RADIOIMMUNOASSAY OF PAPAIN IN BEER

A radioimmunoassay (RIA) of papain is described which is capable of detecting 0·2 μg of papain/ml in beer, a level approximately 1% of that normally used for chillproofing. Changes in incubation conditions significantly accelerates the papain determination with only slight loss of accuracy. Apart from the high sensitivity it has been shown that the method is highly specific and distinguishes papain from the other chillproofing enzymes used.     

A VERY SENSITIVE METHOD FOR ASSAYING PROTEOLYTIC ACTIVITIES IN BEER

A very sensitive method for assaying chillproofing enzymes in beer is described using denatured ¹²⁵I-labelled human serum albumin as a substrate (40°C, pH 6.0 and 30 min reaction time). Less than 0.1 ug of papain in 1 ml of beer has been determined by this method. The results achieved by the proposed method were in good correlation with immunochemical and ‘hide power azure’ (HPA) methods.     

紫菜酶解物的制备及其特性

坛紫菜用纤维素酶和木瓜蛋白酶分别或联合处理,考察了提取物的得率、DPPH自由基清除率、氨基酸组成、蛋白溶解性和乳化性等性质。结果发现,利用木瓜蛋白酶对紫菜进行酶解时,固形物提取率稍微高于纤维素酶,利用这两种酶进行复合酶解时虽然可以提高紫菜的固形物得率,但不会改变蛋白提取率。紫菜酶解物的DPPH自由基清除率与蛋白含量之间存在显著的正相关关系,可能与提取物中具有抗氧化活性的氨基酸含量增加有关。另一方面,紫菜经复合酶酶解4h以上,提取物的蛋白溶解性在任一pH下都接近100%。而且,紫菜酶解物的乳化性在pH9最好,但乳化稳定性在pH7最低。      

SELECTED p-NITROANILIDES AS SUBSTRATES FOR SENSITIVE PAPAIN ASSAY

N-acetyl-Leu-Leu-Gly-p-nitroanilide and N-succinyl-Ala-Ala-(S-benzyl) Cys-p-nitroanilide were found to be very sensitive papain substrates, making it possible to detect 0.5 or 0.7 μg of technical papain in 1 mL of beer in a short time. This detection limit is about 10 times lower than the usual papain level used for chillproofing. The estimated kinetic constants (k_cat, K_m) for papain, bromelain and trypsin permit certain conclusions about papain specificity for oligopeptides, which will help to prepare even more specific p-nitroanilide substrates.     

INACTIVATION OF PAPAIN PROTEOLYTIC ACTIVITY IN THE PRESENCE OF ASCORBIC ACID AND CU++ IONS

The simultaneous presence of ascorbic acid, Cu⁺⁺ ions and oxygen causes reversible inactivation of proteolytic activity of papain. Low concentrations of ascorbic acid and Cu⁺⁺ ions have no effect on their own. Ascorbic acid at concentrations higher than 2 × 10⁻⁴ m inactivates papain irreversibly in the presence of Cu⁺⁺ ions. Papain inactivation is not accompanied by the destruction of papain molecules. Irreversible papain inactivation does not occur in solutions free of oxygen and the intermediates of ascorbic acid oxidation, i.e. dehydroascorbic acid and hydrogen peroxide, do not cause papain inactivation. The irreversible inactivation of papain is most probably due to free radicals formed during ascorbic acid oxidation. Owing to the possible occurrence of both ascorbic acid and Cu⁺⁺ ions in beer, partial irreversible inactivation of papain can be expected even at very low oxygen concentrations.     

蛋白酶处理对鸡骨泥营养组成的影响

研究了蛋白酶处理对鸡骨泥营养组成的影响。研究结果表明,木瓜蛋白酶适合用来水解鸡骨泥以提高游离氨基酸及钙的含量,其最优水解条件为:60℃,pH 6.5条件下,取浓度为50%的粗鸡骨泥,按照加酶量3000 U/g加入木瓜蛋白酶,水解5 h后,鸡骨泥水解液中游离氨基酸及游离钙含量分别可以达到2.0365 mg/g和0.9856 mg/g。适当微波加热预处理可以改善木瓜蛋白酶对粗鸡骨泥的水解作用。     

