennary N-glycans in edible bird's nest[J]. Carbohyd Res,2008,343:1373-1377.
[20]崔慧娥,李昌模,张燕,等.燕窝糖蛋白纯化及多克隆抗体的制备[J].食品工业科技,2009,30(7):307-309.