Analysis of Urea in Petfood Matrices: Comparison of Spectro-Colorimetric, Enzymatic and Liquid Chromatography Electrospray Ionization High Resolution Mass Spectrometry Methods

Adulteration may consist in non authorized source of nitrogen addition to increase the protein content of some raw materials. Urea which is authorized for feed is a non nutritional source of nitrogen in food and pet food. Adulteration of food or pet food raw material by urea is thus monitored by manufacturer and governmental authorities with official methods which are either enzymatic (Association of Official Agricultural Chemists, AOAC) or spectro-colorimetric (European Community, EC). Each method gives results which are not comparable and spectro-colorimetric methods may result in false-positive urea detection. Liquid chromatographic (LC/UV-DAD) analysis of extracts from spectro-colorimetric method indicates that presence of free amino-acid may interfere with colorimetric detection of urea in the EC method with pet food samples. Liquid chromatography electrospray ionization high resolution mass spectrometry (LC/ESI-HRMS) has allowed to quantify low content (<0.01%) of urea in pet food water extracts for samples which resulted in significant urea detection with colorimetric method and in content below the detection threshold with enzymatic method. This study demonstrates the EC colorimetric method is not applicable to pet food and also food samples which have a complex composition with significant levels of free amino acids. On the other hand we clearly evidenced by means of the LC/ESI-HRMS results that the AOC Enzymatic method is applicable to urea quantification in pet food samples and gives reliable results.

A new enzymatic method for determination of E. coli

beta-Galactosidase, a kind of endoenzyme in E. coli cells. can be released by the pore-forming action of colicin El. and E. coli can be detected rapidly by enzymatic method.

Kinetic method for enzymatic analysis by predicting background with uricase reaction as model

Objective:To investigate the reliability for kinetic assay of substance with background predicted by the integrated method using uricase reaction as model. Methods: Absorbance before uricase action （A0） was estimated by extrapolation with given lag time of steady-state reaction. With Km fixed at 12.5 μmol/L, background absorbance （Ab） was predicted by nonlinearly fitting integrated Michaelis-Menten equation to Candida utilis uricase reaction curve. Uric acid in reaction solution was determined by the difference （ΔA） between A0 and Ab. Results:Ab usually showed deviation <3% from direct assay with residual substrate <one-fifth of initial substrate for analysis. ΔA showed CV <5% with resistance to common interferences except xanthine, and it linearly responded to uric acid with slope consistent to the absorptivity of uric acid. The lower limit was 2.0 μmol/L and upper limit reached 30 μmol/L in reaction solution with data monitored within 8 min reaction at 0.015 U/ml uricase. Preliminary application to serum and urine gave better precision than the direct equilibrium method without the removal of proteins before analysis. Conclusion:This kinetic method with background predicted by the integrated method was reliable for enzymatic analysis, and it showed resistance to common interferences and enhanced efficiency at much lower cost.

Technology of Extracting Kiwi Fruit Seed Oil with Ultrasonic-Assisted Enzyme and Response Surface Method

Taken kiwi fruit as raw material, this paper extracted kiwi fruit seed oil with ultrasonic-assisted enzyme, researched the influence of factors such as liquid-to-solid ratio, granularity, type of enzyme, ultrasonic power, treating time, enzymolysis temperature, enzymolysis time, pH and enzyme additive on oil extraction, and optimized the extracting technology of kiwi fruit seed oil with response surface method. The result shows that the best technical parameter is: material granularity: 60, liquid-to-solid ratio: 1:10 (g/mL), ultrasonic power: 400 W, treating time: 30 min, enzyme amount: 2.50%, pH: 9.2, enzymolysis temperature: 53&deg;C, enzymolysis time: 2.80 h;and the extracting ratio under such condition is 92.57%.

Synthesis of CCK-8 Tetrapeptide Fragment by Enzymatic Method

The enzymatic synthesis of a tetrapeptide Phac-Met-Gly-Trp-Met-OEt, a fragment of the cholecystokinin C-terminal octapeptide CCK-8, was reported This fragment was synthesized by coupling Phac-Met-OEt with Gly-OMe, Trp-OMe and Met-OEt successively These three steps were catalyzed by α-chymotrpsin, Papain and α-chymotrpsin respectively The results of FAB-MS showed that all the products had the correct molecular mass

Enzymatic Aqueous Extraction of Soybean Oil

The procedure of enzymatic aqueous extraction of soybean oil was assessed when two-step controlled enzymatic hydrolysis was applied. With aqueous extraction of soybean oil-containing protein, the highest yield of oil was 96.1% at the optimized conditions studied. Soybean oil-containing protein was hydrolyzed and resulted in releasing part of oil. The separated protein that contained 40% oil was enriched due to its adsorption capacity of released oil, the average oil extraction yeild reached 93.5%. Then the high oil content protein was hydrolyzed again to release oil by enzyme, the oil extraction yeild was 80.4%. As a result, high quality of soybean oil was obtained and the content of total oil yield was 74.4%.

