离子对反相高效液相色谱法同时检测水产品中6种ATP关联化合物

建立了水产品中6种ATP关联化合物的离子对反相高效液相色谱(IP-RPLC)检测方法,确定了缓冲液的最佳pH、流速和浓度,并对方法的准确度、精确度和检测限进行了测定.采用10％的高氯酸分离提取样品,用KOH溶液中和后进行HPLC分析.采用Waters 2695色谱系统,Thermo ODS-2 Hypersil(250 mm ×4.6 mm,5μm)色谱柱,柱温为25℃；以10％甲醇,90％ 0.05 mol/L磷酸盐溶液为流动相,磷酸盐溶液含9 mmol/L四丁基氢氧化氨(TBA),并用1 mol/L的磷酸调pH至6.5,采用等度洗脱,流速为0.9 mL/min；检测波长为254 nm.结果表明:6种ATP关联化合物在13 min内完全分离；在2～400 μg/mL范围内线性关系良好,相关系数在0.9994～1.0000之间；方法最低检测限(信噪比S/N =3)在0.1～0.4 μg/mL之间；加标平均回收率为82.2％ ～110.0％,相对标准偏差为4.2％ ～9.5％.本方法可用于水产品中ATP、ADP、AMP、IMP、Hx和HxR等6种ATP关联化合物的同时测定和分析.     

鱼类保鲜新法

日本的专家认为,鱼的新鲜程度降低并不是细菌分解引起的,而是鱼体肌肉的生化变化造成的。鱼体肌肉中的三磷酸腺苷(ATP)分解过程如下:ATP(三磷酸腺苷)一ADP(二磷酸腺苷)一AMP(一磷酸腺苷)一IMP(肌苷—磺酸)一HxR(肌苷)一Hx(hypoxthantine)。据此,日本研究了一种实用的生化测定方法来测定鱼类鲜度指数,即K值,K值是HxR+Hx对ATP分解物的总量的百分比。日本已研制成一种简便而又廉价的K值测量     

鳙和棕点石斑鱼冷藏过程中ATP关联化合物的变化

为分析鳙和棕点石斑鱼贮藏过程中ATP关联产物含量的变化,本研究以高氯酸溶液作为蛋白沉淀剂,运用高效液相色谱法(HPLC),分析(4±1)°C和(0±1)°C贮藏条件下鳙和棕点石斑鱼体内6种ATP关联化合物含量的变化。结果显示,(4±1)°C条件下,2种鱼体内的ATP都快速降解;ADP、AMP含量则始终较低;IMP含量呈先快速上升后缓慢下降的趋势;鳙体内Hx R含量相对较高,棕点石斑鱼体内Hx R含量较少;Hx在4 d后即开始大量生成。在(0±1)°C条件下,2种鱼肉中ATP同样很快降解,ADP、AMP含量少;IMP降解速率变缓;2种鱼中Hx R生成和分解速率皆变缓;Hx生成速率较为平稳。鳙以IMP、Hx R为限速步骤,棕点石斑鱼以IMP为限速步骤。研究表明,不同的贮藏温度对ATP的降解影响较小,但对其关联产物的变化速率有较大影响,且不同鱼种间ATP关联产物降解规律也有所不同。     

超高压处理对凡纳滨对虾冷藏期间ATP降解途径及其关联产物蓄积的影响

近年超高压技术在虾类的加工与保鲜的研究较多,但有关超高压处理对虾类冷藏期间ATP的降解及其关联产物的蓄积影响却少有报道。为研究超高压处理对冷藏期间凡纳滨对虾中ATP关联产物变化的影响,以不同超高压条件(0、100、200、400 MPa)对凡纳滨对虾处理后4℃贮藏。结果显示,鲜活凡纳滨对虾体内ATP关联产物以ATP和ADP为主,其含量分别为8.10和2.11 μmol/g,占总ATP关联物总含量的73.51 %和19.14 %；100和200 MPa超高压处理后,虾肉中ATP、ADP含量迅速下降,AMP含量显著升高；400 MPa超高压处理后, ATP、ADP 含量较高,在贮藏1 d后都迅速下降。在贮藏期间,各处理组AMP、IMP、HxR 都呈先上升后下降趋势,Hx、AdR 基本都呈线性上升趋势,均未检测到Ad；其中400 MPa超高压处理组ATP关联产物降解最慢。超高压处理不会改变凡纳滨对虾ATP的降解途径,但会影响ATP关联产物的降解速率。本研究为利用超高压技术辅助虾类保鲜,最大限度地保持虾类风味和品质提供一定的理论指导。     

