超高压结合酶法消减南美白对虾虾仁的致敏性

以南美白对虾虾仁为原料,研究超高压结合酶法对虾仁致敏性的消减效果。将南美白对虾去头、尾、壳、肠线后,采用超高压法和超高压结合酶法消减其致敏性,用间接酶联免疫吸附法检测致敏性消减效果,确定消减条件。结果表明：采用超高压法处理最佳条件为压力600 MPa、温度40 ℃、保压时间30 min,此条件下消减率为45.66%;超高压结合胰蛋白酶法最佳条件为压力200 MPa、温度40 ℃、保压时间30 min、胰蛋白酶加酶量3 000 U/mg,此条件下消减率为95.27%。由此可见,超高压法对虾仁致敏性有消减作用,超高压结合酶法对虾仁致敏性消减的效果更好。     

高压处理诱发虾原肌球蛋白结构变化与致敏性的关系

以凡纳滨对虾原肌球蛋白为原料,研究不同高压条件下引发构象的变化与致敏性的关系。采用不同高压条件(压力0.1~800.0 MPa,处理时间10~40 min,温度10~37℃)处理原肌球蛋白(tropomyosin,TM),用圆二色谱法检测二级结构变化,荧光光度法检测三级结构变化,间接酶联免疫吸附法检测致敏性。结果显示,超高压处理能够引发TM的致敏性和三级结构变化,而对二级结构无显著影响;高压条件下TM致敏性的变化与其三级结构的改变有显著相关性。在实验压力范围内,20℃、300 MPa处理40 min TM疏水性氨基酸暴露程度最小,且致敏性最低(OD_(492 nm)为0.210±0.005);700 MPa条件下暴露程度最高,且致敏性最高(OD_(492 nm)为0.328±0.004)。因此,TM致敏性与其三级结构有关,与二级结构无关。     

高压结合酶法消减南美白对虾虾仁致敏性

以南美白对虾虾仁为原料,研究高压结合酶法对虾仁过敏原致敏性的消减作用。将南美白对虾去头去尾去壳去肠线后,采用超高压法和高压结合酶法消减其致敏性,用间接酶联免疫吸附法检测致敏性消减效果,确定消减条件。结果表明：采用超高压法处理,在压力100 MPa、温度25 ℃、保压时间15 min的条件下,虾仁过敏原的致敏性降低了67.09%,且与处理过程中蛋白溶出量有关;采用高压结合酶法处理,在盐水质量浓度1 g/100 mL、酶与虾仁质量比1∶130、压力450 MPa、温度40 ℃、保压时间55 min的条件下,虾仁过敏原的致敏性降低了86.58%。由此可见,高压处理对虾仁过敏原的致敏性有消减作用,高压结合酶法的消减效果更好。     

超高压引发虾仁致敏性及感官品质的变化

以南美白对虾为原料,研究超高压处理引发虾仁致敏性及感官品质的变化。将南美白对虾虾仁采用不同压力(0.1~800 MPa)处理,用间接酶联免疫吸附法检测致敏性消减效果,确定消减条件,并分别用电子眼、电子鼻和电子舌检测其颜色、气味和滋味的变化。结果表明:在压力600 MPa、温度40℃的条件下处理30 min,能够较好地消减虾仁致敏性(消减率为45.66%)。另外,高压处理也引发虾仁感官品质的变化,与未处理虾仁相比,随着压力的升高,超高压处理的虾仁的主色色号及占比逐渐增大;主要气味成分乙酰吡嗪的含量逐渐降低,且二甲基三硫类化合物消失;咸味和酸味逐渐增加,而鲜味增加了32~34倍,并且出现了甜味和苦味;在相同压力下,甜味和苦味的强度值相近。因此,超高压处理对虾仁致敏性有消减作用,同时,会引起虾仁色、香、味等感官品质的变化。     

超高压结合酶法消减南美白对虾蛋白过敏原研究

以南美白对虾为研究对象,提取水溶性虾蛋白,采用超高压法、超高压处理后再用木瓜蛋白酶水解、超高压下直接酶解等方法消减其过敏原蛋白,用间接酶联免疫吸附法检测过敏原消减效果,确定消减过敏原的条件。结果表明：采用超高压法处理,其最佳条件为压力150MPa、温度45℃、保压时间35min,产物抗原抗体反应的OD492nm值为0.1997;高压结合木瓜蛋白酶水解法最佳条件为：350MPa、温度45℃、保压时间20min,产物抗原抗体反应的OD492nm值为0.0492;超高压下直接用木瓜蛋白酶处理,其最佳条件为：压力300MPa、温度45℃、保压时间35min,产物抗原抗体反应的OD492nm值为0.05。由此可见,高压对过敏原蛋白有消减作用,先高压再水解和超高压下直接酶处理对过敏原的消减效果更好。     