Enzymatic Synthesis of a CCK-8 Tetrapeptide Fragment

The enzymatic synthesis of a tetrapeptide Phac-Met-Gly-Trp-Met-OEt is reported. It was synthesized by coupling Phac-Met-OEt with Gly-OMe ·HC1, Trp-OMe and Met-OEt successively, catalyzed by α-chymotrypsin, papain and α-chymotrypsin respectively. The results of FAB-MS showed that the products had the correct molecular mass.

Extraction Hydrolysates from Larimichthys Polyactis Swim Bladder Using Enzymatic Hydrolysis

As a kind of biopolymer,hydrolysates of fish swim bladder,safer than those of land mammals,are widely used in food,cosmetics as well as pharmaceutical and biomedical fields for their biocompatibility,biodegradability,and weak antigenicity.To enhance hydrolysate production,in this paper,the papain and alcalase hydrolysis processes of larimichthys polyactis swim bladder were optimized with orthogonal experiments.With 89.5%hydrolysate yield,the optimal processing conditions for alcalase were solid-liquid ratio of 1:30,enzyme concentration of 0.7%,and extraction time of 6 h.As for papain,under the optimal processing conditions:solid-liquid ratio of 1:20,enzyme concentration of 0.5%,and extraction time of 8 h,the hydrolysate yield was 65.1%.To obtain higher hydrolysate yields,the ultrasonic pretreatments were implemented before the optimal enzyme hydrolysis processes.With ultrasonic waves of 100 W for 50 min,the hydrolysate yields were increased 2.1%(alcalase)and 4.5%(papain),respectively.The Fourier Transform Infrared(FTIR)spectroscopic analysis revealed that the hydrolysates extracted by papain exist in triple-helical forms.The Ultra-Violet(UV)absorption spectra indicated that the aromatic amino acids in the hydrolysates had strong absorptions in the wavelength range of 240 nm–300 nm.The results of this research demonstrate that the alcalase hydrolysates have better solubility in water and the solution is more stable under ambient temperature.However,the hydrolysates extracted by papain have a gel property and are insoluble in weak acid at room temperature,which is more suitable for applications in feedstock of biomedical.

熵权-正交法优化赤苍藤中多糖、多酚和黄酮的提取工艺

优化赤苍藤中多糖、多酚和黄酮的提取工艺,为综合评价其质量提供参考。以赤苍藤茎为研究对象,采用酶解法,选取适宜的酶质量分数、酶解p H、酶解温度及酶解时间为考察因素,以多糖、多酚、黄酮和浸膏提取率为评价指标。基于熵权法客观赋权结合L_9(3~4)正交试验,优选赤苍藤的多指标提取工艺。最佳提取工艺条件为:酶质量分数4%,酶解p H值6.0,酶解温度50℃,酶解时间45 min。考察多指标后,确认优选的提取工艺稳定可行,可为赤苍藤的质量评价提供实验依据。

酶解法制备禾虫多肽液的工艺优化及其脱腥工艺探究

目的应用单因素和响应面实验优化禾虫的酶解工艺,结合模糊数学法探究一种有效的脱腥方法。方法以中性蛋白酶为酶解剂,禾虫为主要原料,研究液料比、酶用量、时间、温度、pH对酶解效果的影响。在单因素实验的基础上,以液料比、酶解温度、酶用量为自变量,禾虫多肽液的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为响应值,通过Box-Behnken响应面法分析不同因素对酶解效果的影响,确定酶解的最优组合,并应用美拉德反应、硅藻土和酵母粉对禾虫多肽液进行脱腥实验,模糊数学法评价3种脱腥处理的效果。结果根据响应面实验得出禾虫的最佳酶解组合为:液料比1:1(g/mL)、酶解温度60℃、中性蛋白酶用量2450 U/g,得到酶解液的DPPH自由基清除率47.25%±0.11%;禾虫多肽液脱腥的最佳处理为:酵母粉添加量0.7%,反应温度35℃,反应时间30 min。结论中性蛋白酶酶解禾虫制备禾虫多肽液并采用酵母粉脱腥效果最佳,为禾虫的精深加工提供借鉴,促进禾虫产业的发展。