外源蛋白酶对黔式腊肉风味的影响

在黔式腊肉加工过程中,通过注射添加木瓜蛋白酶和中性蛋白酶,促进蛋白质降解,以利于风味物质的形成.结果表明:黔式腊肉最优酶解条件为木瓜蛋白酶添加量0.001％、中性蛋白酶添加量0.001％、食盐添加量4％、酶解时间48h;添加蛋白酶样品中非蛋白氮(NPN)、氨基态氮(AN)、挥发性盐基氮(TVBN)的含量以及游离氨基酸(FAA)种类和总量均高于未加酶样品;经GC-MS检测,添加蛋白酶样品中有65种挥发性风味物质,比未添加蛋白酶样品多10种,风味物质种类除烃类和羰基类化合物外,都有所增加,烃类、酚类和醇类相对含量增加,酯类相对含量减少.     

木瓜蛋白酶嫩化鹿肉方法的研究

对木瓜蛋白酶嫩化鹿肉的工艺参数进行研究。采用不同浓度的酸性蛋白酶（0.02%、0.03%、0.04%）、不同处理时间（30、60、90min）对鹿肉进行嫩化,研究其嫩化效果。对酶法嫩化鹿肉的剪切力、咀嚼力、弹性、p H、游离氨基酸含量进行测定。结果表明,木瓜蛋白酶法嫩化鹿肉后,剪切力值、咀嚼力值显著降低（p<0.01）,其降幅几乎都在50%以上;p H升高,且极显著（p<0.01）;游离氨基酸含量显著增加,且极显著（p<0.01）。最佳嫩化条件为:酶浓度0.04%,处理时间60min。咀嚼力值与剪切力值存在正相关关系（r=0.9721）,游离氨基酸含量与剪切力值之间存在负相关关系（r=-0.9356）。      

双醛氧化纤维素固定化木瓜蛋白酶及酶学性质的研究

以双醛氧化纤维素为载体固定化木瓜蛋白酶,研究了固定化酶的制备条件、微观结构及酶学性质。结果表明:固定化时间4h,固定化温度4℃,酶/载体=1:3000(g:g)时,固定化酶的活力最高为50.9U/g。红外光谱和扫描电镜对固定化酶的微观结构研究表明,双醛氧化纤维素的醛基与木瓜蛋白酶的氨基发生共价反应形成固定化酶。与游离酶相比,木瓜蛋白酶经过固定化后,热稳定性和耐酸性增强,与底物酪蛋白的亲和力降低,固定化酶重复使用5次后,相对酶活力为55.1%。      

酶法提高白葡萄酒质量稳定性的研究

利用木瓜蛋白酶水解白葡萄酒中非稳定蛋白,通过大量试验,确定了酶反应最佳条件为：加酶量为0．6％（w／v）,酶反应pH6．5,在45℃反应38h。此条件下,白葡萄酒中的蛋白含量降低为0．31g／L,氨基酸含量升高至6．7mg／L。     

纤维素酶对蚕蛹蛋白酶解的促进作用

在木瓜蛋白酶酶解蚕蛹蛋白过程中,加入纤维素酶可提高水解液中9%～11%氨态氮含量,与不加纤维素酶的对照处理之间有明显的差异显著性(p<0.05)。其作用效果与纤维素酶加入的先后顺序有关。双酶作用的酶解液中,氨基酸与蚕蛹总蛋白比值与木瓜蛋白酶单酶酶解液中的相比提高了8.28%。     

固定化木瓜蛋白酶的研究进展

木瓜蛋白酶具有水解蛋白质能力强的特点,但游离的木瓜蛋白酶稳定性差,难以回收利用;固定化木瓜蛋白酶既保持了酶的催化性质,又克服了游离酶的不足,具有广泛的发展前景。     

Quantitative gas-liquid chromatography of amino acids in enzymic hydrolysates of food proteins

A method is described for analysing amino acids released from food proteins by the combined but sequential action of papain and pronase E. Amino acids liberated in this way were analysed by gas-liquid chromatography (g.l.c.) as their N-trifluoroacetyl n-butyl esters. Prior to proteolysis, presolubilisation of the proteins was achieved in 0.05MHCl. Of over 30 different food proteins analysed, more than 90% of the total nitrogen was recovered, showing that the process achieved virtually complete enzymic hydrolysis with little destruction of amino acids. The amino acid data obtained by g.l.c. analysis of enzymic hydrolysates were compared to those of cation-exchange (CIE) analysis and to profiles obtained from acid, performic acid and alkaline hydrolytic procedures. Intraclass repeatability analysis was used to test reproducibility of each amino acid value within the three procedures used to obtain the profile. Although individual amino acid values differed somewhat, on the whole, there was good agreement (r=0.742) between values from g.l.c., CIE analysis of enzymic hydrolysates and CIE analysis of acid, performic acid and alkaline hydrolysates.     