牙鲆在保藏过程中影响ATP关联化合物降解的因素

为了深入了解牙鲆体内ATP关联化合物的变化,尤其是肌苷酸(IMP)降解这一限速步骤,利用高效液相色谱法(high efficiency liquid chromatography,HPLC)测定不同保藏条件下牙鲆体内ATP关联化合物和K值变化,并探讨了影响其变化的因素。结果显示,0°C保藏条件下牙鲆体内ATP在1 d内迅速降解,产生IMP作为初期的主要积累物质,蓄积一段时间(10 d)后迅速降解为次黄嘌呤(Hx),IMP的降解过程呈现二相性;鱼肉制成鱼糜形态保藏,ATP 0 d时几乎降解完全,但IMP的降解过程仍旧呈现二相性;加入抗菌素的鱼肉中微生物的生长得到有效抑制,此时IMP降解缓慢并且呈线性下降,原本的蓄积过程消失,K值的增长速率也随之减慢;加入Tritonx-100破坏细胞膜结构发现,牙鲆体内IMP的降解速率加快,同时K值的增长速率也随之加快。结果表明,牙鲆的保藏形态影响ATP的降解速率,但几乎不影响其他关联产物的降解变化;保藏后期外源微生物产生的酶与内源酶的叠加作用是导致保藏后期IMP加速降解、从而呈现二相性的原因,微生物是影响ATP关联化合物降解的关键因素;表面活性剂通过改变细胞膜结构也能够影响IMP的降解过程。     

用FTP法测定鱼类鲜度判定系数、K值的精度与问题

FTP 是一种简便测定 Hxr Hx(肌甙 次黄嘌呤)量的试纸。对照根据每种鱼制成的 K 值换算色标可以测定鱼的新鲜度。一、测定的原理1、用 FTP 测定 HxR Hx 由于核甙磷酸化酶和黄嘌呤氧化酶的作用,HxR 和 Hx 在尿酸中被氧化。发生这个反应时,如果氧化还原色素和碘硝四唑鎓共存就呈现还原的红紫色。把红紫色与标     

青鱼背肉、腹肉和尾肉不同风味成分的比较

淡水鱼类不同部位肉的化学组成有较大差异,为了更深入地探究青鱼背肉、腹肉和尾肉风味差异,研究了青鱼(Mylopharyngodon piceu)不同部位(背肉、腹肉、尾肉)之间风味成分的异同,比较了青鱼3种部位肉水溶性滋味物质及挥发性成分的差异,为青鱼的水产加工工艺方法及加工装备研制等提供参考。使用高效液相技术和氨基酸分析仪研究了青鱼不同部位肉水溶性滋味物质,并采用电子舌技术对青鱼不同部位肉进行区别分析,使用顶空固相微萃取-气相色谱-质谱联用技术(HS-SPME-GC-MS)研究比较了青鱼不同部位肉的挥发性成分。结果显示:电子舌分析可以有效区分青鱼背肉、腹肉与尾肉之间的差别。青鱼3个部位肉滋味成分差异显著,青鱼腹肉总游离氨基酸含量显著高于背肉,而尾肉含量最低。K值一般用来评价水产品的新鲜度,定义为HxR(次黄嘌呤核苷)和Hx(次黄嘌呤)占ATP(三磷酸腺苷)所有关联化合物总量的百分含量,背肉的K值为5.01%,显著低于腹肉(10.04%)和尾肉(12.87%),说明利用K值比较鱼肉鲜度应注明取样部位。鱼肉EUC值结果表明腹肉呈味优于背肉,背肉优于尾肉。对青鱼肉气味贡献较大的物质有己醛、1-辛烯-3-醇、壬醛、正己醇、庚醛、(E)-2-辛烯醛、戊醛等。青鱼3个部位肉水溶性滋味成分差异显著,挥发性成分也有些许差异。青鱼腹肉与背肉风味优于尾肉,在研究青鱼风味与新鲜度时应注意区分青鱼背肉、腹肉与尾肉。     

高效液相色谱法测定水产品中β-阿朴-8′-胡萝卜素醛的含量

建立了水产品中β-阿朴-8′-胡萝卜素醛的高效液相色谱检测方法.样品采用抗氧化剂焦性没食子酸保护,酸化乙腈提取,二氯甲烷萃取,旋转蒸干后乙腈定容,高效液相色谱-紫外检测器检测,外标法定量.流动相乙腈+0.1％甲酸溶液=97+3,检测波长457 nm,色谱柱ZORBAX Eclipse XDB-C 18柱(4.6mm×150 mm,粒度5 μm).实验结果表明β-阿朴-8′-胡萝卜素醛在0.02～10.0 μg/mL范围内线性良好(r=0.999 95);检出限0.01 mg/kg,平均回收率62％～107％之间,平行样品的标准偏差≤6.1％.     