玉米赤霉烯酮毒素生物降解酶研究进展

玉米赤霉烯酮(ZEN)及其衍生物是由镰刀菌产生的具有类雌激素效应及细胞毒性的次级代谢产物,广泛存在于受镰刀菌侵染的谷物及其制品中。ZEN不仅影响粮食的品质和产量,也会对人畜生命健康造成威胁。ZEN脱毒方法主要包括物理法、化学法和生物法。生物法特别是生物酶法因反应条件温和、特异性好、脱毒效率高、环保、便捷等优势而具有较高的应用价值。本研究对近年来报道的ZEN降解酶的挖掘、鉴定及其实际应用等方面进行了综述,旨在为ZEN的生物酶法脱毒研究提供参考。

酶法快速测定植物油中游离甾醇

目的建立固相萃取(solid phase extraction,SPE)结合酶法测定植物油中游离甾醇含量的快速检测方法。方法采用SPE结合酶法40 min内实现植物油中的游离甾醇含量的快速测定,并应用于植物油中游离甾醇含量测定。结果方法学验证结果显示,植物甾醇含量在浓度范围0.3~7.5 mg/m L之间线性良好,检出限为0.018 mg/g,日内精密度为7.94%,日间精密度为6.88%,回收率在84.75%~99.50%之间。实际样品的检测结果显示,酶法与气相色谱-质谱法(gas chromatography-mass spectrometry,GC-MS)的相对误差在±20%以内,且酶法的检测效率比GC-MS提高3倍。常见的几种植物油中,游离甾醇含量较高的是玉米油和菜籽油,分别为1.53~3.66 mg/g和1.98~3.02 mg/g。结论本研究建立的SPE结合酶法检测植物油中游离甾醇的快速检测方法稳定性、重现性好、灵敏可靠,可满足植物油中游离甾醇的现场批量快速检测。

小麦淀粉的酶法改性研究进展

小麦淀粉是小麦粉的主要成分,其特性对小麦粉工艺性能具有显著影响,同时小麦淀粉作为一类大宗淀粉产品,其食品用途十分广泛。在当前“清洁标签产品”浪潮下,采取非化学改性来扩展小麦淀粉及小麦粉等原料的加工用途已成为该领域的重要手段和研究热点之一。无论是内源酶还是外源酶制剂,它们会显著影响小麦粉中或分离纯化的小麦淀粉的性能以及以小麦淀粉为主要成分或配料的食品的品质。该文在介绍淀粉改性用酶、酶的作用方式的基础上,着重论述酶处理影响小麦淀粉组成结构及理化性质的相关研究进展,同时对当前酶法改性小麦淀粉的食品应用进行综述,以期为酶法改性小麦淀粉或其他来源淀粉的研究与应用提供参考。

超声-微波预处理协同复合酶法制备壳寡糖的工艺优化及其抗氧化活性

为了实现壳寡糖的高效制备,本研究以壳聚糖为原料,利用超声-微波预处理协同复合酶法制备低聚合度壳寡糖。以还原糖含量为指标,通过响应面设计法优化酶解工艺参数,并评估壳寡糖的抗氧化活性。结果表明:α-淀粉酶和壳聚糖酶1:1的复合酶组合效果最佳。最佳制备工艺参数为:微波功率200 W、超声功率200 W、超声-微波预处理时间5 min、底物浓度1%、pH5.8、温度53.5℃、复合酶添加量0.24%,在此条件下酶解5.7 h,还原糖含量达到最大值8.10 mg/mL。所制备的壳寡糖为2~6糖,平均分子量为993.5 Da。抗氧化活性结果表明壳寡糖具有较强的自由基清除活性,其对DPPH自由基和ABTS+自由基的IC50分别为0.274、0.127 mg/mL,铁离子还原能力在1.5 mg/mL时达到最大值24.56 mmol/L。本实验结果表明超声-微波预处理协同复合酶法在壳寡糖制备方向具有较好的应用前景。

花鲈肠上皮细胞原代培养及鉴定方法的探讨研究

为探究建立稳定可靠的花鲈肠上皮细胞原代培养方法,通过组织块法和酶消化法培养花鲈肠上皮细胞,确定酶消化法的最佳消化液及消化时间,所得细胞悬液于L-15培养液中进行培养。利用形态学观察、透射电镜观察和碱性磷酸酶染色方法对细胞进行鉴定。结果显示:组织块法细胞迁出情况不稳定,且迁出时间较长;酶消化法的最佳消化液为胶原酶Ⅰ、Ⅳ联合消化液,最佳消化时间为45 min;胶原酶Ⅰ、Ⅳ联合消化法获得的细胞连接紧密、排列整齐、呈上皮细胞典型的“铺路石”状,细胞相对独立、界限清晰。透射电镜观察和碱性磷酸酶染色进一步证实所培养的细胞为肠上皮细胞。总之,经胶原酶Ⅰ、Ⅳ联合消化液消化45 min、培养48 h可得到初始条件一致、生长旺盛的花鲈原代肠上皮细胞。

