2011_12_17_Prozesse_pH_

REVIEW ARTICLEEffect of pH on Color and Texture of Food ProductsA.Andre´s-Bello •V.Barreto-Palacios •P.Garcı´a-Segovia •J.Mir-Bel •J.Martı´nez-Monzo ´Received:1February 2013/Accepted:29April 2013/Published online:9May 2013ÓSpringer Science+Business Media New York 2013Abstract Color and texture are important quality char-acteristics and major factors affecting sensory perception and consumer acceptance of foods.pH has an important effect on pigments (e.g.,chlorophyll,carotenoids,antho-cyanins,etc.)responsible for fruit color,vegetables and meat color.Also pH has a great impact on water-holding capacity and tenderness of muscle foods that are improved at acidic conditions below the typical pH of post-mortem.Moreover,during processing of food,the pH value affects many phenomena and processes such as protein properties as denaturizing,gelification,enzymatic activities,growth and mortality of microorganisms,germinating or inacti-vation of bacterial spores and chemical reactions such as the Maillard reaction.Thus,knowledge of pH effects and its control during processing is necessary to produce safe,high-quality and value-added products.The goal of this paper is to identify the effect of pH on color and texture of food products to show the importance of control this parameter.Keywords pH ÁVegetables ÁMeat ÁFish ÁColor ÁTextureIntroductionFrom 1980s to the present day,the food industry is driven by consumers’desire for high-quality,minimally pro-cessed,additive-free,shelf stable,convenient and safe food products [151].Hence,a challenge to food processors is to prevent or at least minimize the color and texture degra-dation to produce high-quality products.In this sense,pH control is very important to prevent color and texture loss.pH,a measurement of the activity of hydronium ions (H 3O ?)in a substance,is a dominant factor that determines the proceeding chemical reaction and hence the quality of the product,particularly in food and biochemical processes [22,32,33,37,60].pH was originally defined by Sørensen [133]in 1909in terms of the concentration of hydrogen ions (in modern nomenclature)as pH =-lg(cH/c °)where cH is the hydrogen ion concentration in mol dm -3,and c °=1mol dm -3is the standard amount concentration.Conventionally,pH is measured electrochemically using a pH-sensitive glass electrode and a reference electrode.There are,however,numerous alternative methods and devices for pH measurement based on electrochemical and non-electrochemical principles.While the practical use of the available methods and devices is mostly limited to atmospheric pressure pH measurement,there have been some attempts at high pressure pH measurement of water and buffer solutions.Samaranayake and Sastry [119]and Min et al.[89]have recently reviewed these investigations.Since food systems exhibit inherently complex chemistry and are often opaque,none of the methods based on reaction volume,electrical conductivity and spectropho-tometry is applicable for high pressure pH measurement of foods.Furthermore,the use of glass electrode assemblies is typically limited to lower pressures (B 150MPa)due to the fragile nature of glass [25].A.Andre´s-Bello ÁV.Barreto-Palacios ÁP.Garcı´a-Segovia ÁJ.Martı´nez-Monzo ´(&)Food Technology Department,Universitat Polite`cnica de Vale `ncia,Camino de Vera,s/n,46022Valencia,Spain e-mail:xmartine@tal.upv.esJ.Mir-BelLaboratory of Vegetal Food,University of Zaragoza,Miguel Servet 177,50013Zaragoza,SpainFood Eng Rev (2013)5:158–170DOI 10.1007/s12393-013-9067-2Accurate measurement and reporting of pH data have been a long-standing problem due to the effects of tem-perature and pressure.An increase in the temperature of any solutions will cause a decrease in its viscosity and an increase in the mobility of its ions in solution.An increase in temperature may also lead to an increase in the number of ions in solution due to the dissociation of molecules(this is particularly true for weak acids and bases).As pH is a measure of the hydrogen ion concentration,a change in the temperature of a solution will be reflected by a subsequent change in pH[7,169].pH reaction is difficult to be described with appropriate mathematical models,as it normally comes with inherent nonlinear dynamics with system uncertainty[6,11].During processing of food,the pH value affects many phenomena and processes,for example,protein properties as denaturizing,gelification,enzymatic activities,the growth and mortality of microorganisms,the germinating or inactivation of bacterial spores and chemical reactions such as the Maillard reaction[136].Color,flavor and texture are important quality charac-teristics and major factors affecting sensory perception and consumer acceptance of foods.The pH of most food products varies between3.5and7.0.pH has an important effect on pigments(e.g.,chlorophyll,carotenoids,antho-cyanins,etc.)responsible for the color of fruits,vegetables and meat.Thus,knowledge of pH is necessary to produce safe,high-quality and value-added products.Foods are subjected to cooking or processing to increase their edibility and palatability.Processing also aims to prolong the shelf life while the original sensory and nutritional properties are maintained as high as possible within the constraints put forward by microbial safety.To achieve the balance between food quality and safety,there is a need to optimize conventional processing techniques currently applied in food industries and to develop novel processing techniques[99].The goal of this paper is to identify the effect of pH on color and texture of food products to show the importance of control this parameter in food processing.The Influence of pH on ColorColor is one of thefirst quality attributes a customer can detect in food products.It is also the most important attribute used by the customer to evaluate the quality of a product(meat,fish,vegetables and fruits)and its possible taste without touching the commodity[142].Moreover,fruits and vegetables are good sources of natural antioxidants for the human diet,containing many different antioxidant components which provide protection against harmful free radicals and have been strongly associated with reduced risk of chronic diseases.These antioxidants include carotenoids,vitamins,flavonoids, other phenolic compounds,dietary glutathione and endogenous metabolites[82].It is common that many vegetables are cooked by boiling and microwave process before use or industrial process(blanching,canning,ster-ilization or freezing).These cooking processes or industrial processes would certainly bring about a number of changes in physical characteristics and chemical composition of vegetables[138]affecting the content,composition,anti-oxidant activity and bioavailability of antioxidants.In addition,operations such as cutting and slicing may induce a rapid enzymatic depletion of several naturally occurring antioxidants as a result of cellular disruption which allows contacts of substrates and enzymes.Generally,the antioxidant concentrations and activities in processed veg-etables were lower than those of the corresponding raw samples.This was caused by their degradation,but also by absorption of water during boiling,which diluted the com-pounds and decreased their content per weight unit[107].pH has an important effect on pigments(e.g.,chloro-phyll,carotenoids,anthocyanins,myoglobin,etc.)respon-sible for the color of fruits,vegetables and meats.The color compounds of processed fruits and vegetables can change during processing due to the presence of microorganism and the inactivation or not of enzymes,which can result in undesired chemical reactions(both enzymatic and non-enzymatic)in the food matrix.ChlorophyllsChlorophylls,the pigments responsible for the character-istic green color of fruits and vegetables,are highly sus-ceptible to degradation during processing,resulting in color changes in food[125].It is well known that the excessive heating of food products causes considerable losses in the organoleptic quality of food.Blanching inactivates chlorophyllase and enzymes responsible for senescence and rapid loss of green color.However,chlorophyll degradation is initiated by damaged tissue during blanching and other processing steps[58,142].There is general agreement that the main cause of green vegetable discoloration during processing is the conversion of chlorophylls to pheophytins by the influence of pH.The green color of vegetables turns to an olive green when heated or placed in acidic conditions[50, 52].During this reaction,hydrogen ions can transform the chlorophylls to their corresponding pheophytins by sub-stitution of the magnesium ion in the porphyrin ring[90]. The conversion of chlorophyll to pheophytin and pheo-phorbide results in a change from bright green to dull olive green or olive yellow,which is ultimately perceived by the consumer as a loss of quality[53,151].Since chlorophyll stability is known to be affected by pH and color is one of the most important quality attributes of vegetable products,numerous studies have been con-ducted to investigate the color changes or degradation of chlorophylls during heating[28,63,126]through the application of pH control[9,54],high-temperature short-time processing[123,137,148]or a combination of high-temperature short-time processing with pH adjustments [18,54,72],and it is reported that the chlorophyll degra-dation follows afirst-order reaction kinetic model[23,57, 134,149].Alkalizing agents in blanch and brine solutions, such as sodium bicarbonate,hexametaphosphate,disodium glutamate,sodium hydroxide,zinc and magnesium hydroxide,have been used to raise the pH of green vege-tables and,therefore,retain chlorophyll after processing[9, 49,80,84].Considering that the green color is one of the major sensory characteristics in determining thefinal quality of thermally processed green vegetables,it is important to prevent or at least minimize chlorophyll degradation during thermal processing in the food industry.Koca et al.[72]studied the kinetic parameters for chlorophyll a,chlorophyll b and visual green color deg-radation as a function of pH in buffered solutions at pH5.5, 6.5and7.5,in blanched green peas at70,80,90and 100°C,by high-performance liquid chromatography and tristimulus colorimetry.They concluded that the rate con-stants of green color loss and chlorophyll degradation decreased with increasing pH,indicating that the green color was retained at higher pH conditions.It was found that chlorophyll a degraded faster than chlorophyll b at all pH values for each temperature applied.The results revealed that chlorophyll a was more susceptible to thermal degradation than chlorophyll b in acidic conditions.At room temperature,chlorophylls a and b exhibit extreme stability but at temperatures higher than50°C treatment affects their stability for example,a significant reduction in the chlorophyll content of broccoli juice(the average ratio of chlorophyll a versus chlorophyll b used in the study was 2.9:1)was observed for treatments that combined high pressure with temperatures exceeding 50°C[19,151].The temperature dependency of the deg-radation rate constant of chlorophyll a is higher than that of chlorophyll b.Van Loey et al.[151]in their study with broccoli juice concluded that chlorophyll a is less ther-mostable as compared to chlorophyll b,and hence chlo-rophyll a,which is the major chlorophyll compound in green plants,degrades more quickly.Ryan-Stoneham and Tong[117]studied the degradation kinetics of chlorophyll in peas as a function of pH.The objective of their investigation was to determine the kinetic parameters of chlorophyll degradation as a function of pH in the constant pH kinetic reactor at pH of5.5,6.2,6.8and 7.5and develop a mathematical model that related the rate constant of chlorophyll as a function of temperature and pH.They concluded that the pH of vegetables during thermal processing is not constant.The magnitude of pH changes is dependent upon the type of vegetable,the initial pH and the time–temperature history of the vegetable of interest(whether high-temperature short-time or low-tem-perature long-time).The prediction of chlorophyll con-centration would be more accurate by treating pH as a variable and by knowing the chlorophyll degradation kinetics as a function of pH.A simpler approach can be applied to improve the accuracy of the prediction assuming the pH of the vegetable remained constant at an average pH value(between the initial andfinal pH values)and the rate constant at that average pH is known for a specified pro-cessing condition and product.Tijkens et al.[142]modelled the effect of pH on the color degradation of blanched broccoli withfive different acids.The fundamental pathways were simplified for the blanched product under study,and attention was solely devoted to the behavior of coloring compounds,expressed as observed color in blanched produce.This simplified model is based on the degradation of chlorophyll and, therefore,the green color,as a function of time and pH. They concluded that color of blanched and thawed broccoli strongly depends on the pH of the surrounding environ-ment.The more acidic,the faster the discoloration.The hydrophobicity of the acids,used to impose a certain pH, had a marked effect on the rate of discoloration:the more hydrophobic,the faster the rate of color change.pH boundaries at the transition between environment and product cannot be neglected.A separate process at neutral and higher pH values gradually takes over the acidic extraction of magnesium ions from the chlorophyll.This latter process is,at least in the pH region studied here, independent of pH.AnthocyaninsAnthocyanins are water-soluble vacuolarflavonoid pig-ments;they are widely distributed in nature,amongflow-ers,fruits and vegetable,and are responsible for their bright colors such as green,red and blue[73,87].Significant properties of anthocyanin extracts are color density and color hue.These can be affected by pH,SO2,heat,light, metals,copigmentation and‘thinfilm’effects and others [86].With increasing pH values anthocyanin color becomes paler,however,if the product being colored contains components capable of acting as copigments, color may be retained and also light stabilized to a certain extent[10].Anthocyanins become paler on heating,because the equilibrium between the four anthocyanin species shifts toward the colorless carbinol base and chalcone forms[15].Total anthocyanin color is developed only in strongly acidic solutions.In isolation,anthocyanins have little color above pH3.5,but in natural media,they become much more colored by copigmentation with other(plant)com-ponents,which may themselves be colorless.Based on observations of some relatively simple anthocyanins in vitro,the following scheme,related to pH changes,is generally accepted[17]:at a pH of approximately3or lower,the orange,red or purpleflavylium cation predom-inates.As the pH is raised,kinetic and thermodynamic competition occurs between the hydration reaction on position2of theflavylium cation and the proton transfer reactions related to its acidic hydroxyl groups.While the first reaction gives colorless carbinol pseudo-bases,which can undergo ring opening to yellow retro-chalcones,the latter reactions give rise to more violet quinonoidal bases. Further deprotonation of the quinonoidal bases can take place at pH between6and7with the formation of more bluish resonance-stabilized quinonoid anions.At the pH values typical for fresh and processed fruits and vegetables, each anthocyanin will thus most probably be represented by a mixture of equilibrium forms.Kouniaki et al.[75]studied the stability of two anthocy-anins and ascorbic acid present in fruit juices made from blackcurrants at high-pressure processing.Products con-taining anthocyanins are susceptible to color changes during processing and storage.These changes are due to anthocy-anin degradation and brown pigment formation.This has been studied by Kouniaki et al.,and results have related anthocyanin stability to the levels of ascorbic acid and phe-nolic compounds found in fruits.They demonstrated that ascorbic acid apart from being an antioxidant also tends to accelerate the degradation of anthocyanins.The rapid loss of ascorbic acid appeared to contribute to the lower rate of anthocyanin loss(e.g.,fridge temperature),whereas the low rate of ascorbic acid loss appeared to result in high antho-cyanin losses(e.g.,room temperature and30°C).Reyes and Cisneros-Zevallos[113]investigated the thermal stability of aqueous anthocyanins from grape, purple carrot,and purple and red-fleshed sweet potato at pH3.The results from their investigations showed higher stability by storing extracts at low pH and temperature conditions.The stability of aqueous anthocyanin extracts to pH(B3)followedfirst-order mercial purple carrot extracts showed the highest stability followed by red-flesh potato extracts,whereas purple-flesh potato and commercial grape were the least stable extracts.Fan et al.[41]studied the color stability of anthocyanins extracted from fermented purple sweet potato culture at pH 2–7.These studies were limited simple aqueous solution models that mainly contain water.Five major anthocyanins were detected by high-performance liquid chromatography (HPLC).Results shown that purple sweet potato anthocyanins are more stable under the acid conditions(pH 2.0–4.0)than the subacid conditions(pH5.0–6.0).Walkowiak-Tomczak and Czapski[158]investigated the effect of pH(3.0,4.0and5.0),time(0,15and30days) and temperature of storage(10,20and30°C),oxygen availability and ascorbic acid(0,100and200mg/100g) on anthocyanin stability and color parameters in red cab-bage colorant preparations.Red cabbage is a rich source of phenolic compounds,with the anthocyanins being the most abundant class[26,35,87,165,166].Walkowiak-Tomc-zak and Czapski[158]found that during the storage of solutions of red cabbage preparations at20mg anthocya-nins/100ml,the content of anthocyanins dropped with increases in pH,ascorbic acid concentration,as storage time and temperature.Moreover,colorant losses were higher in samples stored under aerobic conditions than under relatively anaerobic ones.Anthocyanin losses,con-siderable at times,were not reflected in a sensory evalua-tion of a10-point scale,as all the samples received high scores of color intensity and only the tone changed. Changes in color parameters were,in many cases,also dependent on pH.Li et al.[83]studied the identification and thermal sta-bility of purple-fleshed sweet potato anthocyanins in aqueous solutions with various pH values and fruit juices. This study aims to identify the constituents of purple-fle-shed sweet potato anthocyanins(PSPAs)and investigates the effect of pH value(2–6)on the thermal stability of PSPAs in aqueous solutions at80,90and100°C.They concluded that a higher stability of PSPAs was achieved at pH3and4.Apple juice and pear juice colored with PSPAs showed the highest stability during heating at80–100°C.Jing et al.[65]have investigated in radish juice (Raphanus sativus L.)(anthocyanin-rich system)thermal and pH stability of anthocyanins and glucosinolates at pH 2.5or5.8for2h thermal treatment at90or100°C.They concluded that thermal treatments at pH5.8resulted in more anthocyanin degradation but less glucosinolate deg-radation than that attainable at pH2.5.Thermal loss of anthocyanins and glucosinolates were sensitive to different pH environments.Radish juices treated at100°C for2h had an additional22.7or15.85%loss in anthocyanins at pH2.5and5.8,respectively,comparing with counterparts treated at90°C.However,treatments at100°C only caused an additional10.18or11.63%loss in glucosino-lates at pH 2.5and 5.8,respectively,comparing with counterparts treated at90°C.Kirca et al.[71]studied the stability of anthocyanins in both pure black carrot juice and concentrates during heating and storage at various tem-peratures,soluble solids and pHs and the stability of black carrot anthocyanins in citrate–phosphate buffer solutions at different pHs during heating.They concluded that increasing solid content,pH and temperature,during bothheating and storage,increased the degradation rates of anthocyanins.To minimize anthocyanin degradation,they recommended that black carrot concentrates be cooled, possibly to refrigeration temperatures,as soon as produced. Compared to the stability of anthocyanins from other sources in the literature,anthocyanins from black carrot showed greater stability to heat and pH changes.Such high stability may be attributed to the diacylation of the anthocyanin structure.Regarding the thermal processing treatments(blanching, boiling and steaming),Volden et al.[156]studied their effects on red cabbage.The investigations of Volden et al. have shown that all manners of processing affected anthocyanins significantly(p\0.05)with reductions of 59,41and29%for blanching,boiling and steaming, respectively.The reductions observed in the processed cabbage were not fully recovered in the processing waters,fitting the notion that anthocyanins are degraded by heat [86].However,the extent of heat degradation might depend on various factors related to anthocyanin structure and the pH value.The more stable red-colored structure exists at pH\3and,as the pH is raised to4–6,the col-orless structures predominate.BetalainsBetalains pigments are also water-soluble and usually localized in a unique organelle of plant cell:the vacuole. So far,betalain synthesis has only been observed in plants belonging to the taxonomic group of Centrospermae(red beet,etc.)[122].The pH-dependent ionization of betalains provided the rationale for the release technique described by Mukundan et al.[94]:Betacyanins and betaxanthins are cations below pH2.0,zwitterions at pH2.0,monoanions between pH2.0and pH3.5and bisanions above pH3.5and 7.5[114].At pH2,the zwitterionic state might permit diffusion and release from the vacuole.This behavior is similar to ion trapping where metabolites,such as alka-loids,are retained within the vacuole as a result of acquiring a net charge at the acidic vacuolar pH[112].The observed release kinetics of betalains extends well beyond the low-pH treatment,which suggests that the observed mechanism involves an alteration in membrane properties and possibly preferential death of the pigment-containing cortical cells.This release kinetics is actually fortuitous in terms of recovery since betalains are most stable between pH 3.5–6.5and have limited stability at pH2[98]. Therefore,the procedure of acidic exposure followed by release of the pigments into B5medium[46]of pH5.5 facilitates recovery under stable conditions.In this regard, the low-pH-mediated release of betalains is substantially different from that reported previously,where alterations of medium pH have been used to facilitate release of secondary metabolites from plant tissue culture[64,118]. For alkaloids in particular,the acid dissociation constant (pKa)is sufficiently close to the physiological pH for the medium pH to be altered without necessarily disrupting the tissue.Under these conditions,concepts of equilibrium partitioning can be useful to describe release[112],and the acidified medium pH must be maintained throughout the release period.Moßhammer et al.[93]investigated the visual appear-ance and pigment stability of fruit juice blends from Opuntia and Hylocereus cacti and of betalain-containing model solutions derived therefrom at pH values ranging from3to7.Results revealed that highest betalain stability at pH5coming close to the pH values of cactus pear(pH 5.8)and purple pitaya juice(pH4.5),respectively.This result is concordant with previousfindings of Cai et al.[20],Cai et al.[21],Castellar et al.[24],Coskuner et al.[31],Huang and von Elbe[62],Pa´tkai and Barta[103], Von Elbe et al.[157]providing evidence of maximum betalain stability at pH values close to the natural pH values of the respective betalain-containing plant tissue.CarotenesCarotenoids are a class of natural pigments mainly found in fruits and vegetables that typically have40-carbon mole-cules and multiple conjugated double bonds[40].Carote-noids are usually divided into two categories:(1)carotenes comprised entirely of carbon and hydrogen,for example, a-carotene,b-carotene and lycopene;and(2)xanthophylls comprised of carbon,hydrogen and oxygen,for example, lutein and zeaxanthin[40].Carotenoids may be beneficial to human health when consumed at appropriate levels[69]. The relatively low bioavailability of carotenoids from natural sources has been attributed to the fact that they exist as either crystals or within protein complexes in fruit and vegetables that are not fully released during digestion within the gastrointestinal tract[164].A number of studies have shown that carotenoid bioavailability depends strongly on the composition and structure of the food matrix in which they are dispersed[39,140,146,147]. Carotenoids are also strongly colored(red/orange/yellow), which limits the types of foods that they can be incorpo-rated into.Finally,carotenoids are highly prone to chemi-cal degradation during food processing and storage due to the effects of chemical,mechanical and thermal stresses [85,97,139,167].Qian et al.[109]studied the physical and chemical stability of b-carotene-enriched nanoemulsions and the influence of pH,ionic strength,temperature and emulsifier type.The emulsion samples were prepared in aqueous buffer solutions,and then,the pH was adjusted to the desiredfinal value(pH3–8)using either NaOH and/or HClsolution.Emulsion samples(20mL)were then transferred into glass tubes,stored in a dark place at ambient tem-perature(&25°C)for5days and total color difference (D E*)was measured each day.They concluded that the rate of color degradation was appreciably faster at pH3 than at higher pH values,since the overall change in color during storage was relatively small in the samples with pH values4–8(D E*\10).Additionally,the rate of color fading increased with increasing storage temperature,was fastest at the most acidic pH value(pH3)and was largely independent of salt concentration(0–500mM NaCl).Pre-vious studies have also found that the rate of carotenoid (lycopene)degradation in O/W emulsions was higher at acidic pH values[12].Studies carried out to determine the mechanism of carotenoid instability in the presence of acids have shown that carotenoids are protonated and then undergo cis–trans isomerisation and additional degradation reactions[91,92].Zechmeister[168]has been reported that a high con-centration of acid can results in isomerization of b-caro-tene.However,in some other papers,pH has been reported to have only a minor effect of b-carotene isomerization in solvents or in food systems[124,128].Apparently,the effect of pH on b-carotene stability can be attributed to time of exposure to acid,concentration of acid,and the system in which b-carotene exists.Chen et al.[29]studied the effect of processing methods on changes of color,carotenoids and vitamin A contents in carrot juice.As carrots are low-acid(pH5.5–6.5)foods,the sterilization of carrot juice under high temperature is often required.However,this treatment can result in great loss of color[135].To minimize loss of color and carotenoid content,the raw carrot juice is often acidified before pro-cessing so that the sterilization can be lowered.Results indicated that after the carrot juice was heated immediately following acidification,the short exposure time of b-car-otene to acid did not result in isomerization.It has been reported that blanching carrots with acid can increase the brightness of carrot juice and decrease the precipitation of carrot juice during processing[135].Sims et al.[131]also demonstrated that blanching carrots with acid can improve the color and turbidity of heated or canned juice. MyoglobinMuscle pH has been shown to be primarily related to the biochemical state of the muscle at time of slaughter and following rigor mortis development.Both of these factors contribute to meat color and the occurrence of meat color defects[42].The appearance of cooked meat can be influenced by pH,meat source,packaging conditions, freezing history,fat content,added ingredients and pres-ervation treatments such as irradiation and pressure.These factors change the ratio of different forms of myoglobin; the main pigments responsible for the ultimate color of meat[70].The amount of ferrihemochrome formation from myo-globin during cooking is affected by initial meat pH.While mammalian muscle has a pH of around7,normal fresh meat has a pH ranging from5.4to5.6[153].The carbohydrate glycogen is held in the muscles,but with post-mortem reduction in oxygen supply,the glycogen is broken down to lactic acid,lowering the pH.This acidification process will continue until either the glycogen is consumed or the low pH inactivates glycolytic enzymes.Where glycogen is limited, such as in animals that are very active,stressed or exposed to cold over a long period prior to slaughter,the ultimate pH of the meat is higher.Meat with a pH above6.2tends to have tightly packed water-retainingfibers that impede oxygen transfer and promote longer survival of oxygen-scavenging enzymes,favoring deoxyMb rather than oxyMb.The purple-red myoglobin combines with the closed structure of the muscle to absorb rather than reflect light,making the meat appear dark.This condition is commonly known as dark,firm,dry(DFD)[1,159].Several studies have identified a correlation between raw beef pH and the amount of undenatured myoglobin remaining in the cooked product,which itself correlates with red color.When compared normal pH beef with high-pH beef the proportion of myoglobin denatured during cooking is less in high-pH beef samples and a more intense red color is measurable[70].In ground beef with the pH artificially adjusted,patties with elevated pH required higherfinal endpoint tempera-tures to denature similar amounts of myoglobin than patties with lower pH[14].Schmidt and Trout[121]demonstrated that even when cooked to the same internal temperature,high-pH beef, pork and turkey muscle(pH[6.0)was redder than low-pH muscle(pH[5.5)and appeared undercooked.These authors suggested that the high-pH muscle reduced the amount of myoglobin denatured at a given temperature. Trout[144]reported that at temperatures from55to83°C, the presence of sodium chloride and sodium tripolyphos-phate increased the percentage of myoglobin denatured. High-pH muscle markedly decreased the percentage of myoglobin denatured producing obvious color differences in the cooked muscles.Trout[144]concluded that the pink color found in fully cooked high-pH meat products appeared to be due to:(1)incomplete myoglobin denatur-ation at low temperatures(\70°C)and(2)formation of a pink hemochrome at higher temperatures([76°C).The effect of pH on meat color is complex.One effect, as noted earlier,is that many of the haem-associated reactions are pH dependent.In addition,muscle pH affects the water binding nature of the proteins and therefore。