超高压处理对凡纳滨对虾原肌球蛋白构象的影响

以凡纳滨对虾的原肌球蛋白（tropomyosin,TM）为原料,研究不同超高压条件下引发的TM构象变化。采用压力0.1～800 MPa,处理时间10～40 min,温度10～37 ℃处理TM,圆二色光谱法检测其二级结构变化,荧光光度法检测其荧光强度变化,DNA Star Protean软件预测可塑性。结果显示,超高压处理对TM的二级结构无显著影响,不同超高压条件下TM三级结构有显著变化。在实验压力范围内,300 MPa处理的TM疏水性氨基酸暴露程度最小;700 MPa处理后暴露程度最高。经预测,TM的可塑性区域占总氨基酸序列的72.9%。因此,超高压条件下TM的二级结构稳定,三级结构变化较大,三级结构的变化可能与其可塑性区域的比例较高有关。     

植物乳杆菌（Lactobacillus plantarum）对过敏原原肌球蛋白免疫活性的消减作用

为探究植物乳杆菌（Lactobacillus plantarum）对过敏原原肌球蛋白（tropomyosin,TM）免疫活性的影响,以富集纯化的TM为研究对象,利用植物乳杆菌的菌体、破碎内容物、菌体碎片、胞内酶提取液,以及去除蛋白、脂肪和脂磷壁酸、羧基酯化、氨基甲基化后的菌体分别水解TM 12～48 h,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白免疫印迹和竞争性酶联免疫吸附剂测定（enzyme linked immunosorbent assay,ELISA）等方法测定免疫活性,利用表位多克隆抗体结合红外光谱技术研究植物乳杆菌对TM二级结构及其致敏表位的影响,以解析植物乳杆菌消减TM免疫活性的作用位点。免疫学分析结果发现,不同提取物水解TM 48 h后,菌体对TM免疫活性的消减率最高（76.9%）,而菌体碎片和氨基甲基化菌体的消减率最低,分别为60.7%、61.7%,表明植物乳杆菌对原肌球蛋白免疫活性有一定消减作用。红外光谱及ELISA结果显示植物乳杆菌各成分提取物会较大程度改变TM的二级结构并破坏其致敏表位,而氨基甲基化菌体和菌体碎片对TM的α-螺旋结构和致敏表位的破坏最小,且TM的折叠化结构较少,对TM免疫活性消减效果最差。此外,研究发现氨基可能是植物乳杆菌消减TM免疫活性的一个重要作用位点。本研究可为食物过敏原的活性控制及低致敏性水产加工制品的开发提供理论参考。     

蛋白溶出与变性结合消减虾仁致敏性

以南美白对虾虾仁为原料,采用高压下蛋白溶出与变性相结合的方法消减虾仁致敏性。在室温下采用不 同压力（0.1～900.0 MPa）处理虾仁,确定适合虾仁蛋白溶出和变性的压力条件,并以虾仁蛋白致敏性的消减效果 为指标确定虾仁的处理条件。结果显示,在200.0 MPa下保压处理30 min,有利于虾仁蛋白的溶出;当压力大于等 于500.0 MPa时,可引起虾仁蛋白的变性;当压力在600.0 MPa时,虾仁蛋白的致敏性最小。将虾仁在200.0 MPa下 处理30 min后,再用600.0 MPa处理30 min,虾仁的致敏性消减率大于80%。因此,高压处理可消减虾仁的致敏性, 采用适合蛋白溶出与变性相结合的压力处理,能够提高虾仁致敏性消减效果。     

超高压对木瓜蛋白酶构象及酶活力的影响

为研究超高压对木瓜蛋白酶构象变化与酶活力的影响,本研究利用红外光谱法和荧光光谱法对木瓜蛋白酶经超高压处理后的结构变化及特定氨基酸微环境变化进行了分析。结果表明：与常压相比,超高压处理对木瓜蛋白酶活力均有显著影响（P＜0.05）。其中,200 MPa、37 ℃、20 min处理时,酶活力在所选处理范围内达到最大,较常压下的酶活力提高了6.8%;红外光谱结果表明：超高压处理后,木瓜蛋白酶二级结构变化与酶活力变化相关性较差;荧光光谱结果显示：200 MPa、37 ℃处理木瓜蛋白酶20 min,228 nm波长激发后木瓜蛋白酶的外源性荧光强度达到最低（2 023）,荧光强度变化与酶活力变化规律有良好的相关性。因此,木瓜蛋白酶活性变化与疏水性氨基酸的暴露程度有关,暴露程度越小,酶活力越大。     