壳聚糖-埃洛石纳米管微球的制备及其对木瓜蛋白酶的固定化

运用交联-吸附法制备壳聚糖-埃洛石纳米管(chitosan-halloysites nanotube,CTS-HNTs)复合微球固定木瓜蛋白酶,并通过傅里叶红外光谱、扫描电子显微镜、荧光标记等方法进行表征。2%CTS+1%HNTs制备的微球对木瓜蛋白酶的固定量最高。固定化条件为木瓜蛋白酶质量浓度1 mg/mL、固定化时间10 h。固定化木瓜蛋白酶最适pH 6.8(游离酶pH 7.2)、最适温度60℃(游离酶50℃),保存30 d该酶相对活性为62%(游离酶27%),使用4次后,木瓜蛋白酶的相对活性仍然保留26.26%。CTS-HNTs微球固定化木瓜蛋白酶耐贮存,操作稳定性强,可以提高酶的利用率,降低酶解的成本,提高生产效率。     

鸡肉蛋白钙离子螯合肽酶解工艺的优化研究

本文研究了不同酶解工艺条件对鸡肉蛋白酶解产物钙螯合力的影响。研究了木瓜蛋白酶酶解时间对钙螯合力的影响,结果表明酶解时间在3 h时,酶解产物有最大钙螯合力。对比了木瓜蛋白酶单酶酶解,木瓜蛋白酶、风味蛋白酶双酶同步酶解,及双酶分步酶解三种酶解方式,发现风味蛋白酶的加入有助于提高产物钙螯合力,且分步酶解法的对产物螯合力提升效果优于同步酶解。针对双酶分步酶解法,研究了木瓜蛋白酶酶解时间、风味蛋白酶酶解时间、木瓜蛋白酶添加量、风味蛋白酶添加量四个因素对酶解产物钙螯合力的影响,并以木瓜蛋白酶酶解时间、木瓜蛋白酶添加量、风味蛋白酶添加量为自变量,酶解产物的钙螯合力为响应值,设计了三因素三水平响应面实验,得到最佳酶解工艺条件为:木瓜酶酶解时间定为3.5 h,风味蛋白酶酶解时间为2.84 h,风味蛋白酶添加量为0.18%。在该条件下酶解产物含有较多的酸性氨基酸及碱性氨基酸,有利于螯合反应的发生。     

Papain hydrolysates of casein: molecular weight profile and encapsulation in lipospheres

Some reaction parameters were tested in the hydrolysis of casein by papain, in order to prepare hydrolysates with high oligopeptide contents, for either dietetic or pharmaceutical purposes. Five casein hydrolysates were prepared and then fractionated by size‐exclusion HPLC. The rapid correct fraction area method was used for quantifying peptides and free amino acids. Among the five reaction conditions tested, three produced similar peptide profiles. However, the use of a temperature of 37°C and an E:S ratio of 2% is probably the most economical condition for use in large‐scale manufacture. With the aim of masking the bitterness of these preparations, a new method, based on the encapsulation in lipospheres, was used. Also, second derivative spectrophotometry was used for the first time to measure the extent of encapsulation of protein hydrolysates, which changed from 50% to 83%. Moreover, the efficiency of this system was evaluated by analysing other parameters, which showed a reduction of hydrophobicity and bitterness of all samples, as well as good chemical stability during 60 days of storage under refrigeration. The electron microscopical analysis of liposheres showed an average size around 5.0 ± 1.0 µm. Copyright © 2004 Society of Chemical Industry     