标准评析皮革中禁用偶氮染料测试标准ISO 17234-1:2020解读■ 邵娟娟〔吉利汽车研究院（宁波）有限公司〕摘 要：为了研究汽车材料中皮革及毛皮制品禁用偶氮染料的测试，本文对皮革中禁用偶氮染料测试国际标准ISO 17234-1:2020进行解读，并对比了标准变更部分以及带来的影响。

具体涉及仪器设备和试剂方面进行技术上的更新、测试程序的优化以改进测试方法，假阳性测试结果的分析说明，为皮革及毛皮制品中禁用偶氮染料测试提供了技术指引和支撑。

关键词：标准，皮革，偶氮染料，假阳性DOI编码：10.3969/j.issn.1002-5944.2023.22.023Interpretation of Test Standard for Prohibited Azo Dyes in Leather in ISO17234-1:2020SHAO Juan-juan（Geely Automobile Research Institute (Ningbo) Co., Ltd.）Abstract:In order to study the test of prohibited azo dyes in leather and fur products in automotive materials, this paper interprets the international standard ISO 17234-1:2020 for the test of prohibited azo dyes in leather, and compares the part of the standard changes as well as the impact. It specifi cally involves the technical update of instruments, equipment and reagents, and the optimization of test procedures to improve the test method and the analysis of false positive test results. It provides technical guidance and support for the testing of prohibited azo dyes in leather and fur products.Keyword: standard, leather, azo dyes, false positives0 引 言禁用偶氮染料是偶氮基两端连接芳基的一类有机化合物，用于多种天然和合成纤维的染色和印花，也用于皮革、塑料、橡胶等制品的着色[1]。

Microsoft Windows 7: Guide de mise en routeConfiguration de Windows 7Votre ordinateur Dell est préconfiguré avec lesystème d'exploitation Microsoft® Windows® 7si vous l'avez sélectionné au moment de l'achat.Pour installer Windows la première fois, suivezles instructions qui s'affichent. Ces étapes sontobligatoires et peuvent prendre un certain temps.Les écrans de configuration Windows vous guidentdans différentes procédures, notammentl'acceptation des contrats de licence, la définitiondes préférences et la configuration d'uneconnexion Internet.PRÉCAUTION: N'interrompez pas leprocessus d'installation du systèmed'exploitation. 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Epson發表內嵌EPD驅動器的低耗電微處理器2010年12月20日影像產品以及半導體解決方案供應商Seiko Epson（”Epson”, TSE:6724）日前宣佈已開發出S1C17F57之樣品，這款內建驅動器的全新16位元微處理器，已針對如E Ink公司所出品的中小尺寸電子紙顯示器（EPD）之性能進行最佳化。

S1C17F57樣品已開始出貨，Epson計劃於2011年三月進行量產。

同時，Epson亦宣布其新開發的S1D14F50系列節能驅動IC已開始出貨，可協助EPD發揮最大性能。

隨著電子閱讀器（e-reader）的需求呈現爆炸性成長，EPD市場正迅速擴大中。

由於EPD具備高對比、高靈活性、影像穩定性與低耗電特性，而成為相關產品的最佳首選。

為了滿足EPD市場逐漸增長的需求，Epson開發出S1C17F57，一款擁有記憶體（ROM與RAM）、計時器及串列式介面等標準功能的微處理器。

此外，這款產品同時擁有許多嵌入式規格如即時時脈(RTC)、推論調整（theoretical regulation）、高效能區段式EPD驅動程式、以及一個溫度感測器。

因此，這個微處理器不僅可驅動顯示器，還可修正容易損害顯示品質的溫度波動，藉以將電子紙顯示器的效能推升至最大。

Epson還運用其專業的低耗電技術，成功達成極低的產品耗電量（於睡眠模式下耗電僅120 nA）。

此外，此微處理器亦能夠以厚度僅有200微米的裸晶形式出貨，對於需要長效電池壽命及纖薄型態的應用如智慧卡或其他小型行動裝置來說，是最完美的選擇。

同時，Epson也與Citizen Seimitsu共同開發一款內嵌S1C17F57的整合式電子紙顯示模組。

該公司已開始出貨其薄膜覆晶封裝（COF, Chip-on-Flex）模組，並內含一個電子紙顯示器，讓製造商更容易針對電子紙產品進行設計。

Epson Microdevices事業部副執行長宮川隆平表示：「Epson長久以來不斷針對各式電池驅動產品，提供低功耗的半導體產品。

HAZOP e Local approach in the Mexican oil &gas industryM.Pérez-Marín a ,M.A.Rodríguez-Toral b ,*aInstituto Mexicano del Petróleo,Dirección de Seguridad y Medio Ambiente,Eje Central Lázaro Cárdenas Norte No.152,07730México,D.F.,Mexicob PEMEX,Dirección Corporativa de Operaciones,Gerencia de Análisis de Inversiones,Torre Ejecutiva,Piso 12,Av.Marina Nacional No.329,11311México,D.F.,Mexicoa r t i c l e i n f oArticle history:Received 3September 2012Received in revised form 26March 2013Accepted 27March 2013Keywords:HAZOPRisk acceptance criteria Oil &gasa b s t r a c tHAZOP (Hazard and Operability)studies began about 40years ago,when the Process Industry and complexity of its operations start to massively grow in different parts of the world.HAZOP has been successfully applied in Process Systems hazard identi fication by operators,design engineers and consulting firms.Nevertheless,after a few decades since its first applications,HAZOP studies are not truly standard in worldwide industrial practice.It is common to find differences in its execution and results format.The aim of this paper is to show that in the Mexican case at National level in the oil and gas industry,there exist an explicit acceptance risk criteria,thus impacting the risk scenarios prioritizing process.Although HAZOP studies in the Mexican oil &gas industry,based on PEMEX corporate standard has precise acceptance criteria,it is not a signi ficant difference in HAZOP applied elsewhere,but has the advantage of being fully transparent in terms of what a local industry is willing to accept as the level of risk acceptance criteria,also helps to gain an understanding of the degree of HAZOP applications in the Mexican oil &gas sector.Contrary to this in HAZOP ISO standard,risk acceptance criteria is not speci fied and it only mentions that HAZOP can consider scenarios ranking.The paper concludes indicating major implications of risk ranking in HAZOP,whether before or after safeguards identi fication.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionHAZOP (Hazard and Operability)studies appeared in systematic way about 40years ago (Lawley,1974)where a multidisciplinary group uses keywords on Process variables to find potential hazards and operability troubles (Mannan,2012,pp.8-31).The basic prin-ciple is to have a full process description and to ask in each node what deviations to the design purpose can occur,what causes produce them,and what consequences can be presented.This is done systematically by applying the guide words:Not ,More than ,Less than ,etc.as to generate a list of potential failures in equipment and process components.The objective of this paper is to show that in the Mexican case at National level in the oil and gas industry,there is an explicit acceptance risk criteria,thus impacting the risk scenarios priori-tizing process.Although HAZOP methodology in the Mexican oil &gas industry,based on PEMEX corporate standard has precise acceptance criteria,it is not a signi ficant difference in HAZOP studies applied elsewhere,but has the advantage of being fullytransparent in terms of what a local industry is willing to accept as the level of risk acceptance criteria,also helps to gain an under-standing of the degree of HAZOP applications in the Mexican oil &gas sector.Contrary to this in HAZOP ISO standard (ISO,2000),risk acceptance criteria is not speci fied and it only mentions that HAZOP can consider scenarios ranking.The paper concludes indicating major implications of risk prioritizing in HAZOP,whether before or after safeguards identi fication.2.Previous workHAZOP studies include from original ICI method with required actions only,to current applications based on computerized documentation,registering design intentions at nodes,guide words,causes,deviations,consequences,safeguards,cause fre-quencies,loss contention impact,risk reduction factors,scenarios analysis,finding analysis and many combinations among them.In the open literature there have been reported interesting and signi ficant studies about HAZOP,like HAZOP and HAZAN differences (Gujar,1996)where HAZOP was identi fied as qualitative hazard identi fication technique,while HAZAN was considered for the quantitative risk determination.This difference is not strictly valid today,since there are now companies using HAZOP with risk analysis*Corresponding author.Tel.:þ525519442500x57043.E-mail addresses:mpmarin@imp.mx (M.Pérez-Marín),miguel.angel.rodriguezt@ ,matoral09@ (M.A.Rodríguez-Toral).Contents lists available at SciVerse ScienceDirectJournal of Loss Prevention in the Process Industriesjou rn al homepage :/locate/jlp0950-4230/$e see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.jlp.2013.03.008Journal of Loss Prevention in the Process Industries 26(2013)936e 940and its acceptance criteria(Goyal&Kugan,2012).Other approaches include HAZOP execution optimization(Khan,1997);the use of intelligent systems to automate HAZOP(Venkatasubramanian,Zhao, &Viswanathan,2000);the integration of HAZOP with Fault Tree Analysis(FTA)and with Event Tree Analysis(ETA)(Kuo,Hsu,& Chang,1997).According to CCPS(2001)any qualitative method for hazard evaluation applied to identify scenarios in terms of their initial causes,events sequence,consequences and safeguards,can beextended to register Layer of Protection Analysis(LOPA).Since HAZOP scenarios report are presented typically in tabular form there can be added columns considering the frequency in terms of order of magnitude and the probability of occurrence identified in LOPA.There should be identified the Independent and the non-Independent Protection Layers,IPL and non-IPL respec-tively.Then the Probability of Failure on Demand(PFDs)for IPL and for non-IPL can be included as well as IPL integrity.Another approach consists of a combination of HAZOP/LOPA analysis including risk magnitude to rank risk reduction actions (Johnson,2010),a general method is shown,without emphasizing in any particular application.An extended HAZOP/LOPA analysis for Safety Integrity Level(SIL)is presented there,showing the quan-titative benefit of applying risk reduction measures.In this way one scenario can be compared with tolerable risk criteria besides of being able to compare each scenario according to its risk value.A recent review paper has reported variations of HAZOP methodology for several applications including batch processes, laboratory operations,mechanical operations and programmable electronic systems(PES)among others(Dunjó,Fthenakis,Vílchez, &Arnaldos,2010).Wide and important contributions to HAZOP knowledge have been reported in the open literature that have promoted usage and knowledge of HAZOP studies.However,even though there is available the IEC standard on HAZOP studies,IEC-61882:2001there is not a worldwide agreement on HAZOP methodology and there-fore there exist a great variety of approaches for HAZOP studies.At international level there exist an ample number of ap-proaches in HAZOP studies;even though the best advanced prac-tices have been taken by several expert groups around the world, there is not uniformity among different consulting companies or industry internal expert groups(Goyal&Kugan,2012).The Mexican case is not the exception about this,but in the local oil and gas industry there exist a national PEMEX corporate standard that is specific in HAZOP application,it includes ranking risk scenarios (PEMEX,2008),qualitative hazard ranking,as well as the two ap-proaches recognized in HAZOP,Cause by Cause(CÂC)and Devia-tion by Deviation(DÂD).Published work including risk criteria include approaches in countries from the Americas,Europe and Asia(CCPS,2009),but nothing about Mexico has been reported.3.HAZOP variationsIn the technical literature there is no consensus in the HAZOP studies procedure,from the several differences it is consider that the more important are the variations according to:(DÂD)or (CÂC).Table1shows HAZOP variations,where(CQÂCQ)means Consequence by Consequence analysis.The implications of choosing(CÂC)are that in this approach there are obtained unique relationships of Consequences,Safeguards and Recommendations,for each specific Cause of a given Deviation. For(DÂD),all Causes,Consequences,Safeguards and Recommenda-tions are related only to one particular Deviation,thus producing that not all Causes appear to produce all the Consequences.In practice HAZOP approach(DÂD)can optimize analysis time development.However,its drawback comes when HAZOP includes risk ranking since it cannot be determined easily which Cause to consider in probability assignment.In choosing(CÂC)HAZOP there is no such a problem,although it may take more time on the analysis.The HAZOP team leader should agree HAZOP approach with customer and communicate this to the HAZOP team.In our experience factors to consider when choosing HAZOP approach are:1.If HAZOP will be followed by Layers of Protection Analysis(LOPA)for Safety Integrity Level(SIL)selection,then choose (CÂC).2.If HAZOP is going to be the only hazard identification study,it isworth to make it with major detail using(CÂC).3.If HAZOP is part of an environmental risk study that requires aConsequence analysis,then use(DÂD).4.If HAZOP is going to be done with limited time or becauseHAZOP team cannot spend too much time in the analysis,then use(DÂD).Although this is not desirable since may compro-mise process safety.Regarding risk ranking in HAZOP,looking at IEC standard(IEC, 2001)it is found that HAZOP studies there are(DÂD)it refers to (IEC,1995)in considering deviation ranking in accordance to their severity or on their relative risk.One advantage of risk ranking is that presentation of HAZOP results is very convenient,in particular when informing the management on the recommendations to be followedfirst or with higher priority as a function of risk evaluated by the HAZOP team regarding associated Cause with a given recommendation.Tables2and3are shown as illustrative example of the convenience of event risk ranking under HAZOP,showing no risk ranking in Table2and risk ranking in Table3.When HAZOP presents a list of recommendations without ranking,the management can focus to recommendations with perhaps the lower resource needs and not necessarily the ones with higher risk.Table1Main approaches in HAZOP studies.Source HAZOP approach(Crowl&Louvar,2011)(DÂD)(ABS,2004)(CÂC)&(DÂD)(Hyatt,2003)(CÂC),(DÂD)&(CQÂCQ) (IEC,2001)(DÂD)(CCPS,2008);(Crawley,Preston,& Tyler,2008)(DÂD),(CÂC)Table2HAZOP recommendations without risk ranking.DescriptionRecommendation1Recommendation2Recommendation3Recommendation4Recommendation5Table3HAZOP recommendations with risk ranking.Scenario risk DescriptionHigh Recommendation2High Recommendation5Medium Recommendation3Low Recommendation1Low Recommendation4M.Pérez-Marín,M.A.Rodríguez-Toral/Journal of Loss Prevention in the Process Industries26(2013)936e940937As can be seen in Tables 2and 3,for the management there will be more important to know HAZOP results as in Table 3,in order to take decisions on planning response according to ranking risk.4.HAZOP standard for the Mexican oil &gas industryLooking at the worldwide recognized guidelines for hazard identi fication (ISO,2000)there is mentioned that when consid-ering scenarios qualitative risk assignment,one may use risk matrix for comparing the importance of risk reduction measures of the different options,but there is not a speci fic risk matrix with risk values to consider.In Mexico there exist two national standards were tolerable and intolerable risk is de fined,one is the Mexican National Standard NOM-028(NOM,2005)and the other is PEMEX corporate standard NRF-018(PEMEX,2008).In both Mexican standards the matrix form is considered for relating frequency and consequences.Fig.1shows the risk matrix in (NOM,2005),nomenclature regarding letters in this matrix is described in Tables 4e 6.It can be mentioned that risk matrix in (NOM,2005)is optional for risk management in local chemical process plants.For Mexican oil &gas industry,there exist a PEMEX corporate standard (NRF),Fig.2,shows the corresponding risk matrix (PEMEX,2008).Nomenclature regarding letters in this matrix is described in Tables 7e 9for risk concerning the community.It is important to mention that PEMEX corporate standard considers environmental risks,business risks,and corporate image risks.These are not shown here for space limitations.The Mexican National Standard (NOM)as being of general applicability gives the possibility for single entities (like PEMEX)to determine its own risk criteria as this company opted to do.PEMEX risk matrix can be converted to NOM ’s by category ’s grouping infrequency categories,thus giving same flexibility,but with risk speci fic for local industry acceptance risk criteria.One principal consideration in ranking risk is to de fine if ranking is done before safeguards de finition or after.This de finition is relevant in:HAZOP kick-off presentation by HAZOP leader,explaining im-plications of risk ranking.HAZOP schedule de finition.Risk ranking at this point takes shorter time since time is not consumed in estimating risk reduction for each safeguard.If after HAZOP a LOPA is going to be done,then it should be advisable to request that HAZOP leader considers risk ranking before safeguards de finition,since LOPA has established rules in de fining which safeguards are protections and the given risk reduction.Otherwise if for time or resource limitations HAZOP is not going to be followed by LOPA,then HAZOP should consider risk ranking after safeguards de finition.Therefore,the HAZOP leader should explain to the HAZOP team at the kick-off meeting a concise explanation of necessary considerations to identify safeguards having criteria to distinguish them as Independent Protection Layers (IPL)as well as the risk reduction provided by each IPL.In HAZOP report there should be make clear all assumptions and credits given to the Protections identi fied by the HAZOP team.Figs.3and 4,shows a vision of both kinds of HAZOP reports:For the case of risk ranking before and after safeguards de finition.In Figs.3Fig.1.Risk matrix in (NOM,2005).Table 5Probability description (Y -axis of matrix in Fig.1)(NOM,2005).Frequency Frequency quantitative criteria L41in 10years L31in 100years L21in 1000years L1<1in 1000yearsTable 6Risk description (within matrix in Fig.1)(NOM,2005).Risk level Risk qualitative descriptionA Intolerable:risk must be reduced.B Undesirable:risk reduction required or a more rigorous risk estimation.C Tolerable risk:risk reduction is needed.DTolerable risk:risk reduction not needed.Fig.2.Risk matrix as in (PEMEX,2008).Table 7Probability description (Y -axis of matrix in Fig.2)(PEMEX,2008).Frequency Occurrence criteria Category Type Quantitative QualitativeHighF4>10À1>1in 10yearsEvent can be presented within the next 10years.Medium F310À1À10À21in 10years e 1in 100years It can occur at least once in facility lifetime.LowF210À2À10À31in 100years e 1in 1000years Possible,it has never occurred in the facility,but probably ithas occurred in a similar facility.Remote F1<10À3<1in 1000years Virtually impossible.It is norealistic its occurrence.Table 4Consequences description (X -axis of matrix in Fig.1)(NOM,2005).Consequences Consequence quantitative criteriaC4One or more fatalities (on site).Injuries or fatalities in the community (off-site).C3Permanent damage in a speci fic Process or construction area.Several disability accidents or hospitalization.C2One disability accident.Multiple injuries.C1One injured.Emergency response without injuries.M.Pérez-Marín,M.A.Rodríguez-Toral /Journal of Loss Prevention in the Process Industries 26(2013)936e 940938and4“F”means frequency,C means consequence and R is risk as a function of“F”and“C”.One disadvantage of risk ranking before safeguards definition is that resulting risks usually are found to be High,Intolerable or Unacceptable.This makes difficult the decision to be made by the management on what recommendations should be carried outfirst and which can wait.One advantage in risk ranking after safeguards definition is that it allows to show the management the risk scenario fully classified, without any tendency for identifying most risk as High(Intolerable or Unacceptable).In this way,the management will have a good description on which scenario need prompt attention and thus take risk to tolerable levels.There is commercial software for HAZOP methodology,but it normally requires the user to use his/her risk matrix,since risk matrix definition represents an extensive knowledge,resources and consensus to be recognized.The Mexican case is worldwide unique in HAZOP methodology, since it uses an agreed and recognized risk matrix and risk priori-tizing criteria according to local culture and risk understanding for the oil&gas sector.The risk matrix with corresponding risk levels took into account political,economical and ethic values.Advantages in using risk matrix in HAZOP are:they are easy to understand and to apply;once they are established and recognized they are of low cost;they allow risk ranking,thus helping risk reduction requirements and limitations.However,some disad-vantages in risk matrix use are:it may sometimes be difficult to separate frequency categories,for instance it may not be easy to separate low from remote in Table7.The risk matrix subdivision may have important uncertainties,because there are qualitative considerations in its definition.Thus,it may be advantageous to update Pemex corporate HAZOP standard(PEMEX,2008)to consider a6Â6matrix instead of the current4Â4matrix.5.ConclusionsHAZOP studies are not a simple procedure application that as-sures safe Process systems on its own.It is part of a global design cycle.Thus,it is necessary to establish beforehand the HAZOP study scope that should include at least:methodology,type(CÂC,DÂD, etc.)report format,acceptance risk criteria and expected results.Mexico belongs to the reduced number of places where accep-tance risk criteria has been explicitly defined for HAZOP studies at national level.ReferencesABS.(2004).Process safety institute.Course103“Process hazard analysis leader training,using the HAZOP and what-if/checklist techniques”.Houston TX:Amer-ican Bureau of Shipping.CCPS(Center for Chemical Process Safety).(2001).Layer of protection analysis: Simplified process risk assessment.New York,USA:AIChE.CCPS(Center for Chemical Process Safety).(2008).Guidelines for hazard evaluation procedures(3rd ed.).New York,USA:AIChE/John Wiley&Sons.CCPS(Center for Chemical Process Safety).(2009).Guidelines for Developing Quan-titative Safety Risk Criteria,Appendix B.Survey of worldwide risk criteria appli-cations.New York,USA:AIChE.Crawley,F.,Preston,M.,&Tyler,B.(2008).HAZOP:Guide to best practice(2nd ed.).UK:Institution of Chemical Engineers.Crowl,D.A.,&Louvar,J.F.(2011).Chemical process safety,fundamentals with ap-plications(3rd ed.).New Jersey,USA:Prentice Hall.Table8Consequences description(X-axis of matrix in Fig.2)(PEMEX,2008).Event type and consequence categoryEffect:Minor C1Moderate C2Serious C3Catastrophic C4 To peopleNeighbors Health and Safety.No impact on publichealth and safety.Neighborhood alert;potentialimpact to public health and safety.Evacuation;Minor injuries or moderateconsequence on public health and safety;side-effects cost between5and10millionMX$(0.38e0.76million US$).Evacuation;injured people;one ormore fatalities;sever consequenceon public health and safety;injuriesand side-consequence cost over10million MX$(0.76million US$).Health and Safetyof employees,serviceproviders/contractors.No injuries;first aid.Medical treatment;Minor injurieswithout disability to work;reversible health treatment.Hospitalization;multiple injured people;total or partial disability;moderate healthtreatment.One o more fatalities;Severe injurieswith irreversible damages;permanenttotal or partial incapacity.Table9Risk description(within matrix in Fig.2)(PEMEX,2008).Risk level Risk description Risk qualitative descriptionA Intolerable Risk requires immediate action;cost should not be a limitation and doing nothing is not an acceptable option.Risk with level“A”represents an emergency situation and there should be implements with immediate temporary controls.Risk mitigation should bedone by engineered controls and/or human factors until Risk is reduced to type“C”or preferably to type“D”in less than90days.B Undesirable Risk should be reduced and there should be additional investigation.However,corrective actions should be taken within the next90days.If solution takes longer there should be installed on-site immediate temporary controls for risk reduction.C Acceptablewith control Significant risk,but can be compensated with corrective actions during programmed facilities shutdown,to avoid interruption of work plans and extra-costs.Solutions measures to solve riskfindings should be done within18months.Mitigation actions should focus operations discipline and protection systems reliability.D ReasonablyacceptableRisk requires control,but it is of low impact and its attention can be carried out along with other operations improvements.Fig.3.Risk ranking before safeguard definition.Fig.4.Risk ranking after safeguards definition.M.Pérez-Marín,M.A.Rodríguez-Toral/Journal of Loss Prevention in the Process Industries26(2013)936e940939Dunjó,J.,Fthenakis,V.,Vílchez,J.A.,&Arnaldos,J.(2010).Hazard and opera-bility(HAZOP)analysis.A literature review.Journal of Hazardous Materials, 173,19e32.Goyal,R.K.,&Kugan,S.(2012).Hazard and operability studies(HAZOP)e best practices adopted by BAPCO(Barahin Petroleum Company).In Presented at SPE middle east health,safety,security and environment conference and exhibition.Abu Dhabi,UAE.2e4April.Gujar,A.M.(1996).Myths of HAZOP and HAZAN.Journal of Loss Prevention in the Process Industry,9(6),357e361.Hyatt,N.(2003).Guidelines for process hazards analysis,hazards identification and risk analysis(pp.6-7e6-9).Ontario,Canada:CRC Press.IEC.(1995).IEC60300-3-9:1995.Risk management.Guide to risk analysis of techno-logical systems.Dependability management e Part3:Application guide e Section 9:Risk analysis of technological systems.Geneva:International Electrotechnical Commission.IEC.(2001).IEC61882.Hazard and operability studies(HAZOP studies)e Application guide.Geneva:International Electrotechnical Commission.ISO.(2000).ISO17776.Guidelines on tools and techniques for hazard identification and risk assessment.Geneva:International Organization for Standardization.Johnson,R.W.(2010).Beyond-compliance uses of HAZOP/LOPA studies.Journal of Loss Prevention in the Process Industries,23(6),727e733.Khan,F.I.(1997).OptHAZOP-effective and optimum approach for HAZOP study.Journal of Loss Prevention in the Process Industry,10(3),191e204.Kuo,D.H.,Hsu,D.S.,&Chang,C.T.(1997).A prototype for integrating automatic fault tree/event tree/HAZOP puters&Chemical Engineering,21(9e10),S923e S928.Lawley,H.G.(1974).Operability studies and hazard analysis.Chemical Engineering Progress,70(4),45e56.Mannan,S.(2012).Lee’s loss prevention in the process industries.Hazard identifica-tion,assessment and control,Vol.1,3rd ed.,Elsevier,(pp.8e31).NOM.(2005).NOM-028-STPS-2004.Mexican National standard:“Norma Oficial Mexicana”.In Organización del trabajo-Seguridad en los procesos de sustancias químicas:(in Spanish),published in January2005.PEMEX.(2008).Corporate Standard:“Norma de Referencia NRF-018-PEMEX-2007“Estudios de Riesgo”(in Spanish),published in January2008. Venkatasubramanian,V.,Zhao,J.,&Viswanathan,S.(2000).Intelligent systems for HAZOP analysis of complex process puters&Chemical Engineering, 24(9e10),2291e2302.M.Pérez-Marín,M.A.Rodríguez-Toral/Journal of Loss Prevention in the Process Industries26(2013)936e940 940。