超高压结合酶法消减南美白对虾虾肉中的过敏原

以虾肉为原料,研究超高压对酶法消减南美白对虾虾肉过敏原的强化作用。将南美白对虾去头、去尾、去壳、去肠线后用匀浆机匀浆,制成虾肉酱,分别采用超高压法、超高压处理后再用木瓜蛋白酶水解、超高压条件下直接酶解的方法消减其过敏原,用间接酶联免疫吸附法检测过敏原消减效果,确定消减过敏原的条件。结果表明：采用超高压法处理,其最佳条件为：压力200Mpa、温度40℃、保压时间35min,此条件下产物与抗体反应的OD492nm值为0.1986;先超高压处理再用木瓜蛋白酶水解,其水解的最佳条件为：底物质量浓度10g/100mL、温度60℃、酶与底物质量比1:200,此条件下产物与抗体反应的OD492nm值为0.0487;超高压条件下直接用木瓜蛋白酶处理,其最佳条件为：压力300Mpa、温度40℃、保压时间35min,产物与抗体反应的OD492nm值为0.0516。由此可见,超高压处理对南美白对虾过敏原有消减作用,先超高压处理再用木瓜蛋白酶水解和超高压条件下直接酶处理对过敏原的消减效果更好。     

Hydrolysis under high hydrostatic pressure as a means to reduce the binding of beta-lactoglobulin to immunoglobulin E from human sera

Cows' milk allergy is the most frequent food allergy in children, and beta-lactoglobulin (beta-Lg) is a major allergen. Milk-based hypoallergenic ingredients are manufactured by enzymatic hydrolysis, a process that could be improved by the application of high-pressure treatments. This study showed that the treatment of beta-Lg dissolved in buffer with chymotrypsin and trypsin under high pressure for relatively short times accelerated proteolysis by leading to a rapid removal of the intact protein. The rapid proteolysis of the beta-Lg substrate under pressure led to the production, in 20 min, of hydrolysates with lower immunoglobulin (Ig) G binding than those produced in 8 h (chymotrypsin) or 48 h (trypsin) at atmospheric pressure. However, those hydrolysates retained some residual IgE-binding properties that could be traced to the preferential release, during the initial stages of proteolysis, of peptides containing IgE epitopes, such as (Val-41-Lys-60), (Leu-149-Ile-162), and (Ser-21-Arg-40). The formation of these fragments was favored when proteolysis was conducted under high pressure due to the preferential hydrolysis of Arg-40 and Arg-148 by trypsin, and Tyr-42 and Leu-149 by chymotrypsin, all located at the dimer interface of beta-Lg or very close to it. Although our results do not support that trypsin and chymotrypsin under high pressure selectively address the allergenic regions of beta-Lg, it is possible to select the conditions that quickly produce hydrolysates with reduced potential allergenicity that could be used in hypoallergenic foods.     

两步酶解法制备低致敏性乳清蛋白及其致敏性评价

目的 探究两步复合酶解工艺处理对牛乳清蛋白(whey protein isolate, WPI)结构和致敏性的影响。方法 实验采用邻苯二甲醛法、体积排阻色谱法分析WPI水解物在不同酶解时间下的水解程度,同时采用酶联免疫吸附法(enzyme-linked immunoassay, ELISA)、细胞模型和小鼠模型对水解物进行体外和体内的致敏性评估,并利用超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)分析水解物中的残留过敏原表位。结果 WPI经碱性蛋白酶和木瓜蛋白酶顺序酶解3.0 h后水解度达到14.19%,在水解2.0~3.0 h期间,WPI水解物主要由小肽(<5 kDa)组成。水解3.0 h后,水解产物与特异性抗体的结合能力降低了97.83%,细胞模型和小鼠模型结果证实,酶解产物的体外、体内致敏性显著降低,与空白对照组无显著性差异。UPLC-MS/MS结果表明WPI水解3.0 h产物中70.59%的肽段不含过敏原表位。结论 复合酶解工艺有效降低了WPI致敏性,本研究构建的致敏性综合评价体系可为低敏乳制品水解工艺的开发提供指导。     

高静压协同酶法处理对白果蛋白抗原性的影响

本文研究了高静压结合酶解处理对白果蛋白抗原性的影响,分别采用4种蛋白酶水解白果蛋白,水解前分别采用不同压力的高静压对白果蛋白进行预处理,酶解产物水解率和分子量采用OPA法和SDS-PAGE测定,致敏性采用western-blotting和ELISA法测定。结果表明,木瓜蛋白酶,碱性蛋白酶或胃蛋白酶为水解酶时,高静压能显著提高白果蛋白的水解率和降低其致敏性;而中性蛋白酶为水解酶时,白果蛋白的水解和脱敏效果很差,即使高压处理也未见明显提高。木瓜蛋白酶或碱性蛋白酶在处理压力为300 MPa时,而胃蛋白酶在400 MPa时,其水解和脱敏效果最好,在此条件下白果蛋白能被水解为分子量小于15 ku的多肽,95%以上的白果蛋白致敏性能被消除,酶解产物中致敏蛋白条带全部消失。因此,高静压处理能明显提高蛋白酶对白果蛋白的水解效率和脱敏效果,但是取决于选择的蛋白酶种类和处理压力的大小。     