板蓝根多糖酶法脱蛋白工艺、组成分析与免疫调节活性研究

目的优化板蓝根多糖脱蛋白工艺,并进一步探讨其免疫调节活性,为板蓝根多糖的开发利用提供科学依据。方法通过单因素结合Box-Behnken响应面法优化酶法脱蛋白的最佳工艺条件;利用紫外可见光谱、傅里叶变换红外光谱、高效凝胶渗透色谱、高效液相色谱和扫描电镜等方法对板蓝根多糖化学组成与结构特征进行分析;采用斑马鱼免疫低下模型探讨脱蛋白板蓝根多糖对斑马鱼体内中性粒细胞、巨噬细胞、IL-1β和IL-6含量的影响。结果酶法脱蛋白最佳工艺为:胰蛋白酶500 U·mL^(-1)、pH 8.0、酶解时间5 h、酶解温度37℃,脱蛋白率为(86.39±0.07)%,综合评分(91.15±0.37)%。紫外、红外光谱扫描和电镜扫描显示酶法可以除去粗多糖中含有的蛋白质,脱蛋白后相对分子量在5.82~60.26 kDa之间,单糖摩尔组成为甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=2.17∶0.96∶2.90∶83.25∶4.88∶5.84。免疫活性评价结果表明,脱蛋白后的板蓝根多糖浓度在50~300μg·mL^(-1)时,能显著增加斑马鱼免疫细胞密度,增加巨噬细胞增殖,降低免疫低下斑马鱼体内IL-1β和IL-6含量,从而发挥免疫调节作用。结论酶法可以有效去除板蓝根粗多糖中的蛋白质,脱蛋白后的板蓝根多糖具有一定的免疫调节作用。

物理场耦合水酶法同步释放植物油和蛋白的研究进展

水酶法同步释放油和蛋白是一种新兴的提取技术,绿色清洁、能满足可持续发展的社会要求,物理场和水酶法耦合作用可促进油和蛋白的释放,逐渐成为研究热点。该文综述了物理耦合水酶法的技术原理和优势、同步释放油和蛋白的研究进展,同时综述了该技术对油料中蛋白特性的影响,并对目前存在的问题以及未来的发展进行了展望。

双酶法优化禄丰香醋糖化工艺

为提高禄丰香醋的生产效率,通过添加α-淀粉酶与糖化酶,对禄丰香醋生产过程中糖化工艺进行优化。糖化工艺包括液化工艺与糖化工艺两部分:液化实验以还原糖为指标;糖化实验以还原糖为主要指标,黄酮类物质、氨基酸态氮为次要指标。在单因素实验的基础上,采用响应面分析法对禄丰香醋糖化过程中的参数进行优化。研究结果表明,禄丰香醋的最佳糖化工艺参数为α-淀粉酶添加量2.272%、液化pH 4.9、液化温度50℃、液化时间43 min,此条件下还原糖含量为5.372 g/100 g。结合实际生产情况,当糖化酶添加量为2.336%、糖化pH为4、糖化温度为55℃、糖化时间为60 min、蒸煮时间为1 h、料液比为1∶3时得到还原糖含量为13.614 g/100 g,黄酮含量为6.867 mg/L,氨基酸态氮含量为0.101724 g/dL。

绿原酸衍生物的制备及其活性研究进展

绿原酸是一种具有生物活性的水溶性酚类衍生物,然而其低脂溶特性限制了它的产业应用。通过分子修饰制备的绿原酸衍生物,能够显著提升其脂溶性且保留绿原酸本体的生物活性。本文综述以酰氯法为代表的化学法以及酯化法、酯交换法等酶法制备绿原酸衍生物的研究进展,讨论绿原酸衍生物常用的分离和纯化手段,并探讨近年来绿原酸衍生物的抗氧化、抗肿瘤、抗真菌和调节脂质代谢等生物活性的相关研究,总结现阶段绿原酸衍生物研究中存在的问题,并展望未来发展趋势和应用。

鲜切苹果物理防褐变技术研究进展

鲜切苹果在加工过程中受到组织损伤与破坏,导致发生褐变、营养物质流失等问题,使其食用价值与商业价值降低,造成损失,因此需要保鲜技术手段加以控制。物理保鲜方式具有无毒无害、操作方便简单等特点,在国内外受到广泛的推广。该文基于鲜切苹果劣变机理,阐述物理防褐变保鲜技术在鲜切苹果中的研究进展,从温度调控、气调贮藏技术、光电方法等方面论述不同的物理方法对鲜切苹果防褐变效果,旨在提供不同的物理保鲜方法对鲜切苹果的应用依据。