A sensitive HPLC method for measuring bacterial proteolysisand proteinase activity in UHT milkT.X.Le,N.Datta,H.C.Deeth*Australian Dairy Industry Centre for UHT Processing,School of Land and Food Sciences,University of Queensland,St.Lucia,Brisbane,Qld 4072,AustraliaReceived 13December 2005;accepted 21March 2006AbstractA sensitive quantitative reversed-phase HPLC method is described for measuring bacterial proteolysis and proteinase activity in UHT milk.The analysis is performed on a TCA filtrate of the milk.The optimum concentration of TCA was found to be 4%;at lower con-centrations,non-precipitated protein blocked the HPLC while higher concentrations yielded lower amounts of peptides.The method showed greater sensitivity and reproducibility than a fluorescamine-based method.Quantification of the HPLC method was achieved by use of an external dipeptide standard or a standard proteinase.Ó2006Elsevier Ltd.All rights reserved.Keywords:Bacterial proteinase;HPLC;Fluorescamine;Proteinase activity;Proteolysis1.IntroductionProteinase activity in UHT milk causes bitterness and gelation problems (Adams,Barach,&Speck,1975;Datta &Deeth,2001).This activity can be due to residual native proteinase (plasmin)or bacterial proteinases.The latter orig-inate from growth of psychrotrophic bacteria in raw milk and survive the UHT heat treatment which destroys the par-ent organisms.As low levels of these enzymes are sufficient to cause undesirable amounts of protein degradation in UHT milk during storage at room temperature (Mitchell &Ewings,1985),sensitive methods for their detection have been sought by the dairy industry.However,to date,no method has been universally adopted for this purpose.The peptides produced in milk by milk plasmin and the proteinases of psychrotrophic bacterial contaminants are different and can be separated by reversed-phase (RP)HPLC into two distinct groups (Lo ´pez-Fandin ˜o,Olano,San Jose,&Ramos,1993).The peptides produced by plas-min are larger and more hydrophobic and therefore elute after the smaller more hydrophilic peptides produced bythe bacterial proteinases.This phenomenon is the basis of a method used for distinguishing between the two enzyme types (Datta &Deeth,2003).Previous research by Haryani,Datta,Elliott,and Deeth (2003)on the development of proteinase activity and prote-olysis in raw milk during storage at different temperatures showed that RP-HPLC of milk TCA filtrates was capable of detecting lower levels of proteolysis and proteinase activity than the fluorescamine method (Chism,Huang,&Marshall,1979).The work reported in this paper further develops the RP-HPLC method as a sensitive quantitative method for determining both proteolysis and proteinase activity in milk.2.Materials and methods ksRaw milk was obtained from three different bulk milk sources.Samples were collected in sterile containers.Pas-teurized milk samples were obtained from three commercial sources.UHT milks (full cream and skim)were collected from three different batches from one commercial source.0963-9969/$-see front matter Ó2006Elsevier Ltd.All rights reserved.doi:10.1016/j.foodres.2006.03.008*Corresponding author.Tel.:+61733469191;fax:+61733651177.E-mail address:h.deeth@.au (H.C.Deeth)./locate/foodresFood Research International 39(2006)823–8302.1.1.Stored milksEach raw and pasteurized milk sample was stored for1 day at7°C plus14days at4°C,and28days at4°C plus1 day at40°C,respectively,in order to produce detectable proteolysis by psychrotrophic bacterial proteinases.The trial was replicated three times.2.2.Proteinases2.2.1.AlcalaseÒAlcalase(generic name subtilisin),a serine endopepti-dase prepared from a strain of Bacillus licheniformis,with a specific activity of2.4AU1gÀ1,was obtained from Novo Nordisk(Bagsvaerd,Denmark).2.2.2.Crude Pseudomonas proteinasePseudomonas aureofaciens,previously isolated from pas-teurized milk(Deeth,Khusniati,Datta,&Wallace,2002), was cultured in UHT skim milk at28°C for3d.Bacterial cells were removed by centrifugation at24,000g for10min at5°C.The resulting supernatants were stored atÀ20°C until used as a crude proteinase source for inoculation into milks.2.3.Preparation of TCAfiltratesMilk(5ml)was mixed with an equal volume of trichlo-roacetic acid(TCA)of various concentrations(2%,4%, 8%,12%,16%,20%,24%w/v).The mixtures were vortexed in a test tube,left at room temperature for30min,andfil-tered through a Whatmanfilter paper No.41and a 0.45l m Milliporefilter.The TCAfiltrates were collected and analysed for peptides by the HPLC andfluorescamine methods.2.4.Preparation of UHT samples with added enzyme2.4.1.With AlcalaseAlcalase(2.4AU gÀ1,10l l)was added to10ml of cold UHT milk to obtain a stock solution(10À3dilution, 0.003AU mlÀ1).Various volumes of the Alcalase stock solution(0,25,50,75,and100l l)were added to UHT milk to makefinal solutions of5ml(0,5,10,15,and20·10À6 v/v)corresponding to0,15,30,45,and 60·10À6AU mlÀ1.2.4.2.With Ps.aureofaciens proteinaseUHT milks(full cream and skim)were inoculated with cell-free Ps.aureofaciens culture supernatant(125,250, 375,500,625l l)to give solutions of2.5ml withfinal con-centrations of5%,10%,15%,20%,and25%v/v.The milk samples containing the proteinases(Ps.aureo-faciens proteinase or Alcalase)were mixed with a vortex mixer and incubated at37°C in a water bath without shak-ing.Sodium azide(BDH,England)was added to the UHT milk samples just before incubation(final concentration, 0.05%w/v)to inhibit bacterial growth.Controls were pre-pared in the same way but without added enzyme,and were incubated under the same conditions.Samples were withdrawn after incubation for0.5,1,2,3,4,6,and8h. Proteolysis was terminated by adding an equal volume of 8%trichloroacetic acid(TCA)and the TCAfiltrate(4%) was prepared as above.The trial was replicated three times.2.5.Preparation of peptide standards in TCAStandard peptide tyrosyl-leucine(Tyr-Leu)(12.5,25, 37.5,50,62.5mg),obtained from Sigma Chemical Co. (Switzerland),was accurately weighed and dissolved in 1ml of4%TCA(BDH Lab Supplies,England).Aliquots of the12.5mg mlÀ1Tyr-Leu solution(10,20,30,40, 50l l)were dissolved in1ml4%TCA to make up0.125, 0.25,0.375,0.5,0.625mg mlÀ1(0.425,0.85,1.27,1.7,and 2.12mmol lÀ1)standard solutions,respectively.2.6.Peptide analysis byfluorescamine methodPeptide analysis of the TCAfiltrates followed thefluo-rescamine method of Beeby(1980)as modified by Kocak and Zadow(1985).All samples were analysed in duplicate. Filtrate(100l l),0.1M phosphate buffer pH8.0(3ml)and fluorescamine solution(0.2mg mlÀ1in acetone;1ml)were pipetted into afluorimeter tube and mixed thoroughly by vortexing.Thefluorescence of the solution was measured after15min in a Turner Model450fluorimeter at excita-tion390nm and emission480nm.Thefluorimeter was ini-tially zeroed using a mixture of phosphate buffer(3.1ml) andfluorescamine solution(1ml).The Relative Fluores-cence Units(RFUs)were read from thefluorimeter and converted to mmol peptide lÀ1by reference to a standard curve prepared with Tyr-Leu.2.7.Peptide analysis by RP-HPLCSeparation of peptides was performed on a Shimadzu Class-VP Chromatograph using a Phenomenex Luna5l-C18(2)reversed-phase column(150·4.6mm)at40°C and a UV/Vis detector at210nm.Two solvents were used: solvent A was0.1%(v/v)trifluoroacetic acid(TFA)in Milli-Q water,while solvent B consisted of0.1%(v/v) TFA(Scharlau Chemie,AC-3141,Spain)in HPLC-grade acetonitrile(LAB-SCAN,code C2502U).Injection volume was50l l.The solvent gradient was formed by increasing the pro-portion of solvent B from20%to35%during thefirst 20min,and increasing solvent B to100%over5min and holding at100%B for the last3min.The column was then washed by decreasing solvent B to0%in0.01min and1One AU(Anson Unit)is the amount of enzyme which digestshaemoglobin under the standard reaction conditions at an initial rate suchthat the amount of trichloroacetic acid-soluble product which is liberatedper min gives the same colour with phenol reagent as1l mol tyrosine.824T.X.Le et al./Food Research International39(2006)823–830holding for20min before returning to20%solvent B for 2min.The column was equilibrated at20%B for5min, in readiness for the next sample injection.Theflow rate was set at1.0ml minÀ1and the maximum pressure limit was17.5MPa.The peptides were quantified by measuring peak areas using the valley-to-valley integration method and expressed as either the sum of the peak areas(Sum PA)or as mmol Tyr-Leu equivalents by reference to a standard curve pre-pared with Tyr-Leu.2.8.Proteinase assayProteinase activity in UHT milk was determined by mea-suring the increase in peptides produced during incubation for24h at40°C.Sodium azide(0.05%w/v)was added to the milk samples just before incubation to inhibit bacterial growth.The amount of peptides was quantified by thefluo-rescamine and HPLC methods,expressed in mmol Tyr-Leu equivalents by reference to standard curves of Tyr-Leu.The enzyme activity was calculated from these concentrations and expressed as mmol Tyr-Leu equivalents lÀ1hÀ1.The enzyme activity was also expressed in terms of Anson Units (AU)with reference to a standard curve for Alcalase.2.9.Statistical analysisThe data were assessed by analysis of variance (ANOVA)using MINITAB(release13);the results were considered significant if the associated p-value was<0.05; Student’s t-tests were employed for paired comparison of means.3.Results and discussion3.1.TCAfiltrate preparation:optimisation of TCA concentrationThe mean concentration of peptides in the TCAfiltrates from the stored raw and pasteurized milks determined by thefluorescamine and HPLC methods decreased with increasing TCA concentration(Fig.1).The concentration in the4%(w/v)TCAfiltrates was significantly higher (p<0.05)than in the6%,8%,10%,and12%(w/v)TCAfil-trates by both methods.This is consistent with the peptide solubilities calculated from the following formula of Yvon, Chabanet,and Pelissier(1989):y¼100=ð1þeð0:596ðxþ:963cÀ37:375ÞÞwhere y is percent solubility;c is TCA concentration(%w/ v);and x is percentage of acetonitrile in the eluent.The cal-culated solubilities of peptides eluting after16min are 71.3%,44%,20%,7.3%,and2.5%in4%,6%,8%,10%, and12%TCA,respectively.The correlation between thefluorescamine and HPLC methods was not high(R2=0.60and0.69for raw and pas-teurized milks,respectively)(Fig.2).This is attributable to the lower sensitivity and repeatability of thefluorescamine method compared with the HPLC method.Furthermore, proteolysis in different milk samples exhibit different pep-tide patterns.Therefore,conversion between the results from the two methods is not reliable.The difference in peptides extracted with the different con-centrations of TCA is illustrated in the HPLC chromato-grams of thefiltrates shown in Fig.3.Three different groups of peptides were observed:those eluting up to 8min,corresponding to626%acetonitrile(Group1);those eluting between8and16min,corresponding to>26–32% acteonitrile(Group2);and those eluting after16min,corre-sponding to>32%acetonitrile(Group3).Group1peptides were present infiltrates of all TCA concentrations and remained fairly constant over the TCA concentrations used. The amount of peptides in Group2,as measured by the peak areas,decreased as the TCA concentration increased,and Group3peptides decreased considerably with increasing TCA concentrations.The percentages of the total peak area of peptides in Groups1,2,and3ranged from7%to27%, 18%to63%,and51%to71%,respectively.The interactions between TCA and peptides include electrostatic and hydrogen bonding with side chains ofT.X.Le et al./Food Research International39(2006)823–830825polar amino acid residues as well as hydrophobic interac-tions(Yvon et al.,1989).It has been demonstrated that the difference in pH is not the reason for the greater precip-itation of large peptides at12%TCA than at lower concen-trations because the pH of TCA-treated milk solutions is similar for all TCA levels.Thisfinding is supported by the work of Sivaraman,Kumar,Jayaraman,and Yu (1997),who showed that acid-induced protein precipitation by TCA differs from that of other strong acids,e.g.HCl and TFA,and weaker acids,e.g.acetic,chloroacetic and dichloroacetic acid.The interactions of the three chlorine atoms in the TCA molecule with the nitrogen atom in the amide bond in peptides induce precipitation of large pep-tides by causing them to unfold and aggregate through interaction between newly exposed hydrophobic regions (Sivaraman et al.,1997).In preliminary experiments,1%and2%(w/v)TCA were also examined for use in preparing peptide extracts from raw milks.A1%TCAfiltrate gave the highest amount of soluble peptides,as determined by thefluorescamine method.However,this increase can be attributed to the presence offluorescamine-reactive b-lactoglobulin(Pearce, 1979).At low TCA concentrations(<3%w/v),about60% (Fox,Holsinger,Posati,&Pallansch,1967)or77%(Green-berg&Shipe,1979)of b-lactoglobulin is soluble.Lieske, Konrad,and Faber(1997)reported some a-lactalbumin also is in a2%TCAfiltrate.The presence of whey proteins in1and2%TCAfiltrates may be responsible for the development of turbidity in thesefiltrates on standing,an observation made in the pres-ent work.This is consistent with thefinding of Sivaraman et al.(1997)that protein CTX III(purified from cobra venom)in3%TCA turned turbid upon standing for more than7d.The author demonstrated that the protein in3% TCA was in an intermediate state with a‘molten globule’or‘A state’structure,which has a compact secondary, but not rigid tertiary structure,and is prone to aggregation.HPLC analysis of the2%TCAfiltrate in this work proved problematic.Under the HPLC conditions used (40°C),the larger peptides and proteins in thefiltrates were826T.X.Le et al./Food Research International39(2006)823–830unstable and became insoluble and blocked the filter on the guard column,preventing entry of the sample to the col-umn.Therefore,use of a 2%TCA filtrate is not recom-mended when the HPLC analysis is performed at an elevated temperature.Based on these optimisation trials,4%TCA was chosen for all further experiments.This percentage gave maximum yield of peptides but did not dissolve large peptides or pro-teins which cause instability problems in 1%and 2%TCA filtrates.3.2.Quantification of Alcalase proteolysis in milke of tyrosyl-leucine (Tyr-Leu)as peptide standard Preliminary tests were carried out with a variety of di-and tripeptides,which could be,or be similar to,hydrolysis products of milk proteins (e.g.Leu-Ala,Glu-Glu,Leu-Gly-Gly,Lys-Trp-Lys,Leu-Leu,Ile-Pro-Ile,Tyr-Leu).Tyr-Leu was selected as an external HPLC standard because it was well resolved in a reasonable time (8min)by the method used.The calibration curve for analysis of Tyr-Leu by the HPLC method (Fig.4(b))was linear in the range 0–200mmol l À1(R 2=0.9998).By comparison,Tyr-Leu anal-ysis by the fluorescamine method was linear in the range 0–2mmol l À1(R 2=0.9998)but curved downwards from 2to 4mmol l À1(Fig.4(a)).At higher concentrations,the fluorescence values leveled out as the Tyr-Leu concentra-tion increased,due to either exhaustion of the reagent or attainment of the limit of fluorescence measurement by the fluorimeter.While previous use of Tyr-Leu as a stan-dard peptide has not been reported,the linear range for Leu-Leu by the fluorescamine method has been found to be 0–0.6mg ml À1(i.e.0–2mmol l À1)(Chism et al.,1979),the same linear range found for Tyr-Leu by the fluoresca-mine method in this work.3.2.2.Linearity with time of reactionUHT milks (both whole and skim)were hydrolyzed with commercial Alcalase (10À4dilution,0.0003AU ml À1)at 37°C at the natural pH of milk for different periods of time (Fig.5a ).The proteolysis curves,as determined by theHPLC method were reasonably linear up to about 2h and leveled offthereafter.By contrast,the curve as mea-sured by the fluorescamine method curved after a short time of incubation,a result consistent with the results dis-cussed above for the Tyr-Leu calibration.The HPLC pro-files of the 2-h hydrolysates were similar to those of 3,4,6,and 8h (Fig.5b ),suggesting the peptides formed after that time were not further degraded.Therefore,a reaction time of 2h was chosen to test the linearity of the reaction with the level of Alcalase.3.2.3.Linearity with concentration of enzymeFor Alcalase in the concentration range 0–9000mAU l À1,the hydrolysis curve for UHT milk (by HPLC)was very steep for the lowest concentrations of enzyme and then leveled offat the higher concentrations (Fig.6(a)).However,in a lower concentration range (0–150mAU l À1),the curve was virtually linear for Alcalase concentration up to 60mAU l À1and then began to curve downward (Fig.6(b)).The range of enzyme activities of 0–60mAU l À1was then used for the replicated trial to establish the linearity of the proteolysis reaction of Alca-lase on UHT skim and whole milk.Fig.7shows the graphs obtained for UHT skim milk by both the fluorescamine and HPLC methods,demonstrating good linearity with R 2values of 0.983and 0.998,respectively.The results for whole milk (not shown)were very similar with correspond-ing R 2values of 0.988and 0.995,respectively.T.X.Le et al./Food Research International 39(2006)823–8308273.2.parison offluorescamine and HPLC methodsAs shown in Fig.7,the proteinase activities calculated as equivalent mmol Tyr-Leu lÀ1hÀ1by the HPLC andflu-orescamine methods are very different.The proteinase activities determined by HPLC were$600times higher than those determined by thefluorescamine method.This is due to the different measurement principles of the two methods.Thefluorescamine method is based on thefluo-rescence intensity of the product between primary amines and the reagent;the more free amino groups the peptides contain,the greater thefluorescence yield,irrespective of the amino acid composition(Beeby,1980).Therefore the fluorescamine result is heavily influenced by the small pep-tides but relatively insensitive to large peptides and pro-teins.In contrast,HPLC measures the absorbance at a given wavelength of separated peptides and amino acids with the response being based approximately on mass.In this case,the sensitivity of detecting larger peptides is greater than for small peptides.A further consequence of this is that at high levels of proteolysis when initially pro-828T.X.Le et al./Food Research International39(2006)823–830duced peptides are being degraded to smaller peptides,the fluorescamine-reactive peptides may increase while the total mass of the hydrolysate determined by HPLC may not be affected.Although thefluorescamine method is fast,it is tedious to obtain an accurate measurement.The HPLC method is more precise,more sensitive,more repeatable,and hence more reliable than thefluorescamine method.In addition, the graphical results give more information about hydroly-sis changes and proteinase specificities.3.3.Application of HPLC method for measuring proteinase activity3.3.1.Alcalase activityThe above section illustrates how the HPLC method can be applied to assaying the activity of a proteinase,in that case ing the method to assay the amount of proteolysis,the reaction was shown to be linear with both time and enzyme concentration over a2-h incubation per-iod.The proteinase activity can be expressed in terms of the amount of peptide produced in equivalent Tyr-Leu units per unit of time per amount of enzyme source.Alterna-tively,the proteinase activity in a source such as milk can be determined in terms of Anson Units by reference to the activity of a standard enzyme such as Alcalase.In this case,Fig.7(b)can act as a calibration curve.3.3.2.Pseudomonas proteinase activityUHT milks(both whole and skim)were hydrolyzed with a cell-free supernatant of a culture of Ps.aureofaciens.A reaction time of4h(at37°C)was chosen as preliminary trials(results not shown)indicated that the proteolysis reaction rate was almost constant for4h but decreased thereafter.For this incubation time,the assay of the crude Pseudomonas proteinase in UHT skim milk was linear with amount of enzyme(Fig.8shows the data for skim milk;the results for whole milk were very similar).4.ConclusionHPLC analysis of the peptides in TCAfiltrates of milk is a sensitive method for measuring the extent of proteolysis in milk by bacterial proteinases.Quantification can be achieved by relating the sum of the HPLC peak areas elut-ing up to25min to the peak area obtained for an external standard,dipeptide Tyr-Leu.The HPLC peptide method can also be used to deter-mine bacterial proteinase activity by analysing the peptides produced in a given time and quantifying them by the external standard method above.Alternatively,the activity can be expressed in terms of enzyme units of a standard proteinase such as Alcalase.Proteolysis by both Alcalase and a crude Ps.aureofaciens proteinase was linear with time up to2h and with concentration up to the equivalent of$1100and200mmol Tyr-Leu lÀ1hÀ1,respectively,cor-responding to$10and60mAU lÀ1Alcalase,respectively.The optimum concentration of TCA in the milkfiltrates was4%.Lower concentrations extracted large peptides and proteins which did not remain in solution during HPLC analysis and blocked the guard columnfilters.TCA conc-entations higher than4%produced lower peptide yields, especially of the larger more hydrophobic peptides.The HPLC method gives more repeatable and accurate results than thefluorescamine method.Furthermore,the two methods were not well correlated.This is due to their different measurement principles:thefluorescamine method measures moles of primary amine groups while the HPLC method measures mass of peptide product. Therefore,results from one method cannot be reliably esti-mated from those of the other.AcknowledgementsThe authors acknowledge thefinancial assistance from the Dairy Australia and Atlantic Philanthropies(for UQ-VNU scholarship).The assistance of Ms Del Greenway with statistical analyses is gratefully acknowledged. ReferencesAdams,D.M.,Barach,J.T.,&Speck,M.(1975).Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.Journal of Dairy Science,58,828–834.Beeby,R.(1980).The use offluorescamine at pH6.0to follow the action of chymosin on k-casein and to estimate this protein in milk.New Zealand Journal of Dairy Science and Technology,15,99–108. 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E.,&Ewings,K.N.(1985).Quantification of bacterial proteolysis causing gelation in UHT-treated milk.New Zealand Journal of Dairy Science and Technology,20,65–76.Pearce,K.N.(1979).Use offluorescamine to determine the rate of release of the casino-macropeptide in rennet-treated milk.New Zealand Journal of Dairy Science and Technology,14, 233–239.Sivaraman,T.,Kumar,T.K.S.,Jayaraman,G.,&Yu,C.(1997).The mechanism of2,2,2-trichloroacetic acid-induced protein precipitation.Journal of Protein Chemistry,16,291–297.Yvon,M.,Chabanet,C.,&Pelissier,J.P.(1989).Solubility of peptides in trichloroacetic-acid(TCA)solutions–hypothesis on the precipitation mechanism.International Journal of Peptide and Protein Research,34, 166–176.830T.X.Le et al./Food Research International39(2006)823–830。