Enzymatic hydrolysis of fish gelatin under high pressure treatment

The effect of high pressure treatment on the hydrolysis of fish skin gelatin and the antioxidant properties of the hydrolysates was evaluated. Hydrolysis was performed by Alcalase, collagenase, trypsin and pepsin both at atmospheric pressure and under high pressure (100 MPa/15 and 30 min, 200 MPa/15 and 30 min, 300 MPa/15 min). The degree of hydrolysis (DH) was determined according to the base consumption via pH‐stat as well as the percentage of soluble nitrogen in trichloroacetic acid (TCA). About 16% of nitrogen in the gelatin was still soluble in 10% TCA, indicative of a significant amount of low molecular weight components. The high pressure treatment increased the DH with all the enzymes used between 5% and 10%. However, in comparison with the hydrolysates obtained at atmospheric pressure after 3 h of digestion under controlled conditions (using a pH‐stat), the radical scavenging capacity of the pressured hydrolysates was only significantly enhanced when Alcalase or collagenase were used. High pressure may be a useful tool for the quick obtaining of gelatin hydrolysates with antioxidant capacity.     

Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

Effects of Enzymatic Hydrolysis Assisted by High Hydrostatic Pressure Processing on the Hydrolysis and Allergenicity of Proteins from Ginkgo Seeds

The purpose of this work was to study the combined effect of high hydrostatic pressure (HHP) and enzymatic hydrolysis treatment on the hydrolysis and allergenicity of ginkgo seed proteins (GSPs). Four food-grade proteases (papain, alcalase, pepsin, and neutrase) were used, and HHP (200, 300, and 400 MPa separately) was applied prior to hydrolysis. The extent of hydrolysis was measured with the o-phthaldialdehyde method, SDS-PAGE, and MALDI-TOF-MS, and the allergenicity was assessed with a Western blot and enzyme-linked immunosorbent assay (ELISA). The results showed that HHP could significantly improve the extent of proteolysis by papain, alcalase, or pepsin and reduce the antigenicity of GSP, whereas neutrase showed poor effects at any pressure. Papain and alcalase showed the highest proteolysis at 300 MPa, followed by pepsin at 400 MPa, and all of the obtained hydrolysates showed molecular weights lower than 10 kDa; furthermore, papain or alcalase at 300 MPa as well as pepsin at 400 MPa reduced antigenicity by more than 95 %, and all of the immunoreactive bands disappeared in the obtained hydrolysates. These results suggest that HHP can enhance the hydrolysis of GSP by certain enzymes and reduce the residual antigenicity of the hydrolysates. The obtained hypoallergenic hydrolysates could be used as a source of peptides for food ingredients.     

Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and high pressure

Soybean (Glycine max) whey was hydrolyzed for 15 min using three food-grade proteases (Alcalase, Neutrase, Corolase PN-L) at atmospheric pressure (0.1 MPa) and under high pressure (HP) at 100, 200, and 300 MPa. All hydrolysates were analyzed by SDS-PAGE and their residual immunoreactivity was assessed by immunoblotting using the sera from children allergic to soybean. As shown in SDS-PAGE, Alcalase, Neutrase, and Corolase PN-L produced different patterns of hydrolysis. Each enzyme showed a similar proteolytic activity at atmospheric pressure and at 100 MPa, while an increased degree of hydrolysis was observed at 200 and 300 MPa. No residual antigenicity was observed in the hydrolysates obtained by Alcalase and Corolase PN-L in all considered conditions of hydrolysis. A positive reaction associated with a band having molecular weight of about 70 kDa was observed in the immunoblotting of the hydrolysates obtained with Neutrase at 0.1, 100, and 200 MPa, while no antigenicity was detected for those samples obtained under high pressure, at 300 MPa. These results suggest that high pressure combined with suitable enzymatic activity could be a useful tool for obtaining hydrolysates with low immunoreactivity to be used in special foods (hypoallergenic foods).     

响应曲面法优化小黄鱼ACE抑制肽的酶解条件

以小黄鱼为原料,利用酶解技术制备高活性的ACE抑制肽。通过单因素及响应曲面分析,确定胰蛋白酶(PTN6.0S)的酶解工艺,利用反相高效液相色谱(RP-HPLC)法测定酶解产物(0.05g/L)的ACE抑制率,研究pH值、温度、时间、底物质量浓度、酶与底物比等因素对ACE抑制肽活性的影响。结果显示：最佳工艺条件为pH7.0、温度50℃、时间16.5h、底物质量浓度5.6g/100mL、酶与底物比9‰,酶解产物ACE抑制率达87.36%。