养殖水产品中氯霉素残留量的测定

建立了一种超高效液相色谱-串联质谱法测定养殖水产品中氯霉素残留量的方法.样品在碱性条件下,乙酸乙酯超声提取,水浴氮吹浓缩,正己烷去脂净化,ZORBAX SB-C18色谱柱分离,甲醇水溶液[V(甲醇)∶V(水)=70∶30]洗脱,负离子多反应监测模式检测,内标法定量.氯霉素在0.1～5.0μg/L内呈现良好的线性关系,相...     

柱前衍生液相色谱法测定贝类中氨基酸含量

采用柱前衍生液相色谱法,测定了3种贝类中天冬氨酸、赖氨酸、亮氨酸等17种氨基酸含量。衍生试剂为6-氨基喹啉基-N-羟基琥珀酰亚胺基甲酸酯(AQC),流动相为醋酸盐-磷酸盐缓冲溶液、乙睛、水,梯度洗脱。色谱柱为AccQ·Tag(Waters)氨基酸分析柱,紫外检测。结果显示,氨基酸浓度与峰面积呈良好的线性关系,线性范围10~80μmol/L,检出限为0·23μmol/L,相关系数均在0·998以上,相对标准偏差RSD在2·1%~4·8%之间,回收率在91·3%~113·2%之间。     

Effect of bleeding on the quality of amberjack Seriola dumerili and red sea bream Pagrus major muscle tissues during iced storage

The effect of bleeding on white muscle quality in amberjack and red sea bream was evaluated by measuring ATP-related compounds, volatile basic nitrogen (VBN), and the trimethylamine (TMA) content. The freshness was evaluated by the K value, and the degree of spoilage was elucidated by VBN and TMA. ATP was rapidly converted to ADP and AMP in the muscle and IMP was the main product of ATP degradation during iced storage. For both species, the IMP content was higher in the muscles of fish that were bled than in those unbled during iced storage. Conversely, the K value and the levels of hypoxanthine (Hx) and VBN were higher in the muscle tissue of unbled fish than in the bled tissue of both species. Similarly, the TMA content was higher in the unbled muscle tissues of both species after a week of storage.     

凡纳滨对虾虾仁在冻藏过程中品质变化研究

以凡纳滨对虾（Penaeus vannamei）为研究对象,经过去头和去壳处理后,于-35℃冷冻过夜,然后置于-18℃条件下进行保藏试验。通过测定解冻损失率和肉色变化,明确冻藏期间虾肉的质量变化规律,结合腺苷三磷酸（ATP）及其降解产物、总挥发性盐基氮（TVB-N）和酸碱度（pH）等鲜度指标的测定结果,分析对虾在冻藏条件下鲜度的变化规律,旨在为其生产加工提供理论依据。研究结果表明,冷冻保藏至第18天时解冻损失率达到最大,同时表面色差（L^＊）也达到最大值41.56。而虾肌肉中的ATP质量摩尔浓度冻藏第1天就降低了71.78%,次黄嘌呤（Hx）和次黄嘌呤核苷（HxR）则一直维持在较低水平。第10天鲜度指标（K值）达到最大值7.18%,并能比较稳定地保持这一水平,结合TVB-N测定结果,发现冻藏至第30天时,虾肌肉中的TVB-N仍未超过腐败临界值。因此,冻藏处理可以保持凡纳滨对虾的鲜度,使各项理化指标维持在较低水平,但会加重解冻损失现象。     

Effect of Freeze-Chilled Treatment on Flavor of Grass Carp (Ctenopharyngodon idellus) Fillets and Soups During Short-Term Storage

ABSTRACT

In order to evaluate the effect of chilled (T1), frozen (T2), and freeze-chilled (T3) treatment on the flavor of grass carp fillets and soups, adenosine triphosphate (ATP)-related compounds, umami-taste amino acids (UTAA), thiobarbituric acid (TBA), peroxide value (PV), and soup sensory score were analyzed. Results showed that the sum of ATP, ADP, and AMP contents in T2 fillets were significantly higher (p < 0.05) than others, but the sum of hypoxanthine riboside (HxR) and hypoxanthine (Hx) contents for T2 fillets and soups were significantly lower (p < 0.05) than others during storage. The T3 fillets and soups had lower inosine 5-monophosphate (IMP) contents than T1 after Day 2, but higher UTAA contents than T1 during storage. The T3 fillets had the highest TBA and PV values among the three groups during storage. According to the total aerobic counts, K value, and sensory score, all the fillets and soups kept good quality during storage. Therefore, freeze-chilled treatment not only can extend fish shelf life but also retain excellent fish flavor.     