刊名题写：张爱萍《火炸药学报》第八届编辑委员会成员特邀顾问：李俊贤（院士）欧阳平凯（院士）刘怡昕（院士）张生勇（院士)主任委员：王泽山（院士）副主任委员：陈小明（院士）侯委员：以姓氏笔划为序）马海霞王占利王伟力冯昊冯长根卢芳云何国强杜仕国余卫博张路遥李念念杨荣杰周晋红庞罗运军郭艳辉曹端林梁争峰X ijiang(英国 $CliveWoodley(英国 $Charles%Kappenstein(法国$晓（院士）<救>(院士）刘：王伯周王利军王晓峰刘艳刘所恩吕龙余文力张炜张同来汪越沙恒肖川姜炜胡林俊赵凤起强洪夫芳彭翠枝Svatopluk Zeman(捷克$Aleksei B.Sheremetev(俄罗斯)M ichael Gozin(以色列）庞思平王晶禹王琼林王煊军吕剑严启龙何卫东张庆华张建国张洪林肖忠良明陈赵其林徐抗震聂福德裴重华谭波樊学忠Luigi T.Deluca(意大利）Thomas M.KlapDetke(德国 $Lemi Tlirker (土耳其）Richard A.Yetter(美国$Ying Zheng(加拿大）AloneGany(以色列）主编：沙恒副主编：赵凤起执行主编：李念念《火炸药学报》第一届青年编委会成员伟王小龙冯晓军刘宁刘伟强吴世曦张文张俊林李伟李李李李国平杨奇杨伟杨伟杨汪营羞沈飞周星庞强武碧栋金波郝嚷子徐郭兆琦陶俊隋欣海生詹发禄火"#学％公开发行1978年创刊双月刊第44卷第！期（总第216期）CHINESE JOURNAL OF EXPLOSIVES&PROPELLANTS (Bim onthly,Started in 1978)Vol.44 No.2 2021 Series No.216主管单位：中国科学技术协会主办单位：中国兵工学会中国兵器工业第204研究所 编辑出版:《火炸药学报》编辑部主 编：沙恒副主编：赵凤起执行主编：李念念责任编辑：刘星印刷：西安创维印务有限公司中国标准连MSSN 1007-7812续出版物号:CN 61-1310/T J Managed by China Association for Science and Technology Sponsored by China Ordnance SocietyThe Research Institute No.204 of the China Ord­nance Industry CorporationEdited by Editorial Board of Journal of Explosives &Propellants (P.O.Box 18,Xi’an,710065,China)E ditor-in-Chief：Sha HengDeputy Editor-in-Chief：Zhao F eng-qiExecutive Editor-in-Chief：Li N ian-nianTel ：(029)82291297 E-m ail：**************h ttp:#w w w. hzyxb. cn 西安市18 号信箱(710065) 2021 年 04 月出版定价:25. 00 元。

序号工具号工具名称车型订货号13220万向扳手奥迪A4253023299V形皮带拆卸杆奥迪A42590-133370钩子奥迪A42520-143387扳手销奥迪A42587-15T10008锁紧盘奥迪A42588-26T10010内六角形扳手奥迪A4807-3.27T40027插装工具奥迪A48801K-88V/158拉具奥迪A41789-229V/159双销扳手奥迪A4258710T40154后桥差速器支架奥迪A5990SLG-1211VAS6416双弯头螺钉起子奥迪A5818-3123079定位工具奥迪A6259413318510mm套筒扳手奥迪A62528-101432478mm套筒奥迪A62527-815325727mm套筒奥迪A6258316335318mm套筒奥迪A62593-18173357内梅花扳手奥迪A62567-16183410套筒奥迪A6990SLG-10193415紧固器奥迪A62540-1203417机油滤清器扳手奥迪A62169213438钩子奥迪A64964-1223017A 减振器扳手奥迪A62593LG-17233122B 火花塞扳手奥迪A64766-1243287A 球头拉具奥迪A61779-2253352A 调整量具奥迪A64850-1263358B雨刮器调整装置奥迪A64851-127Hazet2171-1机油滤清器扳手奥迪A62171-128KUKKO17/1 分离器(12-75mm)奥迪A61775N-7529KUKKO17/2分离器(12-115mm)奥迪A61775N-11530KUKKO21/1拉具奥迪A61788N-1531KUKKO22/1拉具支架奥迪A61788N-83532T10001成套减振器用工具奥迪A64910/1333T10007折射计奥迪A64810B-CN 34T10058加长内六方扳手奥迪A62527-535T10070专用加长扳手奥迪A6Sep-7936T10127调节工具奥迪A64850-137T40001拉具奥迪A62510-1+2510-838T40010专用工具奥迪A61779-339V/146.1活塞环夹紧钳70~100mm 奥迪A6794U-340V/146.10拉具奥迪A61775N-1241V/175A 套筒扳手头奥迪A6900S-1542V/5活塞环卡钳50~100mm 奥迪A6790-143v/76球头拉具奥迪A61779-1844VAG1275软管卡箍钳奥迪A61847-145VAG1331扭力扳手奥迪A66290-1CT 46VAG1331/1扳手头奥迪A66403-1Hazet 工具与大众专用工具号对应关系表Hazet工具与大众专用工具号对应关系表序号工具号工具名称车型订货号Hazet工具与大众专用工具号对应关系表序号工具号工具名称车型订货号。