分子动力学模拟抗坏血酸对木瓜蛋白酶活性的影响

为探究抗坏血酸对木瓜蛋白酶活性的影响，以实验结果为基础，利用分子对接和分子模拟技术对二者的结合机制进行分析。结果表明：3种浓度(0.1、0.2、0.3 mmol/L)的抗坏血酸均能促进木瓜蛋白酶酶活性，平均激活率分别为10%、21%、28%，抗坏血酸浓度越高，激活作用越明显；分子模拟结果表明，木瓜蛋白酶与抗坏血酸结合后均方根误差呈略微上升的趋势，木瓜蛋白酶内部的氢键数量降低，结合前氢键数量平均值为146，而加入抗坏血酸后为143，总可及表面积增加2.38 nm²，且3个活性位点(Asp158、His159、Cys25)的均方根波动程度均增大，发挥主要催化作用的25号位半胱氨酸与2个辅助氨基酸的间距缩小。因此，木瓜蛋白酶的激活可能是由于抗坏血酸的加入引起了蛋白活性中心结构的变化，从而使木瓜蛋白酶更有利于发挥催化作用。     

Effects of papain,Lactiplantibacillus plantarum 1-24-LJ and their combinations on bacterial community changes and flavour improvement in Suanzhayu,a Chinese traditional fish

Suanzhayu is a traditional Chinese fermented fish that often faces a long fermentation time (about 1 month) and unstable quality. This study evaluated the combined effects of papain and Lactiplantibacillus plantarum 1-24-LJ, a selected starter culture, on the quality of Suanzhayu. The addition of L. plantarum and/or papain increased the content of alcohols, esters, ketones and umami amino acids (UAAs), but decreased the content of bitter amino acids (BAAs). Besides, Lactobacillus might play a role in reducing harmful bacteria and BAAs content and increasing UAAs content, while Lactococcus and Weissella contributed to the characteristic flavour of Suanzhayu. Overall, no significant improvement in sensory quality was observed with the addition of papain alone, while better quality and faster fermentation processes were obtained with the addition of L. plantarum 1-24-LJ alone or in combination with papain. The results contribute to quality control in Suanzhayu production and the selection of starter cultures.     

高效液相色谱法检测食品中17种游离氨基酸

目的使用异硫氰酸苯酯衍生-高效液相色谱法对食品中17种游离氨基酸进行检测。方法对17种游离氨基酸及其苯胺硫甲酰基衍生物稳定性的分析,研究游离氨基酸的最佳衍生条件。同时,对盐沉淀法和C_(18)固相萃取(solid phase extraction, SPE)小柱法2种样品除杂方法的加标回收率进行测定。结果胱氨酸加热干燥5h后,回收率为68.46%±4.01%,热稳定性差;其它16种氨基酸加热1～5h后回收率均在90%～110%之间,热稳定性较好。胱氨酸的苯胺硫甲酰基衍生物,6h后回收率仅为11.04%±1.65%,稳定性差;其它氨基酸的苯胺硫甲酰基衍生物24 h后回收率均大于90%,比较稳定。另外,前处理中使用C_(18) SPE除杂时,样品中游离氨基酸的加标回收率均大于70%,而使用水杨酸和三氯乙酸除蛋白后苯胺硫甲酰基衍生物降解严重。结论除胱氨酸外,其余16种氨基酸的热稳定性均较好,且生成的苯胺硫甲酰基衍生物比较稳定,可以通过异硫氰酸苯酯衍生进行定量分析。相比于水杨酸和三氯乙酸沉淀法, C_(18) SPE柱是游离氨基酸除杂方法的首选,而无机盐影响异硫氰酸苯酯与氨基酸衍生,因此此方法不适用于含盐样品的衍生。     

Debittering Mechanism in Bitter Peptides of Enzymatic Hydrolysates from Milk Casein by Aminopeptidase T

The bitter peptide fraction present in casein hydrolysates obtained by using three proteases (subtilisin, papain and trypsin) was treated with aminopeptidase T from Thermus aquaticus YT-1. The bitterness of the bitter peptide fraction could be decreased, and it sometimes disappeared completely, with an increase in free amino acids. The percentages of total free amino acids released from each bitter peptide fraction (subtilisin, papain and trypsin) by aminopeptidase digestion for 20 hr were approximately 11%, 8.7%, and 6.5%, respectively. Bitter peptide (α_s1-CN f91-100) was isolated from a tryptic hydrolysate of casein by HPLC, its threshold value of bitterness being 2.9 ppm (w/v). The peptide (α_s1-CN f96-100) obtained from the amino peptidase digestion of this bitter peptide showed no bitterness.