Validation of Liquid Chr omatography Analysis of ATP-Related Compounds in Sardines

Abstract

A liquid chromatography (LC) method was tested for simultaneously determining ATP-related compounds in sardines. The methodology consisted in extracting ATP and its degradation products; ADP, AMP, IMP, inosine, and hypoxantine from muscle tissue by perchloric acid solution. The mixture was homogenized, centrifuged, its pH adjusted, and the supernatant was analyzed by LC. An isocratic elution system was used and the mobile phase was a mixture of 0.01 M potassium dihydrogen phosphate with 0.001 M potassium chloride. ATP derivatives were thoroughly separated in less than 15 min of chromatographic elapsed time. This method was validated by assessing the following aspects: specificity, linear ity, r ecovery, limit of detection (LOD), limit of quantification (LOQ), UV detection wavelengths, and stability of nucleotides under frozen storage. The results demonstrated that ATP catabolites from sardines can be evaluated by using this methodology.     

超高效液相色谱-电喷雾串联质谱法测定水产品中亚甲基蓝及代谢物

建立了超高效液相色谱-电喷雾串联质谱法同时检测水产品中亚甲基蓝及其代谢物的分析方法。经过条件优化,试样用10 mL乙腈提取3次,并在体系中添加盐酸羟胺、对甲苯磺酸等提高提取效率,提取液经对丙磺酸固相萃取小柱净化,按V（乙腈）：V（乙酸铵,1 mol·L-1）=1：1洗脱,采用Thermo Hypersil C18色谱柱,以甲醇和0.2%甲酸为流动相进行梯度洗脱分离,电喷雾离子源（ESI）,正离子模式,采用选择反应监测（SRM）扫描模式。结果表明,亚甲基蓝及代谢物（天青A、天青B、天青C）在0.50-100.00 μg·L-1范围内线性关系良好,相关系数R〉0.99,在10 min内实现分离,3个加标水平（2 μg·kg-1、4 μg·kg-1、10 μg·kg-1）的平均回收率为74.23%-94.40%（n=6）,相对标准偏差（RSD）为1.13%-10.28%,检测限（LOD）为0.40~0.60 μg·kg-1,定量限（LOQ）为2.00 μg·kg-1。该方法简便快捷、准确度高,易于推广应用,可作为快速测定水产品中亚甲基蓝及其代谢物的有效方法。     

Nucleotide Degradation Profiles of Iced Barramundi (Lates calcarifer) and Nile Perch (Lates niloticus) Muscle

Adenine nucleotides and their related compounds were determined in muscle extracts from two species of fish that were stored in ice after thawing. The fish were the closely related species, Australian barramundi (Lates calcarifer) and Kenyan Nile perch (Lates niloticus) which had different process histories. For all samples, adenine nucleotides did not exceed 6% of the total nucleotide pool. Inosine monophosphate (IMP) decreased steadily with storage. Hypoxanthine (Hx) was the major product of adenosine triphosphate (ATP) degradation in both barramundi and Nile perch, showing a steady increase with days of iced storage. The Hx level did not reach a maximum during the 9d storage period. The K-value also increased regularly with time of storage and for the later stages (i.e., 7 and 9d) and was significantly different (P < 0.01) for the species. The iced storage life of these typical samples of barramundi and Nile perch was estimated to be 3d after thawing using a K-value of < 30% to indicate excellent quality. Despite the differences in process history the nucleotide profiles were remarkably similar during storage. This precludes the use of nucleotide levels as a means of differentiating between these species.     