Pro filing of phenolic and other polar compounds in zucchini (Cucurbita pepo L.)by reverse-phase high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometryIhsan Iswaldi,Ana María Gómez-Caravaca,Jesús Lozano-Sánchez,David Arráez-Román,Antonio Segura-Carretero ⁎,Alberto Fernández-GutiérrezDepartment of Analytical Chemistry,Faculty of Sciences,University of Granada,Avenida Fuentenueva s/n,18071Granada,SpainResearch and Development Functional Food Center (CIDAF),Health-Science Technological Park,Avenida del Conocimiento 3,18100Granada,Spaina b s t r a c ta r t i c l e i n f o Article history:Received 31July 2012Accepted 18September 2012Keywords:Cucurbita pepo L.ZucchiniPhenolic compounds Polar compounds HPLCQ-TOF MSPolyphenolic compounds are widely distributed in vegetables.Zucchini (Cucurbita pepo L.)has a high nutritional value and a low amount of calories.Nevertheless,few studies in the literature focus on the separation and iden-ti fication of phenolic and other polar compounds in whole zucchini fruit.Therefore,the present study character-izes phenolic and other polar compounds in three kinds of zucchini (Verde,Redondo,and organic Verde)bought in Almería (Spain).For this purpose,an extraction method using methanol:water (80:20w/w)has been applied in order to extract the polar fraction from the samples studied.Afterwards,the extracts were injected into the RP-HPLC system and separated by a C 18column.Q-TOF-MS detection was conducted using a micrOTOF-Q equipped with an electrospray ionization (ESI)interface.The combined use of RP-HPLC coupled to DAD and Q-TOF-MS has proved useful for characterizing numerous phenolic and other polar compounds in zucchini.A total of 57compounds were identi fied with the present methodology.In fact,to the best of our knowledge,41out of 57identi fied compounds are being reported here for the first time in whole zucchini fruit.©2012Elsevier Ltd.All rights reserved.1.IntroductionThe daily intake of polyphenols has received much attention due to the health bene fits from their antioxidant/anti-radical,anti-carcinogenic,anti-in flammatory,antiviral,and antimicrobial activities.This group of chemical compounds is widely distributed in vegetables and can be classi-fied based on their chemical structure,ranging from simple molecules such as phenolic acids,to highly polymerized compounds such as tannins (Pietta,Minoggio,&Bramati,2003).The zucchini (Cucurbita pepo L.),of the Cucurbitaceae family,is low in calories and contains high nutritional (National Food Institute,2009)and medical value (Menéndez et al.,2006;Shokrzadeh,Azadbakht,Ahangar,Hashemi,&Saeedi Saravi,2010).These fruits can be found in many shapes,from spherical to elongated,and they vary in skin color from dark to light green,sometimes with fine white mottling or stripes.Most of the data reported in this sphere concerns the composition of pumpkin (Cucurbita moschata )rge amounts of two triterpenes esterifed with p -aminobenzoic acid have been reported (Appendino,Jakupovic,Belloro,&Marchesini,1999).Iso flavones such as daidzein and genistein and also secoisolariciresinol have beenfound in pumpkin seeds (Adlercreutz &Mazur,1997).Later,lariciresinol as the second lignin in this kind of sample has also been reported (Sicilia,Niemeyer,Honig,&Metzler,2003).Cucurbitosides A to E and cucurbitosides F to M as acylated phenolic glycosides have been identi fied in the seed of C.moschata (Koike,Li,Liu,Hata,&Nikaido,2005)and C .pepo (Li et al.,2009),respectively.Furthermore,pumpkin-seed oil has been demonstrated to possess high antioxidative capacity in methanol –water extract due to its content in polar phenolic compounds (Fruhwirth &Hermetter,2007,2008),but the compounds which are responsible for this antioxidant capacity have not been identi fied.Chlorogenic acid,caffeic acid,p -coumaric acid,and syringic acid were identi fied in pumpkins (Dragovic-Uzelac,Delonga,Levaj,Djakovic,&Pospisil,2005).Besides caffeic acid and p -coumaric acid,ferulic acid and vanillic acid were also reported in zucchini after alkaline and acid hydrolysis (Mattila &Hellström,2007).Little knowledge is available on the phenolic compounds present in the extract of whole zucchini fruit.Therefore,in the present study,polyphenolic compounds in zucchini extract were studied using HPLC coupled with two different de-tection systems:diode array (DAD)and quadrupole time-of-flight (Q-TOF)mass spectrometry.The UV/visible range has proved valuable in identifying the family of these phenolic compounds while MS and MS/MS have enabled accurate mass measurements and complementary structural information.Other polar compounds that could be character-ized using this method have also been reported.Food Research International 50(2013)77–84⁎Corresponding author at:Research and Development Functional Food Center (CIDAF),Health-Science Technological Park,Avenida del Conocimiento 3,18100Granada,Spain.Tel.:+34958243296;fax:+34958249510.E-mail address:ansegura@ugr.es (A.Segura-Carretero).0963-9969/$–see front matter ©2012Elsevier Ltd.All rights reserved./10.1016/j.foodres.2012.09.030Contents lists available at SciVerse ScienceDirectFood Research Internationalj o u rn a l ho m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /f o o d re s2.Materials and methods2.1.Zucchini samplesTwo zucchini varieties were analyzed:“Verde”(elongated in shape with light-green skin and light-grayish mottling)and“Redondo”(more spherical and with dark-green skin with sharp longitudinal stripes and whitish speckles).Also,the organically grown“organic Verde”(cultivated chemical free)was studied.One sample(constituted by5kg of zucchini)of each variety of zucchini was bought at local mar-kets and stored at4°C until used.2.2.Sample preparationEach sample(whole fruit)was ground,frozen at−20°C,and then placed on a lyophilizer(Christ Alpha1-2LD Freeze dryer,Shropshire, UK)shelf,which had been pre-cooled to−50°C at1mbar for1h.Af-terwards,500mg of lyophilized zucchini were extracted using16mL of 80:20(v/v)methanol/H2O and sonicated at room temperature for 30min.The mixture was centrifuged at4000rpm for15min and the supernatant was collected.After centrifugation,the solvent was evapo-rated to dryness using a rotary evaporator under vacuum,and the residue was dissolved in500μL of80:20(v/v)methanol/H2O.Finally, the supernatant wasfiltered with a polytetrafluoroethylene(PTFE) syringefilter(0.2μm pore size)and stored at−20°C until analyzed (Abu-Reidah et al.,2012).2.3.ChemicalsAcetic acid was from Fluka,Sigma-Aldrich(Steinheim,Germany) and the organic solvents methanol and acetonitrile were from Lab-Scan(Gliwice,Sowinskiego,Poland).Distilled water with a resis-tance of18.2MΩwas deionized using a Milli-Q system(Millipore, Bedford,MA,USA).A G560E Vortex-Genie2(Scientific Industries, Bohemia,NY,USA)was used as a vortex mixer.2.4.HPLC conditionsLC analyses were made with an Agilent1200series rapid-resolution LC system(Agilent Technologies,Palo Alto,CA,USA) equipped with a binary pump,an autosampler and a diode array detector(DAD).Separation was carried out with a Zorbax Eclipse Plus C18analytical column(150mm×4.6mm,1.8μm particle size). Gradient elution was conducted with two mobile phases consisting of acidified water(0.5%acetic acid v/v)(phase A)and acetonitrile (phase B)according to gradient methods described previously (Abu-Reidah et al.,2012)with some modifications:0–20min,linear gradient from0%B to20%B;20–30min from20%B to30%B;and 30–35min from30%B to50%B,35–45min from50%B to100%B. Finally,the initial conditions were maintained for10min.Theflow rate used was set at0.80mL/min.The effluent from the HPLC column was split using a T-type phase separator before being introduced into the mass spectrometer(split ratio=1:4).Thus,in this study theflow which arrived into the ESI-Q-TOF-MS detector was0.2mL/min.The column temperature was maintained at25°C and the injection volume was10μL.UV data were collected using DAD set at240and 280nm.2.5.ESI-Q-TOF-MS conditionsQ-TOF-MS was conducted using a micrOTOF-Q™(Bruker Daltonics GmbH,Bremen,Germany),an orthogonal-accelerated Q-TOF mass spec-trometer equipped with an electrospray ionization source(ESI).Analysis parameters were set using a negative ion mode with spectra acquired over a mass range of m/z50–1100.The optimum values of ESI-MS pa-rameters were:capillary voltage,+4000V;dry gas temperature,190°C;dry gasflow,9.0L/min;nebulizer pressure,2.0bar;and spectra rate1Hz.Moreover,automatic MS/MS experiments were performed adjusting the collision energy values as follows:m/z100,20eV;m/z 500,30eV;m/z1000,35eV,and using nitrogen as a collision gas.The accurate mass data of the molecular ions were processed through the software Data Analysis4.0(Bruker Daltonics,Bremen, Germany),which provided a list of possible elemental formula by using the Smart Formula Editor.During the development of the HPLC method,external instrument calibration was performed using a Cole Palmer syringe pump(Vernon Hills,Illinois,USA)directly connected to the interface,passing a solution of sodium acetate clus-ter containing5mM sodium hydroxide in the sheath liquid of0.2% acetic acid in water/isopropanol1:1(v/v).With this method,an exact calibration curve was drawn based on numerous cluster masses,each differing by82Da(NaC2H3O2).Due to the compensa-tion of temperature drift in the micrOTOF-Q,this external calibration provided accurate mass values(better than5ppm)for a complete run without the need for a dual sprayer setup for internal mass calibration.3.Results and discussion3.1.Chromatographic profile and compound characterizationMetabolite profiling compounds were successfully separated and characterized using RP-HPLC-ESI-DAD-Q-TOF-MS.The Q-TOF-MS base peak chromatogram(BPC)of“Verde”,“Redondo”,and“organic Verde”zucchini extracts are depicted in Fig.1(a,b,c,respectively),where the compounds are identified by number from1to62.In the variety Verde,48compounds were identified and most were also identified in both Redondo and organic Verde pounds which were not identified in Verde zucchini were subsequently numbered in the two other kinds of zucchini analyzed.The metabolite assignments were identified by interpreting the mass spectra determined via their MS and MS/MS(Q-TOF),taking into account all the data provided by the literature.Table1provides a summary of all the compounds stud-ied,including retention times,experimental and calculated m/z,molec-ular formula,error(deviation between the measured mass and theoretical mass),sigma value(an exact numerical comparison be-tween the theoretical and measured isotope patterns),MS/MS frag-ments,as well as putative compounds.3.1.1.Phenolic acids and derivativesPeak14(RT 6.28min)with a precursor ion at m/z163.0401 presented a daughter ion at m/z119.0501[M–H–CO2]−.This ion was assigned tentatively to p-coumaric acid,which has previously been reported in pumpkin(Dragovic-Uzelac et al.,2005;Peričin,Krimer, Trivić,&Radulović,2009).Ferulic acid is the other hydroxycinnamic acid detected in the three zucchini studies.It was eluted at RT 21.80min(peak38)with m/z193.0506and presented two product ions at m/z178.0268and m/z134.0372,which corresponded to the loss of CH3and was followed by CO2,respectively.This compound has been previously reported in hull-less pumpkin seed,skin,and oil-cake meal,as well as in the dehulled kernel and the hull itself(Peričin et al.,2009).Six other hydroxycinnamic acids were identified but only in “organic Verde”zucchini.The ion found at m/z311.0417was assigned to caftaric acid or O-caffeoyltartaric acid(peak50)(RT11.90min), which presented a product ion at m/z149.0098corresponding to the loss of caffeoyl acid moiety(162Da).Two other fragment ions at m/z179.0353and135.0456were caffeic acid derived.These results are consistent with thefindings of a previous study in lettuce varieties and escarole(Llorach,Martínez-Sánchez,Tomás-Barberán,Gil,& Ferreres,2008).MS spectrum showed a molecular mass of m/z 353.0891for peak51(RT15.52min),which was assigned to chlorogenic acid(3-O-caffeoylquinic acid)with the fragmentation pattern at m/z191.0575,corresponding to the loss of the caffeoyl acid78I.Iswaldi et al./Food Research International50(2013)77–84moiety (162Da)from the main fragment (Abu-Reidah et al.,2012;Dragovic-Uzelac et al.,2005).Caffeic acid (peak 52)was identi fied at RT 17.38min (m/z 179.0363)with a product ion at m/z 135.0455[M –H –CO 2]−.This compound has been previously mentioned in pumpkin (Dragovic-Uzelac et al.,2005;Peri čin et al.,2009).The pres-ence of 2-O-caffeoylmalic acid (peak 55)was identi fied at RT 19.35min with m/z 295.0476and two product ions at m/z 179.0355and 133.0173,which corresponded to the [caffeic acid –H]−and [malic acid –H]−fragment ions,respectively,indicating the presence of caffeic and malic acids in its structure.The presence of this com-pound has previously been mentioned in lettuce (Lactuca sativa L.)(Ribas-Agustí,Gratacós-Cubarsí,Sárraga,García-Regueiro,&Castellari,2011).Peak 58(RT 22.23min)showed a parent ion at m/z 473.0743.It presented predominant product ions at m/z 311.0407[M –H –caffeoyl moiety]−,m/z 149.0099[M –H –162–162]−(loss of a second caffeic acid moiety),m/z 293.0315and m/z 179.0360.This compound was ten-tatively proposed as chicoric acid (dicaffeoyltartaric acid)based on the fragment patterns and identical UV spectra at 244,305(sh:shoulder)and 328(Schütz,Kammerer,Carle,&Schieber,2005).Peak 60(RT 25.73min)with precursor ion at m/z 341.0720was assigned to dicaffeic acid.It gave product ions at m/z 281.2489,179.0387,and 161.0270,corresponding to the loss of acetic acid (CH 3COOH)group,and deprotonated and dehydrated caffeic acid,respectively (Dugo et al.,2009).Another phenolic acid with its glycoside conjugate was iden-ti fied at RT 17.43min (peak 31)with m/z 385.1157and a product ion at m/z 223.0564,corresponding to the loss of hexose moiety from the pre-cursor ion.This was tentatively assigned to sinapic acid hexoside (Abu-Reidah et al.,2012).Nine hydroxybenzoic acids and derivatives were identi fied in both Verde and Redondo zucchini,and five were identi fied in all the zucchini analyzed.A signal at m/z 153.0201(peak 21)was detected at 11.30min.This compound,with a molecular formula of C 7H 5O 4and fragments at m/z 109.0302,corresponding to the loss of CO 2,was tentatively identi-fied as protocatechuic acid (Peri čin et al.,2009).The ion found at RT 15.12min (peak 28)was suggested to be 3,4,5tri-O -galloylquinic acid with the precursor and product ions at m/z 647.2772and 323.1370[M –2H]2−,respectively.Galloylquinic acid derivatives have been iden-ti fied in other plants (Moore et al.,2005).At RT 15.00min,a signal with m/z 137.0239(peak 27)was tentatively identi fied as p -hydroxybenzoic acid,which has previously been reported in pumpkin seed (Peri čin et al.,2009).Peaks 29(RT 16.15min)and 33(RT 18.11min)showed the same parent ions at m/z 121.0286/121.0288and gave the samemolecularFig.1.Base peak chromatograms of Verde (a),Redondo (b)and organic Verde (c)zucchinis.79I.Iswaldi et al./Food Research International 50(2013)77–84Table1Phenolic and other polar compounds characterized in three zucchini samples by HPLC-ESI-Q-TOF-MS.Peak RT(min)SelectedionMolecularformulam/z Error(ppm)mSigmavalueFragments Proposed compound Verde Redondo OrganicVerde Experimental CalculatedPhenolic acids and derivativesHydroxycinnamic acids and derivatives14 6.28[M–H]−C9H7O3163.0401a163.04010.07.1119.0501p-Coumaric acid+++3821.80[M–H]−C10H9O4193.0506a193.05060.18.4178.0268;134.0372Ferulic acid+++ 5011.90[M–H]−C13H11O9311.0417b311.0409 2.7 1.4135.0456;149.0098;179.0353Caftaric acid d––+5115.52[M–H]−C16H17O9353.0891b353.0878 3.7 5.1191.0575Chlorogenic acid d––+ 5217.38[M–H]−C9H7O4179.0363b179.03507.5 1.1135.0455Caffeic acid––+ 5519.35[M–H]−C13H11O8295.0476b295.0459 5.5 4.0179,0355;133.01732-O-Caffeoylmalic acid d––+ 5822.23[M–H]−C22H17O12473.0743b473.0725 3.723.6311.0407;293.0315;179.0360;149.0099Chicoric acid d––+6025.73[M–H]−C18H13O7341.0720b341.066715.718.7281.2489;179.0387;161.0270Dicaffeic acid d––+3117.43[M–H]−C17H21O10385.1157a385.1140 4.433.6223.0564Sinapic acid hexoside d++–Hydroxybenzoic acids and derivatives2111.30[M–H]−C7H5O4153.0201a153.0193 5.021.4109.0302Protocatechuic acid+++ 2815.12[M–H]−C26H47O18647.2772a647.27680.6 6.8323.13703,4,5Tri-O-galloylquinic acid++–2715.00[M–H]–C7H5O3137.0239a137.0244 4.143.9–p-Hydroxybenzoic acid+++ 2916.15[M–H]−C7H5O2121.0286a121.02957.18.3–p-Hydroxybenzaldehyde+++ 3318.11[M–H]−C7H5O2121.0288a121.0295 6.1 3.0–Benzoic acid d+++ 3017.01[M–H]−C8H7O4167.0338a167.0350 6.97.3–Vanillic acid+++ 2011.00[M–H]−C14H17O9329.0874a329.0878 1.220.0167.0353Vanillic acid glycoside(isomer1)d++-2312.81[M–H]−C14H17O9329.0882a329.0878 1.210.9167.0335Vanillic acid glycoside(isomer2)d++–2211.47[M–H]−C13H15O8299.0768a299.0772 1.413.0137.0272Hydroxybenzoic acid hexose d++–FlavonoidsFlavones and glycosides6226.50[M–H]−C21H19O11447.0947b447.0933 3.122.2285.0418Luteolin O-glucoside d––+ Flavonols and glycosides3419.75[M–H]−C33H39O20755.2065a755.2040 3.3 2.4301.0330Quercetin3-O-rhamnosyl-rhamnosyl-glucoside d++–3922.45[M–H]−C27H29O16609.1493a609.1461 5.213.1301.0353Quercetin3-rutinoside d+++ 4223.35[M–H]−C21H19O12463.0886a463.08820.97.1301.0342Quercetin O-glucoside d+++ 3721.53[M–H]−C34H42O20769.2237a769.2197 5.211.8315.0501;623.1567Isorhamnetin3-rutinoside-7-rhamnoside d++–4123.03[M–H]−C28H31O16623.1630a623.1618 2.07.7315.0509Isorhamnetin O-rutinoside(isomer1)d++–4424.76[M–H]−C28H31O16623.1665a623.1618 6.111.8315.0497Isorhamnetin O-rutinoside(isomer2)d++–3621.34[M–H]−C33H39O19739.2122a739.2091 4.211.2285.0387Robinin d++–4022.83[M–H]−C27H29O15593.1517a593.15120.9 5.8285.0381Kaempferol rutinoside(isomer1)d++–4324.27[M–H]−C27H29O15593.1534a593.1512 3.77.3285.0415Kaempferol rutinoside(isomer2)d++–4525.56[M–H]−C21H19O11447.0942a447.0933 2.016.9285.0404Kaempferol O-glycoside(isomer1)d+−+ 4626.00[M–H]−C21H19O11447.0923a447.0933 2.2 5.8285.0404Kaempferol O-glycoside(isomer2)d+–+ 4727.10[M–H]−C23H21O12489.1017a489.1038 4.515.8285.0370Kaempferol O-(6″-O-acetyl-glucoside d+++ 4828.09[M–H]−C21H19O10431.0992a431.0984 1.828.0285.0397Kaempferol O-α-L-rhamnoside d++–5923.57[M–H]−C26H27O15579.1361b579.13550.912.9447.0938;285.0411Kaempferol O-sambubioside d––+ 6126.18[M–H]−C21H17O12461.0741b461.0725 3.413.7285.0412Kaempferol O-glucuronide d––+ Organic acids and derivatives3521.21[M–H]−C6H5O6173.0100a173.0092 4.87.3–t-Aconitic acid d++–2 2.16[M–H]−C6H11O7195.0513a195.0510 1.4 6.2–Gluconic/galactonic acid d+++4 2.51[M–H]−C4H3O4115.0038a115.0037 1.4 2.6–Fumaric acid d+++5 2.88[M–H]−C4H5O5133.0149a133.0149 5.0 5.2115.0034Malic acid d+++7 3.90[M–H]−C6H7O7191.0198a191.01970.411.9173.0111,111.0102Isocitric acid d+++8 4.25[M–H]−C6H7O7191.0197a191.01970.3 3.7173.0122;111.0081Citric acid d+++10 5.48[M–H]−C4H5O4117.0191a117.0193 2.3 2.5–Succinic acid d+++11 5.61[M–H]−C5H7O5147.0305a147.0299 4.29.6–Citramalic acid d+++2514.53[M–H]−C7H11O5175.0616a175.0616 2.5 5.7115.0400Isopropylmalic acid d+++ Aminoacids and derivatives1 2.11[M–H]−C4H6NO4132.0297a132.0302 3.7 4.2–Aspartic acid++–3 2.20[M–H]−C3H6NO3104.0339a104.035313.89.2–Serine++–6 3.82[M–H]−C5H10NO2116.0713a116.0717 3.69.8–Valine+++12 6.13[M–H]−C9H10NO3180.0664a180.0666 1.28.5163.0398Tyrosine+++189.36[M–H]−C9H10NO2164.0721a164.0717 2.7 1.2147.0447Phenylalanine+++ 2412.92[M–H]−C11H11N2O2203.0832a203.0826 2.87.7–Tryptophan+++9 4.42[M–H]−C5H6NO3128.0350a128.0353 2.731.9–Pyroglutamic acid d+++157.05[M–H]−C12H22NO7292.1416a292.1402 4.811.6202.1095;130.0879Fructosyl leucine/isoleusin d+++ 199.61[M–H]−C15H20NO7326.1256a326.1245 3.3 6.9236.0940;164.0725Fructosyl phenylalanine d+++ Nucleosides13 6.25[M–H]−C9H11N2O6243.0630a243.0623 2.8 2.4200.0572;111.0207Uridine d+++167.85[M–H]−C10H12N5O4266.0904a266.0895 3.37.8134.0470Adenosine d+++ 178.07[M–H]−C10H12N5O5282.0854a282.0844 3.4 3.6150.0420Guanosine d+++ Other metabolites3217.66[M–H]−C10H12NO4210.0777a210.0772 2.5 4.3124.03933,4-Dihydroxyphenyl-1-methylester-carbamic acid d+++ 5317.56[M–H]−C6H11O3131.0719b131.0714 4.1 3.8–Ethyl3-hydrobutanoate d––+ 80I.Iswaldi et al./Food Research International50(2013)77–84formula according to Smart Formula Editor.Based on the literature and using reversed-phase liquid chromatography,p-hydroxybenzaldehyde was eluted just before benzoic acid.Therefore,these compounds were tentatively identified as p-hydroxybenzaldehyde(Peričin et al.,2009) and benzoic acid(Gómez-Romero,Segura-Carretero,&Fernández-Gutiérrez,2010),respectively.Peak30(RT17.01min),with precursor ion at m/z167.0350,corresponded to vanillic acid(Dragovic-Uzelac et al.,2005;Peričin et al.,2009).Meanwhile,vanillic acid glycoside iso-mers(peaks20and23)were identified at RT11.00and12.81min. Found at m/z329.0874and329.0882,they presented the same two prod-uct ions at m/z167.0353and167.0335,respectively,corresponding to the loss of the sugar moiety.This compound has been identified in cucumber (Abu-Reidah et al.,2012).The presence of hydroxybenzoic acid hexose, identified at RT11.47min(peak22)with ion found at m/z299.0768, presented one main product ion at m/z137.0272,corresponding to the loss of hexose moiety(Gómez-Romero et al.,2010).3.1.2.FlavonoidsPeak62(RT26.50min and m/z477.0947)was tentatively identi-fied as luteolin O-glucoside,showing a fragment at285.0414,which is the characteristic fragment mass of luteolin.This compound,the onlyflavone identified in“organic Verde”zucchini,has been isolated from other Cucurbitaceae plants(Mallavarapu&Row,1979).The ion found at m/z755.2065was assigned to quercetin3-O-rhamnosyl-rhamnosyl-glucoside(peak34)(RT19.75min),which presented a product ion at m/z301.0330corresponding to quercetin (Fig.2a).Two other quercetin–sugar conjugates identified in zucchini (peaks39and42)proved identical to those reported previously in Cucurbitaceae(Abu-Reidah et al.,2012).Peaks39(RT22.45min)and 42(RT23.35min),with ions found at m/z609.1493and m/z 463.0886,both presented the same fragment at m/z301.0353and 301.0342,respectively,corresponding to the loss of the sugar moieties, rutinoside and glucose,respectively.Therefore,these peaks were suggested as quercetin rutinoside(Fig.2b)and glucoside,respectively.Peak37gave a molecular mass at m/z769.2226and a product ion at m/z315.0501[M–H–146–146–162]−,which corresponded to a complete loss of the sugar moieties.The fragment ion at m/z623[M–H–146]−was also detected but with very low relative intensity(below5%),indicating a loss of rhamnose from C-7.Since this fragmentation behavior is consis-tent with a previous report(Rösch,Krumbein,Mügge,&Kroh,2004) and was corroborated with the same UV absorbance spectra at255,266 (sh)and354nm,on the basis of such features,it was assigned to isorhamnetin3-rutinoside-7-rhamnoside(Fig.2c).Peaks41(RT 23.03min)and44(RT24.76)gave the same molecular formula C27H29O15and m/z623.1630and623.1665,respectively.Both of them presented the same product ion at m/z315.0509and315.0497,which corresponds to isorhamnetin after the loss of the rutinose sugar moiety. The UV absorbances of both compounds are the same at264,303(sh) and350nm.Therefore,we could not distinguish between them and ten-tatively proposed as isorhamnetin O-rutinoside isomers.Nine kaempferol glycosides(peaks36,40,43,45,46,47,48,59and 61)were tentatively identified for thefirst time in zucchini.They were corroborated by their UV spectra at264,300(sh)and350nm,which are specific forflavonol glycosides.All the kaempferol–sugar conjugates showed product ions at m/z285corresponding to kaempferol.Peak36, presenting a precursor ion at m/z739.2122with molecular formula C33H39O19,was tentatively identified as robinin(kaempferol-3-O-robinoside-7-O-rhamnoside).It has been previously reported in fruit juices(Abad-García,Berrueta,Garmón-Lobato,Gallo,&Vicente,2009) (Fig.2d).The identities of the peaks39,40,59,42,43(Fig.2e),45,46 (Fig.2f)and47of this study agree with thefindings of previous article (Abu-Reidah et al.,2012)in cucumber whole-fruit extract.Peak48has been previously reported in endive(Cichorium endivia),as well as peak 61which was the predominant form in that extract(DuPont,Mondin, Williamson,&Price,2000).Although allflavonols and glycosides above are known,as far as we are aware this is thefirst time that they are reported in whole zucchini fruit.3.1.3.Other polar compoundsBesides polyphenolic compounds,other polar compounds could also be characterized in the whole zucchini anic acids, amino acids and their derivatives eluted as a large peak with signals overlapping.It is due to the very polar compounds that could not be properly separated by C18reversed-phase chromatography(Gómez-Romero et al.,2010).Eight organic acids and derivatives were tentatively identified in all zucchini with the exception of peak35(RT21.21min).This peak with an ion found at m/z173.0100was tentatively identified as t-aconitic acid.Peak2(RT2.16min),which gave a molecular mass of m/z195.0513,was tentatively assigned to gluconic/galactonic acid according to the molecular formula provided for its accurate mass C6H11O7.Fumaric acid with molecular mass at m/z115.0038 was identified at RT2.51(peak4).Peak5(RT2.88min)with m/z 133.0149was suggested as malic acid and presented fragment ion at m/z115.0034corresponding to the loss of a molecule of water from the precursor ion(Dunn,Overy,&Quick,2005).Peaks7and 8presented isocitric acid and citric acid,with an ion found at m/z 191.0198and191.0187,respectively.Both isomers showed the same fragmentation pattern at m/z173.0111and173.0122,which correspond to[M–H–H2O]−,while111.0102and111.0081were related to the loss of[M–H–CO2–2H2O]−(Dunn et al.,2005; Gómez-Romero et al.,2010).Succinic and citramalic acids were identified at RT5.48and5.61min(peaks10and11)and with pre-cursor ions at m/z117.0191and m/z147.0305,respectively(Dunn et al.,2005;Gómez-Romero et al.,2010).At RT14.53min(peak 25),the ion found at m/z175.0616presented a product ion at m/z 115.0400[M–H–2CHOH]−,which was tentatively proposed as isopropylmalic acid(Gómez-Romero et al.,2010).With regard to amino acids and derivatives,these were detected at RT from2.11to12.92min.Six amino acids namely aspartic acidTable1(continued)Peak RT(min)SelectedionMolecularformulam/z Error(ppm)mSigmavalueFragments Proposed compound Verde Redondo OrganicVerde Experimental CalculatedUnknown compounds2614.63[M–H]−C20H29O12461.1667a461.16640.6 3.8–++–4917.28[M–H]−C21H31O12475.1829c475.1821 1.77.4––+–5418.36[M–H]−C17H21O7337.1313c337.1293 6.0 3.9215.1081––+ 5620.76[M–H]−C21H31O9427.2025c427.197412.016.5202.1095––+ 5721.14[M–H]−C29H13O7473.0724c473.066712.023.9202.1096––+a m/z experimental and calculated,sigma value and error were from Verde sample.b m/z experimental and calculated,sigma value and error were from organic Verde sample.c m/z experimental and calculated,sigma value and error were from Redondo sample.d Compounds which have been reported in zucchini fruit for thefirst time.81I.Iswaldi et al./Food Research International50(2013)77–84(peak 1),serine (peak 3),valine (peak 6),tyrosine (peak 12),phenylal-anine (peak 18),and tryptophan (peak 24)which have been previously found in zucchini by the National Food Institute were also identi fied inthe extracts analyzed.Only peak 9(RT 4.42min)was proposed as pyroglutamic acid with a molecular mass at m/z 128.0350,the only one that has not been reported in this database.257355300(sh)301.0330755.2067020406080100Intens.[%]200300400500600700800m/z2040Intens.[mAU]240260280300320340360380[nm](a)[M-H]_[M-H-146-162]_301.0353609.148620406080100Intens.[%]200300400500600700800m/z[M-H]_[M-H-rutinoside]_255355306(sh)(b)100200Intens.[mAU]225250275300325350375400[nm]200300400500600700800m/z315.0501769.2234020406080100Intens.[%][M-H-146-146-162]_2040Intens.[mAU]220240260280300320340360380[nm]255266 (sh)354(c)[M-H]_285.0415739.2120020406080100Intens.[%]200300400500600700800m/z[M-H]_[M-H-146-146-162]_2040Intens.[mAU]220240260280300320340360[nm]266347313(d)Fig.2.MS/MS and UV spectra of the most representative flavonoids:quercetin 3-O -rhamnosyl-rhamnosyl-glucoside,peak 34(a),quercetin 3-rutinoside,peak 39(b),isorhamnetin 3-rutinoside-7-rhamnoside ,peak 37(c),robinin,peak 36(d),kaempferol rutinoside (isomer 2),peak 43(e),kaempferol O -glycoside (isomer 2),peak 46(f).Note:sh=shoulder.82I.Iswaldi et al./Food Research International 50(2013)77–84。

专利名称：PRODUCTION OF COATED GLASS发明人：SANDERSON, Kevin David,LINGL,Gerhard,LEITL, Hans-Eckhard,NELSON,Douglas M,SCHARNAGL, Franz Michael Josef 申请号：GB2009050479申请日：20090507公开号：WO09/136201P1公开日：20091112专利内容由知识产权出版社提供摘要：A process for coating a ribbon of float glass is disclosed. It comprises the steps of forming a glass ribbon, depositing a first transparent conductive coating upon a major surface of the ribbon which does not extend to the edges of the ribbon whilst the ribbon is at an elevated temperature, cooling said coated ribbon under controlled conditions in an annealing lehr and cutting off the edges of the ribbon so as to produce a ribbon having a uniform coating extending across the full width of the cut ribbon which is characterised in that a second conductive coating is deposited upon the uncoated edges of the ribbon whilst that edge is at a temperature which is above the ambient temperature. The invention finds particular application in the production of coated glass products where t;ήe thickness of the glass ribbon is at least 8 mm and most particularly where the thickness of the glass is at least 10 mm.申请人：SANDERSON, Kevin David,LINGL, Gerhard,LEITL, Hans-Eckhard,NELSON, Douglas M,SCHARNAGL, Franz Michael Josef地址：Prescot Road St Helens Merseyside WA10 3TT GB,5 Dewberry Fields, Upholland Skelmersdale, Lancashire WN8 0BQ GB,Fichtenstraße 11 92729 WeiherhammerDE,Rehbühlstr. 20 92637 Weiden DE,8717 Seaman Rd Curtice, Ohio 43412 US,Parksteiner Strasse 16 92637 Weiden DE国籍：GB,GB,DE,DE,US,DE代理机构：PETTET, Nicholas Edward更多信息请下载全文后查看。