溶解无机氮加富对海带养殖水体无机碳体系的影响

通过室内模拟实验,研究了在海带养殖水体中添加不同浓度的无机氮(NO-3-N和NH+4-N)对海水无机碳体系的影响。结果表明,无机碳体系各组分的变化趋势与无机氮添加浓度和无机氮形态有关。当NO-3-N和NH+4-N浓度范围分别在(4.73~52.78)μmol/L和(2.56~34.66)μmol/L时,DIC、HCO-3和pCO2均随着营养盐浓度的增加呈下降趋势,其中以NO-3-3和NH+4-3组变化最为明显,均达到最低值,分别为2 054、2 112μmol/L,1 776、1 869μmol/L,86、114μatm;而当NO-3-N和NH+4-N浓度范围分别为(52.78~427.29)μmol/L、(34.66~268.33)μmol/L时,DIC、HCO-3和pCO2随着营养盐浓度的增加,其下降幅度逐渐减弱,但实验结束时DIC、HCO-3和pCO2仍低于对照组。NO-3-N对海带养殖水体无机碳体系的影响较NH+4-N明显,加NO-3-N组对水体的固碳能力显著高于加NH+4-N组。当NO-3-N和NH+4-N浓度分别为52.78μmol/L、34.66μmol/L时,海带的光合固碳能力达到最大,过高或者过低均会降低海带对水体无机碳的吸收固定。     

乌鳢抽出物成分的研究

分析了乌鳢(Channaargus)即杀后背肉、腹肉、尾部肉、肝脏、生殖腺中ATP关联化合物、游离氨基酸、多胺、糖元及糖酵解中间代谢物、有机酸等的含量。ATP关联化合物在肌肉中的总量为7.5～8.0μmol·g-1。ATP在背肉含量为3.9μmol·g-1,腹肉为4.1μmol·g-1,尾部肉为4.7μmol·g-1。运动激烈的尾部肉ATP占63%,含量相当高。肝脏中有少量的腺苷与肌苷酸一起被检出,据此可认为ATP的分解存在两个途径。游离氨基酸总量在背肉中为436.0mg·(100g)-1,腹肉中为405.0mg·(100g)-1,尾部肉中为356.3mg·(100g)-1。牛磺酸和甘氨酸为主要氨基酸,占68%～73%。丙氨酸和谷氨酸也相当高的含量检出。肌肉中的多胺检出为精胺和亚精胺,肝脏和生殖腺中有较高浓度的腐胺及亚精胺和精胺检出。即杀后糖元的量约占肌肉的0.5%,还有相当多的葡萄糖和6磷酸葡萄糖的糖酵解中间代谢物以及其最终产物乳酸的大量检出。     

Changes in Biochemical Compositions and Quality of White Shrimp (Litopenaeus vannamei) During Storage

ABSTRACT

To understand biochemical characteristics, storage stability, and freshness indicators of white shrimp (Litopenaeus vannamei), changes in extractable nitrogenous compounds, microbial count, and sensory rating of white shrimp during storage at 25 and 4°C were investigated. Free amino acids showed a slow increase during storage at 25°C, but no obvious change was found at 4°C. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were found at initial stage and decreased rapidly after storage. Both inosine 5′-monophosphate (IMP) and adenosine monophosphate (AMP) increased and then decreased during storage. Inosine, hypoxanthine, and the K-value gradually increased with time. The levels of total volatile basic nitrogen (TVB-N), NH₃, and trimethylamine (TMA) of white shrimp increased with storage time at 25 and 4°C. The TVB-N, NH₃, TMA, inosine, hypoxanthine, and K-value could be considered as freshness indicators of white shrimp during storage. However, the total plate count did not corroborate the acceptability recommended limits for white shrimp during storage. The sensory evaluation, associated with TVB-N, TMA, and K-value, showed the quality was unacceptable after 6 h storage at 25°C and 7 days at 4°C.     

冷藏和冰藏条件下虹鳟鱼鱼片品质变化研究

为了科学地了解虹鳟鱼(Oncorhynchus mykiss)鱼片贮藏过程中的品质变化规律,以建立高效的品质控制技术,研究了虹鳟鱼鱼片在冷藏(3±1)℃和冰藏(0±1)℃条件下挥发性盐基氮(TVB-N)、色泽、腺苷三磷酸(ATP)关联物、K值及菌落总数(TAC)等指标的变化,评价不同温度对虹鳟鱼片品质的影响。结果显示:虹鳟鱼片TVB-N的增长速度在冷藏条件下显著高于冰藏条件,冷藏至第9天为20.72 mg/100g,冰藏至第15天为25.76 mg/100g;冷藏至第6天TAC为7.40 lg cfu/g,冰藏至第12天TAC为8.27 lg cfu/g;与冰藏相比,冷藏条件下虹鳟鱼片K值较高;肌苷酸(IMP)含量分别在冷藏8 h(8.36μmol/g)和冰藏24 h(8.70μmol/g)达到最大值,即冷藏和冰藏虹鳟鱼片的最佳食用时间分别是宰杀后的8 h和24 h。结合各项指标变化,冷藏和冰藏虹鳟鱼片的货架期分别为6 d和12 d。