Issue 1 August 2009Men’s & Women’sApparelIssue 1 August 2009Issue 1 August 2009CONTENTSIntroductionMinimum Performance RequirementsTable Product1. Woven Tailored and Soft (Trousers, Skirts, Dresses, Blouses & Shirts)2. Denim (Cotton & Cotton / Elastane)3. Outerwear (Coats & Jackets)Jerseywear4. Knitted5. Knitwear6. Swimwear & Beachwear (Knitted & Woven)7. Lining & Pocketing (Knitted & Woven)8. Lingerie & Underwear (Knitted & Woven)9. Socks & Hosiery10. Nightwear & Robes11. Leather & Suede12. SpecialClaimsPerformance13. Coated Fabrics (Pu & Pvc)14. Zips, Fastenings & TrimsIHTM In House Test MethodsPD 1c Calculation of SpiralityPD 5a Fibre Shedding of ‘Long Pile’ FibresNapStability8PDDurabilityWash10CDCD 10a Durability Wash – Swimwear & LingeriePD 20 Chenille Pile LossRatePDWicking33PD 37 Appearance Assessment for Washable & Dry Cleanable GarmentsPD 38 Bra Wire Casing Punch Through TestAppendicesAppendix A Wash Program and Drying MethodsAppendix B Lingerie Fibre Composition GuidelinesAppendix C Glossary for Testing and Fabric TermsAppendix D Restricted SubstancesIntroductionThe 2009 testing manual update for men’s and women’s apparel replaces the use of tables 1-19 on the B2B website.The tables have been simplified, and each test has a clear indication of when it is needed, and on what fabric/ end use.Wash instructions have been removed from the manual, and therefore testing will be done to a standard method specific to fabric type unless otherwise specified.PD37 tests will rely on the supplier providing the correct care label details in order for the test to be completed.Recommended care instructions for Womenswear and Lingerie products are available from the appropriate Debenhams Technologist, as well as B2B website.Any further changes to the manual will be updated via the B2B website, please continue to check on a monthly basis.Suppliers must pay all charges relating to testing. Testing tables represent the minimum requirement, Debenhams reserve the right to request additional tests at suppliers cost.•All products must be tested in full to meet the Debenhams Minimum Performance Standards.•All reports to be tested to Debenhams standards, at Independent laboratories unless specific lab approval has been given in writing. Mill reports/ other retailers reports will onlybe accepted as indication of performance at development stage.•It is the supplier's responsibility to complete the testing and ensure that this covers the necessary requirements.•The approval of test reports is a crucial part of the Green Seal process. Only when all testing is fully approved should product be considered approved for delivery.•All testing should be held for a minimum of 2 years, and should be readily available on request. •Where testing is inconclusive, or where new fabrics are being used, wearer trials may be requested to ascertain commerciality.•All testing relating to product safety or current legislation must be carried out at a nationally accredited independent test laboratory. Copies of the approved reports must besent to the appropriate Debenhams Technologist.•Where applicable, products must conform to the quality and marketing requirements of any trademark/registered name used e.g. Dupont Lycra, Teflon, Woolmark etc. as well asmeeting Debenhams minimum standards.Procedure for Empowered suppliers:Empowered suppliers are responsible for delivering quality products that meet Debenhams minimum performance standards. All testing should be completed by green seal stage.Self assessment and approval of tests reports is part of the supplier empowerment, where results achieve minimum requirements.All failed testing should be sent to the appropriate Debenhams Technologist for review. Full set of reports must be submitted with a completed test cover sheet and specimens where applicable. One week should be allowed for Debenhams approval of test submissions.The audit process will continue to be used to assess supplier performance.All empowered suppliers must be able to provide testing for any style within 48 hours. Where Debenhams require a copy of a test report this should be in an electronic format and sent via email to the relevant Debenhams Technologist and Buying Team.If procedures are not followed supplier empowerment for test report approval will be removed.Any changes to empowerment status will be notified in writing by the Debenhams Technologist.Suppliers are responsible for selecting and approving the Wash Care instructions, using the recommended care instruction tables for guidelines where applicable.Procedure for new and non empowered suppliers:All new suppliers and any existing suppliers who have had their self empowered status removed must submit all test reports for approval.All submissions must be sent to the appropriate Debenhams Technologist and buying team, with test cover sheet.New suppliers should expect to be empowered within 6-12 months, providing procedures are adopted correctly. Once granted empowerment, suppliers will be audited to monitorperformance.If supplier has empowerment status retracted, performance will be reviewed and empowerment will be reinstated once issues are resolved. Time scale to be reviewed with the Debenhams Technologist. Any changes to empowerment status will be notified in writing by the Debenhams Technologist.Test submissions:All submissions require a completed test cover sheet.If base report is sent:•Submit with test cover sheet.•Where applicable base test for components should be submitted together, to enable Technologist to review product as a whole.•Base test should be within 12 months old, and completed on production quality fabric.If bulk report is sent:•Submit with base test and test cover sheetIf results on bulk do not meet requirements, automatically send physical test specimens for review. Test all base and bulk on minimum number of reports to simplify submissions, reduce cost, and time. Any performance issues must be rectified by the supplier unless otherwise agreed in writingwith a Debenhams Technologist. Do not ask for approval of a fabric you do not believe to be commercial.Repeat orders should follow the below procedure, unless otherwise specified:•They should be fully tested every 12 months•Any amendments to the product, fabric, components, ingredients or processes within 12 months will require full testing.•At Debenhams discretion, repeat orders that occur more frequently may require additional testing for each delivery.Table 1Woven - Tailored and Soft(Trousers, Skirts, Dresses, Blouses & Shirts)Test Method Requirement Base Bulk CommentsPhysical TestsFibre Composition 73/44/EEC & 96/73/EC AS AMENDED BY 2006/2/EC ± 3%(Single fibre no tolerance)√ Required for all fabrics with more than 1 fibre. Single fibrecompositions to be tested unless accompanied with documentation from supplierPilling BS EN ISO 12945-1:2000 Grade 4 @ 18,000 Revs √ Only required for fabrics containing Wool, Acrylic, Spun Nylonor Polyester.Seam Slippage BS EN ISO 13936-1:2004 6mm @ 8Kg (soft) 6mm @ 12kg (Tailored) √ For fabrics containing contrast warp/weft use 3mm seamopening Seam Slippage (Stretch Fabrics) BS EN ISO 13936-2:2004 6mm @ 8Kg (soft)6mm @ 12kg (Tailored) √ Only required for fabrics containing elastomeric yarns. For fabrics containing contrast warp/weft use 3mm seam opening Seam Strength BS EN ISO 13935-2:1999 12 kg (soft) 15 kg (Tailored) √ Tensile Strength BS EN ISO 13534-2:1999 12 kg (soft) 15 kg (Tailored) √Tear Strength BS EN ISO 13937-1:2000 1000g Trousers750g Other Garments √ Not required on 100% Nylon or Polyester (except micro fibres).Residual Extension BS EN ISO 14704-1:2005 5% Max√ Only required for trouser fabrics containing 3% or more of elastomeric yarns.Test in direction of elastane only Abrasion BS EN ISO 12947-2:1999 15,000 rubsShade Change @ 5,000 Grade 4√ Only required for trousers.Not required for 100% Nylon or Polyester. SnaggingBS 8479:2008 Grade 3-4 @ 2,000 Revs √ Only required for fabrics containing Filament Yarns. Accelorotor Pile Loss AATCC 93: 2005 16% Max Weight LossVisual Assessment Negligible √ Only required for cut pile fabrics E.g. Velvet & Corduroy.Fibre Shedding PD 5a√ To be carried out for Fur and Long Pile Fabrics Nap Stability PD 8Moderate @ 4000 rubs √ To be carried out on all nap fabricsSurface Flash BS 4569 (Modified)No Surface Flash√To be carried out on all fabrics/trims with pile length equal to or greater than 3mmTo be carried out conditioned and bone dryStability Tests Stability to Washing BS EN ISO 6330: 2001 (See Appendix A) ± 3% √ √ Required for Washable garments. Comment on any adverse changes in appearance.Stability to Dry CleanCOMMERCIAL PROCESS ± 3% √ √ Required for dry cleanable garments. Comment on any adverse changes in appearance.Stability to Steam BS 4323:1979 ± 3%√Required for dry cleanable garments, tailoring and occasional wearGarment Appearance AssessmentPD 37No Significant Change√Applies to Washable and Dry cleanable Womenswear garments.Garment to be tested on all styles containing multi colours, multiple fabric components, embellishments or trimsColour Fastness Tests CF to Washing BS EN ISO105-C06:1997 A2S Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5 √ Required for Washable Garments. CF to Water BS EN ISO 105-E01:1996 Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5 √ Not required on solid white or cream fabrics.CF to Perspiration BS EN ISO 105-E04 1996 Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5 √ Required for Fabrics containing > 20% Wool, Nylon or Silk CF to Dry CleanBS EN ISO105-D01:1995+ MFS Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5√ Required for Dry Cleanable GarmentsCF to Rubbing BS EN ISO 105-X12:2002 Dry Grade 4√ Not required on solid white or cream fabrics. CF to Light BS EN ISO 105-B02:1999 Shade Change Grade 4 √Durability WashCD 10No breakdown of the print. Shade Change Grade 4√Womens Wear - Only required on Prints, for foil and glitter use PD37 testMenswear – Required on Prints and embellishmentsNotesLined Garments refer to Table 7All Garments with special performance claims e.g. shower proof, breathable etc must meet the relevant requirements of Table 12Table 2 Denim (Cotton & Cotton / Elastane) Test Method RequirementBase Bulk Comments Physical TestsFibre Composition 73/44/EEC & 96/73/ECAS AMENDED BY2006/2/EC± 3%(Single fibre no tolerance)√Required for all fabrics with more than 1 fibre. Singlefibre compositions to be tested unless accompaniedwith documentation from supplierSeam Slippage BS EN ISO13936-1:20046mm @ 8Kg - Lightweight6mm @ 12Kg - Medium /Heavyweight√Seam Slippage (Stretch Fabrics) BS EN ISO13936-2:20046mm @ 8Kg - Lightweight6mm @ 12Kg - Medium /Heavyweight√Seam Strength BS EN ISO13935-2:199912Kg - Lightweight18Kg - Medium /Heavyweight√Tensile Strength BS EN ISO13534-2:199915 Kg – Lightweight20 Kg – Medium /Heavyweight√Tear Strength BS EN ISO13937-1:20001000g - Lightweight1200g - Medium /Heavyweight√Lightweight < 8ozMedium / Heavyweight > 8ozResidual Extension BS EN ISO14704-1:20055% Max √Only required for trouser fabrics containing 3% or moreof elastomeric yarns.Test in direction of elastane onlyAbrasion BS EN ISO12947-2:199915,000 rubsShade Change @ 5,000Grade 3-4√Stability TestsStability to Washing BS EN ISO 6330: 2001(See Appendix A)± 3% √√Required for Washable garments. Comment on anyadverse changes in appearance.GarmentAppearance Assessment PD 37 No Significant Change √Applies to Washable and Dry cleanable Womensweargarments.Garment to be tested on all styles containing multicolours, multiple fabric components, embellishments ortrimsColour Fastness TestsCF to Washing BS EN ISO105-C06:1997 A2SStaining Grade 4Shade Change Grade 4Cross staining grade 4-5√Required for Washable GarmentsCF to Water BS EN ISO105-E01:1996Staining Grade 4Shade Change Grade 4Cross staining grade 4-5√Not required on solid white or cream fabricsCF to Rubbing BS EN ISO105-X12:2002Dry Grade 4 √Not required on solid white or cream fabricsCF to Light BS EN ISO105-B02:1999Shade Change Grade 4 √Durability Wash CD 10 No breakdown of the print.Shade Change Grade 4√Womens Wear - Only required on Prints, for foil andglitter use PD37 testMenswear – Required on Prints and embellishmentsTable 3 Outerwear (Coats and Jackets)Test Method Requirement Base Bulk CommentsPhysical TestsFibre Composition 73/44/EEC & 96/73/EC AS AMENDED BY 2006/2/EC ± 3% (Single fibre no tolerance)√ Required for all fabrics with more than 1 fibre. Single fibrecompositions to be tested unless accompanied with documentation from supplierPilling BS EN ISO 12945-1:2000 Grade 4 @ 18,000 Revs √ Only required for fabrics containing Wool, Acrylic, Spun Nylon or Polyester. Seam Slippage BS EN ISO 13936-1:2004 6mm @ 12 Kg √ For fabrics containing contrast warp/weft use 3mm seam openingSeam Slippage (Stretch Fabrics) BS EN ISO 13936-2:2004 6mm @ 12 Kg √ Only required for fabrics containing elastomeric yarns. For fabrics containing contrast warp/weft use 3mm seam openingSeam Strength BS EN ISO13935-2:199915 Kg √ Tensile Strength BS EN ISO13534-2:199915 Kg √ Tear Strength BS EN ISO 13937-1:2000 1,000g √ Not required on 100% Nylon or Polyester (except microfibres).Abrasion BS EN ISO12947-2:199915,000 rubsShade Change @ 5,000 Grade 4 √ Not required on 100% Nylon or Polyester. SnaggingBS 8479:2008Grade 3-4 @ 2,000 Revs √ Only required for fabrics containing Filament Yarns Accelorotor Pile Loss AATCC 93: 2005 16% Max Weight LossVisual Assessment Negligible √ Only required for cut pile fabrics E.g. Velvet & Corduroy.Fibre Shedding PD 5a Grade 4√To be carried out on Fur and Long Pile Fabrics Nap Stability PD 8Moderate at 4000 rubs √To be carried out on all nap fabrics Fibre Percolation EN 12132-1:1998 Max 6 Fibres @ 6,000 Revs √Polyester, Feather & Down Filled Garments Cleanliness of Filling BS EN 1162:1997 Meets the requirement √ Required on all real feather and down fillingSurface Flash BS 4569 (Modified)No Surface Flash√To be carried out on all fabrics/trims with pile length equal to or greater than 3mmTo be carried out conditioned and bone dryStability Tests Stability to Washing BS EN ISO 6330: 2001 (See Appendix A) ± 3% √ √ Required for Washable garments. Comment on any adverse changes in appearance.Stability to Dry Clean COMMERCIAL PROCESS ± 3% √ √ Required for dry cleanable garments. Comment on any adverse changes in appearance. Stability to Steam BS 4323:1979 ± 3%√Required for dry cleanable garmentsGarment Appearance AssessmentPD 37No Significant Change√Applies to Washable and Dry cleanable Womenswear garments.Garment to be tested on all styles containing multi colours, multiple fabric components, embellishments or trimsColour Fastness Tests CF to WashingBS EN ISO105-C06:1997 A2S Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5√ Required for Washable GarmentsCF to Water BS EN ISO 105-E01:1996 Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5 √ Not required on solid white or cream fabricsCF to Perspiration BS EN ISO 105-E04 1996 Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5√ Only required for unlined garments containing > 20% Wool, Nylon or SilkCF to Dry Clean BS EN ISO105-D01:1995 + MFS Staining Grade 4Shade Change Grade 4 Cross stain grade 4-5 √ Required for Dry Cleanable Garments CF to Rubbing BS EN ISO 105-X12:2002 Dry Grade 4 Wet Grade 3-4 √ Not required on solid white or cream fabrics CF to Light BS EN ISO 105-B02:1999 Shade Change Grade 4√Durability WashCD 10No breakdown of the print. Shade Change Grade 4√Womens Wear - Only required on Prints, for foil and glitter use PD37 testMenswear – Required on Prints and embellishmentsNotesLined Garments refer to Table 7All Garments with special performance claims e.g. shower proof, breathable etc must meet the relevant requirements of Table 12Table 4 Knitted JerseywearTest Method RequirementBase Bulk Comments Physical TestsFibre Composition 73/44/EEC & 96/73/ECAS AMENDED BY2006/2/EC± 3%(Single fibre no tolerance)√Required on all fabrics with more than 1 fibre. Single fibrecompositions to be tested unless accompanied withdocumentation from supplierPilling BS EN ISO12945-1:2000Grade 4 @ 11,000 Revs √Only requested on fabrics containing Wool, Acrylic, SpunNylon or Polyester.Bursting Strength BS EN ISO13938: 1999250 kPa175 kPa Lace & Mesh√Only required on fabrics containing <10% elastaneResidual Extension BS EN ISO14704-1:200510% Max √Only required on fabrics containing 5% or moreelastomeric yarnsSnagging BS 8479:2008 Grade 3-4 @ 2,000 Rubs √Only required on fabrics containing Filament Yarns.Accelorotor Pile Loss AATCC 93: 200516% Max Weight LossVisual AssessmentNegligible√Only required on cut pile fabricsE.g. Velvet & Corduroy.Surface Flash BS 4569 (Modified) No Surface Flash √To be carried out on all fabrics/trims with pile length equal to or greater than 3mmTo be carried out conditioned and bone dryStability TestsStability to Washing BS EN ISO 6330: 2001(See Appendix A)100% Viscose / ViscoseElastane +3% / -7%Other Blends +3% / -5%√√Required for Washable garments. Comment on anyadverse changes in appearance.Spirality PD 1c 5% Max √Single Jersey Fabrics OnlyStability to Dry Clean COMMERCIALPROCESS± 3% √√Only required if garment is dry cleanable or contains silkor wool.Comment on any adverse changes in appearance.Stability to Steam BS 4323:1979 ± 3% √Required for dry cleanable garments GarmentAppearance Assessment PD 37 No Significant Change √Applies to Washable and Dry cleanable Womensweargarments.Garment to be tested on all styles containing multicolours, multiple fabric components, embellishments ortrimsColour Fastness TestsCF to Washing BS EN ISO105-C06:1997 A2SStaining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required for Washable GarmentsCF to Water BS EN ISO105-E01:1996Staining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Not required on solid white or cream fabricsCF to Perspiration BS EN ISO105-E04 1996Staining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required on Fabrics containing > 20% Wool, Nylon orSilkCF to Dry Clean BS EN ISO105-D01:1995 + MFSStaining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required for Dry Cleanable GarmentsCF to Rubbing BS EN ISO105-X12:2002Dry Grade 4 √Not required on solid white or cream fabricsCF to Light BS EN ISO105-B02:1999Shade Change Grade 4 √Durability Wash CD 10 No breakdown of the print.Shade Change Grade 4√Womens Wear - Only required on Prints, for foil andglitter use PD37 testMenswear – Required on Prints and embellishmentsNotesAll Garments with special performance claims e.g. shower proof, breathable etc must meet the relevant requirements of Table 12Table 5 KnitwearTest Method RequirementBase Bulk Comments Physical TestsFibre Composition 73/44/EEC & 96/73/ECAS AMENDED BY2006/2/EC± 3%(Single fibre no tolerance)√Required on all fabrics with more than 1 fibre. Singlefibre compositions to be tested unless accompaniedwith documentation from supplierPilling BS EN ISO12945-1:2000Grade 4 @ 11,000 RevsGrade 3 @ 11,000 Revs.(See Comments)√√Not required on 100% filament yarnsGrade 3 required for Lambs wool, Angora, Mohair andCashmere rich blendsBursting Strength BS EN ISO13938: 1999250 kPa √Only required on fabrics containing <10% elastaneResidual Extension BS EN ISO14704-1:200510% Max √Only required on fabrics containing 5% or moreelastomeric yarnsSnagging BS 8479:2008 Grade 3-4 @ 2,000 Rubs √Only required on fabrics containing Filament Yarns.Accelorotor Pile Loss AATCC 93: 200516% Max Weight LossVisual AssessmentNegligible√Only required on cut pile fabricsE.g. Velvet & Corduroy.Chenille Pile Loss PD 20 Weight loss 35 mg maxVisual assessment negligible√Only required on fabrics containing chenille yarnsSurface Flash BS 4569 (Modified) No Surface Flash √To be carried out on all fabrics/trims with pile length equal to or greater than 3mmTo be carried out on all chenille yarnsTo be carried out conditioned and bone dryStability TestsStability to Washing BS EN ISO 6330: 2001(See Appendix A)’+ 3% / - 5% √√Required for Washable garments. Comment on anyadverse changes in appearance.Spirality PD 1c 5% Max √Single Jersey Fabrics OnlyStability to Dry Clean COMMERCIALPROCESS± 3% √√Only required if garment is dry cleanable. Comment onany adverse changes in appearance.Stability to Steam BS 4323:1979 ± 3% √Required for dry cleanable garments, GarmentAppearance Assessment PD 37 No Significant Change √Applies to Washable and Dry cleanable Womensweargarments.Garment to be tested on all styles containing multicolours, multiple fabric components, embellishments ortrimsColour Fastness TestsCF to Washing BS EN ISO105-C06:1997 A2SStaining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required for Washable GarmentsCF to Water BS EN ISO105-E01:1996Staining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Not required on solid white or cream fabricsCF to Perspiration BS EN ISO105-E04 1996Staining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required on Fabrics containing > 20% Wool, Nylon orSilkCF to Dry Clean BS EN ISO105-D01:1995 + MFSStaining Grade 4Shade Change Grade 4Cross Staining Grade 4-5√Required for Dry Cleanable GarmentsCF to Rubbing BS EN ISO105-X12:2002Dry Grade 4 √Not required on solid white or cream fabricsCF to Light BS EN ISO105-B02:1999Shade Change Grade 4 √Durability Wash CD 10 No breakdown of the print.Shade Change Grade 4√Womens Wear - Only required on Prints, for foil andglitter use PD37 testMenswear – Required on Prints and embellishmentsTable 6 Swimwear & Beachwear (Knitted & Woven) Test Method RequirementBase Bulk Comments Physical TestsFibre Composition 73/44/EEC & 96/73/EC ASAMENDED BY 2006/2/EC± 3%(Single fibre no tolerance)√Womens swimwear – Required on all fabrics including gussetliner – report gusset liner individuallyAll other swim + beachwear - Required on all fabrics withmore than 1 fibre. Single fibre compositions to be testedunless accompanied with documentation from supplierSeam Slippage (Woven only) BS EN ISO13936-1:20046mm @ 8 Kg √For fabrics containing contrast warp/weft use 3mm seamopeningSeam Slippage (Stretch Fabrics) (Woven only) BS EN ISO13936-2:20046mm @ 8 Kg √Only required on fabrics containing elastomeric yarns. Forfabrics containing contrast warp/weft use 3mm seam openingSeam Strength (Woven only) BS EN ISO13935-2:199912 Kg √Tensile Strength (Woven only) BS EN ISO13534-2:199912 Kg √Tear Strength (Woven only) BS EN ISO13937-1:20001000g (Trousers)750g (All other Garments)√Not required on 100% Nylon or Polyester (except microfibres).Residual Extension BS EN ISO14704-1:20055% (Woven)10% (Knitted)√Required on knitted fabrics containing 5% or more elastaneTest in direction of elastane only on woven fabrics2KgLoad @ 40%Extension (Knitted)√For reference only4Kg Load @ 10%Extension (Woven)√For reference onlyExtension/Modulus BS4952:19923.6Kg Load @ 40% Extension (Control Fabrics) √Light Control = 150g -250gMedium Control = 250g - 500gFirm Control = 500g - 750gStability TestsStability to Washing BS EN ISO 6330: 2001 (SeeAppendix A)± 3% √√Required on all fabricsBra Wire CasingPunch ThroughPD 38 15kg √All wire casing to be tested annuallyStability to Washing (Bra Wire Casing) BS EN ISO 6330: 20014A 50o C Tumble dry-2.5% (No Extension) √All wire casing to be tested annuallyGarmentAppearance Assessment PD 37 No Significant Change √Womens swimwear- Required on all hand wash styles – to betested as a full garmentWomen’s Beachwear - Garment to be tested on all stylescontaining multi colours, multiple fabric components,embellishments or trims.Colour Fastness TestsCF to Washing BS EN ISO105-C06:1997 A2SStaining Grade 4Shade Change Grade 4Cross stain grade 4-5√Required on all fabricsCF to Water BS EN ISO105-E01:1996Staining Grade 4Shade Change Grade 4Cross stain grade 4-5√Not required on solid white or cream fabricsCF to Perspiration BS EN ISO105-E04 1996Staining Grade 4Shade Change Grade 4Cross stain grade 4-5√Only required on Fabrics containing > 20% Nylon or SilkCF to Sea Water BS EN ISO105-E02: 1996Staining Grade 4Shade Change Grade 4Cross stain grade 4-5√Required on all fabricsCF to Chlorinated Water BS EN ISO105-03: 1997Shade Change Grade 4 √Swimwear 50 mg/lBeachwear 20 mg/lCF to Rubbing BS EN ISO105-X12:2002Dry Grade 4Wet Grade 3-4√Not required on solid white or cream fabricsCF to Light BS EN ISO105-B02:1999Shade Change Grade 4 √Required on all fabricsDurability Wash CD 10 No breakdown of the print.Shade Change Grade 4√Women’s Beachwear – only required on printsMen’s swim and beachwear – Required on prints andembellishmentsDurability Wash CD 10a No ChangeShade Change Grade 4√Womens swimwear- Required on all machine washable styles- to be tested as a full garmentNotesBase Testing to be carried out on one colour per fabric quality. Bulk Testing to be carried out on all colours. For additional swimwear information see lingerie care label and composition guidelines document.Table 7 Lining & Pocketing (Knitted & Woven) Test Method RequirementBase Bulk Comments Physical TestsFibre Composition 73/44/EEC & 96/73/ECAS AMENDED BY2006/2/EC± 3%(Single fibre no tolerance)√Required on all fabrics with more than 1 fibre. Singlefibre compositions to be tested unless accompaniedwith documentation from supplierPilling BS EN ISO12945-1:2000Grade 4 @ 18,000 Revs √Only required on fabrics containing Wool, Acrylic, SpunNylon or Polyester.Seam Slippage (Woven only) BS EN ISO13936-1:20046mm @ 8 Kg √For fabrics containing contrast warp/weft use 3mmseam openingSeam Slippage (Stretch Fabrics) (Woven only) BS EN ISO13936-2:20046mm @ 8 Kg √Only required on fabrics containing elastomeric yarns.For fabrics containing contrast warp/weft use 3mmseam openingSeam Strength (Woven only) BS EN ISO13935-2:199912 Kg √Tensile Strength (Woven only) BS EN ISO13534-2:199915 Kg √Tear Strength (Woven only) BS EN ISO13937-1:2000750g (Lining)1200g (Pocketing)√Not required on 100% Nylon or Polyester (except microfibres).Abrasion BS EN ISO12947-2:199930,000 rubsShade Change @ 5,000Grade 4√Only required on woven pocket liningsSnagging BS 8479:2008 Grade 3-4 @ 2,000 Revs √Only required on fabrics containing Filament Yarns.Bursting Strength (Knitted only) BS EN ISO13938: 1999250 kPa175 kPa Lace & Mesh√Only required on fabrics containing <10% elastaneFibre Shedding PD 5a Grade 4 √To be carried out on Fur and Long Pile FabricsTo be carried out on all fabrics/trims with pile length equal to or greater than 3mmStability TestsStability to Washing BS EN ISO 6330: 2001(See Appendix A)± 3% (Woven)+3% / -5% (Knitted)√√Required for Washable garments. Comment on anyadverse changes in appearance.Stability to Dry Clean COMMERCIALPROCESS± 3% (Woven)+3% / -5% (Knitted)√√Required for dry cleanable garments. Comment on anyadverse changes in appearance.Stability to Steam BS 4323:1979 ± 3% √Required for dry cleanable garments Colour Fastness TestsCF to Washing BS EN ISO105-C06:1997 A2SStaining Grade 4Shade Change Grade 4Cross stain grade 4-5√Required for Washable GarmentsCross Staining to be carried out against the outer fabricCF to Water BS EN ISO105-E01:1996Staining Grade 4Shade Change Grade 4Cross stain grade 4-5√Not required on solid white or cream fabricsCross Staining to be carried out against the outer fabricCF to Perspiration BS EN ISO105-E04 1996Staining Grade 4Shade Change Grade 4Cross stain grade 4-5√Required on Fabrics containing > 20% Wool, Nylon orSilk.Cross Staining to be carried out against the outer fabricCF to Dry Clean BS EN ISO105-D01:1995 + MFSStaining Grade 4Shade Change Grade 4Cross stain grade 4-5√Required for Dry Cleanable GarmentsCross Staining to be carried out against the outer fabricCF to Rubbing BS EN ISO105-X12:2002Dry Grade 4Wet Grade 3-4√Not required on solid white or cream fabricsWet Rub only required on garments where outer fabricis tested for wet rub also.Durability Wash CD 10 No breakdown of the print.Shade Change Grade 4√Womens Wear - Only required on Prints, for foil andglitter use PD37 testMenswear – Required on Prints and embellishments。

Cytoskeleton-Associated Large RNP Complexes in Tobacco Male Gametophyte (EPPs)Are Associated with Ribosomes and Are Involved in Protein Synthesis,Processing,and LocalizationDavid Honys,*,†,‡,#David Ren ˇa ´k,†,§,#Jana Fecikova ´,†Petr L.Jedelsky ´,|Jana Nebesa ´r ˇova ´,⊥Petre Dobrev,∇and Ve ˇra C ˇapkova ´†Laboratory of Pollen Biology,Institute of Experimental Botany ASCR,v.v.i.,Rozvojova ´263,16502Prague 6,Czech Republic,Department of Plant Physiology,Faculty of Science,Charles University in Prague,Vinic ˇna ´5,12844Prague 2,Czech Republic,Department of Plant Physiology and Anatomy,Faculty of Biological Sciences,University of South Bohemia,Branis ˇovska ´31,37005C ˇeske ´Bude ˇjovice,Czech Republic,Laboratory of MassSpectrometry,Faculty of Science,Charles University in Prague,Vinic ˇna ´7,12844Prague 2,Czech Republic,Laboratory of Electron Microscopy,Biology Centre of the ASCR,v.v.i.,Branis ˇovska ´31,37005C ˇeske ´Bude ˇjovice,Czech Republic,and Laboratory of Hormonal Regulations in Plants,Institute of ExperimentalBotany ASCR,v.v.i.,Rozvojova ´263,16502Prague 6,Czech RepublicReceived November 14,2008The progamic phase of male gametophyte development involves activation of synthetic and catabolicprocesses required for the rapid growth of the pollen tube.It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation.On the contrary,germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs.Here,we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs)in tobacco male gametophyte.These complexes are formed in immature pollen where they contain translationally silent mRNAs.Although massively activated at the early progamic phase,they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region.Moreover,EPPs were shown to contain ribosomal subunits,rRNAs and a set of mRNAs.Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage,transport and subsequent controlled activation resulting in protein synthesis,processing and precise localization.Such an organization is extremely useful in fast tip-growing pollen tube.There,massive and orchestrated protein synthesis,processing,and transport must take place in accurately localized regions.Moreover,presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too.Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis ,the model species with advanced tricellular pollen.Keywords:Nicotiana tabacum •male gametophyte •pollen •model plant •translation regulation •mRNA storage •cytoskeleton •mRNP •ribonucleoprotein particle1.IntroductionThe functional (progamic)phase of male gametophyte development is initiated upon the pollen hydration on a stigma and involves the activation of processes required for the rapid growth of the pollen tube.To achieve this explosive growth,the pollen grain accumulates and stores large amounts of both RNA and protein reserves.It is a well-documented fact that both transcription and translation play an important role in global and specific gene expression patterns during the pollen maturation.On the contrary,germination of many pollen species has been shown to be largely independent of transcrip-tion but vitally dependent on translation.1,2Translationally inactive mRNAs have frequently been found in association with a number of proteins forming stored messenger ribonucleoprotein particles (stored mRNPs).The accumulation of newly synthesized mRNAs in the form of mRNPs was first described in animal cells and such form of translational regulation of gene expression was later docu-mented in a broad spectrum of cell types,from fish and urchin embryos,3spermatic cells,4oocytes to neuronal cells.5-9Trans-lational regulation in plants has been studied to less extent than in animal systems.Stored mRNP complexes were identified in*To whom correspondence should be addressed:Laboratory of Pollen Biology,Institute of Experimental Botany ASCR,Rozvojova ´263,16502Prague 6,Czech Republic.Tel.:+420225106450.Fax:+420225106456.E-mail:honys@ueb.cas.cz.†Laboratory of Pollen Biology,Institute of Experimental Botany ASCR.‡Department of Plant Physiology,Faculty of Science,Charles University in Prague.§University of South Bohemia.#These authors contributed equally to this work.|Laboratory of Mass Spectrometry,Faculty of Science,Charles University in Prague.⊥Biology Centre of the ASCR.∇Laboratory of Hormonal Regulations in Plants,Institute of Experimental BotanyASCR.10.1021/pr8009897CCC:$40.752009American Chemical SocietyJournal of Proteome Research 2009,8,2015–20312015Published on Web01/30/2009wheat embryos.10In alfalfa,differences in mRNP accumulation were described in zygotic and somatic embryos,11whereas stage-specific mRNPs during embryogenesis were identified by Pramanik et al.12Segregation of seed-storage-protein mRNA was described in rice endosperm cells.13,14The presence of specific nonpolysomal mRNPs has also been reported in heat stressed carrot15and tomato cells16and in water-stressed Tortula ruralis gametophytes.17Moreover,the occurrence of polysomes in cytoskeleton and membrane-bound fractions was intensively studied and the association of ribosomes with cytoskeleton was repeatedly proven.18-21Stored mRNPs were found to be a component of successfully exploited mechanism driving the ontogenetic differentiation of polarized and/or transcriptionally quiescent cells.Moreover, mRNPs are organized into higher-order complexes.22Such complexes often contain various components of translational apparatus,both mRNAs and proteins.5,23Translational control of individual mRNAs facilitates precise regulation of their trafficking,localization,and storage.Their activationfinally requires also exact spatial and temporal expression patterns of regulatory proteins.Mechanisms regulating the complex process of mRNA translational repression and activation within such structures are far from being understood,but RNA-binding proteins and cytoskeleton play a major role in this process.The heterogeneous group of proteins binding to cis-elements within RNA has been recently classified into several families.6Microtubules are often mentioned as a cytoplasmic railway for translocation of many RNPs,while actin seems to be connected with anchoring and translational activation of RNPs.However,its role in RNP translocation has been de-scribed as well.23,24Cytoskeleton-dependent localization signals were detected in3-UTR of several mRNAs.25,26The identifica-tion of proteins associated with both RNP proteins and cytoskeletal structures suggests the association of RNPs and cytoskeleton in a sequence-independent but protein size-and conformation-dependent manner.23,25,27,28Tobacco male gametophyte represents a well-characterized model of translational regulation.Pollen specific gene NTP303 encodes p69,29,30a major pollen tube wall-specific glycoprotein, which is massively synthesized in germinating pollen and growing pollen tubes.1,31On the contrary,ntp303mRNA is intensively synthesized during the pollen maturation32and is stored in the form of EPP complexes.33These mRNP complexes containing translationally silent ntp303mRNAs are resistant to polysomal destabilizing components and co-sediment with polysomes.33Here,we analyzed the size and structure of large ribonucleoprotein particles(EPP)complexes by means of the size exclusion chromatography and electron microscopy.The association of EPPs with cytoskeleton was determined by fluorescence microscopy and Western blot immunodetection. We also present evidence that EPPs comprise whole ribosomal subunits,a number of translationally silent mRNAs supple-mented with a set of various proteins.It documents EPPs’complex role in RNA metabolism and precisely regulated protein synthesis.2.Material and Methods2.1.Plant Material.Tobacco plants(Nicotiana tabacum L. cv.Samsun)were grown in a greenhouse.Young pollen grains were collected at the middle stage of pollen maturation,when the vegetative cell isfilled with cytoplasm and the starch deposition begins.Pollen free of microbial contamination was obtained by surface sterilization of buds on the day of anthe-sis.34The pollen was cultivated under sterile conditions as a shaken suspension at26°C and pollen density of0.5mg/mL in sugar-mineral medium supplemented with MES-KOH buffer pH5.9(SMM-MES)35and casein hydrolysate1mg/mL.Culti-vation intervals were30min,4or24h.2.2.Isolation of EPP Complexes.EDTA/puromycin-resis-tant particles were isolated as described previously.33Briefly, immature pollen from20anthers or100mg of mature pollen grains or pollen tubes was homogenized in sterile HSEP buffer (200mM Tris-HCl,pH9.0,500mM mM KCl,1mM Mg-acetate, 2mM DTT,0.5mM PMSF,1%PTE,50mM EDTA,pH8.0,0.2 mM puromycin,and250mM sucrose)and postmitochondrial supernatant was centrifuged through the30%,40%,50%or60% sucrose cushion(Beckman L-70,NVT90,50000rpm,3h and 20min,4°C)in HSEP gradient buffer(40mM Tris-HCl,pH 8.5,200mM KCl,1mM Mg-acetate,2mM DTT,0.5mM PMSF, 50mM EDTA,pH8.0,and0.2mM puromycin).Pelleted EPP complexes or polysomes were washed with deionized water and subjected to further analyses.Alternatively,we employed size exclusion chromatography described below in order to isolate EPP complexes directly from postmitochondrial supernatant.2.3.Size Exclusion Chromatography.FPLC A¨kta Explorer-900(GE Healthcare Bio-Sciences,Uppsala,Sweden)with computer controlled via UNICORN ver.4.11.software was used with following characteristics:column hardware XK16/70, internal diameter16mm,length70cm,maximal operating pressure4bar(72psi,0.5MPa),maximal volume120mL, maximal sample load volume5-10mL.Superdex200prep grade(GE Healthcare Bio-Sciences,Uppsala,Sweden)separa-tion range10-600kDa and Sephacryl S-400HR(GE Healthcare Bio-Sciences,Uppsala,Sweden)separation range20-8000kDa were equilibrated with sample buffer(40mM Tris-HCl,pH8.5, 200mM KCl,1mM Mg-acetate,2mM DTT,0.5mM PMSF, and50mM EDTA,pH8.0).Running conditions:temperature 10°C,injection1or10mL,flow rate0.200mL/min,UV detection280nm.The chromatographic performance of SEC columns was determined by injecting a mixture of six proteins used as molecular weight markers in the range29-669kDa (Sigma-Aldrich,St.Louis,MO).Good linearity of the calibration curve(r2)0.991)over the tested range was obtained.The calibration curve was used for the calculation of the molecular weight of sample components.Two sample types were applied to the column:(A)fractions of EPP complexes pelleted through the60%,50%,40%or30% sucrose cushion and resuspended in HSEP homogenization buffer or(B)postmitochondrial supernatant including complete EPP fraction.Five milliliters or10mL fractions were collected, dialyzed,lyophilized or desalted,concentrated using Vivapore Q10(Vivascience,Hannover,Germany)m and precipitated with 3vol of ethanol.2.4.RNA and Protein Isolation.Total proteins and RNA were extracted from EPP complexes and polysomes with TRI-Reagent(Sigma-Aldrich,St.Louis,MO)according to the manufacturer’s instructions.Isolated RNA was dissolved in sterile deionized water and its yield and concentration were determined spectrophotometrically.Proteins were dissolved in SDS-PAGE buffer36or2-D sample buffer(8M urea,2%urea, 50mM DTT,2%100×Bio-Lyte;BioRad,Hercules,CA).The yield was determined according to Schaffner et al.372.5.Protein Electrophoresis.Proteins in EPP complexes were separated by2-D PAGE on a Protean IEF Cell(Bio-Rad, Hercules,CA;strip size11cm,ampholyte pH range3-10).The second dimension was performed on a Biometra(Whatman-research articles Honys et al. 2016Journal of Proteome Research•Vol.8,No.4,2009Biometra,Go¨ttingen,Germany)apparatus using12%resolving gel.Protein patterns were visualized by silver staining according to Sammons et al.38For immunodection,EPP proteins were separated using1-D SDS-PAGE and transferred to nitrocellulose membrane(NC45,Serva,Heidelberg,Germany)by blotting. Cytoskeletal proteins were detected using commercially avail-able monoclonal antibodies developed against actin(clone C4, MP Biomedicals,Irvine,CA)and tubulin(TU01,ExBio,Prague, Czech Republic).2.6.RT-PCR.Total RNA was extracted from EPP and poly-somal pellets using the RNeasy plant kit(Qiagen,Valencia,CA) according to the manufacturer’s instructions.The yield and RNA purity were determined spectrophotometrically.Samples of1µg of total RNA were reverse transcribed in a20-µL reaction using the ImProm-II Reverse Transcription System(Promega, Madison,WI)following the manufacturer’s instructions except that the oligo(dT)15primer was accompanied with custom-synthesized18S-R and28S-R primers in order to reverse transcribe18S and28S rRNAs together with mRNAs.For PCR amplification,1µL of50×diluted RT mix was used.The PCR reaction was carried out in25µL with0.5units of Taq DNA polymerase(MBI Fermentas,Vilnius,Lithuania),1.2mM MgCl2, and20pmol of each primer.The PCR program was as follows: 2min at95°C,33cycles of15s at94°C,15s at the optimal annealing temperature(55-58°C),and30s at72°C,followed by10min at72°C.As a negative control,complete reverse transcription reaction without reverse transcriptase was used. All primers(Supplementary Table1in Supporting Information) were designed using Primer3software(http://frodo.wi.mit. edu/).2.7.Electron Microscopy.EPP suspension was dropped on grids with Formvar support layer coated with carbon.Excessive liquid was removed usingfilter paper.The grid was then negative stained with2%uranyl acetate.Electron microscopy was performed with JEOL TEM1011electron microscope using 80kV.2.8.Fluoroscence Analyses of EPPs and Polysomal Complexes.Polysomal and EPP pellets were resuspended in PIPES buffer(50mM PIPES,2mM EGTA,and2mM MgSO4) by vortexing for6h at4°C.The suspension was tested separately for the presence of actin and tubulin using rhodamine-phalloidin and FITC-labeled IgG,respectively.Actin and tubulin were visualized using epifluorescence illumination under a Nikon Eclipse TE2000-E microscope and captured images were processed using Lucia G4.70software(Laboratory Imaging,Prague,Czech Republic,www.lim.cz).For actin detec-tion,100µL of protein suspension was mixed with5µL of rhodamine-phalloidin(Sigma-Aldrich,St.Louis,MO)and the fluorescence was observed after10min incubation.For tubulin detection,100µL of protein suspension was gently centrifuged at2500g for90s,the supernatant was discarded,and the pellet was preblocked with100µL of2%bovine serum albumin(BSA; Sigma-Aldrich,St.Louis,MO)in phosphate buffered saline (PBS:138mM NaCl, 2.7mM KCl,10mM NaH2PO4,and K2HPO4,pH7.4,Sigma-Aldrich,St.Louis,MO)for30min. Protein pellet was further incubated with the primary antibody (anti-R-tubulin,clone B-5-1-2,Sigma-Aldrich,St.Louis,MO) at thefinal dilution of1:1000for2h and washed three times with PBS.A2-h treatment with the secondary antibody(anti-mouse IgG-FITC,Sigma-Aldrich,St.Louis,MO)atfinal dilution of1:128was followed by the last wash with PBS.After each step,the suspension was centrifuged at2500g for90s in a microcentrifuge tube and the supernatant was replaced with the appropriate solution.2.9.Mass Spectrometric Analyses.SDS-PAGE gel was cut to40slices which were covered with100µL of50mM ammonium bicarbonate(ABC)buffer in50%acetonitrile(ACN) with50mM dithiotheritol(DTT)and subjected to sonication in an ultrasonic cleaning bath for5min.After15min,the supernatant was discarded and gel was covered with100µL of 50mM ABC/50%ACN with50mM iodoacetamide and soni-cated for5min and then incubated another25min.Superna-tant was discarded and exchanged with100µL of50mM ABC/ 50%ACN with50mM DTT and sonicated5min to remove any excess iodoacetamide.Supernatant was discarded and samples were sonicated for5min in100µL of HPLC water. Water was discarded and samples were sonicated for another 5min in100µL of ACN.ACN was discarded and samples were let open for several minutes to evaporate the rest of ACN.Five nanograms of trypsin(Promega)in10µL of50mM ABC was added to the gel.Samples were incubated at37°C overnight. TFA and ACN were added to reach afinal concentration of1% TFA and30%ACN to wash out peptides.Eluate was diluted with water(1:2)and subjected to LC-MALDI analyses per-formed using Ultimate3000HPLC system(Dionex)coupled with Probot microfraction collector(Dionex).Tryptic peptides were loaded onto a PepMap100C18RP column(3µm particle size,15cm long,75µm internal diameter;Dionex)and separated with a gradient of5%(v/v)ACN,0.1%(v/v)trifluo-roacetic acid to80%(v/v)ACN,0.1%(v/v)trifluoroacetic acid over a period of45min.The eluate was mixed1:3with matrix solution(20mg/mL R-cyano-4-hydroxycinnamic acid in80% ACN)prior to spotting onto a MALDI target.Spectra were acquired on4800Plus MALDI TOF/TOF analyzer(Applied Biosystems/MDS Sciex)equipped with a Nd:YAG laser(355nm,firing rate200Hz).All spots werefirst measured in MS mode and then up to10strongest precursors were selected for MS/ MS which was performed with1kV collision energy and operating pressure of collision cell set to10-6Torr.Peak lists from the MS/MS spectra were generated by GPS Explorer v.3.6(Applied Biosystems/MDS Sciex)and searched by local Mascot v.2.1(Matrix Science)against nonredundant protein database NCBI(,as of June10th 2008).Database search criteria were as follows:enzyme,trypsin; taxonomy,tobacco(actually searched2049sequences);fixed modification,carbamidomethylation;variable modification, methionine oxidation;peptide mass tolerance,120ppm;al-lowed one missed cleavage;MS/MS tolerance,0.2Da;maxi-mum peptide rank,1;minimum ion score C.I.(peptide),95%(E e0.05for individual MS/MS).3.Results3.1.EPPs Are Present in Growing Pollen Tubes.The presence of EPPs in immature and mature pollen was dem-onstrated previously.33During the progamic phase,EPPs were detectable in activated pollen grains after30min imbibition in the cultivation medium as well as in growing pollen tubes cultivated in vitro for4h and even24h(Figure1).2-D SDS-PAGE gels were loaded with proteins isolated from an equal amount of mature dry pollen(40mg).This documents gradual but significant3-fold decrease in EPP abundance during the pollen tube growth.It dropped from2.4µg of EPP proteins/1 mg pollen in mature pollen(0.24%fresh weight of mature pollen)to0.8µg/1mg pollen in24h-old tubes(0.08%). Moreover,this quantitative decline in EPP abundance was notEPP Characterization in Tobacco Male Gametophyte research articlesJournal of Proteome Research•Vol.8,No.4,20092017accompanied by any visible changes in their protein composi-tion,although after 24h of cultivation only the major protein spots were detectable.However,the qualitative stability of EPP complexes during the whole progamic phase was further documented by comparison of 2-D SDS-PAGE patterns,when an equal amount of 150µg of proteins was applied per sample (data not shown).3.2.Size of EPP Complexes.EPP complexes were further analyzed by Size Exlusion Chromatography (SEC).Pollen grains were homogenized in HSEP buffer,which causes efficient dissociation of polysomes by high salt concentration in com-bination with EDTA and puromycin.Under these conditions,ultracentrifugation of postmitochondrial supernatant through 60%sucrose cushion led to the sedimentation of pure EPP complexes.33To determine the apparent molecular weight of EPPs,Superdex 200prep grade gel with range 10-600kDa was initially used.Fractions were collected across the separation range of SEC column and the presence of EPPs was investigated by UV absorbance at 280nm.EPPs were not retained at all,suggesting that they were of molecular weight over 600kDa.Taking this into account,a second gel type Sephacryl S-400HR with wider separation range (20-8000kDa)was packed into the SEC column.EPPs,however,were eluted in the void volume of this gel too.This indicated that EPPs have molecular weight equal to,or higher than,8MDa (Figure 2).With regard to a possible quantitative correlation between the yield of EPPs and ultracentrifugation conditions,SEC was used to evaluate different isolation protocols.The parameters compared included the ultracentrifugation speed and duration as well as various sucrose concentrations in the cushion.InFigure 1.2D-SDS-PAGE of EPP fraction.EPPs were isolated by HSEP buffer from mature pollen (A)and from pollen tubes cultivated for 30min (B),4h (C)and 24h (D)in SMM-MES medium.Important proteins present in EPPs throughout the whole cultivation period are marked witharrowheads.Figure 2.SEC chromatograms of the EPP fractions isolated from mature pollen grains.Samples applied onto the chromatography column:EPP fraction pelleted though 60%sucrose cushion (A),the whole postmitochondrial supernatant (B)and EPP fraction pelleted though 30%sucrose cushion (C).research articlesHonys et al.2018Journal of Proteome Research •Vol.8,No.4,2009comparison with the original protocol (60%sucrose cushion;Figure 2A),33no changes in the EPP profile and yet a higher yield were obtained after 3h and 20min sedimentation though 30%sucrose under 60000rpm (Figure 2C).Alternatively,SEC was employed for the direct isolation of EPP complexes from postmitochondrial supernatant without presedimentation through sucrose cushion.This modification led to approxi-mately 10times higher increase in yield.Moreover,the purity and homogeneity of mature pollen EPP complexes isolated by various protocols was tested by comparison of their 2-D SDS-PAGE protein profiles.Image analysis (LUCIA G,ver.4.70)showed no qualitative changes in protein patterns of pollen EPPs (Figure 1A,other data not shown)sedimented through 30%and 60%sucrose cushion compared to those obtained directly by SEC.Hence,the procedure of SEC-based direct EPP purification from the postmitochondrial supernatant without ultracentrifugation step was successfully used for further biochemical analyses to increase the yield of EPP fraction (Figure 2B).3.3.EPPs Contain Ribosomal Subunits.When the column chromatography revealed the supramolecular character of EPPs,electron microscopy was applied to provide more information about their size and structure.Samples for electron microscopy were prepared from 4-h pollen tubes,in which the store of EPPs was still sufficiently high (Figure 1C).Ultrastruc-tural study displayed the EPP pellet as a heavy ribonucleopro-tein complex.Its structure contained sets of heterogeneous particles,ranging from 20to 60nm in diameter,rather than uniform components (Supplementary Figure 1in Supporting Information).Considering their size,they were considered as ribosomal subunits.To confirm this hypothesis,presence of both major riboso-mal rRNAs in EPP complexes was verified by RT-PCR.Total RNA isolated from polysomes and SEC-purified EPPs was reverse-transcribed using primers specifically designed against tobacco 18S and 28S rRNAs.After reverse transcription,comple-mentary rDNA fragments corresponding to both 18S and 28S rRNAs were PCR-amplified from polysomal and EPP fractions (Figure 4).To exclude the amplification of genomic rDNA,total RNA extracted from EPPs was used as a negative control in PCR reaction without preceding reverse transcription.The ultimate proof was obtained at the proteomic level.LC-MALDI MS analyses identified 13ribosomal proteins from both small (3proteins)and large (10proteins)subunits (Table 1,Supple-mentary Table 2in Supporting Information,for details see below).3.4.EPPs Are Associated with Cytoskeleton.The cyto-skeleton has repeatedly been found in association with mRNP particlesandvariouscomponentsoftranslationalapparatus.20,21,28This led us to analyze the potential presence of actin and tubulin in EPPs and polysomes.EPPs were isolated in HSEP buffer,whereas polysomal fraction was extracted with LS buffer.33The application of LS buffer preserved polysomal structures but also retained EPP complexes in the polysomal fraction.The proportion of EPPs in polysomal pellet was much smaller than that of EDTA/puromycin-sensitive polysomes.The quantity of proteins comprised in EPPs in 4-h pollen tubes represented about 1/10of the whole polysomal pellet.In the EPP fraction,a weak fluorescence was detected in the stage 3of immature pollen when the formation of EPP complexes is initiated,33but a markedly stronger signal was observed in the mature pollen grains and pollen tubes (Supple-mentary Figure 2A,B in Supporting Information).This finding clearly indicated high levels of actin associated with the EPP fraction in later stages of pollen development.In the polysomal fraction,no signal was observed in the stage 3of immature pollen.Actin signal in polysomal pellet from mature pollen and pollen tubes was stronger and comparable to that in the EPP fraction of appropriate developmental stages.In all experi-ments,images had typical nongranular uniform signal through-out all particles.The distribution of tubulin followed a similar pattern as that of actin (Supplementary Figure 2C,D in Supporting Informa-tion).Tubulin fluorescence in the EPP fraction in immature pollen at the developmental stage 3was slightly higher in comparison to the autofluorescence of a control sample.However,this result still supported positive immunofluores-cence.The strong signal in the EPP fraction of mature pollen grain and pollen tube clearly demonstrated the abundance of tubulin in these stages.On the contrary,the polysomal fraction in immature pollen at the stage 3showed no significant fluorescence.Nevertheless,tubulin was highly detected in polysomal pellets in mature pollen and pollen tubes with signal intensity similar to that of the EPP fraction.To eliminate the effect of autofluorescence,the presence of cytoskeletal proteins was verified by Western blot immunode-tection within proteins isolated from SEC fractions (Figure 5).It revealed a similar pattern of distribution of both cytoskeletal proteins in the EPP and polysomal fractions as did the immunoflpared to polysomes,actin is likely to be a preferable component of EPP complexes in course of EPP formation in immature pollen grains as well as during the pollen tube growth.These results were confirmed and further extended by proteomic analyses that led to the identification of R -tubulin,profilin-3and eight actin clones associated with EPPs (Table 1,Supplementary Table 2in Supporting Informaiton,for details see below).3.5.EPPs Contain Different mRNAs.EPPs were previously shown to contain translationally repressedpollen-specificFigure 3.RT-PCR of mRNAs isolated from EPP complexes revealed broad spectra of mRNAs naturally integrated into EPP complexes.mRNAs encoding NTP303,profiling,actin-depoly-merizing factor 1(ADF1),eukaryotic initiation factor (eIF4E)and its isoform (eIFiso4E)and alpha-tubulin A1(tubA)wereanalyzed.Figure 4.RT-PCR of rRNAs.One microgram of total RNA extracted from EPP complexes (EPP)and polysomes (PS)was reverse transcribed using primers specifically designed against 18S and 28S rRNAs.Negative control reaction was performed without reverse transcriptase (-RT).EPP Characterization in Tobacco Male Gametophyteresearch articlesJournal of Proteome Research •Vol.8,No.4,20092019。